Optical Biosensors: Present and Future [1 ed.] 0444509747, 9780444509741

Citation preview

PREFACE

Since the birth of the field of optical biosensors, the pace of evolution of this field has been swift. While myriad reports have appeared describing applications and advancements in optical biosensor technology, few existing volumes are dedicated to a synopsis of this field. Since the development of optical biosensors mirrors the advances in the rapidly evolving telecommunications industry, we deemed the time to be ripe for such an opus. In order to catch the wave of this rapidly developing technology, we endeavored to focus both on the current state of the art and on technologies that will influence tommorrow's state of the art. We hope that this particular compendium of concepts will trigger new synapses to foma in the brains of our readers and yield even more innovation in the years to come. The history sections are included in order to recognize the contributions of the giants upon whose shoulders we stand--and we thank them for their creativity and pioneering spirit. These sections are comparatively short, not so as to minimize such contributions, but so that this book actually gets published in a single volume. According to the thematic focus on Present and Furore technology, the book is divided into two parts. In the first part, we compiled a list of the most outstanding optical biosensor technologies, while in the second part, the editors used their crystal ball to select the science we deem exciting and promising in terms of potential impact on biosensors. The optical biosensor technologies include two very different fiber optic biosensors, planar waveguides, and the displacement flow sensors, as well as sensors based on time-resolved fluorescence, electrochemiluminescence, surface plasmon resonance, resonant mirrors, and interferometry. The science for future technology development includes four different methods for producing new recognition elements (genetic engineering of proteins, chemical synthesis, combinatorial selection of nucleotide-based receptors, and molecular imprinting), two methods for immobilizing receptors on biosensors (sol gels and semi-synthetic membranes), two methods for producing very bright signals (PEBBLES and quantum dots), and soft lithography for surface patterning and microfluidics. We have asked leaders in each field to provide our readers with as thorough and objective a chapter as possible; they and their colleagues have been very patient with our nagging and nit picking and, as will be obvious to you, have put inordinant amounts of time into providing a conscientious review of their field. We tasked the authors to describe the underlying principles behind each technology, enumerate the types of applications for which it has been tested, provide their opinions about the advantages and disadvantages of their favorite vii

Preface biosensor (and the objectivity each has provided is admirable!), and philosophize on the future developments using that particular biosensor. The last section is intended to be fun for the readers as well as the authors; however, it is available for any clever venture capitalist to peruse as well. Finally, the editors intend this book to be a gift of gratitude to our colleagues in this rapidly expanding field. We appreciate the open sharing of ideas, the encouragement, and the competition that motivates us to greater effort. To work in the field of optical biosensors, one must be curious about biochemistry, chemistry, physics, and engineering and the possibilities ever present in the cracks between the disciplines. While information overload is a serious threat, boredom never is. Since it is absolutely impossible to be expert in all these fields, it behooves us to join forces with those who are. But even more than the ideas and accomplishments of our fellows, we delight in their personalities and camaraderie. Sincerely, Fran and Chris

viii

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 9 2002 Elsevier Science B.V. All fights reserved

CHAPTER 1

OPTRODE-BASED FIBER OPTIC BIOSENSORS (BIO-OPTRODE) ISRAEL BIRAN, PH.D. AND DAVID R. WALT, PH.D.

The Max Tishler Laboratory for Organic Chemistry Department Of Chemistry Tufts University, Medford, MA 02155 USA

Optrode-based fiber optic biosensors (bio-optrodes) are analytical devices incorporating optical fibers and biological recognition molecules. Optical fibers are small and flexible "wires" made out of glass or plastic that can transmit light signals, with minimal loss, over long distances. The light signals are generated by a sensing layer, which is usually composed of biorecognition molecules and dyes, coupled to the fiber end. Light is transmitted through the optical fibers to the sensing layer where different optical phenomena such as absorption or luminescence are used to measure the interactions between the analyte and the sensing layer. Bio-optrodes can be used for remote analytical applications including clinical, environmental, and industrial process monitoring. In the last decade, due to the rapidly growing use of fiber optics for telecommunication applications, new fiber optic technologies have been developed resulting in high-quality and inexpensive optical fibers that can be used for bio-optrode applications. Recent advancements in bio-optrode technologies include the development of nanoscale bio-optrodes, enabling measurements inside single living cells, and the development of multi-analyte and reagentless bio-optrodes. Although currently no biooptrodes are commercially available, it is expected that the development of advanced bio-optrode technologies will lead to commercially available devices for various analytical applications.

Biran and Walt

Figure 1. Schematic diagram of optrode system.

I. Principle of Operation The word "optrode" is a combination of the words "optical" and "electrode" and refers to a fiber optic based analytical device that can measure the concentration of a specific chemical or a group of chemicals in a sample of interest. The basic design of an optrode system is shown in Figure 1. The main components of an optrode are: (a) a light source; (b) an optical fiber to both transmit the light and act as the substrate for (c) the sensing material, which is usually immobilized to the surface of the end face of the fiber; and (d) a detector to measure the output light signal. Computers or microprocessors are used to control the optrode instrumentation and are employed to analyze the output signals. The "heart" of the optrode is the sensing element. When the sensing element interacts with the analyte, it undergoes physico-chemical transformations that change its optical properties. This transduction mechanism generates optical signals that can be correlated to the analyte concentration. The optical signals are measured by launching light from the light source through the optical fiber to the fiber end, where the sensing element is immobilized. The same fiber (Figure 1), or a different fiber (Figure 6), is used to guide the output light to the detector

Optrode-based Fiber Optic Biosensors

Core(nl) Cladding(n2)

Jaclket

Figure 2. Schematic diagram of an optical fiber shows core and clad structure.

(e.g., spectrophotometer, fluorometer) where the reflected, emitted or absorbed light is measured. Optrode biosensors or bio-optrodes are optrodes in which the sensing elements are of biological origin. Biological sensing elements, such as enzymes, nucleic acids, antibodies and cells, are immobilized on optical fibers and used for specific recognition of many different analytes (Cunningham, 1998; Kuswandi et al., 2001; Mehrvar et al., 2000; Wolfbeis, 2000). Since most biological sensing elements and most analytes do not possess intrinsic spectral properties, the biorecognition events are transduced to optical signals (e.g. changes in fluorescence or absorbance) by coupling optically responsive reagents to the sensing elements. For example, fluorescent dyes are used to label nucleic acids and convert the biorecognition interaction between two complementary DNA strands into a fluorescence signal. In another example, an indicator dye, which is optically sensitive to changes in H + concentrations, is used to transduce enzymatic activity that consumes or releases H § into an optical signal. The signals are generated on the fiber optic face and transmitted by the optical fiber to a remote measurement device. The small dimensions of bio-optrodes allow measurement in very small sample volumes, which make them suitable for various clinical applications (Meadows, 1996; Vo-Dinh and Cullum, 2000). Biooptrodes are also useful for different sensing applications in the industrial and environmental fields (Rogers and Mascini, 1998; Rogers and Poziomek, 1996; Marose et al., 1999; Mulchandani and Bassi, 1995; Scheper et al., 1996). In this section, optical fibers, their basic characteristics, and the optical methods used to transduce a biorecognition event to an optical signal are described. The instrumentation employed in optrode biosensors, the biological sensing elements, and the methods to immobilize them on the fiber optic surfaces are summarized.

Biran and Walt

l

n~ L:

n2

'

.

7

[Cladding ( a )

Cladding

(b)

Core

(c) [

"-

-

n~

"

Acceptance cone

Core

Figure 3. Propagation of light through the optical fiber occurs when the total internal reflection condition exists at the interface between the core, (nl), and clad, (n2) such that nl > n2. (a) Light entering the fiber is totally internally reflected (TIR), if the light angle is greater than the critical angle (pc. (b) Light will be partially reflected and partially refracted, if the light angle is less then the critical angle ~oc.(c) Light will propagate in TIR only when the entering light angle is within the acceptance cone angle (~0m)range.

1.1. O p t i c a l f i b e r c h a r a c t e r i s t i c s a n d use in b i o - o p t r o d e s

Optical fibers are small and flexible "wires" made out of glass or plastic that can transmit light signals, with minimal loss, for long distances. Optical fibers are remarkably strong, flexible and durable and therefore can be used in harsh and hazardous environments. Optical fibers are non-electrical, which make them highly suitable for applications where the presence of electric current is detrimental (e.g., in-vivo monitoring inside a patient body). In the last decade, due to the rapidly growing use of fiber optics for telecommunication applications, new fiber optic technologies have been developed resulting in high-quality and inexpensive optical fibers that can also be used for sensing applications. Optical fibers can transmit multiple optical signals simultaneously, thereby offering multiplexing capabilities for sensing.

Optrode-based Fiber Optic Biosensors Optical fibers consist of a core with a refractive index, n~, surrounded by a cladding with a lower refractive index, n2 (Figure 2). The difference in the refractive indices between the core and the cladding enables the core-clad interface to effectively act as a mirror such that a series of internal reflections transmits the light from one end of the fiber to the other as shown in Figure 3 (a). Light undergoes total internal reflection (TIR) at the core-clad interface if two basic conditions are fulfilled: (a) The light strikes the cladding at an angle greater than the critical angle, (Pc,(Figure 3 (a) and 3 (b)). The critical angle is defined by the ratio between the clad and the core refractive indices, as shown in Equation (1): sin (p~ - n 2 / n 1 (1) (b) The angles of the light entering the fiber should be within the acceptance cone as shown in Figure 3 (c). The acceptance cone angle, (am, depends on the refractive indices of the core and the clad and also on the refractive index of the medium from which the light enters the fiber, no.

sin(Pm =

(2) l't o

Another important parameter that defines the fiber's light collection efficiency is the numerical aperture (NA). This parameter is related to the acceptance cone's angle and is given by: NA = n0sinfo m

(3)

A high NA indicates a wide acceptance cone and better light gathering capabilities of the fiber. A typical NA value for a high quality glass fiber is 0.55, but fiber NAs as high as 0.66 or as low as 0.22 have been used for sensing. Optical fibers are usually made out of plastic and glass and have many different configurations, formats, shapes, and sizes. Glass fibers are the most commonly used fibers in optrode biosensors. Glass optical fibers can transmit light in the visible and near-infrared regions of the optical spectrum (400 n m < ~, < 700 nm) and are therefore suitable for measuring fluorescence signals generated by most fluorescent dyes. For applications in which light in the UV region is required, quartz (pure silica) is used as the fiber's core material and doped silica (with a lower refractive index) is used as the cladding material. For most fiber opticbased biosensors, optical fibers with diameters ranging from 50 to 500 ~tm are employed.

Biran and Walt

Figure 4. Optical fiber bundle fabrication and its use for imaging. (a) Fiber bundles are constructed from thousands of individual single fibers that are fused together. (b) Coherent bundles can be used for imaging (Pantano and Walt, 1995). Reprinted with permission from the American Chemical Society.

Recently, fiber optic bundles (Figure 4(a)) comprising thousands of identical single fibers each with a diameter of a few micrometers, were employed for biooptrodes. The fibers can be bundled in a coherent or random fashion. In coherent fiber bundles, the position of each fiber on one end is identical to its position on the other end. These fibers were originally designed for imaging applications as shown in Figure 4(b) and are also often called "optical imaging fibers". Imaging fibers are suitable for multi-analyte optrode biosensor design (Healey and Walt, 1995; Healey et al., 1997a; Michael et al., 1998; Steemers and Walt, 1999; Walt, 2000) since each small individual fiber in the bundle can carry its own light signal from one end of the bundle to the other. Moreover, optical imaging fiberbased biosensors can be used for sensing and imaging simultaneously, providing remote spatial sensing capabilities (Walt, 1998).

1.2. Optical phenomena employed for biosensing in bio-optrodes In bio-optrodes, dyes are coupled to the biological sensing element and transduce the biorecognition events to an optically detectable signal. Different optical 10

Optrode-based Fiber Optic Biosensors phenomena, including fluorescence, luminescence and absorption, are employed for monitoring these optical changes. In this section, the basic principles of these phenomena and their use in bio-optrodes are described.

Fluorescence is commonly used in bio-optrodes. Fluorescence occurs when molecules are excited at a specific wavelength and re-emit radiation at a lower energy, i.e., a longer wavelength. The absorption of the excitation light promotes the molecule's energy from its ground state to a higher energy state. The molecule emits fluorescent light when it returns to the ground state. Each fluorescent molecule has a unique fluorescence spectrum since the excitation and emission occur only at distinct energy levels corresponding to particular wavelengths. The characteristic fluorescence spectrum of particular molecules allows multiple fluorescent dyes to be used simultaneously in a single analytical assay. In fluorescence-based bio-optrodes, the fluorescence signals are measured by transmitting the excitation light through an optical fiber and measuring the light emission using a detector. Usually the increase or decrease in fluorescence intensity is measured and then correlated to the analyte concentration. For example, when a fluorescent-labeled antibody is used as the sensing element, the fluorescence intensity is proportional to the amount of antigen (analyte) bound to the optical fiber. One method for measuring fluorescence lifetime is frequencydomain. In this method, sinusoidally modulated light is used to excite the fluorescent molecule. The resulting emission light also oscillates at the same frequency. The emission light is phase shifted (delayed) and demodulated with respect to the excitation light because of the finite lifetime of fluorescence. The phase shift is expressed as a phase angle from which the lifetime can be determined using simple relationships between the modulation frequency and the degree of demodulation. The concentration of analyte that induces changes in the molecule's fluorescence lifetime can be determined by measuring phase angle values (Thompson et al., 1996). A decrease in fluorescence intensity due to quenching can also used for sensing. In this case, the biorecognition event causes a decrease in fluorescence (quenching) of the fluorescent reporter molecule. The fluorescence decrease is related to the analyte concentration. For example, a dye that undergoes fluorescence quenching when the pH decreases can be coupled to an enzymatic reaction that converts a substrate into an acidic product and results in a pH drop. Thus, the decrease in fluorescence can be correlated to the analyte concentration (see also Section 1.4.1). Fluorescence quenching is also one manifestation of another fluorescence phenomenon used for sensing in bio-optrodes -fluorescence resonance energy transfer (FRET). This phenomenon occurs when two distinct fluorophores are present. If the emission spectrum of one fluorophore overlaps with the excitation spectrum of a second fluorophore, and the two fluorophores are in proximity (I1:

CA ] / - N 6 7 C - ABD ( T )

EXC.430nm, 9

.

8

rn n~

~2 z w bz w

o

z w U m

o

0,.O_o \\

x~,

~

w 2 0 _J tl.

,

4

I

-13

I

-12

!

-11

,O..r

!

-10

I

, I

I

I

-9

-8

-7

-6

"

,I

-5

LOG [ M = * ] , M O L A R

Figure 4. Fluorescence intensities of ABD-t-labeled N67C-apo carbonic anhydrase as a function of free Cu(II) (open diamonds), Zn(II) (filled diamonds), Cd(II) (open circles), Ni(II) (triangles), and Co(II) (filled circles) concentration. the fluorophore to deexcite the latter. Occasionally, the analyte will have chemical features which can be exploited for quenching-based sensors: for instance, the iodine atoms in the hormone triiodothyronine are efficient quenchers by the so-called heavy atom effect. In general, the fluorophore will be chosen to maximize the efficiency of the particular quenching process. Thus, the efficiency of quenching by electron transfer depends critically on the standard oxidation potential of the fluorophore with respect to the reducing/oxidizing species (Rehrn and Weller, 1970). If the analyte itself is unlikely to be a good quencher, it may be possible to have it compete for binding to the receptor with a modified form of the analyte which is. Among quenching mechanisms, the most useful for lifetime biosensing is Ft~rster transfer, which is a dipolar coupling of excited donor and acceptor molecules if they have a good match (overlap) between the donor emission and the acceptor absorption (Forster, 1948). Like other dipole couplings, the efficiency of the transfer increases as the inverse sixth power of the distance; the propensity of a given donor to transfer to a given acceptor is thus a function of distance, spectral

148

Fluorescence Lifetime Biosensors

3.2i

~ 2_...8

39 5 0 M H z

5


(.z3 z

LLJ

T ==4.5

ns

f'-

MHz

60

_

,

I

O

,,.

I

TIME ~.~ "~ 2 ~" • TO= m

=

m

,

.

.

.

I

-

30

NANOSECONDS

( MODULATION

(-l'~n .d ~

.

20

10

FREQUENCY

) --- 2 ~ ' f

)I~

Err'fISSION

/ rrt E X C I T A T I O N

~

'

A I C 'B / C -

9 I

TO--.rm "r e < ' r "r

FOR m

SHOULD

MONOEXPONENTIAL

FOR

MULTIEXPONENTIA APPROXIMATELY

L EQUAL

1/~,~

Figure 6. Concept of frequency domain lifetime measurement. The excitation (solid line) is sinusoidally modulated at some frequency in the megahertz range; the emission is phase shifted and demodulated to a degree determined by the lifetime or lifetimes of the sample.

In other cases, binding of the analyte to the receptor does not result in quenching of a fluorophore at all, but rather an increase in intensity and (sometimes) lifetime. Thus some of the saccharide biosensors based on bacterial periplasmic binding proteins exhibit substantial increases in intensity (Li and Cass, 1991), and likely, lifetime. One of the fluorescent-labeled carbonic anhydrase variants (apo-N67C-ABD) also displays a nine-fold increase in intensity upon zinc binding (Thompson et al., 1999), but a fifty percent increase in fluorescence lifetime, which is quite usable for sensing purposes. The disparity between intensity increase and lifetime increase upon zinc binding indicates the presence of a static quenching component when the protein is in the apo-form that is somehow negated when zinc binds. Certain fluorophores such as ANS display enhanced emission intensity and lifetime in hydrophobic solvents and in putatively hydrophobic environments when bound to proteins (Slavik, 1982), but reliably coupling the effect to binding of an analyte is difficult. 1.2. Measurement of lifetimes A precondition for a lifetime-based sensor is obviously the ability to measure the lifetime, or some parameter related to the time-dependent fluorescence decay. While a thorough review of lifetime measurement is beyond the scope of this chapter, suffice it to say that there are two main approaches: time and frequency 150

Fluorescence Lifetime Biosensors

I- - - " - - /

-

9~

#J ~" 1 i

0

/ 213o r ~ I 50

"

I

~: -~.--~::

-.

PERCENT "~ ..-..x~,, -,,, t.sns 4.ons '~-',,'~,",

x\ x x

100

70 50

s;~ ULATEO

1 ~'0 /

| x

~'~-

0.8

,,:,,. ',/.,~~

7

',

'd,','~,////

1

" \"

,

'

5

\

="

_

~:

',, , "

g - 0.2

0

0.0

1

to

Ioo

FREQUENCY, MHz

Figure 7. Frequency-dependent phase shifts (solid lines) and modulations (dashed lines) for 4.0 nsec mono-exponential decay (curve 1), 1.5 nsec (curve 5), and mixtures thereof (curves 2, 3, and 4). domain; only the latter is widely used in lifetime biosensing. The time domain measurement embodies determining the time-dependence of the fluorescence emission following a brief flash of excitation, as illustrated in Figure 1. While many time domain methods have been described, the most popular and by far the most accurate and sensitive is time-correlated single photon counting (TCSPC) (Birch and Imhof, 1991). TCSPC has found little favor in sensing because, unless pulsed diode lasers or extremely expensive mode-locked lasers are used for excitation, acquisition of a single decay curve can take hours. By comparison, frequency domain instruments (phase fluorometers) provide rapid readout of two lifetime-related parameters, the phase and modulation. In frequency-domain fluorometry, the sample is excited with light whose amplitude is sinusoidally modulated at a frequency close to the reciprocal of the lifetime: typically, about 1 - 300 MHz. The fluorescence emission is perforce also modulated at the same frequency, but it is phase-delayed and demodulated to a degree that (for a single exponential) is a simple function of the lifetime (Figure 6). If the phase and modulation are measured over a hundred-fold range of modulation frequencies and plotted as a function of the logarithm of the frequency, one obtains a data set like those depicted in Figure 7. The curves in Figure 7 are depicted for a 1.5 nsec monoexponential decay, a 4 nsec monoexponential decay, and three different mixtures of the 1.5 and 4 nsec decays, corresponding to partial saturation of the transducer binding site. The phase angles increase monotonically from zero with frequency, asymptotically 151

Thompson

FRACTION 1.0 7O

0.8

1.5nsec 0.6

0.4

COMPONENT 0.2

0.0 0.9

.' ..... ;.......

70MHz

! 0.8

tO Ld t.d LM C3

J_J

t.9

50

_

"-,,

-J

0.7

tn ,


D + hv

Oxalate is often referred to as an "oxidative-reductive" coreactant due to its ability to form a strong reducing agent upon electrochemical oxidation. Unlike annihilation schemes where a double potential step (e.g., oxidation followed by reduction) is required to generate the highly energetic precursors, in coreactant ECL the electrode typically only oxidizes or reduces the reagents in a single 181

Richter potential step. For example, in the oxalate system the electrode oxidizes both the oxalate and the ECL reactant D; the reducant, CO2"- is then generated upon bond cleavage of oxalate via Equation (27). This strategy is used in most analytical and biotechnology applications, with the reactant D being Ru(bpy)32+. This methodology has allowed the generation of ECL in aqueous solution, a great advantage in terms of analytical applications. Without this ability, it is doubtful whether ECL would have moved beyond the laboratory phase. Another example of an "oxidative-reductive" system is the commercially important Ru(bpy)32+/TPrA system (TPrA = tri-n-propylamine). As with the oxalate system, this involves the production of a strong reductant (presumably TPrA ~ by an initial oxidation sequence (Leland and Powell, 1991; McCord and Bard, 1991). Ru(bpy)32+- e

~

TPrA - e ---) [TPrA'] +

Ru(bpy)33+

(31)

--~ T P r A ' + H §

Ru(bpy)33+ + TPrA" ---) Ru(bpy)32+* +

products

Ru(bpy)32+" ---) Ru(bpy)32+ + hv

(32) (33) (34)

ECL is produced upon concomitant oxidation of Ru(bpy)32§ and TPrA (Figure 2). Electrochemical studies of various aliphatic amines have indicated a possible reaction pathway for the oxidation of TPrA (Smith and Mann, 1969). Upon oxidation, the short lived TPrA radical cation (TPrA ~ is believed to lose a proton from an c~-carbon to form the strongly reducing intermediate TPrA ~ This radical can then reduce Ru(bpy)33§ to Ru(bpy)32+*. Other reaction mechanisms for production of the excited state have also been proposed. For example, reduction of Ru(bpy)32§ to Ru(bpy)31§ by TPrA', followed by annihilation: Ru(bpy)31+ + Ru(bpy)33+

~ Ru(bpy)32+ + Ru(bpy)32+*

(35)

Although the details of the coreactant ECL mechanism (Equations 31 - 34) to generate light emission are still under study (Zu and Bard, 2000; Kanoufi et al., 2001) the origin of the light emission from Ru(bpy)32+ has been well documented (Glass and Faulkner, 1981; Faulkner and Glass, 1982; Leland and Powell, 1991; McCord and Bard, 1991). Since the photoluminescent and ECL spectra are nearly identical, the emission process in ECL involves the MLCT state of Ru(bpy)32+. This state may be formed if the reducing agent (i.e., TPrA ~ transfers an electron to the rt* orbital of one of the bipyridine ligands. Ru(bpy)32+* can then decay to the ground state, producing the same luminescence as obtained from photoluminescence spectroscopy. Solution phase co-reactant ECL using TPrA

182

Electrochemiluminescence and Ru(bpy)32§ is quite sensitive, with sub-picomolar detection limits achieved (Leland and Powell, 1991; Blackburn et al., 1991). Other systems use coreactants that are reduced to generate reactive species (i.e., "reductive-oxidative" coreactants). For example, in the case of peroxydisulfate (SZO82-), reduction produces the strong oxidant SO4~ that then undergoes an electron-transfer reaction with an ECL luminophore like Ru(bpy)3 z+ to generate light (White and Bard, 1982; Bolletta et al., 1981) as shown below. Ru(bpy)32+ + e ----~Ru(bpy)3 +

(36)

SzO82- + e ----~ SO4" + SO4z

(37)

Ru(bpy)3 + + SO4" ---~ Ru(bpy)32+* + SO4z-

(38)

As well of being of practical interest, ECL reactions of this type also demonstrate the intermediacy of species such as TPrA ~ and CO2". However, the mechanisms of these reactions are still not well understood.

2. History The first detailed studies on ECL were begun in the mid-1960s but interest in light emitted during electrolysis was generated much earlier. In 1927 Dufford and co-workers (Dufford et al., 1927) observed emission at an anode by applying between 500 and 1500 V to a cathode in a solution of Grignard compounds in anhydrous ether. The reaction conditions in these experiments were not very well defined and it is doubtful whether this process was actually ECL. At such high potentials, electroluminescence (the direct injection and removal of charge with the formation of an electron-hole pair), or electrode processes were probably responsible for the observed ligh t emission. However, this initial report was followed two years later when Harvey published experiments on luminol (2,3aminophthalhydrazide) (Figure 1) in aqueous/alkaline solution (Harvey, 1929). The potentials used to generate anodic light emission were much lower than those used by Dufford and coworkers (2.8 V vs 500-1500 V, respectively). Several groups followed up on this (Bemanose et al., 1947; Vojir, 1954; Kuwana et al., 1963), and in fact the luminol system continues to generate interest (Vitt et al., 1991; Haapakka, 1982; Haapakka and Kankare, 1980; van Dyke and Cheng, 1989). Practical applications of the luminol system are hampered by a number of factors, most notably the high non-specific ECL (i.e., background) - possibly due to the formation of oxygen at the anode in aqueous solution, followed by chemiluminescent reactions involving o x y g e n - and the extremely basic conditions (pH > 11) that are needed to generate sufficient light emission.

183

Richter In the mid 1960s several research groups (Hercules, 1964; Chandross and Visco, 1964; Santhanam and Bard, 1965; Bader and Kuwana, 1965) decided to study in detail the luminescence generated during electrolysis of polyaromatic hydrocarbons (e.g., anthracene, diphenylanthracene, thianthrene, rubrene) in aprotic media, Figure 1. In essence, they wished to see if excited states could be generated electrochemically as well as photochemically. It was observed, both visually and spectroscopically, that the radiation emitted by sweeping to both negative and positive potentials (annihilation pathway, Equations 1 - 4 ) was often identical to that generated during photoluminescence, indicating formation of the excited singlet state. Throughout the sixties and seventies work continued on the polyaromatic hydrocarbons, and was eventually extended to other systems, most notable among them the ruthenium chelates. Since the discovery that Ru(bpy)32§ is photoluminescent (Paris and Brandt, 1959), a large body of literature has appeared aimed at understanding both the ground and excited state properties of Ru(bpy)32§ Os(bpy)32§ and their polyazine derivatives (Demas and Crosby, 1971; Sutin and Creutz, 1978; Meyer, 1978; Barigelletti et al, 1991; Roundhill, 1994). Therefore, it is not surprising that these compounds have also played an important role in the development of ECL. The first report of ECL in a metal chelate was in 1972 (Tokel and Bard, 1972), in which the excited state of Ru(bpy)32§ was generated in aprotic media by annihilation of the reduced, Ru(bpy)3 l§ and oxidized, Ru(bpy)33§ species (Equations 11 - 14). The original coreactant, and thus the first report of ECL in aqueous solution, was oxalate ion (C2042; equations 27 - 29) (Rubinstein and Bard, 1981). Subsequently, other species were shown to act as coreactants, among them peroxydisulfate ($208~; Equations 3 6 - 38) and tri-n-propylamine (TPrA; Equations 31 - 34). The discovery of TPrA (Leland and Powell, 1991) allowed efficient ECL not only in aqueous media, but also at physiological pH. Following the first report on TPrA, other species containing amine groups were proposed, among them many biologically important analytes (e.g., alkylamines, NADH, antibiotics, L,D-tryptophan, glucose, erthromycin, valine, HIV- gag gene). The list is quite extensive, and compilations up to 1998 have been published (Knight and Greenway, 1994; Knight, 1999). To date, ECL has found use in studying the properties of both organic and inorganic systems (Faulkner and Bard, 1977; Knight and Greenway, 1994). These include polyaromatic hydrocarbons (Faulkner and Bard, 1977; Faulkner and Glass, 1982; Richards and Bard, 1995), exciplexes (Hemingway, Park and

184

Electrochemiluminescence ......

2+

O

~~

Ru

X Figure 3. Ru(bpy)32§ NHS ester for ECL labeling of proteins and nucleic acids.

Bard, 1975; Prieto et al., 2001), polymer assemblies (Rubinstein et al, 1983; Downey and Niemann, 1992; Richter et al., 1994), transition metal complexes incorporating such metals as Ru, Os and Pt (Faulkner and Glass, 1982; Knight and Greenway, 1994; Tokel and Bard, 1972; Vogler and Kunkeley, 1984; Kim et al., 1985; Richter et al., 1998) as well as rare earth chelates (Hemingway et al., 1975; Richter and Bard, 1996), to name a few. Ru(bpy)32§ is perhaps the most thoroughly studied ECL active molecule (Tokel-Takvoryan et al., 1973; Wallace and Bard, 1979; Itoh and Honda, 1979; Luttmer and Bard, 1981; Glass and Faulkner, 1981) and, as with other ECL systems, there was particular emphasis on characterizing the nature of the excited state, discerning the mechanisms by which these states were formed and determining the efficiency of excited state formation. Various techniques were used and are still being used, including detailed electrochemical studies, spectroscopic and spin-resonancemeasurements as well as magnetic field effects (Faulkner and Bard, 1977, 2001; Faulkner and Glass, 1982). In the early 1980s Bard and Whitesides (Bard and Whitesides, 1993; Bard and Whitesides, 1994) developed a method for the binding of Ru(bpy)32§ to biological molecules of interest (e.g., antibodies, proteins, nucleic acids). The interest in using Ru(bpy)32§ stems from its rather unique properties. Namely, it emits and is soluble at room temperature in aqueous, fluid, solution, and undergoes reversible one-electron transfer reactions at easily attainable potentials. Also, the ligands provide synthetic versatility. For example, by attaching N-hydroxysuccinimide (NHS) ester to one of the bipyridine ligands (Figure 3), the ECL label can bind to 185

Richter substances containing free amino groups. The amino acid will attack the carboxylate ester, leading to displacement of N-hydroxysuccinimide. IGEN International, Inc. began developing ECL for use in biosensor analyses in the early 1980s. In the early 1990s, the prototype ORIGEN | Analyzer was introduced by IGEN International, Inc. The ORIGEN is an ECL-based immunoassay system, incorporating Ru(bpy)32+-tagged antibodies and is engineered to be a biomedical research tool for immunoassays and DNA probes. The ORIGEN instrument is semi-automated, and incorporates a flow injection system to allow rapid and reproducible determinations of single samples. The detector is a photomultiplier tube positioned above the working electrode, and light from the electrode is recorded and integrated for each measurement. Roche Diagnostics licensed the ECL-Ru(bpy)32§ technology from IGEN International, Inc. in 1992, and subsequently produced the first fully automated instruments (Elecsys | 1010 and 2010), built upon the same flow cell design as its predecessor. The Etecsys systems were launched in Europe in 1996 for use in clinical and reference laboratories (those handling large volumes of samples), and in the U.S. starting in January of 1997 following FDA approval. Assays that have been developed for these systems include alpha-fetoprotein, digoxin, thyrotopin, protein and steroidal hormones, cytokines, and various antibodies, to name a few.

3. State of the Art 3.1. Analytical applications of ECL Coreactant ECL has been used in a wide range of analytical applications (Knight and Greenway, 1994; Bard et al., 2000). Since ECL emission intensity is usually proportional to the concentration of the emitter (Cruser and Bard, 1967) or coreactant (Leland and Powell, 1991), ECL can be used in the analysis of various species. For example, the system of interest is introduced into an electrochemical cell, a voltage is applied to an electrode and the light intensity and/or ECL spectrum is measured. ECL in such systems is very sensitive since photoncounting methods can be used to measure very low light levels. For example, ECL from Ru(bpy)32§ has been used to measure the concentrations of coreactants such as oxalate and peroxydisulfate to levels as low as 10-13 M (M = mol L 1) (Ege et al., 1984). In fact, the ability of Ru(bpy)32+ to undergo "oxidativereductive" ECL in the presence of coreactants has led to the selective determination of oxalate in synthetic urine samples (Rubinstein et al., 1983), and Ru(bpz)32+ (where bpz = bipyrazine) has been used for the determination of peroxydisulfate with nanomolar (nM) detection limits (Yamashita et al., 1991). Since the intensity of ECL is a function of both the coreactant and the emitter, ECL can be used to analyze for both. In these examples, ECL was measured in the presence of high, pre-determined concentrations of ECL emitters. These 186

Electrochemiluminescence types of experiments can then be used as a means to assay for compounds that act as coreactants including a variety of amines (Noffsinger and Danielson, 1987; Knight and Greenway, 1994; Bard et al., 2000). ECL assays for amines find many applications since amine groups are prevalent in numerous biologically and pharmacologically important compounds including alkyl-amines, antibiotics, antihistamines, opiates, nicotinamide, and the reduced form of NADH (i.e., adenine dinucleotide) (Danielson et al., 1989; Knight and Greenway, 1994; Knight and Greenway, 1996). In general, these compounds contain no chromophore and therefore cannot undergo luminescence unless an ECL-active compound such as Ru(bpy)32§ is present. As a general rule, the ECL signal from alkylamine coreactants follows the order: 3~176 1~ (Leland and Powell, 1991). Primary amines have been detected using Ru(bpy)3 2§ coreactant ECL after prior derivatization with divinylsulfone (CH2-CH-SO2-CH=CH2). The primary amines undergo a cycloaddition reaction resulting in the formation of acyclic tertiary amines (Uchikura et al., 1993) that then act as efficient coreactants. Other examples of cyclic amines that undergo ECL include nictone, atropine and sparteine (Uchikura and Kirisawa, 1991). It is also possible to quantitatively measure amino acids, peptides and proteins such as proline and valine. In fact, detection limits of 20 pM for proline (He et al., 1990) and 30 pM for valine (Brune and Bobbitt, 1991) using flow injection techniques have been achieved. Although the ability of numerous amines to act as coreactants makes ECL a versatile technique for their detection it also makes selectivity for the presence of a specific amine problematic. More recently, Xu and Dong have obtained high selectivity for the measurement of chlorpromazine, a commonly prescribed dopamine inhibitor. Using Ru(bpy)32+ as the ECL luminophore and chlorpromazine as an oxidative-reductive coreactant, selectivity was achieved by preconcentration of the chlorpromazine at a lauric acid-modified carbon paste electrode with a detection limit of 3.1 x 10.9 M (Xu and Dong, 2000). ECL has also been used to monitor enzymatic reactions. In such systems, the reaction is often coupled to the generation or consumption of an ECL coreactant. A good example is the coenzyme nicotinamide adenine dinucleotide (NADH). NADH contains an amine moiety that acts as a coreactant for Ru(bpy)32§ However, the oxidized form (NAD+) is not a coreactant (Downey and Nieman, 1992). Since numerous NADH-dependent enzymes are known, this allows for the detection of a variety of analytes including glucose (Jameison et al., 1996). Another application of ECL is the detection of ~-lactamase activity (Liang et al., 1996). Pencillin and its derivatives do not act as coreactants with Ru(bpy)3 z§ to produce ECL. However, 13-1actamase catalyzed hydrolysis of pencillin forms a molecule with a secondary amine that can act as a coreactant. The efficiency of the ECL process has been increased by covalent attachment of a ~-lactamase substrate to a Ru(bpy)32§ derivative (Liang et al., 1996). The ECL of aminopeptidase and esterase cleavage products have also been reported by covalently attaching such species as ligands to bis(bipyridine)ruthenium (II). 187

Richter (bpy)zRu 2+ has little to no intrinsic ECL, but attachment of a third ligand leads to enhanced ECL (Dong and Martin, 1996). Similar methods have been used in detector cells for high-performance-liquidchromatography (HPLC). These also involve the ECL of Ru(bpy)3 z+ for the detection of species that act as coreactants, such as amino acids, amines, and NADH (Jackson and Bobbitt, 1994). One technique that has acheived picomole detection limits uses post-column ECL detection. A solution of Ru(bpy)32+ is steadily injected into the solution stream containing separated species coming from the HPLC column. The mixed stream flows into an electrochemical flow cell where the ECL reaction occurs and emission can be measured (Holeman and Danielson, 1994). Ru(bpy)32+ can also be immobilized in a thin film of polymer (e.g., Nation) deposited on the working electrode (Rubinstein and Bard, 1980). This eliminates the need for a constant stream of Ru(bpy)32+. In this technique, ECL results when a species that can act as a coreactant is in the solution coming from the HPLC column and reacts with the immobilized Ru(bpy)32+ in the detector cell (Downey and Nieman, 1992). ECL in flowing streams has also provided information about the hydrodynamics in the detector cell (Schultz et al., 1996). In the methods described above ECL was measured in the presence of high, predetermined concentrations of ECL emitters. ECL can also be used to analyze an emitting species (eg., Ru(bpy)32+) that often serves as a label on a molecule of interest. The coreactant, typically TPrA, is present in high concentrations so the amount of luminescence depends on the concentration of the ECL emitter present in the assay. Since the emitters are bound to the analyte of interest, the amount of luminescence can be correlated with the concentration of the analyte. The most frequently used ECL-active label is Ru(bpy)32+ for reasons discussed in preceding sections. Also, the emission is intense, fairly stable and the emission intensity is proportional tc concentration over several orders of magnitude (e.g., 10 -7 to 1013M) (Ege et al., 1984; Leland and Powell, 1991). By attachment of a suitable group to the bipyridine moieties (Figure 3), Ru(bpy)32+ can be linked to biologically interesting molecules, such as antibodies or DNA, were it serves as a label for analysis in an analogous manner to radioactive or fluorescent labels (Bard and Whitesides, 1993; Blackburn et al., 1991). The most common and, arguably, the most important commercial application to date for ECL is its use in diagnostic assays. These applications typically use

188

Electrochemiluminescence

Figure 4. Representation of ECL "Sandwich" (antibody-antigen/analyte-antibody) assay.

ECL emitters as labels in affinity binding assays that attach the ECL emitter to the analyte of interest (Blackburn et al., 1991). The label is physically linked to one of the binding partners in the assay and provides the means for detecting the coupling of the binding partner to the analyte. Several classes of binding partners 189

Richter are used including antibody/antigen, enzyme/inhibitor, carbohydrate/lectin, and nucleic acid/complementary nucleic acid (Wild, 1994). Commercial instruments are available for ECL assays of antibodies, antigens, and DNA (Blackburn et al., 1991; Yang et al., 1994; Hoyle, 1994) and are currently based on the use of magnetic bead technology. The use of magnetic beads for immunomagnetic separations are well known and has been thoroughly reviewed elsewhere (Uhlen et al., 1994; Olsvik et al., 1994; Safarikova and Forsythe, 1995; Bruno, 1998a). In the context of ECL, the use of magnetic beads allows for the separation of the analyte and ECL label onto a solid support (i.e., the bead) followed by collection of the labeled beads on an electrode surface. Most magnetic beads used in ECL systems are paramagnetic (i.e., magnetic only in the presence of an external magnetic field) and consist of a core of magnetite (Fe304) surrounded by a polystyrene shell. These micron-sized particles may be purchased (e.g., Dynal Corp., Lake Success,NY) with preconjugated streptavidin or a variety of surface immobilization chemistries including amines, hydrazides and long chain alkyl linkers, to name a few. Since the "sandwich assay" format is often used for ECL affinity binding assays our discussion will center on it. The principles of a typical sandwich assay for an antigen are outlined in Figure 4. Magnetic beads modified by attaching an antibody for a particular antigen of interest (e.g., prostrate specific antigen, PSA), the sample of interest, and Ru(bpy)32+ - labeled antibodies are mixed. If the antigen of interest is present it acts as a bridge to form the "sandwich" structure, and the antibody labeled with ECL luminophore becomes attached to the magnetic bead. If no antigen is present, the labeled antibody does not attach to the bead. These labeled and unlabeled beads are then flushed into an ECL flow cell, where they are captured on the working electrode by positioning a magnet beneath the working electrode. The beads are washed to remove any unattached Ru(bpy)32§ - labeled antibodies as well as other reaction components, and a solution of the appropriate composition containing a coreactant (usually TPrA) is pumped into the cell. The concentration of the coreactant is kept constant and high (e.g., > 50 mM) to maximize the sensitivity of the detection and to prevent fluctuations in concentration of the coreactant from changing the ECL. The electrode is then swept to positive potentials to initiate ECL and the intensity of the emitted light is measured with a photomultiplier tube. In these measurements, the number of ECL labels on the solid phase is directly proportional to the concentration of analyte. The magnetic beads are then washed from the cell, which is cleaned and made ready for the next sample. Currently, more than forty assays, including those for tumor and cardiac markers, analytes relevant to infectious diseases, fertility therapies, and thyroid diseases, are commercially available (Bard et al., 2000).

190

Electrochemiluminescence 2+

Ho~O~i/O~

,,~

T

N

Figure 5. Ru(bpy)32§ - phosphoramadite linker for ECL labeling of nucleic acids.

ECL coupled with magnetic bead separation has also been used to develop assays for a variety of biotoxoids that are important for both food industry and military applications. For example, assays for bovine leutenizing hormone show greater sensitivity with ECL than standard radioimmunoassays (Deaver, 1995). Also, several authors have reported extremely sensitive ECL assays for bacterial species in a variety of matrices. Species such as such as anthrax (Bacillus anthracis) (Gatto-Menking et al., 1995; Bruno and Yu, 1996; Bruno and Kiel, 1999), Escherichia coli O157 and Salmonella typhimurium (Yu and Bruno, 1996; Yu, 1996) have been reported with limits of detection and assay sensitivity equal to or greater than conventional assays using flow cytometry, enzyme linked immunosorbant assays (ELISA) and radioallergosorbent test (RAST). Most ECL assays reported in the literature have used an N-hydroxysuccinimide (NHS) ester linked Ru(bpy)32§ conjugate ("TAG NHS Ester" available from IGEN International Inc., Figure 3) or a Ru(bpy)32+ phosphoramidite - conjugate (Figure 5)) to label analytes. Using these labels, the ECL of HW 1 gag gene has been reported with detection limits of < 10 to 30 gene copies (Blackburn et al., 1991; Kenten et al., 1992). Coupling ECL with polymerase chain reaction (PCR) amplification has lowered the detection limit of HIV 1 gag DNA to less than five copies (Schutzbank and Smith, 1995). Other assays and applications incorporating both PCR and ECL for nucleic acid based analyses have been 191

Richter reported (Stem, H.J et al., 1995; Heroux and Szczepanik, 1995; Wilkinson et al., 1995; Motmans et al., 1996; Gudibande et al., 1992; Van Gemen et al., 1994) including the quantitation of Varicella zoster DNA in whole blood, plasma and serum (De Jong et al., 2000) and the detection of viable oocysts of Cryptosporidium parvum (Baeumner et al., 2001). This will undoubtedly continue to be an area of intense research activity. Although Ru(bpy )3 2+ is the most widely used ECL luminophore, assays have also been developed for a range of antioxidants that quench anthracene sensitized ECL upon electrolysis of sodium citrate, methanol and dissolved oxygen (Chmura and Slawinski, 1994). Indole and tryptophan have also been shown to generate ECL upon electrolysis in the presence of hydrogen peroxide, with detection limits of 0.1/,tM for both indole and tryptophan (Chen et al., 1997) and sensors have been reported for a range of alcohols and saccharides since hydroxyl compounds have been shown to generate ECL directly (Egashira et al., 1996). Diaminotoluene isomers form weakly electrochemiluminescent compounds in the presence of Au § and Cu 2§ ions (Bruno and Cornette, 1997). Since aminoaromatic compounds like diaminotoluene are often associated with the degradation of explosives such as TNT, this approach may find use in military applications. Surprisingly, ECL can also be obtained from blood extracted from tunicates ("sea-squirts"), a class of marine invertebrates (Bruno et al., 1997). Although the exact source of the low-level ECL is not known, it is believed to emanate from metal-ion tunichrome complexes since synthetic analogues of the tunichrome chromophore showed a 10 fold ECL enhancement when complexed to Hg 2+. 3.2. Instrumentation

Experiments focused on annihilation ECL of radical ions are carried out in fairly conventional electrochemical apparatus. However, cells, electrodes and experimental procedures must be modified to allow electrogeneration of two reactants, rather than one, while taking into account constraints imposed by optical measurement equipment and the exclusion of stray light (i.e., "light-tight" experiments). In addition, one must pay scrupulous attention to the purity of the s01vent/supporting electrolyte system, especially with organic systems (e.g., polyaromatic hydrocarbons). Water and oxygen are particularly harmful to these experiments since they can quench ECL. Thus, cells and electrodes are constructed to allow transfer of solvent and degassing on high-vacuum lines or in inert-atmosphere ("glove") boxes. Electrochemical apparatus for coreactant ECL are, in many instances, identical to those used in annihilation ECL (Knight and Greenway, 1994). However, the 192

Electrochemiluminescence constraints of working with nonaqueous systems (e.g., vacuum lines) are alleviated. The earliest experiments were carried out in electrochemical batch cells designed to fit into optical spectrophotometer chambers (Rubinstein and Bard, 1981). As the development of coreactant ECL for use in diagnostics and for flow injection and liquid chromatographic applications increased, many ECL flow cell configurations were developed (Hill et al., 1986; Downey and Nieman, 1992; Sakura and Imai, 1988) . Electrode configurations, cells incorporating them and experimental details for both annihilation and coreactant systems have been thoroughly reviewed (Knight and Greenway, 1994; Faulkner and Bard, 1977; Bard and Faulkner, 1980). The ORIGEN TM analyzer (IGEN International, Inc) was the first commercial instrument that used ECL (Carter and Bard, 1990; Blackburn et al., 1991; Kenten et al., 1991; Kenten et al., 1992). This analyzer provides highly sensitive and precise assays in an automated format. It employs a flow injection system that allows rapid and reproducible deter~nations of sequential samples. The detector is a photomultiplier tube positioned directly above the working electrode so that light from the electrode can be recorded and integrated during each measurement. Typically, the assays use magnetic beads as a solid support and Ru(bpy)32+frPrA as the label and coreactant, respectively. However, the instrument may also be operated without the use of beads or other solid supports. A personal computer controls the instrument and aids in the processing and storage of data. The sample and assay reagents are combined in plastic tubes and agitated in a carousel to mix the sample and reagents and allow assay binding reactions to go to completion. The sample solution containing magnetic beads is then flushed automatically into an ECL cell, where they are captured on the working electrode by applying a magnetic field. The beads are washed, a solution containing coreactant is pumped into the cell and ECL is induced. Following light measurement, the beads are washed from the cell and the cell cleaned in preparation for the next measurement. A typical read and clean cycle requires approximately 1 minute. Roche Diagnostics, a licensee of IGEN Intemational, Inc.'s technology, has developed the ELECSYS TM instrument for conducting immunoassays in centralized hospital and reference laboratories. The fundamental technology and operation of this highly automated instrument is similar to the ORIGEN TM analyzer. The instrument can operate in random access mode and has the capability to produce Short Turn .Around Time (STAT) samples. Several other instrument configurations have been developed (Rozhitskii, 1992) including one that incorporates an RIU)E for both electrochemical and ECL analyses. ECL instrumentation has also followed the trend that has developed in the past decade towards smaller cells and electrodes (i.e., microfabrication). For example, an ECL system approximately 1/20 th the size of the original ORIGEN TM instrument has been developed (the TRICORDER | detection system, IGEN International, Inc.). This system is self-contained and has an accuracy and 193

Richter sensitivity equal to the ORIGEN TM analyzer. A miniaturized ECL cell has also been developed based on the ECL of Ru(bpy)3 ~§ for the determination of peptides having proline at the amino terminal (Egashira et al., 2000). Several other cells and devices have also been reported. For example, a thin layer flow cell using a planar optical waveguide coated with an indium-tin oxide (ITO) layer was developed. This ITO layer was then modified by covalently attaching glucose oxidase, and ECL measured from luminol at the end of the waveguide at the cell's edge (Kremeskotter et al., 1995). The generation of Ru(bpy)32§ ECL on interdigitated gold microelectrodes mounted above a photodiode has been observed, with a limit of detection for Ru(bpy)32§ of 0.5 txM (Fiaccabrino et al., 1998a). An ECL cell with gold and optically transparent indium tin oxide coated glass electrodes incorporating a photodiode has also been fabricated (Hsueh et al., 1996) for use in the quantification of DNA labeled with Ru(bpy)32+. Reported limits of detection were 1 nM, with a cell volume of 85 ktL. Cell volumes as low as 100 nanoliters have also been reported (Arora et al., 1997) with Ru(bpy)32+ ECL (detection limit 5 x 10"13 M) using a cell composed of a sandwich of poly(methylmethacrylate) layers containing two platinum thin film electrodes, connected to a conventional flowing systems using photomultiplier tube detection. The ultimate goal of much of this work is the development of miniaturized analytical systems, or the so called "lab on a chip" technology (Haswell, S.J., 1997). Fundamental studies aimed at the development of sensors and probes have also been reported. An especially active area has been the development of fibre optic probes for ECL analyses (van Dyke and Cheng, 1989; Kuhn et al., 1990; Egashira et al., 1992). For example, a gold-coated fibre optic probe for the measurement of Ru(bpy)32§ in the presence of peroxydisulfate has been developed (Kuhn et al., 1990). Also, a miniaturized fibre optic sensor has been developed and applied to the determination of oxalate using Ru(bpy)32+ (Egashira et al., 1990) in real urine samples with a limit of detection of 3 x 10.5 M. The ultimate goal of many of these studies is the development of portable devices for use in point-of-care clinical analyses (e.g., in hospital examining rooms) (Bard et al., 2000) and environmental applications (Bruno, 1998a).

4. Advantages and Limitations As with other measurements based on the emission of light (e.g., photoluminescence, chemiluminescence), ECL labels have distinct advantages over detection methods such as radioactivity. For example, they are sensitive, nonhazardous, inexpensive, diagnostic of the presence of a particular label, linear over a wide range and incorporate simple and relatively inexpensive equipment. When compared to such light emission techniques as photoluminescence (PL) and chemiluminescence (CL), ECL also displays certain desirable qualities. In PL, excited state formation occurs upon absorption of electromagnetic radiation: 194

Electrochemiluminescence R + hv --> *R

(39)

The versatility of this technique lies in the number of species able to luminesce, the quantum efficiency of emission, and the ability to incorporate these molecules into a wide variety of formats. Unfortunately, this versatility also leads to limitations. For example, in clinical situations, typical biological fluids containing analyte may also contain a large number of potential luminophores. In ECL, for a complex to emit it must meet several stringent criteria, including stable redox chemistry and the ability to undergo energetic electron or energy transfer. Of course, this advantage of ECL is also a potential limitation, in that the number of efficient ECL labels is diminished. CL involves the generation of excited states due to an energetic chemical reaction. In a typical CL reaction, reagents are pumped separately to the reaction site. In ECL, on the other hand, production of reagents occurs electrochemically in-situ from passive precursors, allowing spatial and temporal localization of the emission near the electrode. This results in enhanced sensitivity since the optics used for light detection can be focused on a relatively small area. Furthermore, amplification is possible in ECL due to the turnover of reactants at or near the electrode surface resulting in sub-picomol detection limits and a linear dynamic range of greater than six orders of magnitude. Ru(bpy)32+ - labels are also extremely stable and can be stored for over 1 year at room temperature in the dark. Often, the small size of the ECL luminophore allows multiple labels to be attached to the same molecule without affecting the stability, immunoreactivity or hybridization of the probes. However, as with any electrochemical process, stringent cleaning of the electrodes is required prior to and after each run to ensure reproducibility.

5. Potential for Expanding Current Capabilities Solution phase co-reactant ECL is quite sensitive, with sub-picomolar detection limits achieved (Leland and Powell, 1991). When the ECL luminophore is bound to a magnetic particle (the particle can then be captured on the surface of the electrode prior to electrochemical stimulation), detection limits as low as 1018M are attainable (Blackburn et al., 1991; Kenten et al., 1991). However, there are many systems where even greater sensitivity is needed, such as in environmental (where pre-concentration of samples is often necessary) and molecular diagnostics applications, where the detection of as few as 10 molecules would eliminate the need for sample amplification (e.g., via the polymerase chain reaction). One approach has been to vary the properties of the ECL luminophore. For example, Ru(bpy)32+ has an ECL efficiency of 0.050 (Rubinstein and Bard, 1981b; Glass and Faulkner, 1981), or ~ 5 % of the Ru(bpy)32+ molecules that undergo electron transfer generate emission. With the goal of increasing the magnitude of ECL emission, and therefore increasing ECL sensitivity and lowering detection limits, the ECL of the bimetallic ruthenium system

195

Richter [(bpy)zRu]z(bphb) 4+ (bphb = 1,4-bis(4'-methyl-2,2'-bipyridin-4-yl)benzene) was studied (Richter et al., 1998). The ligand bphb is capable of binding two independent metal centers through a "bridging ligand" framework. This bimetallic species produced more intense emission ( 2 - 3 fold) than Ru(bpy)32+ in aqueous and nonaqueous solution using annihilation and coreactant methods. A key point to this study was that for enhanced ECL to be possible in multimetallic assemblies, there must be small electronic coupling between metal centers via the bridging ligand so that the metal centers are electronically isolated or "valence trapped" (Robin and Day class I Systems; Robin and Day, 1967). This work has recently been extended to dendrimeric systems containing eight Ru(bpy)3 z+ units at the periphery (Zhou and Roovers, 2001) of a carbosilane dendrimer platform. Preliminary experiments indicated that the ECL of the Ru(bpy)3 z+ dendrimer is five times that of the reference monometallic species. As with the bimetallic study, spectroscopic and electrochemical studies show that the Ru(bpy)32+ units don't interact in either the ground or excited state showing that ECL (and photoluminescence) emission can be amplified by using multimetallic species. Multimetallic compounds such as these show much promise for use in analytical applications. However, it has yet to be shown whether these types of labels will change nucleic acid hybridization or affinity binding of antigens and antibodies in diagnostic applications. Another approach to improving ECL sensitivity that has met with limited success has been to vary the nature of the coreactant. Numerous amine-based coreactants have been studied, including primary, secondary and tertiary systems (Leland and Powell, 1991), and attempts made to understand the electron donating and withdrawing properties that might lead to optimum coreactant efficiency (Knight and Greenway, 1996). To date TPrA still provides the optimum ECL in the Ru(bpy)32+ system (Leland and Powell, 1991; McCord and Bard, 1991). Yet another approach is to add a species to the solution that will facilitate excited state formation and/or lead to increased quantum yields for emission. For example, the ECL intensity of Ru(bpy)32+ increased slightly (< 5%) in the presence of benzene (Dixon et al., 1993). The reason for the increase is unclear, but the excited states of ruthenium and osmium polypyridyl systems are sensitive to the nature of the environment and are able to detect subtle changes in solution composition (Meyer, 1978). The presence of benzene may lead to decreased interactions between the hydrophobic luminophore and the solvent media, resulting in increased ECL efficiency. Although Ru(bpy)32+ has many properties that make it an ideal ECL luminophore for sensitive and selective analytical methods, it would be useful to have other ECL labels that span a wide range of wavelengths so that simultaneous determination of several analytes in a single sample is possible. For example, Ru(bpy)32+ has a broad emission spectrum stretching from about 500 to 700 nm (Lr~x " 620 nm), and this can be a disadvantage in applications where an ECL internal standard or multianalyte determinations are desired. The ECL of a series of europium chelates, cryptates and mixed-ligand chelate/cryptate complexes were studied (Richter and Bard, 1996) since many trivalent 196

Electrochemiluminescence lanthanides display high photoluminescence efficiencies, large Stokes' shifts (-300 nm) and narrow emission spectra (Crosby et al., 1961; Sinha, 1971). ECL appears to occur by a different mechanism than transition metal systems via a "ligand-sensitization" route, where ECL occurs in the organic ligands with subsequent transfer to the f-orbitals of the metal centers. Although it was clear from this work (Richter and Bard, 1996) that the ligands play an integral role in rare earth ECL, very low ECL efficiencies were observed in nonaqueous solvents with little to no ECL observed in aqueous media. ECL in aqueous solution has been observed for the polyaromatic hydrocarbons 9,10-diphenylanthracene-2sulfonate (DPAS) and 1- and 2-thianthrenecarboxylic acid using TPrA as a coreactant and for DPAS using peroxydisulfate as a reductive-oxidative coreactant (Equation 37) (Richards and Bard, 1995). These complexes emit in the blue and green regions of the spectrum (e.g., ~,max(DPAS) = 430 nm) making them attractive as complementary labels to Ru(bpy)32+. ECL is not only limited to the visible region of the spectrum. Recently, the first report of near-infrared electrochemiluminescence was described (Lee et al., 1997) for a heptamethine cyanine in acetonitrile using coreactants. Undoubtedly, work will continue in these areas to find new molecules and coreactants to improve and expand ECL past its current capabilities. Solubilization of Ru(bpy)32§ in aqueous nonionic surfactant solutions leads to significant, and potentially useful, changes in the electrochemiluminescence (ECL) properties (McCord and Bard, 1991; Workman and Richter, 2000). For example, increases in both ECL efficiency (>8-fold) and duration of the ECL signal were observed in surfactant media upon oxidation of Ru(bpy)32§ and TPrA (Workman and Richter, 2000). However, the mechanism of the surfactant effect is still unclear. The effect of surfactants on Ru(bpy)32+/TPrA (Workman and Richter, 2000; Zu and Bard, 2001) and other ECL systems (e.g., Ru(dp-bpy)32§ and Ru(dp-phen)32§ (dp-bpy = 4,4'-biphenyl-2,2'-bipyridyl and dp-phen = 4,7diphenyl-l,10-phenanthroline) (McCord and Bard, 1991), Os(bpy)32§ (Ouyang and Bard, 1988), and the heptamethine cyanine dye IR-144 (C56H73NsOsS2)) (Lee et al., 1998) were attributed to strong hydrophobic interactions between the ECL luminophore and micellized surfactant. However, recent work (Zu and Bard, 2001) on the effects of electrode hydrophobicity on ECL indicate that adsorption of Triton X-100 (polyoxyethylene(10) isooctylphenyl ether) on Pt and Au electrodes renders the surface more hydrophobic, facilitating coreactant oxidation and leading to increased ECL intensities in the Ru(bpy)32+/TPrA system. A recent study of the effects of nonionic chain lengths on Ru(bpy)32+ / TPrA ECL (Factor et al., 2001) confirm these results. Although the effects of micelles and discrete complexation of the surfactants with Ru(bpy)32+ and TPrA cannot be ruled out, these studies indicate that increases in ECL intensity are probably due to changes in electrode hydrophobicity upon formation of a surfactant adsorption layer and less likely due to micelle interactions (Zu and Bard, 2001; Factor et al., 2001). The precise mechanism of the surfactant effect is still under study, but the dramatic increases in ECL intensity, coupled with work 197

Richter on more efficient ECL labels and coreactants may have profound impacts on the sensitivity of ECL for a variety of applications. Ultrasonic enhancement of ECL has also been investigated. Ultrasonic irradiation on coreactant and annihilation ECL of Ru(bpy)32§ in aqueous oxalate solutions and in acetonitrile solutions, respectively, increases the electrochemiluminescence yield over 100%, results in highly stable and reproducible ECL signals and leads to less electrode fouling (Walton et al., 1992; Malins et al., 1997). The dramatic increases appear to be due to agitation of the system, leading to greater mass transport across the electrode double layer and less dependence on diffusion to get material to the electrode surface. Also, it is speculated that the degassing effects of sonication reduce the aggregation of gas bubbles at the electrode surface and prevent the formation of passivating films. The mechanisms of ECL under sonification are the same as conventional conditions, but the high reproducibility of the signal has allowed the measurement of ECL quenching via oxygen to be measured with greater precision than previously possible (Malins et al., 1997). Environmental applications for ECL are also being explored. For example, the increased ECL emission of Ru(bpy)32+ in the presence of benezene has led to the proposal of using it to detect aromatic hydrocarbon pollutants (Dixon et al., 1993). Other environmental applications include the detection of toxic metal ions (Bruno, 1998b; Taverna et al., 1998), metal ions bonded to aminoaromatics (Bruno and Cornette, 1997), and environmentally important ethoxylate surfactants containing amine groups (Alexander and Richter, 1999). Quenching of Ru(bpy)32+/TPrA electrochemiluminescence in the presence of phenols, hydroquinones, catechols and benzoquinones (McCall et al., 1999; McCall and Richter, 2000) has opened up the possibility of,using ECL to detect these environmentally, biologically and pharmacologically important classes of compounds. The generation of ECL at micro- and ultramicroelectrodes has been known for some time (Collinson and Wightman, 1993; Maness and Wightman, 1995). In fact, an ultramicroelectrode was used to observe individual reaction events of DPA annihilation ECL in nonaqueous solution (Collinson and Wightman, 1995) showing the extreme sensitivity possible with ECL. ECL with microelectrodes has recently been coupled to Scanning Probe techniques such as Scanning Electrochemical Microscopy (SECM) to image surfaces (Fan et al., 1998; Maus et al., 1999). High frequency ECL has also been used to image the surfaces of microelectrodes (Wightman et al., 1998). More recently, ECL has been used as a light source for near-field scanning optical microscopy using ultramicroelectrodes with effective diameters from 1 ~m to less than 100 nm (Zu et al., 2001). This technique was used to image an interdigitated array with resolution comparable to that observed via near-field scanning optical microscopy (NSOM). Using ECL for near-field imaging appears to have several 198

Electrochemiluminescence advantages over NSOM. In NSOM, a metal-coated fibre optic probe is used and this leads to fundamental resolution limits due to the finite skin depth of the metal coating. In ECL, tip preparation is easier since standard techniques can be used to generate nanometer sized electrodes (Zu et al., 2001). In addition, ECL does not require a laser so there is no heating of samples and tip from absorption of light on metal coatings. Therefore, this approach looks promising for nearfield optics in solution. As mentioned above, many recently developed methods of ECL analysis include chromatographic or other separation steps and the use of Ru(bpy)32+ ECL in HPLC has been reviewed (Lee, 1997). More recently, ECL has been coupled with capillary electrophoresis (CE) (Dickson et al., 1997; Forbes et al., 1997; Tsukagoshi et al., 1997; Bobbitt and Jackson, 1997; Gillman et al., 1994). Since biochemical applications of ECL have rapidly expanded in the past decade, coupling ECL with CE should provide rapid, efficient and versatile methods of separating and detecting biochemicals using extremely small sample volumes. Annihilation ECL of Ru(bpy)32+ has recently been reported in aqueous solution containing no electrolyte (Fiaccabrinno et al., 1998b). This is possible using a microfabricated interdigitated carbon dual-electrode system. Each electrode is biased to form the reduced, Ru(bpy)3 +, or oxidized, Ru(bpy)33+, species. The electrodes are in close enough proximity (2 lxm width and spacing) that the simultaneously produced reactants can diffuse together and undergo annihilation (Equation (13)). Also, carbon is used as the electrode material to prevent formation of water oxidation and reduction products (e.g., oxygen) that tend to quench ECL emission. Annihilation ECL has been used to study aluminum quinolate/triarylamine and related organic complexes used as light emitting diodes (Gross et al., 2000; Anderson et al., 1998), sol-gel composites containing Ru(bpy)3 z+ (Sykora and Meyer, 1999), and diode-like chemiluminescence in frozen concentrations gradients of the ruthenium polymer poly-[Ru(vbpy)3](PF6)2 (Maness et al., 1996). ECL in sol-gel derived glasses (Collinson et al., 2000), nation-silica composite films (Khramov and Collinson, 2000) and gel-entrapped Ru(bpy)32+ (Collinson et al., 1999) using coreactants has also been observed with the potential for using both coreacant and annihilation ECL in display device technology. Although commercial applications for these systems have yet to materialize, they have opened up fascinating areas for both fundamental and applied studies. This chapter has centered on the background and history of ECL, and its development into a biomedical research and clinical diagnostic tool. However, ECL is a versatile detection methodology and is being developed as a sensor and probe for other applications. Since the first detailed studies, over 1000 papers, patents and book chapters have appeared on ECL, ranging from the very applied to a focus on the underlying science. With the interest in using ECL reactions as the basis for highly sensitive and selective analysis, the prediction made by 199

Richter Faulkner and Glass that "Continued research in this area will probably stress the development of ECL as a probe rather than as an end in itself' (Faulkner and Glass, 1982) has come to fruition. One wonders what the next 30 years hold for ECL but whatever the improvements and new aspects of ECL that emerge, ECL will continue to show promise for optical biosensing and in other areas of science and technology.

6. References

Alexander, C. and M.M.Richter, 1999, Anal. Chim. Acta 402, 105. Anderson, J.D., E.M. McDonald, P.A. Lee, M.L. Anderson, E.L., Ritchie, H.K. Hall, T. Hopkins, E.A. Mash, J. Wang, A. Padias, S. Thayumanavan, S. Barlow, S.R. Marder, G.E. Jabbour, S. Shaheen, B. Kippelen, N. Peyghambarian, R.M. Wightman and N. R. Armstrong, 1998, J. Am. Chem. Soc. 120, 9646. Arora, A., A.J. DeMello and A. Manz, 1997, Anal. Commun. 34, 393. Bader, J.M. and T. Kuwana, 1965, J. Electroanal. Chem. 10, 104. Baeummer, A.J., M.C. Humiston, R.A. Montagna and R.A. Durst, 2001, Anal. Chem. 73, 1176. Bard, A.J., J.D. Debad, J.K. Leland, G.B. Sigal, J.L. Wilbur and J.N. Wohlstadter, 2000, Encyclopedia of Analytical Chemistry, Ed. R.A. Meyers, Wiley, Chichester, pp 9842-9849. Bard, A.J. and L.R. Faulkner, 1980, Electrochemical Methods, Wiley, New York, pp 624-626. Bard, A.J. and L.R. Faulkner, 2001, Electrochemical Methods Fundamentals and Applications Second Edition, Wiley, New York, pp 736 - 745. Bard, A.J. and C.M.Whitesides, 1993, U.S. Patent 5,21,605. Bard, A.J. and C.M. Whitesides, 1993, U.S. Patent 5,238,808. Bard, A.J. and G.M. Whitesides, 1994, U.S. Patent 5,310,687. Barigelletti, F., L. De Cola, V. Balzani, R. Hage, J.G. Haasnoot, J. Reedijk and J.G. Vos, 1991, Inorg. Chem. 30, 641. Bartelt, J.E., S.M. Drew and R.M. Wightman, 1992, J. Electrochem. Soc. 139, 70. Bernanose, A., T. Bremer and P. Goldfinger, 1947, Bull. Soc. Chim. Belg., 56, 269. B irks, J.B., 1970, Photophysics of Aromatic Molecules, Wiley, New York. Blackburn, G.F., H.P. Shah, J.H. Kenten, J. Leland, R.A. Kamin, J. Link, J. Pterman, M.J. Powell, A. Shah, D.B. Talley, S.K. Tyagi, E. Wilkins, T-G Wu and R.J. Massey, 1991, Clin. Chem. 37, 1626. Bobbit, D.R. and W.A. Jackson, 1997, US Patent 5,614,073. Bolletta, F., A. Rossi and V. Balzani, 1981, Inorg. Chim. Acta 53, L23-L24. Brilmyer, G.H. and A.J. Bard, 1980, J. Electrochem. Soc. 127, 104. Brune, S.N. and D.R. Bobbitt, 1991, Talanta 38, 803.

200

Electrochemiluminescence Bruno, J.G., 1998a, Recent Research Developments in Microbiology. Vol. 1, Ed. S.G. Pandalai, Trivandrum Publishing, India, pp. 25-46. Bruno, J.G., 1998b, J. Biolumin. Chemilumin. 13, 139. Bruno, J.G., S.B. Collard and A.R.J. Andrews, 1997, J. Biolumin. Chemilumin. 12, 155. Bruno, J.G., S.B. Collard, D.J. Kuch and J.C. Comette, 1996, J. Biolumin. Chemilum 11,193. Bruno, J.G. and J.C. Comette, 1997, Microchem. J. 56, 305. Bruno J.G. and J.L. Kiel, 1999, Biosens. Bioelectron. 14, 457. Bruno, J.G. and H. Yu, 1996, Appl. Environ. Microbiol. 62, 3474. Carter, M. and A.J. Bard, 1990, Bioconj. Chem. 1,257. Caspar, J.V., T.J. Meyer, 1983, J. Am. Chem. Soc. 105, 5583. Chandross, E.A. and R.E. Visco, 1964, J. Am. Chem. Soc. 86, 5350. Chen, G.N., R.E. Lin, Z.F. Zhao, J.P Duan and L. Zhang, 1997, Anal. Chim. Acta 341, 251. Chmura, J. and J. Slawinski, 1994, J. Biolum. Chemilumin. 9, 1. Collinson, M.M., B. Novak, S.A. Martin and J.S. Taussig, 2000, Anal. Chem. 72, 2914. Collinson, M.M., J.S. Taussig and S.A. Martin, 1999, Chem. Mater. 11, 2594. Collinson, M.M. and R.M. Wightman, 1993, Anal. Chem. 65, 2576. Collinson, M.M. and R.M. Wightman, 1995, Science 268, 1883. Crosby, G.A., R.E. Whan and R.M. Allire, 1961, J. Chem. Phys. 34, 743. Cruser, S.A. and A.J. Bard, 1967, Anal. Lett. 1, 1I. Danielson, N.D., L. He, J.B. Noffsinger and L. Trelli, 1989, J. Pharm. Biomed. Anal. 7, 1281. Deaver, D.R., 1995, Nature 377, 758. De Jong, M.D., J.F.L. Weel, T. Schuurman, P.M.E. Wertheim-van Dillen and R. Boom, 2000, J. Clin. Microbiol. 38, 2568. Demas, J.N. and G.A. Crosby, 1968, J. Mol. Spectrosc. 26, 72. Demas, J.N. and G.A. Crosby, 1971, J. Am. Chem. Soc. 93, 2841. Dickson, J.A., M.M. Ferris and R.E. Milofsky, 1997, J. High Resolut. Chromatogr. 20, 643. Dixon, S.B., J. Sanford and B.W. Swift, 1993, Principles and Practices for Petroleum Contaminated Soils, Eds. E.J. Calabrese and P.T. Kosteki, Lewis Publishers, pp 85-99. Dong, L. and M.T. Martin, 1996, Anal. BioChem. 236, 344. Downey, T.-M. and T.A. Nieman, 1992, Anal. Chem. 64, 261. Dufford, R.T., D. Nightingale and L.W. Gaddum, 1927, J. Am. Chem. Soc. 49, 1858. Egashira, N., N. Kondoh, Y. Kurauchi and K. Ohga, 1992, Denki Kagaku 60, 1148. Egashira, N., H. Kumasako and K. Ohga, 1990, Anal. Sci. 6, 903. Egashira, N., Y. Nabeyama, Y. Kurauchi and K. Ohga, 1996, Anal. Sci. 12, 793. Egashira, N., J. Piao, E. Hifumi and T. Uda, 2000, Bunseki Kag 49, 1029. Ege, D., W.G. Becker and A.J. Bard, 1984, Anal. Chem. 56, 2413. 201

Richter

Factor, B., G. Muegge, S. Workman, E. Bolton, J. Bos and M.M. Richter, 2001, Anal. Chem. submitted. Fan, F.-R.F., D. Cliffel and A.J. Bard, 1998, Anal. Chem. 70, 2941. Faulkner, L.R. and A.J. Bard, 1977, Electroanalytical Chemistry Vol. 10, Ed. A.J. Bard, Marcel Dekker, New York, pp 1-95. Faulkner, L.R. and R.S. Glass, 1982, Chemical and Biological Generation of Excited States; Eds. A. Waldemar, and C. Giuseppe, Academic Press, New York, ch. 6. Fiaccabrino, G.C., N.F. de Rooij and M. Koudelka-Hep, 1998, Anal. Chim. Acta 359, 263. Fioccabrino, G.C., M. Koudelka-Hep, Y-T Hsueh, S.D. Collins and R.L. Smith, 1998, Anal. Chem. 70, 4157. Forbes, G.A., T.A. Nieman and J.V. Sweedler, 1997, Anal. Chim. Acta, 347, 289. Gatto-Menking, D.L., H. Yu, J.G. Bruno, M.T. Goode, M. Miller, and A.W. Zulich, 1995, Biosens. Bioelectron. 10, 501. Gilman, S.D., C.E. Silverman and A.G. Ewing, 1994, J. Microcolumn Sep., 6, 97. Glass, R.S. and L.R. Faulkner, 1981, J. Phys. Chem. 85, 1160. Gross, E.M., J.D. Anderson, A.F. Slaterbeck, S. Thayumanavan, S. Barlow, Y. Zhang, S.R. Marder, H.K. Hall, M. Flore Nabor, J.-F. Wang, J.-F., E.A. Mash, N.R. Armstrong and R.M. Wightman, 2000, J. Am. Chem. Soc. 122, 4972. Gudibande, S., J.H. Kenten, J. Link, K. Friedman and R.J. Massey, 1992, J. Molec. Cell. Probes 6, 495. Haapakka, K.E., 1982, Anal. Chim. Acta 139, 229. Haapakka, K.E. and J.J. Kankare, 1980, Anal. Chim. Acta 118, 333. Harvey, N., 1929, J. Phys. Chem. 33, 1456. Haswell, S.J., 1997, Analyst 122, R 1. He, L., K.A. Cox and N.D. Danielson, 1990, Anal. Lett. 23, 195. Hemingway, R.E., S.-M. Park and A.J. Bard, 1975, J. Am. Chem. Soc. 95,200. Hercules, D.M., 1964, Science 143, 808. Heroux, J.A. and A.M. Szczepanik, 1995, PCR Methods Appl. 4, 327. Hill, E., E. Humphreys and D.J. Malcolme-Lawes, 1986, J. Chromatogr. 370, 427. Holeman, J.A. and N.D. Danielson, 1994, J. Chromatogr. 679, 277. Hoyle, N.R., 1994, J. B iolumin. Chemilumin. 9, 289. Hsueh, Y.T., R.L. Smith and M.A. Northrup, 1996, Sens. Actuators B Chem. 33, 110. Itoh, I. and K. Honda, 1979, Chem. Lett. 99. Jackson, W.A. and D.R. Bobbitt, 1994, Anal. Chim. Acta. 285,309. Jameison, F., R.I. Sanchez, L. Dory, J.K. Leland, D. Yost and M.T. Martin, 1996, Anal. Chem. 68, 1298. Kanoufi, F., Y. Zu and A.J. Bard, 2001, J. Phys. Chem. B. 105, 210. 202

Electrochemiluminescence Kenten, J.H., J. Casadei, J. Link, S. Lupold, J. Willey, M.J. Powell, A. Rees and R.J. Massey, 1991, Clin. Chem. 37, 1626. Kenten, J.H., S. Gudibande, J. Link, J.J. Wiley, B. Curfman, E.O Major and R.J. Massey, 1992, Clin. Chem. 38, 873. Khramov, A.N. and M.M. Collinson., 2000, Anal. Chem. 72, 2943. Kim, J., F.-R.F. Fan, A.J. Bard, C.-M. Che and H.B. Gray, 1985, Chem. Phys. Lett. 121,543. Knight, A.W., 1999, Trends in Anal. Chem. 18, 47. Knight, A.W. and G.M. Greenway, 1994, Analyst 119, 879. Knight, A.W. and G.M. Greenway, 1996, Analyst 121,101R. Kremeskotter, J., R. Wilson, D.J. Schiffrin, B.J. Luff and J.S. Wilkinson, 1995, Meas. Sci. Technol. 6, 1325. Kuhn, L.S., A. Weer and S.G. Weber, 1990, Anal. Chem. 62, 1631. Kuwana, T., B. Epstein and E.T. Seo, 1963, J. Phys. Chem. 67, 2243. Leland, J.K. and M.J. Powell, 1991, J. Electroanal. Chem. 318, 91. Lee, S.K., M.M. Richter, L. Strekowski and A.J. Bard, 1997, Anal. Chem. 69, 4126. Lee, W.Y., 1997, Mikrochim. Acta 127, 19. Liang, P., L. Dong and M.T. Martin, 1996, J. Am. Chem. Soc. 118, 9198. Luttmer, J.D. and A.J. Bard, 1981, J. Phys. Chem. 85, 1155. Malins, C., R. Vandeloise, D. Walton and E. VanderDonckt, 1997, J. Phys. Chem. 101, 5063. Maloy, J.T., K.B. Prater, K.B. and A.J. Bard, 1971, J. Am. Chem. Soc. 93, 5959. Maloy, J.T. and A.J. Bard, 1971, J. Am. Chem. Soc. 93, 5968. Maness, K.M., R.H. Terrill, T.J. Meyer, R.W. Murray and R.M. Wightman, 1996, J. Am. Chem. Soc. 118, 10609. Maness, K.M. and R.M. Wightman, 1995, J. Electroanal. Chem. 396, 85. Maus, R.G., E.M. McDonald and R.M. Wightman, 1999, Anal. Chem. 71, 4944. McCall, J., C. Alexander and M.M. Richter, 1999, Anal. Chem. 71, 2523. McCall, J. and M.M. Richter, 2000, Analyst 125,545. McCord, P.M. and A.J. Bard, 1991, J. Electroanal. Chem. 318, 91. Meyer, T.J., 1978, Acc. Chem. Res. 11, 94. Motmans, K., J. Raus and C. Vandevyer, 1996, J. Immunol. Meth. 190, 107. Noffsinger, J.B. and N.D. Danielson, 1987, Anal. Chem. 59, 865. Noffsinger, J.B. and N.D. Danielson, 1987, J. Chromatogr. 387, 520. Olsvik, O., T. Popovic, E. Skjerve, K.S. Cudjoe, E. Homes, J. Ugelstad and M. Uhlen, 1994, Clin. Microbiol. Rev. 7, 43. Ouyang, J. and A.J. Bard, 1988, Bull. Chem. Soc. Jpn. 1988, 61. Paris, J.P. and W.W. Brandt, 1959, J. Am. Chem. Soc. 81, 5001. Parker, C.A., 1968, Photoluminescence of Solutions, Elsevier, Amesterdam. Prieto, I., J. Teetsov, M.A. Fox, D.A. Vanden Bout and A.J. Bard, 2001, J. Phys. Chem. A., 105,520. Richards, T.C. and A.J. Bard, 1995, Anal. Chem. 67, 3140. Richter, M.M. and A.J. Bard, 1996, Anal. Chem. 68,2641.

203

Richter Richter, M.M., A.J. Bard, W. Kim and R.H. Schmehl, 1998, Anal. Chem. 70, 310. Richter, M.M., F.-R.F. Fan, F. Klavetter, A.J. Heeger and A.J. Bard, 1994, Chem. Phys. Lett. 226 115. Robin, M.B. and P. Day, 1967, Adv. Inorg. Chem. RadioChem. 10, 247. Roundhill, D.M., 1994, Photochemistry and Photophysics of Coordination Complexes, Plenum, New York, ch. 5. Rozhitskii, N.N., 1992, J. Anal. Chem. USSR, 47, 1288. Rubinstein, I. and A.J. Bard, 1980, J. Am. Chem. Soc. 102, 6641. Rubinstein, I. and A.J. Bard, 1981, J. Am. Chem. Soc. 103,512. Rubinstein, I., C.R. Martin and A.J. Bard, 1983, Anal. Chem. 55, 1580. Safarikova, S.M. andS.J. Forsythe, 1995, J. Appl. Bacteriol. 78, 575. Saji, T. and A.J. Bard, 1977, J. Am. Chem. Soc. 99, 2235. Sakura, S. and H. Imai, 1988, Anal. Sci. 4, 9. Santhanam, K.S.V. and A.J. Bard, 1965, J. Am. Chem. Soc. 87, 139. Schultz, L.L., J.S. Stoyanoff and T.A. Nieman, 1996, Anal. Chem. 68, 349. Schutzbank, T.E. and J. Smith, 1995, J. Clin. Microbiol. 33, 2036. Sinha, A.P.B., 1971, Spectrosc. Inorg. Chem. 2, 255. Smith, P.J. and C.K. Mann, 1969, J. Org. Chem. 34, 1821. Stem, H.J., R.D. Carlos and T.E. Schutzbank, 1995, Clin. Biochem. 28,470. Sutin, N. and C. Creutz, 1978, Adv. Chem. Ser. 168, 1. Sykora, M. and T.J. Meyer, 1999, Chem. Mater. 11, 1186. Taverna, P.J., H. Mayfield and A.R.J. Andrews, 1998, Anal. Chim. Acta 373, 111. Tsukagoshi, K., K. Miyamoto, E. Saito, T. Nakajima, K. Hara and K. Fujinaga, 1997, Anal. Sci. 13, 639. Tokel, N. and A.J. Bard, 1972, J. Am. Chem. Soc. 94, 2862. Tokel-Takvoryan, N.E., R.E. Hemingway and A.J. Bard, 1973, J. Am. Chem. Soc. 95, 6582. Uchikura, K. and M. Kirisawa, 1991, Anal. Sci. 7, 803. Uchikura, K., M. Kirisawa and A. Sugii, 1993, Anal. Sci. 9, 121. Uhlen, M., E. Homes and O. Olsvik, Eds. 1994, Advances in Biomagnetic Separation Eaton Publishing. van Dyke, D.A. and H.Y. Cheng, 1989, Anal. Chem. 61,633. Van Gemen, B., R. Van Beuningen, A. Nabbe, V. Van Strijp, S. Jurriaans, P. Lens, R. Schoones and T. Kievits, 1994, J. Virol. Meth. 49, 157. Van Houten, J. and R.J. Watts, 1976, J. Am. Chem. Soc. 98, 4853. Vitt, J.E., D.C. Johnson and R.C. Engstrom, 1991, J. Electrochem. Soc. 138, 1637. Vogler, A. and H. Kunkeley, 1984, Ang. Chem. Int. Ed. Engl. 23,316. Vojir, V., 1954, Collect. Czech. Chem. Comun.19, 872. Wallace, W.L. and A.J. Bard, 1979, J. Phys. Chem. 83, 1350. Walton, D.J., S.S. Phull, D.M. Bates, J.P. Lorimer and T.J. Mason, 1992, Ultrason. 30, 186. White, H.S. and A.J. Bard, 1982, J. Am. Chem. Soc. 104, 6891. 204

Electrochemiluminescence Wightman, R.M., C.L. Curtis, P.A. Flowers, R.G. Maus and E.M. McDonald, 1998, J. Phys. Chem. B, 102, 9991. Wild, D.Ed., 1994, The Immunoassay Handbook, Macmillan Press Ltd.. Wilkinson, E.T., S. Cheifetz and S.A. De Grandis, 1995, PCR Methods Appl. 4, 363. Workman, S. and M.M. Richter, 2000, Anal. Chem. 72, 5556. Xu. G. and S. Dong, 2000, Anal. Chem. 72, 5308. Yamashita, K., S. Yamazaki-Nishida, Y. Harmia and A. Segawa, 1991, Anal. Chem. 63, 872. Yang, H., Y.K. Leland, D. Yost and R.J. Massey, 1994, Bio/Technol. 12, 193. Yu, H., 1996, J. Immunol. Meth. 192, 63. Yu, H. and J.G. Bruno, 1996, Appl. Environ. Microbiol., 62, 587. Zhou, M. and J. Roovers, 2001, Macromolec. 34, 244. Zu, Y. and A.J. Bard, 2000, Anal. Chem. 72, 3223. Zu, Y. and A.J. Bard, 2001, Anal. Chem., in press. Zu, Y., Z. Ding, J. Zhou, Y. Lee and A.J. Bard, 2001, Anal. Chem. 73, 2153.

205

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 92002 Elsevier Science B.V. All rights reserved

CHAPTER 7

SURFACE PLASMON RESONANCE BIOSENSORS

JIOAHOMOLA, PH.D., SINCLAIRS. YEE, PH.D., ANDDAVID MYSZKA, PH.D.* Department of Electrical Engineering, University of Washington, Seattle, WA 98105, USA Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA

Surface plasmon resonance (SPR) biosensors exploit special electromagnetic w a v e s surface plasmon-polaritons - to probe changes in refractive index at surfaces of metals. SPR biosensors can therefore be used to monitor the interaction between an analyte in solution and its biospecific partner immobilized on the metal surface without the use of labels. Major application areas include detection of low levels of biological analytes and study of biomolecular interactions. In the past ten years, SPR biosensor technology has been commercialized and SPR biosensors have become a central tool for characterizing and quantifying biomolecular interactions both in life science and pharmaceutical research.

1. Technical Concept Surface plasmon resonance (SPR) biosensors use surface plasma waves to probe biomolecular interactions occurring at the surface of a sensor. This chapter introduces surface plasma waves, presents methods for their excitation and interrogation, and discusses the concept of SPR optical biosensors.

1.1. Surface plasma waves I. I. 1. Surface plasmon-polaritons at a plane interface between a semi-infinite dielectric and a metal. Surface plasmon-polaritons or surface plasma waves

occur at the surfaces of metals, which behave like nearly free electron plasmas. 207

Homola, Yee, and Myszka Let us consider a plane interface composed of two semi-infinite homogeneous, isotropic media and choose the coordinate system so that the metal (dielectric function aM) occupies the region z0 (Figure 1). Electromagnetic field modes supported by this geometry can be found by solving Maxwell's equations in each medium and applying boundary conditions at the interface. We are looking for modes guided by the interface, and thus the magnitudes of all their field vectors have to be decreasing with increasing distance from the interface. In general, stratified media with plane boundaries can support transverse electric (TE) modes (in which the direction of propagation and magnetic and electric intensity vectors form an orthogonal triad and the electric vector is parallel to the interface) and transverse magnetic (TM) modes (where the magnetic vector is perpendicular to the direction of propagation of the wave and parallel to the plane of interface) (Figure 1). TE-polarized surface modes can not exist in this geometry if the materials involved are non-magnetic (Boardman, 1982). Therefore, only TMmodes can be supported by the metal-dielectric interface. In complex notation, electric and magnetic intensity vectors of a TM-polarized mode are: Dielectric" 2

E = (E~,O,E z) = 1 , 0 ,

Aexp[-croz + i(flx-cot)],

(1)

r H = (O,H 0) = (0,1,0) -/Aa)e~ exp[-croz + i ( f l x - cot)] Y' c'%/~ Metal: E = (E~,0,E.). =ll,O,-ifl)Aexp[crMz+crMj i(flx-cot)], r

H = (O,H,,O) = (0,1,0 9

c

o~u/z

,4, - :

-

e.

(2)

exp[crM z + i(flx - cot)]

where co is the angular frequency, fl is the propagation constant, c is the speed of light in vacuum, /~ is the permeability of vacuum, and A is a normalization constant proportional to the energy carried by the mode (Boardman, 1982). Application of boundary conditions requiring continuity of tangential components of electric and magnetic intensity vectors (Hy and Ex) at the interface yields an equation for the propagation constant (Boardman, 1982):

co,[ trMe~

(3)

f l = c ~t eM + e o

208

Surface Plasmon Resonance Biosensors

~Z

E

Dielectric

Y

Metal Figure 1. Transverse magnetic (TM) wave at a metal-dielectric interface.

The propagation constant is generally a complex number because the dielectric function of metal em is a complex function of the angular frequency. Equations (1), (2), and (3) represent a true surface plasma wave (SPW) that propagates along the interface and decays exponentially in a direction perpendicular to the direction of propagation, if the real part of eM is negative and its absolute value is smaller than Eo. At optical wavelengths, this condition is fulfilled for several metals of which gold and silver are the most commonly used (Figure 2). The real part of the propagation constant is related to the effective refractive index N - the quantity commonly used in waveguide optics - in the following manner:

N =CRe{fl}=

l~emeMe~ eD}

(4)

where Re{ } denotes the real part of a complex number. The imaginary part of the propagation constant is related to the modal attenuation b (in dB/cm if [3 is given in l/m): b = Im{fl} In 0.2 10 = Im

e~e~

0.2co

[~e~, + e o j clnlO

(5)

where Im{ } denotes the imaginary part of a complex number. Spectral dependencies of the effective refractive index and mode attenuation for SPWs supported by silver and gold surfaces are shown in Figure 3. As follows 209

Homola, Yee, and Myszka

~

.,---- Re{~)

-40 (9

---

" "" "" " -,..,,.,...,, .,, ,.. ,,..

-20

-80

8 6

- "-~

4E

-60

tr

Gold Silver

"

~

600

,...,

0 1 m { e 4 " - ' .-.7. ~ . ' - .: 2

700

800

9O0

1000

Wavelength [nm]

Figure 2. Dielectric function of gold and silver. Data taken from Palik (1985) and Ordal et al. (1983).

a)

b) f

S P W on gold S P W on silver

~) 1.7

~ ....

S P W on gold S P W on silver

105

1.6

{ ,o.

"~ 1.5

>*,

g

1.4 10 s

1.3

i

600

.

.

.

.

|

700

.

.

.

.

.

i

.

.

.

.

800 W a v e l e n g t h

i

900

.

.

.

.

i

!

1000

600

700

800

900

1000

W a v e l e n g t h [nm]

[nm]

Figure 3. Surface plasmon-polaritons: effective refractive index and attenuation. The effective refractive index (a) and attenuation (b) of a surface plasmon-polariton as a function of the wavelength for a surface plasmon-polariton propagating along the interface between a metal (gold or silver) and a non-dispersive dielectric (refractive index = 1.32).

from Figure 3, the effective index of an SPW increases with decreasing wavelength. The effective refractive index of the SPW supported by a gold surface is larger that that of the SPW supported by silver because the real part of the dielectric constant of gold is smaller than that of silver. The mode attenuation of the SPW supported by a gold boundary is larger than that of the SPW supported by a silver surface as the imaginary part of the dielectric constant of gold is larger than that of silver. The field profile of a surface plasmon-polariton is illustrated in Figure 4. 210

Surface Plasmon Resonance Biosensors

a)

b) 1.0

.

.~ 0.8

.

.

.

.

.

.

.

.

.

.

.

.

~

~

1.0

~ 0.8

~"~

0.6

~~

0.4

t--

0.4

0.2

"0

0.2

-

0.6

._z,

~ i.: !~_~~L _ _ _ _ - ' ~ _ _ _ - ' . . . .

0.0

c~

Metal -0.2

-0.2

Dielectric

i

0.0

0.2

0.4

~.~,=o.o

,

~

O)

0.6

z-coordinate ,[p.m]

0.8

1.0 ~

-0.2

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

z-coordinate [p.m]

Figure 4. Surface plasmon-polariton: field pattern. Spatial distribution of the magnetic intensity for a surface plasmon-polariton at the interface between gold and a nondispersive dielectric (refractive index = 1.32) in the direction perpendicular to the interface, calculated for the wavelength of 630 nm (a) and 850 nm (b).

The field decay in the direction perpendicular to the direction of propagation may be characterized by means of the penetration depth, Lp. The penetration depth is defined as the distance from the interface at which the amplitude of the field has fallen to 1/e of its value at the surface and is related to field parameters CtD and ctM as follows:

Lt,o = 1/Re{ere } ,

LoM : 1/Re(a M}

(6)

Similarly, the attenuation of an SPW in the direction of propagation can be characterized by means of the propagation length, which is defined as the distance in the direction of propagation at which the energy of the wave decreases by a factor of 1/e.

L = l/[2Im{ fl}]

(7)

Characteristics of SPWs supported by surfaces of gold and silver are given in Table 1. As follows from Equation (7), Table 1, and Figure 3, SPWs propagating along the surface of silver are less attenuated than those propagating along the surface of gold. SPWs propagate with high attenuation and the attenuation increases with decreasing wavelength. As follows from Figure 4, the electromagnetic field of an SPW is distributed in a highly asymmetric fashion and majority of the field (usually more than 90 per cent) is concentrated in the dielectric medium.

211

Homola, Yee, and Myszka Table 1. Major characteristics of surface plasmon-polaritons at the interface between metal (silver or gold) and a non-dispersive dielectric (refractive index = 1.32) for two different wavelengths.

t

Metal Wavelength [nm] Penetration depth into metal [nm] Penetration depth into dielectric [nm] Propagation length [~m] "'

......

Silver 630 24 219 19

f

Gold 85()

850 23 443

630

29

25 4oo

57

2

24

-

1.1.2. Surface plasmon-polaritons on a thin metal film surrounded by dielectric media. A thin metal film, surrounded by dielectric media may support surface plasmon-polaritons at both the interfaces. For thin metal films there is coupling between surface plasmon-polaritons associated with each boundary, giving rise to mixed m o d e s - symmetric and antisymmetric surface plasmon-polaritons. These modes are found as solutions of Maxwell's equations in each medium, which satisfy boundary conditions at both the interfaces (Burke et al., 1986). Figure 5 shows the effective refractive index and attenuation of these two modes as a function of the thickness of the metal film. Clearly, if the metal film is rather thick (-- 100 nm), the propagation constants of the two modes are almost identical. As the metal thickness decreases, the two modes become more different. The symmetric surface plasmon-polariton exhibits an effective refractive index and attenuation which both decrease with decreasing metal film thickness, while the effective refractive index and attenuation of the antisymmetric surface plasmon-polariton increase with decreasing thickness of the metal film. Thus, the symmetric surface plasmon-polariton exhibits a lower effective refractive index and attenuation than its antisymmetric counterpart. Therefore, the first mode is often referred as a long-range surface plasmonpolariton while the other is referred as a short-range surface plasmon-polariton, a reference to their relative attenuation and propagation lengths. The dispersion properties of symmetric and antisymmetric surface plasmonpolaritons (Figure 6) are rather different from those of the traditional SPWs supported by a single interface (Figure 3). It should be noted that the effective refractive index of the symmetric mode exhibits much lower 'dispersion' (dependence on the wavelength) than that of the antisymmetric mode. This is because the symmetric surface plasmon exhibits a much weaker electromagnetic field in the metal film, which is a strongly dispersive medium, Figure 7. As illustrated in Figure 7, the distribution of magnetic intensity (and the transverse electric intensity) is symmetric and antisymmetric with respect to the

212

Surface Plasmon Resonance Biosensors

10= '

Antiaymmelric SPW

~

10~ .Q

o

g

103

10t 10~

/I

~ y m m a ~ r i e SPW

10.2 Z 1.60

|

~

1.40

Symmetric SPW 1..._~

D

-

.

. ~, - - - - r - - " T - ' ~ .

.

,

. .

i

2D

=

.

9

40

9

~

,

,

r

.

.

--_'.

.

. ,

. ,

80

SO

~o0

Film thickness [nmJ

Figure 5, Surface plasmon-polaritons on a thin metal film, Effective refractive index and mode attenuation as a function of the metal fiim thickness for symmetric and antisymmetric surface plasmon-polaritons propagating along a thin gold film embedded between two identical non-dispersive dielectrics (refractive index = 1.32); wavelength = 800 nm. Note the drop in the attenuation for the symmetric bound mode as the metal thickness approaches zero.

~ i~~----..__~ ...... 48,

7OO

nO0

g00

1000

WavelonO~la,-nl

Figure 6. Surface plasmon-polaritons on a thin metal film. Effective refractive index and mode attenuation as a function of the wavelength, calculated for symmetric and antisymmetric surface plasmon-polaritons propagating along a thin gold film embedded between two identical non-dispersive dielectrics (refractive index = 1.32), gold film thickness = 20 nm.

center of the metal film for symmetric and antisymmetric surface plasmonpolaritons, respectively. The field of the symmetric SPW penetrates deeper into the dielectric media than that of the antisymmetric SPW.

213

Homola, Yee, and Myszka

a) ,--,

1.0

~

0.8

b) 1.0

0.8 0.6

,_...

"~ 0.4

: E ~"

0.6

c..~_ "o ,~

0.4

.~

.~ tt~

0.2

Im{Hy}

~ o.o-

0.2

~

-0.2

~

-0.4

~

-0.6

ImlHy}

O.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-0.8 -0.2

-1 .o

. . . .

i

-o5

. . . .

i

. . . .

0.o

z-coordinate

I

O.S

. . . .

, -1.0 -I .0

1.o

[,p.m]

,

,

,

l

-0.5

. . . .

l

. . . .

0.0 z-coordinate

,

0.5

. . . .

1.0

[~m]

Figure 7. Surface plasmon-polaritons on a thin metal film: field patterns. Spatial distribution of the magnetic intensity of a) symmetric and b) antisymmetric surface plasmon-polaritons propagating along a thin gold film embedded between two identical non-dispersive dielectrics (refractive index = 1.32), gold film thickness = 20 nm, wavelength = 800 nm.

1.2. Optical excitation of surface waves A light wave can couple to a surface plasma wave at a metal-dielectric interface if the component of light's wavevector that is parallel to the interface matches that of the surface plasma wave. As the propagation constant of a surface plasma wave at a metal-dielectric interface (Eq. 3) is larger than that which can be provided by the component of the wavevector of light in the dielectric, SPWs cannot be excited directly by light incident onto a smooth metal surface. In order to allow for excitation of a SPW by a light wave, the light's wavevector needs to be enhanced to match that of the surface plasma wave. The light's wavevector can be increased by passing the light through a medium with a refractive index higher than that of the dielectric medium at the boundary at which the SPW is to be excited, or by roughness of the metal surface (statistical or regular).

1.2.1. Excitation of surface plasmon-polaritons using prism couplers. The enhancement of a light wave's wavevector by passing light through an optically denser medium is illustrated in Figure 8. The effective refractive index of an SPW at the metal (gold) - dielectric (water) interface ranges from 1.35 (at a wavelength of 1000 nm) to 1.68 (at a wavelength of 600 nm) while the effective refractive index of light in the dielectric does not exceed 1.335. A light wave propagating in the dielectric with a higher refractive index, such as BK7 glass, can provide a component of the normalized wavevector parallel to the metal surface with a value between 0 and about 1.52, depending on the angle between the direction of propagation of the light wave and the metal surface. Therefore, 214

Surface Plasmon Resonance Biosensors

1.7 x| "o 9 1.6 .c_

Surlace plasma wave at Id-water interface

1.5

V / / / ~

./~CC//.////

;////.kieht wave in ~ (

fllass

i,. .> 1.4

1.:3 600

9

700

800

9

. ight wave in water;

900

1000

W a v e l e n g t h [nm]

Figure 8. Effective refractive index of a surface plasmon-polariton as a function of the wavelength for a surface plasmon-polariton propagating along the interface between a gold and water. Normalized wavenumbers provided by light waves in a B K7 glass and water shown for comparison.

the condition for coupling light into the SPW can be fulfilled for wavelengths long than 630 nm. The coupling between a light wave in the high refractive index dielectric medium and an SPW at the metal - low-refractive index dielectric interface can be established by the total internal reflection method. A light wave passes through a high refractive index prism and is totally reflected at the prism base generating an evanescent wave penetrating a metal film in the Kretschmann configuration (Figure 9a) or a dielectric layer in the Otto configuration (Figure 9b). This evanescent wave propagates along the interface with the propagation constant, which can be adjusted to match that of the SPW by controlling the angle of incidence. Thus, the matching condition can be fulfilled allowing the evanescent wave to be coupled into the SPW. Assuming that the prism has only a minor influence on the propagation constant of the SPW at the interface of a metal and a low refractive index dielectric, the coupling condition may be expressed as"

(8)

~-Tsin(O) = Rel, ]- e~t''e~ l L~/e,, + eo J

where 0 denotes the angle of incidence, ep, cMand co denote dielectric functions of the prism, the metal film and the dielectric medium (ep > eo). As discussed in Section 1.2.4, this condition is fulfilled for 'thick' metal films. (For gold at optical wavelengths, in this approximation the 'thick' gold films are 50 nm or more). 215

Homola, Yee, and Myszka

Figure 10. Excitation of surface plasma waves by guided modes of optical waveguides, a) excitation of SPW on the inner boundary of a thin metal film, and b) excitation of SPW on the outer metal boundary through a dielectric buffer layer.

1.2.2. Excitation of surface plasmon-polaritons using optical waveguides. Similarly, a surface plasma wave can be excited by a light wave guided by an optical waveguide. This approach is illustrated in Figure 10. Light propagates in a waveguide in the form of guided modes. The electromagnetic field of a guided mode is concentrated in the waveguiding layer. A fraction of the optical energy propagates in the form of an evanescent wave in the low-refractive index medium surrounding the waveguiding layer. In the section of the waveguide containing an SPW-active metal film, this evanescent wave can excite an SPW at the outer (in Figure 10a) or inner (Figure 10b) surface of the metal film. Assuming that the waveguide influences the propagation constant of the SPW only slightly, the coupling condition for a guided mode and a surface plasma wave can be expressed as follows: (9)

216

Surface Plasmon Resonance Biosensors

Figure 11. Excitation of surface plasma waves at the surface of a diffraction grating.

where Ney denotes the effective refractive index of the waveguide mode, and ~v~ and CDdenote dielectric functions of the metal and the dielectric medium.

1.2.3. Excitation of surface plasmon-polaritons using grating couplers. A surface plasma wave may also be excited by a light wave with its wavevector increased by the wave's interaction with surface roughness of the metal film. Most commonly used configurations for SPW excitation on rough metal surfaces are based on diffraction gratings (Figure 11). ff a light wave is made incident on a periodically distorted surface of a diffraction grating, a series of waves directed away from the surface at different angles is produced (Hutley, 1982). The components of the wavevector of these diffractiongenerated light waves parallel to the interface are:

k x + mG =km

(1 O)

where m is the diffraction order (integer), kx is the component of the wavevector of the incident light along the grating surface, G is the grating wavevector, and k~m is the wavevector of the diffracted light wave. In case of shallow gratings, the coupling condition may be expressed as: e ~ o sin(0)+ m-- = +Re A [~/eM +eo

(Ii)

where 0 is the angle of incidence of the light wave, A denotes the pitch of the grating (A= 2n/G), eM and eo denote dielectric functions of the metal and the dielectric medium, and ~ denotes the free-space wavelength. 217

Homola, Yee, and Myszka

1.2.4. Excitation of surface plasmon-polaritons: energy transfer. In the process of optical excitation of surface plasma waves, a portion of the energy of the light wave is transferred into the energy of a surface plasma wave and dissipated in the metal film. Assuming IRe(eM)l>>eoand IRe(eM)l>>Im(eM),the reflectivity for the Kretchmann geometry of the attenuated total reflection method may be expressed as (Raether, 1983): R(k x) = 1 -

4FiFr~a ~kx - fl+~2 + ( F i +

1-',..d

)2

'

(12)

where

k.,. = ~

co sin(O), r+ = Re {/3 + Aft}, F, = Im {fl}, Fr,,a = Im {Aft}. (13) s

0 is the angle of incidence, cv is the dielectric constant of the prism, co is the angular frequency, c is the velocity of light in vacuum, kx is the component of the wavevector of the light wave which is parallel with the interface, and fl is the propagation constant of the SPW as given by Equation (3)./Yi characterizes the attenuation of the surface plasma wave at the metal-dielectric interface due to the dissipation in the metal. The complex quantity Aft characterizes the influence of the prism on the propagation constant of the surface plasma wave. Its imaginary part, Frad, characterizes the attenuation of the surface plasma wave due to the coupling of the SPW to optical radiation in the prism. As follows from Equation (12), the reflectivity exhibits a Lorentzian dip located at the angle Om~ given by the following condition: --sin(0,~,,) =/3+.

(14)

r

In the approximation of thick metal films, Re{fl}>>Re{dfl} and the right-hand side of Equation (14) is equal to the propagation constant of the surface plasmon at the metal-dielectric interface. This suggests that the reflectivity minimum occurs when the component of the wavevector of the incident light wave parallel to the interface matches the propagation constant of the SPW. The depth of the reflectivity dip can theoretically range from 0 to 100 per cent. The reflectivity minimum reaches zero if ~ = ~ , indicating that all the energy of the incident light wave can be lost due to the SPW's excitation if the parameters of the structure and incident light wave are chosen properly. It can be shown by the examination of Equation (12) that the width of the dip is proportional to ~ + ~ad and thus to the total loss the surface plasma wave exhibits. Therefore the excitation of surface plasma waves on a metal with low loss gives rise to a narrow dip in the TM-wave reflectivity and vice versa. For a given metal, the reflectivity minimum and the width of the dip cannot be minimized at the same time, as ~ decreases with decreasing metal film thickness while ~,d exhibits an opposite trend. In order to produce deep and narrow reflectivity dips, one has to 218

Surface Plasmon Resonance Biosensors

a) 1.0 ;

b' ......

SPW..

....

0.8

270 240

1.0

210 180

0.8

150

150,-~ 120 .~

~'9~"0.6

180

120 90

. ~ 0.6

"13

6o ~' 30

13-

0

0.2

-30

0

0.2 -3o

-60 0.0 - '

, 50

.

.

.

.

.

.

.

.

.

55

-90 60

0.0

~

49.0

Angle of incidence [deg]

,-

'~-

49.2

.

.

.

.

.

49.4

.

.

.

.

.

49.6

.

.

.

.

.

49.8

.

.

60

50.0

A n g l e of i n c i d e n c e [deg]

Figure 12. Reflectivity for a transverse magnetic wave in: a) Kretschmann (three-layer) geometry consisting of an SF14 glass prism (refractive index = 1.65), a gold layer (thickness = 50 nm), and a low refractive index dielectric medium (refractive index = 1.32), and b) four-layer geometry consisting of an SF14 glass prism, a low refractive index buffer (refractive index = 1.32, thickness = 1200 nm), a thin gold layer (20 nm), and a low-refractive index dielectric medium (refractive index- 1.32), wavelength - 800 nm. Note the difference in the x-axis scale.

use very thin metal films (13': 219

Homola, Yee, and Myszka

Figure 13. Surface plasma waves probing (a) a homogeneous medium and (b) a thin dielectric layer.

k2 IIn a(z) - n2(z)]H~(z)dz P'BB+~-, 2fl iHZy(z)d z

(15)

where k is the free-space wavenumber and Hy(z) is the spatial dependence of magnetic field of the surface plasma wave. Using Equation (15), one can show that a small change in the refractive index of a homogeneous dielectric medium, An, (An 50 nnl).

Keeping the issues of target analyte affinity and acceptable fluorophore or chromophore wavelengths in mind, the chemosensor designer should proceed to consider binding selectivity, optical signaling mechanism, and the method to be used for immobilizing or delivering the chemosensor. These considerations are discussed in the following sections.

1.2. Molecular recognition A chemosensor must recognize its target analyte, much in the manner of picking out a familiar face in a crowd. More importantly, it must respond quickly and specifically (e.g., Hi there, Fred!). This selection process can result from selective binding or selective response, but in the latter case interfering substances will competitively inhibit optical response to the desired analyte. Having said this, complete specificity for a single potential guest is not necessary because the chemosensor needs to pick out its guest only from the substances typically present in the analyte solution. For example, a chemosensor for measuring sodium or potassium in blood need not discriminate against transition metals, unless the test is intended for patients who are already deceased! How can the molecular structure of a chemosensor be engineered to specifically bind the target analyte? Factors affecting molecular recognition in designed host-guest complexes have been the subject of intense scrutiny over the last three decades (Cram and Cram, 1978; Cram, 1988; Lehn, 1988, 1990; Rebek, 1988, 1990; Schneider, 1991). While there is no reliable way to predict host-guest 334

Artificial Receptors for Chemosensors selectivity, research in this field and in the broader area of supramolecular chemistry (V/3gtle, 1991; Lehn, 1995; Steed and Atwood, 2000) has identified several intermolecular forces that play important roles. The fundamental electrostatic (ion-ion, ion-dipole and dipole-dipole), hydrogen bonding, and van der Waals interactions are of course important, but more subtle forces have also been examined, including n-stacking (Hunter and Sanders, 1990; Hunter, 1993), cation-n interaction (Ma and Dougherty, 1997), CH-rc interaction (Laatikainen et al., 1995; Cloninger and Whitlock, 1998) and solvophobic effects. How can these intermolecular forces be marshaled and controlled to effect molecular recognition? Cram put this task most succinctly in two simple terms: complementarity and preorganization. The principle of complementarity is that: "to complex, hosts must have binding sites which cooperatively contact and attract binding sites of guests without generating strong nonbonded repulsions" (Cram, 1988). Clearly, the number and type of binding sites in the host must match those in the guest to produce an optimally stable complex. This principle alone, however, is insufficient for the design of a host that must select between similar guests, such as alkali metal cations differing only in size. Here we need guidance from the principle of preorganization: "the more highly hosts and guests are organized for binding and low solvation prior to their complexation, the more stable will be their complexes" (Cram, 1986). Excellent selectivities can be achieved in the alkali metal series with highly preorganized hosts, justifying the description of preorganization as the "central determinant of binding power" (Cram, 1986; Reinhoudt, 1988). While host preorganization leads to stronger and more selective guest binding, it also increases rigidity. In chemosensors, rigidity can hinder access of the analyte to the binding site, slowing equilibration (Eq. 1) considerably. This can produce unacceptable delays in equilibrium measurements, simultaneously retarding kinetic measurements, as well. The rate of complex dissociation (kout) is the factor limiting equilibration time, but remember that the binding rate (k~) is proportional according to: kin =

Kako~

(5)

Even when Ka is large, kt~ may be too small for kinetic measurements to be made within the 1-2 minute time frame required in certain sensor applications. Therefore, chemosensor rigidity must be balanced with flexibility. Preorganization does not need to be completely sacrificed, as evidenced by the various degrees of complex stabilization attending the chelate, cryptate and macrocyclic effects (Hancock and Martell, 1988). Indeed, a certain degree of chemosensor flexibility may be desired in order to produce an optical response by an "induced fit" mechanism, which leads us to the next consideration in chemosensor design. 335

Bell and Hext

Intrinsic

h~

Extrinsic

\\1

//

Figure 2. Cartoon showing different optical responses of intrinsic and extrinsic chromophores or fluorophores in chemosensor.

1.3. Optical signaling We have considered the desirable binding and optical characteristics of chemosensors. What about the mechanism coupling the binding event with signal transduction via the chromophore or fluorophore? It makes sense that the binding site and the optical reporter should be structurally integrated as much as possible in order to maximize this communication. In this context, it is useful to draw a distinction between intrinsic and extrinsic fluorophores or chromophores (Bell et al., 1993; Lakowicz, 1999), as shown in Figure 2. Intrinsic optical reporters are structurally integrated with the analyte binding site to maximize the influence of the bound guest on the optical properties of the chemosensor. Here, chemosensors have a profound advantage over biosensors. It is much easier to build a chromophore or fluorophore into a chemosensor binding site during its synthesis than to modify or introduce an optical reporter into the active site of an enzyme or the recognition site of an antibody. Such modifications of biological molecules usually damage their molecular recognition capabilities, so the optical reporter must be conjugated extrinsically to their binding sites. During chemosensor synthesis, optical and other properties 336

Artificial Receptors for Chemosensors (e.g., pKa) of the chromophore or fluorophore can also be fine-tuned in order to optimize performance. Whether the optical reporter is intrinsic or extrinsic to the molecular recognition site, the molecular mechanism for optical response should be considered during chemosensor design. While the mechanism of many known sensors, especially fluorescent chemosensors and biosensors, may not be well understood, Table 1 lists many mechanisms that have been identified and incorporated into chemosensor design. Here an important distinction is made between guest binding effects on chromophores vs. fluorophores. Useful absorbance effects generally result from changes in molecular structure, including proton transfer, other chemical reactions, and isomerization. Fluorescence is much more sensitive to subtle changes in the geometry and electronic structure of the ground state, as well as the electronic excited state. It is uniquely responsive to physical processes affecting depopulation of the emissive excited state (Lakowicz, 1999), such as conformational restriction occurring upon analyte complexation (McFarland and Finney, 2001; Mello and Finney, 2001). As indicated in Table 1, fluorescent chemosensors can utilize several photophysical processes, in addition to all of the structural mechanisms available to chromophoric chemosensors. The structural changes listed in Table 1 for chromophore signaling generally change the polarity or degree of electronic delocalization (conjugation) within the host chromophore. T h e chromophore protonation state can change when a neutral host ionizes during binding of a cationic guest, or when binding drives proton transfer between host and guest or within the host (tautomerization). Guest binding can also change electron distribution in the ground state of the chromophore and the energy of the locally excited (LE) state. The resulting "polarization" mechanism (Table 1) operates in a manner that is similar to solvatochromism (Reichardt, 1979). The LE states of most chromophores are more polar than their ground states, so polar solvents stabilize them more than the ground state. The resulting decrease in the energy difference between ground and excited states causes the )~maxto shift to longer wavelength as solvent polarity increases (positive solvatochromic effect). A polar guest can, in principle, cause a bathochromic shift in the absorption of a chemosensor by the same mechanism, or a specific charge or dipole interaction could cause the opposite effect, a hypsochromic shift. Fluorescence wavelengths are much more sensitive to solvent polarity and are subject to other effects, as illustrated in Figure 3 (Lakowicz, 1999). Solvent (and host-guest) interactions do not have time to adjust immediately to photon absorption and concomitant electronic excitation, occurring on the time scale of 10~5 seconds. Specific interactions and general solvent relaxation involving partial orientation of solvent dipoles stabilize the polar excited state. Relaxation

337

Bell and Hext Table 1. Optical Response Mechanisms.

Proton transfer Tautomerism Skeletal isomerism

Chromophore

Host-guest reaction Polarization Solvent displacement Quenching by guest

Fluorophore

Internal charge transfer (ICT) Twisted internal charge transfer (TICT) Resonance energy transfer (RET) Photoinduced electron transfer (PET)

of polar solvents has a larger stabilizing effect on the excited state than solvation of the LE state, producing larger bathochromic shifts of fluorescence emission bands. Clearly, guest binding can strongly influence the energy of the emissive state of a chemosensor, either by displacing solvent or by introducing new electrostatic interactions in the complex. Moreover, many fluorophores can form an internal charge transfer (ICT) state, also shown in Figure 3, involving transfer of electron density from electron,donating to electron-accepting groups. Interactions of the solvent or the bound guest molecule with these groups will determine the energy of the ICT state and also determine which state has the lowest energy. The twisted intramolecular charge transfer (TICT) state (Rettig, 1994) provides even greater opportunities as a fluorescence sensing mechanism in chemosensors. Formation of the TICT state involves rotation of donor and acceptor groups of the fluorophore. As stated earlier (Section 1.1), a large difference between absorption and fluorescence wavelengths is desirable in chemosensor design, and TICT emission can produce Stokes' shifts in excess of 100 nm. Because formation of the TICT state requires conformational mobility, rigidification of the chemosensor upon guest complexation can profoundly influence the intensity of long-wavelength TICT fluorescence. Resonance energy transfer (RET), involving through-space jumping of electronic excitation energy from a donor fluorophore to an acceptor fluorophore or a quencher, has been described as "the most general and valuable phenomenon for

338

Artificial Receptors for Chemosensors LE state At

~V

~v r Specific interactionS------"

~~~

Solvent relaxation _ ,, (10 1~ s) , ......ICT state _

> OA

h~F

~h~;

k'~ h~'F

~|_n~F iii

(lo ~s s)

So-

~v

f

~V

Figure 3. Jabtofiski diagram showing effects of specific solvent-fluorophore interactions, general solvent relaxation and formation of internal charge transfer (ICT) states (adapted from Lakowicz, 1999).

fluorescence sensing" (Lakowicz, 1999). Because RET operates over macromolecular distances, it can be used to detect major conformational changes of nucleotides or association between biomolecules, as in immunoassays. RET is not sensitive to changes in donor-acceptor separation in the subnanometer range, so it has not been found to be useful for detecting changes in the conformation of synthetic chemosensors upon guest binding. On the other hand, photoinduced electron transfer (PET) is a very useful sensing mechanism in fluorescent chemosensors (Bissell et al., 1993; Czarnik, 1993a,b, 1994; Desvergne and Czarnik, 1997; Granda-Vald6s et al., 2000). PET quenching of fluorescence occurs when an electron-rich group, such as an amino or phenoxide group, donates an electron to the fluorophore excited state. PET is different from ICT in that the electron donor group is not in direct conjugation with the rr system of the fluorophore. Binding of an electron-deficient guest to the donor group increases fluorescence emission by stabilizing and decreasing the mobility of donor electrons. It is also possible that some chemosensors may operate by PET quenching involving electron transfer from the guest to the fluorophore upon complexation.

1.4. Immobilization Chemosensors, as molecular devices, do not need to be tied down and integrated with optics and electronics components of sensing instruments. When employed as free agents, they are more commonly termed indicators, reagents, or molecular 339

Bell and Hext probes. Indeed, fluorescent probes of the intracellular concentrations of metals and other analytes have proven to be extremely useful in biomedical research (Lakowicz, 1999). Also, when used to fabricate disposable or reusable sensing materials for sensing instruments, chemosensors may not need to be covalently attached to a surface or other substrate. Often they can be hydrophobically adsorbed to a nonpolar surface layer or dissolved in the plasticizer of a polymer film or membrane. Covalent immobilization of chemosensors on surfaces or in materials is often used to improve sensor stability and avoid migration of the chemosensor into the analyte solution. Immobilization is usually a late-stage activity in sensor development, but the convenience of adding functional groups or side chains as covalent linkage sites should be considered while planning chemosensor structure and synthesis. Incorporation of a tether that does not interfere with binding or optical response can also be used to apply the techniques of polymer supported synthesis and combinatorial chemistry that are now crucial to pharmaceutical development. It has been pointed out that both drug discovery and chemosensor development are host-guest research and that bead-based screening of combinatorial libraries should be easily accomplished with fluorescent chemosensors (Czarnik and Yoon, 1999).

2. History As artificial molecular recognition systems, chemosensors have their roots in coordination chemistry of metals, the lock-and-key model for enzyme action, and the biomolecular receptor. These concepts were introduced by Alfred Werner in 1893, Emil Fischer in 1894, and Paul Ehrlich in 1904, respectively. Studies in the first half of the 20 th century on hydrogen bonds, clathrates, inclusion compounds, and rr donor-acceptor complexes set the stage for the appearance in the second half of the century of the discipline alternately termed host-guest chemistry (Cram and Cram, 1978; Cram, 1988), supramolecular chemistry (Lehn, 1988, 1990, 1995; V6gtle, 1991; Steed and Atwood, 2000), or molecular recognition (Rebek, 1988, 1990). Macrocyclic ligands for transition metals (Hancock and Martell, 1988) played a special role in this drama, and organic chemists began to take notice in 1967 when Charles Pedersen discovered crown ethers capable of mimicking the alkali metal transport properties of ionophore antibiotics (Pedersen, 1984; Gokel, 1991; Bradshaw et al., 1996; Bradshaw and Izatt, 1997). The design and synthesis of host compounds was recognized as an established field of research by the award of the 1987 Nobel prize in chemistry to Pedersen, Cram and Lehn. Earlier studies in the field of supramolecular chemistry revolved around the complexation of metal cations. Incorporation of dyes and fluorophores into the structures of crown ethers gave chemosensors for alkali metal and alkaline earth 340

Artificial Receptors for Chemosensors metal ions (Takagi and Ueno, 1984; Lbhr and V6gtle, 1985). At about the same time, chelation-based fluorescent probes for intracellular calcium and other metals were developed (Tsien, 1993). Chromogenic reagents for alkali metals based on cryptands, cryptaspherands, and spherands have also been devised (Helgeson et al., 1989; Chapoteau et al., 1993; Dolman et al., 1996). Such reagents are of practical utility in the determination of sodium, potassium, and calcium in blood (Kumar et al., 1988; Chapoteau et al., 1993). More recently, there has been much interest in fluorescent chemosensors for transition metals, mainly involving polyamine ligands (Fabbrizzi et al., 1997a, 1998; Bargossi et al., 2000; Prodi et al., 2000). Supramolecular chemistry of neutral molecules and anions (Schreeder et al., 1996; Bianchi et al., 1997; Schmidtchen and Berger, 1997) has been explored more recently, and here is where many current challenges for chemosensors lie. Many organic analytes of interest in biological systems exist as neutral molecules or anions (e.g., carboxylates and phosphates). Such relatively complex guest molecules present problems in the design of both the molecular recognition and optical response functions of the chemosensor. Therefore, the examples in the following section of this chapter are drawn from recent work on artificial receptors and chemosensors for neutral organic molecules, as well as organic and inorganic anions.

3. State of the A r t - Chemosensors for Organic Analytes The examples of chemosensors in the following section show that a wide range of analytes can be detected by this approach. In selecting these examples, we have used the Czarnik (1994) definition of a chemosensor consisting of a molecule incorporating a binding site, a chromophore or fluorophore, and a mechanism for communicating between the two. This excludes reagent approaches involving irreversible formation of colored or fluorescent products (Davis et al., 1999; Lewis et al., 2000). Also excluded by this definition are receptor-based sensing strategies in which the analyte competes for binding and displaces a fluorophore from the receptor, though elegant work has been done in this area (Lavigne and Anslyn, 2001; Wiskur and Anslyn, 2001; Springsteen and Wang, 2001; Cabell et al., 2001). A key aspect of chemosensor architecture is the attachment or conjugation of a reporter chromophore or fluorophore to a synthetic molecule that is responsible for recognizing the analyte. Recently, there have been important advances in developing sensors for organic analytes based on molecularly imprinted polymers (Subrahmanyanet al., 2000; Appleton and Gibson, 2000), but these materials do not fit our chemosensor concept. Effective sensing materials (optodes) that are not molecular devices can also be prepared by incorporating ionophores and ionizable chromophores or fluorophores in permeable polymers 341

Bell and Hext (Bakker et al., 1997; Murkovic and Wolfbeis, 1997; Spichiger-Keller, 1997; Krause et al., 1999). Finally, cyclodextrins have been used extensively as the recognition component of fluorescent sensor molecules (Ueno, 1993; de Jong et al., 2000; Wang and Ueno, 2000; Narita et al., 2001), but these examples are excluded because cyclodextrins are natural, rather than designed or artifical receptors. As described in the previous section, chemosensors for metal cations have been studied for many years and have been extensively reviewed (Takagi and Ueno, 1984; L/3hr and V/Sgtle, 1985; Czarnik, 1993a,b; Bissel et al., 1993; Fabbrizzi and Pogi, 1995; Dolman et al., 1996; Desvergne and Czamik, 1997; de Silva et al., 1997; Kimura and Koike, 1998a; Fabbrizzi et al., 1998b, 2000; Granda-Vald6s et al., 2000; Yamauchi and Hayashita, 2000; Prodi et al., 2000; Baragossi et al., 2000). While innovative work on chemosensors for metals continues (e.g., Hayashita et al., 2000; Bronson et al., 2001; Baxter, 2001; Raker and Glass, 2001; McFarland and Finney, 2001; Mello and Finney, 2001), the relative maturity of this field of research can be seen from the use of chromogenic chemosensors in clinical chemistry (e.g., Kumar et al., 1988; Chapoteau et al., 1993) and application of fluorescent chemosensors as intracellular probes for metals (e.g., Tsien, 1993; Zalewski et al., 1994; Lakowicz, 1999). The following examples have been selected from the recent literature to illustrate the chemosensor approach to detecting neutral and anionic organic analytes, as well as inorganic anions. Analytes of biological interest, such as carbohydrates, phosphates and blood metabolites, are highlighted. In most cases, performance characteristics including sensitivity, selectivity, and absorption or emission wavelengths must be improved to enable practical application. Nevertheless, these challenges can be met by tuning chemosensor structure by means of the power of organic synthesis.

3.1. Chemosensors for carbohydrates Development of chemosensors for biologically important carbohydrates has become a target for many research groups over the last decade. In general, there have been two approaches to the problem. The first approach is based on a rapid and reversible covalent bond formation process (James et al., 1996a,b; Shinkai and Takeuchi, 1996; Shinkai et al., 2000). The second approach utilizes hydrogen-bonding interactions to recognize the carbohydrate (Davis and Wareham, 1999). Monoboronic acids covalently interact with saccharides in aqueous solutions by formation of the corresponding boronate ester, as shown in Figure 4 with ethylene glycol representing the 1,2-diol unit of saccharides. With phenylboronic acid, this only occurs under basic conditions because esterification is favored when the hydroxyboronate anion is produced. However, if the aromatic ring is 342

Artificial Receptors for Chemosensors

OH

HO

O~

OH

B\

B' ] _

OH

\O I

OH e

/ \

e OH

HO

\ OH

OH

~

e O'~ -

OH

Figure 4. Reversible reaction of phenylboronic acid with 1,2-diols. made sufficiently electron-deficient (lowering the pKa of the boronic acid), hydroxyboronate anion formation can occur at neutral pH. Alternatively, an ortho-anfinomethyl substituent can be attached to the ring, resulting in a Lewis acid-base, boron-nitrogen interaction which significantly lowers the pKa of the boronic acid (Wulff, 1982). The order of affinity of arylboronic acids for monosaccharides is D-fructose > Darabinose > D-mannose > D-glucose. However, for many biological applications a greater degree of selectivity and sensitivity is required, as well as a different specificity. O n e way this can be obtained is by employing suitably designed diboronic acids, with the sugar bridging the space between the two boronic acid groups. This has led to receptors that can sense glucose, for example, with good selectivity and sensitivity. Two glucose chemosensors that have been developed by the groups of Shinkai and Norrild are given as examples, as they are considered by these authors to be significant advances in the field. Anthracene receptors 1 (James et al., 1994) and 2 (Eggert et al., 1999) both give optical responses on complexing monosaccharides at neutral pH (Figure 5). In unbound receptor 1, a PET process quenches the fluorescence of the anthracene moiety by electron transfer from the amino group to the anthracene excited state. When boronic acids form cyclic boronate esters, the Lewis acidity of the boronic acid is enhanced (Lorand and Edwards, 1959). Thus for receptor 1, monosaccharide binding increases the boron-nitrogen interaction, resulting in suppression of the PET process, leading to enhanced fluorescence. For receptor 2, fluorescence of the anthracene moiety is enhanced by saccharide binding, but the reason is unclear. Receptor 1 affords greater increase in fluorescence (7-fold 343

Bell and Hext

)2 N/CH3

HOOH ~B~ .CH3

H;C

1[

B(OH)2

0

HOOH H3C. ~E]/

B;, jCH3

H3C% .B

1 9D-glucose

HO .OH "B'~.OH

HQ.../OH HO--Be

,OH -BeI + glucose

,.-

2 9D-glucose

Figure 5. Two receptors for glucose, and structures of their predicted complexes

2-fold) and greater selectivity for binding of glucose over other monosaccharides, when compared to receptor 2. At pH 7.77 (33.3 % methanol buffer), the stability constants for 1 are: D-glucose (logKa = 3.6); D-allose (logKa = 2.8); D-fructose (logKa - 2.2); D-galactose (logK~ - 2.2). At pH 7.4 the only stability constant reported for 2 is for D-glucose (logK~ = 3.4). However, receptor 2 has the advantage of being water soluble, which is required for many applications. In fact, it has been predicted that a receptor similar to 2 could be used to construct a blood glucose sensor. As shown in Figure 5, the proposed structures of the complexes of these glucose receptors involve the pyranose form for 1 (James et al., 1994) and the furanose form for 2 (Eggert et al., 1999). Norrild has proposed that the optical responses of both receptors involve binding glucose in the furanose form (Bielecki et al., 1999), and there is additional evidence to support this case (Cooper and James, 1998). vs

A "sugar tweezer" that was designed from a boronic-acid-appended ~xoxobis[porphinatoiron(III)] (3, Figure 6) was shown to have high selectivity, as well as the highest known association constant for glucose (Takeuchi et al., 1996a, 1998). Unfortunately, the absorption spectrum of 3 is not significantly affected by saccharide addition, so an optical response would have to be introduced to enable its use as a sensor. The binding of saccharides was 344

Artificial Receptors for Chemosensors

r 8

--

B(OH)3 e

N. ":"' '~/"

(HO

(HO)3B e

3

4

Figure 6. Boronic acid receptors selective for D-glucose (3) and for D-lactulose (4).

observed by circular dichroism spectroscopy, a technique that would be difficult to adapt to a sensor. Also, it would be necessary to lower the pK, of the boronic acid groups in 3 so studies could be performed at physiological pH. Alteration of the distance between two boronic acid groups has also led to receptors for small saccharides (James et al., 1997), as well as di- and trisaccharides. An example of a receptor which selectivity binds D-lactulose in methanol is tetraarylporphyrin 4, also shown in Figure 6 (Kijima et al., 1998). Receptors which discriminate between the two enantiomers of a sugar have also been synthesized, though overall the selectivity between two different sugars is not great (Takeuchi et al., 1997a; Mizuno et al., 1999, 2000). Most carbohydrate receptors based on the hydrogen-bonding approach have been studied by either NMR spectroscopy or circular dichroism (Davis and Wareham, 1999). As sugar binding was apparently not amenable to study by either UVvisible or fluorescence spectroscopy, these receptors will not be discussed in this chapter. However, in the case of four receptors, two of which are shown in Figure 7, the binding of carbohydrates was monitored by UV-visible spectroscopy (Rusin and Kr~d, 1999; Kr~il et al., 2001), and for one of these (receptor 5) fluorescence spectroscopy was used as well. These receptors show good affinity for some saccharides, even in polar solvents, though overall the selectivity is poor. In DMSO (containing 5% methanol) as measured by fluorescence spectroscopy, some association constants for 5 are: o-fructose (log/ 106 M "1) causes an enhancement in fluorescence intensity (X~m= 336 nm). Also in acetonitrile, bis(2-hydroxynaphthalimides) 30 and 31 show fluorescence enhancement upon binding acetate, HzPO4 or 1:r (Yoshida et al., 2000), but negligible response to CI' Br, I or C104-. The response mechanism apparently involves deprotonation of one naphthol unit. Again, fluorescent chemosensors 28-31 require UV excitation (e.g., 314 nm for 28).

356

Artificial Receptors for Chemosensors 0

NH 0

0

HN

0

26

H~'N O / ~/H N/ H

CH3 I s , ~ N~H

~

H'N~--~O HN

N~.H

27

28

,.)

H N~R ====/ \

s

29

OH 0 OH 0

R = 1-naphthyl 31 R = CH2[CH2OCH2]CH2

30

Figure 18. Hydrogen bonding fluorescent chemosensors. Ion pairing with anions can affect the electronic properties of cationic metal complexes, and Beer (1998) has demonstrated that pendant hydrogen bond donors can produce electrochemical and fluorescence sensors having a degree of selectivity (Beer et al., 1996). Of particular significance is the calix[4]arenebridged ruthenium(H) tris(bipyridyl) complex 32 (Figure 19), which binds H2PO4 (Ka = 2.8 x 104 M -I) in DMSO selectively over CI (1.6 x 103 M 1) or Br (3.6 x 10z M I) (Szemes et al., 1996). The metal to ligand charge transfer emission is shifted by H2PO4 from 640 to 622 nm and the fluorescence quantum yield is increased, apparently as a consequence of rigidification. Also shown in Figure 19 is a unique chemosensor (33) that combines metal ligand coordination with recognition by hydrogen bond donor sites (Cabell et al., 2001). Prior 357

Bell and Hext

t-Bu

t-Bu

t-Bu t-Bu 5"

0

0 0 H H

( " 0

N--H

I

O

)

H--

0

~ 0

cu(ll)

H\N HH

_

H--N

H\ H'

33

32

Figure 19. Fluorescent chemosensors consisting of metal complexes with hydrogen bond donor sites for anion recognition.

complexation of Cu(II) causes metal-induced quenching of the 1,10phenanthroline fluorescence centered at 365 nm. Binding of citrate partially restores fluorescence and model studies were done to show that this effect is not caused by stripping of Cu(II) from the phenanthroline moiety. The fluorosensor shows linear response to aqueous citrate concentrations in the #M range and can be used to measure citrate in commercial beverages. Macrocyclic polypyrroles, such as expanded porphyrins (Sessler et al., 1999) and calixpyrroles (Miyaji et al., 1999; Anzenbacher et al., 2000a,b), bind anions and have been investigated as optical chemosensors by the Sessler group. For example, enhancement of the long wavelength (ca. 680 nm, ~x = 450 nm) emission of sapphyrin 34 (Figure 20) can be used for aqueous phosphate measurement in the mM range (Sessler et al., 1999). Fluorinated calix[4]pyrrole 35 (Figure 20) binds H2PO4 more strongly than its nonfluorinated analog, and fluorinated dipyrrol-2-ylquinoxaline 36 can be used as a chromogenic or fluorescence quenching sensor for F-, CI and H2POs in dichloromethane (Anzenbacher et al., 2000b). Perhaps of greater significance are a series of "second generation" calixpyrrole chemosensors (37, Anzenbacher et al., 2000a) that display higher anion affinities than previous systems bearing anthracene 358

Artificial Receptors for Chemosensors

E

F

F F

F

~"~'/"'~ ~

NH

F 0

OH

36

OH

35

i--t,,,NtFI

OH OH 34

FI = fluorophore 37

Figure 20. Polypyrrole receptors and chemosensors for anions.

fluorophores (Miyaji et al., 1999). Calix[4]pyrroles of type 37 with sulfonamidelinked dansyl, lissamine-rhodamine B, or thiourea-linked fluorescein fluorophores (F1) showed fluorescence quenching with F , CI, H2PO4 and HP2073. They all show high selectivity for phosphate vs. chloride, and the thiourea-linked fluorescein system binds phosphate with high affinity in 96:4 acetonitrile/water at pH 7. Adenosine 5'-triphosphate (ATP) is an anion of biological relevance, and several approaches have been used to devise optical chemosensors for measuring ATP concentrations in water. Examples are a linear polyamine attached to anthracene (Albelda et al., 1999), cyclam appended to a ruthenium(H) bis(terpyridyl) luminophore (Padilla-Tosta et al., 2000) and the corresponding Cu(II)/cyclam complex (Padilla-Tosta et al., 2001). Nishizawa et al. (1999) have used pyrophosphate (P2074) complexation of a simple guanidinium-substituted pyrene to influence the intensity of excimer emission, and this approach could be applied to ATP. Another interesting approach involves the displacement of an anionic fluorophore tethered to a cationic site similar to that in 33 (Figure 19). Combinatorial chemistry was used to vary the structure of the peptide tether in 359

Bell and Hext order to enhance ATP binding in water (Ka = 3.4 x 103 M-a; Schneider et al., 2000). It is clear that efforts will continue to improve affinity, selectivity and optical response in chemosensors for this important analyte.

4. Advantages and Limitations of Chemosensors When compared to biosensors, chemosensors potentially offer many advantages. A receptor can be tailored for non-biological analytes, examples being 21 and 22, (Figure 16) which have been shown to sense aromatic carboxylates and azide respectively (Fabbrizzi et al., 1998a, 1999). As stated in Section 1, chemosensors are generally more robust than biosensors toward chemical and thermal degradation. They are stable indefinitely and can operate under diverse conditions. Properties of the receptor, including affinity, selectivity, optical response, and absorption and emission wavelengths, can be tuned for the application desired. For example, Shinkai and co-workers (James et al., 1994) prepared a sensor that can detect glucose at physiological levels (in blood, 0.3 1.0 mM), which is often the primary aim of many researchers. However, lowering selectivity and sensitivity by altering the structure of the receptor gave a chemosensor that can detect the overall saccharide concentration at a higher level (ca. 10 mM, Linnane et al., 1995). This may be of use when confectionery is formulated in industry. Modifying the receptor also can allow the chemist to alter solubility, kinetics of binding, and provide a site to attach a tether required for immobilization. Incorporation of an intrinsic chromophore or fluorophore provides a sensor with good communication of the binding event. As stated previously in Section 1.3, modification of a biosensor so that the binding site includes an optical reporter usually damages its molecular recognition capabilities. Finally, a chemosensor can work in a non-aqueous environment, even the gas phase, in principle. The main limitation of chemosensors is that they often do not possess sufficient sensitivity or selectivity for the application desired. The design of artificial receptors having high affinity and selectivity toward the target analyte is not reliable and requires a process of trial and error. Synthesis of each target chemosensor is also a tedious process.

5. Potential for Expanding Current Capabilities As stated in the previous section, affinities and selectivities of chemosensors (particularly in water) generally need to be improved, especially for biological applications. When combined with the fact that carefully designing and synthesizing a target receptor may not produce a potential chemosensor for the target analyte, it is clear that a better process for predicting the performance of designed chemosensors is desirable. Many aspects of chemosensor design might 360

Artificial Receptors for Chemosensors be addressed through computer modeling. The ideal situation would be to input the analyte of interest, along with characteristics required for the chemosensor, and the program would then design an optimum compound. Obviously, this level of predictability will not be achieved in the near future. However, the development of computer programs to screen various hypothetical receptors at a higher level of theory than simple molecular mechanics (Burkert and Allinger, 1982) may be feasible in the near future. Another possible solution would be to employ the power of combinatorial chemistry, which has revolutionized drug discovery (Seneci, 2000). Then a vast array of receptors could be generated and tested against the analyte of interest (Leipert et al., 1999). Fortunately, a lot of progress is being made in the synthesis of non-peptide molecules using the combinatorial approach, but the range of molecular structures that can be synthesized on solid supports need to be expanded. As noted in Section 3.2, reverse logic employing this technology was used to design chemosensor 17 for a tripeptide sequence (Chen et al., 1998). The approach of Suslick (Rakow and Suslick, 2000), in which the response of an array of chemosensors was shown to be unique for a chemical class of compounds, may also prove useful. This strategy may overcome problems with poor selectivities of individual chemosensors, but affinities available from current technology would have to be improved. Ultimately, this leads to the concept of a system similar to the "artificial tongue" (Lavigne and Anslyn, 2001) or nose (Dickinson et al., 1996). Currently, a lot of interest is being shown in the development of sensors to detect the vapors emitted from landmines (Yang and Swager, 1998), and this technology may prove adaptable for other classes of analytes. Significant progress in the employment of chemosensors in research and industrial settings has occurred, particularly in the area of detecting metal cations (Section 2). Recently the detection of neutral organic molecules and anions has been explored, as described in this chapter. Of particular current interest is the development of a chemosensor for monitoring blood glucose levels in diabetes patients. Two target applications are in vivo monitoring of glucose, which would allow automatic injection of insulin from a small portable device carried on the person (type I diabetes), and regular checking of blood/urine in vitro in type II diabetes patients. Note, as the level of glucose in the two cases are different (0.31.0 vs 10 mM), two different chemosensors are desired (James et al., 1994; Linnane et al., 1995). The examples in Section 3 of this chapter illustrate the range of analyte structures and chemosensor designs that have already been addressed with the artificial receptor approach. Overall, the sensitivities and selectivities of these chemosensors are weaker than biosensors, when available for the same analytes. On the other hand, the wide variability of the molecular structures of artificial 361

Bell and Hext receptors and of the mechanisms for optical response indicate that artificial chemosensors can effectively compete with biosensors for many applications. By and large, the work published to date in this field emanates from academic laboratories and focuses on conceptual, rather than practical, advances. In particular, more attention needs to be directed to the problems of water compatibility, absorption and emission wavelengths, and methods for immobilization to produce practical sensors. The design of optical chemosensors is still a relatively new concept, and much work lies ahead in the application of these molecules to detection and quantitation of analytes in biomedical, environmental, and industrial fields of interest.

6. References

Albelda, M. T., M.A. Bernardo, E. Garcfa-Espafia, M.L. Godino-Salido, S.V. Luis, M.J. Melo, F. Pina and C. Soriano, 1999, J. Chem. Soc. Perkin Trans. 2, 2545. Anzenbacher, P., Jr., K. Jurs~owi and J.L. Sessler, 2000a, J. Am. Chem. Soc. 122, 9350. Anzenbacher, P., Jr., A.C. Try, H. Miyaji, K. Jursikowi, V.M. Lynch, M. Marquez and J.L. Sessler, 2000b, J. Am. Chem. Soc. 122, 10268. Appleton, B. and T.D. Gibson, 2000, Sens. Actuators B 65, 302. Bakker, E., P. Btihlmann and E. Pretsch, 1997, Chem. Rev. 97, 3083. Balzani, V., A. Credi, F.M. Raymo and J.F. Stoddart, 2000, Angew. Chem. Int. Ed. 39, 3348. Bargossi, C., M.C. Fiorini, M. Montalti, L. Prodi and N. Zaccheroni, 2000, Coord. Chem. Rev. 208, 17. Baxter, P. N. W., 2001, J. Org. Chem. 66, 4170. Beer, P. D., 1998, In Molecular Recognition and Inclusion, Ed., A.W. Coleman, Kluwer, Dordrecht, p. 97. Beer, P. D., S.W. Dent, N.C. Fletcher and T.J. Wear, 1996, Polyhedron 15, 2983. Bell, T. W. and Z. Hou, 1997, Angew. Chem. Int. Ed. 36, 1536. Bell, T. W., D.L. Beckles, P.J. Cragg, J. Liu, J. Maioriello, A.T. Papoulis and V.J. Santora, 1993, In Fluorescent Chemosensors for Ion and Molecule Recognition, Chap. 7, Ed., A.W. Czamik, ACS Symposium Series 538, American Chemical Society, Washington, DC, pp. 85-103. Bell, T. W., Z. Hou, Y. Luo, M.G.B. Drew, E. Chapoteau, B.P. Czech and A. Kumar, 1995, Science 269, 671. Bianchi, A., K. Bowman-James and E. Garcfa-Espafia, Eds., 1997, Supramolecular Chemistry of Anions, John Wiley Sons, New York. Bielecki, M., H. Eggert and J.C. Norrild, 1999, J. Chem. Soc. Perkin Trans. 2, 449. Bissell, R. A., A.P. de Silva, H.Q.N. Gunaratne, P.L.M. Lynch, G.E.M. Maguire, C.P. McCoy and K.R.A.S. Sandanayake, 1993, In Topics in Current Chemistry, Vol. 168, Ed., J. Mattay, Springer-Verlag, Berlin, p. 223. 362

Artificial Receptors for Chemosensors Bradshaw, J. S. and R.M. Izatt, 1997, Acc. Chem. Res. 30, 338. Bradshaw, J. S., R.M. Izatt, A.V. Bordunov, C.Y. Zhu and J.K. Hathaway, 1996, In Comprehensive Supramolecular Chemistry, Vol. 1, Pergamon, p. 35. Bronson, R. T., J.S. Bradshaw, P.B. Savage, S. Fuangswasdi, S.C. Lee, K.E. Krakowiak and R.M. Izatt, 2001, J. Org. Chem. 66, 4752. Burkert, U. and N.L. Allinger, 1982, Molecular Mechanics, ACS Monograph 177, American Chemical Society, Washington, DC, 339 pp. Cabell, L. A., M.D. Best, J.J. Lavigne, S.E. Schneider, D.M. Perreault, M.-K. Monahan and E.V. Anslyn, 2001, J. Chem. Soc. Perkin Trans. 2, 315. Chapoteau, E., B.P. Czech, W. Zazulak and A. Kumar, 1993, Clin. Chem. 39, 1820. Chawla, H.M. and K. Srinivas, 1994, J. Chem. Soc. Chem. Commun. 2593. Chen, C.-T., H. Wagner and W.C. Still, 1998, Science 279, 851. Cloninger, M. J. and H.W. Whitlock, 1998, J. Org. Chem. 63, 6153. Cooper, C. R. and T.D. James, 1997, J. Chem. Soc. Chem. Commun. 1419. Cooper, C. R. and T.D. James, 1998, Chem. Lett., 883. Cram, D. J. and J.M. Cram, 1978, Acc. Chem. Res. 11, 8. Cram, D. J., 1986, Angew. Chem. Int. Ed. Engl., 25, 1039. Cram, D. J., 1988, Science, 240, 760. Czarnik, A. W., Ed., 1993a, Fluorescent Chemosensors for Ion and Molecule Recognition, ACS Symposium Series 538, American Chemical Society, Washington, DC. Czarnik, A. W., 1993b, In Advances in Supramolecular Chemistry, Vol. 3, Ed., G.W. Gokel, JAI Press, Greenwich, Connecticut, p. 131. Czarnik, A. W., 1994, Acc. Chem. Res., 27, 302. Czarnik, A. W. and J. Yoon, 1999, In Supramolecular Technology, Chap. 4, Ed., D.N. Reinhoudt, Wiley Sons, Chichester. Davis, A. P. and R.S. Wareham, 1999, Angew. Chem. Int. Ed. 38, 2978. de J ong, M. R., J.F.J. Engbersen, J. Huskens and D.N. Reinhoudt, 2000, Chem. Eur. J. 6, 4034. De Santis, G., L. Fabbrizzi, M. Licchelli, A. Poggi and A. Taglietti, 1996, Angew. Chem. Int. Ed. Engl. 35, 202. de Silva, A. P., H.Q.N. Gunaratne, C. McVeigh, G.E.M. Maguire, P.R.S. Maxwell and E. O'Hanlon, 1996, J. Chem. Soc. Chem. Commun. 2191. de Silva, A. P., H.Q.N. Gunaratne, T. Funnlaugsson, A.J.M. Huxley, C.P. McCoy, J.T. Rademacher and T.E. Rice, 1997, Chem. Rev. 97, 1515. Desvergne, J. P. and A.W. Czamik, Eds., 1997, Chemosensors of Ion and Molecule Recognition, NATO ASI Series C: Mathematical and Physical Sciences, Vol. 492, Kluwer, Dordrecht. Deviprasad, G. R. and F. D'Souza, 2000, Chem. Commun. 1915. Deviprasad, G. R., B. Keshavan and F. D'Souza, 1998, J. Chem. Soc. Perkin Trans. 1, 3133. Dickinson, T. A., J. White, J.S. Kauer and D.R. Walt, 1996, Nature, 382, 697. Dolman, M., A.J. Mason, K.R.A.S. Sandanayake, A. Sheridan, A.F. Sholl and I.O. Sutherland, 1996, Analyst 121, 1775. 363

Bell and Hext D'Souza, F., 1996, J. Am. Chem. Soc. 118, 923. D'Souza, F. and G.R. Deviprasad, 2001, J. Org. Chem. 66, 4601. D'Souza, F., G.R. Deviprasad and Y.-Y. Hsieh, 1997, J. Chem. Soc. Chem. Commun. 533. Eggert, H., J. Frederiksen, C. Morin and J.C. Norrild, 1999, J. Org. Chem. 64, 3846. Fabbrizzi, L. and A. Poggi, 1995, Chem. Soc. Rev. 24, 197. Fabbrizzi, L., G. Francese, M. Licchelli, P. Pallavicini, A. Perotti, A. Poggi, D. Sacchi and A. Taglietti, 1997a, In Chemosensors of Ion and Molecule Recognition, Eds., J.P. Desvergne and A.W. Czarnik, Kluwer, The Netherlands, p. 75. Fabbrizzi, L., G. Francese, M. Licchelli, A. Perotti and A. Taglietti, 1997b, Chem. Commun. 581. Fabbrizzi, L., I. Faravelli, G. Francese, M. Licchelli, A. Perotti and A. Taglietti, 1998a, Chem. Commun. 971. Fabbrizzi, L., M. Licchelli, L. Parodi, A. Poggi and A. Taglietti, 1998b, J. Fluoresc. 8, 263. Fabbrizzi, L., M. Licchelli, L. Parodi, A. Poggi and A. Taglietti, 1999, Eur. J. Inorg. Chem. 35. Fabbrizzi, L., M. Licchelli, G. Rabaioli and A. Taglietti, 2000, Coord. Chem. Rev. 205, 85. Geddes, C. D., 2000, J. Photochem. Photobiol. A Chem. 137, 145. Geddes, C. D., K. Apperson, J. Karolin and D.J.S. Birch, 2001, Anal. Biochem. 293, 60. Gokel, G. W., 1991, Crown Ethers and Cryptands, The Royal Society of Chemistry, Cambridge. Goodman, M. S., A.D. Hamilton and J. Weiss, 1995, J. Am. Chem. Soc. 117, 8447. Grady, T., S.J. Harris, M.R. Smyth and D. Diamond, 1996, Anal. Chem. 68, 3775. Grady, T., T. Joyce, M.R. Smyth, S.J. Harris and D. Diamond, 1998, Anal. Commun. 35, 123. Granda-Vald6s, M., R. Badfa, G. Pina-Luis, M.E. Dfaz-Garcfa, 2000, Qufmica Analftica 19 [Suppl. 1], 38. Hancock, R. D. and A.E. Martell, 1988, Comments Inorg. Chem. 6, 237. Hayashita, T., S. Taniguchi, Y. Tanamura, T. Uchida, S. Nishizawa, N. Teramae, Y.S. Jin, J.C. Lee and R.A. Bartsch, 2000, J. Chem. Soc. Perkin Trans. 2, 1003. Helgeson, R. C., B.P. Czech, E. Chapoteau, C.R. Gebauer, A. Kumar and D.J. Cram, 1989, J. Am. Chem. Soc. 111, 6339. Hennrich, G., H. Sonnenschein and U. Resch-Genger, 2001, Tetrahedron Lett. 42, 2805. Huber, C., T. Werner, C. Krause, I. Klimant, O.S. Wolfbeis, 1998, Anal. Chim. Acta 364, 143. Hulanicki, A., S. Glab and F. Ingman, 1991, Pure Appl. Chem. 63, 1247. 364

Artificial Receptors for Chemosensors Hunter, C. A., 1993, Angew. Chem. Int. Ed. Engl. 32, 1584. Hunter, C. A. and J.K.M. Sanders, 1990, J. Am. Chem. Soc. 112, 5525. Iorio, E. J. and W.C. Still, 1999, Bioorg. Med. Chem. Lett. 9, 2145. James, T. D., K.R.A.S. Sandanayake and S. Shinkai, 1994, Angew. Chem. Int. Ed. Engl. 33, 2207. James, T. D., K.R.A.S. Sandanayake and S. Shinkai, 1996a, Angew. Chem. Int. Ed. 35, 1910. James, T. D., P. Linnane and S. Shinkai, 1996b, J. Chem. Soc. Chem. Commun. 281. James, T. D., H. Shinmori and S. Shinkai, 1997, J. Chem. Soc. Chem. Commun. 71. Kato, R., S. Nishizawa, T. Hayashita and N. Teramae, 2001, Tetrahedron Lett. 42, 5053. Kijima, H., M. Takeuchi and S. Shinkai, 1998, Chem. Lett. 781. Kimura, E. and T. Koike, 1998a, Chem Soc. Rev. 27, 179. Kimura, E. and T. Koike, 1998b, Chem. Commun. 1495. Kr~il, V., O. Rusin and F.P. Schmidtchen, 2001, Org. Lett. 3, 873. Krause, C., T. Werner, C. Huber, O.S. Wolfbeis and M.J.P. Leiner, 1999, Anal. Chem. 71, 1544. Kubo, Y., 1998, J. Inclusion Phenom. Mol. Recognit. Chem. 32, 235. Kubo, Y., 1999, Synlett 161. Kubo, Y., S. Maruyama, N. Ohhara, M. Nakamura and S. Tokita, 1995, J. Chem. Soc. Chem. Commun. 1727. Kubo, Y., S. Maeda, S. Tokita and M. Kubo, 1996, Nature 382, 522. Kubo, Y., S. Maeda, S. Tokita and M. Kubo, 1997, Enantiomer 2, 287. Kubo, Y., N. Hirota, S. Maeda and S. Tokita, 1998, Anal. Sci. 14, 183. Kubo, Y., M. Tsukahara, S. Ishihara and S. Tokita, 2000, J. Chem. Soc. Chem. Commun. 653. Kumar, A., E. Chapoteau, B.P. Czech, C.R. Gebauer, M.Z. Chimenti, O. Raimondo, 1988, Clin. Chem. 34, 1709. Laatikainen, R., J. Ratilainen, R. Sebastian and H. Santa, 1995, J. Am. Chem. Soc. 117, 11006. Lakowicz, J. R., 1999, Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic / Plenum, New York. Lavigne, J. J. and E.V. Anslyn, 2001, Angew. Chem. Int. Ed. 40, 3118. Lee, D. H., K.H. Lee and J.-I. Hong, 2001a, Org Lett. 3, 5. Lee, K. H. and J.-I. Hong, 2000, Tetrahedron Lett. 41, 6083. Lee, K. H., H.-Y. Lee, D.H. Lee and J.-I. Hong, 2001b, Tetrahedron Lett. 42, 5447. Lehn, J.-M., 1995, Supramolecular Chemistry: Concepts and Perspectives, VCH, Weinheim. Lehn, J.-M., 1988, Angew. Chem. Int. Ed. Engl. 27, 89. Lehn, J.-M., 1990, Angew. Chem. Int. Ed. Engl. 29, 1304. Leipert, D., J. Mack, R. Ttinnemann and G. Jung, 1999, Comb. Chem. 335. 365

Bell and Hext Lewis, P. T., C.J. Davis, L.A. Cabell, M. He, M.W. Read, M.E. McCarroll and R.M. Strongin, 2000, Org. Lett. 2, 589. Linnane, P., T.D. James, S. Imazu and S. Shinkai, 1995, Tetrahedron Lett. 36, 8833. L/3hr, H.-G. and F. V/3gtle, 1985, Acc. Chem. Res. 18, 65. Lorand, J. P. and J.O. Edwards, 1959, J. Org. Chem. 24, 769. L/3wik, D. W. P. M., M.D. Weingarten, M. Broekema, A.J. Brouwer, W.C. Still and R.M.J. Liskamp, 1998, Angew. Chem. Int. Ed. 37, 1846. Lustenberger, P., R. Welti and F. Diederich, 1998, Helv. Chim. Acta 81, 2190. Ma, J. C. and D.A. Dougherty, 1997, Chem. Rev. 97, 1303. McFarland, S. A. and N.S. Finney, 2001, J. Am. Chem. Soc. 123, 1260. Mei, M. and S. Wu, 2001, New J. Chem. 25, 471. Mello, J. V. and N.S. Finney, 2001, Angew. Chem. Int. Ed. 40, 1536. Metzger, A. and E.V. Anslyn, 1998, Angew. Chem. Int. Ed. 37, 649. Miyaji, H. and J.L. Sessler, 2001 Angew. Chem. Int. Ed. 40, 154. Miyaji, H., P. Anzenbacher, Jr., J.L. Sessler, E.R. Bleasdale and P.A. Gale, 1999, Chem. Commun. 1723. Mizuno, T., M. Takeuchi and S. Shinkai, 1999, Tetrahedron 55, 9455. Mizuno, T., M. Yamamoto, M. Takeuchi and S. Shinkai, 2000, Tetrahedron 56, 6193. Mohr, G. J. and O.S. Wolfbeis, 1995, Anal. Chim. Acta 316, 239-246. Mohr, G. J. and O.S. Wolfbeis, 1996a, Sens. Actuators B 37, 103. Mohr, G. J. and O.S. Wolfbeis, 1996b, Analyst 121, 1489. Mohr, G. J. and U.E. Spichiger-Keller, 1997, Anal. Chim. Acta 351, 189. Mohr, G. J., D. Citterio and U.E. Spichiger-Keller, 1998a, Sens. Actuators B 49, 226. Mohr, G. J., C. Demuth and U.E. Spichiger-Keller, 1998b, Anal. Chem. 70, 3868. Mohr, G. J., I. Murkovic, F. Lehmann, C. Haider and O.S. Wolfbeis, 1997, Sens. Actuators B 38-39, 239. Mohr, G. J., U.E. Spichiger-Keller, W. Jona and H. Langhals, 2000, Anal. Chem. 72, 1084. Murkovic, I. and O.S. Wolfbeis, 1997, Sens. Actuators B 38-39, 246. Narita, M., S. Mima, N. Ogawa and F. Hamada, 2001, Anal. Sci. 17, 379. Niikura, K. and E.V. Anslyn, 1999, J. Chem. Soc. Perkin Trans. 2, 2769. Nishizawa, S., R. Kato, T. Hayashita and N. Teramae, 1998a, Anal. Sci. 14, 595. Nishizawa, S., H. Kaneda, T. Uchida and N. Teramae, 1998b, J. Chem. Soc. Perkin Trans. 2, 2325. Nishizawa, S., Y. Kato and N. Teramae, 1999, J. Am. Chem. Soc. 121, 9463. Padilla-Tosta, M. E., J.M. Lloris, R. Martfnez-M~ifiez, T. Pardo, J. Soto, A. Benito and M.D. Marcos, 2000, Inorg. Chem. Commun. 3, 45. Padilla-Tosta, M. E., J.M. Lloris, R. Martfnez-M~ifiez, T. Pardo, F. Sancen6n, J. Soto and M.D. Marcos, 2001, Eur. J. Inorg. Chem. 1221. Parker, D., K. Senanayake and J.A.G. Williams, 1997, Chem. Commun. 1777. Pedersen, C. J., 1984, Science 241,536. 366

Artificial Receptors for Chemosensors Pina, F., M.A. Bemardo and E. Garcia-Espafia, 2000, Eur. J. Inorg. Chem. 2143. Prodi, L., F. Bolletta, M. Montalti and N. Zaccheroni, 2000, Coord. Chem. Rev. 2O5, 59. Raker, J. and T.E. Glass, 2001, J. Org. Chem. 66, 6505. Rakow, N. A. and K.S. Suslick, 2000, Nature 406, 710. Rebek, J., Jr., 1988, In Topics in Current Chemistry, Vol. 149, Ed., E. Weber, Springer-Verlag, Berlin, p. 189. Rebek, J., Jr., 1990, Acc. Chem. Res. 23, 399. Reichardt, C., 1979, In Monographs in Modem Chemistry, Vol. 3,Verlag Chemie, Weinheim, Chap. 6. Reinhoudt, D. N. and P.J. Dijkstra, 1988, Pure Appl. Chem. 60, 477. Rettig, W., 1994, In Topics in Current Chemistry, vol. 169, Ed., J. Mattay, Springer-Verlag, Berlin, p. 253. Rothman, J. H. and W.C. Still, 1999, Bioorg. Med. Chem. Lett. 9, 509. Rusin, O. and V. Kr~il, 1999, J. Chem. Soc. Chem. Commun. 2367. Ryan, K. and W.C. Still, 1999, Bioorg. Med. Chem. Lett. 9, 2673. Ryan, K., L.J. Gershell and W.C. Still, 2000, Tetrahedron 56, 3309. Sauvage, J.-P., Ed., 2001, Molecular Machines and Motors, Springer-Verlag, Berlin. Scheerder, J., J.F.J. Engbersen and D.N. Reinhoudt, 1996, Rec. Trav. Chim. Pays-Bas 115,307. Schmidtchen, F. P. and M. Berger, 1997, Chem. Rev. 97, 1609. Schneider, H.-J., 1991, Angew. Chem. Int. Ed. Engl. 30, 1417. Schneider, H.-J. and A. Yatsimirsky, 2000, Principles and Methods in Supramolecular Chemistry, John Wiley Sons, Chichester. Schneider, S. E., S.N. O'Neil and E.V. Anslyn, 2000, J. Am. Chem. Soc. 122, 542. Sen, A. and K.S. Suslick, 2000, J. Am. Chem. Soc. 122, 11565. Seneci, P., 2000, Solid-Phase Synthesis and Combinatorial Technologies, WileyInterscience, New York, 637 pp. Sessler, J. L., N.A. Tvermoes, J. Davis, P. Anzenbacher, Jr., K. Jursl'kov~i, W. Sato, D. Seidel, V. Lynch, C.B. Black, A. Try, B. Andrioletti, G. Hemmi, T.D. Mody, D.J. Magda and V. Kr~il, 1999, Pure Appl. Chem. 71, 2009. Shinkai, S. and M. Takeuchi, 1996, Trends Anal. Chem. 15,418. Shinkai, S., M. Takeuchi and A. Ikeda, 2000, In Polymer Sensors and Actuators, Eds. Osada, DeRossi and Danilo, Springer, Berlin, Chap. 6. Spichiger-Keller, U.E., 1997, Sens. Actuators B 38-39, 68. Springsteen, G. and B. Wang, 2001, Chem. Commun. 1608. Steed, J. W. and J.L. Atwood, 2000, Supramolecular Chemistry, John Wiley Sons, Chichester. Subrahmanyam, S., S.A. Piletsky, E.V. Piletska, B. Chen, R. Day and A.P.F. Turner, 2000, Adv. Mater. 12, 722. Szemes, F., D. Hesek, Z. Chert, S.W. Dent, M.G.B. Drew, A.J. Goulden, A.R. Graydon, A. Grieve, R.J. Mortimer, T. Wear, J.S. Weightman and P.D. Beer, 1996, Inorg. Chem. 35, 5868. 367

Bell and Hext Takagi, M. and K. Ueno, 1984, In Topics in Current Chemistry, Vol. 121: Host Guest Complex Chemistry m, Eds., F. Voegtle and E. Weber, SpringerVerlag, Berlin, p. 39. Takeuchi, M., T. Imada and S. Shinkai, 1996a, J. Am. Chem. Soc. 118, 10658. Takeuchi, M., M. Taguchi, H. Shinmori and S. Shinkai, 1996b, Bull. Chem. Soc. Jpn. 69, 2613. Takeuchi, M., S. Yoda, T. Imada and S. Shinkai, 1997a, Tetrahedron 53, 8335. Takeuchi, M., M. Yamamoto and S. Shinkai, 1997b, J. Chem. Soc. Chem. Commun. 1731. Takeuchi, M., T. Imada and S. Shinkai, 1998, Bull. Chem. Soc. Jpn. 71, 1117. Tsien, R. Y., 1993, In Fluorescent Chemosensors for Ion and Molecule Recognition, Ed., A . W . Czamik, American Chemical Society, Washington, DC, Chap. 9. Ueno, A., 1993, In Fluorescent Chemosensors for Ion and Molecule Recognition, Ed., A.W. Czarnik, American Chemical Society, Washington, DC, Chap. 6. Vt~gtle, F., 1991, Supramolecular Chemistry: An Introduction, John Wiley Sons, Chichester. Wang, J. and A. Ueno, 2000, Macromol. Rapid Commun. 21,887. Wiskur, S. L. and E.V. Anslyn, 2001, J. Am. Chem. Soc. 123, 10109. Wulff, G., 1982, Pure Appl. Chem. 54, 2093. Xie, H., S. Yi, X. Yang and S. Wu, 1999, New J. Chem. 23, 1105. Yamamoto, M., M. Takeuchi and S. Shinkai, 1998, Tetrahedron 54, 3125. Yamauchi, A. and T. Hayashita, 2000, Bunsen Kagaku 49, 75 (in Japanese). Yang, J.-S. and T.M. Swager, 1998, J. Am. Chem. Soc. 120, 5321. Yoshida, H., K. Saigo and K. Hiratani, 2000, Chem. Lett., 116. Zalewski, P. D., I.J. Forbes, R.F. Seamark, R. Borlinghaus, W.H. Betts, S.F. Lincoln and A.D. Ward, 1994, Chem. Biol. 1,153.

368

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 92002 Elsevier Science B.V. All fights reserved

CHAPTER 12

NUCLEIC ACIDS FOR REAGENTLESS BIOSENSORS

MANJULA RAJENDRAN AND ANDREW D. ELLINGTON, PH.D.

Department of Chemistry and Biochemistry University of Texas at Austin, Austin, TX 78712 USA

In vitro selection has yielded a range of nucleic acid binding species (aptamers) and catalysts (ribozymes) whose ligand-binding affinities and activation parameters rival those of proteins. Precisely because functional nucleic acids can be engineered based largely on an appreciation of their secondary structures and chemically synthesized in bulk, it has proven remarkably easy to incorporate aptamers and ribozymes into reagentless biosensors that directly transduce ligand recognition to optical signals. Aptamers undergo conformational changes upon interaction with their cognate ligands, and, by appending fluorophores to aptamers, it has proven possible to generate 'signaling aptamers.' Ribozymes can act on fluorescent substrates to generate fluorescent signals; any molecules or processes that affect ribozyme catalysis can therefore be reported. Aptamers can also be appended to ribozymes to generate aptazymes, or effector-activated ribozymes, that transduce molecular recognition to ribozyme catalysis. Aptazymes have proven remarkably plastic and can be activated by metals, small organic molecules,~peptides and proteins. By simply cleaving apart or adjoining fluorophores and quenchers on oligonucleotide substrates, one can potentially construct reagentless ribozyme or aptazyme chips that could simultaneously report the concentration of multiple different analytes. Prototypes of such chips have now been made. While reagentless nucleic acid biosensors may ultimately prove less sensitive or robust than reagentless protein biosensors, it is nonetheless likely that nucleic acid biosensors will prove much more amenable to generation by high-throughput selection methods, and thus may be the best vehicle for developing chips that can acquire organismal proteomes and metabolomes.

369

Rajendran and Ellington 1. The Concept of a Reagentless Biosensor Reagentless biosensors are sensors which can detect a target analyte in a homogenous format in solution; that is, without the addition of reagents other than the sample. A reagentless biosensor has several advantages over conventional diagnostic assays. The first and most obvious advantage of a reagentless biosensor is its simplicity and ease of use. Reagentless biosensors should prove to be intrinsically practical in a variety of settings, from the detection of hazardous biological agents in the field to monitoring blood glucose levels in a hospital setting to providing real-time readouts of cellular states in a research lab. Second, since the detection process does not require the addition of reagents, the system remains unperturbed throughout the course of the assay. This is of particular importance for the in vivo detection and quantitation of analytes. Third, because reagentless biosensors directly transduce the sensitivity and specificity of biomolecular detection to the production of a signal, they can potentially be integrated with any number of detection platforms. Finally, as a consequence of their ease of use, utility, and adaptability, it is expected that reagentless biosensors may prove to be significantly more cost-effective than conventional diagnostic assays. As alluded to above, reagentless biosensors can be constructed by integrating a signaling or reporter component with a biological macromolecule, allowing the biomolecule to directly transduce the molecular recognition event into a detectable signal (e.g., an optical or electrochemical signal). The simplest reagentless optical biosensors can be envisioned as fluorescentlylabeled macromolecules. For example, many proteins undergo ligand-induced conformational changes. Fluorophores incorporated at specific sites on such molecules can be used to sensitively transduce molecular recognition events; as an example, the maltose binding protein has been derivatized with fluorescent dyes, and the hinge-bending motion that occurs upon interaction with maltose can be used to quantitate maltose levels in solution (Hellinga and Marvin, 1998; Marvin et al., 1997). The reason such sensors work is that fluorophores are frequently sensitive to minute changes in their chemical environments, and such changes are reported as changes in fluorescence intensity, wavelength, or anisotropy. Alternately, changes in fluorescence-resonance energy transfer between pairs of fluorophores, or between fluorophores and quenchers, can be used to detect binding. Similarly, reagentless electrochemical biosensors have been made by electrostatic self-deposition of redox polyelectrolyte mediators and enzymes (Leech and Daigle, 1998; Mulchandani and Pan, 1999; Narvaez et al., 2000). The enzymes turnover analytes (substrates) and produce electrons that are in turn coupled to electrodes via the adjacent mediators. Such integrated molecular 370

Nucleic

Acids for Reagentless Biosensors

sensors are not only inherently more practical, but have enhanced sensitivity and speed of response.

2. H i s t o r y - In Vitro Selection

Aptamers are single stranded nucleic acids (RNA, ssDNA, modified RNA or ssDNA), capable of binding tightly and specifically to their targets. They are isolated from combinatorial oligonucleotide libraries by a process known as in vitro selection. In vitro selection mimics the process of natural evolution inthat a pool of nucleic acids are sieved for a desired functional property, such as the ability to bind to a target or catalyze a reaction. Once functional species have been isolated, they are preferentially amplified via conventional molecular biology techniques, such as reverse transcription, polymerase chain reaction, and in vitro transcription. Over multiple rounds of selection and amplification, quite large populations (> 1013 different sequences) can be sieved and those few, "fittest" nucleic acid species isolated. The in vitro evolution of nucleic acids was first reported by Sol Spiegelman in the 1960's. Spiegelman and his co-workers established a cell-free system in which the genomic RNA of bacteriophage Q/3 could be evolved. For example, in a serial dilution experiment using purified Qfl replicase the genomic RNA was evolved to replicate more quickly (Mills et al., 1967; Spiegelman, 1971). While other phenotypes, such as resistance to ethidium bromide (Spiegelman , 1971) were also evolved, these in vitro selection methods may have ultimately been limited in scope because the diversity of the RNA population was dictated solely by the error rate of the polymerase. More practical approaches to in vitro selection became possible in the 1990's with the advent of solid phase DNA synthesis, and the invention of the polymerase chain reaction (PCR). Using solid phase DNA synthesis, synthetic oligonucleotides with randomized regions could be generated and large nucleic acid libraries (up to 10~5 species) that contained extremely diverse sequences could be obtained. In 1989, Kevin Struhl and co-workers reported the identification of double-stranded DNA binding species from a random library (Oliphant et al., 1989). In 1990, two separate groups reported the in vitro selection of RNA binding species. Tuerk and Gold (1990) started with a library derived from the natural RNA substrate of T4 DNA polymerase and returned both wild-type and non-wild-type winners. Ellington and Szostak (1990) evolved nucleic acid ligands for targets with no previously known nucleic acid affinity, starting from a library with 100 randomized positions. The first selection of a ribozyme from a randomized population was reported by the Joyce group in the same year (Robertson and Joyce, 1990); they evolved a natural ribozyme, the Tetrahymena self-splicing intron, to carry out a novel DNA cleavage reaction. 371

Rajendran and Ellington

Figure 1. Scheme for in vitro selection. A chemically synthesized, single-stranded DNA pool is PCR amplified and transcribed in vitro to generate an RNA pool. Target molecules are incubated with the pool. Those sequences and shapes that can interact with the target are sieved from the population by one of a variety of methods (affinity chromatography, filter capture). The captured sequences are eluted, amplified, and subjected to additional rounds of selection and amplification. 372

Nucleic Acids for Reagentless Biose.nsors

-[I

......! !!

! I i i 1~

-.-/ Molecular Beacon

Target

Hybrid

Figure 2. Molecular Beacon. A stem-loop structure contains a fluorophore (red) and a quencher (blue). Sequence-specific interactions between the loop and a target nucleic acid results in the formation of an extended helix that pries apart the fluorophore and quencher, resulting in a target-specific increase in fluorescent signal. Contemporary in vitro selection experiments begin with the chemical synthesis of single-stranded DNA libraries. The inclusion of constant sequence regions flanking the random sequence core allows the amplification of such libraries via the polymerase chain reaction, and the conversion of libraries to different chemical forms, for example the conversion of a double-stranded DNA template to single-stranded RNA via in vitro transcription. DNA or RNA libraries can be sieved for a variety of functions, the simplest of which is binding. By passing an RNA library over an affinity column or by collecting protein:RNA complexes via filtration on modified cellulose filters, binding species can be sieved from nonbinding species. Eluted binding species can then be amplified by a combination of reverse transcription, PCR, and in vitro transcription. Multiple cycles of selection and amplification generally result in the purification of those binding species (also known as 'aptamers') that have the highest affinity and specificity for a given target. Aptamers possess many properties, which make them potential candidates for biosensor applications (for recent reviews, see Brody and Gold, 2000; Famulok et al., 2000; Hesselberth et al., 2000a; Jayasena, 1999; Wilson and Szostak, 1999). They are the only reagents that rival antibodies in their universal molecular recognition properties and have been selected against an amazingly wide range of targets, ranging from metal ions (Ciesiolka and Yams, 1996; Kawakami et al., 2000) to complex cellular structures such as the ribosome (Ringquist et al., 1995) and even to whole cells (Morris et al., 1998). In fact, aptamers have been identified which bind to viral particles (Pan et al., 1995) and live pathogenic protozoa (Homann and Goringer, 1999). Also, since aptamers are selected in vitro, they can potentially be raised against pathogens, toxins (e.g., ricin, Hesselberth et al., 2000b), and purportedly to biological warfare agents, targets which frequently prove problematic for in vivo immunization procedures. In addition to their comprehensive binding properties, aptamers have high binding affinities and also remarkable specificities. The binding affinities of aptamers are typically in the nanomolar to picomolar range for protein targets, and in the micromolar to nanomolar range for small organic targets. Aptamers 373

Rajendran and Ellington can discriminate between targets on the basis of subtle differences such as single amino acid changes in protein targets (Conrad et al., 1994; Hirao et al., 1998), or the presence or absence of a methyl (Hailer and Samow, 1997; Jenison et al., 1994) or a hydroxyl group (Mannironi et al., 1997; Sassanfar and Szostak, 1993) in small organic targets. Finally, since aptamers can frequently be minimized to relatively small (30-50 nucleotide) oligonucleotides, they can be chemically synthesized in bulk and modified during chemical synthesis for conjugation or sensor function. The primary limitation on the use of aptamers as recognition and/or transduction elements in biosensors has been the perception that they are unstable and highly susceptible to degradation in biological media. However, the incorporation of modified nucleotides either pre- or post-selection can protect aptamers from nuclease degradation (Eaton and Pieken, 1995; Green et al., 1995; Jellinek et al., 1995; Lin et al., 1994; Pagratis et al., 1997), and the conjugation of aptamers to supermolecular carriers such as PEG or liposomes (Tucker et al., 1999; Willis et al., 1998) can greatly increase their stability and retention in biological fluids. Nucleic acid pools can also be sieved for catalytic function (for a review, see Wilson and Szostak, 1999). For example, nucleic acid pools can be immobilized on columns, and catalytically active species will cleave themselves from the column and can be collected in the eluate (Breaker and Joyce, 1994). Conversely, ribozyme ligases can be selected from random sequence pools based on their ability to append particular sequences to themselves that can be captured on affinity columns and used as primer-binding sites for PCR amplification (Bartel and Szostak, 1993). A variety of other ribozymes have been selected based on variations on these two themes" cleave away or add to. Alkyl transferase ribozymes have been selected that can add an activated biotin to themselves (Wilson and Szostak, 1995), tRNA synthetase-like ribozymes have been selected that can aminoacylate themselves (Illangasekare et al., 1995), and amide synthases have been selected by selectively modifying the 5' end of a pool with an amine and then identifying those ribozymes that can conjugate an activated carboxylate to themselves (Lohse and Szostak, 1996). It has also proven possible to meld aptamer and catalytic selections. Just as catalytic antibodies can be selected by identifying antibody variants that bind transition state analogues of a given reaction, aptamers that bind transition state analogues have proven to have catalytic activity. Peter Schultz and coworkers initially isolated ribozymes that could catalyze the isomerisation of a biphenyl compound to its diastereomer by using a transition state analogue as a target (Prudent et al., 1994). Similarly, using N-alkylated porphyrin transition state analogues, both catalytic RNA (Conn et al., 1996) and catalytic DNA (Li and Sen, 1996) have been selected that catalyze porphyrin metalation. More recently, an aptamer having cholesterol esterase activity was isolated by in vitro selection of RNA using a phosphate ester transition-state analogue of cholesterol ester hydrolysis as a target (Chun et al., 1999). 374

Nucleic Acids for Reagentless Biosensors 3. State of the Art 3.1. Signaling aptamers

Nucleic acid biosensors that can directly transduce the molecular recognition of other nucleic acids into optical signals have previously been described. K_ramer and his co-workers originally designed 'molecular beacons' (Figure 2) that juxtaposed fluorophores and quenchers in a stem structure (Tyagi et al., 1998; Tyagi and Kramer, 1996). A loop capping the stem was complementary to some RNA or DNA target; upon interaction with the target, the formation of a new, stable helical structure resulted in the original stem being pried apart, which in turn freed the fluorophore from the adjacent quencher, resulting in a strong optical signal. Molecular beacons have now become mainstays in the diagnostics industry, and have been adapted to a variety of applications, including allele discrimination in real-time PCR assays of genomic DNA (Tyagi et al., 1998), detection of target genes (for example, detection of drug resistance in Mycobacterium tuberculosis (Piatek et al., 1998)), as sensitive DNA biosensors (Liu et al., 2000; Liu and Tan, 1999), for studying protein-DNA interactions (Bar-Ziv and Libchaber, 2001; Fang et al., 2000; Tan et al., 2000), and in antisense research for the real time detection of DNA-RNA hybridization in living cells (Sokol et al., 1998). While sequence recognition alone can potentiate a wide range of diagnostic and other applications, the utility of nucleic acids as reagentless biosensors would be greatly expanded if they could also signal the presence of non-nucleic acid analytes, such as proteins or small organics. To this end, there are several different schemes that can be imagined for converting aptamers to biosensors or 'signaling aptamers' (Figure 3). Each of these models has been realized in practice, and will be described in turn. Just like the reagentless protein biosensors described above, aptamers can be adapted to signal the presence of non-nucleic acid analytes. Structural studies have shown that aptamers frequently undergo small but significant conformational changes or reorganizations upon binding their cognate ligands (Hermann and Patel, 2000; Patel and Suri, 2000). By incorporating a fluorophore into a conformationally labile region of an aptamer, the binding event can lead to a change in the chemical environment of the fluorophore and hence to a change in fluorescence intensity (Figure 3a). Based on the known three-dimensional structures of anti-adenosine RNA and DNA aptamers (Dieckmann et al., 1996; Jiang et al., 1996; Lin and Patel, 1997), fluorescent dyes were introduced in the proximity of the adenosine binding site. The resultant signaling aptamers not

375

Rajendran and Ellington

Figure 3. Strategies for signaling aptamers. (a) Exploitation of small conformational changes. (b) Exploitation of larger secondary structural conformational changes. (c) Exploitation of tertiary structural conformational changes. (d) Exploitation of quartemary structural conformational changes. In the case of (a), the conformational change may be inherent to the aptamer. In (b) - (d), the conformational changes have been engineered into the aptamer by altering its secondary, tertiary, and / or quarternary structure.

only showed an ATP-dependent increase in fluorescence, they were also selective for ATP relative to other nucleotides and could track ATP concentrations in solution (Jhaveri et al., 2000a). Interestingly, while the sites of fluorophore insertion were chosen to interfere with ATP binding as little as possible, the apparent Kd of the designed signaling aptamers (-30 lxM for the DNA signaling aptamer a n d - 300 ~M for the RNA signaling aptamer) was much higher than that of the parental aptamers (Huizenga and Szostak, 1995; Sassanfar and Szostak, 1993) (~6 ~M for the anti-adenosine DNA aptamer and 6-8 ~tM for the anti-adenosine RNA aptamer). This may indicate that the design process must be greatly refined, or it may be that there is an inherent loss of binding affinity during conformational transduction. While the conformational changes that the anti-adenosine aptamers underwent were relatively small, much larger conformational changes are possible. For example, the ligand-dependent organization of aptamer secondary structure can be envisaged. A secondary structure could be poised so that it would be largely unstructured in the absence of analyte, but substantially stabilized upon analyte binding (Figure 3b). An anti-cocaine DNA aptamer has been converted into a signaling aptamer using this strategy (Stojanovic et al., 2001). One of the stems 376

Nucleic Acids for Reagentless Biosensors of a three-way junction that constituted the cocaine binding region was destabilized by truncation. At the same time, the stem was labeled with a 5' fluorophore and a 3' quencher. Ligand binding stabilizes the engineered stem and the aptamer goes from a fluorescent, unliganded form to a quenched, ligandbound form. The signaling aptamer was not only able to measure cocaine concentration in the concentration range from 10 r to 2.5 mM, but was robust enough to report cocaine concentrations in serum. The aptamer appeared selective for cocaine relative to cocaine derivatives, such as benzoyl ecgonine, but it was possible that the observed selectivity was for hydrophobicity rather than for a defined chemical structure. Signaling aptamers that rely on tertiary structural transitions can also be engineered. In this instance, an extant secondary structural element is not merely destabilized, but instead an entirely new conformation is pre-engineered into the aptamer (Figure 3c). A short, anti-thrombin aptamer that was known to form a quadruplex structure (Bock et al., 1992; Macaya et al., 1993; Schultze et al., 1994) served as the starting point for the design of a simple 'aptamer beacon.' Sequences were added to the 5' terminus of the anti-thrombin aptamer that were complementary to critical residues within the thrombin-binding structure (Hamaguchi et al., 2001). In consequence, the dominant structure in solution was not a quadruplex, but rather was a hairpin stem. A fluor was added to the 5' end of the hairpin, while a quencher was added to the 3' end. The addition of thrombin shifts the equilibrium from the quenched stem-loop form to the thrombin-bound form, resulting in dequenching of the fluorophore and the creation of an optical signal. The thrombin 'beacon' could detect thrombin concentrations as low as 5nM and was able to discriminate against other serine proteases, such as factors IX and Xa. An advantage of this tertiary structural rearrangement strategy as opposed to the secondary structural rearrangement strategy related above is that the signal is a dequenched increase in fluorescence, rather than a quenched decrease in fluorescence. While there are numerous analytes in a complex mixture or biological sample that might lead inadvertently to fluorescence quenching, there should be relatively few compounds other than the target analyte that should lead to an increase in fluorescence intensity. Finally, aptamer quaternary structure can also be engineered to yield analytedependent changes in optical signals (Figure 3d). In this case, aptamers are split into separate pieces that can self-assemble in the presence of a cognate ligand. Again, fluorophores are used to label each of the aptamer pieces. In the absence of the target ligand, the two oligomers exist as individual units in solution, but target binding brings the oligomers together and leads to ternary complex stabilization, ultimately resulting in a quenched optical signal (or in a fluorescence resonance energy transfer signal). Anti-cocaine and anti-rATP aptamers have been converted into signaling aptamers using this strategy (Stojanovic et al., 2000). The signaling aptamers were again not only reproducibly sensitive, but also selective for their cognate ligands. Within the 377

Rajendran and Ellington concentration ranges of 10 #M to lmM ATP and 10-150 pM cocaine, it proved possible to simultaneously report the concentrations of the two analytes using the two signaling aptamers via fluorescence changes. In a similar set of experiments, an aptamer that binds the Tat protein of HIV has been converted into a signaling aptamer by this method. In this case, one of the two oligomers was designed to be a molecular beacon construct, which opens up on ligand binding to generate a signal. The obvious advantage of this method is again that it allows a 'positive' optical signal to be generated, rather than looking for a 'negative' signal against a highly fluorescent background. The anti-Tat aptamer had an extremely low Kd for Tat (---120 pM), and the adapted biosensor could quantitate Tat samples as low as 100 nM (Yamamoto et al., 2000). In all the above examples, the generation of signaling aptamers required a prior knowledge of the secondary or even tertiary structure of the aptamer; they were all 'designed' signaling aptamers. Obviously, the need to understand the detailed structure of an aptamer may limit the applicability of these methods, especially in the development of large-scale sensor arrays (see also Section 3.4). Therefore, it is important to determine whether other methods might be developed that would directly couple aptamer selection to signal transduction. In other words, can methods be devised for the direct selection of signaling aptamers? As a first attempt, we have incorporated modified, fluorescent nucleotides directly into selection experiments (Jhaveri et al., 2000b). A single stranded DNA pool was synthesized that contained largely A, G, and C, and only a small fraction of T. In vitro transcription was used to generate an RNA pool that completely incorporated a uridine analogue, fluorescein UTP, at all positions that would have normally contained uridine. The random region in the pool was skewed and the uridine ratio was kept low to avoid an intrinsic background which could mask signaling by selected aptamers. Aptamers were isolated that could bind to the target analyte ATP. Several different families emerged from the selection, and all contained a relatively small number of (or even no) uridine residues. Individual families were then screened for their ability to signal the presence of ATP by a ligand-dependent change in fluorescence intensity. One family showed excellent signaling abilities; the best signaling aptamer contained only one uridine, could sense ATP concentrations as low as 25 txM, was selective for ATP relative to other nucleotides, and was stable enough in complex mixtures to quantitate ATP. The presence of signaling aptamers in the selected population suggested that fluors may be present during the selection of aptamers, rather than added later. 3.2. Nucleic acid catalysts as biosensors

Aptamers function as reagentless biosensors because their ability to signal is embedded within the receptor itself. This concept can also be carried over to catalytic nucleic acids (ribozymes) by simply embedding the ability to signal 378

Nucleic Acids for Reagentless Biosensors within the catalyst, its substrate, or some aspect of the catalytic mechanism. For example, John Burke and his co-workers have generated variants of the hairpin ribozyme that cleave an RNA substrate containing both a fluorophore and a quencher (Vitiello et al., 2000). In the presence of the ribozyme, a fluorescent signal is produced and the kinetics of the ribozyme can be readily followed. Michael Famulok and his co-workers have developed a similar signaling system for the hammerhead ribozyme (Jenne et al., 2001), and Krupp and his co-workers have followed the kinetics of the Group I self-splicing ribozyme by monitoring the ligation-mediated release of a fluorescent dye and concomitant changes in fluorescence polarization (Singh et al., 2000). In each instance, the system is homogenous and tracks ribozyme activity. However, since ribozyme activity is itself dependent upon a number of cofactors, notably metals, these reactions can also be viewed as biosensors for any of the reaction components that lead to catalysis. In this respect, it is interesting to note that the metal-dependence of ribozymes and deoxyribozymes can be altered or de n o v o engineered, seemingly at will. For example, it has long been known that yeast tRNA (Phe) undergoes site-specific cleavage in the presence of lead ions. Pan and Uhlenbeck (1992) exploited this property to select for variants that were even better 'leadzymes'. One variant looked completely unlike the original tRNA, yet still used lead hydroxide to cleave the phosphodiester bond, generating a 2', 3' cyclic phosphate, which was in turn hydrolyzed (Pan et al., 1994). The structure of this leadzyme has now been solved (Wedekind and McKay, 1999), and it appears as though lead coordinates to the 2' hydroxyl of the scissile residue, a mechanism that has previously been seen for protein enzymes. Breaker and Joyce (1994) were able to select a lead-dependent deoxyribozyme from a random sequence pool that could cleave a substrate with a single ribotide. The same technique was further generalized to the selection of deoxyribozymes that were dependent on other ions, such as magnesium, manganese, and zinc (Breaker and Joyce, 1995). Li et al. (2000) repeated Breaker and Joyce's experiments in order to better describe zinc-binding and catalytic motifs, but obtained sequences similar to those that had already been found by Breaker and Joyce. Li and Lu (2000) have taken advantage of the ability to track ribozyme kinetics using fluorescent reporters by developing a lead-sensing ribozyme biosensor. Substrate cleavage could be monitored in real-time using kinetic fluorescence spectroscopy, and there was a modest specificity for lead (80-fold relative to other divalent ions). Amazingly, the chemistry of nucleic acid catalysts can also be extended beyond simple metal-dependence. Since most catalysis is pH dependent, it is not surprising that ribozymes can be evolved to respond to different pH optima (Jayasena and Gold, 1997). Other variables that affect catalysis can also be probed by selection. Breaker and his co-workers have selected a deoxyribozyme that relies upon copper and peroxide to cleave a DNA substrate (Carmi et al., 1996). The radical cleavage induced by the 'DNAzyme' is mechanistically 379

Rajendran and Ellington identical to nucleic acid scission mediated by other radical-generating reagents, such as iron:EDTA complexes. However, the deoxyribozyme locally produces radicals and directs their attack at a restricted set of sites on the substrate. Similarly, Roth and Breaker (1998) have shown that a ribose moiety embedded within a DNA strand can be cleaved by a deoxyribozyme selected in the presence of histidine. The imidazole ring apparently functions as a general base. As a biosensor, the deoxyribozyme is surprisingly specific, eschewing a variety of histidine analogues, including such closely related compounds as D-histidine and 3-methyl-L-histidine. Ribozymes can also report on the presence of inhibitors. Most recently, Famulok and co-workers (Jenne et al., 2001) have used the fluorescence-based assay they originally developed to screen hammerhead ribozymes for novel inhibitors. Such technologies could potentially even be used to identify inhibitors of other RNA sequences that were appended to or activated by ribozymes.

3.3.

Aptazymes

Another variable that clearly impinges on catalytic mechanism is ribozyme structure. While ribozyme structures can be subtly altered by metals or pH or other conditions, more global alterations can be induced by changes in basepairing. Lizardi engineered an allosteric hammerhead ribozyme that initially folded into an inactive conformation which was in turn relieved by the addition of an oligonucleotide effector (Porta and Lizardi, 1995). This strategy is most similar to that shown in Figure 3c for signaling aptamers. However, allosteric activation was only about 10-fold. More recently, Taira and coworkers have generated novel allosteric ribozymes, called maxizymes, which form active quaternary structures following oligonucleotide recognition (Kuwabara et al., 2000a, 2000b; Warashina et al., 2000). This strategy is most similar to that shown in Figure 3d. A hammerhead ribozyme dimer was broken into two pieces such that both ends present 'arms' that can hybridize to a specific nucleic acid sequence. Once the ribozyme is brought together by hybridization of the substrate-binding 'arms' at one end to a mRNA sequence, a second mRNA or another portion of the same mRNA can hybridize to the substrate-binding 'arms' at the other end, leading to cleavage at both sites. Alternatively, a hammerhead heterodimer can be dissected such that one end of stem-loop II serves as a binding (but not cleavage) site for a particular sequence, while the other end still cleaves a desired target. In this way, the maxizyme can serve as a biosensor. It has also proven possible to construct ribozymes that are structurally responsive to effectors other than oligonucleotides. As we have seen in earlier sections, functional nucleic acids undergo conformational changes upon interactions with their cognate ligands. It therefore seemed reasonable to suppose that by appending nucleic acid aptamers to nucleic acid catalysts (ribozymes), it might 380

Nucleic Acids for Reagentless Biosensors prove possible to alter the conformation, and hence the catalytic activity, of a nucleic acid catalyst in a ligand-dependent fashion. Ron Breaker and his coworkers were the first to attempt this feat by swapping an anti-adenosine aptamer with a stem of the hammerhead ribozyme whose sequence was known to be relatively unimportant for catalysis, yet was juxtaposed with the catalytic core (Tang and Breaker, 1997) (Figure 4a). The activity of the resultant chimeric 'aptazyme' was in fact modulated by ATP. While there were various mechanisms by which it could be imagined that the conformational change of the aptamer regulated the activity of the ribozyme, one likely hypothesis was that the joining region between the aptamer and the ribozyme strongly affected the structure of the catalytic core. In order to further probe this hypothesis, Soukup and Breaker (1999) randomized the joining region and selected for ribozymes that were either activated or inhibited in the presence of FMN (Figure 4b). The generality of the selection procedure (Figure 3) was such that both types of aptazymes could be derived from the same random sequence pool. In one instance, ribozymes that reacted prior to the addition of FMN were removed from the population, then the FMN-dependent ribozymes were harvested; in the other instance, FMN was initially added, those ribozymes that reacted were removed, and the remaining catalytically active population was then harvested. The best aptazymes that were selected by these procedures had activities that were modulated by hundreds of fold in the presence of FMN. Moreover, it was found that the 'communication modules' that emerged from the randomized joining regions could actually intermediate between different ligandbinding domains and the hammerhead ribozyme (Figure 4c). For example, when an anti-theophylline aptamer was joined to the hammerhead ribozyme via a communication module originally selected for its ability to transduce FMNbinding to catalysis, the cleavage activity of the hammerhead was again activated, only this time by theophylline. Koizumi et al. (1999) went beyond the modular joining of aptamers and catalysts by randomizing the entire allosteric domain (Figure 4d). A random sequence pool of 25 residues in length was appended to the catalytic core of the ribozyme via the same stem that had previously proven useful for the addition of aptamers, and a selection for ribozymes whose activities were modulated by cyclic mononucleotides was initiated. All four cyclic mononucleotides were included in the selection, and after 16 rounds of selection and amplification the population was observed to be somewhat dependent upon cGMP for activity. This effector was then removed from the population and the selection was continued. Ultimately, ribozymes were found that were dependent upon three of the four cyclic mononucleotides; no cUMP-specific ribozymes were ever discovered. Individual aptazymes could be activated up to 5,000-fold by their cognate effectors.

381

Rajendran and Ellington

Figure 4. Strategies for adapting the hammerhead ribozyme to be an allosteric enzyme. Top: Sequence and structure of the hammerhead ribozyme. (a) Appending an aptamer to the hammerhead ribozyme in place of a non-essential stem. (b) Randomization of the joining region between the catalytic core of the hammerhead ribozyme and the appended aptamer. Selection is for inhibition or activation by the aptamer's cognate ligand, and typically involves coupled negative and positive selection steps. (c) Changing the effector-binding domain on an allosteric ribozyme. The pre-identification of a 'communication module' by the method described in (b) sometimes assists in this process. (d) Complete randomization of the effector-binding domain and selection for inhibition or activation of the ribozyme. 382

Nucleic Acids for Reagentless Biosensors Interestingly, the selected allosteric domains have many of the same characteristics as selected binding domains. A designed, theophylline-dependent hammerhead ribozyme was partially randomized, and variants that could be activated by other effectors were selected (Soukup et al., 2000). A single mutation altered the specificity of the aptamer from theophylline to 3methylxanthine, which differs from theophylline by the absence of a single methyl group. In fact, selected allosteric domains can be detached from ribozymes and function as aptamers in their own right (Soukup et al., 2001). The cNMP binding domains originally selected by Koizumi et al. (1999b) were probed by random mutagenesis and re-selection (Koizumi et al., 1999a). Based on the relative degree of sequence conservation and variation at different positions, it proved possible to predict which residues were involved in effector binding and which were more likely involved in catalysis. The predicted allosteric domains were separately synthesized, and largely retained the ligandbinding properties that they exhibited within the aptazymes. While the hammerhead ribozyme has obviously proven to be an excellent platform for the design and selection of aptazymes, it was originally unclear whether this ribozyme and the design principles that were built around it were unique. Our lab therefore undertook similar experiments with a ribozyme ligase (L1) that had been selected from a random sequence pool (Robertson and Ellington, 1999). The L1 ligase initially (and fortuitously) proved to be highly dependent (10,000-fold activation) on an oligonucleotide effector that was present during the selection, and thus it seemed likely that it might be adapted to other types of effector-dependence as well. To this end, anti-adenosine, antitheophylline, and anti-flavin aptamers were adjoined to the ligase in place of a stem structure that was relatively unimportant for catalysis, yet was adjacent to the catalytic core (Robertson and Ellington, 2000). As with the hammerhead ribozyme, these L1 ligase chimeras proved to be ATP- and theophyllinedependent. However, flavin-dependence was initially minimal, but was readily optimized by randomization and selection of the 'communication module' connecting the aptamer and ribozyme. We were most interested in generating aptazymes that were protein-dependent. However, the design principles that had previously proven effective in identifying aptazymes that were modulated by small organic effectors did not readily yield protein-dependent aptazymes. Anti-protein aptamers directly conjoined to the L1 ligase did not impart protein-dependence, nor did randomization and selection of the communication module, nor did randomization of the entire allosteric domain followed by a coupled negative and positive selection for protein-dependence (Figure 5). However, when both the allosteric domain and a portion of the catalytic domain were randomized and protein- dependence was selected for, it proved possible to identify aptazymes that were highly dependent on their cognate proteins (Robertson and Ellington, 383

Rajendran and Ellington 2001). For example, one aptazyme was isolated that was 75,000-fold dependent on tyrosyl tRNA synthetase from Neurospora mitochondria (Cytl 8), and another was found to be 3,500-fold dependent on hen egg white lysozyme. The proteindependent aptazymes had many of the characteristics previously observed for protein-dependent aptamers, in that they could readily distinguish between cognate and non-cognate proteins, including between the native and denatured states of the same protein. Given that the design principles originally elaborated by Breaker and his coworkers seemed to be generalizable, it is easy to imagine the development of a wide variety of aptazymes with a wide variety of catalytic functionalities. For example, by mounting two effector domains on the same ribozyme it should be possible to make aptazymes that are doubly-dependent on their effectors and that function as molecular 'and' gates (Figure 6). This concept was originally put into practice by Breaker and his co-workers (Jose et al., 2001), who mounted two aptamers on the hammerhead ribozyme in series. The resultant aptazyme was in fact dependent upon both ligands for full activity, and appeared to exhibit cooperative interactions between the effector-binding domains. We have similarly mounted two aptamers on the L1 ligase, only in this instance the aptamers were appended in parallel, rather than in series. Nonetheless, the resultant aptazyme was again dependent upon both ligands for full activity (Michael Robertson, personal communication). 3.4. Nucleic acid biosensor chips In the initial discussion of reagentless biosensors, it was clear that the utility of such sensors was in their ability to be adapted to multiple types of detection platforms. For reagentless biosensors based on nucleic acids, the ease of in vitro selection procedures offers the possibility that multiple different aptamers, ribozymes or aptazymes could be selected and modularly adopted to a single type of detector. Ultimately, it may be possible to develop chip arrays of nucleic acid biosensors that would be suitable for 'large' biological problems such as the acquisition of information about whole proteomes, metabolomes, or environmental dispositions of organisms and compounds (Brody et al., 1999).

384

Nucleic Acids for Reagentless Biosensors

,

/

/( ,1Incubation t-l,ncuua,

Affinity ~ ----------capture Eluani

"~

)/'(+) Incubation//

1. Reversetranscription

21S:lgeCtleVeatiqCRcR 4. Transcription

~ ~ /

p /

I

! ~o

_I

Figure 5. In vitro selection of protein-dependent aptazyme ligases. This scheme is similar to that shown for the isolation of effector-dependent hammerhead ribozymes (Figure 3d). However, in this instance, ligation of a substrate oligonucleotide to a random sequence population allows the capture of active catalysts via two mechanisms: binding to an oligonucleotide affinity column, and preferential amplification via PCR. It is interesting to note that to achieve the selection of protein-dependent aptazymes both the effector-binding domain and the catalytic core of the ribozyme had to be randomized.

385

Rajendran and Ellington

Figure 6. Different 'dual effector' aptazymes. (a) A dual effector hammerhead aptazyme designed by Breaker and co-workers. The two aptamers are joined in series. Stabilization of one ligand-binding domain results in stabilization of the adjacent domain, and overall stabilization of this structure results in stabilization of the catalytic core. (b) A dual effector ligase aptazyme designed by Michael Robertson and co-workers. The two aptamers are mounted in parallel on different stems of the L1 ligase. The responsivity profile as a function of effector concentration is shown at right. Maximal activity of the nucleic acid 'and gate' is only seen when both effectors are present at high concentrations. In the presence of single effectors, activation is minimal. Initial work on adapting aptamers to optical biosensors was modeled after antibody diagnostics. Aptamers have been used in a sandwich ELISA-!ike format (called ELONA, enzyme-linked oligonucleotide assay) to quantitate human vascular endothelial growth factor (VEGF) in serum samples (Drolet et al., 1996). The ELONA assay was found to be reproducible, could be used to measure VEGF concentrations as low as 25 pg/ml, and had a dynamic range of over three orders of magnitude. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has been used to sensitively detect IgE in solution using fluorescently labeled anti-IgE aptamer as a selective fluorescent probe (German et al., 1998). The method was highly sensitive, with a mass 386

Nucleic Acids for Reagentless Biosensors detection limit of 37 zmol of IgE, and a dynamic range of 105. Aptamers have also been used as probes in flow cytometry: a fluoresceinated anti-human neutrophil elastase (HNE) aptamer has been compared with an anti-HNE antibody in detecting HNE coated on beads (Davis et al., 1996). Similarly, aptamers against human CD4 have been conjugated to different fluorophores and used to stain human CD4 expressed on cells by flow cytometry (Davis et al., 1998). These somewhat simplistic antibody substitutions have been followed up by more sophisticated attempts to adapt compact, easily labeled aptamers to more system-specific analytic detection techniques and instrumentation. A fiber-optic microarray biosensor has been developed for thrombin by immobilizing antithrombin DNA aptamer at the distal tip of an imaging fiber coupled to a modified epifluorescence microscope system (Lee and Walt, 2000). The system has a detection limit of 1 nM and was used to measure thrombin concentrations in the range from nanomolar to low micromolar. Other examples include an aptamer biosensor for L-adenosine based on total internal reflection fluorescence detection which Could detect L-adenosine in the submicromolar range (Kleinjung et al., 1998) and one for thrombin based on evanescent wave-induced fluorescence detection (Potyrailo et al., 1998). The thrombin sensor had a dynamic range of three orders of magnitude, and was highly sensitive with a detection limit of 0.7 amol in a 140 pL volume. While these systems and adaptations have variable potential for the eventual development of chip arrays, the very fact that aptamers are readily adapted across such diverse platforms bodes well for the eventual use of aptamers in virtually any detection modality that comes to the fore. The fact that aptazymes directly transduce molecular recognition into catalysis may allow them to function as reagentless biosensors in simple but robust chip arrays. As an example, Seetharaman et al. (2001) immobilized various radioactive hammerhead aptazymes on a gold surface via a 5' phosphorothioate moiety. Following the addition of effectors or effector mixtures, appropriatelyactivated hammerheads cleaved themselves away from the surface and could be detected by simply transferring the supernatant to a new microtitre plate. Most remarkably, a cAMP-sensing aptazyme could be used to accurately quantitate the amount of cAMP in the culture medium of various E. coli strains that had alterations to adenosine metabolism. We have developed similar chips based on the L1 ligase. In this manifestation, however, activated aptazyme ligases conjugate themselves to substrates and coimmobilize radiolabels onto a surface (Figure 7). One of the advantages of aptazymes for chip-based applications is that they are already known to recognize a chemically diverse set of substrates. The ligase chip simultaneously detects the presence of oligonucleotide, small organic molecules, peptides, and proteins, a feat that would not be possible in a conventional ELISA assay. 387

Rajendran and EUington

Figure 7. Aptazyme ligase chip. Radiolabeled aptazymes derived from the L1 ligase (see ligand specificities at left) were incubated with biotinylated substrates. Following reaction, the mixtures were incubated with a streptavidin microtitre plate and unbound material was washed away. While this is not a reagentless application, the figure above does show the ability of aptazymes to specifically detect small molecules (FMN, ATP, theophylline), oligonucleotides (18.90, the effector for the original L1 ligase, see Figure 6, sequence in italics), peptides (the arginine-rich motif of Rev), and proteins (Lys = lysozyme). '-' is the no effector control, and '+' is all effectors mixed together.

4. Advantages and Limitations While the use of signaling aptamers and aptazymes in biosensors holds great potential, it is nonetheless true that nucleic acids are generally not as effective as proteins both as binding reagents and as catalysts. While aptamers typically bind their ligands in the nanomolar to micromolar range, antibodies can generally bind ligands well into the femtomolar range. Similarly, the few protein-sensing aptazymes developed so far can sense proteins into the nanomolar range, whereas ELISA assays can sense as few as 108 molecules in a sample (Rogers, 2000). 388

Nucleic Acids for Reagentless Biosensors Thus, an important question is whether the seemingly superior recognition elements, proteins, can function in a reagentless system in much the same way that aptamers and aptazymes can. Reagentless optical biosensors based on fluorescently labeled proteins are known (Brennan, 1999; Hellinga and Marvin, 1998). Many bacterial periplasmic binding proteins such as the phosphate binding protein (Brune et al., 1994), maltose binding protein (Gilardi et al., 1994; Marvin et al., 1997), glucose/galactose binding protein (Marvin and Hellinga, 1998), and glutamine binding protein (Dattelbaum and Lakowicz, 2001), have been converted into receptors for reagentless biosensors by the incorporation of single fluorophores that report ligand-dependent conformational changes. Such protein-based biosensors are usually generated by incorporating fluorophores (via covalent coupling to site specific single point cysteine mutations) either in close proximity to the ligand binding site, so that they can directly report ligand binding, or in distal regions of the protein such that the fluor can give an indirect readout based on domain movements in proteins which involve an allosteric coupling mechanism. This strategy is much the same as we have described above for signaling aptamers. As an example, when reporter fluors were incorporated into different positions in E. coli glucose binding protein, two of the variants were found to signal well

(Marvin and Hellinga, 1998). One of the variants had the fluorescent label in an allosterically-linked site and showed a 2-fold decrease in fluorescence upon ligand binding with minimal effect on the sugar binding constant [Ka (glc) !ncreased by a factor of 2, and Ka (gal) by a factor of--1.5]. A variant with the reporter fluor in the ligand-binding pocket showed a 4-fold increase in fluorescence, but with a much larger associated loss of binding [K4 (glc) increased--100 fold, and Ka (gal)--500 ~fold]. While these glucose binding proteins can potentially be used t o measure glucose concentrations in the micromolar range, a composite maltose biosensor obtained by mixing four similarly engineered maltose binding proteins measured maltose concentrations over a range of 0.1 jam - 20mM with an accuracy of 5% (Marvin et al., 1997). The best maltose binding protein had a fluorophore incorporated in an allosteric site and showed a greater than 4-fold increase in fluorescence. The remaining three were made by mutating residues in the binding pocket known to interact with maltose; this decreased the affinity for maltose without effecting signaling ability. These latter results suggest that molecular recognition and allosteric signal transduction can be independently-manipulated in protein-based biosensors. Therefore, it should be possible to change the binding specificity of a protein without affecting signaling. Hellinga's group recently converted a maltose sensor into a zinc biosensor by changing the specificity of the maltose binding protein using a rational design strategy (Marvin and Hellinga, 2001). By 389

Rajendran and Ellington

employing an iterative progressive design strategy, they were able to increase the zinc affinity of the maltose binding protein (one of their final constructs had a Ka -- 350 nM for zinc); the mutant also showed a greater than 17-fold increase in fluorescence upon zinc binding, indicating an alteration of the initial structure. Protein biosensors based on other metalloproteins such as zinc finger peptides (Godwin and Berg, 1996; Walkup and Imperiali, 1996; Walkup and Imperiali, 1997) and carbonic anhydrase (Elbaum et al., 1996) have also been made in a similar manner by incorporating environmentally sensitive fluors. Alternately, the intrinsic fluorescence of reporter proteins, such as the green fluorescence protein (GFP), has been exploited through the introduction of engineered analyte-binding sites. Calcium biosensors have been made by fusing two variants of GFP with calmodulin, and calmodulin-binding peptide. The introduction of Ca 2+ induced the binding of calmodulin around the calmodulinbinding peptide, ultimately resulting in a fluorescence energy transfer between the flanking GFPs (Miyawaki et al., 1997). This construct showed a 70% increase in the ratio of ultraviolet-excited emissions at 510 and 445 nm (1.8-fold change in the maximal ratio), and with the introduction of mutations into calmodulin, was able to report calcium concentrations over a very wide range from 10.8 to 10.2 M. In another study, a GFP-based calcium sensor was made by again joining together two GFP variants using the calmodulin-binding peptide (Romoser et al., 1997). This sensor showed a six-fold decrease in the ratio of emissions at 505 nm:440 nm and was able to report calcium concentrations from 50 nM to 1 gM. Peter Schultz and coworkers have developed a totally different strategy for modulating protein-protein and protein-nucleic acid interactions using small molecules (Guo et al., 2000). The method is based on first creating a cavity at a protein-protein interface such that it results in a reduction in binding affinity, and then screening libraries of small molecules to identify ligands that can bind the cavity and restore the interaction. This method is similar to one of the strategies for aptazyme construction (Figure 4d). However, rather than adapting the randomized allosteric domain to a particular effector, a random set of effectors is adapted to a particular allosteric domain. Two amino acids at the interface between human growth hormone (hGH) and the hGH receptor were mutated to glycines, creating a cavity at the interface and decreasing the binding affinity between the two by a factor of 106. By screening a library of indole derivatives, a ligand was identified which increased the affinity of mutated hGH for its mutated receptor more than 1000-fold. 5. Potential for Improving Performance

Although proteins with their wider array of functional groups may be inherently better biopolymer binding species and catalysts than nucleic acids, they have so 390

Nucleic Acids for Reagentless Biosensors far not proven to be much better for reagentless biosensors. For example, initially designed signaling aptamers showed approximately 25-45% increase in fluorescence upon interaction with ATP, while selected signaling aptamers showed an 80% increase (Jhaveri et al., 2000b); these results are similar to those that have been exhibited by the designed protein based biosensors described above. The anti-thrombin aptamer formulated as a signaling aptamer showed a sensitivity of 5 nM in solution (Hamaguchi et al., 2001), while the same aptamer mounted on a glass platform also showed a sensitivity of 5 nM (the biosensor could detect 0.7 amol of thrombin in a 140 pL volume) (Potyrailo et al., 1998). The values are the same or perhaps even better than those exhibited by the reagentless protein biosensors we have examined. Finally, we have seen that aptazymes can be activated over a thousand-fold by their cognate effectors, results that are as good as the engineered allostery that Schultz and co-workers achieved with hGH and its receptor (Guo et al., 2000). Thus, at the current time, nucleic acid and protein biosensors seem to be equivalant with regards to function, and the relative newness of aptamers, ribozymes, and aptazymes in the sensor arena means that virtually any new experiment can potentially result in greatly improved performance. Overall, nucleic acids for use in reagentless biosensors are essentially only as good as three inherent parameters: binding affinity, binding specificity, and signaling relative to background. Binding affinity can potentially be improved by augmenting the chemically simple complement of canonical nucleotides with modified nucleotides. For example, would the inclusion of a uridine residue that contained a branched, hydrophobic (isoleucine-like) group at the 5 position enable the selection of structures that could better recognize more hydrophobic epitopes and small molecules? The binding specificities exhibited by aptamers and aptazymes have so far been quite good, and can likely be greatly improved by a continued focus on negative selection experiments with highly related targets. One of the great advantages of nucleic acid selection relative to in vivo immunization is the ability to discretely and exactly control selection stringencies and parameters in order to deliver up molecules with just the right functional properties. As indicated above, it is the activation parameters of nucleic acid receptors in reagentless biosensors that truly shine relative to that of protein counter parts, and it is likely that sly manipulations of nucleic acid conformational changes by design or selection will continue to tweak these numbers upwards for sometime to come. Paradoxically, though, increases in activation seem to come at a cost in sensitivity: signaling aptamers show higher apparent Ka's for their ligands than do their parental aptamers; aptazymes show higher K4's for their effectors than do corresponding aptamers. In both instances, it is not unreasonable to suspect that ligand-binding energy is transduced into conformational changes, and that the greater the conformational change, the more ligand-binding energy must be diverted to that conformational change. In turn, the more ligand-binding energy that is diverted to a conformational change, the lower the intrinsic affinity of a given signaling aptamer or aptazyme will be for 391

Rajendran and Ellington its cognate ligand. The question thus becomes whether there is an experimental resolution of this seeming thermodynamic paradox; is it possible to design o r (more likely) select reagentless nucleic acid biosensors that simultaneously have both higher affinities and higher activation parameters? The answers to this question will likely become available shortly, but in the interim Ron Breaker has found that it is indeed possible to select for increasingly lower apparent K4'sfor cyclic nucleotide activated hammerhead aptazymes without loss of activation (Koizumi et al., 1999b). The greatest advantage and potential that aptamers and aptazymes currently have relative to protein reagents is the prospect for the high-throughput generation of multiple different receptors against multiple different targets. The ability to generate large numbers of nucleic acid recognition elements at will should make it possible to plumb the constitution and functionality of organismal proteomes and metabolomes. We have recently developed automated methods for the selection of both aptamers and nucleic acid enzymes (Cox and Ellington, 2001; Cox et al., 1998).

6. References

Barrel, D. P. and J. W. Szostak, 1993, Science 261, 1411. Bar-Ziv, R. and A. Libchaber, 2001, Proc. Natl. Acad. Sci. USA 98, 9068. Bock, L. C., L. C. Griffin, J. A. Latham, E. H. Vermaas and J. J. Toole, 1992, Nature 355, 564. Breaker, R. R. and G. F. Joyce, 1994, Chem. Biol. 1,223. Breaker, R. R. and G. F. Joyce, 1995, Chem. Biol. 2, 655. Brennan, J. D., 1999, J. Fluoresc. 9, 295. Brody, E. N. and L. Gold, 2000, Rev. Mol. Biotechnol. 74, 5. Brody, E. N., M. C. Willis, J. D. Smith, S. Jayasena, D. Zichi and L. Gold, 1999, Mol. Diagn. 4, 381. Brune, M., J. L. Hunter, J. E. Corrie and M. R. Webb, 1994, Biochem. 33, 8262. Carmi, N., L. A. Shultz and R. R. Breaker, 1996, Chem. Biol, 3, 1039. Chun, S. M., S. Jeong, J. M. Kim, B. O. Chong, Y. K. Park, H. Park and J. Yu, 1999, J. Am. Chem. Soc. 121, 10844. Ciesiolka, J. and M. Yams, 1996, RNA 2, 785. Conn, M. M., J. R. Prudent and P. G. Schultz, 1996, J. Am. Chem. Soc. 118, 7012. Conrad, R., L. M. Keranen, A. D. Ellington and A. C. Newton, 1994, J. Biol. Chem. 269, 32051. Cox, J. C. and A. D. Ellington, 2001, Bioorg. Med. Chem. 9, 2525. Cox, J. C., P. Rudolph and A. D. Ellington, 1998, Biotechnol. Prog. 14, 845. Dattelbaum, J. D. and J. R. Lakowicz, 2001, Anal. Biochem. 291, 89. Davis, K. A., B. Abrams, Y. Lin and S. D. Jayasena, 1996, Nucleic Acids Res. 24, 702. 392

Nucleic Acids for Reagentless Biosensors Davis, K. A., Y. Lin, B. Abrams and S. D. Jayasena, 1998, Nucleic Acids Res. 26,3915. Dieckmann, T., E. Suzuki, G. K. Nakamura and J. Feigon, 1996, RNA 2, 628. Drolet, D. W., L. Moon-McDermott and T. S. Romig, 1996, Nature Biotechnol. 14, 1021. Eaton, B. E. and W. A. Pieken, 1995, Annu. Rev. Biochem. 64, 837. Elbaum, D., S. K. Nair, M. W. Patchan, R. B. Thompson and D. W. Chri.stianson, 1996, J. Am. Chem. Soc. 118, 8381. Ellington, A. D. and J. W. Szostak, 1990, Nature 346, 818. Famulok, M., G. Mayer and M. Blind, 2000, Acc. Chem. Res. 33, 591. Fang, X., J. J. Li and W. Tan, 2000, Anal. Chem. 72, 3280. German, I., D. D. Buchanan and R. T. Kennedy, 1998, Anal. Chem. 70, 4540. Gilardi, G., L. Q. Zhou, L. Hibbert and A. E. Cass, 1994, Anal. Chem. 66, 3840. Godwin, H. A. and J. M. Berg, 1996, J. Am. Chem. Soc. 118, 6514. Green, L. S., D. Jellinek, C. Bell, U A. Beebe, B. D. Feistner, S. C. Gill, F. M. Jucker and N. Janjic, 1995, Chem. Biol. 2, 683. Guo, Z., D. Zhou and P. G. Schultz, 2000, Science 288, 2042. Haller, A. A. and P. Sarnow, 1997, Proc. Natl. Acad. Sci. USA 94, 8521. Hamaguchi, N., A. Ellington and M. Stanton, 2001, Anal. Biochem. 294, 126. Hellinga, H. W. and J. S. Marvin, 1998, Trends Biotechnol. 16, 183. Hermann, T. and D. J. Patel, 2000, Science 287, 820. Hesselberth, J., M. P. Robertson, S. Jhaveri and A. D. Ellington, 2000a, Rev. Mol. Biotechnol. 74, 15. Hesselberth, J. R., D. Miller, J. Robertus and A. D. Ellington, 2000b, J. Biol. Chem. 275, 4937. Hirao, I., M. Spingola, D. Peabody and A. D. Ellington, 1998, Mol. Divers. 4, 75. Homann, M. and H. U. Goringer, 1999, Nucleic Acids Res. 27, 2006. Huizenga, D. E. and J. W. Szostak, 1995, Biochem. 34, 656. Illangasekare, M., G. Sanchez, T. Nickles and M. Yarus, 1995, Science 267, 643. Jayasena, S. D., 1999, Clin. Chem. 45, 1628. Jayasena, V. K. and L. Gold, 1997, Proc. Natl. Acad. Sci. USA 94, 10612. Jellinek, D., L. S. Green, C. Bell, C. K. Lynott, N. Gill, C. Vargeese, G. Kirschenheuter, D. P. McGee, P. Abesinghe, W. A. Pieken, 1995, Biochem. 34, 11363. Jenison, R. D., S. C. Gill, A. Pardi and B. Polisky, 1994, Science 263, 1425. Jenne, A., J. S. Hartig, N. Piganeau, A. Tauer, D. A. Samarsky, M. R. Green, J. Davies and M. Famulok, 2001, Nature Biotechnol. 19, 56. Jhaveri, S., R. Kirby, R. Conrad, E. J. Maglott, M. Bowser, R. T. Kennedy, G. Glick and A. D. Ellington, 2000a, J. Am. Chem. Soc. 122, 2469. Jhaveri, S., M. Rajendran and A. D. Ellington, 2000b, Nature Biotechnol. 18, 1293. Jiang, F., R. A. Kumar, R. A. Jones and D. J. Patel, 1996, Nature 382, 183. Jose, A. M., G. A. Soukup and R. R. Breaker, 2001, Nucleic Acids Res. 29, 1631. Kawakami, J., H. Irnanaka, Y. Yokota and N. Sugimoto, 2000, J. Inorg. Biochem. 82, 197. 393

Rajendran and Ellington Kleinjung, F., S. Klussmann, V. A. Erdmann, F. W. Scheller, J. P. Fuerste and F. F. Bier, 1998, Anal. Chem. 70, 328. Koizumi, M., J. N. Kerr, G. A. Soukup and R. R. Breaker, 1999a, Nucleic Acids Symp. Ser. 275. Koizumi, M., G. A. Soukup, J. N. Kerr and R. R. Breaker, 1999b, Nature Struct. Biol. 6, 1062. Kuwabara, T., M. Warashina and K. Taira, 2000a, Trends Biotechnol. 18,462. Kuwabara, T., M. Warashina and K. Taira, 2000b, Curr. Opin. Chem. Biol. 4, 669. Lee, M. and D. R. Walt, 2000, Anal. Biochem. 282, 142. Leech, D. and F. Daigle, 1998, Analyst 123, 1971. Li, J. and Y. Lu, 2000, J. Am. Chem. Soc. 122, 10466. Li, J., W. Zheng, A. H. Kwon and Y. Lu, 2000, Nucleic Acids Res. 28, 481. Li, Y. and D. Sen, 1996, Nature Struct. Biol. 3,743. Lin, C. H. and D. J. Patel, 1997, Chem. Biol. 4, 817. Lin, Y., Q. Qiu, S. C. Gill and S. D. Jayasena, 1994, Nucleic Acids Res. 22, 5229. Liu, X., W. Farmerie, S. Schuster and W. Tan, 2000, Anal. Biochem. 283, 56. Liu, X. and W. Tan, 1999, Anal. Chem. 71, 5054. Lohse, P. A. and J. W. Szostak, 1996, Nature 381,442. Macaya, R. F., P. Schultze, F. W. Smith, J. A. Roe and J. Feigon, 1993, Proc. Natl. Acad. Sci. USA 90, 3745. Mannironi, C., A. Di Nardo, P. Fruscoloni and G. P. Tocchini-Valentini, 1997, Biochem. 36, 9726. Marvin, J. S., E. E. Corcoran, N. A. Hattangadi, J. V. Zhang, S. A. Gere and H. W. Hellinga, 1997, Proc. Natl. Acad. Sci. USA 94, 4366. Marvin, J. S. and H. W. Hellinga, 1998, J. Am. Chem. Soc. 120, 7. Marvin, J. S. and H. W. Hellinga, 2001, Proc. Natl. Acad. Sci. USA 98, 4955. Mills, D. R., R. L. Peterson and S. Spiegelman, 1967, Proc Natl Acad Sci USA 58,217. Miyawaki, A., J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. II~ra and R. Y. Tsien, 1997, Nature 388, 882. Morris, K. N., K. B. Jensen, C. M. Julin, M. Weil and L. Gold, 1998, Proc. Natl. Acad. Sci. USA 95, 2902. Mulchandani, A. and S. Pan, 1999, Anal. Biochem. 267, 141. Narvaez, A., G. Suarez, I. C. Popescu, I. Katakis and E. Dominguez, 2000, Biosens. Bioelectron. 15, 43. Oliphant, A. R., C. J. Brandl and K. Struhl, 1989, Mol. Cell Biol. 9, 2944. Pagratis, N. C., C. Bell, Y. F. Chang, S. Jennings, T. Fitzwater, D. Jellinek and C. Dang, 1997, Nature Biotechnol. 15, 68. Pan, T., B. Dichtl and O. C. Uhlenbeck, 1994, Biochem. 33, 9561. Pan, T. and O. C. Uhlenbeck, 1992, Biochem. 31, 3887. Pan, W., R. C. Craven, Q. Qiu, C. B. Wilson, J. W. Wills, S. Golovine and J. F. Wang, 1995, Proc. Natl. Acad. Sci. USA 92, 11509. Patel, D. J. and A. K. Suri, 2000, Rev. Mol. Biotechnol. 74, 39. 394

Nucleic Acids for Reagentless Biosensors Piatek, A. S., S. Tyagi, A. C. Pol, A. Telenti, L. P. Miller, F. R. Kramer and D. Alland, 1998, Nature Biotechnol. 16, 359. Porta, H. and P. M. Lizardi, 1995, Biotechnology (N Y), 13, 161. Potyrailo, R. A., R. C. Conrad, A. D. Ellington and G. M. Hieftje, 1998, Anal. Chem. 70, 3419. Prudent, J. R., T. Uno and P. G. Schultz, 1994, Science 264, 1924. Ringquist, S., T. Jones, E. E. Snyder, T. Gibson, I. Boni and L. Gold, 1995, Biochem. 34, 3640. Robertson, D. L. and G. F. Joyce, 1990, Nature 344, 467. Robertson, M. P. and A. D. Ellington, 1999, Nature Biotechnol. 17, 62. Robertson, M. P. and A. D. Ellington, 2000, Nucleic Acids Res. 28, 1751. Robertson, M. P. and A. D. Ellington, 2001, Nature Biotechnol. 19, 650. Rogers, K. R., 2000, Mol. Biotechnol, 14, 109. Romoser, V. A., P. M. Hinkle and A. Persechini, 1997, J. Biol. Chem. 272, 13270. Roth, A. and R. R. Breaker, 1998, Proc. Natl. Acad. Sci. USA 95, 6027. Sassanfar, M. and J. W. Szostak, 1993, Nature 364, 550. Schultze, P., R. F. Macaya and J. Feigon, 1994, J. Mol. Biol. 235, 1532. Seetharaman, S., M. Zivarts, N. Sudarsan and R. R. Breaker, 2001, Nature Biotechnol. 19, 336. Singh, K. K., T. Rucker, A. Hanne, R. Parwaresch and G. Krupp, 2000, Biotechniques 29, 344. Sokol, D. L., X. Zhang, P. Lu and A. M. Gewirtz, 1998, Proc. Natl. Acad. Sci. USA 95, 11538. Soukup, G. A. and R. R. Breaker, 1999, Proc. Natl. Acad. Sci. USA 96, 3584. Soukup, G. A., E. C. DeRose, M. Koizumi and R. R. Breaker, 2001, RNA 7, 524. Soukup, G. A., G. A. Emilsson and R. R. Breaker, 2000, J. Mol. Biol, 298, 623. Spiegelman, S., 1971, Q Rev Biophys 4, 213. Stojanovic, M. N., P. de Prada and D. W. Landry, 2000, J. Am. Chem. Soc. 122, 11547. Stojanovic, M. N., P. de Prada and D. W. Landry, 2001, J. Am. Chem. Soc. 123, 4928. Tan, W., X. Fang, J. Li and X. Liu, 2000, Chemistry 6, 1107. Tang, J. and R. R. Breaker, 1997, Chem. Biol. 4, 453. Tucker, C. E., L. S. Chen, M. B. Judkins, J. A. Farmer, S. C. Gill and D. W. Drolet, 1999, J. Chromatogr. B Biomed. Sci. Appl. 732, 203. Tuerk, C. and L. Gold, 1990, Science 249, 505. Tyagi, S., D. P. Bratu and F. R. Kramer, 1998, Nature Biotechnol. 16, 49. Tyagi, S. and F. R. Kramer, 1996, Nature Biotechnol. 14, 303. Vitiello, D., D. B. Pecchia and J. M. Burke, 2000, RNA 6, 628. Walkup, G. K. and B. Imperiali, 1996, J. Am. Chem. Soc. 118, 3053. Walkup, G. K. and B. Imperiali, 1997, J. Am. Chem. Soc. 119, 3443. Warashina, M., T. Kuwabara and K. Taira, 2000, Structure Fold Des. 8, R207. Wedekind, J. E. and D. B. McKay, 1999, Nature Struct. Biol. 6, 261. 395

Rajendran and EUington Willis, M. C., B. D. Collins, T. Zhang, L. S. Green, D. P. Sebesta, C. Bell, E. Kellogg, S. C. Gill, A. Magal!anez, S. Knauer, R. A. Bendele, P. S. Gill, N. Janjic and B. Collins, 1998, Bioconjug. Chem. 9, 573. Wilson, C. and J. W. Szostak, 1995, Nature 374, 777. Wilson, D. S. and J. W. Szostak, 1999, Annu. Rev. Biochem. 68, 611. Yamamoto, R.~ T. Baba and P. K. Kumar, 2000, Genes Cells 5,389.

396

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 92002 Elsevier Science B.V. All rights reserved

CHAPTER 13

NEW MATERIALS BASED ON IMPRINTED POLYMERS AND THEIR APPLICATION IN OPTICAL SENSORS

SERGEY A. PILETSKY, PH.D* AND ANTHONY P.F. TURNER, PH.D., D.Sc.

Institute of BioScience and Technology, Cranfield University, Silsoe, Bedfordshire, MK45 4DT, UK.

Molecular imprinting is the process of template-induced formation of specific recognition sites (binding or catalytic) in a material where the template directs the positioning and orientation of the material's structural components by a self-assembling mechanism. Synthetic receptors prepared using molecular imprinting possess a unique combination of properties, such as high affinity, specificity, low price and robustness, which make them an attractive alternative to natural receptors, enzymes and antibodies used in biosensors. This review gives a brief overview of the technology with specific emphasis on the mechanisms underlying the ability of imprinted polymers to perform highly selective functions such as recognition and transformation of a binding event into a detectable optical signal. The problems associated with the application of molecularly imprinted polymers (MIPs) in sensors are highlighted. Possible solutions to these problems are discussed and recommendations made about where commercial application of imprinted sensors seems most feasible in the near future.

1. Molecular Imprinting The molecular imprinting approach exploits the formation of a complex between a template molecule and functional monomers, which is fixed by copolymerisation with cross-linker into a growing polymer network (Figure 1). Following removal of the template, binding sites are left in the polymers, which have the shape and orientation of functional groups, complementary to those of the template molecule (Wulff, 1995; Mayes and Mosbach, 1997). 397

Piletsky and Turner

Figure 1. Scheme of molecularly imprinted polymerization.

1.1. Different formats used for design of imprinted materials The typical recipe for MIP preparation includes mixing together target compound -template with corresponding functional monomer (most f r e q u e n t l y methacrylic acid) and cross-linker (e.g., ethylene glycol dimethacrylate) in appropriate solvent (chloroform, acetonitrile) and polymeristion of this mixture using UV or chemical initiation (O'Shannessy et al., 1989). The template can be extracted from the polymer by washing or by electrophoresis (Piletsky et al., 1992a). Subsequent polymer grinding and washing yields the polymer particles with receptor sites on the accessible surface. Other formats of molecular imprinting include: 1. Polycondensation of silica acid in the presence of template (Katz and Davis, 2000); 2. Electropolymerisation (Malitesta et al., 1999); 3. Formation of 2-dimensional templated monolayers onto a SIO2, metal oxide or gold surface (Starodub et al., 1992; Mirsky et al., 1999); 4. Grafting of imprinted polymers to the inert solid surface (Dhal et al., 1995; Piletsky et al., 2000a) (Figure 2); 5. Templating of a pre-formed polymer structure by precipitation or crosslinking in the presence of template (Braco et al., 1990; Peissker and Fischer, 1999); 6. Formation of imprinted poly/oligomers (e.g. peptides) in the presence of template (Giraudi et al., 2000; Piletska et al., 2000). 398

Materials Based on Imprinted Polymers

Figure 2. Scheme of MIP synthesis via surface photografting onto porous polymeric substrate (Piletsky et al., 2000a) Historically, formation of imprinted silica gels was the first example of molecular imprinting (Polyakov, 1931). Despite the fact that this specific technique reached its peak in the sixties and is now in the process of gradual decline, due to limited flexibility of the method, it still remains the most popular choice for the preparation of specific zeolites (Dong et al., 2000). Electropolymerisation faces the same type of problem as silica imprinting due to limited number of polymerisable functional monomers available, which are selected mainly from the group of aniline, phenol, pyrrole and thiophene (Panasyuk et al., 1999; B lanchard et al., 2000). Electropolymerisation retains its attraction, however, because it provides a means for precise deposition of a sensitive layer on an electrode surface, which is extremely important for microand multisensor production. Electropolymerised MIPs have been used almost exclusively in potentiometric (Boyle et al., 1989; u et al., 1990) and amperometric sensors (Piletsky et al., 1994a). Two-dimensional MIPs or imprinted monolayes were developed and used in optical sensors by Andersson and co-authors (1988). They used Tabushi's method (Tabushi et al., 1987) to ~immobilise octadecylchlorosilane in the presence of inert template hosts (n-hexadecane) onto a silicon oxide surface. 399

Piletsky and Turner After the extraction of hosts, vitamin K1 was detected by ellipsometry. Expanding this method for the preparation of monolayers, imprinted with watersoluble templates, we developed materials selective for amino and nucleic acids (Piletsky and Starodub, 1992b). This approach involves two steps" first, adsorption of the template on the surface of SiO2 or metal oxide; and second, treatment of the surface with adsorbed template by trimethyl chlorosilane from the gas phase. In another similar approach, a gold surface was imprinted with a cholesterol-specific monolayer using co-adsorption of the template with hexadecylmercaptane (Piletsky et al., 1999a). Despite some advantages, such as fast sensor response and easy preparation, these systems, however, suffer from lack of stability. The lateral mobility of the components of imprinted monolayer is responsible for steady decrease in the specificity of imprinted cavities. An essential improvement of the sensor stability was achieved by co-immobilisation of the template in imprinted layer. Using a new approach called "spread bar architecture design," it was possible to develop stable monolayers, consisting of template - thiobarbituric acid - and functional monomer- hexadecylmercaptane. A depression in the hexadecylmercaptane layer formed by the template was able to accommodate barbituric acid, changing electrode capacitance in the binding proces (Mirsky et al., 1999). This two-dimensional format for MIP design is particularly attractive for evanescence-wave sensing, e.g. surface plasmon resonance. Several reports on the preparation of MIPs by surface grafting have appeared, where a thin imprinted layer, most frequently a monolayer, is formed on a solid support (Dhal et al., 1995; Lele et al., 1999; Piletsky et al., 2000a). Grafting can be performed using chemical, UV or plasma initiation (Shi et al., 1999; Piletsky et al., 2000b). The advantage of this approach lies in the possibility of modifying very inert surface (polystyrene, polypropylene, etc.) with specific polymers. The additional attraction of electropolymerisation and grafting methods is their convenient format, which does not require an additional processing step. MIP synthesis and immobilisation is performed as a one-step procedure, directed by applied potential or by exposing the monomer mixture-coated detector to UV light (Figure 2). A further approach, frequently called "bioimprinting", involves precipitation or cross-linking of biological molecules (proteins) in the presence of template (Braco et al., 1990; Peissker and Fischer, 1999). The conformation adopted by interacting biopolymer around the template remains fixed after template extraction with an appropriate solvent. Although the authors are unaware of any examples where bioimprinting has been used for sensor design, this technique could potentially be useful for introducing either additional recognition sites into enzymes or catalytic sites into antibodies. These chimeric molecules might possess the combined characteristics of antibodies and enzymes and, in this way, be useful for the development of new, label-free types of assays and sensors. 400

Materials Based on Imprinted Polymers The last format of molecular imprinting is template-directed synthesis. This process includes the formation of a new substance by a chemical modification of the substrate, or by the coupling of two or more molecules, in the presence of a template to serve as a pattern for the formation of a new structure. The most well known example of this process is gene replication. An important issue is that the synthesised molecule always has a structure, complementary to that of template, which can be exploited for the synthesis of biospecific ligands or to obtain information about the structure and properties of the template molecule. This approach is actively pursued in molecular biology (gene sequencing) and in DNA sensors where complementary DNA or RNA chains are synthesised using transcription facilitated by enzymes such as DNA polymerase or reverse transcriptase. Unfortunately, a similar technique does not exist for the analysis of molecules other than DNA, such as proteins and polysaccharides. It is possible, however, to produce a complementary ligand for a target molecule using a synthetic approach (Giraudi et al., 2000; Piletska et al., 2000). The method involves the formation of oligomers, e.g., peptides, in the presence of template. Prior to the initiation of polymerisation, and during polymerisation, the monomers, which could be amino acids or nucleotides, spatially distribute themselves around the template molecules in accordance with the size, polarity and functionality of the template. The monomers are polymerised into linear, water-soluble oligomers specific for the template. The advantage of this approach is the possibility of obtaining watersoluble ligands, which can be treated in the same manner as antibodies and other natural receptors. Not withstanding the new methods detailed above, traditional bulk polymerisation remains the most popular choice for the preparation of molecularly imprinted polymers for theoretical study and practical application in separation and sensing.

1.2. Mechanism of template recognition by imprinted polymer Three major factors determine the recognition process" the quantity of the functional groups participating in the interaction, their correct arrangement within the cavity, and the shape of the cavity itself. The types of interactions explored in molecular imprinting include reversible covalent bonds (Wulff and Haarer, 1991), electrostatic interactions (ionic and hydrogen bonds) (Piletsky et al., 1990a; Nicholls et al., 1995), van der Waals (Dickert et al., 1998), hydrophobic interactions (Yu et al., 1997), and metal chelation (Matsui et al., 1996) (Figure 3). The shape of the cavity alone can provide specificity (Yoshizako et al., 1998) although the specificity is substantially better when the

401

Piletsky and Turner

OH) 2 +

~

+ H20

(a)

H

coo. § R.H ~

oO'U..~" .rid ~;H

(b)

+ ~N.,.R

0

~H__R

Figure 3. Different types of interactions explored in molecular imprinting: (a) reversible covalent bond formation; (b) electrostatic interactions; (c) metal chelation.

template interacts with one or more properly oriented functional monomers (Ramstrom et al., 1993). The required strength of monomer-template interaction varies depending on the size and the structure of the template. For a small template molecule, the presence of strong interactions, preferably ionic and/or hydrogen bonds, is critically important. For a large molecule such as a protein or nucleic acid, successful results can be achieved with a combination of multiple weak interactions (Hjerten et al., 1997). The choice of solvent depends on the type of interaction. Thus if template recognition depends on hydrogen bond formation, better results can be achieved if both polymer synthesis and re-binding takes place in a hydrophobic solvent, where hydrogen bonds are stronger (Andersson, 1996; Yu and Mosbach, 2000). The equilibrium dissociation constants (Kd) for the binding of ligands to their corresponding polymers have been estimated by Scatchard plot analysis of binding data. Mostly, non-linear plots were obtained because of multiple Kd values, varying in range in the majority of cases from micromolar to nanomolar. In a similar way to polyclonal antibodies, imprinted polymers contain a heterogeneous population of binding sites (Wulff, 1995). One of the important components of the recognition mechanism observed in MIP systems is the conformational change in the polymer induced by template interaction (Piletsky et al., 1992a; Watanabe et al., 1998; Wolfbeis et al., 1998). Depending on experimental conditions (solvent, temperature, and types of the monomer-template and monomer-monomer interactions), the polymer matrix can 402

Materials Based on Imprinted Polymers Table 1. Comparison of natural antibodies and receptors with MIPs. ..

..,.

,,,

Property

,

Natural Biomolecules .,

Stai~iiity

Low

Cost

High

Integrati& into multisensor unit

Compatibility with micromaching technology/.miniaturisation Spectrum of analytes

i

|,|

,

n

1=,.

i

-

MIPs

,

' Difficult due to integration OiY" natural biomolecules in multisensor unit is difficult due to different operational requirements of these molecules (pH, ionic strength, temperature, subs tsr,ate) Poor

Stable at low/high pHs, pressure and temperature . Inexpensive and easy preparation Flexible MIP design allows preparation of MIPs against many combinations of analytes Fully Compatible Practicaliy unlimited

Limited

i

ii

shrink or swell in the presence of template. The mechanism is similar to "induced fit" observed for natural enzymes and receptors (Koshland, 1995; Agmon, 2000). The importance of this effect for sensor technology lies in the possibility of use for measuring template concentration (Piletsky et al., 1998). An additional factor contributing to MIP recognition properties is the presence of nanopores in the polymer structure with specificity for the template molecules (Piletsky et al., 1990b; MathewKrotz and Shea, 1996). Membranes prepared by molecular imprinting possess selective permeability for the imprinted species and can be used for purification of desirable analytes or removal of potential interfering compounds. MIPs are capable of recognising small variations in the structure of the template and the specificity of imprinted polymers under optimised conditions is often equal to or even superior to that of natural enzymes and receptors (Andersson et al., 1995). However, quite often MIPs demonstrate a high level of non-specific binding. Although they quite often referred to in this way, it would be a mistake to see imprinted polymers as "plastic" antibodies or receptors. They are different materials with their own advantages and disadvantages and thus should be considered as additive, complementary systems rather than substitutes (Table 1).

403

Piletsky and Turner 1.3. "Pluses" and "minuses" in MIP technology and their comparison with natural enzymes and receptors

Being purely synthetic materials, it is natural that the imprinted polymers have a much higher stability than enzymes and receptors. The reason for this lies, first of all, in the high level of cross-linking, which provides adequate protection for binding sites created in the polymer by imprinting. Imprinted polymers can withstand harsh treatment with acidic and basic solutions or with organic solvent. They are stable under both high and low pressure, and, as well as at extreme temperatures (Kriz and Mosbach, 1995; Svenson and Nicholls, 2001). Imprinting polymerisation is a very inexpensive procedure for the development of artificial receptors. In the majority of cases, the price of a MIP depends almost entirely on the price of the template used. Furthermore, if the templates themselves are expensive, it maybe possible to recover the template and use it again. Alternatively, inexpensive template analogues can be used for the preparation of MIPs. Generally speaking, MIP preparation is three-to-four orders less expensive than production of the equivalent natural receptor, and this makes the technology very competitive. The possibility of using MIPs in organic solvents opens new areas of application such as biomimetic sensing and catalysis in chemical and pharmaceutical manufacturing. Quality control and on-line monitoring of manufacturing processes are particularly attractive. One of the most challenging problems associated with development of multisensors is related to the significant differences in the performance of natural enzymes and receptors. These biological materials all have different stability, activity and sensitivity; in many cases, they require different substrates and buffers with different ionic strengths and pHs. Due to such factors, the integration of naturally occurring bio-molecules in one single unit maybe problematic. Since a MIP's design is flexible and variety of monomers are available for their preparation, it is possible to develop a set of polymers specific for a range of templates which will have almost identical operational requirements (solvent, temperature, pH, etc.). An additional benefit comes from the possibility of processing MIPs in the same way as traditional photoresist materials. MIPs can be immobilised at precise spots on the detector surface using masks and photopolymerisation. The compatibility of MIPs with micromachining technology makes MIP-based multisensors feasible. Last, but not least, is the ability to develop MIPs for practically any type of compound. Examples of templates producing MIPs successful include inorganic ions, drugs, nucleic acids, proteins and even cells (Table 2). Although antibodies 404

Materials Based on Imprinted Polymers Table 2. Examples of templates used in molecular imprinting. i|

Ill

i

i

Template

Application

Reference

Amino acids and derivatives

Separation, sensors

Kempe and Mosbach, 1995; Vidiasankar et al., 1997; Piletsky et al., 1998

Aniline, phenol, derivatives

Sensing

Vinokurov and Grigoreva, 1990; Morita et al., 1997

Drugs

Separation, sensing

Levi et al., 1997; Wang et al., 1997; Mirsky et al., 1999; Andersson, 2000

Flavanoids

Sensing

SuS.rez-Rodriguez and Diaz-Garcia, 2000

Herbicides

Separation, sensing

Kroger et al., 1999; Sergeeva et al., 1999, 2001

Inorganic ions

Separation and sensing

Hutchins and Bachas, 1995; Yoshida et al., 2000; Kimaro et al., 2001.

Microorganisms

Recognition

Alexander and Vulfson, 1997; Dickert et al., 2001

Nucleic acids and derivatives

Separation, sensing

Piletsky et al., 1990a, 1990b; MathewKrotz and Shea, 1996

Polynuclear aromatic hydrocarbons

Sensing

Dickert et al., 1998

Proteins

Separation, recognition

Hjerten et al., 1997; Shi et al., 1999

Steroids

Separation, detection

Hishiya et al., 1999; Rachkov et al., 2000

Sugars, sugar derivatives

Separation, sensing

Wulff and Haarer, 1991; Piletsky et al., 1998

Toxins and narcotics

Separation, sensing

Kriz and Mosbach, 1995; Matshui et al., 1996; Takeuchi et al., 2001.

Volatile compounds

Sensing

Ji et al., 200; Dickert et al., 2001.

i

405

Piletsky and Turner can also be prepared for a broad range of analytes, they have two disadvantages when compared to MIPs. Firstly, small compounds often have to be derivatised in order to generate the antibodies. This necessitates an additional synthetic step, which can sometimes drastically change the recognition characteristics. Secondly, flexibility in antibody preparation is limited to twenty naturally occurring amino acids. In the case of MIPs, the large number of synthetic monomers available make it possible to engineer binding sites with a variety and flexibility unmatched by nature. As any other technology, molecular imprinting has shortcomings. Among them are: (i) absence of a general technology for MIP design; (ii) poor performance of MIPs in aqueous environments; (iii) high level of non-specific binding which produces too low a signal-to-noise ratio in sensors; (iv) poor processability of MIPs; and (v) difficulty in transforming binding events into electrical signals. Several attempts have been made in the past to develop a general procedure for the rational design of imprinted polymers with predictable properties (Nicholls, 1995; Whitcombe et al., 1998; Takeuchi et al., 1999; Lanza and Sellergren., 1999). In the best examples, workers have produced rules or hints, indicating how MIPs should be made in order to possess a certain level of specificity. The most important conclusion is that the stability of the monomer-template complex formed during polymerisation determines the affinity of the resulting polymer. Thus it is known that polymerisation should be performed in a hydrophobic solvent in order to produce a material able to interact with template through electrostatic interactions. At the same time, the choice of the monomer, solvent and polymerisation conditions generally depends on common knowledge, one's personal experience, or available information describing the behaviour of the similar systems. Recently we developed a method that is believed to be a general solution for MIP design (Piletsky et al., 2000c, 2001). The method involves computational screening of a virtual library of functional monomers against a target molecule. The monomers giving the best score in virtual binding experiments are then brought into the contact with template and left to equilibrate. The composition of the monomer shell surrounding the template after equilibration provides the information on the type and quantity of monomers, and should be used for polymer design. Commercially available software permits calculations to be performed using different dielectric constants, reflecting the polarity of the environment (solvent) where the polymers are prepared and used. Polymers designed using this computational approach have proved to have excellent affinity and specificity for the target compound, surpassing those of polyclonal antibodies (Table 3). The possibility of tailoring MIPs for specific target analytes and specific operational conditions is very attractive since it permits polymers to be 406

Materials Based on Imprinted Polymers Table 3. Affinity and sensitivity range of computationally designed molecularly imprinted polymer in comparison with antibodies for the template-- microcystine-LR. Receptor

Ka, (nM)

Sensitivity range (/xg 1~)

Computational MIP

0.3 + 0.08

0.1-100

Monoclonal antibody

0.03 _+0.004

0.025-5

Polyclonal antibody

0.5 +_0.07

0.05-10

i

iii

developed with optimised characteristics and shortens the time needed for design, preparation and testing of the polymers. The computer simulation and molecular modelling approaches could also help to solve a second major problem associated with MIPs - their poor performance in aqueous environments. The majority of monomers used so far in polymer design form hydrogen and ionic bonds in the process of template recognition. These interactions are less effective in polar solvents and as a result, the use of such MIPs is restricted mainly to hydrophobic solvents such as chloroform, toluene, and acetonitrile. Although MIPs capable of forming hydrophobic and van der Waals interactions with the template under aqueous conditions have been developed (Dickert et al., 1999), the design of such polymers is much more difficult than the design of MIPs which exploit electrostatic interactions. The reason lies in the complex nature of factors contributing to hydrophobic and van der Waals interactions. Computer simulation and molecular modeling can, in principle, solve this problem and help to select the monomers ideally suited for the recognition of the template in water. Typically the ratio of functional monomer:template used in molecular imprinting is 4:1 to 10:1. Therefore, the resulting polymer contains large amounts of monomers outside of the specific binding sites; these are capable of non-specific interaction with the molecules other than the template. Additionally the crosslinker itself can interact with variety of analytes in aqueous media. A combination of these factors, together with the large surface area (80-200 m2/g) of the polymer, is responsible for a high level of non-specific binding, which hinders the development of MIP-based affinity materials and sensors. It is possible to overcome this problem however, by further optimisation of the polymerisation procedure and by rational selection of monomers capable of forming stoichiometric complexes with templates (Lubke et al., 2000).

407

Piletsky and Turner

Figure 4. Three principal types of MIP sensors: (a) Affinity sensor, where response is produced by accumulation of template on MIP surface; (b) Receptor sensor where response is generated by changes in polymer characteristics, induced by its inl:eraction with template; (c) Enzyme-mimicking sensor responding to the change in the environment induced by MIP-mediated catalytic reaction.

Detection of binding can be achieved with the help of optical devices, if the template has, for example, fluorescent properties. At least three general characteristics of MIPs can be used for the design of MIP-based sensors (Figure 4)' First, by substituting MIPs for the antibodies in immunosensors (affinity sensors); Second, by exploring of the receptor properties of the imprinted polymers (receptor sensors); Third, by combining MIPs possessing catalytic properties with traditional electrochemical or optical transducers (catalytic sensors). The majority of biosensors produced to date use enzymes as biorecognition element (Turner, 1999). The reason for this lies in the amplification effect achieved as result of multiple turnover of catalytic processes. Many of the unique characteristics of enzymes are connected with their polymeric nature and this fact attracts attention to the methods of development of the MIP catalysts (Srikovsky et al., 2000). The application of catalytically-active MIPs for sensor development seems to be promising and attractive as the most direct way of achieving the replacement of current biosensors by more stable devices. Nevertheless, no practical examples exist of the integration of MIPs that mimic natural enzymes into sensors. Although essential progress has been made in MIP catalysis, imprinted polymers still have properties inferior to natural enzymes 408

Materials Based on Imprinted Polymers e.g., much lower activity and turnover. Success in the preparation of more effective MIPs-based catalysts, with high turnover and reacting with watersoluble and practically important analytes, will change this situation. Due to the very limited information available on the development of MIP-based catalytic sensors, this review will concentrate on the development of the two remaining types of sensors: affinity and receptor-min'ticking devices. Integration of imprinted polymers with detectors remains a difficult issue. Despite significant improvement in MIP technology, the processability of these materials remains challenging. The high level of cross-linking, necessary for maintaining the polymer's specificity, makes them extremely hard, solid, and fragile materials. One solution to this problem is the use of plasticisers, such as oligourethane acrylates (Sergeeva et al., 1999). As a result, polymers can be made in the form of thin and stable membranes which can be used directly in sensors. Alternatively, imprinted polymers can be grafted (Mirsky et al., 1999) or electropolymerised (Boyle et al., 1989) onto the detector surface.

2. Development of MIP-based Optical Sensors The majority of published papers related to MIP sensors deal with electrochemical or piezoelectric devices. We believe, however, that optical and, in particular, fluorescent sensors will play a major role in the future. This confidence is based on the great flexibility which fluorescent detection offers to MIP technology. The account below highlights current achievements and prospects for the development and commercialisation of MIP sensors, which use optical detection for template recognition.

2.1. Affinity sensors The most common type of MIP sensor is the affinity, immunosensor-type device. The detection principle here is based on the measurement of the concentration of template adsorbed by MIP immobilised on the detector surface. The first example of this type of device was the development of a two-dimensional MIP sensor for vitamin K1 (Andersson et al., 1988). Ellipsometry was used for the measurement of template concentration. Although this work was very preliminary and suffered from lack of appropriate controls, it demonstrated the possibility for direct detection of a template adsorbed by an imprinted mono!ayer. Steinke and co-authors, proposed an interesting variant of an optical sensor device based on MIPs (Steinke et al., 1996). The completely transparent imprinted polymer prepared for their experiments had anisotropic properties and provided a particular orientation of bound template molecules. The polymers therefore showed a pronounced dichroism in UV light, which enabled specific

409

Figure 5. Selectivity pattern of pyrene detection by fluorescence, using polyurethanes imprinted with polyaromatic hydrocarbons of different sizes (Dickert and Tortschanoff, 1999).

binding to be recognised. This work could be applicable in particular for the detection of optical isomers. An optical sensor specific for the fluorescent substance dansyl-L-phenylalanine was developed using a dansyl-L-phenylalanine-imprinted polymer and a fibreoptic sensing device (Kriz et al., 1995). Accumulation of fluorescent template in the polymer matrix resulted in an increase in fluorescence that could be used to detect 10 mg/1 of substrate within 4 h. In another example, fluorescent polycyclic aromatic hydrocarbons were selectively enriched and detected using optical sensors based on imprinted polyurethanes (Figure 5) (Dickert and Tortschanoff, 1999). A problem associated with broadening the scope of this method, is the limited quantity of fluorescent substances, which are practically important and can be used as templates in the preparation of MIPs for sensor technology. To overcome this problem, sensors can be developed that operate in a competitive 410

Materials Based on Imprinted Polymers

0.12 0.1

< J c3

0.08

Triazine

/

~

0.06

e

r~

O

0.04 0.02

Simazine

ill

_

1.E-10

I

t,..x

1.E-08

1.E-06

1.E-04

Log C, M Figure 6. Displacement of fluorescein-triazine from triazine-imprinted polymer by analytes (Piletsky et al., 1997).

mode. The important question was whether the binding sites in imprinted polymers are capable of recognising template molecules that are labeled with a fluorescent dye or enzyme. Successful demonstration of this possibility was performed for polymer imprinted with triazine (Piletsky et al., 1997). The competition between fluorescein-labeled and unlabeled template was used to measure 10s - 10.5 M concentrations of free template dissolved in ethanol (Figure 6). The polymer was able to discriminate the template from other triazines (e.g., atrazine) and triazinone (simazine). Later, competitive assays withenzyme-labeled templates were developed for epinephrine (Piletsky et al., 2000b) and 2,4-dichlorophenoxyacetic acid (Surugiu et al., 2001). The displacement format has been used for the development of an MIP sensor for chloramphenicol (CA) (Levi et al., 1997). The sensor included an HPLC column with CA-specific MIPs. A constant flow of dye-labeled CA (chloramphenicolMethyl Red) at a concentration of 0.5 ~tg/ml was run through the column under equilibrium conditions. When analyte containing free CA was injected, it displaced the adsorbed conjugate, giving a peak with an area proportional to CA concentration (Figure 7). Successful analysis of chloramphenicol was achieved in model and real samples (blood serum). 411

Piletsky and Turner

_

_

/


OH N

OSCH2 O I

|

CH2

|

OH

0H O

Figure 11. Schematic representation of polymer binding site for cAMP with signalling trans-4-[p-(N,N-dimethylamino)styryl]-N-vinylbenzylpyridinium monomer (Turkewitsch et al., 1998).

CMe

Me

CH2--CHCOO

OOCCH=CH2

Strong quenching of fluorescent emission induced by hydrogen bonding might be used for recognition of polar templates. Similarly betaine dyes with strong hypsochromic effect for protic solvents were used for gas phase analysis (Figure 10) (Dickert et al., 2000). Environmentally-sensitive dye has been used in design of a fluorescent sensor for cAMP detection (Turkewitsch et al., 1998). In this case a fluorescent dye, trans4-[p-(N,N-dimethylamino)styryl]-N-vinylbenzylpyridinium chloride, was copolymerised with cross-linker and template. The resulting polymer displayed two functions simultaneously: template recognition and sensing (Figure 11). 416

Materials Based on Imprinted Polymers A new type of proximity scintillation assay has been developed for (S)propranolol (Ye and Mosbach, 2001). A scintillation monomer, 4hydroxymethyl-2,5-diphenyloxazole acrylate (II), has been covalently incorporated into MIP microparticles during the imprinting reaction. This monomer is capable of transforming 13-radiation from the bound tritium-labeled template into a fluorescent signal. The small size of the particles (0.6-1 ~tm) guarantees that the reporter group, randomly distributed throughout the polymer matrix, is located in close proximity to the MIP binding site for signal generation.

.CH2OCOCH==CH2 II

A reverse scenario can be used, in principle, for the quantification of the concentration of environment-sensitive templates (Matsui et al., 2000). Fluorescent spectra of the cinchona alkaloids exhibit a characteristic shift through binding to these polymer particles, containing acidic m o n o m e r - 2(trifluoromethyl)acrylic acid (TFMAA). The authors demonstrated the possibility of using TFMAA-based imprinted polymers as polymer reagents for analysis of the cinchona alkaloid bound to the polymers without bound/free separation (Figure 12).

0

0

III

Fluorescent polymers containing monomer (III) were imprinted with various N abenzylidene pyridine-2-carboxamidrazones (Rathbone and Ge, 2001). Dramatic quenching of fluorescence approaching background levels was observed for most

417

Piletsky and Turner | .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

,,

.

.=,

1

..g

5:000

h

4O0O

po

:C o C~

30.00

(-)-cinchonidine

0

=

2:000

(9

> r

1000

340

390

440

490

540

5.90

Wav etength (rim) Figure 12. Fluorescence spectra of MIP suspension incubated with cinchonidine in chloroform/acetonitrile at concentrations of (mM): a) 0; b) 0.005; c) 0.02; d) 0.05; e) 0.10; f) 0.15; g) 0.25; h) 0.50. (~,ex=330nm) (Matsui et al., 2000).

cases where the "empty" MIP was re-exposed to its template. The authors claim that this approach is suitable for high throughput screening. Jenkins and coauthors developed a very sensitive lanthanide-based luminescent sensor for sarin and soman with a detection limit of 7 ppt (Jenkins et al., 1998). The sensor functions by selectively and reversibly binding the phosphonate hydrolysis product of this agent to a MIP containing a coordinatively bound Eu 3§ ion. This binding leads to the appearance of a narrow luminescence band in the 610-nm region of the Eu 3§ spectrum, which can be monitored using a miniature spectrometer. A high degree of selectivity is obtained by combining both chemical and spectroscopic selectivities. Very promising combinations of group-specific fluorescent reporters with template-specific MIPs were reported for sugars (Wang et al., 1999), carboxylic 418

Materials Based on Imprinted Polymers acids (Zhang et al., 2001), and primary amines (Subrahmanyam et al., 2000). In the first two cases, the anthracene reporting group was modified in order to introduce polymerisable and recognition functionalities. The interactions of boronic acid (IV) with cis-dioles and guanidine (V) with carboxylic acids is non-specific by its nature. Nevertheless they could be made specific by incorporating these monomers into specific binding sites created by imprinting.

0

\

//

_ / N\

B(OH)2 IV

Similarly non-specific interaction between thioacetale and primary amines, which leads to formation of fluorescent isoindole complex, was made specific for creatine by imprinting in the presence of methylated analogue of the template (Subrahmanyam et al., 2000) (Figure 13).

/

/~

H

H2N

u

Diode lasers are instruments of choice for the development of fluorescent sensors: their light flux is coherent and allows better integration with waveguides, and they are inexpensive and small. However, diode lasers necessitate polymerisable fluorescent markers with long wave adsorption and emission, which at present are scarce. A further problem associated with the development of MIP-based optical sensors is light scatter due to heterogeneity in polymer structure.

419

Piletsky and Turner

S

~

S OH H

+

H2N-R

O

Figure 13. Complex formation between polymerisable thioacetale and primary amine, and recognition of analytes by Blank polymer and polymer imprinted with methylated creatine analogue (Subrahmanyam et al., 2000).

One way to overcome this problem is optimisation of the polymerisation conditions (first of all by choice of solvent and polymerisation temperature) which will lead to synthesis of optically transparent and homogeneous materials. Another way is to measure the decay time of luminescence, rather than its intensity. This approach is highly advantageous because measurements of decay time are less affected by light scattering, analyte concentration and detector sensitivity.

420

Materials Based on Imprinted Polymers 3. Market Potential of MIP Sensors and Future Prospects

Three particular properties make commercial application of MIP sensors attractive: (i) polymers are highly stable and can be autoclaved; (ii) they are fully compatible with microfabrication technology, and (iii) the low cost of the materials and easy processes of polymer preparation in comparison with natural and other artificial receptor systems. The most promising areas of MIP sensor applications are: 1. chemical and pharmaceutical manufacturing: using MIP sensors in extreme conditions (high and low pH's, toxic solvents and high temperature, pressure, and radiation); 2. medicine and pharmaceuticals: application of MIPs mimicking natural receptors for drug screening and for in vivo monitoring; 3. environment: remote sensing, continuous emissions sensors and pointsource monitors; 4. defence: rapid detection of chemical and biological warfare agents under battlefield and civil conditions; 5. deep ocean and space exploration: sensors for analysis of extreme environments. Several key problems associated with MIP development need to be addressed, however, before the successful commercialisation can commence. The issues include: 1. development and validation of a general protocol for MIP design; 2. development of MIPs capable of effective functioning in water; 3. the need for a substantial increase in polymer affinity and improvement of the ratio between specific and non-specific binding; 4. development of effective immobilisation protocols. With further progress in polymer science and engineering we can expect to see the appearance of a new generation of MIP sensors which will gradually replace traditional biosensors and chemical sensors in many areas of biotechnology and pharmacology, environmental, clinical and food analysis.

4. References Agmon, N., 2000, J. Phys. Chem. B 104, 7830. Alexander, C. and E. N. Vulfson, 1997, Adv. Mater. 9, 751. Andersson, L. I., R. Muller, G. Vlatakis and K. Mosbach, 1995, Proc. Natl. Acad. Sci USA, 92, 4788. Andersson, L. I., 1996, Anal. Chem. 68, 111. Andersson, L. I., C. F. Mandenius and K. Mosbach, 1988, Tetrahydron Lett. 29, 5437. 421

Piletsky and Turner Andersson, L. I., 2000, J. Chromatogr. B 739, 163. B lanchard, P., L. Huchet, E. Levillain and J. Roncali, 2000, Electrochem. Commun. 2, 1. Boyle, A., E. M. Genies and M. Lapkowski, 1989, Synth. Metals. 28, C769. Braco, L., K. Dabulis and A. M. Klibanov, 1990, Proc. Natl. Acad. Sci. USA 87, 274. Cooper, M. E., B. P. Hoag and D. L. Gin, 1997, Polym. Prepr. 38, 209. Dhal, P. K., S. Vidyasankar and F. H. Arnold, 1995, Chem. Mater. 7, 154. DickeR, F. L., H. Besenbock and M. Tortschanoff, 1998, Adv. Mater. 10, 149. Dickert, F. and M. Tortschanoff, 1999, Anal. Chem. 71, 4559. Dickert, F., U. Geiger, P. Lieberzeit and U. Reumer, 2000, Sens. Actuators B 70, 263. Dickert, F. L., O. Hayden and K. P. Halikias, 2001, Analyst 126, 766. Dong, J., Y. S. Lin, M. Z.-C. Hu, R. A. Peascoe and E. A. Payzant, 2000, Micropor. Mesopor. Mat. 34, 241. Giraudi, G., C. Giovannoli, C. Tozzi, C. Baggiani and L. Anfossi, 2000, Chem. Commun. 1135. Hishiya, T, M. Shibata, M. Kakazu, H. Asanuma and M. Komiyama, 1999, Macromolecules 32, 2265. Hjerten, S., J. L. Liao, K. Nakazato, Y. Wang, G. Zamaratskaia and H. X. Zhang, 1997, Chromatograph. 44, 227. Hutchins, R. S. and G. Bachas, 1995, Anal. Chem. 67, 1654. Jenkins, A. L., O. M. Uy and G. M. Murray, 1998, Anal. Chem. 71,373. Ji, H. S., S. McNiven, K. H. Lee, T. Saito, K. Ikebukuro and I. Karube, 2000, Biosens. Bioelectron. 15,403. Katz, A. and M. E. Davis, 2000, Nature 403,286. Kempe, M. and K. Mosbach, 1995, J. Chromatogr. A, 691, 317. Kimaro, A., L. A. Kelly and G. M. Murray, 2001, Chem. Commun. 1282. Koshland, D. E., 1995, Angew. Chem. Int. Ed. 33, 2375. Kriz, D. and K. Mosbach, 1995, Anal. Chim. Acta 300, 71. Kriz, D., O. Ramstrom, A. Svensson and K. Mosbach, 1995, Anal. Chem. 67, 2142. Kroger, S., A. P. F. Tutner, K. Mosbach and K. Haupt, 1999, Anal. Chem. 71, 3698. Lakowicz, J. R., W. Wiczk, I. Gryczynsky, M. Fishman and M. L. Johnson, 1993, Macromolecules 26, 349. Lanza F. and B. Sellergren, 1999, Anal. Chem. 71, 2092. Lele, B. S., M. G. Kulkarni and R. A. Mashelkar, 1999, React. Functional Polym. 39, 37. Levi, R., S. McNiven, S. A. Piletsky, S.-H. Cheong, K.Yano and I. Karube, 1997, Anal. Chem. 69, 2017. Lubke, C., M. Lubke, M. J. Whitcombe and E. N. Vulfson, 2000, Macromolecules, 33, 5098. Malitesta, C., I. Losito and P. G. Zambonin, 1999, Anal. Chem. 71, 1366. MathewKrotz, J. and K. J. Shea, 1996, J. Am. Chem. Soc. 118, 8154. 422

Materials Based on Imprinted Polymers

Matsui, J., I. A. Nicholls, T. Takeuchi, K. Mosbach and I. Karube, 1996, Anal. Chim Acta 335, 71. Matsui J., H. Kubo and T. Takeuchi, 2000, Anal. Chem. 72, 3286. Mayes, A. G. and K. Mosbach, 1997, TrAC 16, 321. Mirsky, V. M, T. Hirsch, S. A. Piletsky and O. S. Wolfbeis, 1999, Angew. Chemie, Intern. Ed. 38/8, 1108. Morita, M., O. Niwa and T. Horiuchi, 1997, Electrochim. Acta 42, 3177. Nicholls, I. A., 1995, Chem. Lett. 1035. Nicholls, I. A., O. Ramstrom and K. Mosbach, 1995, J. Chromatogr. A, 691,349. O'Shannessy, D. J., B. Ekberg and K. Mosbach, 1989, Anal. Biochem. 177, 144. Panasyuk, T. L., V. M. Mirsky, S. A. Piletsky and O. S. Wolfbeis, 1999, Anal. Chem. 71, 4609. Peissker, F. and L. Fischer, 1999, Bioorg. Med. Chem. 7, 2231. Piletska E. V., S. A. Piletsky, S. Subrahmanyam, I. A. Nicholls and A. P. F. Turner, 2000, Proc. 1st Int. Workshop on Molecular Imprinting, Cardiff, UK, 2000, 87. Piletsky, S. A., D. M. Fedoryak and V. P. Kukhar, 1990a, Dokl. Acad. Sci. Ukraine B. 4, 53 (in Russian). Piletsky, S. A., I. Ya. Dubey, D. M. Fedoryak and V. P. Kukhar, 1990b, Biopolym. Cell 6, 55 (in Russian). Piletsky, S. A., I. A. Butovich and V. P. Kukhar, 1992a, Zh. Anal. Khim. 47, 1681 (in Russian). Piletsky, S. A. and N. F. Starodub, 1992b, Zh. Anal. Khim. 47, 623 (in Russian). Piletsky, S. A., Ya. I. Kuris', A. E. Rachkov and A. V. Erskaya, 1994a, Russ. J. Electrochem. 30, 1090 (in Russian). Piletsky, S. A., Yu. P. Parhometz, T. L. Panasyuk and A. V. El' skaya, 1994, Sens. Actuators B. 18/19, 629. Piletsky, S. A., E. V. Piletska, K. Yano, A. Kugimiya, A. V. Elgersma, R. Levi, U. Kahlow, T. Takeuchi, I. Karube, T. L. Panasyuk and A. V. El' skaya, 1996, Anal. Lett. 29, 157. Piletsky, S. A., E. V. Piletska, A. V. El' skaya, R. Levi, K. Yano and I. Karube, 1997, Anal. Lett. 30, 445. Piletsky, S. A., E. V. Piletskaya, T. L. Panasyuk, A. V. El' skaya, R. Levi, I. Karube and G. Wulff, 1998, Macromolecules 31, 2137. Piletsky, S. A., E. V. Piletskaya, T. A. Sergeeva, T. L. Panasyuk and A. V. El' skaya, 1999a, Sens. Actuators B 60, 216. Piletsky, S. A., E. Terpetschnig, H. S. Andersson, I. A. Nicholls and O. S. Wolfbeis, 1999b, Fresenius J. Anal. Chem. 364, 512. Piletsky, S. A., H. Matuschewski, U. Schedler, A. Wilpert, E. V. Piletska, T. A. Thiele and M. Ulbricht, 2000a, Macromolecules 33, 3092. Piletsky, S. A. E. V. Piletska, B. Chen, K. Karim, D. Weston, G. Barrett, P. Lowe and A. P. F. Turner, 2000b, Anal. Chem. 72, 4381. Piletsky, S. A., R. M. Day, B. Chen, S. Subrahmanyam, O. Piletska and A. P. F. Turner, 2000c, UK patent application 0001513.1. 423

Piletsky and Turner Piletsky, S. A., K. Karim, E. V. Piletska, C. J. Day, K. W. Freebairn, C. Legge and A. P. F. Turner, 2001, Analyst, in press. Polyakov, M. V., 1931, Zhur. Fiz. Khim. 2, 799 (in Russian). Rachkov, A., S. McNiven, A. V. Erskaya, K. Yano and I. Karube, 2000, Anal. Chim. Acta 405, 23. Ramstrom, O., L. I. Andersson and K. Mosbach, 1993, J. Org. Chem. 58, 7562. Rathbone, D. L., D. Su, Y. Wang and D. C. B illington, 2000, Tetrahedron Lett. 41, 123. Rathbone, D. L. and Y. Ge, 2001, Anal. Chim. Acta 435,129. Sergeyeva, T. A., S. A. Piletsky, A. A. Brovko, E. A. Slinchenko, L. M. Sergeeva and A. V. Erskaya, 1999, Anal. Chim. Acta 392, 105. Sergeyeva, T. A., H. Matuschewski, S. A. Piletsky, J. Bendig, U. Schedler and M. Ulbricht, 2001, J. Chromatogr. A 907, 89. Shi, H. Q., W. B. Tsai, M. D. Garrison, S. Ferrari and B. D. Ratner, 1999, Nature 398, 593. Starodub, N. F., S. A. Piletsky, N. V. Lavryk and E. V. El' skaya, 1992, Sens. Actuators B 13-14, 708. Steinke, J. H. G., I. R. Dunkin and D. C. Sherrington, 1996, Macromolecules 29, 407. Strikovsky, A. G., D. Kasper, M. Grtin, B. S. Green, J. Hradil and G. Wulff, 2000, J. Am. Chem. Soc. 122, 6295. Su~irez-Rodrfguez, J. L. and M. E. Dfaz-Garcia, 2000, Anal. Chim. Acta, 405, 67. Subrahmanyam, S., S. A. Piletsky, E. V. Piletska, B. Chen, R. Day and A. P. F. Turner, 2000, Adv. Mater. 12, 722. Surugiu, I., B. Danielsson, L. Ye, K. Mosbach and K. Haupt, 2001, Anal. Chem. 73,487. Svenson, J. and I. A. Nicholls, 2001, Anal. Chim. Acta 435, 19. Tabushi, I., K. Kurihara, K. Naka, K. Yamamura and H. Hatakeyama, 1987, Tetrahedron Lett. 28, 4299. Takeuchi, T., D. Fukuma and J. Matsui, 1999, Anal. Chem. 71,285. Takeuchi, T., A. Seko, J. Matsui and T. Mukawa, 2001, Instrum. Sci. Technol. 29, 1. Turner, A. P. F., 1999, Biosensor: McGraw-Hill Yearbook of Science and Technology, McGraw-Hill, New York. Vidiasankar, S., M. Ru and F. H. Arnold, 1997, J. Chromatogr. A, 775, 51. Vinokurov, I. A. and M. A. Grigoreva, 1990, Zh. Anal. Khim. 45, 1009 (in Russian). Wang, H. Y., T. Kobayashi, T. Fukaya and N. Fujii, 1997, Langmuir 13, 5396. Wang, W., S. Gao and B. Wang, 1999, Org. Lett. 1, 1209. Watanabe, M., T. Akahoshi, Y. Tabata and D. Nakayama, 1998, J. Am. Chem. Soc. 120, 5577. Whitcombe, M. J., L. Martin and E. N. Vulfson, 1998, Chromatogr. 47, 457. Wolfbeis, O. S., E. Terpetschnig, S. A. Piletsky and E. Pringsheim, 1998, In Iimprinted Polymers: Applied Fluorescence in Chemistry, Biology and

424

Materials Based on Imprinted Polymers Medicine, Eds. W. Rettig, B. Strehmel, S. Schrader, H. Seifert, Springer, Berlin-Heidelberg. Wulff, G. and J. Haarer, 1991, Makromol. Chem. 192, 1329. Wulff, G., 1995, Angew. Chem. Int. Ed. Engl. 34, 1812. Ye, L. and K. Mosbach, 2001, J. Am. Chem. Soc. 123, 2901. Yoshida, M., Y. Hatate, K. Uezu, M. Goto, S. Furusaki, 2000, J. Polym. Sci. Pol. Chem. 38, 689. Yoshizako, K., K. Hosoya, Y. Iwakoshi, K. Kimata, N. Tanaka, 1998, Anal. Chem. 70, 386. Yu, C., O. Ramstrom, K. Mosbach, 1997, Anal. Lett. 30, 2123. Yu, C., K. Mosbach, 2000, J. Chromatogr. A 888, 63. Zhang, H., W. Verboom, D. N. Reinhoudt, 2001, Tetrahedr. Lett. 42, 4413.

425

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 92002 Elsevier Science B.V. All rights reserved

CHAPTER 13

NEW MATERIALS BASED ON IMPRINTED POLYMERS AND THEIR APPLICATION IN OPTICAL SENSORS

SERGEY A. PILETSKY, PH.D* AND ANTHONY P.F. TURNER, PH.D., D.Sc.

Institute of BioScience and Technology, Cranfield University, Silsoe, Bedfordshire, MK45 4DT, UK.

Molecular imprinting is the process of template-induced formation of specific recognition sites (binding or catalytic) in a material where the template directs the positioning and orientation of the material's structural components by a self-assembling mechanism. Synthetic receptors prepared using molecular imprinting possess a unique combination of properties, such as high affinity, specificity, low price and robustness, which make them an attractive alternative to natural receptors, enzymes and antibodies used in biosensors. This review gives a brief overview of the technology with specific emphasis on the mechanisms underlying the ability of imprinted polymers to perform highly selective functions such as recognition and transformation of a binding event into a detectable optical signal. The problems associated with the application of molecularly imprinted polymers (MIPs) in sensors are highlighted. Possible solutions to these problems are discussed and recommendations made about where commercial application of imprinted sensors seems most feasible in the near future.

1. Molecular Imprinting The molecular imprinting approach exploits the formation of a complex between a template molecule and functional monomers, which is fixed by copolymerisation with cross-linker into a growing polymer network (Figure 1). Following removal of the template, binding sites are left in the polymers, which have the shape and orientation of functional groups, complementary to those of the template molecule (Wulff, 1995; Mayes and Mosbach, 1997). 397

Piletsky and Turner

Figure 1. Scheme of molecularly imprinted polymerization.

1.1. Different formats used for design of imprinted materials The typical recipe for MIP preparation includes mixing together target compound -template with corresponding functional monomer (most f r e q u e n t l y methacrylic acid) and cross-linker (e.g., ethylene glycol dimethacrylate) in appropriate solvent (chloroform, acetonitrile) and polymeristion of this mixture using UV or chemical initiation (O'Shannessy et al., 1989). The template can be extracted from the polymer by washing or by electrophoresis (Piletsky et al., 1992a). Subsequent polymer grinding and washing yields the polymer particles with receptor sites on the accessible surface. Other formats of molecular imprinting include: 1. Polycondensation of silica acid in the presence of template (Katz and Davis, 2000); 2. Electropolymerisation (Malitesta et al., 1999); 3. Formation of 2-dimensional templated monolayers onto a SIO2, metal oxide or gold surface (Starodub et al., 1992; Mirsky et al., 1999); 4. Grafting of imprinted polymers to the inert solid surface (Dhal et al., 1995; Piletsky et al., 2000a) (Figure 2); 5. Templating of a pre-formed polymer structure by precipitation or crosslinking in the presence of template (Braco et al., 1990; Peissker and Fischer, 1999); 6. Formation of imprinted poly/oligomers (e.g. peptides) in the presence of template (Giraudi et al., 2000; Piletska et al., 2000). 398

Materials Based on Imprinted Polymers

Figure 2. Scheme of MIP synthesis via surface photografting onto porous polymeric substrate (Piletsky et al., 2000a) Historically, formation of imprinted silica gels was the first example of molecular imprinting (Polyakov, 1931). Despite the fact that this specific technique reached its peak in the sixties and is now in the process of gradual decline, due to limited flexibility of the method, it still remains the most popular choice for the preparation of specific zeolites (Dong et al., 2000). Electropolymerisation faces the same type of problem as silica imprinting due to limited number of polymerisable functional monomers available, which are selected mainly from the group of aniline, phenol, pyrrole and thiophene (Panasyuk et al., 1999; B lanchard et al., 2000). Electropolymerisation retains its attraction, however, because it provides a means for precise deposition of a sensitive layer on an electrode surface, which is extremely important for microand multisensor production. Electropolymerised MIPs have been used almost exclusively in potentiometric (Boyle et al., 1989; u et al., 1990) and amperometric sensors (Piletsky et al., 1994a). Two-dimensional MIPs or imprinted monolayes were developed and used in optical sensors by Andersson and co-authors (1988). They used Tabushi's method (Tabushi et al., 1987) to ~immobilise octadecylchlorosilane in the presence of inert template hosts (n-hexadecane) onto a silicon oxide surface. 399

Piletsky and Turner After the extraction of hosts, vitamin K1 was detected by ellipsometry. Expanding this method for the preparation of monolayers, imprinted with watersoluble templates, we developed materials selective for amino and nucleic acids (Piletsky and Starodub, 1992b). This approach involves two steps" first, adsorption of the template on the surface of SiO2 or metal oxide; and second, treatment of the surface with adsorbed template by trimethyl chlorosilane from the gas phase. In another similar approach, a gold surface was imprinted with a cholesterol-specific monolayer using co-adsorption of the template with hexadecylmercaptane (Piletsky et al., 1999a). Despite some advantages, such as fast sensor response and easy preparation, these systems, however, suffer from lack of stability. The lateral mobility of the components of imprinted monolayer is responsible for steady decrease in the specificity of imprinted cavities. An essential improvement of the sensor stability was achieved by co-immobilisation of the template in imprinted layer. Using a new approach called "spread bar architecture design," it was possible to develop stable monolayers, consisting of template - thiobarbituric acid - and functional monomer- hexadecylmercaptane. A depression in the hexadecylmercaptane layer formed by the template was able to accommodate barbituric acid, changing electrode capacitance in the binding proces (Mirsky et al., 1999). This two-dimensional format for MIP design is particularly attractive for evanescence-wave sensing, e.g. surface plasmon resonance. Several reports on the preparation of MIPs by surface grafting have appeared, where a thin imprinted layer, most frequently a monolayer, is formed on a solid support (Dhal et al., 1995; Lele et al., 1999; Piletsky et al., 2000a). Grafting can be performed using chemical, UV or plasma initiation (Shi et al., 1999; Piletsky et al., 2000b). The advantage of this approach lies in the possibility of modifying very inert surface (polystyrene, polypropylene, etc.) with specific polymers. The additional attraction of electropolymerisation and grafting methods is their convenient format, which does not require an additional processing step. MIP synthesis and immobilisation is performed as a one-step procedure, directed by applied potential or by exposing the monomer mixture-coated detector to UV light (Figure 2). A further approach, frequently called "bioimprinting", involves precipitation or cross-linking of biological molecules (proteins) in the presence of template (Braco et al., 1990; Peissker and Fischer, 1999). The conformation adopted by interacting biopolymer around the template remains fixed after template extraction with an appropriate solvent. Although the authors are unaware of any examples where bioimprinting has been used for sensor design, this technique could potentially be useful for introducing either additional recognition sites into enzymes or catalytic sites into antibodies. These chimeric molecules might possess the combined characteristics of antibodies and enzymes and, in this way, be useful for the development of new, label-free types of assays and sensors. 400

Materials Based on Imprinted Polymers The last format of molecular imprinting is template-directed synthesis. This process includes the formation of a new substance by a chemical modification of the substrate, or by the coupling of two or more molecules, in the presence of a template to serve as a pattern for the formation of a new structure. The most well known example of this process is gene replication. An important issue is that the synthesised molecule always has a structure, complementary to that of template, which can be exploited for the synthesis of biospecific ligands or to obtain information about the structure and properties of the template molecule. This approach is actively pursued in molecular biology (gene sequencing) and in DNA sensors where complementary DNA or RNA chains are synthesised using transcription facilitated by enzymes such as DNA polymerase or reverse transcriptase. Unfortunately, a similar technique does not exist for the analysis of molecules other than DNA, such as proteins and polysaccharides. It is possible, however, to produce a complementary ligand for a target molecule using a synthetic approach (Giraudi et al., 2000; Piletska et al., 2000). The method involves the formation of oligomers, e.g., peptides, in the presence of template. Prior to the initiation of polymerisation, and during polymerisation, the monomers, which could be amino acids or nucleotides, spatially distribute themselves around the template molecules in accordance with the size, polarity and functionality of the template. The monomers are polymerised into linear, water-soluble oligomers specific for the template. The advantage of this approach is the possibility of obtaining watersoluble ligands, which can be treated in the same manner as antibodies and other natural receptors. Not withstanding the new methods detailed above, traditional bulk polymerisation remains the most popular choice for the preparation of molecularly imprinted polymers for theoretical study and practical application in separation and sensing.

1.2. Mechanism of template recognition by imprinted polymer Three major factors determine the recognition process" the quantity of the functional groups participating in the interaction, their correct arrangement within the cavity, and the shape of the cavity itself. The types of interactions explored in molecular imprinting include reversible covalent bonds (Wulff and Haarer, 1991), electrostatic interactions (ionic and hydrogen bonds) (Piletsky et al., 1990a; Nicholls et al., 1995), van der Waals (Dickert et al., 1998), hydrophobic interactions (Yu et al., 1997), and metal chelation (Matsui et al., 1996) (Figure 3). The shape of the cavity alone can provide specificity (Yoshizako et al., 1998) although the specificity is substantially better when the

401

Piletsky and Turner

OH) 2 +

~

+ H20

(a)

H

coo. § R.H ~

oO'U..~" .rid ~;H

(b)

+ ~N.,.R

0

~H__R

Figure 3. Different types of interactions explored in molecular imprinting: (a) reversible covalent bond formation; (b) electrostatic interactions; (c) metal chelation.

template interacts with one or more properly oriented functional monomers (Ramstrom et al., 1993). The required strength of monomer-template interaction varies depending on the size and the structure of the template. For a small template molecule, the presence of strong interactions, preferably ionic and/or hydrogen bonds, is critically important. For a large molecule such as a protein or nucleic acid, successful results can be achieved with a combination of multiple weak interactions (Hjerten et al., 1997). The choice of solvent depends on the type of interaction. Thus if template recognition depends on hydrogen bond formation, better results can be achieved if both polymer synthesis and re-binding takes place in a hydrophobic solvent, where hydrogen bonds are stronger (Andersson, 1996; Yu and Mosbach, 2000). The equilibrium dissociation constants (Kd) for the binding of ligands to their corresponding polymers have been estimated by Scatchard plot analysis of binding data. Mostly, non-linear plots were obtained because of multiple Kd values, varying in range in the majority of cases from micromolar to nanomolar. In a similar way to polyclonal antibodies, imprinted polymers contain a heterogeneous population of binding sites (Wulff, 1995). One of the important components of the recognition mechanism observed in MIP systems is the conformational change in the polymer induced by template interaction (Piletsky et al., 1992a; Watanabe et al., 1998; Wolfbeis et al., 1998). Depending on experimental conditions (solvent, temperature, and types of the monomer-template and monomer-monomer interactions), the polymer matrix can 402

Materials Based on Imprinted Polymers Table 1. Comparison of natural antibodies and receptors with MIPs. ..

..,.

,,,

Property

,

Natural Biomolecules .,

Stai~iiity

Low

Cost

High

Integrati& into multisensor unit

Compatibility with micromaching technology/.miniaturisation Spectrum of analytes

i

|,|

,

n

1=,.

i

-

MIPs

,

' Difficult due to integration OiY" natural biomolecules in multisensor unit is difficult due to different operational requirements of these molecules (pH, ionic strength, temperature, subs tsr,ate) Poor

Stable at low/high pHs, pressure and temperature . Inexpensive and easy preparation Flexible MIP design allows preparation of MIPs against many combinations of analytes Fully Compatible Practicaliy unlimited

Limited

i

ii

shrink or swell in the presence of template. The mechanism is similar to "induced fit" observed for natural enzymes and receptors (Koshland, 1995; Agmon, 2000). The importance of this effect for sensor technology lies in the possibility of use for measuring template concentration (Piletsky et al., 1998). An additional factor contributing to MIP recognition properties is the presence of nanopores in the polymer structure with specificity for the template molecules (Piletsky et al., 1990b; MathewKrotz and Shea, 1996). Membranes prepared by molecular imprinting possess selective permeability for the imprinted species and can be used for purification of desirable analytes or removal of potential interfering compounds. MIPs are capable of recognising small variations in the structure of the template and the specificity of imprinted polymers under optimised conditions is often equal to or even superior to that of natural enzymes and receptors (Andersson et al., 1995). However, quite often MIPs demonstrate a high level of non-specific binding. Although they quite often referred to in this way, it would be a mistake to see imprinted polymers as "plastic" antibodies or receptors. They are different materials with their own advantages and disadvantages and thus should be considered as additive, complementary systems rather than substitutes (Table 1).

403

Piletsky and Turner 1.3. "Pluses" and "minuses" in MIP technology and their comparison with natural enzymes and receptors

Being purely synthetic materials, it is natural that the imprinted polymers have a much higher stability than enzymes and receptors. The reason for this lies, first of all, in the high level of cross-linking, which provides adequate protection for binding sites created in the polymer by imprinting. Imprinted polymers can withstand harsh treatment with acidic and basic solutions or with organic solvent. They are stable under both high and low pressure, and, as well as at extreme temperatures (Kriz and Mosbach, 1995; Svenson and Nicholls, 2001). Imprinting polymerisation is a very inexpensive procedure for the development of artificial receptors. In the majority of cases, the price of a MIP depends almost entirely on the price of the template used. Furthermore, if the templates themselves are expensive, it maybe possible to recover the template and use it again. Alternatively, inexpensive template analogues can be used for the preparation of MIPs. Generally speaking, MIP preparation is three-to-four orders less expensive than production of the equivalent natural receptor, and this makes the technology very competitive. The possibility of using MIPs in organic solvents opens new areas of application such as biomimetic sensing and catalysis in chemical and pharmaceutical manufacturing. Quality control and on-line monitoring of manufacturing processes are particularly attractive. One of the most challenging problems associated with development of multisensors is related to the significant differences in the performance of natural enzymes and receptors. These biological materials all have different stability, activity and sensitivity; in many cases, they require different substrates and buffers with different ionic strengths and pHs. Due to such factors, the integration of naturally occurring bio-molecules in one single unit maybe problematic. Since a MIP's design is flexible and variety of monomers are available for their preparation, it is possible to develop a set of polymers specific for a range of templates which will have almost identical operational requirements (solvent, temperature, pH, etc.). An additional benefit comes from the possibility of processing MIPs in the same way as traditional photoresist materials. MIPs can be immobilised at precise spots on the detector surface using masks and photopolymerisation. The compatibility of MIPs with micromachining technology makes MIP-based multisensors feasible. Last, but not least, is the ability to develop MIPs for practically any type of compound. Examples of templates producing MIPs successful include inorganic ions, drugs, nucleic acids, proteins and even cells (Table 2). Although antibodies 404

Materials Based on Imprinted Polymers Table 2. Examples of templates used in molecular imprinting. i|

Ill

i

i

Template

Application

Reference

Amino acids and derivatives

Separation, sensors

Kempe and Mosbach, 1995; Vidiasankar et al., 1997; Piletsky et al., 1998

Aniline, phenol, derivatives

Sensing

Vinokurov and Grigoreva, 1990; Morita et al., 1997

Drugs

Separation, sensing

Levi et al., 1997; Wang et al., 1997; Mirsky et al., 1999; Andersson, 2000

Flavanoids

Sensing

SuS.rez-Rodriguez and Diaz-Garcia, 2000

Herbicides

Separation, sensing

Kroger et al., 1999; Sergeeva et al., 1999, 2001

Inorganic ions

Separation and sensing

Hutchins and Bachas, 1995; Yoshida et al., 2000; Kimaro et al., 2001.

Microorganisms

Recognition

Alexander and Vulfson, 1997; Dickert et al., 2001

Nucleic acids and derivatives

Separation, sensing

Piletsky et al., 1990a, 1990b; MathewKrotz and Shea, 1996

Polynuclear aromatic hydrocarbons

Sensing

Dickert et al., 1998

Proteins

Separation, recognition

Hjerten et al., 1997; Shi et al., 1999

Steroids

Separation, detection

Hishiya et al., 1999; Rachkov et al., 2000

Sugars, sugar derivatives

Separation, sensing

Wulff and Haarer, 1991; Piletsky et al., 1998

Toxins and narcotics

Separation, sensing

Kriz and Mosbach, 1995; Matshui et al., 1996; Takeuchi et al., 2001.

Volatile compounds

Sensing

Ji et al., 200; Dickert et al., 2001.

i

405

Piletsky and Turner can also be prepared for a broad range of analytes, they have two disadvantages when compared to MIPs. Firstly, small compounds often have to be derivatised in order to generate the antibodies. This necessitates an additional synthetic step, which can sometimes drastically change the recognition characteristics. Secondly, flexibility in antibody preparation is limited to twenty naturally occurring amino acids. In the case of MIPs, the large number of synthetic monomers available make it possible to engineer binding sites with a variety and flexibility unmatched by nature. As any other technology, molecular imprinting has shortcomings. Among them are: (i) absence of a general technology for MIP design; (ii) poor performance of MIPs in aqueous environments; (iii) high level of non-specific binding which produces too low a signal-to-noise ratio in sensors; (iv) poor processability of MIPs; and (v) difficulty in transforming binding events into electrical signals. Several attempts have been made in the past to develop a general procedure for the rational design of imprinted polymers with predictable properties (Nicholls, 1995; Whitcombe et al., 1998; Takeuchi et al., 1999; Lanza and Sellergren., 1999). In the best examples, workers have produced rules or hints, indicating how MIPs should be made in order to possess a certain level of specificity. The most important conclusion is that the stability of the monomer-template complex formed during polymerisation determines the affinity of the resulting polymer. Thus it is known that polymerisation should be performed in a hydrophobic solvent in order to produce a material able to interact with template through electrostatic interactions. At the same time, the choice of the monomer, solvent and polymerisation conditions generally depends on common knowledge, one's personal experience, or available information describing the behaviour of the similar systems. Recently we developed a method that is believed to be a general solution for MIP design (Piletsky et al., 2000c, 2001). The method involves computational screening of a virtual library of functional monomers against a target molecule. The monomers giving the best score in virtual binding experiments are then brought into the contact with template and left to equilibrate. The composition of the monomer shell surrounding the template after equilibration provides the information on the type and quantity of monomers, and should be used for polymer design. Commercially available software permits calculations to be performed using different dielectric constants, reflecting the polarity of the environment (solvent) where the polymers are prepared and used. Polymers designed using this computational approach have proved to have excellent affinity and specificity for the target compound, surpassing those of polyclonal antibodies (Table 3). The possibility of tailoring MIPs for specific target analytes and specific operational conditions is very attractive since it permits polymers to be 406

Materials Based on Imprinted Polymers Table 3. Affinity and sensitivity range of computationally designed molecularly imprinted polymer in comparison with antibodies for the template-- microcystine-LR. Receptor

Ka, (nM)

Sensitivity range (/xg 1~)

Computational MIP

0.3 + 0.08

0.1-100

Monoclonal antibody

0.03 _+0.004

0.025-5

Polyclonal antibody

0.5 +_0.07

0.05-10

i

iii

developed with optimised characteristics and shortens the time needed for design, preparation and testing of the polymers. The computer simulation and molecular modelling approaches could also help to solve a second major problem associated with MIPs - their poor performance in aqueous environments. The majority of monomers used so far in polymer design form hydrogen and ionic bonds in the process of template recognition. These interactions are less effective in polar solvents and as a result, the use of such MIPs is restricted mainly to hydrophobic solvents such as chloroform, toluene, and acetonitrile. Although MIPs capable of forming hydrophobic and van der Waals interactions with the template under aqueous conditions have been developed (Dickert et al., 1999), the design of such polymers is much more difficult than the design of MIPs which exploit electrostatic interactions. The reason lies in the complex nature of factors contributing to hydrophobic and van der Waals interactions. Computer simulation and molecular modeling can, in principle, solve this problem and help to select the monomers ideally suited for the recognition of the template in water. Typically the ratio of functional monomer:template used in molecular imprinting is 4:1 to 10:1. Therefore, the resulting polymer contains large amounts of monomers outside of the specific binding sites; these are capable of non-specific interaction with the molecules other than the template. Additionally the crosslinker itself can interact with variety of analytes in aqueous media. A combination of these factors, together with the large surface area (80-200 m2/g) of the polymer, is responsible for a high level of non-specific binding, which hinders the development of MIP-based affinity materials and sensors. It is possible to overcome this problem however, by further optimisation of the polymerisation procedure and by rational selection of monomers capable of forming stoichiometric complexes with templates (Lubke et al., 2000).

407

Piletsky and Turner

Figure 4. Three principal types of MIP sensors: (a) Affinity sensor, where response is produced by accumulation of template on MIP surface; (b) Receptor sensor where response is generated by changes in polymer characteristics, induced by its inl:eraction with template; (c) Enzyme-mimicking sensor responding to the change in the environment induced by MIP-mediated catalytic reaction.

Detection of binding can be achieved with the help of optical devices, if the template has, for example, fluorescent properties. At least three general characteristics of MIPs can be used for the design of MIP-based sensors (Figure 4)' First, by substituting MIPs for the antibodies in immunosensors (affinity sensors); Second, by exploring of the receptor properties of the imprinted polymers (receptor sensors); Third, by combining MIPs possessing catalytic properties with traditional electrochemical or optical transducers (catalytic sensors). The majority of biosensors produced to date use enzymes as biorecognition element (Turner, 1999). The reason for this lies in the amplification effect achieved as result of multiple turnover of catalytic processes. Many of the unique characteristics of enzymes are connected with their polymeric nature and this fact attracts attention to the methods of development of the MIP catalysts (Srikovsky et al., 2000). The application of catalytically-active MIPs for sensor development seems to be promising and attractive as the most direct way of achieving the replacement of current biosensors by more stable devices. Nevertheless, no practical examples exist of the integration of MIPs that mimic natural enzymes into sensors. Although essential progress has been made in MIP catalysis, imprinted polymers still have properties inferior to natural enzymes 408

Materials Based on Imprinted Polymers e.g., much lower activity and turnover. Success in the preparation of more effective MIPs-based catalysts, with high turnover and reacting with watersoluble and practically important analytes, will change this situation. Due to the very limited information available on the development of MIP-based catalytic sensors, this review will concentrate on the development of the two remaining types of sensors: affinity and receptor-min'ticking devices. Integration of imprinted polymers with detectors remains a difficult issue. Despite significant improvement in MIP technology, the processability of these materials remains challenging. The high level of cross-linking, necessary for maintaining the polymer's specificity, makes them extremely hard, solid, and fragile materials. One solution to this problem is the use of plasticisers, such as oligourethane acrylates (Sergeeva et al., 1999). As a result, polymers can be made in the form of thin and stable membranes which can be used directly in sensors. Alternatively, imprinted polymers can be grafted (Mirsky et al., 1999) or electropolymerised (Boyle et al., 1989) onto the detector surface.

2. Development of MIP-based Optical Sensors The majority of published papers related to MIP sensors deal with electrochemical or piezoelectric devices. We believe, however, that optical and, in particular, fluorescent sensors will play a major role in the future. This confidence is based on the great flexibility which fluorescent detection offers to MIP technology. The account below highlights current achievements and prospects for the development and commercialisation of MIP sensors, which use optical detection for template recognition.

2.1. Affinity sensors The most common type of MIP sensor is the affinity, immunosensor-type device. The detection principle here is based on the measurement of the concentration of template adsorbed by MIP immobilised on the detector surface. The first example of this type of device was the development of a two-dimensional MIP sensor for vitamin K1 (Andersson et al., 1988). Ellipsometry was used for the measurement of template concentration. Although this work was very preliminary and suffered from lack of appropriate controls, it demonstrated the possibility for direct detection of a template adsorbed by an imprinted mono!ayer. Steinke and co-authors, proposed an interesting variant of an optical sensor device based on MIPs (Steinke et al., 1996). The completely transparent imprinted polymer prepared for their experiments had anisotropic properties and provided a particular orientation of bound template molecules. The polymers therefore showed a pronounced dichroism in UV light, which enabled specific

409

Figure 5. Selectivity pattern of pyrene detection by fluorescence, using polyurethanes imprinted with polyaromatic hydrocarbons of different sizes (Dickert and Tortschanoff, 1999).

binding to be recognised. This work could be applicable in particular for the detection of optical isomers. An optical sensor specific for the fluorescent substance dansyl-L-phenylalanine was developed using a dansyl-L-phenylalanine-imprinted polymer and a fibreoptic sensing device (Kriz et al., 1995). Accumulation of fluorescent template in the polymer matrix resulted in an increase in fluorescence that could be used to detect 10 mg/1 of substrate within 4 h. In another example, fluorescent polycyclic aromatic hydrocarbons were selectively enriched and detected using optical sensors based on imprinted polyurethanes (Figure 5) (Dickert and Tortschanoff, 1999). A problem associated with broadening the scope of this method, is the limited quantity of fluorescent substances, which are practically important and can be used as templates in the preparation of MIPs for sensor technology. To overcome this problem, sensors can be developed that operate in a competitive 410

Materials Based on Imprinted Polymers

0.12 0.1

< J c3

0.08

Triazine

/

~

0.06

e

r~

O

0.04 0.02

Simazine

ill

_

1.E-10

I

t,..x

1.E-08

1.E-06

1.E-04

Log C, M Figure 6. Displacement of fluorescein-triazine from triazine-imprinted polymer by analytes (Piletsky et al., 1997).

mode. The important question was whether the binding sites in imprinted polymers are capable of recognising template molecules that are labeled with a fluorescent dye or enzyme. Successful demonstration of this possibility was performed for polymer imprinted with triazine (Piletsky et al., 1997). The competition between fluorescein-labeled and unlabeled template was used to measure 10s - 10.5 M concentrations of free template dissolved in ethanol (Figure 6). The polymer was able to discriminate the template from other triazines (e.g., atrazine) and triazinone (simazine). Later, competitive assays withenzyme-labeled templates were developed for epinephrine (Piletsky et al., 2000b) and 2,4-dichlorophenoxyacetic acid (Surugiu et al., 2001). The displacement format has been used for the development of an MIP sensor for chloramphenicol (CA) (Levi et al., 1997). The sensor included an HPLC column with CA-specific MIPs. A constant flow of dye-labeled CA (chloramphenicolMethyl Red) at a concentration of 0.5 ~tg/ml was run through the column under equilibrium conditions. When analyte containing free CA was injected, it displaced the adsorbed conjugate, giving a peak with an area proportional to CA concentration (Figure 7). Successful analysis of chloramphenicol was achieved in model and real samples (blood serum). 411

Piletsky and Turner

_

_

/


OH N

OSCH2 O I

|

CH2

|

OH

0H O

Figure 11. Schematic representation of polymer binding site for cAMP with signalling trans-4-[p-(N,N-dimethylamino)styryl]-N-vinylbenzylpyridinium monomer (Turkewitsch et al., 1998).

CMe

Me

CH2--CHCOO

OOCCH=CH2

Strong quenching of fluorescent emission induced by hydrogen bonding might be used for recognition of polar templates. Similarly betaine dyes with strong hypsochromic effect for protic solvents were used for gas phase analysis (Figure 10) (Dickert et al., 2000). Environmentally-sensitive dye has been used in design of a fluorescent sensor for cAMP detection (Turkewitsch et al., 1998). In this case a fluorescent dye, trans4-[p-(N,N-dimethylamino)styryl]-N-vinylbenzylpyridinium chloride, was copolymerised with cross-linker and template. The resulting polymer displayed two functions simultaneously: template recognition and sensing (Figure 11). 416

Materials Based on Imprinted Polymers A new type of proximity scintillation assay has been developed for (S)propranolol (Ye and Mosbach, 2001). A scintillation monomer, 4hydroxymethyl-2,5-diphenyloxazole acrylate (II), has been covalently incorporated into MIP microparticles during the imprinting reaction. This monomer is capable of transforming 13-radiation from the bound tritium-labeled template into a fluorescent signal. The small size of the particles (0.6-1 ~tm) guarantees that the reporter group, randomly distributed throughout the polymer matrix, is located in close proximity to the MIP binding site for signal generation.

.CH2OCOCH==CH2 II

A reverse scenario can be used, in principle, for the quantification of the concentration of environment-sensitive templates (Matsui et al., 2000). Fluorescent spectra of the cinchona alkaloids exhibit a characteristic shift through binding to these polymer particles, containing acidic m o n o m e r - 2(trifluoromethyl)acrylic acid (TFMAA). The authors demonstrated the possibility of using TFMAA-based imprinted polymers as polymer reagents for analysis of the cinchona alkaloid bound to the polymers without bound/free separation (Figure 12).

0

0

III

Fluorescent polymers containing monomer (III) were imprinted with various N abenzylidene pyridine-2-carboxamidrazones (Rathbone and Ge, 2001). Dramatic quenching of fluorescence approaching background levels was observed for most

417

Piletsky and Turner | .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

,,

.

.=,

1

..g

5:000

h

4O0O

po

:C o C~

30.00

(-)-cinchonidine

0

=

2:000

(9

> r

1000

340

390

440

490

540

5.90

Wav etength (rim) Figure 12. Fluorescence spectra of MIP suspension incubated with cinchonidine in chloroform/acetonitrile at concentrations of (mM): a) 0; b) 0.005; c) 0.02; d) 0.05; e) 0.10; f) 0.15; g) 0.25; h) 0.50. (~,ex=330nm) (Matsui et al., 2000).

cases where the "empty" MIP was re-exposed to its template. The authors claim that this approach is suitable for high throughput screening. Jenkins and coauthors developed a very sensitive lanthanide-based luminescent sensor for sarin and soman with a detection limit of 7 ppt (Jenkins et al., 1998). The sensor functions by selectively and reversibly binding the phosphonate hydrolysis product of this agent to a MIP containing a coordinatively bound Eu 3§ ion. This binding leads to the appearance of a narrow luminescence band in the 610-nm region of the Eu 3§ spectrum, which can be monitored using a miniature spectrometer. A high degree of selectivity is obtained by combining both chemical and spectroscopic selectivities. Very promising combinations of group-specific fluorescent reporters with template-specific MIPs were reported for sugars (Wang et al., 1999), carboxylic 418

Materials Based on Imprinted Polymers acids (Zhang et al., 2001), and primary amines (Subrahmanyam et al., 2000). In the first two cases, the anthracene reporting group was modified in order to introduce polymerisable and recognition functionalities. The interactions of boronic acid (IV) with cis-dioles and guanidine (V) with carboxylic acids is non-specific by its nature. Nevertheless they could be made specific by incorporating these monomers into specific binding sites created by imprinting.

0

\

//

_ / N\

B(OH)2 IV

Similarly non-specific interaction between thioacetale and primary amines, which leads to formation of fluorescent isoindole complex, was made specific for creatine by imprinting in the presence of methylated analogue of the template (Subrahmanyam et al., 2000) (Figure 13).

/

/~

H

H2N

u

Diode lasers are instruments of choice for the development of fluorescent sensors: their light flux is coherent and allows better integration with waveguides, and they are inexpensive and small. However, diode lasers necessitate polymerisable fluorescent markers with long wave adsorption and emission, which at present are scarce. A further problem associated with the development of MIP-based optical sensors is light scatter due to heterogeneity in polymer structure.

419

Piletsky and Turner

S

~

S OH H

+

H2N-R

O

Figure 13. Complex formation between polymerisable thioacetale and primary amine, and recognition of analytes by Blank polymer and polymer imprinted with methylated creatine analogue (Subrahmanyam et al., 2000).

One way to overcome this problem is optimisation of the polymerisation conditions (first of all by choice of solvent and polymerisation temperature) which will lead to synthesis of optically transparent and homogeneous materials. Another way is to measure the decay time of luminescence, rather than its intensity. This approach is highly advantageous because measurements of decay time are less affected by light scattering, analyte concentration and detector sensitivity.

420

Materials Based on Imprinted Polymers 3. Market Potential of MIP Sensors and Future Prospects

Three particular properties make commercial application of MIP sensors attractive: (i) polymers are highly stable and can be autoclaved; (ii) they are fully compatible with microfabrication technology, and (iii) the low cost of the materials and easy processes of polymer preparation in comparison with natural and other artificial receptor systems. The most promising areas of MIP sensor applications are: 1. chemical and pharmaceutical manufacturing: using MIP sensors in extreme conditions (high and low pH's, toxic solvents and high temperature, pressure, and radiation); 2. medicine and pharmaceuticals: application of MIPs mimicking natural receptors for drug screening and for in vivo monitoring; 3. environment: remote sensing, continuous emissions sensors and pointsource monitors; 4. defence: rapid detection of chemical and biological warfare agents under battlefield and civil conditions; 5. deep ocean and space exploration: sensors for analysis of extreme environments. Several key problems associated with MIP development need to be addressed, however, before the successful commercialisation can commence. The issues include: 1. development and validation of a general protocol for MIP design; 2. development of MIPs capable of effective functioning in water; 3. the need for a substantial increase in polymer affinity and improvement of the ratio between specific and non-specific binding; 4. development of effective immobilisation protocols. With further progress in polymer science and engineering we can expect to see the appearance of a new generation of MIP sensors which will gradually replace traditional biosensors and chemical sensors in many areas of biotechnology and pharmacology, environmental, clinical and food analysis.

4. References Agmon, N., 2000, J. Phys. Chem. B 104, 7830. Alexander, C. and E. N. Vulfson, 1997, Adv. Mater. 9, 751. Andersson, L. I., R. Muller, G. Vlatakis and K. Mosbach, 1995, Proc. Natl. Acad. Sci USA, 92, 4788. Andersson, L. I., 1996, Anal. Chem. 68, 111. Andersson, L. I., C. F. Mandenius and K. Mosbach, 1988, Tetrahydron Lett. 29, 5437. 421

Piletsky and Turner Andersson, L. I., 2000, J. Chromatogr. B 739, 163. B lanchard, P., L. Huchet, E. Levillain and J. Roncali, 2000, Electrochem. Commun. 2, 1. Boyle, A., E. M. Genies and M. Lapkowski, 1989, Synth. Metals. 28, C769. Braco, L., K. Dabulis and A. M. Klibanov, 1990, Proc. Natl. Acad. Sci. USA 87, 274. Cooper, M. E., B. P. Hoag and D. L. Gin, 1997, Polym. Prepr. 38, 209. Dhal, P. K., S. Vidyasankar and F. H. Arnold, 1995, Chem. Mater. 7, 154. DickeR, F. L., H. Besenbock and M. Tortschanoff, 1998, Adv. Mater. 10, 149. Dickert, F. and M. Tortschanoff, 1999, Anal. Chem. 71, 4559. Dickert, F., U. Geiger, P. Lieberzeit and U. Reumer, 2000, Sens. Actuators B 70, 263. Dickert, F. L., O. Hayden and K. P. Halikias, 2001, Analyst 126, 766. Dong, J., Y. S. Lin, M. Z.-C. Hu, R. A. Peascoe and E. A. Payzant, 2000, Micropor. Mesopor. Mat. 34, 241. Giraudi, G., C. Giovannoli, C. Tozzi, C. Baggiani and L. Anfossi, 2000, Chem. Commun. 1135. Hishiya, T, M. Shibata, M. Kakazu, H. Asanuma and M. Komiyama, 1999, Macromolecules 32, 2265. Hjerten, S., J. L. Liao, K. Nakazato, Y. Wang, G. Zamaratskaia and H. X. Zhang, 1997, Chromatograph. 44, 227. Hutchins, R. S. and G. Bachas, 1995, Anal. Chem. 67, 1654. Jenkins, A. L., O. M. Uy and G. M. Murray, 1998, Anal. Chem. 71,373. Ji, H. S., S. McNiven, K. H. Lee, T. Saito, K. Ikebukuro and I. Karube, 2000, Biosens. Bioelectron. 15,403. Katz, A. and M. E. Davis, 2000, Nature 403,286. Kempe, M. and K. Mosbach, 1995, J. Chromatogr. A, 691, 317. Kimaro, A., L. A. Kelly and G. M. Murray, 2001, Chem. Commun. 1282. Koshland, D. E., 1995, Angew. Chem. Int. Ed. 33, 2375. Kriz, D. and K. Mosbach, 1995, Anal. Chim. Acta 300, 71. Kriz, D., O. Ramstrom, A. Svensson and K. Mosbach, 1995, Anal. Chem. 67, 2142. Kroger, S., A. P. F. Tutner, K. Mosbach and K. Haupt, 1999, Anal. Chem. 71, 3698. Lakowicz, J. R., W. Wiczk, I. Gryczynsky, M. Fishman and M. L. Johnson, 1993, Macromolecules 26, 349. Lanza F. and B. Sellergren, 1999, Anal. Chem. 71, 2092. Lele, B. S., M. G. Kulkarni and R. A. Mashelkar, 1999, React. Functional Polym. 39, 37. Levi, R., S. McNiven, S. A. Piletsky, S.-H. Cheong, K.Yano and I. Karube, 1997, Anal. Chem. 69, 2017. Lubke, C., M. Lubke, M. J. Whitcombe and E. N. Vulfson, 2000, Macromolecules, 33, 5098. Malitesta, C., I. Losito and P. G. Zambonin, 1999, Anal. Chem. 71, 1366. MathewKrotz, J. and K. J. Shea, 1996, J. Am. Chem. Soc. 118, 8154. 422

Materials Based on Imprinted Polymers

Matsui, J., I. A. Nicholls, T. Takeuchi, K. Mosbach and I. Karube, 1996, Anal. Chim Acta 335, 71. Matsui J., H. Kubo and T. Takeuchi, 2000, Anal. Chem. 72, 3286. Mayes, A. G. and K. Mosbach, 1997, TrAC 16, 321. Mirsky, V. M, T. Hirsch, S. A. Piletsky and O. S. Wolfbeis, 1999, Angew. Chemie, Intern. Ed. 38/8, 1108. Morita, M., O. Niwa and T. Horiuchi, 1997, Electrochim. Acta 42, 3177. Nicholls, I. A., 1995, Chem. Lett. 1035. Nicholls, I. A., O. Ramstrom and K. Mosbach, 1995, J. Chromatogr. A, 691,349. O'Shannessy, D. J., B. Ekberg and K. Mosbach, 1989, Anal. Biochem. 177, 144. Panasyuk, T. L., V. M. Mirsky, S. A. Piletsky and O. S. Wolfbeis, 1999, Anal. Chem. 71, 4609. Peissker, F. and L. Fischer, 1999, Bioorg. Med. Chem. 7, 2231. Piletska E. V., S. A. Piletsky, S. Subrahmanyam, I. A. Nicholls and A. P. F. Turner, 2000, Proc. 1st Int. Workshop on Molecular Imprinting, Cardiff, UK, 2000, 87. Piletsky, S. A., D. M. Fedoryak and V. P. Kukhar, 1990a, Dokl. Acad. Sci. Ukraine B. 4, 53 (in Russian). Piletsky, S. A., I. Ya. Dubey, D. M. Fedoryak and V. P. Kukhar, 1990b, Biopolym. Cell 6, 55 (in Russian). Piletsky, S. A., I. A. Butovich and V. P. Kukhar, 1992a, Zh. Anal. Khim. 47, 1681 (in Russian). Piletsky, S. A. and N. F. Starodub, 1992b, Zh. Anal. Khim. 47, 623 (in Russian). Piletsky, S. A., Ya. I. Kuris', A. E. Rachkov and A. V. Erskaya, 1994a, Russ. J. Electrochem. 30, 1090 (in Russian). Piletsky, S. A., Yu. P. Parhometz, T. L. Panasyuk and A. V. El' skaya, 1994, Sens. Actuators B. 18/19, 629. Piletsky, S. A., E. V. Piletska, K. Yano, A. Kugimiya, A. V. Elgersma, R. Levi, U. Kahlow, T. Takeuchi, I. Karube, T. L. Panasyuk and A. V. El' skaya, 1996, Anal. Lett. 29, 157. Piletsky, S. A., E. V. Piletska, A. V. El' skaya, R. Levi, K. Yano and I. Karube, 1997, Anal. Lett. 30, 445. Piletsky, S. A., E. V. Piletskaya, T. L. Panasyuk, A. V. El' skaya, R. Levi, I. Karube and G. Wulff, 1998, Macromolecules 31, 2137. Piletsky, S. A., E. V. Piletskaya, T. A. Sergeeva, T. L. Panasyuk and A. V. El' skaya, 1999a, Sens. Actuators B 60, 216. Piletsky, S. A., E. Terpetschnig, H. S. Andersson, I. A. Nicholls and O. S. Wolfbeis, 1999b, Fresenius J. Anal. Chem. 364, 512. Piletsky, S. A., H. Matuschewski, U. Schedler, A. Wilpert, E. V. Piletska, T. A. Thiele and M. Ulbricht, 2000a, Macromolecules 33, 3092. Piletsky, S. A. E. V. Piletska, B. Chen, K. Karim, D. Weston, G. Barrett, P. Lowe and A. P. F. Turner, 2000b, Anal. Chem. 72, 4381. Piletsky, S. A., R. M. Day, B. Chen, S. Subrahmanyam, O. Piletska and A. P. F. Turner, 2000c, UK patent application 0001513.1. 423

Piletsky and Turner Piletsky, S. A., K. Karim, E. V. Piletska, C. J. Day, K. W. Freebairn, C. Legge and A. P. F. Turner, 2001, Analyst, in press. Polyakov, M. V., 1931, Zhur. Fiz. Khim. 2, 799 (in Russian). Rachkov, A., S. McNiven, A. V. Erskaya, K. Yano and I. Karube, 2000, Anal. Chim. Acta 405, 23. Ramstrom, O., L. I. Andersson and K. Mosbach, 1993, J. Org. Chem. 58, 7562. Rathbone, D. L., D. Su, Y. Wang and D. C. B illington, 2000, Tetrahedron Lett. 41, 123. Rathbone, D. L. and Y. Ge, 2001, Anal. Chim. Acta 435,129. Sergeyeva, T. A., S. A. Piletsky, A. A. Brovko, E. A. Slinchenko, L. M. Sergeeva and A. V. Erskaya, 1999, Anal. Chim. Acta 392, 105. Sergeyeva, T. A., H. Matuschewski, S. A. Piletsky, J. Bendig, U. Schedler and M. Ulbricht, 2001, J. Chromatogr. A 907, 89. Shi, H. Q., W. B. Tsai, M. D. Garrison, S. Ferrari and B. D. Ratner, 1999, Nature 398, 593. Starodub, N. F., S. A. Piletsky, N. V. Lavryk and E. V. El' skaya, 1992, Sens. Actuators B 13-14, 708. Steinke, J. H. G., I. R. Dunkin and D. C. Sherrington, 1996, Macromolecules 29, 407. Strikovsky, A. G., D. Kasper, M. Grtin, B. S. Green, J. Hradil and G. Wulff, 2000, J. Am. Chem. Soc. 122, 6295. Su~irez-Rodrfguez, J. L. and M. E. Dfaz-Garcia, 2000, Anal. Chim. Acta, 405, 67. Subrahmanyam, S., S. A. Piletsky, E. V. Piletska, B. Chen, R. Day and A. P. F. Turner, 2000, Adv. Mater. 12, 722. Surugiu, I., B. Danielsson, L. Ye, K. Mosbach and K. Haupt, 2001, Anal. Chem. 73,487. Svenson, J. and I. A. Nicholls, 2001, Anal. Chim. Acta 435, 19. Tabushi, I., K. Kurihara, K. Naka, K. Yamamura and H. Hatakeyama, 1987, Tetrahedron Lett. 28, 4299. Takeuchi, T., D. Fukuma and J. Matsui, 1999, Anal. Chem. 71,285. Takeuchi, T., A. Seko, J. Matsui and T. Mukawa, 2001, Instrum. Sci. Technol. 29, 1. Turner, A. P. F., 1999, Biosensor: McGraw-Hill Yearbook of Science and Technology, McGraw-Hill, New York. Vidiasankar, S., M. Ru and F. H. Arnold, 1997, J. Chromatogr. A, 775, 51. Vinokurov, I. A. and M. A. Grigoreva, 1990, Zh. Anal. Khim. 45, 1009 (in Russian). Wang, H. Y., T. Kobayashi, T. Fukaya and N. Fujii, 1997, Langmuir 13, 5396. Wang, W., S. Gao and B. Wang, 1999, Org. Lett. 1, 1209. Watanabe, M., T. Akahoshi, Y. Tabata and D. Nakayama, 1998, J. Am. Chem. Soc. 120, 5577. Whitcombe, M. J., L. Martin and E. N. Vulfson, 1998, Chromatogr. 47, 457. Wolfbeis, O. S., E. Terpetschnig, S. A. Piletsky and E. Pringsheim, 1998, In Iimprinted Polymers: Applied Fluorescence in Chemistry, Biology and

424

Materials Based on Imprinted Polymers Medicine, Eds. W. Rettig, B. Strehmel, S. Schrader, H. Seifert, Springer, Berlin-Heidelberg. Wulff, G. and J. Haarer, 1991, Makromol. Chem. 192, 1329. Wulff, G., 1995, Angew. Chem. Int. Ed. Engl. 34, 1812. Ye, L. and K. Mosbach, 2001, J. Am. Chem. Soc. 123, 2901. Yoshida, M., Y. Hatate, K. Uezu, M. Goto, S. Furusaki, 2000, J. Polym. Sci. Pol. Chem. 38, 689. Yoshizako, K., K. Hosoya, Y. Iwakoshi, K. Kimata, N. Tanaka, 1998, Anal. Chem. 70, 386. Yu, C., O. Ramstrom, K. Mosbach, 1997, Anal. Lett. 30, 2123. Yu, C., K. Mosbach, 2000, J. Chromatogr. A 888, 63. Zhang, H., W. Verboom, D. N. Reinhoudt, 2001, Tetrahedr. Lett. 42, 4413.

425

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 92002 Elsevier Science B.V. All rights reserved

CHAPTER 14

OPTICALLY BASED SOL-GEL BIOSENSOR MATERIALS

JENNA L. RICKUS 1, BRUCE DUNN, PH.D. 2, AND JEFFREY I. ZINK, PH.D. 3 Xlnterdepartmental Program for Neuroscience, Neuroengineering Program 2Department of Materials Science and Engineering 3Department of Chemistry And Biochemistry University Of California, Los Angeles USA

The sol-gel process is a chemical technique for synthesizing a silicate matrix around a biomolecule that can function as the recognition and signaling element for a sensor. Within the past decade, biologically doped sol-gel glasses have surfaced as having great potential in optical biosensor applications. The materials are transparent in the UV and visible spectra allowing for transmission of optical signals. The glass is porous such that small analyte molecules can diffuse through the matrix and reach the large biomolecule that is physically trapped. Biological molecules including heme proteins, enzymes, and antibodies can remain active within the porous sol-gel glass. The flexibility of the method has allowed the encapsulation of a wide range of biomolecules and cells, resulting in sensor materials able to detect small molecules in both gases and in liquids.

1. Technical Concept 1.1. Introduction Solid state optical biosensors must fulfill stringent criteria that often are mutually incompatible. The first obvious criterion is that the material must allow the optical signal to pass in and out, i.e., it must be transparent in the desired wavelength region that may range from the near UV to the near IR. The material must be chemically stable and inert and must be able to function in both liquids and gases. Ideally, it should be compatible with existing optical technologies such as fiber optics and waveguides. This same material must not interfere with 427

Figure 1. Large protein molecules are trapped within the pores of the glass matrix. The small analyte can diffuse into the pore and bind to the protein.

the function of the recognition and signaling molecules. Furthermore, in the operating environment, the material must allow the analyte access to the transduction molecules without loss of the latter's functionality. The usual approach for solid state optical biosensors is to immobilize the recognition and signaling biomolecules (commonly proteins that transduce the recognition event into an optical signal) on solid supports by physical adsorption, covalent attachment, or physical entrapment (Shuler and Kargi, 1992). However, the development of a support material that meets all of the above criteria has been problematic. Sol-gel silica materials that have been developed over the past decade meet most of these criteria. Sol-gel encapsulation methods are a new and promising alternative to the more traditional immobilization techniques. It is a physical encapsulation process that eliminates desorption issues and does not require covalent modifications of the biomolecule. The solid matrix is transparent in the ultraviolet and visible spectral regions allowing for spectroscopic analysis (Avnir et al., 1994; Dave et al., 1994). Silica is a mechanically, thermally, and chemically stable material. Its hydrophilic properties and high porosity provide water-filled pores to house and stabilize signaling and transduction biomolecules. A key feature of the sol-gel material is its nanodimensional porosity. Although relatively large biomolecules can be immobilized within the inorganic network, small ions and molecules are able to diffuse into and out of the network. In this way it is possible to use the biomolecule to respond to chemical changes in its environment. Figure 1 illustrates the geometric properties of the matrix. One of the major advantages of the sol-gel encapsulation strategy is its versatility. Since the report of a sol-gel procedure for protein encapsulation in transparent silica in 1992 (Ellerby et al., 1992), there has been steady growth of bio-doped sol-gel materials. Table 1 presents some examples. With only minor 428

Optically Based Sol-Gels procedural variations, many biomolecules including enzymes and antibodies can be encapsulated. The matrix most likely forms around large molecules, thus stretching the upper limits on dopant size (Dave et al., 1997; Dunn and Zink, 1997). Large globular proteins and even cells have been successfully encapsulated (Chia et al., 2000; Pope et al., 1997; Rickus et al., 2001). Under proper conditions, the function of the biological dopant is maintained. Enzymes retain their catalytic ability, antibodies retain their binding affinity, and cells remain viable. In addition to their chemical and physical properties, sol-gel materials have a number of advantages for sensor development. Encapsulation procedures are fast, simple, and flexible. Flexibility of sensor form is provided by the variety of geometries that can be formed including powders, bulk materials, thin films and fibers. Despite the proliferation of bio-doped sol-gel materials with biosensor potential, only a few functional sensors have been realized. Most of the studies have investigated dosimeters rather than continuous sensors. In spite of the important distinction between continuous sensors and dosimeters, most of the reports in the sol-gel literature loosely use the term sensor for both. In this review, we will generically use the term 'sensor' even though the reported material was functioning as a dosimeter. This chapter will first describe the fundamentals of sol-gel encapsulation and the history of these materials. Next, we will review the effects of sol-gel encapsulation on biomolecules. Then the current state of the art will be reviewed using specific examples of sol-gel sensors and potential applications. We conclude with a discussion of the advantages and limitations of sol-gel optical biosensors.

1.2. Sol-gel chemistry The sol-gel process is a chemical synthesis technique for producing amorphous inorganic solids. A colloidal sol forms from the hydrolysis and polycondensation of a metaUo-organic precursor. Metal alkoxides are common precursors because they are easy to hydrolyze (Brinker and Scherer, 1990). Tetramethoxysilane (TMOS) and tetraethoxysilane (TEOS) are typically used because of the ability to carry out well-controlled hydrolysis and condensation reactions with these precursors (Dave et al., 1994). During biomolecule encapsulation, the silica glass matrix forms around the biomolecule from a silicon alkoxide precursor.

429

Rickus, Dunn, and Zink Table 1. Examples of Sol-Gel Encapsulated Biomolecules and Cells Biomoiecule/Cell Heine Proteins Cytochrome c Cytochrome cdl Hemoglobin Myogl0bin Enzymes Alkaline phosphatase Cholinesterase i

Nitrite reductase Glucose oxidase Glutamate dehydrogenase Horseradish peroxidase Lipase Oxalate oxidase Superoxide dismutase Tr)/psin Antibodies Anti-antrazine Anti -D dimer Anti -fluorescein Anti -TNT Other Proteins Bacteriorhodopsin Cells Bacteria Pancreatic islets Parasitic protozoa Plant cells Yeast

.... i

R'eference'

mm

-

ill

9

Ellerby et al., 1992; Wu et al., 1994 Ferretti et al., 2000 Wu et al., 1994; Khan et al., 2000 Chung et aL, 1995; Wu et al., 1994 Braun et al., 1990 Akbarian et al., 1997; Diaz and Peinado, 1997 Ferretti et al., 2000 Chen et al., 1998; Yamanaka et al., 1992 Husing et al., 1999; Rickus et al., 2001 Wu et al., 1994; Diaz et al., 1998 Reetz et al., 1996 Yamanaka et al., 1996 Ellerby et al., 1992 Braun et al., 1992 Bronshtein et al., 1997; Turniansky et al., 1996 Grant and Glass, 1999 Jordan et al., 1996; Wang et al., 1993 Lan et al., 2000 Weetall, 1996; Wu et al., 1993 Fennouh et al., 1999; Livage et al., 1996 Pope et al., 1997 Barreau et al., 1994;Livage et al., 1996 Campostrini et al., 1996 AI-Sara) et al., 1999; Chia et al., 2000

Hydrolysis is the first reaction to occur (Figure 2, top). The result is the formation of silanol (Si - OH) groups by reaction of the alkoxy (- OR, where R is - C H 3 , - C H 2 C H 3 , etc.) with water. Alcohol is released into solution. The hydrolysis is usually acid-catalyzed rather than base-catalyzed because of the preferred matrix structure. Gels formed under acidic conditions tend to form continuous, transparent polymeric structures that can be made into various

430

Optically Based Sol-Gels Hy drolysis OCHs

I

H+

C H 3 0 - - Si--OCH3 + 4H20

~

OH

I

HO--Si--OH

+ 4HOCH3

Water Condensation

OH

OH

l

OH

I

HO--Si--OH

+

HO--Si--OH

I

~-

--~

OH

I

H O - - S i - - O - - S i - - O H + H2C OH

OH

Alcohol Condelasation OH

OClls

OCH3

I

I

I

HO--Si--OH

I

OH

+

CH30--Si--OCH34-'---~

I

OCHs

OH

I

H3CO--Si--O---Si--OH + HOCH

I

OCHa

I

OH

Figure 2: The hydrolysis and polycondensation reactions for the production of the SiO2 matrix using TMOS as the precursor metal alkoxide. The condensation reactions release water and methanol into solution as the matrix forms.

optical components including lenses (Brinker and Scherer, 1990; Hench and West, 1990). Hydrolysis is followed by condensation of the silanol (Si -- OH) groups to release water (Figure 2, middle) and condensation of the silanol and alkoxy groups to release an alcohol and form siloxane (Si - O - Si) groups (Figure 2, bottom). As the condensation reactions occur, a three dimensional network of silica forms. The structural character of the final matrix, including pore size distribution, depends on the relative rates of the three reactions (hydrolysis, water condensation and alcohol condensation) (Brinker and Scherer, 1990). The gelation point is defined as the instant that the silica rrmtrix forms a continuous solid throughout its container. The material is now composed of two distinct phases, amorphous silica particles (5 - 10 nm in diameter) with an interstitial liquid phase. The gelation time can vary from a few seconds to many days depending on the synthesis conditions used. After gelation, the sol-gel 431

Rickus, Dunn, and Zink material remains a dynamic structure; condensation reactions continue to occur as long as remaining hydroxy and alkoxy groups are close enough to react with one another. Anytime after gelation, the gel can be dried under ambient conditions to form a xerogel. The pores of the silica matrix collapse as solvent is removed during drying. As a result, xerogels typically shrink to - 10-15% of their original volume and have a pore size of "

t"

/ , 500

-

, ......... 9 550

", 600

. . . . . 850

9 700

o.o

'

~e' . . . . . ". . . . . .

-0

,0,

2'o

3'0

go

Oxygen Concentration (ppm)

Wavelength ( n m )

Figure 11. Left. Aqueous phase emission spectra of sol-gel oxygen PEBBLEs excited at 488 nm. Top line: PEBBLE solution purged with N2; middle line: PEBBLE solution purged with air; bottom line: PEBBLE solution purged with 02. Right. Stern-Volmer plot of relative fluorescence intensity ratios for ratiometric sol-gel oxygen PEBBLEs in aqueous phase.

(Ormosil) films using methyltriethoxysilane (MTEOS) and ethyltriethoxysilane (ETEOS) as the precursors. The success of the Ormosil films in raising the QDO ratio to 70-80% is largely attributed to the increased hydrophobicity of the film which reduced the water solubility in the film and enhanced the partitioning of oxygen out of solution and into the film (McDonagh et al., 1998). It is thought that the high QDO response of the TEOS sol-gel PEBBLEs might be caused by the PEG content of the sensing matrix, PEG playing a role analogous to the Ormosil precursors and thus partitioning the oxygen preferentially into the solgel PEBBLEs. It is well known that oxygen has a higher solubility in organic liquids than in water (Merck, 1996), so it should dissolve much better in an organic phase compared to an aqueous phase. In summary, doping the sol-gel PEBBLEs with PEG adds organic components to the sensing matrix, thus encouraging the partitioning of oxygen into the matrix and increases the accessibility of oxygen to the entrapped indicator dye molecules. This is in addition to the role PEG plays in preventing particle aggregation during PEBBLE sensor fabrication. Figure 11 (right) shows the Stern-Volmer plot of fluorescence intensity ratios to oxygen concentrations. Although the performance of the sol-gel PEBBLEs is slightly reduced in the aqueous phase, as opposed to the gas phase, the sensors still demonstrate good reversibility and reproducibility (Xu et al., 2001). The dashed line in Figure 11 (right) shows the extent of the biologically relevant regime of oxygen concentrations. We note that in this regime (from 0 to ~30 ppm oxygen), the Stem-Volmer plot is quasi-linear (r2 = 0.988). The sensors showed at least 95% recovery each time that the sensing environments were changed among air-, O2-, or N2-saturated sensor solutions. 523

Brasuel, Kopelman, Philbert, et al. 4. Advantages and Limi,tations of PEBBLE Sensors

As mentioned, the main advantages of the PEBBLE sensor are first, protection of the cell from the sensing elements and protection of the sensing elements from cellular interferents, and second, the ability to combine components to accomplish complex sensing schemes. The greatest advantage, from a sensing standpoint, is that all of this is accomplished in a sensor with nanometer dimensions! The advantage gained by protecting the cell from the sensing elements is selfevident, especially when the selective sequestration of some dyes into cellular organelles is considered. What may be less intuitive is the interference of cellular components, especially protein, with the function of sensing dyes (Graber et al., 1986). The comparison of the function of free dyes to both acrylamide and sol-gel PEBBLEs utilizing the dyes as sensing elements clearly demonstrates the advantage of preventing macro-molecule interaction with sensing components. One example of the effect of protein binding is demonstrated with 5- (and 6-) carboxynaphthofluorescein (CNF) pH dye. CNF is a highly photostable, ratiometric dye for pH, which is not used for intracellular applications because of the error induced by macromolecule binding. However, protected in an acrylamide matrix, CNF becomes a viable tool for intracellular pH study. Figure 12 illustrates the benefit of entrapping CNF in an acrylamide matrix (PEBBLE). Incubation of the free dye with as little as 0.01% albumin induces alterations in emission ratios of almost 90% (pH was maintained constant as measured with a standard pH electrode), which is an error equivalent to 1 pH unit. The same dye, protected in the PEBBLE, shows minimal perturbation by albumin, with the resulting error equivalent to about 0.01 pH units (Clark et al., 1999b). Another example of the benefit of sensing element protection by the acrylamide matrix is found when comparing the Zn PEBBLE (based on Newport Green) to naked Newport Green in bovine serum albumin (BSA) solution. Aliquots of the BSA solution were added to either a 3 mg/mL PEBBLE suspension or a 125 nM Newport Green dye solution and the resulting fluorescence was monitored. Although Newport Green has good selectivity over intracellular ions, the dye itself is prone to artifacts resulting from non-specific binding of proteins, such as (BSA), as shown in Figure 12. Monitoring the peak of Newport Green at 530 nrn, there is a substantial increase in the peak intensity with each successive addition of BSA. In addition to the increase of intensity, there is also a 4 nm shift in peak wavelength. The PEBBLEs containing the Newport Green dye, however, are unaffected by the additions of BSA, which is confirmed by the peak wavelength remaining the same. As little as 0.02% BSA causes a major change in the Newport Green dye intensity (i.e. >200% increase) but the intensity of the

524

PEBBLE Nanosensors

3.5

160 CNF

140 ~-

o :=

dye~

3.0

120

2.5

100 ,..o

n"

._o

,c

o

20

N

o

CNF P E S e L E , ~ .'-'r"-e,'-":

ooo

o.ol

........;

0.02

::-

/

o;3

o.~

9

o os

% Albumin

/

-.-o

O.S ~ 9 ~

4r . . . . . .

/

1.o

,,o

~ .........

,,&/-"

2.0

1.5

...,

~

....

~ - - - ~ - - - ~ - - - ~ - - - o

-~..o,,,..,,~.,'~.,,~s

o.o ~ l , T , , . 0

....

.... 0.02

-.-N.~., o ,

-. . j. . . . . . . . . . . .

,,

0.04

0.1

0.06 0.08 BSA ( w / v %)

,-~,., 0,12

Figure 12. Effect of protein on sensors: Left: Adding as little as 0.01% albumin to a solution of CNF dye molecules causes almost a 90% change in the fluorescence intensity ratio of this pH-sensitive dye, even though the pH of the solution remains constant. Under the same conditions the PEBBLEs containing the CNF dye are not affected by the addition of albumin. Right: Peak emission intensity of Newport Green, 530-nm, monitored on a fluorometer. Spectra are acquired after each successive aliquot of a 10% (w/v) bovine serum albumin solution. The BSA concentrations are plotted versus the peak intensities. As little as 0.02% BSA causes over a 200% increase in Newport Green dye intensity, but the intensity of the Newport Green embedded in acrylamide PEBBLE remains unchanged, even at BSA concentrations above 0.10%. Newport Green embedded in the sensor remains unchanged, even at BSA concentrations above 0.10% (Sumner et al., 2001). As expected, it has been found that the sol-gel matrix of PEBBLE sensors also prevents macromolecules such as proteins, from diffusing through the matrix. The matrix thus protects the entrapped dyes from the intracellular environment, preventing interference with the fluorescent properties of the dyes. Without this shielding of a dye, its fluorescence would behave unpredictably inside a given cell, making calibration of even ratiometric dyes difficult or impossible. As with the acrylamide PEBBLEs the effects of non-specific protein binding have been investigated by the addition of bovine serum albumin. The results show that adding as little as 0.14% BSA to a solution containing [Ru(dpp)3] 2§ and Oregon Green 488-dextran dye (at the same molar ratio in solution as inside the PEBBLEs) changes the fluorescence intensity ratio of the two dyes by a factor of more than 2.3 (i.e., an increase of over 130%). This change is mostly due to the change in the fluorescence intensity of [Ru(dpp)3] 2§ after the addition of BSA, while the intensity of Oregon Green remains basically unchanged. However, under the same conditions, the PEBBLE sensors containing these two dyes are not affected by the addition of BSA and a change in fluorescence intensity ratio of at most 4% is observed when even an increased concentration of BSA (0.23%) is added, demonstrating the same protection as given to dyes in the acrylamide matrix. The susceptibility of the sol-gel PEBBLEs to heavy metal ions (Hg 2§ and Ag § and to one of the notorious collisional quenchers (I) were also examined. 525

Brasuel, Kopelman, Philbert, et al. Hg(NO3)2, Ag(NO)3 and KI were added to a PEBBLE solution and to a free dye solution of [Ru(dpp)3] 2§ (same concentrations as in PEBBLEs) up to a concentration of about 200 p2Pm. There was a 5-10% decrease in the fluorescence intensity of the [Ru(dpp)3] § free dye each time, while for the PEBBLEs no measurable effect could be observed (Xu et al., 2001). There is not a similar "naked" dye/PEBBLE-protected dye comparison for liquid polymer DMA-based PEBBLEs. The hydrophobic components can not be used "naked" in aqueous solution. The separate phase (the liquid polymer) is essential for the decyl methacrylate sensing mechanism and allows for the complex sensing schemes that effect the ion-exchange and co-extraction mechanisms (Buhlmann et al., 1998). The nanometer dimensions of the PEBBLEs of all three matrices give a useful advantage over traditional, monolithic optodes in terms of response time. In order to follow biological perturbations in real time, a fast response is required from the PEBBLE sensors. Most PEBBLE sensors depend on bulk-equilibrium, between sensor and solution phase (the oxygen sensor depends on steady-state). The diffusion of analyte inside acrylamide and sol-gel should be similar to that in aqueous solution phase, while the diffusion of analyte in the hydrophobic decyl methacrylate matrix is much slower. In all cases, the small size of the PEBBLE sensors gives a rapid response time, despite the need for bulk equilibrium. Figure 13 shows the response times of the various matrices. A reliable method for determining the response time of acrylamide sensors to Ca 2§ used an Olympus IX50 inverted microscope equipped with a mercury arc lamp and a PMT. Calcium selective probes were premixed with a caged calcium ion, and this solution was inserted into a quartz capillary. The calcium was uncaged with a pulse of UV light from a Quanta-Ray 10 ns Nd/Yag laser (Quanta-Ray, Mountain View, CA) equipped with a frequency tripler and coupled into an optical fiber positioned over the capillary. In Figure 13A the response time of the PEBBLE sensors was compared to that of the free dye (no polymer matrix), so as to separate the diffusion time through solution from the diffusion time through the matrix. As can be seen from Figure 13A, the 90 % response time of the PEBBLE sensor to the increase in free calcium is on the order of o n e m i l l i s e c o n d o r less. Theoretically, with an approximate diffusion constant of 10 -6 cm2/sec, the average diffusion time should be about ten microseconds for a 100 nm radius sensor, and 100 nanoseconds for a 10 nm radius sensor (Clark et al., 1999b). The measured transition times for sol-gel (Figure 13C) are on the order of 20 to 30 s, but these times are much longer than the intrinsic response time of the PEBBLEs, due to the significant contribution of the time used to saturate the solution with 02 or N2. It is difficult to measure the exact response time, because changing between oxygenated and deoxygenated PEBBLE solutions takes time. 526

PEBBLE Nanosensors

41 B

I/I

9

t~ /n

~. 30 ,~20 0 Ig

15

._o

0

17

,

,

,

5

10

15

19

. . . . . . . . ,

20

25

Time in S e c o n d s

0.7 O

C

0.6. G:

o

o.5 0.4

II111-11

i.--Im

tl-| ....

Time

,

(minute)

Figure 13. (A) Response time of acrylamide PEBBLE sensors. Calcium was released using a single 10 ns UV pulse from a Nd/Yag laser which photolysed the cage, releasing free calcium into a solution of PEBBLEs. The observed response time, less than 1 msec, was indiscernible from that of the corresponding dye not entrapped in a polymer matrix. (B) Response time of K§ PEBBLEs to added KC1 and added buffer solutions. It can be seen that response in the forward direction is about 0.5 s (0-40 mM KC1, A) and the reverse (see inset) slightly longer (40-20 mM KC1, B), 0.8s. (C) Reversibility of sol-gel PEBBLE sensor response to dissolved oxygen. 527

Brasuel, Kopelman, Philbert, et al. The measured transition times (including the time of saturating the solution) are only an upper limit of the response time. The PEBBLE sensors should intrinsically have shorter response times than previously reported thin film and fiber optic sol-gel sensors (on the order of seconds or minutes) simply because of the smaller sizes of these sensors. According to the Einstein diffusion equation, where X2=2D'c, a shorter diffusion length X (which is directly related to the size of the sensor) results in a much shorter time for oxygen molecules to diffuse through the sensing matrix (which is basically the response time). A lower limit can thus be estimated, using D=2xl0 9 m2/sec (diffusion constant of oxygen in water) and X = 3 x l 0 7 m , giving x=20xl0 6 sec, i.e., a response time in the microsecond range. An upper limit can be estimated considering that the PEBBLE sensor dimensions are 10-100 times smaller than thin film sensors, and have a spherical shape. This should give the PEBBLE sensors a response time in the millisecond range (Xu et al., 2001). For decyl methacrylate based K + sensors, using BME-44 as ionophore, ETH5350 as the chromoionophore and KTFPB as the ionic additive, the ratio of the protonated chromoionophore to free base was analyzed vs. time in response time measurements. It was found (Figure 13B) that in going from log aK+/aH+ = 3.6 to 5.7, the response time (10%-90% signal change) was about 0.5 s (for a concentration change of over 2 decades). In the reverse direction, the response was about 0.8 s (Figure 13B). This fast, sub-second response time of the PEBBLEs is a direct result of their small size. Diffusion in decyl methacrylate is in the range of 108 cm2/s, with small variations that depend on cross-linker content (Ambrose and Meyerhoff, 1996, 1997). Thus, for a PEBBLE radius of about 300 nm, one expects a diffusion time of about 10 -3 S. This is consistent with the experimental values, which are again upper limit values due to the solution mixing times (Brasuel et al., 2001). To summarize, using acrylamide or sol-gel PEBBLEs, one can monitor changes with response times on the microsecond timescale or, using the decyl methacrylate PEBBLEs, one can monitor changes on the 0.5s time scale. Leaching of dye molecules out of the PEBBLE matrix is the greatest limitation of PEBBLE sensors; it is a major concern and is very dependent on the dye and matrix combination. Factors such as the molecular size of the dye (small dyes can more readily diffuse through the pores and leak out of the matrix) and the solubility of the dye in the matrix and in water play a significant role. Assays of the leaching of CDMF and calcium green from polyacrylamide PEBBLEs (each containing the sulforhodamine 101 reference dye) show that less than 50% of the dye leaches from the PEBBLEs in a 48 hr time period. On the time scale of the current single cell experiments, mostly a couple of hours, the dye loss is acceptable. With comparable leaching rates for indicator and 528

PEBBLE Nanosensors reference dye, the problem is minimized due to the ratiometric sensing scheme used with these PEBBLEs (Clark et al., 1999b). Decyl methacrylate-based potassium sensors have a lifetime of 30 minutes, due to component leaching from the liquid polymer membrane. This is consistent with the lifetime of PVC-based optodes of the same composition (Shortreed et al., 1997). After 30 minutes, the sensor response can deviate up to 7% from the initial calibration data at lower K § concentration. After 90 minutes, the deviation is up to 13% at lower K § concentrations. The deviations are smaller at larger K § concentrations (Brasuel et al., 2001). Sol-gel supports provide excellent stability with respect to dye leaching (Ingersoll and Bright, 1997). In particular, ruthenium complexes often have excellent stability inside the sol-gel matrix and in agreement with previous reports (Klimant et al., 1999; McDonagh et al., 1998; Ingersoll and Bright, 1997; Bossi et. al., 1999; Murtagh et al., 1998) the indicator dye [Ru(dpp)3] 2§ shows no signs of leaching. For the reference dye, the large size of the dextran molecular backbone to which the Oregon Green dye molecules are bound should greatly reduce leaching. According to the dilution factor, a rough estimate provides an upper limit of 1% for the amount of dye molecules leached out of the sensing matrix over a three-day period (Xu et al., 2001). In summary, what has been determined for three matrices is that, at the short end of the lifetime scale, the decyl methacrylate PEBBLEs have a lifetime of about half an hour, at midrange the acrylamide PEBBLEs have a lifetime of about 24 hours, and the sol-gel PEBBLEs have a lifetime of more than three days. However, as the PEBBLEs are single-use sensors made for quick measurements inside in vitro cells that survive only a short period of time, this is acceptable. It should also be noted that such well documented steps as covalently attaching the dye to the matrix polymer backbone and adding cages or lipophilic tails (i.e., using dextran with hydrophilic polymers or adding lipophilic tails to lipophilic dyes) can be accomplished to increase PEBBLE sensor lifetime when needed. As PEBBLEs get smaller and smaller, the concentration of dye should remain the same, but fewer and fewer dye molecules are available for generating signal or for participating in equilibrium-based thermodynamic sensing schemes. It is known that with currently available intensified CCD (Charge Coupled Device) technology, at least 5 dye molecules per PEBBLE are required to image a single PEBBLE. This limit has already been reached with our acrylamide-based PEBBLEs. Calculations based on the dye concentration present in the acrylamide emulsion predict that for an acrylamide PEBBLE of 20 nm, on average, there is less than one dye molecule per PEBBLE. We do not attempt to image these particles singly, but rather use an ensemble of acrylamide PEBBLEs for imaging. For single acrylamide PEBBLE work, we have used 200 nm PEBBLEs, which contain approximately 100 dye molecules. In the case of liquid 529

Brasuel, Kopelman, Philbert, et al. polymer (DMA) PEBBLEs, there is a minimum number of required molecules for the thermodynamic equilibrium sensing scheme to be useful. It is not yet known what the minimum size of decyl methacrylate PEBBLEs is, but it will be obtained as the size of the liquid polymer PEBBLEs is decreased. The PEBBLE sensing technology is fairly new and the flexibility in design provided by the available matrices and components has a long way to go before all possibilities for this technology are discovered.

5.

The Future of PEBBLE Technology

The obvious next step in PEBBLE technology is to apply the PEBBLE techniques currently used to other biologically interesting ions and work continues on all three matrices to develop sensors for all ions of biological relevance. Further developments in progress are given below. As an example of an enzyme-based sensor, oxidase enzymes have been incorporated into the acrylamide PEBBLEs along with the oxygen sensing components. In this manner, enzyme selectivity is used to detect molecules of interest while the steady state oxygen sensor gives the means to indirectly monitor the analyte concentration based on the rate of enzyme activity. Glucose-sensitive PEBBLEs have been designed to be ratiometric by entrapping a reference fluorescent dye (Oregon Green 488-dextran), in addition to the oxygen sensitive dye Ru[(dpp)SO3Na]3 and the glucose oxidase enzyme, within a polyacrylamide (PAA) matrix. The common glucose sensing scheme involves the employment of glucose oxidase, which catalyzes the oxidation of glucose according to the following equations, and oxygen is consumed during the process:

d - glucose + 02 cJ.~___> d - gluconolactone + H202

(8)

d- gluconolactone + HzO --->d-gluconic acid

(9)

The measurement of the reduced oxygen level by the oxygen-sensitive dye Ru[(dpp)SO3Na]3, when glucose is oxidized by the enzyme, serves as an indirect indication for the glucose concentration (Figure 14). The calibrations of the glucose PAA PEBBLEs were taken on a FluoroMax-2 spectrofluorometer (ISA Jobin Yvon-Spex, Edison, NJ), slits set to 2 nm for both the emission and excitation. During the calibration, aliquots of a glucose solution 530

PEBBLE Nanosensors

u

1600 g

":" 1400

W r

11

1200

I m

_= looo

8 ~

O

".

0.0

'1

,

~,

L

0.5

|,

1.0

~

,.

!

1.S

...... .

,

!

--

2.0

Glucose added (raM)

Figure 14. Plot showing the response of PAA glucose PEBBLEs to the increasing glucose concentrations. were added to a cuvette that contained a PAA glucose PEBBLE solution. The changes in the ratios between the emission peak intensifies of Ru[(dpp)SO3Na]3 and Oregon Green 488-dextran, due to the changes in the glucose concentration in the PEBBLE solution, were recorded for data analysis. Modifications of the PEBBLE outer shell can serve as both a platform for targeting the PEBBLEs to specific biological locations (using antibodies) or for the development of unique sensing schemes of species too reactive to enter the polymer membrane. The attachment of antibodies to the surface of acrylamide PEBBLEs is facilitated through the use of biotin-avidin binding. This strategy involves biotinylating the PEBBLEs and the antibodies and then using avidin to bridge the two. The use of avidin as a bridging molecule is possible because of its multiple sites that bind biotin with great affinity. In order to biotinylate the acrylamide PEBBLEs, primary amine groups are introduced to the PEBBLE surface through an N-(3aminopropyl)methacrylamide copolymer, in order to produce aminefunctionalized polyacrylic acid (AFPAA) PEBBLEs. Under mildly basic conditions, the AFPAA PEBBLEs can simply be introduced to a succinimidyl ester conjugate of biotin, and the primary amines react with the ester to form a covalent bond. A similar strategy is used to produce acrylamide PEBBLE sensors for hydroxyl radical. The hydroxyl radical is one of the most reactive and potentially damaging species found in biological systems. It is primarily formed by reactions involving superoxide and/or hydrogen peroxide and copper or iron ions. A challenge in creating a sensor for the hydroxyl radical is its high reactivity. Our current detection scheme for hydroxyl radical is a PEBBLE-based assay 531

Brasuel, Kopelman, Philbert, et al. CCA

7-OH-CCA o

~~0

0-F o~H 385 nm HO"

0 ~

"0"

~0

Figure 15. Conversion of non-fluorescent CCA to the fluorescent species 7-OH-CCA by reaction with the hydroxyl radical (Maneyich et al., 1997).

using the molecule coumarin-3-carboxylic(CCA) to detect OH. CCA is a nonfluorescent aromatic compound that reacts with OH to produce 7-OH-CCA, which is highly fluorescent and emits around 450 nm (Figure 15). In most of the PEBBLEs, the dye is entrapped inside the matrix, but for the hydroxyl radical probe, CCA is attached to the outside of the PEBBLE. This is done because of the extreme reactivity of the hydroxyl radical. As with the biotinylated PEBBLEs, CCA-coated PEBBLEs are produced by using AFPAA PEBBLEs and a succinimidyl ester of CCA (coumarin-3-carboxylic acid succinimidyl ester (SECCA)). The succinimidyl ester in SECCA reacts with the primary amine groups on the PEBBLE to produce an amide bond, effectively coating the PEBBLE with CCA. Initial experimentation has shown that the functionalized CCA PEBBLEs increase fluorescence in the presence of hydroxyl radical producing reactions. The conjugation of multiple dyes (20-40+ per PEBBLE) allows for the monitoring of OH production over time in a localized area and may allow for the imaging of single PEBBLEs. This has a great advantage in the measurement of OH radical, because of the localized production and the short diffusion radius of the OH radical in biological systems. Work continues on increasing the scope of analytes that PEBBLEs can detect. PEBBLEs are a new technology. Their possible applications and scope of impact are just beginning to be realized.

532

PEBBLE Nanosensors

Color plate 2, Figure 6. Confocal images of rat C6 glioma cells loaded with sol-gel PEBBLEs by gene-gun injection. (A) Nomarski illumination overlaid with Oregon Green fluorescence of PEBBLEs inside cell. (B) Nomarski illumination overlaid with [Ru(dpp)3] 2§ fluorescence of PEBBLEs inside cells. Right Fluorescence spectra of a typical ratiometric sensor measurement of molecular oxygen inside rat C6-glioma cells; bottom line: cells (loaded with sol-gel PEBBLEs) in air-saturated DPBS; middle line: cells in N2-saturated DPBS, 25 seconds after replacing the air-saturated DPBS; top line: cells in N2-saturated DPBS, after 2 minutes.

533

Brasuel, Kopelman, Philbert, et al.

6. Acknowledgments The authors thank Steve Parus for instrumentation expertise, Susan Barker for technical assistance, Professor Mark E. Meyerhoff and Theresa M. Ambrose for help in the initial stages of utilizing DMA and Maria J. Moreno for assistance with the data normalization formalism for DMA PEBBLEs, Rhonda Lightle and Chris Edwards for transmission electron microscopy, the University of Michigan Transgenic Core for technical assistance, and University of Michigan Electron Microbeam Analysis Laboratory (funded in part by NSF grant EAR-9628196) for use of the SEM.. We also gratefully acknowledge NIH Grants 2R01-GM50300 (Kopelman) and R01-ES08846 (Philbert) for funding.

7. References Albert, K. J. and D.R. Walt, 2000, Anal. Chem. 72, 1947. Ambrose, T. M. and M.E. Meyerhoff, 1996, Electroanal. 8, 1095. Ambrose, T. M. and M.E. Meyerhoff, 1997, Anal. Chem. 69, 4092. Ammann, D., 1986, Ion-Selective Microelectrodes, Springer, Berlin, 346. Bakker, E. and W. Simon, 1992, Anal. Chem. 64, 1805. Barker, S. L. R., M.R. Shortreed and R. Kopelman, 1997, Anal. Chem. 69, 990. Barker, S. L. R., B.A.Thorsrud and R. Kopelman, 1998, Anal. Chem. 70, 100. Bossi, M. L., D., E. Marta and P.F. Aramendia, 1999, J. Photochem. Photobiol. A 120, 15. Brasuel, M., R. Kopelman, T.J. Miller, R. Tjalkens and M.A. Philbert, 2001, Anal. Chem. 73, 2221. Buhlmann, P., E. Pretsch and E. Bakker, 1998, Chem. Rev 98, 1593. Clark, H. A., S.L.R. Barker, R. Kopelman, M. Hoyer and M.A. Philbert, 1998, Sens. Actuators B Chem. 51, 12. Clark, H. A., M. Hoyer, S. Parus, M. Philbert and R. Kopelman, 1999a, Mikrochim. Acta 131, 121. Clark, H. A., M. Hoyer, M. Philbert and R. Kopelman, 1999b, Anal. Chem. 71, 4831. Clark, H. A., R. Kopelman, R. Tjalkens and M.A. Philbert, 1999c, Anal. Chem. 71, 4837. CohenKashi, M., M. Deutsch, R. Tirosh, H. Rachmani and A. Weinreb, 1997, Spectrochim. Acta A Mol. Spectrosc. 53, 1655. Daubresse, C., C. Granfils, R. Jerome and P. Teyssie, 1994, J. Colloid Interface Sci. 168, 222. Demas, J. N. and B. A. DeCrraff, 1991, Anal. Chem. 63, 829A. Dourado, S. and R. Kopelman, 1996, Proc. SPIE 2836, 2. Emmi, A., H.J. Wenzel and P.A. Schwartzkroin, 2000, J. Neurosci. 20, 3915. Graber, M. L., D.C. DiLillo, B.L. Friedman and E. Pastoriza-Munoz, 1986, Anal. Biochem. 156, 202. 534

PEBBLE Nanosensors Haugland, R. P., 1993, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc., Eugene, OR, 679. Ingersoll, C. M. and F.V. Bright, 1997, Chemtech, 27, 26. Jung, S.-K., W. Gorski, C.A. Aspinwall, L.M. Kauri and R.T. Kennedy, 1999, Anal. Chem. 71, 3642. Klimant, I., F. Ruchruh, G. Liebsch, A. Stangelmayer, and O. S. Wolfbeis, 1999, Mikrochim. Acta, 131, 35. Kurihara, K., M. Ohtsu, T. Yoshida, T. Abe, H. Hisamoto and K. Suzuki, 1999, Anal. Chem. 71, 3558. Maneyich, Y., K.D. Held, J.E. Biaglow, 1997, Radiation Res. 148, 580-591. McDonagh, C., B. D. MacCraith and A.K.McEvoy, 1998, Anal. Chem. 70, 45. McEvoy, A. K., C. McDonagh and B.D. MacCraith, 1996, Analyst, 121,785. McNamara, K. P., T. Nguyen, G. Dumitrascu, J. Ji, N. Rosenzweig and Z. Rosenzweig, 2001, Anal. Chem. 73, 3240. McNamara, K. P. and Z. Rosenzweig, 1998, Anal. Chem. 70, 4853. The Merck Index, 12th edition, 1996, Merck & Co, NJ. Mohr, G. J., F. Lehmann, R. Ostereich, I. Murkovic and O.S. Wolfbeis, 1997a, Fresenius J. Anal. Chem. 357,284. Mohr, G. J., I. Murkovic, F. Lehmann, C. Haider and O.S. Wolfbeis, 1997b, Sens. Actuators B Chem. 39, 239. Morelle, B., J.M. Salmon, J. Vigo and P. Viallet, 1994, Cell Biol. Toxicol. 10, 339. Morf, W. E., K. Seiler, B. Lehmann, C. Behringer, K. Hartman and W. Simon, 1989, Pure Appl. Chem. 61, 1613. Murtagh, M. T., M. R. Shahriari and M. Krihak, 1998, Chem. Mater. 10, 3862. Overly, C. C., K.D. Lee, E. Berthiaume and P.J. Hollenbeck, 1995, Proc. Nat. Acad. Sci.~USA 92, 3156. Peper, S., I. Tsagkatakis and E. Bakker, 2001, Anal. Claim. Acta 442, 25-33. Rosenzweig, Z. and R. Kopelman, 1995, Anal. Chem. 67, 2650. Rosenzweig, Z. and R. Kopelman, 1996, Anal. Chem. 68, 1408. Ross, W. N., 1993, Biophys. J. 64, 1655. Sasaki, K., Z.-Y. Shi and R. Kopelman, 1996, Chem. Lett. 2, 141. Seitz, W. R., M.T.V. Rooney, E.W. Miele, H. Wang, N. Kaval, L. Zhang, S. Doherty, S. Milde and J. Lenda, 1999, Anal. Chim. Acta 400, 55. Shortreed, M., E. Bakker and R. Kopelman, 1996, Anal. Chem. 68, 2656. Shortreed, M. R., S. Dourado and R. Kopelman, 1997, Sens. Actuators B Chem. 38-39, 8. Sumner, J. P., J.W. Aylott, E. Monson and R. Kopelman, 2002, Analyst, 127, Advance Article. Suzuki, K., H. Ohzora, K. Tohda, K. Miyazaki, K. Watanabe, H. Inoue and T. Shirai, 1990, Anal. Chim. Acta 237, 155. Tan, W., R. Kopelman, S.L.R. Barker and M.T. Miller, 1999, Anal. Chem. 71, 606A. Tan, W., Z.-Y. Shi, S. Smith, D. Birnbaum and R. Kopelman, 1992, Science 258, 778. 535

Brasuel, Kopelman, Philbert, et al. Tsagkatakis, I., S. Peper and E. Bakker, 2001, Anal. Chem. 73, 315. Uhlmann, D. R., G. Teowee and J. Boulton, 1997, J. Sol-Gel Sci. Technol. 8, 1083. Xu, H., J.W. Aylott, R. Kopelman, T.J. Miller and M.A. Philbert, 2001, Anal. Chem. 73, 4124.

536

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 92002 Elsevier Science B.V. All fights reserved

CHAPTER 17

COLLOIDAL SEMICONDUCTOR QUANTUM DOT CONJUGATES IN BIOSENSING

1 HEDI MATTOUSSI, PH.D., ~M. KENNETH KUNO,

PH.D., 2ELLENR. GOLDMAN,PH.D., 2GEORGEP. ANDERSON, PH.D. AND 2j. MATTHEW MAURO, PH.D.

~Division of Optical Sciences 2Center for Bio/Molecular Science and Engineering U.S. Naval Research Laboratory Washington, DC 20375 USA

This chapter reviews progress in bio-related applications of luminescent colloidal quantum dots (QDs). The material reviewed undoubtedly represents only the prologue of an unfolding story, as quantum dots are a relatively recent discovery and their biological applications are newer still. Nonetheless, a significant body of research literature exists pointing the way toward future advances. We begin with a basic introduction to quantum dots, including their synthesis and some characteristic physical properties, then follow with a review of bio-related work involving semiconductor nanocrystals published todate. Work involving preparation and use of QD-protein conjugates in cellular imaging, quantitative immunoassays, and in early-stage energy transfer applications is reviewed, as well as uses of QD-DNA conjugates as nanoscale building blocks. A listing of early patents in this area is also included for those who contemplate utilizing these materials in the commercial arena. Advantages and limitations in bio-related applications are presented based on the current state-of-the-art in QD technology.

537

Mattoussi, Kuno, Goldman, et al.

1. Principles of Operation 1.1. Introduction Once purely a novelty in the realm of low dimensional semiconductor physics, quantum dots (QDs) have now come of age. The last decade has seen tremendous scientific interest and progress in understanding these semiconductor nanoparticle materials as well as initial attempts to develop and commercialize various applications (Yoffe, 1993, 2001; Alivisatos, 1996; Efros et al., 2000; Murray et al., 2000). Prompting this trend has been a growing realization of the technological importance of understanding the chemistry, physics and biology of materials at the nanometer scale, an area collectively known as nanoscience. The drive for expanding our understanding of semiconductor nanocrystals has also been spearheaded by potential applications for these materials in devices such as absorption filters (Borrelli et al., 1987, Hall et al., 1988), light emitting diodes (Colvin et al., 1994; Dabboussi et al., 1995; Schlamp et al., 1997; Mattoussi et al., 1998a), and photovoltaic cells (Greenham et al., 1996). QD bioconjugates, materials comprised of luminescent colloidal QDs conjugated with biomolecules, can be used in applications such as detection and quantitation of soluble substances, in bioimaging, and potentially, in a range of diagnostics applications. Successful integration of these promising materials into these and other emerging biotechnological areas will necessitate a thorough understanding of the properties of these hybrid bioinorganic systems, requiring multidisciplinary and coordinated efforts in chemistry, physics and materials sciences. Biological tagging using fluorophores is useful in many biotechnological applications, including immunoassays, disease diagnosis, drug development, and cell and tissue imaging in both single and multiplex approaches (Schrock et al., 1996;, Hermanson, 1996). For instance, recent flow cytometry work (Roederer et al., 1997) using a multi-laser excitation system and a multi-color labeling scheme, allowed concurrent observation of ten parameters involving cellular antigens, demonstrating the high level of sophistication possible using dye labels. Furthermore, microarray-based gene analysis using multiple fluorescent probes has become a critical technology in the burgeoning genomics field (Lobenhofer et al., 2001). Virtually all available organic light-emitting dyes, however, have inherent functional limitations such as narrow excitation bands and broad redtailing photoluminescence spectra, low resistance to photodegradation, and the necessity for individually tailoring synthesis and conjugation procedures for each fluorophore. Thus, there remains a need for new and improved types of fluorescent labeling materials. Semiconductor nanocrystals (e.g., CdSe-ZnS core-shell QDs) represent a promising alternative in certain bioanalytical and imaging applications (Bruchez et al., 1998; Gerion et al., 2001). These very bright photoluminescent materials have readily tunable spectral properties, high 538

Quantum Dot Bioconjugates for Biosensing photobleaching thresholds, and biocompatibility. Colloidal QDs made of ZnSe, CdS, CdSe, CdTe, and HgSe emit light over a wide range of wavelengths in the visible and near IR (Hines et al. 1998; Henglein et al., 1982; Weller et al., 1986; Rosetti et al., 1983, 1984; Murray et al., 1993; Rogach et al., 1997; Mikulec, 1999; Eychmialler et al., 2000). In addition, their essentially continuous absorption envelope allows simultaneous excitation of several different colors of QDs with a single wavelength, making them naturally suitable for multiplexing applications. In this chapter, we first describe some basic features and unique properties of colloidal semiconductor QDs, followed by a short history outlining some of the most important early bio-related studies. We then present the current status of known research efforts that involve using luminescent colloidal QD bioconjugates in biosensing and bioimaging.

1.2. Chemistry and physics of semiconductor quantum dots 1.2.1. Description. Colloidal semiconductor quantum dots are small, spherical, crystalline particles of a given material consisting of hundreds to thousands of atoms. They are neither atomic nor bulk semiconductors, but may best be described as artificial atoms. Their properties originate from their physical size, which ranges from 10 to ~ 100 A in radius and is often comparable to or smaller than the bulk Bohr exciton radius (Woggon, 1997; Gaponenko 1998; Yoffe 2001; Efros et al., 2000). As a consequence, QDs no longer exhibit their bulk parent optical or electronic properties. Instead, they exhibit novel electronic properties due to what are commonly referred to as quantum confinement effects. These effects originate from the spatial confinement of intrinsic carriers (electrons and holes) to the physical dimensions of the material rather than to bulk length scales. One of the better-known confinement effects is the increase in semiconductor band gap energy with decreasing particle size; this manifests itself as a sizedependent blue shift of the band edge absorption and luminescence emission with decreasing particle size (Figure 1). This size-dependent absorption can be understood by using a simple analogy to a quantum mechanical particle in a one-dimensional box of length L. In this model, a carrier is localized within a potential minimum between two infinite barriers. Due to this spatial confinement, the energies of the carriers are quantized to discrete values, proportional to the inverse of the square of the length of the box (En 0r n2/L2, with n = 1,2,3 .... ). An extension to QDs is achieved by considering a three dimensional box (or sphere) where the potential minimum represents the QD and the barrier to escape originates from the abrupt termination of the QD at its surface.

539

Mattoussi, Kuno, Goldman, et al.

Figure 1. Photoluminescence spectra for five different core sizes of CdSe-ZnS quantum dots in water solutions. All samples were excited at 350 nm. Core radii were extracted from small angle x-ray scattering (SAXS) data (Mattoussi et al., 1998b).

For a spherical QD with an infinite potential barrier one obtains the following expression for the electron and hole energy levels in the particle: ~12~'~"

(1)

2me,ha Here ~,, is the nth root of the spherical Bessel function of order l, me.h is the effective mass of the electron (e) or hole (h) and a is the radius of the QD. One therefore predicts discrete quantized electron-hole transitions, an increase in effective band gap (or HOMO-LUMO transition in molecular terms) with decreasing particle size and conversely a decrease in spacing between states with increasing size. It should be noted that the Coulomb interaction between the confined electron and hole alters these energies, but since this term scales as 1/a, it is essentially a small perturbation to Equation 1, which varies more strongly with size (i.e., 1/a2). A number of excellent review articles exist on the subject, particularly those by Yoffe (1993, 2001) Gaponenko (1998) and Efros (Efros et al., 2001); the interested reader is referred to them for more information.

1.2.2. Nanocrystal synthesis. QDs were first discovered in doped silicate glasses by Ekimov and Onuschenko (Ekimov et al., 1980, 1981, 1982, 1983, 1984, 1985a,b, 1993, 1996). In their,seminal work, a supersaturated solution of copper and chlorine compounds in glass was heated at high temperatures to cause the controlled precipitation of CuC1. Additional heating of the melt allowed them to 540

Quantum Dot Bioconjugates for Biosensing systematically create collections of small crystalline CuC1 particles ranging in size from tens to hundreds of angstroms, initially denoted as quantum droplets. The particles have since become known as quantum dots, although alternative names exist in the literature, including nanoparticles, nanocrystals, nanocrystallites and Q-dots. Today, a wide variety of methods such as e-beam lithography, x-ray lithography, molecular beam epitaxy (MBE), ion implantation, sonochemistry, and growth in size-restricted environments are available for making small nanocrystallites not only of semiconductors but also of metals. Some of the more common techniques are outlined below (and in Table 1) with particular emphasis on preparations yielding colloidal QDs that have surface capping/passivating molecules (ligands). Using these ligands allows tailoring of QD solubility in a variety of solvents, permits facile solution processing and can, in some cases, make them amenable to biological manipulations. Growth of QDs in glass melts is achieved by doping the melt with salts of the desired material (Ekimov et al., 1980, 1982, 1986; Borrelli et al., 1987). The temperature of the glass is then rapidly dropped to generate small nuclei of the semiconductor. The glass then undergoes a secondary heat treatment over temperatures ranging from 400 to 1000~ to induce the nuclei to grow, forming small spherical crystalline particles of semiconductor dispersed in amorphous glass matrices. Advantages of this technique include h!ghly crystalline particles and the ability of the glass host to support very large (hundreds of angstroms) QDs. A serious disadvantage is that the QDs cannot be easily manipulated after their synthesis. They remain trapped in the glass and there are few possibilities for treating the material once made, for example, to alter surface chemistry or improve their size distribution. In parallel with the discovery of QD growth in glasses, it was found that semiconductor nanoparticles could be grown within inverse micelles (Figure 2) (Henglein, 1982; Rosetti et al. 1983,1984; Kotov et al., 1993,1994; Pileni et al., 1992). This technique exploits natural geometrical structures created by waterin-oil mixtures upon adding an amphiphilic surfactant such as sodium dioctyl sulfosuccinate (AOT). By varying the water content of the mixture, it was shown that the size of the water droplets suspended in the oil phase could be varied systematically. This led to the idea of using these self-enclosed water pools as micro-reactors for carrying out nanoscale sustained chemical reactions. In the case of QDs, it was found that adding metal salts to the water pools could cause nucleation and growth of colloidal nanocrystalline particles. Advantages of this technique include reactions carried out at room temperature and, more importantly, the ability to isolate the QDs after their synthesis. The inverse micelle preparation was therefore a significant advance in the development of QDs, giving researchers access to the surface chemistry of the particles for additional functionalization and manipulation.

541

Mattoussi, Kuno, Goldman, et al. Table 1. Major known types of Colloidal Group II-VI semiconductor QDs, method of preparation and representative literature citation. Some of these materials have been used in bio-related experiments.

Nanocrystal type CdS

Preparation meti~od Silica glass

Polymer and high temperature coordinating solutions Sol-gel glass

Ekimov et al., 1985; Borrelli et al., 1987; Gaponenko et al., 1993 Murray et al., 1993

Silica glass High temperature coordinating solutions Polycrystalline films

CdTe

ZnSe

i

Nogami et al., 1990; Minti et al., 1991; Spanhel et al., 1992; Mathieu et al., 1995 ;Wang et al., 1989; Herron et al., 1989 Gurevich et al., 1992

Semiconductor-glass composite films

CdSeovercoating

i

Ekimov et al., 1985; Potter et al., 1988; Liu et al., 1990, Persans et al., 1989; Zhao et al., 1991 Rosetti et al. 1983, 1984, Weller et al., 1986; Misawa et al., 1991; Woggon et al., 1993 Murray et al., 1993; Artmeyev et al., 1995

Aqueous solutions, inverted micelles

CdSe

Literature citation

Hodes et al., 1987

ZnSe using hybrid micelle/organometallic ZnS using high temperature coordinating solutions CdS using high temperature coordinating solutions

Kortan et al., 1990

Silicate glass Semiconductor-glass Composite films High temperature coordinating solutions

Potter et al, 1988; Liu et al., 1991 Ochoa et al., 1996

High temperature coordinating solutions

Chestnoy et al., 1986; Hines et al., 1998

Hines et al., 1996; Dabbousi et al., 1997 Peng et al., 1997

Murray et al., 1993; Mikulec et al., 1999

542

Quantum Dot Bioconjugates for Biosensing

/~ = AOT CdCI2 + H2S Water/Isooctane Room Temperature

Figure 2. Growth of CdS quantum nanoparticles in inverse micelles. Other materials such as CdSe and CdTe have also been prepared using the inverse micelles approach.

In the early 1990's it was shown by the Bawendi group (Murray et al., 1993) and confirmed shortly thereafter (Bowen Katatri et al., 1994) that an organometallic synthesis based on pyrolysis of metal-organic precursors could yield CdSe QDs with a size distribution of 8-10% as made, with distributions that could be improved during post-reaction processing to values as small as 5 % (Murray et al., 1995). This preparation followed on the early micelle advances, yielding highly crystalline particles which were significantly improved in terms of their fluorescence quantum yield (QY). Colloidal QDs could now be made with room temperature quantum yields on the order of 5-10% (and low temperature QYs near unity), making fluorescence-based applications of QDs viable for the first time. This technique is widely used to generate QDs used in bio-related applications, and is described here (Figure 3). In general, a solution of dimethylcadmium (CdMez) and trioctylphosphine selenide (TOPSe), diluted in trioctylphosphine (TOP), is rapidly injected into a hot stirring solution of trioctylphosphine oxide (TOPO). The rapid introduction and concomitant temperature drop resulting from adding these reagents result in discrete temporal nucleation of CdSe seeds. After reagent injection, the temperature of the solution is raised to 280-300~ in order to grow the particles. The high temperature growth promotes highly crystalline QD cores. Growth is monitored through UV/visible spectroscopy and when the desired size is reached (as monitored by the peak wavelength of the first absorption feature), the temperature is dropped below 100~ to arrest the growth. A more detailed description of a typical laboratory scale organornetallic preparation of QDs is provided in the three steps described below: 543

Mattoussi, Kuno, Goldman, et al.

TOPO CdMe 2 + TOPSe

300-350 ~

Figure 3. High temperature organometallic growth of colloidal CdSe quantum nanocrystals, as described first by Murray et al. (1993).

1. In a glovebox, under nitrogen, a 1M stock solution of trioctylphosphine selenide (TOPSe) is prepared by adding 7.9 grams of amorphous Se (99.99%) shot to 100 ml of trioctylphosphine (TOP, 90-95%). An injection solution is formulated by adding 170-200 ~1 CdMe2 and 3.5-4 ml 1M TOPSe to ~15 ml of TOP. The reagents are mixed and loaded into a syringe equipped with a large gauge needle for rapid injection. 2. On a Schlenk line, a 100 ml three-neck flask is loaded with -20-30 grams of TOPO (90%) and heated to 150-180~ for 2-3 hours under vacuum while stirring in order to remove water. When dry, the flask is backf'llled with inert gas (typically N/) and the temperature is raised to 300-350~ in preparation for precursor injection. The loaded syringe is removed from the glovebox and its contents quickly injected into the flask. Upon injection, there is a vigorous evolution of gas followed by a rapid color change of the solution to light yellow. The temperature falls to ~ 250~ and an absorption spectrum shows sharp features with the peak of the first transition usually located between 470 and 490 rim.

3. The temperature is raised to 290-300~ to allow for growth and annealing of the QDs. During growth, samples are periodically removed and their UV/visible absorption spectra taken. The peak position of the first absorption feature is noted, as well as the relative width of the transition, which gives a measure of a sample's size distribution. Occasionally there is a decrease in the growth rate accompanied by an increase in the relative size distribution. To overcome this growth bottleneck, the temperature is raised by several degrees. When the peak of the first absorption feature reaches a wavelength maximum indicative of a desired size, the temperature is dropped below 100~ to arrest crystal growth. 544

Quantum Dot Bioconjugates for Biosensing Recently, Peng and coworkers have developed a modified organometallic synthesis that is less dependent on the purity of the TOPO and avoids the use of pyrophoric CdMe2 precursor (Peng et al., 2001a, Qu et al., 2001). In their synthesis, high purity TOPO and controlled amounts of cadmium coordinating ligands, e.g., hexylphosphonic acid (HPA) or tetradecylphosphonic acid (TDPA), are combined in the preparation flask. Cadmium compounds such as cadmium oxide (CdO) or cadmium acetate [Cd(Ac)2] are added at a relatively low temperature (e.g., at 140~ and the mixture heated to generate Cd 2§ ions before addition of TOPSe results in nanocrystal nucleation and growth. This procedure is promising; however, additional work is still needed before reproducible high quality CdSe and CdSe-ZnS QDs can be routinely prepared. To obtain material with low size dispersity, growth of CdSe QDs is often followed by size selective precipitation (Murray et al., 1993). This involves adding a "bad" solvent for the TOP/TOPO-capped nanocrystals, such as methanol, to a preparation of QDs whereupon larger particles in the mixture precipitate first due to preferential Van der Waals interactions. Smaller particles remain in solution until enough MeOH is added to drive most of the QDs out of solution. Use of size-dependent QD precipitation enriches for populations of desired nanocrystal size. Repeated precipitations can reduce the overall size distribution of QD synthetic mixtures to values of ~5%. This method is widely employed for polymers and colloids to reduce polydispersity after synthesis. Size and size distribution measurements are usually carried out using transmission electron microscopy (TEM) (Murray et al., 1993) and/or small angle x-ray scattering (SAXS). TEM tends to provide slightly smaller values for the inorganic core than SAXS because TEM does not take into account the amorphous outermost atomic layer on the nanocrystal surface (Mattoussi et al., 1998b). It was discovered in the mid-1990's that passivating QDs with an additional thin layer made of a wider band gap semiconductor could improve the surface quality of the particles (by providing a better passivation of surface states), resulting in dramatic enhancements of the fluorescence quantum yield. Although the principle was previously known from semiconductor bandgap engineering (Steigerwald et al., 1988; Kortan et al., 1990), the optimal set of conditions for creating strongly fluorescent overcoated QDs was not realized until the seminal work of Hines and Sionnest (Hines et al., 1996), when they showed that overcoating CdSe QDs with ZnS improved quantum yields to values of 30% or greater. This was shortly followed by other studies that described additional characterization of CdSe QDs overcoated with ZnS (Dabbousi et al., 1997) and CdS (Peng et al., 1997). In brief, the procedure for overcoating colloidal QDs with another semiconductor involves the following steps. A dilute solution of QDs is made up in an appropriate coordinating solvent (TOPO, for instance). The temperature of the 545

Mattoussi, Kuno, Goldman, et al.

CdSe QDs

ZnEt 2, (TMS)2S ...... TOPO, 140-180 ~

Figure 4. Overcoating of CdSe quantum dots with ZnS using high temperature solution route.

solution is raised to --150~ but kept lower than 200~ to prevent further growth of the QDs. A dilute solution of Zn (or Cd) and S precursors is then slowly introduced into the hot stirring QD solution. The high dilution and relatively low temperature of the mixture prevent separate nucleation of ZnS or CdS quantum dots. Once the precursors have been added, the temperature is lowered to ~ 80~ and the reaction vessel left undisturbed for several hours. Noticeable improvements in the PL quantum yield are apparent after several hours of heat annealing (Figure 4). A typical laboratory scale ZnS overcoating process for CdSe QDs includes the following steps (Dabbousi et al., 1997): Size-selected CdSe particles dispersed in hexane are added to 5-10 grams of dried, degassed TOPO at -70~ Inside the glovebox, equimolar amounts of diethylzinc (or CdMe2) and hexamethydisilathiane are mixed with -5 mls of TOP. The amount of Zn and S precursors added varies depending on the size of the CdSe QD and is calculated to yield a 2-3 (or more) atomic monolayer coverage on the particle surface. Once this solution has been prepared, the temperature of the QD/TOPO solution is raised to a value between 140~ and 180~ The Zn (Cd) and S precursor solution is then brought out of the glovebox and introduced at a rate of ~ 0.5 ml/min through a separate addition funnel attached to the flask holding the QD/TOPO mixture. Once the addition is complete, the pot temperature is lowered to ~80~ and the mixture is left undisturbed for several hours. The overcoated QDs are subsequently precipitated with methanol prior to further processing.

546

Quantmn Dot Bioconjugates for Biosensing

1.3. Semiconductor nanocrystal properties Initial optical studies of QDs in the late 1980's aimed at correlating sizedependent spectral shifts in absorption with quantum confinement effects. Today, the absorption properties of CdSe QDs are relatively well understood with up to ten excited states in the absorption assigned and theoretical avoided crossings observed (Norris et al., 1996). However, the origin of the band edge emission in CdSe QDs was not immediately understood due to the inability of the same theory to explain unusual size-dependent features in QD fluorescence spectra. This presented a serious challenge to the above model, and it was not until the mid 1990' s, when Chamarro (Chamarro et al., 1995, 1996) realized the importance of electron hole exchange interaction in QD materials that a more comprehensive understanding of CdSe QDs was achieved. Modifications by Efros to that theory led to understanding of previously unrecognized "dark exciton" effects, which explained many unusual features in the emission spectra such as the size-dependent ~ and "global" Stokes shift observed in fluorescence line narrowing and global excitation experiments (Nirmal et al., 1995; Efros et al., 1996; Kuno et al., 1997). In 1996 Nirmal et al. conducted the first single-particle fluorescence studies of isolated TOP/TOPO-capped CdSe and CdSe-ZnS QDs. They discovered that QDs underwent intermittent on/off emission (so-called "blinking") under continuous excitation (Nirmal et al., 1996). Unlike single fluorescent molecules, this behavior could not be attributed to a commonly known effect referred to as quantum jumps (Cook and Kimble, 1985). Instead, the on/off intermittency in QD emission was attributed to Auger ionization of the QD (Chepic et al., 1990; Nirmal et al., 1996; Efros et al., 1997). The blinking effect is still not fully understood, however.

2. History of Bio-Related Applications using QD Bioconjugates The amount of published research involving bio-related uses of semiconductor nanocrystals has expanded rapidly since the initial reports from the laboratories of Alivasatos (Bruchez et al., 1998) and Nie (Chan and Nie, 1998) first appeared. This section begins with a description of early work on the preparation of protein-derivatized water-compatible quantum dots, preliminary QD-protein conjugate characterization and their use in imaging cellular structures. It is followed by description of a simple and useful electrostatically controlled conjugation method developed in our laboratory (Mattoussi et al., 2000). We conclude by describing briefly the original work involving formation and use of QD-DNA conjugates as performed in the Mirkin laboratory (Mitchell et al., 1999).

547

Mattoussi, Kuno, Goldman, et al.

Figure 5. Surface treatment of overcoated quantum dots. A) Silica shell with reactive amine functions; B) Mercaptoacetic acid coverage; C) Capping with dihydrolipoic acid [DHLA]. D) Direct binding of thiol-terminated DNA.

2.1. Preparation and use of QD bioconjugates in cellular imaging Water-soluble nanocrystals derivatized with the actin-binding protein, phalloidin, were used for the first attempts at intracellular imaging in fixed mouse fibroblasts (Bruchez et al., 1998). For conjugating phalloidin with nanocrystal surfaces, the authors started with CdSe-CdS core-shell QDs that were capped with a thin amine-derivatized silica shell to render them both reactive and water-compatible (Figure 5A). The silica-encapsulated, red-emitting CdSe-CdS core-shell particles (4 nm diameter core size and emission maximum at 630 nm) were subsequently biotinylated at their exposed reactive amine sites. Streptavidin was then used effectively as a bridge between actin-bound biotinylated phalloidin and the biotinylated red-emitting QDs in cell imaging work. Simultaneous nuclear staining was achieved using green-emitting QDs coated with an anionic silane, in a process driven by electrostatic and hydrogen binding interactions. Light from a mercury lamp with a 488 nm excitation filter and a single long-pass emission filter were used to image both red (actin-bound) and green (nucleus-bound) QDs at the same time. Importantly, it was also demonstrated with sequential scans using laser scanning confocal microscopy that the red nanocrystal bioconjugates were dramatically more photostable than fluorescein-labeled phalloidin bound to actin fibers under essentially identical conditions. A detailed description of the process used for preparation of silica encapsulated reactive QDs, together with more extensive characterization of their properties, has since been published (Gerion et al., 2001). 548

Quantum Dot Bioconjugates for Biosensing

Figure 6. Schematic of engineered two-domain protein electrostatically complexed with a DHLA-capped CdSe-ZnS quantum dot (based on Mattoussi et al., 2000).

A contemporaneous imaging study employed bio-compatible nanocrystals conjugated with human transferrin to conduct intracellular staining of fixed HeLa cells (Chan and Nie, 1998). For conjugating transferrin with nanocrystal surfaces, the authors started with CdSe-ZnS core-shell dots that had been capped by ligand-exchange with mercaptoacetic acid in order to render them both watercompatible and reactive (Figure 5B). Mercaptoacid-capped red-emitting CdSeZnS core-shell particles (2.1 nm radius and emission maximum of 560 nm) were subsequently coupled with transferrin using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide) hydrochloride (EDC)-promoted condensation chemistry. The conjugates preserved the absorption and photoluminescence properties of the nanocrystals as well as the activity of the transferrin. For QD-transferrin conjugates, functionality was demonstrated by exposing the conjugates to cultured HeLa cells overnight followed by extensive washing and imaging using an epifluorescence microscope equipped with a CCD camera. Imaging revealed that luminescent QDs had entered the cells, presumably via receptor-mediated endocytosis. In the absence of transferrin, i.e., using capped 2.1 nm core radius QDs without a protein coating, only weak cellular autofluorescence was observed. These authors also prepared human IgG-QD conjugates using the same methods. Reaction with human IgG-specific polyclonal antibody resulted in extensive aggregation as observed using fluorescence imaging, undoubtedly due to inter-dot bridging among rnultiply-derivatized QDs.

2.2. Preparation of QD-protein conjugates using a non-covalent strategy An alternative method for preparing functional QD-protein conjugates has been developed in our laboratory at NRL. This system employs "bidentate" dihydrolipoic acid (DHLA) groups to coat QDs (Figure 5C) in combination with two-domain proteins engineered to interact electrostatically with negatively 549

Mattoussi, Kuno, Goldman, et al. charged QD surfaces. In a model system that employed engineered variants of E. coli maltose binding protein (MBP) and DHLA-capped CdSe-ZnS nanocrystals, stable self-assembled QD-protein complexes formed in an efficient and controlled manner (Mattoussi et al., 2000). In order to promote the self-assembly process, a modular MBP-basic leucine zipper chimeric protein was designed and prepared in a recombinant system (MBP-zb) (Figure 6). The strongly positivelycharged C-terminal "tail" present in the novel MBP-zb variant resulted in rapid formation of QD-MBP conjugates that retained both the optical QD properties and the active folded state of the MBP protein. Based on incremental increases in fluorescence quantum yield that occurred upon titration of QDs with increasing amounts of MBP-zb, and from steric considerations, it was estimated that for a nanoparticle of-- 70 A, (core-plus-shell) diameter an average of 12-15 engineered protein molecules could be packed around each nanocrystal (Mattoussi et al., 2001). That number can presumably increase or decrease for larger or smaller QDs, respectively. In the first known use of QD-bioconjugates in a quantitative fluoroassay, a functional assay for maltose was developed that monitored displacement of QDMPB-zb conjugates bound to a cross-linked amylose affinity matrix as various amounts of dissolved maltose were injected into the flowing buffer upstream of the affinity column. Maltose concentrations of injected samples were determined by integrating PL intensity (QD emission) versus time as QD-MBP-zb conjugates were displaced from the column by maltose; limits of detection were on the order of 10 pmol of maltose (Mattoussi et al., 2001). In order to prepare reagents for use in fluoroimmunoassays, an analogous recombinant construct was developed based on the IgG-binding domain of protein G from Staphylococcus. In this construct, the engineered dimeric form was critical in providing two points of attachment for each IgG. The PG-zb protein serves as a very effective bridge between the DHLA quantum dot surface and any type of IgG antibody, resulting in reagents that can be used in general fluorimmunoassay protocols. QD-antibody complexes made with this strategy were used in fluoroimmunoassays in analysis of the protein toxin staphylococcal enterotoxin B (SEB) (Tran et al., 2001; Goldman et al., 2001, 2002a) and in analysis of low levels of 2,4,6-trinitrotoluene (TNT) and hexahydro-l,3,5trinitro-l,3,5-triazine (RDX) dissolved in water (Goldman et al., 2002a,b). In the sandwich-assay format for SEB, the limit of detection was about 200 pmol of the protein toxin. A competition assay performed in microtiter plates for TNT and RDX allowed facile quantitation of the dissolved explosives, with detection limits of 1 ng and 0.2 ng of the explosives, respectively (Goldman et al., 2002a).

2.3. QD-DNA conjugates Sequence-dependent hybridization of deoxyoligonucleotides bound to CdSe-ZnS QDs was first demonstrated in the Mirkin laboratory (Mitchell et al., 1999). Two 550

Quantum Dot Bioconjugates for Biosensing populations of nanoparticles were prepared using either 3' or 5' alkyl thiolterminated 22-mer oligonucleotides for QD surface attachment (Figure 5D). Upon addition of a 24-mer "bridging" or "capture" oligonucleotide designed to be complementary to the outer 12-met sequence of both types of DNA-modified nanoparticle, a cross-linked network of specifically hybridized particles was formed. Aggregation was demonstrated to be specific by control experiments with non-complementary capture DNA in which no evidence of multi-center cluster formation was observed. Cluster formation by specific hybridization resulted in a decrease in integrated fluorescence intensity of 26 + 6%; excimer formation between the DNA linked dots was cited as a possible explanation. These researchers also broke new ground by forming and characterizing mixed aggregates composed of both QD-DNA and Au-DNA conjugates. Temperatureinduced melting of the hybridized DNA present within both QD-oligonucleotide complexes and mixed QD-Au nanoparticle-DNA complexes was studied by monitoring light absorption versus temperature. A sharp duplex melting temperature transition was observed, suggesting that cooperativity effects operate within the complexes due to multiple DNA links per particle. Melting of mixed QD-Au-DNA particles ("A-B" structures) could be observed at one tenth the concentration of a pure QD-DNA system due to the very large molar extinction coefficient associated with the plasmon band of the DNA-derivitized 13 nm Au particles used (2.8 x 108 M 1 crnl). Construction of more complex multicomponent nanostructured materials might be possible using these types of building blocks.

2.4. Seminal patents Although applications of quantum dots to bio-related issues is a relatively new area, several U.S. patents have already been issued for processes involving synthesis and use of quantum dots within the biochemical realm. Table 2 presents eight seminal patents in the area. All have been issued within the last two years. A wide range of areas is covered in these patents; interested readers should consult the U.S. Patent and Trademark Office Home Page (http:!/www.uspto.gov/) for viewing the complete patents.

3. State of the Art 3.1. Types of QDs and their optical properties Colloidal semiconductor nanocrystal quantum dots are in general considered to be spherical in shape. They can be dispersed in a solid matrix such as those prepared by annealing at high temperature in glasses (referred to as quantum droplets), where the nanocrystal growth is driven by precipitation/nucleation, or 551

Mattoussi, Kuno, Goldman, et al. Table 2. Seminal patents describing applications involving the use of QD-conjugates.

Patent Number

Issue Date

Patent Title

Oct.

6,309,701

2001

6,207,392

2001

6,319,607 6,306,610

2001 Oct. 2001

Fluorescent nanocrystal-labeled microspheres for Fluorescence analysis Semiconductor nanocrystal probes for biological applications and process for making and using such probes Purification of functionalized fluorescent nanocrystals Biological applications of quantum dots

6,274,323

Aug. 2001

Mar.

NOV.

Nov.

6,319,426

2001 Nov.

5,990,479

1999

6,114,038

Sept. 2000

Authors/Assignees

E. Barbera-Guillem/ Bio-Pixels, Ltd. S. Weiss, M. Bruchez, P. Alivisatos/ Univ. of California

E. Barbera-Guillem/ Bio-Pixels, Ltd. M. Bawendi, F. Mikulec, V. Sundar/ MIT Method of detecting an analyte M. Bruchez, R. Daniels, S. in a sample using semiconductor nanocrystals as a Empedocles, V. Phillips, E. Wong,, D. detectable label Zehnder/ Quantum Dot Corp. M. Bawendi, F. Water-soluble semiconductor Mikulek, J. Lee/ nanocrystals MIT S. Weiss, M. Bruchez, Organo Luminescent P. Alivisatos/ semiconductor nanocrystal Univ. of California probes for biological applications and process for making and using such probes Functionalized nanocrystals and S. Castro and E. Barbera-Guillem/ their use in detection systems BioCrystal Ltd.

surface functionalized with organic ligands to make them soluble in a variety of organic solvents to make colloidal dispersions. The latter are usually grown using inverted micelles or high temperature solution chemistry routes. Recently, preparation of anisotropic semiconductor nanocrystals made of CdSe (quantum rods) using the organometallic synthesis route was reported. Control of the type of cadmium ligands used (e.g., HPA or TDPA) and the concentration of the cadmium complexes formed are the key elements to growing anisotropic

552

Quantum Dot Bioconjugates for Biosensing nanocrystals (Peng et al., 2000, 2001b.). These quantum rods were reported to have linearly polarized luminescence emission, a property that may have a potential use in biological tagging applications (Hu et al. 2001). The common optical property that characterizes QDs is the size-dependence of their spectroscopic properties (e.g., absorption and phohtoluminescence), which results from quantum confinement of charge carriers within a volume smaller or comparable to the Bohr exciton radius. Within the family of II-VI compounds the range of absorption and emission peaks depends on the materials; ZnS and ZnSe QDs have absorption and emission spectra limited to the UV and blue regions, whereas nanocrystals made of heavier atoms such CdTe or HgSe or hybrids composed of PbSe (III-VI compounds, Murray et al. 2001b) have useful optical properties that extend into the near IR region of the spectrum. For biological labeling, colloidal CdSe-ZnS QDs have been the most widely used in published studies. Experiments using CdS QDs, CdS-overcoated CdSe nanocrystals or CdTe QDs have also been described in a few instances (Mahtab et al., 1996, Bruchez et al., 1998, Mamedova et al., 2001).

3.2. Nanocrystal surface treatment

3.2.1. Inorganic overcoating. For colloidal nanocrystals, organic ligands tend to provide only partial surface passivation, which translates into rather modest photoluminescence yields. Overcoating the native core with a wider band gap semiconducting material provides additional surface passivation and reduces leakage of excitons outside the core. Optimal passivation of the surface states occurs when the growth of the overcoating layer is near-epitaxial, i.e., the lattice mismatch between the core and the shell material is as small as possible. On the other hand, using an overcoating material that has a closely matching lattice structure in order to promote epitaxial growth produces a leakage of the exciton to the overcoating layer, which in turn results in a pronounced red shift of absorption and emission spectra in comparison with the native nanocrystals. This pronounced red shift has an additional disadvantage as it moves the range of useful wavelengths further into the red. For example, when using a CdSe core, overcoating with CdS produces a higher PL yield than the one measured with ZnS. However, minimal red shift of the band edge absorption and emission are measured for CdSe-ZnS QDs compared with CdS-overcoated nanocrystals. Thus, only ZnS-overcoated QDs allow a broad range of wavelengths in the visible to be produced including emission in the blue region of the optical spectrum. 3.2.2. Capping ligandsfor bio-compatibility and conjugateformation. Protecting the nanocrystals' physical and chemical integrity in aqueous media, while simultaneously providing sufficiently reactive surface sites to allow bioconjugation, has been challenging. Several means of accomplishing this have been devised, some of which have already been alluded to. Capping with 553

Mattoussi, Kuno, Goldman, et al. mercaptoacids imparts water solubility and provides carboxyl groups for the condensation chemistry necessary for further covalent modification (Chan and Nie, 1998) (Figure 5B). It has been suggested that the "bidentate" type of interaction of dihydrolipoic acid (DHLA) (Figure 5C) (Mattoussi et al., 2000) or dithiothreitol (Pathak et al., 2001) with the inorganic QD surface results in more water-stable nanocrystals, but no systematic study has been performed. The charged surface provided by DHLA-capped CdSe-ZnS QDs drives electrostatic self-assembly of QD-protein conjugates that, once formed, are surprisingly stable even in high salt solutions (Mattoussi et al., 2000). Porous silica shells have been used for passivation and placement of hydroxyl, amino, thiol, and phosphonate groups in position for bioconjugate formation (Gerion et al., 2001) (Figure 5A). Attachment of thiolated DNA directly to QDs by replacement of surface mercaptoacids has been successfully accomplished (Figure 5D) (Mitchell et al., 1999), and similarly, CdS nanoparticles have been grown in the presence of thiolated peptides, resulting in derivatized fluorescent particles able to recognize cellular receptors (Winter et al., 2001).

3.3. Bioassay work involving quantum dots Quantum dot bioconjugates can function as fluorescent reporters in recognitionbased quantitative and qualitative bioassays. QDs conjugated to both proteins and DNA have been used in a limited number of applications, as described below.

3.3.1. Fluoroimmunoassays using QD-antibody conjugates. Immunoassays utilizing CdSe-ZnS QD-conjugates formed by electrostatically driven selfassembly have been developed (Goldman et al., 2001, 2002a, b; Tran et al., 2001). QD-antibody complexes for use in bioassays have been formed using adaptor proteins as bridges to link QDs with antibodies. Either naturally occurring protein bridges (e.g., avidin) or engineered recombinant protein bridges can be used in this capacity. In practice, mixed-surface QD conjugates have been made with both the antibody-binding adaptor protein and an engineered maltose binding protein derivative (MBP-zb) bound to their surface (Figure 7A). The mixed recognition elements on the particles allow separation of QD-antibody complexes from unbound antibody using affinity chromatography. After saturation of antibody binding sites with IgG (or biotinylated IgG when using the avidin bridge) and purification on a cross-linked amylose column to remove excess unbound IgG, various QD-antibody conjugates have been demonstrated to bind antigen directly adsorbed to microtiter plates. Sandwich- or competitivetype assays were then performed in model systems for analysis of staphylococcal enterotoxin B (SEB) and for detecting low levels of the explosives TNT and RDX dissolved in water (Figure 7B and Figure 8).

554

Quantum Dot Bioconjugates for Biosensing

Figure 7. QD antibody conjugates prepared using molecular bridges. A. Mixed surface conjugate after purification by cross-linked amylose affinity chromatography. B. Schematic of competitive assay for the explosive RDX dissolved in water (Goldman et al., 2002a,b).

140 120 .,..,

el

"" 100

~

~

80

~

60

E

o L_

10 20 )

9 '

O. 1

i

i,i

i i l l l

i

1

i

i , , | , 1 1

i

10

i , I

, , I I I L ,

1 O0

i

,

L

i

i

!

i/,

1000

Concentration of RDX (ng/ml)

Figure 8. Results of a quantitative competitive assay for RDX using QD-anti-R.DX antibody conjugates, where inhibition of QD-conjugates to BSA-RDX by free RDX was investigated. 100% indicates signal in the absence of RDX. 555

Mattoussi, Kuno, Goldman, et al. Laser scanning confocal microscopy has also been used as the means of detection in sandwich immunoassays utilizing QD-antibody conjugates (Sun et al., 2001). In these experiments, rabbit anti-human antibody was covalently conjugated to CdSe-ZnS core-shell QDs using EDC chemistry; unconjugated antibody was removed using repeated cycles of washing and centrifugation. Rabbit antihuman IgG was immobilized on a glass slide to form the capture layer, followed by exposure to a mixture of human IgG and goat IgG. After incubation and rinsing unbound antibodies, the surface was exposed to a solution of QDs conjugated with rabbit-anti-human antibodies. Reading the fluorescence signal of the glass slide showed a linear increase of the signal with human IgG concentration, indicating that the QD-conjugates specifically recognized immobilized human IgG. IgG samples containing about 2 ~tg/ml antibody could be detected. Comparison against antibody labeled with a conventional fluorophore (fluorescein isothiocyanate) in the same system gave a limit of detection of about 25 ng/ml. Inappropriate selection of the excitation wavelength with respect to the absorption and emission of the nanocrystal employed in the assay may have contributed to reduced assay sensitivity with the QD conjugates. CdS QDs prepared with amino-derivatized polysaccharides (aminodextrans, Amdex) have been conjugated with mouse monoclonal anti-T4 antibody for use in flow cytometry (Sondi et al., 2000). The QD-Amdex-antibody complexes were purified from unconjugated antibody by size exclusion chromatography. Samples of whole blood were lysed and the anti-T4 antibody-Amdex-QD reagent tested for the detection of T4 positive lymphocytes. For unknown reasons, direct cytometric detection using QD fluorescence was not achieved. Signal was seen, however, when sheep-anti-mouse antibody conjugated to phycoerythrin was added to the sample containing anti-T4 antibody-Amdex-QDs, suggesting that the purified QD conjugates were effectively binding to receptor sites on the lymphocytes. 3.3.2. DNA-based systems using QDs as reporters. QDs have been used as probes of DNA structure (Mahtab et al., 1996; Gearheart et al., 2001). Luminescence from CdS QDs was monitored as DNA with different sequences (implying different local structures) was adsorbed to QD surfaces. Adsorption of increasing amounts of DNA to QD surfaces resulted in systematic decreases in emission intensity. DNA sequences with intrinsically kinked structures were found to bind preferentially to nanoparticles rich in surface Cd z§ sites as well as to neutral, mercaptoethanol-capped CdS QDs. DNA molecules lacking [-finks, however, bound to the CdZ§ surface of CdS QDs and not to mercaptoethanolcapped QDs. DNA methylation affected the interaction of Cd2+-rich surfaces of CdS QDs with DNA. Addition of DNA containing hypermethylated triplet repeats d(mCmCG)7 to QDs produced less quenching of the particle luminescence than equal amounts of d(mCGG)7 DNA, even though the binding constants for the two types of DNA were essentially identical.

556

Quantum Dot Bioconjugates for Biosensing In another demonstration of the use of QD-DNA conjugates, DNA conjugated to CdSe-ZnS QDs have been used for the detection of specific chromosome sequences in human sperm cells using fluorescence in situ hybridization (FISH) (Pathak et al., 2000). Prior to DNA conjugation, the nanoparticles were treated with dithiothreitol, presumably resulting in stable "bidentate" interactions of each capping molecule with the ZnS overcoating. Surface hydroxyl groups made available by this process were then activated with carbonyl diimidazole (CDI) and reacted with 5'aminated oligonucleotides to give stable carbamate linkages. QD-oligonucleotide conjugates with sequences specific for the Y-chromosome were used in FISH assays on human sperm cells. Half the cells are expected to contain a Y chromosome and hybridize to the probe, while the other half should contain the X chromosome and should not hybridize with the probe. As predicted, about half the cells showed the fluorescent signal. Only background emission (less than 5% of positive signal) was seen when identical experiments were performed using unconjugated QDs or QDs conjugated to an oligonucleotide having a non-relevant sequence, i.e., a sequence not present in the human genome.

3.4. Bio-imaging applications using quantum dots The two pioneering efforts described in the History section of this review have demonstrated in a "proof-of-principle" mode the utility of using quantum dot bioconjugates as histochemical imaging reagents (Bruchez et al., 1998; Chan and Nie, 1998). In a logical extension of initial cell imaging work, specific sites on the surface of living cultured neuronal cells have been labeled with CdS quantum dots derivatized with two types of recognition molecules (Winter, et al., 2001). In the first case, primary antibodies directed toward the ~ subunit of the tx~l]~integrin that studs the surface of SK-N-SH neuroblastoma cells were treated with secondary antibody-QD complexes. Specific attachment of QDs was verified by bright field and fluorescence optical microscopy. In an effort to reduce the distance between QDs and the cell body, CdS dots were prepared in the presence of synthetic peptide CGGGRGDS, which includes the RGD (Arg-Gly-Asp) sequence known to bind to ~13~ and ~[33 integrins as well as a terminal cysteine residue for interaction with exposed surface atoms of the nanocrystal. Preparation of these peptide-derivatized nanoparticles was performed by singlestep arrested precipitation in the presence of 1" 10 peptide:mercaptoacetic acid; fluorescence anisotropy studies of the prepared particles strongly suggested that peptide attachment had in fact occurred. Microscopy of the cells following exposure to the RGD dots showed a yellow/orange layer of CdS dots coating the blue autofluorescent cells. The control with a non-binding peptide sequence was negative for the staining. Although the thrust of this work involved demonstration of specific binding to living cells, long-range goals involve 557

Mattoussi, Kuno, Goldman, et al. preparation of nerve cell-semiconductor interfaces for use in future nanodevices and sensors. Time-gated imaging of mouse fibroblast cells has been accomplished using silanized 1.8 nm radius (575 nm peak emission) CdSe-ZnS quantum dots (Dahan et al., 2001). In preliminary experiments, the normalized fluorescence decay for the particles used was fit with a triple exponential with components of 3.4, 16.1, and 35.6 ns, corresponding to 1, 50, and 48 percent, respectively, of the emitted photons. Mouse 3T3 fibroblasts incubated overnight with 1 0 - 100 nM nanocrystals were washed and fixed prior to image collection using a custom built stage-scanning time-correlated single-photon counting confocal microscope. Images constructed using all detected photons (i.e., no time gating) showed dense non-specific autofluorescence throughout the cells, while images constructed with photons from the 35-65 ns window exhibited very low backgrounds with isolated bright clusters of QDs. These aggregated QDs were possibly taken up by the growing cells via endocytosis and stored in lysozomes. Time gating resulted in a 15-fold enhancement in signal-to-noise over the ungated data. Enhanced image constrast will likely be crucial to observing single-nanocrystals in cellular environments.

3.5. Energy transfer and quenching studies Solution phase protein-ligand binding models have been studied using CdSe-ZnS QDs conjugated to proteins by monitoring fluorescence resonance energy transfer (FRET) between a QD energy donor conjugated to one binding partner and an organic acceptor dye attached to the other binding partner (Willard et al., 2001). Biotinylated bovine serum albumin (bBSA) was conjugated to the QDs by introduction of free sulfhydryl groups onto the protein; the new thiol groups served as attachment points for the protein to the QD surface. Unconjugated bBSA was removed by filtration through a 100 kD cutoff spin column. Introduction of increasing concentrations of streptavidin labeled with tetramethylrhodamine (TMR) to purified QD-bBSA conjugates resulted in decreases in the QD fluorescence emission. A concurrent increase in the TMR emission was observed, suggesting that energy transfer was occurring. Energy transfer between a fluorescent protein donor (BSA) emitting in the near UV and CdTe QDs was studied in aqueous solutions of QD-BSA conjugates (Mamedova et al., 2001). BSA emission is centered at 340 nm and originates from naturally occuring tryptophan residues. CdTe nanocrystals, prepared using arrested precipitation, were capped with L-cysteine and conjugated with BSA using a one-step linkage procedure employing glutaric dialdehyde, which forms a bridge between the amino groups on the L-cysteine and the lysine moieties on the BSA. The coupling procedure preferentially yielded a 1:1 stoichiometery BSAQD conjugates with a small fraction of 2:1 (BSA:QD) complexes. When the BSA-CdTe conjugate solutions were excited at 290 nm, where both protein and 558

Quantum Dot Bioconjugates for Biosensing QD absorb, emission from the BSA was completely quenched, while that of the QDs showed a two-fold increase with respect to unconjugated nanocrystals. In controls using unconjugated QDs (i.e., unlinked with protein) BSA emission persisted, but only ~ 1/3 of its value measured in the absence of QDs, while QD emission was unaffected. The decrease in the BSA emission was attributed to changes in the pH and presence of Cd 2§ ions, which alters the luminescence efficiency of tryptophan residues. Excitation at longer wavelengths, where BSA absorption is negligible, showed only emission from the nanocrystals. The authors attributed the above observation to resonance energy transfer from BSA to CdTe QD in the QD-BSA complexes. Solution-phase fluorescence quenching assays have been carried out using a dyelabeled variant of the two-domain maltose binding protein (MBP), MBP-tb A75C, bound to DHLA-capped CdSe-ZnS quantum dots. The protein variant, which contained a single cysteine, was specifically labeled at that residue with the nonemitting quencher dye QSY-7 (Tran et al., 2002). Conjugation of MBP-tb and DHLA-capped QDs was driven by electrostatic self-assembly. QSY-7 was chosen as a quenching chromophore due to the overlap between its absorption spectrum and the emission spectrum of the QDs employed (core radius of 21.5,~ and emission maximum centered at 590 nm). Increased quenching of the nanocrystal emission was observed with increasing amounts of quencher-labeled protein bound; the nanocrystals lost about 90% of their signal when 60% of the MBP-tb QD-bound molecules were QSY-7 labeled (Figure 9). When all the conjugated proteins (ca. 5 per QD) were labeled with QSY-7, the nanocrystal emission was nearly fully quenched. These results were attributed to radiationless energy transfer occurring between the QDs and bound MBP-tb/QSY -7. In a second system, the emission from surface-tethered QDs was monitored as QSY-7-1abeled antibodies were bound to immobilized nanocrystals using an antibody-specific molecular adaptor PG-zb (Tran et al., 2002). DHLA-capped QDs were first immobilized on poly-L-lysine-treated glass slides, followed by incubation with PG-zb to form QD-PG-zb conjugates. The surface-bound QDPG-zb complex has a high selectivity for the Fr region of antibodies, and introduction of the QSY-7-1abeled antibodies onto these surfaces resulted in formation of labeled QD-PG-zb-IgG conjugates. Fluorescence quenching occurred systematically as the proportion of QSY-7 labeled IgGs conjugated to surface-bound QDs increased, until all available QD surfaces had been saturated with labeled antibody (Tran et al., 2002). 3.6. QDs in polymerized microspheres for use as micro-barcodes

B io-related applications of microsphere-encapsulated quantum dots have recently been investigated (Han et al., 2001). CdSe-ZnS nanocrystals were embedded in cross-linked polymeric beads (1-2 ~tm diameter size) formed by emulsion 559

Mattoussi, Kuno, Goldman, et al.

6000 5OOO

/5

4000

3000 E

E Q Q t...n

2000 1000 0 500

__

525

550

_

575

600

625

_ - U ~ - - " ~ ". . . . . .

650

Wavolen.qth (nm) Figure 9. PL spectra of solutions in a 2 mm optical path cell containing 30 pmol QDs and increasing molar ratio of QSY-7-1abeled MBP-tb to QD. The total numbers MPB-tb proteins per QD remained fixed (protein to QD ratio of 5). The highest PL intensity was measured in solution containing unlabeled QD-MPB-tb conjugates, and the signal decreased systematically with increasing fraction of labeled proteins, with QSY-7-MBPtb varying from 0 (unlabeled proteins) to 5 (100% QSY-7-1abeled proteins) in the above data.

polymerization of styrene, divinylbenzene and acrylic acid. The process of embedding the nanocrystals involved swelling the beads in a solution of chloroform and butanol in the presence of one or more populations of nanocrystals. Nanoparticles that had migrated into the swelled spheres were trapped upon removal of solvent. The relatively large size of the polymer spheres allowed embedding of a large number of QDs in each bead. Using a single dot size (thus one color), a range of intensity-coded beads could be prepared. Embedding two, three, or more populations of QDs per bead allowed control of the emission intensity and detailed spectral characteristics of the QDbeads, resulting in sets of "color and intensity barcode" polymer beads. A model DNA hybridization system using oligonucleotide probes conjugated to the QD-encoded beads was designed and tested in biological assays to detect target DNA sequences. Target DNA molecules to be quantified were labeled with a fluorescent dye (Cascade Blue). The dye was chosen to have an absorption band that allows its excitation simultaneously with the codes within the QD-beads, while having emission distinguishable from the coding signal (i.e., no overlap between the dye and bead emissions). Assays were performed at the single bead level, yielding both the DNA identity (based on the QD barcode) as 560

Quantum Dot Bioconjugates for Biosensing well as its abundance (based on Cascade Blue emission). The DNA sequence was identified by the coding signal (i.e., optical code of the bead defined by the spectral definition of the emission and the relative intensity of each embedded QD color). Signal from the dye attached to the DNA target accounted for the amount of the target material present in the assayed sample.

3.7. Biomaterials applications of QDs DNA-mediated assembly of CdS quantum dots into carefully designed layered arrays attached to electrode surfaces has been demonstrated, followed by generation of photoinduced current by the layered arrays in the presence of a sacrificial electron donor (Willner et al., 2001). Microemulsion-grown CdS nanoparticles capped with cystamine/thioethanesulfonate were derivatized with an estimated 20-24 thiolated oligonucleotides per nanoparticle. These DNAmodified QDs were tethered by hybridization to a gold electrode surface previously derivatized with thiolated 13-mer capture DNA, forming a first layer of DNA-QDs. Subsequent layers (up to four total) were built up by hybridization using additional oligo-DNA modified nanoparticles; the layering process could be observed using quartz crystal microgravimetry, absorbance and fluorescence methods. Current flow was observed upon illumination, and photocurrent amplitude correlated closely with absorbance spectra of the arrays. The photocurrent could be switched on and off by cycled illumination. Photocurrent generation likely involved the photoejection of conduction-band electrons of CdS particles in contact with or within tunneling distance of the electrode. Improved photocurrent generation (approximately 2-fold) could be obtained by treating arrays with 5 x 10.6 M [Ru(NI--I3)6]3+, which presumably electrostatically interacts with the DNA network. Multiple bound ruthenium complexes may form a conduit for delivery of electrons to the electrode. Finally, a unique assay for DNA was shown, in which specific hybridization of an oligonucleotide (109 M lower limit) was detected by changes in photocurrent in a two-layer CdS-DNA cross-linked array.

4. Advantages and Limitations Colloidal semiconductor quantum dots have a number of important advantages over conventional organic fluorophores. The QD absorption spectrum extends into the UV, regardless of size, making it possible to excite multiple sized (colored) particles with one excitation wavelength; organic fluorophores, on the other hand, often have discrete, widely spaced, singlet transitions. In addition, QDs have large extinction coefficients, which translate to absorption crosssections on the order of 1015 cm2. By contrast, many organic dyes have absorption cross-sections nearly an order of magnitude smaller than 1016 cm2. These properties, as well as the color variation made possible by simply varying the physical size of the particle (as opposed to synthesizing new analogs or 561

Mattoussi, Kuno, Goldman, et al. derivatives of conventional organic dyes) represent major advantages of QDs, especially in light of potential multiplexing applications. With respect to their emission, the quantum yields (QYs) of QDs can be comparable to those of organic dyes with values close to 30%. Although they are not as bright as some of the best organic laser dyes such as Rhodamine 6G (QY ~ 95%), they outperform organic fluorophores in two ways. First, the colloidal QDs have narrow (~30-40 nm full width at half maximum) and symmetric emission spectra. Organic dyes, on the other hand, often have broad, asymmetric spectra with a distinct phonon progression to the red. This is a limiting factor in the case of multiplexing applications due to undesirable spectral cross talk among different detector channels. Second, organic dyes suffer from rapid irreversible photodegradation effects, a process often referred to as "photobleaching". While the causes of this effect are not completely understood, photooxidation and other types of degradative photochemistry effectively destroy dye molecules or quench their emission (Eggeling et al., 1999). Semiconductor QDs are much less susceptible to photobleaching since they are made of inorganic materials. This dramatic difference in photostability has been observed at the ensemble level and at the single particle level, where a single CdSe QD has been observed to emit 108 photons with no evidence of photobleaching. That value decreases to ~ 105106 photons for organic dyes (Kuno et al., 2001). Fluorescence resonance energy transfer (FRET), a phenomenon that involves nonradiative transfer of excited state energy from a donor to an acceptor, has an efficiency that depends on the degree of overlap between the donor emission spectrum and the absorption spectrum of the acceptor. Early experiments reported efficient F6rster energy transfer between neighboring closely packed colloidal QDs of different sizes (Kagan et al., 1996). The tunable and narrow PL spectra of QDs make them potentially very suitable for biosensing applications based on energy transfer, where dye-labeled receptors conjugated to colloidal QDs can report binding events. Experiments demonstrating that QD luminescence can be quenched by surface-bound acceptor dyes in QD-protein conjugates in solution and in solid-phase formats have been described (Willard et al., 2001; Tran et al., 2002). Substantial difficulties are associated with making QDs water-soluble and derivatizable. No consistent protocol for achieving QD water-compatibility has been devised that can be applied to a wide range of QD materials. As discussed above, a number of methods exist for coating CdSe, CdSe-ZnS, CdS, or CdTe nanocrystals. Most published works to date have focused on the use of CdSe-ZnS QDs, with a few exceptions where CdS, CdSe-CdS, or CdTe QDs have been used (Mahtab et al. 1996; Bruchez et al., 1998; Mamedova et al., 2001). However, at this time, materials with cores made of materials other than CdSe tend to have low quantum yields and poor resistance to degradation in aqueous environments. 562

Quantum Dot Bioconjugates for Biosensing In our experience, although CdSe-ZnS QDs capped with DHLA are relatively stable and easy to handle, they have a quite limited functional pH range. For instance, DHLA-capped QDs are stable in basic solutions at pH > 7, but aggregation, often accompanied by loss of luminescence, takes place even in "mildly" acid solutions. Surface functionalization using a porous silica shell is reported to provide stable water-compatible QDs, but the coating process is tedious and tends to result in small amounts of material having a low quantum yield. QD conjugation to proteins has often been carried out using EDC condensation. However, even though this type of chemistry is well established for labeling biomacromolecules with organic dyes, conjugation to QDs can produce irreversible aggregation that may be either immediate or can develop with time (Mattoussi et al., 2000). Non-covalent, electrostatically driven QDbioconjugate self-assembly can ameliorate aggregation problems in some cases, but it requires the use of surface-charged nanoparticles and oppositely charged proteins with the appropriate biological activity. Other potential problems that may complicate analysis and understanding of experiments using QD-conjugates derive from the size dependence of their optical properties (e.g., emission) and from the stoichiometry of the QDbiomolecule complexes. Thus, particles emitting further in the red are larger and thus have different diffusion characteristics than smaller ones. This may, for example, complicate experiments where diffusion and dynamics of particle movement within a cell are important. In addition, QDs conjugated with multiple protein receptors or DNA oligomers per dot will have different mobility and likely experience different avidity effects than 1:1 QD:biomolecule conjugates. This may limit their usefulness in some applications.

5. Potential for Use of Quantum Dots in Bio-related Applications Semiconductor nanocrystals continue to be viewed as potentially extremely useful materials in the realm of biotechnology (Niemeyer, 2001), and the work performed so far in a number of laboratories reaffirms this expectation. Nonetheless, although great strides have been made in the short period of time since the introduction of QDs in biocompatible forms, the state of our knowledge, both in terms of basic science and of nanoengineering technology, is far from mature. Substantial opportunities will exist for new contributions in this dynamic area for the foreseeable future, particularly in the areas of DNA and protein microarray technology, fluorescence-based imaging, and high-throughput drug candidate screening. The major near-term focus will likely be on uses of QD bioconjugates as photostable substitutes for organic fluorophores as well as in multi-color barcode applications. In the former case, expanded usefulness will come when functional problems related to nanocrystal surface chemistry are solved. Remaining 563

Mattoussi, Kuno, Goldman, et al. obstacles in this area include surface oxidation, long-term stability in physiological environments, passivation methods needed to reduce non-specific interactions, and potential toxicity issues related to in vivo use. Further expansion of usefulness would also accrue from development of robust biocompatible near IR and IR-emitting materials. Linked to the these obstacles is the fact that there currently exists no acceptable way to mass-produce biocompatible QDs and QD conjugates inexpensively, a situation which will surely hinder wider usage of these materials in new applications until it is overcome. In the case of potential use of QDs and mixtures of QDs as molecular barcode elements, most likely encapsulated within polymer spheres, an obvious advantage is the inherent multiplex nature of the materials due to their nearly infinite flexibility with respect to excitation wavelengths. This flexibility should translate into simpler and less expensive optical platforms used in barcode applications. In fact, QDs embedded into microbeads have already been tested in this scenario (Han et al., 2001). It should be noted, however, that organic dyebased microsphere technology is quite advanced and may be expected to present considerable competition for analogous QD-based applications. A case in point is the high throughput flow cytometer and associated dye-labeled microsphere reagents commercialized by Luminex (Austin, TX). The system is capable of identifying 100 or more different bead types by the dye ratio contained in each. The amount of fluorescent analyte or antibody bound to each bead is simultaneously measured. In this manner very rapid multiple homogeneous assays can be performed (Kettman et al., 1998; Vignali, 2000; Iannone et al., 2001, Ye et al., 2001). Although semiconductor nanocrystals as replacements for organic fluorophores will likely account for the bulk of their near-term uses, exploitation of some of their other unique properties in bio-related scenarios is possible. As an example, experiments involving QD bioconjugates binding to neural receptors point to the possibility of utilizing the optoelectronic properties of nanocrystals in less obvious, more sophisticated ways (Winter et al., 2001). Finally, understanding the behavior of the nanocrystals and their bioconjugates at the single particle level will contribute to as yet undiscovered applications. We anticipate that fuller understanding of single-dot phenomena such as intermittent blinking, spectral shifts, effects of crystal lattice defects and surface traps, etc., will lead to development of new types of nanosensors and provide materials for additional bio-related applications.

564

Quantum Dot Bioconjugates for Biosensing

6. Acknowledgements We thank Professor M.G. Bawendi from MIT and Drs. B.L. Justus and F.S. Ligler from NRL for the fruitful discussions and useful suggestions. Financial support from the Office of the Naval Research (ONR, Dr. K. Ward), grants # N0001499WX30470, # N0001400WX20094 and # N0001401WX20854, is highly appreciated. The views, opinions, and/or findings described in this report are those of the authors and should not be construed as official Department of the Navy positions, policies or decisions.

7. References Alivisatos, A.P., 1996, Science 271,933. Artmeyev, M.V., S.V. Gaponenko, I.N. Germanenko and A.M. Kapitonov, 1995, Chem. Phys. Lett. 243, 450. BorreUi, N.F., D.W. Hall, H.J. Holland and D.W. Smith, 1987, J. Appl. Phys. 61, 5399. Bowen Katari, J.E., V.L. Colvin and A.P. Alivisatos, 1994, J. Phys. Chem. 98, 4109. Bruchez, M., Jr., M. Moronne, P. Gin, S. Weiss and A.P. Alivisatos, 1998, Science 281, 2013. Chamarro, M., C. Gourdon, P. Lavallard, O. Lublinskaya and A.I. Ekimov, 1995, Nuovo. Cim. Soc. Ital. Fis. D 17, 1407. Chamarro, M., C. Gourdon, P. Lavallard, O. Lublinskaya and A.I. Ekimov, 1996, Phys. Rev. B 53, 1336. Chan, W.C.W. and S. Nie, 1998, Science 281, 2016. Chepic, D.I., A1.L.Efros, A.I. Ekimov, M.G. Vanov, V.A. Kharchenko, I.A. Kudriavtsev and T.V. Yazeva, 1990, J. Luminesc. 47, 113. Chestnoy, N., R. Hull and L.E. Brus, 1986, J. Chem. Phys. 85, 2237. Colvin, V.L., M.C. Schlamp and A.P. Alivisatos, 1994, Nature 370, 354. Cook, R.J. and H.J. Kimble, 1985, Phys. Rev. Lett. 54, 1023. Dabbousi, B.O., M.G. Bawendi, O. Onitsuka and M.F. Rubner, 1995, Appl. Phys. Lett. 66, 1316. Dabbousi, B.O., J. RodriguezViejo, F.V. Mikulec, J.R. Heine, H. Mattoussi, R. Ober, K.F. Jensen and M.G. Bawendi, 1997, J. Phys. Chem. B 101, 9463. Dahan, M., T. Laurence, F. Pinaud, D.S. Chemla, A.P. Alivisatos, M. Sauer and S. Weiss, 2001, Optics Lett. 26, 825. Efros, A.L., M. Rosen, M. Kuno, M. Nirmal, D.J. Norris and M.G. Bawendi, 1996, Phys. Rev. B54, 4843. Efros, A.L. and M. Rosen, 1997, Phys. Rev. Lett. 78, 1110. Efros, A.L. and M. Rosen M., 2000, Ann. Rev. Mater. Sci. 30, 475.

565

Mattoussi, Kuno, Goldman, et al. Eggeling, C., J. Widengren, R. Rigler and C.A.M. Seidel, 1999, In Applied Fluorescence in Chemistry, Biology and Medicine, W. Rettig, B. Strehmel, S. Schrader, H. Seifert, Eds., Springer, Berlin, pp. 193. Ekimov, A.I., A.A. Onuschenko and V.A. Tsekhomskii, 1980, Fiz. Khim. Stekla 6,511. Ekimov, A.I. and A.A. Onuschenko, 1981, JETP Lett. 34, 345. Ekimov, A.I. and A.A. Onuschenko, 1982, Sov. Phys. Semicond. 16, 775. Ekimov, A.I. and A.A. Onuschenko, 1984, JETP Lett. 40, 337. Ekimov, A.I., A.A. Onuschenko, A.G. Pluhkin and A.L. Efros, 1985, JETP Lett. 88, 1490. Ekimov, A.I., A.L Efros and A.A. Onuschenko, 1985, Sol. Stat. Comm. 56, 921. Ekimov, A.I., F.Hache, M.C. Schanneklein, D. Ricard, C. Flytzanis, I.A. Kudryavtsev, T.V. Yazeva, A.V. Rodina and A.L. Efros, 1993, J. Opt. Soc. Am. B-Opt. Phys. 10, 100. Ekimov, A.I., 1996, J. Luminesc. 70, 1. Gaponenko, S.V., U. Woggon, M. Saleh, W. Langbein, A. Uhrig, M. Muller and C. Klingshirn, 1993, J. Opt. Soc. Am. B-Opt. Phys. 10, 1947. Gaponenko, S.V., 1998, Optical Properties of Semiconductor Nanocrystals, Cambridge University Press, Cambridge, 260 pp. Gaponik, N.P., D.V. Talapin, A.L. Rogach, A. Eychmuller, 2000, J. Mat. Chem. 10,2163. Gearheart, L., K.K. Caswell and C.J. Murphy, 2001, J. B iomed. Optics, 6, 111. Goldman, E.R., E.D. Balighian, M.K. Kuno, S. Labrenz, P.T. Tran, G.P. Anderson, J.M. Mauro and H. Mattoussi, 2002, Phys. Stat. Sol., in press. Goldman, E.R., G.P. Anderson, P.T. Tran, H. Mattoussi, P.T. Charles and J.M. Mauro, 2002, Anal. Chem., in press. Goldman, E.R., H. Mattoussi, P.T. Tran, G.P. Anderson and J.M. Mauro, 2001, In Semiconductor Quantum Dots, Ed, S. Fafard, D. Huffaker, R. Leon and R. Noetzel, Mat. Res. Soc. Pro., Pittsburgh, 642, J2.8.1. Greenham, N.C., X.G. Peng. and A.P. Alivisatos, 1996, Phys. Rev. B54, 17628. Gurevich, S.A., A.I. Ekimov, I.A. Kudryavtsev, A.V. Osinskii, V.I. Skopina and D.I. Chepik, 1992, Sov. Phys. Semicond. 26, 57. Hall, D.W. and Borrelli N.F., 1988, J. Opt. Soc. Am. B-Opt. Phys. 5,1650. Han, M., X. Gao, J.Z. Su and S. Nie, 2001, Nature Biotech.19, 631. Henglein, A., 1982, Ber. Bunsen Phys. Chem. 86, 301. Hermanson, G.T., 1996, B ioconjugate Techniques, Academic Press: London. Herron, N, Y. Wang, M.M. Edd, G.D. Stucky, D.E. Cox, K. Moiler and T. Bein, 1989, J. Am. Chem. Soc. 111,530. Hines, M.A. and P. Gnyot-Sionnest, 1996, J. Phys. Chem. B 100, 468. Hines, M.A. and P. Guyot-Sionnest, 1998, J. Phys. Chem. B 102, 3655. Hodes, G., A. Albuyaron, F. Decker and P. Motisuke, 1987, Phys. Rev. B 36, 4215. Hu, J.T., L.S. Li, W.D. Yang, L. Manna, L.W. Wang and A.P. Alivisatos, 2001, Science 292, 2060. 566

Quantum Dot Bioconjugates for Biosensing Iannone, M.A., T.G. Consler, K.H. Pearce, J.B. Stimmel, D.J. Parks and J.G. Gray, 2001, Cytometry 44, 326. Kagan C.R., C.B. Murray and M.G. Bawendi, 1996, Phys. Rev. B 54, 8633. Kettman, J.R., T.Davies, D. Chandler, K.G. Oliver and R.J. Fulton, 1998, Cytometry 33, 234. Kortan, A.R., R. Hull, R.L. Opila, M.G. Bawendi, M.L. Steigerwald, P.J. Carrolla and L.E. Brus, 1990, J. Am. Chem. Soc. 112, 1327. Kotov, N.A., K. Putyera, J.H. Fendler, E. Tombacz and I. Dekany, 1993, Coll. Surf. A-Phys. Eng. Aspects 71, 317. Kotov, N.A., F.C. Meldrum, C. Wu and J.H. Fendler, 1994, J. Phys. Chem. 98, 2735. Kuno, M., J.K. Lee, B.O. Dabbousi, F.V. Mikulec and M.G. Bawendi, 1997, J. Chem. Phys. 106, 9869. Kuno, M., D.P. Fromm, H.F. Hamann, A. Gallagher and D.J. Nesbitt, 2001, J. Chem. Phys. 115, 1028 Liu, L.C. and S.H. Risbud, 1990, J. Appl. Phys. 68, 28. Liu, L.C., M.J. Kim, S.H. Risbud and R.W. Carpenter, 1991, Philosoph. Mag. B 63,769. Lobenhofer E.K., P.R. Bushel, C.A. Afshari and H.K. Hamadeh, 2001, Env. Health Pers. 109, 881. Mahtab, R., J.P. Rogers, C.P. Singleton and C.J. Murphy, 1996, J. Am. Chem. Soc., 118, 7028. Mamedova, N.N., N.A. Kotov, A.L. Rogach and J. Studer, 2001, Nano Lett. 1, 281. Mathieu, H., T. Richard, J. Allegre, P. Lefebvre and G. Arnaud, 1995, J. Appl. Phys. 77, 287. Mattoussi, H., A.W. Cumming, C.B. Murray, M.G. Bawendi, and R. Ober, 1998-11, Phys. Rev. B 58, 7850. Mattoussi, H., L.H. Radzilowski, B.O. Dabbousi, E.L. Thomas, M.G. Bawendi and M.F. Rubner, 1998, J. Appl. Phys. 83, 7965. Mattoussi, H., J.M. Mauro, E.R. Goldman, G.P. Anderson, V.C. Sundar, F.V. Mikulec and M.G. Bawendi, 2000, J. Am. Chem. Soc. 122, 12142. Mattoussi, H., J.M. Mauro, E.R. Goldman, R.M. Green, G.P. Anderson, V.C. Sundar and M.G. Bawendi, 2001, Phys. Stat. Sol. 224, 277. Meyer, M., C. W allberg, K. Kurihara and J.H. Fendler, 1984, J. Chem. Soc. Chem. Commun. 90, 90. Mikulec, F.V., 1999, Ph.D. Dissertation, Massachusetts Institute of Technology. Minti, H., M. Eyal, R. Reisfeld and G. Berkovic, 1991, Chem. Phys. Lett. 183, 277. Misawa, K., H. Yao, T. Hayashi and T. Kobayashi, 1991, Chem. Phys. Lett. 183, 113. Misawa, K., H. Yao, T. Hayashi and T. Kobayashi, 1991, J. Chem. Phys. 94, 4131. Mitchell, G.P., C.A. Mirkin and R.L. Letsinger, 1999, J. Am. Chem. Soc. 121, 8122. 567

Mattoussi, Kuno, Goldman, et al. Murray, C.B., S. Sun, W. Gaschler, H. Doyle, T.A. Betley and C.R. Kagan, 2001, IBM J. Res. Dev. 45, 47. Murray, C.B., C.R. Kagan and M.G. Bawendi, 2000, Ann. Rev. Mater. Sci. 30, 545. Murray, C.B., D.J. Norris and M.G. Bawendi, 1993, J. Am. Chem. Soc. 115, 8706. Niemeyer, C.M., 2001, Angew. Chem. Int. Ed. 40, 4218. Nirmal, M., D.J. Norris, M. Kuno, M.G. Bawendi, A.L. Efros and M. Rosen, 1995, Phys. Rev. Lett. 75, 3728. Nirmal, M., B.O. Dabbousi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris and L.E. Brus, 1996, Nature 383, 802. Nogami, M., K. Nagasaka and M. Takata, 1990, J. Non-Cryst. Sol. 122, 101. Norris, D.J., N. Yao, F.T. Chamock and T.A. Kennedy, 2001, Nano Lett. 1, 3. Ochoa, R.O., C. Colajacomo, E.J. Witkowski, J.H. Simmons and B.G. Potter, 1996, Sol. Stat. Comm. 98, 717. Pathak, S., S.K. Choi, N. Amheim and M.E. Thompson, 2001, J. Am. Chem. Soc. 123, 4103. Peng, X., M.C. Schlamp, A.V. Kadavanich, U. Banin and A.P. Alivisatos, 1997, J. Am. Chem. Soc. 119, 7019. Peng, X.G., L. Manna, W.D. Yang, J. Wickham, E. Scher, A. Kadavanich and A.P. Alivisatos, 2000, Nature 404, 59. Peng, Z.A. and X.G. Peng, 2001, J. Am. Chem. Soc. 123, 183. Peng, Z.A. and X.G. Peng, 2001, J. Am. Chem. Soc. 123, 1389. Persans, P.D., A. Tu, Y.I. Wu and M. Lewis, 1989, J. Opt. Soc. Am. B-Opt. Phys. 6, 818. Pileni M.P., L. Motte and C. Petit, 1992, Chem. Mater. 4, 338. Potter, B.G. and J.H. Simmons, 1988, Phys. Rev. B 37, 10838. Potter, B.G. and J.H. Simmons, 1988, J. Appl. Phys. 68, 1218. Qu, L., Z.A. Peng and X.G. Peng, 2001, Nano Lett. 1,333. Rodriguez-Viejo, J., H. Mattoussi, J.R. Heine, M.K. Kuno, J. Michel, M.G. Bawendi, and K.F. Jensen, 2000, J. Appl. Phys. 87, 8526. Roederer, M., S. DeRosa, R. Gerstein, M. Anderson, M. Bigos, R. Stovel, T. Nozaki, D. Parks, L. Herzenberg and L. Herzenberg, 1997, Cytometry 29, 328. Rogach, A.L., L. Katsikas, A. Kornowski, D. Su, A. Eychmuller and H. Weller, 1997, Ber. Bunsen. Phys. Chem. 101, 1668. Rossetti, R., S. Nakahara and L.E. Brus, 1983, J. Chem. Phys. 79, 1086. Rossetti, R., J.E. Ellison, J.M. Gibson and L.E. Brus, 1984, J. Chem. Phys. 80, 4464. Schlamp, M.C., X.G. Peng and A.P. Alivisatos, 1997, J. Appl. Phys. 82, 5837. SchrOck, E., S. du Manoir, T. Veldman, B. Schoell, J. Wienberg, M.A. FergusonSmith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini and T. Ried, 1996, Science, 273,494. Spanhel L., E. Arpac and H. Schmidt, 1992, J. Non-Crys. Sol. 147, 657. Sondi, I., O. Siiman, S. Koester and E. Matijevic, 2000, Langmuir 16, 3107. 568

Quantum Dot Bioconjugates for Biosensing Steigerwald, M.L., A.P. Alivisatos, J.M. Gibson, T.D. Harris, R. Kortan, A.J. Muller, A.M. Thayer, T.M. Duncan, D.C. Douglass and L.E. Brus, 1988, J. Am. Chem. Soc. 110, 3046. Steigerwald, M.L. and L.E. Brus, 1989, Ann. Rev. Mat. Sci. 19, 471. Sun, B., W. Xie, G. Yi, D. Chen, Y. Zhou, and J. Cheng, 2001, J. Irranunol. Meth., 249, 85. Tedeschi, C.; F. Caruso, H. MOhwald and S. Kirstein, 2000, J. Am. Chem. Soc. 122, 5841. Tran, P.T., E.R. Goldman, H. Mattoussi, G.P. Anderson and J.M. Mauro, 2001, Proc. SPIE 4258, 1. Tran, P.T., E.R. Goldman, G.P. Anderson, J.M. Mauro and H. Mattoussi, 2002, Phys. Stat. Sol., In Press. Trindade T., P. O'Brien, N.L. Pickett, 2001, Chem. Mat. 13, 3843. Vignali, D.A.A., 2000, J. Immunol. Methods 243, 243. Wang, Y, N. Herron, W. Mahler and A. Suna, 1989, J. Opt. Soc. Am B-Opt. Phys. 6, 808. Weller, H, H.M. Schmidt, U.Koch, A. Fojtik, S. Baral, A. Henglein, W. Kunath, and K. Weiss, 1986, Chem. Phys. Lett. 124, 557. Willard, D.M., L.L. Carillo, J. Jung and A.Van Orden, 2001, Nano Lett. 1,469. Willner, I., F. Patolsky and J. Wasserman, 2001, Ang. Chem. Intl. Ed. 40, 1861. Winter, J.O., T.Y. Liu, B.A. Korgel and C.E. Schmidt, 2001, Adv. Mater. 13, 1673. Woggon, U., S.V. Bogdanov, O. Wind, K.-H., Schlaad, H. Pier, C. Klingshim, P. Chatziagorastou and H.P. Fritz, 1993, Phys. Rev. B48, 11979. Woggon; U., 1997, Optical Properties of Semiconductor Quantum Dots, Springer-Verlag, Berlin, 251 pp. Ye, F., M.S. Li, J.D. Taylor, Q. Nguyen, H.M. Colton, W.M. Casey, M. Wagner, M.P. Weiner and J. Chen, 2001, Hum. Mutat. 17, 305. Yoffe, A.D., 1993, Adv. Physics, 42, 173. Yoffe, A.D., 2001, Adv. in Physics 50, 1. Zhao, X.S., J. Schroeder, P.D. Persans and T.G. Bilodeau, 1991, Phys. Rev. B 43, 12580.

569

Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 92002 Elsevier Science B.V. All rights reserved

CHAPTER 18

SOFT LITHOGRAPHY AND MICROFLUIDICS

RAVI S. KANE ~ PH.D ABRAHAMD STROOCK1 NOO LI JEON 1 PH.D. DONALD E. INGBER2 PH.D. AND GEORGE M. WHITESIDES1 PH.D 9

9

1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 0213 8 2Departments of Pathology and Surgery, Children's Hospital and Harvard Medical School Boston, MA 02115

Optical biosensors necessarily involve an interface between synthetic materials and biological systems. This chapter describes the application of soft lithography to create and control this interface. Soft lithography is a set of techniques that includes 1) methods of fabricating microstructures in polymers, especially elastomers, 2) uses of these methods in combination with organic surface chemistry to generate micron-scale patterns on synthetic surfaces, and 3) uses of microfluidic systems to pattern the composition of the fluid medium adjacent to a surface. These techniques allow the immobilization of biomolecules and cells at surfaces with micron-scale resolution, and for the control of the subsequent interaction of these species with liquid media. These techniques are compatible both with optical and electronic materials and with biological systems. This review focuses on the use of soft lithography to fabricate microfluidic systems and to position and manipulate living cells on surfaces.

1. Technical Concept 1.1. Introduction This chapter describes the use of a set of non-conventional (i.e., not based on photolithography) microfabrication techniques known as soft lithography (Xia 571

Kane, Stroock, Jeon, et al. and Whitesides, 1998; Whitesides et al., 2001) to create and control the interfaces between synthetic materials and biological systems. The term, "soft lithography," describes an integrated set of techniques for fabricating microstructures in an elastomeric material, for modifying the chemical properties of surfaces, and for controlling flows of fluid adjacent to surfaces. In the context of the interface between synthetic materials and biological systems, soft lithography makes use of elastomeric stamps, membranes, and microfluidic channels to deposit small molecules, biological molecules, and living cells on synthetic substrates with micron-scale spatial resolution. Microfluidic channels made with soft lithography provide an environment for cell culture in which reagents and analytes can be delivered non-destructively to cells with sub-cellular precision (Duffy et al., 1998; Takayama et al., 1999). These soft lithographic techniques are inexpensive, procedurally simple, and do not require stringent control of the laboratory environment (i.e., a cleanroom is not required). They provide greater flexibility and convenience than photolithography for patterning organic and biological materials. Soft lithographic techniques offer a means for tailoring the interface between the "optical" and the "bio" components of optical biosensors. Figure 1 illustrates this interface schematically. The characteristics of the interface that must be controlled are 1) the position (on the scale of microns) of biological elements with respect to sensing elements, 2) the chemical interaction (both attractive and repulsive) between the surfaces of the materials used in the biosensors and biomolecules and cells, and 3) the local environment of the biological system, in such a way that the biological components remain active. The materials used in creating this interface must also be compatible with both the optical system (that is, they must be transparent, have the correct index of refraction, and form adequate mechanical seals) and the biological system (that is, they must be nontoxic, be selective in their molecular recognition, and have appropriate surface composition). These criteria are met by the materials used in soft lithography. The examples that we use in this chapter concentrate on systems of the class that is shown in Figure 1 in which molecules, cells, and media can all, in principle, be patterned. These systems--instrumented micro-cell culture systems~are immediately useful in fundamental studies of cell biology. They also have the potential to act as sophisticated biosensors and analytical systems. Both cellbased assays, which require repeated examination of individual cells, and biosensors, which rely on the collective observation of multiple cells, will benefit from accurate control of cell location. B iosensors and combinatorial screens may also require surfaces that display specific ligands in a surface that otherwise minimizes non-specific interactions with proteins or cells. Control of the bio/materials interface is also key to solving an important, long-term problem: the design of hybrid systems that combine (or allow direct communications between) living and non-living systems.

572

Soft Lithograpy and Microfluidics

Figure 1. Controlling the interactions of a cell with its environment using soft lithography. Most of the environmental features sensed by the cell can be patterned using soft lithography or devices fabricated using soft lithography: the surface on which proteins adsorb or that presents ligands (patterning by microcontact printing); the identity of neighboring cells (membrane-based patterning or patterning using three-dimensional microfluidic systems); the composition of the extracellular medium (laminar flow patterning in microchannels).

1.2. Soft lithographic methods of microfabrication Controlling the environment experienced by individual cells or groups of cells requires control over the composition of both the surface and the medium on relevant length scales (micrometers for single cells and millimeters to centimeters for groups of cells). We have developed a set of techniques that we call "soft lithography" that is an alternative to photolithography, and that can be used to create microstructures, and to control the surface chemistry of synthetic materials, with a spatial resolution of microns. Soft lithographic techniques are inexpensive, procedurally simple, and do not require stringent control over the laboratory environment. These techniques can be used to pattern both planar and non-planar substrates, and also to pattern the cell culture medium. In soft lithography, elastomeric stamps, microfluidic channels, and membranes prepared by casting or spin coating the liquid prepolymer of an appropriate elastomer against a master that has a patterned relief structure (Figure 2). Most of the research based on soft lithography has used poly(dimethylsiloxane) 573

Kane, Stroock, Jeon, et al.

Figure 2. Schematic diagrams of the soft lithographic approach of fabricating microfluidic channels (A), membranes, and stamps (B). A) A transparency prepared on a high resolution printer (5000 dpi) is used as a photomask. Epoxy photoresist is spun onto a silicon wafer, exposed, and developed to create a master structure. Many (> 100) negative copies of the structure on the master can be formed by molding the structure into poly(dimethylsiloxane) (PDMS), an elastomeric polymer. To form a closed channel, the PDMS mold is sealed to a flat surface either covalently by oxidizing the surfaces in a low temperature plasma or non-covalently by applying pressure. B) Fabrication of a membrane (left) and a stamp (right) from a master. To form a membrane, PDMS is spun onto the master in a thin layer such that the features on the master create holes that tranverse the entire thickness of the layer. On a stamp, the negative of the features on the master are molded in bas-relief on one surface (McDonald et al., 2000; Xia and Whitesides, 1998; Whitesides and Stroock, 2001). (PDMS) as the elastomer, because PDMS is biocompatible, permeable to gases (and can thus be used for cell culture), and inexpensive. PDMS also has good optical characteristics; the cured polymer is transparent from 235 nm to the near infrared (Wu and Whitesides, 2001) and can make tight, weakly scattering seals around embedded optical elements such as optical fibers (Chabinyc et al., 2001). The interfacial properties of PDMS can be readily modified by plasma oxidation and silanization (Chaudhury and Whitesides, 1991). PDMS structures can often be used many times in transferring patterns (we have used the same PDMS stamp in microcontact printing approximately 100 times over a period of several months without any noticeable degradation in its performance), and each master can be used to make a large number of stamps or membranes. The access to photolithographic equipment required (to fabricate masters) in soft lithography is therefore minimal. 574

Soft Lithograpy and Microfluidics

An advantage of soft lithography as a method for patterning cells is that, at the feature sizes required for this application (typically, 2-500 /zm), it is often possible to make photomasks using procedures that are significantly more rapid and less expensive than those commonly used to make chrome masks for conventional photolithography. For the fabrication of masters having feature sizes greater than or equal to 20 /~m, masks can be generated by the highresolution laser printing of patterns (generated using computer programs such as Freehand or AutoCAD) onto flexible transparencies (Qin et al., 1996). The masks can be made in a few hours at a cost as low as $0.25 per square inch. For feature sizes between 10/zm and 20 #m, the optical reduction of images printed onto transparencies generates patterns in microfiche (Deng et al., 1999). Microfiche is then used as the photomask. For feature sizes between 2/xm and 20 #m, a relatively inexpensively approach is to use commercial laser writing to fabricate masters from which PDMS stamps can be molded (Grzybowski et al., 1998). For feature sizes between a few hundred nanometers and 20 ~tm, there are more specialized techniques (Wu and Whitesides, 2001; Love et al., 2001b). The capability to produce features larger than 20/zm rapidly and inexpensively allows researchers to prototype and produce small numbers of simple microstructures and microsystems. This capability, which we call "rapid prototyping", has minimized the barriers to the use of lithographic techniques by biochemists. Soft lithography also facilitates the fabrication of complex structures such as three-dimensional (3D) networks of channels (Love et al., 2001a; Anderson et al., 2000). Figure 3 shows an example of a multi-level network of microchannels made by stacking a stamp and a membrane (Chiu et al., 2000). The elastomeric character of PDMS enables the simple integration of a variety of thin organic materials such as filters and dialysis membranes (Ismagilov et al., 2001; Chiu et al., 2001). 1.3. Molecular control of interfaces

A necessary ingredient for the control of the bio-material interface is a versatile strategy for adding organic functionality to synthetic substrate. We have made extensive use of self-assembled monolayers (SAMs) for this purpose (Dubois and Nuzzo, 1992; Whitesides and Gorman, 1995; Whitesides et al., 1996; Wilbur and Whitesides, 1999; Folch and Toner, 2000; Mrksich, 2000). SAMs are organized organic monolayer films that provide molecular-level control over the composition and properties of the interface. Most studies of SAMs have involved monolayers of alkanethiolates on gold and silver. A benefit of working with SAMs of thiols on thin metal layers is compatibility with surface plasmon resonance (SPR) techniques (Sigal et al., 1996, 1997; Lahiri et al., 1999b; Chapman et al., 2000). SAMs of alkanethiolates on gold and silver are also

575

Kane, Stroock, Jeon, et al.

Figure 3. Three-dimensional microfluidic network for applying reagents to a surface in a discontinuous pattern, a technique called 3D MIMIC. A) Scheme for the fabrication of 3D-microfluidic stamp. The slab and the membrane are formed with different masters (cf. Figure 2B). The membrane contains segments of channel that will be in contact with the surface that is to be patterned. Vertical vias in the membrane connect the lower channels (in contact with surface) to the upper layer of channels in the slab. The aligned stack of the slab and the membrane are sealed (non-covalenfly) to the surface that is to be patterned. B) Schematic illustration of the fluid paths in the 3D network. C) Silicon oxide surface that has been etched with a network of channels such as in (A) and (B) in which different concentrations of etchant (hydrofluoric acid) were run through the three independent channels. Different shades of the etched regions correspond to different thicknesses of the oxide after etching (Chiu et al., 2000).

compatible with microcontact printing, a powerful technique for creating chemical patterns with micron-scale resolution (cf. Section 1.4) (Wilbur et al., 1994; Xia and Whitesides, 1998). Figure 4 outlines three methods developed in our group for attaching ligands covalently to surfaces with controlled orientation and density (Lahiri et al., 1999a, 1999b; Roberts et al., 1998; Yan et al., 1997). Control over the density of groups presented at a surface can be achieved by forming a SAM from a solution containing a mixture of alkanethiols, although phase segregation in the monolayer might affect the surface properties of certain mixed SAMs. In Figures 4A and 4B, the ligand is presented in a background of SAMs terminated with oligomers of ethylene glycol (OEG); SAMs that present OEG resist the nonspecific adsorption of protein (Prime and Whitesides, 1991); such surfaces are called "inert". The presentation of ligands in an inert background is important for performing quantitative, low-noise binding assays at surfaces (Lahiri et al., 1999b).

576

Soft Lithograpy and Microfluidics A. Direct method

B. C o m m o n intermediate method

C. Anhydride method

(CF3CO}~O, Et3N,

DMF, 20 rain, rl

[~

~ 1)SHCIIHr~EGQ

~,

t,AU,

t,

t I

2) 5 HCIIHtzE ~C HzCONH"v--.~>

,

I

s

L

s,

,s~

AU

s,

s

.s

]

!

,

.,

AU

,

~

!

[

,

,

, ,,

Au

,

,__

I

. . . . .

Au

!

Figure 4. Generation of SAMs presenting specific ligands: A) Immersion of a gold substrate in a solution containing a mixture of an alkanethiol terminating in oligo(ethylene glycol) groups (OEG), and a second alkanethiol terminating in a ligand of interest, generates a surface presenting the desired ligand (Roberts et al., 1998). B) A mixed SAM is formed by the immersion of a gold substrate in a solution containing a mixture of EG3OH-terminated and EG6-OCH2COOH-terminated alkanethiols. Activation of the carboxylic acid groups using N-hydroxysuccinimide (NHS) and 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), followed by a reaction with an amine-terminated ligand, generates a surface presenting that ligand (Lahiri et al., 1999a, 1999b). C) A surface presenting interchain carboxylic acid anhydrides is generated by treating SAMs that present terminal carboxylic acid groups with trifluoroacetic anhydride. Reacting these activated SAMs with amine-terminated ligands results in a surface presenting a 1:1 mixture of the desired ligand and carboxylic acid groups (Yan et al., 1997).

1.4. Patterning molecules and cells on synthetic surfaces The types of microstructures (Section 1.2) and surface chemistry (Section 1.3) described i n the proceeding sections can be combined to form powerful techniques for patterning organic chemical functionality, biomolecules, and even living cells on surfaces with micron-scale precision. In this section, we outline the use of three such techniques: microcontact printing (lxCP) (Wilbur et al., 1994; Xia and Whitesides, 1998), membrane patterning (MEMPAT)(Ostuni et al., 2000), and three-dimensional micromolding ha capillaries (3D MIMIC)(Chiu et al., 2000). Microcontact printing is a technique that uses the relief pattern on the surface of an elastomeric PDMS stamp to form patterns of SAMs on the 577

Kane, Stroock, Jeon, et al.

Figure 5. Procedure for patterning SAMs by microcontact printing: A stamp is inked with an alkanethiol and placed on a gold (or silver) surface; the pattern on the stamp is transferred to the gold by the formation of a SAM on the regions that contacted the substrate. The bare areas of the gold are exposed to a different alkanethiol to generate a surface patterned with a SAM that presents different chemical functionalities in different regions (Xia and Whitesides, 1998). surfaces of substrates (Figure 5). Patterned SAMs generated by microcontact printing can be used to control the adsorption of proteins on surfaces. L6pez et al. (1993) first used microcontact printing to pattern gold surfaces into regions presenting oligo(ethylene glycol) groups in a background of methyl groups. Immersion of the patterned SAMs in solutions of proteins resulted in the adsorption of proteins only on the methyl-terminated regions. These systems have subsequently been extended to other experiments in cell biology (see Section 3.2) (Chen et al., 1997; Mrksich et al., 1997; Mrksich, 2000). While microcontact printing is a technique that has sufficient resolution to allow the patterning of single cells, in its simplest configuration, it does not allow any changes in the pattern or shape of adsorbed cells. MEMPAT and 3D MIMIC are complementary techniques that not only allow the patterning of cells~individually or in groups---on arbitrary substrates, but also allow studies of the spreading or migration of cells from their initial pattern. MEMPAT makes use of elastomeric membranes~free-standing PDMS films that have through-membrane pores---to pattern proteins and cells on a variety of substrates including plastics and glass (Figure 6) (Folch and Toner, 2000; Ostuni et al., 2000). Bringing an elastomeric membrane into contact with a substrate restricts access of a solution of protein or a suspension of cells to those regions of the substrate exposed through the pores. The deposition of proteins, or the attachment of cells, is therefore restricted to these exposed regions of the substrate. The patterned cells are constrained by the walls of the pores in the membrane. The ability to remove the constraints imposed by the membrane by peeling it away from the substrate, and to observe the subsequent spreading or 578

Soft Lithograpy and Microfluidics

Figure 6. Schematic diagram that describes the use of MEMPAT for plating cells onto a substrate with well defined position and shape. The surface of the membrane and the walls of its holes are coated with bovine serum albumin (BSA). The membrane is placed on a clean surface (e.g., Petri dish) and exposed to a solution of fibronectin (FN). After rinsing with a solution of a phosphate buffer solution, the membrane and the substrate are covered with a suspension of ceils for 24 h. The membrane can be removed without damaging the cells and the protected areas of the substrate can be modified by the adsorption of an adhesive protein that allows the patterned cells to spread (Ostuni et al.,

2000).

Figure 7. Schematic diagram of a "T-Sensor" based on the diffusional mixing between three laminarly flowing streams in a microchannel (Weigl and Yager, 1999). The analyte of interest (e.g., an enzyme) is introduced in the sample stream (bottom). A solution of indicator (e.g., a substrate that becomes fluorescent upon interaction with the enzyme) flows in the detection stream (center). The third stream contains a reference solution. Diffusive mixing of the solutions occurs at the interface of the streams. The product of this mixing (e.g., a fluorophore) will be localized in the diffusively mixed region between the streams. migration of the cells as a function of the composition of the substrate and the cellular environment, may be exploited in several areas of cell biology. 3D 579

Kane, Stroock, Jeon, et al. MIMIC allows the generation of arbitrary and discontinuous patterns of proteins or cells on planar substrates (Figure 3). We had previously developed a soft lithographic technique called MIMIC (micromolding in capillaries) (Kim et al., 1995) for fabricating three-dimensional structures by allowing solutions to flow into microfluidic channels. The use of MIMIC was limited to relatively simple, continuous patterns. To overcome some of the limitations of MIMIC, we developed an analogous technique that makes use of three-dimensional microfluidic systems (Anderson et al., 2000) for patterning (3D MIMIC). Figure 3C illustrates the use of 3D MIMIC to create a discontinuous pattern of etched regions in a silicon oxide layer on silicon. The same methods can be used with solutions of proteins and suspensions of cells to deliver these elements to surfaces in complex, discontinuous patterns (cf. Section 3.2.2).

1.5. Laminar flow in microfluidic systems Networks of microchannels can be used to control the location of fluids on the micron scale. This spatial control can be extended by taking advantage of the laminar character of flows in microchannels (Takayama et al., 1999; Kenis et al., 1999). Since microfluidic channels are small (100 /.tm in cross-sectional dimension), the flow of liquids in them is usually laminar (Bird, 1960); adjacent streams of different composition mix into one another only by diffusion. This characteristic of laminar flows has been exploited by Weigl and Yager (1999) for continuous chemical analysis of solutions flowing in a microchannel (Figure 7). Laminar flows in microchannels also allow the chemical composition of solutions and surfaces to be patterned on scales smaller than the channel itself; we refer to this method of patterning as "laminar flow patterning" (Kenis et al., 2000). Figure 8 demonstrates the use of laminar flow patterning for the electroless deposition of a silver wire on the center of the floor of a microchannel (Kenis et al., 1999). Laminar flow patterning can be extended to biological systems and :allows for control of both cells and the cellular environment---qhat is, it allows control over the nature of molecules that are deposited on the substrate, the nature and position of neighboring cells, and the composition of the extracellular medium. Laminar flow patterning makes it possible to pattern the fluid culture medium itself (cf. Section 3.2.3). Networks of microchannels can be used to generate complicated patterns in the composition of flowing solutions. A strategy for forming gradients in the concentration of solutes in a flow of buffer in a microchannel is shown in Figure 9: A small number (six in the case shown) of solutions of different composition are injected into a network of channels; in the network, streams of the solutions are allowed to divide and mix into one another to form a larger number (24 in the case shown) of streams of solutions of intermediate composition; at the outlet of the network, the multiple streams are allowed to recombine in single channel across which there is, as a result of this "combination", a gradient in the 580

Soft Lithograpy and Microfluidics

Figure 8. Demonstration of patterned surface chemistry achieved with laminar fowing streams in a microchannel. A silver wire deposited in a zigzag channel at the laminar flow interface between solutions containing the components of an electroless silver plating solution (Kenis et al., 1999).

Figure 9. Gradients of solute made with a microfluidic network, a) Schematic diagram of network that transforms 6 input solutions into 24 output solutions that contain intermediate concentrations of solute in the incoming streams. The 24 streams flow laminarly in a broad outlet channel (2160/ma wide), b-d) Demonstration of three types of gradients that can be formed in the outlet channel using fluorescein solutions. On top are fluorescent micrographs of streams flowing in the outlet channel. Below, plots show the corresponding fluorescence intensity profile across the outlet channel at the beginning of the channel (L1, white dotted line) and 800/an downstream from the junction (L2, white dotted line). The fluorescein concentration of the solutions introduced into the inlets of the microfluidic device is shown above the individual gradients (Dertinger et al., 2001).

concentration of the solutes from the original solutions; under laminar flow conditions, this gradient propagates along the channel with only diffusional 581

Kane, Stroock, Jeon, et al. broadening of the gradient profile (Jeon et al., 2000; Dertinger et al., 2001). The gradient in the combined stream can be used to study cell behaviors such as chemotaxis. In the case in which the solutes adsorb on walls of the channel, we have found that the gradient in the combined stream led to gradients of qualitatively similar form in the concentration of the species that are bound to the wall (unpublished results).

2. History 2.1. Self assembled monolayers The history of ordered molecular monolayers is long, but only in the past few decades have convenient methods emerged that allow for the formation of high quality films on solid substrates. Early work on monolayers was based on the Langmuir-Blodgett method in which the film is formed at the liquid-air interface and subsequently transferred to a solid support (Ulman, 1991). This method can lead to films with a high degree of molecular ordering, but the process is complicated and prone to errors. Silanes offer a flexible way to bring organic functional groups to solid surfaces that present hydroxyl groups (Grushka, 1974). The molecules in silane layers are only partially ordered, and silane films are prone to degradation in aqueous buffer due to the hydrolysis of silicon-oxygensilicon bonds. In 1985, Allara and Nuzzo discovered the self-assembly of disulphides on metal surfaces (Allara and Nuzzo, 1985); this discovery led to work with thiols on metals. Long-chain (greater than 10 carbons) alkane thiols form SAMs with crystalline order that are very stable in aqueous medium. SAMs of thiols have been extensively characterized by our group and others (Dubois and Nuzzo, 1992; Whitesides and Gorman, 1995).

2.2. History of patterned surface chemistry Until recently, there were few methods available to pattern the chemical groups presented on a surface with micron-scale resolution: photo-labile groups presented a surfaces could be patterned with exposure through a photomask (Wollman et al., 1994); photolithography could also be used to define patterns in photoresist that would act as a mask for the deposition of metals, oxides, and organics (Muller and Kamins, 1986). The development of SAMs of thiols on metals opened the possibility of using traditional patterning methods such as writing and stamping to create patterns of well ordered molecular layers with sub-micron-scale resolution (Kumar et al., 1992, 1994; Wilbur et al., 1994; Piner et al., 1999).

582

Soft Lithogravy and Microfluidics

2.3. History of microfluidics The initial development of microfluidics (Manz et al., 1991) used fabrication techniques adapted from the electronics industry. With these techniques, channel structures are formed in hard materials such as glass and silicon using photolithography followed by etching; the channels are typically sealed by anodic bonding (Kovacs, 1998). These steps are slow, expensive, and require a cleanroom environment. Hard plastics are also used (see for example, Micronics, www.micronics.net). Most of the early work in microfluidics focused on using electroosmotic flow (Harrison et al., 1993; Jacobson et al., 1994). Over the past few years, we and others have been developing alternative methods based on soft lithography to fabricate microfluidic devices (Delamarche et al., 1997; Duffy et al., 1998; Beebe et al., 2000; Quake and Scherer, 2000) (Figures 2 and 3). These methods, which use PDMS as the principle material, are simple, are inexpensive, and can be performed in a standard laboratory environment. The mechanical flexibility of PDMS makes it appropriate for the fabrication of the movable components that are often required for the control of pressure-driven flows (Unger et al., 2000).

2.4. History of patterning cells Early work on patterning cells on synthetic substrates was done using silanes and photolithography (Kleinfeld et al., 1988); this process required multiple steps. The development of ~tCP simplified the process of patterning cells. We have employed ~tCP to pattern extracellular matrix proteins and to control the position, shape, and function of single living cells (Singhvi et al., 1994; Mrksich et al., 1997; Chen et al., 1997; Dike et al., 1999; Kane et al., 1999; Takayama et al., 2000). Others working in the area of cell patterning include the groups of Toner (Folch and Toner, 2000) and Shakesheff (Patel et al., 1998). The use of cells in sensors has been developed by the groups of Stenger and Kovacs (Jung et al., 1998; Pancrazio et al., 1998).

3. State of the Art 3.1. Microfluidic systems fabricated using soft lithography 3.1.1. Microfluidic channels and components. Soft lithography provides a method for fabricating almost any system of channels that might be needed for microfluidics (Figure 2, of. Section 1.2) (McDonald et al., 2000). For example, channels made in PDMS using soft lithography support electroosmotic flow; these channels can be used for capillary electrophoresis (Duffy et al., 1998). Soft

583

Kane, Stroock, Jeon, et al.

Figure 10. Rotary pump made with soft lithography in PDMS. A) Schematic diagram of a peristaltic pump based on pneumatic valves that take advantage of the elastomeric character of PDMS. The air pressure in each upper channels is controlled independently; high pressure in one of the upper channels locally deforms the lower channel and restricts flow. When actuated sequentially, the three valves act as a peristaltic pump. B) Flow ring. Six valves are activated sequentially to drive fluid around the circle when the input and output channels are blocked. The recirculating flow can be used to increase the residence time of flow in the channel for mixing or for the completion of a slow chemical reaction (Unger et al., 2000; Quake and Scherer, 2000).

lithography also simplifies the fabrication of three-dimensional channels (Figure 3) (Anderson et al., 2000); the Beebe group used soft lithography to fabricate a active components, such as pumps and valves, that are required in microfluidic systems. Chou and Quake have designed a valve and a peristaltic pump using a multilayer structure made from PDMS (Figure 10) (Unger et al., 2000; Quake and Scherer, 2000). Soft lithographic methods make the registration and integration of multiple layers (at the 50 ktm scale) simple (Love et al., 2001b). The elastomeric character of PDMS allows the valve to be actuated with small changes in the pressure in the gas-filled channels.

3.1.2. Integrated microfluidic devices. Soft lithographic methods also facilitate the integration of multiple materials (e.g., PDMS, glass, organic membranes, polymer tubing, and metal films) and non-fluidic components (e.g., lenses, optical fibers, and electrodes) into a single device. An important integration process that is simplified by soft lithography is the connection of external tubing with on-chip microchannels for the introduction and collection of samples. With soft lithography, these connections are made by simply boring holes in the PDMS and press-fitting tubing into these holes; this press-fit seal can withstand several bars of pressure (McDonald et al., 2000).

584

Soft Lithograpy and Microfluidics

Figure 11. Parallel detection using an array of crossing microfluidic channels. A) Schematic drawing of a lxl array of microfluidic channels in which a poly(carbonate) membrane (0.1 ktm pores) separates the two channels at the point at which they cross. The bottom channel contains a substrate entrapped in a gel matrix. B) Detection of enzymatic activity using fluorometric (top) and colorimetric (bottom) methods in a 5x5 array of crossing channels. Substrates for enzymes were immobilized in a 1.25% agarose gel in the lower (horizontal) set of channels. Solutions of different enzymes were allowed to flow by gravity in the upper (vertical) set of channels. The ELF-97-1inked substrates release a fluorescent molecule upon cleavage by an enzyme; these products are visualized under UV illumination (top). BCIP/NBT and X-gal form precipitates upon cleavage; these products are visualized by optical adsorption (bottom).

Figure 11 shows a simple optical biosensor that allows for five tests to be performed on five solutions in parallel. In this device, a poly(carbonate) membrane is sealed between two layers of PDMS, each of which contains a set of microchannels; the membrane allows for diffusive (not convective) exchange of molecules between the channels in the regions in which they cross (Ismagilov et al., 2001). The bottom channels contain substrates for the enzymes of interest in an agarose gel. The sample solutions are allowed to flow in the upper channels. An enzyme is detected when it diffuses from an upper channel into the lower channel and acts on one of the substrates to form either a precipitate or a fluorescent molecule. This type of hybrid system (PDMS-thin film-PDMS) is easy to fabricate using soft lithography because PDMS conforms and seals around the intervening layer. Figure 12 shows an integrated microfluidic device for the separation and detection of fluorescently-labeled proteins; an optical fiber, optical filter, and micro avalanche photo diode (ktAPD) are integrated with the microchannel (Chabinyc et al., 2001). The fiber is molded into the slab of PDMS that contains 585

Kane, Stroock, Jeon, et al.

Figure 12. Microfluidic chip in PDMS for capillary electrophoresis with integrated optical fiber and micro avalanche photo diode (~APD) detector. A) Schematic diagram of experimental setup. The excitation light was provided by a blue LED off-chip. This light was coupled into an optical fiber that was molded into the PDMS chip. Both the excitation and the detection light were filtered with inexpensive polymeric optical filters. The chip has three levels: a slab of PDMS that contains the channel and the fiber, a polymeric optical filter, and a array of laAPDs embedded in a slab of PDMS. The signal from the I.tAPD was processed off-chip. B) Micrograph of microchannel and optical fiber. The serpentine microchannel was filled with fluorescein. The image shows the size of the detection volume (--25 nL) and that the light is coupled from the fiber into the PDMS with minimal scattering (Chabinyc et al., 2001).

the channel. Figure 11B illustrates the clean optical coupling that is achieved between the fiber and the PDMS. A sheet of polymeric filter is sealed (noncovalently) between the slab of PDMS and the lxAPD that is embedded in PDMS; the filter eliminates stray excitation light. The PDMS makes conformal contact with the filter so the interface between the PDMS and the filter is optically smooth.

3.2. Controlling the cellular environment using soft lithography

3.2.1. Spatially constrained cell culture using IzCP. The ability to pattern SAMs by microcontact printing, and the resulting control over the adsorption of adhesive proteins (for example, the extracellular matrix proteins fibronectin, vitronectin, and laminin), enables the patterning of cells on substrates (Figure 13). Mrksich et al. (1997) used microcontact printing to pattern gold substrates into regions comprising SAMs capped with oligo(ethylene glycol) groups that resist the adsorption of proteins and regions comprising SAMs capped with methyl groups that adsorb proteins. After immersing the substrates in a solution of fibronectin, bovine capillary endothelial cells were found to attach only to the methyl-terminated, fibronectin-coated regions of the patterned SAMs.

586

Soft Lithograpy and Microfluidics

Figure 13. Patterned cells on a surface patterned with micro-contact printing. A) A gold surface was patterned into regions of hexadecanethiolate and undecanethiolate terminated with tri(ethylene glycol). Fibronectin (bright) adsorbed on the hydrophobic squares of hexadecanethiolate but not on the tri(ethyleneglycol)-terminated alkanethiolate (dark). Patterned substrates were soaked in a solution of fibronectin, fixed using paraformaldehyde, and immersed in a solution of anti-human fibronectin IgG and then rinsed. The substrates were then placed in contact with a solution of Texas Red| goat anti-rabbit IgG and mounted in fluoromount-G. B) Bovine capillary endothelial (BCE) cells patterned by culturing on a substrate presenting hydrophobic squares of varying sizes that were coated with fibronectin prior to incubation with cells using the procedure described in (A)) (Chen et al., 1997).

The ability to engineer the properties of the interface between mammalian cells and their substrates using microcontact printing has been useful in understanding the effect of cell shape on cell behavior. Singhvi et al. (1994) used the ability to control cell shape by microcontact printing to investigate the effect of cell shape on cell function. They plated primary hepatocytes on substrates (patterned by microcontact printing) presenting square and rectangular islands of laminin surrounded by non-adhesive regions. Cells attached preferentially to the laminincoated regions, and in most cases, conformed to the shape of the island. The size and shape of cells could therefore be manipulated by changing the size and shape of the adhesive islands, without changing the density of the adhesive protein laminin. The synthesis of DNA was highest on unpatterned surfaces, where the cells could spread without restriction, and a decrease in the size of the cells led to a progressive reduction in DNA synthesis. For the smallest islands (< 1600 /zmZ), less than 3 % of the adherent cells entered the DNA synthesis phase of the cell cycle. The size of the cells also affected the differentiated function of hepatocytes, as reflected by the concentration of secreted albumin in the culture supematant: albumin secretion rates increased as the size of the adhesive island was decreased. This study demonstrated that cell shape could influence cell growth and protein secretion independent of any changes in the density of the adhesive protein laminin. Studies of ceils grown on micropatterned substrates 587

Kane, Stroock, Jeon, et al.

Figure 14. A) Optical micrographs of bovine capillary endothelial cells patterned using MEMPAT on a bacteriological petri dish (see Figure 6 for experimental details). The top left frame shows the state of the cells just after the membrane was removed (7 hours after cells were plated). The other frames show the spreading of the unconstrained cells in the hours following the removal of the membrane. (Ostuni et al., 2000) B) Schematic representation of the channel network used to pattern cells by 3D MIMIC (see Figure 3 for generic structure of channels). C) Fluorescence micrograph of human bladder cancer cells (ECVs; labeled (bright) with 5-chloromethylfluorescein diacetate) and bovine capillary endothelial cells (BCEs; labeled (dim) with 1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine); the cells were patterned on alternating squares of a checkerboard motif. The cells were cultured for 42 h before the fluorescence micrograph was taken. The channel structure is still on the surface. D) A picture of a confluent layer of cells before the 3D microfluidic stamp was removed. E-F) Pictures taken in fluorescence (E)and in phase contrast (F) show the spreading and growth of the two cell types after the removal of the PDMS stamp. The pictures in (E) and (F) were taken 20 h after removal of the stamp. The three images in D-F are registered; the dotted lines show the relative orientation of the patterns. The BCEs spread more rapidly than the ECVs by a factor of 2-3 (Chiu et al., 2000).

have also indicated that the constrained size and shape of a cell influences whether it lives or dies (undergoes apoptosis) (Chen et al., 1997) as well as its differentiated state (Singhvi et al., 1994; Dike et al., 1999).

3.2.2. Patterned cell culture with variable spatial constraints. M E M P A T and 3D MIMIC offer the possibility of plating cells in regions of well defined size, shape, and location (cf. Section 1.4). Furthermore, with both methods the spatial constraint on the cell(s) imposed by the membrane or channel can be released by peeling the membrane (MEMPAT) or channel network (3D MIMIC) away from the substrate to allow the cells to spread and migrate. Figure 14A shows the movement of bovine capillary endothelial cells across the surface after their release from the regions defined by the holes in a PDMS membrane (see Figure 6 for experimental details) (Ostuni et al., 2000). The rate and degree of the spreading of cells could be used as a simple indicator of the state of cells as they interact with their environment in an cell-based effect sensor. 588

Soft Lithograpy and Microfluidics

Figure 15. Patterned plating of cells from laminar flow. A) Patterning different cell types on the floor of a single microchannel. Chick erythrocytes and E. coli were deposited selectively in their designated lanes by patterned flow of cell suspensions. Adherent ceils were visualized with a fluorescent nucleic acid stain (Syto 9). B) Patterning the delivery of a stain to bovine capillary endothelial cells in a microchannel. A suspension of bovine capillary endothelial cells was introduced into channels that were pre-treated with fibronectin and allowed to attach and spread. After removing non-adherent cells by washing with medium, Syto 9 was allowed to flow through one of the inlets as medium was allowed to flow through the other two (Takayama et al., 1999).

3D MIMIC makes it possible to deposit different cell types in close proximity (~ 200 btm) to one another. This technique may therefore be useful in exploring interactions between different types of cells, and valuable in exploring processes such as morphogenesis, angiogenesis, and differentiation. In the experiment shown in Figures 14B-F, 3D MIMIC is used to create a culture of two cell types (cancer cells and capillary endothelial cells) that are relevant for the study of angiogenesis (Chiu et al., 2000). To achieve an alternating pattern of the two types of cell (Figure 14C), suspensions of cells were allowed to fill the two orthogonal sets of channels in the 3D microfluidic network that is shown schematically in Figure 14B (cf. also Figure 3). The cells were allowed to settle and attach to the substrate for 42 hours with the microfluidic network still sealed against the surface. Figures 14D-F show the evolution of cells after the microfluidic network was removed. This type of experiment could be useful for studying the effect of angiogenic factors released by the cancer cells.

3.2.3. Controlling the total environment of a cell with laminar flow patterning. Laminar flow patterning allows for the patterned deposition of cells and the patterned delivery of reagents to cells that are already present on the walls of a microchannel. Figure 15A illustrates patterning of multiple cell types (erythrocytes and E.coli in this case) on the floor of a microchannel (Takayama et al., 1999). In this experiment, streams of the suspensions of the different types of cell were introduced into the channel through independent inlets. As these streams join in the main channel, they flow laminarly along side one another; the populations of the cells remain segregated in streams and they attach to the floor in the distinct regions covered by the different streams. This technique is a 589

Kane, Stroock, Jeon, et al.

Figure 16. PARTCELL. Patterned flow of trypsin over a bovine capillary endothelial cell on the floor of a microchannel. A) The scheme shows the geometry of the channel. B) Micrograph that shows the cell before treatment with trypsin. C) Micrograph that shows the partially detached cell after treatment (Takayama et al., 1999).

simple alternative to 3D MIMIC to plate different cell types in close proximity to one another for studies of cell interaction. Figure 15B illustrates patterning of the culture medium itself. In this experiment, a single type of cell was plated uniformly on the floor of the microchannel by allowing a suspension to flow through all three inlets. Subsequently, a stream of medium containing a fluorescent stain (Syto-9) was allow to flow through only one of the three inlets of the channel as regular media was allowed to flow through the other inlets. In the main channel, the stain remained localized to a third of the width of the channel as the streams of media flowed laminarly along side one another. As is seen in the micrograph, the only cells that were stained were those covered by the stream that carded the stain. This experiment demonstrates the selective delivery of a reagent to only part of a small population of cells. In this experiment, the untreated cells could act as an internal control or the other inlets could be used to selectively deliver another reagent to a distinct or overlapping sub-population of the cells. For large cells (~ 100 ~tm), laminar flow patterning can be used to deliver reagents selectively to parts of a single cell; we call this technique PARTCELL (Figure 16) (Takayama et al., 1999). In the experiment shown, a suspension of cells was allowed to flow through the entire channel and attach to its floor. Subsequently, distinct streams of medium containing trypsin (a protease that cleaves the attachments of the cells to the surface) and medium without trypsin were allowed to flow through the two inlets of the channel. Figures 16B and 16C show a cell that spanned the interface between the two laminarly flowing streams. In the region covered by the stream that contained trypsin, the attachments that held the cell to the surface were cleaved; the cell retracted from this region. PARTCELL provides the new capability of interacting with a cell on the cellular scale in a non-destructive way (Takayama et al., 2001). 590

Soft Lithograpy and Microfluidics

4. Advantages and Limitations The central advantages of soft lithography as compared to conventional microfabrication methods are its simplicity and its broad compatibility with both organic and inorganic systems. The methods described in this chapter can be used with a minimum of specialized equipment and in a typical laboratory environment (not a cleanroom). The abilities to make many copies of a microstructure by molding, and copies of a chemical pattern by stamping, are useful outcomes of soft lithography. The use of SAMs of thiols allows chemical modification of substrates that are compatible with both electrical (e.g., electrochemical) and optical (e.g., SPR and microscopy) measurements. Soft lithography offers a simple means of fabricating complicated microstructures that integrate organic (filters, membranes, gels), inorganic (glass and silicon surfaces, optical elements, electrodes), and biological elements (medium, proteins, living cells). PDMS, the core material in these systems, has a number of useful properties including low cost, low toxicity, transparency from the visible into the near ultraviolet, chemical inertness, versatile surface chemistry, mechanical flexibility, and durability. PDMS forms conformal seals with most smooth surfaces and can be covalently sealed to itself and to glass after a short (one minute) oxidation step in a low temperature plasma. Soft lithographic methods facilitate the fabrication of microfluidic systems in PDMS. The use of flows in these microfluidic devices to deliver reagents to surfaces with micron-scale resolution is particularly interesting for work with living cells; the flows are non-destructive and the walls of the channels are gas permeable. One limitation of soft lithographic methods comes from the incompatibility of PDMS with many organic solvents; solvents such as dichloromethane and tetrahydrofuran penetrate PDMS and cause it to swell. The use of micro-contact printing has been most successful with thiols on metals. Some work has been done with silanes on silicon oxide, but the procedure is more complicated and the resolution is not as high as with thiols (Jeon et al., 1997). PDMS structures might not be durable enough for certain industrial applications; the low cost of these devices means that they can often serve in "one-time-use" applications.

5. Potential for Improving Biosensor Performance Soft lithographic methods can be used in both the fabrication and the operation of biosensors. Many of the structural, mechanical, and optical elements of biosensors could be fabricated simply and inexpensively in PDMS using soft lithographic techniques. For example, an array of micro-lenses could be molded into one side of a thin slab of PDMS that contains a network of microchannels in relief on the other side. If aligned with the channels, the array of lenses could be used to focus the light from a flood illumination source onto small regions of the 591

Kane, Stroock, Jeon, et al. underlying fluidic network (Wu and Whitesides, 2001). Furthermore, this slab could be aligned and sealed to another slab of PDMS in which an array of optical detectors was embedded. Waveguides and photonic structures could also be fabricated and integrated into sensors using soft lithographic methods (Schueller et al., 1999; Yang et al., 2000). Waveguides can act as highly sensitive detectors of binding at surfaces: the evanescent field of the light in a waveguide can excite fluorescence in molecules bound to the surface of the guide; the emission light is then coupled back into the guide with an efficiency that depends, in part, on the geometry of the guide (Golden et al., 1992). Using soft lithography, this geometry could be controlled precisely. Self-assembled monolayers of thiols on gold-coated glass are an attractive system for use with SPR detection. SAMs of thiols are particularly useful for assays of specific binding of proteins to ligands at the surface. Mixed SAMs of thiols that are terminated with a ligand of interest and thiols that are terminated with oligo(ethylene glycol) offer a effective platform for binding assays; in this system, the degree of specific binding can be tuned by changing the concentration of ligand-terminated thiol and non-specific binding is very low. These are both important characteristics in applications in biosensing. We believe that soft lithographic techniques will be particularly important for the development of cell-based effect sensors in which the detection of an agent is based on the response of a living cell. For example, microcontact printing or MEMPAT could be used to plate cells in an initial condition (e.g. spatially constrained) from which they evolve in a known way as a function of their environment. Laminar flow patterning could be used to treat a single culture of cells or even a single cell in parallel with different solutions of interest. This method has the advantage of having a built-in control.

6. Acknowledgments Work from both the GMW group and the Ingber group was support by NSF DMR 9809363 (MRSEC). The Whitesides group was also supported by DARPA, NIH GM 30367, and NSF ECS 9729405. The Ingber group was also supported by NIH CA 45548 and NIH CA 55833. A number of our co-workers have been responsible for the work reported here. Although they are listed in the references, we are especially grateful to Milan Mrksich, Shu Takayama, Robert Chapman, Emanuele Ostuni, Chris Chen, Lin Yan, and Joydeep Lahiri for major contributions to the program.

592

Soft Lithograpy and Microfluidics 7. References

Allara, D. L. and R. G. Nuzzo, 1985, Langmuir 1, 45. Anderson, J. R., D. T. Chiu, R. J. Jackman, O. Cherniavskaya, J. C. McDonald, H. Wu, S. H. Whitesides and G. M. Whitesides, 2000, Anal. Chem. 72, 3158. Beebe, D. J., J. S. Moore, Q. Yu, R. H. Liu, M. L. Kraft, B. H. Jo and C. Devadoss, 2000, Proc. Natl. Acad. Sci. USA 97, 13488. Bird, R. B., 1960, Transport Phenomena, Wiley, New York. Chabinyc, M. L., D. T. Chiu, J. C. McDonald, A. D. Stroock, J. F. Christian, A. M. Karger and G. M. Whitesides, 2001, Anal. Chem. 73,4491. Chapman, R. G., E. Ostuni, S. Takayama, R. E. Holmlin, L. Yan and G. M. Whitesides, 2000, J. Am. Chem. Soc. 122. Chaudhury, M. K. and G. M. Whitesides, 1991, Langmuir 7, 1013. Chen, C. S., M. Mrksich, S. Huang, G. M. Whitesides and D. E. Ingber, 1997, Science 276, 1425. Chiu, D. T., N. L. Jeon, S. Huang, R. Kane, C. J. Wargo, I. S. Choi, D. E. Ingber, and G. M. Whitesides, 2000, Proc. Natl. Acad. Sci. USA 97, 2408. Chiu, D. T., E. Pezzoli, H. Wu, A. D. Stroock, and G. M. Whitesides, 2001, Proc. Natl. Acad. Sci. USA 98, 2961. Delamarche, E., A. Bernard, H. Schmid, B. Michel and H. Biebuyck, 1997, Science 276, 779. Deng, T., J. Tien, B. Xu and G. M. Whitesides, 1999, Langmuir 15, 6575. Dertinger, S. K. W., D. T. Chiu, N. L. Jeon and G. M. Whitesides, 2001, Anal. Chem. 79, 1240. Dike, L. E., C. S. Chen, M. Mrksich, J. Tien, G. M. Whitesides and D. E. Ingber, 1999, In Vitro Cell. Dev. Biol.-Anita. 35, 441. Dubois, L. H. and R. G. Nuzzo, 1992, Annu. Rev. Phys. Chem. 43,437. Duffy, D. C., J. C. McDonald, O. J. A. Schueller and G. M. Whitesides, 1998, Anal. Chem. 70, 4974. Folch, A. and M. Toner, 2000, Annu. Rev. Biomed. Eng. 2, 227. Golden, J. P., L. C. Shriver-Lake, G. P. Anderson, R. B. Thompson and F. S. Ligler, 1992, Opt. Eng. 31, 1458. Grushka, E., 1974, Bonded Stationary Phases in Chromatography, Ann Arbor Science Publication, Ann Arbor, 937 pp. Grzybowski, B. A., R. Haag, N. Bowden and G. M. Whitesides, 1998, Anal. Chem. 70, 4645. Harrison, D. J., K. Fluri, K. Seiler, Z. Fan, C. S. Effenhauser and A. Manz, 1993, Science 261,895. Ismagilov, R. F., J. M. K. Ng, P. J. A. Kenis, and G. M. Whitesides, 2001, Anal. Chem. 73, 5207. Jacobson, S. C., R. Hergenroder, L. B. Koutny and J. M. Ramsey, 1994, Anal. Chem. 66, 1114. Jeon, N. L., S. K. W. Dertinger, D. T. Chiu, I. S. Choi, A. D. Stroock and G. M. Whitesides, 2000, Langmuir 16, 8311. 593

Kane, Stroock, Jeon, et al. Jeon, N. L., K. Finnie, K. Branshaw and R. G. Nuzzo, 1997, Langmuir 13, 3382. Jo, B. H., L. M. Van Lerberghe, K. M. Motsegood and D. J. Beebe, 2000, J. Microelectromech. Syst. 9, 76. Kane, R. S., S. Takayama, E. Ostuni, D. E. Ingber and G. M. Whitesides, 1999, Biomaterials 20, 2363. Kenis, P. J. A., R. F. Ismagilov, S. Takayama, G. M. Whitesides, S. Li and H. S. White, 2000, Acc. Chem. Res. 33, 841. Kenis, P. J. A., R. F. Ismagilov and G. M. Whitesides, 1999, Science, 285, 83. Kim, E., Y. Xia and G. M. Whitesides, 1995, Nature 376, 581. Kleinfeld, D., K. H. Kahler and P. E. Hockberger, 1988, J. Neurosci. 8, 4098. Kovacs, G. T. A., 1998, Micromachined Transducers Sourcebook, CB/McGrawHill, Boston, 911 pp. Kumar, A., H. A. Biebuyck, N. L. Abbott, and G. M. Whitesides, 1992, J. Am. Chem. Soc. 114, 9188. Kumar, A., H. A. Biebuyck and G. M. Whitesides, 1994, Langmuir 10, 1498. Lahiri, J., L. Isaacs, B. Grzybowski, J. D. Carbeck and G. M. Whitesides, 1999a, Langmuir 15, 7186. Lahiri, J., L. Isaacs, J. Tien and G. M. Whitesides, 1999b, Anal. Chem. 71,777. L6pez, G. P., H. A. B iebuyck, R. H~irter, A. Kumar and G. M. Whitesides, 1993, J. Am. Chem. Soc. 115, 10774. Love, J. C., J. R. Anderson and G. M. Whitesides, 200 l a, MRS Bull. 26, 523. Love, J. C., D. B. Wolfe, H. O. Jacobs and G. M. Whitesides, 200 l b, Langmuir 17, 6005. Manz, A., D. J. Harrison, E. M. J. Verpoorte, J. C. Fettinger, H. Ludi and H. M. Widmer, 1991, Chimia 45, 103. McDonald, J. C., D. C. Duffy, J. R. Anderson, D. T. Chiu, H. Wu and G. M. Whitesides, 2000, Electrophoresis 21, 27. Mrksich, M., 2000, Chem. Soc. Rev. 29, 267. Mrksich, M., L. E. Dike, J. Tien, D. E. Ingber and G. M. Whitesides, 1997, Exp. Cell Res. 235,305. Muller, R. S. and T. I. Kamins, 1986, Device Electronics for Integrated Circuits, 2nd ed., Wiley, New York, 524 pp. Ostuni, E., R. Kane, C. S. Chen, D. E. Ingber and G. M. Whitesides, 2000, Langmuir 16, 7811. Pancrazio, J. J., P. P. Bey, D. S. Cuttino, J. K. Kusel, D. A. Borkholder, K. M. Shaffer, G. T. A. Kovacs and D. A. Stenger, 1998, Sens. Actuators BChem. 53, 179. Patel, N., R. Padera, G. H. W. Sanders, S. M. Cannizzaro, M. C. Davies, R. Langer, C. J. Roberts, S. J. B. Tendler, P. M. Williams and K. M. Shakesheff, 1998, Faseb J. 12, 1447. Piner, R. D., J. Zhu, F. Xu, S. Hong and C. A. Mirkin, 1999, Science 283, 661. Prime, K. L. and G. M. Whitesides, 1991, Science 252, 1164. Qin, D., Y. Xia and G. M. Whitesides, 1996, Adv. Mater. 8,917. Quake, S. R. and A. Scherer, 2000, Science 290, 1536.

594

Soft Lithograpy and Microfluidics Roberts, C., C.S. Chen, M. Mrksich, V. Martichonok, D.E. Ingber and G.M. Whitesides, 1998, J. Am. Chem. Soc. 120, 6548. Schueller, O. J. A., X.-M. Zhao, G.M. Whitesides, S.P. Smith and M. Prentiss, M., 1999, Adv. Mater. 11, 37. Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger and G.M. Whitesides, 1996, Anal. Chem. 68, 490. Sigal, G. B., M. Mrksich and G.M. Whitesides, 1997, Langmuir 13, 2749. Singhvi, R., A. Kumar, G.P. Lopez, G.N. Stephanopolous, D.I.C. Wang, G.M. Whitesides and D.E. Ingber, 1994, Science 264, 696. Takayama, S., R.G. Chapman, R. Kaneand G.M. Whitesides, Eds., 2000, Patterning of Cells and Their Environment, Academic Press, San Diego, 209 pp. Takayama, S., J.C. McDonald, E. Ostuni, M.N. Liang, P.J.A. Kenis, R.F. Ismagilov and G.M. Whitesides, 1999, Proc. Natl. Acad. Sci. USA 5545. Takayama, S., E. Ostuni, P. LeDuc, K. Naruse, D.E. Ingber and G.M. Whitesides, 2001, Nature 411, 1016. Ulman, A., 1991, An Introduction to Ultrathin Organic Films Langmuir-Blogett to Self-Assembly, Academic Press, Boston, 442 pp. Unger, M. A., H. Chou, T. Thorsen, A. Schererand S.R. Quake, 2000, Science 288, 113. Weigl, B. H. and P. Yager, 1999, Science 283, 346. Whitesides, G. M., A.J. Black, P.F. Nealey and J.L. Wilbur, 1996, In The Robert A. Welch Foundation 39th Conference on Chemical Research on Nanophase Chemistry, Houston, TX, 109 pp. Whitesides, G. M. and C.B. Gorman, Eds., 1995, Self-Assembled Monolayers: Models for Organic Surface Chemistry, CRC Press, Boca Raton, 713 pp. Whitesides, G. M., E.S. Ostuni, S. Takayama, X. Jiang and D.E. Ingber, 2001, Ann. Rev. Biomed. Eng. 3, 335. Whitesides, G. M. and A.D. Stroock, 2001, Physics Today 54, 42. Wilbur, J. L., A. Kumar, E. Kim and G.M. Whitesides, 1994, Adv. Mater. 6, 600. Wilbur, J. L. and G.M. Whitesides, 1999, In Nanotechnology, Ed., G. Timp, Springer-Verlag, New York, 331 pp. Wollman, E. W., D. Kang, C.D. Frisbie, I.M. Lorkovic and M.S. Wrighton, 1994, J. Am. Chem. Soc. 116, 4395. Wu, M. H. and G.M. Whitesides, 2001, Appl. Phys. Lett. 78, 2273. Xia, Y. and G.M. Whitesides, 1998, Angew. Chem. Int. Ed. Engl. 37, 550. Yan, L., C. Marzolin, A.Terfort, A. and G.M. Whitesides, 1997, Langmuir 13, 6704. Yang, P., G. Wirnsberger, H. Huang, S.R. Cordero, M.D. McGehee, B. Scott, T. Deng, G.M. Whitesides, B.F. Chmelka, S.K. Buratto and G.D. Stucky, 2000, Science 287,465.

595

Index absorption .... 5, 11, 14, 62, 144, 149, 195, 200, 242, 279, 291, 321, 326, 333-334, 338-339, 343, 345, 348, 354, 356, 361, 363, 435, 438, 444, 467, 539-540, 544-545, 548, 550, 552, 554, 557, 560-563 acceptance cone angle .................. 8, 9 acetate .................... 23, 355-357,546 acetylcholine receptor (AChR) ........ 70, 74-75, 79, 116, 269 acetylcholinesterase ....... 18, 40, 49, 7278, 117 actin ................................. 549, 264 aequorin ........................... 313, 316 affinity...18, 40, 49, 68, 72-72, 79, 88, 101, 114, 127, 161, 190-191, 197, 226, 240, 242, 245, 261-272, 299, 326, 332, 334-335, 344, 346, 351, 356, 360-361, 372-377, 386, 390392, 398, 407-413, 422, 429, 439, 442, 515, 532, 551,555-556 affinity chromatography ....... 270, 299, 373, 555-556 aged gels ................................. 433 aggregation...78, 199, 442, 451, 524, 550, 564 alcohol...193, 271, 345-350, 354, 356, 412, 416, 432-437, 451, 461-466, 472, 483, 489, 511, 517, 522, 546547 AlexaFluor ........................ 167, 168 alkaline phosphatase ..... 13, 21, 34, 436, 438,440 alkaloids atropine .............................. 188 sparteine .............................. 188 alkanethiol ........ 461-463, 466, 467, 578-579

amino acid...44, 101, 187-189, 264, 308-309, 314, 318-320, 332, 375, 391,402, 407, 414 amino acids glutamic acid .... 45, 46, 160, 351, 448-452 glutamine..45, 46, 159-161,315, 390 histidine .................. 103, 355,381 proline .......................... 188, 195 tryptophan ......... 185, 193,314, 559 valine ........................... 185, 188 aminopentanoic acid ................... 350 aminopropanoic acid .................. 350 annihilation ECL ..... 180, 193, 199, 200 ANS ................................ 151,159 anthracene...344, 347, 354-355, 359, 360, 420 antibiotics .......... 29, 45, 185, 188, 341 polymyxin B ..................... 74, 79 valinomycin .......................... 71 antibody ..... 7, 11-13, 16, 19-21, 25-29, 35-36, 39, 42-46, 49-51, 69-78, 83, 86-88, 96, 101-118, 124-134, 137, 139, 141, 150, 158, 186-191, 197, 236-242, 259-263, 270, 271, 288289, 297-300, 332, 337, 374-375, 387-389, 398-409, 428, 429, 433, 436-437, 445-448, 452, 458, 460, 471-480, 483, 532, 550, 551, 555560, 565 antimony ................................. 323 aptamer..23, 49, 116, 141,332, 369-396 DNA ................ 116, 376-377,388 RNA ........................ 88, 116, 377 aptamer beacon ......................... 378 aptazyme .... 116, 375,382-289, 392, 393 aptazyme ligases ......... i ........ 386, 388 arsenic .................................... 323 artificial receptor ...... 86, 331-368, 405, 422 artificial tongue ......................... 362

aluminum ...... 13, 21, 34, 200, 323,444 amine...25, 185, 188, 197, 199, 236, 258, 299, 349, 375, 421, 532, 533, 549, 578 597

Index assay configurations competition assay ...... 20, 36, 74-75, 238, 239,271,272, 551 direct assay ...................... 71, 101 displacement assay ...... 80, 102, 125137,445 sandwich assay ...... 21, 36, 72, 74, 88, 101, 103, 191,237,239, 240, 300, 742, 479,557 association constant(Ka)...333-336, 345361 ATP ..... 14, 38-39, 271, 313, 322, 360, 377-384, 389, 392 atrazine ........... 109, 238,300, 412, 414 attenuated total reflectance ...... 96, 225227 attenuated total reflection method...218, 224-229 avidin...25, 34-35, 69, 71, 81, 102-108, 114, 116, 259, 264, 299, 532, 555 neutravidin ..................... 105,267 streptavidin...88, 1 0 7 , 116, 191, 262-268, 318, 389, 472, 476, 487, 488,492, 559 azide ................................. 355, 361,490 Bacillus anthracis ............. 110-112, 192 Bacillus globigii ............................... 110 bacteria .... 12, 14, 23, 29, 66, 73-76, 96, 240, 243, 307, 312-317, 323-326, 450, 458, 460, 468,470 B a c i l l u s a n t h r a c i s ........ 110-112, 192 B a c i l l u s g l o b i g i i .......................... 110

bicarbonate ....................................... 356 bilayer lipid membrane ............. 457-496 binding constant...86, 1 3 2 , 266-267, 299, 301,350-351,354, 390, 557 binding protein...71, 79, 88, 161, 263269, 308,308, 322-326 protein G ............................ 551 acetylcholine receptor .............. 70, 74, 79, 116, 269, DNA-binding ........................ 79 fluorophore-labeled ......... 307,324 glucose/galactose binding protein .................... 159, 390 glutamine binding protein...315,390 maltose binding protein...38, 315, 371,390, 551,555,560 periplasmic binding protein ....... 151, 160, 390 binding site .... 20, 25, 49, 75, 85, 88, 101-104, 114, 133, 148, 152, 159, 166, 238, 242, 264, 265, 308, 318, 321, 332-337, 342, 351, 366, 375376, 390, 391, 398, 403-408, 412, 417-420, 433, 445, 448, 460, 465, 555 biocompatibility .... 443, 447, 453, 499, 540 bioconjugate .... 278, 279, 298-301,555, 564 biological warfare ............ 274, 374, 422 bioluminescence ... 13, 41, 68, 313, 314 biomolecular interaction analysis kinetics...69, 72, 102, 132, 140, 145, 178, 241,242, 263-269-273, 361, 380, 414, 433, 438, 440, 451, 452463, 464, 492 rate constants ....................... 241,263 biotic receptor .................................. 332 biotin...24, 35, 69-71, 81, 104-108, 114-116, 258-268, 299, 315, 318, 375,469,471,472, 487,492, 532 blood...29, 41-43, 77-79, 96, 107, 111, 129-131, 156, 159-161, 193, 207244, 256, 260, 264-267, 270, 301, 344, 345, 361, 371, 378, 387, 442, 443, 447, 450, 454, 461, 464, 471,

B r u c e l l a a b o r t u s .................. 11 O- 112

40-41, 49, 77, 159-160, 240, 323,388,390, 493, 551,590 F r a n c i s e l l a t u l a r e n s i s .... 83, 110-112 H e l i c o b a c t e r p y l o r i ................ 80, 268 L i s t e r i a ................................ 240, 482 S a l m o n e l l a ..... 88, 152, 240, 300, 301 Y e r s i n i a p e s t i s ................ 77, 110, 111 bacteria-based sensing .............. 312-317 barcode ............................. 561,564-565 benzene ............................... 85,197,324 benzoate .................................... 354, 355 E. c o l i . . . 1 9 ,

598

Index charge coupled device (CCD)...30, 33, 48, 64, 99, 113, 114, 228, 273, 294, 452, 504-507, 530, 550 chemiluminescence... 12, 13, 17, 21, 40, 46, 174, I79, 195-196, 199, 200, 313, chemosensor ............................. 331-368 chip arrays ................................ 385, 388 chloramphenicol transferase ............ 313 chloride ...................... 70, 126, 360, 506 chromophore... 188, 193,313,321,332342, 356, 361,415,416, 560 citrate ............................... 193, 354, 359 CLE-ASV ........................................ 168 CMOS camera ..................... 48, 89, 118 cocaine...74, 83, 128, 134, 138, 377, 378 cofactors...18, 39, 101,266, 314, 332, 380, 351,352, 380 colloid ...... 430, 538-546, 553,554, 563 complementarity .............................. 335 complex reversible ............................. 147 confocal microscopy... 170, 549, 557, 559 conformation...12, 273, 309, 319, 340, 378, 381, 382, 401, 414, 438-442, 458.466, 470, 471,473, continuous sensors ........................... 429 copper...165-168, 317, 323, 380, 438, 532, 541, coreactant ECL ......... 182-183, 188, 193 cotinine ............................................ 353 creatinine ................................. 353, 354 critical angle ................ 8, 9, 58, 97, 245 C r y p t o s p o r i d i u m .............................. 193 cyan fluorescent protein (CFP) ...... 316, 321,322 cytochrome c ...................... 26, 433, 451 d-d absorbance bands ......... 37, 165, 166 D-dimer...43, 110, 111,431,437, 447, 448 dendrimers ....................................... 197 deoxyribozyme ................................ 380

474-475, 488-489, 505, 525-526, 557, 559, 575,580, 592 blue fluorescent protein (BFP) ...... 311, 316, 320-324 Brucella abortus ........................ 110-112 cadmium ................... 317, 323, 546, 553 calcium...24, 169, 264, 269, 318-320, 341, 391, 459, 500, 505, 515, 527, 529 calmodulin .......... 38, 264, 315-322, 391 capacitance ....... 401,467, 483-487, 492 Helmholtz capacitance ................. 484 capillary electrophoresis...200, 387, 584, 587 carbohydrate...ll4, 151,159, 191,265, 271,343-347, 361,390, 450 allose ............................................ 345 arabinose ...................... 317,323,344 cyclodextrins ............................... 342 fructose ................................ 344-347 galactose ........................ 31,265, 345 glucosamine ......................... 347-348 glucose .... 17, 25, 31, 42, 45, 117, 158-160, 185, 188, 195, 315, 323344, 347, 361,362, 371,390, 415,480, 498,522, 531,532 lactulose ....................................... 346 maltotriose ........................... 346-347 mannose ............................... 344, 415 monosaccharide ........................... 344 trehalose .............................. 346, 491 trisaccharide ................................. 347 carbonate .................................. 453, 586 carbonic anhydrase...37, 149-151, 154, 160-166, 241,391 carboxylicacid...351, 353, 420, 533, 578 cells...5, 7, 13-19, 26-29, 40, 41, 45, 49, 50, 88, 96, 108-109, 115, 165, 169, 189, 193-194, 243, 253, 256, 258, 264-267, 300, 310, 313-316, 320-326, 374-376, 388, 405, 428, 429, 431, 451, 454, 482, 492, 498509, 513, 530, 539, 550, 558, 559, 572-581,584, 587-593 599

Index diagnostics...41, 43, 96, 109, 174, 175, 189, 194, 196-200, 270, 271, 371, 376, 387,447,458, 461,473,539 dicarboxylic acid ...................... 350-351 diffusion...24, 43, 47, 86, 88, 115, 165, 181, 199, 236, 243, 301, 303, 323, 415, 5435, 439, 451,466, 492, 523, 527,529, 533,564, 580-582 digoxin ........................ 36, 187,479-482 dimethylaminobenzoate .................... 354 diphenylanthracene (DPA)...175, 178, 180, 185, 198, 199 dissociation rate...132, 133, 241, 261263,266, 267, 271,272, DNA .... 7, 22, 29, 33-36, 71-75, 79-83, 88, 96, 102-105, 109, 115-118, 174, 187, 189-192, 195, 236, 259, 265267, 271, 299-300, 109, 312, 317, 321-326, 372-377, 380, 388, 402, 458,473, 480538, 548-552, 555-558, 561-564, 588 double-stranded DNA...22, 73, 372, 374 single-stranded DNA...36, 266, 267, 372-374, 379 triplex DNA ..................... 75, 80, 266 dopants..,429, 436, 437,440, 431,453, 454, dosimeters ........................ 429, 444, 452 drug screening ................. 115, 135,422, drugs ......................... 130, 135, 138, 188 cocaine74, 83, 128, 134, 138, 377, 378 digoxin ................... 36, 187, 479-482 methamphetamine ............... 238,239 theophylline...36, 109, 382, 384, 389, 483, dynamic range...48, 144, 164, 169, 196, 314, 320, 387, 388, 487, 508, 513, 520 E. coli...19, 40-41, 49, 77159-160, 240, 323, 388,390, 551,590 ELECSYS ........................................ 194 electrochemical detection ................. 450 electrochemiluminescence ....... 173-206

annihilation ECL... 180, 193, 199, 200 electron transfer...148, 149, 174-180, 186, 196, 339, 340, 344, 351, 356357, 465 electrostatic...23-24, 262, 335, 339, 371, 402, 403, 407, 408, 549, 555, 560 ellipsometry ..................... 401,467,487 ELONA ............................................ 387 encapsulation...9, 86, 428-430, 436453, 503,510, 516, 549,560, 565, endoscopes ................................... 41, 49 energy transfer... 11, 26, 36, 43, 68, 148, 150, 158, 164-166, 169, 196, 218, 310, 311, 324, 339, 371, 378, 391,416, 538,559, 560, 563 F/Srster ................................. 149, 170 environmental applications...39, 40, 195, 199 enzyme-linked immunosorbent assay 109, 112-114, 192, 263, 271, 300, 387-389, 473 enzyme-linked oligonucleotide assay (ELONA) ............................. 387 enzymes...7, 13-21, 25, 29, 39, 78, 86, 117, 118, 188, 266, 371, 380, 393, 398-405, 409, 428, 429, 433, 438, 439, 440-443, 448, 451-453, 522, 531,586 acetylcholinesterase... 18, 40, 49, 7278, 117 alkaline phosphatase... 13, 21, 34, 436, 438, 440 carbonic anhydrase...37, 149-151, 154, 160-166, 241,391 chloramphenico! acetyltransferase .................... 313 galactosidase ........... 13, 19, 313,450 glucoseoxidasel7, 25, 31, 42, 195439, 522, 531 glutamate dehydrogenase... 449, 552 horseradish peroxidase ....... 431,437 lactamase ..................................... 188 oxidase... 17, 18, 431,437,439, 453, 522, 531 600

Index polarization ......................... 380, 441 fluorescent resonant energy transfer ...11, 36, 38, 42, 46,, 68, 307, 310311, 314-316, 320-326, 415, 416, 559, 563 fluoride ............................. 354, 356 fluoroimmunoassay ................ 57-142 fluorophores... 11, 35, 36, 57-73, 77, 81, 87, 96-98, 106, 114, 117, 126-130, 144-155, 165-166, 307-326, 332342, 347, 353-354, 360-361, 370371, 374-380, 388, 390, 440, 509, 523, 539, 557,562-565, 580 ABD ..................... 149-151,154, 166 acrylodan ..................... 160, 161, 318 Alexa Fluor ......................... 167, 168 coumarin ............................. 101, 533 CPM ............................................ 319 cyanine...69, 99-102, 111-112, 126, 198, dansylamide ......................... 162-164 fluorescein...17, 29, 35, 43, 49, 69, 78-79, 99-102, 105, 117, 126, 164, 316, 319, 322, 360, 379,412, 415,416, 431,444, 447,500, 510, 549, 557,582, 587 lanthanide ions .................... 146, 419 MDCC ................................. 319, 320 MLCT probe ............................... 159 Oregon green... 166, 508, 522-523, 526, 530-532, POPOP ........................................ 146 pyrene ........... 27, 146, 357,360, 411 rhodamine...69, 101, 316, 322, 360, 437, ruthenium...17, 42, 117, 156, 158, 181, 185, 188, 196, 200, 358,360, 530, 562 Francisella tularensis .......... 83, 110-112 frequency synthesizer ...................... 154 galactosidase ................ 13, 19, 313, 450 green fluorescent protein (GFP) ...... 12, 49, 103 gelation .................................... 432-435, gene gun ................... 502, 507,508, 511

reductase ......................................431 serine proteases ............................ 378 superoxide dismutase ................... 438 estrogen ........................................74, 79 estrogen receptor ..........................74, 79 ethanol...348-350, 354, 412-416, 461466, 472, 483, 489, 511,517, 522 evanescent field . . .57-122, 215, 216, 243, 253, 254, 258, 267, 272, 278282, 287-289, 293, 296, 300-303, 388,593 exciplexes ......................................... 185 explosives...77, 124, 126-136, 193, 551,555 RDX...77, 83, 125, 129, 130, 134137,551,555,556 TNT...77, 83, 128-131, 134-136, 193, 431,437,445,447, 551,555 ferritin ............................... 474-476, 488 flow immunosensor .................. 123-142 fluidics...64-68, 82, 84, 87-89, 112, 116, 124, 132, 140, 286, 301, 571597 fluorescence .... 7-22, 28-43, 50, 59-89, 96-117, 126-128, 132, 139, 141, 144-169, 242, 288, 308-325, 332360, 371, 376-392, 411-418, 435, 437, 440-449, 500, 504-509, 516518, 523-533, 544-552, 557-564, 582, 589, 593 emissive state ............... 175, 179, 339 excited state...12, 14, 144, 147, 174186, 195-197, 338-340,.344, 354, 548,563 exponential decay...98, 1 4 5 , 152, 253,254, 258, ground state...ll, 12, 14, 147, 176, 178, 183, 338, intensity...ll, 32, 68, 80, 81, 106, 117,128, 139, 144-147, 159-160, 168, 308, 325, 356, 357, 371, 376-379, 448, 449, 505, 508, 518, 524, 526, 562, 582 lifetime ............ 11, 69, 144-169, 500 microscopy .......................... 164, 169 multiexponential decay ................ 145 601

Index genetic engineering...16, 19, 38, 71, 114, 140, 307-330 glass...5, 8, 9, 20-24, 70, 100-107, 112, 126-127, 195, 214-215, 219, 222, 228-229, 233, 253, 259, 281, 282, 295, 296, 299, 392, 428, 430, 453, 467, 511, 516, 517, 521, 541-543, 557, 560, 579, 584-585, 592, 593 glucose...17, 25, 31, 42, 45, 117, 158160, 185, 188, 195, 315, 323344, 347, 361, 362, 371, 390, 415, 480, 498,522, 531,532 glucoseoxidase...17, 25, 31, 42, 195439, 522, 531 glucose/galactose binding protein... ...................................... 159,390 glutamate dehydrogenase ......... 449, 552 glutamine binding protein ........ 315, 390 glycoproteins .................................... 261 ricin ................................ 83,265,374 gold film...209, 213-215, 236, 388, 399, 401,458-490, 579-588 graded index (GRIN) lens .................. 99 grarnicidin...458, 459, 466, 467, 470493 grating couplers ................ 217-231,279 green fluorescent protein (GFP)...12, 19, 49, 103, 311,313-316, 319-326, 391 hapten ............... 263, 272, 300, 479-482 Helicobacter pylori ..................... 80, 268 heine proteins ................................... 428 hemoglobin ........................... 43, 44, 442 hexylamine ....................................... 350 homogeneous assay .................... 36, 565 hormones... 109, 149, 192, 236, 391, 474 chorionic gonadotropin ................ 109 diethylstilbestrol ............................ 79 estradiol ......................................... 79 estriol ............................................. 79 estrogen ................................... 74, 79 serotonin ...................................... 103 thyroid stimulating hormone .......................... 473, 374,482

horseradish peroxidase ............. 431,437 host/guest chemistry ........................ 333 HPLC...124, 130, 131, 135, 137, 270, 412-413, 189, 200, 412, 413 human immunodeficiency virus (HIV) ............................. 185, 192, 379 hybrid bilayer membrane .............. 466 hydrogen bonding...104, 335,341,351, 353,354,402, 403,417,441,470, hydrolysis... 19-24, 188, 298, 313, 375, 419, 430-436, 468,522, 583 hydroquinone ................... 199, 351,352 ICP-MS ............................................ 161 immobilization...23-26, 47, 49, 58, 62, 70, 75, 87, 88, 96, 102-110, 114-116, 126, 127, 156, 191, 236, 245, 324, 341, 361, 363, 429, 444, 450, 452, 572 immunoassay...21, 60, 64, 73-76, 86, 101, 111, 113, 124, 134-141, 150, 158, 174, 187, 194, 340, 437, 538539 immunornagnetic separation ............ 191 impedance...458, 561, 466-468, 483486 in vitro selection ............... 372-375,386 indicator...7, 17, 23, 30, 31, 39, 42, 47, 49, 158-164, 169, 413, 435, 444, 453, 499-500, 506, 513, 516-519, 524, 529, 530, 580, 589 ink jet printing .................................. 108 inositol triphosphate ......................... 354 integrated optical waveguide (IOW) ..... 100, 104, 115, 116, 233,234 internal charge transfer (ICT)...33, 340, 465 internal reflection element ............ 100 inverse rnicelle ......................... 542, 544 ion channel...458-461, 466, 471, 473, 476, 484-487,493,507 alamethecin gramicidin...458, 459, 466, 467, 470-493 valinomycin .................................. 71

602

Index mass transport .......... 132, 199,256, 474 matrix effects...96, 113, 114, 118, 136, 138, 140, 325,479, 487, 489 membrane-based biosensor ....... 457-496 membranes...23, 28, 67, 96, 126, 127, 146, 315, 354, 410, 414, 458, 460, 463, 466, 467, 487, 489, 492, 493, 503,572-576, 579, 585,592 membrane-spanning lipid...463, 468, 487, 488 mercury .................... 317, 323,527, 549 metal ions Ca(II)...161264, 313-322, 391,498, 501,513, 516, 527 Cd(II)...149, 161, 166, 546, 557, 560 Co(II)...37, 149, 154, 161,165, 166, 315,322, Mg(II) ......................................... 161 Ni(II)...105, 149, 161, 165, 166, 322 Zn(II)...37, 149, 161,166, 267, 315, 316, 321,498,515, 516 methamphetamine .................... 238, 239 micelle .............................. 198,542-544 microarray ................ 109, 388,539, 564 microenvironment ..... 30, 439,443,453, microfabrication...194, 326, 322, 444, 572, 574, 592 microsphere .......... 33-35, 510, 560, 565 microstructures...435, 492572-578,592 microviscosity miniaturization...26, 46, 48, 76, 82, 96, 236, 245,274, 443,444, 491,516 m-nitrobenzoate ............................... 354 ModE repressor protein ................... 266 modulator acousto-optic device .................... 154 Pockels cell ................................. 154 molecular beacon...12, 23, 35, 36, 81, 86, 88, 110, 111,117, 376, 379 molecular device .............. 333,340, 342 molecular imprinted polymer (MIP) ...................................... 397-426 molecular machine ........................... 333

kinetics...69, 72, 102, 132, 140, 145, 178, 241, 242, 263-266, 269, 273, 361, 380, 414, 433, 438, 440, 451, 452, 463, 464, 492 Michaelis-Menten ................ 438,439 lactamase ........................................... ! 88 laminin .............................. 261,587,588 Langmuir-Blodgett films ............ 71,583 lead...5, 28, 48, 114, 117, 118, 197, 244, 273, 274, 376-380, 421, 442, 453,510, 565,583 lectin ................................................. 191 ligand fishing .................... 259, 270, 271 light detectors ....... .............................. 48 light sources...15, 46, 48, 64, 85, 154, 160, electroluminescent source ........... 154 halogen lamp ........................... 63, 82 lasers diode...48, 64, 76, 82, 89, 99, 152, 154, 166, 167, 420 titanium sapphire .................... 169 LED ....................................... 154, 87 xenon lamp .................................... 64 lipid vesicles ..................................... 466 lipopolysaccharide .................. 74, 77, 79 liposomes...259, 268, 269, 375, 459, 492, 503,505, 510 liquid crystal display ........................ 113 Listeria ...................................... 240, 482 low density lipoprotein (LDL) .......... 263 luciferase...13, 19, 39-41, 313, 323, 324 luminescent ..... 175,436, 538-540, 550 luminol ......................... 12, 40, 184, 195 luminophore... 174, 176, 181, 184, 188, 191,193, 196-198 Lutheran glycoproteins ..................... 261 macrophages ............. 498, 502-505, 510 magnetic beads ......................... 191, 194 maltose binding protein...38, 315, 371, 390, 551,555,560 mass spectrometry (MS)... 124, 130, 134, 239, 265,272 603

Index molecular probe ................ 340, 440, 499 molecular recognition...87, 96, 101, 104, 124, 138, 140, 335-338, 341342, 350, 354, 361, 370-376, 388, 390, 573 monolith ...........................................437 multianalyte...5, 10, 26, 29-36, 46-52, 76, 82, 83, 86, 89, 96, 109-112, 115, 197,234, 235,326, 452 multiphoton microscopy ................... 169 Na+/H§ exchanger regulatory factor .............................................260 NADH .......... 14, 18, 185, 188, 189, 449 nano bio-optrodes ......................... 26-28 nanocrystal ........ 546-553,557-560, 564 nanoparticles...510, 539, 542, 544, 551552, 555-558, 562, 564 nanosensor ........................................499 near field optics ................................444 near-field scanning optical microscopy .............................................. 199 nebulin ...............................................264 Neisseria meningitides ..................... 262 neural networks .................................. 52 nicotinamide ..................................... 188 nicotine .........................79, 83,352, 353 nitrate ................................................354 nitrite ........................................354, 506 nucleic acid...7, 12, 13, 16, 19, 22, 25, 36, 46, 51, 76, 79, 80, 86, 96, 101, 116, 186, 191, 192, 197, 259, 266, 370-381, 385, 393, 401-405, 414, 590 nucleic acid hybridization...22.29, 75, 79-81, 86, 87, 101, 102, 109, 196, 197, 288, 376, 381, 551, 558, 561, 562 nucleotide...29, 30, 80, 81, 102, 117, 266, 370, 372, 381, 384-388, 460, 480, 482, 552, 558,561-562 numerical aperture...9, 61, 65, 157, 508, organometallic ...24, 472, 543-546, 553 ORIGEN ................................... 187, 194 overcoating ............... 543-547, 554, 558

oxalate ...................... 181, 187,195, 199 oxidation...17, 70, 107, 149, 175-183, 198, 200, 298-299, 444, 462, 463, 531,565,575,592 oxidation potential ........................... 149 oxygen...13, 17, 19, 42, 117, 158, 184, 193, 199, 200, 308, 442, 444, 445, 461-464, 508, 509, 515, 522-524, 527-531,583 oxygen sensors .......... 442-444, 527, 531 panning ............................................ 263 PEBBLE ................................... 497-536 acrylamide...500, 506, 508, 511512, 514-516, 526, 530-532 calcium ........................................ 505 decyl methacrylate...508, 516, 520, 529-531 oxygen ................................. 508,523 pH .............................................. 513 sol-gel ........... 508,522-526, 529-530 Peltier ....................................... 257,302 peptides...88, 141, 188, 195, 259, 263, 370, 388-391, 399, 402, 452, 493, 555 pesticides... 18, 19, 39, 40, 109, 130, 137, 141 pH .... 1, 12, 17, 18, 23, 26, 30-32, 42, 46, 51, 72, 75, 78, 104, 117, 141, 164, 184, 185, 266, 269, 298, 300, 325, 333-350, 354, 355, 360, 380, 381, 404-405, 422, 433-437, 440444, 451, 453, 491, 500, 505-520, 525,560, 564 phage display ............................... 49, 88 phenols...40, 199, 349, 354, 356, 400, 406, 416 phenylalaninol .................................. 349 phenylethylamine ............................. 349 phenylglycinol ................................. 349 phloretin ............................................. 71 phosphate...38, 103, 105, 309, 315, 319, 354, 359, 360, 375, 380, 390, 447,491,511,580 phosphorescence ....................... 146, 158 photoablation .................................... 106 604

Index prism coupling... 100, 214, 215, 218, 219, 222, 224, 227-231, 253, 254, 259, 284, 285,293,294, 298 propanal ........................................... 348 protein A .................................... 71, 114 protein G ................ 71, 79, 88, 114, 551 protein stability ................ 435,440, 442 proteinase K ..................................... 261 proteomics ............................... 273,298 protozoa ....................... 74, 76, 374, 431 Cryptosporidium ......................... 193 Giardia .................................... 77, 83 pyridine ............................ 266, 350, 418 pyrolysis ........................................... 544 pyrophosphate .................................. 360 Q-dot ......................................... 537-570 quantum dot ........................ 89, 537-570 quantum efficiency... 12, 69, 101, 146, 150, 179, 196-197, 326, 358, 544, 546, 547,551,563, 564, quaternary structure ................. 378, 381 quenchers...35, 36, 81, 117, 146-149, 339,353,374-380, 560 quenching...ll, 36, 42, 43, 68, 69, 81, 103, 117, 126, 146-154, 158, 165, 166, 193, 199, 200, 308, 340, 347, 354-360, 370, 371, 376-378, 417, 418, 437, 445-447, 500, 501, 523, 526, 557, 559, 560, 563 collisional ..... ............................... 147 dynamic ....................................... 147 quenching constant ..................... 146 static .................................... 147, 151 quenching mechanisms electron exchange... 148, 174-180, 186, 196 electron transfer 1...48, 149339, 340, 344, 351,356, 357,465 paramagnetic ............... 148, 19i, 451 proton transfer ............. 148,338, 356 spin-orbit coupling ...................... 148 unpaired electrons ....................... 148 quinones ........................................... 351 Rad51 protein ................................... 266

photobleaching...47, 61, 68, 69, 87, 435, 540, 563 photodiode .................... 64, 69, 195, 228 photoinduced electron transfer (PET) 339, 340, 344, 347, 351, 351, 354357 photolithography... 106, 108, 280, 292, 572-576, 583-584 photoluminescence 1...76-185, 195-198, 539, 547,551-554, 561-563 photomultiplier tube... 16, 28, 48, 64, 99, 155, 187, 191, 194, 195, 323, 527 photopolymerization ....... ........ 29, 31, 32 physiological markers acetylcholine...18, 40, 49, 50, 70, 74, 79, 116.117,269, 459 cardiac troponin I ......................... 111 D-dimer... 110, 111, 431, 437, 447, 448 myoglobin ........................ 42-44, 111 thrombin ..... 116, 378,388, 392, 444 pKa ................... 164, 337,344, 346, 443 polarization...116, 144, 222, 223, 227, 235,295,296, 338, 356, 440 pollutants .......................... 199, 323, 324 poly(methylmethacrylate) (PMMA)... ................................ 62, 111,195 polydimethyl siloxane...108, 111, 575579, 584-589, 592 polyethylene glycol...70, 107, 299, 375, 516, 517, 522, 524 polymerase chain reaction (PCR) .... 22, 79, 80, 192, 196, 203, 206, 312, 372-376, 386 polymyxin B ................................. 74, 79 polystyrene...62, 67, 70, 88, 104, 108, 191,300, 401,510 pore-matrix interface ........................ 441 porosity...429,432, 433,451,452, 521, potassium...281, 335, 342, 350, 506510, 516-520, 530 preorganization ......................... 335,336

605

Index ratiometric imaging .................. 499, 500 reagentless biosensors...307, 370, 371, 376, 379, 385,388, 392 bio-optrode ...................... 5, 26, 36 real-time measurements...48, 86, 114, 128, 138, 140, 161, 167, 168, 241, 242, 246, 253, 260, 272, 371, 376, 380, 498,504, 527 receptors...17, 19, 69, 72, 74, 78, 88, 103, 116, 245, 246, 260, 268, 303, 333, 334, 343-347, 351-354, 360363, 390-393, 398, 402-405, 422, 460, 480, 555,563-565 acetylcholine receptor...70, 74-75, 79, 116, 269 estrogen receptor ..................... 74, 79 ganglioside ................... 103, 105,270 serotonin receptor ........................ 103 reduction...18, 79, 147, 148, 175-184, 200, 262, 391, 439, 449, 461-464, 468,493,523,576, 588 reference dye .... 499, 508, 513, 529, 530 refractive index...9, 57-62, 96-100, 113, 207-224, 228-237, 242-244, 253-257, 271-273, 278-289, 295, 298, 301,302, 436 regeneration of surfaces... 18, 75, 79, 87, 102, 116, 125, 262, 272, 274, 296 resonant angle ........................... 254, 255 resonant mirror ................. 242, 253-276 response time...24-27, 43, 44, 47, 80, 270, 272, 324, 325, 348, 433, 435, 443,476, 482, 499, 509, 527, 529 reverse transcriptase polymerase chain reaction ................................. 312 reverse transcription ................. 372, 374 ribozymes ................. 370, 375,379-385 allosteric .............................. 381,383 hammerhead ........................ 380-386 RM cuvette...159, 227, 254-259, 262264, 267, 271,532 rotational mobility ............ 438, 441-442 rubrene .............................................. 185 Salmonella . . . . . . . . . . 88, 152, 240, 300, 301

scanning electrochemical microscopy ..................................... 199,492 selectivity... 19, 37, 51, 124, 161, 169, 174, 188, 253, 260, 314, 322-326, 332, 335, 343-351, 356-361, 378, 414, 419, 506, 512, 513, 516, 519, 520, 525,531,560 semiconductor...48, 155,232, 280, 282, 295, 298, 538-543, 546, 548, 552553,559, 562, 565 serine protases .................................. 378 sialic acid ......................... 268, 414, 415 signaling aptamers...370, 376-381,389392 sodium...102, 193, 281,335, 342, 350, 444, 490, 491, 506, 511, 517, 519, 520, 542 sol-gel...24, 40, 43, 69, 200, 282, 286, 302, 427-456, 498, 502, 508, 511, 521-530, sol-gel dopants cx-naphtholphthalein .................... 443 Ru(II) 4, 7-diphenyl 1, 10phenanthroline ..................... 437 seminaphthorhodamine- 1 carboxylate ..................... 437, 443 solubility165, 179, 361, 523-524, 529, 542,555 solvatochromism .............................. 338 stability...16, 24, 46, 48, 72, 87, 88, 101-102, 113, 176, 180, 196, 225, 261, 264, 266, 269, 273, 287, 288, 291,295, 341,345, 354, 375,, 401, 405, 407, 438-443, 451, 453, 459, 460, 463,489, 490, 530, 565 Sterne-Volmer equation strokes ...................................... 443,447 succinimidyl esters .... 186, 258, 532533 sugar tweezer ................................... 345 sulfonamide ............................. 164, 165 dansylamide ......................... 162-164 superoxide dismutase ....................... 438 supramolecular chemistry ........ 335, 341

606

Index bungarotoxin ..................... 74, 79, 83 cholera toxin...83, 110-112, 268-270 Naja naja toxin ....................... 79, 83 neurotoxins ........... 40, 74, 78, 79, 83 ricin ............... 83, 111, 112, 265, 374 SEA ............................................. 271 SEB...83, 88, 110, 111, 238, 239, 551,555 transcription...312-314, 318, 325, 372, 374, 379,402 transition metals ............... 335, 341,342 transition state analogs ..................... 375 triethylphosphite .............................. 350 tri-n-propylamine (TPrA) ....... 182-185, 189, 191,194, 197-199 tripeptide .................................. 353, 362 twisted internal charge transfer...339, 355 urea .... 75, 102, 116, 299, 300, 353-356 urine...42, 74, 77-78, 111, 130, 131, 187, 195, 261,362, 461 vaccines ........................................... 270 valinomycin ................................. 74, 79 vascular endothelial growth factor (VEGF) ................................ 387 vibro-stirring ............................ 256, 257 viruses .............................................. 270 virus-like particles (VLP) ................ 270 V-number .......................... 59-62, 66, 87 whole-cell sensing...313-314, 317, 325, 326 xerogel ............................................. 433 yellow fluorescent protein (YFP)...315, 316, 319, 321,322, 325 Yersinia pestis .................... 77, 110, 111 zinc finger ........................ 316, 322, 391

surface plasmon resonance (SPR) .... 68, 96, 113-116, 207-252, 253,254, 297, 298,401,436, 576, 592, 593 surfactants...198, 199, 269, 453, 511, 542 synthesis...326, 337, 341, 343, 362, 372, 374, 275, 400-403, 421, 430, 432, 435, 450, 467, 526, 538-546, 552,553,588 Tamm-Horsfall protein (THP) .......... 261 T-dependent immunogen .................. 262 tethered lipid ..... 458,460, 463,473, 484 tethered lipid bilayer ......................... 484 theophyUine...36, 109, 382, 384, 389, 483 thyroid stimulating hormone (TSH) ....................... ...... 473,474, 482 time-correlated single photon counting .............................................. 152 time-resolved fluorescence lifetime phase...ll, 37, 101, 131, 138, 140, 148-168, 183, 191, 196, 219-223, 226, 227, 235, 236, 241, 244, 255, 278, 282-98, 302, 350, 353, 361, 372, 401, 417, 463, 483-486, 499, 523, 524, 527, 542, 559-560, 563, 577, 588, 589 phase fluorometers .......... 152, 155 frequency ................ 151,152, 157 TCSPC .................................... 152 titin ................................................... 264 TM modes ................................ 273, 295 TMPD ....................................... 179, 180 toluene .............................. 317, 353, 408 total internal reflection (TIR)...8, 9, 96117,215,254, 280, 388 toxins...79, 83, 127,265,269, 274, 317, 324, 452, 493, 498, 505, 551 botulinum toxin ................... 110, 112

607