A method for detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy

Abstract

The present invention discloses a method for detecting the novel coronavirus N protein based on surface-enhanced Raman spectroscopy, comprising the following steps: S1 preparing immune Raman signal gold nanoparticles of Au@IR808@antibody; S2 preparing biotinylated antibody Ab2; S3 preparing an immune sandwich complex; S4 magnetically attracting and washing with a washing solution and finally concentrating and diluting to a small volume; S5 dropping the small-volume liquid onto a surface-enhanced Raman chip for Raman detection. The present invention utilizes the high binding efficiency of immune Raman signal gold nanoparticles with SARS-CoV-2 N protein and biotinylated antibody Ab2 in the liquid phase to achieve specific recognition and capture of N protein to form a "double antibody" immune complex. Then, by virtue of the high affinity between streptavidin and biotin, the immune complex formed in the previous step is captured by magnetic beads modified with streptavidin to form an immune sandwich complex, enabling ultrasensitive, stable and rapid detection of SARS-CoV-2 virus N-protein within 15 minutes.

Description

Technical Field

[0001] The present invention belongs to the field of bio-immunological analysis and detection, and particularly relates to a method for detecting the nucleocapsid protein (N) of the novel coronavirus based on surface-enhanced Raman spectroscopy, which is used for detecting the nucleocapsid protein gene (nucleocapsid protein, N) of the novel coronavirus. Background Art

[0002] The etiological and serological examinations in the detection methods of the novel coronavirus SARS-CoV-2 mainly include nucleic acid detection, antibody detection, and antigen detection. Among them, nucleic acid detection is the "gold standard" for SARS-CoV-2 detection, but it has the disadvantages of long time consumption and inability to perform on-site detection. Antibody detection cannot be used alone for the diagnosis of the novel coronavirus, and it cannot rule out infection or explain the infection status. Antigen detection is a very important supplement to nucleic acid detection. Through antigen detection, positive infected persons can be detected earlier, isolated as soon as possible, and nucleic acid reexamination can be carried out, so as to achieve early detection, early reporting, and early isolation to the greatest extent.

[0003] Existing detection methods, for example: CN202011431411.8 discloses a kit for detecting the novel coronavirus and its mutants, which is characterized by including the following parts: (1) a pre-substrate A chain and a pre-substrate B chain; (2) Cas 13a protein; (3) crRNA or its DNA template; (4) nucleic acid ligase; (5) a light-up RNA aptamer or its DNA template; (6) in vitro transcription reagents. CN202110353115.9 discloses a novel coronavirus neutralizing antibody detection kit prepared by an immunochromatography method labeled with Pt-Pd alloy nanoparticles, its detection method and application.

[0004] Compared with nucleic acid detection, antigen detection is faster and more convenient to operate. As a supplementary means, it can be used for the screening of specific populations, which is beneficial to improving the ability of "early detection". Moreover, it has a lower cost and faster speed, and can obtain results within 10-20 minutes. Therefore, antigen detection is more suitable for early large-scale screening and can corroborate with nucleic acid detection, antibody detection, and CT detection. However, existing rapid antigen detection kits have problems such as insufficient detection sensitivity leading to false negatives and low detection rates. Therefore, there is an urgent need for a faster, more sensitive and stable rapid antigen detection method. Summary of the Invention

[0005] The object of the present invention is to overcome the defects existing in the prior art, make up for the deficiencies of the existing technologies for detecting novel coronavirus, and provide a method for rapidly and sensitively detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy. The present invention utilizes the high binding efficiency of immunological Raman signal gold nanoparticles, SARS-CoV-2 N protein, and biotinylated antibody Ab2 in liquid phase to achieve specific recognition and capture of the N protein to form a "double antibody" immune complex. Then, by virtue of the high affinity between streptavidin and biotin, the immune complex formed in the previous step is captured by magnetic beads modified with streptavidin, forming an immune sandwich complex with the structure of "immunological Raman signal gold nanoparticles - N protein - biotinylated antibody - streptavidinylated magnetic beads". The Raman spectrometer can achieve ultrasensitive, stable, and rapid detection of the N protein of SARS-CoV-2 virus within 15 minutes.

[0006] To achieve the above object, one of the technical solutions of the present invention is: A method for detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy, specifically including the following steps:

[0007] S1: Couple Raman signal molecules and the antibody Ab1 of SARS-CoV-2 virus N protein to gold nanoparticles respectively to prepare immunological Raman signal gold nanoparticles of Au@IR808@antibody;

[0008] S2: Couple biotin with antibody Ab2 to prepare biotinylated antibody Ab2 (Bio-Ab2);

[0009] S3: Mix and incubate the immunological Raman signal gold nanoparticles prepared in step S1, the biotinylated antibody Ab2 prepared in step S2, and the dilution of the N protein of novel coronavirus, and then mix and incubate with streptavidinylated magnetic beads to obtain an immune sandwich complex;

[0010] S4: Magnetically adsorb and wash the immune sandwich complex in step S3 with a washing solution several times, and finally concentrate and make up the volume to a small volume;

[0011] S5: Drop the small-volume liquid in S4 onto a surface-enhanced Raman chip for Raman detection.

[0012] Preferably, the immunolabeled Raman signal gold nanoparticles in step S1 are prepared by the following method: Mix gold nanoparticles with Raman signal molecules under stirring, incubate at room temperature, centrifuge, discard the supernatant, wash once with buffer and make up the volume, add EDC ((1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solutions for activation, centrifuge and wash, make up the volume with buffer, add the SARS-CoV-2 virus N protein antibody Ab1, incubate overnight for binding, centrifuge, discard the supernatant, add the blocking solution for incubation, wash and centrifuge, discard the supernatant, and finally redisperse in the preservation solution to obtain the immunolabeled Raman signal gold nanoparticles.

[0013] Furthermore, the buffer is BB buffer, which is prepared with borax and sodium hydroxide and has a pH of 7.2 - 7.6.

[0014] Furthermore, the particle size of the gold nanoparticles is 30 - 80 nm.

[0015] Furthermore, the reaction activation time of adding EDC and NHS is 10 - 60 min, the activation temperature is room temperature, the incubation binding time of adding the antibody is 10 - 24 h, and the incubation temperature is 2 - 8 °C.

[0016] Furthermore, the components of the blocking solution include PBS salt solution, bovine serum albumin, casein, and skim milk powder, and the components of the preservation solution include PBS salt solution, bovine serum albumin, casein, glycine, and skim milk powder.

[0017] Furthermore, the blocking time of adding the blocking solution is 2 - 12 h, and the blocking temperature is room temperature.

[0018] Preferably, the immunolabeled Raman signal gold nanoparticles in step S1 have Raman signal expression and specific recognition antibodies for the novel coronavirus SARS-CoV-2 N protein.

[0019] Preferably, the SARS-CoV-2 virus N protein antibody Ab1 in step S1 is the novel coronavirus N protein antibody SARS-CoV-2NP-mAb.

[0020] Preferably, the Raman signal molecule in step S1 is the near-infrared indole-based cyanine dye IR808, but it is not limited to this signal molecule. Other Raman molecules such as Nile blue A and indocyanine green (ICG) are also within the range, and the incubation time with gold nanoparticles is 5 - 30 min.

[0021] Preferably, the preparation method of the biotinylated antibody in step S2 is to mix the antibody with biotin for 2 - 6 h, and then dialyze in PBS solution for 15 - 30 h.

[0022] Preferably, the antibody Ab2 added to the biotinylated antibody in step S2 is the neutralizing antibody SARS-CoV-2NP-mAb against the novel coronavirus.

[0023] Preferably, the composition of the dilution solution of the novel coronavirus N protein in step S2 is 10 mM PBS, 0.01%-1% Triton X-100, 0.05%-5% EDTA, and 0.05-5% Tween-20.

[0024] Preferably, the biotinylated antibody in steps S2 and S3 is an antibody modified with biotin.

[0025] Preferably, the streptavidinylated magnetic beads in step S3 have magnetism and are modified with streptavidin.

[0026] A method for detecting the novel coronavirus N protein based on surface-enhanced Raman spectroscopy, the steps of which include: mixing and incubating the immunological Raman signal gold nanoparticles prepared with Au@IR808@ antibody, the novel coronavirus SARS-CoV-2 N protein, and the biotinylated antibody Ab2 to form a "double antibody" immunocomplex, then adding streptavidinylated magnetic beads to capture the complex, further forming an immunological sandwich complex, washing several times with PBS-T after magnetic separation, concentrating and fixing the volume to a small volume, and directly performing Raman detection to obtain the Raman spectroscopy result of rapid detection of the novel coronavirus N protein within 15 minutes.

[0027] Preferably, the washing solution in step S4 is PBS-T, and the fixing solution is PBS. PBS-T is prepared by adding Tween-20 with a concentration of 0.01%-1% to the PBS solution.

[0028] Preferably, the number of washing times in step S4 is 1-5 times, and the small volume is 3-30 ul.

[0029] Preferably, the surface-enhanced Raman chip in step S5 is a silicon wafer coated with a layer of gold film.

[0030] Preferably, the Raman detection instrument in step S5 is a portable Raman spectrometer, a confocal Raman spectrometer, a handheld Raman spectrometer, etc., and the excitation wavelength is 785 nm.

[0031] Preferably, a method for detecting the novel coronavirus N protein based on surface-enhanced Raman spectroscopy specifically includes the following steps:

[0032] (1) Preparation of immunolabeled Raman signal gold nanoparticles: Mix 2 - 20 ml of gold nanoparticles with 10 - 100 μl of 0.5 mM IR808 Raman signal molecules under stirring, incubate at room temperature for 2 - 60 min, centrifuge at 5000 - 8000 rpm, discard the supernatant, wash once with 2 - 20 ml of BB buffer and make up the volume. Add 50 - 100 μl of 5 - 50 mM EDC ((1 - ethyl - 3 - [3 - dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N - hydroxysuccinimide) solutions for activation, wash with 2 - 20 ml of BB buffer after centrifugation, and make up the volume with 1 - 20 ml of BB buffer. Add 1 - 100 μg of SARS - CoV - 2 virus N protein antibody Ab1, incubate overnight at 2 - 8 °C for binding, centrifuge, discard the supernatant, add 1 - 20 ml of blocking solution and incubate for 1 - 12 h, wash and centrifuge, discard the supernatant, and finally redisperse in 2 - 10 ml of storage solution to obtain immunolabeled Raman signal gold nanoparticles.

[0033] (2) Preparation of biotinylated antibody: Mix 10 - 500 μl of N protein antibody Ab2 at 1 - 10 mg / ml with 1 - 20 ml of 50 mM biotin for 1 - 10 h, then dialyze through a dialysis membrane with a molecular weight of 1000 - 10000 in PBS (pH 7.2) solution for 10 - 24 h, and then make up the volume with PBS solution to a concentration of 1 - 50 mg / ml.

[0034] (3) Preparation of immunocomplex sandwich structure: Mix 10 - 100 μl of immunolabeled Raman signal gold nanoparticles prepared with Au@IR808@antibody, 100 - 1000 μl of SARS - CoV - 2 N protein at different concentrations (0.1 pg / ml, 1 pg / ml, 5 pg / ml, 50 pg / ml) prepared with different concentration diluents, and 1 - 50 μl of biotinylated antibody Ab2 and incubate for 1 - 10 min to form a "double - antibody" immunocomplex. Then add 10 - 100 μl of streptavidin - coated magnetic beads at 1 - 20 mg / ml to capture the complex and incubate for 1 - 10 min to construct an immunocomplex sandwich. After magnetic separation, wash with PBS - T for 1 - 5 times, concentrate and make up the volume to a small volume of 3 - 30 μl.

[0035] (4) Detection of SARS - CoV - 2 N protein: Take out the small - volume immunocomplex sandwich solution and drop it on a silicon wafer coated with a gold film, and directly perform Raman detection with a portable Raman spectrometer to obtain the Raman spectrum results for rapid detection of SARS - CoV - 2 N protein within 15 min.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] 1. The present invention prepares immune Raman signal gold nanoparticles of Au@IR808@antibody. The IR808 Raman signal molecule itself contains -COOH groups, which can be directly connected to the antibody after activation by EDC and NHS. That is, it serves as both a Raman signal group and a linker for binding the antibody, avoiding the complex modification process of the antibody on the surface of the gold nanoparticles. Moreover, IR808 is in a resonance state under a 785nm laser, and has a higher signal intensity compared to other Raman signal molecules, greatly improving the detection sensitivity.

[0038] 2. The present invention utilizes the high binding efficiency of immune Raman signal gold nanoparticles, SARS-CoV-2 N protein, and biotinylated antibody Ab2 in the liquid phase to achieve specific recognition and capture of the N protein to form a "double antibody" immune complex. Then, relying on the high affinity between streptavidin and biotin, the immune complex formed in the previous step is captured by magnetic beads modified with streptavidin, forming an immune sandwich complex with the structure of "immune Raman signal gold nanoparticles - N protein - biotinylated antibody - streptavidinylated magnetic beads". This reaction method can improve the sensitivity by nearly an order of magnitude compared to the "Raman signal gold - antigen - magnetic bead" sandwich complex formed by a simple reaction.

[0039] 3. The detection method provided by the present invention is used for the detection of the novel coronavirus SARS-CoV-2 N protein. The detection limit for the recombinant N protein of the novel coronavirus under standard buffer is 0.1 pg / ml, and the detection limit for the simulated throat swab sample is 1 pg / ml. Compared with the novel coronavirus antigen detection kit (gold nanoparticle method) (the detection limit for N protein is 10 - 100 pg / ml), the sensitivity is increased by 20 - 50 times, which well solves the problem of insufficient sensitivity of the current rapid antigen detection method. This detection method can also specifically detect the novel coronavirus SARS-CoV-2 N protein in nasopharyngeal swabs, and only requires within 15 minutes using a portable Raman spectrometer, which can meet the needs of on-site detection. Description of the Drawings

[0040] Figure 1 is a schematic diagram of the detection principle of the present invention;

[0041] Figure 2 is an SEM image of the structure of the immune sandwich complex in Example 1 of the present invention;

[0042] Figure 3 is a surface-enhanced Raman spectroscopy image for detecting different concentrations of SARS-CoV-2 N protein in the detection diluent;

[0043] Figure 4 is a surface-enhanced Raman spectroscopy image for detecting different concentrations of SARS-CoV-2 N protein in the throat swab collection fluid;

[0044] Figure 5 It is a schematic diagram of the structural principle of the immune sandwich complex of the "Raman signal gold - antigen - magnetic bead" reaction mode in Comparative Example 1 of the present invention;

[0045] Figure 6 It is a surface - enhanced Raman spectroscopy diagram of detecting different SARS - CoV - 2 N protein concentrations in the dilution solution by the "Raman signal gold - antigen - magnetic bead" reaction method in Comparative Example 1;

[0046] Figure 7 It is a surface - enhanced Raman spectroscopy diagram of detecting different SARS - CoV - 2 N protein concentrations in the throat swab collection fluid by the "Raman signal gold - antigen - magnetic bead" reaction method in Comparative Example 2. Specific Embodiments

[0047] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0048] The detection method of the present invention utilizes the high binding efficiency of immune Raman signal gold nanoparticles, SARS - CoV - 2 N protein, and biotinylated antibody Ab2 in the liquid phase to achieve specific recognition and capture of N protein to form a "double - antibody" immune complex. Then, by virtue of the high affinity between streptavidin and biotin, the immune complex formed in the previous step is captured by magnetic beads modified with streptavidin, forming an immune sandwich complex with the structure of "immune Raman signal gold nanoparticles - N protein - biotinylated antibody - streptavidin - modified magnetic beads".

[0049] A method for detecting novel coronavirus N protein based on surface - enhanced Raman spectroscopy specifically includes the following steps:

[0050] S1: Coupling Raman signal molecules and SARS - CoV - 2 virus N protein antibody Ab1 to gold nanoparticles respectively to prepare immune Raman signal gold nanoparticles of Au@IR808@antibody;

[0051] S2: Coupling biotin with antibody Ab2 to prepare biotinylated antibody Ab2 (Bio - Ab2);

[0052] S3: Mixing and incubating the immune Raman signal gold nanoparticles prepared in step S1, the biotinylated antibody Ab2 prepared in step S2, and the dilution solution of novel coronavirus N protein, and then mixing and incubating with streptavidin - modified magnetic beads to obtain an immune sandwich complex;

[0053] S4: Magnetically separating and washing the immune sandwich complex in step S3 several times with a washing solution, and finally concentrating and fixing the volume to a small volume;

[0054] S5: Drop the small-volume liquid in S4 onto the surface-enhanced Raman chip for Raman detection.

[0055] Figure 1 This is a schematic diagram of the detection principle of the present invention. The immunological Raman signal gold nanoparticles prepared with Au@IR808@antibody, the SARS-CoV-2 N protein of the novel coronavirus, and the biotinylated antibody Ab2 are mixed and incubated to form a "double antibody" immunocomplex. Then, streptavidin-coated magnetic beads are added to capture the complex, further forming an immunocapture complex.

[0056] In the following examples, the nanoparticles used are the ultra-high concentration gold nanoparticle solution with a particle size of 40-60 nm developed by Zhengzhou Lingsi Biotechnology Co., Ltd.; the SARS-CoV-2 virus N protein antibody Ab1 is the novel coronavirus N protein antibody SARS-CoV-2NP-mAb with the product number C00154 developed by Jiangsu Dongkang Biomedical Technology Co., Ltd.; the antibody Ab2 added to the biotinylated antibody is the novel coronavirus neutralizing antibody SARS-CoV-2NP-mAb with the product number C00172 developed by Jiangsu Dongkang Biomedical Technology Co., Ltd.

[0057] Example 1

[0058] (1) Preparation of immunological Raman signal gold nanoparticles: Mix 10 ml of gold nanoparticles with 20 μl of 0.5 mM IR808 Raman signal molecule under stirring, incubate at room temperature for 10 min, centrifuge at 6000 rpm, discard the supernatant, wash once with 10 ml of BB buffer and make up the volume. Add 50 μl of 5 mM EDC ((1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution for activation, wash with 10 ml of BB buffer after centrifugation, and make up the volume with 5 ml of BB buffer. Add 50 μg of the SARS-CoV-2 virus N protein antibody Ab1, incubate at 4 °C overnight for binding, centrifuge, discard the supernatant, add 10 ml of blocking solution and incubate for 1 h, wash and centrifuge, discard the supernatant, and finally redisperse in 10 ml of preservation solution to obtain the immunological Raman signal gold nanoparticles.

[0059] (2) Preparation of biotinylated antibody: Take 250 μl of 5 mg / ml N protein antibody Ab2 and mix it with 2 ml of 50 mM biotin for 5 h, then dialyze through a dialysis membrane with a molecular weight cut-off of 5000 in PBS (pH 7.2) solution for 20 h, and then make up the volume to a concentration of 1 mg / ml with PBS solution.

[0060] (3) Preparation of the immune sandwich complex structure: Mix 20 μl of the immune Raman signal gold nanoparticles for preparing Au@IR808@antibody, 300 μl of the SARS-CoV-2 N protein at different concentrations (0.1 pg / ml, 1 pg / ml, 5 pg / ml, 50 pg / ml) prepared with different concentration diluents, and 10 μl of the biotinylated antibody Ab2, and incubate for 4 min to form a "dual antibody" immune complex. Then add 40 μl of 1 mg / ml streptavidin-coated magnetic beads to capture the complex and incubate for 4 min to construct an immune sandwich complex. After magnetic separation, wash twice with PBS-T, concentrate and fix the volume to a small volume of 10 μl. Figure 2 It is the SEM image of the immune sandwich complex structure.

[0061] (4) Detection of the SARS-CoV-2 N protein: Take out the small-volume immune sandwich complex solution and drop it on a silicon wafer coated with a gold film, and directly perform Raman detection through a portable Raman spectrometer. The rapid detection Raman spectrum result of the SARS-CoV-2 N protein appears in 15 min, as Figure 3 .

[0062] From Figure 3 it can be seen that: the detection limit for detecting the recombinant N protein of SARS-CoV-2 in a standard buffer (standard system) is 0.1 pg / ml, and the sensitivity is increased by 100 - 1000 times compared with the SARS-CoV-2 antigen detection kit (gold nanoparticle method) (the detection limit of N protein is 10 - 100 pg / ml).

[0063] Example 2

[0064] (1) Preparation of immune Raman signal gold nanoparticles: Mix 10 ml of gold nanoparticles with 20 μl of 0.5 mM IR808 Raman signal molecules under stirring, incubate at room temperature for 10 min, centrifuge at 6000 rpm, discard the supernatant, wash once with 10 ml of BB buffer and fix the volume. Add 50 μl of 5 mM EDC ((1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solutions for activation, wash with 10 ml of BB buffer after centrifugation, and fix the volume with 5 ml of BB buffer. Add 50 μg of the SARS-CoV-2 virus N protein antibody Ab1, incubate overnight at 4 °C for binding, centrifuge, discard the supernatant, add 10 ml of blocking solution and incubate for 1 h, wash and centrifuge, discard the supernatant, and finally redisperse in 10 ml of preservation solution to obtain immune Raman signal gold nanoparticles.

[0065] (2) Preparation of biotinylated antibody: Take 250 μl of N protein antibody Ab2 at 5 mg / ml and mix it with 2 ml of 50 mM biotin for 5 h. Then dialyze it through a dialysis membrane with a molecular weight cut-off of 5000 in PBS (pH 7.2) solution for 20 h, and then make the volume up to 1 mg / ml concentration with PBS solution.

[0066] (3) Preparation of the immune sandwich complex structure: Use a throat swab sampling stick to fully wipe the oral pharynx, then stir it in 3 ml of N protein diluent to obtain a throat swab sampling solution, and add N protein to prepare throat swab sample solutions of different SARS-CoV-2 N proteins (0.1 pg / ml, 1 pg / ml, 5 pg / ml, 50 pg / ml). Mix 20 μl of the immunological Raman signal gold nanoparticles for preparing Au@IR808@ antibody, 300 μl of the throat swab sampling solutions of different concentrations of SARS-CoV-2 N protein (1 pg / ml, 5 pg / ml, 50 pg / ml), and 10 μl of biotinylated antibody Ab2 and incubate for 5 min to form a "double antibody" immune complex. Then add 40 μl of 1 mg / ml streptavidin-coated magnetic beads to capture the complex and incubate for 5 min to construct an immune sandwich complex. After magnetic separation, wash it 3 times with PBS-T and concentrate and make the volume up to a small volume of 10 μl.

[0067] (4) Detection of the throat swab simulated sample of SARS-CoV-2 N protein: Take out the solution of the small-volume immune sandwich complex and drop it on a silicon wafer coated with a gold film, and directly perform Raman detection with a portable Raman spectrometer. The Raman spectrum results of the rapid detection of SARS-CoV-2 N protein can be achieved within 15 min, as Figure 4 .

[0068] From Figure 4 it can be seen that the detection limit of the throat swab simulated sample for detecting SARS-CoV-2 is 1 pg / ml, and the sensitivity is improved by 10 - 100 times compared with the SARS-CoV-2 antigen detection kit (gold nanoparticle method) (the detection limit of N protein is 10 - 100 pg / ml).

[0069] Comparative Example 1

[0070] (1) Preparation of immunolabeled Raman signal gold nanoparticles: Mix 10 ml of gold nanoparticles with 20 μl of 0.5 mM IR808 Raman signal molecule under stirring, incubate at room temperature for 10 min, centrifuge at 6000 rpm, discard the supernatant, wash once with 10 ml of BB buffer and make up the volume. Add 50 μl of 5 mM EDC ((1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solutions for activation, wash with 10 ml of BB buffer after centrifugation, and make up the volume with 5 ml of BB buffer. Add 50 μg of SARS-CoV-2 virus N protein antibody Ab1, incubate at 4 °C overnight for binding, centrifuge, discard the supernatant, add 10 ml of blocking solution and incubate for 1 h, wash and centrifuge, discard the supernatant, and finally redisperse in 10 ml of preservation solution to obtain immunolabeled Raman signal gold nanoparticles.

[0071] (2) Preparation of immunomagnetic beads: Incubate 100 μl of 10 mg / ml carboxylated magnetic beads with 50 μg of SARS-CoV-2 virus N protein antibody Ab2 at 4 °C overnight for binding, centrifuge, discard the supernatant, add 10 ml of blocking solution and incubate for 1 h, wash and centrifuge, discard the supernatant, and finally redisperse in 1 ml of preservation solution to obtain immunolabeled Raman magnetic beads.

[0072] (3) Preparation of immunocomplex sandwich structure: Mix 20 μl of the immunolabeled Raman signal gold nanoparticles prepared with Au@IR808@antibody, 300 μl of SARS-CoV-2 N protein (0.1 pg / ml, 1 pg / ml, 5 pg / ml, 50 pg / ml) prepared with different concentration diluents, and immunomagnetic beads, incubate for 5 min to form a "Raman signal gold - antigen - magnetic bead" immunocomplex. After magnetic separation, wash twice with PBS-T, concentrate and make up the volume to a small volume of 10 μl. Figure 5 It is the schematic diagram of the immunocomplex sandwich structure of the "Raman signal gold - antigen - magnetic bead" reaction mode.

[0073] (4) Detection of SARS-CoV-2 N protein: Take out the small volume immunocomplex sandwich solution and drop it on a silicon wafer coated with a gold film, and directly perform Raman detection with a portable Raman spectrometer. The Raman spectrum results of the rapid detection of SARS-CoV-2 N protein can be obtained in about 15 min, as Figure 6 .

[0074] From Figure 6It can be seen that the detection limit of the "Raman signal gold - antigen - magnetic bead" reaction mode for detecting the recombinant N protein of the novel coronavirus in a standard buffer solution (standard system) is 1 pg / m. Compared with the results of Example 1, the detection limit in Example 1 is 0.1 pg / ml, and in Comparative Example 1 it is 1 pg / ml. Example 1 is one order of magnitude higher than Comparative Example 1.

[0075] Comparative Example 2

[0076] (1) Preparation of immunized Raman signal gold nanoparticles: Mix 10 ml of gold nanoparticles with 20 μl of 0.5 mM IR808 Raman signal molecules under stirring, incubate at room temperature for 10 min, centrifuge at 6000 rpm, discard the supernatant, wash once with 10 ml of BB buffer and make up the volume. Add 50 μl of 5 mM EDC ((1 - ethyl - 3 - [3 - dimethylaminopropyl] carbodiimide hydrochloride) and NHS (N - hydroxysuccinimide) solutions for activation, wash with 10 ml of BB buffer after centrifugation, and make up the volume with 5 ml of BB buffer. Add 50 μg of SARS - CoV - 2 virus N protein antibody Ab1, incubate at 4°C overnight for binding, centrifuge, discard the supernatant, add 10 ml of blocking solution and incubate for 1 h, wash and centrifuge, discard the supernatant, and finally redisperse in 10 ml of storage solution to obtain immunized Raman signal gold nanoparticles.

[0077] (2) Preparation of immunomagnetic beads: Incubate 100 μl of 10 mg / ml carboxylated magnetic beads with 50 μg of SARS - CoV - 2 virus N protein antibody Ab2 at 4°C overnight for binding, centrifuge, discard the supernatant, add 10 ml of blocking solution and incubate for 1 h, wash and centrifuge, discard the supernatant, and finally redisperse in 1 ml of storage solution to obtain immunized Raman magnetic beads.

[0078] (3) Preparation of the immunocomplex sandwich structure: Mix 20 μl of the immunized Raman signal gold nanoparticles prepared with Au@IR808@antibody, 300 μl of the SARS - CoV - 2 N protein in different concentrations of throat swab sampling fluid (0 pg / ml, 10 pg / ml, 100 pg / ml), and immunomagnetic beads, incubate for 5 min to form a "Raman signal gold - antigen - magnetic bead" immunocomplex. After magnetic separation, wash twice with PBS - T, concentrate and make up the volume to a small volume of 10 μl.

[0079] (4) Detection of the throat swab simulated sample of the SARS - CoV - 2 N protein: Take out the small - volume immunocomplex sandwich solution and drop it on a silicon wafer coated with a gold film, and directly perform Raman detection with a portable Raman spectrometer. The rapid detection Raman spectrum result of the SARS - CoV - 2 N protein appears in 15 min, as Figure 7 .

[0080] From Figure 7 It can be seen that the detection limit of the "Raman signal gold-antigen-magnetic bead" reaction method for detecting the recombinant N protein of the novel coronavirus in a simulated throat swab sample of the novel coronavirus is 10 pg / ml. Compared with the results of Example 2, the detection limit of Example 2 (simulated throat swab sample) is 1 pg / ml, which is one order of magnitude more sensitive than Comparative Example 2.

[0081] The above embodiments are only the optimized implementation methods of the present invention, used to exemplarily illustrate the principles and effects of the present invention, rather than to limit the present invention. It should be noted that for any person skilled in the art, without departing from the spirit and scope of the present invention, modifications to the above embodiments should also be regarded as within the protection scope of the present invention.

Claims

1. A method for detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy for non-diagnostic and non-therapeutic purposes, characterized in that, it comprises the following steps: S1: Coupling Raman signal molecules and the SARS-CoV-2 virus N protein antibody Ab1 to gold nanoparticles respectively to prepare immune Raman signal gold nanoparticles of Au@IR808@antibody, and the Raman signal molecule is a near-infrared indocyanine dye IR808; S2: Coupling biotin with antibody Ab2 to prepare biotinylated antibody Ab2; S3: Mixing and incubating the immune Raman signal gold nanoparticles prepared in step S1 with the biotinylated antibody Ab2 prepared in step S2 and the dilution of the N protein of novel coronavirus, and then mixing and incubating with streptavidin-coated magnetic beads to obtain an immune sandwich complex; S4: Magnetically separating and washing the immune sandwich complex in step S3 with a washing solution, and finally concentrating and fixing the volume to a small volume; S5: Dropping the small-volume liquid in S4 onto a surface-enhanced Raman chip for Raman detection.

2. A method for detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy for non-diagnostic and non-therapeutic purposes according to claim 1, characterized in that, step S1 includes: mixing gold nanoparticles with Raman signal molecules under stirring, incubating at room temperature, centrifuging, discarding the supernatant, washing once with a buffer solution and fixing the volume, adding EDC and NHS solution for reaction activation, centrifuging and washing, fixing the volume with a buffer solution, adding the SARS-CoV-2 virus N protein antibody Ab1, incubating overnight for binding, centrifuging, discarding the supernatant, adding a blocking solution for incubation, washing and centrifuging, discarding the supernatant, and finally redispersing in a preservation solution to prepare immune Raman signal gold nanoparticles.

3. A method for detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy for non-diagnostic and non-therapeutic purposes according to claim 2, characterized in that, the buffer solution is prepared from borax and sodium hydroxide, with a pH of 7.2 - 7.6; the particle size of the gold nanoparticles is 30 - 80 nm.

4. A method for detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy for non-diagnostic and non-therapeutic purposes according to claim 2, characterized in that, the reaction activation time of EDC and NHS is 10 - 60 min, the activation temperature is room temperature, the incubation binding time is 10 - 24 h, and the incubation temperature is 2 - 8 °C; the components of the blocking solution include PBS salt solution, bovine serum albumin, casein, and skim milk powder; the components of the preservation solution include PBS salt solution, bovine serum albumin, casein, glycine, and skim milk powder; the blocking time of the blocking solution is 2 - 12 h, and the blocking temperature is room temperature.

5. A method for detecting the N protein of novel coronavirus based on surface-enhanced Raman spectroscopy for non-diagnostic and non-therapeutic purposes according to claim 1, characterized in that, In step S1, the immunological Raman signal gold nanoparticles have Raman signal expression and specific recognition antibodies for the SARS-CoV-2 N protein; in step S1, the Raman signal molecule is one of near-infrared indocyanine dyes IR808, Nile blue A dye, and indocyanine green, and the incubation time with gold nanoparticles is 5 - 30 min.

6. A method for detecting the SARS-CoV-2 N protein based on surface-enhanced Raman spectroscopy for non-disease diagnosis and treatment purposes as described in claim 1, characterized in that, in step S1, the SARS-CoV-2 virus N protein antibody Ab1 is the SARS-CoV-2 NP-mAb, and in step S2, the antibody Ab2 added to the biotinylated antibody is the SARS-CoV-2 neutralizing antibody SARS-CoV-2 NP-mAb.

7. A method for detecting the SARS-CoV-2 N protein based on surface-enhanced Raman spectroscopy for non-disease diagnosis and treatment purposes as described in claim 1, characterized in that, the preparation method of the biotinylated antibody in step S2 is to mix the antibody with biotin for 2 - 6 h and then dialyze in PBS solution for 5 - 30 h; the composition of the SARS-CoV-2 N protein diluent in step S3 is 10 mM PBS, pH 7.2 - 7.4, 0.01% - 1% Triton X-100, 0.05% - 5% EDTA, 0.05 - 5% Tween-20.

8. A method for detecting the SARS-CoV-2 N protein based on surface-enhanced Raman spectroscopy for non-disease diagnosis and treatment purposes as described in claim 1, characterized in that, the biotinylated antibody in steps S2 and S3 is an antibody with biotin-modifying function; the streptavidinylated magnetic beads in step S3 have magnetism and are modified with streptavidin.

9. A method for detecting the SARS-CoV-2 N protein based on surface-enhanced Raman spectroscopy for non-disease diagnosis and treatment purposes as described in claim 1, characterized in that, the fixed solution in step S4 is PBS, and the washing solution is PBS-T prepared by adding 0.01% - 1% Tween-20 to the PBS solution; the number of washing times in step S4 is 1 - 5 times, and the small volume is 3 - 30 ul.

10. A method for detecting the SARS-CoV-2 N protein based on surface-enhanced Raman spectroscopy for non-disease diagnosis and treatment purposes as described in claim 1, characterized in that, the surface-enhanced Raman chip in step S5 is a silicon wafer coated with a layer of gold film; the Raman detection instrument in step S5 is one of a portable Raman spectrometer, a confocal Raman spectrometer, and a handheld Raman spectrometer, and the excitation wavelength is 785 nm.

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