Antibodies can be generated by injecting the antigen of interest into the animal or a recombinant antibody can be prepared using the DNA sequence of the antigen. Several sensing systems have been demonstrated for influenza using suitable antiinfluenza antibodies. The binding of the antibody and the target (antigen) from the influenza virus generates a signal that can be used to identify the influenza viruses. As stated above, to detect a whole influenza virus, the antigen should be either hemagglutinin or neuraminidase (Fig. 2.2). Gopinath et al.^4,5 used HA antibody to detect the influenza viruses and discriminated between HA of human and bird influenza viruses. Again in the case of anti-HA antibody, this can be either anti-HA1 or anti-HA2 antibodies. Researchers have used enzyme linked immunosorbent assay (ELISA) to detect influenza viruses with the HA1 antibodies. In most cases, using antibodies means that we can only differentiate types of influenza viruses. Because of its larger size, in several cases we cannot differentiate subtypes. To substitute antibodies, researchers are currently looking at using aptamers as a probe to detect and differentiate influenza viruses.

Aptamer is an artificial chemical antibody that is generated from the randomized nucleic acid library by three simple steps: binding, separation, and amplification. The selected aptamer has a high binding affinity with the target molecule.^25–30 In most cases, aptamer was found to be better than antibodies. Additionally, aptamers have more advantages, such as being easy to prepare, having no variation with different preparations, higher sensitive, they are easy to modify, and they are nonimmunogenic. In the past, several generated aptamers against influenza viruses have been reported to have high affinity. Moreover, since aptamer binds with only a few bases on the target molecule, it has a higher chance to differentiate subtypes of influenza viruses. Among the different target/antigens from influenza viruses, HA is a suitable target for designing the detection strategy because of its high abundance (Fig. 2.4b). HA accounts for 80% of the influenza surface proteins; next to HA, NA molecules are higher in number on influenza surface. Gopinath et al.^10,11 have selected the aptamers against both influenza A (H3N2) and B (Johannesburg) viruses, using either HA or whole virus. The same team also generated the aptamer against H1N1; the selected aptamer has a high binding affinity with the HA protein.¹⁴ Lakshmipriya et al.³¹ have selected the aptamers that can be used against intact influenza B virus (B/Tokio) and HA protein of influenza B virus (B/Jilin).

The initiation of viral infection is highly dependent on the presence of receptor molecules on the host cells and it is mediated predominantly by either α-2,6 or α-2,3 linked sialic acid (glycan) chains in human and bird (avian) influenza, respectively³² (Fig. 2.4c). However, following the emergence of different strains in the past several decades, the specificities of these glycans have become varied. Some exceptional cases have been reported, irrespective of glycan specificities, due to special viral adaptations. Furthermore, the avian influenza virus could also infect humans when a close physical touch between human and bird occurred. Different amino acids from the HA protein have been found to be specifically involved in the host interaction. Different sensing strategies have previously been formulated based on glycan to diagnose and discriminate influenza viruses.^33,34 The main advantage of sialic acid based sensing is to discriminate avian and human influenza viruses.

Using the above probes, several sensing methods have been reported and have achieved different levels by both qualitative and quantitative detection. With qualitative detection, immunochromatographic test (ICT) has been demonstrated to be reliable. ICT operates based on the lateral flow of solutions, with different set-ups of pad. This system immobilized with gold nanoparticle (GNP)-conjugated antiinfluenza antibody. Upon the interaction of the GNP-conjugated antibody and influenza virus, there will be an accumulation of this complex, which causes the appearance of red colored lines that indicate an influenza positive sample. At the same time, it displays a control red-line and no color formation with a negative line (influenza A or B), depending on the samples to be analyzed (Fig. 2.5). However, this system does not predominantly demonstrate for subtyping (within influenza A or B) purposes. Furthermore, physicians have claimed that ICT can only detect influenza when the patient has attained a high fever. This means that it could not detect an influenza virus in the earlier stages and it needs the higher viral count that happens with the on-set of high fever. Even though ICT has been successful in several instances, for quantitative analyses it is necessary to look for established sensors. The following section will discuss sensors that can detect the target from influenza virus in a quantitative manner.

Surface Plasmon Resonance (SPR) is the favorite sensor system to analyze biomolecular interactions on the sensing surface in the lower femtomolar range. In an SPR system, light hits a prism and the substrate is then reflected. The angle of the reflected light will be changed depending on the molecules binding on the substrate. Changes in the reflection are considered as the real binding of the ligand and analyte. Using the SPR based principle, various kinds of sensor have been proposed to detect the target against its appropriate partner, such as antibody or aptamer. Among SPR based sensors, BIACORE is the most well established sensing system and it is used to detect biomolecules with higher sensitivity. In the BIACORE system, various sensing surfaces are readily available. Basically, the entire gold surface in this system was modified with different linkers, which are suited for different biomolecular interactive analyses. Using the BIACORE system, Gopinath and colleagues have analyzed the interactions of influenza and aptamers in several different studies with human influenza A viruses.^10,11,14,33 With these aptamers, they could reach a picomolar level sensitivity during analysis of influenza belonging to H3N2 and influenza B. With a further step, they attained a femtomolar level sensitivity during analysis of aptamer and H1N1 (swine flu) virus. Similarly, Suenaga et al. have made different studies with avian influenza viruses on SPR.^34–37

Surface plasmon resonance fluorescence spectroscopy (SPFS) works on a similar principle to SPR, the only difference is that SPFS uses fluorescence to detect the analyte. Lakshmipriya et al.³¹ have detected intact influenza B virus and HA protein of influenza B using SPFS by their appropriate aptamer or antibody. They compared the interaction between antibody and aptamer against influenza viruses and showed an improved detection level. Furthermore, they found that aptamer behaves better than antibody and that it could discriminate influenza types and subtypes. Comparative studies between SPFS and radio isotope labeling revealed the better performance of SPFS. The authors attested to the higher success rate of the operation of SPR, SPFS, and other similar systems, such as waveguide mode sensors, which are is based on Kretschmann configuration.

A waveguide mode sensor is used to detect analyte molecules in a solution and has been used to diagnose influenza virus. Even though the principles of SPR and a waveguide mode sensor are similar, in the waveguide mode system the light that hits the prism passes through the surface as a guiding mode and not as a surface mode.^38,39 Depending on the molecular binding on the substrate environment, the reflected light will be changed, and changes in the reflection are considered as the real binding. Gopinath et al.^10,11 have demonstrated the use of the binding events for the detection of an influenza virus against antiinfluenza antibody. In their study, they used an antibody raised in the laboratory against H3N2 influenza and had better discriminating ability than commercial antiinfluenza antibody. This anti-H3N2 antibody is able to discriminate types and subtypes of influenza viruses among H3N2, H1N1, and influenza B. To improve the sensitivity of their waveguide mode sensor, they conjugated GNP on the surface of influenza viruses. They were able to attain a sensitivity level of 8 × 10³ PFU/mL. They also demonstrated waveguide mode detection of influenza in the absence of GNP and in the presence of dye materials.⁴⁰

GNP-based colorimetric assay has been formulated to detect different kinds of molecule with the naked eye. This assay is more suitable for controlled assembly and disassembly of aptamers or antibodies on the GNP in the presence (or absence) of target molecules. Gold has naturally no charge, but GNP will get a surface charge, which may be either positive or negative charge depending on the reagents that are used. Prepared GNP is usually in a dispersed condition (red colored solution) and when we add charged ions they induce aggregation (blue/purple-colored solution).¹⁹ Usually monovalent or divalent ions can be used to induce the aggregation. Sodium chloride (NaCl) has predominantly been used to induce the aggregation. When aptamer or antibody is used with dispersed GNP, it keeps the same status (dispersed red solution), even in the presence of NaCl, and the aptamer/antibody will bind to GNP by electrostatic interaction or chemical reaction. However, in the presence of appropriate target molecules to the aptamer/antibody, GNP will be aggregated (blue solution) in the presence of NaCl. Under this condition, a complex of aptamer/antibody target will be formed and this will induce the aggregation. Lee et al.⁴¹ have developed a colorimetric assay for influenza detection, they used glycan (sialic acid) as the probe and reached a sensitivity of up to 512 HA titer (for influenza B/Victoria and B/Yamagata). In this case, the assay is slightly modified, but this is not like controlled assembly and disassembly. They formulated that in the presence of influenza virus, the complex will be formed with sialic acid-conjugated GNP and this will induce virus mediated aggregation. Gopinath et al.¹⁹ have revealed the nonfouling effect of HA protein on the GNP and they compared their results with other molecules.

As mentioned above, sensing platforms have been reported to be appropriate systems for detection of influenza virus and other molecules. However, most of the sensing designs are suitable for analyzing a limited number of samples. A sensing platform/system suitable for high-throughput screening with the facility to analyze higher number of samples is highly appreciated. The disc platform is suitable for high-throughput sampling. The disc platform is basically designed based on a commercial compact disc (CD) or digital versatile disc (DVD). Varma et al.⁴² have shown CD-based detections of biomolecules with antigen–antibody interactions. Later, Gopinath et al.^43–45 have shown the progression with the DVD platform and analyzed different samples, including influenza viruses. These two platforms have spiral tracks on the surface that can accommodate several thousands of samples. The cost of the sensing surface is cheaper and easier for surface functionalization. In most cases, gold was used as the top substrate layer and it can be easily linked to biomolecules through thiol groups. Gopinath et al.⁴⁴ have used five layer structures, with a top gold layer, for biomolecular interactive analyses. For the interactive analysis of influenza virus-A against its antibody, they attained a dissociation constant to the lower nanomolar level. This level is comparable to the detection level of BIACORE with the same molecules.

In most cases, these platforms are defined as label-free, except for SPFS. However, other fluorescent labelling techniques are also used for the diagnosis of influenza. For example, fluorescent labelling has the advantage of higher sensitivity, although it has been argued that it is difficult to label the molecule due to the unavailability of the site for labelling or because the labelling site may be hidden. Several different labelling materials, such as rhodamine, cyanine, Alexa Flour, and fluorescein, are routinely used for different bio-labelling strategies (Fig. 2.6a,b). In the SPFS method for influenza virus detection, Lakshmipriya et al.³¹ used cyanine as the fluorescent material to label the aptamer. Nomura et al.⁸ used Alexa Flour 700 for the detection of influenza virus. They used a new kind of SPR system without involving the use of a prism (prism-free). A CCD camera system has been used to capture the fluorescent signaling upon interaction of influenza virus and antiinfluenza H3N2 antibody. With this system, the authors have shown a clear discrimination between influenza subtypes using antibody.

ELISA is the gold standard method to detect a wide range of target molecules assisted with appropriate partner molecules. ELISA is used not only for the detection but also for the basic screening of many important diseases, such as HIV, influenza, and so on. Gopinath et al.¹⁴ have demonstrated ELISA based detection of influenza virus using anti-H3N2 antibody and discriminated against other influenza viruses. Due to its high sensitivity and selectivity, ELISA helps to identify the target molecules, even in the human crude samples (serum, urine, and saliva). Until now, antibodies have commonly been used as a probe to detect biomolecules in ELISA. In general, the binding of the target (antigen) and the probe (antibody) on the ELISA plate was detected by the enzyme (eg, horseradish peroxidase (HRP), alkaline phosphatase) conjugated with a secondary antibody and detected by chromogenic substrates. Biotin-streptavidin conjugation has also been used to improve the ELISA methods. In this case, biotinylated secondary antibody was detected by streptavidin-conjugated enzyme. Various kinds of pattern are used in the ELISA method to improve the detection methods, such as direct, indirect, sandwich, and competitive ELISA. In most of the cases, antibody was used as the probe due its strong binding, stability, and selectivity. The sandwich ELISA is usually conducted by the monoclonal or polyclonal antibody of the specific target (Fig. 2.7). In some cases, only the Fc region of the antibody has been used as the capture molecule on the ELISA plate. After aptamer generation, some researchers have used aptamer as the probe instead of antibody. This method is called aptamer linked immunosorbent assay (ALISA).⁴⁶ Since aptamer has a higher sensitivity than the antibody, it is possible to increase the limit of detection when aptamer is used as the probe. In addition, there is a possibility to do a sandwich assay with two different aptamers for the same target. Since both aptamer and antibody are suitable as detection molecules, there is a higher possibility of increasing the limit of detection with the sandwich patterns using aptamer and antibody.