# Surface-enhanced Raman scattering of CoV-SARS-2 viral proteins in a strong coupling regime

K Mochalov, P Samokhvalov, G Nifontova, T Tsoi, A Sukhanova, I Nabiev

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K Mochalov, P Samokhvalov, G Nifontova, T Tsoi, A Sukhanova, et al.. Surface-enhanced Raman scattering of CoV-SARS-2 viral proteins in a strong coupling regime. Journal of Physics: Conference Series, 2021, 4th International Symposium and School for young scientists on "Physics, Engineering and Technologies for Bio-Medicine" (PhysBioSymp 2019) 26-30 October 2019, Moscow, Russia, 2058, pp.012020. 10.1088/1742-6596/2058/1/012020. [https://hal-03500981](https://hal.science/hal-03500981)

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# Surface-enhanced Raman scattering of CoV-SARS-2 viral proteins in a strong coupling regime

To cite this article: K Mochalov et al 2021 *J. Phys.: Conf. Ser.* **2058** 012020

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# Surface-enhanced Raman scattering of CoV-SARS-2 viral proteins in a strong coupling regime

K Mochalov<fnref n="1" />,https://orcid.org/0000-0001-8157-9613, P Samokhvalov<fnref n="1" />,https://orcid.org/0000-0002-2878-8376,

G Nifontova<fnref n="1" />,https://orcid.org/0000-0002-6956-5654, T Tsoi<fnref n="1" />,https://orcid.org/0000-0002-5201-4725, A

Sukhanova<fnref n="2" />,https://orcid.org/0000-0003-2796-7898, and I Nabiev<fnref n="1" />,<fnref n="2" />,https://orcid.org/0000-0002-8391-040X

**Abstract.** Fast, sensitive, high-throughput detection of coronavirus antigens at physiologically relevant levels is essential for population screening that could prevent epidemics such as the current COVID-19 global pandemic. Optical methods based on surface-enhanced Raman scattering (SERS) spectroscopy are promising for this purpose because they ensure quick detection of even single biological molecules in a sample. For achieving such a high sensitivity, it is crucial to design SERS-active systems concentrating incident radiation into small sample volumes. Here, metal-dielectric cavities have been obtained through interaction of protein sulfhydryl groups with a SERS-active silver surface. The concentration of light in these cavities allows the differential detection of spike glycoprotein and nucleocapsid protein of SARS-COV-2, which are its key antigens, at physiologically relevant concentrations. The cavity Q-factor can be increased by additionally covering the dielectric protein film with a silver shell to form an ultrathin cavity, which provides an at least tenfold enhancement of the detection signal. The results could be used to design high-throughput systems for specific and sensitive detection of viral antigens and quick diagnosis of viral infections.

## 1. Introduction

This century has already witnessed three coronavirus epidemics causing deadly pneumonia in humans: severe acute respiratory syndrome (caused by SARS-CoV) [1], Middle-East respiratory syndrome (MERS-CoV) [2], and COVID-19 (SARS-CoV-2) [3]. The entry of these viruses into host cells is determined by the transmembrane spike glycoprotein (S-glycoprotein) forming homotrimers that protrude from the virus surface [4]. S-glycoprotein consists of the distal S1 and membrane-anchored S2 subunits responsible, respectively, for binding to cell surface receptors and for fusion of the viral and cellular membranes (Figure 1). The S1 subunit includes the receptor-binding domain (RBD), which stabilizes the prefusion state of the fusion machinery contained in the S2 subunit [5]. The location and function of S-glycoprotein makes it the main target of neutralizing antibodies (Abs) and the focus of therapy and vaccination. N-linked glycans extensively covering the S-glycoprotein trimers serve for the proper folding of the glycoprotein and are targets for the modulation of the glycoprotein accessibility to host proteases and neutralizing Abs [6,7]. The effect of S230 anti-SARS-CoV Abs is determined by functionally mimicking the attachment to the receptor and promoting conformational rearrangements of S-glycoprotein determining the characteristic pattern of the activation of the coronavirus membrane fusion [6]. Deeper insight into the specific characteristics of the interaction between S-glycoprotein and

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Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.

<footnote n="1">¹National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Laboratory of Nano-Bioengineering, 115409 Moscow, Russia</footnote>

<footnote n="2">²Université de Reims Champagne-Ardenne, Laboratoire de Recherche en Nanosciences, LRN-EA4682, 51100 Reims, France</footnote>

<footnote>E-mail: igor.nabiev@univ-reims.fr</footnote>
its cell surface receptor could make it possible to develop better vaccines ensuring the protection against all the three pathogens, SARS-CoV-2, SARS-CoV, and SARSr-CoV. It should be noted that coronaviruses are assumed to use conformational masking of epitopes and glycan shielding, thereby limiting their recognition by the host's immune system [6,8].

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Overall structure of SARS-CoV-2 receptor-binding protein (RBD). The SARS-CoV-2 RBD core is shown in cyan and receptor-binding motif (RBM) part of RBD-protein, interacting with the ACE-receptor, in shown in red. Disulfide bonds in the SARS-CoV-2 RBD are shown as sticks and indicated by arrows. Figure is adapted from Lan et al. Nature 581, 215–220 (2020), doi: 10.1038/s41586-020-2180-5.</figcaption></figure>

Another problem is that serological assays detecting S-glycoprotein trimers are not sensitive enough to differentiate between SARS-CoV-2 and other SARSr-CoVs; hence, more specific methods should be developed. The available immune and molecular assays [9,10], including ELISA [11] and PCR [12], are sufficiently sensitive for precise identification of each coronavirus, but they require prior knowledge of the specific strain. The methods of deep sequencing [13], which are capable of detecting mutations in a viral genome and providing data on the genetic diversity of a virus population are highly efficient for virus surveillance provided that the sequence coverage is sufficient. However, low virus titres in most samples result in that sequence reads are typically dominated by the host's nucleotide sequences and contain little viral material. This entails time-consuming processing of clinical samples, the use of expensive reagents, and the necessity of technical expertise. A highly sensitive and specific optical method for the detection of SARS-CoV-2, as well as other coronaviruses, would both accelerate the detection and reduce its cost.

The sensitivity of surface-enhanced Raman scattering (SERS) spectroscopy, a method of vibrational spectroscopy, is sufficiently high for detecting even single biomolecules [14-17]. Specifically, it has been demonstrated that SERS signals from viral protein antigens can be detected at antigen concentrations close to those in single virions [18]. This makes it possible to solve the task of detecting coronaviruses within a few minutes. The results of recent research and technological developments offer the opportunity of engineering a novel system for highly sensitive and specific quick detection of viruses on the basis of spectroscopic identification of the characteristic conformational patterns and glycosylation profiles of their proteins.

## 2. Results

In this study, we have obtained the first known Raman and SERS spectra of the key SARS-CoV-2 antigens, its nucleocapsid and S-glycoprotein. We have developed an approach to fabricating metal-
dielectric cavities (Figure 2) by making use of the interaction of the viral protein sulfhydryl groups and a silver surface in order to obtain the SERS spectra of viral protein antigens at concentrations sufficiently low for their detection at physiologically relevant concentrations (lower than 1 pM).

We have also demonstrated that the SERS signal can be increased by an order of magnitude by coating the dielectric protein film with a silver film a few nanometres wide. This forms an ultra-narrow nano-cleft and increases the cavity Q-factor. Thus, the SERS-based approach using metal-dielectric and metal-metal cavities developed here allows the detection of the key SARS-CoV-2 antigens at concentrations tenfold lower than those at which the virus is detectable in biological media. This offers the opportunity to develop a high-throughput optical method for the detection and study of SARS-CoV-2.

<figure><img src="image_5.png" /><figcaption>**Figure 2.** The SARS-CoV-2 receptor-binding protein (RBD) located in the proximity from the Ag-surface of the SERS-active substrate. The distances between the surface of the SERS-active substrate and the Trp amino acid residues of RBD protein which are located in an interior of the protein globule are shown.</figcaption></figure>

## Acknowledgements

The work on the preparation of the dielectric-metallic resonators was supported by the Russian Science Foundation, grant no. 21-79-30048. The work on the CoV-SARS-2 viral proteins preparation, handling and analysis was supported by the Russian Foundation for Basic Research, grant no. 20-04-60440.

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