High-Performance Graphene-Modified Sensing Chip for SARS-CoV-2 Detection

Summary

The present protocol describes the fabrication of low-cost biosensing prototypes based on useful nanosystems for accurately detecting viral proteins (at the Fg level). Such a tiny sensor platform allows for point-of-care applications that can be integrated with the Internet of Medical Things (IoMT) to meet telemedicine objectives.

Abstract

This sensing prototype model involves the development of a reusable, twofold graphene oxide (GrO)-glazed double inter-digitated capacitive (DIDC) detecting chip for detecting severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) specifically and rapidly. The fabricated DIDC comprises a Ti/Pt-containing glass substrate glazed with graphene oxide (GrO), which is further chemically modified with EDC-NHS to immobilize antibodies (Abs) hostile to SARS-CoV-2 based on the spike (S1) protein of the virus. The results of insightful investigations showed that GrO gave an ideal engineered surface for Ab immobilization and enhanced the capacitance to allow higher sensitivity and low sensing limits. These tunable elements helped accomplish a wide sensing range (1.0 mg/mL to 1.0 fg/mL), a minimum sensing limit of 1 fg/mL, high responsiveness and good linearity of 18.56 nF/g, and a fast reaction time of 3 s. Besides, in terms of developing financially viable point-of-care (POC) testing frameworks, the reusability of the GrO-DIDC biochip in this study is good. Significantly, the biochip is specific against blood-borne antigens and is stable for up to 10 days at 5 °C. Due to its compactness, this scaled-down biosensor has the potential for POC diagnostics of COVID-19 infection. This system can also detect other severe viral diseases, although an approval step utilizing other virus examples is under development.

Introduction

A viral pandemic caused by a new beta coronavirus¹ (i.e., 2019-nCoV), which was later named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)² (hereafter predominantly referred to as the virus), involving a pneumonic cluster and severe acute respiratory distress, emerged in the Wuhan city, China, at the end of 2019³. Owing to its fast worldwide human-to-human transmission, high infection rate, high mortality rate, and serious life-threatening adverse effects⁴, during the pandemic, virology research⁵ evolved quickly to identify the virus' genomic organization and structure^5,6. The symptoms of COVID-19^7,8 include a high fever, a dry cough, and generalized pain⁹. Importantly, different serotypes of the virus lead to differing disease severities¹⁰. Moreover, asymptomatic carriers can potentially spread the virus. Usually, under the microscope, COVID-19 virus particles show club-like projections formed by spike proteins¹¹. Therefore, to control the spread of this new pathogen, the detection of cases must be timely and efficient. Thus, the ultra-sensitive, rapid, and selective detection of the virus at the early stages of viral infection has become crucial^2,11. Social/physical distancing is needed to avoid the transmission¹² of the virus. Health agencies are emphasizing the development of smart diagnostic tools and nano-systems¹³. Indeed, as suggested by health agencies, targeted and mass testing^14,15 are required and are still in demand.

In principle, ongoing biological diagnosis methods like reverse-transcription polymerase chain reaction (RT-PCR) are the best means for the mass identification of SARS-CoV-2, as with the Middle East respiratory syndrome-related coronavirus (MERS-CoV)¹⁶ and SARS-CoV-1¹⁷. In this context, the current standard identification of SARS-CoV-2 contamination depends on the enhancement of infection-specific characteristics^18,19. Additionally, the variation in SARS-CoV-2 infection according to the area, age, race, and gender should be taken into account. With the ultimate goal of saving lives, it is crucial to build fast diagnosis tools for point-of-care (POC)^20,21 use.

In this context, regular strategies like fluorescence in situ hybridization (FISH), protein immunosorbent examination (ELISA), microsphere-based methods, electrochemical tests, and MRI, PET, and NIRFOI²² have low sensitivity to low virus levels, low selectivity, and low reuse capacity; additionally, such procedures have disadvantages, including costly biosensing diagnostic systems, non-reusable reagents, and the requirement for a highly skilled workforce²³. Therefore, these insightful techniques cannot be viewed as fast, reasonable, exceptionally specific, or sensitive POC methods^24,25. Of note, there are different kinds of DNA and immunizer-based biosensors that utilize compound, capacitive, and electrical techniques^18,26,27,28. As an example, electrical DNA biosensors, which have high responsiveness, can be scaled down simply, and are tunable^29,30, have been produced for the detection of Ebola³¹, Zika, MERS-CoV, and SARS-CoV^32,33,34. Similarly, a field-impact semiconductor (FET) biosensor for detecting the spike protein of the virus utilizing certain antibodies (monoclonal) immobilized onto graphene-glazed devices has been effectively created^35,36. Nonetheless, this new strategy is less sensitive than RT-PCR. Furthermore, more recently, an on-aerosol jet nanoparticle-diminished graphene oxide (GrO)-covered 3D terminal-based detecting framework for the virus has been developed, which has a low limit of identification (2.8 × 10⁻¹⁵ M); in any case, the proposed complex biosensor structure³⁵ has been tested with regard to POC use and compared with other existing biosensor strategies that are utilized for the detection of the virus^35,37,38.

In this study, we designed and fabricated a scaled-down and reusable GrO-based DIDC biosensor for identifying the virus spike protein without the limitations depicted above for other biosensors. This biosensor permits detection at the femtogram (fg) level within 3 s^18,27 of response time. To accomplish this research, GrO nanoflakes were chosen for better responsiveness and selectivity, which means low concentrations of the virus antigen protein from oropharyngeal or nasopharyngeal swabs can be detected. GrO is an appropriate, synthetically dependable, consistent, and conductive material that can be beneficially utilized for biosensing applications^2,39,40,41. Additionally, a monoclonal IgG antibody label-free hybridization approach was utilized, focusing on the virus spike S1 protein. The fabricated SARS-CoV-2-GrO-DIDC biosensor is reusable after advanced treatment and cleaning with piranha solution. This ultrafast, sensitive, selective, label-free, and reusable biosensor can be utilized for clinical sample biosensing and personalized healthcare applications^26,42,43,44.

Protocol

1. Cleaning of the DIDC sensing chip

1. At the beginning of the experiment, clean the DIDC chip surface²⁶ with piranha solution (H₂SO₄:H₂O₂in a 3:1 ratio), and place it on the hot plate at 80 °C for 15 min. Next, rinse the sensor surface with distilled water drop by drop using a pipette to remove the cleaning reagents completely. To ensure the complete removal of the reagent, rinse the surface with four to five drops of ethyl alcohol.
   NOTE: The DIDC chip was fabricated following a previously published report²⁶.
2. Then, dry the sensor surface at room temperature for the complete removal of the reagents to obtain a hydrophilic sensor surface. This chip can be used for further fabrication of the graphene oxide layer on the chip (step 2).
3. Cover the clean sensor chip electrode pads with polyimide tape.

2. Fabrication of the thin layer of graphene oxide on the DIDC sensing chip

1. Place the chip on the center of the spin-coating machine in the horizontal position, and add 4 µL of an aqueous solution of commercially available single-layer graphene oxide (GO) (see Table of Materials) onto the chip surface. Then, close the spin-coating chamber, and run for 2 min at 1,300 rpm.
2. For the annealing of the fabricated GO chip, keep the chip on the hot plate horizontally for 40 min at 80 °C.

3. Cross-linking and functionalization of the GO-glazed DIDC sensing chip

1. Perform cross-linking of the N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and NHydroxysuccinimide (NHS) with the thin-film GO chip.
   1. Add 4 µL (0.4 M and 0.1 M, respectively) of EDC-NHS (see Table of Materials) to the thin-film GO chip for generating the covalent conjugation of amine and carboxylic groups via amide bond formation²⁶.

4. Antibody preparation and immobilization on the chip for protein sensing

1. For binding the functionalized GO-DIDC chip with the antibody, dissolve commercially available anti-SARS-CoV-2 Abs (reproduced by rabbit mAb anti-S1 protein, see Table of Materials) using the dilution buffer (0.01 M PBS containing 0.1% BSA [bovine serum albumin] and 0.86% NaCl).
   1. To 1 µg of purified antibody, add 1 mL of diluted PBS. Then, drop cast 4 µL of the antibody solution onto the cross-linked activated GO-DIDC chip. Leave the chip in the closed chamber for 2 h to bind the Abs onto functionalized chip surface at room temperature.
      NOTE: The Fab region of the Abs usually consists of abundant reactive amine and carboxylic groups due to its polar nature²⁶; therefore, the subsequent specific immobilization leads to robust covalent "tail-on" Ab-specific orientation.
2. Once the antibody immobilization on the sensor surface is done, drop-cast 4 µL of bovine serum albumin (BSA) onto the chip to block the non-specific sites of the immuno-capacitive sensing chip. Place the chip horizontally in the closed chamber for 20 min at room temperature.
3. Wash the immuno-capacitive sensing chip with DI water, and then continue drying at room temperature.
   NOTE: After drying, the DIDC-based capacitive immunosensor (SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO2-DIDCs) is ready to perform the serial detection of the virus spike antigen.
4. For further sensing of the virus spike protein, prepare different concentrations from 1.0 mg to 1.0 fg to obtain a wide detection limit.

Results

Here, a protocol is presented for sensing the S1 protein of the SARS-CoV-2 virus using a graphene oxide-glazed double inter-digitated capacitive (DIDC) sensing chip. Figure 1 shows a schematic representation (fabrication with the circuit layout) of the extremely sensitive and recyclable graphene oxide-modified double interdigitated capacitive (DIDC) sensing chip. The detailed stepwise fabrication process is shown in Figure 2. Figure 3 represents the surface functionality characterization via atomic force microscopy (AFM). A typical top-view of the fabricated chip, the FTIR spectroscopic characterization, and the surface contact angle are shown in Figure 4. Figure 5 denotes the biomolecules of the S1 antigen disturbing the electric field between the metal electrodes and the projected prototype, the sensitivity of the DIDC chip, the selectivity of the DIDC chip, the concentration versus capacitance graph for linearity (18.56 nF/g), and the response times of multiple concentrations. Figure 6 shows the assessment of constancy and reproducibility with respect to time (in hours) and days. Figure 7 shows the reusability test results of the DIDC chip after the regeneration. Figure 8 presents the Nyquist plots of the recyclable DIDC chips in terms of the capacitance versus the virus spike (S1) antigen-sensing performance. The stepwise fabrication process was analyzed by AFM and SEM characterization, and chemical bonding and functionalization were analyzed by FTIR.

The schematic of our projected biosensor is depicted in Figure 1. The created SARS-CoV-2 S1 protein biosensor represented in Figures 1A and B exhibited the twofold GrO-based double interdigitated capacitive (DIDC) chip characteristics with the abovementioned components; the identical circuit format of the manufactured twofold interdigitated capacitor (DIDC) chip is displayed in Figure 1C. CL represents the capacitors for the major anodes, and CS represents the capacitors for little focal terminals linked in series; the width and length of the chip are 4.1 mm and 5 mm, respectively. The GrO-based DIDC detecting chip with immobilized anti-SARS-CoV-2 Abs restricting to the virus S1 antigen protein (as the designated analyte) is shown in Figure 1D. The morphological examination of the anti-SARS-CoV-2 Abs implanted in the GrO at a 1 µm scale through electron microscopy (SEM) is shown in Figure 1E; the inset picture from the AFM shows that the graphene oxide flakes were distributed similarly, with a level of 25 nm. Figure 1F shows the scanning electron microscope image of an uncovered chip, and the estimation of two metal fingers (i.e., 20 µm) is presented in Figure 1G.

Importantly, the biosensor fabrication for the DIDC-based detection of virus S1 protein is reliant on certain key elements, such as the dielectric constant, conductivity, and charge distribution^18,45. The detecting protein is positioned onto the DIDC biosensor, and the key element with the immunizer and antigen is modified as far as detecting boundaries compared to the DIDC capacitive reactance. A schematic portrayal of the directed exploration work process is shown in Figure 2A-E. The DIDC was assessed utilizing an LCR meter (handheld, 0.2 GΩ, 200 pF, 1 kHz, 2 kH, U1730C series) with a PC for data analysis in Figure 2A. A 0 V DC voltage with a 1 kHz frequency was applied across the DIDC biosensor. Firstly, the sensor space was set up with cleaning with a piranha solution with a 3:1 proportion (H₂SO₄:H₂O₂) at 80 °C, and afterward, the space was cleaned with DI water to eliminate the hydrophilic reagents, as detailed in previous research^46,47. After the actuation interaction, the surface was fabricated with GrO (4 µL) at 1,300 rpm by means of a spin-coater (Figure 2B). Then, the graphene oxide on the DIDC chip was strengthened by placing the chip on a hot plate for 1 h at 80 °C. After the tempering process, EDC-NHS chemistry (4 µL; 0.4M and 0.1 M,) was performed⁴⁸, utilizing an upgraded cycle to create the covalent formation of amine and carboxylic groups through amide bond³⁵development, as addressed in Figure 2C. Then, a reconstitution buffer was used with a 0.1 M reactant buffer and a 1 µg/mL decontaminated neutralizer. For the formation of the virus Abs (replicated by rabbit mAbs hostile to the S1 protein), a droplet of 4 µL was added onto the NHS-enacted graphene oxide (Figure 2D). Of note, the Fab area of the antibodies generally comprises many receptive amine and carboxylic groups because of its polar nature; in this manner, the progressive explicit immobilization results in robust covalent "tail-on" Ab-specific orientation²⁶, as characterized in Figure 2E. Significantly, treatment with bovine serum albumin (BSA) was utilized with shaking for 20 min at room temperature to block the non-specific sites of the sensing chip. After drying, the capacitive immunosensor^49,50 based on DIDC (SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-Glass-DIDCs) was prepared to sequentially detect the virus spike antigen.

To assess the surface morphology of the DIDC chip, bare chip, or with regard to the graphene oxide, EDC-NHS activation, hostile to the virus S1 Abs and the virus spike antigen, was performed using AFM and SEM. The process of surface functionalization is presented in Figure 3A-D. The successive surfaces showed various morphologies and levels at the micro-scale. Standardized 25 nm graphene oxide morphology was seen on the DIDC chip (Figure 3A), while on the GrO-functionalized DIDC chip, immobilization of the anti-SARS-CoV-2 antibodies onto the EDC-NHS-GrO-Pt/Ti DIDC chip was observed, and a further level expansion up to 400 nm was seen subsequently (Figure 3B). The EDC-NHS cross-linker was dispersed at a level of 40 nm (Figure 3C), affirming that the antibodies against SARS-CoV-2 were immobilized into the functionalized DIDC chip. Besides, after the expansion of the virus spike antigen, a sharp peak at 600 nm was observed due to the spike (S1) protein, as shown in Figure 3D, indicating "tail-on" antibody-antigen orientation.

Typical top-view SEM pictures are displayed in Figure 4A-D. Figure 4A at 500 µm presents a successful photoresist-covered SiO₂wafer and a Ti/Pt terminal-exposed DIDC chip; additionally, the inset at 20 µm is the distance between two metal fingers. The fabricated DIDC chip surface depicts a homogeneous and smooth surface morphology; the part of the chip that is graphene oxide-functionalized, the part that is EDC-NHS-enacted, the part that is immunized to be hostile to the virus, and the virus protein-spiked chips are displayed in panels (A), (B), (C), and (D), respectively. The peeled graphene oxide drops provide the chip with a hydroxyl group (-OH) and a carboxylic group (-COOH), which enhances the detection in terms of the sensitivity and selectivity. Figure 4A shows the dissemination of the homogenous GrO flakes at 1 µm. Figure 4B shows the EDC-NHS coupling, with an empty, round, hollow construction that is hostile to the virus at 3 µm. Figure 4C shows a spiked antibody implanted in the graphene oxide-EDC-NHS functionalized detecting chip at 1 µm. Figure 4D shows the virus-spiked antigen-like pool structure reinforced with the spiked antibody at 10 µm. The development of the expected bonds during the stepwise manufacture of the GrO-DIDC biosensor was demonstrated by FTIR spectroscopy, as displayed in Figure 4E(i-iv). The FTIR results²⁶ were as follows: (i) a 2,976 cm⁻¹ C-H bond peak caused by the antibodies on the chip surface; (ii) EDC-NHS functionalization, with an N-O peak at 1,567 cm⁻¹ indicating the presence of the nitro group; (iii) a peak at 1,038 cm⁻¹ suggesting the presence of C-O bonds (fragrant ester) and aromatic rings in the setting graphene oxide immobilized onto the DIDC chip; and (iv) the exposed DIDC chip showing primary alcohol (i.e., C-O bonds) with a weak intensity at 1,060 cm⁻¹.

The proposed recyclable DIDC bionic chip was designed and manufactured to target the virus spike S1 protein through immobilizing the explicit monoclonal antibodies onto a graphene oxide-EDC-NHS binding surface. As the chip includes a bioengineered surface and layout for the formation of anti-SARS-CoV-2 Abs, the capacity of the cross-linkers to enhance the charge move rate, as indicated by the electrical capacitance, can be used to confirm the effect of the chip (Figure 5A). Surprisingly, the immunosensor showed a minimum LOD of 1.0 fg/mL, which is lower than the current biosensing systems for detecting the virus. Critically, the proposed electrical capacitive immunosensor additionally has better contrast, and other electrochemical proclivity biosensors depicted such a long way for the location of the virus protein, with regards to responsiveness, reaction time, selectivity, and reusability.

To test the sensitivity of the created SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO2-DIDCs immuno-stage, we took 13 progressive amounts of the virus S1-His protein (from 1.0 × 10⁻³ g to 1.0 × 10⁻¹⁵ g), and the drop-projecting technique was used to add the protein to the DIDC chips and quantify the capacitance with regard to the various antigen fixations; subsequently, the spectra were contrasted with control spectra (Figure 5B). Furthermore, we likewise estimated the selectivity to the virus antigens (Figure 5C) in order to examine the potential effects of interfering substances in natural examples; for example, PSA and amyloid β_1-42 proteins may be contaminants. The concentration of the interfering protein was 1.0 µg/mL for both PSA and amyloid β_1-42. The selectivity test worked in the presence and absence of interfering proteins. Testing was conducted with 10 distinct sensors; specifically, the first to the fifth sensors were tested when the interfering protein was present, and the rest of the sensors (sixth to tenth) were tested without the interfering protein. There were no differences noticed between these conditions. From the first to the fifth sensor, the capacitance changed from 286.9 nf to 275.5 nf, and from the sixth to the tenth sensor, the capacitance ranged between 271.9-273.0 nF. Every one of the samples was prepared in the 0.1 M PBS cradle arrangement (pH 7.4). The tests were conducted utilizing approximately 1.0 µg/mL virus S1 protein for the conditions both with and without the presence of the non-target protein biomolecules. After a single incubation step, this capacitive immunosensing chip gave quantitative outcomes with regard to the 5 µL solution samples in a short period of time; the capacitance changed in 3 s with regard to the virus antigens, with no significant cross-reactivity with the non-specific analytes. The sensitivity estimation of the virus antigen protein showed a straight-line relationship between the capacitance versus fixation (i.e., y = mx + c). Here, y is an element of x, m is the inclination, the point of the line to the x-pivot, and c is a block on the y-hub. In factual estimation, each estimation has played out a base multiple times, and a typical worth was utilized; the responsiveness was characterized as 18.56 nF/g (Figure 5D). To check the reaction condition of the created SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO₂-DIDCs immuno-stage, three specific antigen concentrations were chosen, including 1.0 × 10⁻¹⁵ g/mL, 1 × 10⁻⁹ g/mL, and 1 × 10⁻³ g/mL. Capacitance estimation becomes closed in no less than 3 s at the level of every focus separately, as shown in Figure 5E.

Then, the durability of the bio-composite (SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO₂-DIDCs) was checked; for this, the chips were placed in a humid chamber at 5 °C for 10 days. Critically, no significant changes were seen in the presentation of the fabricated DIDC chip with incremental increases in the storage time Figure 6A. Moreover, the reproducibility of the DIDC-based sensor was additionally assessed, as shown in Figure 6B. The capacitance values of the SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO2-DIDCs were assessed for 10 h. Significantly, the capacitance values were steady, without any more than ±5% deviation in the values between the support points. Moreover, following the durability results above, we likewise evaluated the reproducibility over 10 days. Of note, these capacitance estimations likewise varied by less than ±5% between the support points. Of note, on the second day, we noticed a little contrast (from 8.8 nF to 7.2 nF), most likely because of moving the chip from room temperature to a low temperature; indeed, from the second day forward, the capacitance values remained steady. Critically, from the first day to the tenth day, the capacitance values shifted from 8.8 nF to 7.2 nF, with no significant changes observed in the reproducibility/repeatability based on the standard deviation (SD), relative standard deviation (RSD), and mean (x̄), as shown in Table 1.

Finally, the reusability of the virus DIDC chip was assessed. To elute the antibodies from the sensor surface, a 0.1 M elution buffer of glycine-HCl at 2.7 pH was utilized; of note, a pH between 7 and 7.4, which is not physiologically possible in the body, was selected to assess the disturbance by immunoaffinity. Significantly, the elution buffer was applied once onto the sensor surface, and after the application, the capacitance value reached a similar level to that found with the functionalized GrO (1.2 nF). A short time later, piranha solution was added to the DIDC sensor at 80 °C, and afterward, the capacitance was shown to be at a level almost equivalent to that of the exposed DIDC chip (0.26 nF); indeed, the DIDC chip recovered 96% of its uncovered qualities. Notably, the outcomes were compared after the first and second recoveries, and no significant changes were identified, as shown in Figure 7A,B and Figure 8A-C.

Figure 1: Schematic representation of the ultra-sensitive and reusable graphene oxide-modified double interdigitated capacitive (DIDC) sensing chip. (A) The structure of the virus S1 protein. (B) An outline of the manufactured twofold digitated capacitive detecting chip (DIDC). (C) The twofold interdigitated capacitor (DIDC) with the circuit (equivalent). CL = the capacitor for the major terminals connected in series; CS = the capacitor for the minor focal electrodes connected in series; the width of the chip is 4.1 mm, and the length is 5 mm. (D) The GrO-glazed twofold digitated capacitive (DIDC) detecting chip with immobilized anti-SARS-CoV-2 Abs specific to the virus S1 antigen (focused on the analyte). (E) Morphological presentation of the anti-virus Abs embedded in the graphene oxide at the 1 µm scale; the inset picture shows the AFM of the graphene oxide flakes, which are dispersed homogeneously at the level of 25 nm. (F) An SEM image of the exposed chip. (G) The distance between two fingers (metal) is defined. Scale bar = 20 µm. This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Figure 2: Stepwise process of DIDC fabrication and evaluation of the surface changes and functionalities via AFM and scanning electron microscopy (SEM). (A) The fabricated DIDC sensing chip with the photoresist-covered SiO₂wafer and exposed Ti/Pt electrode. (B) The fabrication of the GrO on the chip via spin-coating at 1,300 rpm. (C) The EDC-NHS chemistry interaction for the twofold GrO-coated interdigitated capacitive (DIDC) detecting chip. (D) The antibody binding responds to the virus S1 protein with "tail-on" Ab alignment due to the EDC-NHS (EDC-NHS-GrO-Ti/Pt-SiO₂-DIDCs). (E) The virus S1 protein binding on the SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO2-DIDCs.This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Figure 3: AFM characterization. Surface clarification of the DIDC chip separately and with regard to (A) graphene oxide, (B) the antibody against the virus spiked protein, (C) EDC-NHS, and (D) the spiked antigen of the virus, as accomplished through atomic force microscopy (AFM). This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Figure 4: SEM and FTIR characterization. (A-D) This shows the typical top-view SEM pictures. The DIDC-covered examples present a complementary and additional standardized surface etiology; (A) the part of the chip functionalized with graphene oxide, (B) the part that is EDC-NHS-enacted, (C) the part functionalized to be hostile to the virus, and (D) the virus protein-spiked chips are displayed. (B) FTIR and contact point estimations. (E) FTIR spectroscopy: (i) hostile to antibodies of the virus, (ii) EDC-NHS coupling, (iii) the DIDC chip coated with graphene oxide, and (iv) the bare DIDC chip. (F) Water contact angle measurement: (i) the bare DIDC chip (82.0° ± 3.0°), (ii) GrO (80.4° ± 3.0°), and (iii) EDC-NHS (75.4° ± 3.0°). Furthermore, the wettability of the various chips was estimated in terms of the contact angle of a 5 µL water droplet. The liquid (water) displayed a higher contact point (around 82.0° ± 3.0°) with regard to the stripped chips in Figure F(i). Of note, this point diminished with the GrO (80.4° ± 3.0°), as in Figure F(ii), and the EDC-NHS (75.4° ± 3.0°), as in Figure F(iii). These surface contact points demonstrate that graphene oxide and EDC-NHS-treated chips were more hydrophilic than the exposed double inter-digitated capacitive electrodes. This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Figure 5: Sensitivity and functional characterization. (A) The Ti/Pt-based DIDC cathodes bound by the antibodies and antigen of the virus biomolecules disrupt the electric field across/between the metal (fingers), and the impact on the capacitance of the manufactured DIDC chip is assessed using the capacitance estimation gadget. (B)The sensitivity of the arranged SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO₂-DIDCs immuno-stage detecting chip covered a wide range (i.e., 1.0 mg/mL to 1.0 fg/mL); capacitance on the top of the uncovered chip, the Chip-GrO, the Chip-Gro-EDC-NHS-SARS-CoV-2-Ab, and the EDC_NHS+ SARS-CoV-2-Ab+ SARS-CoV-2-Ag. (C) The selectivity of the arranged SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-SiO₂-DIDCs immuno-stage biosensor with and without the presence of an interfering protein. (D) The linearity was 18.56 nF/g for an extensive variety of concentrations, and there was a strong direct relapse line; the slant and relapse coefficient (R) was determined through fitting the information. (E) The response time (3 s) of the 1.0 × 10⁻¹⁵ g/mL, 1 × 10⁻⁹ g/mL, and 1 × 10⁻³ g/mL concentrations versus the capacitance values after applying the protein samples. This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Figure 6: Assessment of the stability and reproducibility of the biosensor. (A) The SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-Glass-DIDCs capacitance versus time diagram from 1-10 h, with a ±5% deviation between the point of support values. (B) The stability results, which evaluated the reproducibility of the arranged SARS-CoV-2-Ab-EDC-NHS-GrO-Ti/Pt-Glass-DIDCs across time, showed a deviation of ±5% over 10 days between the support point values. This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Figure 7: Reusability of the DIDC chip based on atomic force microscopy and scanning electron microscopy images. (A) A 3D AFM picture of the DIDC chip after one trial; thickening of the chip is plainly apparent up to 250 nm. (B) A high-resolution (HR) SEM picture after one trial of the virus spike antigen detection at the 1 mm scale; in the center, pictures of the Pt/Ti cathodes at 50 µm, the metal electrode finger HR picture at 10 µm, and the distance between the two fingers (i.e., 20 µm) are presented. In both the AFM and SEM pictures, no imperfections or breaks were noticed, and the same chip was utilized for one more analysis trial. This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Figure 8: Capacitance v/s SARS-CoV-2 spike (S1) antigen sensing at different concentrations and the regeneration and reusability of the DIDC chip: (A) Nyquist plots of the graphene oxide-coated DIDC bionic electrode measured by an impedance analyzer; capacitance method without and with the spike S1 antigens at concentrations of 1.0 mg/mL to 1.0 fg/mL in PBS solution. (ii,iii) Nyquist plots corresponding to those in A. (B) After one successful sensor recovery at a low pH (2.7) using an elution buffer (0.1 M) of glycine-HCl and piranha cleaning. The recovery was achieved in 20 min or less. For all focuses, the sign in (B,C) was 96% of that in (A). (C) after two sequential sensor regenerations. For every one of the estimations, the charge moves for the graphene oxide-coated DIDC bionic anodes; the 0.1 M PBS buffer arrangement (pH 7.4) was utilized as a control. Three successive readings were acquired for different concentrations of the virus spike (S1) antigen. A 1 kHz frequency was applied to collect this data. This figure has been modified from Sharma et al.²⁶. Please click here to view a larger version of this figure.

Time (in hrs)	Chip + Gro + EDC- NHS + Ab	Error	No. of Days	Chip + Gro + EDC- NHS + Ab	Error
1	9.8	0.49	1	8.8	0.44
2	9.7	0.485	2	7.2	0.36
3	9.8	0.49	3	9.7	0.485
4	9.5	0.475	4	9.1	0.455
5	9.9	0.495	5	8.4	0.42
6	9.9	0.495	6	9.1	0.455
7	9.7	0.485	7	7.4	0.37
8	9.4	0.47	8	7.7	0.385
9	9.9	0.495	9	8.5	0.425
10	9.8	0.49	10	8.3	0.415
Mean (x̄)	9.74			8.42
SD (±)	0.171269768			0.801110479
RSD (%)	1.758416506			9.514376238

Table 1: Evaluation of the reproducibility/repeatability with the mean (x̄), standard deviation (SD), and relative standard deviation (RSD) values.

Discussion

For fashioning a productive DIDC chip-based biosensor, the charge distribution, conductivity, and dielectric constant of the DIDC are extremely important. Significantly, the improvements in these detection boundaries relate to the capacitive reactance of the DIDC^18,26,27. In this study, a capacitance immunosensor was fabricated that is hostile to the virus Abs and functionalized by EDC-NHS coupling on the graphene oxide-DIDC-based SiO₂substrate²⁷. Additionally, EDC-NHS cross-linking was added by employing covalent binding onto the GrO substrate to functionalize against the virus Abs.

With the painstakingly planned procedure that ensured sensor surface readiness, anti-SARS-CoV-2 Abs immobilization, and binding with the virus S1 protein, an equilibrium was found among the contending elements of the simplicity of immobilization, the explicitness, and the general capacitance. The advancement of the GrO-regulated DIDC biochip was checked utilizing electrical and optical surface examination. The large surface area of graphene-based materials causes the adsorption of various analytes. Additionally, this super-sensitive and ultra-fast GrO-DIDC biochip represents a simple and suitable system for POC testing. In this way, the detailed biosensor is a cutting-edge technology. Firstly, the sensor could create a capacitance change in excess of 70 nF because the exposed GrO-coated DIDC chip has a low convergence of 1.0 fg/mL. The extent of the capacitance change increases in increments with the target virus antigen protein concentration within the range of 1.0 mg/mL to 1.0 fg/mL. The identification limits of this biosensor are among the lowest to date for a non-faradaic biosensor (capacitive). Second, the sensor explicitness was shown by the variation in the method of capacitance differential among the targeted and non-target biomarkers (protein). For non-target proteins, the change in capacitance was fundamentally not the same as for the objective virus S1 antigen target protein, and there was no considerable change in capacitance with non-target protein fixation increments. Third, the sensor accomplished a wide detection range from 1.0 mg/mL to 1.0 fg/mL with great capacitance linearity (i.e., 18.56 nF/g). On one hand, the quick identification of the virus S1 antigen allows key personal contacts to be detected quickly, expands the options for treatment, and supports designated treatment; furthermore, this identification helps to curb the spread of the illness, which is necessary for combating the pandemic in a timely manner. The most crucial part of this complicated and constrained technology is optimizing the fabrication process and chip functionality. Every step of the fabrication process needs to be managed. The fabrication results could differ and be limited depending on how it is handled.

Conclusion and viewpoint
This paper presents a designed and fabricated ultra-fast and recyclable graphene oxide-altered twofold interdigitated capacitive (DIDC) detecting chip for identifying virus S1 proteins specifically at the fg level. The explorations in this work uncovered that using a GrO-functionalized DIDC capacitive chip further develops the sensing limits, thus enhancing the responsiveness and selectivity of the biosensing device. The restricted line shape and the changes in the electrical properties (impedance coupling) caused capacitance changes for an array of identification ranges (1.0 × 10⁻³ gm to 1.0 × 10⁻¹⁵ gm). The functioning of the DIDC capacitive sensor was comprehensively tested in profoundly weakened arrangements containing BSA biomolecules and spike proteins. These studies demonstrated that the proposed DIDC capacitive sensor could be used to detect virus S1 proteins at low levels in a short time (~5-6 s). Therefore, the proposed painless, non-contact, and quick procedure in this work can detect the virus S1 protein at the beginning phases of the illness with high sensitivity.

Later on, the plan is to identify virus spike proteins at POC to empower patient-driven treatment and to upgrade all the device parts (i.e., the detecting chip, developing full coordination of the model-based DIDC capacitive sensor with genuine patient examples [simultaneously]). Eventually, COVID-19 could be detected by utilizing cell phone-based technology. Nonetheless, the discussed and introduced biosensor has not yet been tried utilizing genuine samples because of an absence of biological sample availability and ethical approval. Serious endeavors are being made to establish cooperation between South Korean virology and infection labs and emergency clinics abroad to obtain bio-liquids from COVID-19-infected patients. The consequences of future investigations into COVID-19 POC diagnostics utilizing our high-quality capacitive sensor will be published elsewhere.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amyloid β1-42 Protein	Merck (Sigma-Aldrich)	107761-42-2
anti-SARS-CoV-2 Spike (S1) monoclonal IgG antibody	SinoBiological	40150-R007
EDC [N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride]	Thermo Fisher Scientific	A35391
Ethyl alcohol (C2H5OH)	Sigma-Aldrich
Hydrogen peroxide (H2O2)
Kapton tape			polyimide tape
NHS (NHydroxysuccinimide, 98+%; C4H5NO3)	Thermo Fisher Scientific	A39269
PBS
Prostate-specific antigen	Sigma-Aldrich	P3338-25UG
SARS-CoV-2 Spike S1-His recombinant protein	SinoBiological	40591-V08H
Single layer Graphene Oxide	Graphene Supermarket
Spin Coater	High Precision Spin Coater (Spin Coating System)		ACE-200
Sulfuric acid (H2SO4)