SERS (Surface Enhanced Raman Scattering) detection method of osteoarthritis marker type II collagen

Abstract

The invention relates to the technical field of biological detection, in particular to an SERS (Surface Enhanced Raman Scattering) detection method of an osteoarthritis marker type II collagen. The SERS detection method of the osteoarthritis marker type II collagen comprises the following five steps: synthesizing gold nanoparticles, functionalizing an SERS substrate, coupling an SERS probe, establishing a detection standard curve and carrying out SERS spectrum detection. According to the method, the SERS signal of the osteoarthritis marker type II collagen is specifically enhanced through the plasmon resonance effect of the functionalized gold nanoparticles, trace detection of the type II collagen is achieved, detection can be achieved only by simply diluting or centrifuging a sample, purification and concentration steps needed by ELISA detection are avoided, a result is obtained within a short time, and the method is suitable for large-scale popularization and application. The kit is suitable for convenient and rapid diagnosis of early osteoarthritis; meanwhile, the invention provides an SERS (Surface Enhanced Raman Scattering) detection kit for the osteoarthritis marker type II collagen, which can be synthesized and stored on a large scale, so that the detection time is comprehensively shortened, and the detection efficiency is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a SERS detection method for a marker of osteoarthritis, collagen type II. BACKGROUND

[0002] Osteoarthritis is a common degenerative joint disease. At present, the early detection of osteoarthritis mainly relies on the clinical symptoms such as joint pain and stiffness of patients combined with imaging examination; however, the method of using clinical symptoms combined with imaging examination is often difficult to find problems in the early stage of the disease: the clinical symptoms appear late and lack specificity; and the structural changes such as joint space narrowing and osteophyte formation visible in imaging usually appear in the middle and late stages of the disease, and once the disease is obvious in imaging, the treatment effect is often significantly reduced.

[0003] The development of osteoarthritis joint tissue lesions is divided into three stages of matrix degradation period, repair response period and repair imbalance period; in the matrix degradation period, the extracellular matrix on the surface of the joint cartilage is degraded, and the main components of the matrix, collagen type II and aggregate proteoglycan are metabolized; in the repair response period, the tissue starts the repair mechanism, which is manifested as the expression level of growth factors is increased; in the repair imbalance period, metalloproteinase inhibitors are overexpressed, which leads to excessive inhibition of matrix metalloproteinase activity, further hindering normal matrix repair and remodeling; as can be seen, the metabolic changes of collagen type II are the key events in the early pathological process of osteoarthritis, and its detection can provide an important basis for the early diagnosis of osteoarthritis. However, the existing collagen type II detection methods have technical limitations such as insufficient sensitivity, which is difficult to effectively reflect the subtle pathological changes of the early osteoarthritis joint microenvironment.

[0004] In view of the above problems, the present application provides a solution. SUMMARY

[0005] The purpose of the present application is to provide a SERS detection method for a marker of osteoarthritis, collagen type II, which has the advantages of high sensitivity and low detection limit.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical scheme: a SERS detection method for a marker of osteoarthritis, collagen type II, includes five steps of gold nanoparticle synthesis, SERS substrate functionalization, SERS probe coupling, detection standard curve establishment and SERS spectrum detection.

[0007] Further, the gold nanoparticle synthesis includes the following steps:

[0008] A1: Soak glassware in aqua regia for 30 min, then remove and rinse 3 times with ultrapure water, after rinsing, transfer to oven, dry at 60°C for 30 min, filter 1% chloroauric acid solution and 1% sodium citrate solution using 0.22um filter screen to remove solid particles;

[0009] A2: Take 100ml ultrapure water into a round-bottom flask, use a magnetic stirrer to assist stirring, set the speed to 500rpm, heat to boiling, after boiling, quickly add 1ml 1% chloroauric acid solution, at this time the solution turns light yellow;

[0010] A3: Keep the solution boiling, quickly add 2ml 1% sodium citrate solution, continue to boil for 15min, the solution turns wine red, then continue to boil for 15min, after boiling is complete, remove the heat source, stir at 500rpm with a magnetic stirrer to cool to room temperature;

[0011] A4: After cooling, transfer to a high-speed centrifuge, centrifuge at 10000rpm for 10min, after centrifugation, remove the supernatant, then add 10ml phosphate buffer solution, resuspend the solid particles using a pipette to obtain a gold nanoparticle resuspension.

[0012] Further, the functionalization of the SERS substrate includes taking 1ml of gold nanoparticle resuspension, adding 10ul of 1mM thiol PEG, then placing in a room temperature environment, setting the shaker to 300rpm, shaking at room temperature for 1h, after shaking is complete, transfer to a high-speed centrifuge, set the centrifuge speed to 10000rpm, centrifuge at room temperature for 10min to remove free thiol PEG, then add 10ml of phosphate buffer solution to resuspend the gold nanoparticles to obtain a functionalized SERS substrate.

[0013] Further, the SERS probe coupling of the osteoarthritis marker collagen type II includes the following steps:

[0014] B1: Add collagen type II specific antibody to the functionalized SERS substrate, transfer to a 4°C refrigerator for 8h, after coupling is complete, add 1% bovine serum albumin solution for 30min, then rinse the functionalized SERS substrate after blocking with phosphate buffer solution.

[0015] B2: Mix the collagen type II specific antibody with dithiothreitol and dinitrophenyl acid in a shaker, set the shaking frequency to 300rpm, mix and shake for 15min, then transfer to a 4°C refrigerator for 2h, after incubation is complete, ultrafiltration to obtain the signal source secondary antibody.

[0016] Further, the collagen type II specific antibody in step B1 is an anti-COL2A1 antibody with a concentration of 10 ug / ml, wherein the mass ratio of the anti-COL2A1 antibody with a concentration of 10 ug / ml to the functionalized SERS substrate is 1:2; the mass ratio of the 1% bovine serum albumin solution to the functionalized SERS substrate in step B1 is 10:1; the collagen type II specific antibody in step B2 is an anti-COL2A1 antibody, wherein the molar ratio of the anti-COL2A1 antibody to dithiothreitol is 1:5;

[0017] Further, the establishment of the standard curve for detecting the osteoarthritis marker collagen type II includes the following steps:

[0018] C1: Gradient concentration recombinant COL2A1 protein solution is prepared using a phosphate buffer solution and recombinant COL2A1 protein, 5 ul of the gradient concentration recombinant COL2A1 protein solution is added to 45 ul of the phosphate buffer solution for dilution, then 50 ul of the functionalized SERS substrate is added and incubated in a 37℃ incubator for 1 h, after the incubation is completed, it is transferred to a 4℃ centrifuge, centrifuged at 10000 rpm for 10 min to remove the unbound protein;

[0019] C2: The incubated functionalized SERS substrate is washed with a phosphate buffer solution, then 50 ul of the signal source secondary antibody is added and incubated in a 37℃ incubator for 30 min, after the incubation is completed, it is washed with a phosphate buffer solution, after the washing is completed, 100 ul of a phosphate buffer solution is added to prepare a standard suspension;

[0020] C3: 20 ul of the standard suspension is dropped on a silicon wafer, the silicon wafer loaded with the test suspension is placed under a confocal Raman spectrometer microscope and focused, the laser wavelength is set to 785 nm, the laser power is 5 mW, the signal collection time is 5 s, and the SERS spectrum is collected to obtain a standard curve.

[0021] Further, the SERS spectrum detection includes the following steps:

[0022] D1: 5 ml of venous blood is extracted on an empty stomach in a blood collection tube without an anticoagulant, after standing for 30 min, the blood sample is placed in a centrifuge, centrifuged at a speed of 1000 rpm / min for 5-10 min, the supernatant is taken to an enzyme-free EP tube, then centrifuged at a speed of 1000 rpm / min for 2-4 min, the supernatant is taken to a new enzyme-free EP tube to obtain a serum sample;

[0023] D2: 5 ul of the serum sample is taken, added to 45 ul of a phosphate buffer solution for dilution, then 50 ul of the functionalized SERS substrate is added and incubated in a 37℃ incubator for 1 h, after the incubation is completed, it is transferred to a 4℃ centrifuge, centrifuged at 10000 rpm for 10 min to remove the unbound protein;

[0024] D3: using phosphate buffer solution to wash the functionalized SERS substrate after incubation, then adding 50ul signal source secondary antibody to incubate in 37℃ incubator for 30min, after incubation, using phosphate buffer solution to wash, after washing, adding 100ul phosphate buffer solution to prepare test suspension;

[0025] D4: taking 20ul standard suspension to drop on silicon wafer, placing the silicon wafer loaded with test suspension under the microscope of confocal Raman spectrometer and focusing, setting laser wavelength as 785nm, laser power as 5mW, signal collection time as 5s, collecting SERS spectrum, then determining the content of osteoarthritis marker type II collagen in serum sample according to standard curve.

[0026] Further, the application provides a SERS detection kit for osteoarthritis marker type II collagen, which comprises the gold nanoparticle resuspension prepared in step A4, 1mM thiol PEG, phosphate buffer solution, anti-COL2A1 antibody, bovine serum albumin, dithiothinitrobenzoic acid and recombinant COL2A1 protein.

[0027] In summary, due to the adoption of the above technical solutions, the application has the following beneficial effects: through the plasmonic resonance effect of functionalized gold nanoparticles, the application specifically enhances the SERS signal of osteoarthritis marker type II collagen, realizes trace detection of type II collagen, and only needs to dilute or centrifuge the sample to detect, avoids the purification and concentration steps required by ELISA detection, obtains results in a short time, and is suitable for convenient and rapid diagnosis of early osteoarthritis; meanwhile, the application provides a SERS detection kit for osteoarthritis marker type II collagen, which can be synthesized and stored in large quantities, comprehensively shortens the detection time, and improves the detection efficiency. DETAILED DESCRIPTION

[0028] In order to make the technical means, creative features, purposes and effects realized by the application easy to understand, the following further describes the application in combination with specific embodiments.

[0029] In the following examples, the experimental methods are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0030] Example 1

[0031] 1: Soak glassware in aqua regia for 30 min, then remove and rinse 3 times with ultrapure water, after rinsing, transfer to an oven, dry at 60°C for 30 min, filter 1% chloroauric acid solution and 1% sodium citrate solution using a 0.22um filter screen to remove solid particles;

[0032] 2: Take 100ml ultrapure water into a round-bottom flask, use a magnetic stirrer to assist stirring, set the speed to 500rpm, heat to boiling, after boiling, quickly add 1ml 1% chloroauric acid solution, at this time the solution turns light yellow;

[0033] 3: Keep the solution boiling, quickly add 2ml 1% sodium citrate solution, continue to boil for 15min, the solution turns wine red, then continue to boil for 15min, after boiling is complete, remove the heat source, stir at 500rpm with a magnetic stirrer to cool to room temperature;

[0034] 4: After cooling, transfer to a high-speed centrifuge, centrifuge at 10000rpm for 10min, after centrifugation, remove the supernatant, then add 10ml phosphate buffer solution, resuspend the solid particles with a pipette to obtain a gold nanoparticle resuspension;

[0035] 5: Take 1ml of gold nanoparticle resuspension, add 10ul of 1mM thiol PEG, then place in a room temperature environment, set the shaker to 300rpm, shake at room temperature for 1h, after shaking is complete, transfer to a high-speed centrifuge, set the centrifuge speed to 10000rpm, centrifuge at room temperature for 10min to remove free thiol PEG, then add 10ml of phosphate buffer solution to resuspend the gold nanoparticles to obtain a functionalized SERS substrate;

[0036] 6: Add 10g of 10ug / ml anti-COL2A1 antibody to 20g of functionalized SERS substrate, transfer to a 4°C refrigerator for 8h, after coupling is complete, add 100g of 1% bovine serum albumin solution to block for 30min, then rinse the blocked functionalized SERS substrate with phosphate buffer solution;

[0037] 7: Mix 2mol of anti-COL2A1 antibody with 10mol of dithiothreitol, set the shaker to 300rpm, mix and shake for 15min, then transfer to a 4°C refrigerator for 2h, after incubation is complete, ultrafiltrate to obtain a signal source secondary antibody;

[0038] 8: Gradient concentration recombinant COL2A1 protein solution was prepared using phosphate buffer solution and recombinant COL2A1 protein, 5ul of different gradient concentration recombinant COL2A1 protein solution was added to 45ul of phosphate buffer solution for dilution, then 50ul of functional SERS substrate was added to 37℃ incubator for incubation for 1h, after incubation, it was transferred to 4℃ centrifuge, centrifuged at 10000rpm for 10min to remove unbound protein;

[0039] 9: The incubated functional SERS substrate was washed with phosphate buffer solution, then 50ul of signal source secondary antibody was added to 37℃ incubator for incubation for 30min, after incubation, it was washed with phosphate buffer solution, after washing, 100ul of phosphate buffer solution was added to prepare standard suspension;

[0040] 10: 20ul of standard suspension was dropped on the silicon wafer, the silicon wafer loaded with test suspension was placed under the confocal Raman spectrometer microscope and focused, the laser wavelength was set to 785nm, the laser power was 5mW, the signal collection time was 5s, and the standard curve was obtained by collecting SERS spectrum;

[0041] 11: Osteoarthritis mice were purchased from Elabio, and after grouping and normal feeding for 90 days, 5ml of fasting venous blood was taken in a blood collection tube without anticoagulant, and after standing for 30min, the blood sample was placed in a centrifuge at a speed of 1000rpm / min for 5min, the supernatant was taken to an enzyme-free EP tube, and then centrifuged at a speed of 1000rpm / min for 2min, the supernatant was taken to a new enzyme-free EP tube to obtain serum sample;

[0042] 12: 5ul of serum sample was taken, 45ul of phosphate buffer solution was added for dilution, then 50ul of functional SERS substrate was added to 37℃ incubator for incubation for 1h, after incubation, it was transferred to 4℃ centrifuge, centrifuged at 10000rpm for 10min to remove unbound protein;

[0043] 13: The incubated functional SERS substrate was washed with phosphate buffer solution, then 50ul of signal source secondary antibody was added to 37℃ incubator for incubation for 30min, after incubation, it was washed with phosphate buffer solution, after washing, 100ul of phosphate buffer solution was added to prepare test suspension;

[0044] 14: 20ul of standard suspension was dropped on the silicon wafer, the silicon wafer loaded with test suspension was placed under the confocal Raman spectrometer microscope and focused, the laser wavelength was set to 785nm, the laser power was 5mW, the signal collection time was 5s, the average value of SERS spectrum result was collected, then the content of osteoarthritis marker type II collagen in serum sample was determined according to the standard curve;

[0045] Table 1, grouping table of osteoarthritis mice

[0046] Group Grouping Days of rearing Experimental group 1 Early osteoarthritis mice 10 90 days Experimental group 2 Late osteoarthritis mice 10 90 days Control group Normal mice 10 90 days

[0047] Table 2, SERS spectrum intensity table of gradient concentration recombinant COL2A1 protein solution of Example 1

[0048] Note: Because the SERS signal intensity and the target molecule concentration are not simply directly linearly related, but are mediated by the adsorption behavior of the functionalized nanomaterial to the marker, in order to ensure that the standard curve is linear, the gradient 4 data in the table is the result of diluting 10 times with phosphate buffer solution;

[0049] Name Concentration I 1330 (a.u.) I 520 (a.u.) I 1330 / I 520 ]]> Gradient 1 0 ng / ml 51 3187 0.016 Gradient 2 1 ng / ml 826 6883 0.12 Gradient 3 10 ng / ml 5121 6245 0.82 Gradient 4 100 ng / ml 5227 6298 0.83

[0050] Standard curve y=495.64x+179.6 (R 2 =0.9977)

[0051] Table 3, SERS detection results of mice

[0052] Note: Because the mice in experimental group 2 are in the late stage of arthritis, the test solution of experimental group 2 is diluted 10 times with phosphate buffer solution before detection;

[0053] Group I 1330 (a.u.) Osteoarthritis marker type II collagen content Experimental group 1 7019 13.8 ng / ml Experimental group 2 3446 65.9 ng / ml Control group 328 0.3 ng / ml

[0054] By analyzing Table 3, it can be seen that the collagen type II content of the mice in different groups calculated according to the standard curve is consistent with the grouping situation.

[0055] Example 2

[0056] 1: Soak the glassware in aqua regia for 30 min, then take it out and rinse it with ultrapure water 3 times, and after rinsing, transfer it to an oven, dry at 60°C for 30 min, filter 1% chloroauric acid solution and 1% sodium citrate solution using a 0.22um filter screen to remove solid particles;

[0057] 2: Take 100ml ultrapure water and add it to a round-bottom flask, use a magnetic stirrer to assist stirring, set the speed to 500rpm, heat to boiling, and after boiling, quickly add 1ml of 1% chloroauric acid solution, at which point the solution turns light yellow;

[0058] 3: Keep the solution boiling, quickly add 2ml of 1% sodium citrate solution, continue boiling for 15min, the solution turns wine red, then continue boiling for another 15min, after boiling is complete, remove the heat source, and stir at 500rpm with a magnetic stirrer to cool to room temperature;

[0059] 4: After cooling, transfer to high-speed centrifuge, 10000 rpm centrifugation for 10 min, remove the supernatant after centrifugation, then add 10 ml of phosphate buffer solution, and use a pipette to resuspend the solid particles to obtain a gold nanoparticle suspension;

[0060] 5: Take 1 ml of gold nanoparticle suspension, add 10 ul of 1 mM thiol PEG, then place it in a room temperature environment, set the shaker to 300 rpm, and shake for 1 hour at room temperature. After shaking, transfer to a high-speed centrifuge, set the centrifuge speed to 10000 rpm, and centrifuge at room temperature for 10 min to remove free thiol PEG. Then add 10 ml of phosphate buffer solution to resuspend the gold nanoparticles to obtain a functionalized SERS substrate;

[0061] 6: Add 10 g of 10 ug / ml anti-COL2A1 antibody to 20 g of functionalized SERS substrate, transfer to a 4°C refrigerator for 8 hours of coupling, then add 100 g of 1% bovine serum albumin solution for 30 minutes of blocking, and then use phosphate buffer solution to rinse the functionalized SERS substrate after blocking is complete;

[0062] 7: Mix 2 mol of anti-COL2A1 antibody with 10 mol of dithiothinitrobenzoic acid in a shaker, set the shaking frequency to 300 rpm, and mix for 15 minutes, then transfer to a 4°C refrigerator for 2 hours of incubation. After incubation, ultrafiltration is performed to obtain a signal source secondary antibody;

[0063] 8: Prepare a gradient concentration recombinant COL2A1 protein solution using phosphate buffer solution and recombinant COL2A1 protein. Take 5 ul of different gradient concentrations of recombinant COL2A1 protein solution and dilute it with 45 ul of phosphate buffer solution, then add 50 ul of functionalized SERS substrate to a 37°C incubator for 1 hour of incubation. After incubation, transfer to a 4°C centrifuge, centrifuge at 10000 rpm for 10 min to remove unbound protein;

[0064] 9: Wash the incubated functionalized SERS substrate with phosphate buffer solution, then add 50 ul of signal source secondary antibody to a 37°C incubator for 30 minutes of incubation. After incubation, wash with phosphate buffer solution. After washing, prepare 100 ul of phosphate buffer solution as a standard suspension;

[0065] 10: Take 20 ul of standard suspension and drop it on a silicon wafer. Place the silicon wafer loaded with the test suspension under a confocal Raman spectrometer microscope and focus. Set the laser wavelength to 785 nm, the laser power to 5 mW, and the signal acquisition time to 5 s. Collect SERS spectrum to obtain a standard curve;

[0066] 11: recruit 5 volunteers, of which the No. 5 volunteer is diagnosed with early osteoarthritis, 5ml of fasting venous blood is taken in a blood collection tube without anticoagulant, after standing for 30min, the blood sample is placed in a centrifuge at a speed of 1000rpm / min for 10min, the supernatant is taken to an enzyme-free EP tube, and then centrifuged at a speed of 1000rpm / min for 4min, the supernatant is taken to a new enzyme-free EP tube to obtain a serum sample;

[0067] 12: take 5ul of the serum sample, add 45ul of phosphate buffer solution for dilution, then add 50ul of functionalized SERS substrate to a 37℃ incubator for incubation for 1h, after incubation, transfer to a 4℃ centrifuge, centrifuge at 10000rpm for 10min to remove unbound proteins;

[0068] 13: wash the incubated functionalized SERS substrate with phosphate buffer solution, then add 50ul of signal source secondary antibody to a 37℃ incubator for incubation for 30min, after incubation, wash with phosphate buffer solution, after washing, add 100ul of phosphate buffer solution to prepare a test suspension;

[0069] Take 20ul of the standard suspension and drop it on a silicon wafer, place the silicon wafer loaded with the test suspension under a confocal Raman spectrometer microscope and focus, set the laser wavelength to 785nm, the laser power to 5mW, and the signal collection time to 5s, take the average value of the collected SERS spectrum, then determine the content of the osteoarthritis marker collagen type II in the serum sample according to the standard curve;

[0070] Table 4, SERS spectrum intensity table of gradient concentration recombinant COL2A1 protein solution of Example 2

[0071] Note: Since the SERS signal intensity is not a simple direct linear relationship with the concentration of the target molecule, but is mediated by the adsorption behavior of the functionalized nanomaterials to the markers, in order to ensure that the standard curve is linear, the data in Table 4 is diluted 10 times with phosphate buffer solution;

[0072] Name Concentration I 1330 (a.u.) I 520 (a.u.) I 1330 / I 520 ]]> Gradient 1 0 ng / ml 52 3467 0.015 Gradient 2 1 ng / ml 841 7008 0.12 Gradient 3 10 ng / ml 5079 6270 0.81 Gradient 4 100 ng / ml 5114 6088 0.84

[0073] y=491.13x+184.12 (R 2 =0.9973)

[0074] Table 5, SERS detection results of different volunteers

[0075] Group I 1330 (a.u.) Osteoarthritis marker type II collagen content Volunteer 1 2443 4.6 ng / ml Volunteer 2 528 0.7 ng / ml Volunteer 3 5979 11.8 ng / ml Volunteer 4 2738 5.2 ng / ml Volunteer 5 6372 12.6 ng / ml

[0076] From the analysis of Table 5, it can be seen that the detection result of volunteer 5 is consistent with the confirmed condition, volunteer 3 is also detected as early osteoarthritis, and volunteers 1 and 4 have obvious early osteoarthritis tendency, and volunteer 2 is relatively healthy.

[0077] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A SERS detection method for type II collagen, a biomarker of osteoarthritis, characterized in that, The process includes five steps: gold nanoparticle synthesis, SERS substrate functionalization, SERS probe coupling, establishment of detection standard curve, and SERS spectral detection.

2. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 1, is characterized in that, The synthesis of the gold nanoparticles includes the following steps: A1: Immerse the glassware in aqua regia for 30 minutes, then remove it and rinse it three times with ultrapure water. After rinsing, transfer it to an oven and dry it at 60°C for 30 minutes. Use a 0.22µm filter sieve to filter 1% chloroauric acid solution and 1% sodium citrate solution to remove solid particles. A2: Add 100ml of ultrapure water to a round-bottom flask, use a magnetic stirrer to assist stirring, set the speed to 500rpm, heat to boiling, and after boiling, quickly add 1ml of 1% chloroauric acid solution. At this time, the solution turns pale yellow. A3: Keep the solution boiling, quickly add 2ml of 1% sodium citrate solution, continue boiling for 15min until the solution turns wine red, then continue boiling for 15min. After boiling, remove the heat source and stir with a magnetic stirrer at 500rpm to cool to room temperature. A4: After cooling, transfer to a high-speed centrifuge and centrifuge at 10,000 rpm for 10 min. After centrifugation, remove the supernatant, then add 10 ml of phosphate buffer solution and resuspend the solid particles using a pipette to obtain a gold nanoparticle resuspension.

3. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 1, is characterized in that... The SERS substrate functionalization process involves taking 1 ml of gold nanoparticle resuspension, adding 10 μL of 1 mM thiol PEG, then placing it at room temperature, setting the shaker frequency to 300 rpm, and shaking at room temperature for 1 hour. After shaking, the mixture is transferred to a high-speed centrifuge, set the centrifuge speed to 10,000 rpm, and centrifuged at room temperature for 10 minutes to remove free thiol PEG. Then, 10 ml of phosphate buffer solution is added to resuspend the gold nanoparticles to obtain the functionalized SERS substrate.

4. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 1, is characterized in that, The SERS probe coupling includes the following steps: B1: Type II collagen-specific antibody was added to the functionalized SERS substrate and transferred to a 4°C freezer for conjugation for 8 hours. After conjugation, 1% bovine serum albumin solution was added for blocking for 30 minutes, followed by rinsing the blocked functionalized SERS substrate with phosphate buffer solution. B2: Mix type II collagen-specific antibody with dithiodinitrobenzoic acid in a shaker, set the shaking frequency to 300 rpm, mix and shake for 15 min, then transfer to a 4°C refrigerator for 2 h incubation. After incubation, ultrafilter to obtain the signal source secondary antibody.

5. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 1, is characterized in that... The establishment of the detection standard curve includes the following steps: C1: Gradient concentration recombinant COL2A1 protein solutions were prepared using phosphate buffer and recombinant COL2A1 protein. 5 μL of recombinant COL2A1 protein solutions of different gradient concentrations were diluted with 45 μL of phosphate buffer, followed by 50 μL of functionalized SERS substrate. The solution was incubated at 37°C for 1 h. After incubation, the solution was transferred to a centrifuge at 4°C and centrifuged at 10,000 rpm for 10 min to remove unbound protein. C2: Wash the functionalized SERS substrate after incubation with phosphate buffer solution, then add 50 μL of signal source secondary antibody and incubate at 37°C for 30 min. After incubation, wash with phosphate buffer solution, and then add 100 μL of phosphate buffer solution to prepare a standard suspension. C3: Take 20 μL of standard suspension and drop it onto a silicon wafer. Place the silicon wafer loaded with the test suspension under a confocal Raman spectrometer microscope and focus it. Set the laser wavelength to 785 nm, the laser power to 5 mW, and the signal acquisition time to 5 s. Acquire the SERS spectrum to obtain the standard curve.

6. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 1, is characterized in that, The SERS spectral detection Includes the following steps: D1: Draw 5ml of fasting venous blood into a blood collection tube without anticoagulant. After standing for 30 minutes, place the blood sample in a centrifuge and centrifuge at 1000rpm / min for 5-10 minutes. Take the supernatant into an enzyme-free EP tube and centrifuge again at 1000rpm / min for 2-4 minutes. Take the supernatant into a new enzyme-free EP tube to obtain the serum sample. D2: Take 5 μL of serum sample, add 45 μL of phosphate buffer solution to dilute, then add 50 μL of functionalized SERS substrate and incubate in a 37°C incubator for 1 h. After incubation, transfer to a 4°C centrifuge and centrifuge at 10,000 rpm for 10 min to remove unbound proteins. D3: Wash the functionalized SERS substrate after incubation with phosphate buffer solution, then add 50 μL of signal source secondary antibody and incubate at 37°C for 30 min. After incubation, wash with phosphate buffer solution, and then add 100 μL of phosphate buffer solution to prepare the test suspension. D4: Take 20 μL of standard suspension and drop it onto a silicon wafer. Place the silicon wafer loaded with the test suspension under a confocal Raman spectrometer microscope and focus it. Set the laser wavelength to 785 nm, the laser power to 5 mW, and the signal acquisition time to 5 s. Acquire the SERS spectrum and then determine the content of type II collagen, a marker of osteoarthritis, in the serum sample according to the standard curve.

7. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 4, is characterized in that, The type II collagen-specific antibody mentioned in step B1 is an anti-COL2A1 antibody with a concentration of 10ug / ml, wherein the mass ratio of the anti-COL2A1 antibody with a concentration of 10ug / ml to the functionalized SERS substrate is 1:

2.

8. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 4, is characterized in that, The mass ratio of the 1% bovine serum albumin solution to the functionalized SERS substrate mentioned in step B1 is 10:

1.

9. The SERS detection method for type II collagen, a biomarker of osteoarthritis, according to claim 4, is characterized in that, The type II collagen-specific antibody mentioned in step B2 is an anti-COL2A1 antibody, wherein the molar ratio of the anti-COL2A1 antibody to dithiodinitrobenzoic acid is 1:

5.

10. A SERS detection kit for type II collagen, a biomarker of osteoarthritis, characterized in that, The kit includes the gold nanoparticle resuspension prepared in step A4, 1 mM thiol PEG, phosphate buffer, anti-COL2A1 antibody, bovine serum albumin, dithiodinitrobenzic acid, and recombinant COL2A1 protein.