Precious metal core-shell-satellite assembly AuNR (at) Ag (at) SiO2-AuNP as well as preparation method and application thereof in detection of vomitoxin

Abstract

The invention discloses a noble metal core-shell-satellite assembly AuNR (at) Ag (at) SiO2-AuNP as well as a preparation method and application thereof in detection of vomitoxin, and belongs to the technical field of nano materials and detection of vomitoxin. According to the nanoparticles, AuNR (at) Ag (at) SiO2-AuNP nanoparticles are obtained by growing AuNP satellites on the surfaces of AuNR (at) Ag (at) SiO2 nanoparticles through a silicon layer surface reduction method. The preparation method has the beneficial effects that the prepared AuNR (at) Ag (at) SiO2-AuNP nanoparticles can be used for detecting vomitoxin, and the unique advantages of SERS and an immunochromatography ICA technology can be fully exerted by combining the SERS and the immunochromatography ICA technology, so that the AuNR (at) Ag (at) SiO2-AuNP nanoparticles have the advantages of high sensitivity, high specificity, convenience in operation, stable performance, low cost and the like in the aspect of detecting DON.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials and deoxynivalenol detection, and in particular to a noble metal core-shell-satellite assembly AuNR@Ag@SiO2-AuNP and a preparation method thereof and application thereof in detecting deoxynivalenol. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, is mainly produced by Fusarium fungi such as Fusarium graminearum, Fusarium oxysporum and Fusarium moniliforme, and is widely present in grains and their processed products. Generally speaking, when people are directly exposed to an environment containing high concentrations of DON, they will suffer from symptoms such as anorexia, vomiting, diarrhea and bleeding. In addition, as the most serious toxin that is easily introduced into the food chain, DON can also cause a variety of immune stress responses such as cytotoxicity, immunotoxicity and reproductive toxicity in the human body through the cumulative effect of the food chain. The nature of DON is relatively stable, and it will maintain strong toxicity during the long-term storage of grains. Due to its high acid and heat resistance, DON can maintain partial stability even under high temperature operating conditions. Therefore, many countries and international organizations have stipulated the maximum allowable concentration of DON. For example, China and the United States set the maximum residue limit of DON in grains at 1 mg / kg. The European Commission sets the maximum residue limit of DON in grains at 750 μg / kg. Although all countries have restricted the content of DON in grains, DON pollution still exists.

[0003] At present, reliable quantitative detection methods for DON are mainly based on instruments, such as high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS / MS). Although these methods have high accuracy and sensitivity, they also have certain limitations. These methods have high requirements on the selection and purity of solvents, and the analyzed samples usually need to be finely extracted, which makes their operation more complicated. In addition, the sample can only be detected after it is dissolved in the mobile phase, and sample separation requires a certain amount of time, so the analysis speed of the instrumental method is also relatively slow. Therefore, it is necessary to develop a fast, reliable and highly sensitive method for detecting DON in grains.

[0004] Immunochromatography (ICA), also known as lateral flow analysis, is one of the most commonly used point-of-care analytical methods. As a promising instant detection platform, ICA has the advantages of simplicity, rapidity, specificity, and low cost, and has been widely used in the detection of mycotoxins. Raman scattering refers to the inelastic scattering phenomenon that occurs when incident light hits the surface of a material. Raman shifts are related to molecular vibration energy and crystal vibrations, and represent a spectral "fingerprint" of the molecular structure. However, under normal conditions, the Raman spectrum of a molecule is relatively weak and difficult to observe. When the object being measured is adsorbed on a rough noble metal surface, the Raman scattering intensity of the object being measured is significantly enhanced. Therefore, this enhancement associated with the rough metal surface is called the surface enhanced Raman scattering (SERS) effect. SERS has the advantages of high specificity, narrow bandwidth, resistance to photobleaching, and multiple detection under single laser wavelength excitation, and the Raman scattering enhancement factor can be as high as 10 10 ~10 11 .

[0005] The Chinese patent application document with publication number CN113156126A discloses an ELISA kit for detecting vomitoxin and its detection method. The kit includes: an ELISA plate coated with a monoclonal antibody to vomitoxin, a standard substance to vomitoxin, a sample diluent, a biotin-labeled competitive antigen to vomitoxin, an enzyme-labeled avidin, a washing solution, a color developing solution, and a stop solution. Compared with the conventional ELISA method, the kit uses avidin and biotin coupling to replace the traditional binding reaction of the primary antibody and the secondary antibody, so that the sensitivity of the detection method has been greatly improved and can reach the ppb level; it has strong specificity and can distinguish other common toxins, such as aflatoxin B1, zearalenone, etc. It has the advantages of high sensitivity and simple operation, and is suitable for the detection of a large number of samples. However, the patent does not involve the precious metal core-shell-satellite nanoparticles of the present invention and its application in the detection of vomitoxin. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to provide a rapid, reliable and highly sensitive method for detecting DON in cereals.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The first aspect of the present invention provides a method for preparing a noble metal core-shell-satellite assembly AuNR@Ag@SiO2-AuNP, comprising the following steps:

[0009] (1) Preparation of gold nanorods:

[0010] S1: Synthesize seed solution: mix cetyltrimethylammonium bromide (CTAB) with ultrapure water, heat to dissolve, add HAuCl4 solution, mix well, add NaBH4 solution, stir, and let stand for use;

[0011] S2: Synthetic growth solution: Dissolve CTAB and 5-bromosalicylic acid (5-BrSA) in ultrapure water, add AgNO3 solution, mix well, and let stand for use;

[0012] S3: Add HAuCl4 to the growth solution, mix well, then add ascorbic acid, mix well, then add the seed solution, mix well, place in a constant temperature water bath, and let stand to obtain a gold nanorod solution;

[0013] (2) Preparation of gold and silver core-shell nanostructures:

[0014] The gold nanorod solution was centrifuged, and the bottom precipitate was taken, and CTAB and AgNO3 solutions were added, mixed, and then AA was added, mixed, and then NaOH solution was added, mixed, and placed in a constant temperature water bath to obtain a gold-silver core-shell nanostructure AuNR@Ag of silver-wrapped gold nanorods;

[0015] (3) Preparation of silicon-coated gold-silver core-shell nanostructures:

[0016] Add 4-MBA to the AuNR@Ag solution, react, centrifuge, disperse, and obtain a 4-MBA-modified AuNR@Ag solution, add polyethylene glycol monomethyl ether (mPEG) solution, stir, centrifuge, resuspend, add ammonia water and tetraethoxysilane (TEOS) solution, centrifuge, resuspend, and obtain AuNR@Ag@SiO2 nanoparticles;

[0017] (4) Preparation of heterojunction silicon-gold-silver bimetallic core-gold nanosphere satellite assembly:

[0018] AuNP satellites were grown on the surface of AuNR@Ag@SiO2 nanoparticles by silicon layer surface reduction method to obtain AuNR@Ag@SiO2-AuNP.

[0019] Beneficial effects: The AuNR@Ag@SiO2-AuNP nanoparticles prepared by the present invention can be used for the detection of vomitoxin. The combination of SERS and immunochromatography ICA technology can give full play to the unique advantages of both technologies, thereby having the advantages of high sensitivity, high specificity, easy operation, stable performance and low cost in the detection of DON.

[0020] Preferably, the molar ratio of CTAB, HAuCl4 and NaBH4 in step S1 is (3-7):(23-28):(4-8), and the more preferred molar ratio is 5:25:6.

[0021] Preferably, the molar ratio of CTAB, 5-BrSA and AgNO3 in step S2 is (230-250):(45-55):(2-7), and the more preferred molar ratio is 240:51:4.

[0022] Preferably, the ratio of the amount of HAuCl4 to the volume of the growth solution and the volume of the seed solution in step S3 is (3-8)×10 -5 mol: 40-60mL: 0.1-0.4mL, more preferably 5×10 -5 mol:50mL:0.2mL.

[0023] Preferably, in step (2), the ratio of the volume of the gold nanorod solution to the amount of CTAB substance is 80-120 mL: 0.005-0.01 mol, and more preferably 100 mL: 0.008 mol.

[0024] Preferably, the ratio of the amount of 4-MBA substance to the volume of AuNR@Ag in step (3) is (5-10)×10 -7 mol: 80-120mL, more preferably 8×10 -7 mol:100mL.

[0025] Preferably, the step (4) of growing AuNP satellites on the surface of AuNR@Ag@SiO2 nanoparticles by silicon layer surface reduction method specifically includes: firstly ammoniating the AuNR@Ag@SiO2 nanoparticles, adding HAuCl4 to the ammoniated AuNR@Ag@SiO2 nanoparticle solution, and then adding sodium citrate solution and NaBH4 solution to obtain.

[0026] Preferably, the volume ratio of the AuNR@Ag@SiO2 to the amount of HAuCl4 is 8-12mL: (0.8-1.5)×10 -6 mol, more preferably 10mL: 1.25×10 -6 mol.

[0027] The second aspect of the present invention proposes AuNR@Ag@SiO2-AuNP nanoparticles prepared by the above preparation method.

[0028] The third aspect of the present invention proposes the application of the above-mentioned AuNR@Ag@SiO2-AuNP nanoparticles in the detection of vomitoxin.

[0029] The advantages of the present invention are:

[0030] SERS-ICA detected different concentrations of DON (0.1fg / mL-1μg / mL), and there was a good linear fitting relationship between the intensity of the characteristic peak of the Raman signal and the logarithm of the DON concentration. This method has a wide linear range for detecting DON, and the detection limit is as low as 0.23fg / mL, indicating its ultra-high sensitivity. Through specificity experiments and stability experiments, the high specificity and long-term storage stability of the determination method were demonstrated. Finally, the SERS-ICA method was used to perform DON spike detection on corn and wheat samples, and the recovery rate was 95.3%-104.6%, and the RSD value was 1.9%-5.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the detection principle of the present invention;

[0032] Figure 2 The morphology analysis diagram of the AuNR@Ag@SiO2-AuNP assembly prepared in Example 1 of the present invention, wherein A is an electron microscope image of the assembly; B is a SEM image and element distribution diagram of the assembly; C is an absorption spectrum diagram of the assembly; and D is a SERS spectrum diagram of the assembly;

[0033] Figure 3 The immunodetection analysis diagram of the immunoprobe prepared in Example 1 of the present invention on vomitoxin, wherein A is the effect diagram of the antibody labeling amount; B is the effect diagram of the concentration of DON-BSA; C is the effect of the amount of SERS immunoprobe; D is the analysis diagram of the pre-incubation time and detection time;

[0034] Figure 4 : is an analysis diagram of the effect of DON concentration on the sensitivity of the SERS-ICA test strip in Example 1 of the present invention, wherein A is an analysis diagram of different DON concentrations on the sensitivity of the test strip, and B is a calibration curve of DON obtained by the relationship between the SERS signal intensity and the logarithm of the DON concentration;

[0035] Figure 5 Specificity and stability analysis diagrams of the SERS-ICA strips for DON analysis and detection in Example 1 of the present invention, wherein A is a specificity analysis diagram and B is a stability analysis diagram. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0038] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0039] Embodiment 1:

[0040] A method for preparing a noble metal core-shell-satellite assembly AuNR@Ag@SiO2-AuNP comprises the following steps:

[0041] (1) Preparation of gold nanorods

[0042] First, synthesize the seed solution: add 0.365g of hexadecyltrimethylammonium bromide (CTAB) to 5mL of ultrapure water and heat to dissolve, then add 2.5mL of chloroauric acid tetrahydrate (HAuCl4) (0.01M) to the CTAB solution and mix well. Then quickly add 0.6mL of sodium borohydride (NaBH4) (0.01M) solution to the above mixed solution (the molar ratio of CTAB, HAuCl4, and NaBH4 is 5:25:6), stir vigorously for 2min, the color of the reaction solution changes from light yellow to dark brown, and place it at a constant temperature of 28℃ for use.

[0043] Next, synthesize the growth solution: fully dissolve 1.8 g CTAB and 0.22 g 5-bromosalicylic acid (5-BrSA) in 50 mL ultrapure water, then add 2.4 mL AgNO3 solution (4 mM) (the molar ratio of CTAB, 5-BrSA, and AgNO3 is 240:51:4), mix well, and let stand at 28°C for 15 min.

[0044] Subsequently, 50 mL of HAuCl4 (1 mM) was added to 50 mL of the above growth solution and mixed slowly. 0.7 mL of ascorbic acid (AA) (0.064 M) was added to the above reaction solution and mixed evenly. The solution gradually changed from light yellow to colorless. Finally, 0.2 mL of the above seed solution was added, mixed thoroughly, and placed in a 30°C water bath for 12 h.

[0045] (2) Preparation of gold and silver core-shell nanostructures

[0046] Take 100mL of gold nanorod solution and centrifuge at 8000rpm for 20min, add the bottom precipitate to 100mL of CTAB (0.08M), then add 7mL of AgNO3 (0.01M) solution, mix well, add 8mL of ascorbic acid (AA) (0.1M), mix well again, add 20mL of NaOH (0.1M) solution, and finally mix well. Place the solution in a 65℃ water bath for 4h to complete the preparation of silver-wrapped gold nanorods.

[0047] (3) Preparation of silicon-coated gold-silver core-shell nanostructures

[0048] 8 mL of 4-mercaptobenzoic acid (4-MBA) (10 -4 M) solution was dropped into 100 mL of AuNR@Ag colloid, and the mixture continued to react at room temperature for 1 h. The 4-MBA-modified AuNR@Ag was first centrifuged at 10,000 rpm for 15 min and then dispersed with 100 mL of ultrapure water.

[0049] Take 25mL of 4-MBA modified AuNR@Ag solution, add 1.25mL of 1mg / mL polyethylene glycol monomethyl ether (mPEG) solution, stir for 1 hour, then centrifuge at 9500rpm for 15 minutes, resuspend in 25mL of a mixed solution of ethanol and water (ethanol: water = 9:2), add 250μL of ammonia water and 25μL of 10% (volume percentage) tetraethoxysilane (TEOS) solution under stirring conditions, stir for 12 hours under sealed conditions, centrifuge three times, and resuspend in ethanol solution to synthesize AuNR@Ag@SiO2 nanoparticles.

[0050] (4) Preparation of heterojunction silicon-gold-silver bimetallic core-gold nanosphere satellite assembly

[0051] AuNP satellites were grown on the surface of AuNR@Ag@SiO2 nanoparticles by silicon layer surface reduction method. First, AuNR@Ag@SiO2 nanoparticles were aminated, and 10μL 3-aminopropyltriethoxysilane (APTES) solution was added to 10mL AuNR@Ag@SiO2 nanoparticle solution, stirred at 65℃ for 4h, and resuspended in aqueous solution after centrifugation. Then, 50μL HAuCl4 (25mM) was added to the aminated AuNR@Ag@SiO2 nanoparticle solution with pH adjusted to 3.2. Subsequently, 0.1mL sodium citrate solution (0.17M) and 1mL ice NaBH4 (0.01M) solution were added to the HAuCl4 / AuNR@Ag@SiO2-NH2 particle solution, and stirred vigorously for 6min to obtain the assembled AuNR@Ag@SiO2-AuNP.

[0052] Preparation of immunoprobes:

[0053] Add 20 μL of anti-DON monoclonal antibody (0.1 mg / mL) to the prepared AuNR@Ag@SiO2-AuNP assembly solution and incubate at room temperature for 1 h. Centrifuge to remove excess antibody in the solution, resuspend in 1 mL of 0.1% BSA solution, react for 0.5 h, and centrifuge again to resuspend in 1 mL of ultrapure water.

[0054] Preparation of test strips:

[0055] The SERS test strip consists of a NC membrane, a sample pad, an absorption pad, and a base plate. DON-BSA-coupled antigen (0.25 mg / mL) and goat anti-mouse secondary antibody (0.4 mg / mL) diluted with PBS buffer (0.01 M, pH 7.4) were dispensed onto the NC membrane at a volume of 20 μL / cm as the test line (T line) and control line (C line). Then, the NC membrane was dried at 37 ° C for 24 hours. The sample pad and the absorption pad were pretreated by soaking in PBS buffer solution for 30 seconds and dried at 37 ° C for 12 hours. Finally, all the above components were assembled on the substrate in sequence and then cut into single strips with a width of 4.0 mm.

[0056] Application of AuNR@Ag@SiO2-AuNP in detection of vomitoxin:

[0057] Testing steps:

[0058] (1) Corn and wheat samples were purchased from a local supermarket and then pretreated according to the following standard protocol. 20 g of sample was weighed, crushed and sieved, and 100 mL of a mixed solution of methanol: water (84:16) was added. Then, the mixture was homogenized at high speed for 3 min. After standing for 10 min, the extract was filtered through filter paper and the filtrate was collected. The collected filtrate was filtered through 0.22 μm polytetrafluoroethylene and diluted for analysis.

[0059] (2) DON standard solution was added to corn and wheat samples to give concentrations of 500 ng / mL, 800 ng / mL, and 1000 ng / mL, and a SERS probe was added and mixed and incubated for 20 minutes.

[0060] (3) The test strip was inserted into the sample solution and incubated for 15 minutes to complete the test. The signal intensity on the T line was measured using a Raman spectrometer.

[0061] Comparative Example:

[0062] The AuNR@Ag@SiO2-AuNP assembly designed in this experiment exhibits a significant Raman enhancement effect, which is mainly attributed to the placement of the Raman signal molecule 4-MBA in the gap between the AuNR@Ag core and the AuNPs satellite where the electric field coupling is strongest. Therefore, even for extremely low concentrations of analytes, its characteristic peaks are relatively obvious. Table 1 shows the detection limit and detection range of DON detection using nanoparticles with different morphologies. Compared with most nanoparticles for detecting DON, the assembled structure has a wider detection range and lower detection limit as a DON probe, and has great potential in the detection of trace amounts of mycotoxins.

[0063] Table 1 Comparison of detection limits and detection ranges of DON by nanoparticles with different morphologies

[0064]

[0065] from Figure 1 In the figure, it can be seen that the principle of SERS-LFIA strip detection of DON is based on competitive immunoassay. In samples without DON, the SERS nanoprobes are captured by the DON-BSA coupled antigen on the T line, and high Raman intensity appears on the T line. In contrast, in samples containing DON, the SERS nanoprobes first bind to DON during the premixing reaction. Therefore, the DON-BSA coupled antigen is able to capture fewer SERS nanoprobes on the T line, which leads to the appearance of low Raman intensity. In addition, as long as the test protocol is followed correctly, excess SERS nanoprobes will migrate and the goat anti-mouse secondary antibody on the C line will be able to capture them.

[0066] from Figure 2 It can be seen that the AuNR@Ag@SiO2-AuNP satellite nanoassembly is composed of the AuNR@Ag core, the ultrathin continuous SiO2 layer and the outer layer of AuNP satellite spheres. The synthesis process is as follows Figure 2As shown in A. First, AuNR is synthesized by the seed growth method, and then NaOH is added to adjust the solution to weak alkalinity, and then silver nitrate and ascorbic acid are added. The reduced Ag atoms will be uniformly deposited on the surface of the gold nanorods to form an AuNR@Ag bimetallic structure. At this time, the lateral absorption peak and the longitudinal absorption peak of the gold nanorods are significantly blue-shifted, and a silver absorption peak appears at 389nm. The gap between the core and the satellite is the strongest electric field, which has strong SERS activity, so the Raman signal molecule 4-MBA is modified on the outside of AuNR@Ag. At this time, the absorption spectrum is red-shifted to 621nm, proving that the Raman signal molecule modification is successful. Next, the AuNR@Ag nanoparticles are coated with silicon. When silica is wrapped on the surface of AuNR@Ag, the refractive index of the silica material is greater than that of water, which leads to an increase in the refractive index near the surface of the metal nanoparticles, and the absorption spectrum is red-shifted. Finally, the AuNP satellite is grown on the surface of the AuNR@Ag@SiO2 nanoparticles by the silicon layer surface growth method. First, the surface of AuNR@Ag@SiO2 nanoparticles was modified with amino groups, and then HAuCl4 was added to adjust the pH value of the solution to acidic. NaBH4 was added, and the reduced Au nanoparticles were deposited on the surface of AuNR@Ag@SiO2 nanoparticles, indicating the successful synthesis of AuNR@Ag@SiO2-AuNP assembly. At this time, the first absorption peak intensity of the AuNR@Ag@SiO2-AuNP assembly became lower, the second absorption peak was red-shifted, and the third absorption peak was blue-shifted. This is because the AuNR@Ag@SiO2 particle surface was connected to the gold satellite ball, which shielded the plasmon excitation of the silver layer, resulting in a decrease in the absorption peak intensity related to silver, and the plasmon coupling between the AuNR@Ag core and the AuNPs satellite caused the red shift of the second absorption peak and the blue shift of the third absorption peak.

[0067] from Figure 3 It can be seen that in immunoassay, a small amount of monoclonal antibody will affect the labeling efficiency, but due to steric hindrance, an excess of antibody will have a negative impact on the sensitivity of the method. Therefore, the number of antibodies coupled to the AuNR@Ag@SiO2-AuNP assembly is particularly important, which can not only protect the probe from aggregation but also improve the stability of the probe. The AuNR@Ag@SiO2-AuNP assembly was coupled with different numbers of anti-DON monoclonal antibodies, such as Figure 3As shown in A, when the amount of DON monoclonal antibody increased from 10 μL to 20 μL, the competitive inhibition rate increased. However, when the amount of DON monoclonal antibody was greater than 20 μL, the inhibition became worse, indicating that the excess monoclonal antibody in the reaction system may have led to the occurrence of nonspecific reactions, thereby reducing the sensitivity of the method. Therefore, the optimal combination amount of AuNR@Ag@SiO2-AuNP assembly and DON monoclonal antibody is 20 μL, and the Raman intensity is also higher at this time.

[0068] In immunoassays, reducing the concentration of immunoreagents can improve the sensitivity of detecting small molecule mycotoxins. Therefore, an appropriate concentration of DON-BSA conjugated antigen can improve the detection sensitivity. Figure 3 As shown in Figure B, the optimal antigen concentration was determined by the competitive inhibition rate and the Raman intensity on the T line. When the concentration of DON-BSA was 0.25 mg / mL, the competitive inhibition rate reached the highest point, and at this time there was also a relatively strong Raman signal on the T line. Therefore, the concentration of 0.25 mg / mL was selected as the optimal concentration of DON-BSA for subsequent experiments.

[0069] Using the appropriate number of SERS immunoprobes not only determines whether the target can be fully bound, but is also important for the sensitivity of the method. Figure 3 As shown in Figure C, the effects of probe dosages of 5, 6, 7, 8, 9, and 10 μL on the competitive inhibition rate and Raman signal intensity were observed. When the probe dosage increased from 5 μL to 8 μL, the competitive inhibition rate also increased; when the probe dosage was too high (>8 μL), the inhibition rate decreased significantly, which may be due to excessive probe binding to the DON-BSA-coupled antigen. When the probe volume was 8 μL, the inhibition rate was the highest, which can be used as the optimal probe dosage for the experiment.

[0070] The immune response is a dynamic process. Figure 3 As shown in D, the horizontal axis plots the reaction time, and the vertical axis plots the Raman intensity of the T line to generate the corresponding immune response curve and determine the optimal immune response time. As the incubation time increases, the Raman intensity of the T line gradually decreases. After 20 minutes, the system tends to be stable. Similarly, as the detection time increases, the Raman intensity of the T line gradually increases. After 15 minutes, the system tends to be stable. Therefore, the pre-incubation time of the first step is 20 minutes, and the immune detection time is 15 minutes.

[0071] from Figure 4As can be seen, the sensitivity of the SERS-ICA test strip was evaluated by detecting different DON concentrations. As shown in 4A, as the DON concentration increased (from 0.1 fg / mL to 1 μg / mL), the SERS peak intensity at 1580 cm-1 on the T line decreased, which is consistent with the principle of competitive immunoassay, that is, there are more DON molecules in the sample, competing with the DON-BSA-coupled antibody on the T line. The calibration curve of DON was obtained using the relationship between the SERS signal intensity at 1580 cm-1 on the T line and the logarithm of the DON concentration ( Figure 4 B). The regression equation is Y = -933X + 8617, where Y in the equation is the SERS signal intensity of 4-MBA at 1580 cm-1, and X is the logarithm of the DON concentration. The calibration curve shows a good linear relationship in the range of 0.1 fg / mL-1 μg / mL (R2 = 0.997). The detection limit (LOD) of the SERS-ICA strip based on DON was calculated to be 0.053 fg / mL.

[0072] from Figure 5 In the figure, it can be seen that in order to evaluate the specificity of SERS-ICA bands for DON analysis and detection, several other common fungal toxins, including AFB1, zearalenone (ZEN), OTA and T-2 toxins, were selected as interfering toxins for anti-interference experiments. The concentration of interfering toxins was 0.5 μg / mL, and the concentration of DON was 0.5 ng / mL, which was 1000 times lower than that of interfering toxins. Raman signal molecules are at 1580 cm -1 The signal strength results at Figure 5 As shown in A, when this method is used to detect DON, only the solution containing DON can significantly reduce the Raman signal intensity. For other interfering toxins, even at high concentrations, the SERS intensity is still similar to that of the blank sample. The results show that the SERS-ICA test strip has satisfactory specificity for DON.

[0073] Stability is an important aspect in evaluating SERS-ICA test strips. The stability of SERS-ICA strips in detecting DON was determined after they were stored at 4°C for different time periods (1, 7, 14, 21, and 28 days). Figure 5 The results of Figure B show that the Raman intensity of the SERS-ICA strip decreased by 4.5% after 28 days of storage, which indicates that the SERS-ICA strip prepared in this study has good stability and can be stored for a period of time.

[0074] Embodiment 2:

[0075] A method for preparing a noble metal core-shell-satellite assembly AuNR@Ag@SiO2-AuNP comprises the following steps:

[0076] (1) Preparation of gold nanorods

[0077] First, synthesize the seed solution: add hexadecyltrimethylammonium bromide (CTAB) to 5mL ultrapure water and heat to dissolve, then add chloroauric acid tetrahydrate (HAuCl4) (0.01M) to the CTAB solution and mix well. Then quickly add sodium borohydride (NaBH4) (0.01M) solution to the above mixed solution (the molar ratio of CTAB, HAuCl4, and NaBH4 is 3:23:4), stir vigorously for 2min, the color of the reaction solution changes from light yellow to dark brown, and place it at a constant temperature of 28℃ for use.

[0078] Next, the growth solution was synthesized: CTAB and 5-bromosalicylic acid (5-BrSA) were fully dissolved in 50 mL of ultrapure water, and then AgNO3 solution (4 mM) was added (the molar ratio of CTAB, 5-BrSA, and AgNO3 was 230:45:2), mixed evenly, and allowed to stand at 28°C for 15 min.

[0079] Subsequently, 30 mL of HAuCl4 (1 mM) was added to 40 mL of the above growth solution and mixed slowly. 0.7 mL of ascorbic acid (AA) (0.064 M) was added to the above reaction solution and mixed evenly. The solution gradually changed from light yellow to colorless. Finally, 0.1 mL of the above seed solution was added, mixed thoroughly, and placed in a 30°C water bath for 12 h.

[0080] (2) Preparation of gold and silver core-shell nanostructures

[0081] Take 80mL of gold nanorod solution and centrifuge at 8000rpm for 20min, add the bottom precipitate to 100mL of CTAB (0.05M), then add 7mL of AgNO3 (0.01M) solution, mix well, add 8mL of ascorbic acid (AA) (0.1M), mix well again, add 20mL of NaOH (0.1M) solution, and finally mix well. Place the solution in a 65℃ water bath for 4h to complete the preparation of silver-wrapped gold nanorods.

[0082] (3) Preparation of silicon-coated gold-silver core-shell nanostructures

[0083] 5 mL of 4-mercaptobenzoic acid (4-MBA) (10 -4 M) solution was dropped into 80 mL of AuNR@Ag colloid, and the mixture continued to react at room temperature for 1 h. The 4-MBA-modified AuNR@Ag was first centrifuged at 10,000 rpm for 15 min and then dispersed with 100 mL of ultrapure water.

[0084] Take 25mL of 4-MBA modified AuNR@Ag solution, add 1.25mL of 1mg / mL polyethylene glycol monomethyl ether (mPEG) solution, stir for 1 hour, then centrifuge at 9500rpm for 15 minutes, resuspend in 25mL of a mixed solution of ethanol and water (ethanol: water = 9:2), add 250μL of ammonia water and 25μL of 10% (volume percentage) tetraethoxysilane (TEOS) solution under stirring conditions, stir for 12 hours under sealed conditions, centrifuge three times, and resuspend in ethanol solution to synthesize AuNR@Ag@SiO2 nanoparticles.

[0085] (4) Preparation of heterojunction silicon-gold-silver bimetallic core-gold nanosphere satellite assembly

[0086] AuNP satellites were grown on the surface of AuNR@Ag@SiO2 nanoparticles by silicon layer surface reduction method. First, AuNR@Ag@SiO2 nanoparticles were aminated, and 10μL 3-aminopropyltriethoxysilane (APTES) solution was added to 8mL AuNR@Ag@SiO2 nanoparticle solution, stirred at 65℃ for 4h, and resuspended in aqueous solution after centrifugation. Then, 32μL HAuCl4 (25mM) was added to the aminated AuNR@Ag@SiO2 nanoparticle solution with pH adjusted to 3.2. Subsequently, 0.1mL sodium citrate solution (0.17M) and 1mL ice NaBH4 (0.01M) solution were added to the HAuCl4 / AuNR@Ag@SiO2-NH2 particle solution, and stirred vigorously for 6min to obtain the assembled AuNR@Ag@SiO2-AuNP.

[0087] Embodiment 3:

[0088] A method for preparing a noble metal core-shell-satellite assembly AuNR@Ag@SiO2-AuNP comprises the following steps:

[0089] (1) Preparation of gold nanorods

[0090] First, synthesize the seed solution: add hexadecyltrimethylammonium bromide (CTAB) to 5 mL of ultrapure water and heat to dissolve, then add chloroauric acid tetrahydrate (HAuCl4) (0.01 M) to the CTAB solution and mix well. Then quickly add sodium borohydride (NaBH4) (0.01 M) solution to the above mixed solution (the molar ratio of CTAB, HAuCl4, and NaBH4 is 7:28:8), stir vigorously for 2 minutes, and the color of the reaction solution changes from light yellow to dark brown. Place it at a constant temperature of 28°C for use.

[0091] Next, the growth solution was synthesized: CTAB and 5-bromosalicylic acid (5-BrSA) were fully dissolved in 50 mL of ultrapure water, and then AgNO3 solution (4 mM) was added (the molar ratio of CTAB, 5-BrSA, and AgNO3 was 250:55:7), mixed evenly, and allowed to stand at 28°C for 15 min.

[0092] Subsequently, 80 mL of HAuCl4 (1 mM) was added to 60 mL of the above growth solution and mixed slowly. 0.7 mL of ascorbic acid (AA) (0.064 M) was added to the above reaction solution and mixed evenly. The solution gradually changed from light yellow to colorless. Finally, 0.4 mL of the above seed solution was added, mixed thoroughly, and placed in a 30°C water bath for 12 h.

[0093] (2) Preparation of gold and silver core-shell nanostructures

[0094] Take 120mL of gold nanorod solution and centrifuge at 8000rpm for 20min, add the bottom precipitate to 100mL of CTAB (0.1M), then add 7mL of AgNO3 (0.01M) solution, mix well, add 8mL of ascorbic acid (AA) (0.1M), mix well again, add 20mL of NaOH (0.1M) solution, and finally mix well. Place the solution in a 65℃ water bath for 4h to complete the preparation of silver-wrapped gold nanorods.

[0095] (3) Preparation of silicon-coated gold-silver core-shell nanostructures

[0096] 10 mL of 4-mercaptobenzoic acid (4-MBA) (10 -4 M) solution was dropped into 120 mL of AuNR@Ag colloid, and the mixture continued to react at room temperature for 1 h. The 4-MBA-modified AuNR@Ag was first centrifuged at 10,000 rpm for 15 min and then dispersed with 100 mL of ultrapure water.

[0097] Take 25mL of 4-MBA modified AuNR@Ag solution, add 1.25mL of 1mg / mL polyethylene glycol monomethyl ether (mPEG) solution, stir for 1 hour, then centrifuge at 9500rpm for 15 minutes, resuspend in 25mL of a mixed solution of ethanol and water (ethanol: water = 9:2), add 250μL of ammonia water and 25μL of 10% (volume percentage) tetraethoxysilane (TEOS) solution under stirring conditions, stir for 12 hours under sealed conditions, centrifuge three times, and resuspend in ethanol solution to synthesize AuNR@Ag@SiO2 nanoparticles.

[0098] (4) Preparation of heterojunction silicon-gold-silver bimetallic core-gold nanosphere satellite assembly

[0099] AuNP satellites were grown on the surface of AuNR@Ag@SiO2 nanoparticles by silicon layer surface reduction method. First, AuNR@Ag@SiO2 nanoparticles were aminated, and 10μL 3-aminopropyltriethoxysilane (APTES) solution was added to 8mL AuNR@Ag@SiO2 nanoparticle solution, stirred at 65℃ for 4h, and resuspended in aqueous solution after centrifugation. Then, 32μL HAuCl4 (25mM) was added to the aminated AuNR@Ag@SiO2 nanoparticle solution with pH adjusted to 3.2. Subsequently, 0.1mL sodium citrate solution (0.17M) and 1mL ice NaBH4 (0.01M) solution were added to the HAuCl4 / AuNR@Ag@SiO2-NH2 particle solution, and stirred vigorously for 6min to obtain the assembled AuNR@Ag@SiO2-AuNP.

[0100] The performance of the assembled AuNR@Ag@SiO2-AuNP prepared in Examples 2 and 3 is similar to that in Example 1.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a noble metal core-shell-satellite assembly AuNR@Ag@SiO2-AuNP, characterized in that: The following steps are involved: (1) Preparation of gold nanorods: S1: Synthesize seed solution: mix cetyltrimethylammonium bromide (CTAB) with ultrapure water, heat to dissolve, add HAuCl4 solution, mix well, add NaBH4 solution, stir, and let stand for use; S2: Synthetic growth solution: Dissolve CTAB and 5-bromosalicylic acid 5-BrSA in ultrapure water, add AgNO3 solution, mix well, and let stand for use; S3: Add HAuCl4 to the growth solution, mix well, then add ascorbic acid, mix well, then add the seed solution, mix well, place in a constant temperature water bath, and let stand to obtain a gold nanorod solution; (2) Preparation of gold and silver core-shell nanostructures: The gold nanorod solution was centrifuged, and the bottom precipitate was taken, and CTAB and AgNO3 solutions were added, mixed, and then AA was added, mixed, and then NaOH solution was added, mixed, and placed in a constant temperature water bath to obtain a gold-silver core-shell nanostructure AuNR@Ag of silver-wrapped gold nanorods; (3) Preparation of silicon-coated gold-silver core-shell nanostructures: Add 4-MBA to the AuNR@Ag solution, react, centrifuge, and disperse to obtain a 4-MBA-modified AuNR@Ag solution, add polyethylene glycol monomethyl ether mPEG solution, stir, centrifuge, resuspend, add ammonia water and tetraethoxysilane TEOS solution, centrifuge, and resuspend to obtain AuNR@Ag@SiO2 nanoparticles; (4) Preparation of heterojunction silicon-gold-silver bimetallic core-gold nanosphere satellite assembly: AuNP satellites were grown on the surface of AuNR@Ag@SiO2 nanoparticles by silicon layer surface reduction method to obtain AuNR@Ag@SiO2-AuNP.

2. The preparation method according to claim 1, characterized in that: The molar ratio of CTAB, HAuCl4 and NaBH4 in step S1 is (3-7): (23-28): (4-8).

3. The preparation method according to claim 1, characterized in that: The molar ratio of CTAB, 5-BrSA and AgNO3 in step S2 is (230-250): (45-55): (2-7).

4. The preparation method according to claim 1, characterized in that: The ratio of the amount of HAuCl4 to the volume of the growth solution and the volume of the seed solution in step S3 is (3-8)×10 -5 mol:40-60mL:0.1-0.4mL.

5. The preparation method according to claim 1, characterized in that: In the step (2), the ratio of the volume of the gold nanorod solution to the amount of CTAB substance is 80-120 mL: 0.005-0.01 mol.

6. The preparation method according to claim 1, characterized in that: The ratio of the amount of 4-MBA substance to the volume of AuNR@Ag in step (3) is (5-10)×10 -7 mol:80-120mL.

7. The preparation method according to claim 1, characterized in that: In the step (4), the AuNP satellite is grown on the surface of AuNR@Ag@SiO2 nanoparticles by silicon layer surface reduction method, which specifically includes: firstly, the AuNR@Ag@SiO2 nanoparticles are aminated, HAuCl4 is added to the aminated AuNR@Ag@SiO2 nanoparticle solution, and then sodium citrate solution and NaBH4 solution are added to obtain.

8. The preparation method according to claim 1, characterized in that: The ratio of the volume of AuNR@Ag@SiO2 to the amount of HAuCl4 is 8-12mL: (0.8-1.5)×10 -6 mol.

9. AuNR@Ag@SiO2-AuNP nanoparticles prepared by the preparation method according to any one of claims 1 to 8.

10. Application of AuNR@Ag@SiO2-AuNP nanoparticles prepared by the preparation method according to any one of claims 1 to 8 in the detection of vomitoxin.

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