A method for identifying new coronavirus strain xbb.1.5 based on surface-enhanced raman spectrum chip

Abstract

The application discloses a new coronavirus strain XBB.1.5 identification method based on a surface-enhanced Raman spectrum chip, and belongs to the technical field of new coronavirus detection. The application identifies the new coronavirus strain XBB.1.5 through the detection method, avoids complex and expensive gene sequencing, can accurately determine the new coronavirus subtype, has low cost, high accuracy, short time and convenient operation.

Description

Technical Field

[0001] This invention relates to a method for identifying the novel coronavirus strain XBB.1.5 based on a surface-enhanced Raman spectroscopy chip, belonging to the field of novel coronavirus detection technology. Background Technology

[0002] Surface-enhanced Raman spectroscopy (SERS) is renowned for its fast response, strong anti-interference capabilities, and high sensitivity. It can enhance the weak Raman spectral signals generated by biological samples adsorbed on the surface of noble metal nanostructures. SERS technology, combined with the narrow characteristic spectra, specificity, and sensitivity of Raman probe molecules, enables the spectroscopic quantification of various pathogens. The applications of SERS technology are wide-ranging, including the detection and analysis of various biological samples.

[0003] Existing technologies can be divided into two categories: labeled detection and unlabeled detection of Raman signals.

[0004] Label-free detection directly amplifies the biological signals of the target (e.g., viral proteins) and determines the composition of the target by analyzing the position and intensity of spectral peaks. This type of technology can intuitively display the unique fingerprint information of a substance. However, due to the complexity of liquid components in practical applications, the vibrational peaks of a large number of lipids, proteins, and various nucleic acid molecules overlap, making it impossible to accurately determine the subtype of SARS-CoV-2 (e.g., XBB.1.5).

[0005] Current inventions involving labeling and detection also involve the specific capture of the target and the design of specially structured Raman signal molecules. However, most existing technologies only design a single specific immune binding reaction and a single Raman signal enhancement process during the capture of target proteins. Simply using nanoparticles as primers to enhance Raman spectral signals has some problems, such as being affected by the "coffee ring effect" and particle aggregation, resulting in uneven signal distribution, which is not conducive to collection and analysis. Summary of the Invention

[0006] This invention provides a method for identifying the COVID-19 strain XBB.1.5 based on a surface-enhanced Raman spectroscopy chip. Specifically, a sandwich structure is constructed with the target antigen as the "core," a silicon wafer with silver nanoparticles bound to antibody A as the "bottom," and an immune probe consisting of multilayer core-shell structured metal nanoparticles bound to antibody C as the "top." The S protein of the COVID-19 strain XBB.1.5 is captured through specific immune coupling of this triple structure. A portable Raman spectrometer is used to collect the SERS spectral signal intensity value at the characteristic peak of 4-MBN in the bound immune probe within 5 seconds. This value shows a good linear relationship with the S protein content of the COVID-19 strain XBB.1.5, thereby achieving the identification of the COVID-19 strain XBB.1.5.

[0007] Preferably, antibody A is R11, and its sequence is as follows: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLRGVAPKLLIYGNNNRPSGVPDRFSGSKSGSSASLAITGLQAEDEADYYCQSYDSSLSGSVFGGGTKLTVLRTGGGGSGGGGSGGGGSQVQLVESGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREIGYSSSGSNYYMDVWGKGTTVTVSS;

[0008] The antibody C is N125, and its sequence is shown below:

[0009] QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNKYVSWYQQFPGTAPKLLIHDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLRTGGGGSGGGGSGGGGSQ VQLVQSGPGLVKPSQTLSLTCTVSGGSINSGSYYWSWIGQPAGKGLEWIGRIYPTGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAPHHMYYYDSSGYWDAFDIWGQGTMVTVSS.

[0010] Preferably, the preparation of the silver nanoparticle-coated silicon wafer bound to antibody A as the immune substrate is specifically as follows:

[0011] Step 1: Preparation of Ag NPs silver nanoparticles:

[0012] Heat 100 mL of AgNO3 (1 mM) solution on a magnetic stirrer until boiling, then quickly add 4 mL of trisodium citrate solution (1% (w / v)), continue stirring for 1 hour, then stop heating and cool to room temperature before use.

[0013] Step 2: Preparation of Si-Ag NPs two-dimensional substrate:

[0014] Take 2 mL of Ag NPs, centrifuge at 8000 rpm for 10 min, remove the supernatant, then disperse it in 2 mL of PVP (1% (w / v)) solution, centrifuge at 8200 rpm for 10 min, remove the supernatant, and then disperse it in 2 mL of ethanol solution for later use; take the treated Ag NPs Mix 200 μL of dichloromethane and 1 mL of ultrapure water in a test tube, shake thoroughly for 40 seconds, and let stand until the bubbles disappear and the liquid completely separates into layers. Then, add 100 μL of n-hexane along the wall of the test tube to the top and gently rotate the test tube to form a dense silver film. Let stand until the silver film shows no change, then remove the upper layer of n-hexane with a pipette. Let stand until the film has a metallic luster, then slightly tilt the test tube and vertically immerse the silicon wafer (4*4 cm2) under the film along the tube wall. Rotate the silicon wafer to a certain angle so that the silver film can completely cover the smooth surface of the cleaned silicon wafer, and then quickly remove the silicon wafer to obtain a two-dimensional SERS substrate with a silver film. Let it air dry naturally and store it in a sealed container at room temperature until use.

[0015] Step 3: Formation of the SERS immune base:

[0016] Immerse the two-dimensional substrate in 20 μL of DSP (5 mM) solution, let it stand at room temperature for 10 h, then wash it with ultrapure water. After the surface is completely dry, add 10 μL of 10 μg / ml antibody A solution to the surface, incubate at 37 °C for 2 h, gently wash off excess antibody with a small amount of ultrapure water, and then dry the surface at 4 °C. Seal and store for later use.

[0017] Preferably, the preparation of the immune probe of the multilayer core-shell structured metal nanoparticles that bind to antibody C specifically involves:

[0018] Step 1: Preparation of Au@4-MBN@Ag NPs:

[0019] ① To prepare core-shell gold nanoparticles (Au NPs), 100 mL of ultrapure water was placed on a magnetic stirrer and heated. First, 1 mL of 1% (w / v) chloroauric acid trihydrate solution was added and stirred until boiling. Then, 1.3 mL of 1% (w / v) trisodium citrate solution was quickly added. The mixture was kept boiling and stirred for 15 min before heating was stopped. After cooling, the solution was clear and wine-red.

[0020] ② Prepare silver-shell-encapsulated gold nanoparticles, with the signal molecule 4-mercaptobenzonitrile encapsulated in the central layer to form Au@4-MBN@Ag NPs; add 10 μL of 4-MBN (1 mM) to 10 mL of Au NPs, incubate at room temperature for 2 h, then centrifuge at 6800 rpm for 10 min. After centrifugation, remove the supernatant, collect the precipitate, and resuspend it in ultrapure water to 10 mL; place the formed Au@4-MBN on a stirrer and add 300 μL of trisodium citrate solution (1% (w / v)), 100 μL of NaOH (0.1 M) solution, 1 mL of AgNO3 (1 mM) solution, and 1 mL of ascorbic acid (10 mM) solution in sequence; stir the reaction for 15 min to obtain the Au@4-MBN@Ag NPs solution, and store it at 4℃;

[0021] Step 2: Bind antibody C onto the metal nanoprobe to form the Au@MBA@Ag@Protein immune probe:

[0022] ① Take 3 mL of Au@4-MBN@Ag, add 2 μL of DSP (5 mM), shake at a constant speed for 3 h, then resuspend in ultrapure water to 0.4 mL, and adjust the pH of the solution to 7.4;

[0023] ② Add 2 μL of antibody C (200 μg / mL) to the above solution, incubate at 37°C for 1.5 h, resuspend in 0.05 mg / mL bovine serum albumin (BSA) to 100 μL, block for 0.5 h, Au@MBA@Ag@Protein immune probe is formed, and store at 4°C.

[0024] Preferably, the synthesis of the sandwich structure specifically involves: incubating the immune substrate in a mixed solution containing 4 μL of the target antigen to be tested and 8 μL of the immune probe for 10 min, washing off the excess solution with ultrapure water, and then using it for SERS detection.

[0025] Preferably, the detection range of the S protein of the SARS-CoV-2 strain XBB.1.5 shows a good linear relationship in the range of 0.01 ng / mL to 1000 ng / mL, with a LOD of 3.3949 pg / mL.

[0026] Beneficial effects:

[0027] 1. This invention employs a dual-stage specific immune binding mechanism. The immune probe and immune substrate are designed as nano-metal ions with different structures, forming a sandwich structure of immune probe-target protein-immune substrate from bottom to top. The signal-amplifying silicon substrate, with Ag NPs, further amplifies the Raman signal molecules inside the metal core-shell nanoparticles of the coupled immune probe. The construction of the bilayer of nano-metal particles enhances the signal by generating strong hotspot signals through surface plasmon resonance. The uniform two-dimensional SERS substrate results in a more uniform SERS spectral signal. The relatively rough surface morphology of the synthesized substrate provides numerous "hot spots," thereby enhancing the SERS spectral signal. This approach captures the target protein more accurately, with higher sensitivity, better specificity, a wider detection linear range, and a lower detection limit.

[0028] 2. This invention enables the construction of an immune sandwich detection structure corresponding to the antigen through different antibody combinations, identifying the SARS-CoV-2S protein and distinguishing subtypes. The detection method of this invention for identifying the SARS-CoV-2 strain XBB.1.5 avoids complex and expensive gene sequencing, accurately determining the SARS-CoV-2 subtype, and is characterized by low cost, high accuracy, short processing time, and convenient operation. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of an integrated detection chip for SARS-CoV-2 SERS.

[0030] Figure 2 This is a diagram illustrating the synthesis process of the sandwich structure.

[0031] Figure 3 Characterization diagram of the synthesized immune probe particles.

[0032] Figure 4 This is a characterization diagram of the synthetic immune base.

[0033] Figure 5 Map for identifying the S protein on a two-dimensional immune sandwich structure of SERS.

[0034] Figure 6 This is a gradient map of the detection of S protein on a two-dimensional immune sandwich structure of SERS.

[0035] Figure 7 The detection process and results are shown for real samples. Detailed Implementation

[0036] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0037] This invention provides a method for identifying the COVID-19 strain XBB.1.5 based on a surface-enhanced Raman spectroscopy chip. Specifically, a sandwich structure is constructed with the target antigen as the "core," a silicon wafer with silver nanoparticles bound to antibody A as the "bottom," and an immune probe consisting of multilayer core-shell structured metal nanoparticles bound to antibody C as the "top." The S protein of the COVID-19 strain XBB.1.5 is captured through specific immune coupling of this triple structure. A portable Raman spectrometer is used to collect the SERS spectral signal intensity value at the characteristic peak of 4-MBN in the bound immune probe within 5 seconds. This value shows a good linear relationship with the S protein content of the COVID-19 strain XBB.1.5, thereby achieving the identification of the COVID-19 strain XBB.1.5.

[0038] Preferably, antibody A is R11, and its sequence is as follows: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLRGVAPKLLIYGNNNRPSGVPDRFSGSKSGSSASLAITGLQAEDEADYYCQSYDSSLSGSVFGGGTKLTVLRTGGGGSGGGGSGGGGSQVQLVESGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREIGYSSSGSNYYMDVWGKGTTVTVSS;

[0039] The antibody C is N125, and its sequence is shown below:

[0040] QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNKYVSWYQQFPGTAPKLLIHDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLRTGGGGSGGGGSGGGGSQ VQLVQSGPGLVKPSQTLSLTCTVSGGSINSGSYYWSWIGQPAGKGLEWIGRIYPTGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAPHHMYYYDSSGYWDAFDIWGQGTMVTVSS.

[0041] Preferably, the preparation of the silver nanoparticle-coated silicon wafer bound to antibody A as the immune substrate is specifically as follows:

[0042] Step 1: Preparation of Ag NPs silver nanoparticles:

[0043] Heat 100 mL of AgNO3 (1 mM) solution on a magnetic stirrer until boiling, then quickly add 4 mL of trisodium citrate solution (1% (w / v)), continue stirring for 1 hour, then stop heating and cool to room temperature before use.

[0044] Step 2: Preparation of Si-Ag NPs two-dimensional substrate:

[0045] Take 2 mL of Ag NPs, centrifuge at 8000 rpm for 10 min, remove the supernatant, then disperse it in 2 mL of PVP (1% (w / v)) solution, centrifuge at 8200 rpm for 10 min, remove the supernatant, and then disperse it in 2 mL of ethanol solution for later use; take the treated Ag NPs Mix 200 μL of dichloromethane and 1 mL of ultrapure water in a test tube, shake thoroughly for 40 seconds, and let stand until the bubbles disappear and the liquid completely separates into layers. Then, add 100 μL of n-hexane along the wall of the test tube to the top and gently rotate the test tube to form a dense silver film. Let stand until the silver film shows no change, then remove the upper layer of n-hexane with a pipette. Let stand until the film has a metallic luster, then slightly tilt the test tube and vertically immerse the silicon wafer (4*4 cm2) under the film along the tube wall. Rotate the silicon wafer to a certain angle so that the silver film can completely cover the smooth surface of the cleaned silicon wafer, and then quickly remove the silicon wafer to obtain a two-dimensional SERS substrate with a silver film. Let it air dry naturally and store it in a sealed container at room temperature until use.

[0046] Step 3: Formation of the SERS immune base:

[0047] Immerse the two-dimensional substrate in 20 μL of DSP (5 mM) solution, let it stand at room temperature for 10 h, then wash it with ultrapure water. After the surface is completely dry, add 10 μL of 10 μg / ml antibody A solution to the surface, incubate at 37 °C for 2 h, gently wash off excess antibody with a small amount of ultrapure water, and then dry the surface at 4 °C. Seal and store for later use.

[0048] Preferably, the preparation of the immune probe of the multilayer core-shell structured metal nanoparticles that bind to antibody C specifically involves:

[0049] Step 1: Preparation of Au@4-MBN@Ag NPs:

[0050] ① To prepare core-shell gold nanoparticles (Au NPs), 100 mL of ultrapure water was placed on a magnetic stirrer and heated. First, 1 mL of 1% (w / v) chloroauric acid trihydrate solution was added and stirred until boiling. Then, 1.3 mL of 1% (w / v) trisodium citrate solution was quickly added. The mixture was kept boiling and stirred for 15 min before heating was stopped. After cooling, the solution was clear and wine-red.

[0051] ② Prepare silver-shell-encapsulated gold nanoparticles, with the signal molecule 4-mercaptobenzonitrile encapsulated in the central layer to form Au@4-MBN@Ag NPs; add 10 μL of 4-MBN (1 mM) to 10 mL of Au NPs, incubate at room temperature for 2 h, then centrifuge at 6800 rpm for 10 min. After centrifugation, remove the supernatant, collect the precipitate, and resuspend it in ultrapure water to 10 mL; place the formed Au@4-MBN on a stirrer and add 300 μL of trisodium citrate solution (1% (w / v)), 100 μL of NaOH (0.1 M) solution, 1 mL of AgNO3 (1 mM) solution, and 1 mL of ascorbic acid (10 mM) solution in sequence; stir the reaction for 15 min to obtain the Au@4-MBN@Ag NPs solution, and store it at 4℃;

[0052] Step 2: Bind antibody C onto the metal nanoprobe to form the Au@MBA@Ag@Protein immune probe:

[0053] ① Take 3 mL of Au@4-MBN@Ag, add 2 μL of DSP (5 mM), shake at a constant speed for 3 h, then resuspend in ultrapure water to 0.4 mL, and adjust the pH of the solution to 7.4;

[0054] ② Add 2 μL of antibody C (200 μg / mL) to the above solution, incubate at 37°C for 1.5 h, resuspend in 0.05 mg / mL bovine serum albumin (BSA) to 100 μL, block for 0.5 h, Au@MBA@Ag@Protein immune probe is formed, and store at 4°C.

[0055] Preferably, the synthesis of the sandwich structure specifically involves: incubating the immune substrate in a mixed solution containing 4 μL of the target antigen to be tested and 8 μL of the immune probe for 10 min, washing off the excess solution with ultrapure water, and then using it for SERS detection.

[0056] Surface scattering Raman spectroscopy (SERS) was used to detect and identify the spike protein (Spikeglycoprotein) of the XBB.1.5 SARS-CoV-2 subtype. The identification process involved constructing six antibody combinations (AB, AC, AD, BC, BD, and CD) on an immune substrate and an immune probe, respectively, based on four antibodies (A, B, C, and D) selected through binding tests. Known ELISA binding test results showed that the XBB.1.5 subtype S protein exhibited good binding to antibodies A and C. The known results were validated using the method described in this invention. Sandwich structures were constructed for the XBB.1.5 SARS-CoV-2 subtype S protein using the AC antibody combination, and SERS signals were acquired and compared using a portable Raman spectrometer. The antibody-antigen binding strength results obtained in this detection method were consistent with the ELISA experimental results.

[0057] In subsequent detection experiments, antibody A was loaded onto the immune substrate, and antibody C was loaded onto the immune probe used to detect XBB.1.5. (SXBB.1.5 protein (Fuzhou Chuangfang Pharmaceutical Technology Co., Ltd.), antibodies A, B, C, and D (Fuzhou Chuangfang Pharmaceutical Technology Co., Ltd.)).

[0058] Gold and silver nanoparticles are common SERS substrates. They are not only relatively simple to fabricate, but also have good Raman enhancement effects, so they have been widely used in the biomedical field.

[0059] The 4-MBN signal molecule was chosen because this marker has clear spectral peaks, with a characteristic peak at 2229 cm⁻¹ in the silent region. It is not affected by other proteins, carbohydrates, or nucleic acid substances. In the non-silent region, the SERS spectral signal intensity of other interfering proteins is much lower than the enhancement effect of the signal marker. The Raman signal of this marker can be used to more accurately and indirectly determine the protein to be tested.

[0060] To achieve better signal enhancement, this scheme specifically optimized the size and coverage of Ag NPs on the immune substrate, the size of Au NPs inside the immune probe, the content of signal probe molecules, the thickness of the outer Ag shell, the overall size and concentration of the immune probe, and the amount bound to the target protein solution. Each step underwent characterization, detection, and signal enhancement comparison to ensure the stable establishment of the sandwich structure, allowing the plasmon thermoelectric effect to better amplify the signal.

[0061] Experimental results: The detection range of the S protein of subtype XBB.1.5 showed good linearity in the range of 0.05 ng / mL to 1000 ng / mL, with a LOD of 4.0530 pg / mL. After mixing with human saliva samples, it also showed good linearity in the range of 0.1 ng / mL to 1000 ng / mL, with a LOD of 2.2345 pg / mL.

[0062] The embodiments described above are merely preferred embodiments of the present invention, and are described in a relatively specific and detailed manner. However, the present invention is not limited to these embodiments. It should be noted that for those skilled in the art, any modifications made without departing from the spirit of the present invention fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A method for identifying new coronavirus strain XBB.1.5 based on surface-enhanced Raman spectroscopy chip, characterized in that, Specifically, a sandwich structure is constructed, with the target antigen to be tested as the "sandwich", the nano-silver ion tiled silicon wafer combined with antibody A as the "bottom", and the multi-layer core-shell structure metal nanoparticle Au@4-MBN@Ag NPs combined with antibody C as the "top" of the sandwich structure. The specific immune coupling of the triple structure captures the S protein of the new coronavirus strain XBB.1.

5. The portable Raman spectrometer collects the SERS spectral signal intensity value of 4-MBN in the bound immune probe at the characteristic peak of 2229 cm-1 on the two-dimensional plane within 5s, which has a good linear relationship with the content of the S protein of the new coronavirus strain XBB.1.5, thereby realizing the identification of the new coronavirus strain XBB.1.

5. The signal amplification silicon substrate paved with Ag NPs will amplify the signal of the Raman signal molecules in the metal core-shell structure nanoparticles in the coupled immune probe again. The construction of double-layer nano-metal particles can produce strong hot spot signal enhancement and amplification through surface plasmon effect; The antibody A is R11, and the sequence is as follows: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLRGVAPKLLIYGNNNRPSGVPDRFSGSKSGSSASLAITGLQAEDEADYYCQSYDSSLSGSVFGGGTKLTVLRTGGGGSGGGGSGGGGSQVQLVESGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREIGYSSSGSNYYMDVWGKGTTVTVSS; The antibody C is N125, and the sequence is as follows: QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNKYVSWYQQFPGTAPKLLIHDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLRTGGGGSGGGGSGGGGSQVQLVQSGPGLVKPSQTLSLTCTVSGGSINSGSYYWSWIGQPAGKGLEWIGRIYPTGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAPHHMYYYDSSGYWDAFDIWGQGTMVTVSS; The preparation of the immune substrate includes: preparation of Ag NPs nano-silver particles; preparation of Si-Ag NPs two-dimensional substrate; formation of SERS immune substrate; The preparation of the immunoprobes includes: preparing gold nanoparticles Au NPs as a core-shell center; preparing a silver shell to wrap the gold nanoparticles, and wrapping a signal molecule 4-mercaptobenzonitrile in the center layer to form Au@4-MBN@Ag NPs; combining an antibody C on the metal nanoprobe to form an Au@MBA@Ag@Protein immunoprobe; The synthesis of the sandwich structure is specifically as follows: incubating the immunosubstrate in a mixed solution containing 4 μL of the target antigen to be detected and 8 μL of the immunoprobes for 10 min, and washing the excess solution with ultrapure water for SERS detection; The detection range of the S protein of the new coronavirus strain XBB.1.5 is 0.01 ng / mL-1000 ng / mL, which presents a good linear relationship, and the LOD is 3.3949 pg / mL.

2. The method for identifying the new coronavirus strain XBB.1.5 based on surface-enhanced Raman spectrum chip according to claim 1, characterized in that, The preparation of the immunosubstrate of the silver ion tiled silicon wafer combined with the antibody A is specifically as follows: Step 1: Preparation of Ag NPs silver nanoparticles: 100 mL of 1 mM AgNO3 solution is heated and stirred on a magnetic stirrer until boiling, 4 mL of 1% (w / v) trisodium citrate solution is quickly added, and the stirring is continued for 1 h after the heating is stopped. After cooling to room temperature, it is used; Step 2: Preparation of Si-Ag NPs two-dimensional substrate: Take 2 mL Ag NPs, centrifugal at 8000 rpm for 10 min, remove supernatant, and then disperse it into 2 mL 1% (w / v) PVP solution, centrifugal at 8200 rpm for 10 min, remove supernatant, and then disperse it into 2 mL ethanol solution for standby; take 200 μL of the treated Ag NPs, mix with 1 mL dichloromethane and 1 mL ultrapure water in a test tube, shake thoroughly for 40 s, stand until the bubbles disappear and the liquid is completely layered, add 100 μL n-hexane on the top along the wall, gently rotate the test tube to form a dense silver film layer, stand until the silver film is unchanged, remove the upper n-hexane with a pipette, slightly tilt the test tube, vertically immerse a 4*4 cm 2 silicon wafer under the film along the tube wall, rotate the silicon wafer to a certain angle so that the silver film can completely cover the smooth surface of the cleaned silicon wafer, quickly take out the silicon wafer, and then a two-dimensional SERS substrate with a silver film silicon plate is obtained, and then naturally air dry and store in a sealed container at room temperature until use; Step 3: Formation of SERS immunosubstrate: The two-dimensional substrate is immersed in 20 μL of 5 mM DSP solution, and after standing at room temperature for 10 h, it is washed with ultrapure water. After the surface is completely dried, 10 μL of 10 μg / ml antibody A solution is added to the surface, and incubation is performed at 37°C for 2 h. After the excess antibody is gently washed with a small amount of ultrapure water, the surface is dried at 4°C, and the sealed storage is used.

3. The method for identifying the new coronavirus strain XBB.1.5 based on the surface-enhanced Raman spectrum chip according to claim 1, characterized in that, The preparation of the immunoprobes of the multi-layer core-shell structure metal nanoparticles combined with the antibody C is specifically as follows: Step 1: Preparation of Au@4-MBN@Ag NPs: ①Preparation of gold nanoparticles Au NPs as a core-shell center, 100 mL of ultrapure water is stirred and heated on a magnetic stirrer; first, 1 mL of 1% (w / v) chloroauric acid trihydrate solution is added, and after stirring to boiling, 1.3 mL of 1% (w / v) trisodium citrate solution is quickly added. The boiling and stirring state is maintained for 15 min, and then the heating is stopped. After cooling, the solution is clear wine red; (ii) Preparation of silver shell wrapped gold nanoparticles, signal molecule 4-mercaptobenzonitrile is wrapped in the center layer to form Au@4-MBN@Ag NPs; 10 mL of Au NPs is added with 10 μL of 1 mM 4-MBN, incubated at room temperature for 2 h, then centrifuged at 6800 rpm for 10 min in a centrifuge, the supernatant is removed after centrifugation, and the precipitate is resuspended in 10 mL of ultrapure water; Au@4-MBN is formed and placed in a stirrer for stirring, and 300 μL of 1% (w / v) trisodium citrate solution, 100 μL of 0.1M NaOH solution, 1 mL of 1 mM AgNO3 solution, and 1 mL of 10 mM ascorbic acid solution are sequentially added; stirring for 15 min can obtain Au@4-MBN@Ag NPs solution and store it in a 4℃ environment; Second step: antibody C is combined on the metal nanoprobe to form Au@MBA@Ag@Protein immune probe: (i) 3 mL of Au@4-MBN@Ag is added with 2 μL of 5 mM DSP, and the reaction is uniformly oscillated for 3 h, then resuspended in 0.4 mL of ultrapure water, and the solution pH is adjusted to 7.4; (ii) 2 uL of 200 μg / mL antibody C is further added to the above solution, incubated at 37 ℃ for 1.5 h, resuspended in 100 μL of 0.05 mg / mL bovine serum albumin (BSA), blocked for 0.5 h, and Au@MBA@Ag@Protein immune probe is formed, which is stored at 4℃.

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