key: cord-0787585-e0djh8x8
authors: Zavyalova, Elena; Ambartsumyan, Oganes; Zhdanov, Gleb; Gribanyov, Dmitry; Gushchin, Vladimir; Tkachuk, Artem; Rudakova, Elena; Nikiforova, Maria; Kuznetsova, Nadezhda; Popova, Liubov; Verdiev, Bakhtiyar; Alatyrev, Artem; Burtseva, Elena; Ignatieva, Anna; Iliukhina, Anna; Dolzhikova, Inna; Arutyunyan, Alexander; Gambaryan, Alexandra; Kukushkin, Vladimir
title: SERS-Based Aptasensor for Rapid Quantitative Detection of SARS-CoV-2
date: 2021-05-25
journal: Nanomaterials (Basel)
DOI: 10.3390/nano11061394
sha: 7244569ba8b3638002c907acc03fc263f5f11d0b
doc_id: 787585
cord_uid: e0djh8x8

During the COVID-19 pandemic, the development of sensitive and rapid techniques for detection of viruses have become vital. Surface-enhanced Raman scattering (SERS) is an appropriate tool for new techniques due to its high sensitivity. SERS materials modified with short-structured oligonucleotides (DNA aptamers) provide specificity for SERS biosensors. Existing SERS-based aptasensors for rapid virus detection are either inapplicable for quantitative determination or have sophisticated and expensive construction and implementation. In this paper, we provide a SERS-aptasensor based on colloidal solutions which combines rapidity and specificity in quantitative determination of SARS-CoV-2 virus, discriminating it from the other respiratory viruses.

In late 2019 in Wuhan (Hubei, China), a new strain of coronavirus SARS-CoV-2 was first detected, which caused a pandemic of acute respiratory disease, named coronavirus disease 2019 . The virus is rapidly spread and has already claimed the lives of more than two million people all over the world. It also made a significant impact on global economy, put pressure on healthcare, and affected social life. The disease is characterized by progressive inflammatory pulmonary cellular infiltration, hypoxemia, and the development of an acute respiratory distress syndrome (ARDS), which could lead to death.

The situation is complicated by the fact that many infected patients have very mild symptoms or are completely asymptomatic during the entire period of infection, but can still transmit the disease to others [1, 2] . Additionally, symptoms of COVID-19 are similar In our previous work, we compared the use of solid-state SERS substrates and colloidal SERS particles to detect influenza A virus. On solid-state SERS substrates, we managed to achieve LOD for influenza A virus of 10 4 VP/mL with less than 15 min for an assay. We applied SERS substrate with immobilized primary DNA aptamer RHA0385 with broad specificity towards to influenza A virus hemagglutinin on silver and secondary labeled aptamer for "sandwich" assembly. This approach allowed to detect whole viral particles with hemagglutination activity. However, the dependence was not monotonous, which allowed only qualitative interpretation of the result [20] .

In our second work of influenza detection, we switched from the solid SERS substrate to the colloidal one with the same "sandwich" approach and aptamers. This allowed us to create a simple and rapid "one-pot" technique with a monotonous dependence but with a modest dynamic range of 2 × 10 5 − 2 × 10 6 VP/mL [21] .

For this work, we used a colloidal SERS-based aptasensor with a new setup including changes in nanoparticle aggregation, aptamer, and target virus, i.e., SARS-CoV-2 virus. The aptamer was RBD-1C, which was shown to have high affinity to receptor binding domain (RBD) of surface S-protein of SARS-CoV-2 [22] .

Preparation of the silver colloids by reducing a silver nitrate solution with hydroxylamine hydrochloride was conducted in concordance with the method [23] . Silver nitrate (AgNO 3 , CAS 7761-88-8), hydroxylamine hydrochloride (NH 2 OH-HCl, CAS 5470-11-1) were of the highest purity available; sodium hydroxide (NaOH, CAS 1310-73-2), sodium chloride (NaCl, CAS 7647- , xanthine (CAS 69-89-6)were analytical-grade and used without further purification. All the products were purchased from Aldrich (Sigma-Aldrich Inc., St. Louis, MO, USA). The stock solutions of reagents and silver sols were prepared with water from a Milli-Q system (18.2 MΩ·cm resistivity at 25 • C).

The AgNPs were prepared at room temperature under vigorous stirring by rapid addition of a small volume of a concentrated silver solution (10 mL) to a large volume of a less-concentrated hydroxylamine hydrochloride/sodium hydroxide solution (90 mL). The concentration of AgNO 3 and NH 2 OH-HCl/NaOH was 10 −3 M and 1.5 × 10 −3 /3 × 10 −3 M, respectively, in the final reaction mixture. It was kept under stirring for 30 min once the addition of silver nitrate was complete. A yellow-brown colored transparent colloidal suspension with no sediment was obtained. The synthesized colloids were stable for several weeks under refrigeration conditions at +6 − +8 • C.

UV-Vis spectra of AgNPs were recorded on a spectrometer Genesis 10S UV-Vis (Thermo-Fisher Scientific, Madison, WI, USA). The morphology of silver nanoparticles was studied by SEM (Scanning Electron Microscopy), performed using a Supra 50VP electron microscope (Zeiss, Germany) with a resolution of 1.5 nm. The mean size and ζ-potential of AgNP were determined by DLS (Dynamic Light Scattering), performed using Zetasizer Nano ZS (Malvern, Worcestershire, UK).

Vero E6 (ATCC CRL-1586) cell line was obtained from the Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products RAS, Russia, and was maintained in complete Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum (FBS, HyClone, Logan, Utah, USA), L-glutamine (4 mM), and penicillin/streptomycin solution (100 IU/mL; 100 µg/mL) (PanEco, Moscow, Russia). SARS-CoV-2 isolate PMVL-5 (GISAID accession EPI_ISL_470899) was isolated in May 2020 from a nasopharyngeal swab specimen taken from a 22-year-old female. The nasopharyngeal swab was inoculated on Vero E6 (non-human primate kidney) cells. The inoculated cells were monitored for cytopathic effects by light microscopy and cytopathic effects were detected at 72 h post inoculation. Virus was passaged three times before the experiments to pile the virus. Viral titer of 4.6 × 10 6 TCID50/mL was determined as Nanomaterials 2021, 11, 1394 4 of 13 TCID50 by endpoint dilution assay. Virus titer was calculated using the Reed and Muench method [24] . All experiments with the live SARS-CoV-2 followed the approved standard operating procedures of the NRCEM biosafety level 3 facility.

Influenza viruses and Newcastle disease virus were provided by the Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of the Russian Academy of Sciences. The following strains were studied: influenza A virus (IvA) A/FPV/Rostock/34 R6p (256 HAU/mL in stock solution); influenza B virus (IvB) B/Victoria/2/1987 (2000 HAU/mL in stock solution); Newcastle disease virus(NDV) (256 HAU/mL in stock solution). Virus stocks were propagated in the allantoic cavity of 10-dayold embryonated specificpathogen-free chicken eggs. Eggs were incubated at 37 • C, cooled at 4 • C 48 h post-infection, and harvested 16 h later. The study design was approved by the Ethics Committee ofthe Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow, Russia (Approval #4 from 2 December 2014). Viruses were inactivated via the addition of 0.05% (v/v) glutaric aldehyde, preserved via the addition of 0.03% (w/v) NaN 3 , and stored at +4 • C.

Adenovirus type 6 (AdV) Strain Tonsil 99 (Bialexa, Russia) and respiratory syncytial virus (RSV) (Bialexa, Russia) were inactivated by treatment with Thimerosal and beta propiolactone. Viral content of RSV was 1 mg/mL (2·10 12 VP/mL); viral content of AdV was 1.9 mg/mL (4·10 12 VP/mL). Viral particle concentrations (VP/mL) were calculated from the protein concentration.

The following oligonucleotides were studied. Biotin-RBD-1C (Biotin-5 -CAGCAC CGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3 from Synthol, Moscow, Russia), Biotin-RBD-1C-sh (Biotin-5 -TTTGGGAGTGCTGGTCCAAGGGCGTTAA TGGACA-3 from Synthol, Moscow, Russia), BDP FL-RBD-1C (Bodipy FL-5 -CAGCACCGA CCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3 from Lumiprobe, Moscow, Russia). To assemble the structure of the aptamers, the following algorithm was used. Biotinylated aptamers were prepared in 2 µM concentrations in the buffer containing 10 mM tris-HCl pH 7.0, 140 mM NaCl, and 10 mMKCl. Bodipy FL labeled aptamer was prepared in 2 µM concentrations in the buffer containing 10 mM PBS. The solutions were heated at 95 • C for 5 min and cooled at room temperature.

A 2 µM aptamer solution was placed in a quartz cuvette with 1 cm path. Circular dichroism (CD) spectra were acquired using CD spectrometer Chirascan (Applied Photophysics, Leatherhead, UK) and a dichrograph MARK-5 (Jobin-Yvon; Montpellier, France) equipped with a thermoelectric temperature regulator. The spectra were acquired in the range of 220-360 nm.

Kinetic constants of association and dissociation of the RBD-S-protein complexes were determined using interferometer Blitz (Forte-Bio, Fremont, CA, USA). Streptavidin biosensors from Forte-Bio (USA) were used. The biosensors were hydrated for 10 min in the buffer. The biotinylated aptamer was loaded on the biosensor from 1 µM solution for 120 s. The binding experiments were performed as following: The experiments on "EVA 2.0" with viruses were performed as following:multilayered glass SiO 2 |Ta 2 O 5 |SiO 2 was silanized with APTES from the one side by hydrolisation. The other side of the glass was treated with oil to the panel of the device with a red laser. A glass cell with hosepipes was installed on top of the glass. A disc pump was connected to the outlet hose. At the beginning of the experiment, bidistilled water was run through the entire system to avoid the bubbles' formation and to elevate of measurement accuracy. The ideal interference pattern obtained could be seen through the video camera set up on the device.

The silanized side of the glass was treated with the following solutions at room temperature:

(1) An amount of 0.1 M NaH 2 PO 4 pH = 6.2 for 2 min.

(2) The activation of -NH 2 groups of APTES by EDC (50 mg/mL) and NHS (50 mg/mL) for 20 min. 

The SERS spectra were acquired using a handheld Raman analyzer RaPort (Enhanced Spectrometry, Inc., San Jose, CA, USA) with a laser wavelength of 532 nm and a working output power of 38 mW. The spectrometer had a spectral resolution of 4-6 cm −1 and a spectral range of 160-4000 cm −1 . The recording time of a single spectrum was 400 ms with 20 averages. The measurements were carried out in glass vials with a volume of 1.5 mL (Akvilon, Moscow, Russia). The focus of the laser beam coincided with the center of the vial.

We took 150; 50; 25; 12.5; 9; 7.5; 6; 4.5; 3; 1.5; 0.75; 0.37; and 0.15 µl of a cell culture medium with SARS-CoV-2, control viruses (RSV, IvA, IvB, NDV, AdV), or a virus-free cell culture medium. The cell culture medium was the same for all viruses for a more objective comparison. The control viruses were prepared in a concentration of 10 8 VP/mL, supposing that the ratio between TCID50/mL and VP/mL is about 1 to 100 as was estimated for fresh influenza viruses by Kramberg et al. [26] . Then, we added 4 µL of 2 µM BDP FL-RBD-1C. For the volumes of 25 µL and less, we added PBS to obtain a total volume of 50 µL before the addition of the labeled aptamer. After 5 min of the incubation, we added 250 µL of PBS and injected the solution to 196 µL of AgNPs (the total volume was 500 µL). SERS spectra was registered at 1 min after the aggregation step.

In this work, we used hydroxylamine-reduced AgNPs [23] as SERS substrate. These silver colloids are stable and highly SERS-active with good reproducibility in the obtained enhancement factors, as well as having the possibility of long-term (about a month) storage without loss of efficiency.

To characterize the morphology of the produced colloids, UV-Vis spectroscopy ( Figure  S1 ) and DLS ( Figure S2 ) were used. The absorption maximum of the measured UV-Vis spectrum of the colloidal solution provides information on the average particle size, whereas its full width at half-maximum (FWHM) can be used to estimate particle dispersion. The absorption maximum was found at 394 nm with a FWHM of approximately 55 nm. The mean size according to the number distribution of DLS is 4.8 nm, whereas the intensity distribution gives the size of 44 nm, being in agreement with the value calculated from spectral characteristics of NP. These results have been confirmed by SEM ( Figure S3 ). The AgNPs are supposed to have no interaction with the aptamer as both have strong negative charge. ζ-potential of NPs is −54 + −18 mV ( Figure S4 ).

Xanthine, purine base, was selected as a test compound to investigate the effectiveness of silver colloids in enhancing SERS spectra. The spectra of the test substance xanthine at different ionic strengths of the solution are shown in Figure S5 .

The structure of the aptamer RBD-1C was supposed by Song et al. [22] ; it consists of two hairpins that are end-to-end stacked ( Figure 1A ). The RNA fold webserver [27] provided the same folding for the sequence of RBD-1C. DNA duplexes are known to have hyperchromic effect during unfolding in UV-spectra at the wavelength of 260 nm [28] . However, no notable changes in UV-spectra were tracked during RBD-1C melting in potassium buffer ( Figure S6 ). Circular dichroism (CD) spectra were recorded to identify the other possible topologies of RBD-1C ( Figure 1B ).

In this work, we used hydroxylamine-reduced AgNPs [23] as SERS substrate. These silver colloids are stable and highly SERS-active with good reproducibility in the obtained enhancement factors, as well as having the possibility of long-term (about a month) storage without loss of efficiency.

To characterize the morphology of the produced colloids, UV-Vis spectroscopy ( Figure S1 ) and DLS ( Figure S2 ) were used. The absorption maximum of the measured UV-Vis spectrum of the colloidal solution provides information on the average particle size, whereas its full width at half-maximum (FWHM) can be used to estimate particle dispersion. The absorption maximum was found at 394 nm with a FWHM of approximately 55 nm. The mean size according to the number distribution of DLS is 4.8 nm,whereas the intensity distribution gives the size of 44 nm, being in agreement with the value calculated from spectral characteristics of NP. These results have been confirmed by SEM ( Figure S3 ). The AgNPs are supposed to have no interaction with the aptamer as both have strong negative charge. ζ-potential of NPs is −54 + −18 mV ( Figure  S4) .

Xanthine, purine base, was selected as a test compound to investigate the effectiveness of silver colloids in enhancing SERS spectra. The spectra of the test substance xanthine at different ionic strengths of the solution are shown in Figure S5 .

The structure of the aptamer RBD-1C was supposed by Song et al. [22] ; it consists of two hairpins that are end-to-end stacked ( Figure 1A) . The RNA fold webserver [27] provided the same folding for the sequence of RBD-1C. DNA duplexes are known to have hyperchromic effect during unfolding in UV-spectra at the wavelength of 260 nm [28] . However, no notable changes in UV-spectra were tracked during RBD-1C melting in potassium buffer ( Figure S6 ). Circular dichroism (CD) spectra were recorded to identify the other possible topologies of RBD-1C ( Figure 1B) . The spectra contained a positive peak at 290 nm, a shoulder at 254 nm, and a slight negative peak at 230 nm. These CD spectra could not be attributed to duplexes [29] ; similar spectra were reported for two-quartet antiparallel G-quadruplex with a diagonal loop ( Figure 1A ) [30] . CD spectra in sodium buffer had a shoulder at 290 nm and major peak at 270-280 nm that correspond to a preferable DNA duplex. Thus, an equilibrium The spectra contained a positive peak at 290 nm, a shoulder at 254 nm, and a slight negative peak at 230 nm. These CD spectra could not be attributed to duplexes [29] ; similar spectra were reported for two-quartet antiparallel G-quadruplex with a diagonal loop ( Figure 1A ) [30] . CD spectra in sodium buffer had a shoulder at 290 nm and major peak at 270-280 nm that correspond to a preferable DNA duplex. Thus, an equilibrium between two conformations was observed. A shortened version of the aptamer was studied to reveal whether G-quadruplex is a recognizing element in this aptamer. Seventeen nucleotides from the 5 -end of the aptamer were excluded (oligonucleotide RBD-1C-sh). This oligonucleotide had CD spectra that are close to RBD-1C. Both oligonucleotides were studied in affinity experiments.

The affinity of RBD-1C to the recombinant protein has been previously estimated by Song et al. [22] . Here, we estimated it once more and compared the affinities of RBD-1C with its truncated variant. The sensorgrams are shown in Figure 2. between two conformations was observed. A shortened version of the aptamer was studied to reveal whether G-quadruplex is a recognizing element in this aptamer. Seventeen nucleotides from the 5′-end of the aptamer were excluded (oligonucleotide RBD-1C-sh). This oligonucleotide had CD spectra that are close to RBD-1C. Both oligonucleotides were studied in affinity experiments.

The affinity of RBD-1C to the recombinant protein has been previously estimated by Song et al. [22] . Here, we estimated it once more and compared the affinities of RBD-1C with its truncated variant. The sensorgrams are shown in Figure 2 . Dissociation constant of RBD-1C was 13±2nM (Table 1) ,which agrees well with the previous value of 5.8 ± 0.8 nM [19] . The truncated variant, RBD-1C-sh, had a dissociation constant of 330 ± 60 nM that corresponds to low specific binding. Thus, the unique spatial structure of RBD-1C is crucial for high affine recognition of the RBD of S-protein. Next, the ability of binding the whole virus was tested for both RBD-1C and RBD-1C-sh. RBD-1C showed excellent curves for both concentrations of the virus; whereas RBD-1C-sh provided low-efficient binding of the viruses (Figure 3 ). It could be concluded that the active aptamer structure is a DNA hairpin of RBD-1C, whereas G-quadruplexes are low-active additives. The content of the virus was estimated in TCID50 that corresponds to virulent particles. Dissociation constant of RBD-1C was 13 ± 2 nM (Table 1) ,which agrees well with the previous value of 5.8 ± 0.8 nM [19] . The truncated variant, RBD-1C-sh, had a dissociation constant of 330 ± 60 nM that corresponds to low specific binding. Table 1 . The parameters of the complexes of RBD of S-protein with aptamer RBD-1C and its shortened version, RBD-1C-sh. Kinetic constants of association (k a ) and dissociation (k d ) as well as equilibrium dissociation constants (K d ) are provided.

Thus, the unique spatial structure of RBD-1C is crucial for high affine recognition of the RBD of S-protein. Next, the ability of binding the whole virus was tested for both RBD-1C and RBD-1C-sh. RBD-1C showed excellent curves for both concentrations of the virus; whereas RBD-1C-sh provided low-efficient binding of the viruses (Figure 3 ). It could be concluded that the active aptamer structure is a DNA hairpin of RBD-1C, whereas G-quadruplexes are low-active additives. The content of the virus was estimated in TCID50 that corresponds to virulent particles. 

For each virus studied, the concentration dependence of the SERS signal was investigated. The cell culture medium was used as a control. In the observed SERS spectra in the experimental and control samples, the intensity of the main Raman peak of the Bodipy FL label 587 cm −1 was studied ( Figure 4A ). As a result, the concentration dependence of the intensity ratio of the SERS line (587 cm −1 ) in the experimental and control samples was constructed ( Figure 4C,D) . 

For each virus studied, the concentration dependence of the SERS signal was investigated. The cell culture medium was used as a control. In the observed SERS spectra in the experimental and control samples, the intensity of the main Raman peak of the Bodipy FL label 587 cm −1 was studied ( Figure 4A ). As a result, the concentration dependence of the intensity ratio of the SERS line (587 cm −1 ) in the experimental and control samples was constructed ( Figure 4C,D) . Figure 4C demonstrates the performance of the system for detecting the inactivated SARS-CoV-2. The specificity was tested for RSV, IvA, IvB, NDV, and AdV. For qualitative comparison, control viruses were reduced to uniform concentrations and concentration curves were constructed for them. Calibration curve for SARS-CoV-2 is ranging from 5.5 × 10 4 to 1.4 × 10 6 TCID50/mL.

Our hypothesis explaining the behavior of the curves for SARS-CoV-2 and for nonspecific viruses ( Figure 4C ) and describing the mechanism of SERS signal generation is as follows:

It is known [31, 32] that at high ionic strength, silver nanoparticles interact well with the surface proteins of the virus, forming aggregates on the virus particles. With an increase in the concentration of proteins, the aggregates of silver nanoparticles create inhomogeneities of the electromagnetic field with a locally high density («hot spots») near their surface. O Due to significant differences in the number of the reporter molecules near nonspecific viral particles and SARS-CoV-2, the SERS signal decreases with increasing concentration of the non-specific virus, and in the case of SARS-CoV-2, it increases. Figure 4C demonstrates the performance of the system for detecting the inactivated SARS-CoV-2. The specificity was tested for RSV, IvA, IvB, NDV, and AdV. For qualitative comparison, control viruses were reduced to uniform concentrations and concentration curves were constructed for them. Calibration curve for SARS-CoV-2 is ranging from 5.5 × 10 4 to 1.4 × 10 6 TCID50/mL.

Our hypothesis explaining the behavior of the curves for SARS-CoV-2 and for non-specific viruses ( Figure 4C ) and describing the mechanism of SERS signal generation is as follows:  It is known [31, 32] that at high ionic strength, silver nanoparticles interact well with the surface proteins of the virus, forming aggregates on the virus particles. With an increase in the concentration of proteins, the aggregates of silver nanoparticles create inhomogeneities of the electromagnetic field with a locally high density («hot spots») near their surface.  Due to significant differences in the number of the reporter molecules near non-specific viral particles and SARS-CoV-2, the SERS signal decreases with in- increases. Figure 5 shows the scheme of the experiment and explains the principle of operation of the SERS-aptasensor. The target virus (SARS-CoV-2 virion, left side) accumulates more labeled aptamers due to the specific interaction mentioned earlier, while control viruses (i.e., influenza A virion, right side) have fewer labeled aptamers for the same enhancement field from AgNPs aggregates. This resultsin an increase of SERS intensity with higher concentrations of SARS-CoV-2 and in a decrease for control viruses. 

SARS-CoV-2 virus has many common symptoms inherent in other respiratory viral infections, therefore, it is often identified only in the late stages of the disease. In this regard, the creation of a simple and cheap method and device for its rapid detection may slow down such a rapid increase in the spread of the pandemic.

There are many different types of SERS substrates [33] [34] [35] [36] and schemes for creating aptasensors based on them [20, [37] [38] [39] [40] . However, to create a quantitative method for the selective detection of SARS-CoV-2 from a group of other respiratory viral infections, this method should be the cheapest, simplest, and most reproducible. The reproducibility of SERS sensors can be achieved only in the case of using either nanoperiodic SERS structures obtained using electron lithography methods [41, 42] (however, in this case, the cost of their production is high and therefore the method cannot compete with ELISA), or colloidal SERS solutions [23, 43] . Colloidal solutions are easy to synthesize, homogeneous, and allow to work on a handheld Raman spectrometer with high laser radiation powers for a long exposure time.

In this paper, we have developed a very simple rapid method for determining SARS-CoV-2, based on the scheme of a direct method for assembling SERS aptasensor using colloidal SERS. The method presented in this article is simple («one-step»), fast (7 min), and has high sensitivity (LOD of 5.5 × 10 4 TCID50/mL) and specificity.

Test specificity is a vital problem in diagnostics and the following treatment. In all existing detection methods, there is a trade-off between sensitivity and specificity. In the ideal situation, both diagnostic sensitivity and diagnostic specificity are 100%. However, the fact is that this situation is rarely reached and intention to increase the diagnostic sensitivity results in a decrease in diagnostic specificity. For tests in which false positives 

SARS-CoV-2 virus has many common symptoms inherent in other respiratory viral infections, therefore, it is often identified only in the late stages of the disease. In this regard, the creation of a simple and cheap method and device for its rapid detection may slow down such a rapid increase in the spread of the pandemic.

There are many different types of SERS substrates [33] [34] [35] [36] and schemes for creating aptasensors based on them [20, [37] [38] [39] [40] . However, to create a quantitative method for the selective detection of SARS-CoV-2 from a group of other respiratory viral infections, this method should be the cheapest, simplest, and most reproducible. The reproducibility of SERS sensors can be achieved only in the case of using either nanoperiodic SERS structures obtained using electron lithography methods [41, 42] (however, in this case, the cost of their production is high and therefore the method cannot compete with ELISA), or colloidal SERS solutions [23, 43] . Colloidal solutions are easy to synthesize, homogeneous, and allow to work on a handheld Raman spectrometer with high laser radiation powers for a long exposure time.

In this paper, we have developed a very simple rapid method for determining SARS-CoV-2, based on the scheme of a direct method for assembling SERS aptasensor using colloidal SERS. The method presented in this article is simple («one-step»), fast (7 min), and has high sensitivity (LOD of 5.5 × 10 4 TCID50/mL) and specificity.

Test specificity is a vital problem in diagnostics and the following treatment. In all existing detection methods, there is a trade-off between sensitivity and specificity. In the ideal situation, both diagnostic sensitivity and diagnostic specificity are 100%. However, the fact is that this situation is rarely reached and intention to increase the diagnostic sensitivity results in a decrease in diagnostic specificity. For tests in which false positives are unacceptable but false negatives may occur, high diagnostic specificity is required. It should be noticed that the more steps, materials, and hard-managed equipment a new method requires, the greater the degree of cross-reactivity in the approach.

Therefore, it is extremely important to develop new methods for selective and rapid detection of other viral respiratory diseases, such as: influenza virus, adenovirus, respiratory syncytial virus, and Newcastle disease virus. The authors of this article are already working on the creation of optical aptasensors for the selective detection of these viruses.

The following are available online at https://www.mdpi.com/article/ 10.3390/nano11061394/s1, Figure S1 : UV-Vis spectroscopy of colloidal silver solutions, Figure S2 : Dynamic Light Scattering (DLS) of colloidal silver solutions, Figure S3 : SEM of AgNPs, Figure S4 : ζ-potential of AgNPs, Figure S5 : Spectra of the test substance (2 mkM xanthine) at different ionic strengths of the solution, Figure S6 : UV-spectra of RBD-1C (during melting in potassium buffer). 

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The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.