2.3. Biosensors; nanotechnology based methods

The first biosensor, invented in 1962 by Clark and Lyons to diagnose the glosses/glasser’s disease, is broadly developed, especially in connection with infectious diseases due to its technological advantages. Various types of viral targets and affinity reagents such as an antibody, aptamer, peptide nucleic acid (PNA) and whole-cell are used extensively in biosensors technology. Antigen-antibody based biosensors (Immunosensors), nucleic acid-based biosensors (Genosensors) and whole cell-based biosensors are the most developed biosensors in the detection of viral infection ^{[6], [8], [36]}. Due to the quick and successful isolation of antibodies in connection with broad analytes, the expansion of immunosensors has become more considerable in recent years ^[37]. Owing to the advantages of electrochemical biosensors, such as high sensitivity and specificity, low cost, and simple structure as well as the ability to miniaturize, electrochemical biosensors are more suitable for diagnosing viral infections ^{[38], [39]}. Changes in conductivity of solutions (conductometry), provide measurable current at variable potential (voltammetry), quantifiable potential without drawing noticeable current (potentiometry) and opposition of a circuit to the current flow (impedance) are the most important bio-recognition procedures in electrochemical biosensors ^[40]. Among the various techniques, voltammetric and impedimetric techniques will be most considered due to their high sensitivity and affinity between targets and probes interaction ^[41].

As shown in Fig. 5 , biosensors are capable of detecting a wide range of biomarkers. Therefore, biosensor technology can be developed to diagnose a wide range of diseases, including infectious diseases, cancers, and various of disorders related to the immune system.

Schematic illustration of biosensors and components on a proposed platform. The figure includes the introduction of various analyzers, bio-receptors and transducers used in biosensors technology.

There is no doubt, that the detection limit (LOD) is one of the most important performance characteristics of an analytical procedure. Progress in analytical chemistry might well be measured by the shift of the LOD towards lower values. Of course, the picture emerging would reflect only part of the progress ^{[42], [43]}. Certainly, many problems in analytical chemistry are problems related to detecting and determining elements or compounds in small amounts of sample (micro-analysis), very low concentrations or small amounts in the larger specimen (trace analysis), or even of determining low concentrations in small samples. In the most cases a method is assumed to be very sensitive when the LOD is low, and the LOD and sensitivity in many cases in point are considered synonymous ^[44]. However, in other divisions of science, the sensitivity is defined as the slope of the curve that is acquired when the result of the measurement is plotted against the amount that is to be determined. In analytical chemistry, the sensitivity defined in this way is equal to the slope of the analytical calibration curve, and the definition of the sensitivity will be used in biosensor technology. The lower LOD is exactly understood as the limit below which detection is impossible ^{[43], [44]}.

Innovative biosensors used to detect RNA-viruses include nucleic-acid based, CRISPR-Cas9 based paper strips, optical biosensor, aptamer-based, antigen-Au/Ag nanoparticles-based electrochemical biosensors, and Surface Plasmon Resonance (SPR), Fig. 6 ^{[11], [45]}. Biosensors might be effective bio-device for rapid, accurate, portable, and more promising diagnosis in the existing pandemic that has exaggerated the world humanity and economies ^{[46], [47]}.

Various type of the biosensors for SARS-CoV-2 virus detection ^[47]. A) CRISPR based nucleic acid (RNA) detection ^[48]. B) FET based biosensor operation ^[49], C) AuNPs based FTO electrode biosensor ^[50], D) Surface Plasmon Resonance (SPR) based biosensor, E) 2D gold Nano-islands (AuNIs) functionalized biosensors ^[47].

In order to develop diagnostic coronavirus biosensors, a dual-functional plasmonic biosensor with the plasmonic photothermal (PPT) effect combined with localized surface plasmon resonance (LSPR) sensing transduction was fabricated as a hopeful solution for the clinical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. The two-dimensional gold nanoislands (AuNIs) functionalized with complementary DNA receptors can perform a sensitive detection of the selected sequences from COVID-19 through nucleic acid hybridization. High sensitivity and specificity are unique features of the developed system ^[51]. Nucleocapsid (N) protein in the SARS-CoV is the most important protein and aids as an analytical marker for ultra-sensitive detection of the virus. Localized surface plasmon coupled fluorescence (LSPCF) fiber-optical bio-sensing tools were fabricated for the detection of nucleocapsid protein of SARS-CoV N in human serum as an appropriate clinical approach. The created platform has a simple structure and operation, with a suitable linear range and limit of detection ^[52]. During experimental studies, the systematic evolution of ligand by exponential enrichment (SELEX) technique was performed for detection of N protein of coronaviruses. Aptamer–antibody hybrid based immunoassay was used as a fast and sensitive method in the detection of SARS-CoV N protein ^[53]. Antibody mimetic proteins (AMPs) are useful polypeptides with high affinity and specificity that bind to their target analytes. Compared to routine methods, these types of peptides can be used in very small amounts. AMPs were applied in a nano-biosensor platform to detect N protein of SARS. Employing of this biosensor, N protein was identified at a low concentration in bovine serum albumin(BSA) ^[54]. A novel surface plasmon resonance (SPR) based biosensor was created for sensitive and specified detection of SARS and common respiratory viruses. This system was developed by immobilizing virus-specific oligonucleotides in an SPR chip. To increase the biosensor sensitivity, biotin and streptavidin were used to label the PCR primer and amplify the signal more after hybridization. Accordingly, the created biosensor can sensitively recognize the SARS and some respiratory viruses ^[55]. A simple and effective technique for assembly of SPR based sensing platforms was established for selective and sensitive recognition of SARS coronaviral surface antigen (SCVme) Fig. 7 ^[56].

Illustration of proposed SPR based biosensor for detection of coronaviral surface antigen (SCVme), AFM images of the sequential binding of GBP-E-SCVme and anti-SCVme on the gold-micro-patterned surface. (A) Bare gold surface, (B) binding of the GBP-E-SCVme fusion proteins onto the gold surface, and (C) subsequent binding of the anti-SCVme antibodies on the GBP-E-SCVme layer. Left, representation plans for the successive binding of GBP-E-SCVme and anti-SCVme on the gold micro-patterns; middle, three-dimensional topological images; right, the cross-sectional contours of samples a–c, sequentially (these are average height differences of the individual scan lines from each area) ^[56].

An electrochemical immunosensor was fabricated for sensitive and selective detection of MERS-CoV in single-step. The created system displayed suitable stability and acceptable, good selectivity compared to other non-specific targets such as the flu virus. The high sensitivity of

the immunosensor is attributed to the application of AuNP modified carbon array electrodes, which leads to improved electron transfer efficacy and electrode surface area. Moreover, the use of a disposable electrode decreases the cost of the assay and allows the multiplexed and simultaneous detection of HCoV and MERS-CoV. In summary, the fabricated immunosensor was effectively applied to detect HCoV and MERS-CoV proteins in spiked nasal samples presenting acceptable recovery percentages Fig. 8 ^[57].

Illustration of immunosensor for detection of HCoV and MERS-CoV proteins, (A) COV immunosensor array chip (B), The immunosensor fabrication steps (C) The detection process of the competitive immunosensor for the virus ^[57].

Luciferase-Based Biosensors were developed for the detection of coronavirus Papain-Like (PLpro) and 3-chymotrypsin-like protease (3C-Like Proteases) properly. PLpro and 3CLpro from MERS-CoV are the two most important proteins which can be applied as therapeutic target biomarkers in the diagnostic approach. Accordingly, in the optimum expression of MERSCoV PLpro and 3CLpro, established luciferase-based biosensor will monitor and identify effective small-molecule inhibitors and protease activity for MERSCoV and other future emerging coronaviruses ^[58]. In the last decade, major technological successes have been made in bio-assay, such as the recognition approaches, transduction devices, and signal amplification. Innovative biosensor technology was established for the improvement of COVID-19 diagnosis in clinical labs through the present greater specificity and sensitivity, with user-friendly design in a short time. This platform can similarly be used to improve monitor devices, for instance by being attached to the severe care ventilators, they may help to reveal the spread route of the SARS-covid-2 viruses ^[59]. Ultra-sensitivity, rapid measurements and being usable with a low quantity of analytes, are the advantages of field-effect transistor (FET)-based biosensors. FET-based biosensors are considered useful devices in clinical diagnosis. Graphene-based FET biosensors are capable of sense surrounding alterations on their surface and therefore offering an ideal sensing environment for

minimum noise detection and high sensitivity. From this point of view, graphene-based FET tools

are suitable for applications associated with the sensitive immunological diagnosis. Accordingly, FET based biosensor functionalized with SARS-CoV-2 spike antibody was established for the detection of coronaviruses. The created system shows acceptable LOD and is used detect of the virus antigen-protein in transport medium from nasopharyngeal swabs from clinical samples ^[60]. The proposed bio-sensing platform illustrated in Fig. 9 .

A field-effect transistor (FET)-based biosensors for detection of SARS-CoV-2 spike antibody, Graphene as a sensing material is selected and SARS-CoV-2 spike an antibody is conjugated on the graphene sheet by 1-pyrenebutyric acid n-hydroxysuccinimide ester, which is an interfacing molecular as a probe linker ^[60].

CRISPR–Cas9 based biosensor on a graphene field-effect transistor was created for the detection of unamplified target genes. Similar work can be considered as a modern bio-sensing tool for the detection of viral targets such as covid-19 nucleic acids ^[61].

The most important challenge associated with Covid-19 disease is the rapid transmission, asymptomatic and long-term incubation in carriers. Due to the simple and low-cost structure, easy operation process, high sensitivity, and specificity, biosensor technology could be the best alternative to conventional methods in the detection of covid-19; however, the development of genosensors should be considered more closely due to their high sensitivity and specificity.

The disadvantages and advantages of routine methods with biosensor technology are summarized in Table 3 .

Comparison of routine methods with biosensor technology.

As revealed in several studies rapid detection is the main feature of biosensors technology compared to routine diagnosis methods. In addition to biosensors, other diagnostic methods are being developed for rapid detection of the COVID-19, for example, rapid antigen diagnostic (RAD) test was developed as an immunoassay approach ^[63]. Although rapid diagnosis is a necessary factor, especially in relation to COVID-19, it should be noted that the sensitivity and specificity of a diagnostic method are more important than a rapid diagnosis. For instance, serological methods are the main rapid diagnostic techniques in virology, but unfortunately its false positive and negative results have limited its use ^[64].