*2.1.1 Labeled SERS technology*

Label-free SERS technology can not only quickly screen and diagnose SARS-CoV-2 carriers, but also further analyze SARS-CoV-2 according to the characteristic spectra of the molecules to be tested (nucleic acid, antigen, antibody, or pathogen), including the differentiation of virus subtypes, the classification of virus variants, and the identification of virus infectiousness. In SARS-CoV-2 detection, the poor reproducibility of SERS spectra is giant challenge, which is mainly due to the different adsorption sites of biomacromolecules on the surface of SERS-active substrate.

Shanghai Institute of Ceramics, Chinese Academy of Sciences has carried out systematic research in the field of accurate capture and detection of SARS-CoV-2 by label-free SERS technology and achieved a series of pioneering research results (**Figure 1a**) [18, 19]. The SERS-based biosensor designed by the team has ACE2 functionalized gold nano "forest" structure, which can selectively capture sars-cov-2, and its detection sensitivity has reached the level of single virus. The SERS-based biosensor designed by the team has ACE2 functionalized Au "virus traps" nanostructure, which can selectively capture SARS-CoV-2, and its detection sensitivity has reached the level of single virus. Due to the special "virus traps" nanostructure and high affinity of ACE2 for SARS-CoV-2 S protein, the ability of the SERS-based biosensor to enrich virus in water has been improved 106 times. Due to the multi-effect SERS enhancement mechanism produced by the specially designed Au nanostructure, the Raman signals of the SERS biosensor are enhanced by 109 times. With the help of machine learning, the identification methods of Raman signals of SARS-CoV-2 are established. The LOD of SARS-CoV-2 is 80 copies/mL, which takes only 5 min. This is of great significance for the pointof-care test (POCT) of SARS-CoV-2. A two-step SERS detection method based on ultrahigh sensitive SnS2 microsphere substrate was creatively proposed, and the SERS signals identification standard of SARS-CoV-2 S protein and RNA was established (**Figure 1b**). This identification standard is used to identify the "life or dead" and infectiousness of SARS-CoV-2 in the environment. It solves the problem of infectious diagnosis of SARS-CoV-2 in the environment that cannot be solved by RT-qPCR technology at present, which is of great significance to avoid misjudgment of the epidemic situation under the current COVID-19 pandemic [20].

Although label-free SERS technology can enhance the Raman signal intensity of the target analyte, the Raman scattering cross section of biomacromolecules is small. Even in the case of enhancement, some vibrational Raman signals of biomacromolecules are still weaker than those of dye molecules. Due to the influence of impurities in different Physiological environment and the different adsorption sites of biomacromolecules on the surface of SERS-active substrate, the Raman band assignments of SARS-CoV-2 have not been systematic. Therefore, the biomarkers of SARS-CoV-2 detected by label-free SERS technology are S protein and intact virus.

#### **Figure 1.**

*a: Schematic diagram of COVID-19 SERS sensor operation procedure. b: Application of SnS2 microspheres for diagnosing the infectiousness of SARS-CoV-2 based on two-step diagnosis method. c: Schematic illustration of the SERS-based immunoassay.*

### *2.1.2 Label-free SERS technology*

Labeled SERS technology can not directly obtain the spectral information of targeted molecules, but this method has good quantitative properties, that is, the Raman intensity of SERS tags has a strong linear relationship with the concentration of the molecules to be measured [21]. Because of its high sensitivity and rapid response, the labeled SERS detection platform has been widely applied to the rapid detection of SARS-CoV-2.

Xiaomin Liu's team of Jilin University [22] used a novel method of oil/water/ oil three-phase liquid-liquid interfaces self-assembly to prepare a double-layer Au nanoparticles films (**Figure 1c**). After the surface of Au nanoparticles films is modified with SARS-CoV-2 antibody, it can be used as an SERS-immune substrate to detect SARS-CoV-2 antigen. The team also designed a labeled SERS-immune detection platform, which is a "sandwich" structure composed of SERS-immune substrate, reported molecules, and Ag nanoparticles modified with antibody (SERS tags). This SERSbased immune platform will not be disturbed by impurities in physiological environment. The LOD of SARS-CoV-2 S protein in untreated saliva can reach 6.07 fg/mL, and the detection platform has excellent specificity and reproducibility. The labeled SERS technology mainly relies on the Raman signals of the reported molecules in the SERS tags to indirectly detect SARS-CoV-2, so the selection of the reported molecules and the construction of the SERS-active substrate are very important. Noble metals with strong electromagnetic enhancement such as Au/Ag nanoparticles/nanostructure are usually used as the SERS-active substrate, and reported molecules containing -SH/-NH2 functional groups such as 4-MBA, 4-ATP, and R6G are selected to form Ag/ Au-S/N bonds with strong binding force through strong electrostatic interaction.

### *2.1.3 Application of nanotechnology in SERS-based biosensors*

At present, most of the SERS-active substrates involved in the detection of SARS-CoV-2 are noble metal substrates, whether labeled or label-free SERS technology. The preparation methods of SERS-active substrate can be roughly divided into two categories: chemical method and physical method. Chemical methods include wet chemical synthesis, liquid-liquid self-assembly, hydrothermal method, etc.; physical methods include ion sputtering, magnetron sputtering, etc. [13, 18, 20, 23–26] Compared with the chemical method, the substrate prepared by the physical method has a more regular array structure, which can build a more uniform "hot spot" structure according to the detection requirements, so as to achieve higher detection sensitivity. In addition, compared with nanomaterials prepared by chemical method, substrates with nanostructures prepared by physical method are easier to be produced on a large scale. Chemical synthesis of SERS substrate has simple steps and low requirements for equipment. In order to inhibit the agglomeration of nanomaterials with high surface energy, various surfactants will be used in the process of preparing SERS-active substrate by chemical method, such as sodium citrate, PEI, PEG, and so on. Surfactants will introduce Raman bands of background, and SERS-active substrates prepared by some physical methods can effectively avoid these bands.

#### **2.2 SPR-based biosensors**

Surface plasmon resonance-based (SPR)-based biosensors realize the detection of target substances through the change of refractive index caused by the interaction between plasma resonance wave and target molecules on the metal surface. It has the advantages of real-time, label-free, high cost-effective, noninvasive nature, good reutilization, and excellent reproducibility [27, 28]. However, the sample volume and power consumption required for SPR-based biosensing detection are still large, and the sensitivity and resolution of SPR-based biosensors or devices still need to be further improved. These shortcomings hinder the application of SPR-based biosensors in biomedical detection [29]. In order to settle these problems, nanomaterials, microfluidic devices, compact and power-free pumps have been tried to be integrated into SPR-based biosensor system.

#### *2.2.1 SPR-based biosensors for SARS-CoV-2 detection*

Researchers from Huazhong University of Science and Technology, Shanghai Public Health Clinical Center affiliated to Fudan University, Liangzhun (Shanghai) Industrial Co. Ltd. [30] have successfully developed a high-sensitivity optical detection system based on a spike protein specific nano-plasmonic resonance sensor, which can quickly and specifically measure the concentration of SARS-CoV-2 virus particles without sample preparation and make it possible to quickly and noninvasively detect the asymptomatic patients of SARS-CoV-2 in the early stage of infection (**Figure 2**). The team has developed an optical nano plasma resonance chip, which has special optical properties caused by the collective oscillation of electron gases in metal metamaterial nanostructures surrounded by dielectric materials. With the help of this SPR-based biosensors, the quantitative analysis of the binding process between protein of SARS-CoV-2 surface and antibody can be completed only with conventional ordinary equipment such as optical microscope or Microplate Reader. The experimental results show that the lowest LOD of the system is 370 vp/ml, which can satisfy the requirements of rapid detection of SARS-CoV-2 in saliva. Taka-aki Yano et al. [31] constructed the "sandwich" structure composed of Au substrate, N protein of SARS-CoV-2, and antibody modified Au nanoparticles, and used SPR technology to detect novel coronavirus with fmol/L detection sensitivity of N protein. The team attributed the excellent sensitivity of the SPR-based biosensors to the coupling of surface plasmon resonance between Au nanoparticles and Au substrate and the use of large particle size Au nanoparticles (about 150 nm). Through experiments and electromagnetic field simulation, they believe that ~150 nm Au nanoparticles are more conducive to the detection of biomacromolecules than Au nanoparticles with tens of nanometers.

#### **Figure 2.**

*a: Schematic diagram of the nanoplasmonic resonance sensor for. determination of SARS-CoV-2 pseudovirus concentration. b: Photograph (Middle) of one piece of Au nanocup array chip with a drop of water on top.*

*Perspective Chapter: Novel Diagnostics Methods for SARS-CoV-2 DOI: http://dx.doi.org/10.5772/intechopen.105912*

#### *2.2.2 Application of nanotechnology for SPR-based biosensors*

The sensing performance of SPR-based biosensor is directly related to the surface plasmon resonance produced by nanomaterials/nanostructures. As a result, the preparation of SPR-based biosensor materials also involves the preparation of a variety of traditional or novel preparation method of nanomaterials and micro/nanofabrication. SPR technology is mainly divided into two categories: transmitted surface plasmon resonance (commonly referred to SPR) and local surface plasmon resonance (LSPR). The kernel of substrate based on transmitted SPR technology is the design of Micro/ Nano Architectures. The main preparation methods include lithography, electron beam lithography method, focused ion beam lithography method, and nanosphere self-assembly. The nanomaterials based on LSPR are related to the material quality, geometry, size, and the distance between nanoparticles [32]. The main methods for preparing LSPRbased substrate are wet chemical method, electrochemical method, and hydrothermal method. Among them, wet chemical method is the most commonly used preparation method. In order to overcome the limitation of low sensitivity of SPR-based biosensors, researchers designed composite SPR-based biosensors combined the transmitted SPR technology and LSPR technology to enable them to couple and excite each other, which greatly improved the sensing performance of the SPR-based biosensors.

#### **2.3 Electrochemical biosensors**

Electrochemical biosensors are a quantitative or semiquantitative sensing technology with high sensitivity and specificity, which works by potentiometric, amperometric, conductometric, polarographic, capacitive, or piezoelectric ways [33]. The effective physical transduce in electrochemical biosensors depends on the working electrode, and the sensitive layer is the interface between the electrode and the analyzed environment [34]. The sensing element of electrochemical biosensor must be a conductor, and the target molecules can be specifically identified and adsorbed after some modification on the conductor surface. Electrochemical biosensors have the characteristics of short detection time, simple device, low cost, and high portability, which has the potential to become a point-of-care detection tool [35, 36].

It is extremely important to select appropriate materials when designing electrochemical biosensors. When an electrochemical reaction occurs, the material must be inert at the current potential. At present, the solid electrodes employed commonly are mainly metals, such as gold, silver, nickel, copper, and so on. Metal electrodes with metal nanoparticles or nanostructure have high specific surface area and are easy to modify and label, which can improve the sensitivity, specificity, and accuracy of electrochemical biosensors. For electrochemical biosensors, suitable surface modification can shorten the detection time. Electrochemical biosensors are mainly divided into four types, including voltammetric/amperometric biosensors, impedance biosensors, potential biosensors, and field effect transistor (FET)-based biosensors.

#### *2.3.1 Voltammetric/amperometric biosensors*

Voltammetric/amperometric biosensors have the advantage of high sensitivity, which makes them the most commonly used electrochemical biosensors [37]. So far, voltammetric/amperometric biosensors have developed a variety of methods, comprising cyclic voltammetry, linear sweep voltammetry, square wave voltammetry, and differential pulse voltammetry [38]. Both voltammetric and amperometric biosensors detect the target molecules through the current generated by electrolysis caused by electrochemical oxidation and reduction on the working electrode. When the potential is applied to the indicator electrode versus the reference electrode, the signals are determined by the mass transfer rate of reactant molecules from the solution to the electrode interface. The potential applied by the working electrode increases progressively at a constant rate in voltammetric biosensors, while the potential is applied at a constant rate in potential biosensors.

Voltammetric biosensor is one of the most commonly used electrochemical biosensors, which is widely employed for the rapid detection of SARS-CoV-2. Fabiani et al. [39] facilitate the detection of S protein and N protein of SARS-CoV-2 by electrochemical biosensors based on magnetic beads and carbon-black-based screen printed electrodes (**Figure 3a**). The electrochemical biosensor can detect the target substance in untreated saliva through the change of voltammetry curve within 30 min with LOD of S protein and N protein being 19 and 8 ng/mL, respectively. Compared with the detection results of RT-qPCR, the detection results of the electrochemical biosensor showed no difference, and the detection time is faster.

#### *2.3.2 Impedimetric biosensors*

Impedimetric biosensors are another commonly used biosensors with high sensitivity and low amplitude, which realize the analysis of targets by electrochemical impedance spectroscopy (EIS) [40]. Electrochemical impedance is the ratio of the increased voltage change to the resulting current change, and the electrochemical impedance spectroscopy can determine the resistive and capacitive components of circuit through a frequency-changed small-amplitude sinusoidal AC excitation signal. When the frequency is quite high, the redox species will be blocked by the target analyte when migrating to the electrode surface and produce a rate limiting, resulting in a frequency-dependent phase lag between the AC voltage and current. Electrochemical impedance spectroscopy can be based on Faradaic and non-Faradaic modes. EIS of Faradaic mode involves charge transfer between electrodes and the adding of redox couples, while EIS of non-Faradaic mode does not need to add additional reagents.

#### **Figure 3.**

*a: The magnetic-beads-based electrochemical assay for SARS-CoV-2 detection in untreated saliva. b: Principle of the proposed electrochemical biosensor for sensitive analysis of SARS-CoV-2 RNA.*

The change of capacitive behavior is generated by charge separation at the electrodeelectrolyte interface in EIS of non-Faradaic mode.

Impedimetric biosensors have also made outstanding contributions to the detection of SARS-CoV-2 antigens, antibodies, and nucleic acids. Peng et al. [41] proposed a high-sensitivity electrochemical biosensor based on impedance and voltammetry to detect the RNA of SARS-CoV-2 with the LOD of 26 fmol/L for RNA (**Figure 3b**). Target RNA will trigger the catalytic hairpin assembly circuit and cause DNA polymerization mediated by terminal deoxynucleotidyl transferase, resulting in the production of a massive of single-stranded DNA. These negatively charged single-stranded DNAs will combine with a large number of positively charged electrochemically active molecules due to electrostatic adsorption, which would amplify the electrochemical signal. The research team based on the proposed electrochemical sensor to detect clinical samples of SARS-CoV-2, which showed a high degree of stability.

#### *2.3.3 Potentiometric biosensors*

The potentiometric biosensors utilize two reference electrodes (mainly ion-selective electrode (ISEs)) to measure the charge accumulation on an electrode [42, 43]. For biological detection, potentiometric biosensors usually use enzymes to catalyze chemical reactions and generate ions near the sensing ISE. Potentiometric biosensor has the advantages of small size, fast response, easy to use, low cost, strong antiinterference ability, independent of sample volume, and has the potential to become an SARS-CoV-2 point-of-care detection tool.

After SARS-CoV-2 invades the human body, it will not only cause various antibodies in the blood, but also have a certain impact on some metabolism and enzymatic reactions. Studies have reported that the level of cholinesterase will decrease in the acute stage of severe SARS-CoV-2 infection. Pershina et al. [44] constructed a carbon-fiber-based potentiometric biosensor using polyelectrolyte multilayers to detect the ion concentration in the human biofluid of patients with SARS-CoV-2. Polyethyleneimine/polystyrene sulfonate complex has hygroscopicity and can retain ion clusters of inorganic salts, which allows the adhesion of hydrophobic ion selective membrane and produces Nernst response in miniature sensor system. This biosensor based on ion selective electrode can detect the changes of Na + or K+ concentration in urine or blood of COVID-19 patients, and then evaluate the course of the disease. The potential biosensor has not been applied to detect the antigen, antibody, and pathogen of SARS-CoV-2 temporarily. However, this method can assess the infection status of patients by detecting the ion balance or enzymatic reaction in patients, then analyze the disease course of patients, treatment methods, and recommend the dosage of drugs.

### *2.3.4 FET-based biosensors*

The biosensor based on field effect transistor (FET) also belongs to a kind of electrochemical biosensor, which is employed to detect the conductivity change in the electric field caused by the accumulation of charged target substances on the biosensor surface [45, 46]. FET-based biosensors have the advantages of label-free, miniaturization, easy-to-batch production, strong universality, and low cost, which are an ideal candidate for the point-of-care test of SARS-CoV-2.

Li et al. [47] constructed a graphene-based field effect transistor modified by Au nanoparticles and then modified the complementary phosphorodiamidate

morpholino oligos (PMO) probe on the surface of Au nanoparticles (**Figure 4**). The FET-based biosensor can perform SARS-CoV-2 RdRp high-sensitivity test within 2 min and process low background signal caused by PMO without charge, and the LOD in throat swab is 2.29 fmol/L. In addition, the biosensor is also employed to test 30 real clinical samples, and the detection results are highly consistent with RT-qPCR. The research team further used the constructed biosensor to successfully distinguish SARS-CoV-2 RdRp and SARS-CoV RdRp. Dacheng Wei's team of Fudan University [48] constructed a electromechanical system assembling the nucleic acid fragment of SARS-CoV-2 and the graphene-based field effect transistor, which can detect the SARS-CoV-2 within 4 min.

Electrochemical biosensors have the characteristics of fast response, simple operation, low cost, and miniaturization of detection equipment, but there are still some challenges in commercialization. First of all, most of the electrochemical biosensors for detecting SARS-CoV-2 are in the laboratory stage, and some of the electrochemical biosensors have poor stability when in the external environment. Secondly, how to ensure that the structure of target molecules such as S protein or N protein will not be polluted and damaged on the unclean electrochemical detection platform, as well as the reliability of the detection results. In addition, the clinical samples are complex. The biological environment of virus/protein/nucleic acid is complicated, and the components of preservation solution will be different. Whether the sensitivity of electrochemical sensor will be affected and whether the stability of the results can be guaranteed is a problem. Of course, how to ensure that the detection personnel are not infected by the targeted virus is also a key problem for all kinds of novel biosensors.
