Nanosensors for the detection of viruses

Chapter 19

Nanosensors for the detection of viruses Sumaira Younis1,2, Ayesha Taj1,3, Rabisa Zia1,3, Hunza Hayat1,3, Ayesha Shaheen1,3, Fazli Rabbi Awan1, Haq Nawaz Bhatti2, Waheed S. Khan1 and Sadia Z. Bajwa1 1

National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, 2Department of Chemistry, University of Agriculture

Faisalabad, Faisalabad, Pakistan, 3Pakistan Institute of Engineering and Applied Sciences, Islamabad, Pakistan

Chapter outline 19.1 19.2 19.3 19.4

Introduction Magnetic nanoparticles for the detection of viral particles Membrane-based sensor for virus detection Electrochemical-based detection of virus

19.1

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19.5 Fluorescence approach for virus detection 19.6 Microfluidic devices for virus detection 19.7 Point-of-care testing for detection of viral infections References

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Introduction

Viruses are very tiny parasites, and they have the ability to affect the physiology and behavior of the host they enter and infect. Their host can include any living species ranging from bacteria to plants, animals, and human beings. The modes of action are varied, but the most serious factor remains is that viruses are adaptable. This is the main reason for the greater number of deaths worldwide, especially by influenza viruses, human immunodeficiency virus (HIV), and most recently Zika viruses (ZIKVs). Most probably, the mutation of either of the existing known viruses may lead to epidemics. When this mutation happens at the level of single nucleic acid, it can be lethal to ease the large masses of human population. Although reliable statistics are not available for showing the gravity of the situation, irrespective of the cause and reasons of spread, such epidemics result in huge financial burden to health-care managers and policy makers. This problem is worse in developing countries where the resources are scarce, and population is not educated enough to have awareness. This situation necessitates the development of fast, cost-efficient diagnostics, especially in the situation of a pandemic. A variety of methods are available to study the viruses and their interaction in developing infection and to detect them. It is worth mentioning that when a disease is spread, detection is the most sought out process to prevent and treat infection. Therefore we can say that new methodologies will be required to detect new mutations of viruses and diseases caused by them. For example, in the disease onset and spread of dengue viruses, the detection of virus is the first step to control the progress of the epidemic, which requires the direct identification of either viral protein or nucleic acids. However, conventional clinical methods are based on western-blot or real-time reverse transcription polymerase chain reaction (RT-PCR). They are usually expensive and tedious, and these factors offer serious limitation when quick screening is required for the large numbers of specimen. Further the storage of the specimen also affects the quality of the assay. Antibody-based methods are generally economical, but sometimes their detection takes a longer time (several weeks or maybe months). The antibodies are produced as the result of the immune response after attaching of the virus. In the case of direct methods such as by protein limitation occurs if any change happens in the conformation of the protein. In this scenario, any methods that can lead to fast and early detection of the virus is highly desired. Therefore new approaches for sensitive and, especially, early detection are highly needed. Biosensors offer the development of low-cost, sensitive, and portable methods, especially for the point-of-care analyses. Since the last two decades, nanotechnology has progressed into a multidisciplinary area. The worth of nanomaterials and the strength of nanotechnology can be augmented to construct detection methods with desirable features, such as low detection limit, cost efficiency, small sample volume, and short analysis time. Fig. 19.1 is describing the general mechanism of sensing. Biosensors used for diagnostic purposes are based on various techniques, such as PCR, enzyme-linked Nanosensors for Smart Cities. DOI: https://doi.org/10.1016/B978-0-12-819870-4.00018-9 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 19.1 General mechanism of the working of biosensor.

FIGURE 19.2 Properties of biosensors.

immunosorbent assay (ELISA), and other detection procedures including gel electrophoresis and fluorescent and radioactive labeling. The typical arrangement of ELISA demonstrates the interaction of analyte molecules toward the immobilized probe (i.e., “sandwich assay” arrangement). This occurs due to the conjugation property of detection proteins, which binds to the molecules of the target analyte [1,2]. Contrary to it, PCR generates amplified signals when template DNA is presented by specifically amplifying the target DNA sequences [3]. The complex multistep protocols, tedious sample preparation, rigorous handling, high analysis costs, and nonavailability of highly expert labor restrict the extensive use of these techniques [4]. Several advanced biosensors are fabricated to overcome the issues related to conventional diagnostic techniques. Generally, they are based on the nanomaterials, such as the sensing platforms or the transducer, for the detection of biomolecules. Here, the inherent characteristics of tunneling and quantum effects associated with the nanomaterials play a vital role in the amplification of the signal [5]. High surface-to-volume ratio has enormous effects on the sensitivity of nanosensors by increasing the active surface area [6]. Molecules of target analytes (virus) exist at nanoscale, making nanosensors attractive choice for analysis [7]. Fig. 19.2 describes the properties of nanosensors. One of the numerous potential benefits of nanobiosensors over conventional detection systems is high sensitivity. It is associated with the interaction and relation of nanomaterials with the analyte molecules [8,9]. These interactions determine the increase in the response of detection system, that is, increase in the amplitude of current signal [10,11]. During detection process, the sensing materials interact with analyte molecules according to their characteristic ability and available surface area, due to which they are sensitive to a large amount of desired analytes. Sensitivity is not only related to the inherent electrical sensitivity of the device but also to the overall exposed surface area of nanomaterials, a binding mechanism of interactions between analyte molecules and sensing moieties as well as other experimental conditions such as temperature, time, pH, and concentration. These factors play a key role in practical applications of biosensors based on nanomaterials [12,13]. Biosensors further offer superior specificity. The ability to respond to the presence of the analyte is not enough for the practical application of a biosensor. It should be able to distinguish analyte of interest from other interfering agents [14]. The specific detection behavior of a sensing element is significantly important to detect a desired analyte especially if it is present in low concentrations in a sample containing a large number of alternative materials [15]. Generally, biosensors use natural, specific binding phenomenon such as antibody
antigen, enzyme and substrate interactions, macromolecules, and biotin and streptavidin reactions. However, artificially manufactured ligands, such as

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aptamers, can also be used to specifically interact with a large number of molecules through their specific binding interactions. Molecularly imprinted strategies are also very popular in fabricating selective sensors. In this procedure, monomers are polymerized round to a template, or print molecular structure, which is then removed by washing through a suitable solvent, and it leaves imprints for specific analyte molecules [16,17].

19.2

Magnetic nanoparticles for the detection of viral particles

Quick and robust response for the early detection of infection demands highly sensitive recognition of infectious moieties. Nanotechnology helps us in developing sensitive, fast, precise, and orderly representative biosensing techniques to detect desired analytes [18]. Nanomaterials having magnetic properties are considered main sources of labels/tags/markers for diverse biosensing applications. They act as attractive choices for the development of multiple designs and biosensing instruments due to precise nanoscale size, composition, and outstanding magnetic properties, which are not found in biological systems. The use of magnetic nanomaterials in advanced instruments is highly beneficial for a variety of applications in point-of-care sensor systems [19]. Superparamagnetic nanoparticles (NPs) have been applied as contrast agents in magnetic resonance imaging (MRI) for immunomagnetic separation of nucleic acids, proteins, cells, and viruses by conjugation with antibodies. Primarily, the synthesis of magnetic nanomaterials is based on water-based processes involving the alkaline precipitation of related iron salts. In order to increase the water stability and surface modification, the iron-oxide nanostructures are coated with specific polymers including polyacrylic acids, dextran, and silica. To make easy conjugations, they can also be modified with important functional groups such as carboxylic acids and amino acids [20]. The working principle of magnetic materials is based on their direct or indirect linkage with viral components, which makes a composite. As a magnetic field is applied, the whole composite is separated with it or can be detected through sensing devices [18]. Due to lack of magnetic properties in biomaterials, magnetic nanomaterials can be utilized as attractive choices for the sensitive detection of specific biomolecules without any interference with the detection signal [21]. Iron-oxide NPs are considered promising NPs used in biomedical investigations and have been utilized in a variety of piezoelectric, optical, electrochemical, and magnetic field sensor systems. Due to their superparamagnetic properties, they are widely used for magnetic-activated cell sorting, where desired cells are labeled with antibodyconjugated magnetic NPs (MNPs). Target cells are then sorted by exposure to external magnetic field. Most commonly used antigen-detection techniques involving MNPs are based on variations occurring in the spin
spin relaxation-time (T2) values of surrounding water molecules. These changes occur due to the clustering of MNPs as they interact with target molecules as shown in Fig. 19.3. The variations in T2 values are computed by conventional nuclear-magnetic-resonance (NMR) relaxometers or resonance-imaging techniques. Such modified devices become more attractive for practical applications due to the highly sensitive response. In a recent study, MNPs were used for protein detection using ultralow-field NMR technique. Results show the lower limit of detection (LOD), that is, 10 pg/mL lower than the LOD of conventionally available techniques such as ELISA. Due to better efficiency, such materials can be utilized to design chip-based detection systems for the recognition of multiple microliter volumes of the analytes. They also play a key role in electrochemical detection of interacting specific ligands through their direct interaction with electrode aided by transfer of electrons generated in redox reactions [22]. Synthetic and biologically important NPs, for example, viruses and metallic NPs act as basic building blocks for the development of three-dimensional supramolecular structures with multivalent interactions. They are used as attractive FIGURE 19.3 Schematic illustration for the clustering of MNPs due to interaction of specific antigen. This clustering of MNPs results in the variations of relaxation-time (T2) values of water molecules, which surrounds the MNPs, which can be observed through magnetic resonance technique for diagnosis. MNPs, Magnetic nanoparticles. Reproduced with permission from M. Shevtsov, L. Zhao, U. Protzer, M. Klundert, Applicability of metal nanoparticles in the detection and monitoring of hepatitis B virus infection, Viruses 9 (7) (2017) 193. Licensed under a Creative Commons Attribution 4.0 License.

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FIGURE 19.4 Formation of magnetic viral nanosensor by immobilization of specific viral antibodies on the surface of magnetic nanoparticles. Reproduced with permission from J. M. Perez, F.J. Simeone, Y. Saeki, L. Josephson, R. Weissleder, Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media, J. Am. Chem. Soc. 125 (34) (2003) 10192
10193. Copyright 2003 American Chemical Society.

choices because they change the magnetic and optical properties of magnetic nanostructures during the formation of viral-induced assembly. During this procedure, magnetic nanostructures behave as magnetic relaxation switches and cause changes in the spin
spin relaxation-time values of water molecules in the surrounding area. The outstanding potential of magnetic nanomaterials has allowed the design of sophisticated detection systems for sensitive, specific, and rapid detection of clinically important viruses, nucleic acids, RNA, DNA as well as proteins by generating their target-induced self-assembly with synthetic magnetic nanomaterials. In a study (Fig. 19.4), viral-induced assemblies of spherical MNPs have been used for sensitive, selective, and rapid detection of a virus by measuring the variations in relaxation-time (T2) values of water molecules, which occur owing to the interaction of MNPs and viral particles in aqueous media. The strategy could recognize five viral particles in 10 µL (25%) solution of protein without using PCR replication. This technique offers many advantages over currently available PCR techniques for the sensitive detection of virus particles. This method does not involve protein removal or separation and further does not depends upon the optical properties of the solution, thus amplifying the scope of enzymatic studies. It can detect viruses in turbid complex media and is more sensitive and faster than conventional ELISA. During this study, MRI and NMR instrumentation were used to detect experimental changes and can be utilized for the recognition of viruses even in complicated biological media as in cell suspension, blood serum, lipid emulsions, culture media, lipid emulsions, and for complete tissue [17].

19.3

Membrane-based sensor for virus detection

Membrane-based devices are commonly utilized in several biomedical applications. Due to nanoporous nature, they are used for the filtration of samples to isolate the cells, bacteria, viruses, RNA, DNA, and detection of proteins [23,24]. They have regular and uniformly spaced nanochannels acting as templates for the incorporation of nanosized materials, such as polymers, metals, semiconductors, and biomolecules imparting novel physicochemical and biological characteristics, which enhance the membrane applications. Nanoporous membranes have high surface areas, nanopores of uniform sizes, high aspect ratio and are easy to prepare and cost effective [25]. Fig. 19.5 shows the application of nanosensor in which a membrane-based electrochemical nanobiosensor was designed to detect whole viral particles. In this sensor system a submicrometer-thick alumina membrane having nanoporous structure was formed over a disk-like platinum electrode. Probe molecules of the antibody are adsorbed physically on the walls of membrane channels. The sensor response was based on monitoring the Faradaic current of electrode against ferrocene methanol, which is highly sensitive for the immunocomplex formation within the nanomembrane. This membrane-based nanosensor was used for the detection of inactivated West Nile viral particle and West Nile virus protein domain III (WNV-DIII) using anti-WNV-DIII immunoglobulin M as recognition probe. The recognition of viral protein and particle is up to 53 pg/mL with a correlation coefficient R2 5 0.99 as well as 50 viral particles for each 100 mL and correlation coefficient R2 5 0.93 was observed at pH 7 with low detection limits of 4 pg/mL comparable to the sensitivities of PCR techniques. The relative standard deviation for the entire detection of viral particles in blood serum was observed to be 6.9%. Moreover, the straightforward development method for biosensor, insignificant arrangement, and short identification time period of about 30 minutes, makes it a fascinating sensor system for wide-scope application of locating the proteins and infections.

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FIGURE 19.5 Schematic diagram of membranebased nanosensors to detect West Nile Viral Protein D III and WNV molecule using IgM binding. IgM, Immunoglobulin M. Reproduced with permission from B.T. Nguyen, G. Koh, H.S. Lim, A.J. Chua, M. M. Ng, C.-S. Toh, Membrane-based electrochemical nanobiosensor for the detection of virus, Anal. Chem. 81 (17) (2009) 7226
7234. Copyright 2009 American Chemical Society.

19.4

Electrochemical-based detection of virus

Nanomaterial-based biosensors have been used for the detection of a wide number of biological molecules. It is an emerging field where electrochemical interrogation is used in the form of current, potential, and impedance. The change in the signal response occurred, which shows biological recognition event with high sensitivity and selectivity [26]. Such detection methods have also been used for the detection of glucose [27]. These methods are used with the combination of analytical techniques, such as voltammetry, potentiometry, and conductometry, with the specificity of desire analyte [28]. Nowadays, biosensors with these outstanding and rapid detection systems as compared to conventional techniques, such as ELISA and RT-PCR, which are highly expensive, require more time, and highly professionally trained personnel are used for the detection of virus in disease diagnosis [29]. There are some recent reports on graphene-based biosensor system that is used for highly sensitive detection of pathogenic virus. Graphene is a 2D material with carbon honeycomb, free-standing conductive thin-film structure that provides a conducive environment to flow the electron in the electrochemical detection system [30,31]. The modification of 2D graphene sheet with different bioreceptors is shown in Fig. 19.6. Different bioreceptors, such as specific antibodies, enzyme, or DNA for a particular analyte that can be ion, nucleic acid molecules, and cells in the case of virus, were used to modify 2D graphene sheet. This was a two-step process; first, graphene sheet was functionalized with bioreceptorspecific functional entities, and second, these receptors anchored the bioreceptor on 2D graphene sheet [32]. In another study, graphene surface functionalized with pyrene derivatives and after that, virus-specific antibodies covalently linked with these functional derivatives. After that, modified graphene structure with rotavirus-specific antibodies was utilized to modify the glassy carbon electrode that gave cyclic voltametric response by strong antibody
antigen interaction. Under optimized conditions the desired 105 and 103 pfu/mL of input cells of rotavirus specifically detected on the graphene-modified electrode with c.30.7% sensitivity and c.1.3% sensitivity. These results demonstrate that graphene-based immunobiosensors can be applied in the electrochemical detection of food safety test, environmental monitoring, and clinical diagnostics. There are serious health threats from viral pathogens around the world. There is a huge need of some diagnostic tools for rapid and sensitive detection of virus, particularly in developing countries where detection system is slow, insufficient, and overburdened by these fetal virus pathogens [28]. A label-free paper-based microfluidic analytical device integrated on Au electrode is shown in Fig. 19.7. The biotinmodified microwires are used to capture the streptavidin-modified NPs. To fabricate the electrode, Au microwires were first modified with bioaffinity agents that drive the binding of desired analyte by bioconjugation process. For this purpose a cleaned 25 µm diameter gold microwire was incubated in 1.0 mM lipoic acid, followed by a virus-specific

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FIGURE 19.6 The modification of 2D graphene thin sheet with bioreceptor that can be a specific antibody, enzyme, or DNA of a particular analyte. Reproduced with permission from J. Pen˜a-Bahamonde, H.N. Nguyen, S.K. Fanourakis, D.F. Rodrigues, Recent advances in graphene-based biosensor technology with applications in life sciences, J. Nanobiotechnol. 16 (1) (2018) 75. Licensed under a Creative Commons Attribution 4.0 License.

FIGURE 19.7 (A) Reaction scheme with (B) representative corresponding cyclic voltammograms and (C) EIS for Au microwire electrodes at different stages of modification with biotin for capture of SA particles on a static ePAD. EIS, Electrochemical impedance spectroscopy; SA, Streptavidin. Reproduced with permission from R.B. Channon, Y. Yang, K.M. Feibelman, B.J. Geiss, D.S. Dandy, C.S. Henry, Development of an electrochemical paper-based analytical device for trace detection of virus particles, Anal. Chem. 90 (12) (2018) 7777
7783. Copyright 2018 American Chemical Society.

envelope protein 4G2 antibody for 16 hours in methanol solution fused in aluminum file
coated container, to provide oxygen and UV-free environment. The Au microwire is rinsed with deionized water three times and after that it is incubated containing 200 µM amine-PEG2-biotin for 4 hours. To get the crosslinking reaction, carbodiimide with 0.10 M solution of ethanolamine was added for 4 hours to increase the carboxyl functional groups on the surface of Au microwire. As a result of these, cross-linkages increase the sensitivity due to larger surface area for the flow of electron transfer. The as-fabricated Au microwires are rinsed with deionized water for three times and dried under nitrogen gas. The resultant modified Au microwires were ready to assemble in microfluidic paper devices (ePAD). The small antibodyfunctionalized microfluidic devices are used to detect the West Nile virus particles by applying electrochemical impedance spectroscopy. The high LOD of 10.2 was obtained to detect virus particles in cell culture media of 50 µL. This

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FIGURE 19.8 Schematic illustration of lateral flow immunoassay for direct and rapid visual test to detect influenza A virus using carbon nanotags. The signal visualization is induced by the accumulation of carbon nanotags if virus is present in the sample. Reproduced with permission from N. Wiriyachaiporn, H. Sirikett, W. Maneeprakorn, T. Dharakul, Carbon nanotag based visual detection of influenza A virus by a lateral flow immunoassay, Microchim. Acta 184 (6) (2017) 1827
1835. Copyright 2017 Springer Nature.

diagnostic motif is rapid, significantly cheaper, and more sensitive as compared to other conventional methods. As a result of these, outstanding properties of sensing motif open up new avenues for the detection of other biological pathogens [33].

19.5

Fluorescence approach for virus detection

Fluorescent-based nanomaterials ranging from 1 to 20 nm in size are known as quantum dots (QDs) that are mostly semiconductor nanocrystals in nature. These nanomaterials possess specific electronic and optical properties, depending upon their surface-to-volume ratio, known as quantum confinement. This outstanding optical property of these nanomaterials can be used for the fluorescent-based detection of different pathogenic viruses [34]. A novel study has been reported for highly sensitive and selective detection of Japanese encephalitis virus (JEV) using virus-imprinted polymers based on fluorescent biosensors. In this study a simple-surface imprinting of virus particles with magnetic silicon nanomaterial was used as a carrier material. Tetraethyl orthosilicate was used as a cross-linkage in imprinted polymer. The resulting imprinted polymer film contained the recognition cavities so that it captures the target virus. Here comes the role of magnetic silicon nanomaterials for visual fluorescent detection and fast magnetic separation of JEV with linear range of 2.5
45 nM (R2 5 0.9948). Finally, this study confirmed that the imprinted polymers along with fluorescent nanomaterials can be used for the rapid and selective detection of other types of viruses [35]. In Fig. 19.8, another lateral flow assay reported for the visual detection of influenza A virus based on carbon nanotag in the form of nanostrings that act as a reporter. When influenza A virus
specific antibodies interact with antigen, an antibody
antigen-based complex is formed and a carbon nanotag accumulates in the test zone, which allows direct visual detection. Allantoic fluid of embryonated chicken eggs was collected after virus inoculation and used for the detection of virus under optimized conditions. A highly encouraging LOD of 50 mL21 was obtained for 350 TCID and no interfering agents, such as other proteins and closely related virus, were found. The sample was collected containing cell lysates with different strains of influenza viruses. It has been found that this method can detect virus pathogens without interfering with biological entities. Thus it proves that this method has a huge potential for rapid and selective detection of influenza A virus [36].

19.6

Microfluidic devices for virus detection

Microfluidic platforms are from a relatively new generation of diagnostic methods, which depend upon miniaturization of steps such as specimen preparation, bioreaction, and further detection into a unique system. The conventional diagnostic approaches require high pathogen titer for isolation and identification, in addition to cell culture requirements. These steps are also time-consuming, for example, from identification to PCR it requires at least half a week [37]. Microfluidic devices are easy to use, fast, sensitive, and accurate for some of the most dangerous viruses such as HIV, hepatitis B virus (HBV), and ZIKV. Microfluidic devices have significantly decreased the time between specific diagnosis and establishment of targeted clinical treatment, which is vital for the survival of the patient [38,39]. Owing to the portable nature of microfluidic kits, these can be advantageous in localities with poor health facilities. Microfluidic technology has also been utilized successfully for the detection of foodborne viruses [40].

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FIGURE 19.9 Illustration of RT-LAMP on chip for the detection of Zika virus. (A) Designing of paper-based chip on which channels were made by wax printer after which it was heated to melt the channel along the depth of the paper. (B) Spiked Zika virus RNA samples were loaded onto the chip, which moved to detection area leaving behind the bulky impurities. (C) Detection portion of the chip was cut from the paper and RT-LAMP reagents were added and heated to 68 C up to 40 min and color change was detected and analyzed by software. Reproduced with permission from K. Kaarj, P. Akarapipad, J.Y. Yoon, Simpler, faster, and sensitive Zika virus assay using smartphone detection of loop-mediated isothermal amplification on paper microfluidic chips, Sci. Rep. 8 (1) (2018) 12438. Licensed under a Creative Commons Attribution 4.0 License.

Kaarj et al. reported a ZIKV detection method with paper-based microfluidic chip employing the real-time loop mediated isothermal amplification (RT-LAMP) on paper. They used a cellulose grade 4 paper over which channel design was printed using a wax printer. The fabrication was completed by heating the printed paper at 120 C for 5 minutes on a hot plate, which created hydrophobic barriers throughout the paper depth. Spiked ZIKV RNA sample (50 µL) was loaded with pipette onto the paper microfluidic channel. Capillary action helped in the flow of solution along the channels, filtering out the bulky molecules and only allowing the flow of viral RNA toward the area of the chip where detection is monitored. While designing the chip, it is important to consider the dimensions of the loading area and sample volume to be loaded and to determine the volume of sample that could travel along certain length of the chip without evaporation. Afterward, the detection area was excised from the paper and an RT-LAMP (15 µL) reaction mixture consisting of colorimetric master mix containing a pH indicator (i.e., phenol red), and primers was added onto the paper sandwiched in glass slides. The assembly was incubated on a hot plate set at 68 C for 30 minutes, and changes on the paper from yellow-red coloration to yellow indicated progression of amplification. Images were acquired by smartphone at different time intervals and analyzed by software to confirm viral amplification. For further confirmation, amplicon was eluted from the paper using TrisHCL and EDTA buffer. Agarose gel of 3% w/v was prepared, and amplification was confirmed [41] (Fig. 19.9). In another study, surface antigen of hepatitis B virus (HBsAg) was identified from human blood using surfaceenhanced Raman scattering (SERS) consisting of a Raman reporter and a SERS substrate lying on Au
Ag-coated Gallium nitride (GaN) integrated into a microfluidic chip. The microfluidic chip was fabricated in 5 mm polycarbonate slab, and channels were made with about 400 mm width and 350 mm depth. A square-shaped detection area containing SERS active substrate was made on the chip. The working of the whole assembly is based upon a three-layered sandwich structure consisting of immobilized anti-HBsAg antibodies onto GaN/Au
Ag surface. Bovine serum albumin in phosphate buffer saline (PBS), blood plasma containing HBsAg, Au nanoflowers labeled with Raman reporter, and PBS were added sequentially in the detection area, where immunocomplexes were sandwiched upon each other. SERS spectrum of basic fuchsin appeared due to the presence of HBsAg in the serum. SERS was measured by Raman system equipped with a diode LASER, which worked as an excitation source over the sample and the Raman scattering light was collected by a charge-coupled device (CCD) detector to acquire the specific Raman spectrum [42]. Since microfluidic chips are more rapid, require less sample and reagent volume, and highly reproducible. These advantages make them more attractive and an ideal candidate for point-of-care (POC) testing (POCT). But microfluidics in virology is in its infancy and needs further exploration. Currently, it could not replace conventional techniques, but it could be used along with system biology to devise new methods of viral study.

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Point-of-care testing for detection of viral infections

POCT is medical diagnostic testing, which the patients can use at their bedside. The POC devices are of two types mainly: the handheld or the benchtop. The handheld devices are used for rapid diagnosis such as checking glucose levels and HIV detection through salivary assay. The POC offers an inexpensive and portable platform for the detection of many diseases, including viral diseases such as influenza, HIV, and hepatitis. This could help identify outbreaks of epidemics in remote settings for timely therapeutic interventions. The paper-based diagnosis is a well-adaptive mode due to low manufacturing cost, ease of use, and disposability. For virus sensors, paper-based and material-based sensors have developed an early market. Paper-based sensors for basic colorimetric detection were replaced by chromatographic, fluorometric, electrochemical, and microfluidics over the course of time [43]. The electrical and optical modalities have been explained in Fig. 19.10. Fig. 19.10A shows that electrical sensing platform with flexible electrodes was used for HIV detection. Magnetic beads coated with streptavidin and conjugated with antibodies were used to capture viruses from whole blood samples. Washing was done to remove electrically conductive solution followed by lysis of viruses using 1% Triton X-100. The viruses release biomolecules in solution, which changes its electrical conductivity. The decrease in impedance of viral lysate is the signal of interest. Fig. 19.10B portrayed the concept about the detection of bacteria on cellulose paper using NP aggregation and colorimetric detection via portable cellphone camera. N-Ethyl-N0 -(3-dimethylaminopropyl) carbodiimide hydrochloride, 11mercaptoundeconoic acid, and N-hydroxy succinimide were used to modify gold NP (AuNP) surfaces. AuNP surfaces FIGURE 19.10 The paper-based electrical and optical sensing platforms. (A) Flexible microchip for virus (HIV) detection, (B) paper-based nanoparticle aggregation concept for bacteria detection, and (C) flexible microchip for cell (CD41) detection. Reproduced with permission from H. Shafiee, W. Asghar, F. Inci, M. Yuksekkaya, M. Jahangir, M.H. Zhang, et al., Paper and flexible substrates as materials for biosensing platforms to detect multiple biotargets, Sci. Rep. 5 (2015) 8719. Licensed under a Creative Commons Attribution 4.0 License.

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FIGURE 19.11 Schematic illustration of SERS-based immunoassay. SERS, surface-enhanced Raman scattering. Reproduced with permission from Y. Sun, L. Xu, F. Zhang, Z. Song, Y. Hu, Y. Ji, et al., A promising magnetic SERS immunosensor for sensitive detection of avian influenza virus, Biosens. Bioelectron. 89 (2017) 906
912. Copyright 2017 Elsevier.

FIGURE 19.12 Schematic illustration of the preparation of aptamer-Ag/SiO2 sensor. Reproduced with permission from Y. Pang, Z. Rong, J. Wang, R. Xiao, S. Wang, A fluorescent aptasensor for H5N1 influenza virus detection based-on the core
shell nanoparticles metal-enhanced fluorescence (MEF), Biosens. Bioelectron. 66 (2015) 527
532. Copyright 2015 Elsevier.

are functionalized to facilitate the detection of bacteria pathogens. Bacteria-spiked samples were added causing accumulation of NPs and color change of solution, which was detected using image analysis via cellphone camera. Fig. 19.10C describes an unaided shadow CD41 T lymphocytes image, which detects and counts the cell from whole blood via a microchannel containing film-based platform. The shadows of captured CD41 T cells were obtained on the substrate [44]. The study of various materials and their properties has led to improvement in sensitivity, binding affinity, and reliability. Sensitivity of paper-based devices is enhanced by using gold, silver, and carbon NPs, along with liposomes, MNPs, nanocomposites, QDs, enzymes, and conjugates [45]. Nanomaterials have played an immense role in the development of these diagnostic devices mainly due to their inherent properties. For instance, portable Raman spectrometer has been used to study magnetic immunosensor based on SERS for the identification of influenza virus H3N2, which is inactivated yet still in contact from human serum and saliva samples. 4mercaptobenzoic acid (4-MBA)-labeled AuNPs and other six assistive substrates were made up of iron-oxide NPs, with high sensitivity to detect H3N2 down to 102 TCID 50/mL [46]. The good linearity, portability, efficiency, and sensitivity pave its way as a potential POC test for diagnostics. Protocol illustrated in Fig. 19.11 is of the SERS-based magnetic immunoassay. The property of antibody
antigen (virus)-specific interaction and magnetic property of the supporting substrates allowed the enrichment and separation of viruses from a complex matrix. The molecules of 4-MBA were spontaneously chemisorbed onto the surface of AuNPs, and it played an important role as Raman reporter due to its intrinsically strong Raman scattering. 4-MBA enabled a simpler preparation process of SERS tags, which was beneficial to the reproducibility and sensitivity of the SERS test, thus resulting in a facile and sensitive method for detection of the influenza viruses [47]. In another study a metal-enhanced fluorescence (MEF)-sensing platform/fluorescence aptasensor based on silver
silicon oxide NPs has been developed for detection of H5N1 influenza virus recombinant hemagglutinin (rHA)

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protein. Thiazole orange (TO) was used as fluorescent tag. The property of surface plasmon resonance improved the fluorescence emission signal (Fig. 19.12). An MEF aptasensor is used for H5N1 influenza virus detection. G-rich anti-rHA protein of H5N1 aptamers was immobilized on core
shell Ag/SiO2 NPs. TO fluorescence signal reporter was involved in the target-aptamer binding. The detection can be done from both aqueous buffer and human serum with a limit of 2 and 3.5 ng/mL, respectively. The POC has been reported in a polyethylene (PE) tube with 30 minutes analysis time. This method is time-efficient as compared to the ELISA test of 3 hours and standard method of 5
7 days [48]. Rapid analysis of HIV infection and its therapy are also an application of POC diagnostics. Europium NPs based sensitive, rapid and non enzymatic microtiter-plate immunoassay was designed to detect target analyte at subpicogram/ ml levels. A benchtop technique was transformed to POC by means of a microchip platform, which caused 4.5-fold reduction of sample/reagent and twofold shortening of the total time required in comparison with microtiter plate assays. There are certain limitations, such as dilution factors, that need to be catered to, yet the results are promising and practical [49].

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