TWI819345B - Method for detecting viruses - Google Patents

Abstract

The method of the present disclosure comprises the following steps: providing a SERS (Surface-Enhanced Raman Scattering)-active substrate and a Raman spectra virus database; applying a virus sample onto the SERS-active substrate; applying an incident light by a Raman spectrometer onto the SERS-active substrate to generate a Raman spectrum of the virus sample; and comparing the Raman spectrum of the virus sample with a Raman spectra virus database to identify the species of the virus sample. Herein, the SERS-active substrate comprises: a support; a dielectric layer disposed on the support, wherein a plurality of cavities are formed on a surface of the dielectric layer; and a plurality of noble metal clusters formed in the plurality of cavities.

Description

Virus detection methods

The present disclosure relates to a virus detection method. More specifically, the present disclosure relates to a virus detection method using Surface-Enhanced Raman Scattering (SERS) technology.

Rapid and accurate detection and identification of dangerous pathogens (pathogens) are extremely helpful for the diagnosis or treatment of early-stage diseases caused by pathogens. Currently, antibody-based bioarrays can be used to monitor viruses, and this technology is such as enzyme-linked immunosorbent assays (ELISA), fluorescent antibody arrays, and serological testing. ). However, these techniques may encounter some clinical problems, such as time consumption, false positives, etc.

In recent years, with the continued development of polymerase chain reaction (PCR), related methods such as single-nucleotide polymorphism (SNP), in which the target DNA sequence is also proliferated, Become an alternative method of virus identification. However, complex purification and isolation steps are required for the target DNA before ELISA or PCR. Therefore, if these complex purification and isolation steps can be eliminated, the identification of the target virus will be simplified and accelerated.

Currently, the main difficulties in virus identification or detection are complex purification and separation, the detectable amount, apparent size and size of the virus, and the requirement for a chemical identification database of specific viruses. related. Generally, it is difficult to achieve rapid screening in the early stages of viral infection because in biomolecule detection using purification procedures, the amount of virus usually cannot reach a detectable amount, that is, exceed 10 ⁶ plaque-forming units (PFU). )/ml. The reagents used in common biomolecule tests are much smaller than the apparent size and size of viruses. Therefore, only a portion of the complete virus can be detected, resulting in incomplete virus information. Furthermore, it is difficult to establish complete chemical identification information for viruses in databases.

Biomolecules or their fragments can be studied using resonating mechanical cantilevers, evanescent wave biosensors, or atomic force microscopy, but these methods can only be used for measurement. number of biomolecules. Alternatively, labeling methods can be used to detect microorganisms by specific functional groups. However, it is difficult to prepare contamination-free samples, or the signal of the labeled functional group easily overlaps with the signal of the target microorganism. Therefore, these technologies consume a lot of time in detection and cannot meet the clinical requirements for rapid detection.

The purpose of this disclosure is to provide a virus detection method using SERS technology.

The method of the present disclosure is accomplished by using a SERS-active substrate, wherein the SERS-active substrate includes: a support; and a dielectric layer disposed on the support, wherein: Cavities are formed on a surface of the dielectric layer; and a plurality of noble metal clusters are formed in the cavities. The disclosed method includes the following steps: Steps: provide the above-mentioned SERS active substrate and a Raman spectrum virus database; apply a virus sample to the cavities of the SERS active substrate; apply an incident light to the cavity through a Raman spectrometer on the noble metal clusters on the SERS active substrate to generate a Raman spectrum of the virus sample; and compare the Raman spectrum of the virus sample with a Raman spectrum virus database to determine the type of the virus sample.

In the method of the present disclosure, the SERS-active substrate includes noble metal clusters formed in the cavities. Specifically, noble metal clusters are formed in each cavity. When the SERS-active substrate of the present disclosure is used with a Raman spectrometer to detect virus samples, the overall hot spots in each cavity are the result of the collection of small hot spots (from the noble metal clusters) generated by the noble metal clusters. . Therefore, the intensity of the Raman signal of a virus sample can be enhanced by hot spots, and viruses housed in cavities with hot spots can be detected.

In the present disclosure, the material of the support member is not particularly limited and may include, for example, quartz, glass, silicon wafer, sapphire, polycarbonate (PC), polyimide (PI), polypropylene (PP), polyparaphenylene Ethylene diformate (PET) or other plastic or polymer materials or combinations thereof, but the present disclosure is not limited thereto.

In the present disclosure, the material of the dielectric layer may be ceramic material. For example, the material of the dielectric layer may be a high-k ceramic material with a dielectric constant (k) ranging from 3.9 to 30. Specific examples of high-k ceramic materials include zirconium oxide (ZrO ₂ ), titanium dioxide (TiO ₂ ), hafnium dioxide (HfO ₂ ), aluminum oxide (Al ₂ O ₃ ), or combinations thereof, but the present disclosure is not limited thereto.

In the present disclosure, cavities formed on a surface of the dielectric layer may be arranged in an array. For example, the cavities can be arranged in an n x m array, where n and m are each an integer of 1 or greater.

In the present disclosure, each of the plurality of cavities may have a bowl-like shape.

In the present disclosure, each of the plurality of cavities may have a depth ranging from 20nm to 300nm, such as 20nm to 280nm, 20nm to 250nm, 20nm to 230nm, 20nm to 200nm, 20nm to 180nm, 20nm to 150nm, 25nm to 150nm. , 25nm to 130nm, 30nm to 130nm, 30nm to 100nm, or 40nm to 100nm. Here, the depth of the cavity can be selected depending on the size and dimensions of the target virus to be detected. Furthermore, the depth of the cavity may be the maximum depth of the cavity.

In the present disclosure, each of the plurality of cavities may have a width in the range of 100nm to 1400nm, such as 100nm to 1300nm, 100nm to 1200nm, 100nm to 1100nm, 100nm to 1000nm, 100nm to 900nm, 100nm to 800nm, 100nm to 700nm , 100nm to 600nm, 100nm to 500nm, 150nm to 500nm, 150nm to 400nm, 200nm to 400nm, or 200nm to 300nm. Here, the width of each cavity can be selected according to the size and dimensions of the target virus to be detected. Furthermore, the width of the cavity may be the maximum width of the cavity.

In the present disclosure, the metals in the plurality of precious metal clusters may be gold, silver or alloys thereof. In one embodiment of the present disclosure, the metal may be gold.

In the present disclosure, each of the plurality of noble metal clusters has a disk-like shape with a thickness of several nanometers. The dish-like shape may be flat. The outline of the dish-like shape is not particularly limited, and may be circular or irregular, for example. In addition, each of the plurality of noble metal clusters may have a width in the range of 0.5 nm to 50 nm, such as 0.5 nm to 45 nm, 0.5 nm to 40 nm, 0.5 nm to 35 nm, 0.5 nm to 30 nm, 1 nm to 30 nm, 1 nm to 25 nm, 2 nm to 25nm, 2nm to 20nm, 3nm to 20nm, 3nm to 15nm, 4nm to 15nm, 4nm to 10nm, or 5nm to 10nm. Here, the width of the noble metal cluster refers to the average width of the disc-like shape of the noble metal cluster, which can be selected according to the size and dimensions of the target virus to be detected. In addition, the average thickness of the disc-like shape of the noble metal clusters can be 0.5 nm to 5 nm. Scope. In addition, a spacing between two adjacent metal clusters may be in the range of 5 nm to 10 nm, and the spacing may refer to an average minimum distance between two adjacent metal clusters.

In the present disclosure, each cavity can accommodate 0-3 viruses depending on the target virus to be detected (eg, the size of the virus particles). For example when a virus sample is applied to a SERS-active substrate, one cavity can hold 1, 2 or 3 viruses, but the other cavity is empty (i.e. 0 viruses). How much virus is contained in the cavity may depend, for example, on the amount of virus in the virus sample.

In the present disclosure, a distance between two adjacent cavities in the plurality of cavities may be in the range of 10 nm to 200 nm, such as 10 nm to 190 nm, 10 nm to 180 nm, 10 nm to 170 nm, 10 nm to 160 nm, 10 nm to 150nm, 10nm to 140nm, 10nm to 130nm, 10nm to 120nm, 10nm to 110nm, 10nm to 100nm, 15nm to 100nm, 15nm to 90nm, 20nm to 90nm, 20nm to 80nm, 25nm to 80nm, 25nm to 70nm , or 30nm to 70nm ; But this disclosure is not limited to this. Here, the distance between two adjacent cavities may be the minimum distance between two adjacent cavities.

In the present disclosure, the wavelength of the incident light provided by the Raman spectrometer can be adjusted according to the size of the cavity (eg, depth), or the virus to be detected (eg, the size and type of virus particles). Therefore, an optimized signal of the SERS effect can be obtained. In addition, the incident optical power provided by the Raman spectrometer can be in the range of 0.3mW to 40mW, such as 0.3mW to 30mW, 0.3mW to 20mW, 0.5mW to 20mW, 0.5mW to 15mW, 1mW to 15mW, 1mW to 10mW, 1.5mW to 10mW, 1.5mW to 5mW, 1.5mW to 4.5mW, 2mW to 4.5mW, 2.5mW to 4.5mW, 2.5mW to 4mW, or 3mW to 4mW. If the power is too high, the virus sample may be degraded.

In this disclosure, the types of viruses to be detected are not particularly limited. For example, the virus may be a virus with a spike protein, such as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) virus or a variation thereof.

Conventional detection methods are known, such as nucleic acid detection (such as polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR)) or serological detection (such as enzyme immunosorbent assay (ELISA) or lateral flow immunoassay) (lateral flow immunoassay, LFIA)) is time-consuming or insufficient. However, once a virus becomes a pandemic, rapid and convincing detection systems or methods are needed to instantly identify the type of virus in patients so that the spread of the disease can be effectively controlled. Here, the method of the present disclosure adopts SERS technology, so it can quickly and accurately identify the type of virus. In addition, since the SERS effect can be enhanced by the noble metal clusters in the cavities of the SERS-active substrate of the present disclosure, the type of virus can still be identified even if the number of viruses in the virus sample is very small.

Other novel features disclosed will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

11:Support

12: Nanoparticles

13: Dielectric layer

131:Cavity

132:Surface

14:Precious metal clusters

RA:region

RB:Region

D: Depth

W1: Width

W2: distance

W3: Width

W4: spacing

1A to 1D show cross-sectional views of preparing a SERS-active substrate according to Embodiment 1 of the present disclosure.

2 shows a perspective view of the support and dielectric layer of the SERS-active substrate according to Embodiment 1 of the present disclosure.

FIG. 3 is an enlarged view of the cavity of the SERS-active substrate according to Embodiment 1 of the present disclosure.

4A to 4D are SEM top views of the SERS-active substrate according to Embodiment 1 of the present disclosure.

Figure 5A shows the SERS spectra of the SARS-CoV-2 S pseudovirus (pseudovirus) obtained five times at the same position on the array.

Figure 5B shows the intensity map of the SERS spectra obtained five times by the SARS-CoV-2 S pseudovirus at the same position on the array.

Figure 6A shows the SERS spectra of SARS-CoV-2 S pseudovirus obtained at 6 different locations on the array.

Figure 6B shows the intensity plot of the SARS-CoV-2 S pseudovirus SERS spectra taken at 6 different locations on the array.

Figure 7 shows the SERS spectrum of SARS-CoV-2 S pseudovirus versus VSV-G pseudovirus.

Figure 8 shows the SERS spectrum of SARS-CoV-2 S pseudovirus against H1N1.

Figure 9 shows the SERS spectrum of SARS-CoV-2 S pseudovirus against H3N2.

Figure 10 is a top view of a SERS-active substrate according to Embodiment 2 of the present disclosure.

Different embodiments of the present invention are provided in the following description. These embodiments are intended to illustrate the technical content of the present invention but are not intended to limit the scope of the present invention. A feature described in one embodiment may be applied to other embodiments through appropriate modification, replacement, combination or separation.

It should be noted that in this specification, unless otherwise stated, when a component is described as having an element, it means that the component may have one or more of the element, but does not mean that the component only has one of the element.

In addition, in this specification, unless otherwise stated, ordinal numbers such as "first" or "second" are used to distinguish multiple components with the same name, and they do not necessarily represent the existence of a hierarchy, order, or execution. Execution order, or the order in which components are manufactured. The "first" component and the "second" component can exist together in the same component, or they can exist in different components. The presence of an element described with a higher ordinal number does not imply the inherent presence of another element described with a lower ordinal number.

In addition, in this specification, terms such as "top", "bottom", "left", "right", "front", "rear", or "middle", as well as "upper", "above", "lower" The terms "under", or "between" are used to describe the relative position between multiple elements, and the described relative position may be interpreted to include their translation, rotation or reflection.

Furthermore, in this specification, when an element is described as being disposed on another element, it does not mean that the element is in contact with the other element, unless stated otherwise. This explanation applies to other situations similar to the "on" situation.

In addition, in this specification, terms such as "preferably" or "advantageously" are used to describe optional or additional elements or features. In other words, this element or feature is not an essential element and in some cases Examples can be ignored.

Additionally, in this specification, when an element is described as being "fitted" or "adapted to" another element, the other element is an example or reference that is helpful in imagining the characteristics or applications of the element, and the other element is not shall be considered to form part of the protected subject matter; similarly, unless otherwise stated, in this specification, when an element is described as "fitting" or "adapting" to a configuration or action, the element shall be deemed to form part of the protected subject matter. The description is for the characteristics or application of the component and does not mean that the configuration has been set or the effect has been performed.

In addition, in this specification, unless otherwise stated, a value (value) may be interpreted as encompassing a range within ±10% of the value, and in particular a range within ±5% of the value; unless otherwise stated, the range ( range) can be interpreted as consisting of multiple subranges defined by smaller endpoints, smaller quartiles, medians, larger quartiles, and larger endpoints.

Example 1 - Preparation of SERS active substrate

1A to 1D show cross-sectional views of the SERS-active substrate prepared according to this embodiment. Figure 2 shows a perspective view of the support and dielectric layer of the SERS-active substrate according to this embodiment. Figure 3 is an enlarged view of the cavity of the SERS-active substrate according to this embodiment.

As shown in FIG. 1A , a support member 11 is provided, wherein the support member 11 is a silicon chip support member. After cleaning the support 11 , a plurality of nanoparticles 12 are formed on the support 11 . In this embodiment, the nanoparticles 12 are polystyrene (PS) nanoparticles, but the disclosure is not limited thereto. In another embodiment of the present disclosure, the nanoparticles 12 may be polyvinyl alcohol (PVA) nanoparticles, polyethylene glycol (PEG) nanoparticles, or other polymer nanoparticles. Furthermore, in this embodiment, three different SERS-active substrates are prepared using nanoparticles 12 of three different diameters (150 nm, 250 nm and 350 nm).

After slight annealing at 50° C. for 10 minutes, the precursor solution of ZrO ₂ was applied to the support 11 by a spin coating step. A precursor solution of _ZrO2 was prepared from a mixture of zirconium tetrachloride ( _ZrCl4 , 98%, Acros Organics, Pittsburgh, PA, USA) and isopropyl alcohol (99.8%, Panreac Applichem, Darmstadt, Germany) and spin-coated Before, the precursor solution of _ZrO2 is allowed to stand for about 24 hours until it reaches gel-like consistency. Next, the support 11 was heated (heating rate: 5°C/min) and maintained at 600°C for 3 hours to perform an annealing step. As shown in Figure 1B, after cooling to room temperature, a dielectric layer 13 formed of _ZrO2 is obtained.

/sec直到值達到50

，接著將沉積速度改成1

/sec，直到儀器參數達到3nm。獲得的類碟狀簇的寬度為 30-60nm，扁平厚度(flatten thickness)為數個奈米(nm)，且相鄰簇之間的間距為5-10nm的範圍內。 As shown in FIG. 1C and FIG. 2 , after the nanoparticles 12 are removed, a cavity 131 having a shape corresponding to the nanoparticles 12 is formed. Next, as shown in FIG. 1D , an electron beam evaporator (VT1-10CE, ULVAC) is used to deposit Au atoms on the dielectric layer 13 and form clusters, where the pressure of the chamber is about It is 7×10 ^-6 Torr. The deposition rate is 0.1

/sec until value reaches 50

, then change the deposition rate to 1

/sec until instrument parameters reach 3nm. The width of the obtained disc-like clusters is 30-60 nm, the flatten thickness is several nanometers (nm), and the spacing between adjacent clusters is in the range of 5-10 nm.

After the above steps, the SERS-active substrate of this embodiment is completed. As shown in Figure 1D, the SERS-active substrate includes: a support 11; a dielectric layer 13 disposed on the support 11, wherein a plurality of cavities 131 are formed on a surface 132 of the dielectric layer 13; and A plurality of noble metal clusters 14 are formed in the cavity 131 . Here, the dielectric layer 13 is a _ZrO2 layer, and the noble metal clusters are Au clusters.

As shown in FIG. 1C and FIG. 2 , the cavities 131 are arranged in a hexagonal array, and each cavity 131 has a bowl-like shape. In the SERS-active substrate prepared using nanoparticles 12 with a diameter of 250 nm, the cavity 131 obtained has a depth D (maximum depth) of 60 nm to 80 nm, and a width W1 (maximum width) of 240 nm to 250 nm. In addition, the distance W2 (minimum distance) between two adjacent cavities 131 is 50 nm. The above characteristics can be confirmed through SEM (scanning electron microscope) and AFM (atomic force microscope) images, as well as the cross-sectional profile of the SERS-active substrate, but the present disclosure is not limited thereto.

In addition, as shown in FIG. 3 , the noble metal clusters 14 have a disc-like shape, the width W3 of the noble metal clusters 14 is estimated to be 30-60 nm, and the noble metal clusters 14 have an average spacing distance W4 of 5-10 nm. The above characteristics can be confirmed by AFM images of SERS-active substrates, but the present disclosure is not limited thereto.

The depth D, width W1 and distance W2 of the cavity, the width W3 of the noble metal clusters 14, and the spacing W4 between adjacent noble metal clusters 14 can be adjusted as needed, and the present disclosure is not limited to the above values.

的電流拍攝的。此外，貴金屬簇的寬度和貴金屬簇之間的間距可以通過AFM(Dimension Icon,Bruker,Karlsruhe,Germany)測量。使用AFM的PF-QNM以獲得SERS-活性基板的圖像，其中，掃描速率為0.3Hz，且掃描線為256。然而，本揭露並不局限於此，且可以使用任何已知的方法來分析獲得的SERS-活性基板的形態和組成。 In this example, a high-resolution thermal field emission scanning electron microscope and an energy dispersive X-Ray spectroscopy (FE-SEM/EDS, JSM-7000 , JEOL, Tokyo) and atomic force microscopy (AFM, Dimension Icon, Bruker, Karlsruhe, Germany) can be used to analyze the morphology and composition of the obtained SERS-active substrate. Images of SERS-active substrates were taken in secondary electron imaging mode at an accelerating voltage of 10 kV and 8 × 10 ^-8

Photographed by the current. In addition, the width of the noble metal clusters and the spacing between the noble metal clusters can be measured by AFM (Dimension Icon, Bruker, Karlsruhe, Germany). Images of SERS-active substrates were obtained using PF-QNM with AFM, where the scan rate was 0.3 Hz and the scan lines were 256. However, the present disclosure is not limited thereto, and any known method may be used to analyze the morphology and composition of the obtained SERS-active substrate.

Figures 4A to 4D are SEM top views of the SERS-active substrate of this embodiment. Figures 4A to 4C are respectively SEM top views of the SERS-active substrate prepared with nanoparticles with diameters of 150nm, 250nm and 350nm. And Figure 4D is an enlarged SEM top view of the cavity of the obtained SERS-active substrate. From the results shown in FIG. 4 , it can be found that the width of the noble metal clusters 14 is estimated to be 30-60 nm, and the noble metal clusters 14 have an average separation distance of 5-10 nm.

It should be noted that one Raman spot can cover several cavities. For example, when the diameter of one Raman spot is 1 μm, one Raman spot can cover about 11 cavities of a SERS-active substrate prepared from nanoparticles with a diameter of 250 nm, and each of the cavities covered by the Raman spot The cavities may or may not contain viral particles.

Example 2 - Detection System

The SERS-active substrate of Example 1 can be used with a Raman spectrometer to form a detection system. Here, the Raman spectrometer can provide an incident laser to the noble metal clusters of the SERS-active substrate to obtain a Raman scattering signal, and then output a Raman spectrum.

test case

This test example uses the SERS-active substrate of Example 1 and the detection system of Example 2. The procedure for virus detection includes the following steps: providing a SERS-active substrate and a Raman spectroscopic virus library; applying a virus sample to a plurality of cavities of the SERS-active substrate: passing an incident light through a Raman spectrometer Light is applied to the noble metal clusters of the SERS-active substrate to generate a Raman spectrum of the virus sample; and the Raman spectrum of the virus sample is compared with a Raman spectrum virus database to determine the type of the virus sample.

Preparation of SARS-CoV-2 S pseudovirus

SARS-CoV-2 S pseudovirus was generated based on the report of Huang et al. with slight modifications (Huang, S.W., Tai, C.H., Hsu, Y.M., Cheng, D., Hung, S.J., Chai, K.M., Wang, Y.F. ,Wang,J.R.,2020.Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development.Biomed.J.43,375-387). The lentiviral vector system was provided by the National RNAi Core of Academia Sinica Taiwan. De novo synthesis was performed to obtain the sequence of the spike protein, which was then cloned into the pMD.G plasmid to express the SARS-CoV-2 S pseudovirus. A total of 40 μg of pCMVdeltaR8.91, pLAS3w and pMD.G (VSV-G pseudo-type lentivirus, or VSV-G pseudovirus) or pcDNA3.1-spike Wuhan plasmid (pcDNA3.1-spike) was used. Wuhan plasmids) (SARS-CoV-2 S pseudovirus) transfect cells.

Preparation of H1N1 virus and H3N2 virus

The samples containing H1N1 or H3N2 viruses used in this example were provided by Dr. Wang, Jen-Ren, Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University School of Medicine, and the virus staining was obtained from the Department of Pathology, National Cheng Kung University Hospital. MOCK cells (Martin-Darby canine kidney cells) (Madin-Darby Canine Kidney cells) were cultured in a 75T culture flask. After obtaining a complete monolayer of cells, remove the original culture medium and wash the cells twice with PBS buffer. After removing the PBS buffer, place the virus liquid into 37°C water to thaw quickly. When infecting cells, add thawed cells to a virus culture tube with a single cell layer, and then shake evenly so that the cell surface can be in complete contact with the virus liquid. Afterwards, place the culture in a 35°C incubator for 1 hour. After adding an appropriate amount of culture containing influenza (influenza) virus, the obtained culture was placed in an incubator at 35°C again. When 75% of the cells have a cytopathic effect (CPE), the virus is divided into several parts and stored.

Detection

SERS experiments were performed using a Raman spectrometer (Confocal Micro and Nano Raman Spectrometer, UniDRON, CL Technology Co. Ltd.) with a 633nm laser light source and a maximum laser power of 35mW. Pseudovirus samples were dropped onto a SERS-active substrate and then covered with a glass coverslip before measurement with a Raman laser. In this experiment, 5 spectra were taken at the same point of detection to explore the effect of exposure to the same area on changes in peak intensity, and 6 different locations were tested on the same SERS-active substrate to see the reproducibility of the results and SERS spectra. Additionally, a laser power measurement of 3.5 mW was used.

result

Figure 5A shows the SERS spectra obtained five times by the SARS-CoV-2 S pseudovirus at the same position on the array. Figure 5B shows the intensity diagram of the SERS spectra obtained five times by the SARS-CoV-2 S pseudovirus at the same position on the array. These results point out that in 5 measurements, the same characteristic peaks 830, 1002, 1355 and 1507 cm ^-1 were found at the same Raman shift, but with the use of the same point (cumulative from the 1st to the 5th time) Increase the intensity and it will decrease. This decrease in intensity is due to damage caused by successive applications of the laser to the array and the analyte to the same spot.

Figure 6A shows the SERS spectra of the SARS-CoV-2 S pseudovirus obtained at 6 different positions on the array, and Figure 6B shows the intensity diagram of the SERS spectra of the SARS-CoV-2 S pseudovirus obtained at 6 different positions on the array. These results indicate that the characteristic peaks marked in Figure 6A are strong regardless of their position, but it should be noted that some peaks are not obvious at certain positions. This may be due to non-uniformity of the microstructure in the array.

From the results shown in Figure 5A to Figure 6B, approximately 4 characteristic peaks 830, 1000-1003, 1355, and 1507 cm ^-1 were found at the same Raman shift in the SERS spectrum of SARS-CoV-2 S pseudovirus.

As shown in FIG. 1C , in the SERS-active substrate used here, each cavity 131 has a bowl-like shape, and the obtained cavity 131 has a depth D (maximum depth) of 60 nm to 80 nm and a width of 240 nm to 250 nm. W1 (maximum width). Therefore, one cavity 131 can accommodate up to 3 SARS-CoV-2 S pseudoviruses with a viral particle size of approximately 100 nm. In addition, when the virus particle is located in the cavity 131, the overall hotspot of the cavity 131 depends on the collection of small hotspots generated by each Au cluster. In addition, the dielectric layer 13 of _ZrO2 also contributes to the chemical enhancement effect.

The above results indicate that the SERS-active substrate of the present disclosure can indeed use SERS technology to distinguish live SARS-CoV-2 S pseudoviruses.

Figure 7 shows the SERS spectra of SARS-CoV-2 S pseudovirus versus VSV-G pseudovirus, in which two SERS-active substrates are used to compare the detection of SARS-CoV-2 and VSV-G pseudovirus. Since the composition of VSV-G pseudovirus has been extensively studied, it can be used as a reference for SARS-CoV-2 pseudovirus to identify peaks belonging to SARS-CoV-2 pseudovirus. The figure shows the SERS spectra of two pseudoviruses and the corresponding subtracted spectra. The subtracted spectrum represents the subtraction of the VSV-G pseudovirus spectrum from the SARS-CoV-2 S pseudovirus spectrum; the positive peaks on the subtracted spectrum correspond to the SARS-CoV-2 S pseudovirus peaks, and the negative peaks are the peaks of VSV-G pseudovirus. The results show that the SERS-active substrate of this embodiment can detect two pseudoviruses.

Figure 8 shows the SERS spectra of SARS-CoV-2 S pseudovirus, H1N1 and their mixtures, and Figure 9 shows the SERS spectra of SARS-CoV-2 S pseudovirus, H3N2 and their mixtures. As shown in Figures 8 and 9, when the sample contains multiple viruses (SARS-CoV-2 pseudovirus and H1N1 in Figure 8, SARS-CoV-2 pseudovirus and H3N2 in Figure 9), the SERS of the present disclosure is used -Active substrate can detect multiple viruses simultaneously.

Example 2 - Preparation of SERS-Active Substrate

10 is a top view of a SERS-active substrate according to Embodiment 2 of the present disclosure, particularly the pattern of the dielectric layer of the SERS-active substrate.

The steps for preparing the SERS-active substrate are similar to those shown in Example 1, except that two different arrays are formed on the dielectric layer 13. Therefore, the dielectric layer 13 of this embodiment includes two areas RA and RB. Here, the width of the cavity 131 in the area RA and the width of the cavity 131 in the area RB are different, which can be achieved by forming the dielectric layer 13 with nanoparticles 12 having different diameters. For example, in the step of forming the dielectric layer 13 , the cavity 131 in the region RA may be formed using nanoparticles 12 having a diameter of 250 nm, and the cavity 131 in the region RB may be formed using nanoparticles 12 having a diameter of 350 nm. to form; but this disclosure is not limited to this.

When detecting a virus sample, the intensity of the SERS spectrum can be obtained at multiple locations on the array. When the characteristic peaks of the virus to be detected are found (for example, the SARS-CoV-2 S pseudovirus is 830, 1000-1003, 1355, and 1507 cm ^-1 ), it means that the virus sample contains the target virus particles. In addition, by analyzing the percentage of characteristic peak positions where the virus is found, the relative number of virus particles contained in the virus sample can be known.

Furthermore, when viral samples are loaded into SERS-active substrates, viral particles can be entrapped within the cavities. When using a Raman spectrometer at a specific wavelength (such as 633nm or 785nm) When the light detects virus samples, the precious metal clusters create hot spots. Therefore, the Raman signal of the virus particles can be enhanced.

Although the present disclosure has been described through the embodiments related thereto, it is to be understood that many other possible modifications and changes can be made without departing from the spirit and scope of the present disclosure as protected by the following claims.

11:Support

13: Dielectric layer

131:Cavity

132:Surface

14:Precious metal clusters

Claims (16)

A virus detection method includes the following steps: providing a SERS active substrate and a Raman spectrum virus database, wherein the SERS active substrate includes: a support member; a dielectric layer disposed on the support member, wherein: A cavity is formed on a surface of the dielectric layer, and each of the cavities has a bowl-like shape; and a plurality of noble metal clusters are formed in the cavities; a virus sample is applied to the on the cavities of the SERS active substrate; applying an incident light to the noble metal clusters of the SERS active substrate through a Raman spectrometer to generate a Raman spectrum of the virus sample; and applying the Raman spectrum of the virus sample The Raman spectrum is compared with a Raman spectrum virus database to determine the type of the virus sample; the virus is a virus with a spike protein, and each of the cavities has a width ranging from 200nm to 300nm. . The method of claim 1, wherein the cavities are arranged in an array. The method of claim 1, wherein each of the cavities has a depth ranging from 20 nm to 300 nm. The method of claim 3, wherein each of the cavities has a depth ranging from 40 nm to 100 nm. The method of claim 4, wherein each of the cavities has a depth ranging from 60 nm to 80 nm. The method of claim 1, wherein one metal in the precious metal clusters is gold, silver or alloys thereof. The method of claim 6, wherein the metal in the precious metal clusters is gold. The method of claim 1, wherein each of the precious metal clusters has a disc-like shape. The method of claim 1, wherein each of the noble metal clusters has a width ranging from 0.5 nm to 50 nm. The method of claim 9, wherein each of the noble metal clusters has a width ranging from 5 nm to 10 nm. The method of claim 1, wherein a distance between two adjacent metal clusters in the precious metal clusters is in the range of 5 nm to 10 nm. The method of claim 1, wherein a distance between two adjacent cavities in the cavities is in the range of 10 nm to 200 nm. The method of claim 12, wherein the distance between two adjacent cavities in the cavities is in the range of 30 nm to 70 nm. The method of claim 1, wherein the dielectric layer includes a ceramic material with a dielectric constant ranging from 3.9 to 30. The method of claim 14, wherein the ceramic material is zirconium oxide, hafnium dioxide, alumina or a combination thereof. The method of claim 1, wherein the virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus or a mutation thereof.

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