SERS biosensor based on C3N4 nanosheets and its preparation method and application

Abstract

The invention belongs to the field of nanobiotechnology, and discloses a SERS biosensor based on carbon three nitrogen four nanosheets and a preparation method and application thereof; the SERS biosensor is composed of an immune probe, an immune substrate and a glycoprotein 125 antigen; the immune probe is obtained by linking a glycoprotein 125 antibody on a functionally labeled magnetic nano molybdenum disulfide; the immune substrate is obtained by linking a glycoprotein 125 antibody on an activated carbon three nitrogen four nanosheet; and the glycoprotein 125 antibody on the immune probe and the immune substrate specifically binds to the glycoprotein 125 antigen to form a SERS biosensor with a sandwich structure. The SERS immunosensor prepared by the invention has good biocompatibility, a simple preparation process, and can realize double recycling of the substrate and the probe; and has excellent photocatalytic performance, which is conducive to the rapid degradation of antigens, antibodies and Raman molecules.

Description

Technical Field

[0001] The present invention relates to the field of nano-biotechnology, and in particular to a SERS biosensor based on carbon three nitrogen four nanosheets and a preparation method and application thereof. Background Art

[0002] Early detection of cancer is the key to improving cure rates. Compared with traditional detection technologies, SERS has become one of the main strategies for detecting biomarkers due to its unique fingerprint, non-destructive data acquisition, and sensitivity at the single molecule level.

[0003] To this end, a type of SERS-based biosensor based on noble metal nanoparticles (Au, Ag, etc.) has been proposed in the prior art. This type of SERS-based biosensor has high sensitivity and high throughput, and its SERS generally occurs on the surface of the noble metal nanoparticles, so that the analyte can be directly detected, and therefore has attracted widespread attention.

[0004] However, both the substrate and probe of this type of SERS-based biosensor need to be made of a large amount of precious metals, which not only has a high preparation cost, but also its precious metal immune substrate generally requires the use of electron beam, ion beam, photolithography and electrodeposition to prepare the precious metal layer (such as Ag layer). The preparation process is complicated and difficult to operate, resulting in poor controllability of the prepared substrate morphology, which in turn leads to poor stability and biocompatibility of the SERS-based biosensor, thereby affecting the detection effect of the SERS-based biosensor.

[0005] To this end, the present invention provides a SERS biosensor based on C3N4 nanosheets and a preparation method and application thereof. Summary of the invention

[0006] In order to solve the above-mentioned deficiencies in the prior art, the present invention provides a SERS biosensor based on C3N4 nanosheets and its preparation method and application. The present invention combines magnetic nano-molybdenum disulfide with C3N4 nanosheets to produce a SERS biosensor that can be used for tumor marker detection.

[0007] The SERS biosensor based on C3N4 nanosheets and its preparation method and application are realized by the following technical solutions:

[0008] The first object of the present invention is to provide a SERS biosensor based on carbon trinitrogen tetranitrate nanosheets, which is composed of an immune probe, an immune substrate and a glycoprotein 125 antigen;

[0009] The immune probe is obtained by linking glycoprotein 125 antibody on functionally labeled magnetic nano-molybdenum disulfide, and the diameter of the magnetic nano-molybdenum disulfide is 100-1000nm;

[0010] The immune substrate is obtained by linking the glycoprotein 125 antibody to a carbon trinitrogen tetranitrate nanosheet, and the diameter of the carbon trinitrogen tetranitrate nanosheet is 50 to 300 nm;

[0011] The immune probe and the glycoprotein 125 antibody on the immune substrate specifically bind to the glycoprotein 125 antigen to form a SERS biosensor with a sandwich structure.

[0012] Furthermore, the functionalized labeled magnetic nano-molybdenum disulfide is obtained by the following steps:

[0013] The magnetic nano-molybdenum disulfide is labeled with Raman molecules to obtain the functionally labeled magnetic nano-molybdenum disulfide;

[0014] The Raman molecule is any one of rhodamine 6G, methylene blue and crystal violet, and the usage ratio of the Raman molecule to the magnetic nano-molybdenum disulfide is 1-3 mmol:10 g.

[0015] Furthermore, the preparation method of the magnetic nano-molybdenum disulfide is as follows:

[0016] The molybdate, the sulfur source and the ferroferric oxide are uniformly dispersed in the solvent B, and subjected to a hydrothermal reaction at a temperature of 180 to 220° C. for 6 to 12 hours. After cooling to room temperature, the mixture is washed and dried to obtain magnetic nano-molybdenum disulfide coated with ferroferric oxide.

[0017] Furthermore, the molybdate is sodium molybdate or ammonium molybdate;

[0018] The sulfur source is thioacetamide or thiourea;

[0019] The molar ratio of the molybdate to the sulfur source and the ferroferric oxide (232) is 1.5-2:10:0.06-0.55;

[0020] The usage ratio of the molybdate to the solvent B is 0.15-0.2 mol:1L.

[0021] Furthermore, the activated carbon trinitrogen tetranitrate nanosheet carbon trinitrogen tetranitrate nanosheet is obtained by the following steps:

[0022] S1, adding C3N4 powder to concentrated hydrochloric acid, and after ultrasonic treatment, reacting at 70-90° C. for 8-20 h to obtain acidified C3N4;

[0023] The ratio of the carbon trinitrogen tetrachloride powder to concentrated hydrochloric acid is 2-3 mg: 1-2 mL;

[0024] S2, dispersing the acidified C3N4 in a water solvent under ultrasound, and then centrifuging, and the obtained supernatant is the C3N4 nanosheet solution;

[0025] Wherein, the usage ratio of the water solvent to the carbon trinitrogen tetrakis powder is 1-2 mg:1-1.5 mL;

[0026] S3, uniformly dispersing the C3N4 nanosheet solution and the imine compound in a phosphate buffered saline solution, and reacting at a temperature of 36 to 38° C. for 1 to 3 hours to obtain an activated C3N4 nanosheet solution;

[0027] Wherein, the imine compound is N-hydroxysuccinimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0028] Furthermore, the dosage ratio of the glycoprotein 125 antibody to the activated C3N4 nanosheet solution is 0.002-0.006 mg: 1-1.5 mL.

[0029] The second object of the present invention is to provide a method for preparing the above-mentioned SERS biosensor, comprising the following steps:

[0030] Step 1, preparation of immune probe:

[0031] Adding glycoprotein 125 antibody to the functionalized labeled magnetic nano-molybdenum disulfide and then performing incubation treatment A to link the glycoprotein 125 antibody to the magnetic nano-molybdenum disulfide labeled with Raman molecules to obtain the immune probe;

[0032] Step 2, preparation of immune substrate:

[0033] After activating the C3N4 nanosheets, adding glycoprotein 125 antibody for incubation treatment B, so as to link the glycoprotein 125 antibody to the activated C3N4 nanosheets to obtain an immune substrate;

[0034] Step 3, preparation of sandwich structure SERS biosensor:

[0035] The immune substrate is dispersed in a phosphate buffer solution A to form an immune substrate solution, and then the glycoprotein 125 antigen is added to perform an incubation treatment C to obtain a mixed solution A;

[0036] The immune probe is dispersed in a phosphate buffer solution B to form an immune probe solution, and then the immune probe solution is added to the mixed solution A for incubation treatment D to obtain a SERS biosensor.

[0037] Furthermore, the mass ratio of the glycoprotein 125 antibody to the functionalized labeled magnetic nano-molybdenum disulfide is 2 to 6:1000;

[0038] The dosage ratio of the immune substrate to the phosphate buffer solution A is 1-2 mg: 1-1.5 mL;

[0039] The dosage ratio of the immune probe to the phosphate buffer solution B is 10 mg: 1-1.5 mL;

[0040] The dosage ratio of the glycoprotein 125 antigen to the immune substrate solution is 0.002-0.006 mg: 1-1.5 mL;

[0041] The dosage ratio of the immune probe solution to the mixed solution A is 1-1.5 mL:1-1.5 mL;

[0042] The concentration of the glycoprotein 125 antigen is 80 to 120 IU / mL.

[0043] Furthermore, the treatment temperature of the incubation treatment A is 3-5°C, and the treatment time is 1.5-2.5h;

[0044] The incubation treatment B has a treatment temperature of 3 to 5° C. and a treatment time of 1.5 to 2.5 h.

[0045] The incubation treatment C has a treatment temperature of 36 to 38° C. and a culture time of 1 to 3 hours;

[0046] The incubation temperature of the incubation treatment D is 36-38° C., and the incubation time is 1-3 hours.

[0047] The third object of the present invention is to provide an application of the above-mentioned SERS biosensor in tumor marker detection.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The SERS biosensor of the present invention is composed of an immune probe, an immune substrate and a glycoprotein 125 antigen, wherein the immune probe is obtained by linking a glycoprotein 125 antibody to a functionally labeled magnetic nano-molybdenum disulfide, and the immune substrate is obtained by linking the glycoprotein 125 antibody to an activated carbon trinitrogen tetrakis nanosheet. The preparation process is simple and easy to operate, and the activated g-C3N4 nanosheet has a larger surface area and more active electrons, showing improved Raman scattering performance. In addition, carbon trinitrogen tetrakis and molybdenum disulfide have a very matching staggered energy band structure and have a synergistic chemical enhancement effect, which greatly promotes the chemical enhancement effect, causes more charge transfer processes, leads to an increase in polarizability, and significantly amplifies the Raman signal of the labeled molecule, thereby being able to cause excellent SERS activity, so that the SERS immunosensor of the present invention has good biocompatibility and greatly improves the detection efficiency.

[0050] The present invention introduces a small amount of magnetic particles of ferroferric oxide in the process of preparing molybdenum disulfide, so that the molybdenum disulfide is coated with ferroferric oxide, thereby promoting the stacking and self-assembly of molybdenum disulfide nanosheets into a flower shape, exposing a higher specific surface area and active sites, which is conducive to molecular adsorption. In the SERS immunosensor prepared by the present invention, the synergistic chemical enhancement of carbon trinitrogen tetracarbon nanosheets and magnetic nano molybdenum disulfide causes more charge transfer processes, resulting in an increase in polarization rate and an improvement in SERS signals, thereby being able to cause excellent SERS activity.

[0051] The SERS immunosensor prepared by the present invention has good biocompatibility, and the introduction of magnetic ferroferric oxide realizes the dual recycling of substrate and probe. At the same time, by virtue of the high carrier mobility of the above two semiconductors, the immunosensor also has excellent photocatalytic performance, which is conducive to the rapid degradation of antigens, antibodies and Raman molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a scanning electron microscope photo of the immune probe prepared in Example 1;

[0053] Figure 2 This is a scanning electron microscope photo of the immune probe prepared in Example 2;

[0054] Figure 3 This is a scanning electron microscope photo of the immune probe prepared in Example 3;

[0055] Figure 4 This is a scanning electron micrograph of the immune substrate prepared in Example 1;

[0056] Figure 5 A scanning electron microscope photograph of the SERS biosensor prepared in Example 1;

[0057] Figure 6 The Raman detection results of Rhodamine 6G by the immune probe and immune substrate prepared in Example 1;

[0058] Figure 7 The Raman detection result of the immune probe prepared in Example 2 on rhodamine 6G;

[0059] Figure 8 The Raman detection result of the immune probe prepared in Example 3 on rhodamine 6G;

[0060] Fig. 9 This is the Raman detection result of the SERS biosensor prepared in Example 1 for glycoprotein 125 in serum. DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0062] The present invention provides a SERS biosensor based on carbon three nitrogen four nanosheets, which is composed of an immune probe, an immune substrate and a glycoprotein 125 antigen, and the preparation method thereof is as follows:

[0063] Step 1, preparation of immune probe:

[0064] 1.1 Preparation of magnetic nano-MoS2

[0065] It should be noted that the present invention preferably uses magnetic flower-shaped nano-molybdenum disulfide as the magnetic nano-molybdenum disulfide. Optionally, the present invention adopts the following steps to prepare the magnetic flower-shaped nano-molybdenum disulfide:

[0066] 1.1.1 Preparation of ferroferric oxide:

[0067] The iron source, stabilizer and surfactant are uniformly dispersed in solvent A in sequence, and subjected to hydrothermal reaction at 200° C. for 10 h to obtain small ferrosoferric oxide particles;

[0068] The present invention is not limited to a specific type of iron source, as long as it can provide Fe, for example, it can be selected from any one of ferrous sulfate heptahydrate, ferrous chloride tetrahydrate and ferric chloride hexahydrate.

[0069] The present invention does not limit the specific type of the stabilizer, as long as it can prevent agglomeration between particles, such as any one selected from sodium acetate, sodium citrate and sodium acetate.

[0070] The present invention is not limited to a specific type of surfactant as long as it can prevent agglomeration between particles.

[0071] The specific type of solution A of the present invention can be any type as long as it can form a uniform solution or suspension with the iron source, stabilizer and surfactant.

[0072] The present invention does not limit the dispersion method of the iron source, stabilizer and surfactant in the solvent A, as long as the iron source, stabilizer and surfactant can be uniformly dispersed in the solvent A.

[0073] 1.1.2 Hydrothermal synthesis:

[0074] The molybdate, the sulfur source and the small particles of ferroferric oxide are uniformly dispersed in the solvent B, and subjected to a hydrothermal reaction at a temperature of 180 to 220° C. for 6 to 12 hours. After cooling to room temperature, the particles are washed and dried to obtain magnetic nano-molybdenum disulfide coated with ferroferric oxide;

[0075] The present invention is not limited to the specific type of molybdate, as long as it can provide molybdenum ions, such as sodium molybdate or ammonium molybdate.

[0076] The present invention is not limited to the specific type of sulfur source, as long as it can provide S, such as thioacetamide or thiourea.

[0077] The present invention does not limit the specific type of solution B, as long as it can form a uniform solution or suspension with molybdate, sulfur source and ferrosoferric oxide.

[0078] The present invention does not limit the dispersion method of the molybdate, the sulfur source and the ferroferric oxide in the solvent B, as long as the molybdate, the sulfur source and the ferroferric oxide can be uniformly dispersed in the solvent B.

[0079] The present invention does not limit the specific manner of washing treatment, as long as the impurities on the surface of the product (such as unreacted substances) can be removed.

[0080] The present invention does not limit the specific method of drying treatment, as long as the residual solvent on the surface of the product can be removed.

[0081] 1.2 Labeling of magnetic nano-MoS2

[0082] In order to facilitate labeling, the present invention prepares the magnetic nano-molybdenum disulfide into a solution before labeling: the magnetic nano-molybdenum disulfide powder obtained above is uniformly dissolved in deionized water under ultrasound to prepare a magnetic nano-molybdenum disulfide solution with a concentration of 5 to 15 mg / mL.

[0083] The Raman molecules are mixed with the magnetic nano-molybdenum disulfide solution and then cultured for treatment A, and then a phosphate buffered saline solution of an imine compound is added for culture treatment B, and after magnetic separation treatment, the mixture is uniformly dispersed in the phosphate buffered saline solution to obtain a magnetic nano-molybdenum disulfide solution labeled with rhodamine 6G;

[0084] It should be noted that the present invention does not limit the specific type of Raman molecules, as long as they can mark the magnetic nano-MoS2 probe, for example, any one of rhodamine 6G, methylene blue and crystal violet can be selected, or other Raman molecules with larger scattering cross sections can be selected.

[0085] The present invention does not limit the specific method of Raman molecule labeling of magnetic nano-molybdenum disulfide, as long as labeling can be achieved.

[0086] In order to facilitate the subsequent treatment of the labeled magnetic nano-molybdenum disulfide, the present invention also performs magnetic separation and cleaning three times before the culture treatment B to remove the unbound Raman molecules during the culture treatment A. It should be noted that during the magnetic separation and cleaning process, ferroferric oxide gives the molybdenum disulfide nano-flowers good magnetic properties, and the magnetic nano-molybdenum disulfide can be firmly adsorbed on the wall of the test tube by a magnet, the solution in the test tube is washed out, and deionized water is added, and the above operation is repeated.

[0087] The present invention does not limit the specific magnetic separation method after the culture treatment B, as long as it can achieve the removal of Raman molecules not adsorbed on the magnetic nano-molybdenum disulfide. Optionally, in this embodiment, the Raman molecules on the magnetic nano-molybdenum disulfide are magnetically separated by magnet adsorption to obtain the mixed material A after magnetic treatment.

[0088] The present invention does not limit the specific method of the culture treatment B, as long as the Raman molecule can be used to label the magnetic molybdenum dioxide nanoflowers for subsequent SERS detection. Preferably, the present invention uses a phosphate buffered saline solution of N-hydroxysuccinimide as the phosphate buffered saline solution of the imide compound, and the phosphate buffered saline solution has salt balance and an adjustable suitable pH buffering effect.

[0089] 1.3 Preparation of immunoprobes

[0090] Adding glycoprotein 125 antibody to the above-mentioned Raman molecule labeled magnetic nano-molybdenum disulfide solution, performing incubation treatment A, then adding bovine serum albumin blocking solution, mixing evenly and performing incubation treatment C to obtain an immune probe;

[0091] It should be noted that the present invention does not limit the specific method of incubation treatment A, as long as it can achieve the linking of antibodies to magnetic nano-molybdenum disulfide for subsequent immune response.

[0092] In order to improve the blocking effect of the bovine serum albumin blocking solution on the nonspecific adsorption sites on the probe, the present invention further performs a magnetic separation treatment on the magnetic nano-molybdenum disulfide solution after incubation treatment A before performing incubation treatment C to remove excess glycoprotein 125 antibodies to obtain a mixed material B.

[0093] The present invention does not limit the specific manner of the culture treatment C, as long as the non-specific adsorption sites on the immune probe can be blocked.

[0094] 1.4 Preparation of immunoprobe solution

[0095] Dispersing the above-obtained immune probe in a phosphate buffered saline solution to obtain an immune probe solution;

[0096] It should be noted that in order to improve the detection effect of the probe, before dispersing the immune probe in the phosphate buffered saline solution, the present invention rinses it with a phosphate buffered saline solution of N-hydroxysuccinimide, and magnetically collects the molybdenum disulfide nanoflowers with a magnet, washes them multiple times to remove excess bovine serum albumin, and then dissolves them in a phosphate buffered saline solution to obtain an immune probe solution.

[0097] It should also be noted that the role of using phosphate buffered saline solution to prepare the immune probe solution in the present invention is to dissolve the protective reagent; the reason is that the phosphate buffer has salt balance and can adjust the appropriate pH buffering effect. Generally, it cannot be replaced by other materials. For example, distilled water does not have a salt balance effect and can destroy the structure and biological properties of biological proteins; physiological saline does not have the function of adjusting pH, and cannot ensure that intact and active substances participate in biological reactions under optimal conditions, so the use of phosphate buffered solution is the first choice.

[0098] Step 2, Preparation of Immunoassay Substrate

[0099] 2.1 Preparation of C3N4 nanosheets

[0100] The present invention does not limit the specific preparation method of C3N4 nanosheets, as long as C3N4 nanosheets with a diameter of 50 to 300 nm can be obtained. Optionally, the C3N4 nanosheets are obtained by the following steps:

[0101] According to the dosage ratio of 2-3 mg of C3N4 powder to concentrated hydrochloric acid: 1-2 mL, C3N4 powder is added to concentrated hydrochloric acid and ultrasonicated at 800 W ultrasonic power for 1-3 hours. Then, the mixed solution is reacted at 70-90 ° C for 8-20 hours. After that, the mixture is washed with a large amount of deionized water until the washing liquid is neutral to remove excess hydrochloric acid and obtain the acidified C3N4. According to the dosage ratio of water solvent to C3N4 powder 1-2 mg: 1-1.5 mL, the acidified C3N4 is dispersed in deionized water under ultrasonic action (ultrasonic power of 800 W, ultrasonic time of 5-10 minutes). The dispersed C3N4 is centrifuged at a speed of 2500-3500 r / min for 5-20 minutes, and the unpeeled large pieces of C3N4 sink to the bottom of the test tube wall, and the protonated C3N4 nanosheets are left in the supernatant, and the supernatant is the obtained C3N4 nanosheet solution.

[0102] 2.2 Preparation of immunomodulatory substrate solution

[0103] 2.2.1 Activated carbon trinitrogen tetrakis:

[0104] The C3N4 nanosheet solution and the imine compound are uniformly dispersed in a phosphate buffered saline solution, and reacted at a temperature of 36 to 38° C. for 1 to 3 hours to activate the C3N4 nanosheet, thereby improving the coupling efficiency of the protein, and obtaining an activated C3N4 nanosheet solution;

[0105] It should be noted that the present invention does not limit the specific dispersion method of the C3N4 nanosheet solution and the imine compound in the phosphate buffered saline solution, as long as the C3N4 nanosheet can be activated by the imine compound to improve the coupling efficiency of the protein.

[0106] 2.2.2 Formation of immune matrix:

[0107] After mixing the glycoprotein 125 antibody with the activated carbon trinitrogen tetraoxide nanosheet solution, an incubation treatment B is performed, and solid-liquid separation is performed, and the obtained solid component is the immune substrate;

[0108] It should be noted that the present invention does not limit the specific method of incubation treatment B in step 2.2.2, as long as it can achieve the linking of the antibody to the C3N4 substrate for subsequent immune response.

[0109] In order to improve the treatment effect of bovine serum albumin, the present invention further centrifuges the mixed material C before adding bovine serum albumin to the mixed material C to remove excess glycoprotein 125 antibodies in the mixed material C.

[0110] The present invention does not limit the specific method of solid-liquid separation. Optionally, this embodiment uses centrifugation for separation, and the product after incubation treatment B is centrifuged and washed twice to remove excess bovine serum albumin to obtain an immune substrate.

[0111] 2.2.3 Preparation of immune substrate solution

[0112] dissolving the immune substrate in a phosphate buffered saline solution under ultrasound to obtain a carbon trioxide tetraoxide immune substrate solution;

[0113] It should be noted that the ultrasonic power of the above ultrasonic action is 800W, and the ultrasonic time is 5 to 10 minutes.

[0114] Step 3: Preparation of sandwich structure SERS biosensor

[0115] Adding glycoprotein 125 antigen to the above-mentioned carbon trioxide tetrakis immune substrate solution, performing incubation treatment C, and obtaining a mixed solution A;

[0116] The immune probe is added to the mixed solution A, and then incubated in a treatment D after mixing, and the immune substrate and the immune probe are combined with the antigen and the antibody to form a sandwich structure, thereby obtaining a SERS biosensor;

[0117] It should be noted that the present invention does not limit the specific method of incubation treatment C, as long as the antigen can be linked to the C3N4 substrate for subsequent immune response.

[0118] In order to prevent the uncaptured antigens in the solution from affecting subsequent processing, the present invention also uses a phosphate buffer solution to perform multiple centrifugal washing treatments on the solid components obtained from incubation treatment C (the solid components sink on the test tube wall, and the unlinked antigens are absorbed in the solution) to remove the uncaptured antigens.

[0119] The present invention does not limit the specific manner of incubation treatment D, as long as the antigen can be linked to the C3N4 substrate for subsequent immune response.

[0120] It should be noted that the SERS biosensor with a sandwich structure prepared in this embodiment is stored in 1 mL of phosphate buffer solution for subsequent SERS detection. After the SERS biosensor with a sandwich structure prepared in this embodiment is subjected to SERS detection, the immune structure is catalyzed by a visible light simulator, and the Raman molecules, antigens and antibodies can be degraded based on the good photocatalytic activity of molybdenum disulfide nanoflowers and carbon trinitrogen tetranitrate nanosheets. After the catalysis is completed, a magnet is used to separate and collect the immune probes and immune substrates, and subsequent tumor marker detection is continued. This is repeated to achieve cyclic immune detection.

[0121] Example 1

[0122] This embodiment provides a SERS biosensor based on C3N4 nanosheets, and the preparation method of the SERS biosensor based on C3N4 nanosheets in this embodiment is as follows:

[0123] Step 1, preparation of immune probe:

[0124] 1.1 Preparation of magnetic nano-MoS2

[0125] 1.1.1 Preparation of ferroferric oxide:

[0126] In this embodiment, ferric chloride hexahydrate is used as the iron source; sodium acetate is used as the stabilizer; polyethylene glycol is used as the surfactant; ethylene glycol is used as the solvent A;

[0127] 1.35 g of ferric chloride hexahydrate was added to solvent A, and then ultrasonicated at an ultrasonic power of 800 W for 10 minutes to dissolve the ferric chloride hexahydrate. Then, 3.6 g of sodium acetate and 1 g of polyethylene glycol were added in sequence, and then ultrasonicated at an ultrasonic power of 800 W for 10 minutes again. Then, the mixture was stirred at a stirring rate of 300-500 r / min for 30 minutes to uniformly disperse the iron source, sodium acetate and polyethylene glycol in the ethylene glycol. Then, the mixture was hydrothermally reacted at a temperature of 200° C. for 10 hours to obtain small particles of ferrosoferric oxide.

[0128] 1.1.2 Hydrothermal synthesis:

[0129] In this embodiment, ammonium molybdate is used as the molybdate, thiourea is used as the sulfur source, and deionized water is used as the solvent B;

[0130] In this embodiment, the dispersion treatment is carried out in an ultrasonic manner. 0.35g of ammonium molybdate, 0.76g of thiourea and 15.7mg of the above-mentioned ferroferric oxide particles are added to 10mL of deionized water, and ultrasonicated for 10min at an ultrasonic power or power of 800W to uniformly disperse the molybdate, sulfur source and ferroferric oxide particles in solvent B; then it is hydrothermally reacted at a temperature of 200°C for 10h, cooled to room temperature, and washed with anhydrous ethanol and deionized water three times in sequence to remove impurities on the surface of the product; then dried at 60°C for 6 to 12h to remove the residual solvent on the surface of the product, and magnetic nano molybdenum disulfide is obtained.

[0131] 1.2 Labeling of magnetic nano-MoS2

[0132] In this embodiment, the prepared magnetic nano-molybdenum disulfide solution has a concentration of 10 mg / mL; and rhodamine 6G is used as the Raman molecule; in this embodiment, 200 μL of rhodamine 6G with a concentration of 10 mmol / L and 1 mL of a magnetic nano-molybdenum disulfide solution with a concentration of 10 mg / mL are weighed, and ultrasonic labeling is performed, and the ultrasonic power is 800 W, the ultrasonic time is 3 min, and then treated at room temperature for 12 h to complete the culture treatment A, so as to achieve the combination of rhodamine 6G molecules and magnetic nano-molybdenum disulfide to obtain labeled magnetic nano-molybdenum disulfide.

[0133] The labeled magnetic nano-molybdenum disulfide was subjected to magnetic separation and washing for 3 times to remove the unbound rhodamine 6G molecules during the culture treatment A. It should be noted that during the magnetic separation and washing process, ferroferric oxide imparts good magnetic properties to the molybdenum disulfide nanoflowers, and the magnetic nano-molybdenum disulfide can be firmly adsorbed on the wall of the test tube with a magnet to magnetically separate the rhodamine 6G molecules on the magnetic nano-molybdenum disulfide, wash out the solution in the test tube, add deionized water, repeat the above operation 3 times, and obtain the labeled magnetic nano-molybdenum disulfide after magnetic treatment, that is, the mixed material A. Because the rhodamine 6G solution is red, the cleaning is considered to be completed when the solution in the test tube does not become transparent.

[0134] Then, a phosphate buffered saline solution of N-hydroxysuccinimide is used as a phosphate buffered saline solution of the imide compound, and the phosphate buffered saline solution has salt balance and an adjustable suitable pH buffering effect. 1 mL of N-hydroxysuccinimide with a concentration of 0.02 mol / L is weighed, and the mixture A obtained above is added and dispersed evenly, and then cultured at a culture temperature of 37° C. for 1 hour, and the culture treatment B is completed to improve the coupling efficiency of the protein, and a rhodamine 6G labeled magnetic nano-molybdenum disulfide solution is obtained.

[0135] 1.3 Preparation of immunoprobes

[0136] In this embodiment, 20 μL of 0.2 mg / mL glycoprotein 125 antibody was weighed and added to the above-mentioned rhodamine 6G labeled magnetic nano molybdenum disulfide solution, mixed and reacted at 4°C for 2 hours, and incubation treatment A was completed to achieve the linking of the antibody to the magnetic nano molybdenum disulfide; then magnetic separation treatment was performed to remove excess glycoprotein 125 antibody to obtain a mixed material B; then, 10 μL of a 3% mass concentration of bovine serum albumin blocking solution was weighed and added to the above-mentioned mixed material B, mixed and reacted at 37°C for 1 hour, and incubation treatment C was completed to achieve the blocking of nonspecific adsorption sites on the immune probe to obtain the immune probe.

[0137] 1.4 Preparation of immunoprobe solution

[0138] In this embodiment, the above-mentioned immune probe is rinsed with a phosphate buffered saline solution of N-hydroxysuccinimide at a concentration of 0.02 mol / L, and the molybdenum disulfide nanoflowers are magnetically collected by a magnet and washed 4 times to remove excess bovine serum albumin, and then dissolved in 1 mL of phosphate buffered saline solution to obtain an immune probe solution to improve the detection effect of the probe.

[0139] The purpose of using phosphate buffered saline to prepare the immune probe solution in this embodiment is to dissolve the protective reagent; the reason is that phosphate buffer has salt balance and can adjust the appropriate pH buffering effect. Generally, it cannot be replaced by other materials. For example, distilled water does not have salt balance and can destroy the structure and biological properties of biological proteins; physiological saline does not have the function of adjusting pH, and cannot ensure that intact and active substances participate in biological reactions under optimal conditions, so the use of phosphate buffered saline is the first choice.

[0140] Step 2, Preparation of Immunoassay Substrate

[0141] 2.1 Preparation of C3N4 nanosheets

[0142] The present invention adopts the following steps to obtain carbon three nitrogen four nanosheets:

[0143] 25 mg of C3N4 powder was added to 15 mL of concentrated hydrochloric acid and ultrasonicated at 800 W for 2 h. Then, the mixed solution was transferred into a 25 mL reactor and reacted at 80 ° C for 12 h. After that, the mixture was washed with a large amount of deionized water until the washing liquid was neutral to remove excess hydrochloric acid. The acidified C3N4 was dispersed in 5 mL of deionized water under ultrasonic action (ultrasonic power of 800 W, ultrasonic time of 5 to 10 min). The dispersed C3N4 was centrifuged at 3000 r / min for 10 min, and the unpeeled large pieces of C3N4 sank to the bottom of the test tube wall, and the protonated C3N4 nanosheets were left in the supernatant. The supernatant was the C3N4 nanosheet solution, and C3N4 nanosheets with a diameter of 50 to 300 nm were obtained.

[0144] 2.2 Preparation of immunomodulatory substrate solution

[0145] 2.2.1 Activated carbon trinitrogen tetrakis:

[0146] Ultrasonic dispersion was performed, with N-hydroxysuccinimide phosphate buffered saline solution as the phosphate buffered saline solution of the imide compound, 1 mL of 0.02 mol / L N-hydroxysuccinimide phosphate buffered saline solution was weighed, and the C3N4 solution obtained in 2.1 was added, and the dispersion was uniformly performed under ultrasonic power of 800 W for 3 min, and the reaction was performed at 37° C. for 1 h to activate the C3N4 nanosheets, thereby improving the coupling efficiency of the protein, and obtaining an activated C3N4 nanosheet solution.

[0147] 2.2.2 Formation of immune matrix:

[0148] Weigh 20 μL of 0.2 mg / mL glycoprotein 125 antibody and add it to the above 1 mL activated C3N4 nanosheet solution, mix well and culture at 4°C for 2 hours to obtain a mixture C; then centrifuge the mixture C at a rate of 8000 r / min for 10 minutes to remove excess glycoprotein 125 antibody in the mixture C; then add 10 μL of 3% bovine serum albumin phosphate buffer solution, then incubate at room temperature for 1 hour to link the antibody to the C3N4 substrate and complete incubation treatment B; then separate the product after incubation treatment B by centrifugation, and centrifuge and wash the product after incubation treatment B twice to remove excess bovine serum albumin to obtain an immune substrate with a concentration of 1 to 2 mg / mL.

[0149] 2.2.3 Preparation of immune substrate solution

[0150] The above immune substrate was ultrasonically treated at an ultrasonic power of 800 W for 5 to 10 minutes to be dissolved in 1 mL of phosphate buffered saline solution to obtain a carbon trioxide tetrakis(III) immune substrate solution.

[0151] Step 3: Preparation of sandwich structure SERS biosensor

[0152] 10 to 30 μL of glycoprotein 125 antigen with an initial concentration of 100 IU / mL was weighed and added to 1 mL of the above-mentioned C3N4 immune substrate solution, and incubated at an incubation temperature of 37°C for 2 hours to link the glycoprotein 125 antigen to the C3N4 substrate to achieve incubation treatment C; subsequently, the solid component obtained by incubation treatment C (the solid component sank on the wall of the test tube, and the unlinked antigen was absorbed in the solution) was centrifuged at a rate of 8000 r / min for 10 minutes using a phosphate buffer solution to remove the uncaptured antigen and obtain a mixed solution A; then, 0.5 to 1.5 mL of the above-mentioned immune probe solution was weighed and added to the above-mentioned mixed solution A, and incubated at a culture temperature of 37°C for 2 hours to complete incubation treatment D to form a SERS biosensor with a sandwich structure based on the specific immune reaction between the antigen and the antibody.

[0153] It should be noted that the SERS biosensor with a sandwich structure prepared in this embodiment is stored in 1 mL of phosphate buffer solution for subsequent SERS detection. After the SERS biosensor with a sandwich structure prepared in this embodiment is subjected to SERS detection, the immune structure is catalyzed by a visible light simulator, and the Raman molecules, antigens and antibodies can be degraded based on the good photocatalytic activity of molybdenum disulfide nanoflowers and carbon trinitrogen tetranitrate nanosheets. After the catalysis is completed, a magnet is used to separate and collect the immune probes and immune substrates, and subsequent tumor marker detection is continued. This is repeated to achieve cyclic immune detection.

[0154] Example 2

[0155] This embodiment provides a SERS biosensor based on C3N4 nanosheets, and the preparation method thereof is different from that of embodiment 1 only in that:

[0156] In step 1 of this embodiment, the amount of ferrosoferric oxide particles used is 62.8 mg.

[0157] Example 3

[0158] This embodiment provides a SERS biosensor based on C3N4 nanosheets, and the preparation method thereof is different from that of embodiment 1 only in that:

[0159] In step 1 of this embodiment, the amount of ferrosoferric oxide particles used is 125.6 mg.

[0160] Example 4

[0161] This embodiment provides a SERS biosensor based on C3N4 nanosheets, and the preparation method thereof is different from that of embodiment 1 only in that:

[0162] In step 1.1.1 of this embodiment: ferrous sulfate heptahydrate is used as the iron source, and sodium citrate is used as the stabilizer;

[0163] In step 1.1.2 of this embodiment:

[0164] Sodium molybdate is used as molybdate, thioacetamide is used as sulfur source, and the molar ratio of molybdate to sulfur source and ferrosoferric oxide is 1.5:10:0.55;

[0165] The temperature of the hydrothermal reaction is 180°C and the reaction time is 12h;

[0166] In step 1.2 of this embodiment:

[0167] Methylene blue was used as the Raman molecule, and the dosage ratio of the Raman molecule to the magnetic nano-molybdenum disulfide was 1mmol:10g;

[0168] The treatment temperature of culture treatment A was 20°C and the treatment time was 14 h;

[0169] The treatment temperature of culture treatment B was 36°C and the treatment time was 2h.

[0170] In step 1.3 of this embodiment:

[0171] The mass ratio of glycoprotein 125 antibody to rhodamine 6G labeled magnetic nano-molybdenum disulfide is 2:1000, that is, 10 μL of glycoprotein 125 antibody with a concentration of 0.2 mg / mL is added to 1 mL of rhodamine 6G labeled magnetic nano-molybdenum disulfide solution;

[0172] The treatment temperature of incubation treatment A was 3°C and the treatment time was 2.5 h;

[0173] The treatment temperature of culture treatment C was 36°C and the treatment time was 2 h.

[0174] In step 1.4 of this embodiment:

[0175] The dosage ratio of molybdenum disulfide nanoflower immune probe to phosphate buffer solution is 10 mg:1.5 mL.

[0176] In step 2.1 of this embodiment:

[0177] The ratio of C3N4 powder to concentrated hydrochloric acid is 2mg:1mL, and the temperature during acidification is 70℃ and the reaction time is 20h;

[0178] The usage ratio of water solvent to C3N4 powder is 1-2 mg:1-1.5 mL.

[0179] In step 2.2.1 of this embodiment:

[0180] The activation treatment temperature was 36°C and the treatment time was 2 h;

[0181] In step 2.2.2 of this embodiment:

[0182] The dosage ratio of glycoprotein 125 antibody to activated carbon trinitrogen tetrakis nanosheet solution was 0.0026 mg:1.5 mL;

[0183] The treatment temperature of incubation treatment B was 3°C and the treatment time was 2.5 h;

[0184] In step 2.2.3 of this embodiment:

[0185] The dosage ratio of immune substrate to phosphate buffer solution was 1 mg:1.5 mL;

[0186] In step 3 of this embodiment:

[0187] The concentration of glycoprotein 125 antigen is 80 IU / mL, and the dosage ratio of glycoprotein 125 antigen to immune substrate solution is 0.002 mg:1.5 mL;

[0188] The treatment temperature of incubation treatment C was 36°C and the incubation time was 3 h;

[0189] The dosage ratio of the immunoprobe solution to the mixed solution A is 1.5 mL:1.5 mL;

[0190] The incubation temperature of incubation treatment D was 36°C and the incubation time was 3 h;

[0191] The phosphate buffered saline solution used in each step is a phosphate buffered saline solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a concentration of 0.07 mol / L.

[0192] Example 5

[0193] This embodiment provides a SERS biosensor based on C3N4 nanosheets, and the preparation method thereof is different from that of embodiment 1 only in that:

[0194] In step 1.1.1 of this embodiment: ferric dichloride tetrahydrate is used as the iron source, and sodium acetate is used as the stabilizer.

[0195] In step 1.1.2 of this embodiment: sodium molybdate is used as molybdate, and the molar ratio of molybdate to sulfur source and ferrosoferric oxide is 2:10:0.06; the temperature of the hydrothermal reaction is 220°C, and the reaction time is 8h;

[0196] In step 1.2 of this embodiment:

[0197] Crystal violet is used as the Raman molecule, and the usage ratio of the Raman molecule to the magnetic nano-molybdenum disulfide is 3mmol:10g;

[0198] The treatment temperature of culture treatment A was 25°C and the treatment time was 8 h;

[0199] The treatment temperature of culture treatment B was 38°C and the treatment time was 0.5h.

[0200] In step 1.3 of this embodiment:

[0201] The mass ratio of glycoprotein 125 antibody to rhodamine 6G labeled magnetic nano-molybdenum disulfide is 6:1000, that is, 30 μL of glycoprotein 125 antibody with a concentration of 0.2 mg / mL is added to 1 mL of rhodamine 6G labeled magnetic nano-molybdenum disulfide solution;

[0202] The treatment temperature of incubation treatment A was 5°C and the treatment time was 1.5 h;

[0203] The treatment temperature of culture treatment C was 38°C and the treatment time was 0.5h.

[0204] In step 1.4 of this embodiment:

[0205] The dosage ratio of molybdenum disulfide nanoflower immune probe to phosphate buffer solution is 10 mg:1.3 mL.

[0206] In step 2.1 of this embodiment:

[0207] The ratio of C3N4 powder to concentrated hydrochloric acid is 3mg:2mL, and the temperature during acidification is 90℃ and the reaction time is 8h;

[0208] The usage ratio of water solvent to C3N4 powder is 2mg:1mL.

[0209] In step 2.2.1 of this embodiment:

[0210] The activation treatment temperature was 38°C and the treatment time was 0.5 h;

[0211] In step 2.2.2 of this embodiment:

[0212] The dosage ratio of glycoprotein 125 antibody to activated carbon trinitrogen tetrakis nanosheet solution was 0.006 mg:1.3 mL;

[0213] The treatment temperature of incubation treatment B was 5°C and the treatment time was 1.5 h;

[0214] In step 2.2.3 of this embodiment:

[0215] The dosage ratio of immune substrate to phosphate buffer solution was 2 mg:1.3 mL;

[0216] In step 3 of this embodiment:

[0217] The concentration of glycoprotein 125 antigen is 120 IU / mL, and the dosage ratio of glycoprotein 125 antigen to immune substrate solution is 0.006 mg:1.3 mL;

[0218] The treatment temperature of incubation treatment C was 38°C and the incubation time was 1 h;

[0219] The dosage ratio of the immunoprobe solution to the mixed solution A is 1.3 mL:1.3 mL;

[0220] The incubation temperature of incubation treatment D was 38°C and the incubation time was 1 h;

[0221] The phosphate buffered saline solution used in each step is a phosphate buffered saline solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a concentration of 0.09 mol / L.

[0222] Experimental part

[0223] (I) Scanning electron microscope test

[0224] The present invention respectively carried out scanning electron microscope tests on the immune probes prepared in Example 1, Example 2 and Example 3, and the immune substrate and SERS biosensor prepared in Example 1, and the results are as follows: Figure 1-5 shown.

[0225] Figure 1 This is a scanning electron microscope photo of the immune probe prepared in Example 1. It can be seen that in the immune probe prepared in Example 1, the molybdenum disulfide nanosheets grow on the surface of the ferroferric oxide particles. Since the added ferroferric oxide particles are relatively small, there are many molybdenum disulfide sheets existing alone.

[0226] Figure 2 This is a scanning electron microscope photograph of the immune probe prepared in Example 2. It can be seen that in the immune probe prepared in Example 2, as more ferrosoferric oxide particles are added, more molybdenum disulfide nanoflowers are formed.

[0227] Figure 3 This is a scanning electron microscope photograph of the immune probe prepared in Example 3. It can be seen that in the immune probe prepared in Example 3, as the number of ferrosoferric oxide particles continues to increase, the ferrosoferric oxide particles are embedded in the molybdenum disulfide nanosheets.

[0228] Figure 4 This is a scanning electron microscope photograph of the immune substrate prepared in Example 1. It can be seen that in the immune substrate prepared in Example 1, the carbon trinitrogen tetraacidization has become smaller and thinner, and has a larger surface area.

[0229] Figure 5 This is a scanning electron microscope photograph of the SERS biosensor prepared in Example 1. It can be seen that in the SERS biosensor prepared in Example 1, due to the specific reaction of the glycoprotein 125 antigen and the antibody, the magnetic nano-molybdenum disulfide and the carbon trinitrogen tetranitride nanosheets are tightly combined together.

[0230] (II) Raman test

[0231] The present invention respectively performs Raman detection of rhodamine 6G on the immune probes and immune substrates prepared in Examples 1, 2 and 3, and the SERS biosensor prepared in Example 1 under a 532 nm laser, and the tests are as follows: Figure 6-9 shown.

[0232] Figure 6 The Raman detection results of the immune probe and immune substrate prepared in Example 1 for rhodamine 6G show that the immune probe and immune substrate prepared in Example 1 have good Raman enhancement effects on rhodamine 6G. -1 The Raman signal intensities at 6913 and 5041 respectively.

[0233] Figure 7 The Raman detection results of the immune probe prepared in Example 2 for rhodamine 6G show that the immune probe prepared in Example 2 has a good Raman enhancement effect on rhodamine 6G. -1 The Raman signal intensity at reached 8246.

[0234] Figure 8 The Raman detection results of the immune probe prepared in Example 3 for rhodamine 6G show that the immune probe prepared in Example 3 has a SERS enhancement effect on rhodamine 6G. -1 The Raman signal intensity at reached 6959.

[0235] Fig. 9 This is the Raman detection result of the SERS biosensor prepared in Example 1 for glycoprotein 125 in serum. It can be seen that the SERS biosensor prepared in Example 1 can achieve ultrasensitive detection of glycoprotein 125 in serum.

[0236] Obviously, the above embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A SERS biosensor based on carbon trioxide tetranitrate nanosheets, characterized in that: It is composed of immune probe, immune substrate and glycoprotein 125 antigen; The immune probe is obtained by linking glycoprotein 125 antibody on functionally labeled magnetic nano-molybdenum disulfide, and the diameter of the magnetic nano-molybdenum disulfide is 100-1000nm; The immune substrate is obtained by linking the glycoprotein 125 antibody to the activated carbon trinitrogen tetranitrate nanosheet, and the diameter of the carbon trinitrogen tetranitrate nanosheet is 50 to 300 nm; The immune probe and the glycoprotein 125 antibody on the immune substrate specifically bind to the glycoprotein 125 antigen to form a SERS biosensor with a sandwich structure; The functionalized labeled magnetic nano-molybdenum disulfide is obtained by the following steps: The magnetic nano-molybdenum disulfide is labeled with Raman molecules to obtain the functionally labeled magnetic nano-molybdenum disulfide; The Raman molecule is any one of rhodamine 6G, methylene blue and crystal violet, and the dosage ratio of the Raman molecule to the magnetic nano-molybdenum disulfide is 1-3 mmol:10 g; The activated C3N4 nanosheets are obtained by the following steps: S1, adding C3N4 powder to concentrated hydrochloric acid, and after ultrasonic treatment, reacting at 70-90° C. for 8-20 h to obtain acidified C3N4; The ratio of the carbon trinitrogen tetrakis powder to concentrated hydrochloric acid is 2-3 mg: 1-2 mL; S2, dispersing the acidified C3N4 in a water solvent under ultrasound, and then centrifuging, and the obtained supernatant is the C3N4 nanosheet solution; Wherein, the usage ratio of the water solvent to the carbon trinitrogen tetrakis powder is 1-2 mg:1-1.5 mL; S3, uniformly dispersing the C3N4 nanosheet solution and the imine compound in a phosphate buffered saline solution, and reacting at a temperature of 36 to 38° C. for 1 to 3 hours to obtain an activated C3N4 nanosheet solution; Wherein, the imine compound is N-hydroxysuccinimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; The preparation method of the magnetic nano molybdenum disulfide is as follows: The molybdate, the sulfur source and the ferroferric oxide are uniformly dispersed in the solvent B, and subjected to a hydrothermal reaction at a temperature of 180 to 220° C. for 6 to 12 hours. After cooling to room temperature, the mixture is washed and dried to obtain magnetic nano-molybdenum disulfide coated with ferroferric oxide; The solvent B is water.

2. The SERS biosensor according to claim 1, wherein The molybdate is sodium molybdate or ammonium molybdate; The sulfur source is thioacetamide or thiourea; The molar ratio of the molybdate to the sulfur source and ferrosoferric oxide is 1.5-2:10:0.06-0.55; The usage ratio of the molybdate to the solvent B is 0.15-0.2 mol:1L.

3. The SERS biosensor according to claim 1, wherein: The dosage ratio of the glycoprotein 125 antibody to the activated carbon three nitrogen four nanosheet solution is 0.002-0.006 mg: 1-1.5 mL.

4. A method for preparing a SERS biosensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, preparation of immune probe: Adding glycoprotein 125 antibody to the functionalized labeled magnetic nano-molybdenum disulfide and then performing incubation treatment A to link the glycoprotein 125 antibody to the functionalized labeled magnetic nano-molybdenum disulfide to obtain the immune probe; Step 2, preparation of immune substrate: Adding glycoprotein 125 antibody to the activated C3N4 nanosheet for incubation treatment B, so as to link the glycoprotein 125 antibody to the activated C3N4 nanosheet to obtain an immune substrate; Step 3, preparation of sandwich structure SERS biosensor: The immune substrate is dispersed in a phosphate buffer solution A to form an immune substrate solution, and then the glycoprotein 125 antigen is added to perform an incubation treatment C to obtain a mixed solution A; The immune probe is dispersed in a phosphate buffer solution B to form an immune probe solution, and then the immune probe solution is added to the mixed solution A for incubation treatment D to obtain a SERS biosensor.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the glycoprotein 125 antibody to the functionalized labeled magnetic nano-molybdenum disulfide is 2 to 6:1000; The dosage ratio of the immune substrate to the phosphate buffer solution A is 1-2 mg: 1-1.5 mL; The dosage ratio of the immune probe to the phosphate buffer solution B is 10 mg: 1-1.5 mL; The dosage ratio of the glycoprotein 125 antigen to the immune substrate solution is 0.002-0.006 mg: 1-1.5 mL; The dosage ratio of the immune probe solution to the mixed solution A is 1-1.5 mL:1-1.5 mL; The concentration of the glycoprotein 125 antigen is 80 to 120 IU / mL.

6. The preparation method according to claim 4, characterized in that: The incubation treatment A has a treatment temperature of 3 to 5° C. and a treatment time of 1.5 to 2.5 h. The incubation treatment B has a treatment temperature of 3 to 5° C. and a treatment time of 1.5 to 2.5 h. The incubation treatment C has a treatment temperature of 36 to 38° C. and a culture time of 1 to 3 hours; The incubation temperature of the incubation treatment D is 36-38° C., and the incubation time is 1-3 hours.

7. Use of the SERS biosensor according to any one of claims 1 to 3 in the preparation of an ovarian tumor marker detection reagent.

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