Perspective Chapter: Novel Diagnostics Methods for SARS-CoV-2

Abstract

A novel coronavirus of zoonotic origin (SARS-CoV-2) has recently been recognized in patients with acute respiratory disease. COVID-19 causative agent is structurally and genetically similar to SARS and bat SARS-like coronaviruses. The drastic increase in the number of coronavirus and its genome sequence has given us an unprecedented opportunity to perform bioinformatics and genomics analysis on this class of viruses. Clinical tests such as PCR and ELISA for rapid detection of this virus are urgently needed for early identification of infected patients. However, these techniques are expensive and not readily available for point-of-care (POC) applications. Currently, lack of any rapid, available, and reliable POC detection method gives rise to the progression of COVID-19 as a horrible global problem. To solve the negative features of clinical investigation, we provide a brief introduction of the various novel diagnostics methods including SERS, SPR, electrochemical, magnetic detection of SARS-CoV-2. All sensing and biosensing methods based on nanotechnology developed for the determination of various classes of coronaviruses are useful to recognize the newly immerged coronavirus, i.e., SARS-CoV-2. Also, the introduction of sensing and biosensing methods sheds light on the way of designing a proper screening system.

1. Introduction

In the twenty-first century, there were three outbreaks caused by massive coronavirus infection, namely Severe Acute Respiratory Syndromes (SARS) in 2003, Middle East Respiratory Syndrome (MERS) in 2012, and Corona Virus Disease 2019 (COVID-19) in 2019. In particular, the outbreak of COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has aroused great concern about this major public health emergency [1, 2]. SARS-CoV-2 is highly infectious and has a high mortality rate. As of April 8, 2022, 490 million people have been infected, 6.17 million people have died, and 231 countries and regions have reported cases of infectiousness (data source: WHO). With the recent outbreak of COVID-19 in multiple waves and countries caused by SARS-CoV-2 variants, the prevention and control tend to be normalized. At present, WHO has defined five “variant of concern” (VOC) of SARS-CoV-2, including alpha (b.1.1.7), beta (b.1.351), gamma (P.1), delta (b.1.617.2), and omicron (b.1.1.529). With Omicron BA.2 subtype appearing particularly, the basic infectiousness coefficient (R0) of BA.2 is 9.1, which is greatly enhanced compared with the wild type (R0 close to 3.0) according to Sutter health’s calculation.

SARS-CoV-2 is mainly transmitted through respiratory droplets and close contacts. It is possible to transmit through aerosol when exposed to high concentration aerosol in a relatively closed environment. Since SARS-CoV-2 can be isolated from feces and urine, it should also be careful that it may cause contact transmission or aerosol transmission to the environment [3]. In order to suppress the spread of SARS-CoV-2 as soon as possible, it is necessary to develop rapid and accurate virus detection technology. The spread of the epidemic can be effectively controlled by screening infected persons and monitoring the pollution of SARS-CoV-2 in the environment to cut off the source and route of transmission timely [4].

In view of the pandemic of COVID-19, new requirements are put forward for the detection technology of SARS-CoV-2. Among the conventional diagnostic methods, enzyme-linked immunosorbent assay (ELISA), real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-qPCR), and loop-mediated isothermal amplification (LAMP) are very important for the discovery of human coronavirus. However, these methods also have limitations. For example, RT-qPCR requires skilled personnel and certain laboratory conditions and can be time-consuming. In addition, the preparation of ELISA reagents requires specific, high-affinity antibodies or expensive recombinant antibodies. In order to solve these challenges, the community of scholars and industrial circles have developed a variety of novel diagnostics methods for SARS-CoV-2 based on the research progress of nanomaterials, nano-sensing technology, and biotechnology.

2. Novel diagnostics methods for SARS-CoV-2

Nano-biosensing technology is the integration of nanotechnology and biosensing technology. The principle of nano-biosensing technology is similar to that of biosensor technology. Taking the substance to be tested as the identification element, the biological reaction is transformed into identifiable physical or chemical signals through sensitive elements (receptors) and transformation elements (transducers). On the one hand, using the unique optical, electrical, magnetic, and surface activity of nanomaterials is conducive to the construction of high-specificity and high-sensitivity biosensors. On the other hand, the size and shape of nanomaterials are easier to adjust, which is more conducive to the load and modification of targets.

2.1 SERS-based biosensors

Raman spectra could characterize the vibration of molecular chemical bonds. However, the Raman signals are weaker because of low Raman scattering cross section [5]. Therefore, surface-enhanced Raman spectroscopy (SERS) was introduced to solve the disadvantage of weak signal inherent in Raman spectroscopy technology. SERS refers to the phenomenon that Raman signals are enhanced on the surface of some rough nanomaterials. The enhancement mechanism of SERS mainly includes electromagnetic enhancement and chemical enhancement, which are caused by localized surface plasmon resonance (LSPR) of SERS-active substrate and photo-induced charge transfer (PICT) between SERS-active substrate and probe molecules, respectively [6, 7]. SERS-based sensors have high sensitivity, and the detection ability of some SERS-based sensors can even reach the level of single molecule. SERS-based sensing technology mainly depends on the performance of nano SERS-active substrate, so the properties of nanomaterials, such as high surface energy, agglomeration and dispersion, surface plasmon resonance, and the preparation technology of nanomaterials will have influence on the activity of SERS substrate [8, 9]. In medical detection, SERS-based sensing technology has been widely applied to cancer detection, virus detection, biological imaging, and other medical fields [10, 11, 12]. Limited by the inherent non-specificity of SERS substrate itself, SERS-based biosensors need to be modified by biomolecules such as proteins, antibodies, and aptamers on the surface of the SERS-active substrate to specifically capture targets to be detected [13, 14]. Because of its fast, low-cost, high sensitivity, and accuracy, SERS-based biosensors have been employed for the rapid detection of SARS-CoV-2 and the diagnosis of virus infectiousness.

SERS technology can be divided into two categories: labeled SERS technology and label-free SERS technology. Labeled SERS technology refers to label reporter molecules with high Raman scattering cross section on the SERS-active substrate. Through the reasonable design of SERS tags and SERS detection system, the Raman signals of the reported molecules are proportional to the concentration of the targeted substance, so as to obtain the concentration information of the targets. The labeled SERS technology is applicable to the molecular vibration of the target molecules, which does not have Raman activity or has weak Raman activity. Therefore, the molecules to be tested can be detected indirectly by labeling the reporter molecules with strong Raman activity. However, because the Raman signals of the molecules to be tested are not directly collected, the molecular structure cannot be analyzed. So the advantage of Raman as fingerprint spectrum is lost. Moreover, the design of SERS tags and detection system in labeled SERS technology is complex, and the stability of substrate performance is difficult to ensure.

Label-free SERS technology is to directly collect the Raman spectra of targets. It can analyze the molecular structure of the substance to be measured by analyzing the corresponding Raman vibration spectra. Especially for the identification of different viruses and variants, we can not only distinguish different viruses from the perspective of spectral vibration, but also further analyze and verify the mutation characteristics of virus nucleic acid and protein. However, for the label-free SERS detection of biological macromolecules, the weak spectral signal intensity and the poor spectral reproducibility caused by different adsorption directions of biomolecules are the major challenges [15, 16, 17].

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 point-of-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].

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.

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.

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 SERS-based 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, 24, 25, 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.

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 LSPR-based 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.

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.

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. The change of capacitive behavior is generated by charge separation at the electrode-electrolyte 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 anti-interference 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.

Figure 4.

Schematic diagram of rapid direct identification of SARS-CoV-2 using the PMO-functionalized G-FET nano-sensors.

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.

2.3.5 Application of nanotechnology in electrochemical biosensors

In electrochemical biosensors, nanomaterials are mainly applied for modifying the sensitive interface of the biosensors or immobilizing biomolecules. Because of its large specific surface area and high surface energy, nanomaterials can become an active electron acceptor or electron donor. The electrode modified by nanomaterials can significantly improve the specific surface area of the electrode, improve the conductivity of the electrode, load more biomolecules, improve the sensitivity and stability of the biosensors, and speed up the response of the biosensor [49].

Nanomaterials commonly used in electrochemical biosensors include noble metal nanomaterials, carbon nanotubes, graphene, magnetic oxide nanoparticles, and so on. Noble metal nanoparticles represented by gold nanoparticles not only have large specific surface area and high surface energy, but also have high catalytic efficiency, strong adsorption, and excellent biocompatibility, which can effectively load and label target biomolecules. In addition, gold nanoparticles have excellent electrochemical activity and can effectively improve the electron transmission efficiency. Au nanoparticles can also form strong covalent binding through Au-S bond and Au-N bond, which is conducive to the coordination of biomolecules containing -SH and -NH2. Carbon-based materials such as carbon nanotubes and graphene have large specific surface area and strong electron conduction ability. The modification of this kind of carbon-based nanomaterials can greatly improve the electrochemical activity of the electrode, improve the detection sensitivity, increase the current signal, and improve the response time of the electrochemical biosensors [50]. Magnetic nanoparticles can be modified on the surface of the electrode to improve the specific surface area of the electrode, which can also be applied for immobilizing biomolecules to improve the selectivity and specificity of electrochemical detection and avoid the interference of other impurities in the biological environment to the target molecules.

2.4 Magnetic biosensors

Magnetic biosensors have attracted extensive attention of researchers in the past two decades. The biosensors can be divided into surface-based and volume-based magnetic biosensors, which are widely used in the detection of viruses, pathogens, and cancer biomarkers [51, 52, 53]. In magnetic biosensors, magnetic nanoparticles modified by suitable antibodies or DNA/RNA probes are usually used as magnetic nanotags, which can skillfully convert the concentration of analytes into magnetic signals [54]. Compared with optical, plasma, and electrochemical biosensors, magnetic biosensors have lower background noise. Because the biological environment of most biomolecules is non-magnetic, the magnetic biosensors will not be disturbed by the biological environment, so as to produce more accurate and reliable detection results [55]. Magnetic biosensors can be roughly divided into three categories: magnetoresistive (MR) biosensors, magnetic particle spectroscopy (MPS) platforms, and nuclear magnetic resonance (NMR) platforms.

2.4.1 Magnetoresistive biosensor

Magnetoresistive biosensor is a surface-based sensing technology, which is very sensitive to the stray field from generated by magnetic nanoparticles close to the sensor surface, so as to convert the binding of magnetic nanoparticles with analytes into readable electrical signals [56, 57]. Magnetic nanotags in magnetoresistive biosensors need to produce high magnetic moment without losing paramagnetic properties.

At present, there are few studies on magnetoresistive biosensors for the detection of SARS-CoV-2, and we infer main reason is that magnetic nanoparticles would inevitably reduce the magnetic moment when their size decreases. Jinhong Guo’s team at the University of Electronic Science and technology of China [58] constructed an LFIA detection platform based on superparamagnetic nanoparticles and giant magnetoresistive sensing system to detect the immunoglobulin IgM and IgG of SARS-CoV-2 at the same time. Among them, the giant magnetoresistive sensing platform can transmit medical data to smart phones through Bluetooth, which is convenient for medical personnel to obtain patient information. Superparamagnetic nanoparticles with an average size of 68 nm were synthesized by a simple and rapid coprecipitation method with excellent dispersion and magnetic properties. This sensing technique has the advantages of low cost, rapidity, easy operation, and high sensitivity, which can simultaneously detect two antibodies of SARS-CoV-2 within 10 min with the LOD of 10 ng/mL for IgM and the LOD of 5 ng/mL for IgG.

2.4.2 Nuclear magnetic resonance (NMR) platform

The nuclear magnetic resonance (NMR) platform uses magnetic nanoparticles as contrast enhancer, which causes the nonuniformity of local magnetic field and disturbs the precession frequency variations of surrounding water protons [59]. Therefore, the development of high-sensitivity NMR platform essentially depends on the application of appropriate magnetic nanoparticles with high transverse relaxation.

Siwei Yang’s team of Shanghai Institute of Microsystems and Information Technology, Chinese Academy of Sciences [60] reported a rapid and highly sensitive detection of SARS-CoV-2 pathogens based on ultralow-field NMR relaxometry (Figure 5a). This method utilizes magnetic graphene quantum dots modified by SARS-CoV-2 antibody as a probe to construct a magnetic relaxation switch to specifically detect novel coronavirus. It is worth noting that closed-tube one-step strategy is safer for experimenters without samples preparation. This one-step detection method has the characteristics of excellent sensitivity and rapid detection with 248 particles/mL within 2 min.

Figure 5.

a: The detection process of SARS-CoV-2 of the magnetic relaxation switches assay with ULF NMR. b: The schematic diagram of the detection of SARS-CoV-2 RNA based on magnetic particle spectroscopy biosensors.

NMR spectroscopy, like infrared spectroscopy and Raman spectroscopy, can analyze the structure of the molecules to be measured. Different from infrared spectroscopy and Raman spectroscopy, which can directly reflect the molecular structure information, NMR spectroscopy obtains the skeleton structure of the molecules to be tested by analyzing the ¹H, ¹³C, and ¹⁵N NMR spectra. Nuclear magnetic resonance spectroscopy is also widely applied to the screening of antibodies of SARS-CoV-2 and the characterization of protein and nucleic acid structures. Magnetic nanoparticles can be used as a signal amplification tags in this technology to improve the sensitivity of detection. Schoenle et al. [61] reported the sequence-specific backbone assignment of the SARS-CoV-2 RBD and proved that biomolecular NMR spectroscopy chemical shift perturbation (CSP) mapping can quickly and successfully identify the molecular epitopes of RBD-specific antibodies. CSP mapping combined with other detection technology of biomolecules could help us accurately recognize the interaction between RBD and antibody, which is of great significance for antibody screening and further vaccine development.

2.4.3 Magnetic particle spectroscopy platform

Magnetic particle spectroscopy platform is a volume-based detection technology, which directly detects the dynamic magnetic responses of magnetic nanoparticles [62, 63]. Therefore, for this kind of biosensor, the properties of excitation magnetic field, saturation magnetization, and anisotropy should be considered. Rosch et al. [64] reported a novel SARS-CoV-2 nucleic acid detection platform based on magnetic response changes of magnetic nanoparticles (Figure 5b). The specific modified magnetic nanoparticles and target molecules mediated assembling will lead to the increase of hydrodynamic radius, which can be measured by the magnetic particles spectrum in alternating magnetic field. This sensing technology has high sensitivity for the detection of SARS-CoV-2 RNA, with LOD of 0.28 nmol/L, and the biological environment such as saliva will not affect the performance of the detection platform.

Compared with optical biosensors, magnetic biosensors have simpler sample processing steps in the detection process, and magnetic tags are safer than electrochemical biosensors. Because of its high sensitivity, accuracy, and specificity, magnetic sensing technology is expected to be employed for on-site detection tools to restrain the spread of SARS-CoV-2.

2.4.4 Application of nanotechnology in magnetic biosensors

The unique magnetic relaxation properties and good biocompatibility of magnetic nanomaterials give many biosensors high sensitivity and selectivity. In the process of designing magnetic biosensors, which rely on magnetic nanoparticles tags, the size of magnetic nanotags is required to match that of analytes [65]. However, with the decrease of the size of magnetic nanoparticles, these magnetic nanotags often have low magnetic moment and uneven particle size distribution. In addition, the serious surface defects on the surface of nanoparticles and the unavoidable magnetocaloric effect will cause the fluctuation of magnetic signal in the detection of low-concentration analytes. Therefore, the development of magnetic biosensors should focus on how to prepare magnetic nanoparticles with uniform size and good dispersion and the point-of-care detection of magnetic biosensors.

3. Conclusions

Up to now, SARS-CoV-2 has continued to diffuse and spread all over the world, and the epidemic of COVID-19 is facing the dilemma of globalization and time sustainability. Especially in view of the continuous variants of SARS-CoV-2 with increasing transmission speed, concealment, and the proportion of immune escape, even new variants still pose a serious and death threat to people with low immunity and the elderly. Therefore, it is necessary to further improve the conventional detection methods and break through the limitations of the original detection methods and develop new methods as a supplement or substitute for future monitoring and detection tools. Especially in some developing countries with a shortage of medical resources, it is particularly important to develop rapid, simple, high-throughput, and intelligent detection methods.

The nanotechnology attached to the novel nano-biosensing technology has developed relatively mature, but the application of biomacromolecules detection still needs to be further improved, such as the biological toxicity of nanomaterials, the modification of biomolecules on the surface of nanomaterials, the large-scale manufacturing of nanomaterials, and so on. For the novel diagnostics methods, the following aspects need to be considered:

1. High-sensitivity detection. At present, the accuracy of various PCR diagnostic kits at home and abroad is acceptable, and the limitation of detection can reach 200 ~ 500 copies/ml. However, false negatives may still occur in the detection of COVID-19 patients with low viral load. In view of the screening and future monitoring needs of the normalized management of SARS-CoV-2, it is still very necessary to develop a highly sensitive detection method that is less affected by the sampling method and sample quality in order to detect the infected person as soon as possible.

2. High-throughput detection. The simultaneous detection of a large number of samples can alleviate the detection pressure caused by the large-scale outbreak of the epidemic.

3. Multi-pathogen detection. Strengthen the ability to identify and detect a variety of pathogens, viruses, and variants. The differential diagnosis of multiple pathogens under normal management can save manual labor, material resources, and time.

4. Detection of environmental virus and identification of virus infectiousness. For the cured cases, virus detection in the environment, air, and freight develops intelligent detection technology that can distinguish the death and life of the virus, which can accurately identify the virus and avoid unnecessary false alarm and huge social cost consumption. At this stage, a new rapid and highly sensitive pathogen detection method that can distinguish the activity of SARS-CoV-2 is developed to fill the gap of current detection methods.

5. On-site detection. For different application scene such as hospitals, customs, communities, and even families, develop portable, economical, and miniaturized instruments to realize on-site rapid detection, which will greatly help to improve the timeliness of monitoring.

6. Automation and integrated detection of multiple technologies. Single detection technology always has its disadvantages. The novel diagnostic methods integrate sample pretreatment, nucleic acid extraction, amplification, and detection to truly realize “sample in-result out.” By integrating the new diagnostic technology with the existing nucleic acid, antibody, and pathogen detection methods and complementing their advantages, the combined use of them is expected to achieve the accurate and rapid detection of SARS-CoV-2. This method not only provides a powerful means for the current outbreak of COVID-19 and the detection of unknown pathogens in the future, but also has important practical significance for the future application in the fields of respiratory disease differential diagnosis, environmental monitoring, food safety assessment, etc.

7. Intelligent detection. Standardize the high-throughput screening results, instrument interpretation results, and analysis results for large population to avoid the lag and subjectivity of manual interpretation. The interpretation results are not only comparable, but also can be output in time for on-site or remote research and judgment.

Conflict of interest

The authors declare no conflict of interest.