**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 photoinduced 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 [10–12]. 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 [15–17].
