Preparation method and application of colorimetric / SERS (Surface Enhanced Raman Scattering) dual-mode biosensor

Abstract

The invention discloses a preparation method and application of a colorimetric / SERS (Surface Enhanced Raman Scattering) dual-mode biosensor, and belongs to the technical field of food safety detection. The preparation method of the sensor comprises the following steps: carrying out amino modification on MIL-101 nanoparticles to obtain aminated MIL-101; taking the aminated MIL-101 and Au-NRs as raw materials, and carrying out reaction to prepare an MIL-101 / Au NRs nano composite material; and mixing the MIL-101 / Au NRs nano composite material with a sodium acetate buffer solution, a TMB (Tetramethylbenzidine) solution and an H2O2 solution, and reacting at the temperature of 40-70 DEG C for 0.2-2 hours to obtain the sensor. According to the colorimetric / SERS dual-mode biosensor constructed by the invention, various analysis and detection of CAP in the same sensor are realized, so that the field requirement is met to the greatest extent, and the complex and variable conditions in the detection field are dealt with.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a preparation method and application of a colorimetric / SERS dual-mode biosensor. Background Art

[0002] Chloramphenicol (CAP) is a class of broad-spectrum antimicrobial antibiotics commonly found in aquatic products derived from Venezuelan Streptomyces cultures, which inhibits bacterial growth by binding to the 50S subunit of the ribosome and preventing protein synthesis. Although CAP can provide protection in aquaculture, its overuse may lead to the accumulation of residues in fish and pose a potential risk to human health systems. The main harmful effect of CAP on the human body is to inhibit the hematopoietic function of the bone marrow, which also affects the metabolic rate of other drugs, thereby exacerbating their toxic effects on the body, which further increases the health risks faced by consumers when consuming contaminated seafood.

[0003] Currently available methods for detecting CAP include high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), mouse bioassay (MBA) and high performance liquid chromatography / mass spectrometry (HPLC-MS / MS). Although most of them can meet the relevant testing standards for food safety and environmental protection, they still have certain limitations. For example, mouse bioassay (MBA) has the disadvantages of being time-consuming and having poor repeatability. High performance liquid chromatography / mass spectrometry (HPLC-MS / MS) has complex pre-treatment and requires experienced testing personnel. Therefore, it is very important to develop new rapid, sensitive and low-cost methods in the field of CAP detection. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of a colorimetric / SERS (surface enhanced Raman scattering) dual-mode biosensor to solve the problems existing in the detection of CAP in the prior art, such as: long detection time, cumbersome detection steps, the need for large-scale instruments, and limited on-site detection.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: a method for preparing a colorimetric / SERS dual-mode biosensor, comprising the following steps:

[0007] The MIL-101 nanoparticles are amino-modified to obtain aminated MIL-101; the aminated MIL-101 and Au-NRs (gold nanoparticles) are used as raw materials to react and prepare a MIL-101 / Au NRs nanocomposite material;

[0008] The MIL-101 / AuNRs nanocomposite material is mixed with sodium acetate buffer, TMB (3,3',5,5'-tetramethylbenzidine) solution and H2O2 solution, and reacted at a temperature of 40-70°C for 0.2-2h. After the reaction, the mixed solution obtained is the colorimetric / SERS dual-mode biosensor.

[0009] The synthesized MIL-101 / Au-NRs nanocomposite material of the present invention not only has peroxidase-like activity, but also has SERS activity, and can be used for signal amplification. The present invention uses TMB as a signal source, and the synthesized MIL-101 / Au-NRs nanocomposite material as a peroxidase-like enzyme, and constructs a colorimetric / SERS dual-mode biosensor based on SERS activity-peroxidase-like MIL-101 / Au-NRs, thereby realizing rapid and sensitive detection of CAP. The specifically designed MIL-101 / Au-NRs has strong peroxidase-like activity, which improves the sensitivity of detection, and in the presence of H2O2, it can catalyze the oxidation of TMB into oxTMB, which will cause the generation and change of both SERS and colorimetric signals, and can simultaneously realize multiple analyses and detections of CAP in the same sensor (detection principle as shown in FIG. 2 ). Figure 1 As shown), it can meet the rapid analysis and detection of CAP to the greatest extent, cope with complex and changeable situations in detection and reduce detection costs.

[0010] Furthermore, the preparation method of the MIL-101 nanoparticles comprises: mixing an iron source, a nickel source, an organic acid, a titanium source and a solvent, and performing a solvothermal reaction to obtain the MIL-101 nanoparticles;

[0011] The iron source is ferric chloride; the nickel source is nickel chloride; the organic acid is terephthalic acid (H2BDC); the titanium source is tetraisopropyl titanate (TTIP); the solvent is DMF; the amount ratio of the iron source, nickel source, organic acid, titanium source and solvent is 3g:3g:4g:125μL:200mL; the temperature of the solvent thermal reaction is 25-200℃; the time is 10-50h. The participation of MIL-101 nanoparticles can make the SERS enhancement effect better.

[0012] Furthermore, the reaction is preferably carried out at a temperature of 50° C. for 0.5-2 h.

[0013] Furthermore, the solvent thermal reaction is carried out under nitrogen protection.

[0014] Furthermore, the ferric chloride is specifically FeCl3·6H2O; and the nickel chloride is specifically NiCl2·6H2O.

[0015] Furthermore, after the iron source, nickel source, organic acid, titanium source and solvent are mixed, an ultrasonic treatment operation is also included; after the solvothermal reaction is completed, the reaction product is ultrasonically washed and dried in sequence with anhydrous ethanol and ultrapure water.

[0016] Furthermore, the amino-modification of the MIL-101 nanoparticles to obtain the aminated MIL-101 specifically comprises: mixing the MIL-101 nanoparticles, the amino-modifying agent and anhydrous ethanol, stirring and reacting for 0.1-10 hours, washing and filtering after the reaction to obtain the aminated MIL-101;

[0017] The amino modifier is 3-aminopropyltrimethoxysilane (APTES); the dosage ratio of the MIL-101 nanoparticles, the amino modifier and anhydrous ethanol is 3 mg:1.5 mL:1 mL.

[0018] Furthermore, the stirring reaction is carried out at room temperature for 0.1-10 h.

[0019] Furthermore, the method of preparing the MIL-101 / Au NRs nanocomposite material by reaction using aminated MIL-101 and Au-NRs as raw materials specifically includes: dispersing the aminated MIL-101 in water to obtain an aminated MIL-101 solution; mixing the aminated solution with the Au-NRs solution, stirring the reaction for 1-10 hours, and obtaining the MIL-101 / Au NRs nanocomposite material.

[0020] Furthermore, the stirring reaction is carried out at room temperature for 1-10 hours.

[0021] Further, the concentration of the aminated MIL-101 solution is 1 g / L; the volume ratio of the aminated MIL-101 solution to the Au-NRs solution is 1:1;

[0022] The preparation method of the Au-NRs solution comprises: mixing HAuCl4 solution, CTAB solution and NaBH4 solution, stirring and reacting for 0.1-10h, and standing for 1-10h after the reaction is completed to obtain a gold seed solution; mixing CTAB (hexadecyltrimethylammonium bromide), NaOL and water, first heating to 40-100°C for reaction for 0.5-2h and then cooling to room temperature, then adding AgNO3 solution, stirring and reacting for 1-5h, then adding HCl solution and ascorbic acid solution, stirring and reacting for 1-2h to obtain an Au-NRs growth solution; mixing the gold seed solution and the Au-NRs growth solution, stirring and reacting for 10-50h to obtain the Au-NRs solution;

[0023] The stirring reactions were all carried out at room temperature.

[0024] Furthermore, the concentration ratio of the HAuCl4 solution, the CTAB solution and the NaBH4 solution is 1:400:2, and the volume ratio is 1:1:1.2; the concentration of the AgNO3 solution is 1-10mM; the concentration of the HCl solution is 20-50wt%; the concentration of the ascorbic acid solution is 1-200mM; the dosage ratio of the CTAB, NaOL, water, AgNO3 solution, HCl solution and ascorbic acid solution is 9g:1.2g:10-500mL:1-50mL:1-50mL:1-100mL; the volume ratio of the gold seed solution and the Au-NRs growth solution is 100μL:270.8mL.

[0025] Further, the mixing of the MIL-101 / Au NRs nanocomposite material with the sodium acetate buffer, the TMB solution and the H2O2 solution specifically comprises: dispersing the MIL-101 / Au NRs nanocomposite material in water to obtain a MIL-101 / Au NRs nanocomposite material solution; then mixing the MIL-101 / Au NRs nanocomposite material solution with the sodium acetate buffer, the TMB solution and the H2O2 solution;

[0026] The concentration of the MIL-101 / Au NRs nanocomposite solution is 0.25 g / L; the pH of the sodium acetate buffer is 3-5, preferably 4, and the concentration is 0.2 M; the concentration of the TMB solution is 4-30 mM, preferably 5 mM; the concentration of the H2O2 solution is 0.2-0.4 M, preferably 0.3 M; the volume ratio of the MIL-101 / Au NRs nanocomposite solution to the sodium acetate buffer, the TMB solution and the H2O2 solution is 2:20:1:1.

[0027] The second technical solution of the present invention: a colorimetric / SERS dual-mode biosensor prepared by the above-mentioned preparation method.

[0028] The third technical solution of the present invention: application of the above colorimetric / SERS dual-mode biosensor in CAP detection.

[0029] Furthermore, the application specifically refers to the application of the above-mentioned colorimetric / SERS dual-mode biosensor in detecting CAP in food.

[0030] The fourth technical solution of the present invention is a method for detecting CAP, comprising the following steps:

[0031] Adding different concentrations of CAP standard solutions into the colorimetric / SERS dual-mode biosensor respectively, reacting for 0.1-5 hours, and obtaining a mixed solution to be tested containing the CAP standard solution;

[0032] Determine the absorbance characteristic value of the mixed solution containing the CAP standard solution at 652nm and the absorbance characteristic value of the mixed solution containing the CAP standard solution at 1009cm under 785nn excitation light. -1 SERS intensity at ;

[0033] Establish a standard curve between the absorbance characteristic value and the logarithm of the corresponding CAP standard solution concentration, that is, the standard curve for CAP content detection in the colorimetric mode; establish a standard curve between the SERS intensity and the logarithm of the corresponding CAP standard solution concentration, that is, the standard curve for CAP content detection in the SERS mode;

[0034] Adding the sample to be tested into the colorimetric / SERS dual-mode biosensor, reacting (i.e., incubating) for 0.1-5 hours to obtain a mixed solution containing the sample to be tested;

[0035] Determine the absorbance characteristic value of the mixed solution containing the sample to be tested at 652nm, substitute it into the standard curve of CAP content detection in the colorimetric mode, and obtain the CAP concentration in the colorimetric mode; or determine the absorbance characteristic value of the mixed solution containing the sample to be tested at 1009cm under 785nm excitation light. -1 The SERS intensity at the position is substituted into the standard curve of CAP content detection under the SERS mode to obtain the CAP concentration under the SERS mode.

[0036] The colorimetric mode can be detected with the help of a variety of instruments such as spectrophotometers and ELISA readers. These instruments have a high penetration rate and are small in size for easy mobility, meeting the needs of convenient detection. The SERS mode is detected with the help of Raman spectrometers. Raman spectrometers have a low penetration rate in laboratories but have good detection performance and can meet the needs of high-sensitivity detection. When the instruments used for detection in the two modes coexist, the concentration detected in the SERS mode is the final concentration.

[0037] Furthermore, the sample to be tested is obtained by pre-processing and extracting food.

[0038] Furthermore, the volume ratio of the CAP standard solution to the colorimetric / SERS dual-mode biosensor is 1:2; the volume ratio of the test sample to the colorimetric / SERS dual-mode biosensor is 1:2.

[0039] Furthermore, the reaction is carried out at room temperature.

[0040] The present invention discloses the following technical effects:

[0041] The present invention provides a colorimetric / SERS dual-mode biosensor, which can realize rapid analysis and detection of CAP in dual modes, thereby meeting the needs of on-site detection to the greatest extent and coping with complex and changeable situations in the detection site.

[0042] The colorimetric / SERS dual-mode biosensor constructed by the present invention can realize the output of two signals in the same detection system, thereby improving the sensitivity and adaptability of the detection technology, and a suitable detection means can be selected according to the detection requirements.

[0043] The colorimetric / SERS dual-mode biosensor constructed by the present invention realizes the specific detection of CAP in food. The detection limits of the colorimetric mode and the SERS mode are 0.1μM and 0.1nM, respectively. In addition, the colorimetric / SERS dual-mode biosensor has been successfully applied to the detection of CAP in real samples. The results show that the quantitative detection of the colorimetric / SERS dual-mode biosensor has good accuracy, stability and adaptability, and can be used as a potential alternative for on-site detection of CAP. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 Schematic diagram of the principle of the colorimetric / SERS dual-mode biosensor for detecting CAP of the present invention;

[0046] Figure 2 Transmission electron microscopy images of the MIL-101 nanoparticles (A) prepared in step 1 of Example 1 and the MIL-101 / AuNRs nanocomposite (B) prepared in step 3;

[0047] Figure 3 The feasibility of detecting CAP in colorimetric mode (A) and the feasibility of detecting CAP in SERS mode (B);

[0048] Figure 4 Optimization screening results for reaction temperature (A), buffer pH (B), H2O2 concentration (C), and TMB concentration (D) of the colorimetric / SERS dual-mode biosensor;

[0049] Figure 5 (A) is the standard curve diagram of CAP content detection in colorimetric mode and (B) is the standard curve diagram of CAP content detection in SERS mode. DETAILED DESCRIPTION

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0052] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0053] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0054] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0055] The present invention provides a method for preparing a colorimetric / SERS dual-mode biosensor, comprising the following steps:

[0056] The MIL-101 nanoparticles are amino-modified to obtain aminated MIL-101; the aminated MIL-101 and Au-NRs (gold nanoparticles) are used as raw materials to react and prepare a MIL-101 / Au NRs nanocomposite material;

[0057] The MIL-101 / AuNRs nanocomposite material is mixed with a sodium acetate buffer, a TMB (3,3',5,5'-tetramethylbenzidine) solution and a H2O2 solution, and reacted at a temperature of 40-70° C., preferably at a temperature of 50° C. for 0.2-2 h. After the reaction, the mixed solution obtained is the colorimetric / SERS dual-mode biosensor;

[0058] As a preferred embodiment of the present invention, the method for preparing the MIL-101 nanoparticles comprises: mixing an iron source, a nickel source, an organic acid, a titanium source and a solvent, and performing a solvothermal reaction to obtain the MIL-101 nanoparticles;

[0059] The iron source is ferric chloride; the nickel source is nickel chloride; the organic acid is terephthalic acid (H2BDC); the titanium source is tetraisopropyl titanate (TTIP); the solvent is DMF; the dosage ratio of the iron source, nickel source, organic acid, titanium source and solvent is 3g:3g:4g:125μL:200mL; the temperature of the solvent thermal reaction is 25-200℃; and the time is 10-50h.

[0060] As a preferred embodiment of the present invention, the solvent thermal reaction is carried out under nitrogen protection.

[0061] As a preferred embodiment of the present invention, the ferric chloride is specifically FeCl3·6H2O; the nickel chloride is specifically NiCl2·6H2O.

[0062] As a preferred embodiment of the present invention, after mixing the iron source, nickel source, organic acid, titanium source and solvent, an ultrasonic treatment operation is also included; after the solvent thermal reaction is completed, the reaction product is ultrasonically washed and dried in sequence with anhydrous ethanol and ultrapure water.

[0063] As a preferred embodiment of the present invention, the amino-modification of MIL-101 nanoparticles to obtain aminated MIL-101 specifically comprises: mixing MIL-101 nanoparticles, an amino-modifying agent and anhydrous ethanol, stirring at room temperature for a reaction of 0.1-10 hours, washing and filtering after the reaction to obtain the aminated MIL-101;

[0064] The amino modifier is 3-aminopropyltrimethoxysilane (APTES); the dosage ratio of the MIL-101 nanoparticles, the amino modifier and anhydrous ethanol is 3 mg:1.5 mL:1 mL.

[0065] As a preferred embodiment of the present invention, the method of preparing the MIL-101 / Au NRs nanocomposite material by reaction using aminated MIL-101 and Au-NRs as raw materials specifically includes: dispersing the aminated MIL-101 in water to obtain an aminated MIL-101 solution; mixing the aminated solution with the Au-NRs solution, stirring the reaction at room temperature for 1-10 hours, and obtaining the MIL-101 / Au NRs nanocomposite material.

[0066] As a preferred embodiment of the present invention, the concentration of the aminated MIL-101 solution is 1 g / L; the volume ratio of the aminated MIL-101 solution to the Au-NRs solution is 1:1;

[0067] The preparation method of the Au-NRs solution includes: mixing HAuCl4 solution, CTAB solution and NaBH4 solution, stirring and reacting at room temperature for 0.1-10 hours, and standing at room temperature for 1-10 hours after the reaction is completed to obtain a gold seed solution; mixing CTAB, NaOL and water, first heating to 40-100°C for reaction for 0.5-2 hours and then cooling to room temperature, then adding AgNO3 solution, stirring and reacting at room temperature for 1-5 hours, then adding HCl solution and ascorbic acid solution, stirring and reacting at room temperature for 1-2 hours to obtain an Au-NRs growth solution; mixing the gold seed solution and the Au-NRs growth solution, stirring and reacting at room temperature for 10-50 hours to obtain the Au-NRs solution.

[0068] As a preferred embodiment of the present invention, the concentration ratio of the HAuCl4 solution, the CTAB solution and the NaBH4 solution is 1:400:2, and the volume ratio is 1:1:1.2; the concentration of the AgNO3 solution is 1-10mM; the concentration of the HCl solution is 20-50wt%; the concentration of the ascorbic acid solution is 1-200mM; the dosage ratio of the CTAB, NaOL, water, AgNO3 solution, HCl solution and ascorbic acid solution is 9g:1.2g:10-500mL:1-50mL:1-50mL:1-100mL; the volume ratio of the gold seed solution and the Au-NRs growth solution is 100μL:270.8mL.

[0069] As a preferred embodiment of the present invention, the mixing of the MIL-101 / Au NRs nanocomposite material with the sodium acetate buffer, the TMB solution and the H2O2 solution specifically comprises: dispersing the MIL-101 / Au NRs nanocomposite material in water to obtain a MIL-101 / Au NRs nanocomposite material solution; then mixing the MIL-101 / Au NRs nanocomposite material solution with the sodium acetate buffer, the TMB solution and the H2O2 solution;

[0070] The dosage ratio of the MIL-101 / Au NRs nanocomposite material to water is 1:1; the pH of the sodium acetate buffer solution is 3-5, preferably 4, and the concentration is 0.2M; the concentration of the TMB solution is 4-30mM, preferably 5mM; the concentration of the H2O2 solution is 0.2-0.4M, preferably 0.3M; the volume ratio of the MIL-101 / Au NRs nanocomposite material solution to the sodium acetate buffer solution, the TMB solution and the H2O2 solution is 2:20:1:1.

[0071] As a preferred embodiment of the present invention, the preparation method of the colorimetric / SERS dual-mode biosensor, more specifically the preparation steps are as follows:

[0072] Step 1: Preparation of MIL-101 nanoparticles:

[0073] FeCl3·6H2O, NiCl2·6H2O, H2BDC and TTIP are dissolved in DMF (the dosage ratio is 3g:3g:4g:125μL:200mL), and a mixed solution is obtained after ultrasonic treatment, and then the mixed solution is added to a reactor to react at 25-200°C for 10-50h (the reaction is carried out under nitrogen protection); after the reaction is completed, the reaction system is cooled to room temperature, filtered to obtain a black powder precipitate, and the black powder precipitate is ultrasonically washed with anhydrous ethanol and ultrapure water in turn, and after the ultrasonic washing is completed, it is placed in a vacuum drying oven for drying to obtain MIL-101 nanoparticles;

[0074] Step 2: Preparation of Au-NRs:

[0075] 1) preparing a gold seed solution: mixing a HAuCl4 solution and a CTAB solution to obtain a mixed solution, and then adding a NaBH4 solution to the mixed solution (the concentration ratio of the HAuCl4 solution, the CTAB solution and the NaBH4 solution is 1:400:2, and the volume ratio is 1:1:1.2), stirring at room temperature for a reaction of 0.1-10 hours, and standing at room temperature for 1-10 hours after the reaction is completed to obtain a gold seed solution;

[0076] 2) preparing Au-NRs growth solution: dissolving CTAB and NaOL in ultrapure water to obtain a mixed solution, heating the mixed solution to 40-100°C in a water bath for reaction for 0.5-2h and then cooling to room temperature; then adding 1-10mM AgNO3 solution to the mixed solution under magnetic stirring for reaction at room temperature for 1-5h, adding 20-50wt% HCl solution and 1-200mM ascorbic acid solution (the dosage ratio of CTAB, NaOL, ultrapure water, AgNO3 solution, HCl solution and ascorbic acid solution is 9g:1.2g:10-500mL:1-50mL:1-50mL:1-100mL) after the reaction at room temperature for 1-2h, and obtaining Au-NRs growth solution after the reaction;

[0077] 3) Preparing Au-NRs: mixing the gold seed solution and the Au-NRs growth solution at a volume ratio of 100 μL:270.8 mL, stirring and reacting at room temperature for 10-50 h, and obtaining an Au-NRs solution after the reaction is completed;

[0078] Step 3: Preparation of MIL-101 / Au NRs nanocomposite materials:

[0079] The MIL-101 nanoparticles prepared in step 1 are mixed with APTES and anhydrous ethanol (the ratio of MIL-101 nanoparticles to APTES and anhydrous ethanol is 3mg:1.5mL:1mL), and the mixed solution is stirred at room temperature for reaction for 0.1-10h. After stirring, the product is dispersed in ultrapure water for washing and separation to obtain MIL-101 nanoparticle solids. The MIL-101 nanoparticle solids are dispersed in ultrapure water to obtain an aminated MIL-101 solution with a concentration of 1g / L; the aminated MIL-101 solution and the Au-NRs solution are mixed in a volume ratio of 1:1, and stirred at room temperature for reaction for 1-10h; after the reaction, the mixture is centrifuged and washed to obtain a MIL-101 / Au-NR nanocomposite material, which is dispersed in ultrapure water to obtain a MIL-101 / Au NRs nanocomposite material solution with a concentration of 0.25g / L.

[0080] Step 4: Preparation of colorimetric / SERS dual-mode biosensor:

[0081] The MIL-101 / Au NRs nanocomposite solution is uniformly mixed with sodium acetate buffer (pH=3-5, preferably 4, concentration of 0.2M), 4-30mM (preferably 5mM) TMB solution, and 0.2-0.4M (preferably 0.3M) H2O2 solution (the volume ratio of the MIL-101 / Au NRs nanocomposite solution to the sodium acetate buffer, the TMB solution and the H2O2 solution is 2:20:1:1), and reacted under heating conditions at 40-70°C (preferably 50°C) in a water bath for 0.2-2h. The mixed solution obtained after the reaction is a colorimetric / SERS dual-mode biosensor.

[0082] The present invention also provides a colorimetric / SERS dual-mode biosensor prepared by the above preparation method.

[0083] The present invention also provides application of the colorimetric / SERS dual-mode biosensor in CAP detection.

[0084] As a preferred embodiment of the present invention, the application specifically refers to the application of the above-mentioned colorimetric / SERS dual-mode biosensor in detecting CAP in food.

[0085] The present invention also provides a method for detecting CAP, comprising the following steps:

[0086] Adding different concentrations of CAP standard solutions into the colorimetric / SERS dual-mode biosensor, respectively, and reacting at room temperature for 0.1-5 hours to obtain a mixed solution to be tested containing the CAP standard solution;

[0087] Determine the absorbance characteristic value of the mixed solution containing the CAP standard solution at 652nm and the absorbance characteristic value of the mixed solution containing the CAP standard solution at 1009cm under 785nn excitation light. -1 SERS intensity at ;

[0088] Establish a standard curve between the absorbance characteristic value and the logarithm of the corresponding CAP standard solution concentration, that is, the standard curve for CAP content detection in the colorimetric mode; establish a standard curve between the SERS intensity and the logarithm of the corresponding CAP standard solution concentration, that is, the standard curve for CAP content detection in the SERS mode;

[0089] Add the sample to be tested into the colorimetric / SERS dual-mode biosensor and react at room temperature for 0.1-5 hours to obtain a mixed solution containing the sample to be tested;

[0090] Determine the absorbance characteristic value of the mixed solution containing the sample to be tested at 652nm, substitute it into the standard curve of CAP content detection in the colorimetric mode, and obtain the CAP concentration in the colorimetric mode; or determine the absorbance characteristic value of the mixed solution containing the sample to be tested at 1009cm under 785nn excitation light. -1 The SERS intensity at the position is substituted into the standard curve of CAP content detection under the SERS mode to obtain the CAP concentration under the SERS mode.

[0091] Schematic diagram of the principle of colorimetric / SERS dual-mode biosensor for detecting CAP Figure 1 As shown, from Figure 1 As can be seen in the figure, the working principle of the colorimetric / SERS dual-mode biosensor is based on the MIL-101 / Au-NRs nanocomposite, which has good SERS activity and catalytic activity. In this dual-mode biosensor system, the MIL-101 / Au-NRs nanocomposite plays a peroxidase-like role, causing H2O2 to produce hydroxyl radicals (OH·), thereby converting the system from TMB to oxidized TMB (oxTMB). The system undergoes a color reaction, a UV absorption peak appears at 652nm, and the SERS signal of oxTMB is detected. In the presence of CAP, the reaction of CAP with OH· effectively inhibits the oxidation process of TMB and affects the yield of oxTMB. Therefore, as the concentration of CAP in the sample being tested increases, the generation of oxTMB decreases. This reduction can be observed by the decrease in the UV absorption peak at 652nm and the decrease in the SERS signal of oxTMB. Therefore, CAP can be detected by the dual-mode (colorimetric / SERS) sensor with sensitivity and specificity to the sample.

[0092] As a preferred embodiment of the present invention, the test sample is obtained by pre-processing and extracting food.

[0093] As a preferred embodiment of the present invention, the volume ratio of the CAP standard solution to the colorimetric / SERS dual-mode biosensor is 1:2; the volume ratio of the test sample to the colorimetric / SERS dual-mode biosensor is 1:2.

[0094] In the specific embodiments of the present invention, all reactions without specifying the reaction temperature are carried out at room temperature.

[0095] The room temperature involved in the specific implementation manner of the present invention specifically refers to 20-30°C.

[0096] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0097] All raw materials used in the following embodiments, test examples and application examples are common commercially available products.

[0098] Example 1

[0099] A method for preparing a colorimetric / SERS dual-mode biosensor, the steps are as follows:

[0100] Step 1: Preparation of MIL-101 nanoparticles:

[0101] 0.6g FeCl3·6H2O, 0.6g NiCl2·6H2O, 0.8g H2BDC and 25μL TTIP were dissolved in 40mL DMF, and a mixed solution was obtained after ultrasonic treatment. Then the mixed solution was added to a reactor and reacted at 120°C for 20h (the reaction was carried out under nitrogen protection). After the reaction was completed, the reaction system was cooled to room temperature, filtered to obtain a black powder precipitate, and the obtained black powder precipitate was ultrasonically washed with anhydrous ethanol and ultrapure water in turn. After the ultrasonic washing, it was placed in a vacuum drying oven and dried to obtain MIL-101 nanoparticles.

[0102] Step 2: Preparation of gold nanoparticles (Au-NRs):

[0103] 1) Preparation of gold seed solution: 5 mL of 0.5 mM HAuCl4 solution (aqueous solution) was mixed with 5 mL of 0.2 M CTAB solution (aqueous solution) to obtain a mixed solution, and then 6 mL of 1 mM NaBH4 solution (aqueous solution) was added to the mixed solution and stirred at room temperature for 5 h. After the reaction was completed, the mixture was allowed to stand at room temperature for 10 h to obtain a gold seed solution.

[0104] 2) Preparation of Au-NRs growth solution: 9g CTAB and 1.2g NaOL were dissolved in 250mL ultrapure water to obtain a mixed solution, which was heated to 60°C in a water bath for 1h and then cooled to room temperature. Then, 18mL 4 mM AgNO3 solution (aqueous solution) was added to the mixed solution under magnetic stirring and reacted at room temperature for 90min. After the reaction, 1.5mL 37wt% HCl solution and 1.3mL 64mM ascorbic acid solution were added and reacted at room temperature for 20min. After the reaction, the Au-NRs growth solution was obtained.

[0105] 3) Preparation of Au-NRs: 100 μL of the gold seed solution prepared in step 1) was added to the entire Au-NRs growth solution (270.8 mL) prepared in step 2), and the reaction was continued with stirring at room temperature for 12 h. After the reaction was completed, an Au-NRs solution was obtained.

[0106] Step 3: Preparation of MIL-101 / Au NRs nanocomposite materials:

[0107] 30 mg of the MIL-101 nanoparticles prepared in step 1 were mixed with 15 mL of APTES and 10 mL of anhydrous ethanol, and the mixed solution was stirred at room temperature for reaction for 6 h. After stirring, the product was dispersed in 30 mL of ultrapure water for washing and separation to obtain a MIL-101 nanoparticle solid. The MIL-101 nanoparticle solid was dispersed in ultrapure water to obtain an aminated MIL-101 solution with a concentration of 1 g / L. 2 mL of the aminated MIL-101 solution was mixed with 2 mL of the Au-NRs solution prepared in step 3) of step 2, and stirred at room temperature for reaction for 4 h. After the reaction was completed, centrifugation and washing were performed to obtain a MIL-101 / Au-NR nanocomposite solid, and the obtained MIL-101 / Au-NR nanocomposite was dispersed in ultrapure water to obtain a MIL-101 / Au NRs nanocomposite solution with a concentration of 0.25 g / L.

[0108] Step 4: Preparation of colorimetric / SERS dual-mode biosensor:

[0109] Add 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M), 50 μL of 5 mM TMB solution, and 50 μL of 0.3 M H2O2 solution to 100 μL of the MIL-101 / Au NRs nanocomposite solution prepared in step 3, mix well, and react in a 50°C water bath for 25 min. The mixed solution obtained after the reaction is the colorimetric / SERS dual-mode biosensor.

[0110] Figure 2The transmission electron micrographs of the MIL-101 nanoparticles (A) prepared in step 1 of Example 1 and the MIL-101 / AuNRs nanocomposite (B) prepared in step 3 are shown in FIG. Figure 2 It can be seen that MIL-101 nanoparticles and MIL-101 / Au NRs nanocomposites were successfully synthesized.

[0111] Test Example 1

[0112] The feasibility of colorimetric / SERS dual-mode biosensor for detecting CAP was investigated. The specific method is as follows:

[0113] Set up five groups of experiments, as follows:

[0114] Group A (H2O2+TMB): The absorbance and SERS intensity of H2O2 solution and TMB solution were measured.

[0115] Group B (MIL-101 / Au-NRs+TMB): The absorbance and SERS intensity of MIL-101 / Au-NRs nanocomposite materials and TMB solution were measured.

[0116] Group C (MIL-101 / Au-NRs+H2O2): The absorbance and SERS intensity of MIL-101 / Au-NRs nanocomposite materials and H2O2 solution were measured.

[0117] Group D (MIL-101 / Au-NRs+TMB+H2O2): The absorbance and SERS intensity of MIL-101 / Au-NRs nanocomposites, H2O2 solution, and TMB solution were measured.

[0118] Group E (MIL-101 / Au-NRs+TMB+H2O2+CAP): The absorbance and SERS intensity of MIL-101 / Au-NRs nanocomposite materials, H2O2 solution, TMB solution, and CAP standard solution were measured.

[0119] The operation of group A is as follows: 50 μL of 5 mM TMB solution, 50 μL of 0.3 M H2O2 solution and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) were mixed, and the mixture was heated in a 50°C water bath for 25 min. After the reaction, the absorbance and SERS intensity of the mixed solution were measured.

[0120] The operation of group B is as follows: 100 μL of LMIL-101 / Au-NRs nanocomposite solution (prepared in step 3 of Example 1), 50 μL of 5 mM TMB solution and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) were mixed, and the mixture was reacted in a 50°C water bath for 25 min. After the reaction, the absorbance and SERS intensity of the mixed solution were measured.

[0121] The operation of group C is as follows: 100 μL of LMIL-101 / Au-NRs nanocomposite solution (prepared in step 3 of Example 1), 50 μL of 0.3 M H2O2 solution and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) were mixed, and the mixture was reacted in a 50°C water bath for 25 min. After the reaction, the absorbance and SERS intensity of the mixed solution were measured.

[0122] The operation of group D is as follows: 100 μL of LMIL-101 / Au-NRs nanocomposite solution (prepared in step 3 of Example 1), 50 μL of 5 mM TMB solution, 50 μL of 0.3 M H2O2 solution and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) were mixed, and the mixture was reacted in a 50°C water bath for 25 min. After the reaction, the absorbance and SERS intensity of the mixed solution were measured.

[0123] The operation of Group E is as follows: 100 μL of MIL-101 / Au-NRs nanocomposite solution (prepared in step 3 of Example 1), 50 μL of 5 mM TMB solution, 50 μL of 0.3 M H2O2 solution, 50 μL of CAP standard solution and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) are mixed, and the mixture is reacted in a 50°C water bath for 25 min. After the reaction, the absorbance and SERS intensity of the mixed solution are measured.

[0124] The results of the five experiments are shown in Figure 3 As shown, A is the absorbance measurement result, and B is the SERS intensity measurement result. Figure 3 It can be seen that when CAP exists, the catalytic activity of MIL-101 / Au-NRs is inhibited due to the action of CAP, so compared with group D, the absorbance at 652nm of other groups decreases, and the SERS intensity decreases. It proves that the dual-mode biosensor can be applied to the detection of CAP.

[0125] Test Example 2

[0126] Optimization screening of conditions

[0127] The absorbance measurement involved in the following condition optimization screening experiment is specifically to measure the absorbance characteristic value of the mixed solution at 652nm, and the SERS intensity measurement is specifically to measure the absorbance characteristic value of the mixed solution at 1009cm under 785nm excitation light. -1 The SERS intensity value at .

[0128] (1) Optimization and screening of reaction temperature

[0129] The specific operation is as follows: add 50 μL of 5 mM TMB solution, 50 μL of 0.3 M H2O2 solution and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) to 100 μL of LMIL-101 / Au NRs nanocomposite material solution (prepared in step 3 of Example 1), mix well, and react for 25 min under heating conditions at 20, 30, 40, 50, 60, and 70°C in a water bath. After the reaction, the absorbance and SERS intensity of the mixed solution are measured.

[0130] The optimization screening results of reaction temperature are as follows Figure 4 As shown in A, it can be seen that when the temperature gradually increases from 20°C to 50°C, the absorbance and SERS intensity of the mixed solution gradually increase, and when the temperature gradually increases from 50°C to 70°C, the absorbance and SERS intensity of the mixed solution gradually decrease. In the temperature range of 40-70°C, the absorbance and SERS intensity of the mixed solution are higher, which is a better reaction temperature, and 50°C is selected as the best reaction temperature.

[0131] (2) Optimization and screening of buffer pH

[0132] The specific operation is as follows: add 50 μL of 5 mM TMB solution, 50 μL of 0.3 M H2O2 solution and 1 mL of sodium acetate buffer with different pH values ​​(pH = 2, 3, 4, 5, 6, 7, 8 or 9, with a concentration of 0.2 M) to 100 μL of MIL-101 / Au NRs nanocomposite material solution (prepared in step 3 of Example 1), mix well, and react in a 50°C water bath for 25 min. After the reaction, measure the absorbance and SERS intensity of the mixed solution.

[0133] The optimization screening results of buffer pH are as follows Figure 4 As shown in B, it can be seen that when the buffer pH is 2-4, the absorbance and SERS intensity of the mixed solution increase with the increase of pH, and when the pH is 4-9, the absorbance and SERS intensity of the mixed solution decrease with the increase of pH. When pH is 3-5, the absorbance and SERS intensity of the mixed solution are higher, which is a better buffer pH, among which pH = 4 is selected as the best pH.

[0134] (3) Optimization and screening of H2O2 concentration

[0135] The specific operation is as follows: add 50 μL of 5 mM TMB solution, 50 μL of H2O2 solutions of different concentrations (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM) and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) to 100 μL of MIL-101 / Au NRs nanocomposite material solution (prepared in step 3 of Example 1), mix well, and react in a 50°C water bath for 25 minutes. After the reaction, the absorbance and SERS intensity of the mixed solution are measured.

[0136] The optimization screening results of H2O2 concentration are as follows Figure 4 As shown in C, it can be seen that within the range of 0.1-1.0M, with the increase of H2O2 concentration, the absorbance and SERS intensity of the mixed solution first increase and then decrease. When the H2O2 concentration is 0.3M, the SERS intensity and absorbance of the mixed solution reach the highest value. When the H2O2 concentration is 0.2-0.4M, the absorbance and SERS intensity of the mixed solution are higher, which is a better H2O2 concentration, among which 0.3M is selected as the best concentration.

[0137] (4) Optimization and screening of TMB concentration

[0138] The specific operation is as follows: add 50 μL of TMB solution of different concentrations (1, 2, 4, 5, 10, 30, 40 or 50 mM), 50 μL of 0.3 M H2O2 solution and 1 mL of sodium acetate buffer (pH = 4, concentration of 0.2 M) to 100 μL of MIL-101 / Au NRs nanocomposite material solution (prepared in step 3 of Example 1), mix well, and react for 25 minutes under heating conditions at 50°C in a water bath. After the reaction, the absorbance and SERS intensity of the mixed solution are measured.

[0139] The optimization screening results of TMB concentration are as follows Figure 4 As shown in D, it can be seen that within the range of 0-50mM, as the TMB concentration increases, the absorbance and SERS intensity of the mixed solution first increase and then decrease. When the TMB concentration is 5mM, the SERS intensity and absorbance of the mixed solution are the highest. When the TMB concentration is 4-30mM, the absorbance and SERS intensity of the mixed solution are high, which is a better TMB concentration, and 5mM is selected as the best concentration.

[0140] Application Example 1

[0141] The colorimetric / SERS dual-mode biosensor prepared in Example 1 was used for the detection of CAP

[0142] Step 1: Establishment of CAP content detection standard curve:

[0143] Prepare CAP standard solutions of different concentrations (0.01, 0.05, 0.1, 0.5, 1.5, 10, 50, 100, 500 μM), add 50 μL of CAP standard solutions of different concentrations to 100 μL of the colorimetric / SERS dual-mode biosensor prepared in Example 1 (the mixed solution obtained in step 4 of Example 1), mix well and react at room temperature for 25 min, to obtain multiple groups of mixed solutions to be tested containing CAP standard solutions;

[0144] First, the absorbance characteristic value (denoted as A) of each group of mixed solutions containing CAP standard solution at 652 nm was determined. 652 ), establish a standard curve between the absorbance characteristic value and the logarithm of the corresponding CAP standard solution concentration, that is, the standard curve for CAP content detection in the colorimetric mode, such as Figure 5 Then, the mixed solution containing CAP standard solution in each group was measured under the excitation of 785nm excitation light at 1009cm -1 The SERS intensity value at the position (denoted as I) is obtained, and a standard curve between the SERS intensity value and the logarithm of the corresponding CAP standard solution concentration is established, that is, the standard curve for CAP content detection under SERS mode, such as Figure 5 As shown in B.

[0145] Depend on Figure 5 As can be seen from the A in the figure, with the continuous increase of CAP concentration, A 652 The standard curve of CAP content detection obtained by linear fitting is A 652 =0.01384log(CAP)+0.5561, correlation coefficient R 2 =0.9905 (where A 652 represents y in the figure, log(CAP) represents x in the figure, i.e. the logarithm of the concentration of the CAP standard solution). The detection limit is 0.1 μM, and the linear range is 0.1-10 μM.

[0146] Depend on Figure 5 As shown in B, with the continuous increase of CAP concentration, the SERS intensity (I) continues to increase; after linear fitting, the standard curve of CAP content detection is I = 16554.94log(CAP) + 2454.61 (where I represents y in the figure, log(CAP) represents x in the figure, that is, the logarithm of the concentration of CAP standard solution), and the correlation coefficient R 2 =0.9954. The detection limit is 0.1 nM and the linear range is 0.0001-1 μM.

[0147] Step 2: Detection of CAP content in food samples

[0148] The preparation of the test samples followed the Chinese National Food Safety Standard with slight modifications (GB5009.206-2016). Specifically, the fish samples (5 g) were homogenized and transferred to centrifuge tubes containing different concentrations of CAP standard solutions, vortexed vigorously for 2 min, and ultrasonicated in a 50°C water bath for 15 min to promote the effective extraction of CAP, and then the solution was centrifuged at 8000 rpm for 5 min at 4°C. The supernatant was collected and purified with an immunoaffinity column to obtain an eluent. The eluent was dried with nitrogen at 45°C, and the residue was finally dissolved in 10 mL of ultrapure water to obtain a test sample solution.

[0149] 50 μL of the sample solution to be tested was added to 100 μL of the colorimetric / SERS dual-mode biosensor prepared in step 4 of Example 1, mixed evenly and reacted at room temperature for 25 min to obtain a mixed solution containing the sample to be tested; first, the absorbance characteristic value of the mixed solution containing the sample to be tested at 652 nm was measured, and then the absorbance characteristic value of the mixed solution containing the sample to be tested at 1009 cm under 785 nm excitation light was measured. -1 The SERS intensity value at the position is substituted into the standard curve of CAP content detection in the colorimetric mode and the standard curve of CAP content detection in the SERS mode obtained in step 1 of this application example, respectively, to obtain the CAP concentration in the colorimetric mode and the SERS mode. At the same time, the GC method in GB 31656.16-2022 is used to detect the test solution as a control. The details are shown in Table 1.

[0150] Table 1

[0151]

[0152]

[0153] As can be seen from Table 1, the spiked recovery rate of CAP in the SERS mode is 98.17-101.2%, and the spiked recovery rate of CAP in the colorimetric mode is 82.95-91.93%. The above data show that the colorimetric / SERS dual-mode biosensor has been successfully applied to the detection of actual samples, and has accuracy and reliability when used for detection.

[0154] The detection accuracy and reliability in the SERS mode are higher, but the detection accuracy and reliability in the colorimetric mode can basically reach the level of the GC method. Therefore, when there is no equipment for SERS detection at the detection site, the CAP concentration measured in the colorimetric mode can also be used as the reference concentration.

[0155] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a colorimetric / SERS dual-mode biosensor, characterized in that: The following steps are involved: The MIL-101 nanoparticles are amino-modified to obtain aminated MIL-101; the aminated MIL-101 and Au-NRs are used as raw materials to react and prepare a MIL-101 / Au NRs nanocomposite material; The MIL-101 / AuNRs nanocomposite material is mixed with sodium acetate buffer, TMB solution and H2O2 solution, and reacted at a temperature of 40-70°C for 0.2-2h. After the reaction, the mixed solution obtained is the colorimetric / SERS dual-mode biosensor.

2. The method for preparing the colorimetric / SERS dual-mode biosensor according to claim 1, characterized in that: The preparation method of the MIL-101 nanoparticles comprises: mixing an iron source, a nickel source, an organic acid, a titanium source and a solvent, and performing a solvothermal reaction to obtain the MIL-101 nanoparticles; The iron source is ferric chloride; the nickel source is nickel chloride; the organic acid is terephthalic acid; the titanium source is tetraisopropyl titanate; the solvent is DMF; the usage ratio of the iron source, nickel source, organic acid, titanium source and solvent is 3g:3g:4g:125μL:200mL; the temperature of the solvent thermal reaction is 25-200℃; and the time is 10-50h.

3. The method for preparing the colorimetric / SERS dual-mode biosensor according to claim 1, characterized in that: The amino-modification of the MIL-101 nanoparticles to obtain the aminated MIL-101 specifically comprises: mixing the MIL-101 nanoparticles, the amino-modifying agent and anhydrous ethanol, stirring and reacting for 0.1-10 hours, washing and filtering after the reaction is completed to obtain the aminated MIL-101; The amino modifier is 3-aminopropyltrimethoxysilane; the dosage ratio of the MIL-101 nanoparticles, the amino modifier and anhydrous ethanol is 3 mg:1.5 mL:1 mL.

4. The method for preparing the colorimetric / SERS dual-mode biosensor according to claim 1, characterized in that: The method of preparing the MIL-101 / Au NRs nanocomposite material by reaction using aminated MIL-101 and Au-NRs as raw materials specifically comprises: dispersing the aminated MIL-101 in water to obtain an aminated MIL-101 solution; mixing the aminated solution with the Au-NRs solution, stirring and reacting for 1-10 hours to obtain the MIL-101 / Au NRs nanocomposite material.

5. The method for preparing the colorimetric / SERS dual-mode biosensor according to claim 4, characterized in that: The concentration of the aminated MIL-101 solution is 1 g / L; the volume ratio of the aminated MIL-101 solution to the Au-NRs solution is 1:1; The preparation method of the Au-NRs solution includes: mixing HAuCl4 solution, CTAB solution and NaBH4 solution, stirring and reacting for 0.1-10 hours, and standing for 1-10 hours after the reaction is completed to obtain a gold seed solution; mixing CTAB, NaOL and water, first heating to 40-100°C for reaction and then cooling to room temperature, then adding AgNO3 solution, stirring and reacting for 1-5 hours, then adding HCl solution and ascorbic acid solution, stirring and reacting for 1-2 hours to obtain an Au-NRs growth solution; mixing the gold seed solution and the Au-NRs growth solution, stirring and reacting for 10-50 hours to obtain the Au-NRs solution.

6. The method for preparing the colorimetric / SERS dual-mode biosensor according to claim 5, characterized in that: The concentration ratio of the HAuCl4 solution, CTAB solution and NaBH4 solution is 1:400:2, and the volume ratio is 1:1:1.2; the concentration of the AgNO3 solution is 1-10mM; the concentration of the HCl solution is 20-50wt%; the concentration of the ascorbic acid solution is 1-200mM; the dosage ratio of the CTAB, NaOL, water, AgNO3 solution, HCl solution and ascorbic acid solution is 9g:1.2g:10-500mL:1-50mL:1-50mL:1-100mL; the volume ratio of the gold seed solution and the Au-NRs growth solution is 100μL:270.8mL.

7. The method for preparing the colorimetric / SERS dual-mode biosensor according to claim 1, characterized in that: The mixing of the MIL-101 / Au NRs nanocomposite material with the sodium acetate buffer, the TMB solution and the H2O2 solution specifically comprises: dispersing the MIL-101 / Au NRs nanocomposite material in water to obtain a MIL-101 / Au NRs nanocomposite material solution; then mixing the MIL-101 / Au NRs nanocomposite material solution with the sodium acetate buffer, the TMB solution and the H2O2 solution; The concentration of the MIL-101 / Au NRs nanocomposite solution is 0.25 g / L; the pH of the sodium acetate buffer solution is 3-5, and the concentration is 0.2 M; the concentration of the TMB solution is 4-30 mM; the concentration of the H2O2 solution is 0.2-0.4 M; the volume ratio of the MIL-101 / Au NRs nanocomposite solution to the sodium acetate buffer solution, the TMB solution and the H2O2 solution is 2:20:1:

1.

8. A colorimetric / SERS dual-mode biosensor prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the colorimetric / SERS dual-mode biosensor as claimed in claim 8 in CAP detection.

10. A method for detecting CAP, characterized in that: The following steps are involved: Adding different concentrations of CAP standard solutions to the colorimetric / SERS dual-mode biosensor according to claim 8, respectively, and reacting for 0.1-5 hours to obtain a mixed solution to be tested containing the CAP standard solution; Determine the absorbance characteristic value of the mixed solution containing the CAP standard solution at 652nm and the absorbance characteristic value of the mixed solution containing the CAP standard solution at 1009cm under 785nn excitation light. -1 SERS intensity at ; Establish a standard curve between the absorbance characteristic value and the logarithm of the corresponding CAP standard solution concentration, that is, the standard curve for CAP content detection in the colorimetric mode; Establishing a standard curve between the SERS intensity and the logarithm of the corresponding CAP standard solution concentration, i.e., the standard curve for CAP content detection under the SERS mode; Adding the sample to be tested into the colorimetric / SERS dual-mode biosensor according to claim 8, reacting for 0.1-5 hours, and obtaining a mixed solution containing the sample to be tested; Determine the absorbance characteristic value of the mixed solution containing the sample to be tested at 652nm, substitute it into the standard curve of CAP content detection in the colorimetric mode, and obtain the CAP concentration in the colorimetric mode; or determine the absorbance characteristic value of the mixed solution containing the sample to be tested at 1009cm under 785nn excitation light. -1 The SERS intensity at the position is substituted into the standard curve of CAP content detection under the SERS mode to obtain the CAP concentration under the SERS mode.

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