A Short Review Comparing Carbon‐Based Electrochemical Platforms With Other Materials For Biosensing SARS‐Cov‐2

Abstract

Due to the 2019 SARS‐CoV‐2 outbreak, low‐cost, fast, and user‐friendly diagnostic kits for biosensing SARS‐CoV‐2 in real samples employing multiple working electrodes are in high demand. Choosing SARS‐CoV‐2 detecting electrodes is difficult because each has advantages and limitations. Carbon‐based electrochemical sensing applications have attracted attention from the electrochemical sensing community because carbon and carbon‐based materials have been a godsend for testing utilizing an electrochemical platform. Carbon working electrode electrochemical platforms are cost‐effective and fast. Covid‐sensors use carbon‐based materials because they can be easily changed (with inorganic and organic functionalities), have quick response kinetics, and are chemically resistant. Covid‐19 sensing materials include graphene and graphite. This review explains how carbon materials have been employed in N and S protein electrochemical detection. Here, we discussed a carbon‐based technology for SARS‐CoV‐2 biosensing. We′ve compared carbon‐based electrochemical sensing to different electrodes.

This review covers the status of carbon‐based electrodes for the detection of SARS‐Cov‐2. Carbon materials should be modified to use as electrode materials for practical applications. The current methodologies to modify carbon surfaces for COVID‐19 sensing applications have been discussed and compared their electrochemical responses with other commonly used electrode materials.

1. Introduction

Covid‐19 emerged in November 2019 in Wuhan city of China. Since then, it has been disrupting the life of people around the globe and has been declared a global pandemic. According to the data shown in Figure 1, on average around 10,000 articles have been published each year indicating extensive research has been carried out to protect humans from this dangerous virus, but rapid mutations and transmissibility have been a great barrier.

Figure 1.

Shows different types of articles published in the last 2.3 years on SARS‐CoV‐2. (Data retrieved from science direct up to 16th March 2022).

Furthermost sensors target spike proteins responsible for the virus's attachment to the host cells (ACE2 receptor) and mutations at those sites can hamper the functioning of materials in the immunogenic response and sensing applications. ^[1] And overviewing the fast transmission rate there is an urgent requirement for fast testing methods to contaminate the spread. As discussed in Table 1, methods like ELISA (Enzyme‐Linked Immunosorbent Assay), RT‐PCR (Reverse transcriptase‐polymerase chain reaction), and various assay techniques consuming which limits their point of care use. Moreover, samples containing the virus cannot be kept for a longer time due to fast transmission and even the concentration of the virus in each sample can vary from very low to very high, so there is a need for a technique that can overcome these issues. ^[2] Electrochemical methods have proven as a boon in providing point of care services; therefore, these methods have been used by many researchers (data in Figure 2).

Table 1.

Advantages and disadvantages of electrochemical methods and gold standards (RT‐PCR and ELISA) for covid‐19 detection.[ ³ , ⁴ , ⁵ , ⁶ , ⁷ ]

Techniques	Advantages	Disadvantages
RT‐PCR	‐high accuracy ‐high sensitivity ‐high specificity	‐require highly skilled labor ‐time‐consuming ‐costly process ‐May give a false negative, as identifies active carriers ‐ requires a large amount of sample
ELISA	‐moderate accuracy ‐higher specificity	‐require highly skilled labor ‐time‐consuming ‐costly process ‐ qualitative results
Electrochemical sensors	‐rapid response ‐ portable ‐easy to use ‐lower detection limit ‐a little amount of sample required	‐false results ‐interferants may affect performance

Figure 2.

Shows electrochemical detection of covid‐19 in recent years. Data retrieved from science direct. (Data retrieved from science direct up to 16th March 2022).

When compared to traditional optical detection methods, electrochemical sensors and biosensors have better selectivity and sensitivity, require a lesser amount of sample, have faster response times, are easier to operate, are more cost‐effective, can multiplex, and can be miniaturized.[ ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ ] Voltammetric methods are based on a three‐electrode system approach (working electrode, reference electrode, and counter electrode). Out of all working electrodes is the most important one as the entire redox process occurs at its surface. ^[14] Electrochemical sensing involves potentiometric, amperometric, and impedimetric approaches to detect nucleic acid and immunological detection. ^[15] But electrochemical methods require an effective material that enables the testing of viruses along with multiple biomarkers with greater sensitivity, reproducibility, and selectivity. Overviewing the growing attention towards carbon‐based materials in electrochemical sensing, we have tried to summarize the recent research on carbon‐based materials as the working electrode and compared them with other materials for sensing Covid‐19. The usage of digital and telehealth approaches for patient treatment has been quickly adopted. The constraints to telehealth were temporarily removed, and rapid growth of remote health care was introduced to offer patients and health care professionals (HCPs) increased safety through social distance. ^[16]

2. Current detection and tracking technologies for COVID‐19

2.1. Molecular method

Reverse transcriptase‐quantitative polymerase chain reaction (RT‐qPCR) detects nucleic acid‐based genetic sequences. ^[17] It detects viruses. Upper and lower respiratory fluid is used for SARS‐CoV2 RT‐qPCR. ^[18] Oropharyngeal and nasal swabs gather fluid. Fluid is filtered to extract virus RNA. This enzyme produces cDNA from viral RNA. Specific sections of cDNA are amplified by polymerase chain reaction, and a DNA probe intended to hybridize within a limited fraction of the cDNA permits real‐time detection. ^[19] DNA probes are tagged with fluorophore and quencher for real‐time detection. Formerly, radioactive isotopes were used to target nucleic acids. Recently, fluorescent tags have taken their place. ^[20]

Real‐time detection of viral cDNA occurs when the DNA polymerase enzyme, while adding nucleotides to a specific area of viral cDNA, comes in touch with double‐stranded DNA (from a DNA probe), causing its exonuclease activity to separate fluorophore and quencher molecules. After a few rounds of polymerase reaction, a large amount of fluorescence signal is formed. If the device is properly calibrated, the fluorescence intensity is proportional to the virus concentration in the infected persons. The sensitivity of RT‐PCR is 500 to 1000 viral RNA copies/ml. ^[21] Viral protein RNA‐dependent RNA polymerase (RdRP), E, and N genes have been found for detecting SARS‐CoV2. SARS‐Cov2 damages the target RNA when it releases its viral capsid. The host‘s immune system may be a factor. It releases small RNA fragments into the circulation, making RT‐PCR difficult to detect. Signal enrichment strategies for separating RNA fragments using CRISPR or nanomaterials like gold nanoparticles and metal‐organic complexes may be the key to overcoming this issue. ^[22]

2.2. Biochemical tests

COVID‐19 is identified by scientific methods such as ELISA, which recognizes viral proteins or antibodies generated by the body in response to SARS‐CoV2 infection. 96‐well microtiter plates are used to detect antibodies discovered through protein‐protein interactions. Colorimetric, fluorescence, or luminescence detection with enzymes intensifies these interactions. Due to fluctuating viral load, it might be difficult to identify low levels of viral protein. ^[23] That's the issue. Antibodies can help determine if vaccines are acting properly. A person infected with a virus for weeks can still be utilized to find the source. It may be the most appropriate diagnostic for informing intervention policymakers about asymptomatic individuals. ^[24]

Technical issues might induce false‐negative antibody testing. Low antibody concentrations in fluid samples; homologous proteins; insensitive detecting technology. Despite low antibody concentrations and homologous proteins, a designed protein that binds to our target antibody may increase biosensor sensitivity. Long‐term, sensitive biological sensors ^[25] are needed.

2.3. Nano‐biosensors

Recent studies have shown that nano‐biosensor tools are a good starting point for developing low‐cost and quick sensors for a wide range of organisms. ^[26] Because of their capacity to conjugate with biological systems and detect SARS‐CoV‐2, nano‐bio sensors have found use in this setting (COVID‐19). Various biosensors have been developed to improve the detection limit to solve the difficulty.

Seo et al. ^[27] constructed a graphene‐oriented nano biosensor to detect SARS‐CoV‐2 and COVID‐19 to forecast the SARS‐CoV‐2 viral identification platform. Coating FET graphene sheets with a protein that can be detected in coronavirus spikes created the immunosensor. Song et al. ^[28] designed an antifouling electrochemical nano‐biosensor for COVID‐19. The sensor used synthetic peptides and PANI nanowires. Zamzami's group ^[29] created a simple, quick, accurate, quantifiable, and cost‐effective CNT‐FET nano biosensor to detect SARS‐CoV‐2 spike protein (S1) in saliva.

Plasmon‐enhanced fluorescence, luminescence, surface‐enhancing reflectance, absorbance, and Raman scattering[ ³⁰ , ³¹ , ³² , ³³ , ³⁴ ] are approaches employed in optical nano‐bio sensors to explore bioreceptor‐analyte interactions. These are the analyte concentration and nanomaterial types. Hadi and Khurshid ^[35] constructed a nano‐biosensor to detect COVID‐19.

Polymethylmethacrylate (PMMA) was employed as the core material for a U‐shaped optical fiber (POF) sensing probe with a 1.49 refractive index. The core was coated with a 1.41‐refractive‐index fluorinated polymer. Using saliva samples, biofunctionalized probes were used to identify SARS‐CoV‐2. This sensor can identify even Omicron, the latest SARS‐CoV‐2 strain. Signal manipulation methods include wavelength division multiplexing, intensity modulation, and frequency multiplexing. Samples are obtained from the nasopharyngeal/oropharyngeal area using a U‐shaped POF probe. Experiments suggest the accurate identification of tiny POFs. SARS‐CoV‐2 can be identified in 15 minutes.

Magnets and aptamers can increase nano‐biosensor sensitivity and selectivity. Magnetic nano‐biosensors produce less background noise than electrochemical or optical nano‐biosensors,[ ³⁶ , ³⁷ ] Magnetic nanoparticle‐based immunosensors were described by Li and Lillehoj. ^[38] This new immunosensor utilizes immunomagnetic signal amplification to reliably detect viral protein. Aptamers are 25‐to‐90‐base single‐stranded oligonucleotides. Due to aptamers′ great combining capacity, their three‐dimensional spatial arrangements help identify viral components. Abrego‐Martinez and his colleagues ^[39] developed a simple, fast, and sensitive aptameric nano‐biosensor to detect SARS‐CoV‐2. Using AuNPs, the aptasensor was immobilized. The detection technique depends on the aptamer‘s binding affinity to SARS‐spike CoV‐2’s RBD (S‐protein). Current nano‐biosensor research must focus on generating more accurate, faster‐responding, less‐processed, recyclable, longer‐lasting, and less expensive sensors. Nano‐biosensors for SARS COV‐2 must increase their binding affinity and specificity for usage in medical emergencies.

2.4. Artificial intelligence‐based detection of other biomarkers

Artificial intelligence may enhance diagnoses and forecasts by eliminating human judgment. AI biomarkers and indicators are crucial when RT‐PCR isn′t enough to diagnose a patient with early symptoms. ^[40] Recent AI‐powered COVID‐19 applications include an imaging platform, lung and infection region segmentation, clinical evaluation and diagnosis, and groundbreaking scientific and clinical research. The AI‐powered imaging workflow includes X‐ray and CT devices with cameras for patient monitoring. These tools make it easy to scan without touching. Live footage from the camera is sent to the control room so technicians may watch the patient. From the camera‘s overhead perspective, it′s hard for the technician to determine the scan range. Using visual sensors such as RGB, TOF pressure imaging, or FIR cameras, AI may determine a patient‘s stance and shape. With this information, scan parameters can be improved.

AI‐powered visual sensors can predict the CT scans start and finish points, or scan range. Find the scan range by looking at the subject‘s joints. Wang and colleagues ^[41] found that an automated procedure may boost scanning productivity and reduce radiation exposure. Such critical spots depict a small sampling of the digital human body‘s 3‐D mesh. In another parameter, AI may infer scanning properties such as ISO‐centering. “ISO‐centering” refers to matching the subject‘s target body area with the scanner‘s ISO center. This assures high‐quality images. If ISO‐centering is improved, radiation dose can be reduced while picture quality is maintained. Georgakis et al. ^[42] suggest using SMPL to recreate human mesh from a single monocular RGB photo. Anatomical key points indicate a poor sampling of the complete 3D mesh in the 3D space that comprises the digital human body, therefore the target body area must be aligned to the ISO. Proper alignment is achieved.

RT‐PCR can detect and target coronavirus pathogens based on their genetic sequence. RT‐PCR is not extensively employed in public health circumstances like COVID‐19 infection due to time‐consuming analysis, expensive equipment, qualified workers, and modern laboratory infrastructure, as well as logistics for reagents and extraction kits. Improper sample collection and handling, viral RNA degradation, PCR inhibitors, and viral shedding might impact RT‐PCR accuracy. Because of these circumstances that restrict analytical sensitivity, RT‐PCR may give false negatives, which can lead to community transmission when unwell individuals return without isolation and treatment. ^[45]

Electrochemistry diagnostics may solve the drawbacks of current approaches for identifying coronavirus‐induced diseases by offering simplicity, cost‐effectiveness, on‐the‐spot diagnosis, and a rapid turnaround time from sample to results.

3. Challenges faced by current diagnostics in coronavirus detection

Coronaviruses′ structural features are employed to build infection detection systems. Significant research has gone into molecular (nucleic acid‐based) and serological (antibody‐based) coronavirus detection approaches. Molecular diagnostics help identify infected people during the acute phase of sickness, whereas serological tests detect viral antibodies. ^[43] ELISA‐based serological diagnostics track seroconversion to determine past infections and viral immunity to design therapies. ELISA tests require specialist staff and labs and take one to five hours to complete. These limitations raise turnaround time and costs, restricting bulk manufacture of diagnostic tests to meet demand. Components requiring laboratories and specialized workers increase the test‘s complexity, making portable on‐site detection by end‐users difficult or impossible. ^[44]

RT‐PCR can detect and target coronavirus‐induced diseases based on their genetic sequence. RT‐PCR isn′t extensively employed in public health circumstances like COVID‐19 infection due to time‐consuming analysis, expensive equipment, qualified workers, and modern laboratory infrastructure, as well as logistics for reagents and extraction kits. Incorrect sample collection and handling, viral RNA degradation, PCR inhibitors, and viral shedding may impact RT‐PCR accuracy. Due to limited analytical sensitivity, RT‐PCR may give false negatives, which might lead to community transmission when ill individuals return untreated. ^[45]

Electrochemistry diagnostics may address the drawbacks of current approaches for identifying coronavirus‐induced diseases by offering simplicity, cost‐effectiveness, on‐the‐spot diagnosis, and a rapid turnaround time from sample to findings.

4. Advantages of carbon materials in electrochemical sensing

When we talk about electrochemical biosensors, carbon and carbon‐based materials are always a fascinating class of substances, be it in zero (Nanodiamonds, fullerene, carbon dots), one (carbon nanotubes, CNT and carbon nanofibers, CNF), or two‐dimensions (graphene),[ ⁴⁶ , ⁴⁷ ] Carbon's property of catenation enables it to form long chains, which in turn is responsible for easy fabrication and functionalization. Of these carbon dots, carbon nanotubes, graphene, and fullerenes are used in the detection of viral infections. Carbon nanotubes possess excellent conductivity, flexibility, and resistance. ^[48] Graphene is a two‐dimensional material with a layered structure and π‐π stacking that allows for faster electron delocalization, making it an ideal material for electrochemical studies, particularly sensing applications. Even graphene bears good viral inhibition capacity that's why has been explored by researchers in combating covid‐19.[ ¹⁵ , ¹⁶ , ¹⁷ ] Carbon dots are a class of 0‐dimensional materials that are water‐soluble, biocompatible, and environmentally friendly with good electrical conductivity, chemical inertness, high specific surface area, low toxicity, low cost, and easy functionalization. ^[52] Carbon nanofibers (CNF) are straight, non‐filamentous carbon materials with sp² hybridization and diameters in the nanoscale range. They have a high surface area to volume ratio, exceptional superstrength, and remarkable flexibility,[ ⁵³ , ⁵⁴ ] The chemical inertness, inexpensiveness, and low toxicity of carbon materials allow their wide applicability in electrochemical biosensing. ^[48] Therefore, carbon materials are capable of providing excellent tensile strength and flexibility along with a wide potential window, good electrical conductivity, and chemical inertness. ^[55] The advantages and disadvantages of carbon‐based electrodes with different electrode materials are given in Table 2.

Table 2.

comparison of carbon‐based electrodes with other different types of electrodes used in electrochemical biosensing.[ ⁴⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ ]

S.No.	Electrode material	Advantages	Disadvantages
1.	Au	large anodic potential range	‐limited cathodic potential range ‐expensive
2.	Pt	‐low corrosion resistance ‐low contact resistance	‐expensive
3.	Hg (dropping mercury, hanging mercury drop, static mercury drops, streaming mercury, and mercury film)	‐high hydrogen overvoltage ‐constantly changing surface area ‐low cost, high sensitivity	‐mechanical instability ‐toxicity
4.	Carbon	‐low cost ‐large specific ‐surface area, ‐high electron mobility ‐reduce the interface resistance ‐excellent tensile strength ‐flexibility	‐low conductivity ‐larger dimensions ‐high fabrication cost ‐low energy density ‐poor dispersity

Possibility of using electrochemistry to overcome the shortcomings of current diagnostic techniques: Electrochemical approaches provide viable alternatives to chemiluminescence, fluorescence, and colorimetric‐based detection, particularly in conditions of financial hardship and scarcity of resources. With nanotechnology‘s remarkable advancements in electrochemical device design and performance, an electrochemical sensor may detect changes in current, potential, conductivity, and impedance in signals owing to the recognition process occurring on the sensing surface with the electrode material functioning as the transducer. Electrochemical sensors and biosensors can provide enhanced selectivity and sensitivity as compared to conventional standard techniques based on optical detection, as well as shorter reaction times, less sample volume required, multiplexing capabilities, and the potential for downsizing. ^[56] Assumed ASSURED (Affordable, Sensitive, Specific, User‐friendly, Rapid and robust, Equipment‐free, and Deliverable to end‐users) diagnostic criteria given by the World Health Organization (WHO) are met by these desired features. Because of this fact, electrochemistry can help find good ways to fight the COVID‐19 pandemic and any other coronaviruses that might show up in the future. ^[57] It can also help get around the problems with the current clinical diagnostics used to find COVID‐19. There have been recent studies using electrochemistry instead of optical PCR, where electroactive indicators or detection labels were utilized to monitor the PCR reaction‘s progress, and the amount of amplified product could be determined by comparing observed oxidation or reduction signals. Electrochemical PCR is a cost‐effective and portable solution for COVID‐19 intense testing without the requirement for fluorescent labeling and optical detection. ^[43]

5. The principle behind electrochemical sensing of COVID‐19

A typical biosensor comprises 4 parts: analyte/sample, bioreceptor, transducer (electrochemical system), and an electronic system. Electrochemical sensors detect analytes by measuring the electric current generated by chemical reactions. These chemical processes are transformed into this detectable electrochemical signal by a recognition element and a transducer of the sensor, which generates an electrical double layer at the electrode interface between the recognition element, and the binding analyte. The chemical recognition system and the physicochemical transducer are the two primary components of an electrochemical sensor, which transform chemical interactions into electrical signals that current electrical instruments can easily detect and display. Conductometric, voltammetric, and potentiometric are the three main categories of electrochemical sensors that may be used to quantify chemical interactions,[ ⁶² , ⁶³ ] In the case of covid‐19, the sample mostly employed is the viral protein or the viral nucleic acid collected from the nasopharyngeal and oropharyngeal swabs of the patient. The receptor mimics the biological systems wherein the antibodies tend to produce specific and non‐specific interactions with the antigens. The non‐specific interactions are suppressed by blocking agents such as BSA (Bovine Serum Albumin). The receptor is usually an electrode surface bounded with an immobilized antibody for the interaction of antigen (viral protein) as shown in Figure 3,[ ⁵ , ⁶⁴ , ⁶⁵ ] Therefore when there is maximum antigen‐antibody interaction, resistance at the surface of the electrode increases because of which there is a current drop is indicative that electrode material is successful in binding the antigen,[ ⁶⁴ , ⁶⁵ ]

Figure 3.

Interaction between antibody and antigen at the electrode surface.

Sensors prepared for covid‐19 detection are usually based on nucleic acid detection, immunological detection, or serological detection (electro)as shown in Figure 4. In nucleic acid detection viral RNA extracted is converted into cDNA and further amplified. While immunological detection is based upon antigen‐antibody interaction in which we detect the proteins that make up the virus, mostly N and S proteins are detected using this method but serological tests are concerned with the past infections and detect immunoglobulins formed as a result of the body's response towards foreign particle (in case of covid‐19 during initial days IgA and IgM will be detected but after few days IgG will be detected and IgG can be detected even after recovery),[ ² , ⁶⁶ ]

Figure 4.

Various detection techniques for COVID sensing.

6. Preparation of transducer element for biosensing of COVID‐19

A carbon‐containing electrode,[ ⁶⁴ , ⁶⁷ , ⁶⁸ ] or electrode modified with carbon material,[ ⁶⁹ , ⁷⁰ ] is subjected to immobilization with an antigen linking agent (probably an antibody solution or immobilization of RBD) and further treated with a blocking agent (mostly bovine serum albumin, BSA) for preventing non ‐specific interactions. Then the fabricated electrode is characterized using various physicochemical methods such as SEM, TEM, UV‐Vis spectroscopy, FTIR, etc. to check the immobilization of antibodies and confirm the preparation of the electrode. Also, the electrode is electrochemically characterized using a potassium ferricyanide solution containing 100 mM KCl.

7. Different types of carbon materials used for sensing SARS‐CoV‐2

As mentioned earlier in the paper, carbon‐based materials possess properties such as good electric conductivity, electric isolation, chemical resistance, fast electron kinetics, a wide potential window, and a low residual current. Also, in terms of sensibility, reproducibility, and stability carbon materials are ideal candidates for biosensing. ^[48]

7.1. Carbon‐Based Screen‐Printed Electrode (SPCE)

Screen‐printed electrodes represent a class of inexpensive electrodes with rapid detection. They are commonly employed for portable biosensing applications. They are usually printed on a substrate (ceramic or self‐made ink) that can be easily modified. Mostly they are used after modification with Au, Ag, Pt, Pd, other metal nanoparticles and carbon nanotubes (CNTs), and graphene‐based inks because modification helps in diverse applications. ^[20]

7.1.1. Magnetic bead modified SPCE

Magnetic bead‐based matrix along with carbon black modified SPE was also employed for detection of Covid‐19. This smart biosensor worked on a three‐step procedure: blocking, immunoassay, and electrochemical steps. The electrochemical studies were carried out using a potentiostat. The sensor was tested for N and S protein in saliva samples and gave a detection limit of 19 n g/mL for S protein and 8 n g/mL for N protein in an untreated saliva sample. For clinical samples of 24 patients, this gave an accuracy of 91.66 %. Further, these results were satisfactory when compared with RT‐PCR results. This magnetic bead‐based biosensor took 30 mins as analysis time. ^[67]

7.1.2. SiO2@UiO‐66 modified SPCE

Zirconium and carboxylate‐based MOF(UiO‐66) along with silicon dioxide and cysteamine were drop cast on an SPE for the detection of SARS‐CoV‐2 S‐protein. This modified electrode surface was then immobilized with antibody solution (ACE2) in presence of glutaraldehyde and treated with BSA. This fully fabricated electrochemical immunosensor was then tested for binding of SARS‐CoV‐2 S‐protein with ACE2 using CV and EIS (electrochemical impedance spectroscopy) (Figure 5). CVs obtained show that an increase in binding between antigen and antibody causes a decrease in current with this modified electrode, indicating that the SiO2@UiO‐66 modified SPE acted as a good biosensor with a detection limit of 100.0 f g/mL. EIS was applied to sense the spike protein in nasal real samples and found that with an increase in binding the impedance increased and recovery greater than 90 % was achieved. ^[65]

Figure 5.

A) CV curves B) Nyquist plots for bare SPE (black plot), SiO₂/MOF modified SPE(red plot), and probe+SiO₂/MOF modified SPE(blue plot). Reproduced from ref., ^[65] Copyright 2022, with permission from Elsevier.

7.1.3. Stencil‐printed carbon electrodes

The electrode was fabricated to capture anti‐N antibodies by carbodiimide coupling and further, the non‐specific interactions were blocked using aged casein solution. Then the sample solution was dropped on this fabricated electrode and further treated with anti‐N‐ horseradish peroxidase (HRP) detection antibody solution. This prepared electrode material after treatment with buffer solution was tested chronoamperometrically. Chronoamperograms shown in Figure 6 were recorded for concentrations ranging from 0 to 110,000 PFU/mL and a maximum drop in current was found in the case of 110,000 PFU/mL concentration, indicating maximum antibody‐antigen interaction. Here the standard deviation was found to be 3 and this electrochemical bioassay gave a detection limit of around 25 cycle threshold value. Even comparing this biosensor with RT‐PCR for real samples good sensitivity and specificity were achieved. ^[64]

Figure 6.

Electrochemical detection mechanism (left side) and chronoamperograms for different concentrations (right side) Reproduced from Ref., ^[64] Copyright 2022, with permission from American Chemical Society.

7.1.4. Antibody modified SPCE/e‐CovSens

The covid‐19 antibody was immobilized on a screen‐printed carbon electrode and tested for Covid‐19 antigens in the concentration range from 1 fM to 1 μM and even compared the results with Au NPs‐F doped tin oxide electrode. Au nanoparticles (NPs) dropped cast on the Fluorine doped tin oxide electrode and were further immobilized with Covid‐19 antibody upon drying. Both electrodes were characterized by FTIR, SEM, UV‐Vis spectroscopy, and electrochemical methods. This modified electrode was evaluated based on various parameters such as pH, the concentration of antibody, temperature, and response time using CV and DPV. This e‐CovSens electrode was sensitive in both the buffer and spiked saliva samples and gave a detection limit of 90 fM while the Au NPs‐F doped tin oxide gave a detection limit of 120 fM. This electrode was further tested for cross‐reactivity with HIV‐antigen and it was found that the electrode was highly sensitive and specific to the Covid‐19 antigen. ^[71]

7.2. Various Combinations of Graphene

Graphene is the most employed electrode material. But to increase its specificity and sensitivity it has been used along with other materials for increased outputs and better results. The improved features of modified graphene materials, such as a large surface area and the presence of functional groups at the surface, qualify them for use in massive electrochemical sensor arrays. Graphene nanoparticles having a wide surface area are able to adsorb and conjugate with functional groups or biomolecules, resulting in their multifunctional sensing application. Every variety of graphene material has diverse and unique adjustable features (physical, chemical, mechanical, electrical, and defect density), making it a good candidate for use as a future hybrid material in electrochemical devices with customizable electrochemical properties. ^[63]

For the detection, covid‐19 graphene has been used in combination with the paper electrode, laser technique, and tin oxide electrode which have been discussed below.

7.2.1. Along with paper substrate

Graphene has been used in combination with paper electrodes for covid‐19 detection by Ehsan and co‐workers. Graphene/carbon ink was screen printed on a paper electrode (working electrode). This electrode was fabricated with a coupling agent,1‐pyrenebutanoic acid succinimidyl ester (PBASE) for immobilization of IgG anti‐SARS‐CoV‐2 spike antibody. Even this graphene‐based paper electrode used ProtA (immunological instrument) for the immobilization of antibodies on the electrode surface. Both the prepared electrodes gave similar results (∼50 μA oxidation peak current with ∼2 kΩ Rct). Then these prepared electrodes were evaluated based on EIS studies using artificially prepared samples (nasopharyngeal sample from a healthy human spiked with varying concentrations of covid‐19 spike protein). It was seen that there was a gradual increase in Rct semicircle as the concentration of spike protein was increased in the sample indicating the immobilization of antigen using an antibody. CV was performed to evaluate the repeatability of the electrode and <10 % relative standard deviation was observed. Thus, the sensor worked on the principle of antigen‐antibody interaction and was able to quantify antigen concentration as low as 0.25 f g/mL and gave good sensitivity (due to the presence of probe) with low response time. The proposed sensor was also employed in the real sample detection using nasopharyngeal swab samples of the infected individuals and satisfying results were obtained. The prepared portable biosensor was superior in comparison to ELISA techniques and simple paper electrodes in terms of detection limit and response time. ^[69]

7.2.2. Laser engraved graphene

Laser engraved graphene electrode gives high sensitivity, selectivity, charge mobility, and high surface area. So, it was employed to detect covid‐19 S‐protein, N‐protein, IgG, IgM as well as inflammatory biomarker C‐reactive protein (CRP). This laser engraved electrode was fabricated with antigen immobilization linker PBA (as it provides functional −COOH groups to interact with ‐NH2 bearing capture receptors of the antigen) and then blocked the remaining active sites by BSA. Further, on analyzing the fabricated electrode electrochemically by DPV and OCP‐EIS it was seen that the peak current decreased on each fabrication step while resistance increased on each step which indicated that the electrode was completely anchored with the linker. This anchorage was further supported by the SEM technique. This fabricated electrode was employed in covid sensing amperometrically using four working electrodes, an Ag/AgCl as reference electrode and graphene as counter electrode in phosphate‐buffered saline (PBS) solutions supplemented with 1.0 % BSA with varying concentrations of S‐protein, N‐protein, IgG, IgM, and CRP. This detection was based upon the sandwich and double sandwich assay technique and the sensor showed an increase in current with an increase in concentration (see Figure 7). So, this graphene‐based biosensor detects simultaneously not only the covid but also detects the severity of the infection. Results of real sample analysis of both infected and non‐infected human serum and saliva using this sensor were as per those provided by RT‐PCR results. ^[5]

Figure 7.

Shows the effect of concentration of A) NP B) CRP C) IgG D) IgM on the current using laser engraved graphene electrode. Reproduced from ref., ^[5] Copyright 2020, with permission from Elsevier.

7.2.3. Graphene‐modified Indium tin oxide (ITO)

ITO electrodes fabricated with gold decorated reduced graphene oxide were also employed in covid‐19 detection. This working electrode was immobilized with anti‐covid‐19 antibodies on its surface as shown in Figure 8. This immobilization was confirmed by Surface Enhanced Raman Spectroscopy (SERS). CV studies for the detection of covid protein gave two peaks (cathodic and anodic at 0.42 V and 0.52 V respectively). SWV was also performed using this modified ITO in 10 nM PBS at a scan rate of 50 mV/s to predict the efficiency of covid‐19 protein detection. In vitro, the sensor gave a detection limit of 39.5 fM while real sample analysis of covid protein in human serum gave a recovery >99 % and a mean relative standard deviation of 1.9. ^[70]

Figure 8.

Shows the fabrication of graphene and Au NPs on the ITO and SWV and SERS plots using this modified electrode. Reproduced from ref., ^[70] Copyright 2022, with permission from Elsevier.

7.2.4. Graphene oxide as an electrode modifier with Au micropillar array electrode

Even reduced graphene oxide was employed to modify screen‐printed electrodes to detect S protein and receptor‐binding domain (RBD). This modified electrode was subjected to antigen functionalization in presence of coupling reagent 1‐ethyl‐3‐(3‐dimethyl aminopropyl) carbodiimide hydrochloride and N‐hydroxysuccinimide in 1 : 1 ratio by volume, further treated with BSA to prevent non‐specific interactions. Using CV analysis this modified 3‐D electrode was compared with the 2‐D type of this electrode. Both the electrodes gave clear oxidation as well as reduction peaks, but 3‐D showed both radials, as well as linear diffusion, and 2‐D, showed only linear diffusion. The resistance of the prepared electrode was evaluated using EIS which showed that upon functionalization the Rct value decreased 30 Ω to 0.894 Ω. For evaluating the electrochemical sensing activity of the sensor EIS pattern was studied at different concentrations for spike protein (ranging from 10⁻¹⁵–10⁻⁹ m) and observed that with the increase in concentration the resistance increased because the antigen‐antibody interactions restricted further reaction at the interface due to blockage of non‐specific interactions and gave a detection limit of 2.8 fM with a sensitivity of 1 pM. This is because of the 3‐D environment of the electrode which provides more interactions with more surface area and porous nature of the electrode, thereby lowering the detection limit. For RBD, similar results were obtained with a detection limit of 16.9 fM and good sensitivity of 1 fM. ^[72]

7.3. Pencil Graphite Electrode

The graphite pencil electrode was functionalized with the aldehydic group using 25.0 % (vol/vol) glutaraldehyde (GA) solution for 1 h at 37 °C. This enabled the anchoring of Au nanoparticles (functionalized with cysteamine) on the electrode surface. It was seen that both the aldehydic group and amine group help in the immobilization of Au NPs. Further, this modified electrode was dipped in a solution having N‐(3‐dimethylamino propyl)‐N‐ethyl carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS) with ACE2 at 37 °C for 30 minutes. Then the remaining active sites on the electrode surface were blocked with bovine serum albumin (BSA) treatment. This prepared electrode material was characterized using UV‐Vis spectroscopy and electrochemical methods (CV and EIS) (see Figure 9). But the biosensor ability of the electrode was evaluated using SWV for the interaction between spike protein and the ACE2 site. It was seen that binding of protein with the electrode site results in a decrease in current due to blockage of the active site which indicates the presence of Covid‐19. This sensor gave a good detection limit of 229 f g/mL with quantification of 0.91 p g/mL and a low response time of 6.5 mins. Real sample analysis using nasopharyngeal swabs and saliva samples gave a sensitivity of 88.7 % and 100 % respectively with an accuracy rate of >87 %. ^[68]

Figure 9.

Shows CV and Nyquist plots for a modified pencil graphite electrode using KCl as the supporting electrolyte at a scan rate of 50 mV s⁻¹ where green colored plots correspond to a fully modified electrode. Reproduced from ref., ^[68] Copyright 2021, with permission from PNAS.

8. Comparing the electrochemical performance of carbon electrodes with other electrodes

Table 3 compares the electrochemical responses of various types of electrodes with and without carbon material. It can be said that the presence of Au provides excellent conductivity and thermal stability, so electrodes in combination with Au and carbon provide excellent results. But using Au NPs and ITO electrodes alone with other combinations gives lower detection limits but takes greater testing time. Overall, the carbon‐based electrodes are cost‐effective and have comparable results with the gold standards of testing (ELISA and PCR) and even other electrodes based on only tin oxide and titanium dioxide. Even paper‐based sensors have achieved 90 % specificity and 100 % sensitivity toward COVID‐19, but the detection limit is not yet determined. ^[73]

Table 3.

Comparison of the carbon‐based electrode based on detection limit and detection time.

S.No.	Type of electrode	Type of antigen	Detection limit	Real sample used	Time taken	References
1.	SPE+magnetic bead	N protein S protein	8 n g/mL 19 n g/mL	Saliva	30 mins	[67]
2.	SiO2@UiO‐66 modified SPE	S protein	100 f g/mL	Nasal sample	5 mins	[65]
3.	Stencil‐printed carbon electrodes	N protein	–	nasopharyngeal swab	70 min.	[64]
4.	Antibody/SPCE(e‐CovSens) Au NPs−F doped tin oxide	S protein	90 fM 120 fM	Spiked saliva samples	10–30 s	[71]
5.	Graphene+paper electrode	–	0.25 f g/mL	nasopharyngeal swab	–	[69]
6.	Laser engraved Graphene	S‐protein, N‐protein, IgG, IgM, CRP	–	human serum & saliva	–	[5]
7.	Graphene modified ITO	–	39.5 fM	human serum	–	[70]
8.	Reduced graphene oxide+3‐D micropillar electrode	S protein RBD	2.8 fM 16.9 fM	–	∼11.5 s	[72]
9.	Functionalized Pencil graphite electrode	S protein	229 f g/mL	nasopharyngeal swabs and saliva	6.5 mins	[68]
10.	Co‐TiO2 NT	RBD	0.7 nM	Nasal and saliva samples	30 s	[74]
11.	Carbon Nanofibre+SPCE	N‐protein	0.8 p g/mL	Nasal samples	3 hours	[75]
12.	Polymerized Au‐coated ITO	Nucleocapsid protein	0.48 f g/mL	artificial nasal secretion	45 mins	[76]
13.	Epoxy‐thiophene ITO	RBD	0.58 f g/mL	Nasal Secretions	45–60 mins	[77]
14.	Au‐mercaptoundecanoic	RBD	0.577 f g/mL	artificial nasal secretion	60 mins	[78]
15.	AuNPs‐modified SPCE	RBD	1.30 pM	HIVNL4‐3Env‐luc+ SARS‐CoV‐2 S‐protein	40 min	[79]

9. Conclusion

Overviewing the efficiency of all the carbon‐based electrodes, we can say that reduced graphene oxide in combination with a gold micropillar electrode gave an excellent detection limit for S‐protein within seconds. In other words, using carbon materials to functionalize metal electrodes can act better when using them directly as an electrode material. Even functionalized pencil graphite electrodes gave good results in comparison to other carbon‐based electrodes. So, it can be said that the fight against the COVID‐19 carbon materials, specifically graphite and graphene, has proved a boon at the testing level as a point of care. Also, the electrochemical methods have provided good sensitivity along with a good detection range. The combination of electrochemistry and graphene resulted in a variety of portable detection devices that eliminated the need for a lengthy, time‐consuming detection process. Overviewing the current trend of the use of these materials in COVID detection, it can be said that if deeply investigated, this approach possesses great potential for contaminating the spread.

Conflict of interest

The authors declare no conflict of interest.

Biographical Information

Isha Soni is a Ph.D. student in the School of Advanced Chemical Sciences, Shoolini University of Biotechnology and Management Sciences, Himachal Pradesh, India. She did her M.Sc. in Chemistry also from Shoolini University in 2021. Her research is focused on developing novel nonmetal electrocatalysts using MOFs for electrochemical processes and their applications in biosensing and energy‐related applications.

Biographical Information

Pankaj Kumar is a doctoral research assistant at the School of Advanced Chemical Sciences, Shoolini University. He received his BSc. from Himachal Pradesh University in 2015, and his M.Sc. from Shoolini University in 2018. His research interest includes the synthesis of ionic liquids and their applications in biosensors, catalysis, and hydrogen evolution reactions.

Biographical Information

Dr. Gururaj Kudur Jayaprakash is working as an Assistant Professor at the Department of Chemistry, Nitte Meenakshi Institute of Technology, Bangalore, Karnataka, India, and as an adjunct faculty at the School of Advanced Chemical Science, Shoolini University, Himachal Pradesh, India. He received a Tsinghua Postdoctoral fellowship from the Department of Chemistry, Tsinghua University, Beijing, P.R. China, to work as a postdoctoral scholar. He has completed his doctoral studies at the Department of Material Science, University of Guadalajara, CUCEI, Guadalajara, Mexico. He has been awarded a CONACyT Ph.D. scholarship for doctoral studies from the Govt. of Mexico. He also worked as a project graduate trainee at the C.S.I.R. National Aerospace Laboratory, Bangalore, India. The main theme of his research group is voltammetric and quantum chemical methods to analyze 2D materials for electronic and electrochemical applications.

Biographical Information

Dr. Anup Pandith finished doctoral studies with Prof. Hong‐Seok Kim's research group at the Dept. of Applied Chemistry, Kyungpook National University, Korea and worked as a post‐doctoral student with Dr. Young Jun Seo, (2017‐2019), he worked as Research Professor in Department of Chemistry and Bioactive Materials, Jeonbuk National University, and Kyunghee University. Currently, he is working as Tenure Track Assistant Professor in the College of Biomedical Engineering, Taipei Medical University, Taiwan.

I. Soni, P. Kumar, G. K. Jayaprakash, A. Pandith, ChemistrySelect 2022, 7, e202202465.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.