Method for detecting aflatoxin B1 in wine mash based on surface enhanced Raman spectroscopy

Abstract

The invention belongs to the technical field of rapid food detection, and particularly relates to a method for detecting aflatoxin B1 in wine mash based on surface enhanced Raman spectroscopy. According to the method, on the basis of sample pretreatment advantages and signal amplification effects of magnetic nanoparticles co-modified by polydopamine and gold nanoflowers, the magnetic nanoparticles and a graphene oxide flexible material which is formed by orderly assembling gold nanoparticles can form a compound system on the basis of base complementary pairing; the aflatoxin B1 in the wine mash is competitively combined with the aptamer on the magnetic nano material, so that the dissociation of the composite system and the reduction of a Raman signal are caused; based on the linear relation between the concentration of aflatoxin B1 and Raman signal decline, the method for detecting aflatoxin B1 in wine mash, which is high in sensitivity, resistant to matrix interference and reliable in result, is established. The method has the characteristics of high sensitivity, high signal repeatability, suitability for a complex matrix environment and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a method for detecting aflatoxin B1 (AFB1) in wine mash based on surface enhanced Raman spectroscopy. Background Art

[0002] Zhenjiang vinegar is an acidic condiment made from high-quality glutinous rice through soaking, steaming, saccharification, alcohol fermentation, acetic acid fermentation, sealing and aging, vinegar decoction and other processes. Among them, alcohol fermentation is the first step in the brewing of Zhenjiang vinegar. It mainly uses the mold and yeast in the wine koji and wheat koji to gradually convert glutinous rice starch into wine mash, providing a material basis for acetic acid fermentation. However, mold is very easy to breed fungal toxin pollution during the fermentation process. Aflatoxin B1, ochratoxin A and other fungal toxins have been detected in the raw materials for making wine mash and the fermented products, and even multiple fungal toxins have been detected at the same time. Among them, aflatoxin has received widespread attention due to its huge threat to human health.

[0003] As a highly sensitive and specific analytical detection technology, surface-enhanced Raman scattering spectroscopy has been successfully used for the sensitive detection of a variety of mycotoxins such as aflatoxin B1 (AFB1), ochratoxin (OTA), and vomitoxin (DON), but the detection objects are mostly peanuts, corn and other grains. When grains are used as the detection object, they can be detected through simple pretreatment, but the viscosity of the wine mash itself and the uneven detection environment make it difficult to enrich the traditional gold and silver nanomaterial probes, and the large amount of organic matter rich in it will produce many interference signals, affecting the accurate quantification of the target toxin AFB1. In addition, the high organic content of the wine mash makes it easy for the nanomaterials modified with aptamers / antibodies to aggregate and the aptamer recognition unit to inactivate, which poses a higher challenge to the accurate detection of mycotoxins in the wine mash; the existing research on the detection of mycotoxins under complex matrix conditions is relatively small, and the constructed Raman detection platform has the defects of insufficient signal intensity and low stability. Summary of the invention

[0004] In view of the above technical problems, the purpose of the present invention is to establish a Raman enhanced spectroscopy detection method that can achieve rapid and accurate quantification of fungal toxins in a complex matrix environment. The method is suitable for inhomogeneous detection conditions, has strong Raman signal intensity and excellent anti-matrix effect ability, and can be used to detect aflatoxin B1 in wine mash. The present invention solves the problems of extremely low dispersion and stability of nanomaterials in the inhomogeneous environment of wine mash, easy interference of detection signals, and insufficient Raman signal intensity and repeatability of fungal toxins restricted by the high salt environment in wine mash.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for detecting the content of aflatoxin B1 in wine mash based on surface enhanced Raman spectroscopy comprises the following steps:

[0007] S1 draws the standard curve of AFB1:

[0008] S11: Preparation of AFB at different concentrations 1 Standard solution, GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with AFB1 aptamer complementary chain, and Fe 3 O 4 Magnetic nanocolloid probe of @PDA@Cys-AuNFs;

[0009] S12: Take the same volume and different concentrations of AFB respectively 1 Standard solution, denoted as V 1 ; respectively with V 2 Volume of AFB1 aptamer complementary chain modified GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate, V 3 Volumetric AFB 1 Aptamer-modified Fe 3 O 4 The @PDA@Cys-AuNFs magnetic nanocolloid probe was mixed to obtain mixed colloids of different concentrations; and then incubated under certain conditions. After incubation, the conjugate was separated by a permanent magnet to obtain the conjugate, which was washed several times with phosphate buffer (PB) and then separated by a permanent magnet. Finally, the conjugate was resuspended in deionized water, where the volume of the resuspended deionized water was recorded as V. 4 ; Finally, a conjugate mixture with different concentrations is obtained;

[0010] S13: Take a certain volume of the combined substance mixture and drop it on the silicon wafer. After the droplet is stable, use a Raman spectrometer to scan the spectrum to obtain a Raman spectrum. －1 The characteristic Raman peak is used as the quantitative reference peak, and the Raman peak intensity value I at the displacement of the quantitative reference peak is measured. 1 , with AFB 1 The concentration of the standard solution and its corresponding Raman peak intensity value I 1 Draw a standard curve;

[0011] S2 Preparation of the sample solution to be tested: Weigh the mash, crush it with a tissue homogenizer, record the mass as x, add acetonitrile aqueous solution, shake and extract at room temperature to obtain a crude extract; centrifuge the crude extract, collect the supernatant as the test solution, and record the volume v 0 ;

[0012] S3 Raman detection of test liquid:

[0013] Take the same volume V as step S12 1 The test solution prepared in step S2 is added to the same volume as in step S12. 2 Volumetric AFB 1 The GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with the complementary chain of the aptamer was added with the same volume of V as in step S12. 3 Volume AFB1 aptamer modified Fe 3 O 4 The magnetic nanocolloid probe of @PDA@Cys-AuNFs was vortexed and mixed evenly and incubated. After incubation, the conjugate was separated by a permanent magnet and washed several times with PB solution. The conjugate was separated by a permanent magnet and resuspended in an aqueous solution to obtain a conjugate mixture, wherein the volume of the resuspended deionized water was recorded as V 4 ; Finally, the combined mixed solution is added dropwise onto the silicon wafer, and after the droplet is stabilized, a Raman spectrometer is used to perform spectral scanning to obtain a Raman spectrum;

[0014] S4 quantitative determination:

[0015] According to the Raman spectrum of the test liquid obtained in step S3, the characteristic Raman peak of DTNB is used as the quantitative reference peak, the Raman peak intensity value at the displacement of the quantitative reference peak is measured, and the concentration of AFB1 in the test liquid according to the AFB1 standard curve obtained in step (1) is respectively recorded as m, and the content x of AFB1 in the wine mash sample is further calculated according to the formula;

[0016]

[0017] x: AFB1 content in wine mash sample, μg / kg;

[0018] m: AFB1 concentration in the test solution obtained according to the standard curve, μg / L;

[0019] v 0 : volume of the liquid to be tested, L;

[0020] w: weight of mash sample, kg;

[0021] Furthermore, in step S11, the graphene oxide substrate of GO@PEI@Au seed@PEI@Au@DTNB modified with the complementary chain of AFB1 aptamer and the Fe 3 O 4 The specific preparation process of the magnetic nanocolloid probe of @PDA@Cys-AuNFs is as follows:

[0022] S111 Preparation of Au colloidal solution: Add tetrachloroauric acid HAuCl 4The solution was diluted with deionized water, placed in a water bath for heating and stirring, and then sodium citrate solution was added for a second heating and stirring. After the heating was completed, the solution was taken out and continued to be stirred and cooled to room temperature to obtain a gold colloidal solution for standby use. Further, the HAuCl 4 The volume ratio of solution, deionized water and sodium citrate solution is 1:10 3 : 6×10 3 ; the HAuCl 4 The concentration of the solution is 0.2-0.5M; the concentration of the sodium citrate solution is 0.03-0.05M; the temperature of the water bath is 100-120°C, and the heating time is 3-5min; the temperature of the second heating water bath is 100-120°C, and the heating time is 25-30min; the concentration of the gold colloid solution is 0.005-0.01mM;

[0023] S112 Preparation of gold seed solution Au seed: HAuCl 4 The solution was diluted with deionized water, placed in a water bath and heated with stirring, and then sodium citrate solution was added under stirring. After the reaction, sodium borohydride (NaBH 4 ) solution and continue stirring at room temperature to obtain a gold seed solution for standby use; further, the HAuCl 4 solution, deionized water, sodium citrate solution, NaBH 4 The volume ratio is 40:3×10 5 : 9×10 3 :1×10 3 ; the HAuCl 4 The concentration of the solution is 0.2-0.5M; the concentration of the sodium citrate solution is 0.03-0.05M; the NaBH 4 The concentration of the solution is 0.01-0.03M; the concentration of the gold seed solution is 0.0001-0.0005mM; the heating temperature in the water bath is 100-120°C for 3-5min; the second stirring time is 1-2min; the stirring time at room temperature is 3-6h;

[0024] S113 Preparation of L-cysteine ​​carboxylated gold nanoflower solution Cys-AuNFs: First, deionized water, HAuCl 4The solution and the sodium citrate solution are mixed and stirred evenly, and the resulting mixed solution is the growth solution, and then the growth solution is added to the gold colloidal solution prepared above, stirred and mixed, and then heated at a constant temperature under stirring conditions, and the hydroquinone solution is quickly added, and the stirring is continued at a constant temperature. After the reaction is completed, it is cooled to room temperature to obtain a sodium citrate-modified gold nanoflower solution, which is recorded as Tr-AuNFs; the Tr-AuNFs are then centrifuged, and the precipitate is redissolved in deionized water, L-cysteine ​​is mixed with the Tr-AuNFs solution in an environment isolated from air, and the mixture is oscillated at a uniform speed at room temperature. After centrifugal washing, it is resuspended in deionized water to obtain an L-cysteine ​​carboxylated gold nanoflower solution, which is recorded as Cys-AuNFs colloidal solution;

[0025] Furthermore, the deionized water, HAuCl 4 The volume ratio of solution and sodium citrate solution is 1×10 4 :18:2.8×10 2 : HAuCl 4 The concentration of the aqueous solution is 0.2-0.5M, the concentration of the sodium citrate solution is 0.03-0.05M; the volume ratio of the gold colloid solution, the growth solution, and hydroquinone is 6:103:28; the dosage ratio of the citric acidified gold nanoflower solution and L-cysteine ​​is 100mL:5mg; the concentration of the hydroquinone solution is 20-30mM; the concentration of the L-cysteine ​​carboxylated gold nanoflower solution is 0.029-0.032mM;

[0026] The water bath is heated at a constant temperature of 40 to 60°C;

[0027] The stirring time after adding the hydroquinone solution is 10 to 15 minutes;

[0028] The centrifugal condition is 7000-8000 r / min, and the centrifugal time is 5-10 min;

[0029] The uniform oscillation time is 20 to 40 minutes;

[0030] S114 Preparation of graphene oxide substrate of GO@PEI@Au seed@PEI@Au@DTNB modified with complementary chain of AFB1 aptamer: After dissolving graphene oxide (GO) in deionized water, centrifugally collect the precipitate and resuspend it in deionized water to obtain a graphene oxide solution, then mix the graphene oxide solution with the PEI solution, ultrasonically treat, centrifugally wash, and resuspend the precipitate in deionized water to obtain a PEI-coated graphene oxide nanosheet solution, recorded as GO@PEI; after mixing GO@PEI with the gold seed solution prepared in step S112, ultrasonically treat, centrifugally wash, and resuspend the precipitate in deionized water to obtain a GO@PEI@Au seed colloidal solution; after mixing the GO@PEI@Au seed colloidal solution with the PEI solution, ultrasonically treat, centrifugally wash, and resuspend the precipitate in deionized water to obtain a GO@PEI@Au seed@PEI colloidal solution; The seed@PEI colloid solution is mixed with the gold colloid solution prepared in step S111, and then the precipitate is taken and resuspended in deionized water after ultrasonic treatment and centrifugal washing to obtain a GO@PEI@Au seed@PEI@Au colloid solution;

[0031] The GO@PEI@Au seed@PEI@Au colloidal solution was mixed with the DTNB solution and then subjected to ultrasonic treatment to obtain a GO@PEI@Au seed@PEI@Au colloidal solution labeled with DTNB. After centrifugal washing, the precipitate was resuspended in deionized water to obtain a GO@PEI@Au seed@PEI@Au@DTNB colloidal solution.

[0032] The GO@PEI@Au seed@PEI@Au@DTNB colloidal solution was mixed with the AFB1 aptamer complementary chain solution and incubated. After incubation, the precipitate was collected by centrifugation, washed by centrifugation with a PB solution, and then the precipitate was collected again and redissolved in an equal volume of PB solution to obtain a GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with an AFB1 aptamer complementary chain.

[0033] Further, in step S114, the weight-to-volume ratio of graphene oxide, deionized water used for dissolution, and deionized water used for resuspension is 4 mg: 20 mL: 20 mL;

[0034] The volume ratio of the GO solution, PEI solution, and deionized water used for resuspension is 2 mL: 8 mL: 2 mL;

[0035] The volume ratio of the GO@PEI solution, gold seed solution, and deionized water used for resuspension is 1:6:1;

[0036] The volume ratio of the GO@PEI@Au seed colloid solution, PEI solution, and deionized water used for resuspension is 1 mL: 4 mL: 1 mL;

[0037] The dosage of the GO@PEI@Au seed@PEI colloid solution, the gold colloid solution, and the deionized water used for resuspension is 1:20:1;

[0038] The dosage ratio of the GO@PEI@Au seed@PEI@Au to the DTNB solution and the deionized water used for resuspension is 200:3:200;

[0039] The dosage ratio of the GO@PEI@Au seed@PEI@Au@DTNB colloidal solution, the AFB1 aptamer complementary chain solution, and the deionized water used for resuspension is 1:10000:1; the concentration of the PEI solution mixed with the GO solution is 0.3-0.5 mg / mL; the concentration of the PEI solution mixed with the GO@PEI@Au seed colloidal solution is 0.1-0.3 mg / mL; the concentration of the DTNB is 8-12 mM; the concentration of the AFB1 aptamer complementary chain solution is 800-1200 nM;

[0040] The solvent of the DTNB solution is a 40-60 mM PBS solution at pH 7; the solvent of the AFB1 aptamer complementary chain solution is a 8-12 mM PB solution at pH 7.4;

[0041] The centrifugal condition is 5000-10000 r / min, 5-10 min;

[0042] The ultrasonic time is 30min to 120min;

[0043] The incubation temperature is 35-40°C and the time is 1-3h;

[0044] Preparation of AFB1 aptamer-modified Fe 3 O 4 Magnetic nanocolloid probe of @PDA@Cys-AuNFs: FeCl 3 6H 2 O, sodium acetate and polyethylene glycol 6000 were stirred and dissolved in ethylene glycol, and heated to react. After the reaction was completed, a permanent magnet was used to separate the obtained precipitate. The obtained precipitate was washed alternately with deionized water and ethanol. After washing, the precipitate was separated again using a permanent magnet. After vacuum drying, the obtained product was Fe 3 O 4 Nanoparticles;

[0045] Fe 3 O 4The nanoparticles were dissolved in Tris buffer and ultrasonically treated after being fully dissolved to form a well-dispersed colloidal solution. Polydopamine was added to the solution and stirred overnight after ultrasonic treatment. The precipitate was then separated by a permanent magnet and washed with deionized water and resuspended in deionized water to obtain Fe 3 O 4 @PDA nanocolloid solution;

[0046] Fe 3 O 4 The PDA nanocolloid solution was mixed with the Cys-AuNFs colloidal solution prepared in step c. After ultrasonic treatment, the mixture was separated into a precipitate using a permanent magnet and washed with deionized water. After washing, the precipitate was resuspended in deionized water to obtain Fe 3 O 4 @PDA@Cys-AuNFs magnetic nanocolloid probe;

[0047] Fe 3 O 4 The @PDA@Cys-AuNFs nanocolloid solution was mixed with the AFB1 aptamer solution and incubated. After incubation, the precipitate was collected by magnetic adsorption, washed with PB solution, and then the precipitate was collected by magnetic adsorption again and redissolved in PB solution to obtain the AFB1 aptamer-modified Fe 3 O 4 Magnetic nanocolloid probe of @PDA@Cys-AuNFs;

[0048] Further, in step S115, the FeCl 3 6H 2 O, sodium acetate, polyethylene glycol 6000 and ethylene glycol have a mass volume ratio of 2.7 g: 5.4 g: 2 g: 80 mL; the Fe 3 O 4 The mass volume ratio of nanoparticles, Tris buffer and polydopamine is 10 mg: 50 mL: 50 mg; the Fe 3 O 4 The volume ratio of the PDA nanocolloid solution, the Cys-AuNFs colloidal solution, and the deionized water used for resuspension was 1:20:1; the AFB1 aptamer solution, Fe 3 O 4 The volume ratio of @PDA@Cys-AuNFs magnetic nanocolloid probe and PB solution used for resuspension was 1:100mL:100;

[0049] The concentration of the AFB1 aptamer solution is 800-1200 nM;

[0050] The pH of the Tris buffer is 8.5, and the concentration is 8-12 mM;

[0051] The solvent of the AFB1 aptamer solution is PB solution, the pH is 7.4, and the concentration is 8-12 mM;

[0052] The heating reaction temperature is 180-220°C, and the time is 4-8 hours; the vacuum drying temperature is -70--50°C, and the time is 6-14 hours; the overnight stirring time is 12-18 hours; the ultrasonic time is 20-40 minutes; the incubation temperature is 35-40°C, and the time is 1-3 hours;

[0053] Furthermore, in step S12, the concentration range of the AFB1 standard solution is 0 to 5 μg / L; 1 、V 2 、V 3 and V 4 The volume ratio is 2:1:1:1; when the conjugate is dissolved in deionized water, the usage ratio of the conjugate to deionized water is 1-3 mg:1 mL; the incubation temperature is 35-40° C., and the time is 0.5-1 h;

[0054] Furthermore, in step S13, the amount of the combined substance mixture added to the silicon wafer is 2-3 μL; the characteristic Raman peak of AFB1 is 1334 cm -1 ;

[0055] Furthermore, in step S2, the shaking conditions are: 150 r / min, the extraction time is 25 to 35 min; the mass volume ratio of the mash to the acetonitrile aqueous solution is 5 to 20 g: 25 to 100 mL; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 84:16; and the centrifugation conditions are: a rotation speed of 6000 to 7000 r / min and a time of 5 to 10 min.

[0056] Furthermore, in step S3, the incubation is carried out at room temperature for 0.5 to 1 h; the amount of the conjugate mixture added dropwise to the silicon wafer is 2 to 3 μL; the V 1 、V 2 、V 3 and V 4 The volume ratio of the conjugate is 2:1:1:1; when the conjugate is resuspended in the PB solution, the mass volume ratio of the conjugate to the PB solution is 1-3 mg:1 mL;

[0057] Furthermore, in step S3, the instrument parameter conditions are as follows: the Raman spectrometer emits a laser wavelength of 785 nm, a fixed laser power of 5 mW, and a scanning range of 500 to 2200 cm -1 , the integration time is 3 to 5 s, and each sample is scanned 6 times to take the average value;

[0058] Compared with the prior art, the advantages of the present invention are:

[0059] (1) The existing AFB1 detection system generally uses nanomaterials such as gold and silver to directly bind to signal molecules for detection, but it is difficult to collect in the heterogeneous system of wine mash. 3 O 4 @PDA@Cys-AuNFs nanocolloids were used as probes, which enabled them to have the ability of rapid separation and signal enrichment in heterogeneous systems through permanent magnets. The magnetic detection probe was prepared by loading L-cysteine ​​carboxylated gold nanoflowers (Cys-AuNFs) modified with AFB1 aptamers on the surface of polydopamine (PDA)-coated magnetic nanomaterials (Fe3O4). At the same time, graphene oxide (GO) was used as a flexible substrate and polyethyleneimine (PEI) was used as a connecting layer. The Raman signal probe was prepared by gradually fixing gold seeds (Au seeds), colloidal gold (Au), 5,5' dithiobis (2-nitrobenzoic acid) (DTNB) signal molecules and AFB1 aptamer complementary chains using a layer-by-layer assembly strategy. The two probes were combined through base complementary pairing. When AFB1 was present, it could compete with the aptamer to cause the probes to separate, resulting in a decrease in the Raman signal. The surface modification of PDA greatly improved the stability of magnetic nanomaterials in the complex environment of non-uniform matrix and weakened the interference of matrix effect on the detection signal. At the same time, the multi-layer ordered and controllable assembly of gold nanospheres on the flexible substrate significantly amplified the full signal intensity.

[0060] (2) Since the mash contains abundant organic matter, and various organic matter will produce many matrix Raman signals, thereby interfering with the detection of the target substance, the present invention uses Au seed and Au nanoparticles to enhance the Raman signal of the substrate, and then uses AuNF nanoflowers to further enhance the Raman signal after the probe and the substrate are combined, so that the system of the present invention has a strong Raman signal and excellent sensitivity.

[0061] (3) Wine mash has the property of matrix complexity, which makes the antibodies / aptamers modified on the nanomaterials discrete and the aptamers themselves inactivated, resulting in the failure of conventional detection methods and the inability to effectively detect. The present invention uses L-cysteine ​​carboxylated gold nanoflowers to modify the complementary chain of the AFB1 aptamer, so that it has a certain tolerance in slightly acidic and high-salt environments, and uses PEI to modify the AFB1 aptamer to make its link more secure. Effective detection of the AFB1 content in wine mash is achieved; the present invention applies Raman technology to the detection of AFB1 in wine mash, with a fast detection speed and more accurate results (the error rate with HPLC results is less than 10%). BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the Raman spectrum of the AFB1 standard.

[0063] Figure 2 This is the standard curve of AFB1.

[0064] Figure 3 This is an example of the Raman spectrum of AFB1 in the test liquid. DETAILED DESCRIPTION

[0065] In order to explain the content of the present invention more clearly, the present invention is further described below in conjunction with specific embodiments.

[0066] Embodiment 1:

[0067] 1. Experimental samples, main reagents and instruments

[0068] Samples: Six different batches of wine mash were selected as test objects (samples were taken from the vinegar factory).

[0069] Reagents: AFB1 standard was purchased from Beijing Bailingwei Technology Co., Ltd. with a purity greater than 98%. All water used in the experiment was deionized water, and all experimental reagents were analytical grade.

[0070] The AFB1 aptamer and its complementary strand were purchased from Shanghai Sangon Biotechnology Co., Ltd.;

[0071] AFB1 aptamer: 5′-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCGTGC CCTTCGCTAGGG CCC-NH 2 -3′;

[0072] AFB1 aptamer complementary strand: 5′-ACAC GTG CCCAACAAAAAA-NH 2 -3′;

[0073] Instrument: Portable Raman spectrometer (SR-510Pro, Shanghai Weihai Optical Instrument Co., Ltd.).

[0074] 2. Draw the AFB1 standard curve

[0075] AFB1 aptamer modified Fe 3 O 4 The specific preparation process of the magnetic nanocolloid probe of @PDA@Cys-AuNFs and the graphene oxide substrate of GO@PEI@Au seed@PEI@Au@DTNB modified with the complementary chain of AFB1 aptamer is as follows:

[0076] a. Preparation of gold colloid solution: HAuCl 4The aqueous solution (0.25M, 50μL) was added to 50mL of deionized water, placed in a water bath and heated at 100℃ with stirring for 5min until the solution boiled, and then sodium citrate solution (0.0388M, 3mL) was added for a second heating and stirring for 30min. After the heating was completed, the solution was taken out and continued to be stirred and cooled to room temperature to obtain a gold colloidal solution (0.0078mM) for use;

[0077] b. Preparation of gold seed solution: HAuCl 4 The aqueous solution (0.25M, 40μL) was added to 50mL of deionized water, placed in a water bath and heated at 100℃ for 5min until the solution boiled, then sodium citrate solution (0.0388M, 1.5mL) was added under stirring, and after reacting for 1min, freshly prepared sodium borohydride solution (0.0198M, 0.5mL) was added and continued to stir at room temperature for 5h to obtain gold seed solution (0.0004mM) for use;

[0078] c. Preparation of L-cysteine ​​carboxylated gold nanoflower solution (Cys-AuNFs):

[0079] First, prepare the growth solution by mixing 100 mL of deionized water, HAuCl 4 The aqueous solution (0.25 M, 0.18 mL) and the sodium citrate solution (0.0388 M, 2.8 mL) were mixed and stirred evenly, and the resulting mixed solution was the growth solution;

[0080] Add 6 mL of gold colloid solution prepared in step a to 103 mL of growth solution, then heat at 50°C under stirring, increase the stirring speed, and quickly add hydroquinone solution (25 mM, 28 mL); continue stirring at 50°C for 10 min. After the reaction is completed, cool to room temperature to obtain sodium citrate-modified gold nanoflowers (Tr-AuNFs) for use;

[0081] 100 mL of sodium citrate gold nanoflowers were centrifuged (8000 r / min, 10 min), and the precipitate was redissolved in 100 mL of deionized water. 5 mg of L-cysteine ​​was mixed with the Tr-AuNFs solution in an air-tight environment, and the mixture was uniformly shaken at room temperature for 30 min. After centrifugal washing (7000 r / min, 10 min), the mixture was resuspended in 10 mL of deionized water to obtain a L-cysteine ​​carboxylated gold nanoflower solution (Cys-AuNFs colloidal solution) (0.031 mM) for later use.

[0082] d. Preparation of graphene oxide substrate of GO@PEI@Au seed@PEI@Au@DTNB modified with complementary chain of AFB1 aptamer: 4 mg GO was completely dissolved in 20 mL deionized water by ultrasonication, and the graphene oxide with smaller flake size was removed by centrifugation (10000 r / min, 10 min), and the graphene oxide solution was obtained by resuspending in 20 mL deionized water for use;

[0083] 2 mL of graphene oxide solution was mixed with PEI (0.4 mg / mL, 8 mL) solution and ultrasonically treated for 30 min, and then the PEI-coated graphene oxide nanosheets were collected by centrifugation (10000 r / min, 10 min), resuspended in 2 mL of deionized water, and repeated twice to obtain a PEI-coated graphene oxide nanosheet solution (GO@PEI);

[0084] 2 mL of PEI-coated graphene oxide nanosheet solution was mixed with 12 mL of gold seed solution prepared in step b and ultrasonicated for 30 min, the supernatant was removed by centrifugation (9000 r / min, 10 min), and the mixture was resuspended in 2 mL of deionized water, and the mixture was repeated twice to obtain a GO@PEI@Au seed colloidal solution;

[0085] 2 mL of GO@PEI@Au seed colloidal solution was mixed with PEI (0.2 mg / mL, 8 mL) solution and ultrasonically treated for 30 min, then centrifuged (8000 r / min, 5 min) to remove the supernatant and resuspended in 2 mL of deionized water. This process was repeated twice to obtain a GO@PEI@Auseed@PEI colloidal solution.

[0086] 2 mL of GO@PEI@Au seed@PEI colloidal solution was mixed with 40 mL of gold colloidal solution prepared in step a and ultrasonically treated for 30 min. The supernatant was removed by centrifugation (8000 r / min, 5 min), and the mixture was resuspended in 2 mL of deionized water. The mixture was repeated twice to obtain a GO@PEI@Au seed@PEI@Au colloidal solution.

[0087] After mixing 2 mL of GO@PEI@Au seed@PEI@Au colloidal solution with DTNB (10 mM, 30 μL) PB (50 mM, pH: 7.0) solution and ultrasonically treating for 2 h, the DTNB-labeled GO@PEI@Au seed@PEI@Au colloidal solution was centrifuged (7000 r / min, 5 min) and washed twice with deionized water, and then resuspended in 2 mL of deionized water to obtain a GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate;

[0088] 2 mL of GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate was mixed with AFB1 aptamer complementary chain solution (1000 nM, 20 μL), incubated at 37°C for 2 h, and then blocked with BSA (2 mL, mass concentration 3%) for 1 h. The supernatant was removed by centrifugation (7000 r / min, 5 min), and resuspended in 2 mL of PB solution. The process was repeated twice to obtain GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with AFB1 aptamer complementary chain.

[0089] e. Preparation of AFB1 aptamer-modified Fe 3 O 4 Magnetic nanocolloid probe of @PDA@Cys-AuNFs: FeCl 3 6H 2 O (2.7 g), sodium acetate (5.4 g) and polyethylene glycol 6000 (2 g) were stirred and dissolved in 80 mL of ethylene glycol, and then transferred to a reactor and heated in an oven at 200 ° C for 6 h. After the reaction was completed, a permanent magnet was used for separation. The obtained isolate was washed alternately with deionized water and ethanol three times, and then separated again using a permanent magnet. After low-temperature vacuum drying at -60 ° C for 6 h, Fe 3 O 4 Nanoparticles;

[0090] Fe 3 O 4 Nanoparticles (10 mg) were dissolved in 50 mL Tris buffer (10 mM, pH: 8.5) and dispersed by constant temperature ultrasonic dispersion for 30 min until a stable and dispersed colloidal solution was formed. 50 mg polydopamine was added to the solution, and mechanical stirring was used overnight after ultrasonic treatment for 30 min. After the reaction was completed, the solution was separated and washed by a permanent magnet and resuspended in 50 mL deionized water to obtain Fe 3 O 4 @PDA colloid solution;

[0091] 2mLFe 3 O 4 The PDA nanocolloid solution was mixed with 40 mL of L-cysteine ​​carboxylated gold nanoflower solution prepared in step c, and after ultrasonic treatment for 30 min, the mixture was separated using a permanent magnet and washed with deionized water, and resuspended in 2 mL of deionized water, and the process was repeated twice to obtain Fe 3 O 4 @PDA@Cys-AuNFs magnetic nanocolloid probe;

[0092] 2 mL Fe 3 O 4The @PDA@Cys-AuNFs magnetic nanocolloid probe was mixed with the AFB1 aptamer solution (1000 nM, 20 μL) and incubated at 37 °C for 2 h. After incubation, BSA (2 mL, mass concentration 3%) was blocked for 1 h. The precipitate was collected by magnetic adsorption, washed with PB solution, and then the precipitate was collected by magnetic adsorption again and redissolved in 2 mL PB solution. This was repeated twice to obtain the AFB1 aptamer-modified Fe 3 O 4 @PDA@Cys-AuNFs magnetic nanocolloid probe;

[0093] (1) Preparation of AFB1 standard sample solution

[0094] 1 mg of AFB1 standard was accurately weighed and dissolved in 2 mL of acetonitrile to prepare a standard stock solution. The standard stock solution was diluted with acetonitrile to prepare a series of AFB1 solutions with different concentrations (50, 100, 200, 500, 1000 ng / L), which were stored at 4 °C in the dark. 2 mL of AFB1 standard solution with different concentrations, 1 mL of GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with AFB1 aptamer complementary chain, and 1 mL of FeO2 modified with AFB1 aptamer complementary chain were added to the solution. 3 O 4 The @PDA@Cys-AuNFs magnetic nanocolloid probe was mixed separately and incubated at room temperature for 30 minutes. After the incubation, the precipitate was collected by magnetic adsorption, washed with PB solution, and then the precipitate was collected by magnetic adsorption again and redissolved in 1 mL PB solution. This was repeated twice. Finally, the combined mixture was dripped on the surface of the silicon wafer and dried at 500-2000 cm －1 A portable Raman spectrometer is used to scan the spectrum within the range to obtain the Raman spectrum, and smoothing and baseline optimization are performed. Figure 1 AFB1 is excited at 785 nm with a wavelength of 1334 cm －1 ±3cm －1 The characteristic Raman peak at is used as the standard curve Raman spectrum of the quantitative reference peak of AFB1. The standard curve of AFB1 is as follows: Figure 2 As shown, the functional relationship of the standard curve in the linear range of 50-1000 ng / L is: y=-11907.5353*x+13815.09, and the correlation coefficient R 2 =0.99243, with a good linear relationship, and can be used for quantitative analysis of AFB1 in wine mash. The portable Raman spectrometer detection working conditions are: laser power: 5mW; integration time: 3s; average times: 3; smoothing parameter: 2.

[0095] (2) Preparation of wine mash sample solution to be tested;

[0096] Weigh 10 g of mash into a conical flask, crush and mix with a tissue homogenizer, add 50 mL of acetonitrile aqueous solution, and extract by shaking at room temperature for 30 min to obtain a crude extract; after shaking, centrifuge again at 6000 r / min for 5 min, collect the supernatant, and refrigerate for later use.

[0097] (3) Raman detection of the test liquid:

[0098] Take 100 μL of GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with complementary chain of LAF1 aptamer, add 200 μL of the test solution prepared in step (2), and then add 100 μL of Fe modified with LAF1 aptamer. 3 O 4 The @PDA@Cys-AuNFs magnetic nanocolloid probe was mixed and incubated at room temperature for 45 minutes, and then the conjugate was separated by a permanent magnet. After centrifugation and washing twice with PB solution, the conjugate was separated by a permanent magnet and resuspended in 100 μL PB solution to obtain a conjugate mixture. Finally, 2.5 μL of the conjugate mixture was added dropwise on the surface of the silicon wafer and heated at 500-2500 cm －1 A portable Raman spectrometer is used to scan the spectrum within the range to obtain the Raman spectrum, and smoothing and baseline optimization are performed. Figure 3 It is the Raman spectrum of the test liquid at an excitation wavelength of 785nm, the abscissa is the Raman shift, and the ordinate is the Raman peak intensity.

[0099] (4) Quantitative determination:

[0100] Based on the detection results of step (3), the Raman spectrum of the test liquid is obtained, with a wavelength of 1334 cm －1 ±3cm －1 The characteristic Raman peak at is used as the quantitative reference peak of AFB1, and the content x of AFB1 in the mash sample is calculated according to the corresponding peak intensity of the spectrum at AFB1.

[0101]

[0102] x: AFB1 content in wine mash sample, μg / kg;

[0103] m: AFB1 concentration in the test solution obtained according to the standard curve, μg / L;

[0104] v 0 : volume of the liquid to be tested, L;

[0105] w: mass of mash sample, kg.

[0106] (5) Validation experiments;

[0107] The HPLC method was used to measure and calculate the AFB1 content in the six batches of mash samples to verify the accuracy of the measurement results of the method of the present invention. The six batches of mash samples were numbered 1-6 in sequence;

[0108] HPLC was used to detect the AFB1 standard and draw the AFB1 standard curve. 10 mL of the test solution was taken and purified and eluted by the immunoaffinity column, and 1.5 mL was filtered through a 0.22 μm filter membrane and then subjected to liquid chromatography detection; the peak with a retention time of 5.89 to 6.58 min was taken as the target peak, and the AFB1 concentration in the test solution was calculated based on the sum of the calculated peak areas and the linear regression equation corresponding to the standard curve; the Raman detection method and the AFB1 determined by HPLC are shown in Table 1. The relative error range between the HPLC determination value of the AFB1 content in different wine mash samples and the Raman spectroscopy method is 1.67% to 7.93%, which is less than 10%. The results show that the AFB1 content in the wine mash sample can be well obtained by Raman spectroscopy, and the comparison of the results with the results of HPLC further illustrates that the detection accuracy of the present invention is high.

[0109] Table 1 Comparison of the results of AFB1 determination by HPLC and Raman spectroscopy

[0110]

[0111] (6) AFB1 recovery and precision spike recovery results

[0112] Select No. 2, 4, and 5 wine mash samples, and according to step (3) of the implementation method, add different amounts of AFB1 standard substances during the ultrasonic extraction process to prepare unspiked test solutions and test solutions containing AFB1 standard substances, and then detect and calculate the AFB1 concentrations in the unspiked and spiked test solutions according to steps (3) (4) (5) of the implementation method. The results are shown in Table 2:

[0113] Table 2 Results of AFB1 spike recovery experiment

[0114]

[0115]

[0116] From the results in Table 2, it can be seen that the recovery rate of AFB1 is between 91.66% and 108.55%. The data results show that the detection method established by the present invention is reliable.

[0117] In summary, the controllable magnetic recovery of magnetic nanomaterials effectively solves the matrix effect caused by the inhomogeneous system of wine mash, and the ordered multilayer assembly of gold nanoparticles efficiently regulates the number and distribution of "hot spots", significantly improving the full signal intensity and signal-to-noise ratio. In addition, the surface enhanced Raman detection system of the present invention is relatively stable under high hydrochloric acid conditions and is suitable for accurate quantitative detection of AFB1 in wine mash.

[0118] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, a person skilled in the art may modify or make equivalent substitutions to the present invention; and all technical solutions and improvements thereof that do not depart from the essential contents of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy, characterized in that: The following steps are involved: S1 draws the standard curve of AFB1: S11: Preparation of AFB1 standard solutions with different concentrations, graphene oxide substrates of GO@PEI@Au seed@PEI@Au@DTNB modified with complementary chains of AFB1 aptamers, and magnetic nanocolloid probes of Fe3O4@PDA@Cys-AuNFs modified with AFB1 aptamers; S12: Take the same volume of AFB1 standard solution with different concentrations, record it as V1; mix it with V2 volume of GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with AFB1 aptamer complementary chain and V3 volume of Fe3O4@PDA@Cys-AuNFs magnetic nanocolloid probe modified with AFB1 aptamer, respectively, to obtain mixed colloids of different concentrations; incubate it under certain conditions, separate it with a permanent magnet after incubation to obtain the conjugate, wash it with phosphate buffer (PB) several times, separate it with a permanent magnet again, and finally resuspend it in deionized water, wherein the volume of resuspending deionized water is recorded as V4; finally, obtain mixed solutions of conjugates of different concentrations; S13: Take a certain volume of the combined substance mixture and drop it on the silicon wafer. After the droplet is stable, use a Raman spectrometer to scan the spectrum to obtain a Raman spectrum. －1 The characteristic Raman peak is used as the quantitative reference peak, and the Raman peak intensity value I1 at the displacement of the quantitative reference peak is measured, and a standard curve is drawn based on the concentration of the AFB1 standard solution and the corresponding Raman peak intensity value I1; S2 Preparation of the sample solution to be tested: weigh the mash, crush it with a tissue homogenizer, record the mass as w, add acetonitrile aqueous solution, shake and extract at room temperature to obtain a crude extract; centrifuge the crude extract, collect the supernatant as the test solution, and record the volume v0; S3 Raman detection of test liquid: Take the test solution prepared in step S2 with a volume V1 equal to the volume of step S12, and add it to the graphene oxide substrate of GO@PEI@Au seed@PEI@Au@DTNB modified with the aptamer complementary chain of AFB1 with a volume V2 equal to the volume of step S12, then add the magnetic nanocolloid probe of Fe3O4@PDA@Cys-AuNFs modified with the AFB1 aptamer with a volume V3 equal to the volume of step S12, vortex mix evenly and incubate, after incubation, separate the conjugate with a permanent magnet to obtain the conjugate, wash it with PB solution several times, separate the conjugate with a permanent magnet and resuspend it in an aqueous solution to obtain a conjugate mixture, wherein the volume of the resuspended deionized water is recorded as V4; finally, take the conjugate mixture and drop it on the silicon wafer, and after the droplet is stable, use a Raman spectrometer to perform spectral scanning to obtain a Raman spectrum; S4 quantitative determination: According to the Raman spectrum of the test liquid obtained in step S3, the characteristic Raman peak of DTNB is used as the quantitative reference peak, the Raman peak intensity value at the displacement of the quantitative reference peak is measured, and the concentration of AFB1 in the test liquid according to the AFB1 standard curve obtained in step (1) is respectively recorded as m, and the content x of AFB1 in the wine mash sample is further calculated according to the formula; x: AFB1 content in wine mash sample, μg / kg; m: AFB1 concentration in the test solution obtained according to the standard curve, μg / L; v0: volume of the liquid to be tested, L; w: mass of mash sample, kg.

2. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In step S11, the specific preparation process of the graphene oxide substrate of GO@PEI@Au seed@PEI@Au@DTNB modified with the complementary chain of the AFB1 aptamer and the magnetic nanocolloid probe of Fe3O4@PDA@Cys-AuNFs modified with the AFB1 aptamer is as follows: S111 Preparation of Au colloidal solution: dilute the tetrachloroauric acid HAuCl4 solution with deionized water, place it in a water bath for heating and stirring, then add the sodium citrate solution for a second heating and stirring, after the heating is completed, take it out and continue stirring and cooling to room temperature to obtain a gold colloidal solution for standby use; further, the volume ratio of the HAuCl4 solution, deionized water, and sodium citrate solution is 1:10 3 : 6×10 3 ; The concentration of the HAuCl4 solution is 0.2-0.5M; The concentration of the sodium citrate solution is 0.03-0.05M; The temperature of the water bath is 100-120°C for 3-5min; The temperature of the second heating water bath is 100-120°C for 25-30min; The concentration of the gold colloid solution is 0.005-0.01mM; S112 Preparation of gold seed solution Au seed: HAuCl4 solution is diluted with deionized water, placed in a water bath for heating and stirring, and then sodium citrate solution is added under stirring, and sodium borohydride (NaBH4) solution is added after the reaction and continued to be stirred at room temperature to obtain gold seed solution for use; further, the volume ratio of HAuCl4 solution, deionized water, sodium citrate solution, and NaBH4 is 40:3×10 5 : 9×10 3 :1×10 3 ; The concentration of the HAuCl4 solution is 0.2-0.5M; the concentration of the sodium citrate solution is 0.03-0.05M; the concentration of the NaBH4 solution is 0.01-0.03M; the concentration of the gold seed solution is 0.0001-0.0005mM; the heating temperature in the water bath is 100-120°C for 3-5min; the second stirring time is 1-2min; the stirring time at room temperature is 3-6h; S113 Preparation of L-cysteine ​​carboxylated gold nanoflower solution Cys-AuNFs: First, deionized water, HAuCl4 solution and sodium citrate solution are mixed and stirred evenly, and the resulting mixed solution is the growth solution, and then the growth solution is added to the gold colloidal solution prepared above, stirred and mixed, and then heated at a constant temperature under stirring conditions, and the hydroquinone solution is quickly added, and the stirring is continued at a constant temperature. After the reaction is completed, it is cooled to room temperature to obtain a sodium citrate-modified gold nanoflower solution, which is recorded as Tr-AuNFs; Tr-AuNFs is then centrifuged, and the precipitate is redissolved in deionized water, L-cysteine ​​is mixed with the Tr-AuNFs solution in an environment isolated from air, and the mixture is oscillated at a uniform speed at room temperature. After centrifugation and washing, it is resuspended in deionized water to obtain an L-cysteine ​​carboxylated gold nanoflower solution, which is recorded as Cys-AuNFs colloidal solution; further, the volume ratio of the deionized water, HAuCl4 solution and sodium citrate solution is 1×10 4 :18:2.8×10 2 : The concentration of the HAuCl4 aqueous solution is 0.2-0.5M, and the concentration of the sodium citrate solution is 0.03-0.05M; the volume ratio of the gold colloid solution, the growth solution, and hydroquinone is 6:103:28; the dosage ratio of the citric acidified gold nanoflower solution and L-cysteine ​​is 100mL:5mg; the concentration of the hydroquinone solution is 20-30mM; the concentration of the L-cysteine ​​carboxylated gold nanoflower solution is 0.029-0.032mM; the temperature of the water bath constant temperature heating is 40-60°C; the stirring time after adding the hydroquinone solution is 10-15min; the centrifugal condition is 7000-8000r / min, and the centrifugal time is 5-10min; the uniform oscillation time is 20-40min; S114 Preparation of GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with AFB1 aptamer complementary chain: S1141: After dissolving graphene oxide (GO) with deionized water, centrifugally collect the precipitate and resuspend it in deionized water to obtain a graphene oxide solution, then mix the graphene oxide solution with a PEI solution, ultrasonically treat, centrifuge and wash, and then resuspend the precipitate in deionized water to obtain a PEI-coated graphene oxide nanosheet solution, which is recorded as GO@PEI; GO@PEI is mixed with the gold seed solution prepared in step S112, and then ultrasonically treat, centrifugally wash, and then resuspend the precipitate in deionized water to obtain a GO@PEI@Au seed colloidal solution; the GO@PEI@Au seed colloidal solution is mixed with a PEI solution, and then ultrasonically treat, centrifugally wash, and then resuspend the precipitate in deionized water to obtain a GO@PEI@Au seed@PEI colloidal solution; The GO@PEI@Au seed@PEI colloidal solution is mixed with the gold colloidal solution prepared in step S111, and then the precipitate is taken and resuspended in deionized water after ultrasonic treatment and centrifugal washing to obtain a GO@PEI@Au seed@PEI@Au colloidal solution; S1142: After mixing the GO@PEI@Au seed@PEI@Au colloidal solution with the DTNB solution, ultrasonic treatment is performed to obtain a GO@PEI@Au seed@PEI@Au colloidal solution labeled with DTNB, and after centrifugation and washing, the precipitate is resuspended in deionized water to obtain a GO@PEI@Au seed@PEI@Au@DTNB colloidal solution; S1143: mixing the GO@PEI@Au seed@PEI@Au@DTNB colloidal solution with the AFB1 aptamer complementary chain solution, incubating, collecting the precipitate by centrifugation after incubation, washing with a PB solution by centrifugation, collecting the precipitate again and redissolving it in an equal volume of PB solution, and obtaining a GO@PEI@Au seed@PEI@Au@DTNB graphene oxide substrate modified with an AFB1 aptamer complementary chain; Further, in step S114, the weight-to-volume ratio of graphene oxide, deionized water used for dissolution, and deionized water used for resuspension is 4 mg: 20 mL: 20 mL; Further, in step S114, the volume ratio of the GO solution, the PEI solution, and the deionized water used for resuspension is 2 mL: 8 mL: 2 mL; Further, in step S114, the volume ratio of the GO@PEI solution, the gold seed solution, and the deionized water used for resuspension is 1:6:1; Further, in step S114, the volume ratio of the GO@PEI@Au seed colloid solution, the PEI solution, and the deionized water used for resuspension is 1 mL: 4 mL: 1 mL; Further, in step S114, the ratio of the GO@PEI@Au seed@PEI colloid solution, the gold colloid solution, and the deionized water used for resuspension is 1:20:1; Further, in step S114, the ratio of the amount of GO@PEI@Au seed@PEI@Au to the DTNB solution and the deionized water used for resuspension is 200:3:200; Further, in step S114, the dosage relationship of the GO@PEI@Au seed@PEI@Au@DTNB colloidal solution, the AFB1 aptamer complementary chain solution, and the deionized water used for resuspension is 1:10000:1; the concentration of the PEI solution mixed with the GO solution is 0.3-0.5 mg / mL; the concentration of the PEI solution mixed with the GO@PEI@Au seed colloidal solution is 0.1-0.3 mg / mL; the concentration of the DTNB is 8-12 mM; the concentration of the AFB1 aptamer complementary chain solution is 800-1200 nM; Furthermore, in step S114, the solvent of the DTNB solution is a 40-60 mM PBS solution at pH 7; the solvent of the AFB1 aptamer complementary chain solution is a 8-12 mM PB solution at pH 7.4; Further, in step S114, the centrifugal condition is 5000-10000 r / min, 5-10 min; Further, in step S114, the ultrasonic time is 30 min to 120 min; Further, in step S114, the incubation temperature is 35-40°C and the time is 1-3h; S115 Preparation of magnetic nanocolloid probe of Fe3O4@PDA@Cys-AuNFs modified with AFB1 aptamer: FeCl3·6H2O, sodium acetate and polyethylene glycol 6000 were stirred and dissolved in ethylene glycol, and heated to react. After the reaction was completed, a permanent magnet was used to separate the obtained precipitate, and the obtained precipitate was washed alternately with deionized water and ethanol. After washing, the precipitate was separated again with a permanent magnet. After vacuum drying, the obtained product was Fe3O4 nanoparticles; S1151: dissolving Fe3O4 nanoparticles in Tris buffer, and performing ultrasonic treatment after being fully dissolved to form a colloidal solution with good dispersion, adding polydopamine to the solution, and stirring overnight after ultrasonic treatment, and then separating the precipitate by using a permanent magnet and washing it with deionized water, and resuspending it in deionized water to obtain a Fe3O4@PDA nanocolloidal solution; S1152: the Fe3O4@PDA nanocolloid solution is mixed with the Cys-AuNFs colloidal solution prepared in step S113, and after ultrasonic treatment, the mixture is separated into a precipitate using a permanent magnet and washed with deionized water, and after washing, the precipitate is resuspended in deionized water to obtain a Fe3O4@PDA@Cys-AuNFs magnetic nanocolloid probe; S1153: The Fe3O4@PDA@Cys-AuNFs nanocolloid solution is mixed with the AFB1 aptamer solution and then incubated. After incubation, the precipitate is collected by magnetic adsorption, washed with PB solution, and then the precipitate is collected by magnetic adsorption again and redissolved in PB solution to obtain the magnetic nanocolloid probe of Fe3O4@PDA@Cys-AuNFs modified with AFB1 aptamer; Further, in step S115, the mass volume ratio of the FeCl3·6H2O, sodium acetate, polyethylene glycol 6000 and ethylene glycol is 2.7g:5.4g:2g:80mL; the mass volume ratio of the Fe3O4 nanoparticles, Tris buffer and polydopamine is 10mg:50mL:50mg; the volume ratio of the Fe3O4@PDA nanocolloid solution, the Cys-AuNFs colloidal solution and the deionized water used for resuspension is 1:20:1; the volume ratio of the AFB1 aptamer solution, the Fe3O4@PDA@Cys-AuNFs magnetic nanocolloid probe and the PB solution used for resuspension is 1:100:100; Further, the concentration of the AFB1 aptamer solution is 800-1200 nM; Furthermore, the pH of the Tris buffer is 8.5 and the concentration is 8-12 mM; Furthermore, the solvent of the AFB1 aptamer solution is PB solution, the pH is 7.4, and the concentration is 8-12 mM; Furthermore, the temperature of the heating reaction is 180-220°C, and the time is 4-8 hours; the temperature of the vacuum drying is -70--50°C, and the time is 6-14 hours; the overnight stirring time is 12-18 hours; the ultrasonic time is 20-40 minutes; the incubation temperature is 35-40°C, and the time is 1-3 hours.

3. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In step S12, the concentration range of the AFB1 standard solution is 0-5 μg / L; the volume ratio of V1, V2, V3 and V4 is 2:1:1:1; when the conjugate is dissolved in deionized water, the dosage ratio of the conjugate to deionized water is 1-3 mg:1 mL; the incubation temperature is 37° C. and the time is 0.5-1 h.

4. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In step S13, the amount of the conjugate mixture added to the silicon wafer is 2-3 μL; the characteristic Raman peak of AFB1 is 1334 cm -1 .

5. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In step S2, the shaking condition is: 100-200 r / min, and the extraction time is 25-35 min.

6. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The usage ratio of the wine mash to the acetonitrile aqueous solution is 5-20 g: 25-100 mL.

7. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 84:

16.

8. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The centrifugal conditions are: a rotation speed of 6000-7000 r / min and a time of 5-10 min.

9. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In step S3, the incubation is carried out at room temperature for 0.5 to 1 h; the amount of the conjugate mixture added dropwise to the silicon wafer is 2 to 3 μL; the volume ratio of V1, V2, V3 and V4 is 2:1:1:1; when the conjugate is resuspended in the PB solution, the mass volume ratio of the conjugate to the PB solution is 1 to 3 mg: 1 mL.

10. The method for detecting aflatoxin B1 content in wine mash based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In step S3, the instrument parameters are as follows: the Raman spectrometer emits a laser wavelength of 785 nm, a fixed laser power of 5 mW, and a scanning range of 500 to 2200 cm -1 The integration time was 3 to 5 s, and each sample was scanned 6 times to take the average value.

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