Paper-Based Medical Diagnostic Devices: As a Part of Bioanalysis-Advanced Materials, Methods, and Devices 9789811587221, 9789811587238

Citation preview

Bioanalysis Series Editor: Tuan Vo-Dinh

Jeong Hoon Lee   Editor

Paper-Based Medical Diagnostic Devices As a Part of Bioanalysis-Advanced Materials, Methods, and Devices

Bioanalysis Advanced Materials, Methods, and Devices Volume 10

Series Editor Tuan Vo-Dinh, Fitzpatrick Institute for Photonics, Duke University, Durham, NC, USA

The book series on BIOANALYSIS: Advanced Materials, Methods, and Devices is intended to serve as an authoritative reference source for a broad, interdisciplinary audience involved in the research, teaching, learning, and practice of bioanalytical science and technology. Bioanalysis has experienced explosive growth due to the dramatic convergence of advanced technologies and molecular biology research, which has led to the development of entirely new ways to probe biomolecular and cellular processes as well as biological responses to implanted biomaterials and engineered tissues. Novel optical techniques using a wide variety of reporter gene assays, ion channel probes, and fluorescent probes have provided powerful bioanalytical tools for cell-based assays. Fluorescent reporters allow the development of live cell assays with the ability for in vivo sensing of individual biological responses across cell populations, tracking the transport of biological species within intracellular environments, and monitoring multiple responses from the same cell. Novel classes of labels using inorganic fluorophors based on quantum dots or surface-enhanced Raman scattering labels provide unique possibilities for multiplex bioanalyses. Laser-based technologies are important in the development of ultrasensitive bioanalytical techniques. Lasers are now used as excitation light sources in a wide variety of molecular bioassays. Today, single-molecule detection techniques using laser excitation provide the ultimate tools to elucidate cellular processes. The possibility of fabricating nanoscale materials and components has recently led to the development of devices and techniques that can measure fundamental parameters at the molecular level. With “optical tweezer” techniques, for example, small particles may be trapped by radiation pressure in the focal volume of a high-intensity, focused laser beam. Ingenious optical trapping systems have also been used to measure the force exerted by individual motor proteins. Whereas the laser has provided a new technology for excitation, the miniaturization and mass production of sensor devices and their associated electronic circuitry has radically transformed the ways detection and imaging of biological species can be performed in vivo and ex vivo. Sensor miniaturization has enabled significant advances in imaging technologies over the last decade in such areas as microarrays and biochips for bioanalysis of a wide variety of species. The miniaturization of high-density optical sensor arrays has also led to the development of advanced high-resolution imaging methods at the cellular or molecular scales. With powerful microscopic tools using near-field optics, scientists are now able to image the biochemical processes and sub-microscopic structures of living cells at unprecedented resolutions. Recently, nanotechnology, which involves research on and development of materials and species at length scales between 1 to 100 nanometers, has been revolutionizing important areas in bioanalysis at the molecular and cellular level. The combination of molecular nanotechnology and various sensing modalities (optical, electrochemical, etc) opens the possibility of detecting and manipulating atoms and molecules using nano-devices, which have the potential for a wide variety of bioanalyses at the cellular level. These new bioanalytical tools are capable of probing the nanometer world and will make it possible to characterize the chemical and mechanical properties of biomolecules and cells, discover novel phenomena and processes, and provide science with a wide range of tools, materials, devices, and systems with unique characteristics. This book series will present the most recent scientific and technological advances in materials, methods and instrumentation of interest to researchers, students, and manufacturers. The goal is to provide a comprehensive forum to integrate the contributions of biophysicists, biomedical engineers, materials scientists, chemists, chemical engineers, biologists, and others involved in the science and technology revolution reshaping molecular biology and biomedicine. More information about this series at http://www.springer.com/series/8091

Jeong Hoon Lee Editor

Paper-Based Medical Diagnostic Devices As a Part of Bioanalysis-Advanced Materials, Methods, and Devices

123

Editor Jeong Hoon Lee Department of Electrical Engineering Kwangwoon University Seoul, Korea (Republic of)

ISSN 2364-1118 ISSN 2364-1126 (electronic) Bioanalysis ISBN 978-981-15-8722-1 ISBN 978-981-15-8723-8 (eBook) https://doi.org/10.1007/978-981-15-8723-8 © Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Contents

Paper-Based Diagnostic Device History and Challenges . . . . . . . . . . . . . Dohwan Lee and Jeong Hoon Lee

1

Basic Paper-Based Microfluidics/Electronics Theory . . . . . . . . . . . . . . . Ali Turab Jafry, Hosub Lim, and Jinkee Lee

7

Novel Materials and Fabrication Techniques for Paper-Based Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seong-Geun Jeong, Reya Ganguly, and Chang-Soo Lee

41

Technical Features and Challenges of the Paper-Based Colorimetric Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dongtak Lee, Insu Kim, Sang Won Lee, Gyudo Lee, and Dae Sung Yoon

69

Paper-Based Nucleic Acid Detection for Point-of-Care Diagnostics . . . . Jongmin Kim and Yong-Ak Song

87

Paper-Based Biosensors with Lateral/Vertical Flow Assay . . . . . . . . . . . 115 Dohwan Lee and Jeong Hoon Lee Paper-Based Applications for Bacteria/Virus . . . . . . . . . . . . . . . . . . . . . 137 Sumin Han, Manika Chopra, Ilaria Rubino, and Hyo-Jick Choi Paper-Based Molecular Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Bhagwan S. Batule, Youngung Seok, and Min-Gon Kim

v

About the Editor

Jeong Hoon Lee is a professor at Electrical Engineering, Kwangwoon University, South Korea. He received the B.S. degree in the department of Ceramic Engineering at Yonsei University, Seoul, South Korea, in 1997. He received the Ph.D. from same department in 2004. He specialized in MEMS/Nanomechanics from 1999 to 2005 at Korea Institute of Science and Technology (KIST) in Seoul, South Korea. Before joining Kwangwoon University in Sep 2008, he was a Postdoctoral Associate at RLE and EECS, Massachusetts Institute of Technology (MIT), USA (2005 to 2008). His current main research is the development of simple and powerful POCT and diagnostic systems based on the integration of electronics and fluidics. He published 85+ papers and +40 patents. Also he is co-founder of CALTH Co. for POCT diagnostics/sample preparation.

vii

Paper-Based Diagnostic Device History and Challenges Dohwan Lee and Jeong Hoon Lee

1 History of Paper-Based Diagnostic Devices Historically, woven cellulosic paper has attracted scientific attention for its unique advantages of porosity, low cost, versatility, disposability, biocompatibility, and inherent capillary flow [1–3]. In the seventeenth century, the invention of litmus paper, a pH indicator paper treated with a natural water-soluble dye obtained from lichens, was the first milestone demonstrating the significant potential of paper as a substrate material in analytical applications [1, 4]. Since the 1940s, paper has been used extensively as an analytical substrate material (Fig. 1). Consden et al. reported a paper-based chromatographic method for the qualitative analysis of proteins in 1944 [5], and West developed a spot test paper for metal ion detection in 1945 [6]. Until this point, liquid-phase samples were simply transported through the analytical papers by capillary action in randomly distributed fiber networks without a uniform direction; therefore, all areas of the paper required treatment with chemicals for analysis. In 1949, Müller and Clegg first suggested a method for guiding the direction of the fluid flow by patterning a fluidic channel on filter paper using hydrophobic paraffin [7]. This approach has become the basis for the fabrication of modern paper-based diagnostic devices because the manipulation of fluid flow in a designated direction provides many advantages: (i) transportation of the sample to specific regions where the analytical reaction occurs; (ii) accessibility to small sample volumes, necessary for limited sample availability (i.e., tears, saliva, sweat, urine from neonates, and blood drops from finger sticks); (iii) distribution of a liquid sample into multiple regions to enable multiplexing or repetition of assays simultaneously on a single device [2]. The first paper-based dipstick tests to measure urinary glucose levels were reported in the 1950s by Comer [8] and Free et al. [9]. In 1956, the concept of the lateral flow assay (LFA), currently the most widely used D. Lee · J. H. Lee (B) Department of Electrical Engineering, Kwangwoon University, Seoul 01897, Republic of Korea e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 J. H. Lee (ed.), Paper-Based Medical Diagnostic Devices, Bioanalysis 10, https://doi.org/10.1007/978-981-15-8723-8_1

1

2

D. Lee and J. H. Lee

Fig. 1 Timeline of paper-based diagnostic devices

form of paper-based diagnostic devices, was introduced by Plotz and Singer [10], who diagnosed rheumatoid arthritis using the latex agglutination test. Inspired by this concept, Leuvering et al. developed a colloidal gold or silver particle-based colorimetric LFA in 1980, called “sol particle immunoassay” (SPIA) [11]. They demonstrated sandwich immunoassays in the LFA format to detect human placental lactogen and chorionic gonadotrophin. Before the concept of microfluidic paper-based analytical devices (µPADs) was presented by the Whitesides Group of Harvard University in 2007 [12], academic and commercial paper-based diagnostic devices were limited to LFA-type products. The Whitesides Group reported a simple method for patterning a chromatographic paper with hydrophobic photoresist via conventional photolithography to create millimeter-scale channels and reaction zones. Using these well-defined regions of the paper, the analysis of glucose and protein was performed without any external equipment (Fig. 2a). Subsequently, in 2008, they proposed a method for fabricating three-dimensional (3D) microfluidic devices by stacking papers that were similarly patterned [13]. These 3D µPADs enabled the distribution of samples into multiple detection reaction zones, thereby simultaneously allowing the generation of calibration curves for the assays and measurement of glucose and protein levels. The development of these µPADs verified that the manipulation of sample distribution, varied analytical functionalization, and multiplex assays could be implemented on paper as a substrate, in addition to exploiting the inherent merits of paper. Therefore, the introduction of µPADs was an innovation suggesting the tremendous potential of paper as point-of-care (POC) diagnostic devices by their integration with today’s advanced understanding of microfabrication and microfluidics.

2 Challenges of Paper-Based Diagnostic Devices The advent of µPADs has inspired many academic and corporate researchers in the fields of microfluidics and POC tests, demonstrating new routes toward the development of advanced paper-based diagnostic devices [14, 15]. However, in the investigation and production of paper-based diagnostic devices, standards were required to select devices with commercial viability or potential for further development. For this reason, fields relating to paper-based diagnostic devices have focused on the “ASSURED” criteria, established and reported by the World Health Organization

Paper-Based Diagnostic Device History and Challenges

3

Fig. 2 First two studies presenting the concept of µPADs by the Whitesides Group of Harvard University. a Patterned chromatographic paper as an analytical platform for quantitation of glucose and protein [12]. Figure panel adapted and reproduced from [12] with permission from Wiley-VCH. b 3D µPADs fabricated with multiple layers of patterned papers and tapes for quantitative detection of glucose and protein [13]. Copyright (2008) National Academy of Sciences, U.S.A

(WHO) in 2004, to specify the capabilities that appropriate POC devices should have (Table 1) [16]; “ASSURED” is an acronym for Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end users. Based on the ASSURED criteria, POC devices should aim to provide same-day results, thus facilitating immediate decision-making, and require user-friendliness with high-throughput operation in resource-limited environments and settings to widen the pool of end users [17]. With ever-increasing demand and markets for paper-based diagnostic devices, many new strategies and studies have been reported to satisfy the ASSURED criteria. Of course, some criteria are sometimes considered less significant than

4

D. Lee and J. H. Lee

Table 1 ASSURED criteria and examples of target specifications for the evaluation of point-of-care devices [17] Characteristic

Target specification

Affordable

Less than US$500 per machine, less than US$10 per test

Sensitive, specific

Lower limit of detection: 500 HIV RNA copies per mL, 350 CD4+ T-cells per µL

User-friendly

1–2 days of training, easy to use

Rapid and robust

0). For a convenient understanding of the working principle throughout the device (Fig. 4b), it was considered as three separate sections of the lower section (bottom part), bridge section (pullulan film), and upper section (top edge of pullulan film and above). The lower section was predicted to follow the modified Lucas–Washburn equation, shown by comparing the classic and modified Lucas–Washburn equations. Equation (8) provides the full model prediction of fluid flow in Whatman Grade 1 filter paper, considering the effects of gravity, evaporation, and the fluid content of the fiber.   2  Bh + Dh + A  1   ln  t =(1 − w%)  2B A   ⎞ ⎛   2Bh + D − √D2 − 4BA D − √D2 − 4BA    D   ⎠ − √ ln⎝  √ √ 2 2B D − 4BA  2Bh + D + D2 − 4BA D + D2 − 4BA  (8) Equation (9) provides the evaporation model where gravitational pull is neglected. h(t) = (1 − w%)

A A exp(2Bt) − B B

(9)

where A=

−m ˙ ev −gρK 2Kδv cos θ ,B = ,D = Rμφ 2d ρφ μφ

(10)

and, h, t, K, θ, R, φ, μ, δ, ρ, g, and δ lv represent the distance traveled, time, permeability, contact angle, average capillary radius, porosity, viscosity, average paper thickness, density, gravitational constant, and surface tension, respectively. The evaporation rate of water at a specific RH is obtained as:

Novel Materials and Fabrication Techniques for Paper-Based Devices

53

Fig. 4 a Flow shutoff system assembly: schematic. Letters “F” and “R” indicate the front and rear sides of the device, respectively. b Flow shutoff by using Pullulan dissolvable bridge. c Graphical data of water flow distance in plain Whatman Grade 1 paper strips as a function of time. The strip size was 8 cm (length) × 2.5 cm (width). Experimental data ( ), Lucas–Washburn equation ( ), evaporation model considering 10% fiber water content ( ), and full model considering 10% fiber water content ( ). d Rate of water capillary rise through the pullulan shutoff system at 21 °C and 48% RH. The evaporation model prediction is represented by the dashed line in the lower (green) and upper (red) sections of the device, and the solid markers show experimental data in the lower section ( ), and in the upper section for the pullulan film thicknesses of 0.30 mm ( ) and 0.25 mm ( ), respectively. The behavior of water flow through the upper section of the device is similar to the rate of capillary rise of the 50% diluted pullulan solution through plain filter paper; this analogy holds until the pullulan bridge connection is dissolved, when the flow approaches its maximum possible height and the upper section is severed from the water source

54

S.-G. Jeong et al.

m ˙ evp = (Pw − PRH ) × (0.089 + 0.0782Vair )/Y

(11)

where Pw , PRH , Y, and V air represent the water-saturated pressure, partial pressure of water vapor, heat of vaporization of water, and air flow velocity in the environment (zero in all cases studied), respectively. When classic, full, and evaporation models were plotted along with the experimental dataset on a graph of the flow distance as a function of time, it was clearly shown that the gravitational factor could be neglected for strips shorter than 10 cm (in other terms, distances shorter than 10 cm) because the deviation in the graph was less than 0.5% between the full and evaporation models (Fig. 4c). Therefore, the upper section of the device also followed the same modified Lucas–Washburn equation. The only parameter that differed in both cases was the viscosity of the fluid because of the dissolution of the pullulan in the traveling liquid while it crossed the bridge section. Further experiments investigating the difference between the capillary rise in the lower and upper sections showed deviations because of the complete dissolution of the pullulan bridge (Fig. 4d). This strategy was demonstrated as an effective regulator to automatically break off connections between the channels as required based on the thickness of the dissolvable pullulan film. This technique in various aspects can overcome the limitation of previously designed devices and inspire new development of complex patterns. In short, for paper-based microfluidic analytical devices, pullulan as a rapidly dissolving polymer can be used to fabricate an automatic flow shutoff system. Some of the paper channel was replaced by an interruptible capillary channel comprising dissolvable film, allowing for automatic flow control through paper-based devices. This technology allowed users to manipulate fluid movement to accommodate timesensitive or multistep reactions and assays.

2.4 Automatic Timed Flow Progress in automatic timed flow was another turning point in the fabrication of paper devices with nearly error-free control over the sequential flow of hydrophilic solutions. Chemical-based flow shutoff has been demonstrated; other approaches include mechanical and geometry-based channel break-off. In mechanical flow actuation, example methods include electromagnetic [32], hygroexpanding [33, 34], manually operated pushbuttons, [35] flaps [36], pop-up [11], folding [12, 37], and sliding strips [38] for valve operation. Geometry-based valving patterns are generally made by changing the dimension of source pad or the length of the channel from the reagent reservoir. Here, we address two approaches in this field of electrotextiles and valves operated by movable paper strips, along with fluid-triggered expanding elements [8].

Novel Materials and Fabrication Techniques for Paper-Based Devices

2.4.1

55

Electrical Textile Valves

Electrotextile-based valves can improve the ability of paper-based devices to perform multiplex experiments that generally incorporate washing steps and multiple reagents. An electrically actuated valve comprising an electroactive mesh of woven fibers has been reported, the wettability of which could be controlled by an applied voltage (Fig. 5a). Fabrication Technique: To characterize electrowetting of electrotextile valves, woven textiles containing metallic or metal-coated plastic fibers were used. All the materials were coated with Parylene-C [39, 40] for electrical insulation between the liquid and the metal, and Teflon-AF was used to increase the hydrophobicity of the fiber surfaces. The water breakthrough pressure was in the range 0.6–1.2 kPa for the materials used. The voltage thresholds for the actuation of different materials using deionized water were then calculated. All tested electrotextiles were activated in the potential range 100–1000 V. The breakthrough pressure of the hydrophobic textiles was sufficiently high to allow the containment of liquids for applications in microfluidics. The working principle of this device was based on the combined action of the Washburn and Lippmann–Young equations because the valves had nonplanar surfaces and operated based on the principle of the penetration of liquids into porous materials assisted by electrowetting. Equation (12) was derived for the activation voltage U a required to trigger fluid flow through the electrotextile:  Ua =

−

2σ di cos θ0 εε0

(12)

Here, θ 0 , ε, d i , and σ represent the stationary contact angle of water on the planar surface of the electrotextile material (Teflon-AF), the dielectric permeability of the layer of insulation on the wires (ε is 3.15 for Parylene-C), the thickness of the insulation layer (di is ~10 µm Parylene-C), and the surface tension of the liquid, respectively. In the experiment, the actuation voltage was decreased with decreasing surface tension, as suggested by Eq. (12). The standard deviation of the actuation voltage decreased monotonically with decreasing surface tension. Equation (12) was thus shown to estimate the voltage for the actuation of different materials and liquids and describe trends; however, it could not provide exact numbers. Hence, to explain electrowetting through textiles, the Lippmann–Young equation was used, replaced with a model fitting 3D microscale geometries. This equation was expected to describe the pressure required for a liquid to penetrate the textile. After establishing the bi-stable valve, it was incorporated in microfluidic devices by stacking pattern-printed paper with electrodes printed on its surface with the electrotextile into a multilayer device (Fig. 5b). Additionally, by adding an electrical valve and a fluid-to-electrical switch, simple integrated circuits could be formed. Such valves in the device circuit enable behavior similar to semiconductor thyristors

56

S.-G. Jeong et al.

Novel Materials and Fabrication Techniques for Paper-Based Devices

57

Fig. 5 Bistable electrical valve principle. a Valve operation schematic, based on the principles of electrowetting on dielectric (EWOD). The gate electrode is a conductive textile covered with layers of insulator and hydrophobic coatings (“electrotextile”) and is impermeable to liquid. On the application of a voltage between the electrotextile and liquid, liquid flows into the paper layer underneath via electrowetting. b–d. Electrofluidic device design and fabrication steps. b Fabrication of 3D printed electrofluidic circuits; schematics. Using wax printing, microfluidic channels are printed, followed by the printing of electrodes (for valves and switches) and electrical wires on the paper layers. Using hot-lamination with polyethylene films, conductive, insulated, and hydrophobic electrotextiles are bonded together. c The figure shows a schematic cross-section of the fluid-toelectrical switch and an integrated valve containing two microfluidic layers and a valve layer. On the application of voltage between the liquid (“source electrode”) and the electrotextile (“gate electrode”), the valve is actuated and liquid can pass from the “liquid source” into the “liquid drain.” d Circuit diagram symbols for printed microfluidic channels, printed wires/electrodes, integrated fluid-electrical switches, and valves (“electrofluidic thyristors”). e, f Parallel connection valves. e Images of the fabrication process. On a single sheet of paper, the device was printed and then folded around the electrotextile and laminated with polyethylene films. f Image captured while operating the device; two aqueous solutions in separate reservoirs (red and green) are injected into a common channel. A light-emitting diode indicates when an electrical pulse is applied to open each valve

(used in AC power electronics), thus permitting liquid and electric signals to control each other interchangeably. The resulting circuit was called an electrofluidic circuit and a circuit diagram symbol was proposed for electrofluidic components (Fig. 5d). This design was integrated to fabricate a switch for a paper microfluidic chip, where two carbon electrodes were printed separated by a microfluidic channel (Fig. 5c). The conductivity of dry paper [41] is insufficient for valve operation; therefore, without conducting fluid in the channel, the switch remains off. With a fluid, it is on, allowing a high current to pass through. The fundamental design given for this device was a sandwiched structure of electrotextile mesh between two wax-printed patterned papers. The top layer was patterned as a liquid reservoir with a carbon electrode as well. The electrode acted as a source electrode for each valve, while the electrotextile in the middle layer was connected to ground as a gate electrode. With an applied voltage pulse to the electrode, the valves were opened, allowing the fluid to penetrate the middle layer. On reaching the bottom layer, the fluid could flow freely through the hydrophilic channel. In addition to showing the controllability of the valve, its feasibility for the timecontrolled addition of multiple solutions and reagents was verified. This experiment is demonstrated in Fig. 5e with a valve integrated in parallel. Two valves were fabricated in the same layer with two separate liquid reservoirs; the design delivered both liquids to a common outlet via a common fluidic channel. This configuration allowed the timely delivery of fluid (Fig. 5f). This innovation could be exploited to design time-sensitive multistep reactions, such as ELISA [37, 42], ECL [43], or nucleic acid biosensing. The electrotextile valves could also be utilized in other microfluidic systems based on thin materials [18, 44] or open-channel paper chips [25, 45, 46].

58

2.4.2

S.-G. Jeong et al.

Valving Kit

The concept of a valving toolkit was intended to utilize movable paper strips and fluidtriggered expanding elements for actuation as switches. Researchers categorized actuation based on fixed time and volume. Some highly desirable features could be achieved, including independence from batteries or electricity in operation, no chemical being necessary for actuation to avoid chemical contamination of the flow path, and pre-programmability could be used to avoid user intervention [34]. Valve timing was mainly controlled by manipulating the channel geometry. However, the switching part was controlled via either the displacement of one end of the paper channel, causing connection or disconnection, or expandable absorbent materials (cellulose, hydrogels, or sponges). The most suitable expandable material for the experiment was reported to be a compressed sponge, which permitted fast actuation and reproducibility. The ability to regulate the program after a fixed time point was achieved by a movable paper channel, while actuation after the passage of a fixed amount of fluid was obtained using a moveable volume-metering pad. Here, we discuss the time- and volume-metered valves separately. Fabrication Technique: The device was constructed by stacking layers of 10mil-thick adhesive-backed Mylar (Fralock, Valencia, CA), slabs of poly(methyl methacrylate) (PMMA; McMaster-Carr, Elmhurst, IL), and paper channels. DraftSight (Dassault Systems HQ, France) was used to draw the designs, and a 42-W CO2 laser cutter (M360, Universal Laser Systems, Scottsdale, AZ) was used to cut all parts (Figs. 6 and 7). Layers of plastic and paper were cut to the desired shapes individually before stacking. The procedure to cut nitrocellulose channels has been described previously [47] . Compressed sponges were also laser-cut to achieve the desired dimensions. The actuator cavity was laser-cut into a 2.5-mm-thick PMMA layer. PMMA of 1.5 or 2 mm in thickness was used to maintain the height difference between flow channels. A cantilever channel was formed by selectively removing the laminate protective adhesive from one end of the Mylar layer, while the other end was not adhered to the surface and left free to move. The moveable end was then attached to an impermeable fluid barrier comprising a flap of 10-mil-thick double-adhesivebacked Mylar. The channel was adhered to the flap on its top surface, whereas the bottom surface of the flap was adhered over the cavity containing the actuator. No adhesive was used to attach the sponge actuator to the glass fiber actuating channel; by reducing the width of the actuating channel by half, in order to expose the adhesive on the surrounding Mylar, good adhesion was achieved between the actuator and the actuation channel. To avoid the tendency of unbaked nitrocellulose to warp when wet, the unbacked nitrocellulose was laminated with adhesive-backed 4-milthick Mylar films wherever the NC did not contact other channels from both sides. For volume-metered valves with displaceable metering pads, the weight and support posts were formed by stacking three 3.8-mm-thick PMMA layers after laser cutting to desired shape. Time-metered Valve: This valve, as explained before, could be operated by controlling the time at which the actuation fluid arrived at the actuator. Two remarkable

Novel Materials and Fabrication Techniques for Paper-Based Devices Fig. 6 a Design of time-metered valve. b On-switch (b, c) and off-switch (d, e) made using time-metered valves. The two valves are made of nitrocellulose flow channels (containing yellow fluid) along with glass fiber actuation channels (containing water), separated by an impermeable fluid barrier. Water is added to the actuation channels manually after a certain period of time to actuate the valves

59

60 Fig. 7 a Design of volume-metered valve with a moveable metering pad. b Volume-metered flow-diversion switch scheme, showing transverse and lateral views. The valve comprises an actuator inserted on the metering pad, which rests on top of a flow channel. By attaching a weight to the pad, firm contact is ensured between the flow channel and metering pad. Until the actuator expands and lifts the pad off, fluid in the channel continues flowing into the metering pad. After actuation, fluid flows downstream of the valve in the flow channel. The schematic of the lateral view shows the valve with two flow channels, a and b, with an adapter channel between. c Top and side views of the valve before (t = 15 min) and after (t = 38 min) actuation. Flow of fluid before actuation into the metering pad (t = 15 min; top view) and after actuation into channel b (t = 38 min; top view). The scale bar is 1 cm. The side view shows the effects before (t = 15 min) and after (t = 38 min) actuation on the sponge actuator. The scale bar is 1 mm

S.-G. Jeong et al.

Novel Materials and Fabrication Techniques for Paper-Based Devices

61

types of valve were presented of on- and off-switches (Fig. 6). To verify the activity of these valves in controlling fluid flow, some experiments were presented to test all on- and off-switches. In all cases, the actuator is attached to an actuation channel that remains separate from the fluid flow channel. Specifically, in the on-switch, the actuator is connected with channel A via a fluid barrier. On actuation, it lifts channel A to connect with channel B. For the off-switch, channel A is connected with channel B along with the actuator; on actuation, channel A is lifted up, which disconnects it from channel B. Both switch types were combined in a diversion switch, in which channel A remained connected with channel B and the actuator; upon actuation, channel A is lifted up, disconnecting it from channel B but connecting it to channel C, thus establishing a new connection among the three channels. Part-by-part assembly of a time-metered on-switch: The individual parts used for the on-switch are depicted in Fig. 6. Here, “AC” represents a single-side adhesivebacked Mylar layer; “ACA” indicates that both sides are adhesive. The adhesivebacked Mylar layers were protected with 1-mil-thick laminate layers. The laminate required removal to expose the adhesive; this was performed in selected regions using a low-power laser cutter to cut through only the laminate. Twelve parts total make up the switch (Fig. 6). Some of the critical points for the proper functioning of these valves include the proper alignment of the actuation channel, actuator, and the ends of the flow channels [35]; good contact between the end of the actuation channel and the actuator, and the securing of the fluid barrier attached to the flow channel on top of the actuator cavity to allow displacement after actuator expansion. The width of the actuation channel where the actuator is located is reduced to half and the laminate from this region is removed to expose the adhesive. Thus, the adhesive ensures stable contact between the end of the actuation channel and the actuator. The actuator is placed under a square hole cut in the 2.5-mm-thick PMMA slab and an additional layer of ACA of 10 mil in thickness with a square hole in the same position is attached above it because the PMMA slab is nonadhesive; this allows the continued stacking of layers. A rectangular rim is marked by laser etching around the actuator cavity on the top surface of the ACA layer (dashed region; Fig. 6). An impermeable fluid barrier comprising ACA is attached over this rim and laminate is removed from the select region on which the nitrocellulose channel was attached. The width of the rim was 0.75 mm, providing sufficient adhesion to hold the nitrocellulose channel before actuation while still allowing the detachment and displacement of the channel on actuation. Volume-metered valves: This valve functions as follows (Fig. 7): on the loading of a fixed sample amount upstream of the valve, the fluid passes through channel A, a volume-metered pad, and the connector pad to the actuator, which is connected to channel A via an impermeable fluid barrier that holds the actuator in position before actuation. When the actuator is sufficiently filled with fluid, it expands, causing disconnection of channel A from the volume-metered pad. Simultaneously, channel A is connected to channel B, which is placed above channel A. This process causes fluid to flow to channel B after a certain volume is attained in the actuator.

62

S.-G. Jeong et al.

Another type of volume-metered switch was presented where channel A and channel B were connected with an adopter channel, but the actuator and metering pad were placed on the top of adopter channel, which caused the fluid coming from either channel A or B to pass into the metering pad before flowing in the opposite direction. Once the actuator attached to the metering pad was filled with the desired volume of fluid, it expanded, pushing the metering pad up. This connected channel A and channel B via the adopter channel, allowing fluid passage without any resistance. Overall, such devices are user-friendly; the valving technique is simple and can be used conveniently by laypersons. In addition to their efficacy, valve-equipped devices maintain the advantage of avoiding pumps and electricity to drive fluid flow.

3 Novel Method to Form Paper-Based Channels 3.1 3D Wax Patterning A simple approach to fabricate 3D-µPAD from a single sheet of paper by doublesided printing and lamination was demonstrated by Jeong et al. [14]. This technique has overcome some of the drawbacks of conventional fabrication methods, avoiding the use of double-sided adhesives, origami clamping, and spray adhesive glues [14, 35, 48, 49]. Such methods require more complex fabrication processes to create more complicated flow channels, including vertical channels that do not exist in 2D-µPADs. However, the technique of double-sided printing and lamination is an adhesive- and alignment-free process. The steps include only the double-sided printing of wax patterns and lamination for heating the wax to penetrate the cellulose fiber. This process forms a 3D microfluidic network in a single sheet of paper. The width and height of the wax pattern can be controlled by manipulating the heating time and speed of the laminator. Here, we discuss this fabrication procedure for better understanding. Fabrication of a 3D-microfluidic network in a single sheet of paper: Hydrophobic wax patterns were designed in image software (Adobe Illustrator CS6) and printed using a solid ink printer set to yield photo-quality prints. The overall process includes two steps of printing the designed wax pattern on both sides of the paper and then heating it with a laminator set at 140 °C for 0.26 s, causing the wax to melt and spread through the thickness of the paper (Fig. 8A). The paper was cooled to room temperature ( Pth . The lower channel is formed by an independent upper-heated wax. H LC is determined by: H LC = Pth − H hf . The lower channel exists when H LC > 0, given by: H hf < Pth . The upper channel is formed by an independent lower-heated wax (Fig. 8C). H UC is given by: H UC = Pth − H hb . An upper channel exists when H UC > 0. Therefore, it can be expressed as in the equation. The experimental results demonstrated the efficacy of utilizing this technique in creating diagnostic tools. The fabrication technology can be applied to a roll-to-roll process for mass production without encountering the defects in fabrication that were mentioned in the introduction (Fig. 9).

64

S.-G. Jeong et al.

Novel Materials and Fabrication Techniques for Paper-Based Devices

65

Fig. 8 A, B Heated height (H h ) and heated width (W h ) change from the vertical and lateral spreading of the printed wax line (W n = 1.0 mm) as a function of the heating time (t). A Spreading process for molten wax in a cross-section of paper is represented schematically; the width and height of the printed wax are represented by W p and H p , respectively, and the width and height of wax lamination by W h and H h , respectively. B Graph shows the increase of H h and W h depending on the heating time. The actual image of printed wax is also depicted after heating at 140 °C. C Schematic representation of the conditions required for the fabrication of a docking wax barrier and lower and upper channels

Fig. 9 Comparison of 3D wax pattering method and conventional method of fabrication for 3DµPADs

66

S.-G. Jeong et al.

4 Conclusion and Perspective In this chapter, recent novel fabrication techniques for paper-based devices are demonstrated to achieve better fluid handling and wicking flow, thereby minimizing the concerns in previous devices of (1) interference with assay, (2) user intervention, and (3) required peripheral equipment. Although the devices show advanced control of wicking fluid that can be applied to automatic multistep assays, challenges for commercialization remain as follows: (1) Vulnerability to changes in humidity. The precise and reproducible control of wicking speed is necessary to obtain reliable results in automatic sequential multistep assays with paper-based microfluidic devices. However, in the demonstrated devices, the speed of wicking flow is easily affected by changes of environmental humidity. Variations in the speed of wicking flow cause errors in the assay. (2) Insufficiently simple fabrication for commercial-scale production. Although the valve system shows remarkable performance for automatic multistep assay, the complex structure is difficult to adapt for mass production. (3) Nonspecific bonding in paper channels. Longer paper channels require longer fluid travel distances. This increases the possibility of analyte loss by nonspecific binding with paper, which reduces the limit of detection (LOD). Paper channels should be sufficiently short to minimize nonspecific binding. (4) The World Health Organization (WHO) and other governmental agencies still seek adequate pricing and reliable devices [52]. Therefore, an ideal fabrication method should be simple enough to allow commercial-scale manufacturing, produce reliable devices that show the same results in automatic multistep assays under different humidity conditions, and should avoid the concern of nonspecific bonding.

References 1. Cate, D.M., Adkins, J.A., Mettakoonpitak, J., Henry, C.H.: Recent developments in paper-based microfluidic devices. Anal. Chem. 87, 19–41 (2015) 2. Consden, R., Gordon, A.H., Martin, A.J.: Qualitative analysis of proteins: A partition chromatographic method using paper. Biochem. J. 38, 224–232 (1944) 3. West, P.W.: Selective spot test for copper. Ind. Eng. Chem. Anal. Ed. 17, 740–741 (1945) 4. Martinez, A.W., Phillips, S.T., Butte, M.J., Whitesides, G.M.: Patterned paper as a platform for inexpensive, low-volume, portable bioassays. Angew. Chem. Int. Ed. 46, 1318–1320 (2007) 5. Yetisen, A.K., Akram, M.S., Lowe, C.R.: Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13, 2210–2251 (2013) 6. Jeong, S.-G., Kim, J., Jin, S.H., Park, K.-S., Lee, C.-S.: Flow control in paper-based microfluidic device for automatic multistep assays: A focused minireview. Korean J. Chem. Eng. 33, 2761– 2770 (2016) 7. Lutz, B., et al.: Dissolvable fluidic time delays for programming multi-step assays in instrumentfree paper diagnostics. Lab Chip 13, 2840–2847 (2013)

Novel Materials and Fabrication Techniques for Paper-Based Devices

67

8. Ainla, A., Hamedi, M.M., Güder, F., Whitesides, G.M.: Electrical textile valves for paper microfluidics. Adv. Mater. 29, 1702894 (2017) 9. Jiang, Y., Hao, Z., He, Q., Chen, H.: A simple method for fabrication of microfluidic paperbased analytical devices and on-device fluid control with a portable corona generator. RSC Adv. 6, 2888–2894 (2016) 10. Guo, T., et al.: UV-driven microvalve based on a micro–nano TiO2 /SiO2 composite surface for microscale flow control. Nanotechnology 25, 125301 (2014) 11. Wang, C.-C., et al.: A paper-based, “pop-up” electrochemical device for analysis of betahydroxybutyrate. Anal. Chem. 88, 6326–6333 (2016) 12. Ding, J., Li, B., Chen, L., Qin, W.: A three-dimensional origami paper-based device for potentiometric biosensing. Angew. Chem. Int. Ed. 55, 13033–13037 (2016) 13. Gong, M.M., Sinton, D.: Turning the page: advancing paper-based microfluidics for broad diagnostic application. Chem. Rev. 117, 8447–8480 (2017) 14. Jeong, S.-G., Lee, S.H., Choi, C.H., Kim, J., Lee, C.S.: Toward instrument-free digital measurements: A three-dimensional microfluidic device fabricated in a single sheet of paper by double-sided printing and lamination. Lab Chip 15, 1188–1194 (2014) 15. Jang, I., Song, S.: Facile and precise flow control for a paper-based microfluidic device through varying paper permeability. Lab Chip 15, 3405–3412 (2015) 16. Darcy, H.P.G.: Exposition et application des principes à suivre et des formules à employer dans les questions de distribution d’eau, etc. In: Dalamont, V. (ed.) Les Fontaines publiques de la ville de Dijon (Paris, 1856) 17. Fu, E., Ramsey, S.A., Kauffman, P., Lutz, B., Yager, P.: Transport in two-dimensional paper networks. Microfluid. Nanofluid. 10, 29–35 (2011) 18. Osborn, J.L., et al.: Microfluidics without pumps: Reinventing the T-sensor and H-filter in paper networks. Lab Chip 10, 2659–2665 (2010) 19. Camplisson, C.K., Schilling, K.M., Pedrotti, W.L., Stone, H.A., Martinez, A.W.: Two-ply channels for faster wicking in paper-based microfluidic devices. Lab Chip 15, 4461–4466 (2015) 20. Meredith, N.A., et al.: Paper-based analytical devices for environmental analysis. Analyst 141, 1874–1887 (2016) 21. Fries, N., Odic, K., Conrath, M., Dreyer, M.: The effect of evaporation on the wicking of liquids into a metallic weave. J. Colloid Interface Sci. 321, 118–129 (2008) 22. Carrilho, E., Martinez, A.W., Whitesides, G.M.: Understanding wax printing: a simple micropatterning process for paper-based microfluidics. Anal. Chem. 81, 7091–7095 (2009) 23. Schilling, K.M., Jauregui, D., Martinez, A.W.: Paper and toner three-dimensional fluidic devices: Programming fluid flow to improve point-of-care diagnostics. Lab Chip 13, 628–631 (2013) 24. Barry, D., Parlange, J.-Y., Lockington, D.A., Wissmeier, L.: Comment on “The effect of evaporation on the wicking of liquids into a metallic weave”. In: Fries, N., Odic, K., Conrath M., Dreyer, M. (eds.) J. Colloid Interface Sci. 336, 374–375 (2009) 25. Renault, C., Li, X., Fosdick, S.E., Crooks, R.M.: Hollow-channel paper analytical devices. Anal. Chem. 85, 7976–7979 (2013) 26. Giokas, D.L., Tsogas, G.Z., Vlessidis, A.G.: Programming fluid transport in paper-based microfluidic devices using razor-crafted open channels. Anal. Chem. 86, 6202–6207 (2014) 27. Yuen, P.K., Goral, V.N.: Low-cost rapid prototyping of flexible microfluidic devices using a desktop digital craft cutter. Lab Chip 10, 384–387 (2010) 28. Hossain, S.M.Z., Luckham, R.E., McFadden, M.J., Brennan, J.D.: Reagentless bidirectional lateral flow bioactive paper sensors for detection of pesticides in beverage and food samples. Anal. Chem. 81, 9055–9064 (2009) 29. Fu, E., Lutz, B., Kauffman, P., Yager, P.: Controlled reagent transport in disposable 2D paper networks. Lab Chip 10, 918–920 (2010) 30. Jahanshahi-Anbuhi, S., et al.: Paper-based microfluidics with an erodible polymeric bridge giving controlled release and timed flow shutoff. Lab Chip 14, 229–236 (2014)

68

S.-G. Jeong et al.

31. Nagar, P., Chauhan, I., Yasir, M.: Insights into polymers: Film formers in mouth dissolving films. Drug Interv. Today 3, 280–289 (2011) 32. Zwanenburg, P., Li, X., Liu, X.: Magnetic valves with programmable timing capability for fluid control in paper-based microfluidics. In: 2013 IEEE 26th International Conference on Micro Electro Mechanical Systems (MEMS). IEEE, Piscataway, NJ (2013) 33. Hamedi, M.M., et al.: Electrically activated paper actuators. Adv. Mater. 26, 2446–2453 (2016) 34. Toley, B.J., et al.: A versatile valving toolkit for automating fluidic operations in paper microfluidic devices. Lab Chip 15, 1432–1444 (2015) 35. Martinez, A.W., et al.: Programmable diagnostic devices made from paper and tape. Lab Chip 10, 2499–2504 (2010) 36. Jahanshahi-Anbuhi, S., et al.: Creating fast flow channels in paper fluidic devices to control timing of sequential reactions. Lab Chip 12, 5079–5085 (2012) 37. Glavan, A.C., et al.: Folding analytical devices for electrochemical ELISA in hydrophobic RH paper. Anal. Chem. 86, 11999–12007 (2014) 38. Connelly, J.T., Rolland, J.P., Whitesides, G.M.: “Paper machine” for molecular diagnostics. Anal. Chem. 87, 7595–7601 (2015) 39. Lu, B., Zheng, S., Quach, B.Q., Tai, Y.-C.: A study of the autofluorescence of parylene materials for µTAS applications. Lab Chip 10, 1826–1834 (2010) 40. Fortin, J.B., Lu, T.-M.: Mass spectrometry study during the vapor deposition of poly-paraxylylene thin films. J. Vac. Sci. Technol., A 18, 2459–2465 (2000) 41. Spicar-Mihalic, P., et al.: CO2 laser cutting and ablative etching for the fabrication of paperbased devices. J. Micromech. Microeng. 23, 067003 (2013) 42. Ramachandran, S., Fu, E., Lutz, B., Yager, P.: Long-term dry storage of an enzyme-based reagent system for ELISA in point-of-care devices. Analyst 139, 1456–1462 (2014) 43. Zhang, M., et al.: Three-dimensional paper-based electrochemiluminescence device for simultaneous detection of Pb2+ and Hg2+ based on potential-control technique. Biosens. Bioelectron. 41, 544–550 (2013) 44. Focke, M., et al.: Lab-on-a-foil: Microfluidics on thin and flexible films. Lab Chip 10, 1365– 1386 (2010) 45. Lutz, S., et al.: Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA). Lab Chip 10, 887–893 (2010) 46. Thuo, M.M., et al.: Fabrication of low-cost paper-based microfluidic devices by embossing or cut-and-stack methods. Chem. Mater. 26, 4230–4237 (2014) 47. Glavan, A.C., et al.: Rapid fabrication of pressure-driven open-channel microfluidic devices in omniphobic RF paper. Lab Chip 13, 2922–2930 (2013) 48. Lewis, G.G., DiTucci, M.J., Phillips, S.T.: Quantifying analytes in paper-based microfluidic devices without using external electronic readers. Angew. Chem. Int. Ed. 51, 12707–12710 (2012) 49. Lewis, G.G., DiTucci, M.J., Baker, M.S., Phillips, S.T.: High throughput method for prototyping three-dimensional, paper-based microfluidic devices. Lab Chip 12, 2630–2633 (2012) 50. Ge, L., Wang, S., Song, X., Ge, S., Yu, J.: 3D origami-based multifunction-integrated immunodevice: Low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device. Lab Chip 12, 3150–3158 (2012) 51. Washburn, E.W.: The dynamics of capillary flow. Phys. Rev. 17, 273 (1921) 52. Fridley, G.E., Le, H., Yager, P.: Highly sensitive immunoassay based on controlled rehydration of patterned reagents in a 2-dimensional paper network. Anal. Chem. 86, 6447–6453 (2014) 53. Güder, F., et al.: Paper-based electrical respiration sensor. Angew. Chem. Int. Ed. 55, 5727–5732 (2016)

Technical Features and Challenges of the Paper-Based Colorimetric Assay Dongtak Lee, Insu Kim, Sang Won Lee, Gyudo Lee, and Dae Sung Yoon

1 Introduction The future of sensing and diagnostic devices for developing countries should be, as described by the World Health Organization (WHO), ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment free, and Deliverable to end-users [1]. Paper-based sensors using colorimetric approaches are significant because they feature all key factors for ideal diagnostic devices. Conventional sensors, such as enzyme-linked immunosorbent assay (ELISA), are bulky and complex, requiring different functional blocks including transducers, processing units, detection units, and so on, which can delay sensor response [2]. Current technology based on colorimetry is concerned with the miniaturization of size, decreasing cost, permitting in situ use, and achieving freedom from additional instruments. Moreover, colorimetric sensors can be used for the instantaneous detection of analytes (e.g., protein, DNA, small molecules, etc.), by showing a visually detectable color change [3]. The color change or intensity is proportional to the analyte concentration, which indicates that colorimetric assays have capacities beyond binary sensing. Recently, paper has been used as a substrate for constructing microfluidic devices to enhance the advantages of colorimetric assay. In detail, by patterning the paper substrate into detection areas of hydrophilic channels separated by hydrophobic barriers (or air), four-basic functions are achieved in the paper-based colorimetric sensor [4]: (i) Distribution of the sample to several spatially separated areas (replicate assay); (ii) Motion of the sample by capillary action (no pump required); D. Lee · I. Kim · S. W. Lee · D. S. Yoon (B) School of Biomedical Engineering, Korea University, Seoul 02841, South Korea e-mail: [email protected] G. Lee (B) Department of Biotechnology and Bioinformatics, Korea University, Sejong 30019, South Korea e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 J. H. Lee (ed.), Paper-Based Medical Diagnostic Devices, Bioanalysis 10, https://doi.org/10.1007/978-981-15-8723-8_4

69

70

D. Lee et al.

(iii) Compatibility with small sample amounts; and (iv) Facile removal of hazardous waste because the sensors can be disposed of by incineration. Because of these advantages, paper-based colorimetric sensors provide versatility and broad applicability. In this chapter, we discuss the development and challenges of colorimetric paperbased sensors and platforms using colorimetric detection strategies. We initially discuss different approaches to generate color on paper-based assays, addressing methods used to improve color generation and homogeneity. A discussion on measuring color changes using multiple reporting systems and a comparison of reporting systems is then presented. Finally, we critically evaluate the current problems facing colorimetric paper-based sensors and discuss general strategies for signal-to-noise improvement. We also include thoughts and insights for future research on this topic that may facilitate the use of low-cost diagnostic devices by those who need them most.

2 Colorimetric Transduction When paper-based colorimetric assays were introduced around 2007 [4, 5], the goal was to perform a colorimetric assay that included the incorporation of bioassays [4, 6] and to explore the colorimetric spot tests established by Feigl [7] with a technological approach, using information technology (IT) communication equipment [8]. In this section, we discuss different strategies used to generate color on paperbased devices through redox indicators, nanoparticle aggregation tests, and other colorimetric transduction methods.

2.1 Redox Indicators Redox indicators work when coupled with paper-based sensors and should have the following characteristics: (i)

Color absence in reduced form or a sharp color change between the oxidized and reduced form; (ii) Remarkable (intense) color in the oxidized form (elevated molar absorptivity); (iii) Sufficient color stability for the time duration of digitalization (complete analysis); and (iv) Low or no toxicity. In real systems, it is difficult to obtain all of these ideal characteristics simultaneously. The final observed coloration is mainly influenced by the contrast between

Technical Features and Challenges of the Paper-Based …

71

the reduced and oxidized colors of the redox indicator, the support material background (in this case, paper), the enzyme colors, and the color of the sample itself. Table 1 presents the most common redox indicators used in conjunction with paperbased sensors. Because most common redox indicators are used with oxidase-based systems, the stability of enzymes (i.e., oxidase) is of significant importance. We discuss strategies to improve the stability of oxidase in Sect. 4.

2.2 Nanoparticles Another sensing method for colorimetric detection on paper-based devices utilizes nanoparticles (NPs). NPs are essential in biomolecule detection because NPs can be labeled with antibodies, antigens, or oligonucleotides, and their optical properties enable naked-eye observation of bio-recognition [14]. The optical properties of metal nanoparticles arise from localized surface plasmon resonance (LSPR) [14], which yields a well-defined absorption band in the ultravioletvisible (UV-vis) spectrum (Fig. 1a). Many properties can affect the LSPR and thus its band intensity and maximum wavelength. The dielectric constant of the surrounding medium, the specific characteristics of the NP compositions, and the sizes and shapes of the NPs can all affect LSPR (Fig. 1b and c). NPs can be prepared from non-metallic sources, including polymeric latex and carbon. Here, we present a comprehensive review of paper-based sensors with NPs, with a few key examples highlighted. Table 2 compares multiple NPs with distinct sources.

2.2.1

Gold Nanoparticles (AuNPs)

Gold nanoparticles (AuNPs) are among the most popular NPs for bioconjugation and colorimetric assays because of their optical properties [19, 20]. AuNPs can be conjugated with antibodies to sense antigens (Fig. 2a) and used unmodified to sense antigens [21] and single-stranded oligonucleotides for tuberculosis diagnosis (Fig. 2b) or even used in Ag(I) analysis of drinking water [22–24]. Furthermore, the in situ growth of AuNPs in paper-based sensors can be used to sense the antioxidant activity of species in different matrices, including teas and wines [25].

2.2.2

Silver Nanoparticles (AgNPs)

The most common color of colloidal suspensions of silver nanoparticles (AgNPs) is yellow, but suspensions can also be orange, red, green, or even grayish in color by controlling the size and shape of the AgNPs [26, 16]. Yen et al. [27] produced multicolored AgNPs to differentially detect the Dengue, Yellow Fever, and Ebola viruses.

[11, 12]

[13]

λAbsorbance = N.A. λemission = 454 nm

λAbsorbance = 289 nm λemission = 417 nm

o-Phenylenediamine (OPD)

[10]

[9]

References

3,3 -Diaminobenzidine (DAB)

λAbsorbance = 340 nm λemission = 414 nm

Absorbance and emission

λAbsorbance = 285 nm λemission = 450 nm

acid) (ABTS)

Molecular structure

3,3 ,5,5 -Tetramethylbenzidine (TMB)

2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic

Indicator

Table 1 Common redox indicators for peroxide assays used in conjunction with oxidase-based systems on paper-based sensors

72 D. Lee et al.

Technical Features and Challenges of the Paper-Based …

73

Fig. 1 a Schematic of localized surface plasmon resonance (LSPR) on metal NPs. LSPR is the collective vibration and resonance of confined free electrons in the conduction band at the incident radiation frequency (ν). Incident radiation excites free electrons from the Fermi level (E F ) to a higher energy surface plasmon (SP) state. Far-field light is localized within a few nanometers of the surface. This localized light enhancement allows the plasmonic NPs to function as an ultrasensitive receiver optical antenna. Molecules near the antenna interact with the localized light field and are excited from the ground state (E 0 ) to the excited state (E 1 ). b Various AuNP shapes for LSPR enhancement, namely nanosphere, nanorod, nanoshell, nanocage, nanostar, nanocrescent, and Au plant viruses. c The LSPR shifts with various shapes and sizes of metal NPs. By varying the relative size and shapes of the metal NPs, the optical resonances can be systematically and precisely tuned over a broad range from the visible to the near-infrared regions. These figures are reproduced with permission from reference [14], © 2018 Nature Publishing Group

A common practice for utilizing Ag is the synthesis of core–shell particles to enhance the plasmonic properties of colloidal Au solutions [28]. This process remarkably enhances the visibility of colloidal Au, which can enhance the detection limit by orders of magnitude (from the nanomolar to picomolar range) [29].

74

D. Lee et al.

Table 2 Advantages and disadvantages of nanoparticles from different sources Type of nanoparticles

Advantages

Disadvantages

References

AuNPs

Instrument-free detection Easy to synthesize Facile surface modification Compatibility with biomolecules

Difficult for quantitative analysis Relatively low sensitivity, without the application of silver enhancement technique

[15]

AgNPs

Instrument-free detection Can be used with other NPs for signal enhancement High compatibility with biomolecules Possibility of synthesizing different colors, thus application in multiplexed assays

More synthetic steps

[16]

Latex

Instrument-free detection Good homogeneity Compatibility Low cost Different commercial particles available for application in multiplexed assays

Difficult synthesis Relatively low sensitivity and detection limit

[15, 17]

SiNPs

Instrument-free detection Good homogeneity Easy surface modification

More synthetic steps

[18]

2.2.3

Silica Nanoparticles (SiNPs)

Silica (SiO2 ) NPs (SiNPs) have various advantages, such as size uniformity and color homogeneity; Evan et al. [18] used SiNPs for multiple measurements of lactate, glucose, and glutamate (Fig. 3). With SiNPs, the signal intensity and homogeneity of paper-based sensors were significantly improved by facilitating the adsorption of selected enzymes and preventing the washing away effect that can create color gradients in colorimetric measurements (Fig. 3A and B). The color could change differently in each detection area (Fig. 3C), which indicated that this paper-based sensor allowed the simultaneous analysis of the three analytes with SiNPs.

2.3 Other Colorimetric Transduction Methods Redox indicators are not the only chromophores that can be used in conjunction with paper-based devices. In fact, most colorimetric spot tests for inorganic and organic analyses presented by Feigl [7] can be performed in a cellulosic matrix. Some such

Technical Features and Challenges of the Paper-Based …

75

Fig. 2 a Schematics of procedures for synthesizing functionalized Au nanoprobes featuring different orders of surface modification. Figures are reproduced with permission from reference [21], © 2018 American Chemical Society. b Schematic of the TB diagnostic method. For all the experiments, double-stranded DNA (dsDNA) with the detection probe single-strand DNA (ssDNA) sequences is mixed, followed by the addition of the AuNPs. If the dsDNA comprises the TB target sequence (extracted from a clinical sample), the ssDNA hybridizes with these strands, and the solution color becomes more blue after the addition of NaCl. On the other hand, if the TB target sequence is absent from the solution (i.e., TB negative), the color of the mixture remains red, even after the addition of NaCl. Figures are reproduced with permission from reference [22], © 2017 American Chemical Society

tests present sharper color changes than others, depending on the concentrations and initial colors of the samples, reagents, and final products. Others cannot be used with paper-based sensors because they require heating or organic solvents that are incompatible with some patterning methods used to fabricate paper-based sensors, including most wax printing [30]. The best-known examples of colorimetric reagents are acid–base indicators, such as 7-hydroxyphenoxazone, the chromophore present on litmus paper, pH sticks, the

76

D. Lee et al.

Fig. 3 A Optical images showing the colorimetric multiple assays for (a) and (b) lactate, (c) and (d) glucose, and (e) and (f) glutamate assays on native (without SiNPs) and SiO2 -modified (with SiNPs) papers. B Calibration curves for lactate, glucose, and glutamate assays performed on colorimetric paper-based sensors containing chemically modified SiNPs. C Optical image showing the analysis of an artificial urine sample spiked with lactate, glucose, and glutamate on the SiNPs-immobilized paper-based sensors. Figures are reproduced with permission from reference [18], © 2014 Royal Chemical Society

first example of paper-based sensors, are used routinely in laboratories to provide rapid estimations of pH without the use of complex equipment. However, the leaching of chromophore agents like pH indicators interferes with signal homogeneity. To minimize this effect, Lopez-Ruiz et al. [31] have introduced an ammonium quaternary salt in their testing zones to act as an ion-pairing agent with the charged forms of pH indicators. A novel approach to colorimetric sensing was proposed by Jang et al. [32]. The signal losses commonly observed during enzyme-mediated colorimetric sensing

Technical Features and Challenges of the Paper-Based …

77

were eliminated, and a pattern-free quantitative analysis of glucose and uric acid was achieved by mixing enzymes and color forming reagents with chitosan oligosaccharide lactate (COL) (Fig. 4A). The results are focused, intense colorimetric signals at

Fig. 4 A Color focusing effects of chitosan and its derivatives; color signals at six detection spots on the membrane treated with (i) nothing, (ii) 1 wt% methylglycol chitosan, (iii) 1 wt% glycol chitosan, (iv) 1 wt% chitosan (141 kDa), and (v) 3 wt% chitosan oligosaccharide lactate (COL). B Flow rate (mm/min) of the chitosan derivative-treated NC membranes. C Schematic of the color focusing effect based on the induced asymmetric flow on the COL film-treated NC membranes. Figures are reproduced with permission from reference [32], © 2019 American Chemical Society. D (i) Protein concentration-dependent response of the original paper-based sensor. (ii) The textdisplaying paper-based sensors comprise paper layers with printed indicators, 3D-printed units, and screening transparent film. (iii) Commercial colorimetric dipstick. Figures are reproduced with permission from reference [33], © 2017 American Chemical Society

78

D. Lee et al.

the detection spot, using asymmetric flow induced by alternating the flow rate of the COL-treated paper (Fig. 4B and C). Recently, Yamada et al. [33] presented a text-displaying paper-based analytical sensor that combined a classical colorimetric indicator system with an additional inert colorant, achieving a versatile text-displaying detection mechanism on a paper-based sensor (Fig. 4D). Entire text-displaying paper-based sensors have been developed based on printing techniques including inkjet printing, wax printing, and 3D printing. The Sikes group at MIT has developed a light-induced polymerization amplification method to enhance the colorimetric readout of paper-based devices [34]. The amplification method by polymerization is based on the covalent bonding between a photoinitiator molecule and a target analyte. With the proper monomers and visiblelight irradiation of the area, a photopolymerization reaction occurs specifically in the location of the photoinitiator (the detection zone). This photoinitiator indicates the presence of the analyte as an innovative reporting method for immunoassays. The use of colored complexes and compounds is another useful method for colorimetric detection on paper-based sensors and has been used for sensing heavy metals (e.g., Ag+ , Hg+ , and Pb2+ ) in different matrices, with limits of detection from parts per million to parts per billion [35–37]. In the presence of the target, a complex reaction occurs to generate the colored product. Another approach is the metal complexing indicator–displacement assay [38], which is useful for sensing highly reactive anions, such as sulfides. In this case, the color change is caused by the release of the previously complexed species from the metallic center; this species then interacts with the analyte of interest.

3 Reporting Systems After performing a colorimetric assay, the information displayed by paper-based sensors must be recorded. This is critical for instrument-dependent quantitative readouts, but it can be performed in many ways including scanning and photography for qualitative and semi-quantitative readouts, depending on the purpose. The information can be obtained by a standard analytical chemical instrumental technique, such as diffuse reflectance spectroscopy, or by IT communication equipment, including scanners and cell phone cameras [39], based on diffuse reflectance spectroscopy. Here, we review available methods for digitizing the results of paper-based assay results.

3.1 Scanners Instead of separating light, scanners use filters for three- or four-specific wavelengths before detecting light with a charge-coupled device (CCD) [40]. Data from scanners and cell phone cameras can be pre-processed by software and interpreted as results.

Technical Features and Challenges of the Paper-Based …

79

Because the results can vary depending on the scanning method, quantitative analysis requires an understanding of the differences between flatbed scanners, CCDs, and contact image sensors (CIS), as well as proper data processing [41]. Moreover, the success of this tool for quantitative analysis coupled with paper-based sensors arises from the conditions under which the test readings are obtained. When using a scanner, the focal length, lighting conditions, and background should be the same for single and continuous scans. This has been demonstrated by the results of Martinez et al. [42], which show that maintaining these conditions yields better reproducibility and performance.

3.2 Cell Phone Cameras Like scanners, mobile phone cameras can be applied to paper-based sensors at low cost. Martinez et al. stated that mobile phone camera-based methods are suitable for use with paper-based sensors because they are cheap, require little training for operation, and can transmit data from the hardware itself to off-site specialists [42]. Moreover, cell phones have broad global reach. Compared to other IT communication equipment, especially flatbed scanners, mobile phone cameras have minor drawbacks regarding performance [42, 43]. This contributes to the variability of internal analysis because of variations in lighting conditions, imaging angles, shadows, and focal lengths. Using unique hardware to device coupling strategies, researchers have worked to address these problems [44, 45]. Because the most common redox indicators are used with oxidase-based systems, the stability of the oxidase is important and is discussed more extensively in Sect. 4.

3.3 Luminosity and Lighting Conditions To avoid ambient stray light and provide a fixed focal length, Salles et al. [43] placed a cell phone in a clear poly (methyl methacrylate) (PMMA) box and illuminated it with four white light-emitting diodes (LEDs) to test for explosive compounds (Fig. 5a). This approach eliminated the inconvenience of cell phone camera detection by improving the reproducibility of the results and analytical methods. However, it reduces the universality of the method because it requires extra materials and some training and is only applicable to the dimensions of a particular mobile phone (in this case, an Apple® iPhone 4S) for the analysis of a specific design of paper-based sensor. These issues can be mitigated with careful engineering in the future. Another approach uses a 3D-printed external device with a flash diffuser attached to the mobile phone, as suggested by Oncescu et al. [46] (Fig. 5b). This device is used for the colorimetric measurement of the pH of saliva and sweat and is more advantageous than the PMMA box because of its portability (pocket size), but it

80

D. Lee et al.

Fig. 5 External devices to control luminosity and lighting conditions. a PMMA box for Apple® iPhone 4S for analyzing explosive compounds using paper-based sensors. This image is reproduced from reference [43] with permission © 2014 Royal Chemical Society. b 3D-printed external device support with flash diffuser attached to Apple® iPhone 4S to analyze biomarkers in sweat and saliva. This image is reproduced from reference [46] with permission © 2013 Royal Chemical Society. c Handheld reader for reflectance measurement using urinalysis paper as a probe. Figures are reproduced with permission from reference [47], © 2011 Royal Chemical Society

presents drawbacks regarding the limitations of extra material, training, and probe size.

3.4 Handheld Readers Ellerbee et al. described the use of a handheld platform for reading the colorimetric output of a paper-based microfluidic sensor [48]. This device measures transmittance rather than reflectance and is limited by its handheld operation, which causes difficulties in matching the refractive index of the paper with the appropriate solvents. Despite these problems, Lee et al. [47] reduced the error of a handheld device using an array reading method and demonstrated wireless communication as a proof-of-concept handheld device (Fig. 5c).

Technical Features and Challenges of the Paper-Based …

81

4 General Strategies to Improve Signal-to-Noise Ratio Paper-based sensors enable relative freedom in design [49] that permits the addition of multiple functionalities. Device modifications include the incorporation of preconcentrators and the addition of reagents in the reactional zone to improve colorimetric readouts [50]. Here, we review strategies such as concentrators, additives, and methods for sample and reagent preparation to improve colorimetric signals. Pre-concentrators in conjunction with paper-based devices have been under development since 2014 [51, 52], using multiple approaches. Wong et al. [51] obtained a 20-fold increase in concentration of a tuberculosis-specific glycolipid in artificial urine using a pre-concentrator system on a heat block. Han et al. [52] reported a 1000fold increase in the concentration of bovine serum albumin (BSA) and fluorescent dyes using the ion concentration polarization effect. The shelf life of paper-based sensors depends on the stability of their components, especially enzymes [42, 53]. Factors including temperature, pH, and humidity can impact enzymatic activity [53] and impair color formation on paper-based sensors. Thus, additives that prevent losses of enzymatic activity are extremely useful for extending the shelf life of these sensors. Sugars, sugar alcohols, and surfactants have been reported as enzyme stabilizers for paper-based sensors [54, 55]. Because of the concentration capabilities of paper, the final buffer concentration can be up to three times higher than the initial concentration [49]. This condition can affect the enzymatic activity and hinder homogeneous color formation on paperbased sensors. The use of a saline buffer, such as phosphate-buffered saline (PBS), is common in assays with antibodies and nanoparticles [56, 57], but care is necessary when working with enzymatic assays, as buffers can leach the enzymes to the borders of the reaction zones, which increases signal heterogeneity.

5 Conclusion The development of paper-based microfluidic dynamics has been underway for over a decade since paper-based sensors were first proposed in 2007. However, there is still room for improvement, and a discussion of colorimetric readings provides a potential pathway for achieving such improvements. Meeting the WHO requirements as specified by the ASSURED challenge requires much creativity and effort, but colorimetric paper-based sensors can meet these standards. To maximize the potential of colorimetric paper-based devices, the future of this research sector depends on colorimetric readers that do not require technology and external equipment. Because many variables can affect the color table results, the reproducibility and accuracy of paper-based sensors are critical. We hope that the insights reviewed here will facilitate solutions to some of these problems and raise new relevant questions about paperbased colorimetric analysis, which can greatly contribute to public health worldwide by improving the functionality and applicability of paper-based sensors.

82

D. Lee et al.

Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No. NRF-2019R1A2B5B01070617, NRF2020R1A2C2102262, and NRF-2018M3C1B7020722). Dr. Gyudo Lee is thankful for the financial support by Korea University Grant. This research was supported by the Korea University Graduate School Junior Fellow Research grant.

References 1. Peeling, R.W., Holmes, K.K., Mabey, D., Ronald, A.: Rapid tests for sexually transmitted infections (STIs): The way forward. Sex. Transm. Infect. 82, v1–v6 (2006). https://doi.org/10. 1136/sti.2006.024265 2. Cate, D.M., Adkins, J.A., Mettakoonpitak, J., Henry, C.S.: Recent developments in paper-based microfluidic devices. Anal. Chem. 87, 19–41 (2015). https://doi.org/10.1021/ac503968p 3. Morbioli, G.G., Mazzu-Nascimento, T., Stockton, A.M., Carrilho, E.: Technical aspects and challenges of colorimetric detection with microfluidic paper-based analytical devices (μPADs)—A review. Anal. Chim. Acta 970, 1–22 (2017). https://doi.org/10.1016/j.aca.2017. 03.037 4. Martinez, A.W., Phillips, S.T., Whitesides, G.M., Carrilho, E.: Diagnostics for the developing world: microfluidic paper-based analytical devices. Anal. Chem. 82, 3–10 (2009). https://doi. org/10.1021/ac9013989 5. Martinez, A.W., Phillips, S.T., Butte, M.J., Whitesides, G.M.: Patterned paper as a platform for inexpensive, low-volume, portable bioassays. Angew. Chem. Int. Ed. 46, 1318–1320 (2007). https://doi.org/10.1002/anie.200603817 6. Carrilho, E., Phillips, S.T., Vella, S.J., Martinez, A.W., Whitesides, G.M.: Paper microzone plates. Analy. Chem. 81, 5990–5998 (2009). https://doi.org/10.1021/ac900847g 7. Feigl, F., Anger, V.: Spot Tests in Inorganic Analysis. Elsevier Science, Amsterdam (2012) 8. Grudpan, K., Kolev, S.D., Lapanantnopakhun, S., McKelvie, I.D., Wongwilai, W.: Applications of everyday IT and communications devices in modern analytical chemistry: a review. Talanta 136, 84–94 (2015). https://doi.org/10.1016/j.talanta.2014.12.042 9. Boschloo, G., Hagfeldt, A.: Characteristics of the iodide/triiodide redox mediator in dyesensitized solar cells. Acc. Chem. Res. 42, 1819–1826 (2009). https://doi.org/10.1021/ar9 00138m 10. Effects of glass matrix on reactivity: Kadnikova, E. N. & Kosti´c, N. M. Oxidation of ABTS by hydrogen peroxide catalyzed by horseradish peroxidase encapsulated into sol–gel glass. J. Mol. Catal. B Enzym. 18, 39–48 (2002). https://doi.org/10.1016/S1381-1177(02)00057-7 11. Hulanicki, A., Gł˛ab, S., Ackermann, G.: Compleximetric indicators: characteristics and applications. Pure Appl. Chem. 55, 1137–1230 (1983). https://doi.org/10.1351/pac198355 071137 12. Stuart, S.: Redox Indicators: Characteristics and Applications. Pergamon, Oxford, UK (1978) 13. Steenken, S., Neta, P.: One-electron redox potentials of phenols. Hydroxy-and aminophenols and related compounds of biological interest. J. Phys. Chem. 86, 3661–3667 (1982). https:// doi.org/10.1021/j100215a033 14. Xin, H., Namgung, B., Lee, L.P.: Nanoplasmonic optical antennas for life sciences and medicine. Nat. Rev. Mater. 3, 228–243 (2018). https://doi.org/10.1038/s41578-018-0033-8 15. Quesada-González, D., Merkoçi, A.: Nanoparticle-based lateral flow biosensors. Biosens. Bioelectron. 73, 47–63 (2015). https://doi.org/10.1016/j.bios.2015.05.050 16. El-Nour, K.M.M.A., Eftaiha, A., Al-Warthan, A., Ammar, R.A.A.: Synthesis and applications of silver nanoparticles. Arabian J. Chem. 3, 135–140 (2010). https://doi.org/10.1016/j.arabjc. 2010.04.008

Technical Features and Challenges of the Paper-Based …

83

17. Lee, S., Mehta, S., Erickson, D.: Two-color lateral flow assay for multiplex detection of causative agents behind acute febrile illnesses. Anal. Chem. 88, 8359–8363 (2016). https:// doi.org/10.1021/acs.analchem.6b01828 18. Evans, E., Gabriel, E.F.M., Benavidez, T.E., Coltro, W.K.T., Garcia, C.D.: Modification of microfluidic paper-based devices with silica nanoparticles. Analyst 139, 5560–5567 (2014). https://doi.org/10.1039/C4AN01147C 19. Elghanian, R., Storhoff, J.J., Mucic, R.C., Letsinger, R.L., Mirkin, C.A.: Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 277, 1078–1081 (1997). https://doi.org/10.1126/science.277.5329.1078 20. Lee, D., Lee, G., Yoon, D.S.: Anti-Aβ drug candidates in clinical trials and plasmonic nanoparticle-based drug-screen for Alzheimer’s disease. Analyst 143, 2204–2212 (2018). https://doi.org/10.1039/c7an02013a 21. Huang, J.-Y., et al.: Signal amplified gold nanoparticles for cancer diagnosis on paper-based analytical devices. ACS Sens. 3, 174–182 (2018). https://doi.org/10.1021/acssensors.7b00823 22. Tsai, T.-T., et al.: Diagnosis of tuberculosis using colorimetric gold nanoparticles on a paperbased analytical device. ACS Sens. 2, 1345–1354 (2017). https://doi.org/10.1021/acssensors. 7b00450 23. Lee, D., et al.: Extremely sensitive and wide-range silver ion detection via assessing the integrated surface potential of a DNA-capped gold nanoparticle. Nanotechnology 30, 085501 (2018). https://doi.org/10.1088/1361-6528/aaf66f 24. Zhang, Y., Zuo, P., Ye, B.-C.: A low-cost and simple paper-based microfluidic device for simultaneous multiplex determination of different types of chemical contaminants in food. Biosens. Bioelectron. 68, 14–19 (2015). https://doi.org/10.1016/j.bios.2014.12.042 25. Choleva, T.G., Kappi, F.A., Giokas, D.L., Vlessidis, A.G.: Paper-based assay of antioxidant activity using analyte-mediated on-paper nucleation of gold nanoparticles as colorimetric probes. Anal. Chim. Acta 860, 61–69 (2015). https://doi.org/10.1016/j.aca.2014.12.025 26. Mulfinger, L., et al.: Synthesis and study of silver nanoparticles. J. Chem. Educ. 84, 322 (2007). https://doi.org/10.1021/ed084p322 27. Yen, C.-W., et al.: Multicolored silver nanoparticles for multiplexed disease diagnostics: distinguishing dengue, yellow fever, and Ebola viruses. Lab Chip 15, 1638–1641 (2015). https://doi. org/10.1039/c5lc00055f 28. Shankar, S.S., Rai, A., Ahmad, A., Sastry, M.: Rapid synthesis of Au, Ag, and bimetallic Au core–Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J. Colloid Interface Sci. 275, 496–502 (2004). https://doi.org/10.1016/j.jcis.2004.03.003 29. Horton, J., Swinburne, S., O’Sullivan, M.: A novel, rapid, single-step immunochromatographic procedure for the detection of mouse immunoglobulin. J. Immunol. Methods 140, 131–134 (1991). https://doi.org/10.1016/0022-1759(91)90135-3 30. Carrilho, E., Martinez, A.W., Whitesides, G.M.: Understanding wax printing: A simple micropatterning process for paper-based microfluidics. Anal. Chem. 81, 7091–7095 (2009). https://doi.org/10.1021/ac901071p 31. Lopez-Ruiz, N., et al.: Smartphone-based simultaneous pH and nitrite colorimetric determination for paper microfluidic devices. Anal. Chem. 86, 9554–9562 (2014). https://doi.org/10. 1021/ac501920 32. Jang, H., Park, J.-H., Oh, J., Kim, K., Kim, M.-G.: Advanced colorimetric paper sensors using color focusing effect based on asymmetric flow of fluid. ACS Sens. 4, 1103–1108 (2019). https://doi.org/10.1021/acssensors.9b00390 33. Yamada, K., Suzuki, K., Citterio, D.: Text-displaying colorimetric paper-based analytical device. ACS Sens. 2, 1247–1254 (2017). https://doi.org/10.1021/acssensors.7b00464 34. Badu-Tawiah, A.K., et al.: Polymerization-based signal amplification for paper-based immunoassays. Lab Chip 15, 655–659 (2015). https://doi.org/10.1039/C4LC01239A 35. Mentele, M.M., Cunningham, J., Koehler, K., Volckens, J., Henry, C.S.: Microfluidic paperbased analytical device for particulate metals. Anal. Chem. 84, 4474–4480 (2012). https://doi. org/10.1021/ac300309c

84

D. Lee et al.

36. Hossain, S.M.Z., Brennan, J.D.: β-Galactosidase-based colorimetric paper sensor for determination of heavy metals. Anal. Chem. 83, 8772–8778 (2011). https://doi.org/10.1021/ac2 02290d 37. Ariza-Avidad, M., et al.: Printed disposable colorimetric array for metal ion discrimination. Anal. Chem. 86, 8634–8641 (2014). https://doi.org/10.1021/ac501670f 38. Ariza-Avidad, M., Agudo-Acemel, M., Salinas-Castillo, A., Capitán-Vallvey, L.: Inkjet-printed disposable metal complexing indicator-displacement assay for sulphide determination in water. Anal. Chim. Acta 872, 55–62 (2015). https://doi.org/10.1016/j.aca.2015.02.045 39. Quesada-González, D., Merkoçi, A.: Mobile phone-based biosensing: An emerging “diagnostic and communication” technology. Biosens. Bioelectron. 92, 549–562 (2017). https://doi.org/10. 1016/j.bios.2016.10.062 40. Poce-Fatou, J., Bethencourt, M., Moreno-Dorado, F., Palacios-Santander, J.: Using a flatbed scanner to measure detergency: a cost-effective undergraduate laboratory. J. Chem. Educ. 88, 1314–1317 (2011). https://doi.org/10.1021/ed100635z 41. Göröcs, Z., Ozcan, A.: Biomedical imaging and sensing using flatbed scanners. Lab Chip 14, 3248–3257 (2014). https://doi.org/10.1039/C4LC00530A 42. Martinez, A.W., et al.: Simple telemedicine for developing regions: camera phones and paperbased microfluidic devices for real-time, off-site diagnosis. Anal. Chem. 80, 3699–3707 (2008). https://doi.org/10.1021/ac800112r 43. Salles, M., Meloni, G., De Araujo, W., Paixão, T.: Explosive colorimetric discrimination using a smartphone, paper device and chemometrical approach. Anal. Methods 6, 2047–2052 (2014). https://doi.org/10.1039/C3AY41727A 44. Mudanyali, O., et al.: Integrated rapid-diagnostic-test reader platform on a cellphone. Lab Chip 12, 2678–2686 (2012). https://doi.org/10.1039/c2lc40235a 45. Chun, H.J., Park, Y.M., Han, Y.D., Jang, Y.H., Yoon, H.C.: Paper-based glucose biosensing system utilizing a smartphone as a signal reader. BioChip J. 8, 218–226 (2014). https://doi.org/ 10.1007/s13206-014-8308-7 46. Oncescu, V., O’Dell, D., Erickson, D.: Smartphone based health accessory for colorimetric detection of biomarkers in sweat and saliva. Lab Chip 13, 3232–3238 (2013). https://doi.org/ 10.1039/c3lc50431j 47. Lee, D.-S., Jeon, B.G., Ihm, C., Park, J.-K., Jung, M.Y.: A simple and smart telemedicine device for developing regions: A pocket-sized colorimetric reader. Lab Chip 11, 120–126 (2011). https://doi.org/10.1039/c0lc00209g 48. Ellerbee, A.K., et al.: Quantifying colorimetric assays in paper-based microfluidic devices by measuring the transmission of light through paper. Anal. Chem. 81, 8447–8452 (2009). https:// doi.org/10.1021/ac901307q 49. Evans, E., Gabriel, E.F.M., Coltro, W.K.T., Garcia, C.D.: Rational selection of substrates to improve color intensity and uniformity on microfluidic paper-based analytical devices. Analyst 139, 2127–2132 (2014). https://doi.org/10.1039/C4AN00230J 50. Cheung, S.F., Cheng, S.K.L., Kamei, D.T.: Paper-Based Systems for Point-of-Care Biosensing. J. Lab. Autom. 20, 316–333 (2015). https://doi.org/10.1177/2211068215577197 51. Wong, S.Y., Cabodi, M., Rolland, J., Klapperich, C.M.: Evaporative concentration on a paperbased device to concentrate analytes in a biological fluid. Anal. Chem. 86, 11981–11985 (2014). https://doi.org/10.1021/ac503751a 52. Han, S.I., Hwang, K.S., Kwak, R., Lee, J.H.: Microfluidic paper-based biomolecule preconcentrator based on ion concentration polarization. Lab Chip 16, 2219–2227 (2016). https://doi. org/10.1039/C6LC00499G 53. Dungchai, W., Chailapakul, O., Henry, C.S.: Use of multiple colorimetric indicators for paperbased microfluidic devices. Anal. Chim. Acta 674, 227–233 (2010). https://doi.org/10.1016/j. aca.2010.06.019 54. Arakawa, T., Prestrelski, S.J., Kenney, W.C., Carpenter, J.F.: Factors affecting short-term and long-term stabilities of proteins. Adv. Drug Deliv. Rev. 46, 307–326 (2001). https://doi.org/10. 1016/s0169-409x(00)00144-7

Technical Features and Challenges of the Paper-Based …

85

55. Guan, L., et al.: A preliminary study on the stabilization of blood typing antibodies sorbed into paper. Cellulose 21, 717–727 (2014). https://doi.org/10.1007/s10570-013-0134-x 56. Nie, J., et al.: Low-cost fabrication of paper-based microfluidic devices by one-step plotting. Anal. Chem. 84, 6331–6335 (2012). https://doi.org/10.1021/ac203496c 57. Fu, E., et al.: Enhanced sensitivity of lateral flow tests using a two-dimensional paper network format. Anal. Chem. 83, 7941–7946 (2011). https://doi.org/10.1021/ac201950g

Paper-Based Nucleic Acid Detection for Point-of-Care Diagnostics Jongmin Kim and Yong-Ak Song

1 Introduction Nucleic acid detection has widely been used in key biosensing applications starting from diagnostics, food and water safety, and forensics. Traditionally operated in the laboratory, it has become an essential method to detect nucleic acids sensitively and reliably. With the increasing need of performing the nucleic acid detection outside of the traditional laboratory settings, the concept of “Lab-on-a-Chip”, colloquially known as microfluidic chips, has emerged to offer a portable platform for the fieldbased diagnostics. Using a fraction of the reagents and short travel length of fluids, it has enabled a rapid progress in point-of-care diagnostics. However, there are still formidable challenges lie ahead. One of the key bottlenecks for a wider acceptance of microfluidics in the main stream diagnostics has been the material of devices and the peripherals required to run the diagnostic device. Currently, polydimethylsiloxane (PDMS) material is a commonly used material for microfluidic applications. Despite its outstanding optical properties and easiness of molding and bonding, this material has intrinsically hydrophobic properties that require a pressure source to drive the sample fluid into the microchannel unless it has been treated prior to filling to become hydrophilic temporarily. The material cost is another prohibiting factor for a widespread use of PDMS.

J. Kim · Y.-A. Song (B) Division of Engineering, New York University Abu Dhabi (NYUAD), P.O. Box 129188, Abu Dhabi, United Arab Emirates e-mail: [email protected] Y.-A. Song Department of Chemical and Biomolecular Engineering, Tandon School of Engineering, NYU, Brooklyn, NY 11201, USA Department of Biomedical Engineering, Tandon School of Engineering, NYU, Brooklyn, NY 11201, USA © Springer Nature Singapore Pte Ltd. 2021 J. H. Lee (ed.), Paper-Based Medical Diagnostic Devices, Bioanalysis 10, https://doi.org/10.1007/978-981-15-8723-8_5

87

88

J. Kim and Y.-A. Song

In contrast, paper offers significant advantages in terms of the following properties. First, it is relatively cheap and easily available compared to polymeric materials. More importantly, it inherently possesses two distinctive physiochemical properties: One is hydrophilicity and another one is porous network playing a role as microchannel. These unique features enable (1) facile fluids transportation driven by capillary force on the paper substrates without an external pump, (2) sample, reagents storage, and filtration through the network structures, and (3) enhanced detection speed resulted by increasing the number of possible reagents in the porous network a with high surface to volume ratio. Therefore, the paper has been utilized partially or entirely for microfluidic analytical devices, known as paperfluidics (µPAD), and it is finding a wide-spread use in the analytical field, especially in nucleic acid-based point-of-care (POC) applications. In this chapter, we review some of the key aspects of paperfluidic devices for nucleic acid-based diagnosis with regard to sample preparation such as extraction and purification, amplification and electrokinetic sample preconcentration, and integration of multiple steps of nucleic acid analysis on chip. Last, but not least, the emerging field of synthetic biology- and CRISPR/Cas-based diagnostics in paperfluidics will be highlighted to show how this groundbreaking biotechnology is going to drive the field of paper-based diagnostics in future.

2 Key Aspects of Paperfluidic Devices for Nucleic Acid Detection 2.1 Sample Preparation/Purification For the decade, there have been numerous studies showing utilization of paperfluidics for nucleic acid analysis including sample preparation/purification [1–5], amplification [6–8], and detection [9–12]. Since the core detection principle is essentially the same across the different platforms, proper sample preparation is the first step to ensure high sensitivity and selectivity. Like any other molecular diagnostic platforms, nucleic acid-based diagnostics on paperfluidics require extensive sample preparations to remove all the non-important molecules and cellular components including proteins, nuclear membrane, and cell membrane that are present in complex samples such as blood, plasma, serum, saliva, urine, and cerebrospinal fluid (CSF). The preparation/purification step facilitates effective accessibility and interaction between the nucleic acids and enzymes (e.g., polymerase) and eventually enables highly sensitive and selective nucleic acid-based diagnosis. Initially, when the first paperfluidic device was proposed by Martinez et al. in 2007, the devices did not incorporate a sample preparation module, but performed necessary sample preparation steps outside the device using commercially available kits and tools. Since then, several paper-based sample preparation/purification approaches have been reported which utilize the surface chemical reactivity on the paper. Specifically, the hydroxyl groups on cellulose paper, one of the most abundant chemical moieties, have been

Paper-Based Nucleic Acid Detection for Point-of-Care …

89

utilized for various chemical reactions based on esterification, oxidation, etherification, amination, and radical copolymerization for bioassays [13, 14] and also enabled a facile binding ability with nucleic acids by strong polar interactions under specific manners [15]. Favored by the outstanding characteristics of paper for surface chemical modification, nucleic acid filtration and extraction, numerous nucleic acid sample preparation tools are made of paper or fibrous nitrocellulose. One of them is the Fast Technology Analysis (FTA) card. It has widely been used for DNA/RNA sample collection, extraction, and storage. These cards are made of paper material and have been chemically modified to lyse cell, denature proteins, prevent bacterial growth, and protect nucleic acids from nucleases, oxidative and UV damage [16]. The starting sample volume for FTA, however, is typically 125 µL that could potentially limit its applicability to some of the samples that are available only in lower amount. Given the same material characteristics shared by the sample preparation tools and paperfluidic devices, it is only a natural course of the technology development to merge them on a single platform. An ingenious way of integration of various sample preparation steps has been proposed inspired by the ancient Japanese paperfolding technique of origami where the sequence of folding of the paper can be aligned with the sample preparation steps. One of such integrated early devices has used guanidinium thiocyanate method that has been executed in sequence by sequential folding [1]. In the final step, DNA was then absorbed on Fusion 5 membrane. Similarly, filtration isolation of nucleic acid (FINA) technology has also been utilized for paper-based sample preparation [17]. This technology is based on alkaline (e.g., NaOH) extraction method to extract DNA. After capturing DNA on Fusion 5 membrane, the lysis debris is simply removed into absorption pad exploiting the vertical flow of chemical buffer between the stacked layers. Finally, for downstream analysis, the adsorbed DNA on paper filter is eluted in an elution buffer. This method allowed to extract 5.6–21.8 ng of DNA from 0.25 to 1 µL of blood higher than what the commercial QIAamp DNA Micro Kits can extract with reported amount of 3.6–13.0 ng. Following them, numerous paperfluidic approaches have been reported for extraction/purification of the nucleic acids from various samples such as blood sample [2], urine [18], and bacteria solutions [3]. For example, Byrnes et al. have developed a novel paperfluidic device based on a porous chitosan membrane, which purifies DNA up to 80% of the input DNA from the most types of the complex samples [2] within approximately 30 min (Fig. 1a). Depending on the pH, electrostatic adsorption between the DNA and the chitosan is simply controlled which enables capturing and elution of the DNA in a single step (Fig. 1b). This approach is not required to use guanidine and isopropanol, which is commonly known as PCR inhibitor [19]. Moreover, the approach has demonstrated simultaneous purification of the DNA from complex samples containing high protein content, excess non-target DNA, and blood. Since the approach can be used on a wide range of samples, it is quite promising and favorable to be coupled with downstream processes, such as qPCR, without further purification. RNA is also one of the most important target analytes in many POC platforms, but stepwise processes for preparation/purification of the RNA often cause unexpected degradation of the RNA due to their instability from hydrolysis by biological and

90

J. Kim and Y.-A. Song

Fig. 1 Purification of DNA and RNA on paperfluidic devices. a A schematic diagram of a chitosanbased paperfluidic device for DNA purification, and time sequence images showing purification of the DNA (green) indicated by white arrows. b A principle showing pH dependent capturing (pH < 6.3) and releasing (pH > 6.3) of DNA on chitosan functionalized paper. c A schematic diagram showing top and side view of the poly;ether sulfone (PES)] based paperfluidic device for purification of RNA. d A bright field image showing the paper/plastic hybrid device for RNA purification and visible blue film (inset) of RNA-glycoblue precipitate on the PES paper after purification step. Figures are adapted and reproduced from Refs. [2, 4] with permission from Royal Chemical Society (RSC) and American Chemical Society (ACS), respectively

environmental RNases. Rodriguez et al. have reported a facile approach based on paperfluidics for purification of RNA from clinical samples, which utilizes poly [ether sulfone (PES)] filter paper with an alcohol precipitation method [4]. Briefly, a nasopharyngeal swab (NPS) sample is mixed with lysis buffer and glycoblue coprecipitant, and then the mixture is loaded onto the PES membrane paper through the extraction setup inlet port (Fig. 1c). Because PES is hydrophilic, the capillary force allows only liquid phase to pass through the PES and eventually to reach the absorbent pad, while the solid phase of RNA-glycoblue precipitate is remained on the PES with visible blue film (Fig. 1d). Finally, the RNA is easily recovered in nucleasefree water due to the low protein/biomolecule adsorption characteristic of the PES. This approach is a simple and one-step purification process of the RNA on the low-cost PES paper, which prune centrifuges or other sample treatment equipment. However, it still requires elution step before downstream processes (i.e., isothermal amplification) for detection of influenza A (H1N1).

Paper-Based Nucleic Acid Detection for Point-of-Care …

91

2.2 Nucleic Acid Amplification 2.2.1

Polymerase Chain Reaction (PCR)

Given the small amount of nucleic acids extracted and inherent sample loss during the multiple sample preparation steps, amplification is mandatory for most of the paperfluidic detection systems. Polymerase chain reaction (PCR) as the major workhorse for nucleic acid detection since its invention in 1983 is clearly the first choice for such application. One of such combined approach has been reported in 2007 [20]. In this study, a lateral flow technique has been combined with PCR for end point detection. Due to its thermal cycling procedure and relatively high instrument cost, this amplification method requires more engineering efforts to combine with paperfluidics. Due to its relatively high cost, even though the price has significantly come down and the size of the instrument has decreased drastically for enhanced portability, it defeats the purpose of using the low-cost paperfluidic devices for POC. For this reason, this amplification approach has not been actively pursued in paperfluidic diagnostics.

2.2.2

Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) Compared to PCR, isothermal amplification methods are clearly offering significant technical advantages with their easiness in temperature control. Among many isothermal amplifications, loop-mediated isothermal amplification (LAMP) working at 60–65 °C [21] has been recognized as one of the most effective approaches, because it provides high specificity for recognition on the target which is resulted by use of six different primers. As a result, LAMP provides 100 times higher production rate for DNA generations than that of PCR. There are several studies showing utilization of papers for LAMP in microfluidics. Liu et al. firstly demonstrated LAMP on papers combined with poly(methyl methacrylate; PMMA) device to detect human immunodeficiency virus [22] (Fig. 2a). In their hybrid device, nucleic acids (DNA/RNA) were purified through Whatman FTA paper incorporated in the reaction chamber (Fig. 2b). Subsequently, LAMP was directly performed without elution of the nucleic acids thereby minimization of complicate flow control in the device with a thermocouple mounted underneath (Fig. 2c). The FTA paper has been further utilized for LAMP assay. Zhang et al. demonstrated LAMP on a microcapillary system prepared by using the FTA paper [23]. The entire microcapillary systems were prepared by just simple integration of a capillary with a small piece of FTA paper without any milling or photolithography processes. Linnes et al. showed a systematical comparison study of LAMP and RTLAMP on each different material such as chromatographic paper, PES, and PC and provided an insight into the choice of materials for the amplification efficiency of target DNA/RNA on paperfluidics [24].

92

J. Kim and Y.-A. Song

Fig. 2 LAMP assay on paper-based hybrid chips. a A schematic diagram of the paper-PMMA hybrid chip composed of three PMMA stacking layers. b The reaction chamber with FTA paper in the paper-PMMA hybrid chip. c The paper-PMMA hybrid chip with a thermocouple mounted underneath for LAMP assay. d The paper-tape hybrid chip composed of stacking of three paper substrate layers (absorbent pad, PES membrane, a lateral flow detection (LFD) strip) and tapes for LAMP assay. e A stepwise procedure including sample (blue), washing solvents (yellow) loading, and discarding the absorbent pad for purification of DNA. f LAMP assay on a LFD test strips sealed by hydrophobic tape. Figures are adapted and reproduced from Refs. [22, 25] with permission from Royal Chemical Society (RSC)

The Klapperich Group at BU showed the detection of cervical cancer via LAMP on the paperfluidic platform composed of three layers of papers [25] (Fig. 2d). It was made of hydrophobic tapes used for sealing and one-by-one alignment of three paper substrates functioning as an absorbent pad, purification filter, and membrane for filtration, respectively. First, a loop-mediated isothermal amplification (LAMP) assay was used for rapid amplification and detection of the HPV 16 E7 gene using primer sequences. An absorbent pad placed underneath a PES membrane to remove all impurities while the purified precipitated DNA remained on the membrane (Fig. 2e). After sample purification, the LAMP reaction mix was placed on the membrane. A cover film was placed on the sample port to prevent evaporation during the heat step (Fig. 2f). After the amplification, a hydrophobic tape barrier between the sample port and the lateral flow assay was removed to perform an assay on the paper strip. All five positive samples resulted in clear positive LFD results, while the five negative samples gave three negative and two false-positive results. These false-positive results indicated that further improvement of the LAMP was required. Like all other amplification

Paper-Based Nucleic Acid Detection for Point-of-Care …

93

methods, any potential impurities can be amplified with the target sequence resulting in false positives just like in the abovementioned application example which emphasizes the importance of sample preparation even more. In sum, a simpler approach based on amplification on paper, a minimized workflow, and fabrication of the paperfluidic heating method would also be necessary, so the high-risk HPV 16 in cervical samples could be detected without the need for sophisticated instrument and highly trained personnel. Recombinase polymerase amplification (RPA) Recombinase polymerase amplification (RPA) is one of the isothermal amplification methods using a recombinase, a strand displacement polymerase, and single-strand binding proteins. Specifically, the RPA runs within between 37 and 42 °C, lower than that of LAMP assay, is tolerant to temperature fluctuation of ±1 °C, and is fast with an operating time of 10–15 min. Rohrman et al. have firstly demonstrated RPA in a paper-plastic-based hybrid chip where target sequences amplify within region of cellulose and glass fiber of the chip [6]. The chip was fabricated by stacking of acetate sheets, double-sided adhesive, glass fiber matrix, and cellulose strip. Key features of the device are a capability of storage of RPA assay reagents (master mix and magnesium acetate) into different paper layers, hydrophobic wax patterns on a sample wick strip for direct sample introduction without pipetting, and facile mixing of reagents achieved by simply folding of the device (Fig. 3a). The commercially available dried blood spot (DBS) DNA extraction and lateral flow assay (LFA) were individually utilized for sample preparation and readout approaches as separate steps. The device showed a performance to amplify 10 copies of HIV DNA to detectable levels within 15 min at a constant temperature of 37 °C. Following up, the same group further developed a paper-plastic chip for detection of malaria [7]. A novel set of RPA primers was utilized which binds to sequences of the four species of Plasmodium. The RPA results were visually detected on a lateral flow dipstick within an hour. A limit of detection of synthetic Plasmodium DNA in the chip was 5 copies/µL (50 total copies) which is comparable to the performance from the benchtop approach. Although total test costs per one-time assay give approximately $8 when commercially available RPA kit and lateral flow strip considered, approximately $1 (only chip) per a chip fabrication can be a remarkable reduction of cost. This is a promising step toward the POC diagnosis for malaria detection on demand. In addition to RPA, reverse transcription-recombinase polymerase amplification (RT-RPA) on a wax-patterned paperfluidic chip has been demonstrated for detection of Ebola virus RNA by Magro et al. [26] (Fig. 3b). In their chip, the RT-RPA mixture was freeze-dried on paper in advance and rehydrated with distilled water or samples followed by heating at 40 °C for amplification. Control experiments were performed experiments at C+ region (positive control) containing preloaded RT-RPA reagents with the target RNA, and at C− region (negative control) containing preloaded RT-RPA reagents only. Patient samples were loaded at ST region (sample test) containing preloaded RT-RPA reagents. The signal was monitored over time by fluorescence, and the limit of detection on the chip was

94

J. Kim and Y.-A. Song

a

b

Fig. 3 RPA and RT-RPA assay on paperfluidic chips. a An image showing the workflow of RPA on a paper-plastic hybrid chip; RPA reagent loading on paper (two white pads), sample loading on waxpatterned sample wick strip, running of RPA in the folded chip, and detection results by LFA. b A scheme (upper) showing reverse transcription-recombinase polymerase amplification (RT-RPA) on the wax-patterned paperfluidic chip, and the fluorescent image (lower) showing detection of Ebola RNA on the chip. Figures are adapted and reproduced from Refs. [6, 26] with permission from Royal Chemical Society (RSC) and Springer Nature, respectively

107 copies/µL. One of the key features of the chip is the capability to store the RTRPA mixture on the chip by freeze-drying for one month without significant change of sensitivity up to 90% which is comparable to the result from conventional gold standard RT-RPA approach for the result obtained from 43 patient samples. Other Isothermal Amplification Methods Other isothermal amplification methods have been also reported on paperfluidic devices such as helicase-dependent amplification (HDA) [18, 27], strand displacement amplification (SDA) [28–32], and nucleic acid sequence-based amplification (NASBA) [33–35]. In the HDA, helicase enzyme is utilized to induce denaturation of double-stranded DNA instead of the conventional thermal denaturation, producing single-stranded

Paper-Based Nucleic Acid Detection for Point-of-Care …

95

templates. Then, the single-stranded templates are utilized for primer hybridization and subsequent primer extension by a DNA polymerase. Linnes et al. showed a simple approach based on chromatography paper incorporated in pipette tips to allow filtration of cell followed by in situ isothermal HDA [18]. The simplified workflow, optimized concentration of reagents, and primers of their approach enable shortening the reaction time to be 30 min then the previously reported paper-based HDA [36] and achieved 100 times higher sensitivity than the conventional immunoassays used for chlamydia diagnosis. Shetty et al. demonstrated HDA-based rapid amplification of Mycobacterium tuberculosis DNA on a chromatography paper substrate [27]. In their work, they showed that all HDA reagents, except the template DNA, can be stored on a single paper in a dried form at room temperature for more than a month without significant change of enzyme stability. This feature offers a promising potential as a point-of-care device that can be used in the field on demand. SDA is a rapid isothermal amplification method employing both a restriction endonuclease with an ability to nick a hemiphosphorothioate form of its recognition site and a DNA exonuclease deficient polymerase to initiate synthesis at a nick and displacing the downstream DNA strand. During each polymerase displacement step, the nicking site is regenerated which results in exponential amplification. This approach enables an amplification factor of 1010−12 from as few as 10 target copies within 10–30 min. Lafleur et al. applied SDA on two-dimensional paper network (2DPN) to detect methicillin-resistant Staphylococcus aureus (MRSA) bacteria [37]. They showed that the estimated sensitivity of ~5 × 103 input genomic copies corresponding ~600 genomic copies in each amplification reaction within less than an hour. NABSA is a sensitive amplification method for RNA, and there are several studies showing amplification based on the cartridges or tubes followed by LFA [33–35]. Compared to other amplification approaches, NABSA directly on paper is rarely reported, possibly due to an inability of NASBA to amplify DNA and requirement of even extra 95 °C denaturation step before amplification makes it less versatile [36, 38].

2.3 Electrokinetic Sample Preconcentration 2.3.1

Ion Concentration Polarization (ICP)

Considering the clinically relevant concentrations range from 10−12 to 10−16 M for most of diseases while the detection limits of LFA are around 10−11 M [39], efficient concentration of nucleic acid samples would be necessary. Even though PCR as well as isothermal amplification methods are more popular in the paperfluidic devices, there have been attempts to incorporate electrokinetic phenomena into concentration/enrichment of target analytes in lieu of PCR. Unlike enzymatic amplification methods, sample preconcentration based on electrokinetic phenomena simply increases the concentration locally at the site of

96

J. Kim and Y.-A. Song

detection by various physical or chemical means without resorting to enzymatic amplification using specifically designed primers and polymerases under heating. There are several types of the electrokinetic sample preconcentration approaches for nucleic acids, which have been applied on paperfluidics such as ion concentration polarization (ICP) [40, 41], faradaic ion concentration polarization (fICP) [44], isotachophoresis (ITP) [42, 43], and field-amplified sample stacking (FASS) [45] (Table 1). ICP is electrokinetic transport of ions through ion-selective membrane/nanostructures. When an external voltage is applied across fluidic channel, the concentration of ion at near the ion-selective membrane is reduced due to the perm-selectivity and surface charge, resulting in formation of a depletion zone. This polarization generates an uneven distribution of electric field in the channel and different migration speeds for the charged analytes at different positions. Eventually, the analytes get accumulated at where the electroosmotic and electrophoretic flow balanced which allows ICP-based preconcentration of target analytes without enzymatic amplification (Fig. 4a) [40]. Gong et al. demonstrated ICP-based preconcentration of hepatitis B DNA on a nitrocellulose paper-based device [40]. By simply depositing Nafion as a cationselective membrane on a wax-patterned paper chip (Fig. 4b), ICP functionality was provided on a wax-patterned paper chip. They showed ICP-based preconcentration, separation, and detection of hepatitis B DNA in human serum within 10 min of single operation (Fig. 4c). The approach achieved an LOD of 150 copies/mL and resolved four specific regions of the HBV genome. Following up, Nosrati et al. showed sperm chromatin integrity analysis on the same paper chips with ICP functionality to quantify both DNA fragmentation and packaging [41]. Under the applying voltage on the chip, high preconcentration of both ssDNA and dsDNA at the depletion achieved by ICP, allowing high signal amplification for efficient detection and downstream analysis for the sperm integrity (Fig. 4d). The percentage of DNA fragmentation index (%DFI) and the percentage of high DNA stability (%HDS) obtained from the paper-based assay strongly correlated (R2 ≥ 0.86) with the result from the conventional sperm chromatin structure assay (SCSA). In all case studies, the paper-based assay showed identical diagnostic information with low cost, and rapid and sensitive analysis compared with the conventional SCSA, and it eventually provides the clinical decision for each patient similar as SCSA.

2.3.2

Isotachophoresis (ITP)

The principle of ITP is based on electrophoretic mobility falling in between that of the leading ions (LE, high electrophoretic mobility) and that of the trailing ions (TE, low electrophoretic mobility). Under electric field, sample ions stack and concentrate between LE and TE zones according to their mobility. However, there is a problem of Joule heating and evaporation when using in paper. Using shallow channels, however, can alleviate the problem by allowing fast heat dissipation [46].

300

≤5

Synthetic DNA

100

≤5

FASS

18

≤4

dsDNA ladder

Synthetic DNA

60

10

E. coli DNA

fICP

ITP

15

Sperm DNA

150

40–140

≤10

Hepatitis B virus DNA

ICP

Voltage (V)

Time (min.)

Target sample

Approaches

Glass fiber

Nitrocellulose, Cellulose

Cellulose

Nitrocellulose

Nitrocellulose

Nitrocellulose

Type

Paper

–

Wax pattern, Stacking

Wax pattern, Origami

Wax pattern

Wax pattern

Wax pattern

Fabrication

Table 1 Electrokinetic sample preconcentration approaches applied for nucleic acids on paperfluidics

1000

264

100

–

–

100

Concentration (fold)

–

–

–

5 pM

–

150 copies/mL

LOD

[45]

[44]

[43]

[42]

[41]

[40]

References

Paper-Based Nucleic Acid Detection for Point-of-Care … 97

98

J. Kim and Y.-A. Song

a

c

b

d

Fig. 4 Ion concentration polarization (ICP) based sample preconcentration on paperfluidic chips. a A scheme showing principle of ICP on cation-selective membrane (Nafion) deposited paperfluidic chips. b A scheme showing wax-patterned paperfluidic chip with ICP functionality used for preconcentration of hepatitis B DNA in human serum and paper-based sperm DNA integrity test. c Preconcentrated hepatitis B DNA achieved by ICP on the paperfluidic chip. d Intact (dsDNA, green fluorescence) and damaged (ssDNA, red fluorescence) DNA, which is preconcentrated on the paperfluidic chip by ICP. Figures are adapted and reproduced from Refs. [40, 41] with permission from American Chemical Society (ACS) and Royal Chemical Society (RSC), respectively

Bercovici et al. have developed an isotachophoresis focusing functionality implemented on a nitrocellulose paperfluidic chip [42]. As a proof of concept of the chip operation, a 200 µL of fluorescent dye sample was concentrated with a factor of 20,000 times in a short paperfluidic channel with a length of 7 mm in approximately 6 min (Fig. 5A). When the chip was applied for amplification-free detection of E. coli-based DNA target with Morpholino as probes, a limit of detection of 5 pM was demonstrated in 10 min (Fig. 5B). In addition, they demonstrated potential of the chip as a multiplexed platform to run 12 assays simultaneously in a 24-well plate format by showing ITP-based concentration of fluorescent dye.

Paper-Based Nucleic Acid Detection for Point-of-Care …

99

Fig. 5 Isotachophoresis (ITP) based sample preconcentration on paperfluidic chips. A A paperfluidic chip used for ITP-based preconcentration of DNA for amplification-free detection of E. coli-based DNA target (an optical image) and demonstration of ITP by preconcentration of fluorescent dye on the paperfluidic chip (a fluorescent image). B A scheme (left) showing principle of the assay for amplification-free detection of DNA, and a quantitative graph (right) showing enhanced signal detection by ICP-based concentration of the DNA. C A Scheme showing a design of paper-based origami device for ITP focusing [oPAD-ITP (above)], and a location of preconcentration (S:red) in the oPAD-ITP (below). D Preconcentration of DNA on the oPAD-ITP shown by the time-resolved fluorescent images and shown by the quantitative data with concentration factor, peak position as a function of time. Figures are adapted and reproduced from Refs. [42, 43] with permission from Royal Chemical Society (RSC)

100

J. Kim and Y.-A. Song

Li et al. developed a paper-based origami device for ITP focusing (oPAD-ITP) by simple folding of the wax-patterned paper [43] (Fig. 5C). Briefly, a piece of predesigned wax-patterned paper is folded with center alignment. During the folding, a plastic slip is inserted into one of the folds, which are sandwiched between reservoirs of trailing electrolyte (TE) and leading electrolyte (LE). After loading the target DNA in the TE solution and LE solution at each reservoir, applying voltage induces ITP-based concentration of the target ssDNA and dsDNA (lengths of up to 1517 bps) with a concentration factor up to 100-fold in less than 4 min (Fig. 5D). Compared to the other lateral flow format for ITP, the oPAD-ITP chip minimizes the overall length for the assay, allowing high electric field generation with low-voltage sources (e.g., two 9 V batteries). This improvement enables portability for field deployment of the ITP-based concentration of targets analytes on paper chips, which is suitable for POC diagnosis on demand.

2.3.3

Other Electrokinetic Sample Preconcentration Approaches

Other electrokinetic sample preconcentration approaches have been also reported on paperfluidic devices such as faradaic ion concentration polarization (fICP) and field-amplified sample stacking (FASS). fICP is quite similar to ICP, but is different electrokinetic sample preconcentration approach in terms of the following major exceptions [47, 48]. Unlike non-faradaic ICP which requires selective charge transport via perm-selective membrane/nanojunction, the fICP uses a bipolar electrode to produce faradaic processes, eventually leading to generation of ion depletion zone (IDZ). Li et al. developed paperfluidic chips with fICP functionality by stacking of nitrocellulose and cellulose papers and printing conductive carbon pastes [44]. By using the chip with fICP, the target DNA was effectively preconcentrated up to 264-fold within 5 min. Compared to other electrokinetic sample preconcentration approaches, the key feature of this approach is elimination of an external circuit connection, a second power supply, and complicated perm-selective nanochannel. FASS utilizes uneven electric field which causes electrophoretic velocity change of target samples. Once ions enter into the boundary of two solutions, their electromigration velocity decreases, which eventually leading to stacking of target samples [45, 49]. During this process, the conductivity of the sample analytes is required to be lower than that of the background electrolyte for preconcentration/enrichment of the samples. Ma et al. firstly demonstrated FASS on paperfluidic chips with combination of a counter electroosmotic flow to improve concentration ability of the target sample [45]. The FASS on the paperfluidic chip enabled over 1000-fold signal enhancement of a fluorescence probe and target DNA within 300 s.

Paper-Based Nucleic Acid Detection for Point-of-Care …

101

2.4 Integration of Sample Preparation and Nucleic Acid Analysis on a Single Paperfluidic Chip In the early stage of the paper-based nucleic acid analysis, paperfluidic chips were utilized as a separated platform for sample preparation/purification, amplification, or terminal analysis, individually. Although paperfluidic chips for individual step of nucleic acids have shown remarkable progress, a fully integrated system of all nucleic acid analysis process on paperfluidic chips (i.e., sample-in-answer-out) is highly needed for a step toward to POC analysis. Ideally, integration of the nucleic analysis in a single paperfluidic chip without or with minimally uses of other materials is essential to meet up the demands such as device simplicity, user-friendly diagnosis, accessibility for resource-limited regions, and affordability. With this growing demand, several types of fully integrated paperfluidic chips for nucleic acid analysis have been developed [25, 50–53]. In 2015, LAMP on FTA paper equipped with a sliding device, so-called paper machine, was reported for integrated analysis of E. coli from human plasma by Connelly et al. [54] In their platform, the entire nucleic acid analysis consists of cell lysis, DNA extraction, and washing, and LAMP amplification was carried out by simple sliding of paper within the magnetic sliding strip device (Fig. 6a). They showed efficient LAMP for E. coli malB gene to proceed with an analytical sensitivity of one double-stranded DNA target copy, and a limit of detection was five cells. This platform is one of the promising cases for the fully integrated nucleic

a

b

c

Fig. 6 Fully integrated paperfluidic platforms for nucleic acid analysis. a The paper machine based on sliding strip. b The integrated paper-based lateral flow format. c The fully disposable and integrated paperfluidic platform with incorporation of battery-powered heating system. Figures are adapted and reproduced from Refs. [54–56] with permission from American Chemical Society (ACS) and Royal Chemical Society (RSC), respectively

102

J. Kim and Y.-A. Song

acid analysis on paperfluidics in consideration of elimination of using benchtop instruments such as centrifuges, pipettes, and vortex mixers, even though it still needs UV light source and imaging equipment for analyzing the result. Next, Choi et al. developed an integrated paper-based lateral flow format for detection of E. coli in spiked samples and Streptococcus pneumonia in blood samples [55] (Fig. 6b). In this platform, Fast Technology Analysis (FTA) card and glass fiber were used as substrates for target nucleic acid extraction and amplification, respectively. In addition, hydrophobic polyvinyl chloride (PVC) was incorporated together, acting as a valve to control fluid flow from extraction to amplification zone and lateral flow strip. This platform enabled successful detection of the target DNA via LAMP and on-site naked-eye detection from spiked drinking water, milk, blood, and spinach, and a limit of detection was as low as 10–1000 CFU/mL. Several efforts shown in this platform for elimination of instruments are quite remarkable steps toward POC such as simple control of fluids using hydrophobic PVC layers, a colorimetric signaling detection achieved by gold nanoparticles, and use of a portable battery-powered heating device. Next, a fully disposable and integrated paperfluidic platform was introduced by Tang et al. [56] (Fig. 6c). The platform is composed of papers playing a role as valves for nucleic acid extraction, sponge as reservoir for sample loading, on-chip integrated battery and heater for HDA amplification, and lateral flow assay strip for colorimetric detection. By using this platform, Salmonella typhimurium DNA, as a model target was detected within an hour. The limit of detection was as low as 102 CFU/mL in wastewater and egg and 103 CFU/mL in milk and juice. Notably, the onchip dried reagents storage and equipment-free amplification achieved by integration of battery-powered heating system are remarkable improvements. One of the challenges of fully integrated POC diagnosis for nucleic acid analysis is to introduce a capability for multiplex assay in a single platform. To this end, a fully integrated 3D origami paperfluidic chip for multiplex malaria diagnostics from whole blood has been developed by Xu et al. [57]. The original chip consisted of five panels folding onto each other and an acetate film cover for LAMP to avoid evaporation (Fig. 7A). The paper was folded after adding the sample of the device in panel 3 (Fig. 7B). That led to the first steps of the assay consisting of cell lysis and DNA extraction for purified DNA on the glass fiber paper. To transfer the DNA from the extraction panel to the amplification panel, the fold S1 was flipped to opposite side for elution of DNA. The eluted sample was then flown to the four holes where species-specific LAMP reagents were printed in advance. After sealing with an acetate film, the amplification was conducted at 63 °C for up to 45 min. on a simple hotplate. The results of species-specific LAMP were visualized with a handheld UV lamp of 365 nm wavelength. By using the 3D origami paperfluidic chip, multiple pathogens such as Plasmodium falciparum (98%), P. vivax (98%), and P. malariae (96%) from either finger-prick fresh blood sample or frozen blood have simultaneously been detected (Fig. 7C). Remarkable feature of the chip is that flow control with sequential steps from DNA extraction to detection, achieved by just simply folding the chip with minimal user intervention.

Paper-Based Nucleic Acid Detection for Point-of-Care …

103

Fig. 7 3D origami paperfluidic chip for multiplex malaria diagnostics from whole blood. A An optical image showing the 3D origami paperfluidic chip composed of five panels before folding (left) and folded chip (right). B Sequential folding steps of the 3D origami paperfluidic chip for LAMP-based nucleic acids assay. C Fluorescent images showing the result of multiplex LAMP amplification; numbers 1–4 indicate different species-specific LAMP reaction, individually: (1) Internal control (IC), (2) Plasmodium pan, (3) P. falciparum, (4) P. vivax.; letters a-d of each image indicate the different positive results: (a) 1 positive (IC), (b) 1 and 2 positive (IC and P. Pan), (c) 1, 2 and 3 positive (IC, P. Pan and P. falciparum), and (d) all positive. Figures are adapted and reproduced from Ref. [57] with permission from Wiley

3 Emerging Technologies of Paperfluidic-Based Diagnostics 3.1 Synthetic Biology-Based Approaches As introduced in the fore sections so far, for the past decades, starting from PCR to other isothermal amplification, numerous nucleic acid-based analysis have also been intensively highlighted as a major assay in life sciences, biosecurity, food safety, and environmental monitoring [58–60]. However, there are still serious threats from newly emerging or reemerging infectious diseases, unprecedented antimicrobial resistance like superbug, environmental pollution, and food contamination, especially in low resource or limited regions. Therefore, there is a significant need regarding improvements of performance in most of the nucleic acids-based diagnosis, especially its sensitivity and specificity for targeted detection. Recently, two

104

J. Kim and Y.-A. Song

emerging fields have been intensively investigated that have promising potential to provide synergetic effects by combination with paperfluidics: One is synthetic biology and another one is CRISPR/Cas-based diagnostics (Table 2). Synthetic biology is a new emerging field in biotechnology whereby manipulating genetic circuitry a new functionality in living organisms can be created. Using this approach, they can be “programmed” to do what they do not naturally do, for instance, producing or detecting molecules that are not occurring naturally. A great example is the reprogramming E. coli to produce insulin that has helped to save lives of millions of diabetic patients. Recently, the synthetic biology has also extended into several powerful synthetic gene networks that is capable of sensing and providing a measurable output [61, 66, 67]. The approach can be applied to create molecular sensors that can interrogate into living organisms without perturbations. One of the pioneers in the field, Collin’s Group at MIT, has developed a programmable molecular sensor, so-called RNA toehold switch that can virtually detect any RNA sequence [61, 62, 67]. Toehold switch is a sort of de-novo-designed prokaryotic riboregulators, composed of two RNA strands referred to as switch and trigger. The switch RNA comprises coding sequence of the gene regulated by both a strong ribosome binding site (RBS) and a start codon. As the trigger RNA (target) hybridizes to the hairpin via a toehold mediated strand displacement, RBS is exposed and subsequently codon starts, eventually initiating translation of the target gene. Upon response to cognate RNAs with arbitrary sequences, the toehold switch activates gene expression (Fig. 8A). To effectively utilize the toehold system as a sensor applicable for any of target nucleic acids, the trigger RNA is systematically designed to have no complimentary base to the RBS the start codon. Based on the mechanism, toehold switch has been utilized as a novel in vitro diagnosis platform. Pardee et al. have demonstrated mRNA quantification on a paper substrate combined with toehold switch [61]. For that, all reagents are freeze-dried on individual spots of paper disks, enabling in vitro cell-free transcription-translation system on a paper (Fig. 8B). Notably, the paper disks with freeze-dried reagents are stable at room temperature for over 1 year, and simple rehydration makes them activated. When the target RNA matched synthetic molecular construction, translation of a repressed gene is activated by ribosome, leading to colorimetric or fluorescent change after two hours of incubation. Using this feature and algorithm, the toehold switch-based paper sensor has shown its versatility in various target detection such as mRNA, antibiotic resistance gene mRNA for spectinomycin, chloramphenicol, kanamycin, ampicillin, and glucose. Remarkably, the toehold switch-based paper sensor with a panel of 24 has sensitively and strain specifically detected target mRNA within a day, which even distinguish differences in length by only three nucleotides between the Sudan and Zaire strains of Ebola. The sensitivity of trigger DNA for both strains was as low as 30 nM [61] (Fig. 8C). Following up, a toehold switch-based paper sensor-enabled detection mRNA of a panel of 10 bacteria related to inflammatory bowel disease (IBD), childhood malnutrition, and cancer immunotherapy [62]. In all these applications, the toehold switch sensors targeted the V3 hypervariable region of the 16S rRNA for each target species.

Zika virus RNA in patient sample

Synthetic Zika virus ssRNA

Synthetic Dengue virus ssRNA

EGFR L858R mutation

SHERLOCK, HUDSON

SHERLOCKv2

SHERLOCKv2

SHERLOCKv2

b Concentration

storage, reaction, and readout substrate of trigger DNA c It was varied depending on the specific toehold switch

a Reagents

Zika virus RNA

Mock Zika virus

CRISPR/Cas9 with Toehold switch

ssRNA 1

SHERLOCK

– ≤2.8 fM

2 aM 2 aM

≤1.5 ≤1.5 ≥2

0.9 aM

≤2

1–3

20 aM

20 fM

2

2

30 aM–3 fMc

3–5

10 bacterial mRNA

SHERLOCK

Cellulose

30 nMb

≤12

Ebola virus RNA (Sudan, Zaire)

CRISPR/Cas

Cellulose

–

Cellulose

HybriDetect 1

HybriDetect 1

HybriDetect 1

HybriDetect 1

Glass fiber

Glass fiber

Cellulose

Type 2

Paper

GFP mRNA, mCherry mRNA

LOD

Toehold switch

Time (h)

Analyte

Approach

Table 2 Emerging technologies combined with paperfluidics for nucleic acid-based analysis

Entire platform

Readout substrate

Readout substrate

Readout substrate

Readout substrate

Entire platform

Entire platform

Entire platform

Entire platform

Entire platforma

Role

[66]

[65]

[65]

[65]

[64]

[63]

[63]

[62]

[61]

[61]

References

Paper-Based Nucleic Acid Detection for Point-of-Care … 105

106

J. Kim and Y.-A. Song

Fig. 8 A toehold switch-based paper sensor for detection of Ebola virus with strain specificity. A A scheme showing principle of toehold switch. B The synthetic gene network with reagents stored by freeze-drying on a paper disk; the network includes genetically encoded tools with trigger, regulatory transducer, and output elements. (C) A composite image (left) showing colorimetric change of the 240 reaction on toehold switch-based paper sensor; yellow indicates control and untriggered toehold sensors and purple indicates activated toehold sensors, and a quantitative graph (right) showing sequence specificity for each of 4 Sudan and 4 Zaire sensors from the original set of 24. Figures are adapted and reproduced from Refs. [61, 67] with permission from Elsevier

Using the toehold switch in combination with NASBA, mRNA of three biomarkers associated with inflammation (calprotectin, CXCL5, and IL-8) and oncostaton M (OSM) could also be detected. The paper-based platform could also provide a method of diagnosing active Clostridium difficile infection (CDI) based on the detection of C. difficile toxin mRNA. The major advantages of this toehold detection platform over RT-qPCR are cost and multiplexing of multiple RNA transcripts. The detection limit for stool RNA ranges between 30 aM and 3 fM.

3.2 CRISPR/Cas-Based Approaches The CRISPR/Cas-system is revolutionizing the world of biology and biotechnology with its unprecedented capability to precisely edit genes. Great potential applications

Paper-Based Nucleic Acid Detection for Point-of-Care …

107

are anticipated in the treatment of various genetic diseases that, however, have raised serious ethical concerns recently. Likewise, it is to make a great impact on diagnostics. CRISPR/Cas-system, originally discovered in bacteria and archaea, is a sort of unique genomic element which plays a role as an adaptive immune system to defend the external invasion of phage or other foreign nucleic acids. In general, the system is mainly composed of a short-repeated DNA array known as the clustered regularly interspaced short palindromic repeats (CRISPR) and a type of CRISPR associated proteins (Cas) expressed by cas genes. Basically, CRISPR/Cas-based biosensing methods transfer the sequence information of target nucleic acids to detectable information (i.e., visual readout) such as fluorescence and colorimetric values. In general, CRISPR/Cas-based biosensing methods employ three major Cas effectors (Cas9, Cas13, and Cas12), which are the enzymatic units with an activity of the targetdependent cleavage. Typically, Cas9 and Cas12 recognize DNA, while Cas13 is RNA targeting enzyme. For the past years, CRISPR/Cas-based biosensing methods have been utilized for detection of various targets such as bacteria, virus, cancer mutations, human genotype, and single nucleotide polymorphisms (SNPs) [63–65, 68, 69]. Some of the studies show CRISPR/Cas-system combined with paper-based lateral flow strip which enables instrument-free assay, showing a potentially applicable for low- or limited resource region as an ideal POC diagnosis [64, 65]. So far, two major CRISPR enzyme-based diagnostic methods have been reported, specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) and DNA endonuclease-targeted CRISPR trans-reporter (DETECTR). Gootenberg et al. firstly demonstrated SHERLOCK based to detect specific strains of Zika and Dengue viruses, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in clinical samples [63]. In their work, RNA-guided Cas13a (previously known as C2c2) has been used as a Cas effector, which exhibits collateral effect of promiscuous ribonuclease (RNase) activity upon target recognition. On recognition of its RNA target, activated Cas13a starts cleaving nearby non-targeted RNAs simultaneously. By combining Cas13a with RPA, the SHERLOCK was able to detect the ssRNA and ssDNA 1 at concentrations down to 2 aM and 0.1 aM, respectively, that is more sensitive than Cas13a alone. It can also identify bacterial pathogens and bacterial genes, even with a single-based mismatch. Even for cancer diagnostics, SHERLOCK can detect low-frequency cancer mutations in cell-free DNA (cfDNA) fragments such as EGFR L858R (L, Leu; R, Arg) and BRAF V600E (V, Val; E, Glu) in mock cfDNA samples with allelic fractions as low as 0.1%. In addition, the SHERLOCK has been applied to paper-based assay showing detection ability for non-amplified ssRNA 1 down to 20 fM and mock Zika virus as low as 20 aM. Notably, SHERLOCK reaction reagents could be stored on paper by freeze-drying and can be readily reconstituted on paper by rehydration, promising for potential field applications. Following up, the second version of SHERLOCK (SHERLOCKv2) combined with lateral flow assay has been reported, enabling visualized detection with multiplex assay. In this approach, four orthogonal CRISPR enzymes (LwaCas13a, CcaCas13b, PsmCas13b, and Cas12a) were used for preferential cleavage at certain di-nucleotide combinations [65]. For readout on a later flow strip, destruction of

108

J. Kim and Y.-A. Song

FAM-biotin reporter was designed, which eventually enables accumulation of antiFAM antibody gold nanoparticle conjugates at the first line and prevention of binding the antibody gold nanoparticle conjugates to protein A at the second line of the strip after the end of reaction. This SHERLOCKv2-based paper sensor has demonstrated the instrument-free detection of Zika virus (ZIKV) or Dengue virus (DENV) ssRNA with sensitivity down to 2 aM within 90 min. Furthermore, the SHERLOCKv2based paper sensor was sensitive to detect single nucleotide mutation (EGFR L858R mutation) in liquid biopsies sample from non-small cell lung cancer patients. In the case of DETECTR-based biosensing approach, Cas12a-mediated transcleavage activity is utilized and is combined with isothermal amplification for one-pot detection. By using DETECTR-based detection, Chen et al. demonstrated rapid detection (≤2 h) with attomolar (aM) sensitivity for human papillomavirus (HPV) DNA [70]. For this approach, Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) is combined with RPA. The DETECTR-based detection identified HPV16 and HPV10 in SiHa cells and HeLa cells, respectively. Furthermore, it also accurately identified between HPV16 (25/25 agreement) and HPV18 (23/25 agreement) in a clinical sample composed of heterogeneous mixture of HPV, which shows good correlation with the results from PCR. There is one great study showing combinatorial utilization of CRISPR/Cas and toehold switch on a paper sensor, demonstrated by Pardee et al. [66]. By combining toehold switch with CRISPR/Cas9 which selectively cleaves the target DNA sequence in the presence of a NGG protospacer adjacent motif (PAM) domain, colorimetric discrimination of viral strains at single-base resolution was demonstrated on the paper sensor (Fig. 9a). To demonstrate the performance of the sensor,

a

b

Fig. 9 Combinatorial utilization of CRISPR/Cas and toehold switch on a paper sensor for discrimination of viral strains at single-base resolution. a A scheme showing mechanism of the combinatorial utilization of the CRISPR/Cas and toehold switch on the paper sensor. b Colorimetric discrimination between American Zika virus (yellow color) and African Zika virus (purple color) on the paper sensor. Figures are adapted and reproduced from Ref. [66] with permission from Elsevier

Paper-Based Nucleic Acid Detection for Point-of-Care …

109

American Zika virus and African Zika virus were used, because there is only a singlebase difference in the trigger regions of the two strains, and a PAM site that only exists in the American lineage sequence is an effective factor. Therefore, in the case of the African strain, full-length RNAs were amplified by NASBA without cleavage that subsequently activated the toehold switch turning the color change to purple. While, the American strain sequences were cleaved into fragments by Cas9, and were subsequently amplified via NASBA, but that had no capability to active the toehold switch, resulting in the color remained yellow (Fig. 9b). This is quite remarkable result showing synergetic effect of the two emerging technologies integrated with paper-based sensing approach. To quantify the detection of the Zika virus, a custom made electronic optical reader has been used. The toehold switch-based paper sensor has successfully detected Zika virus RNA with concentration at 2.8 fM (1.7 × 106 ) in plasma samples from an infected rhesus macaque which is clinically relevant level validated by qRT-PCR.

4 Conclusion and Perspective As introduced in this chapter, nucleic detection has been successfully implemented on paperfluidics. Due to the fact that paper is a natural fibrous material, suitable for filtration and extraction, and moreover, easily foldable, makes an integration of several sample preparation steps on the paper substrate possible. While paper has already been used for sample preparation, the possibility of folding to perform several steps in sequence makes it a unique design element of paperfluidics. The trend is going toward integration of all the sample preparation steps including detection on chip. Using an intelligent design of sliding of the sample layer or sliding of the barrier layer to open the vertical flow, it is possible to program a sequence of steps starting from cell lysis, nucleic acid extraction, purification, amplification, or concentration which can be performed on chip before optical or less frequently electrochemical detection. However, while sample preparation is crucial for integrated analysis when dealing with real-world samples, complete integration has still not been achieved. The ideal paperfluidics would enable quick answers from clinical samples with improved reagent storage capability, highly efficient nucleic acid extraction with minimal loss. Another exciting development is taking place in the areas of synthetic biology-based and CRISPR/Cas-based approaches. While synthetic biology is geared toward developing genetic circuit that can initiate a cascade of steps for detection, CRISPR/Cas allows detection based on the single sequence, enabling high sensitivity. The great promise of CRISPR/Cas lies in the fact that the sequence of polymerase can finely be tuned to target the matching sequence specifically and do the sensitive detection. In future, more highly sensitive CRISPR/Cas-based diagnostic devices implemented on paperfluidics are on the horizon. By taking together of advantages from paperfluidics and the newly emerged technologies, it is greatly feasible to improve sensing performance in terms of sensitivity, selectivity, and specificity, while keeping the excellences such as availability, field deployability, and cost-effectiveness available

110

J. Kim and Y.-A. Song

from using paper compared to other materials. Therefore, it is highly expected for emergence of revolutionary next-generation diagnosis platforms with outstanding performance.

References 1. Govindarajan, A.V., Ramachandran, S., Vigil, G.D., Yager, P., Bohringer, K.F.: A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami. Lab Chip 12, 174–181 (2012) 2. Byrnes, S.A., Bishop, J.D., Lafleur, L., Buser, J.R., Lutz, B., Yager, P.: One-step purification and concentration of DNA in porous membranes for point-of-care applications. Lab Chip 15, 2647–2659 (2015) 3. Fronczek, C.F., Park, T.S., Harshman, D.K., Nicolini, A.M., Yoon, J.Y.: Paper microfluidic extraction and direct smartphone-based identification of pathogenic nucleic acids from field and clinical samples. RSC Adv. 4, 11103–11110 (2014) 4. Rodriguez, N.M., Linnes, J.C., Fan, A., Ellenson, C.K., Pollock, N.R., Klapperich, C.M.: Paperbased RNA extraction, in situ isothermal amplification, and lateral flow detection for low-cost, rapid diagnosis of influenza A (H1N1) from clinical specimens. Anal. Chem. 87, 7872–7879 (2015) 5. Tang, R.H., Yang, H., Choi, J.R., Gong, Y., Hu, J., Feng, S.S., Pingguan-Murphy, B., Mei, Q.B., Xu, F.: Improved sensitivity of lateral flow assay using paper-based sample concentration technique. Talanta 152, 269–276 (2016) 6. Rohrman, B.A., Richards-Kortum, R.R.: A paper and plastic device for performing recombinase polymerase amplification of HIV DNA. Lab Chip 12, 3082–3088 (2012) 7. Cordray, M.S., Richards-Kortum, R.R.: A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA. Malaria J. 14 (2015) 8. Fang, X.E., Guan, M., Kong, J.L.: Rapid nucleic acid detection of Zaire ebolavirus on paper fluidics. RSC Adv. 5, 64614–64616 (2015) 9. Rigano, L.A., Malamud, F., Orce, I.G., Filippone, M.P., Marano, M.R., do Amaral, A.M., Castagnaro, A.P., Vojnov, A.A.: Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick. BMC Microbiol 14 (2014) 10. Kersting, S., Rausch, V., Bier, F.F., von Nickisch-Rosenegk, M.: Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malaria J. 13 (2014) 11. Crannell, Z., Castellanos-Gonzalez, A., Nair, G., Mejia, R., White, A.C., Richards-Kortum, R.: Multiplexed recombinase polymerase amplification assay to detect intestinal protozoa. Anal. Chem. 88, 1610–1616 (2016) 12. Chen, Y.T., Cheng, N., Xu, Y.C., Huang, K.L., Luo, Y.B., Xu, W.T.: Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor. Biosens. Bioelectron. 81, 317–323 (2016) 13. Cunha, A.G., Gandini, A.: Turning polysaccharides into hydrophobic materials: a critical review. Part 1. Cellulose. Cellulose 17, 875–889 (2010) 14. Credou, J., Berthelot, T.: Cellulose: from biocompatible to bioactive material. J. Mater. Chem. B 2, 4767–4788 (2014) 15. Ali, N., Rampazzo, R.D.P., Costa, A.D.T., Krieger, M.A.: Current nucleic acid extraction methods and their implications to point-of-care diagnostics. Biomed. Res. Int. (2017) 16. Tang, R.H., Yang, H., Choi, J.R., Gong, Y., Feng, S.S., Pingguan-Murphy, B., Huang, Q.S., Shi, J.L., Mei, Q.B., Xu, F.: Advances in paper-based sample pretreatment for point-of-care testing. Crit. Rev. Biotechnol. 37, 411–428 (2017)

Paper-Based Nucleic Acid Detection for Point-of-Care …

111

17. Jangam, S.R., Yamada, D.H., McFall, S.M., Kelso, D.M.: Rapid, point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from Whole Blood for Detection by Real-time PCR. J. Clin. Microbiol. 47, 2363–2368 (2009) 18. Linnes, J.C., Fan, A., Rodriguez, N.M., Lemieux, B., Kong, H., Klapperich, C.M.: Paper-based molecular diagnostic for Chlamydia trachomatis. RSC Adv. 4, 42245–42251 (2014) 19. Hagan, K.A., Reedy, C.R., Uchimoto, M.L., Basu, D., Engel, D.A., Landers, J.P.: An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents. Lab Chip 11, 957–961 (2011) 20. Aveyard, J., Mehrabi, M., Cossins, A., Braven, H., Wilson, R.: One step visual detection of PCR products with gold nanoparticles and a nucleic acid lateral flow (NALF) device. Chem. Commun. (Camb), pp. 4251–4253 (2007) 21. Niemz, A., Ferguson, T.M., Boyle, D.S.: Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 29, 240–250 (2011) 22. Liu, C.C., Geva, E., Mauk, M., Qiu, X.B., Abrams, W.R., Malamud, D., Curtis, K., Owen, S.M., Bau, H.H.: An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases. Analyst 136, 2069–2076 (2011) 23. Zhang, L., Zhang, Y., Wang, C.Y., Feng, Q.A., Fan, F., Zhang, G.J., Kang, X.X., Qin, X.Z., Sun, J.S., Li, Y.G., Jiang, X.: Integrated microcapillary for sample-to-answer nucleic acid pretreatment. Amplification, Detect. Anal. Chem. 86, 10461–10466 (2014) 24. Linnes, J.C., Rodriguez, N.M., Liu, L., Klapperich, C.M.: Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics. Biomed. Microdevices 18 (2016) 25. Rodriguez, N.M., Wong, W.S., Liu, L., Dewar, R., Klapperich, C.M.: A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples. Lab Chip 16, 753–763 (2016) 26. Magro, L., Jacquelin, B., Escadafal, C., Garneret, P., Kwasiborski, A., Manuguerra, J.C., Monti, F., Sakuntabhai, A., Vanhomwegen, J., Lafaye, P., Tabeling, P.: Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics. Sci. Rep-Uk 7 (2017) 27. Shetty, P., Ghosh, D., Singh, M., Tripathi, A., Paul, D.: Rapid amplification of Mycobacterium tuberculosis DNA on a paper substrate. RSC Adv. 6, 56205–56212 (2016) 28. Chen, J.H., Zhou, S.G., Wen, J.L.: Disposable strip biosensor for visual detection of Hg2+ based on Hg2+ -triggered toehold binding and exonuclease III-assisted signal amplification. Anal. Chem. 86, 3108–3114 (2014) 29. Huang, K.J., Shuai, H.L., Zhang, J.Z.: Ultrasensitive sensing platform for platelet-derived growth factor BB detection based on layered molybdenum selenide-graphene composites and exonuclease III assisted signal amplification. Biosens. Bioelectron. 77, 69–75 (2016) 30. Deng, X.L., Wang, C., Gao, Y., Li, J.W., Wen, W., Zhang, X.H., Wang, S.F.: Applying strand displacement amplification to quantum dots-based fluorescent lateral flow assay strips for HIV-DNA detection. Biosens. Bioelectron. 105, 211–217 (2018) 31. Phillips, E.A., Moehling, T.J., Bhadra, S., Ellington, A.D., Linnes, J.C.: Strand displacement probes combined with isothermal nucleic acid amplification for instrument-free detection from complex samples. Anal. Chem. 90, 6580–6586 (2018) 32. Toley, B.J., Covelli, I., Belousov, Y., Ramachandran, S., Kline, E., Scarr, N., Vermeulen, N., Mahoney, W., Lutza, B.R., Yagera, P.: Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-carediagnosis. Analyst 140, 7540–7549 (2015) 33. Rohrman, B.A., Leautaud, V., Molyneux, E., Richards-Kortum, R.R.: A lateral flow assay for quantitative detection of amplified HIV-1 RNA. Plos One 7 (2012) 34. Liu, H.X., Xing, D., Zhou, X.M.: Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor. Proc. Spie 9230 (2014) 35. Lee, H.H., Dineva, M.A., Chua, Y.L., Ritchie, A.V., Ushiro-Lumb, I., Wisniewski, C.A.: Simple amplification-based assay: a nucleic acid based point-of-care platform for HIV-1 testing. J. Infect. Dis. 201, S65–S72 (2010)

112

J. Kim and Y.-A. Song

36. de Paz, H.D., Brotons, P., Munoz-Almagro, C.: Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics. Expert. Rev. Mol. Diagn. 14, 827–843 (2014) 37. Lafleur, L.K., Bishop, J.D., Heiniger, E.K., Gallagher, R.P., Wheeler, M.D., Kauffman, P., Zhang, X.H., Kline, E.C., Buser, J.R., Kumar, S., Byrnes, S.A., Vermeulen, N.M.J., Scarr, N.K., Belousov, Y., Mahoney, W., Toley, B.J., Ladd, P.D., Lutz, B.R., Yager, P.: A rapid, instrumentfree, sample-to-result nucleic acid amplification test. Lab Chip 16, 3777–3787 (2016) 38. Craw, P., Balachandran, W.: Isothermal nucleic acid amplification technologies for point-ofcare diagnostics: a critical review. Lab Chip 12, 2469–2486 (2012) 39. Giljohann, D.A., Mirkin, C.A.: Drivers of biodiagnostic development. Nature 462, 461–464 (2009) 40. Gong, M.M., Nosrati, R., San Gabriel, M.C., Zini, A., Sinton, D.: Direct DNA analysis with paper-based ion concentration polarization. J. Am. Chem. Soc. 137, 13913–13919 (2015) 41. Nosrati, R., Gong, M.M., Gabriel, M.C.S., Zini, A., Sinton, D.: Paper-based sperm DNA integrity analysis. Anal. Methods 8, 6260–6264 (2016) 42. Rosenfeld, T., Bercovici, M.: Amplification-free detection of DNA in a paper-based microfluidic device using electroosmotically balanced isotachophoresis. Lab Chip 18, 861–868 (2018) 43. Li, X., Luo, L., Crooks, R.M.: Low-voltage paper isotachophoresis device for DNA focusing. Lab Chip 15, 4090–4098 (2015) 44. Li, X., Luo, L., Crooks, R.M.: Faradaic ion concentration polarization on a paper fluidic platform. Anal. Chem. 89, 4294–4300 (2017) 45. Ma, B., Song, Y.Z., Niu, J.C., Wu, Z.Y.: Highly efficient sample stacking by enhanced field amplification on a simple paper device. Lab Chip 16, 3460–3465 (2016) 46. Rosenfeld, T., Bercovici, M.: 1000-fold sample focusing on paper-based microfluidic devices. Lab Chip 14, 4465–4474 (2014) 47. Anand, R.K., Sheridan, E., Knust, K.N., Crooks, R.M.: Bipolar electrode focusing: faradaic ion concentration polarization. Anal. Chem. 83, 2351–2358 (2011) 48. Sheridan, E., Hlushkou, D., Anand, R.K., Laws, D.R., Tallarek, U., Crooks, R.M.: Label-free electrochemical monitoring of concentration enrichment during bipolar electrode focusing. Anal. Chem. 83, 6746–6753 (2011) 49. Tu, Y.Y., Hsieh, M.M., Chang, S.Y.: Sensitive detection of piperazinyl phenothiazine drugs by field-amplified sample stacking in capillary electrophoresis with dispersive liquid-liquid microextraction. Electrophoresis 36, 2828–2836 (2015) 50. Choi, J.R., Yong, K.W., Tang, R.H., Gong, Y., Wen, T., Li, F., Pingguan-Murphy, B., Bai, D., Xu, F.: Advances and challenges of fully integrated paper-based point-of-care nucleic acid testing. Trac-Trend Anal. Chem. 93, 37–50 (2017) 51. Choi, J.R., Tang, R.H., Wang, S.Q., Abas, W.A.W., Pingguan-Murphy, B., Xu, F.: Paperbased sample-to-answer molecular diagnostic platform for point-of-care diagnostics. Biosens. Bioelectron. 74, 427–439 (2015) 52. Hongwarittorrn, I., Chaichanawongsaroj, N., Laiwattanapaisal, W.: Semi-quantitative visual detection of loop mediated isothermal amplification (LAMP)-generated DNA by distancebased measurement on a paper device. Talanta 175, 135–142 (2017) 53. Park, B.H., Oh, S.J., Jung, J.H., Choi, G., Seo, J.H., Kim, D.H., Lee, E.Y., Seo, T.S.: An integrated rotary microfluidic system with DNA extraction, loop-mediated isothermal amplification, and lateral flow strip based detection for point-of-care pathogen diagnostics. Biosens. Bioelectron. 91, 334–340 (2017) 54. Connelly, J.T., Rolland, J.P., Whitesides, G.M.: “Paper Machine” for molecular diagnostics. Anal. Chem. 87, 7595–7601 (2015) 55. Choi, J.R., Hu, J., Tang, R.H., Gong, Y., Feng, S.S., Ren, H., Wen, T., Li, X.J., Abas, W.A.W., Pingguan-Murphy, B., Xu, F.: An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care. Lab Chip 16, 611–621 (2016) 56. Tang, R.H., Yang, H., Gong, Y., You, M.L., Liu, Z., Choi, J.R., Wen, T., Qu, Z.G., Mei, Q.B., Xu, F.: A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection. Lab Chip 17, 1270–1279 (2017)

Paper-Based Nucleic Acid Detection for Point-of-Care …

113

57. Xu, G.L., Nolder, D., Reboud, J., Oguike, M.C., van Schalkwyk, D.A., Sutherland, C.J., Cooper, J.M.: Paper-origami-based multiplexed malaria diagnostics from whole blood. Angew. Chem. Int. Edit. 55, 15250–15253 (2016) 58. Mumford, R.A., Macarthur, R., Boonham, N.: The role and challenges of new diagnostic technology in plant biosecurity. Food Secur 8, 103–109 (2016) 59. Zhao, Y.X., Chen, F., Li, Q., Wang, L.H., Fan, C.H.: Isothermal amplification of nucleic acids. Chem. Rev. 115, 12491–12545 (2015) 60. Scheler, O., Glynn, B., Kurg, A.: Nucleic acid detection technologies and marker molecules in bacterial diagnostics. Expert Rev. Mol. Diagn. 14, 489–500 (2014) 61. Pardee, K., Green, A.A., Ferrante, T., Cameron, D.E., DaleyKeyser, A., Yin, P., Collins, J.J.: Paper-based synthetic gene networks. Cell 159, 940–954 (2014) 62. Takahashi, M.K., Tan, X., Dy, A.J., Braff, D., Akana, R.T., Furuta, Y., Donghia, N., Ananthakrishnan, A., Collins, J.J.: A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nat. Commun. 9 (2018) 63. Gootenberg, J.S., Abudayyeh, O.O., Lee, J.W., Essletzbichler, P., Dy, A.J., Joung, J., Verdine, V., Donghia, N., Daringer, N.M., Freije, C.A., Myhrvold, C., Bhattacharyya, R.P., Livny, J., Regev, A., Koonin, E.V., Hung, D.T., Sabeti, P.C., Collins, J.J., Zhang, F.: Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017) 64. Myhrvold, C., Freije, C.A., Gootenberg, J.S., Abudayyeh, O.O., Metsky, H.C., Durbin, A.F., Kellner, M.J., Tan, A.L., Paul, L.M., Parham, L.A., Garcia, K.F., Barnes, K.G., Chak, B., Mondini, A., Nogueira, M.L., Isern, S., Michael, S.F., Lorenzana, I., Yozwiak, N.L., MacInnis, B.L., Bosch, I., Gehrke, L., Zhang, F., Sabeti, P.C.: Field-deployable viral diagnostics using CRISPR-Cas13. Science 360, 444–448 (2018) 65. Gootenberg, J.S., Abudayyeh, O.O., Kellner, M.J., Joung, J., Collins, J.J., Zhang, F.: Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439–444 (2018) 66. Pardee, K., Green, A.A., Takahashi, M.K., Braff, D., Lambert, G., Lee, J.W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N.M., Bosch, I., Dudley, D.M., O’Connor, D.H., Gehrke, L., Collins, J.J.: Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 165, 1255–1266 (2016) 67. Green, A.A., Silver, P.A., Collins, J.J., Yin, P.: Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014) 68. Qiu, X.Y., Zhu, L.Y., Zhu, C.S., Ma, J.X., Hou, T., Wu, X.M., Xie, S.S., Min, L., Tan, D.A., Zhang, D.Y., Zhu, L.Y.: Highly effective and low-cost MicroRNA detection with CRISPR-Cas9. ACS Synth. Biol. 7, 807–813 (2018) 69. Zuo, X.L., Fan, C.H., Chen, H.Y.: BIOSENSING CRISPR-powered diagnostics. Nat. Biomed. Eng. 1 (2017) 70. Chen, J.S., Ma, E.B., Harrington, L.B., Da Costa, M., Tian, X.R., Palefsky, J.M., Doudna, J.A.: CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018)

Paper-Based Biosensors with Lateral/Vertical Flow Assay Dohwan Lee and Jeong Hoon Lee

1 History and Market of the Lateral Flow Assay The lateral flow assay (LFA), which is a simple paper-based device for the detection and analysis of disease, is now the most predominant diagnostic platform in pointof-care (POC) markets [1–4]. This market-dominating power stems from the unique characteristics of the LFA: low cost, portability, versatility, easy use, free of other instruments, and results within 10–20 min [5–7]. In addition, the LFA is compatible with various biological samples such as whole blood, plasma, serum, urine, sweat, and saliva and can be used for the detection of bacteria, viruses, protozoa, nucleic acids, proteins, toxins, and heavy metals [2, 7, 8]. Therefore, the LFA is an ideal POC platform. The concept of the LFA technique was first introduced by Plotz and Singer in 1956, using the latex agglutination test to diagnose rheumatoid arthritis [1]. The most common form of the LFA at present is a gold nanoparticle (AuNP)-based colorimetric LFA, first presented by Leuvering et al. in 1980, called the “sol particle immunoassay” (SPIA) [1, 2, 4]. The first LFA product to be commercialized was ClearbluTM launched by Unipath Ltd. in 1988; it was developed for individuals to diagnose pregnancy at home. Because the level of human chorionic gonadotropin (hCG) hormones rapidly increase in the urine of a pregnant woman, this LFA can detect such an increase, which appears as two blue lines [1, 4]. In the 30 years since then, many LFA techniques have been developed and reported to show improved sensitivity and specificity, with reduced rates of false positives or negatives, and many new LFA products are now developed and marketed for a wide variety of purposes, including medical diagnosis, monitoring animal and plant health, agriculture, industry, identification of drug abuse, and detection of contamination and food toxicity (Fig. 1) [1, 5]. As the demand for POC tests in these fields has increased, the global market size for LFAs is expected to reach US$6284.9 million by 2024, D. Lee · J. H. Lee (B) Department of Electrical Engineering, Kwangwoon University, Seoul 01897, Republic of Korea e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 J. H. Lee (ed.), Paper-Based Medical Diagnostic Devices, Bioanalysis 10, https://doi.org/10.1007/978-981-15-8723-8_6

115

116

D. Lee and J. H. Lee

Fig. 1 Applications of the LFA technique. a Potential markets for LFA [2]. b Commercial LFA products. (i) DetermineTM HIV 1/2 Ag/Ab Combo. © 2013 Alere. All rights reserved. (ii) DetermineTM TB LAM Ag test. © 2013 Alere. All rights reserved. (iii) QuickVue Influenza A + B test. Courtesy of Quidel Corporation. (iv) Clearview® Malaria P.f. Test. © 2013 Alere. All rights reserved. (v) DirectigenTM EZ Flu A + B. Courtesy of Beckton Dickinson. (vi) ICON HP. Courtesy of Beckman Coulter. (vii) ImmunoCard STAT!® E. coli O157 Plus. Courtesy of Meridian Bioscience. (viii) A multiplex lateral-flow assay. RAIDTM 5 for the determination of biological threat agents. Courtesy of Alexeter Technologies [5]. Figure panels adapted and reproduced from Refs. [2, 5] with permission from The Royal Society of Chemistry

from US$4572.9 million in 2019 at a compounded annual growth rate (CAGR) of 6.6% [9, 10].

2 Basic Structure and Operation of the LFA The basic structure of the LFA consists of multiple segments combined together, including a sample pad, a conjugate pad, a reaction membrane-containing test and control line, and an absorbent pad (Fig. 2a) [5]. All segments, which comprise different types of porous membranes with specific functions, are serially connected to drive continuous capillary flow from the sample pad to the absorbent pad. The functions and typical materials of each segment are summarized in Table 1. To run a test with the LFA, first, a liquid sample (analyte) such as blood, urine, sweat, or saliva is applied to the sample pad (Fig. 2b, i), which absorbs the amount of the sample volume that the LFA is designed to analyze and thereby controls the flow rate of sample transfer to the conjugate pad, preventing sample overflow [1, 7]. In the conjugate pad, nano- or microparticles that are conjugated with recognition elements (e.g., antibodies or antigens) to the target analyte are positioned in dried form, typically as antibody-conjugated AuNPs. Thus, when the sample is transferred from the sample pad to the conjugate pad, the analyte in the sample is captured by the recognition element, forming analyte–particle complexes (Fig. 2b, ii) [3]. Because the sensitivity and specificity of the LFA are significantly affected by these recognition

Paper-Based Biosensors with Lateral/Vertical Flow Assay

117

Fig. 2 Schematic view of a components and basic structure of the LFA. Courtesy of Merck Millipore Corporation [5]. Figure panel adapted and reproduced from Ref. [5] with permission from The Royal Society of Chemistry. b Operation procedures of a typical LFA

Table 1 Functions and materials for each component of the LFA [1, 5, 6] Components

Material

Function

Sample pad

Cellulose, cotton linter, and glass fiber

Absorbs a specific volume of the sample and distributes the sample in a controlled flow rate to the next stage

Conjugate pad

Glass fiber, polyester, and fused glass

Preserves the dried conjugated particles until the test is complete. Capturing of analytes by the conjugate particles occurs in this pad

Reaction membrane

Nitrocellulose, nylon, and polyvinylidene fluoride

Enables confirmation of test results by producing visible bands when analytes are present or absent in the sample. Test and control lines are in this pad

Absorbent pad

Cellulose and cotton linter

Serves as a waste reservoir to drain off the remaining fluid after the test, preventing wicking towards the reaction membrane

probes, the use of the correct probe is necessary in determining the performance of the LFA [4]. The analyte–particle complexes and the excess particles that did not form complexes continue migrating to the next reaction membrane by capillary flow. In the reaction membrane, other recognition probes (e.g., antibodies, antigens, or biotins) have been immobilized on the test and control line as bands. The analyte– particle complexes and excess particles are captured by these probes on the test and control line, respectively (Fig. 2b, iii). The nano- or microparticles emit signals in colorimetric or fluorescence form, yielding visual results from the test and control line that are visible to the naked eye or by using a LFA readout device [1, 3]. Most LFAs are adequate for qualitative or semi-quantitative analysis, but some LFA kits and readers can perform quantitative measurements based on signal intensities. The

118

D. Lee and J. H. Lee

remaining reagent, after passing through the test and control line, is absorbed by the absorbent pad as waste.

3 Formats of the LFA In terms of results interpretation, the LFA can be divided into two main formats: sandwich (i.e., direct) and competitive (i.e., inhibitive) assays (Fig. 3). The sandwich format is typically used for the detection of high-molecular-weight analytes with multiple antigenic sites (e.g., hCG, Dengue antibody or antigen, or HIV) [3]. The signals in both the test and control lines indicate that the LFA was operated correctly and that the sample contains the target analytes. If the analytes are not present in the sample, the signal only appears on the control line. The competitive format is typically applied for the detection of low-molecular-weight analytes with single antigenic sites (e.g., pesticides or toxins) [1, 3]. In such assays, the analytes are pre-immobilized on the test line to compete with the analyte in the sample for binding with the detection antibody. The absence of a signal on the test line indicates a positive result, that is, the presence of the analyte in the sample. Obviously, if the signal does not appear on the control line, whether it is a sandwich or competitive assay, it indicates the malfunction of this LFA and the result is unreliable. These two formats are the most common, but other formats exist, such as the vertical flow assay, which follows the same principle as the LFA but the fluid flows vertically. Other formats have certain advantages (e.g., faster assay times or greater specificity); however, they often require multiple extra steps such as sample loading, washing, and the addition of gold conjugates [5]. Thus, compared to the LFA technique, these are less commonly accepted by users because of their specialized skill requirements or troublesome use.

Fig. 3 Two main formats of the LFA. a Sandwich (direct) and b competitive (inhibitive) assay

Paper-Based Biosensors with Lateral/Vertical Flow Assay

119

4 LFA Applications for the Detection of Various Analytes Since ClearbluTM was launched commercially in 1988, the LFA technique has inspired many researchers and companies to employ it for specific purposes. Because the LFA can be the most effective solution for fields where POC testing and selfor home diagnosis are required, many modified LFA have been developed for applicability to different types of analytes, such as proteins, bacteria, viruses, protozoa, heavy metals, and drugs. The overview of LFA applications for various analytes is summarized in Table 2.

5 Recent Advances in LFA Techniques 5.1 Architecture Tuning for Flow Control in the LFA Once users begin an assay by dropping a liquid sample onto the sample pad, the operation of a typical LFA is initiated. The dropped reagent sample continues to flow until it saturates the absorbent pad. By tuning the device architecture, fluidic flow of the reagents required for the LFA operation can be controlled. The individual fluidic flow control of a sample containing target analytes, the signaling nano- or microparticles (e.g., antibody-AuNPs), and the reagent for signal amplification (e.g., gold enhancers) can improve the sensitivity and specificity of the LFA. Lutz et al. [35] presented a method for creating programmable flow delays in a paper network by applying dissolvable sugar to paper. Different concentrations of sucrose solution (10–70%) have differences in sucrose dissolution time, thus creating programmable delays in fluidic flow (Fig. 4a). Four sugar-treated branches with different concentrations enable the timed sequential delivery of the dyed PBST solution, with the resident time of each dyed solution controlled by the volume (11 μL); the yellow dye flows first, then light blue, red, and dark blue dyes sequentially (Fig. 4b). Based on this strategy, they designed a simple folding card format to detect a malaria diagnostic biomarker (PfHRP2) in fetal bovine serum. The four-step fluidic process required for the LFA-based immunoassay of PfHRP2 was automated into a single step; the sample of PfHRP2 antigen mixed with anti-PfHRP2-AuNPs, washing buffer, and gold enhancement reagent were sequentially flowed to the detection zone (Fig. 4c). This sequential delivery of each solution enabled sensitive malaria detection with 2.6-fold signal amplification (Fig. 4d, initial signal: ~0.07 and amplified signal: ~1.8). Toley et al. [36] reported a similar concept for the detection of PfHRP2. Their concept delayed fluid progress through a porous channel by diverting fluid into an absorbent pad-based shunt, which was placed in contact with a nitrocellulose paper channel (Fig. 5a). By modulating the length and thickness of the shunt, the flow delay time was controlled (Fig. 5b). They designed a three-legged paper device in which each leg had a shunt of a different length and was connected to a different

120

D. Lee and J. H. Lee

Table 2 Overview of the LFA applications for various analytes [1, 2, 8] Applications

Target

Sample

Sensitivity

Analysis time

References

Clinical analysis

hCG

Urine

~50 IU/L

1–10 min

[11]

PSA

Serum

1 ng/mL

20 min

[12]

Thrombin

Plasma

2.5 nM

10 min

[13]

Troponin I

Serum

0.01 ng/mL

10 min

[14]

Human IgG

PBS buffer

200 pg/mL

~20 min

[15]

Escherichia coli O157:H7

Milk

5 × 103 CFU/mL 20 min

Salmonella enteritidis

Raw eggs 107 cells/mL

20 min

[17]

Staphylococcus aureus

Food samples

0.9 CFU/g

10 min

[18]

Bacillus anthracis

Milk powder and soil

2 × 104 (milk powder) and 1.3 × 104 (soil) CFU/mL

30 min

[19]

Listeria monocytogenes

Milk and cheese

20 CFU/mL

15 min

[20]

Influenza A (H3N2)

HEPES buffer

2 × 106 virus particles

15 min

[21]

HIV

Serum and plasma

30 pg/mL

40 min

[22]

Ebola

Serum

200 ng/mL

15 min

[23]

Norovirus

PBS buffer

107

virus-like particles per mL

20 min

[24]

Zika

Blood

Single copy

35 min

[25]

Malaria

Plasma

10 ng/mL

>20 min

[26]

Leishmaniasis

Dog blood

1 parasite/100 μL 10 min of DNA sample

[27]

Bacteria

Virus

Protozoa

Heavy metal

Drug

[16]

Schistosomiasis

Serum

30 pg/mL

2h

[28]

Pb2+

Paints

0.5 μM

15 min

[29]

Hg2+

Water

0.1 nM

30 min

[30]

Cd2+

Water

0.4 ppb

10 min

[31]

Morphine

Urine

2.5 ng/mL

10 min

[32]

Chloramphenicol

Milk

10 ng/mL

10 min

[33]

Ofloxacin

Food samples

30 ng/mL

10 min

[34]

Paper-Based Biosensors with Lateral/Vertical Flow Assay

121

Fig. 4 Programmable fluidic flow delays based on sucrose dissolution time [35]. a Different concentrations of sucrose yield different flow delays. b Timed sequential fluid delivery with this strategy. The yellow dye flows first, then light blue, red, and dark blue sequentially. c A card format to detect malaria using the signal-amplified LFA-based immunoassay. The four-step process required for the test was automated into a single step by this strategy. d The signal for malaria detection was amplified. Figure panels adapted and reproduced from Ref. [35] with permission from The Royal Society of Chemistry

fluid source (Fig. 5c, green dotted line, first leg: no shunt, second leg: 5.1-mm shunt, and third leg: 20.3-mm shunt). According to the intended delay, three different dyed solutions (red, yellow, and pink) flowed sequentially into the detection zone and the resident time of each solution in the detection zone was controlled by the solution volume (12, 20, and 80 μL of red, yellow, and pink, respectively). By replacing each fluid source area with a solution for malaria immunoassay, they demonstrated the automated and signal-amplified LFA-based immunoassay of PfHRP2 (Fig. 5d). Toley et al. also developed a toolkit of paper-based microfluidic valves automatically actuated by movable paper strips and fluid-triggered expanding elements [37]. The introduction of fluids to the device automatically triggered valve actuation after a certain period of time, determined by the passage of a certain volume of fluid. For the fully automated and signal-amplified LFA-based immunoassay of PfHRP2, the only required steps were the application of a 30 μL sample and adding 2 mL water. Shin et al. [38] suggested another method for flow control by pressing a specific region of the paper (or polypropylene sheet), which increased the resistance to fluid

122

D. Lee and J. H. Lee

Fig. 5 Tunable delay of fluidic flow using absorbent pad-based shunts [36]. a Fluidic flow through a porous channel in the absence (control) and presence of a shunt. b The parameters for tuning the delay are the length and thickness of the shunt. c The three-legged paper device where each leg has a shunt of a different length, which enables the sequential flow of the red-, yellow-, and pink-dyed solutions. d Demonstration of the signal-amplified LFA-based immunoassay of malaria with this strategy. Figure panels adapted and reproduced from Ref. [36] with permission from the American Chemical Society

flow in this region by reducing the pore size and permeability (Fig. 6a). Depending on the pressure, the flow rate could be modulated (Fig. 6b, fluid front at 30 s) and slowed by 740%. The Y-shaped merging design, where different pressures were applied on three branches, allowed the sequential loading of dyed water into the merging region (Fig. 6c); the red dye flowed first, then yellow and blue dyes sequentially. With this concept, they simplified the signal-amplified LFA-based immunoassay, which required the sequential loading of a sample, antibody-AuNPs, and gold enhancer, into one step and demonstrated the detection of progesterone receptors with 4.3-fold signal intensification (Fig. 6d). As other approaches, Rivas et al. [39] proposed a simple and facile method to improve the LFA sensitivity by patterning hydrophobic barriers on the LFA with a wax printer. Several wax pillar patterns were printed on the nitrocellulose reaction membrane; these delayed fluid flow and generated pseudo-turbulence in the pillar zone, thus increasing the chance of binding between the analyte and labeled antibodies to increase immunocomplex formation. The effect of the wax pillar-modified LFA was evaluated by the detection of HIgG protein; the pillar modification improved the sensitivity by almost three-fold. Choi et al. [40] suggested a similar strategy incorporating a paper-based shunt and a polydimethylsiloxane (PDMS) barrier to achieve optimal fluidic delays for LFA signal enhancement. With the optimal shunt size and the number of PDMS barrier droplets, they achieved a 10-fold signal enhancement over an unmodified LFA with highly sensitive detection of the Hepatitis B virus of ~102 IU/mL, comparable to laboratory-based assays.

Paper-Based Biosensors with Lateral/Vertical Flow Assay

123

Fig. 6 Programmed liquid delivery by paper pressurization [38]. a A paper strip compressed at a specific region where fluid flow is delayed. b The flow rate can be modulated by modifying the amount of applied pressure. c Three-branched paper channels with regions under different amounts of pressure (channel 1: no pressure, channel 2: 15.7 MPa, and channel 3: 31.4 MPa). Three different dyed solutions sequentially flow in the order of red, yellow, and blue. d Demonstration of the signal-amplified LFA-based immunoassay for the detection of progesterone receptors. Figure panels adapted and reproduced from Ref. [38] with permission from AIP Publishing

5.2 Different Nanomaterials for Labeling Conjugates Conjugate labeling is the component of the LFA that generates colorimetric or fluorescent signals. Because conjugate labeling significantly contributes to the sensitivity of the LFA, several types of nanomaterials have been reported as candidate conjugates for highly sensitive LFA, including AuNPs, colored latex beads, magnetic particles, quantum dots (QDs), liposomes, and carbon particles or carbon nanotubes (CNPs/CNTs). The most widely used nanomaterial in commercial LFAs is AuNPs because they are easy to synthesize and label with recognition elements (e.g., antibodies or aptamers), size-tunable, biocompatible, and intensely red in color to allow naked-eye detection [41]. In addition, the chemically inert nature of gold enables AuNPs to maintain exceptional stability against degradation for extended periods of time. The size range of AuNPs that can be used for LFA systems is 5–150 nm, but the size, sensitivity, and colloidal stability should be well-balanced to achieve the best performance [42]. NanoHybrids Inc. provides selection guidelines for AuNPs on their website. In general, larger AuNPs provide better sensitivity because of the higher magnitude of light absorption and surface area for antibody conjugation, but they also experience reduced contrast against the white nitrocellulose reaction membrane in the test strip because of their absorption of longer wavelengths of light. The most common size of AuNPs for LFA systems is 40 nm (Fig. 7a, SEM images) because particles of this size feature an optimal combination of high-contrast color (absorption ~523 nm) and surface area (minimal wasted targeting biomolecules).

124

D. Lee and J. H. Lee

Fig. 7 a SEM images of 40-nm AuNPs. b Absorbance spectra of 30-, 40-, and 60-nm AuNPs. Reprinted with permission by Dr. Jason Cook and NanoHybrids, Inc., 3913 Todd Lane, Suite 310, Austin, TX 78,744 [42]

For applications where the cost of the conjugate antibody is low and samples contain abundant target analyte, the use of 30-nm AuNPs is recommended. Meanwhile, 60nm AuNPs are recommended for applications with low target analyte concentration in samples or high-cost conjugate antibodies. Recent advances in nanomaterial synthesis have led many researchers to replace AuNPs to improve the detection limits of LFAs and achieve more quantitative analysis. QDs, which are semiconducting nanoparticles in the size range 1–10 nm comprising GaN, GaP, ZnO, ZnS, CdS, or CdSe [43], show very high brightness, size-tunable fluorescent emission, narrow spectral line widths, large absorption coefficients, and excellent stability against photobleaching [44]. In addition, they are water-soluble and easily conjugated with biomolecules because of their biocompatibility and similar dimensions, which are essential for application to LFA systems; therefore, QDs have emerged to substitute AuNPs. Wu et al. have developed a QD-based LFA system for the quantitative detection of C-reactive protein (CRP) [44]. The structure of the QD-based LFA is the same as the sandwich format, but the CRP antibody was conjugated with a hydrophobic CdSe–ZnS core–shell QD. As the CRP concentration was increased (0, 2.5, 25, 250, 500, and 1000 ng/mL), increasing fluorescence intensities appeared on the test line after 3 min (Fig. 8a) and a four-parameter logistic fitting curve was established based on these results (Fig. 8b). T/C indicates the ratio of fluorescence intensity between the test and control lines. Compared to the Roche latex-enhanced immunoturbidimetry assay, which is a gold standard test for in vitro diagnostics, the proposed LFA system showed good concordance for 135 CRP patient samples, verifying its capacity for highly sensitive and quantitative analysis of CRP (Fig. 8c). Similar concepts were suggested by Bai et al. [45]. They synthesized a novel silica nanoparticle/CdTe QD composite conjugated with antibodies and applied it to an LFA system for a-fetoprotein (AFP) detection. The proposed QD-based LFA showed results at least 10 times more sensitive than those of the conventional AuNP-based LFA. To amplify the fluorescence signal of QDs and achieve higher sensitivity, Li et al. [46] fabricated encapsulated QDs with a modified tri-copolymer [poly(tert-butyl acrylatecoethylacrylate-co-methacrylic acid)] and used them as conjugate nanoparticles for hCG LFA (Fig. 8d). The detection limit of the encapsulated QD-based LFA was

Paper-Based Biosensors with Lateral/Vertical Flow Assay

125

Fig. 8 Quantum dot (QD)-based LFA systems [44, 46]. a A QD-based LFA for quantitative detection of CRP (0, 2.5, 25, 250, 500, and 1000 ng/mL) and b four-parameter fitting curve between T/C (the ratio of test and control line intensities) and CRP concentration. c Compared to standard assay, the proposed LFA shows good concordance for 135 patient samples. d Fluorescence images of a QD-based hCG LFA (0, 1, 5, 10, 100, 500, and 1000 IU/L). Calibration curves for hCG quantification by (e) the encapsulated QD-based LFA and f the nonmodified QD-based LFAs. Figure panels adapted and reproduced from Ref. [44] with permission from Elsevier and from Ref. [46] with permission from Springer

lowered to 0.016 IU/L, representing an enhancement by ~38.5 times compared to that of the nonmodified QD-based LFA. Figure 8e and f show calibration curves for hCG quantification by the encapsulated QD-based LFA and the nonmodified QDbased LFAs, respectively. In addition to these, various QD-based LFA studies have been reported for the detection of pathogens [47], puerarin [48], chloramphenicol [49], nitrated ceruloplasmin [50], and tetanus antibodies [51]. CNPs or CNTs are also good alternative nanomaterials because they are lower in manufacturing cost; in addition, their dark colors provide higher contrast against the white reaction membrane than reddish AuNPs [1, 41]; therefore, many CNP- and CNT-based LFA systems have been reported to improve LFA sensitivity. Koets et al. [52] have developed CNP-based LFA for the detection of fungal alpha-amylase, which is an enzyme causing bakers’ allergy and occupational asthma. Colloidal CNPs were conjugated with monoclonal anti-alpha-amylase antibody (IX3c4) and applied to their LFA system. The proposed CNP-based LFA had no conjugate pad; instead, IX3c4-conjugated CNPs were dried at the bottom of the sample tube, thereby binding with the amylase when the sample was loaded (Fig. 9a). The detection limit of the LFA is ~0.32 ng/mL in buffer (Fig. 9b, concentration is 0.015–31 ng/mL). In trial assays at various bakery workstations, the LFA showed positive results in locations exposed to high levels of the amylase (e.g., near the dough mixer) and

126

D. Lee and J. H. Lee

Fig. 9 CNP and CNT-based LFA systems [52–54]. a The CNP-based LFA for detection of fungal alpha-amylase shows b the detection limit of ~0.32 ng/mL in buffer and c positive results at places exposed to high levels of the amylase. d Comparison of the detection limit of Dengue virus NS1 protein shows that the CNP-based LFA had 10 times greater sensitivity than the AuNP-based LFA. e A CNT-based LFA biosensor for DNA sequence detection showed the detection limit of 0.1 nM target DNA and f linearity between the black color intensity and the logarithmic concentration of the target DNA. Figure panels adapted and reproduced from Ref. [52] with permission from The Royal Society of Chemistry and from Refs. [53, 54] with permission from Elsevier

negative results in locations where the level should have been minimal (e.g., in the bakery office) (Fig. 9c). A comparison of the detection limit between CNPs and other nanoparticles (AuNPs, silver-enhanced AuNPs, and blue latex beads) was performed by Linares et al. [53]. The detection limit of biotinylated-BSA with CNPs was 0.01 μg/mL, remarkably lower than the detection limits of 0.1, 1, and 1 mg/mL for AuNPs, silver-enhanced AuNPs, and blue latex beads, respectively. They also designed and compared the detection sensitivity of Dengue virus NS1 protein with CNP- and AuNP-based LFAs (Fig. 9d), and demonstrated that the detection limit of the CNP-based LFA (57 ng/mL) was 10 times lower than that of AuNP-based LFA (575 ng/mL). As an application of CNTs, Qui [54] developed a CNT-based LFA biosensor for the rapid detection of DNA sequences. Amine-modified DNA detection probes conjugated with shortened multi-walled CNTs (MWCNTs) could hybridize with target DNA on the LFA and then the captured MWCNTs on the test and control zones would produce clear black bands. Based on the CNT-based LFA, 0.1 nM target DNA was identified without any instrumentation (Fig. 9e). A quantitative analysis showed that the black color intensities versus the logarithm of the target DNA concentration were linear over the 0.1–20 nM range with a detection limit of 0.04 nM (Fig. 9f), which is 12.5 times lower than that of an AuNP-based LFA [55]. Many other target analytes, such as bacteria DNA [56], schistosome circulating

Paper-Based Biosensors with Lateral/Vertical Flow Assay

127

anodic antigens [57], anti-HIV-1, 2 antibodies [58], methiocarbs [59], and influenza A virus [60] were also analyzed by CNP- or CNT-based LFA systems.

5.3 Different Types of Recognition Elements Selecting proper recognition elements is the most critical step in determining the overall performance of the LFA because the capture of the target analytes by the labeling conjugate and the test and control lines is dominated by the response of the recognition element. The most widely accepted recognition elements in the current LFA market are antibodies. Monoclonal antibodies are preferred over polyclonal for LFA systems because of the easy production of large quantities of specific antibodies [3, 61]. Monoclonal antibodies can originate from mice, rats, rabbits, guinea pigs, and humans; polyclonal antibodies can be obtained from rabbits, goats, sheep, donkeys, and some other animals [4]. However, polyclonal antibodies can provide comparable results in many cases; in addition, commercial and antigen-dependent issues must be considered in choosing the proper antibody. The key considerations and issues in the choice of these antibodies for LFA systems (Table 3) were described by O’Farrell [3]. The limitation of LFAs employing antibodies as the recognition elements is that these require well-controlled temperature and humidity for optimal stability and Table 3 Key consideration and issues for choosing monoclonal vs polyclonal antibodies for LFA [3] Key considerations

Issues

• • • • •

Whether to use monoclonals or polyclonals How to select and screen antibodies How best to conjugate them How to immobilize antibodies onto a membrane How to optimize the characteristics of the antibodies

Monoclonals

• • • • • •

Well-defined reagent Easy to prepare pure antibody Take longest to prepare Typically more expensive May not provide the highest affinities Can achieve higher antibody loading on particles and capture lines

Polyclonals

• • • • • •

Fastest to produce in high quantity Relatively inexpensive to produce Can provide very high affinities Can vary between animals and overtime May require extensive purification Low percentage of total antibody is target specific

128

D. Lee and J. H. Lee

storage [3, 62]. High temperatures and humidity deteriorate the stability of antibodies in the conjugate pad and the test and control lines, causing significant performance degradation. Because LFAs have the largest commercial uses in health care in developing countries and POC applications in the fields of forensics, agriculture, biowarfare, and pandemic preparedness, this restriction must be solved for the general use and globalization of LFAs. In this regard, aptamer-based LFAs are good alternatives. An aptamer is a short single-stranded DNA or RNA oligonucleotide or a peptide molecule with high temperature and pH stability; therefore, it is relatively independent of environmental conditions. By forming specific three-dimensional conformations, an aptamer can specifically bind to target analytes with high affinity in a manner similar to that of antibodies [62]. The screening and selection process to find target-specific aptamer sequences are called systematic evolution of ligands by exponential enrichment (SELEX), which was first developed by Tuerk and Gold in 1990 [63]. After this process was introduced, various aptamer sequences with high specificities to target molecules have been discovered and exploited to develop aptamer-based LFAs. Wu et al. [64] developed an aptamer-based LFA for the simple and sensitive detection of E. coli O157:H7. Two different aptamers specific to the outer membrane of E. coli were employed; one was pre-labeled with a biotin moiety for magnetic bead enrichment (red aptamers in Fig. 10a). The other was intended as a signal reporter for E. coli (blue aptamers in Fig. 10a), which was amplified by isothermal strand displacement amplification (SDA) and further detected by the LFA. When these two aptamers were incubated with the E. coli sample, they were anchored to the outer membrane of E. coli. Then, streptavidin-coated magnetic beads were added and washed three times to retain only the aptamers attached to the outer

Fig. 10 Aptamer-based LFA systems [64, 65]. a–c Simple and sensitive detection of E. coli O157:H7 with d sensitivity of 10 CFU/mL. e, f Aptamer-based fluorometric LFA for the detection of creatine kinase MB with the sensitivity of 0.63 ng/mL. Figure panels are adapted and reproduced from Ref. [64] with permission from Elsevier and from Ref. [65] with permission from Springer

Paper-Based Biosensors with Lateral/Vertical Flow Assay

129

membranes (Fig. 10a). The aptamers enriched by magnetic separation (blue) were used as templates for SDA to produce single-stranded DNA amplicons (Fig. 10b). After amplification, the amplicons were loaded into the sample pad with a running buffer; these were captured by probe-AuNP conjugates through a complementary reaction. This yielded complexes between the amplicons and probe-AuNPs. In the test line, the other complementary nucleic acid probes were immobilized; thus, the complexes were captured and produced clear reddish colorimetric signals (Fig. 10c). With this LFA, 10 CFU/mL of E. coli O157:H7 were detected, even without a DNA extraction step (Fig. 10d). Zhang et al. [65] developed an aptamer-based fluorometric LFA for the detection of creatine kinase MB (CKMB), which is a biomarker for the diagnosis of acute myocardial infarction. Single-stranded DNA aptamers (C.Apt.30) specific to CKMB were selected by SELEX and conjugated with fluorescent microspheres through streptavidin–biotin integration (FM-C.Apt.30). Another aptamer to CKMB (C.Apt.21) and DNA probes complementary to C.Apt.30 were immobilized on the test and control lines, respectively (Fig. 10e). In the presence of CKMB in the sample (positive), CKMB was captured by FM-C.Apt.30, producing complexes of CKMB and FM-C.Apt.30. The complexes were then captured by the C.Apt.21 aptamers on the test line and the unreacted FM-C.Apt.30 were captured by the DNA probes on the control line, producing fluorescence signals in both bands. A fluorescence signal of 5 ng/mL CKMB could be detected by the test strip reader (Fig. 10f) and the calibration curve between the fluorescence intensities in the test and control lines and the CKMB concentrations showed the detection limit of CKMB in a serum sample of 0.63 ng/mL, which is lower than the cutoff value of CKMB (5 ng/mL). In addition, aptamer sequences have been discovered and applied to LFA techniques for the detection of ochratoxin A in Astragalus membranaceus [66], cortisol in sweat [67], cholera toxins [68], and cocaine serum [69]. In addition to linear aptamers, special hairpin-structured DNA aptamers, or molecular beacons, have also been used as recognition elements in LFA. Typically, molecular beacons comprise 18–30 base pairs in the loop region with a sequence complementary to that of the target analyte with 5–7 base pairs in the double-stranded stem. The ends of the molecular beacon are labeled with a fluorophore and its quencher [8]. Therefore, the beacon initially has no fluorescence signal because the fluorophore is quenched. When the molecular beacon is hybridized with the target analytes, which have sequences complementary to the beacon loop, the double-stranded stem is opened, thus separating the fluorophore and the quencher and producing a fluorescent signal. Because of their high sensitivity and selectivity, molecular beacons have been used in many aptamer-based LFA devices [70–73].

5.4 Other Efforts to Improve LFA Performance For the purpose of improving LFA performance, we have discussed studies of different nanomaterials for labeling conjugates and recognition elements, which

130

D. Lee and J. H. Lee

Fig. 11 Benchtop-scale and portable LFA readout devices. a ESEQuant™. Courtesy of Qiagen Lake Constance. b HRDR-200. Courtesy of Holomic LLC. c SkanFlexi X500 and SkanEasy Courtesy of Skannex AS. d RDS-2500 and iPhone rapid diagnostic lateral flow. Courtesy of Detekt Biomedical LLC. e Google glass-based LFA reader platform [74]. Figure panel adapted and reproduced from Ref. [74] with permission from the American Chemical Society (https://pubs.acs.org/ doi/10.1021/nn500614k, further permissions related to the material excerpted should be directed to the ACS)

are key components contributing to the LFA performance. In addition to these, many other approaches independent of internal modifications of the LFA have been suggested to improve the performance and practicality of LFA techniques. The most commonly used and commercialized approach is to employ an external reader based on scanning optics to overcome the limitation of naked-eye detection and thus achieve better sensitivity with reliable quantitative analysis. Many LFA and biotech companies have developed benchtop-scale reader and readout devices that can be incorporated with smartphones or are otherwise portable; examples include the ESEQuant™ lateral flow reader from Qiagen Lake Constance (Fig. 11a), HRDR200 from Holomic LLC (Fig. 11b), SkanFlexi X500 and SkanEasy from Skannex AS (Fig. 11c), and RDS-2500 and iPhone rapid diagnostic lateral flow readers from Detekt Biomedical LLC (Fig. 11d). In the more advanced technology, Feng et al. [74] developed a Google Glass-based LFA reader platform for qualitative and quantitative analysis (Fig. 11e). In this custom-written mobile application, digital images of LFAs with custom-designed QR codes are acquired by the camera built into Google Glass via a hands-free and voice-controlled interface. The images are transmitted to a server for digital processing that yields quantitative analysis. The results are then stored on a central server with the QR code and other related information, and then returned to the Google Glass user. With this strategy, qualitative (i.e., yes/no) HIV and quantitative PSA tests were demonstrated. Such a wearable device-based LFA platform can provide benefits for the real-time spatiotemporal tracking and mapping of various diseases, personal medical conditions, and epidemiologic and pandemic control.

Paper-Based Biosensors with Lateral/Vertical Flow Assay

131

Fig. 12 Studies for improvement of LFA performance in different perspectives [75–77]. a The LFA platform incorporating SERS for detection of PA. b As PA concentration increases (from 0 to 100), the SERS intensities are gradually decreased. c The LFA device combining electrochemical and colorimetric methods for 8-OHdG detection. d The LFA device incorporating with battery-operated ICP preconcentration. e The preconcentration enables detection of 150 ng/mL of β-hCG, which is a non-detectable level without preconcentration; thus, the sensitivity is enhanced 2.69-fold. Figure panels adapted and reproduced from Ref. [75] with permission from American Chemical Society and from Refs. [76, 77] with permission from The Royal Society of Chemistry

Efforts have also been made to improve LFA performance in different perspectives. Li et al. [75] showed a novel LFA platform incorporating surface-enhanced Raman scattering (SERS) for the ultrasensitive and quantitative analysis of phenylethanolamine A (PA). They employed the competitive format; if PA was present in the sample, signals on both the test and control lines would appear (negative result), and otherwise only one band would be present as the control line (positive result) (Fig. 12a). The SERS scattering intensity of the test line was measured by a portable Raman analyzer. As the PA concentration increased, the SERS intensities at 1074 cm−1 were gradually decreased (Fig. 12b). The limit of detection of this system was 0.32 pg/mL, between one and three orders of magnitude lower than that of other immunoassays. Zhu et al. [76] developed a novel LFA device for the electrochemical and colorimetric measurement of 8-hydroxy-20-deoxyguanosine (8-OHdG), which is a DNA oxidative damage biomarker. They also employed the competitive format, where the captured 8-OHdG on the control line was chronoamperometrically measured by CNT paper as the working electrode; the color intensity on the test line was measured by a scanner and analyzed by ImageJ software (Fig. 12c). The detection limit of 8-OHdG in a urine sample was 5.76 ng/mL for the colorimetric method and 8.85 ng/mL for the electrochemical method. By integrating these two methods, they achieved double confidence on the same assay to avoid false results. Kim et al. [77] developed an enhanced LFA device combined with battery-operated ion-concentration polarization (ICP) for sample preconcentration (Fig. 12d). The preconcentration of the target analyte (β-hCG) on the sample pad allowed the visual detection of 150 ng/mL of β-hCG, which is a non-detectable level

132

D. Lee and J. H. Lee

without preconcentration (Fig. 12e). With this approach, β-hCG on the sample pad was preconcentrated ~15-fold by a 9-V battery and the sensitivity was enhanced by 2.69-fold.

6 Conclusions As discussed in this chapter, LFA techniques can be exploited for many applications in various fields because of their simplicity and high sensitivity; in addition, many routes exist to improve LFA performance in various ways. LFA techniques hold many advantages and provide an ideal platform for POC use, such as cost-efficiency, disposability, portability, rapid analysis, and user-friendliness. In addition, the versatile nature of LFA facilitates collaboration with existing nano- and biotechnologies, as discussed above. Despite these universal advantages, LFA techniques can still suffer from poor reproducibility, low sensitivity, and occasional false-positive or -negative results compared to immunoassay and molecular diagnostic methods. By compensating for these drawbacks and improving the reliability of LFA, future research can facilitate the commercialization of LFA kits for the investigation of various analytes.

References 1. Rivas, L., de la Escosura-Muñiz, A., Pons, J., Merkoçi, A.: Lateral flow biosensors based on gold nanoparticles. Compr. Anal. Chem. 66, 569–605 (2014). https://doi.org/10.1016/B978-0444-63285-2.00014-6 2. Banerjee, R., Jaiswal, A.: Recent advances in nanoparticle-based lateral flow immunoassay as a point-of-care diagnostic tool for infectious agents and diseases. Analyst 143, 1970–1996 (2018). https://doi.org/10.1039/c8an00307f 3. O’Farrell, B.: Lateral flow immunoassay systems: evolution from the current state of the art to the next generation of highly sensitive, quantitative rapid assays. In: Wild, D. (ed.) The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques, 4th edn, pp. 89–107. Elsevier, Oxford, UK (2013) 4. van Amerongen, A., et al.: Lateral flow immunoassays. In: Vashist, S., Luong, J. (eds.) Handbook of Immunoassay Technologies, pp. 157–182. Academic Press, Cambridge, MA (2018) 5. Yetisen, A.K., Akram, M.S., Lowe, C.R.: Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13, 2210–2251 (2013). https://doi.org/10.1039/C3LC50169H 6. Juntunen, E.: Lateral Flow Immunoassays with Fluorescent Reporter Technologies. University of Turku, Turku, Finland (2018) 7. Posthuma-Trumpie, G.A., Korf, J., van Amerongen, A.: Lateral flow (immuno) assay: Its strengths, weaknesses, opportunities and threats. A literature survey. Anal. Bioanal. Chem. 393, 569–582 (2009). https://doi.org/10.1007/s00216-008-2287-2 8. Sajid, M., Kawde, A.-N., Daud, M.: Designs, formats and applications of lateral flow assay: A literature review. J. Saudi Chem. Soc. 19, 689–705 (2015). https://doi.org/10.1016/j.jscs.2014. 09.001 9. Lateral Flow Assay Market. Available at: https://www.marketsandmarkets.com/Market-Rep orts/lateral-flow-assay-market-167205133.html. Accessed: 17 Dec 2019

Paper-Based Biosensors with Lateral/Vertical Flow Assay

133

10. Lateral Flow Assay Market Size to surpass 6.6%+ CAGR up to 2024. Available at: https://www.marketwatch.com/press-release/lateral-flow-assay-market-size-to-surpass-66cagr-up-to-2024-2019-06-03. Accessed: 17 Dec 2019 11. Butler, S.A., Khanlian, S.A., Cole, L.A.: Detection of early pregnancy forms of human chorionic gonadotropin by home pregnancy test devices. Clin. Chem. 47, 2131–2136 (2001) 12. Fernández-Sánchez, C., et al.: One-step immunostrip test for the simultaneous detection of free and total prostate specific antigen in serum. J. Immunol. Methods 307, 1–12 (2005). https:// doi.org/10.1016/j.jim.2005.08.014 13. Xu, H., et al.: Aptamer-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for protein analysis. Anal. Chem. 81, 669–675 (2008). https://doi.org/10.1021/ac8 020592 14. Choi, D.H., et al.: A dual gold nanoparticle conjugate-based lateral flow assay (LFA) method for the analysis of troponin I. Biosens. Bioelectron. 25, 1999–2002 (2010). https://doi.org/10. 1016/j.bios.2010.01.019 15. Parolo, C., de la Escosura-Muñiz, A., Merkoçi, A.: Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes. Biosens. Bioelectron. 40, 412–416 (2013). https:// doi.org/10.1016/j.bios.2012.06.049 16. Wang, J.-Y., et al.: Development of colloidal gold immunochromatographic signal-amplifying system for ultrasensitive detection of Escherichia coli O157:H7 in milk. RSC Adv. 5, 62300– 62305 (2015). https://doi.org/10.1039/C5RA13279G 17. Seo, K.-H., Holt, P.S., Stone, H.D., Gast, R.K.: Simple and rapid methods for detecting Salmonella enteritidis in raw eggs. Int. J. Food Microbiol. 87, 139–144 (2003). https://doi. org/10.1016/S0168-1605(03)00053-9 18. Huang, S.-H.: Gold nanoparticle-based immunochromatographic assay for the detection of Staphylococcus aureus. Sens. Actuators B Chem. 127, 335–340 (2007). https://doi.org/10. 1016/j.snb.2007.04.027 19. Wang, D.-B., et al.: Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system. Biosens. Bioelectron. 42, 661–667 (2013). https://doi.org/10.1016/j.bios.2012.10.088 20. Liu, H., et al.: Visual and sensitive detection of viable pathogenic bacteria by sensing of RNA markers in gold nanoparticles based paper platform. Biosens. Bioelectron. 62, 38–46 (2014). https://doi.org/10.1016/j.bios.2014.06.020 21. Le, T.T., et al.: Dual recognition element lateral flow assay toward multiplex strain specific influenza virus detection. Anal. Chem. 89, 6781–6786 (2017). https://doi.org/10.1021/acs.ana lchem.7b01149 22. Workman, S., et al.: Rapid detection of HIV-1 p24 antigen using magnetic immunochromatography (MICT). J. Virol. Methods 160, 14–21 (2009). https://doi.org/10.1016/j.jvi romet.2009.04.003 23. Brangel, P., et al.: A serological point-of-care test for the detection of IgG antibodies against Ebola virus in human survivors. ACS Nano 12, 63–73 (2018). https://doi.org/10.1021/acsnano. 7b07021 24. Hagström, A.E.V., et al.: Sensitive detection of norovirus using phage nanoparticle reporters in lateral-flow assay. PLoS ONE 10, e0126571 (2015). https://doi.org/10.1371/journal.pone.012 6571 25. Lee, D., et al.: Simple and highly sensitive molecular diagnosis of Zika virus by lateral flow assays. Anal. Chem. 88, 12272–12278 (2016). https://doi.org/10.1021/acs.analchem.6b03460 26. Nash, M.A., Waitumbi, J.N., Hoffman, A.S., Yager, P., Stayton, P.S.: Multiplexed enrichment and detection of malarial biomarkers using a stimuli-responsive iron oxide and gold nanoparticle reagent system. ACS Nano 6, 6776–6785 (2012). https://doi.org/10.1021/nn3 015008 27. Rivas, L., et al.: Triple lines gold nanoparticle-based lateral flow assay for enhanced and simultaneous detection of Leishmania DNA and endogenous control. Nano Res. 8, 3704–3714 (2015). https://doi.org/10.1007/s12274-015-0870-3

134

D. Lee and J. H. Lee

28. van Dam, G.J., et al.: A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen. Exp. Parasitol. 135, 274–282 (2013). https://doi.org/10.1016/j.exppara.2013.06.017 29. Mazumdar, D., Liu, J., Lu, G., Zhou, J., Lu, Y.: Easy-to-use dipstick tests for detection of lead in paints using non-cross-linked gold nanoparticle–DNAzyme conjugates. Chem. Commun. 46, 1416–1418 (2010). https://doi.org/10.1039/B917772H 30. He, Y., et al.: Visual detection of Hg2+ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes. Biosens. Bioelectron. 26, 4464–4470 (2011). https://doi. org/10.1016/j.bios.2011.05.003 31. López Marzo, A.M., Pons, J., Blake, D.A., Merkoçi, A.: High sensitive gold-nanoparticle based lateral flow Immunodevice for Cd2+ detection in drinking waters. Biosens. Bioelectron. 47, 190–198 (2013). https://doi.org/10.1016/j.bios.2013.02.031 32. Gandhi, S., Caplash, N., Sharma, P., Raman Suri, C.: Strip-based immunochromatographic assay using specific egg yolk antibodies for rapid detection of morphine in urine samples. Biosens. Bioelectron. 25, 502–505 (2009). https://doi.org/10.1016/j.bios.2009.07.018 33. Byzova, N.A., Zvereva, E.A., Zherdev, A.V., Eremin, S.A., Dzantiev, B.B.: Rapid pretreatmentfree immunochromatographic assay of chloramphenicol in milk. Talanta 81, 843–848 (2010). https://doi.org/10.1016/j.talanta.2010.01.02 34. Byzova, N.A., et al.: Rapid immunochromatographic assay for ofloxacin in animal original foodstuffs using native antisera labeled by colloidal gold. Talanta 119, 125–132 (2014). https:// doi.org/10.1016/j.talanta.2013.10.054 35. Lutz, B., et al.: Dissolvable fluidic time delays for programming multi-step assays in instrumentfree paper diagnostics. Lab Chip 13, 2840–2847 (2013). https://doi.org/10.1039/c3lc50178g 36. Toley, B.J., et al.: Tunable-delay shunts for paper microfluidic devices. Anal. Chem. 85, 11545– 11552 (2013). https://doi.org/10.1021/ac4030939 37. Toley, B.J., et al.: A versatile valving toolkit for automating fluidic operations in paper microfluidic devices. Lab Chip 15, 1432–1444 (2015). https://doi.org/10.1039/c4lc01155d 38. Shin, J.H., Park, J., Kim, S.H., Park, J.-K.: Programmed sample delivery on a pressurized paper. Biomicrofluidics 8, 054121 (2014). https://doi.org/10.1063/1.4899773 39. Rivas, L., Medina-Sánchez, M., de la Escosura-Muñiz, A., Merkoçi, A.: Improving sensitivity of gold nanoparticle-based lateral flow assays by using wax-printed pillars as delay barriers of microfluidics. Lab Chip 14, 4406–4414 (2014). https://doi.org/10.1039/c4lc00972j 40. Choi, J.R., et al.: Polydimethylsiloxane-paper hybrid lateral flow assay for highly sensitive point-of-care nucleic acid testing. Anal. Chem. 88, 6254–6264 (2016). https://doi.org/10.1021/ acs.analchem.6b00195 41. Quesada-González, D., Merkoçi, A.: Nanoparticle-based lateral flow biosensors. Biosens. Bioelectron. 73, 47–63 (2015). https://doi.org/10.1016/j.bios.2015.05.050 42. Gold Nanoparticle Labels Custom Designed for Lateral Flow Assays. Available at: https://nan ohybrids.net/pages/gold-nanoparticles-for-lateral-flow-assays. Accessed 17 Dec 2019 43. Hildebrandt, N.: Biofunctional quantum dots: Controlled conjugation for multiplexed biosensors. ACS Nano 5, 5286–5290 (2011). https://doi.org/10.1021/nn2023123 44. Wu, R., et al.: Quantitative and rapid detection of C-reactive protein using quantum dot-based lateral flow test strip. Anal. Chim. Acta 1008, 1–7 (2018). https://doi.org/10.1016/j.aca.2017. 12.031 45. Bai, Y., et al.: A sensitive lateral flow test strip based on silica nanoparticle/CdTe quantum dot composite reporter probes. RSC Adv. 2, 1778–1781 (2012). https://doi.org/10.1039/C2RA00 976E 46. Li, X., et al.: Ultrasensitive lateral-flow assays based on quantum dot encapsulations with signal amplification. J. Nanopart. Res. 20, 139 (2018). https://doi.org/10.1007/s11051-018-4241-3 47. Morales-Narváez, E., Naghdi, T., Zor, E., Merkoçi, A.: Photoluminescent lateral-flow immunoassay revealed by graphene oxide: highly sensitive paper-based pathogen detection. Anal. Chem. 87, 8573–8577 (2015). https://doi.org/10.1021/acs.analchem.5b02383 48. Qu, H., et al.: Rapid lateral-flow immunoassay for the quantum dot-based detection of puerarin. Biosens. Bioelectron. 81, 358–362 (2016). https://doi.org/10.1016/j.bios.2016.03.008

Paper-Based Biosensors with Lateral/Vertical Flow Assay

135

49. Berlina, A.N., Taranova, N.A., Zherdev, A.V., Vengerov, Y.Y., Dzantiev, B.B.: Quantum dotbased lateral flow immunoassay for detection of chloramphenicol in milk. Anal. Bioanal. Chem. 405, 4997–5000 (2013). https://doi.org/10.1007/s00216-013-6876-3 50. Li, Z., et al.: Rapid and sensitive detection of protein biomarker using a portable fluorescence biosensor based on quantum dots and a lateral flow test strip. Anal. Chem. 82, 7008–7014 (2010). https://doi.org/10.1021/ac101405a 51. Wang, J., et al.: Quantum dot-based lateral flow test strips for highly sensitive detection of the tetanus antibody. ACS Omega 4, 6789–6795 (2019). https://doi.org/10.1021/acsomega. 9b00657 52. Koets, M., Sander, I., Bogdanovic, J., Doekes, G., van Amerongen, A.: A rapid lateral flow immunoassay for the detection of fungal alpha-amylase at the workplace. J. Environ. Monit. 8, 942–946 (2006). https://doi.org/10.1039/b605389k 53. Linares, E.M., Kubota, L.T., Michaelis, J., Thalhammer, S.: Enhancement of the detection limit for lateral flow immunoassays: evaluation and comparison of bioconjugates. J. Immunol. Methods 375, 264–270 (2012). https://doi.org/10.1016/j.jim.2011.11.003 54. Qiu, W., et al.: Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence. Biosens. Bioelectron. 64, 367–372 (2015). https://doi.org/10.1016/j.bios. 2014.09.028 55. Mao, X., et al.: Disposable nucleic acid biosensors based on gold nanoparticle probes and lateral flow strip. Anal. Chem. 81, 1660–1668 (2009). https://doi.org/10.1021/ac8024653 56. Noguera, P., et al.: Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli. Anal. Bioanal. Chem. 399, 831– 838 (2011). https://doi.org/10.1007/s00216-010-4334-z 57. van Dam, G.J., et al.: Diagnosis of schistosomiasis by reagent strip test for detection of circulating cathodic antigen. J. Clin. Microbiol. 42, 5458–5461 (2008). https://doi.org/10.1128/ JCM.42.12.5458-5461.2004 58. Rayev, M., Shmagel, K.: Carbon–protein covalent conjugates in non-instrumental immunodiagnostic systems. J. Immunol. Methods 336, 9–15 (2008). https://doi.org/10.1016/j.jim.2008. 03.005 59. Blažková, M., Micková-Holubová, B., Rauch, P., Fukal, L.: Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water. Biosens. Bioelectron. 25, 753–758 (2009). https://doi.org/10.1016/j.bios.2009.08.023 60. Wiriyachaiporn, N., Sirikett, H., Maneeprakorn, W., Dharakul, T.: Carbon nanotag based visual detection of influenza A virus by a lateral flow immunoassay. Microchim. Acta 184, 1827–1835 (2017). https://doi.org/10.1007/s00604-017-2191-6 61. https://fnkprddata.blob.core.windows.net/domestic/download/pdf/IBS_A_guide_to_lateral_ flow_immunoassays.pdf 62. Chen, A., Yang, S.: Replacing antibodies with aptamers in lateral flow immunoassay. Biosens. Bioelectron. 71, 230–242 (2015). https://doi.org/10.1016/j.bios.2015.04.041 63. Tuerk, C., Gold, L.: Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990). https://doi.org/10.1126/ science.2200121 64. Wu, W., et al.: A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification. Anal. Chim. Acta 861, 62–68 (2015). https://doi.org/10.1016/j.aca.2014.12.041 65. Zhang, J., et al.: Aptamer-based fluorometric lateral flow assay for creatine kinase MB. Microchim. Acta 185, 364 (2018). https://doi.org/10.1007/s00604-018-2905-4 66. Zhou, W., et al.: An aptamer based lateral flow strip for on-site rapid detection of ochratoxin A in Astragalus membranaceus. J. Chromatogr. B 1022, 102–108 (2016). https://doi.org/10. 1016/j.jchromb.2016.04.016 67. Dalirirad, S., Steckl, A.J.: Aptamer-based lateral flow assay for point of care cortisol detection in sweat. Sens. Actuators B Chem. 283, 79–86 (2019). https://doi.org/10.1016/j.snb.2018.11.161 68. Frohnmeyer, E., et al.: Aptamer lateral flow assays for rapid and sensitive detection of cholera toxin. Analyst 144, 1840–1849 (2019). https://doi.org/10.1039/C8AN01616J

136

D. Lee and J. H. Lee

69. Liu, J., Mazumdar, D., Lu, Y.: A simple and sensitive “dipstick” test in serum based on lateral flow separation of aptamer-linked nanostructures. Angew. Chem. Int. Ed. 45, 7955–7959 (2006). https://doi.org/10.1002/anie.200603106 70. Javani, A., Javadi-Zarnaghi, F., Rasaee, M.J.: A multiplex protein-free lateral flow assay for detection of microRNAs based on unmodified molecular beacons. Anal. Biochem. 537, 99–105 (2017). https://doi.org/10.1016/j.ab.2017.09.005 71. Beni, V., Hayes, K., Lerga, T.M., O’Sullivan, C.K.: Development of a gold nano-particle-based fluorescent molecular beacon for detection of cystic fibrosis associated mutation. Biosens. Bioelectron. 26, 307–313 (2010). https://doi.org/10.1016/j.bios.2010.08.043 72. Mao, X., Xu, H., Zeng, Q., Zeng, L., Liu, G.: Molecular beacon-functionalized gold nanoparticles as probes in dry-reagent strip biosensor for DNA analysis. Chem. Commun. 21, 3065–3067 (2009). https://doi.org/10.1039/b822582f 73. Ying, N., et al.: Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction. PLoS ONE 12, e0185091 (2017). https://doi.org/10.1371/journal.pone.0185091 74. Feng, S., et al.: Immunochromatographic diagnostic test analysis using Google Glass. ACS Nano 8, 3069–3079 (2014). https://doi.org/10.1021/nn500614k 75. Li, M., et al.: Ultrasensitive and quantitative detection of a new β-agonist phenylethanolamine A by a novel immunochromatographic assay based on surface-enhanced Raman scattering (SERS). J. Agric. Food Chem. 62, 10896–10902 (2014). https://doi.org/10.1021/jf503599x 76. Zhu, X., Shah, P., Stoff, S., Liu, H., Li, C.Z.: A paper electrode integrated lateral flow immunosensor for quantitative analysis of oxidative stress induced DNA damage. Analyst 139, 2850–2857 (2014). https://doi.org/10.1039/c4an00313f 77. Kim, C., et al.: Battery operated preconcentration-assisted lateral flow assay. Lab Chip 17, 2451–2458 (2017). https://doi.org/10.1039/C7LC00036G

Paper-Based Applications for Bacteria/Virus Sumin Han, Manika Chopra, Ilaria Rubino, and Hyo-Jick Choi

1 Introduction 1.1 Relevance of Paper-Based Point-of-Care (POC) Devices The World Health Organization (WHO) defines an infectious disease as an illness “caused by pathogenic microorganisms, such as bacteria, viruses, parasites, or fungi” (World Health Organization n.d.). Infectious diseases have been present throughout human history, with notable cases including the Black Death in the Middle Ages and the Spanish Flu pandemic in 1918, which killed up to 50 million people worldwide. In 2016, the WHO ranked lower respiratory infections, diarrheal diseases, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), malaria, and tuberculosis among the top ten causes of death in low-income countries. All of these conditions arise from the transmission of pathogenic organisms, such as bacteria and viruses (Baylor College of Medicine n.d.). The emergence, mutation, and spread of such life-threatening diseases necessitate advancements in diagnosis and sensing technologies to rapidly identify and respond to infectious diseases. Therefore, the development of low cost, easy-to-use diagnostic devices is important in global health to prevent and control infectious diseases in a timely manner. Currently, for diagnostic testing, about 40,000 products are available as traditional laboratory tests or POC diagnostics. Traditional laboratory tests require specialized technicians or facilities for the processing and analysis of samples [24]. Meanwhile, POC tests are performed at the point of patient care and provide test results in short periods of time, allowing for immediate diagnosis [29]. In addition, POC tests are vital in the management of infectious diseases because they offer efficient care, lower costs, and no required trained personnel [24, 29]. With the prevalence of infectious S. Han · M. Chopra · I. Rubino · H.-J. Choi (B) Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 J. H. Lee (ed.), Paper-Based Medical Diagnostic Devices, Bioanalysis 10, https://doi.org/10.1007/978-981-15-8723-8_7

137

138

S. Han et al.

diseases in low-income countries, the WHO established the so-called ASSURED criteria that can be used in selecting diagnostic tests. ASSURED is an acronym for Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment free, and Deliverable to end users, criteria which are all usually met by POC tests [24]. The first lateral flow-based POC immunoassay technique adapted from latex agglutination assays [28] was made commercially available in the 1990s for the diagnosis of malaria. To implement POC technologies, top-down or bottom-up designs were used to generate diagnostic devices, such as lateral flow assays (LFAs) and electrochemical analytical devices for use in low-income countries [49]. In topdown approaches, existing technologies are simplified by removing certain functional components for adaptation to resource-limited settings, but this often causes the loss of certain features. In bottom-up designs, application-specific functionalities are built on a basic component [51].

1.2 Paper Properties The benefits of paper-based devices include easy mass production and distribution, portability, elimination of hazardous waste generation thanks to disposal by incineration, and low cost. The inherent hydrophilic nature and capillary action of paper allow for intrinsic fluid flow without requiring pumps. In addition, paper can be easily modified with printed materials or patterns, enabling diverse device fabrication strategies [18, 42]. So far, two different types of paper (i.e., cellulose fiber and nitrocellulose) have been used for POC tests. Cellulose fibers with a minimum cellulose content of 98% are often used as substrates that can be further functionalized with biomolecules [41]. Nitrocellulose is produced from the esterification of cellulose with a nitrating acid, such as sulfuric or nitric acid. The hydroxyl groups (OH) in cellulose are replaced by nitro groups (NO2 ); the degree of nitration determines the solubility and flammability of the resulting nitrocellulose [42]. Attractive aspects of nitrocellulose include its binding capabilities with biomolecules (such as proteins) and superior retention of reagents, which enables a longer reaction time within the device [41]. Despite these advantages, nitrocellulose lacks structural integrity and must be coupled with plastic supports within devices [39]. The key physical attributes defining the optimum paper type for a specific application are the surface area, capillary flow rate, pore size, porosity and thickness. The surface area decreases nonlinearly with pore size and increases nonlinearly with porosity, and increases linearly with thickness. Modulation of the surface area, to control for instance the sample evaporation rate, cannot be done without consideration of the dependence on the pore size and porosity, which affect the capillary effect mainly used to move analytes on paper. The correlation of the surface area with these other characteristics of paper influences the sensitivity of the tests. Capillary flow is the speed of migration of the sample along the paper, which ultimately affects the concentration of the analyte present and the performance of the assay. The pore size determines the maximum size of particles that can pass through the membrane; its

Paper-Based Applications for Bacteria/Virus

139

distribution determines the capillary flow rate. Porosity represents the void volume in the membrane, which also affects the capillary flow rate. Lastly, the thickness of the paper is an important factor in determining the amount of absorbed analyte. In a thick piece of paper, the applied sample diffuses both downwards and outwards, whereas in a thin piece of paper, fluid flows mainly outwards. Thus, the analyte concentration varies according to the thickness of the paper. Furthermore, the signal visibility (i.e., detection) is affected by the thickness [42].

1.3 Modifications of Paper-Based Technologies Advancements in paper fabrication technologies have enabled diverse applications through chemical and biological functionalizations. As an example of chemical modification, the addition of wax or SU-8 photoresist causes a hydrophilic-to-hydrophobic property change, which can be applied to create specified channels in which the fluid flows only in hydrophilic regions. Another approach is to coat the entire surface with a layer of polystyrene, followed by channel definition using toluene [35]. The creation of hydrophilic channels allows the production of multiple channels within a device and prevents cross-contamination between channels. Chemical additives, such as cationic polyacryl amide (CPAM), can be added as retention aids during the manufacturing process [39]. Biological modifications with antibodies can be used to separate targeted proteins and allow for enzyme-linked immunoabsorbent assay (ELISA) for detection purposes. ELISA uses an antibody to detect the presence of the target antigen; the antigen can then be detected by an enzyme-labeled secondary antibody or gold nanoparticles (AuNPs) [38]. These methods have provided additional functionality to paper-based devices, such as the ability to include the dimension of time with the addition of hydrophobic regions and signal amplification with AuNPs, as discussed in the upcoming sections.

2 Technologies for Bacteria and Virus Diagnosis As discussed in Sect. 1, successful POC tests require diverse characteristics, such as rapid and easy-to-use processes, high sensitivity with small sample size and costeffectiveness. To meet these demands, various technologies have been developed utilizing the advantageous properties of paper. The core technologies that have been used for the fabrication of paper-based devices in the literature can be categorized into paper plates, lateral flow assays (LFAs), and three-dimensional slip pads (3D slip pads), which are discussed below.

140

S. Han et al.

2.1 Paper Plates Paper plates are likely the most basic available form of paper diagnostic tests. They were developed based on dot-immunobinding assays, in which a circular test zone consisting of nitrocellulose that is 5 mm in diameter is used, as seen in Fig. 1. The reagents, depending on the method being used (i.e., ELISA), are localized within the circular test zone, and the analyte solution is added perpendicular to this surface. Because of the small size of the test zone, minimal amounts of reagents and analytes are required for the paper plates, which is advantageous compared to other devices. Both the bottom and top sides of the test zone are exposed to the atmosphere; therefore, the analytes can be detected on the surface, and excess solution can pass through without affecting the test zone. The reagents avoid diffusing through a large distance within the paper because the analyte is applied directly to the surface of the reagents, unlike in LFAs and 3D slip pads. A common method used with paper plates is paperbased ELISA (P-ELISA), which is explained further below. Because paper plates use fairly basic concepts, their sensitivity is typically lower than that of other devices, which can be a disadvantage [9]. Paper plates produce strong color contrasts during detection reactions due to the high surface-to-volume ratio, which also enables a rapid evaporation. This property allows for easy nucleic acid or protein detection via colorimetric reactions using AuNPs [47].

Fig. 1 Schematic of paper plate

Paper-Based Applications for Bacteria/Virus

141

Fig. 2 Schematic of lateral flow assay (LFA) and sample flow from the sample pad to absorption pad

2.2 Lateral Flow Assays (LFAs) LFAs detect analytes in a test sample by allowing the fluid to flow parallel to the surface of the paper. The fibers in the paper filter contaminants in the analyte solution, such as soil, dust or algae, as higher molecular weight and structural differences deter them from traveling to the test zone [33]. LFA devices are treated chemically or biologically to detect biomolecules, i.e. proteins and nucleic acids. Furthermore, the fluid flow allows the addition of multiple reagents, such as nanoparticles followed by biomolecules, at various points throughout the fluid path. In general, LFAs are comprised of five sections: the sample pad, conjugation pad, test zone, control zone and absorption pad, as seen in Fig. 2. The test sample is loaded onto the device at the sample pad and flows through the paper to the conjugation pad, where the particles for detection are embedded. These particles can be nanoparticles of gold or silver, as discussed in further detail in the detection methods section. In the test zone, biomolecules are immobilized that react with the sample solution, and nanoparticles provide a signal in the presence of a pathogen. The control zone provides a visual signal to ensure that the fluid is flowing correctly through the device and has reached past the test zone. Any excess fluid from the device is collected at the absorbent pad to ensure that the fluid does not backflow into the control zone. LFAs can detect the presence of pathogens in samples of blood, plasma, serum and saliva [22, 23, 34].

2.3 Three-Dimensional Slip Pads (3D Slip Pads) Several layers of paper make up 3D slip pads to allow the fluid to flow in both vertical and lateral directions, unlike in LFAs, as seen in Fig. 3. Wax barriers create hydrophobic zones to control the fluid flow at and within layers, and the sliding of the “slip” top allows for fluid flow. This device can be used to enhance the detection of a pathogen, as each layer can contain a different reagent, and the mixing of these reagents can induce a chemical reaction for amplification. Another function of 3D slip pads is the integration of two different fluids within the device. Multiple fluid inlets can be formed by creating wax barriers between the inlets and sequential

142

S. Han et al.

Fig. 3 Schematic of three-dimensional paper-based slip device. As the sample is injected and the top layer is closed, the sample flows through the top and bottom layers. The black and white areas in the layers indicate hydrophobic barriers and hydrophilic zones, respectively. Source Han et al. [16]

paper layers. By sliding the “slip” layer, the two fluids can mix and flow through the device, allowing a chemical reaction that can also be used for amplification [16]. The incubation time can be controlled, as the slip layer can be moved at a specific time, allowing fluid flow as required for the reaction. Overall, the main goal of the 3D slip pad is user-friendliness, as multiple functions can be integrated within the device to enhance the detection without requiring multiple steps or equipment [10, 26].

3 Detection of Pathogens The devices described previously use protein and nucleic acids detection methods to identify viruses or bacteria from a sample. The two methods have significant differences, which are described below.

Paper-Based Applications for Bacteria/Virus

143

3.1 Protein Extensive research efforts have focused on utilizing protein antigens in bacteria and viruses as targets for the diagnosis of infectious diseases. Proteins from bacteria or viruses can act as bio-recognition sites, and specific bio-receptors can be used to target these proteins [2]. For example, antibodies are a type of glycoprotein produced by immune cells in the host as a response to foreign substances, such as antigens. An antibody can therefore be used as a bio-receptor for the detection of the presence of antigenic surface proteins, as they have high specificity to each other [25]. There are five classes of antibodies, but the most commonly used is IgG, the general structure of which is shown in Fig. 4. Either polyclonal or monoclonal antibodies can be used for detection. Polyclonal antibodies are produced by infecting an animal with an antigen and collecting different classes of antibodies from the blood. For monoclonal antibodies, a single cell from the spleen of an infected animal is collected and then cultured in tissue to produce cells identical to the parent cell. The cells cloned in the tissue also produce antibodies, which are all of the same class. Monoclonal antibodies are specific and therefore preferred, but also expensive to produce. The specificity of monoclonal antibodies can sometimes be a concern, as the numerous variants of viral strains cannot necessarily be detected [7]. The antigen from the sample of infectious agent can then bind with the antibody at the end of the fragment antigen-binding region, as seen in biological detection processes [20]. One concern with using protein antigens for the detection of bacteria or viruses is the structural stability of the biomolecules (i.e., antibodies and antigens). Because proteins are unstable at room temperature, cold chain transport, storage, and distribution are required to ensure thermal stability. This increases the cost of the devices. Furthermore, the sensitivity of the device largely depends on the antibody quality, which causes difficulties in meeting minimum quality standards; cross-reactivity can also cause inaccurate diagnosis, as antigens can react with un-targeted molecules with similar structural regions [30]. Fig. 4 Structure of IgG antibody

144

S. Han et al.

3.2 Nucleic Acid Nucleic acid detection has received considerable attention as a potential solution to overcome the limitations of protein detection. Nucleic acids are the macromolecules that store and transfer genetic information in cells. There are two types of nucleic acids: deoxyribonucleic acid (DNA), which contains information for all cell functions, and ribonucleic acid (RNA), which is required for protein synthesis [3]. Thus, DNA and RNA collected from cells after lysis can be used to detect pathogens and determine the stage of infection [43]. Nucleic acids exhibit higher thermal stability than proteins, enabling convenient supply, transportation, storage and handling of the device without requiring cold chain. In addition, nucleic acid detection methods lack cross-reactivity concerns. In typical laboratory procedures, nucleic acid analysis is time-consuming since the nucleic acid is through a lysis step, and the sequence is amplified using a polymerase chain reaction (PCR) at a precisely controlled temperature [52]. Millions of copies of the required DNA sequence are generated by PCR, which consists of three different thermal steps: denaturing, annealing and extending [14]. The double-helix DNA sequence is separated into single strands at 94–98 °C. In the annealing step, the primer (i.e., a sequence complementary to the targeted DNA) is added to initiate DNA synthesis at 37–65 °C. The extension of the sequence occurs at 72 °C using DNA polymerase, which adds nucleotides next to the primer [14]. To operate the PCR equipment, electric power, a laboratory setting and highly trained technicians are required. Additionally, for the detection of nucleic acids at POC, multiple steps such as extraction, amplification and detection, should be integrated into a single analytical system. Some efforts have focused on the development of a device equipped to perform the amplification, extraction and detection steps to minimize the need for additional equipment and procedures. In the patented fast technology analysis (FTA), the paper in the device can be used to extract and store nucleic acids [11– 13]. The extracted sequences are treated with reagents for loop-mediated isothermal amplification (LAMP) to replicate the targeted sequence. LAMP has six primers that amplify DNA with a loop-shaped structure. The prototype reverse transcriptionrecombinase polymerase amplification (RT-RPA) can also be used to replicate the targeted sequences. This amplification method employs three enzymes: recombinase, single-stranded DNA-binding protein, and strand-displacing polymerase. Firstly, the recombinase attaches the primer to the targeted sequence. Then, the single-stranded DNA-binding protein binds and stabilizes the single strain, followed by the addition of nucleotides using the strand-displacing polymerase. The amplified nucleic acid is dyed using SYBR Green I or eluted on an LFA strip to produce a visible signal. Although these potential substitutions to PCR have been proposed, their general application has been significantly limited because of the complexity of the design and operation [11, 13, 37].

Paper-Based Applications for Bacteria/Virus

145

4 Detection Methods Two main approaches are used to detect the presence of infectious diseases, classified into colorimetric and electrochemical techniques.

4.1 Colorimetric Colorimetric detection enables the rapid and simple analysis of the presence of a pathogen. A reaction-initiated color change can be detected by the naked eye, meaning it does not require highly trained personnel or expertise for detection. Because of the simplicity of colorimetric detection, this method is the most widely used in POC devices for qualitative results. The reaction that causes the color change can be biological, as in the case of ELISA, or chemical, as with chromogenic substrates. Moreover, a combination of ELISA and AuNPs can be used to further enhance the colorimetric signal.

4.1.1

Enzyme-Linked Immunosorbent Assay (ELISA)

There are two types of ELISA: antigen ELISA and antibody ELISA. As described previously, antigen ELISA uses antibodies to quantify the targeted antigens via a specific conjugation between the antibody and antigen, thus diagnosing bacterial or viral infections. Similarly, in antibody ELISA, antigens are used to detect the presence of the targeted antibody, which can indicate whether the host was in contact with the virus or bacteria (Biobest Laboratories Ltd. n.d.). Therefore, an antigen can be used to detect the presence of antibodies in the sample, but the opposite is also possible, where an antibody is used to detect the presence of an antigen. The procedures for both types of detection are the same, but to explain the four main steps used in each method, we will use antigen ELISA as an example. As shown in Fig. 5, the four steps are antigen immobilization, blocking, antibody complexing, and signal amplification. Firstly, the antigen is immobilized on the test surface. Next, the surface can be blocked by a buffer containing other proteins or compounds to reduce interference by remaining binding surfaces. The antibody is then placed on the test surface; unbound antibodies are removed by a washing step. Finally, the signal is generated by an enzyme reaction. Frequently used enzymes include alkaline phosphatase (ALP) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT). The ALP-labeled antibody is conjugated to the antigen, and then, BCIP/NBT is added to the paper to interact with the enzyme in order to generate a color, which indicates the presence of a pathogen [9]. Paper-based ELISA (P-ELISA) uses the same methodology as ELISA but with paper plates, which has gained increasing attention because of its importance in

146

S. Han et al.

Fig. 5 Schematic of ELISA procedure. Antigen-immobilized paper is blocked to deter interaction with other biomolecules. The ALP-conjugated antibody is captured by the antigen. The interaction of ALP and BCIP/NBT produces a purple precipitate

POC diagnosis. For P-ELISA, 12 μL of the sample is required for all the aforementioned steps of ELISA, which is significantly smaller than the 300 μL required by conventional ELISA. In terms of the reaction time, P-ELISA is significantly quicker than conventional ELISA at 51 minutes versus 213 minutes for all four steps [9]. Despite the advantages of smaller sample volume and shorter reaction time with PELISA, its low sensitivity compared to conventional ELISA is concerning; this can be attributed to i) non-specific interactions between the antibody and paper and ii) the use of a small sample volume. According to one report, the sensitivity of P-ELISA is ten times lower than that of conventional ELISA on a 96-well plate; the detection limits of P-ELISA and conventional ELISA were measured as 54 fmol/zone and 4 fmol/zone, respectively, for the detection of rabbit immunoglobulin G (IgG) [9]. However, there have been reports of P-ELISA being successfully used to detect human immunodeficiency virus (HIV) and T7 bacteriophages [9, 48]. Sandwich ELISA is another ELISA method that requires a secondary antibody to detect the presence of a pathogen. In sandwich ELISA, there is an additional step to conventional ELISA wherein a capture antibody is applied to paper to immobilize the antigen, and a detection antibody conjugated with an enzyme/AuNP is used to label the antigen [45, 48]. Carbon compounds can also be used to produce signals after capturing the antibody or antigen because carbon particles themselves have color. A carbon compound was reportedly used to capture the nucleic acid of Enterobacter sakazakii [6].

Paper-Based Applications for Bacteria/Virus

4.1.2

147

Gold Nanoparticles (AuNPs)

AuNPs are well known to exhibit (i) biocompatibility with diverse molecules such as protein, DNA, and RNA, and (ii) long-term stability as they can be stored for up to 20 days in the dark at 4 °C [21]. In addition, AuNPs can be used for colorimetric detection. Depending on the size or shape of the AuNPs, the wavelength absorbed by AuNPs changes because of surface plasmon resonance (SPR). That is, the electrons on AuNPs oscillate upon exposure to light; therefore, AuNPs of 5–20 nm can absorb light of 520–530 nm. However, as the AuNPs are aggregated, the absorption wavelength is shifted to 620 nm. This shift in the absorption wavelengths changes the reflection from blue to red, as a so-called red shift [48]. In addition, AuNPs can be functionalized with proteins and nucleic acids to detect targeted biomolecules, which is one of the most important properties for paper devices [15, 32].

4.1.3

AuNPs with ELISA Experiments

As mentioned previously, ELISA uses antibody–antigen binding abilities to detect the presence of pathogens. AuNPs can be combined with the ELISA methodology to utilize the properties of both methods and provide a colorimetric signal visible to the naked eye. For example, in LFAs, AuNPs can act as carriers for the detection antibody or antigen and then aggregate under antibody–antigen binding at the test line. This aggregation yields a change in color, as mentioned previously. LFAs that employ AuNP and ELISA have been used to detect pathogenic antibodies from influenza virus [34], Escherichia coli O157:H7 and Salmonella typhimurium [31, 32]. Two surface antigens are targeted for the detection of the influenza virus: hemagglutinin (HA) and neuraminidase (NA). Thus, antibodies against the HA or NA are conjugated with AuNPs to identify the virus [34]. Similarly, polyclonal antibodies against E. coli O157:H7 and S. typhimurium were used for the detection of these respective bacterial pathogens [32]. However, even with the addition of AuNPs, a gold enhancer solution containing gold ions may be necessary to achieve a detectable signal [31]. The gold ions are deposited on the AuNPs, thereby increasing their physical size, which can amplify the visual signal [16]. Similar to protein detection, AuNPs can be used to detect the presence of nucleic acids. A lower detection limit was achieved by the addition of AuNPs with a streptavidin coating. The complementary DNA to the targeted virus (hepatitis B) was functionalized on the AuNP surfaces, along with another single-stranded DNA that was biotinylated. Biotin has strong non-covalent interactions with streptavidin, therefore acting as an additional probe to detect the streptavidin-coated AuNPs and enhancing signal detection. The test sample, DNA-functionalized AuNPs and streptavidincoated AuNPs were added to the sample pad and allowed to migrate toward the test line. The targeted virus DNA was sandwiched at the test line by complementary DNA probes immobilized at the line and the complementary DNA functionalizing the AuNPs. The streptavidin-coated AuNPs further aggregated here via biotin bonding,

148

S. Han et al.

resulting in a colorimetric detection and a lower detection limit for the hepatitis B virus [15].

4.1.4

Chromogenic Substrate

Chromogenic substrates are another type of colorimetric diagnosis method. The basic principle is to use specific enzymes produced by the targeted bacteria to cause a reaction with a chromogenic substance printed on the device. More specifically, the chromogenic substrate is cleaved by an enzyme and the product causes a color change. As colorimetric substrates, 5-bromo-4-chloro-3-iindolyl-B-D-glucuronide sodium salt (XG), chlorophenol red B-galactopyranoside (CPRG), 5-bromo-4-chloro-3indolyl-myo-inositol phosphate (X-InP), and 5-bromo-6-chloro-3-indolyl caprylate (magenta caprylate) have been used [17, 19]. This method has demonstrated selectivity between pathogenic and non-pathogenic E. coli because of the different enzymes produced by each strain. For instance, CPRG and XG can be printed on paper to detect the presence of E. coli. β-galactosidase (β-gal) is produced from coliforms and cleaves CPRG, changing the color from yellow to red [1, 17, 19, 44]. However, only non-pathogenic E. coli produces β-glucuronidase (GUS), which reacts with XG to produce a blue color. Therefore, the presence of a red color without a blue color indicates the existence of pathogenic E. coli. Similarly, Listeria monocytogenes, Salmonella enterica and the influenza virus were also detected using this methodology with different substrates [19, 27].

4.2 Electrochemical Detection Electrochemical devices can be used to investigate the amount of detected samples to diagnose the presence of an infectious disease by the conversion of biological and chemical events into electrical signals. Electrochemical sensors consist of three electrodes printed on the device, i.e., the counter electrode (CE), working electrode (WE) and reference electrode (RE). For example, to detect the antibodies from HIV and HCV, the CE and WE are made of carbon ink, and the RE is made of Ag/AgCl ink. ELISA is first conducted on the reaction zones, followed by the addition of p-aminophenyl phosphate (pAPP) to produce a current. The antibody from ELISA catalyzes the pAPP, which is then oxidized by the electrochemical potential between the WE and CE. The RE maintains a consistent current, which is used as a control. The current produced is monitored as the output signal, which is proportional to the amount of antibodies in the sample [53] Nucleic acids can also be used as target biomolecules in an electrochemical pad comprising four layers, as in a 3D slip pad, with silver nanoparticles (AgNPs). First, the targeted DNA sandwich (three-strand DNA comprised of the targeted DNA, label DNA and capture DNA complimentary to the targeted DNA) is conjugated with AgNPs, followed by injection into the 3D slip pad. The first layer features electrodes

Paper-Based Applications for Bacteria/Virus

149

and an inlet/outlet for sample transportation. The second layer has a hollow channel for sample flow, while the third layer has an oxidant (i.e., KMnO4 ) to oxidize the sample. Lastly, the bottom layer consists of a sink and a hydrophilic hemichannel to enable the sample to flow through the hollow channel above. Within 20 s, the KMnO4 oxidizes the AgNPs to Ag+ , which is deposited onto the WE. The number of attached ions is measured by the anodic stripping voltammetry (ASV) detection method. The sensitivity of this device is increased by 250,000 times, as each AgNP contains 250,000 Ag atoms for detection at the WE [26]. The data from electrochemical devices can be transmitted to a smartphone, allowing for quantitative results [53].

5 Future Prospects Major progress has occurred in the development of POC diagnostics for detecting infectious diseases, from paper plates to LFAs and 3D slip pads. However, further research is needed in various areas to fully integrate these devices with the ASSURED criteria. The first step for using a POC device is the collection of the sample (i.e., blood, urine, or saliva) to be tested, enabling self-collection of samples. A finger stick, for instance, is a device originally designed for glucose monitoring, but finger stick collection may also be used for nucleic acid analysis [21]. Following collection, the separation of the collected samples remains an area requiring development. For example, blood contains two main components: plasma, containing the analyte needed for diagnostics, and red blood cells, which can block diagnostic devices from functioning properly. The traditional separation of blood requires a laboratory setting for centrifugation, which is unreasonable for POC tests. To address this demand, there have been developments in the nanofiltration of blood, but challenges such as membrane clogging and cost increases must be addressed before application [21]. One of the biggest challenges with POC tests is the stability of paper diagnostic devices. In particular, the thermal stability of devices, and especially for enzymes, antigens, and antibodies, is an inherent challenge, as the shipping and storage conditions often differ in resource-limited settings. Biomolecules require refrigeration during transport and storage to prevent denaturation, which is a concern because of its additional costs [40], working against the affordability aspect of the ASSURED criteria from the WHO. Some reports have shown that microbial enzymes have higher thermal stabilities than those sourced from animals or plants; this can be further investigated for POC applications [36]. The sensitivity of the tests is also a concern as the limit of detection of analytes must always be improved because this ultimately allows for a faster treatment plan and minimization of the progress of the infectious disease [40]. Furthermore, specificity is important to minimize the amount of false diagnosis, especially with tests that qualitatively detect the bacteria or virus. False diagnosis can be minimized by improvements in technologies to decrease the cross-reactivity. It is difficult to determine the sensitivity and reproducibility of the tests if visual detection is used, as human error is significantly higher [8]. Along with human error, paper often becomes

150

S. Han et al.

yellow or brown in color over time depending on its storage conditions, which affects visual detection [42]. Smartphones can be incorporated to limit human error, as they can minimize variability in colorimetric detection. Smartphones can filter out the optical signals arising from the nonhomogeneous cellulose fibers, enabling more accurate detection, but they also increase the cost of the test [40]. The integration of smartphone technology with healthcare is an ongoing process, amounting to a separate field of its own. The ability to test for multiple markers simultaneously is another area with great potential [8]. A handful of devices can achieve the detection of more than one analyte, but these remain in the process of further performance optimization [31]. If a single device can replace multiple devices for the detection of various diseases, the costs and waste of diagnostic devices would likely decrease, further helping the progress of POC devices.

6 Summary As discussed in this chapter, POC technology is of great importance for the utilization of diagnostic devices in resource-limited settings where traditional laboratories and equipment are not feasible. Paper has entered use in the medical field to improve the lives of many, as it fits the POC criteria desired for low-income countries. Paper can be used in different formats to produce diagnostic devices, with the most common being paper plates, lateral flow assays and three-dimensional slip pads. These devices can be combined with chemical or biological processes to detect the presence of proteins or nucleic acids from pathogens. The processes used for detection include ELISA, NPs, and electrochemistry or a combination of these. The detection of a pathogen is often confirmed colorimetrically by the naked eye, but the integration of smartphones for analysis and documentation is gaining interest, especially with electrochemical processes. AuNPs are often used because of their stability, biocompatibility and ability to aggregate, yielding the colorimetric detection of a pathogen. However, concerns regarding various aspects of POC paper diagnostic devices remain, including issues with stability, sensitivity, specificity, and sample collection and separation. This is an evolving field where research continues to address these concerns to ensure that paper diagnostic devices meet the ASSURED criteria established by the WHO. Acknowledgements This work was supported by Alberta Innovates Technology Futures [AIF200900279]; University of Alberta [startup fund for H.-J.C]. The authors have no conflicts of interest to declare.

Paper-Based Applications for Bacteria/Virus

151

References 1. Adkins, J., et al.: Colorimetric and electrochemical bacteria detection using printed paper- and transparency-based analytic devices. Anal. Chem. 89, 3613–3621 (2017). https://doi.org/10. 1021/acs.analchem.6b05009 2. Ahmed, A., Rushworth, J., Hirst, N., Millner, P.: Biosensors for Whole-Cell Bacterial Detection. Clin. Microbiol. Rev. 27, 631–646 (2014). https://doi.org/10.1128/CMR.00120-13 3. Bailey, R.: Learn about nucleic acids and their function. Available at: https://www.thoughtco. com/nucleic-acids-373552. Accessed 18 Dec 2019 4. Baylor College of Medicine: Introduction to infectious diseases. (n.d). Available at: https://www.bcm.edu/departments/molecular-virology-and-microbiology/emerging-infect ions-and-biodefense/introduction-to-infectious-diseases. Accessed 18 Dec 2019 5. Biobest Laboratories Ltd.: ELISA-How does the test work? (n.d.). Available at: http://www. biobest.co.uk/diagnostics/techniques/elisa-how-does-the-test-work.html. Accessed 18th Dec 2019 6. Blažková, M., Jav˚urková, B., Fukal, L., Rauch, P.: Immunochromatographic strip test for detection of genus Cronobacter. Biosens. Bioelectron. 26, 2828–2834 (2011). https://doi.org/10. 1016/j.bios.2010.10.001 7. Campbell, A.: Production and purification of antibodies. In: Diamandis, E., Christopoulos, T. (eds.) Immunoassay, pp. 95–115. Academic Press, Cambridge, MA (1996) 8. Carrell, C., et al.: Beyond the lateral flow assay: A review of paper-based microfluidics. Microelectron. Eng. 206, 45–54 (2019). https://doi.org/10.1016/j.mee.2018.12.002 9. Cheng, C.-M., et al.: Paper-Based ELISA. Angew. Chem. Int. Ed. 49, 4771–4774 (2010). https://doi.org/10.1002/anie.201001005 10. Chen, P., et al.: Paper-based vertical flow immunoassay (VFI) for detection of bio-threat pathogens. Talanta 191, 81–88 (2019). https://doi.org/10.1016/j.talanta.2018.08.043 11. Choi, J.R., et al.: An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care. Lab Chip 16, 611–621 (2016). https://doi.org/10.1039/C5LC01388G 12. Choi, J.R., et al.: Paper-based sample-to-answer molecular diagnostic platform for point-of-care diagnostics. Biosens. Bioelectron. 74, 427–439 (2015). https://doi.org/10.1016/j.bios.2015. 06.065 13. Connelly, J.T., Rolland, J.P., Whitesides, G.M.: “Paper machine” for molecular diagnostics. Anal. Chem. 87, 7595–7601 (2015). https://doi.org/10.1021/acs.analchem.5b00411 14. Elrich, H.A., Gelfand, D., Sninsky, J.J.: Recent advances in the polymerase chain reaction. Science 252, 1643–1651 (1991). https://doi.org/10.1126/science.2047872 15. Gao, Y., et al.: Ultrasensitive paper based nucleic acid detection realized by three-dimensional DNA-AuNPs network amplification. Biosens. Bioelectron. 92, 529–535 (2017). https://doi. org/10.1016/j.bios.2016.10.068 16. Han, K.N., Choi, J.S., Kwon, J.: Three-dimensional paper-based slip device for one-step pointof-care testing. Sci. Rep. 6, 25710 (2016). https://doi.org/10.1038/srep25710 17. Hossain, S.M., et al.: Multiplexed paper test strip for quantitative bacterial detection. Anal. Bioanal. Chem. 403, 1567–1576 (2012). https://doi.org/10.1007/s00216-012-5975-x 18. Hu, J., et al.: Advances in paper-based point-of-care diagnostics. Biosens. Bioelectron. 54, 585–597 (2014). https://doi.org/10.1016/j.bios.2013.10.075 19. Jokerst, J.C., et al.: Development of a paper-based analytical device for colorimetric detection of select foodborne pathogens. Anal. Chem. 84, 2900–2907 (2012). https://doi.org/10.1021/ ac203466y 20. Ju, H., Lai, G., Yan, F.: Introduction. In: Immunosensing for Detection of Protein Biomarkers, pp. 1–30. Elsevier Ltd., Oxford, UK (2017) 21. Karlikow, M., Pardee, K.: The many roads to an ideal paper-based device. In: Land, K. (ed.) Paper-based Diagnostics, pp. 171–201. Springer, Cham, Switzerland (2018) 22. Kim, H., Chung, D.R., Kang, M.: A new point-of-care test for the diagnosis of infectious diseases based on multiplex lateral flow immunoassays. Analyst 144, 2460–2466 (2019). https://doi.org/10.1039/c8an02295j

152

S. Han et al.

23. Koczula, K.M., Gallotta, A.: Lateral flow assays. Essays Biochem. 60, 111–120 (2016). https:// doi.org/10.1042/EBC20150012 24. Kosack, C., Page, A., Klaster, P.: Policy and Practice (2017). Available at: https://www.who. int/bulletin/volumes/95/9/16-187468/en/. Accessed July 2019 25. Lipman, N.S., Jackson, L.R., Trudel, L.J., Weis-Garcia, F.: Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J. 46, 258–268 (2005). https://doi.org/10.1093/ilar.46.3.258 26. Li, X., Scida, K., Crooks, R.M.: Detection of Hepatitis B virus DNA with a paper electrochemical sensor. Anal. Chem. 87, 9009–9015 (2015). https://doi.org/10.1021/acs.analchem. 5b02210 27. Murdock, R.C., et al.: Development of a point-of-care diagnostic for influenza detection with antiviral treatment effectiveness indication. Lab Chip 17, 332–340 (2017). https://doi.org/10. 1039/c6lc01074a 28. O’Farrell, B.: Evolution in lateral flow–based immunoassay systems. In: Wong, R., Tse, H. (eds.) Lateral Flow Immunoassay, pp. 1–33. Humana Press, New York, NY (2008) 29. Pai, N.P., Vadnais, C., Denkinger, C., Engel, N., Pai, M.: Point-of-care testing for infectious diseases: diversity, complexity, and barriers in low- and middle-income countries. PLOS Med. 9, 1–7 (2012). https://doi.org/10.1371/journal.pmed.1001306 30. Pardee, K., Green, A.A., Takahashi, M.K., et al.: Rapid, low-cost detection of zika virus using programmable biomolecular components. Cell. 2016 165(5), 1255– 1266. https://www.sciencedirect.com/science/article/pii/S0092867416305050. https://doi.org/ 10.1016/j.cell.2016.04.059 31. Park, J., Shin, J.H., Park, J.-K.: Pressed paper-based dipstick for detection of foodborne pathogens with multistep reactions. Anal. Chem. 88, 3781–3788 (2016). https://doi.org/10. 1021/acs.analchem.5b04743 32. Park, T.S., Li, W., McCracken, K.E., Yoon, J.Y.: Smartphone quantifies Salmonella from paper microfluidics. Lab Chip 13, 4832–4840 (2013). https://doi.org/10.1039/c3lc50976a 33. Park, T.S., Yoon, J.-Y.: Smartphone detection of Escherichia coli from field water samples on paper microfluidics. IEEE Sens. J. 15, 1902–1907 (2015). https://doi.org/10.1109/JSEN.2014. 2367039 34. Peng, F., et al.: Development of an immunochromatographic strip for rapid detection of H9 subtype avian influenza viruses. Clin. Vaccine Immunol. 15, 569–574 (2008). https://doi.org/ 10.1128/CVI.00273-07 35. Rivet, C., Lee, H., Hirsch, A., Hamilton, S., Lu, H.: Microfluidics for medical diagnostics and biosensors. Chem. Eng. Sci. 66, 1490–1507 (2011). https://doi.org/10.1016/j.ces.2010.08.015 36. Robinson, P.K.: Enzymes: Principles and biotechnological applications. Essays Biochem. 59, 1–41 (2015). https://doi.org/10.1042/bse0590001 37. Rodriguez, N.M. et al.: Paper-based RNA extraction, in situ isothermal amplification, and lateral flow detection for low-cost, rapid diagnosis of influenza A (H1N1) from clinical specimens. Anal. Chem. 87, 7872–7879 (2015). https://doi.org/10.1021/acs.analchem.5b01594 38. Sakamoto, S., et al.: Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. J. Nat. Med. 72, 32–34 (2018). https://doi.org/10.1007/ s11418-017-1144-z 39. Schabel, S., Biesalski, M.: The role of paper chemistry and paper manufacture in the design of paper-based diagnostics. In: Land, K. (ed.) Paper-based Diagnostics, pp. 23–46. Springer, Cham, Switzerland (2018) 40. Sher, M., Zhuang, R., Demirci, U., Asghar, W.: Paper-based analytical devices for clinical diagnosis: recent advances in the fabrication techniques and sensing mechanisms. Expert Rev. Mol. Diagn. 17, 351–366 (2017). https://doi.org/10.1080/14737159.2017.1285228 41. Singh, A.T., et al.: Paper-based sensors: Emerging themes and applications. Sensors 18, 2838 (2018). https://doi.org/10.3390/s18092838 42. Smith, S., Korvink, J., Mager, D., Land, K.: The potential of paper-based diagnostics to meet the ASSURED criteria. RSC Adv. 8, 34012–34034 (2018). https://doi.org/10.1039/C8RA06 132G

Paper-Based Applications for Bacteria/Virus

153

43. Song, Y., et al.: Point-of-care technologies for molecular diagnostics using a drop of blood. Trends Biotechnol. 32, 132–139 (2014). https://doi.org/10.1016/j.tibtech.2014.01.003 44. Srisa-Art, M., Boehle, K.E., Geiss, B.J., Henry, C.S.: Highly sensitive detection of Salmonella typhimurium using a colorimetric paper-based analytical device coupled with immunomagnetic separation. Anal. Chem. 90, 1035–1043 (2018). https://doi.org/10.1021/acs.analchem.7b04628 45. Stanker, L.H., Hnasko, R.M.: A double-sandwich ELISA for identification of monoclonal antibodies suitable for sandwich immunoassays. InELISA 2015 (pp. 69–78). Humana Press, New York, NY 46. Tsai, T.T., et al.: Diagnosis of tuberculosis using colorimetric gold nanoparticles on a paperbased analytical device. ACS Sens. 2, 1345–1354 (2017). https://doi.org/10.1021/acssensors. 7b00450 47. Veigas, B., et al.: Gold on paper–paper platform for Au-nanoprobe TB detection. Lab Chip 12, 4802–4808 (2012). https://doi.org/10.1039/C2LC40739F 48. Wang, C.C., Wu, S.M., Li, H.W., Chang, H.T.: Biomedical applications of DNA-conjugated gold nanoparticles. ChemBioChem Rev. 17, 1052–1062 (2016). https://doi.org/10.1002/cbic. 201600014 49. Whitesides, G.: TR:10 Paper Diagnostics [Interview] (24 Feb 2009) 50. World Health Organization: Infectious diseases [Online] (n.d.). Available at: https://www.who. int/topics/infectious_diseases/en/. Accessed 18 Dec 2019 51. Yetisen, A.K., Akram, M.S., Lowe, C.R.: Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13, 2210–2251 (2013). https://doi.org/10.1039/C3LC50169H 52. Zhang, W., Guo, S., Carvalho, W.S.P., Jiang, Y., Serpe, M.J.: Portable point-of-care diagnostic devices. Anal. Methods 8, 7847–7867 (2016). https://doi.org/10.1039/C6AY02158A 53. Zhao, C., Liu, X.: A portable paper-based microfluidic platform for multiplexed electrochemical detection of human immunodeficiency virus and hepatitis C virus antibodies in serum. Biomicrofluidics 10, 024119 (2016). https://doi.org/10.1063/1.4945311

Paper-Based Molecular Diagnostics Bhagwan S. Batule, Youngung Seok, and Min-Gon Kim

1 Introduction 1.1 Nucleic Acid Testing (NAT) Nucleic acids have been widely recognized as ideal biomarkers in various applications, such as microorganism detection, [50] medical diagnostics [15, 24, 25], food safety [9, 54], environmental monitoring [73], and science forensics [53]. In these areas, nucleic acid testing (NAT) is a widely used analytical tool, as the direct detection of nucleic acid is a highly sensitive and specific technology among early diagnostic methods [56]. NAT is also called molecular diagnostics or genetic testing, which refers to the detection of specific DNA or RNA sequences in the target samples. Based on its outstanding sensitivity [30], NAT has become the standard diagnostic method for the detection of harmful microorganisms, [13] representing a significant index recorded in many types of market reports. NAT ranked fourth in market share but showed top-level growth in all diagnostics technology. This point represents the high potential value of the NAT system in the market. Increases in the market share of NAT can be easily expected in the near future. Figure 1a shows various applications of NAT in medical diagnostics and the market size. According to 2017 data, the global NAT market size is almost 9.1 billion dollars. Medical diagnosis of infectious diseases has the largest market size and highest growth rate of all the fields of NAT (Fig. 1b). NAT for infectious diseases has powerful advantages in sensitivity and specificity, able to detect pathogens in early stages [55]. Moreover, NAT can be applied to all types of pathogens, including viruses and bacteria, because all target pathogens have unique sequences of DNA or RNA. B. S. Batule · Y. Seok · M.-G. Kim (B) Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Gwangju 500-712, Republic of Korea e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 J. H. Lee (ed.), Paper-Based Medical Diagnostic Devices, Bioanalysis 10, https://doi.org/10.1007/978-981-15-8723-8_8

155

156

B. S. Batule et al.

Fig. 1 a Molecular diagnostic market: segment growth analysis, 2012–2019. Source Frost & Sullivan analysis. b Molecular diagnostics market share (Source KISTI Market report)

1.2 Technologies for Nucleic Acid Testing: Extraction, Amplification, and Detection NAT generally comprises the following processes: sample collection, extraction, amplification, and detection (Fig. 2). At present, several specialized techniques have been well developed for each step of NAT, as mentioned before. For the sample collection and extraction processes, silica columns are the most widely used platform for nucleic acid isolation [12, 52]. For nucleic acid amplification and detection, the polymerase chain reaction (PCR) [16, 33] method is the gold standard. The PCR technique has been widely utilized for the detection of infectious diseases, genetic sequences, and other types of diseases. PCR is performed in three steps: the denaturation of the target double-stranded DNA, annealing, and the extension of

Paper-Based Molecular Diagnostics

157

Fig. 2 Overall process of nucleic acid testing (NAT)

primers by controlling the reaction temperature. PCR-based nucleic acid detection is highly sensitive and selective; however, large and specialized equipment is required for nucleic acid detection. To address these issues, several isothermal amplification techniques have been utilized to perform nucleic acid amplification at a constant temperature. In isothermal amplification, the role of temperature control in PCR is successfully replaced by the shape of the primer or another enzyme. For example, loop-mediated isothermal amplification (LAMP) was reported as a sensitive and specific diagnostic technique [70] based on the loop primer conformation during the nucleic acid amplification process. Recombinase polymerase amplification (RPA) achieved sensitive detection results by combination with polymerase and recombinase. Helicase-dependent amplification (HDA) can be utilized to detect nucleic acids using the helicase enzyme, which can unwind double-stranded DNA into singlestranded DNA. Rolling-circle amplification has been reported for the detection of single-stranded DNA without a primer. In this method, single-stranded DNA or RNA acts as a primer for circle DNA and performs continuous amplification through strand displacement at constant room temperature. A comparison of PCR, LAMP, RPA, HDA, and RCA data is summarized in Table 1 [71, 86]. Isothermal nucleic acid amplification techniques are simple, fast, sensitive, and selective for the detection of nucleic acid [2]. Table 1 Comparison of nucleic acid amplification techniques for PCR, LAMP, RPA, HDA, and RCA PCR

LAMP

RPA

HDA

RCA

Target

DNA or RNA

DNA or RNA

DNA or RNA

DNA or RNA

RNA

Reaction time

2–3 h