SERS detection system and preparation method and application thereof

Abstract

The invention relates to an SERS detection system and a preparation method and application thereof. The preparation method of the SERS detection system comprises the following steps: pretreating a silicon wafer, and culturing in a PDA-containing MIF antibody solution to prepare a PDA chip; the HAuCl4 solution is added into the NaBH4 solution in batches and stirred, and gold nanoparticles AuNPs are prepared; the preparation method comprises the following steps: mixing ascorbic acid, a silver nitrate solution, a Tris-HCl buffer solution, 4-aminobenzenethiol and a bovine serum albumin solution, and adding gold nanoparticles AuNPs to prepare Au-Ag nanoparticles with Au as a core and Ag as a shell; the MIF antibody solution and the PDA solution are mixed and then react with the Au-Ag nanoparticles, and the SERS sensor is prepared; and the PDA chip and the SERS sensor jointly form an SERS detection system. The SERS detection system disclosed by the invention can be used for rapidly and accurately detecting the MIF in the clinical PRP sample at low cost.

Description

Technical Field

[0001] The invention relates to a SERS detection system and a preparation method and application thereof. The SERS detection system can quickly detect macrophage migration factor in platelet-rich plasma and belongs to the field of novel nano functional material preparation and SERS technology. Background Art

[0002] Osteoarthritis (OA) is a common chronic degenerative disease that usually affects the knee joint, causing joint pain, stiffness, and swelling. It has become one of the most common joint diseases in the elderly and can lead to disability. Recently, biological therapy has become a promising method for treating musculoskeletal diseases in the elderly, and platelet-rich plasma (PRP) technology has become an important form of autogenous therapy to promote tissue repair and regeneration. However, improper preparation of PRP may lead to the extraction of concentrated red blood cells, which may cause a large release of the inflammatory factor macrophage migration inhibitory factor (MIF) and aggravate pain and osteoarthritis symptoms. Although red blood cell filtration devices have been developed, they are expensive and cannot completely filter blood at one time. In addition, traditional techniques such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are costly and take a long time to analyze, which may affect the efficacy of PRP.

[0003] Surface-enhanced Raman scattering (SERS) is an analytical technique for characterizing substances adsorbed on rough noble metal surfaces at the molecular level. It is mainly based on the abnormal optical enhancement effect of nanoscale rough surfaces. It has the advantages of high sensitivity, short detection time, low water interference, direct in-situ analysis, wide detection range, and no need for complex pre-treatment processes. It has aroused widespread interest among scholars in many fields such as biomedicine, food safety and environmental analysis. In recent years, with the development of fiber optic sensing technology and the miniaturization of Raman spectrometers, it has become possible to use SERS technology for rapid on-site detection. Based on the advantages of high sensitivity and good signal light stability for the detection of extremely small volume samples, SERS technology has gradually developed into one of the most promising technologies in the field of environmental analysis. For example, the invention patent application with application number 201710855689.X and application publication number CN107727633A discloses a surface enhanced Raman spectroscopy detection platform, including a polydopamine-modified substrate chip (denoted as PDA-chip) and a polydopamine-modified SERS probe (denoted as SERS-PDA Tag); during detection, the exosomes are dripped onto the surface of the PDA-chip and rinsed with a buffer after the reaction, the SERS-PDA Tag is dripped and rinsed with a buffer after the reaction, and then a micro-Raman spectrometer is used for detection. However, there is no rapid detection method for the concentration of MIF in PRP in the existing SERS technology. Therefore, if a technical method for detecting the concentration of MIF in PRP based on SERS technology with low cost, rapidity and high sensitivity can be established, it is possible to avoid the need for expensive red blood cell filtration equipment and maximize the effectiveness of PRP. Summary of the invention

[0004] The main purpose of the present invention is to overcome the problems existing in the prior art and provide a method for preparing a SERS detection system. The obtained SERS detection system can quickly and accurately detect the content of MIF in an ultra-small volume PRP sample, which helps to reduce the side effects of PRP and improve the quality and clinical efficacy of PRP. At the same time, the SERS detection system and related applications are also provided.

[0005] The technical solution of the present invention to solve the technical problem is as follows:

[0006] A method for preparing a SERS detection system comprises the following steps:

[0007] In the first step, the silicon wafer is pre-treated and then cultured in a MIF antibody solution containing PDA to obtain a PDA chip.

[0008] In the second step, the HAuCl4 solution was added to the NaBH4 solution in batches and stirred to obtain a gold nanoparticle AuNPs suspension.

[0009] The third step is to mix the ascorbic acid solution, silver nitrate solution, Tris-HCl buffer, 4-aminobenzenethiol solution, and bovine serum albumin solution, and then add the gold nanoparticle AuNPs suspension. After the reaction, Au-Ag nanoparticles with Au as the core and Ag as the shell are prepared.

[0010] Step 4: Mix the MIF antibody solution with the PDA solution and react with Au-Ag nanoparticles to obtain core-shell nanoparticles with a PDA protective shell and connected to antibodies, which is the SERS sensor; the PDA chip and the SERS sensor together constitute a SERS detection system.

[0011] In the SERS detection system prepared by the preparation method, the PDA chip can capture MIF from the PRP sample, the SERS sensor can be used as a probe for detection, and the strong SERS signal of the Raman reporter molecule is used to perform qualitative and quantitative analysis on the MIF in the PRP.

[0012] The technical solution further improved by the present invention is as follows:

[0013] Preferably, the specific process of the first step is as follows:

[0014] The silicon wafer is soaked in an alkaline tank for 2 to 3 hours, washed with deionized water, then soaked in a piranha solution for 2 to 3 hours, and washed with deionized water; the obtained silicon wafer is cultured in a MIF antibody solution containing PDA for 1 to 2 hours to prepare a PDA chip; the PDA chip is washed with deionized water and stored at 0°C to 4°C for future use.

[0015] More preferably, the solution contained in the alkaline tank is piranha solution; the piranha solution is a mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide solution in a volume ratio of 3±0.5:1; the PDA concentration in the MIF antibody solution containing PDA is 33±5mM.

[0016] After adopting the above preferred scheme, the PDA chip can be better prepared. Among them, the use of a self-assembled dopamine layer for wrapping can increase the density of the captured antibody and maintain its biological activity.

[0017] Preferably, the specific process of the second step is as follows:

[0018] The HAuCl4 solution was added in batches to the continuously stirred ice-cold NaBH4 solution for reaction; after the reaction, the mixture was boiled for 1 to 2 hours to decompose the excess NaBH4; the final volume was adjusted to 5±0.5 times the volume of the above HAuCl4 solution; and a gold nanoparticle AuNPs suspension was prepared.

[0019] More preferably, the concentration of HAuCl4 solution is 5±0.5×10 -3 M, the concentration of NaBH4 solution is 2±0.5×10 - 3 M, the volume ratio of HAuCl4 solution to NaBH4 solution is 1:3±0.5.

[0020] After adopting the above preferred scheme, the specific process of the second step can be further optimized.

[0021] Preferably, the specific process of the third step is as follows:

[0022] Ascorbic acid solution, silver nitrate solution, pH 8.0 Tris-HCl buffer, 4-aminobenzenethiol and bovine serum albumin solution were mixed evenly, and then the gold nanoparticle AuNPs suspension prepared in the second step was added; after reacting for 30 to 60 minutes, the mixture was centrifuged at 5800 to 6500 rpm for 15 to 30 minutes, and the precipitate was resuspended with pH 8.0 Tris-HCl buffer; Au-Ag nanoparticles with Au as the core and Ag as the shell were prepared.

[0023] More preferably, the concentration of the ascorbic acid solution is 0.1±0.01M, the concentration of the silver nitrate solution is 0.096±0.005mM, the concentration of the pH8.0 Tris-HCl buffer is 1±0.1M, the concentration of 4-aminobenzenethiol is 9.6±0.5mM, and the mass concentration of the bovine serum albumin solution is 1±0.1%; the volume ratio of the ascorbic acid solution, the silver nitrate solution, the pH8.0 Tris-HCl buffer, the 4-aminobenzenethiol solution, the bovine serum albumin solution, and the gold nanoparticle AuNPs suspension is 0.2±0.05:0.05±0.01:1±0.1:1±0.1:2±0.2:6±0.5.

[0024] After adopting the above preferred scheme, the specific process of the third step can be further optimized.

[0025] Preferably, the specific process of the fourth step is as follows:

[0026] The MIF antibody solution and the PDA solution were mixed evenly, and the Au-Ag nanoparticles prepared in the third step were added to react for 1 to 2 hours to prepare a SERS sensor; the solution was resuspended after centrifugation and stored in a refrigerator at 0°C to 4°C.

[0027] More preferably, the concentration of the MIF antibody solution is 15±0.5 μg / mL, the concentration of the PDA solution is 5-20 mg / mL; and the volume ratio of the MIF antibody solution to the PDA solution is 1±0.1:1.

[0028] After adopting the above preferred scheme, the specific process of the fourth step can be further optimized. Among them, after the Au-Ag nanoparticles are successfully connected to the antibody, the sensor with the SERS tag is successfully prepared; by making a PDA protective shell of a certain thickness to protect the SERS tag, the activity of the antibody can be protected and the non-specific adsorption of the antibody can be prevented; by adjusting the concentration of the PDA solution (such as 5 mg / mL, 10 mg / mL, 20 mg / mL), PDA protective shells of different thicknesses can be prepared.

[0029] The present invention also provides:

[0030] The SERS detection system was prepared by the preparation method described above.

[0031] The SERS detection system described above is used to detect the content of MIF in PRP samples.

[0032] The present invention develops a SERS detection system for quickly and accurately detecting the content of MIF in ultra-small volume PRP samples. Although Ag nanoparticles (AgNPs) have high SERS activity, they have the disadvantages of uncontrollable size and short-term stability. In order to achieve the stability of SERS tags and the strongest signal amplification, the inventor's research group prepared a gold (core)-silver (shell) structure using the easily controllable size distribution and long-term stability of gold nanoparticles. Polymeric dopamine (PDA) can protect metal nanoparticles from oxidation while maintaining SERS activity due to its good biocompatibility and ability to stabilize SERS signals. MIF is captured from PRP samples by enriching MIF-specific antibodies embedded in PDA on the surface of a Si substrate, and then MIF antibody-gold (shell)-silver (core) nanoparticles labeled with Raman reporter molecules are used as SERS probes. The type and concentration of the antigen can be determined. The strong SERS signal of the Raman reporter molecule is used to qualitatively and quantitatively analyze MIF in PRP. Therefore, the present invention provides a rapid, highly sensitive, and low-cost technical means for detecting MIF in PRP, which helps to reduce the side effects of PRP and improve the quality and clinical efficacy of PRP.

[0033] Compared with the expensive ELISA method, the SERS detection system of the present invention has almost the same accuracy. Without considering the instrument cost, the SERS detection system of the present invention can be economically controlled within the range of US$0.40-0.70, showing a huge economic advantage. In terms of detection time, the detection time required for the SERS detection system of the present invention is only 10s, and since the detection threshold of the ELISA method is very small, it is usually necessary to pre-test the sample dilution. In contrast, Raman detection does not require such pretreatment, thereby establishing its advantages in clinical applications in terms of simplicity and rapidity. In short, the research results of the inventor's research group show that the SERS detection system of the present invention can detect MIF in clinical PRP samples quickly and accurately at low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a STEM image of the Au (core)-Ag (shell) nanoparticles of Example 1 of the present invention.

[0035] Figures 2 to 5 They are respectively the EDS elemental images of the Au (core) -Ag (shell) nanoparticles of Example 1 of the present invention.

[0036] Figure 6 This is a schematic diagram of ELISA detection in Example 2 of the present invention.

[0037] Figure 7 It is a comparative schematic diagram of Example 2 of the present invention. DETAILED DESCRIPTION

[0038] A method for preparing a SERS detection system specifically implemented in the present invention comprises the following steps:

[0039] In the first step, the silicon wafer is pre-treated and then cultured in a MIF antibody solution containing PDA to obtain a PDA chip.

[0040] The specific process is as follows:

[0041] The silicon wafer is soaked in an alkaline bath for 2 to 3 hours, washed with deionized water, then soaked in a piranha solution for 2 to 3 hours, and washed with deionized water; the obtained silicon wafer is cultured in a MIF antibody solution containing 33±5mM PDA for 1 to 2 hours to prepare a PDA chip; the PDA chip is washed with deionized water and stored at 0°C to 4°C for future use.

[0042] The solution contained in the alkaline tank is piranha solution; the piranha solution is a mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide solution in a volume ratio of 3±0.5:1.

[0043] In the second step, the HAuCl4 solution was added to the NaBH4 solution in batches and stirred to obtain a gold nanoparticle AuNPs suspension.

[0044] The specific process is as follows:

[0045] 5±0.5×10 -3 M HAuCl4 solution was added in portions into a continuously stirred ice-cold 2±0.5×10 -3 The reaction was carried out in MNaBH4 solution, wherein the volume ratio of HAuCl4 solution to NaBH4 solution was 1:3±0.5; after the reaction, the mixture was boiled for 1 to 2 hours to decompose excess NaBH4; the final volume was adjusted to 5±0.5 times the volume of the above HAuCl4 solution; and a gold nanoparticle AuNPs suspension was obtained.

[0046] The third step is to mix the ascorbic acid solution, silver nitrate solution, Tris-HCl buffer, 4-aminobenzenethiol solution, and bovine serum albumin solution, and then add the gold nanoparticle AuNPs suspension. After the reaction, Au-Ag nanoparticles with Au as the core and Ag as the shell are prepared.

[0047] The specific process is as follows:

[0048] 0.1±0.01M ascorbic acid solution, 0.096±0.005mM silver nitrate solution, 1±0.1M pH8.0 Tris-HCl buffer, 9.6±0.5mM 4-aminobenzenethiol and 1±0.1% bovine serum albumin solution were mixed evenly, and then the gold nanoparticle AuNPs suspension prepared in the second step was added; wherein, the ascorbic acid solution, the silver nitrate solution, the pH8.0 The volume ratio of Tris-HCl buffer, 4-aminobenzenethiol solution, bovine serum albumin solution, and gold nanoparticle AuNPs suspension is 0.2±0.05:0.05±0.01:1±0.1:1±0.1:2±0.2:6±0.5; after reacting for 30-60 minutes, centrifuge at 5800-6500 rpm for 15-30 minutes, and re-suspend the precipitate with pH8.0 Tris-HCl buffer; Au-Ag nanoparticles with Au as the core and Ag as the shell are obtained.

[0049] Step 4: Mix the MIF antibody solution with the PDA solution and react with Au-Ag nanoparticles to obtain core-shell nanoparticles with a PDA protective shell and connected to antibodies, which is the SERS sensor; the PDA chip and the SERS sensor together constitute a SERS detection system.

[0050] The specific process is as follows:

[0051] Mix 15±0.5 μg / mL of MIF antibody solution and 5-20 mg / mL of PDA solution evenly, wherein the volume ratio of MIF antibody solution to PDA solution is 1±0.1:1; add Au-Ag nanoparticles prepared in the third step and react for 1-2 hours to prepare a SERS sensor; resuspend after centrifugation and store in a refrigerator at 0°C to 4°C.

[0052] The present invention is specifically implemented using the SERS detection system prepared by the above preparation method.

[0053] The SERS detection system specifically implemented in the present invention is used to detect the content of MIF in PRP samples.

[0054] The present invention will be described in further detail below with reference to the accompanying drawings and in combination with embodiments, but the present invention is not limited to the examples given.

[0055] Example 1

[0056] This embodiment is to prepare a SERS detection system, which can perform qualitative and quantitative analysis on macrophage migration inhibitory factor MIF in platelet-rich plasma PRP.

[0057] The specific contents of this embodiment are as follows:

[0058] 1. Materials:

[0059] HAuCl4·3H2O, trisodium citrate, ascorbic acid (AA), silver nitrate (AgNO3), 4-aminothiophenol (pATP), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, USA. Concentrated sulfuric acid, sodium hydroxide, and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. Phosphate buffered saline (PBS); Tris-HCl, pH 8.0 was purchased from ThermoFisherScientific, USA. Rat MIF antibody was purchased from Arigo Biological Laboratory, China. Human VEGF165, human PDGF-BB, and human EGFR were purchased from Novoprotein, China. The remaining materials were all common commercial products.

[0060] 2. Preparation process:

[0061] (1) Preparation of PDA chip: The silicon wafer was soaked in an alkaline bath (containing piranha solution) for 2 hours, washed with deionized water, and then soaked in piranha solution (98% concentrated sulfuric acid and 30% hydrogen peroxide solution mixed in a volume ratio of 3:1) for 2 hours, and then washed again with deionized water. In order to increase the density of the captured antibody and maintain its biological activity, a self-assembled dopamine layer was used for wrapping, that is, the silicon wafer was incubated in a MIF antibody solution containing 33mM PDA for 1 hour, and the resulting PDA chip was washed with deionized water and stored at 4°C for later use.

[0062] (2) Synthesis of gold nanoparticles (AuNPs): 5×10 -3 M HAuCl4 solution (100 mL) was added portionwise into 300 mL of vigorously stirred ice-cold 2×10 -3 M NaBH4 solution. The mixture was boiled for 1 hour to decompose the excess NaBH4. The final volume was adjusted to 500 mL to obtain the AuNPs suspension.

[0063] (3) Synthesis of SERS substrate: 200 μL 0.1 M ascorbic acid solution, 50 μL 0.096 mM silver nitrate solution, 1 mL 1 M Tris-HCl buffer (pH 8.0), 1 mL 9.6 mM pATP solution and 2 mL 1% bovine serum albumin (BSA) solution were added to 6 mL of AuNPs suspension to synthesize Au (core)-Ag (shell) nanoparticles. After reacting for 30 min, the mixture was centrifuged at 5800 rpm for 15 min and the precipitate was resuspended with 5 mL Tris-HCl buffer.

[0064] (4) In order to protect the activity of the antibody and prevent nonspecific adsorption of the antibody, it is necessary to make a PDA shell of a certain thickness to protect the SERS tag. PDA shells of different thicknesses were prepared by adjusting the concentration of PDA (5 mg / mL, 10 mg / mL, 20 mg / mL). 1 mL of 15 μg / mL MIF antibody solution, 1 mL of 5 mg / mL, 10 mg / mL, 20 mg / mL PDA solution were reacted with the nanoparticles obtained above for 1 hour, resuspended after centrifugation, and stored in a refrigerator at 4°C. After the core-shell nanoparticles were successfully connected to the antibody, the SERS sensor was successfully produced.

[0065] The PDA chip and SERS sensor thus obtained together constitute a SERS detection system.

[0066] The test results for Au (core)-Ag (shell) nanoparticles are shown in the figure below. Figures 1 to 5 As shown. Among them, Figure 1The STEM image of Au (core)-Ag (shell) nanoparticles reveals a clear core-shell nanostructure, confirming the formation of the silver shell. Figures 2 to 5 The EDS elemental map of Au (core) -Ag (shell) nanoparticles shows a clear core-shell geometric structure, indicating that there is an Ag shell layer around the Au core, proving that Au (core) -Ag (shell) nanoparticles were successfully synthesized.

[0067] Example 2

[0068] This example is a test in clinical RPR samples.

[0069] In recent years, many studies have found that the MIF level in the synovial fluid of knee patients is related to the occurrence and development of knee osteoarthritis. In view of the above outstanding characteristics, the actual detection efficiency of the SERS detection system of the present invention is evaluated by detecting the concentration of MIF in clinical PRP samples.

[0070] Since it takes a certain amount of time from blood sample collection to PRP preparation and then to laboratory testing, MIF may increase with the occurrence of hemolysis, which will inevitably cause errors. The SERS detection system prepared in Example 1 was used to detect PRP samples of 40 patients with knee osteoarthritis using a Raman spectrometer, and each sample was tested at least 3 times. The basic detection process is: the PRP sample is dripped onto the surface of the PDA chip and rinsed with buffer after the reaction, a SERS sensor is added and rinsed with buffer after the reaction, and then a Raman spectrometer is used for detection to obtain a Raman spectrum. Select 1076cm -1 As a characteristic peak, the MIF concentration of 40 PRP samples was calculated to be 2.6±0.6 ng / mL. After excluding the deviation from the maximum value (concentrated red blood cells can be extracted), the reference detection result range of MIF is 2.0-3.6 ng / mL. It can be concluded that when the Raman signal is greater than 975.2 (au), red blood cells can be extracted in theory, and the samples need to be screened for secondary processing and re-centrifuged under the same conditions.

[0071] In order to verify the actual measurement accuracy of the SERS detection system of the present invention, this example uses a human MIF enzyme-linked immunosorbent assay kit to measure the above 40 PRP samples using the ELISA method.

[0072] Note: Human MIF ELISA kit, model ARG81294, was purchased from Arigo Biolabs, China. The assay was performed according to the instructions of the kit.

[0073] The relationship between the OD value read at 450 nm and the MIF concentration is as follows: Figure 6As shown. The actual concentration of MIF was calculated based on the curve. The results of the SERS detection system were compared with the results of the ELISA test. Figure 7 As shown, Mann-Whitney U test analysis showed that the overall distribution of the two groups of MIF concentration data was the same, and there was no difference between the data groups, that is, there was no significant difference in the MIF concentrations measured by the two methods.

[0074] It can be concluded that the accuracy of the SERS detection system of the present invention is almost the same as that of the ELISA method. Therefore, the SERS detection system of the present invention can be used to detect the MIF content in PRP samples.

[0075] In addition to the above embodiments, the present invention may also have other implementation modes. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A method for preparing a SERS detection system, comprising the following steps: The first step is to pre-treat the silicon wafer and then culture it in a MIF antibody solution containing PDA to prepare a PDA chip; Step 2: Add the HAuCl4 solution into the NaBH4 solution in batches and stir to obtain a gold nanoparticle AuNPs suspension; Step 3: After mixing the ascorbic acid solution, silver nitrate solution, Tris-HCl buffer, 4-aminobenzenethiol solution, and bovine serum albumin solution, the gold nanoparticle AuNPs suspension is added to react to obtain Au-Ag nanoparticles with Au as the core and Ag as the shell; In the fourth step, the MIF antibody solution is mixed with the PDA solution and then reacted with Au-Ag nanoparticles to obtain core-shell nanoparticles with a PDA protective shell and connected with antibodies, which is a SERS sensor; the PDA chip and the SERS sensor together constitute a SERS detection system.

2. The method for preparing a SERS detection system according to claim 1, characterized in that: The specific process of the first step is as follows: The silicon wafer is soaked in an alkaline tank for 2 to 3 hours, washed with deionized water, then soaked in a piranha solution for 2 to 3 hours, and washed with deionized water; the obtained silicon wafer is cultured in a MIF antibody solution containing PDA for 1 to 2 hours to prepare a PDA chip; the PDA chip is washed with deionized water and stored at 0°C to 4°C for future use.

3. The method for preparing a SERS detection system according to claim 2 is characterized in that, in the first step, the solution contained in the alkaline tank is a piranha solution; the piranha solution is a mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide solution in a volume ratio of 3±0.5:1; the PDA concentration in the MIF antibody solution containing PDA is 33±5mM.

4. The method for preparing a SERS detection system according to claim 2, characterized in that: The specific process of the second step is as follows: The HAuCl4 solution was added in batches to the continuously stirred ice-cold NaBH4 solution for reaction; after the reaction, the mixture was boiled for 1 to 2 hours to decompose the excess NaBH4; the final volume was adjusted to 5±0.5 times the volume of the above HAuCl4 solution; and a gold nanoparticle AuNPs suspension was prepared.

5. The method for preparing a SERS detection system according to claim 4, characterized in that: In the second step, the concentration of the HAuCl4 solution was 5±0.5×10 -3 M, the concentration of the NaBH4 solution is 2±0.5×10 -3 M, the volume ratio of the HAuCl4 solution to the NaBH4 solution is 1:3±0.

5.

6. The method for preparing a SERS detection system according to claim 4, characterized in that: The specific process of the third step is as follows: Ascorbic acid solution, silver nitrate solution, pH 8.0 Tris-HCl buffer, 4-aminobenzenethiol and bovine serum albumin solution were mixed evenly, and then the gold nanoparticle AuNPs suspension prepared in the second step was added; after reacting for 30 to 60 minutes, the mixture was centrifuged at 5800 to 6500 rpm for 15 to 30 minutes, and the precipitate was resuspended with pH 8.0 Tris-HCl buffer; Au-Ag nanoparticles with Au as the core and Ag as the shell were prepared.

7. The method for preparing a SERS detection system according to claim 6, characterized in that: The specific process of the fourth step is as follows: The MIF antibody solution and the PDA solution were mixed evenly, and the Au-Ag nanoparticles prepared in the third step were added to react for 1 to 2 hours to prepare a SERS sensor; the solution was resuspended after centrifugation and stored in a refrigerator at 0°C to 4°C.

8. The method for preparing a SERS detection system according to claim 7, characterized in that: In the third step, the concentration of the ascorbic acid solution is 0.1±0.01M, the concentration of the silver nitrate solution is 0.096±0.005mM, the concentration of the pH8.0 Tris-HCl buffer is 1±0.1M, the concentration of the 4-aminobenzenethiol is 9.6±0.5mM, and the mass concentration of the bovine serum albumin solution is 1±0.1%; the volume ratio of the ascorbic acid solution, the silver nitrate solution, the pH8.0 Tris-HCl buffer, the 4-aminobenzenethiol solution, the bovine serum albumin solution, and the gold nanoparticle AuNPs suspension is 0.2±0.05:0.05±0.01:1±0.1:1±0.1:2±0.2:6±0.5; In the fourth step, the concentration of the MIF antibody solution is 15±0.5 μg / mL, the concentration of the PDA solution is 5-20 mg / mL; and the volume ratio of the MIF antibody solution to the PDA solution is 1±0.1:

1.

9. A SERS detection system prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the SERS detection system according to claim 9 for detecting the content of MIF in a PRP sample.

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