Nanosilver-porous silicon-based simultaneous sers detection of multiple mycotoxins biochip sensor, preparation method and application thereof

Abstract

The application discloses a kind of based on nano-silver-porous silicon simultaneously SERS detection variety of mycotoxin biochip sensor and its preparation method and application, the sensor includes SERS substrate and SERS label two parts compositions, SERS substrate is by porous silicon surface magnetron sputtering on silver nanoparticles and silver nanoparticles surface connection three kinds of mycotoxin artificial antigen composition, SERS label is connected with nile blue and corresponding mycotoxin antibody gold nanoparticles.The application can be combined by each mycotoxin and porous silicon silver surface mycotoxin artificial antigen mycotoxin antibody on SERS label, by detecting the Raman signal of NBA on SERS label surface, realize three kinds of mycotoxin simultaneous quantitative analysis, detection speed is fast, sensitivity is high, while various toxins in gualou, malt and kudzu root recovery rate is all greater than 75%, the coefficient of variation of same batch is less than 5.2%, and the coefficient of variation of different batches is less than 7.8%.

Description

Technical Field

[0001] This invention pertains to food safety testing, specifically relating to a biochip sensor based on nano-silver-porous silicon for simultaneous SERS detection of multiple fungal toxins, its preparation method, and its application. Background Technology

[0002] Mycotoxins are secondary metabolites produced by fungi. The sclerotia of fungi form toxic alkaloids that can cause infectious diseases and death in humans and other animals. Currently, more than 400 types of mycotoxins are known, with common ones including aflatoxin, ochratoxin, vomitoxin, and alkaloids. Ochratoxin A (OTA) has been found to be a contaminant in various animal and human foods, including traditional Chinese medicine and its products, grains, beer, and wine. OTA also has immunosuppressive, teratogenic, genotoxic, and carcinogenic effects, and affects blood clotting and carbohydrate metabolism. OTA has been found in the blood, bile, and urine of humans and animals after consuming contaminated food and traditional Chinese medicine, and is considered one of the causes of Balkan endemic kidney disease. Aflatoxin is a secondary metabolite produced by toxin-producing strains such as Aspergillus flavus and Aspergillus parasiticus, and is a highly toxic substance. Aflatoxin B1 is the most common naturally contaminated food and is one of the most potent known chemical carcinogens. In 1993, the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) classified AFB1 as a Group 1 carcinogen for humans. In some countries and regions, AFB1 is widely believed to play an important role in the formation of primary hepatocellular carcinoma. Aflatoxin is present in all aspects of our lives, especially in hot and humid environments. From peanuts, corn, and soybeans that we eat, to soil, plants, and animals, all are susceptible to aflatoxin contamination. Deoxynivalenol (DON) is a common foodborne fungal toxin widely present in nature. When humans ingest food contaminated with DON, it can cause acute poisoning symptoms such as loss of appetite, vomiting, diarrhea, fever, unsteady gait, and lethargy. In severe cases, it can damage the hematopoietic system and cause death. Because the proportion of grains in the traditional Chinese diet is much higher than in the West, the harm caused by DON is more pronounced. Therefore, countries and regions around the world have established strict standards to protect the safety of agricultural products, food, medicinal materials, and animal feed.

[0003] Common methods for detecting mycotoxins include thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and enzyme-linked immunosorbent assay (ELISA). These methods all have drawbacks, such as complex sample pretreatment, expensive equipment, and high detection costs. Therefore, establishing a simple, highly specific, and low-cost detection method capable of simultaneously and effectively detecting and quantifying three mycotoxins (OTA, AFB1, and OTA) is of great significance. Summary of the Invention

[0004] Objective of the Invention: To address the problems existing in the prior art, this invention provides a biochip sensor based on nano-silver-porous silicon for simultaneous SERS detection of multiple mycotoxins. This invention utilizes three mycotoxins (OTA, AFB1, DON) and three artificial mycotoxin antigens (OTA-BSA, DON-BSA, AFB1-BSA) to competitively bind to three mycotoxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) on the SERS tag. The SERS sensor prepared by this invention overcomes the limitation of traditional ELISA kits, which can only detect one mycotoxin at a time, and achieves simultaneous quantitative analysis of three mycotoxins.

[0005] The second objective of this invention is to provide a method for preparing the SERS sensor that can simultaneously detect three fungal toxins (OTA, AFB1, and DON); the third objective of this invention is to provide the application of the above-mentioned SERS sensor in the simultaneous detection of three fungal toxins (OTA, AFB1, and DON).

[0006] Technical Solution: To achieve the above objectives, the present invention provides a biochip sensor for simultaneous SERS detection of multiple mycotoxins based on nano-silver-porous silicon. The sensor comprises two parts: a SERS substrate and a SERS tag. The SERS substrate consists of silver nanoparticles magnetron sputtered onto a porous silicon surface and three mycotoxins connected to the surface of the silver nanoparticles. The SERS tag is connected to gold nanoparticles with Nile blue and corresponding mycotoxin antibodies. The three mycotoxin artificial antigens are ochratoxin A artificial antigen, vomitoxin artificial antigen, and aflatoxin B1 artificial antigen (OTA-BSA, DON-BSA, AFB1-BSA), and the corresponding mycotoxin antibodies are ochratoxin A antibody, vomitoxin antibody, and aflatoxin B1 antibody (OTA-Ab, DON-Ab, AFB1-Ab).

[0007] The SERS tag is constructed by first physically adsorbing NBA onto the surface of gold nanoparticles, then modifying them with methoxy polyethylene glycol thiol and thiol polyethylene glycol carboxyl groups, respectively, and finally covalently linking fungal toxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) under the activation of EDC and NHS. The SERS substrate is prepared by electrochemical anodizing of porous silicon after cleaning of single-crystal silicon, and then sputtering silver nanoparticles onto the surface by magnetron sputtering. The surface of the silver nanoparticles covalently adsorbs fungal toxin artificial antigens (OTA-BSA, DON-BSA, AFB1-BSA). The SERS substrate and the SERS tag are specifically linked by antigen and antibody.

[0008] The artificial mycotoxin antigen includes any one or more of OTA-BSA, DON-BSA, and AFB1-BSA, and the mycotoxin antibody includes any one or more of OTA-Ab, DON-Ab, and AFB1-Ab.

[0009] The method for fabricating a biochip sensor based on nano-silver-porous silicon for simultaneous SERS detection of multiple fungal toxins, as described in this invention, includes the following steps:

[0010] (1) P-type monocrystalline silicon wafers are cleaned and dried for later use;

[0011] (2) Prepare porous silicon from the silicon wafers processed in step (1), clean and dry them for later use;

[0012] (3) After evacuating the porous silicon prepared in step (2), silver nanoparticles are sputtered onto the surface of the porous silicon; the porous silicon is cooled to room temperature and then held under an inert gas to obtain porous silicon.

[0013] (4) The porous silicon-silver nanoparticles prepared in step (3) are physically adsorbed on the surface of three fungal toxin artificial antigens to obtain (OTA-BSA, DON-BSA, AFB1-BSA) SERS substrates.

[0014] (5) Add chloroauric acid solution and trisodium citrate aqueous solution to boiling water in sequence, stir and heat until purple-red and no longer change color, cool to room temperature to obtain colloidal gold, and set aside.

[0015] (6) Nile blue was added to the colloidal gold solution prepared in (5), and after shaking at room temperature, SH-PEG-COOH and SH-PEG-SH were added in sequence. After centrifugation to remove the supernatant and concentration, EDC and NHS were added for activation treatment. Then, three fungal toxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) were covalently linked to obtain the SERS tag.

[0016] In step (1), the resistivity of the P-type monocrystalline silicon wafer is 0.0008-0.00012Ω.

[0017] In step (2), the processed silicon wafer is pressed and assembled into an electrochemical etching instrument, an electrolyte is added, and porous silicon is prepared by electrochemical anodic oxidation. After cleaning with ethanol, it is dried with nitrogen for later use. The electrolyte is prepared by hydrofluoric acid and anhydrous ethanol. Porous silicon is prepared by electrochemical etching. The electrochemical etching current is 300-500mA and the etching time is 10-30S.

[0018] Preferably, the electrochemical etching current is 400mA and the etching time is 20s.

[0019] Preferably, the electrolyte is prepared by mixing 49% hydrofluoric acid and anhydrous ethanol in a 3:1 ratio.

[0020] In step (3), the prepared porous silicon is fixed in a reactive magnetron sputtering apparatus, a silver target is loaded, and a vacuum of 5 × 10⁻⁶ is applied. -4 After Pa, silver nanoparticles were sputtered onto the porous silicon surface; cooled to room temperature and stored in nitrogen gas.

[0021] The magnetron sputtering conditions are: voltage 45-55W, sputtering time 22-26 min, and annealing 15-20 min. In this invention, silver nanoparticles are sputtered onto a porous silicon surface using a magnetron sputtering target, and the silver layer thickness is controlled by adjusting the sputtering conditions (voltage and time).

[0022] Preferably, the magnetron sputtering conditions are 50W voltage, 24min sputtering time, and 15min annealing, resulting in a silver layer thickness of 200nm for the prepared nano-silver-porous silicon.

[0023] In step (4), artificial antigens are added to the substrate, and three artificial antigens, OTA-BSA, DON-BSA, and AFB1-BSA, are added to the SERS substrate; in step (6), NBA solution, SH-PEG-SH solution, SH-PEG-COOH solution, EDC solution, and NHS solution are added to the colloidal gold solution; and three antibodies, OTA-Ab, DON-Ab, and AFB1-Ab, are added.

[0024] Further, in step (4), antigen is dropped onto the substrate. Each SERS substrate (a sputtered silver nanoporous silicon wafer is called a SERS substrate, approximately 1.5cm x 1.5cm in size) is a silver nanoporous silicon wafer, and the dropping method is as described in the principle. Figure 1 As shown, three antigens were added, three drops of each antigen (OTA-BSA, DON-BSA, AFB1-BSA) were added each time, with each addition being 0.3 μL, and the concentrations being 60 μg / mL, 80 μg / mL, and 80 μg / mL, respectively.

[0025] Further, in step (5), 0.25 mL of 2% chloroauric acid solution and 0.35 mL of 1% trisodium citrate solution are added sequentially to every 50 mL of boiling ultrapure water. The concentrations of the three antibodies OTA-Ab, DON-Ab, and AFB1-Ab added are 80 μg / mL, 40 μg / mL, and 60 μg / mL, respectively.

[0026] Further, in step (6), 2.5 mL of 50 μM NBA solution, 0.75 mL of 10 μM SH-PEG-SH solution, 5 mL of 50 μM SH-PEG-COOH solution, 6 μL of 40 mg / mL EDC solution, and 6 μL of 110 mg / mL NHS solution are added to every 22.5 mL of colloidal gold. The concentrations of the three antibodies OTA-Ab, DON-Ab, and AFB1-Ab added are 80 μg / mL, 40 μg / mL, and 60 μg / mL, respectively.

[0027] Preferably, the method for fabricating the biochip sensor for simultaneous SERS detection of multiple fungal toxins based on nano-silver-porous silicon includes the following steps:

[0028] (1) P-type monocrystalline silicon wafers are ultrasonically cleaned in sequence with ultrapure water, acetone, ethanol and ultrapure water, and then dried with nitrogen gas for later use.

[0029] (2) The silicon wafer processed in step (1) is pressed and assembled into an electrochemical etching instrument, an electrolyte is added, and porous silicon is prepared by electrochemical anodic oxidation. After cleaning with ethanol, it is dried with nitrogen for later use.

[0030] (3) The porous silicon prepared in step (2) is fixed in a reactive magnetron sputtering apparatus, a silver target is loaded, and a vacuum is drawn to 5 × 10⁻⁶. -4 After Pa, silver nanoparticles were sputtered onto the porous silicon surface; cooled to room temperature and stored in nitrogen.

[0031] (4) The porous silicon-silver nanoparticles prepared in step (3) are used to physically adsorb three fungal toxin artificial antigens (OTA-BSA, DON-BSA, AFB1-BSA) on their surface to complete the preparation of the SERS substrate.

[0032] (5) Add chloroauric acid solution and trisodium citrate aqueous solution to boiling water in sequence, and heat at 100°C with stirring until it turns purple-red and no longer changes color. After cooling to room temperature, set aside.

[0033] (6) Add NBA to the colloidal gold solution prepared in (5), shake at room temperature, and then add SH-PEG-COOH and SH-PEG-SH in sequence. After centrifugation to remove the supernatant and concentration, add EDC and NHS for activation treatment, and then covalently link three fungal toxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) to obtain the SERS tag.

[0034] The biochip sensor based on nano-silver-porous silicon for simultaneous SERS detection of multiple fungal toxins described in this invention is an application in the simultaneous quantitative detection and analysis of multiple fungal toxins.

[0035] As a preferred embodiment, the present invention describes the application of a biochip sensor based on nano-silver-porous silicon for simultaneous SERS detection of multiple mycotoxins in the quantitative detection and analysis of three mycotoxins (OTA, AFB1, DON). Specifically, the aforementioned SERS sensor based on nano-silver-porous silicon is mainly used for the simultaneous detection of three mycotoxins (OTA, AFB1, DON) in food and pharmaceuticals.

[0036] The specific process of quantitative detection and analysis is as follows: buffer solutions of different concentrations of mycotoxins (OTA, DON, AFB1) are added to the SERS-tagged solution, so that the three mycotoxins (OTA, DON, AFB1) and the three mycotoxin artificial antigens (OTA-BSA, DON-BSA, AFB1-BSA) on the SERS substrate compete for binding to the three mycotoxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) on the SERS tag. The quantitative analysis of the three mycotoxins is achieved by detecting the intensity of the NBA characteristic peak signal bound to the SERS tag.

[0037] Furthermore, buffer solutions containing different concentrations of mycotoxins (OTA, DON, AFB1) were added to the SERS-tagged solution. This allowed the three mycotoxins (OTA, DON, AFB1) and three artificial mycotoxin antigens (OTA-BSA, DON-BSA, AFB1-BSA) on the SERS substrate to competitively bind to three mycotoxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) on the SERS tag. Quantitative analysis of the three mycotoxins was achieved by detecting the intensity of the NBA characteristic peak signal bound to the SERS tag. The competitive reaction time was 1 hour, the reaction temperature was 37°C, and the Raman shift of the NBA Raman characteristic peak was 591 cm⁻¹. -1 .

[0038] In the preparation of SERS tags, this invention involves adding Nile Blue (NBA) solution for signal detection to a colloidal gold solution. Under oscillation conditions, NBA is enriched on the surface of gold nanoparticles through physical adsorption. Simultaneously, methoxy polyethylene glycol thiol solution and thiol polyethylene glycol carboxyl solution are added during the reaction, specifically CH3O-PEG5000-SH and SH-PEG5000-COOH with molecular weights of 5000. The thiol groups form Au-S bonds with gold atoms, thus fixing them to the surface of the gold nanoparticles. PEG is a large polymer with long molecular chains, which, after being linked, act as a support to prevent aggregation. The addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activates the carbonyl groups at the outer ends of SH-PEG5000-COOH, which then covalently bind to the amino groups on the chains of the three mycotoxin antibodies, thereby obtaining gold nanoparticles simultaneously modified with NBA and the three mycotoxin antibodies.

[0039] In the SERS substrate preparation process, a clean, diced silicon wafer is placed at the anode. After applying an electric current, electrochemical etching is performed in the electrolyte to form a porous silicon layer on the wafer surface. This process involves oxidation with four electrons: two from the electrode and two from SiF2. Si atoms, under the nucleophilic attack of F-, detach as SiF4, forming pores. SiF4 then reacts with F- in the solution to form SiF62-, ultimately creating a porous structure. The porous silicon is then placed in a magnetron sputtering apparatus and loaded with a silver target. A layer of silver is sputtered onto the porous silicon surface. The process is as follows: electrons, under the influence of an electric field E, collide with argon atoms as they fly towards the porous silicon, ionizing them to produce Ar ions and new electrons. The new electrons fly towards the porous silicon, while the Ar ions, under the influence of the electric field, accelerate towards the cathode target and bombard the silver target surface with high energy, causing silver to be sputtered. Neutral silver atoms are deposited on the porous silicon in the sputtered particles, forming silver nanoparticles. Three fungal toxin antigens (OTA-BSA, DON-BSA, and AFB1-BSA) were dropped onto the surface of a silver layer and adsorbed onto the silver nanoparticles via covalent bonds.

[0040] The principle of this invention is as follows Figure 1As shown, this invention employs an immune competitive reaction: the three mycotoxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) linked to the SERS tag specifically bind to the corresponding mycotoxin artificial antigens (OTA-BSA, DON-BSA, AFB1-BSA) on the SERS substrate, while another portion binds to the corresponding mycotoxins. If no mycotoxin is added, the mycotoxin antibodies on the SERS tag bind first to the mycotoxin antigens on the SERS substrate, exhibiting a high NBA characteristic peak Raman signal. If a mycotoxin is added to the system, the mycotoxin antibodies linked to the SERS tag bind first to the mycotoxin, and the remaining mycotoxin antibodies on the SERS tag bind to the mycotoxin antigens on the SERS substrate, resulting in a decrease in the Raman signal. Therefore, the final detected NBA characteristic peak Raman signal value reflects the concentration of the added mycotoxin. Thus, by establishing a standard curve for each toxin, detection curves can be established. Due to the specificity of competitive binding, three mycotoxins (OTA, AFB1, DON) in substances such as traditional Chinese medicine can be detected simultaneously. Specific determination is as per the principle. Figure 1 As shown above, different antigens are dropped onto different positions on the SERS substrate. To obtain a specific mycotoxin, the SERS tag solution and the corresponding sample solution are dropped onto the corresponding antigen position. This invention utilizes the competitive binding of various mycotoxins and artificial mycotoxin antigens on the porous silicon silver surface to the mycotoxin antibodies on the SERS tag. By detecting the Raman signal of NBA on the SERS tag surface, simultaneous quantitative analysis of three mycotoxins—OTA, DON, and AFB1—is achieved. The detection is fast and highly sensitive, with recoveries of all toxins greater than 75% in Poria cocos, malt, and kudzu root. The coefficient of variation within the same batch is less than 5.2%, and the coefficient of variation between different batches is less than 7.8%.

[0041] This invention presents a novel biochip sensor based on nano-silver-porous silicon for simultaneous SERS detection of multiple mycotoxins. The SERS substrate fabrication process improves overall stability, particularly the use of magnetron sputtering, which significantly enhances stability and reduces errors compared to spin coating and deposition. Simultaneous detection of three mycotoxins reduces time costs and significantly decreases the amount of antigen and antibody required compared to ELISA kits. The sensor fabricated in this invention achieves simultaneous detection through competitive reactions and the specificity of antigen-antibody binding. The competitive reaction of this invention requires only 1 hour, and sample determination takes only 0.3 seconds. It can simultaneously detect three mycotoxins, while ELISA kits can only detect one. Currently, ELISA kits require adding 50 µL of antibody and 50 µL of artificial antigen to a 96-well plate for each assay, using 50-500 µg of antigen and antibody per sample, far exceeding the amounts used in this invention. The market price for 1 mg of artificial mycotoxin antigen and antibody is approximately 2000 to 4000 yuan. This invention effectively reduces the amount of antigen and antibody used, thereby lowering the price.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0043] (1) This invention utilizes electrochemical etching to form porous silicon, and the network structure increases the specific surface area, providing more hot spots; (2) Magnetron sputtering technology is used to fix silver nanoparticles on the porous silicon surface, which has high stability, low error, and simple preparation. More than 30 SERS substrates can be prepared in a single magnetron sputtering, which can be mass-produced and can be stored for a long time in nitrogen, thus meeting the conditions for long-term market storage and circulation; (3) The sample only requires 0.024 μg OTA-BSA or 0.018 μg AFB1-BSA or 0.024 μg DON-BSA, and 0.024 μg OTA-Ab and 0.012 μg DON-BSA connected to the SERS label. Compared with the traditional enzyme-linked immunosorbent assay (ELISA), AFB1-Ab and 0.018 μg DON-Ab require less artificial antigen and antibody, reducing the cost of a single sample; (4) Compared with traditional chromatography or enzyme-linked immunosorbent assay (ELISA), the detection method using the sensor of this invention has the advantages of high sensitivity, good specificity, no damage to the sample, and simultaneous detection of multiple toxins; (5) Using the sensor and detection method of this invention, three fungal toxins (OTA, AFB1, DON) can be detected simultaneously and quantitatively analyzed, and each sample detection only requires 0.3S (the Raman instrument laser only requires 0.3S to elicit a peak), meeting the market demand for rapid and efficient detection. (6) The SERS sensor prepared by this invention can be mass-produced, has a large specific surface area, high stability, long storage time, and can simultaneously detect three fungal toxins. Attached Figure Description

[0044] Figure 1 This is a schematic diagram illustrating the principle of simultaneous detection of three fungal toxins (OTA, AFB1, and DON) based on nano-silver-porous silicon surface-enhanced Raman spectroscopy.

[0045] Figure 2 Electron micrograph of the SERS substrate;

[0046] Figure 3 A diagram illustrating the enhancement effect of NBA as a probe molecule on SERS signal;

[0047] Figure 4 SERS signal diagrams for sputtering different targets onto porous silicon surfaces;

[0048] Figure 5 This is a graph showing the relationship between Raman intensity and fungal toxin concentration.

[0049] Figure 6 This is a linear range graph for OTA detection.

[0050] Figure 7 Linear range plot for AFB1 detection;

[0051] Figure 8 Linear range graph for DON detection;

[0052] Figure 9 This is a graph showing the specificity of OTA analysis.

[0053] Figure 10 A graph showing the specificity of AFB1;

[0054] Figure 11 This is a chromatogram showing the specificity of DON.

[0055] Figure 12 The spiked recovery rate of the method of the present invention in kudzu root, poria cocos and malt;

[0056] Figure 13 The ELISA method represents the spike recovery rate of kudzu root, poria cocos, and malt. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0058] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0059] Preparation of PB buffer: Weigh 27.6g NaH2PO4, dissolve in water, and bring the volume to 1000mL to prepare solution A. Weigh 71.6g Na2HPO4·12H2O, dissolve in water, and bring the volume to 1000mL to prepare solution B. Take 23mL of solution A and 77mL of solution B, mix well, and add 200mL of distilled water to obtain 0.1mol / mL PB buffer with pH 7.4.

[0060] The main instruments used in this invention are as follows: a laser confocal Raman microscope, model OEPRO, manufactured by Hangzhou Pulei Optoelectronic Technology Co., Ltd.; a reactive magnetron sputtering instrument, model SKY, manufactured by Shenyang Scientific Instruments Co., Ltd., Chinese Academy of Sciences; an electrochemical etching instrument, model NJ320B, manufactured by Nanjing Hanlong Experimental Equipment Co., Ltd.; a benchtop low-speed automatic balancing centrifuge, model TDZ5-WS, manufactured by Changsha Xiangzhi Centrifuge Instrument Co., Ltd.; an ultrasonic cleaner, model KQ-300B, manufactured by Kunshan Ultrasonic Instrument Co., Ltd.; a multifunctional microplate reader, model Infinite200, manufactured by Diken (Shanghai) Trading Co., Ltd.; a shaking incubator, model ZQTY-79, manufactured by Shanghai Zhichu Instrument Co., Ltd.; a vortex mixer, model QL-861, manufactured by Haimen Qilinbei Instrument Manufacturing Co., Ltd.; and a traditional Chinese medicine pulverizer, model FW177, manufactured by Shangsha Changhong Pharmaceutical Machinery Equipment Factory.

[0061] Artificial mycotoxin antigens (OTA-BSA, DON-BSA, AFB1-BSA) were all purchased from Shandong Landu Biotechnology Co., Ltd.; mycotoxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) were all purchased from Shandong Landu Biotechnology Co., Ltd.

[0062] The silver sputtering target was purchased from Qijin New Materials Co., Ltd., and is of analytical grade with a purity of 99.99%.

[0063] P-type monocrystalline silicon wafers were purchased from Lijing Electronics Co., Ltd., with a resistivity of 0.0008-0.00012Ω and a thickness of 500-550μm.

[0064] Example 1

[0065] Preparation of nano-gold-porous silicon SERS substrate

[0066] (1) Cleaning of monocrystalline silicon wafers: P-type monocrystalline silicon wafers with resistivity of 0.0008-0.00012Ω are cut into 1.5cm×1.5cm pieces and cleaned sequentially with ultrapure water for 10min, acetone for 10min, anhydrous ethanol for 10min, and ultrapure water for 10min three times. After cleaning, they are dried with nitrogen gas for later use.

[0067] (2) Preparation of porous silicon: After treatment, the silicon wafer is placed in an electrochemical etching device. An electrolyte is prepared by mixing 49% hydrofluoric acid and anhydrous ethanol in a 3:1 ratio. 1 mL of electrolyte is dropped into the groove of the electrochemical etching device. The waveform of the electrochemical etching instrument is selected as square wave, the current is selected as 400 mA, the duration is selected as 20 s, and the number of jumps is selected as no jump. The instrument is started, and porous silicon is prepared by electrochemical etching. After repeated cleaning with ethanol, it is dried with nitrogen gas for later use.

[0068] Scanning electron microscope image of the prepared porous silicon is shown below. Figure 2 Figure a shows that the prepared porous silicon has a network structure. The porous silicon prepared by current 400mA has a pore size of 70-85nm. The network is relatively regular and the network structure increases the specific surface area, which can provide good hot spots and signal strength.

[0069] (3) Magnetron sputtering process: The porous silicon sample prepared in step (2) is fixed on the sample target and placed in the SKY reactive magnetron sputtering equipment. A silver target (purity 99.99%) is loaded. The pre-sputtering voltage is set to 50W and the time to 30S. The sputtering voltage is set to 50W and the time to 24min. The vacuum is then reduced to 5×10⁻⁶. -4 After Pa, the instrument is started, and silver nanoparticles are sputtered onto the porous silicon surface. The silver layer thickness of the nano-silver-porous silicon is 200 nm. After cooling for 15-20 min, the sample is removed and stored in nitrogen.

[0070] Electron micrographs after magnetron sputtering are shown below. Figure 2 Figure b, compared to Figure a, shows that the porous silicon surface is sputtered with densely packed silver particles, and the substrate provides a good hotspot. Figure 3 It can be seen that the SERS signal of nano-silver-porous silicon is significantly enhanced compared to that of porous silicon.

[0071] (4) Addition of artificial antigens: Three drops each of 80 μg / mL OTA-BSA, 60 μg / mL LAFB1-BSA, and 80 μg / mL DON-BSA were added to the nano-silver-porous silica surface, with each drop containing 0.3 μg. The specific procedures were the same as those in principle. Figure 1 Let it stand at 37℃ for 20 minutes; after that, add 0.3 μL of PB solution to each spot to wash away excess antigen, and repeat the washing process three times.

[0072] SERS substrate electron microscopy image and surface image are shown below. Figure 2 In Figures b and c, the surface images before and after antigen immobilization with nano-silver-porous silica are not very obvious. Further observation of the cross-sectional images reveals the difference. Figure 2 Figures d and e show that, compared to figure b, the artificial antigen of fungal toxin is firmly bound to the surface of nano-silver-porous silicon. In summary, this demonstrates the feasibility of the SERS substrate prepared by this invention.

[0073] like Figure 4 It can be seen that by sputtering SERS substrates prepared with different materials on the porous silicon surface, and comparing their SERS signal intensity, it was found that the SERS signal intensity of sputtering silver target on the porous silicon surface is higher than that of sputtering titanium target and sputtering titanium target first and then silver target (sputtering method is the same as step 3). Therefore, the present invention selects nano-silver-porous silicon as SERS substrate.

[0074] Example 2

[0075] Preparation of SERS tags

[0076] (1) Preparation of gold nanoparticles: Boil 50 mL of double-distilled water, and add 0.25 mL of chloroauric acid solution (2%, w / w) and 0.375 mL of trisodium citrate aqueous solution (1%, w / w) in sequence under vigorous stirring. Continue stirring and heating at 100 °C. After the solution changes from dark blue to purplish red for about 10-15 min, the solution color no longer changes. Stop heating and cool to room temperature under continuous stirring to obtain colloidal gold solution.

[0077] (2) Add 2.5 mL of 50 μM NBA solution to 22.5 mL of colloidal gold, shake to mix at room temperature, then add 0.75 mL of 10 μmol / L SH-PEG5000-COOH solution, shake at room temperature (150 r / min) for 20 min, then add 5 mL of 50 μmol / L SH-PEG5000-SH solution, shake at room temperature for 3 h. Remove excess PEG by repeated centrifugation (10000 r / min, 15 min) three times, and dissolve the precipitate in 1 mL of PB solution to obtain Au@NBA@PEG nanosolution. Simultaneously add 6 μL of freshly prepared 40 mg / mL EDC and 6 μL of 110 mg / mL NHS to the obtained Au@NBA@PEG nanosolution to activate the carboxyl groups, so that the carboxylic acid functional groups on the surface of these nanoparticles can be coupled with antibodies. After reacting vigorously at room temperature for 20 minutes, excess EDC and NHS were removed by centrifugation and washing. The precipitate was then resuspended in 1 mL of PB and stored at 4°C for later use.

[0078] (3) Antibody fixation: Add the activated colloidal gold solution from step (2) to each centrifuge tube, 450 μL per tube, and simultaneously add 100 μL each of OTA-Ab (80 μg / mL), AFB1-Ab (40 μg / mL), and DON-Ab (60 μg / mL) solutions. The ratio of colloidal gold to antibody (v / v) is 1.5:1. Incubate overnight at 4°C with shaking at 150 r / min. After the reaction, centrifuge and wash 3-4 times to remove excess antibody, and resuspend the precipitate in 450 μL of PB solution.

[0079] (4) Colloidal gold blocking: Add 300 μL of 1% BSA solution to the colloidal gold solution with the antibody fixed in step (3), react at 37°C for 1 h. After the reaction, centrifuge and wash 3-4 times, and resuspend the precipitate in 450 μL of BSA solution to obtain the SERS tag solution.

[0080] Example 3

[0081] Establishment of OTA Standard Curve

[0082] (1) Prepare a series of OTA solutions with different concentration gradients: Prepare OTA solutions with concentration gradients of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000 and 10000 ng / mL, and prepare 50 μL of OTA solution for each concentration gradient.

[0083] (2) Competitive reaction: 0.3 μL of OTA solution with 9 concentration gradients and 0.3 μL of SERS tag solution prepared in Example 2 were added dropwise to the OTA-BSA position on the 9 nano-silver-porous silicon (prepared in Example 1). The reaction was carried out at 37°C for 1 h. After the reaction was completed, 0.3 μL of LPB solution was added dropwise to the reaction position for cleaning. The operation was repeated 3-4 times.

[0084] (3) Surface-enhanced Raman spectroscopy determination: The Raman spectral intensity of the NBA characteristic peaks was measured to determine the optimal antigen concentration. The intensity of the NBA Raman characteristic peaks at each point was detected using a laser confocal Raman microscope under the following conditions: 785 nm, 550 mA, integration time 3 s, and peak elution time 0.3 s.

[0085] Depend on Figure 5 It can be seen that as the concentration of OTA increases, the Raman signal gradually decreases, showing a linear relationship, and OTA detection curves are established accordingly; from Figure 6 It is known that the linear detection range of OTA is 0.01–100 ng / mL, and the limit of detection is 2.87 pg / mL.

[0086] Example 4

[0087] Establishment of the AFB1 standard curve

[0088] The specific implementation steps are the same as in Example 3, except that OTA in step (1) is replaced with AFB1, and OTA-BSA in step (2) is replaced with AFB1-BSA.

[0089] Depend on Figure 5 It can be seen that as the concentration of AFB1 increases, the Raman signal gradually decreases, showing a linear relationship, and OTA detection curves are established accordingly; from Figure 7 It is known that the linear detection range of OTA is 0.01–100 ng / mL, and the limit of detection is 0.39 pg / mL.

[0090] Example 5

[0091] Establishment of the DON standard curve

[0092] The specific implementation steps are the same as in Example 3, except that OTA in step (1) is replaced with DON, and OTA-BSA in step (2) is replaced with DON-BSA.

[0093] Depend on Figure 5 It can be seen that as the DON concentration increases, the Raman signal gradually decreases, showing a linear relationship, and OTA detection curves are established accordingly; from Figure 8 It is known that the linear detection range of DON is 0.001–10 ng / mL, and the limit of detection is 1.86 pg / mL.

[0094] Example 6

[0095] OTA-specific detection

[0096] The specific implementation steps are the same as in Example 3, except that in step (1), a series of OTA solutions with different concentration gradients are prepared: three mixed fungal toxin solutions are prepared respectively: OTA-AFB1-DON, OTB-AFB1-DON, and AFB1-DON, each toxin at 10 ng / mL, and these three mixed toxins are used to replace the series of OTA solutions with different concentration gradients. The rest of the steps are the same.

[0097] Depend on Figure 9 It can be seen that, compared with the mixed mycotoxins containing OTA, the Raman intensity of the NBA characteristic peak is significantly enhanced when the mixed mycotoxins do not contain OTA, indicating that AFB1 and DON do not react with OTA-Ab in the system and have good specificity; when OTA is replaced with the structural analog OTB, the Raman intensity of the NBA characteristic peak does not change significantly, indicating good specificity for the detection of OTA.

[0098] Example 7

[0099] AFB1 specificity detection

[0100] Similar to Example 4, except that step (1) involves preparing a series of AFB1 solutions with different concentration gradients: four mixed fungal toxin solutions are prepared respectively: AFB1-OTA-DON, AFG1-OTA-DON, and AFG2-OTA-DON, each toxin at 10 ng / mL. These four mixed toxins are used to replace the series of AFB1 solutions with different concentration gradients. The rest of the steps are the same.

[0101] Depend on Figure 10It can be seen that, compared with the mixed mycotoxins containing AFB1, the Raman intensity of the NBA characteristic peak is significantly enhanced when the mixed mycotoxins do not contain AFB1, indicating that OTA and DON do not react with AFB1-Ab in the system and have good specificity; when AFB1 is replaced with structural analogs AFG1 and AFG2, the Raman intensity of the NBA characteristic peak does not change significantly, indicating that the detection specificity for AFB1 is good.

[0102] Example 8

[0103] DON specific detection

[0104] Similar to Example 5, except that step (1) involves preparing a series of DON solutions with different concentration gradients: preparing three mixed fungal toxin solutions: DON-AFB1-OTA, ZEN-AFB1-OTA, and AFB1-OTA, each toxin at 10 ng / mL, replacing the series of DON solutions with these three mixed toxins, and the rest of the steps are the same.

[0105] Depend on Figure 11 It can be seen that, compared with the mixed mycotoxins containing DON, the Raman intensity of the NBA characteristic peak is significantly enhanced when the mixed mycotoxins do not contain DON, indicating that AFB1 and OTA do not react with DON-Ab in the system and have good specificity; when DON is replaced with the structural analog ZEN, the Raman intensity of the NBA characteristic peak does not change significantly, indicating good specificity for the detection of OTA.

[0106] Example 9

[0107] Determination of spiked recovery rates of three mycotoxins in traditional Chinese medicine

[0108] (1) Sample preparation: Malt, kudzu root, and poria cocos were pulverized using a traditional Chinese medicine pulverizer and passed through a 20-mesh sieve. 15g of each sample was weighed and divided into three 5g portions, each placed in a 100mL Erlenmeyer flask. The OTA-AFB1-DON mixed toxin solution was diluted with 100% methanol to prepare 0, 0.1, 1, and 10 ng / mL solutions (four concentrations of the three toxins, each toxin being at the above concentration). 2500μL of each concentration of the mixed toxin solution was added to 5g of each of the three traditional Chinese medicine samples. After thorough mixing, the samples were placed in a ventilated area until the solvent evaporated completely. 15mL of extraction solvent (methanol:water = 7:3) was added, and the mixture was extracted for 2 hours at 150r / min in a shaker. The extract was filtered through Whatman filter paper and then through a 0.45μm filter membrane. The filtrate was the sample to be tested.

[0109] (2) Competitive Immunoassay: The extracted Chinese herbal medicine sample and the SERS tag from Example 2 were combined in equal proportions and reacted at 37°C for 20 min. The mixture was then dropped onto the corresponding SERS substrate prepared in Example 1 (e.g., to determine the recovery rate of OTA, it was dropped onto the OTA-BSA position on the SERS substrate), 0.3 μL per drop, and reacted at 37°C for 1 h. The intensity of the NBA Raman characteristic peak at each point was detected using a laser confocal Raman microscope at 785 nm, 550 mA, integration time 3 s, and peak elution time 0.3 s. The recovery rate was calculated based on the NBA Raman characteristic peak intensity value. The recovery rate calculation formula is as follows:

[0110]

[0111] Depend on Figure 12 It can be seen that the recoveries of OTA, AFB1, and DON in Poria cocos, kudzu root, and malt determined by this method are between 76.59±7.24% and 91.02±5.38%; indicating that the nano-silver-porous silica SERS detection system prepared by this invention can effectively detect the three fungal toxins in grains, with low detection limits and good detection effects.

[0112] Example 10

[0113] ELISA method for detecting the spiked recovery rate of three fungal toxins in traditional Chinese medicine

[0114] (1) Sample preparation: Weigh 20g of pulverized and sieved malt, kudzu root, and poria cocos samples, and add 1mL of OTA-AFB1-DON mixed toxin solution (1ng / mL, 5ng / mL, and 10ng / mL from ELISA kits, respectively) to each sample. Place the mixture in a 250mL stoppered conical flask, add 30mL of petroleum ether and 100mL of methanol-water (7:3) solution, shake for 30min, filter with rapid qualitative filter paper and a separatory funnel, and after the lower methanol-water solution has separated, release the methanol-water solution into another conical flask. Take 20mL of methanol-water solution (equivalent to 4g of sample) and place it in another 125mL separatory funnel, add 20mL of chloroform, shake for 2min, and let it stand to separate the layers. If emulsification occurs, methanol can be added dropwise to promote separation. The chloroform layer was released and filtered through a quantitative slow-speed filter paper containing approximately 10g of anhydrous sodium sulfate pre-moistened with chloroform into a 50mL evaporating dish. Then, 5mL of chloroform was added to a separatory funnel, and the extraction was repeated by shaking. The chloroform layer was filtered through the evaporating dish until dry. Finally, the filter was washed with a small amount of chloroform, and the washings were added to the evaporating dish. The evaporating dish was then dried in a fume hood at 65°C in a water bath. After cooling to room temperature, the solution was thoroughly dissolved in 40mL of methanol-water (35:65) to obtain the test solution.

[0115] (2) Before use, remove the OTA, DON, and AFB1 enzyme-linked immunosorbent assay kit from the refrigerator and allow it to equilibrate at room temperature for 15-20 minutes. Carefully read the instructions and follow them. Use a multi-functional microplate reader to detect the absorbance at 492 nm and calculate the spiked recoveries of OTA, AFB1, and DON in the traditional Chinese medicines Poria cocos, Pueraria lobata, and malt.

[0116] Depend on Figure 13 It can be seen that the recovery rates of ELISA in Poria cocos, kudzu root and malt are between 70.78±6.13% and 89.05±3.21%, which are not only lower than the method of the present invention, but also the ELISA kit can only detect one fungal toxin at the same time. Compared with the method of the present invention, the nano-silver-porous silicon SERS sensor prepared by the present invention can effectively detect OTA, DON and AFB1 in grains, with good detection effect, high detection sensitivity, and can detect three fungal toxins at the same time. The detection time is shorter and the sample processing is simpler. This further shows that the present invention meets the market demand for rapid, efficient and accurate detection of multiple fungal toxins and has broad market prospects.

Claims

1. An application of a nano-silver-porous silicon SERS-based biochip sensor for the simultaneous quantitative detection and analysis of three mycotoxins, OTA, AFB1, and DON; the specific process of the quantitative detection and analysis is as follows: adding buffer solutions of different concentrations of mycotoxins OTA, DON, and AFB1 to the SERS-tagged solution, so that the three mycotoxins and the three mycotoxin artificial antigens on the SERS substrate compete for binding to the three corresponding mycotoxin antibodies on the SERS tag; and simultaneously achieving quantitative analysis of the three mycotoxins by detecting the intensity of the NBA characteristic peak signal bound to the SERS tag. The sensor consists of two parts: a SERS substrate and a SERS tag. The SERS substrate is composed of silver nanoparticles magnetron sputtered onto a porous silicon surface and three artificial mycotoxin antigens connected to the surface of the silver nanoparticles. The SERS tag is composed of gold nanoparticles connected to Nile blue and corresponding mycotoxin antibodies. The three artificial mycotoxin antigens are ochratoxin A artificial antigen, vomitoxin artificial antigen, and aflatoxin B1 artificial antigen, and the corresponding mycotoxin antibodies are ochratoxin A antibody, vomitoxin antibody, and aflatoxin B1 antibody. The SERS tag is made by first physically adsorbing NBA onto the surface of gold nanoparticles, then modifying them with methoxy polyethylene glycol thiol and thiol polyethylene glycol carboxyl groups respectively, and finally covalently linking three mycotoxin antibodies under the activation of EDC and NHS, thereby obtaining gold nanoparticles simultaneously modified with NBA and three mycotoxin antibodies; the SERS substrate is made by cleaning single-crystal silicon, preparing porous silicon by electrochemical etching, and sputtering silver nanoparticles onto the surface by magnetron sputtering, with three mycotoxin artificial antigens covalently linked on the surface of the silver nanoparticles; the SERS substrate and the SERS tag are specifically linked by antigen and antibody.

2. The application according to claim 1, characterized in that, The method for fabricating the biochip sensor based on nano-silver-porous silicon for simultaneous SERS detection of multiple fungal toxins includes the following steps: (1) Clean and dry the P-type monocrystalline silicon wafers for later use; (2) Prepare porous silicon from the silicon wafers processed in step (1), clean and dry them for later use; (3) After evacuating the porous silicon prepared in step (2), silver nanoparticles are sputtered onto the surface of the porous silicon; the porous silicon is cooled to room temperature and then held under an inert gas to obtain porous silicon. (4) Physically adsorb three fungal toxin artificial antigens OTA-BSA, DON-BSA and AFB1-BSA onto the surface of the silver nanoparticles in the porous silicon silver prepared in step (3) to obtain the SERS substrate. (5) Add chloroauric acid solution and trisodium citrate aqueous solution to boiling water in sequence, stir and heat until purple-red and no longer change color, cool to room temperature to obtain colloidal gold, and set aside; (6) Nile blue was added to the colloidal gold solution prepared in (5), and after shaking at room temperature, SH-PEG-COOH and SH-PEG-SH were added in sequence. After centrifugation to remove the supernatant and concentration, EDC and NHS were added for activation treatment. Then, three fungal toxin antibodies (OTA-Ab, DON-Ab, AFB1-Ab) were covalently linked to obtain the SERS tag.

3. The application according to claim 2, characterized in that, In step (1), the resistivity of the P-type single crystal silicon wafer is 0.00012-0.0008Ω.

4. The application according to claim 2, characterized in that, In step (2), the processed silicon wafer is pressed and assembled into an electrochemical etching instrument, an electrolyte is added, and porous silicon is prepared by electrochemical anodic oxidation. After cleaning, it is dried for later use. The electrolyte is prepared by hydrofluoric acid and anhydrous ethanol. Porous silicon is prepared by electrochemical etching. The electrochemical etching current is 300-500mA and the etching time is 10-30S.

5. The application according to claim 2, characterized in that, In step (3), the prepared porous silicon is fixed in a reactive magnetron sputtering apparatus, a silver target is loaded, and a vacuum of 5 × 10⁻⁶ is applied. -4 After Pa, silver nanoparticles were sputtered onto the porous silicon surface; cooled to room temperature and stored in nitrogen.

6. The application according to claim 5, characterized in that, The magnetron sputtering conditions are: voltage 45-55V, sputtering time 22-26min, annealing 15-20min. The thickness of the silver layer is controlled by controlling the sputtering conditions.

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