Nanobiosensors 1588830624

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Encyclopedia of Nanoscience and Nanotechnology

www.aspbs.com/enn

Nanobiosensors Tuan Vo-Dinh Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

CONTENTS 1. Introduction 2. Near-Field Optics and Nanofibers 3. Biosensors 4. Nanosensor Fabrication 5. Experimental Protocol and Instrumental System 6. Applications 7. Conclusion Glossary References

1. INTRODUCTION Biology has entered a new era with the recent advances in nanotechnology, which have recently led to the development of biosensor devices having nanoscale dimensions that are capable of probing the inner space of single living cells. Nanosensors provide new and powerful tools for monitoring in vivo processes within living cells, leading to new information on the inner workings of the entire cell. Such a systems biology approach could greatly improve our understanding of cellular function, thereby revolutionizing cell biology. Fiber-optic sensors provide useful tools for remote in-situ monitoring. Fiber-optic sensors can be fabricated to have extremely small sizes, which makes them suitable for sensing intracellular/intercellular physiological and biological parameters in microenvironments. A wide variety of fiber-optic chemical sensors and biosensors have been developed in our laboratory for environmental and biochemical monitoring [1–8]. Submicron fibers have been developed for use in near-field optics [9, 10]. Tapered fibers with submicron tip diameters have also been developed for near-field scanning optical microscopy (NSOM). NSOM was used to achieve subwavelength 100 nm spatial resolution in Raman detection [11–13]. Tan and co-workers developed and used chemical nanosensors to perform measurements of calcium and nitric oxide, among other physicochemicals in single cells [14, 15]. Vo-Dinh and co-workers

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developed nanobiosensors with antibody probes to detect biochemical targets inside living single cells [16–23]. This chapter presents an overview of the principle, development, and applications of fiber-optic nanosensors.

2. NEAR-FIELD OPTICS AND NANOFIBERS Nanoscale optical fibers were first developed for use in nearfield optical microscopy, which is a relatively recent technique involving light sources or detectors that are smaller than the wavelength of light [9]. The first method developed for performing these experiments was to place a pinhole in front of the detector, thus effectively reducing the detector size. In a later variation to these pinholes, an excitation probe with dimensions smaller than the wavelength of the light was used for sample interrogation. Betzig and Chichester reported the development of one such probe capable of obtaining measurements with a spatial resolution of approximately 12 nm [10]. The probe was constructed by using a micropipet puller to pull a single-mode optical fiber to a tip diameter of 20 nm, and then coating the walls of the fiber with 100 nm of aluminum to confine the excitation radiation to the tip. With this nanoprobe, images of a pattern were reconstructed from a raster scan performed in the illumination mode, with the probe acting as a localized light source. Due to its extremely high spatial resolution (subwavelength), near-field microscopy has received great interest, and has been used in many applications [9]. For example, a relatively new technique known as near-field surfaceenhanced Raman spectroscopy (NF–SERS) has been used for the measurement of single-dye and dye-labeled DNA molecules with a resolution of 100 nm [11–13]. In this work, DNA strands labeled with the dye brilliant cresyl blue (BCB) were spotted onto a SERS-active substrate that was prepared by the evaporation of silver on a nanoparticle-coated substrate. The silver-coated nanostructured substrates are capable of inducing the SERS effect, which can enhance the Raman signal of the adsorbate molecules up to 108 times [24]. NF–SERS spectra were collected by illuminating the sample using the nanoprobe, and detecting the SERS signals

Encyclopedia of Nanoscience and Nanotechnology Edited by H. S. Nalwa Volume 6: Pages (53–60)

54 using a spectrometer equipped with a charge-coupled device (CCD). Raster scanning the fiber probe over the sample and normalizing for surface topography using Rayleigh-scattered light produced a two-dimensional SERS image of the DNA on the surface of the substrate with subwavelength spatial resolution. Near-field optical microscopy promises to be an area of growing research that could potentially provide an imaging tool for monitoring individual cells, and even biological molecules. Single-molecule detection and imaging schemes using nanofibers could open new possibilities in the investigation of the complex biochemical reactions and pathways in biological and cellular systems.

Nanobiosensors

Target Analyte Molecule

Antibody Probe

Molecular Binding At Nanoprobe

3. BIOSENSORS 3.1. Biosensor Principle A biosensor is generally defined as a measurement system that consists of a probe with a biological recognition element, often called a bioreceptor, and a transducer [3, 18, 22]. Two fundamental operating principles of a biosensor are: (1) “biological recognition,” and (2) “sensing.” Therefore, a biosensor can be generally defined as a device that consists of two basic components connected in series: (1) a biological recognition system, often called a bioreceptor, and (2) a transducer. The basic principle of a biosensor is to detect this molecular recognition, and to transform it into another type of signal using a transducer. The main purpose of the recognition system is to provide the sensor with a high degree of selectivity for the analyte to be measured. The interaction of the analyte with the bioreceptor is designed to produce an effect measured by the transducer, which converts the information into a measurable effect, such as an electrical signal. Biosensors can be classified based on the transduction methods they employ. Transduction can be accomplished through a large variety of methods. Most forms of transduction can be categorized in one of three main classes: (1) optical detection methods, (2) electrochemical detection methods, and (3) mass-based detection methods. Other detection methods include voltaic and magnetic methods. New types of transducers are constantly being developed for use in biosensors. Each of these three main classes contains many different subclasses, creating a large number of possible transduction methods or combinations of methods. Special emphasis will be placed on the description of optical transducing principles, which is the focus of this chapter. Figure 1 illustrates the conceptual principle of the biosensing process using an antibody as the bioreceptor probe and fluorescence as the detection method.

3.2. Bioreceptors Bioreceptors are the key to specificity for biosensor technologies. They are responsible for binding the analyte of interest to the sensor for the measurement. These bioreceptors can take many forms, and the different bioreceptors that have been used are as numerous as the different analytes that have been monitored using biosensors. However, bioreceptors can generally be classified into five different major categories: (1) antibody/antigen, (2) enzymes, (3) nucleic

Laser-Excitation And Fluorescence

Optical Detection

Information Recording

Figure 1. Operating principle of biosensor systems.

acids/DNA, (4) cellular structures/cells, and (5) biomimetic. This chapter deals with biosensor systems using antibody probes, often called immunosensors, with optical detection. The antigen–antibody (Ag–Ab) binding reaction, which is a key mechanism by which the immune system detects and eliminates foreign matter, provides the basis for specificity of immunoassays. Antibodies are complex biomolecules, made up of hundreds of individual amino acids arranged in a highly ordered sequence. Antibodies are produced by immune system cells when such cells are exposed to substances or molecules, which are called antigens. The antibodies appearing following antigen exposure have recognition/binding sites for specific molecular structures (or substructures) of the antigen. The way in which an antigen and an antigen-specific antibody interact is analogous to a lock and key fit, in which specific configurations of a unique key enable it to open a lock. In the same way, an antigen-specific antibody fits its unique antigen in a highly specific manner, so that the three-dimensional structures of antigen and antibody molecules are complementary. Due to this threedimensional shape fitting, and the diversity inherent in individual antibody make up, it is possible to find an antibody that can recognize and bind to any one of a large variety of molecular shapes. This unique property of antibodies is the key to their usefulness in immunosensors; this ability to recognize molecular structures allows one to develop antibodies that bind specifically to chemicals, biomolecules, microorganism components, and so on. One can then use such antibodies as specific probes to recognize and bind to an analyte of interest that is present, even in extremely small amounts, within a large number of other chemical substances.

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In the 1980s, advances in spectrochemical instrumentation, laser miniaturization, biotechnology, and fiber-optics research provided opportunities for novel approaches to the development of sensors for the detection of chemicals and biological materials of environmental and biomedical interest. Since the first development of a remote fiber-optics immunosensor for in-situ detection of the chemical carcinogen benzo[a]pyrene [1], antibodies have become common bioreceptors used in biosensors today.

4. NANOSENSOR FABRICATION 4.1. Fabrication of Nanofiber Probes This section discusses the protocols and instrumental systems involved in the fabrication of fiber-optic nanoprobes. Two methods are generally used for preparing the nanofiber tips. The so-called “heat-and-pull” method is the most commonly used. This method consists of local heating of a glass fiber using a laser or a filament, and subsequently pulling the fiber apart. The shape of the nanofiber tips obtained depends on controllable experimental parameters such as the temperature and the timing of the procedure. The second method, often referred to as “Turner’s method,” involves the chemical etching of glass fibers. In a variation of the standard etching scheme, the taper is formed inside the polymer cladding of the glass fibers. The description of these fabrication methods is given in the following section. The fabrication of nanosensors requires techniques capable of making reproducible optical fibers with a submicron-size diameter core. Figure 2 illustrates the experimental procedures for the fabrication of nanofibers using the heat-and-pull procedure [21]. Since these nanoprobes are not commercially available, investigators have to fabricate them in their own laboratories. Our laboratory uses the heatand-pull procedure, which consists of pulling a larger silica optical fiber to produce the tapered nanotip fiber using a special fiber-pulling device (Sutter Instruments P-2000). This method yields fibers with submicron diameters. One end of a 600
m silica/silica fiber is polished to a 0.3
m finish with an Ultratec fiber polisher. The other end of the optical fiber is then pulled to a submicron length using a fiber puller. A scanning electron microscopy (SEM) photograph of one of the

fiber probes fabricated for studies is shown in Figure 3. The distal end of the nanofiber is approximately 30 nm. To prevent light leakage of the excitation light on the tapered side of the fiber, the sidewall of the tapered end is then coated with a thin layer of metal, such as silver, aluminum, or gold (100 nm thickness) using a thermal evaporation metal coating device. The coating procedure is schematically illustrated in Figure 4 [21]. The metal coating is only for the sidewall, and leaves the distal end of the fiber free for subsequent binding with bioreceptors. The fiber probe is attached on a rotating plate inside a thermal evaporation chamber [3, 19, 21]. The fiber axis and the evaporation direction formed an angle of approximately 45
. While the probe is rotated, the metal is allowed to evaporate onto the tapered side of the fiber tip to form a thin coating. The tapered end is coated with 300–400 nm of silver in a Cooke Vacuum Evaporator system using a thermal source at 10−6 torr. Since the fiber tip is pointed away from the metal source, it remains free from any metal coating. With the metal coating, the size of the probe tip is approximately 250–300 nm (Fig. 5). Chemical etching using HF is the basis of the second method for fabricating optical nanofibers. There are two variations of the HF etching method: one method involving the use of a mixture of HF acid and organic solvent, known as Turner etching [25], and the second using only HF, known as tube etching [26–28]. In the Turner method, a fiber is placed in the meniscus between the HF and the organic overlayer, and over time, a small tip is formed, with a smooth, large-angled taper. This large taper angle provides much more light at the tip of the fiber, which in turn greatly increases the sensitivity of the nanosensors. The reproducibility of the Turner method is strongly affected by environmental parameters such as temperature and vibration because of the dual chemical nature of the etching process. To avoid this problem, a variation of the etching method was developed, which involves a tube-etching procedure. In this procedure, an optical fiber with a silica core and an organic cladding material is paced in an HF solution. The HF slowly dissolves the silica core, producing a fiber with a large taper angle and nanometer-sized tip. The HF begins first to dissolve the fiber’s silica core, while not affecting

Laser Beam

Laser

Optical Fiber (A) Pulling

Clamps

Laser Heating

Nanofiber Tip Diameter ~ 40 nm

Pulling

Tapered Fiber (B) Pulling

Pulling

100 nm

(C)

Nanofiber Tip

Figure 2. The “heat-and-pull” method for the fabrication of nanofibers.

Figure 3. Scanning electron photograph of an uncoated nanofiber. The size of the fiber tip diameter is approximately 40 nm.

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Nanobiosensors (A)

Quartz tip

(C) Tapered end

Metal coating

Quartz tip

(B) Turning plate

Thermal evaporation

Metal source

Motor

Heater

Figure 4. Procedure for coating a fiber with silver. (A) Uncoated fiber tip. (B) Fiber coating using metal evaporation over the rotating tip. (C) Coated fiber. Adapted from [21], T. Vo-Dinh et al., Nature Biotechnol. 18, 76 (2000). © 2000, Nature Publishing Group.

the organic cladding material. This unaffected cladding creates localized convective currents in the HF solution, which causes a tip to be formed. After some time, more of the silica core is dissolved, until it emerges above the surface of the HF solution. At this juncture, the HF is drawn up the cladding walls via capillary action, and runs down the silica core to produce a nanometer-sized tip. By varying the time of HF exposure and the depth to which the fiber is submerged in the HF solution, one can control the size of the fiber tip and the angle of the taper. Once the tip has been formed, the protruding cladding can be removed either with a suitable organic solvent or by simply burning it off. Nanotips fabricated using etching procedures, which can be designed to have sharp tips [13], have been used in NSOM studies to detect SERS-labeled DNA molecules on solid substrates at the subwavelength spatial resolution [11–13].

4.2. Antibody Immobilization The next step in the preparation of the nanobiosensor probes involves the covalent immobilization of receptors onto the nanofiber tip. For antibody binding, several strategies can be used to retain the antibody at the sensing probe.

Silver-coated Tip 250 nm

20 kV

X 50,000

0.5 µm

155330

Figure 5. Scanning electron photograph of a nanofiber having the sidewall coated with silver.

Whatever procedure is involved, one requirement is that the antibody should retain its antigen-binding activity as much as possible. Perhaps the easiest procedure involves enclosure of the antibody in solution, within a semipermeable membrane cap, which fits over the end of the sensor [2]. However, this design is more complicated, and would increase the size of the tip of the nanosensor. Antibodies can be immobilized onto the nanofiber probes by using a chemical immobilization method. The fiber is derivatized in 10% GOPS in H2 O (v/v) at 90
C for 3 h. The pH of the mixture is maintained below 3 with concentrated HCl (1 M). After derivatization, the fiber is washed in ethanol, and dried overnight in a vacuum oven at 105
C. The fiber is then coated with silver as described previously. The derivatized fiber is activated in a solution of 100 mg/mL 1,1′ carbonyldiimidazole (CDI) in acetonitrile for 20 min, followed by rinsing with acetonitrile and then phosphatebuffered saline (PBS). The fiber tip is then incubated in a 1.2 mg/mL antibody solution (PBS solvent) for four days at 4
C, and then stored overnight in PBS to hydrolyze any unreacted sites. The fibers are then stored at 4
C, with the antibody-immobilized tips stored in PBS. This procedure has been shown to maintain over 95% antibody activity [21].

5. EXPERIMENTAL PROTOCOL AND INSTRUMENTAL SYSTEM 5.1. Experimental Procedure An application that is truly unique to nanosensors involves monitoring living single cells in vivo [18–23]. Examples of this application in molecular biology will be discussed later. This section provides a description of the procedures for growing cell cultures for analysis using the nanosensors. Cell cultures were grown in a water-jacketed cell culture incubator at 37
C in an atmosphere of 5% CO2 in air. Clone 9 cells, a rat liver epithelial cell line, were grown in Ham’s F-12 medium (Gibco), supplemented with 10% fetal bovine serum and an additional 1 mM glutamine (Gibco). In preparation for an experiment, 1 × 105 cells in 5 mL of medium were seeded into standard dishes (Corning Costar Corporation). The growth of the cells was monitored daily by microscopic observation, and when the cells reached a state of confluence of 50–60%, the analyte solution was added and left in contact with the cells for 18 h (i.e., overnight). This procedure is designed to incubate the cells with the analyte molecules for subsequent monitoring using the nanosensors. The growth conditions were chosen so that the cells would be in log-phase growth during the chemical treatment, but would not be so close to confluence that a confluent monolayer would form by the termination of the chemical exposure. The analyte solution was prepared as a 1 mM stock solution in reagent grade methanol, and further diluted in reagent grade ethanol (95%) prior to addition to the cells. Following chemical treatment, the medium containing the analyte was aspirated and replaced with standard growth medium, prior to the nanoprobe procedure. Monitoring target analyte molecules in single cells was then performed using antibody nanoprobes in the following way. A culture dish of cells was placed on the prewarmed microscope stage, and the nanoprobe, mounted on the

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micropipette holder, was moved into position (i.e., in the same plane of the cells), using bright field microscopic illumination, so that the tip was outside the cell to be probed. The total magnification was usually 400×. Under no room light and no microscope illumination, the laser shutter was opened to illuminate the optical fiber for excitation of the analyte molecules bound on the antibodies at the fiber tip. Usually, if the silver coating on the nanoprobe was appropriate, no light leaked out of the sidewall of the tapered fiber. Only a faint glow of laser excitation at the tip could be observed on the nanoprobe. A reading was first taken with the nanoprobe outside the cell and the laser shutter closed. The nanoprobe was then moved into the cell, inside the cell membrane, and extending into the cellular compartments of interest. The laser was again opened, and readings were then taken and recorded as a function of time during which the nanoprobe was inside the cell.

6. APPLICATIONS 6.1. Nanofiber Probes for Intracellular Fluorescence Measurements

5.2. Instrumentation The optical measurement system used for monitoring single cells using the nanosensors is schematically illustrated in Figure 6 [18–23]. Laser excitation light, either the 325 nm line of an HeCd laser (Omnichrome, 8 mW laser power) or the 488 nm line of an argon ion laser (Coherent, 10 mW), was focused onto a 600
m delivery fiber, which was connected to the nanofiber through an SMA connector. The nanofiber was secured to a micromanipulator on the microscope. The experimental setup used to probe single cells was adapted for this purpose from a standard micromanipulation/microinjection apparatus. A Nikon Diaphot 300 inverted microscope (Nikon, Inc.) with a Diaphot 300/Diaphot 200 Incubator, to maintain the cell cultures at ∼37
C on the microscope stage, was used for these experiments. The micromanipulation equipment used consisted of MN-2 (Narishige Company, Ltd.) Narishige three-dimensional manipulators for coarse adjustment, and Narishige MMW-23 three-dimensional hydraulic micromanipulators for final movements. The optical-fiber nanoprobe was mounted on a micropipette holder (World Precision Instruments, Inc.). The fluorescence emitted from the cells was collected by the microscope objective, and passed through an appropriate long-pass dichroic mirror to Optics

Laser

eliminate the laser excitation scatter light. The fluorescence beam was then focused onto a photomultiplier tube (PMT) for detection. The output from the PMT was passed through a picoammeter, and recorded on a strip-chart recorder or a personal computer (PC) for further data treatment. To record the fluorescence of analyte molecules binding to antibodies at the fiber tip, a Hamamatsu PMT detector assembly (HC125-2) was mounted in the front port of the Diaphot 300 microscope, and fluorescence was collected via this optical path (80% of available light at the focal plane can be collected through the front port). A charge-coupled device mounted onto another port of the microscope could be used to record images of the nanosensor monitoring single cells.

Imaging Port

Optical nanofiber probes without antibody probes have been fabricated and used to monitor fluorescence emission from chemical species inside living single cells [17]. In this study, mouse epithelial cells were incubated with a fluorescent dye by incubating the cells in the dye solution and allowing membrane permeabilization to take place. Another procedure for loading cells with fluorophors involved the method called “scrape loading.” In the scrape loading procedure, a portion of the cell monolayer was removed by mechanical means, and cells along the boundary of this “scrape” were transiently permeabilized, allowing the dye to enter these cells. The dye was subsequently washed away, and only permeabilized cells retained the dye molecules, as they were not internalized by cells with intact membranes. Following incubation, the fluorescence signal of fluorescent dye molecules in single cells was detected using the optical nanofibers. A photograph of a nanosensor used to monitor clone 9 single cells is shown in Figure 7. Micromanipulators were used to move the optical fiber into contact with the cell membrane of a cell to be monitored. The fiber tip was then gently inserted just inside the cell membrane for fluorescence measurements. An argon–ion laser beam was transmitted through the optical nanofiber, and used for excitation. The dye molecules inside the cell were excited,

Mirror

Coupling Fiber Micromanipulator

2-D Display System Nanosensor

Single Cell Inside Holder

Inverted Microscope PMT Dtector Optical Filter

Figure 6. Instrumental system for fluorescence measurements of single cells using nanosensors.

Figure 7. Photograph of single-cell sensing using a fiber-optic nanosensor.

58 and their fluorescence emission was collected and detected using the microscope instrumental system described previously. Background measurements were performed with cells that were not loaded with the fluorophores. Fluorescence signals were successfully detected inside the fluorophoreloaded cells, and not inside nonloaded cells. As another control, the optical-fiber probe was then moved to an area of the specimen where there were no cells, and the laser light was again passed down the fiber for excitation. No visible fluorescence (at the emission wavelength) was detected for this control measurement, thus demonstrating the successful detection of the fluorescent dye molecules inside single cells. These results demonstrated the capability of optical nanofibers for measurements of fluorophores in intracellular environments of single living cells.

6.2. Nanobiosensors for Monitoring Subcompartments of Single Cells Nanosensors having antibody-based probes for use in measuring fluorescent targets inside a single cell have been demonstrated [18–23]. Since cells have very small sizes (1– 10
m), the success of intracellular investigations depends on several factors, including the sensitivity of the measurement system, the selectivity of the probe, and the small size of the nanofiber probes. The smallest cells to be nondestructively probed with a fiber-optic nanobiosensor were reported by Vo-Dinh and co-workers [21]. In that work, the antibody probe was targeted against benzopyrene tetrol (BPT), an important biological compound, which was used as a biomarker of human exposure to the carcinogen benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon of great environmental and toxicological interest because of its mutagenic/carcinogenic properties and its ubiquitous presence in the environment. Benzo[a]pyrene has been identified as a chemical carcinogen in laboratory animal studies [29]. The small size of the probe allowed manipulation of the nanosensor at specific locations within the cells. The cells were first incubated with BPT prior to measurements using the experimental procedures described previously. Interrogation of single cells for the presence of BPT was then carried out using antibody nanoprobes for excitation and a photometric system for fluorescence signal detection. Nanobiosensors for BPT were used for the measurement of intracellular concentrations of BPT in the cytoplasm of two different cell lines: (1) human mammary carcinoma cells, and (2) rat liver epithelial cells, following treatment of the culturing media with an excess of BPT. Figure 8 shows a digital image of the nanosensor actually being inserted into a single human mammary carcinoma cell. The measurements were performed on rat liver epithelial cells (Clone 9) used as the model cell system. The cells had been previously incubated with BPT molecules prior to measurements. The results demonstrated the possibility of in-situ measurements of BPT inside a single cell. In this study, the nanosensors employed single-use bioprobes because the probes were used to obtain only one measurement at a specific time, and could not be reused due to the strong association constant of the antibody–antigen binding process. The antibody probes, however, could be regenerated using ultrasound methods. Our laboratory has

Nanobiosensors

Figure 8. Digital image of a nanosensor inserted into a single human mammary carcinoma cell (the small size of the probe allowed manipulation of the nanoprobe at specific locations within a single cell).

successfully developed a method using ultrasound to noninvasively release antigen molecules from the antibodies, and therefore to regenerate antibody-based biosensors [30]. The results of the measurements with antibody against breast cancer antigen illustrate the effectiveness and potential of the regenerable immunosensor. A 65% removal of the antigens bound to the monoclonal antibodies immobilized on the fiber surface is attained after ultrasound regeneration. The ultrasound regeneration scheme is a nondestructive approach that has a great potential to be applied to nanosensors. The results demonstrate the effectiveness of this innovative ultrasound-based approach for biosensor regeneration, that is, releasing the antigen from the antibody probe. Multiple (e.g., five) recordings of the fluorescence signals could be taken with each measurement using a specific nanoprobe. We have made a series of calibration measurements of solutions containing different BPT concentrations in order to obtain a quantitative estimation of the amount of BPT molecules detected. For these calibration measurements, the fibers were placed in petri dishes containing solutions of BPT with concentrations ranging from 156 × 10−10 to 156 × 10−8 M. By plotting the increase in fluorescence from one concentration to the next versus the concentration of BPT, and fitting these data with an exponential function in order to simulate a saturated condition, a concentration of 96 ± 02 × 1011 M was determined for BPT in the individual cell investigated [23]. Detection of BaP transport inside single cells is of great biomedical interest since it can serve as a means for monitoring BaP exposure, which can lead to DNA damage [29]. In another study, nanosensors were developed for in-situ measurements of the carcinogen BaP [31]. In order to perform these measurements, it was necessary to use antibodies targeted to BaP. The fluorescent BaP molecules were bound by interaction with the immobilized antibody receptor, forming a receptor–ligand complex. Following laser excitation of this complex, a fluorescence response from BaP provided a basis for the quantification of BaP concentration in the cell being monitored. The fluorescence signal generated allows for a high sensitivity of detection. The intracellular measurements of BaP depend on the reaction times involved. The reaction time established in this study for antibody– BaP complexing was 5 min. This was used as a standard

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time to enable calibration from fiber to fiber. Additionally, the nanosensors were calibrated using standard analytical procedures using measurements of known concentration of reference solutions.

7. CONCLUSION Dynamic information of signaling processes inside living cells is important to a fundamental biological understanding of cellular processes. Many traditional microscopy techniques involve incubation of cells with fluorescent dyes or nanoparticles, and examining the interaction of these dyes with compounds of interest. However, when a dye or nanoparticle is incubated into a cell, it is transported to certain intracellular sites that may or may not be where it is most likely to stay, and not to areas where the investigator would like to monitor. The fluorescence signals which are supposed to reflect the interaction of the dyes with chemicals of interest is generally directly related to the dye concentration as opposed to the analyte concentration. Only with optical nanosensors can excitation light be delivered to specific locations inside cells. An important feature of nanosensors is the minimal invasiveness of the monitoring process. A cell survival study was previously performed whereby an investigation was made to determine whether penetration of the cell by the nanosensor resulted in intracellular or membrane damage of such a nature as to compromise cellular viability. It was determined that the process of mitosis continued normally, and that nanosensor insertion and withdrawal did not affect the life cycle of the cell. Nanosensors are an important technology that can be used to measure biotargets in a living cell, and that does not significantly affect cell viability. Combined with the exquisite molecular recognition of antibody probes, nanosensors could serve as powerful tools capable of exploring biomolecular processes in subcompartments of living cells. They have a great potential to provide the necessary tools to investigate multiprotein molecular machines of complex living systems, and the complex network that controls the assembly and operation of these machines in a living cell. Future developments would lead to the development of nanosensors equipped with nanotool sets that enable tracking, assembly, and disassembly of multiprotein molecular machines and their individual components. These nanosensors would have multifunctional probes (antibody as well as DNA probes) that could measure the structure of biological components in single cells. Until now, scientists have been limited to investigating the workings of individual genes and proteins by breaking the cell apart and studying its individual components in vitro. The advent of nanosensors will hopefully permit research on entire networks of genes and proteins in an entire living cell in vivo.

GLOSSARY Bioreceptor A biological recognition element, such as antibody, DNA, enzyme, etc., used to bind selectively to a target analyte compound. Biosensor A measurement system that consists of a probe with a bioreceptor, and a transducer.

Nanobiosensor Biosensor having nanoscale-dimension probe. Near-field scanning optical microscopy (NSOM) Microscopy technique using scanning probe providing subwavelength spatial resolution. Surface-enhanced Raman scattering (SERS) Enhanced Raman scattering of compounds adsorbed on nanostructures metallic materials.

ACKNOWLEDGMENTS The author acknowledges the contribution of G. D. Griffin, J. P. Alarie, B. M. Cullum, and P. Kasili. This research was sponsored by the LDRD Project (Advanced Nanosensors), and by the Office of Biological and Environmental Research and the National Nuclear Safeguard Agency (NN-20 Program), U.S. Department of Energy under Contract DEAC05-00OR22725 managed by UT-Battelle, LLC.

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