Nanomaterials‐based sensors for the detection of COVID‐19: A review

Abstract With the threat of increasing SARS‐CoV‐2 cases looming in front of us and no effective and safest vaccine available to curb this pandemic disease due to its sprouting variants, many countries have undergone a lockdown 2.0 or planning a lockdown 3.0. This has upstretched an unprecedented demand to develop rapid, sensitive, and highly selective diagnostic devices that can quickly detect coronavirus (COVID‐19). Traditional techniques like polymerase chain reaction have proven to be time‐inefficient, expensive, labor intensive, and impracticable in remote settings. This shifts the attention to alternative biosensing devices that can be successfully used to sense the COVID‐19 infection and curb the spread of coronavirus cases. Among these, nanomaterial‐based biosensors hold immense potential for rapid coronavirus detection because of their noninvasive and susceptible, as well as selective properties that have the potential to give real‐time results at an economical cost. These diagnostic devices can be used for mass COVID‐19 detection to understand the rapid progression of the infection and give better‐suited therapies. This review provides an overview of existing and potential nanomaterial‐based biosensors that can be used for rapid SARS‐CoV‐2 diagnostics. Novel biosensors employing different detection mechanisms are also highlighted in different sections of this review. Practical tools and techniques required to develop such biosensors to make them reliable and portable have also been discussed in the article. Finally, the review is concluded by presenting the current challenges and future perspectives of nanomaterial‐based biosensors in SARS‐CoV‐2 diagnostics.

considered the most contagious period. 3 Due to the inability to analyze and quantify this viral infection rate, the degree of the pandemic remains uncertain. 4 Apart from causing health distress, the virus has also caused significant havoc in the financial and social lives of millions of people around the globe. 5 Therefore, the rapid diagnosis is highly critical to reduce the rate at which the virus is transmitting.
The unprecedented time calls for more research to understand its epidemiology to create better-targeted diagnostics and therapeutics.
While the conventional molecular diagnostics and microscopybased detection of virus infections have existed for a long time, sensing of analytes using electrochemical, colourimetric, or chemiluminescence methods are a great cost-effective alternative for rapid detection of the SARS-CoV-2 with high sensitivity and selectivity. 6 Several metal, nonmetal, and carbon-based materials can be used to develop such biosensors. 7 Among several materials available for their biosensing applications, nanomaterials are highly promising as they impart high selectivity and sensitivity to the sensor electrodes owing to their larger surface area that presents a large number of active sites for trapping or reacting with the analytes. 8,9 Nanomaterials like graphene and its derivatives, 10 carbon nitrides, 11 and gold 12 can be effectively employed to develop biosensors for coronavirus detection.
In addition, such sensors have the potential to be miniaturized and be more user friendly. Therefore, nanomaterial-based sensors can serve as beneficial point of care devices and give reliable results even outside laboratory settings, thus also benefiting people in secluded areas.
The National Institutes of Health strategic plan for COVID -19 research calls for the need "to improve the basic understanding of SARS-CoV-2 and COVID 19 and develop the necessary tools and approaches to diagnose, prevent, and treat this disease," highlighting the impetus on the rapid and reliable diagnosis of the coronavirus. 13 Thus, in the past couple of years, a lot of work has been conducted on rapidly detecting COVID-19 using novel biosensors. 14 The maximum confirmed COVID-19 cases had been diagnosed in America (69,131,242), followed by Europe (54,828,356), and South-East Asia (33,213,135

| Test methods for COVID-19 detection and their challenges
Considering the danger associated with the COVID-19 pandemic and its contagious nature, the research on the detection and the cure for this dangerous disease has been gaining momentum. There are many existing options available for the detection of viral infection, as shown in Figure 2. Cell culture techniques are conventional methods that can detect viruses; however, this method does not have high specificity and involves several time-consuming steps. 31 On the other hand, the electron microscopy technique is an essential viral diagnostic tool and helps overcome any inconsistencies observed during the virus detection process. However, drawbacks like relatively lower detection sensitivity, extremely time consuming, and costly instrument dependence make this option problematic for the detection of COVID-19. Likewise, due to similar drawbacks, methods like next-generation gene sequencing and enzyme-linked immunosorbent assay have not gained much popularity for virus detection.  Currently, the nucleic acid test is the principal methodology employed for coronavirus testing. 45 RT-PCR is popular in the testing kits currently available to diagnose coronavirus in suspected patients. The basic process behind SARS-CoV-2 testing through this kit is the reverse transcription of the virus RNA into its complementary DNA using specific enzymes. After this, particular regions of the complementary DNA are amplified to detect the virus's existence. However, the currently available PCR testing techniques require sophisticated laboratory and apparatus that are usually not available everywhere. 46 Consequently, the transportation of the nasal swab or other COVID-19-related samples becomes necessary. Hence, in many instances, it may take up to 3 or more days to produce results, even though the actual testing of the sample may only take a few hours.
Moreover, due to the number of steps involved in sample handling and transportation at various levels, there is a considerable risk of sample contamination or accidental spread of the virus. Additionally, the viral nucleic acid presence is not a direct indication of the severity of the disease, 47,48 an essential factor in the clinical diagnosis of a patient needing further medical attention.
Another added challenge to micro-PCR testing is interfering molecules that sometimes give false-positive or false-negative results.
Since over 30% of confirmed cases had been observed to be symptomless, 49,50 a high false-negative rate will rapidly spread the disease within the community. Such false results are mainly attributed to unfavorable conditions of sample handling and transportation. 51,52 The samples usually collected from the patients are in the form of nasopharyngeal, anterior nasal, and midturbinate swabs. 53 Recently, there have also been reports 54 that suggest the possibility of iatrogenic CSF leakage due to nasal swab testing for COVID-19. These limitations are the primary driving force toward finding other alternatives for nasal screening, especially in individuals with a history of skull base defects, surgery, or erosions, or even in patients with a history of sinuses. Studies suggest a risk of about 5%-10% of tested individuals who may have the case of epistaxis followed by a nasal swab test. 55 Another study has reported an increased risk of epistaxis in nursing home residents. As many as 50% of people were affected, they were treated with oral anticoagulants. 56 This triggers a significant attention toward less invasive testing methods like saliva sampling or midturbinate testing.
To reduce the severity of nasal swab testing for SARS-CoV-2 detection, saliva-based tests have also been encouraged. 57 The saliva collection process is noninvasive and does not produce aerosols.
Moreover, the testing does not require professionals, and the testing can be done in simple and easy steps, even in remote areas. However, studies also indicate that there may be lower detection rates of COVID-19 from saliva samples (63%) when compared to the samples of bronchoalveolar lavage fluid (93%). 58 Hence, saliva-based testing F I G U R E 4 Schematic structure of SARS-Cov-2 and its possible target sites that can be used for biosensing and diagnosis for SARS-CoV-2 diagnosis will need further testing to confirm the patient's infection status.
As discussed above, the currently popular RT-PCR and related techniques provide delayed results and disallow on-site diagnosis.
Other shortcomings include their efficiency to diagnose cases in the early stages, cumbersome sample preparation and purification process that are time-consuming. 59 Most of the procedures require many complex apparatuses, skilled laboratory technicians, and additional requirements that increase the overall expense of these methods.
Hence, better and more efficient methodologies are quickly needed to detect and analyze the virus and its antibodies, taking the diversity and the viral replication niches into account. Also, these diagnosis methods must be user-friendly for early detection of the viral infection before the first signs of the symptoms set in to reduce the spread of the viral infection. These issues can be overcome by biosensorsbased detection as they can be far more effective than traditional methods due to their ability to give rapid results with high sensitivity, selectivity, and specificity even in very low sample concentration. 60

| NOVEL BIOSENSORS FOR THE DETECTION OF COVID-19
Sensors can play a fundamental role in reducing the time taken to get coronavirus testing results, especially during this pandemic. Biosensors are devices that are integrated with the transducer and detector recognize biomolecules like enzymes, nucleic acids, and antibodies.

| Loop-mediated isothermal amplificationbased sensors
The viral genomic RNA, spike glycoproteins, and membrane proteins, when bound to the host ACE-2 receptors, elicit rapid humoral immune response 67 mediated by IgM and IgG antibodies. These antibodies can be used to identify the foreign COVID-19 infection prevailing inside the body. 68 The drawbacks posed by the conventional qRT-PCRbased assays are combated using the reverse transcription loopmediated isothermal amplification (RT-LAMP) method. [69][70][71] This was demonstrated in work put forward by Zhu et al. who studied the single-step RT-LAMP-assisted nanoparticles-based biosensors (NBS), creating the RT-LAMP-NBS assay for the specific and quick detection of COVID-19 72 using the F1ab LAMP primer sets. The nucleoproteins of coronavirus were multiplied and analyzed in a single step through the NBS. Their work demonstrated that the RT-LAMP-NBS detected SARS CoV-2 with high specificity and selectivity. The assay's sensitivity was observed to be 12 copies per reaction, which made the amplification process far more efficient and reduced the chances of errors during diagnosis. The authors also concluded that the assay had 100% sensitivity against coronavirus detection in clinical samples and took less than an hour for the results. 72 Thus, this sensor has the translational potential for detecting and monitoring coronavirus in clinical settings.
In another study by Amaral et al., developed a single tube test based on RT-LAMP that allowed visual detection of less than 100 SARS-CoV-2 genome copies within 30 min. 73  LAMP reaction thus provides a time-efficient pathway that successfully overcomes the intricate and laborious processes in traditional detection techniques like PCR (Table 2). Under isothermal conditions, this technique rapidly multiplies nucleic acid with high efficiency and specificity and is more favorable for creating point-of-care devices for COVID-19 detection. 74 Thus, LAMP-based sensors can be potentially applied for clinical applications toward the SARS-CoV-2 detection and monitoring.

| Clustered regularly interspaced short palindromic repeats associated proteins-based biosensors
The relatively new CRISPR system can also be used for SAR-CoV-2 detection and diagnosis. 75 This method effectively spots bacteria, cancer mutations, and microRNAs by substituting target-specific crRNA/ sgRNA (the cas unit). Nanomaterials, because of their versatile properties, have been used alongside the system to create CRISPR-based biosensors that can detect respiratory viruses. This was observed in the work of Hajian et al., 76 who developed a CRISPR-chip biosensor associated with the graphene-based field-effect transistor (FET) to digitally detect a given target sequence present inside a genome. This CRISPR chip made the use of deactivated CRISPR-Cas 9 complex linked to a specific single-guide RNA and was attached to a transistor resulting in a label-free nucleic acid sensing device whose signals  77 The protein concentrations analyzed using this bioassay were further cross-checked using respiratory RNA extract swabs of several dozen virus-infected patients. This particular assay was reported to show high sensitivity at shallow levels (10 copies/μl input) and could exhibit a high selectivity of 95%-100% for noninfected individuals.
Called the DETECTR assay, this method also works similar to the qRT-PCR technique and has shown to have high accuracy like the PCR technique but at the same time has disadvantages synonymous with the qRT-PCR such as availability of chemicals, reagents, and isolation and extraction kits. Thus, the CRISPR-based assay using Cas-12 genes allows high selectivity and can be used to successfully detect SARS-CoV-2 particles in human clinical samples.

| Electrochemical sensors
Electrochemical sensors have proven to be a highly suitable diagnostic tool for SARS-CoV-2 detection. 44 These devices can analyze target molecules at picomolar concentrations. 93 Electrochemical sensors can be used in many configurations for the detection of a molecule, such as a three-electrode configuration depicted in Figure 8. At present, disposable immune-sensing chips can be used in electrochemical biosensors to cut down the total cost of biosensors. Nanoparticles modified substrate and interdigitated electrodes can enhance the efficiency of the biosensors for coronavirus detection. 44 These functionalized biosensors also amplify signals to allow room for a low detection limit and enable a more comprehensive biosensing range.
Such a biosensing chip can be linked to a small potentiostat interfaced to a smartphone to give rapid, on-site coronavirus results. Gold nanosystems have exceptional physicochemical properties that enable them to play a wide variety of roles, primarily as signal transducers in biosensors. 95 Mahari et al. reported an electrochemical biosensor to detect spike S1 protein antigen of COVID-19. 94 The sensor was   As noted from the studies mentioned above, such paper-based assays can detect respiratory viruses. Therefore, these paper-based biosensors can be potentially used to detect COVID-19 viruses. More details about the paper-based biosensors for detection of coronaviruses is covered in a recent review. 107 As noted from the studies mentioned above, such paper-based assays can detect respiratory viruses. Therefore, these paper-based biosensors can be potentially used to detect COVID-19 viruses.

| Nanoparticles-based electrochemical biosensors
In electrochemical biosensors, the substance used for making the electrode surface plays a key role because it determines the sensor's performance. For instance, the double-layered capacitance determines the detection limit of the sensor, while the electron transfer rate influences its sensitivity and time lag before the results. At the same time, the detection limit was found to be between 10 2 and 10 6 EID 50 /ml.

| NANOMATERIALS-BASED SENSORS AND THEIR COMPARISON WITH THE CONVENTIONAL SENSORS
The recent nanobiotechnology developments have resulted in better methodologies and sensing techniques for diagnosing SARS-CoV-2 infection. [116][117][118][119] The unique properties of nanoparticles, like their high surface area-to-volume ratio, increased reactivity, and high adsorption, are essential for creating efficient biosensing techniques. Furthermore, the diameter and morphology of nanoparticles can be efficiently modified through covalent or noncovalent interactions to give distinct sensing properties, including low detection limit, increased sensitivity, selectivity, and rapid response. 93 These attributes make nanomaterials-based biosensors significantly different from conventional sensors.
One of the significant challenges for designing a highly sensitive and selective biosensor is to confer them to capture signals even with minute amounts. For this, the sensor must be susceptible to meager quantities of the target analytes. Nanomaterials are capable of amplifying signals at concentrations that are significantly low but are high enough to be detected and recorded owing to a large number of active sites on the surface of nanomaterials. 42 Additionally, many metal nanoparticles like AuNPs, AgNPs or quantum dots like Cd, Pb label the target analyte by attaching to them. 65,120 With nano-labeling, any electrochemical biosensor transforms into a highly selective labeled biosensor (Table 3). 65 Thus, in comparison to conventional biosensors, nanoparticlebased biosensors present various advantages as noted in Table 4.

Conventional biosensors have been used to detect viral infections
through the virus nucleic acid, antibody, aptamer, or antigen dependence. 136 However, they lack the unique physicochemical properties of nanomaterials like their magnetic, optical, electrical, and optomagnetic properties. Traditional biosensors often require extensive specialized instruments for their fabrication and application, posing a challenge to its portability. Moreover, technologies involved in conventional biosensors require further steps for the samples to be processed at several levels before being analyzed. This thereby makes the whole process a little cumbersome and slower as compared to detection using nanomaterials-based biosensors. 42 The impressive reduction in the size of the electrodes since the advent of nanotechnology has not only improved the biosensing properties but has also significantly increased their clinical applicability. In contrast to conventional technologies, nanoparticle-based biosensors are time efficient and inexpensive. However, further work can be done to create nanomaterial-based sensors such that analytes like a complementary single-stranded nucleic acid aptamer can be linked.
Aptamer-based bifunctional biosensors are highly specific in targeting viral surface spike proteins S1, influencing the enzymatic processes to give electrochemical signals. 137 shows promising advantages over other existing coronavirus testing options.

| Internet of medical things integrated biosensors
The SARS-CoV-2 pandemic has proven that this infection creates several complications in the body and affects the function of many organs. Though a lot of effort has been put recently toward developing rapid COVID testing kits, studies are still under process to better understand the possible therapeutic options to treat the condition. 142 To combat any challenges posed during sensing and therapies, artificial intelligence (AI) and internet of medical things integrated sensors (IoMT) can prove to be very beneficial to detect SARS-CoV-2 and to study the individual data and compare with a more extensive profile for a better and intelligent healthcare management system. 131 contamination of bioreceptors is a critical factor that hinders the sensor sensitivity, thereby yielding false results. Characteristic features of antigen, protein, antibody, nanomaterial type, and other factors can also affect the sensitivity and selectivity of the biosensor.
Hence, alternatives like CRISPR can be coupled with nanomaterialbased biosensors to increase their selectivity in all kinds of samples like urine, blood, sputum, and nasopharyngeal swab. 145 However, a plethora of false positive results from the improper nasopharyngeal swab testing calls for an effective and reliable sampling of the swabs. For starters, only synthetic fiber swabs with a flexible shaft must be used for specimen collection. The nasopharyngeal swab must be taken with utmost precaution. The non-toxic synthetic fiber swab must be put deep inside the nasal cavity to obtain the clean samples. Then necessary safety precautions must be taken to process the samples accurately during the testing. 146 Nanostructures like plasmonics have shown promising potential to be used in electrochemical biosensors to produce reliable and reproducible results during such assessments for COVID-19 detection. 61 147 The Ag-NPs hybridization techniques in quartz crystal microbalance DNA-QCM sensing system can also be used to spot the coronavirus. 148 Besides, techniques like localized surface plasmoncoupled fluorescence (LSPCF) fiber optic biosensors can be used to detect SARS-CoV-2. 123 Unique thiolated DNA capture probe sequences can be immobilized on screen-printed electrodes surfaces and further linked to biotinylated target strand DNA. This was previously seen in the work of Ilkhani and the team for Ebola virus detection, 149 and a similar strategy can be applied for COVID-19 detection.
Thus it is clear that nanomaterial-based biosensors offer several advantages for the detection of viral infections as opposed to the conventional testing methods. As discussed in this review, biosensors make the detection process more effective by increasing the sensitivity and selectivity of the sensors, reducing the response time, and can be easily miniaturized in the form of a portable point of care devices. 150   Writingreview and editing (lead).

CONFLICT OF INTEREST
There are no conflicts of interest to declare.

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study being a review.