A method for detecting NDKA protein by a kind of immunosorbent SERS sandwich assay

Abstract

The present invention provides a method for detecting NDKA protein by an immunosorbent SERS sandwich method, which is as follows: Sodium citrate aqueous solution is added to water, and then HAuCl4 aqueous solution and AgNO3 aqueous solution are added. After incubation, the mixture is stirred and refluxed at 100 °C to obtain a mixed solution A containing Au 20 NPs; After adding chloroform to the mixed solution A containing Au 20 NPs and shaking, acetone is added, and a gold film is self-assembled on the liquid surface and transferred to an amino-functionalized silicon wafer. Then, it is immersed in a solution containing NDKA protein and polydopamine to obtain a SERS active substrate; A mixed solution B containing Au 45 NPs is prepared, added to 4-mercaptobenzoic acid and centrifuged to obtain Au@4-MBA; NDKA antibody is added to Au@4-MBA, incubated and centrifuged. After the precipitate is blocked and centrifuged, the precipitate is dispersed in ultrapure water to obtain a SERS label; First, the test NDKA protein solution is dropped on the SERS active substrate, and then the SERS label is dropped, and Raman spectrometer detection is carried out. The present invention can specifically detect NDKA protein, with high sensitivity and simple operation.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein detection, and particularly relates to a method for detecting NDKA protein by an immunosorbent SERS sandwich method. Background Art

[0002] Colorectal cancer (CRC) is one of the most common cancers, with more than 1.3 million new cases worldwide each year, and it is a growing public health problem. Research shows that early diagnosis of colorectal cancer can significantly improve the survival rate of CRC patients and reduce complications. Currently, colonoscopy is considered the most accurate test for early detection of cancer and clinical adenomas, but due to the unbearable pain during the examination, many people cannot adhere to colonoscopy, resulting in a low survival rate for this cancer.

[0003] Therefore, it is urgent to develop a rapid, convenient, non-invasive and accurate detection method to improve the survival rate and quality of life of colorectal cancer patients.

[0004] To effectively reduce the death rate of CRC, early detection of tumor markers is of great significance for early detection, efficacy monitoring and prognosis judgment of tumors. Currently, there is a lack of specific tumor markers for CRC diagnosis. By analyzing the tumor database Oncomine database, we found that the content and changes of members of the Nucleosides Diphosphate Kinase (NDK) family can be very important tumor markers for CRC diagnosis.

[0005] Research has confirmed that CRC tumor tissues have higher expression levels of NDKA protein and mRNA than normal tissues. The higher the degree of CRC tumor differentiation, the higher the expression level of NDKA. NDKA can be a new clinically valuable marker for CRC. In CRC, by detecting the ratio of the expression levels of each member of the NDK family, the change process of colon cancer can be dynamically predicted. Therefore, the change in the content of NDKA can be an important tumor marker for early screening, prediction, dynamic monitoring and diagnosis of CRC.

[0006] In the existing technologies, the methods commonly used for detecting proteins mainly include enzyme immunoassay, chemiluminescence immunoassay and radioimmunoassay. Firstly, these methods have poor specificity and sensitivity; secondly, the operation is cumbersome and accurate quantification cannot be achieved; the content and changes of multiple family members cannot be analyzed simultaneously; moreover, the cost is relatively high; in addition, there are many interfering factors in the detection process, such as temperature and reaction time, which are likely to cause result distortion. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting NDKA protein by an immunosimilar SERS sandwich method in view of the deficiencies of the above-mentioned prior art. The detection limit of this method is 1 pg / mL, which is 5 orders of magnitude higher than the label-free detection method. Secondly, the immunosimilar SERS sandwich method can specifically detect NDKA protein and has no specificity for other proteins in serum such as gamma-globulin and serum albumin. In addition, the detection method of the present invention is rapid, convenient and simple to operate. Only by dropping the sample on the detection substrate and then dropping the SERS label and incubating them together, Raman detection can be carried out.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is: a method for detecting NDKA protein by an immunosimilar SERS sandwich method, and the method is as follows:

[0009] S1. Add an aqueous solution of sodium citrate with a mass fraction of 1% to water, then add an aqueous solution of HAuCl4 with a mass fraction of 1% and an aqueous solution of AgNO3 with a mass fraction of 0.1%. After incubating for 4 min to 5 min, a mixed solution A is obtained. Stir and reflux the mixed solution A under the condition of a water bath at a temperature of 100 °C for 30 min to 35 min. The color of the solution changes from dark purple to wine red, and a mixed solution A containing gold nanoparticles is obtained. The average particle size of the gold nanoparticles in the mixed solution A containing gold nanoparticles is 20 nm. The gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au 20 NPs; the volume ratio of the aqueous solution of sodium citrate with a mass fraction of 1%, water, the aqueous solution of HAuCl4 with a mass fraction of 1% and the aqueous solution of AgNO3 with a mass fraction of 0.1% is: 0.9:0.85:0.75:0.0425;

[0010] S2. Add chloroform to the mixed solution A containing gold nanoparticles obtained in S1. After shaking, add acetone along the tube wall, and self-assemble to form a gold film on the liquid surface. Transfer the gold film to an aminated silicon wafer to obtain a silicon wafer loaded with Au 20 NPs;

[0011] The treatment method of the aminated silicon wafer is as follows: First, ultrasonically clean a 5 mm × 5 mm silicon wafer with acetone for 10 min, then treat it with a plasma cleaner for more than ten seconds to make its surface hydrophilic, and finally perform an oil bath with a 1% APTES alcohol solution for 6 h to obtain an aminated silicon wafer;

[0012] S3. Immerse the silicon wafer loaded with Au 20 NPs obtained in S2 in a solution containing NDKA protein and polydopamine, soak for 4 h to 5 h, take it out and dry it naturally to obtain a SERS active substrate;

[0013] The NDKA protein is commercially available and purchased from Huamei Biological Engineering Co., Ltd.

[0014] S4. Add an aqueous solution of sodium citrate with a mass fraction of 1% to water, then add an aqueous solution of HAuCl4 with a mass fraction of 1% and an aqueous solution of AgNO3 with a mass fraction of 0.1%. After incubating for 5 min to 6 min, a mixed solution B is obtained. Stir and reflux the mixed solution B under the condition of a water bath at a temperature of 100 °C for 30 min to 35 min to obtain a mixed solution B containing gold nanoparticles; the average particle size of the gold nanoparticles in the mixed solution B containing gold nanoparticles is 45 nm, and the gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au 45 NPs; the volume ratio of the aqueous solution of sodium citrate with a mass fraction of 1%, water, the aqueous solution of HAuCl4 with a mass fraction of 1%, and the aqueous solution of AgNO3 with a mass fraction of 0.1% is: 0.5:0.5:0.5:0.0425;

[0015] S5. Add the mixed solution B containing gold nanoparticles obtained in S4 to 4-mercaptobenzoic acid. After stirring evenly at room temperature and centrifuging, ultrasonically disperse the precipitate in water, and the obtained substance is named Au@4-MBA;

[0016] S6. Add the NDKA antibody to the Au@4-MBA obtained in S5. After incubating at a temperature of 4 °C for 5 h to 6 h and centrifuging, block the precipitate with a 5% bovine serum albumin solution, then centrifuge again, and disperse the precipitate in ultrapure water to obtain a SERS tag;

[0017] The NDKA antibody is commercially available and purchased from Huamei Biological Engineering Co., Ltd.;

[0018] S7. First, drop the test NDKA protein solution onto the SERS active substrate obtained in S3, and then drop the SERS tag obtained in S6 to form a "sandwich-like" immune sandwich structure among the SERS immunosubstrate (SERS active substrate), the target protein (test NDKA protein solution), and the SERS tag. Perform Raman spectrometer detection, and qualitatively and semi-quantitatively detect the type and content of NDKA protein in the test NDKA protein solution according to the signal intensity of the Raman reporter molecule.

[0019] Preferably, the volume ratio of the mixed solution A containing gold nanoparticles, chloroform, and acetone in S2 is 1:5:1.

[0020] Preferably, the solution containing NDKA protein and PDA in S3 is composed of NDKA protein, polydopamine, and water with a dosage ratio of 1 mg:5 mg:10 mL.

[0021] Preferably, the volume ratio of the mixed solution B containing gold nanoparticles to 4-mercaptobenzoic acid in S5 is 1:9; the dosage ratio of the precipitate to water in S5 is 5 mg:1 mL.

[0022] Preferably, the rotation speed of centrifugation in S5 and S6 is 6000 rpm to 8000 rpm, and the centrifugation time is 10 min to 15 min.

[0023] Preferably, the volume ratio of the NDKA antibody to Au@4-MBA in S6 is 3:100.

[0024] Preferably, the dosage ratio of the precipitate used in the SERS tag to ultrapure water in S6 is 1 mg:1 mL.

[0025] Preferably, the detection limit of the protein solution of NDKA to be detected in S7 is 1 pg / mL.

[0026] The present invention has the following advantages compared with the prior art:

[0027] 1. The present invention adopts an immunolike sandwich technique with labeled molecules. The detection substrate is a gold film coated with polydopamine. The signal of NDKA protein is detected through the signal of the Raman reporter molecule on the SERS tag. It can not only specifically detect NDKA protein, but also be used for semi-quantitative detection of the concentration change of NDKA protein molecules.

[0028] 2. The immunolike SERS sandwich method for detecting NDKA protein in the present invention has high sensitivity, and the detection limit is 1 pg / mL, which is 5 orders of magnitude higher than the label-free detection method; secondly, the immunolike SERS sandwich method can specifically detect NDKA protein and has no specificity for other proteins in serum such as γ-globulin and serum albumin; in addition, the detection method of the present invention is fast, convenient and simple to operate. Only need to drop the sample on the detection substrate, and then drop the SERS tag and incubate together, then Raman detection can be carried out.

[0029] The present invention will be further described in detail below with reference to the drawings and embodiments. Description of the Drawings

[0030] Figure 1 It is a schematic diagram for preparing the SERS tag of Embodiment 1 of the present invention.

[0031] Figure 2 It is a schematic diagram of the immunolike sandwich detection process of Embodiment 1 of the present invention.

[0032] Figure 3 It is a Raman spectrogram for detecting NDKA in the serum of Embodiment 1 of the present invention.

[0033] Figure 4It is a specific detection diagram of other proteins and NDKA protein in serum in Embodiment 1 of the present invention. Detailed implementation mode

[0034] Embodiment 1

[0035] The method for detecting NDKA protein by the immunological SERS sandwich method in this embodiment is as follows:

[0036] S1. Add an aqueous solution of trisodium citrate with a mass fraction of 1% to water, then add an aqueous solution of HAuCl4 with a mass fraction of 1% and an aqueous solution of AgNO3 with a mass fraction of 0.1%. After incubation for 4 min, a mixed solution A is obtained. Stir and reflux the mixed solution A under the condition of a water bath at 100 °C for 30 min. The color of the solution changes from dark purple to wine red, and a mixed solution A containing gold nanoparticles is obtained. The average particle size of the gold nanoparticles in the mixed solution A containing gold nanoparticles is 20 nm. The gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au 20 NPs; the volume ratio of the aqueous solution of trisodium citrate with a mass fraction of 1%, water, the aqueous solution of HAuCl4 with a mass fraction of 1%, and the aqueous solution of AgNO3 with a mass fraction of 0.1% is: 0.9:0.85:0.75:0.0425;

[0037] S2. Add chloroform to the mixed solution A containing gold nanoparticles obtained in S1. After shaking, add acetone along the tube wall, and self-assemble to form a gold film on the liquid surface. Transfer the gold film to an aminated silicon wafer to obtain a silicon wafer loaded with Au 20 NPs; the volume ratio of the mixed solution A containing gold nanoparticles, chloroform, and acetone is 1:5:1;

[0038] The treatment method of the aminated silicon wafer is as follows: First, ultrasonically clean a 5 mm × 5 mm silicon wafer with acetone for 10 min, then treat it with a plasma cleaner for more than ten seconds to make its surface hydrophilic, and finally perform an oil bath with a 1% APTES alcohol solution for 6 h to obtain an aminated silicon wafer;

[0039] S3. Immerse the silicon wafer loaded with Au 20 NPs obtained in S2 in a solution containing NDKA protein and polydopamine for 4 h. After taking it out, let it dry naturally to obtain a SERS active substrate; the solution containing NDKA protein and PDA is composed of NDKA protein, polydopamine, and water with a dosage ratio of 1 mg:5 mg:10 mL;

[0040] The NDKA protein is commercially available and purchased from Huamei Biological Engineering Co., Ltd.;

[0041] S4, adding a 1% sodium citrate aqueous solution to water, then adding a 1% HAuCl4 aqueous solution and a 0.1% AgNO3 aqueous solution, incubating for 5 minutes, obtaining a mixed solution B, stirring and refluxing the mixed solution B in a water bath at a temperature of 100° C. for 30 minutes, to obtain a mixed solution B containing gold nanoparticles; the average particle size of the gold nanoparticles in the mixed solution B containing gold nanoparticles is 45 nm, and the gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au. 45 NPs; the volume ratio of the 1% trisodium citrate aqueous solution, water, 1% HAuCl4 aqueous solution and 0.1% AgNO3 aqueous solution is: 0.5:0.5:0.5:0.0425;

[0042] S5. Add the mixed solution B containing gold nanoparticles obtained in S4 to 4-mercaptobenzoic acid, stir evenly at room temperature, centrifuge at a speed of 6000 rpm for 15 minutes, and ultrasonically disperse the precipitate into water. The obtained substance is named Au@4-MBA; the volume ratio of the mixed solution B containing gold nanoparticles to 4-mercaptobenzoic acid is 1:9; the dosage ratio of the precipitate to water is 5 mg:1 mL;

[0043] S6, adding NDKA antibody to Au@4-MBA obtained in S5, incubating at 4°C for 5-6 hours, centrifuging at 6000 rpm for 15 minutes, blocking the precipitate with 5% bovine serum albumin solution, and then centrifuging again, dispersing the precipitate in ultrapure water to obtain a SERS tag; the volume ratio of the NDKA antibody to Au@4-MBA is 3:100; the amount ratio of the precipitate to ultrapure water used in the SERS tag is 1 mg:1 mL;

[0044] The NDKA antibody was purchased from Huamei Bioengineering Co., Ltd.; ( Figure 1 Schematic diagram of SERS tag preparation, i.e. S1-S6)

[0045] S7, first drip the NDKA protein solution to be tested onto the SERS active substrate obtained in S3, and then drip the SERS label obtained in S6, so that a "sandwich-like" immune sandwich structure is formed between the SERS immune substrate (SERS active substrate), the target protein (NDKA protein solution to be tested) and the SERS label, and Raman spectrometer detection is performed, and the type and content of the NDKA protein in the NDKA protein solution to be tested are qualitatively and semi-quantitatively detected according to the signal intensity of the Raman reporter molecule; the detection limit of the NDKA protein solution to be tested is 1pg / mL. ( Figure 2 Schematic diagram of the immune sandwich detection process, i.e. S7)

[0046] Figure 3 is the Raman spectrum of the detection of NDKA in the serum of this example. It can be seen from the figure that in the concentration range of 1 pg / mL to 1 mg / mL, as the concentration of NDKA protein increases, the detected SERS intensity also increases, and there is a certain linear relationship at the Raman characteristic peak of 1077 cm -1 .

[0047] Figure 4 is the specific detection diagram of other proteins and NDKA protein in the serum of this embodiment of the present invention.

[0048] Experimental content and method: Drop the γ-globulin solution, serum protein solution and the to-be-detected NDKA protein solution on the SERS active substrate obtained in S3, and then drop the SERS label obtained in S6 to form a "quasi-sandwich" immune sandwich structure among the SERS immune substrate (SERS active substrate), the target protein (to-be-detected NDKA protein solution) and the SERS label, and perform Raman spectrometer detection.

[0049] As Figure 4 can be seen, even at a concentration of 100 pg / ml, the NDKA protein still has a strong SERS signal. For γ-globulin and serum protein, even at a concentration of 10 μg / ml, the SERS signal is still very weak. This indicates that the construction of the SERS-based immune sandwich structure fails, and at the same time, it can also be seen that the proposed SERS-based biosensor has high specificity and selectivity for the NDKA protein.

[0050] The sensitivity of detecting NDKA protein by the quasi-immune SERS sandwich method in this example is high, and the detection limit is 1 pg / mL, which is 5 orders of magnitude higher than the label-free detection method; secondly, the quasi-immune SERS sandwich method can specifically detect the NDKA protein and has no specificity for other proteins in the serum such as γ-globulin and serum protein; in addition, the detection method of this example is fast, convenient and simple to operate. Only need to drop the sample on the detection substrate, then drop the SERS label and incubate together, and then Raman detection can be carried out.

[0051] Example 2

[0052] The method for detecting NDKA protein by the quasi-immune SERS sandwich method in this example is as follows:

[0053] S1. Add an aqueous solution of trisodium citrate with a mass fraction of 1% to water, then add an aqueous solution of HAuCl4 with a mass fraction of 1% and an aqueous solution of AgNO3 with a mass fraction of 0.1%. After incubating for 5 min, a mixed solution A is obtained. Stir and reflux the mixed solution A under the condition of a water bath at a temperature of 100 °C for 35 min. The color of the solution changes from dark purple to wine red, and a mixed solution A containing gold nanoparticles is obtained. The average particle size of the gold nanoparticles in the mixed solution A containing gold nanoparticles is 20 nm. The gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au 20 NPs; The volume ratio of the aqueous solution of trisodium citrate with a mass fraction of 1%, water, the aqueous solution of HAuCl4 with a mass fraction of 1%, and the aqueous solution of AgNO3 with a mass fraction of 0.1% is: 0.9:0.85:0.75:0.0425;

[0054] S2. Add chloroform to the mixed solution A containing gold nanoparticles obtained in S1. After shaking, add acetone along the tube wall, and self-assemble to form a gold film on the liquid surface. Transfer the gold film to an aminated silicon wafer to obtain a silicon wafer loaded with Au 20 NPs; The volume ratio of the mixed solution A containing gold nanoparticles, chloroform, and acetone is 1:5:1;

[0055] The treatment method of the aminated silicon wafer is as follows: First, ultrasonically clean a 5 mm × 5 mm silicon wafer with acetone for 10 min, then treat it with a plasma cleaner for more than ten seconds to make its surface hydrophilic, and finally carry out an oil bath with a 1% APTES alcohol solution for 6 h to obtain an aminated silicon wafer;

[0056] S3. Immerse the silicon wafer loaded with Au 20 NPs obtained in S2 in a solution containing NDKA protein and polydopamine for 5 h. After taking it out, let it dry naturally to obtain a SERS active substrate; The solution containing NDKA protein and PDA is composed of NDKA protein, polydopamine, and water with a dosage ratio of 1 mg:5 mg:10 mL;

[0057] The NDKA protein is commercially available and purchased from Huamei Bio-Engineering Co., Ltd.;

[0058] S4. Add an aqueous solution of trisodium citrate with a mass fraction of 1% to water, then add an aqueous solution of HAuCl4 with a mass fraction of 1% and an aqueous solution of AgNO3 with a mass fraction of 0.1%. After incubating for 6 min, a mixed solution B is obtained. Stir and reflux the mixed solution B under the condition of a water bath at a temperature of 100 °C for 35 min to obtain a mixed solution B containing gold nanoparticles. The average particle size of the gold nanoparticles in the mixed solution B containing gold nanoparticles is 45 nm. The gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au 45NPs; The volume ratio of the 1% sodium citrate aqueous solution, water, the 1% HAuCl4 aqueous solution, and the 0.1% AgNO3 aqueous solution is: 0.5:0.5:0.5:0.0425;

[0059] S5. Add the obtained mixture B containing gold nanoparticles in S4 to 4-mercaptobenzoic acid. After stirring evenly at room temperature, centrifuge at 8000 rpm for 10 min, and then ultrasonically disperse the precipitate in water. The obtained substance is named Au@4-MBA; The volume ratio of the mixture B containing gold nanoparticles and 4-mercaptobenzoic acid is 1:9; The dosage ratio of the precipitate and water is 5 mg:1 mL;

[0060] S6. Add the NDKA antibody to the Au@4-MBA obtained in S5. After incubating at 4 °C for 5 h to 6 h, centrifuge at 8000 rpm for 10 min. Block the precipitate with a 5% bovine serum albumin solution, then centrifuge again, and disperse the precipitate in ultrapure water to obtain the SERS label; The volume ratio of the NDKA antibody and Au@4-MBA is 3:100; The dosage ratio of the precipitate used in the SERS label and ultrapure water is 1 mg:1 mL;

[0061] The NDKA antibody is commercially available and purchased from Huamei Biological Engineering Co., Ltd.;

[0062] S7. First, drop the test NDKA protein solution on the SERS active substrate obtained in S3, and then drop the SERS label obtained in S6 to form a "sandwich-like" immune sandwich structure among the SERS immunosubstrate (SERS active substrate), the target protein (test NDKA protein solution), and the SERS label. Perform Raman spectrometer detection, and qualitatively and semi-quantitatively detect the type and content of NDKA protein in the test NDKA protein solution according to the signal intensity of the Raman reporter molecule; The detection limit of the test NDKA protein solution is 1 pg / mL.

[0063] The above is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change, and equivalent change made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting NDKA protein by a quasi-immuno SERS sandwich assay, characterized in that, The method is: S1. Add an aqueous solution of trisodium citrate with a mass fraction of 1% to water, then add an aqueous solution of HAuCl4 with a mass fraction of 1% and an aqueous solution of AgNO3 with a mass fraction of 0.1%. After incubating for 4 min to 5 min, obtain a mixed solution A. Stir and reflux the mixed solution A under the condition of a water bath at a temperature of 100 °C for 30 min to 35 min to obtain a mixed solution A containing gold nanoparticles; the average particle size of the gold nanoparticles in the mixed solution A containing gold nanoparticles is 20 nm, and the gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au 20 NPs; the volume ratio of the aqueous solution of trisodium citrate with a mass fraction of 1%, water, the aqueous solution of HAuCl4 with a mass fraction of 1%, and the aqueous solution of AgNO3 with a mass fraction of 0.1% is: 0.9:0.85:0.75:0.0425; S2. Add chloroform to the mixed solution A containing gold nanoparticles obtained in S1. After shaking, add acetone along the tube wall, and perform self-assembly on the liquid surface to form a gold film. Transfer the gold film to an aminated silicon wafer to obtain a silicon wafer loaded with Au 20 NPs; S3. Immerse the silicon wafer with Au 20 NPs loaded in S2 into a solution containing NDKA protein and polydopamine, soak for 4 h to 5 h, take it out and air-dry naturally to obtain a SERS active substrate; S4. Add an aqueous solution of trisodium citrate with a mass fraction of 1% to water, then add an aqueous solution of HAuCl4 with a mass fraction of 1% and an aqueous solution of AgNO3 with a mass fraction of 0.1%. After incubating for 5 min to 6 min, a mixed solution B is obtained. Stir and reflux the mixed solution B under the condition of a water bath at a temperature of 100 °C for 30 min to 35 min to obtain a mixed solution B containing gold nanoparticles. The average particle size of the gold nanoparticles in the mixed solution B containing gold nanoparticles is 45 nm, and the gold nanoparticles in the mixed solution containing gold nanoparticles are denoted as Au 45 NPs; The volume ratio of the aqueous solution of trisodium citrate with a mass fraction of 1%, water, the aqueous solution of HAuCl4 with a mass fraction of 1% and the aqueous solution of AgNO3 with a mass fraction of 0.1% is: 0.5:0.5:0.5:0.0425; S5, adding the mixed solution B containing gold nanoparticles obtained in S4 to 4-mercaptobenzoic acid, stirring evenly at room temperature, centrifuging, and then ultrasonically dispersing the precipitate into water. The obtained substance is named Au@4-MBA; S6, adding NDKA antibody to the Au@4-MBA obtained in S5, incubating at 4°C for 5-6 hours and then centrifuging, blocking the precipitate with 5% bovine serum albumin solution, and then centrifuging again, dispersing the precipitate in ultrapure water to obtain a SERS tag; S7. First, drop the NDKA protein solution to be tested onto the SERS active substrate obtained in S3, and then drop the SERS label obtained in S6, and perform Raman spectrometer detection. According to the signal intensity of the Raman reporter molecule, the type and content of the NDKA protein in the NDKA protein solution to be tested are qualitatively and semi-quantitatively detected.

2. The method for detecting NDKA protein by a kind of immunosorbent SERS sandwich method according to claim 1, wherein, The volume ratio of the mixed solution A containing gold nanoparticles, chloroform and acetone in S2 is 1:5:

1.

3. The method for detecting NDKA protein by a kind of immune SERS sandwich method according to claim 1, characterized in that The solution containing NDKA protein and PDA described in S3 is composed of NDKA protein, PDA and water in a dosage ratio of 1 mg:5 mg:10 mL.

4. The method for detecting NDKA protein by a kind of immune SERS sandwich method according to claim 1, characterized in that, The volume ratio of the mixed solution B containing gold nanoparticles and 4-mercaptobenzoic acid in S5 is 1:9; the dosage ratio of the precipitate and water in S5 is 5 mg:1 mL.

5. The method for detecting NDKA protein by a kind of immune-like SERS sandwich method according to claim 1, wherein, The centrifugal speed in S5 and S6 is 6000 rpm to 8000 rpm, and the centrifugal time is 10 min to 15 min.

6. The method for detecting NDKA protein by a kind of immune-like SERS sandwich method according to claim 1, wherein, The volume ratio of NDKA antibody and Au@4-MBA described in S6 was 3:

100.

7. A method for detecting NDKA protein by a kind of immune-like SERS sandwich method according to claim 1, characterized in that The amount ratio of the precipitate and ultrapure water used in the SERS tag described in S6 is 1 mg:1 mL.

8. The method for detecting NDKA protein by a kind of immune-like SERS sandwich method according to claim 1, wherein, The detection limit of the NDKA protein solution to be tested described in S7 is 1 pg / mL.

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