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CN-116660513-B - Biochip sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS and preparation method and application thereof

CN116660513BCN 116660513 BCN116660513 BCN 116660513BCN-116660513-B

Abstract

The invention discloses a biochip sensor for simultaneously detecting various mycotoxins based on nano silver-porous silicon and SERS, a preparation method and application thereof, the sensor comprises a SERS substrate and a SERS label, the SERS substrate is formed by magnetron sputtering silver nano particles on the surface of porous silicon and connecting three mycotoxin artificial antigens on the surface of the silver nano particles, and a SERS label is connected with gold nano particles of nile blue and corresponding mycotoxin antibodies. According to the invention, mycotoxin artificial antigens on the surfaces of mycotoxins and porous silicon silver can be competitively combined with mycotoxin antibodies on the SERS label, and three mycotoxins are simultaneously quantitatively analyzed by detecting Raman signals of NBA on the surface of the SERS label, so that the detection speed is high, the sensitivity is high, meanwhile, the recovery rate of each toxin in poria cocos, malt and radix puerariae is more than 75%, the variation coefficient of the same batch is lower than 5.2%, and the variation coefficient of different batches is lower than 7.8%.

Inventors

  • ZHENG TIESONG
  • LI HAO
  • LI JIANLIN
  • LV GUANGPING
  • LI QIANJIN

Assignees

  • 南京师范大学

Dates

Publication Date
20260508
Application Date
20230510

Claims (6)

  1. 1. The specific process of quantitative detection and analysis comprises the steps of adding buffer solutions of different mycotoxin OTA, DON, AFB 1 samples with different concentrations into SERS label solutions, enabling three mycotoxins and three mycotoxin artificial antigens on a SERS substrate to compete for combining three corresponding mycotoxin antibodies on the SERS label, and simultaneously realizing quantitative analysis of the three mycotoxins by detecting NBA characteristic peak signal intensity combined on the SERS label; The sensor consists of a SERS substrate and a SERS label, wherein the SERS substrate consists of three mycotoxin artificial antigens which are formed by sputtering silver nano particles on the surface of porous silicon in a magnetron way and are connected with the surface of the silver nano particles, and the SERS label is connected with Nile blue and gold nano particles of corresponding mycotoxin antibodies, wherein the three mycotoxin artificial antigens are ochratoxin A artificial antigen, vomitoxin artificial antigen and aflatoxin B 1 artificial antigen, and the corresponding mycotoxin antibodies are ochratoxin A antibody, vomitoxin antibody and aflatoxin B 1 antibody; The SERS label is characterized in that NBA is firstly physically adsorbed on the upper surface of gold nanoparticles, methoxy polyethylene glycol sulfhydryl and sulfhydryl polyethylene glycol carboxyl are respectively modified, and finally three mycotoxin antibodies are connected through covalent bonds under the activation of EDC and NHS, so that gold nanoparticles with NBA and three mycotoxin antibodies simultaneously modified are obtained, the SERS substrate is prepared by electrochemical etching after monocrystalline silicon is cleaned, nano silver particles are sputtered on the surface of the SERS substrate through magnetron sputtering, three mycotoxin artificial antigens are combined on the surface of the nano silver particles through covalent bonds, and the SERS substrate and the SERS label are connected through antigen-antibody specificity.
  2. 2. The use according to claim 1, wherein the preparation method of the sensor based on simultaneous SERS detection of multiple mycotoxins by nano silver-porous silicon comprises the following steps: (1) Cleaning a P-type monocrystalline silicon piece and drying for later use; (2) Preparing porous silicon from the silicon wafer treated in the step (1), cleaning and drying for later use; (3) Sputtering silver nano particles on the surface of the porous silicon after vacuumizing the porous silicon prepared in the step (2), cooling to room temperature to obtain the porous silicon silver inert gas, and keeping; (4) The surface of silver nano particles in the porous silicon silver prepared in the step (3) is physically adsorbed with three mycotoxin artificial antigens of OTA-BSA, DON-BSA and AFB1-BSA to obtain a SERS substrate; (5) Sequentially adding chloroauric acid solution and trisodium citrate aqueous solution into boiling water, stirring and heating until the mixture is purple red and does not change color, and cooling to room temperature to obtain colloidal gold for later use; (6) Adding Nile blue into the colloidal gold solution prepared in the step (5), oscillating at room temperature, sequentially adding SH-PEG-COOH and SH-PEG-SH, centrifuging to remove supernatant, concentrating, adding EDC and NHS, activating, and connecting three mycotoxin antibodies (OTa-Ab, DON-Ab and AFB 1-Ab) through covalent bonds to obtain the SERS label.
  3. 3. The use according to claim 2, characterized in that the P-type monocrystalline silicon piece in step (1) has a resistivity of 0.00012 to 0.0008 Ω.
  4. 4. The application of the method according to claim 2, wherein in the step (2), the processed silicon wafer is assembled into an electrochemical etcher by tabletting, electrolyte is dripped into the wafer, the wafer is anodized by electrochemistry to prepare porous silicon, the porous silicon is cleaned and dried for standby, the electrolyte is prepared by hydrofluoric acid and absolute ethyl alcohol, the porous silicon is prepared by electrochemistry etching, the electrochemistry etching current is 300-500mA, and the etching time is 10-30S.
  5. 5. The method according to claim 2, wherein the porous silicon prepared in the step (3) is fixed in a reaction magnetron sputtering device, a silver target is filled, the vacuum is pumped to 5 x 10 -4 Pa, silver nano particles are sputtered on the surface of the porous silicon, and the porous silicon is cooled to room temperature and stored in nitrogen.
  6. 6. The method according to claim 5, wherein the magnetron sputtering conditions are a voltage of 45-55V, a sputtering time of 22-26min, an annealing time of 15-20min, and the thickness of the silver layer is controlled by controlling the sputtering conditions.

Description

Biochip sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS and preparation method and application thereof Technical Field The invention belongs to food safety detection, and particularly relates to a biochip sensor for simultaneously detecting various mycotoxins based on nano silver-porous silicon and SERS, and a preparation method and application thereof. Background Mycotoxins are secondary metabolites produced by fungal organisms, toxic alkaloids are formed in the sclerotium of the mycotoxins, which can cause infectious diseases and death of humans and other animals, and more than 400 mycotoxins are known at present, and commonly include aflatoxins, ochratoxins, vomitoxins, alkaloids and the like. Ochratoxin A (Ochratoxin A) is found as a contaminant in a variety of animal and human foods, including traditional Chinese medicine and traditional Chinese medicine products, grains, beer, wine, etc., OTA also has immunosuppressive, teratogenic, genotoxic and carcinogenic properties and affects blood clotting and carbohydrate metabolism, and OTA is found in blood, bile and urine after eating contaminated foods and traditional Chinese medicines, etc., and is considered to be one of the causes of baldry endemic nephropathy. Aflatoxin is a secondary metabolite produced by virulent strains such as aspergillus flavus and aspergillus parasiticus, and is a highly toxic substance. Of naturally contaminated foods, aflatoxin B 1(Aflatoxin B1) is most common and is one of the strongest chemical carcinogens currently known. The cancer research institute of the World Health Organization (WHO) in 1993 demarcates AFB 1 as a class I carcinogen to humans. AFB 1 is widely considered to play an important role in the formation process of primary hepatocellular carcinoma in some countries and regions, aflatoxins exist in aspects of our lives, are particularly easy to occur in humid and hot environments, are as small as peanuts, corns and soybeans eaten by us, are as large as soil, animals and plants, and are possibly polluted by aflatoxins. Vomitoxin (Deoxynivalenol) is a common mycotoxin in foods and is widely found in nature, and when people ingest food contaminated by DON, acute poisoning symptoms such as anorexia, emesis, diarrhea, fever, unstable standing, slow response and the like can be caused, and when serious, the hematopoietic system is damaged to cause death. Because the proportion of cereal grains in the traditional Chinese eating habit is greatly higher than that in the western world, the harm of vomitoxin is more prominent. Based on this, strict standards are established in various countries and regions of the world to protect the safety of agricultural products, foods, medicinal materials, feeds and the like. Common detection methods for mycotoxins include Thin Layer Chromatography (TLC), gas Chromatography (GC), liquid chromatography (HPLC), capillary Electrophoresis (CE), enzyme-linked immunosorbent assay (ELISA), and the like. All the common methods have some defects such as complex sample pretreatment, expensive instruments, high detection cost and the like, so that the establishment of a detection method which is simple to operate, high in specificity and low in cost can effectively detect and quantitatively analyze three mycotoxins (OTA, AFB 1 and OTA) at the same time is quite significant. Disclosure of Invention Aiming at the problems in the prior art, the invention provides a biochip sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS, which overcomes the limitation that one reagent kit can only detect one mycotoxin at the same time in the traditional ELISA reagent kit, and realizes simultaneous quantitative analysis on three mycotoxins by utilizing three mycotoxins (OTA, AFB1, DON) and three mycotoxin artificial antigens (OTA-BSA, DON-BSA, AFB 1-BSA) to compete for binding with three mycotoxin antibodies (OTa-Ab, DON-Ab, AFB 1-Ab) on a SERS label. The second object of the invention is to provide a preparation method of the SERS sensor capable of simultaneously detecting three mycotoxins OTA, AFB1 and DON, and the third object of the invention is to provide an application of the SERS sensor in synchronous detection of three mycotoxins (OTA, AFB1 and DON). According to the technical scheme, the sensor for detecting multiple mycotoxins based on simultaneous SERS of nano-silver-porous silicon comprises a SERS substrate and a SERS label, wherein the SERS substrate consists of three mycotoxins formed by magnetron sputtering silver nanoparticles on the surface of the porous silicon and connecting the surfaces of the silver nanoparticles, the SERS label is connected with gold nanoparticles of nile blue and corresponding mycotoxin antibodies, the three mycotoxin artificial antigens are ochratoxin A artificial antigen, vomitoxin artificial antigen and aflatoxin B 1 artificial antigen (OTA-BSA, DON-BSA and AFB 1-BSA), and the c