New crown virus strain BF.7 identification method based on surface enhanced Raman spectrum chip

Abstract

The invention discloses a new crown virus strain BF.7 identification method based on a surface enhanced Raman spectrum chip, and belongs to the technical field of new crown virus detection. By constructing an immune sandwich detection structure corresponding to the antigen, the novel coronavirus Sars-CoV-2S protein is identified, and subtypes are distinguished. The novel crown virus strain BF.7 is identified through the detection method, complex and expensive gene sequencing is avoided, the type of the novel crown virus strain BF.7 can be accurately judged, the cost is low, the accuracy is high, the used time is short, and the operation is convenient.

Description

Technical Field

[0001] The present invention relates to a method for identifying the new coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip, and belongs to the technical field of new coronavirus detection. Background Art

[0002] Surface enhanced Raman spectroscopy (SERS) is known for its fast response speed, strong anti-interference and high sensitivity. It can enhance the weak Raman spectral signals generated by biological samples adsorbed on the surface of precious metal nanostructures. SERS technology combines the narrow characteristic spectrum, specificity and sensitivity of Raman probe molecules, making spectral quantification of various pathogens possible. SERS technology has a wide range of applications, including the detection and analysis of various biological samples.

[0003] The existing technologies can be divided into two categories, namely, label detection and label-free detection of Raman signals.

[0004] Label-free detection is to directly amplify the biological signal of the target to be tested (such as viral protein) and determine the composition of the target by the spectral peak position and intensity. This type of technology can intuitively display the unique fingerprint information of the substance, but due to the complex composition of the liquid to be tested in actual application, the vibration spectrum peaks of a large number of lipids, proteins, and various nucleic acid molecules overlap with each other, and it is impossible to accurately determine the type of new coronavirus subtype (such as BF.7).

[0005] The label detection involved in the existing inventions is also accompanied by the specific capture of the detection target and the design of Raman signal molecules with special structures. However, most of the existing technologies only design a single specific immune binding reaction and a single Raman signal enhancement process in the process of capturing the target protein. There are some problems with simply using nanoparticles as primers to enhance Raman spectroscopy signals. For example, it will be affected by factors such as 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] The present invention provides a method for identifying the new coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip, specifically: constructing a sandwich structure with a target antigen to be tested as a "sandwich", a nanosilver ion flat silicon wafer combined with antibody A as an immune substrate as a "bottom", and an immune probe of a multi-layer core-shell structured metal nanoparticle combined with antibody D as a "top", capturing the S protein of the new coronavirus strain BF.7 through the specific immune coupling of the triple structure, and using a portable Raman spectrometer to collect the SERS spectral signal intensity value at the characteristic peak of 4-MBN in the immune probe bound on a two-dimensional plane within 5 seconds, which has a good linear relationship with the S protein content of the new coronavirus strain BF.7, thereby achieving the identification of the new coronavirus strain BF.7.

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

[0008] The antibody D is N36, and its sequence is as follows:

[0009] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYAVHWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKTSASLAITGLQAEDEADYYCQSYDSSLSVFGGGTKLTVLRTGGGGSGGGGSGGGG SEVQLLESGGGLVQPGGSLRLSCAASGFSFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISSRDNAKNSLYLQMNSLRAEDTAVYYCAREETVRGYFYYYGMDVWGQGTTVTVSS.

[0010] Preferably, the preparation of the nano silver ion tiled silicon wafer combined with antibody A as the immunosubstrate is specifically as follows:

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

[0012] Take 100 mL of AgNO3 (1 mM) solution and heat and stir it on a magnetic stirrer until it boils. Quickly add 4 mL of trisodium citrate solution (1% (w / v)). Continue stirring for 1 h, then stop heating and cool to room temperature before use.

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

[0014] Take 2 mL of Ag NPs, centrifuge at 8000 rpm for 10 min, remove the supernatant, and disperse it in 2 mL of PVP (1% (w / v)) solution, centrifuge at 8200 rpm for 10 min, remove the supernatant, and disperse it in 2 mL of ethanol solution for later use; take the treated Ag NPs 200 μL of the solution was mixed with 1 mL of dichloromethane and 1 mL of ultrapure water in a test tube, and the solution was shaken thoroughly for 40 seconds. After the bubbles disappeared and the liquid was completely separated, 100 μL of n-hexane was added along the wall and at the top. The test tube was gently rotated to form a dense silver film layer. The solution was allowed to stand until the silver film did not change. The upper n-hexane was removed with a pipette. After the solution was allowed to stand until the film had a metallic luster, the test tube was slightly tilted, and a silicon wafer (4*4 cm2) was vertically immersed under the film along the tube wall. The silicon wafer was rotated to a certain angle so that the silver film could completely cover the smooth surface of the cleaned silicon wafer. The silicon wafer was quickly taken out to obtain a two-dimensional SERS substrate with a silver film silicon plate. The substrate was naturally dried and sealed at room temperature until use.

[0015] Step 3: Formation of SERS immunosubstrate:

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

[0017] Preferably, the preparation of the immunoprobe of multilayer core-shell metal nanoparticles combined with antibody D is specifically as follows:

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

[0019] ① To prepare the gold nanoparticles Au NPs in the core-shell center, take 100 mL of ultrapure water on a magnetic stirrer, stir and heat; first add 1 mL of 1% (w / v) chloroauric acid trihydrate solution, stir until boiling, then quickly add 1.3 mL of 1% (w / v) trisodium citrate solution, keep boiling and stirring for 15 minutes, then stop heating. After cooling, the solution is clear wine red;

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

[0021] Step 2: Bind antibody D to the metal nanoprobe to form Au@MBA@Ag@Protein immunoprobe:

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

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

[0024] Preferably, the sandwich structure is synthesized by incubating the immunosubstrate in a mixed solution containing 4 μL of the target antigen to be detected and 8 μL of the immunoprobe for 10 min, and washing the excess solution with ultrapure water for SERS detection.

[0025] Preferably, the detection range of the S protein of the new coronavirus strain BF.7 shows a good linear relationship between 0.01ng / mL and 1000ng / mL, and the LOD is 4.0530pg / mL.

[0026] Beneficial effects:

[0027] 1. The present invention designs two specific immune bindings; the immune probe and immune substrate are designed as nano-metal ions with different structures, which form a sandwich structure of immune probe-target protein-immune substrate from bottom to top with the target protein to be detected. The signal amplification silicon substrate laid by Ag NPs will further amplify the signal of the Raman signal molecule inside the metal core-shell structure nanoparticles in the coupled immune probe. The construction of double-layer nano-metal particles can generate strong hot spot signal enhancement amplification through the surface plasmon effect. The uniform two-dimensional SERS substrate makes the collected SERS spectral signal more uniform. The surface morphology of the synthetic substrate is relatively rough, which can provide a large number of "hot spots" to enhance the SERS spectral signal. This scheme can more accurately capture the target protein to be detected, with higher sensitivity, better specificity, a wide detection linear range, and a low detection limit.

[0028] 2. Through different antibody combinations, an immune sandwich detection structure corresponding to the antigen can be constructed to identify the S protein of the new coronavirus Sars-CoV-2 and distinguish subtypes. The detection method of the present invention is used to identify the new coronavirus strain BF.7, avoiding complex and expensive gene sequencing, and can accurately determine the subtype of the new coronavirus, with low cost, high accuracy, short time and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the SARS-CoV-2 SERS integrated detection chip.

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

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

[0032] Figure 4 This is a synthetic immune substrate characterization diagram.

[0033] Figure 5 To identify the S protein image on the SERS two-dimensional immune sandwich structure.

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

[0035] Figure 7 The detection process and result diagram of the real sample. DETAILED DESCRIPTION

[0036] The embodiments of the present invention are described below in conjunction with the accompanying drawings.

[0037] The present invention provides a method for identifying the new coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip, specifically: constructing a sandwich structure with a target antigen to be tested as a "sandwich", a nanosilver ion flat silicon wafer combined with antibody A as an immune substrate as a "bottom", and an immune probe of a multi-layer core-shell structured metal nanoparticle combined with antibody D as a "top", capturing the S protein of the new coronavirus strain BF.7 through the specific immune coupling of the triple structure, and using a portable Raman spectrometer to collect the SERS spectral signal intensity value at the characteristic peak of 4-MBN in the immune probe bound on a two-dimensional plane within 5 seconds, which has a good linear relationship with the S protein content of the new coronavirus strain BF.7, thereby achieving the identification of the new coronavirus strain BF.7.

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

[0039] The antibody D is N36, and its sequence is as follows:

[0040] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYAVHWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKTSASLAITGLQAEDEADYYCQSYDSSLSVFGGGTKLTVLRTGGGGSGGGGSGGGG SEVQLLESGGGLVQPGGSLRLSCAASGFSFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISSRDNAKNSLYLQMNSLRAEDTAVYYCAREETVRGYFYYYGMDVWGQGTTVTVSS.

[0041] Preferably, the preparation of the nano silver ion tiled silicon wafer combined with antibody A as the immunosubstrate is specifically as follows:

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

[0043] Take 100 mL of AgNO3 (1 mM) solution and heat and stir it on a magnetic stirrer until it boils. Quickly add 4 mL of trisodium citrate solution (1% (w / v)). Continue stirring for 1 h, then stop heating and cool to room temperature before use.

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

[0045] Take 2 mL of Ag NPs, centrifuge at 8000 rpm for 10 min, remove the supernatant, and disperse it in 2 mL of PVP (1% (w / v)) solution, centrifuge at 8200 rpm for 10 min, remove the supernatant, and disperse it in 2 mL of ethanol solution for later use; take the treated Ag NPs 200 μL of the solution was mixed with 1 mL of dichloromethane and 1 mL of ultrapure water in a test tube, and the solution was shaken thoroughly for 40 seconds. After the bubbles disappeared and the liquid was completely separated, 100 μL of n-hexane was added along the wall and at the top. The test tube was gently rotated to form a dense silver film layer. The solution was allowed to stand until the silver film did not change. The upper n-hexane was removed with a pipette. After the solution was allowed to stand until the film had a metallic luster, the test tube was slightly tilted, and a silicon wafer (4*4 cm2) was vertically immersed under the film along the tube wall. The silicon wafer was rotated to a certain angle so that the silver film could completely cover the smooth surface of the cleaned silicon wafer. The silicon wafer was quickly taken out to obtain a two-dimensional SERS substrate with a silver film silicon plate. The substrate was naturally dried and sealed at room temperature until use.

[0046] Step 3: Formation of SERS immunosubstrate:

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

[0048] Preferably, the preparation of the immunoprobe of multilayer core-shell metal nanoparticles combined with antibody D is specifically as follows:

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

[0050] ① To prepare the gold nanoparticles Au NPs in the core-shell center, take 100 mL of ultrapure water on a magnetic stirrer, stir and heat; first add 1 mL of 1% (w / v) chloroauric acid trihydrate solution, stir until boiling, then quickly add 1.3 mL of 1% (w / v) trisodium citrate solution, keep boiling and stirring for 15 minutes, then stop heating. After cooling, the solution is clear wine red;

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

[0052] Step 2: Bind antibody D to the metal nanoprobe to form Au@MBA@Ag@Protein immunoprobe:

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

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

[0055] Preferably, the sandwich structure is synthesized by incubating the immunosubstrate in a mixed solution containing 4 μL of the target antigen to be detected and 8 μL of the immunoprobe for 10 min, and washing the excess solution with ultrapure water for SERS detection.

[0056] The spike glycoprotein of the BF.7 coronavirus subtype was detected and identified using the surface scattering Raman spectroscopy (SERS) method. Identification process: 4 antibodies A, B, C, and D screened through binding tests were constructed on immune substrates and immune probes, respectively, to form 6 detection antibody combinations AB, AC, AD, BC, BD, and CD. Known Elisa binding test results: BF.7 subtype S protein has good binding to antibodies A and D. The known results were verified by the scheme pointed out in the present invention, and the AD antibody combination was used to build a sandwich structure for the S protein of the BF.7 coronavirus subtype, and the SERS signals were collected and compared using a portable Raman spectrometer. The antibody-antigen binding strength results obtained in this detection scheme are consistent with the Elisa experimental results.

[0057] In the subsequent detection experiments, antibody A was loaded on the immune substrate, and the immune probe used to detect BF.7 type was loaded with antibody D. (SBF.7 protein (Fuzhou Chuangfang Pharmaceutical Technology Co., Ltd.), antibodies A, B, C, D (Fuzhou Chuangfang Pharmaceutical Technology Co., Ltd.)).

[0058] Gold and silver nanoparticles are relatively common SERS substrates. Not only are their preparation methods relatively simple, but they also have good Raman enhancement effects. Therefore, they have been widely used in the biomedical field.

[0059] The 4-MBN signal molecule was selected because this marker has a clear spectral peak and a characteristic peak at 2229cm-1 in the silent zone. It will not be affected by other proteins, sugars, and nucleic acids. In the non-silent zone, 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 indirectly judge the protein to be tested.

[0060] In order to obtain better signal enhancement effect, this scheme specifically optimizes 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 external Ag shell, the size and concentration of the overall immune probe, and the amount of binding with the target protein solution to be tested. Each step is characterized, tested and compared with signal enhancement to achieve the stable establishment of the sandwich structure, and the plasmon thermoelectric effect can better amplify the signal.

[0061] Experimental results: The detection range of S protein of subtype BF.7 showed a good linear relationship in the range of 0.01ng / mL-1000ng / mL, and the LOD was 4.0530pg / mL. After mixing human saliva samples, it was able to show a good linear relationship in the range of 0.1ng / mL-1000ng / mL, and the LOD was 8.5703pg / mL.

[0062] The above-described embodiments only express the preferred embodiments of the present invention, and the description thereof is relatively specific and detailed, but the present invention is not limited to these embodiments. It should be noted that it is obvious to those skilled in the art. Without departing from the purpose of the present invention, any improvement made shall fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for identifying the new coronavirus strain BF.7 based on a 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 nanosilver ion flat silicon wafer bound to antibody A as the immune substrate as the "bottom", and the immune probe of multi-layer core-shell structured metal nanoparticles bound to antibody D as the "top". The S protein of the new coronavirus strain BF.7 is captured through the specific immune coupling of the triple structure, and the SERS spectral signal intensity value at the characteristic peak of 4-MBN in the bound immune probe on the two-dimensional plane is collected within 5 seconds by a portable Raman spectrometer. It has a good linear relationship with the S protein content of the new coronavirus strain BF.7, thereby realizing the identification of the new coronavirus strain BF.

7.

2. According to claim 1, a method for identifying the new coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip is characterized in that: The antibody A is R11, and its sequence is as follows: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLRGVAPKLLIYGNNNRPSGVPDRFSGSKSGSSASLAITGLQAEDEADYYCQSYDSSLGSVFGGGTKLTVLRTGGGG SGGGGSGGGGSQVQLVESGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREIGYSSSGSNYYMDVWGKGTTVTVSS; The antibody D is N36, and its sequence is as follows: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYAVHWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKTSASLAITGLQAEDEADYYCQSYDSSLSVFGGGTKLTVLRTGGGGSGGGGSGGGG SEVQLLESGGGLVQPGGSLRLSCAASGFSFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISSRDNAKNSLYLQMNSLRAEDTAVYYCAREETVRGYFYYYGMDVWGQGTTVTVSS.

3. According to claim 1, a method for identifying the new coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip is characterized in that: The preparation of the nano silver ion tiled silicon wafer combined with antibody A as the immunological substrate is specifically as follows: Step 1: Preparation of Ag NPs nanoparticles: Take 100 mL of AgNO3 (1 mM) solution and heat and stir it on a magnetic stirrer until it boils. Quickly add 4 mL of trisodium citrate solution (1% (w / v)). Continue stirring for 1 h, then stop heating and cool to room temperature before use. Step 2: Preparation of Si-Ag NPs 2D substrate: Take 2 mL of Ag NPs, centrifuge at 8000 rpm for 10 min, remove the supernatant, and disperse it in 2 mL of PVP (1% (w / v)) solution, centrifuge at 8200 rpm for 10 min, remove the supernatant, and disperse it in 2 mL of ethanol solution for later use; take the treated AgNPs 200 μL of the solution was mixed with 1 mL of dichloromethane and 1 mL of ultrapure water in a test tube, and the solution was shaken thoroughly for 40 seconds. After the bubbles disappeared and the liquid was completely separated, 100 μL of n-hexane was added along the wall and at the top. The test tube was gently rotated to form a dense silver film layer. The solution was allowed to stand until the silver film did not change. The upper n-hexane was removed with a pipette. After the solution was allowed to stand until the film had a metallic luster, the test tube was slightly tilted, and a silicon wafer (4*4 cm2) was vertically immersed under the film along the tube wall. The silicon wafer was rotated to a certain angle so that the silver film could completely cover the smooth surface of the cleaned silicon wafer. The silicon wafer was quickly taken out to obtain a two-dimensional SERS substrate with a silver film silicon plate. The substrate was naturally dried and sealed at room temperature until use. Step 3: Formation of SERS immunosubstrate: Immerse the two-dimensional substrate in 20 μL DSP (5 mM) solution, let it stand at room temperature for 10 hours, then wash it with ultrapure water. After the surface is completely dry, add 10 μL 10 μg / ml antibody A solution on its surface and incubate it at 37°C for 2 hours. After gently washing the excess antibody with a small amount of ultrapure water, store it at 4°C until the surface is dry, and then seal and store for later use.

4. According to claim 1, a method for identifying the new coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip is characterized in that: The preparation of the immunoprobe of multilayer core-shell metal nanoparticles combined with antibody D is specifically as follows: Step 1: Preparation of Au@4-MBN@Ag NPs: ① To prepare the gold nanoparticles Au NPs in the core-shell center, take 100 mL of ultrapure water on a magnetic stirrer, stir and heat; first add 1 mL of 1% (w / v) chloroauric acid trihydrate solution, stir until boiling, then quickly add 1.3 mL of 1% (w / v) trisodium citrate solution, keep boiling and stirring for 15 minutes, then stop heating. After cooling, the solution is clear wine red; ②Prepare a silver shell to wrap the gold nanoparticles, and wrap the signal molecule 4-mercaptobenzonitrile in the central layer to form Au@4-MBN@Ag NPs; take 10mL Au NPs and add 10μL 4-MBN (1mM), incubate at room temperature for 2h, place it in a centrifuge and centrifuge it at 6800rpm for 10min, remove the supernatant after centrifugation, collect the precipitate and resuspend it to 10mL with ultrapure water; place the formed Au@4-MBN on a stirrer and stir, and add 300μL trisodium citrate solution (1% (w / v)), 100μL NaOH (0.1M) solution, 1mL AgNO3 (1mM) solution, and 1mL ascorbic acid (10mM) solution in sequence; stir the reaction for 15min, and the Au@4-MBN@Ag NPs solution can be obtained and stored at 4℃; Step 2: Bind antibody D to the metal nanoprobe to form Au@MBA@Ag@Protein immunoprobe: ① Add 2 μL DSP (5 mM) to 3 mL Au@4-MBN@Ag and shake at a constant speed for 3 h, then resuspend to 0.4 mL with ultrapure water and adjust the solution pH to 7.4; ② Add 2uL of antibody D (200μg / mL) to the above solution, incubate at 37°C for 1.5h, resuspend to 100μL with 0.05mg / mL bovine serum albumin (BSA), block for 0.5h, and the Au@MBA@Ag@Protein immune probe is formed, which is stored at 4°C.

5. According to claim 1, a method for identifying the new coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip is characterized in that: The synthesis of the sandwich structure is as follows: the immune substrate is incubated in a mixed solution containing 4 μL of the target antigen to be detected and 8 μL of the immune probe for 10 minutes, and the excess solution is washed with ultrapure water for SERS detection.

6. The method for identifying the novel coronavirus strain BF.7 based on a surface enhanced Raman spectroscopy chip according to claim 1, characterized in that: The detection range of the S protein of the new coronavirus strain BF.7 showed a good linear relationship between 0.01ng / mL and 1000ng / mL, and the LOD was 4.0530pg / mL.

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