Point‐of‐care detection of Japanese encephalitis virus biomarker in clinical samples using a portable smartphone‐enabled electrochemical “Sensit” device

Abstract Japanese encephalitis (JE), a neglected tropical zoonotic disease prevalent in south‐east Asian and western pacific countries, caused by the flavivirus JE virus (JEV), has a dearth of electrochemical point‐of‐care (PoC) diagnostic tools available to manage endemic breakouts. To overcome this, we have developed a screen‐printed carbon electrode (SPCE) immunosensor for rapid PoC detection of JEV nonstructural 1 (NS1) antigen (Ag), found circulating in serum of infected individuals using a smartphone based portable “Sensit” device. The modification of SPCE surface with JEV NS1 antibody (Ab) was confirmed via observation of globular protein structures via scanning electron microscopy (SEM), increase in electrode surface hydrophilicity via contact angle measurement and decrease in current via differential pulse voltammetry (DPV). The fabrication and testing parameters were optimized based on highest current output obtained using DPV. The SPCE was tested for detection limit of target JEV NS1 Ag ranging from 1 fM to 1 μM, which was determined as 0.45 fM in spiked serum. The disposable immunosensor was also found to be highly specific in detecting JEV NS1 Ag over other flaviviral NS1 Ag. Finally, the modified SPCE was clinically validated by testing 62 clinical JEV samples using both a portable miniaturized electrochemical “Sensit” device coupled with a smartphone and a laboratory‐based potentiostat. The results were corroborated with gold‐standard RT‐PCR and showed 96.77% accuracy, 96.15% sensitivity, and 97.22% specificity. Hence, this technique may further be developed into a one‐step rapid diagnostic tool for JEV, especially in rural areas.

recurring JEV outbreaks, early-stage sensitive detection techniques are essential to overcome conventional diagnostic limitations. These include requirement of skilled labor, expensive instrumentation, low sensitivity or specificity, high sample volume and not a rapid method. The laboratorybased methods, which display the above listed drawbacks, include reverse transcriptase polymerase chain reaction (RT-PCR), whole virus isolation, plaque reduction neutralization test, and hemagglutination test. 6 Research has shown that infectious disease diagnostics 7-10 using electrochemical sensors is advantageous due to a lower limit of detection. [11][12][13][14] Furthermore, they remain unaffected by clinical sample absorbance or turbidity when compared to optical assays such as enzyme-linked immunosorbent assays (ELISA). 15 However, due to the required laboratory setup for signal current detection, there is an absence of on-field electrochemical sensing devices for JEV. 16 Currently, the only commercial point-of-care (PoC) diagnostic assays available for JEV are enzyme-based immunoassays and lateral flow assay (LFA) based kits which target IgM/IgG antibodies (Ab) against JEV. 17,18 The drawback of antibody targeted detection is that Abs develop in the body after Day 4/5 of infection unlike the whole virus and/or viral antigen (Ag) present from Day 1 19 resulting in delayed diagnosis. A recent PoC immunochromatic LFA has been developed for the detection of JEV NS1 Ag in clinically infected JEV serum samples. 20 However, the major limitation of existing optical-based immunoassay detection kits is the detection limit, and hence electrochemical sensors are essential for rapid detection of even trace quantities of pathogenic viral biomarkers 21,22 at early-stage infections, for better management and therapeutic care.
Out of the three structural and seven nonstructural proteins of JEV, 23,24 we have selected nonstructural 1 (NS1) as the prospective biomarker since it is a known immunogen, [25][26][27][28] and found secreted in the serum of infected patients. 29 Studies have shown that the virus takes 2-4 weeks to seroconvert in the amplifying host animal. 30 Hence, screening for the NS1 biomarker in serum of sentinels can help in surveillance and minimize the risk of an epidemic outbreak in humans. 31 Previously, there have been reports of ultra-sensitive electrochemical sensors [32][33][34][35] developed for the detection of different JEV antigen, which include screen-printed carbon electrode (SPCE)-based sensors. 36,37 However, they are not portable, on-field/bedside PoC devices, and require laboratory based detectors. The advantage of using SPCE is that precise surface area, thickness, composition (e.g., catalysts such as graphene oxide can directly be incorporated into the screen-printing paste/ink), and accurate relative position of the three electrodes can be designed in a controlled manner. [38][39][40] Moreover, in comparison to the traditional cell-based three electrode system, the sample volume required for SPCE is minimal for electrochemical analysis.
To overcome the scarcity of bedside PoC electrochemical sensors for JEV, we have fabricated a disposable SPCE immunosensor and used a portable "Sensit" device for detection of signal currents. The "Sensit"-based SPCE sensor has demonstrated a lower detection limit, 41 is easier to fabricate, 33 does not require additional signal enhancing nanomaterial, 32,34 has portable PoC field applicability, 35 and has been validated on clinical serum samples as compared to the earlier laboratory-based electrochemical biosensor developed for JEV diagnostics. Herein, the graphene oxide (GO) working electrode surface of the SPCE was modified with in-house generated JEV NS1 specific polyclonal antibody as a bioreceptor using carbodiimide chemistry. The bioconjugation was confirmed via observation of globular protein structures via scanning electron microscopy (SEM), increase in electrode surface hydrophilicity via contact angle measurement and decrease in current via differential pulse voltammetry (DPV). Out of various bioreceptors applicable for protein detection 42 such as monoclonal antibodies, 43 aptamers 44 and peptides, 45,46 we have selected polyclonal antibodies as they are easy to generate, less expensive, and show higher binding affinity resulting in increased sensitivity due to multiple epitope recognition for higher chances of detecting trace/mutating antigens. 47 The fabrication and testing parameters were optimized based on highest current output obtained using DPV. The developed electrode displayed an ultra-sensitive detection limit of 0.45 fM for JEV NS1 Ag in spiked serum ranging from 1 fM to 1 μM. Hence, the electrode can measure the minimum concentration of circulating NS1 protein required to cause infection ranging from 3.5 to 284 ng/mL as reported in similar flaviviral infections. 48,49 The SPCE sensor was designed to specifically detect JEV NS1 Ag and did not cross-react against other closely related flaviviral NS1 Ag. It showed 3 weeks of storage stability after fabrication when stored in a dry environment at 4 C. Finally, the modified electrode was clinically validated by testing 62 clinical JEV samples using both a portable miniaturized electrochemical "Sensit" device coupled with a smartphone and a laboratorybased potentiostat. The results were corroborated with gold-standard RT-PCR and showed 96.77% accuracy, 96.15% sensitivity, and 97.22% specificity. Despite miniaturization resulting in lower range of current, the novel portable "Sensit-"based sensor is comparable in terms of accuracy, sensitivity, and specificity to laboratory-based electrochemical sensors and is the first electrochemical PoC sensing technique reported for JEV diagnostics with clinical sample validation. Hence, the fabricated immunosensor combined with the PoC electrochemical "Sensit" device and a smartphone may further be developed into a novel on-site diagnostic screening assay for rapid, sensitive, and specific detection of JEV.

| Fabrication and optimization of testing parameters for maximum efficiency of fabricated SPCE
In order to fabricate the SPCE, the electrode was subjected to 20 cyclic voltametric scans with the potential range from 0.3 to 1.5 V.
The SPCE surface was washed using 50 mM phosphate buffer (PB, pH 7.4), air dried and later carbodiimide activated at room temperature (RT). The activation was performed by incubating equimolar ratio of EDC:NHS (both 5 μL) on the working electrode. The electrode surface was again rinsed with PB, air dried and JEV NS1 Ab (5 μL) was added at 4 C. The unbound sites of the electrode surface were blocked using 0.1% BSA in PB (5 μL) and incubated for 1 h at RT. BSA is one of the most commonly used blocking agents as a routine practice to block excess active sites for nonspecific binding of other antigen with the electrode surface that may cause false current fluctuations. 50 The advantages of BSA are that is a compatible protein, is inexpensive, and can be easily stored. Finally, the fabricated electrode was washed using PB, air dried and stored at 4 C in a dust-free environment until further experimentation.
The surface morphology of the working electrode surface before and after fabrication was studied by capturing images via scanning electron microscopy at 10.00 kV and 1.6 kx magnification. Also, the increase in hydrophilicity of the electrode surface by the introduction of oxygen functionalities with each fabrication step was determined.
This was done by measuring the contact angle of a water droplet on the working electrode surface at each stage using a goniometer to confirm modification. Finally, JEV NS1 Ag was added to the electrolytic buffer and allowed to bind to the immobilized Ab on the fabricated immunosensor. Electrochemical characterization of each modification and testing step was performed via DPV, and sweeping the potential from À0.5 to 0.5 V. For maximum electrode current output efficiency, fabrication and testing parameters were optimized using DPV. This included concentration of Ab coated on the working electrode surface (0.25-1.5 μg), stable response time (5-30 s), rate of each scan (0.01-0.1 V/s), buffer pH (6.0-8.0), and buffer temperature (4 C, RT, 37 C, 60 C). Following the optimized parameters, the SPCE sensors were fabricated and further tested in spiked serum.

| Analytical performance of optimized SPCE for JEV NS1 Ag
To determine the efficiency of the fabricated SPCE, JEV NS1 Ag was

| Optimization of testing parameters of fabricated SPCE immunosensor
For efficient testing, various parameters were standardized for the developed SPCE by studying maximum current output via DPV.
The major reason for selecting the maximum peak currents for hexacyanoferrate in DPV for all the optimization experiments is to give a maximum current range. Since the current decreases on detection of target antigen, the higher the blank current reading (i.e., more the current range), greater is the resolution to measure even a slight decrease in current upon the addition of target antigen which in turn lowers   the detection limit. In case of antibody immobilization concentration, highest current was observed at 1 μg (Figure 2a) and selected as the optimum amount since beyond this concentration, the active binding sites on the electrode surface reached saturation. Upon addition of sample, the response was observed at 5 s intervals between 5 and 30 s, and a stable response was obtained at 20 s and beyond (Figure 2b), which was selected as the optimal time required for the antigen-antibody interaction to complete. The testing was also carried out at variable scan rates from 0.1-0.01 V/s and maximum current was obtained at 0.1 V/s (Figure 2c). From the linear plot of scan rate versus peak current (Figure 2d), it was found that with increasing scan rate, the current also increased. Finally, environmental testing parameters were standardized by changing the pH (6-8) and temperature (4-60 C) with most favorable results obtained at physiological pH 7.5 ( Figure 2e) and room temperature (Figure 2f). The acceptable range for Ab-Ag interaction is pH 6-8 53 as antibodies undergo fragmentation (degradation) at very low or high pH 54  T A B L E 2 Comparative analysis of clinical samples using the portable "Sensit," laboratory-based potentiostat and gold standard RT-PCR.

| Clinical validation of fabricated SPCE immunosensor
The  Table 1.
The LOD is also lower than the minimum concentration of circulating NS1 protein reported in other flaviviral infections which ranges between 3.5 and 284 ng/mL. 48,49 Furthermore, nonspecific binding was studied by measuring the peak current of the immunosensor upon addition of 1 μM NS1 Ag of JEV, WNV, YFV, and DENV spiked in serum. It was found that while a minimization in current by 50% was observed due to binding of target JEV NS1 Ag in comparison to the negative control serum, no such decrease in current was observed for the other closely related flaviviral NS1 Ag (Figure 3c).
This confirmed that the developed electrode was highly sensitive for JEV detection. Also, after fabrication, the storage stability of the SPCE was determined by read-through the current output at 7 days intervals. It was perceived that SPCE may be stored at 4 C in a dry and dust-free environment for up to 3 weeks without any substantial decrease in electrode performance (Figure 3d).  Conceptualization (lead); funding acquisition (lead); project administration (lead); supervision (equal); writingreview and editing (lead).