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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 structure5,6. The symptoms of COVID-197,8 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 crucial2,11. 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 testing14,15 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 characteristics18,19. 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 methods24,25. Of note, there are different kinds of DNA and immunizer-based biosensors that utilize compound, capacitive, and electrical techniques18,26,27,28. As an example, electrical DNA biosensors, which have high responsiveness, can be scaled down simply, and are tunable29,30, have been produced for the detection of Ebola31, Zika, MERS-CoV, and SARS-CoV32,33,34. 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 created35,36. 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 virus35,37,38.

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 s18,27 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 applications2,39,40,41. 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 applications26,42,43,44.

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

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 DIDC18,26,27. 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-bas...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was upheld to some extent by the Basic Science Research Program through the National Research Foundation of Korea (NRF) sponsored by the Ministry of Education under Grant 2018R1D1A1A09083353 and Grant 2018R1A6A1A03025242, somewhat by the GCS Group Association Ltd., and by the Korea Ministry of Environment (MOE) Graduate School invested huge energy in Integrated Pollution Prevention and Control Project and a Research Grant of Kwangwoon University in 2022.

E.M. would like to acknowledge the support from the National Institute of Biomedical Imaging and Bioengineering (5T32EB009035).

Materials

NameCompanyCatalog NumberComments
Amyloid β1-42 ProteinMerck (Sigma-Aldrich)107761-42-2
anti-SARS-CoV-2 Spike (S1) monoclonal IgG antibody SinoBiological40150-R007
EDC [N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride]Thermo Fisher ScientificA35391
Ethyl alcohol (C2H5OH)Sigma-Aldrich
Hydrogen peroxide (H2O2)
Kapton tapepolyimide tape
NHS (NHydroxysuccinimide, 98+%; C4H5NO3)Thermo Fisher ScientificA39269
PBS
Prostate-specific antigen Sigma-AldrichP3338-25UG
SARS-CoV-2 Spike S1-His recombinant proteinSinoBiological40591-V08H
Single layer Graphene OxideGraphene Supermarket
Spin CoaterHigh Precision Spin Coater (Spin Coating System)ACE-200 
Sulfuric acid (H2SO4)

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