CN116660513A - Biochip sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS and preparation method and application thereof - Google Patents

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

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CN116660513A
CN116660513A CN202310524882.0A CN202310524882A CN116660513A CN 116660513 A CN116660513 A CN 116660513A CN 202310524882 A CN202310524882 A CN 202310524882A CN 116660513 A CN116660513 A CN 116660513A
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sers
porous silicon
ota
mycotoxin
silver
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郑铁松
李�浩
李建林
吕广萍
李前进
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Nanjing Normal University
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Abstract

The invention discloses a sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon and SERS, and a preparation method and application thereof. 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%.

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, and toxic alkaloids are formed in the sclerotium of mycotoxins, which can lead to infectious diseases and death in humans and other animals; the currently known mycotoxins include more than 400 kinds, and common mycotoxins include aflatoxin, ochratoxin, vomitoxin, alkaloid 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, cereals, beer and wine, and the like; OTA also has immunosuppressive, teratogenic, genotoxic and carcinogenic properties, and affects blood clotting and carbohydrate metabolism; OTA is found in blood, bile and urine after people and animals eat contaminated foods and traditional Chinese medicines, etc., and is considered to be one of the causes of bardry endemic nephropathy. Aflatoxin is a secondary metabolite produced by virulent strains such as aspergillus flavus and aspergillus parasiticus, and is a highly toxic substance. Aflatoxin B in naturally contaminated foods 1 (Aflatoxin B 1 ) Most commonly, one of the strongest chemical carcinogens is currently known. World health organization (W)HO) cancer research institute will AFB 1 Are defined as class I carcinogens to humans. In some countries and regions, AFB 1 Are generally considered to play an important role in the primary hepatocellular carcinoma formation process; aflatoxins exist in aspects of our life, are particularly easy to appear in damp-heat 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, is widely found in nature, and can cause acute poisoning symptoms such as anorexia, emesis, diarrhea, fever, unstable standing, slow response and the like after people ingest food polluted by DON, and damages the hematopoietic system to cause death when serious. 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. These common methods all have some drawbacks such as: the sample pretreatment is complex, the instrument is expensive, the detection cost is high, and the like, so that a detection method with simple operation, high specificity and low cost is established, and three mycotoxins (OTA and AFB) can be simultaneously detected 1 OTA) is of great interest for efficient detection and quantitative analysis.
Disclosure of Invention
The invention aims to: aiming at the problems existing 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 a traditional ELISA kit can only detect one mycotoxin at the same time by using three mycotoxins (OTA, AFB1, DON) and three mycotoxin artificial antigens (OTA-BSA, DON-BSA, AFB 1-BSA) to compete for combining with three mycotoxin antibodies (OTa-Ab, DON-Ab, AFB 1-Ab) on a SERS label.
A second object of the present invention is to provide a method for preparing the SERS sensor capable of simultaneously detecting three mycotoxins OTA, AFB1, DON); a third object of the present invention is to provide the use of the SERS sensor described above for simultaneous detection of three mycotoxins (OTA, AFB1, DON).
The technical scheme is as follows: in order to achieve the above purpose, the sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS 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, and 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 antigens (OTA-BSA, DON-BSA, AFB 1-BSA), corresponding mycotoxin antibodies are ochratoxin A antibody, vomitoxin antibody and aflatoxin B 1 Antibodies (OTa-Ab, DON-Ab, AFB 1-Ab).
Wherein, the SERS label is formed by physically adsorbing NBA on the upper surface of gold nano particles, respectively modifying methoxy polyethylene glycol sulfhydryl and sulfhydryl polyethylene glycol carboxyl, and finally connecting mycotoxin antibodies (OTa-Ab, DON-Ab and AFB 1-Ab) through covalent bonds under the activation of EDC and NHS; the SERS substrate is prepared by preparing porous silicon through electrochemical anodic oxidation after cleaning monocrystalline silicon, and sputtering nano silver particles on the surface through magnetron sputtering, wherein mycotoxin artificial antigens (OTA-BSA, DON-BSA and AFB 1-BSA) are adsorbed on the surface of the nano silver particles through covalent bonds; the SERS substrate and the SERS label are specifically connected through antigen-antibody.
Wherein the mycotoxin artificial antigen comprises any one or more of OTA-BSA, DON-BSA and AFB1-BSA, and the mycotoxin antibody comprises any one or more of OTa-Ab, DON-Ab and AFB1-Ab.
The preparation method of the biochip sensor for simultaneously detecting various mycotoxins based on nano silver-porous silicon SERS 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 prepared in the step (2) after vacuumizing the porous silicon; cooling to room temperature to obtain porous Si-Ag inert gas and maintaining;
(4) The porous silicon silver prepared in the step (3) and three mycotoxin artificial antigens are physically adsorbed on the surfaces of silver nano particles to obtain a (OTA-BSA, DON-BSA, AFB 1-BSA) 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.
Wherein, the resistivity of the P-type monocrystalline silicon piece in the step (1) is 0.0008-0.00012 omega.
The processed silicon wafer is pressed into pieces and assembled into an electrochemical etching instrument, electrolyte is dripped into the piece, the piece is subjected to electrochemical anodic oxidation to prepare porous silicon, and the porous silicon is cleaned by ethanol and dried by nitrogen for later use; the electrolyte is prepared from hydrofluoric acid and absolute ethyl alcohol, porous silicon is prepared by electrochemical etching, the electrochemical etching current is 300-500mA, and the etching time is 10-30S.
Preferably, the electrochemical etching current is 400mA, and the etching time is 20S.
Preferably, the electrolyte is 49% hydrofluoric acid and absolute ethyl alcohol 3:1 are arranged in proportion.
Wherein, in the step (3), the prepared porous silicon is fixed in a reaction magnetron sputtering device, a silver target is filled, and the vacuum is pumped to 5 multiplied by 10 -4 Sputtering silver nano particles on the surface of porous silicon after Pa; cooling to room temperature, and storing in nitrogen;
wherein the magnetron sputtering condition is that the voltage is 45-55W, the sputtering time is 22-26min, and the annealing time is 15-20 min. According to the invention, the silver target material is sputtered onto the surface of porous silicon by magnetron sputtering, and the thickness of the silver layer is controlled by controlling sputtering conditions (voltage and time).
Preferably, the magnetron sputtering condition is that the voltage is 50W, the sputtering time is 24min, and the thickness of the silver layer of the nano silver-porous silicon prepared by annealing for 15min is 200nm.
Wherein, in the step (4), artificial antigens are dripped on the substrate, and three artificial antigens, namely OTA-BSA, DON-BSA and AFB1-BSA, are dripped on the SERS substrate; adding NBA solution, SH-PEG-SH solution, SH-PEG-COOH solution, EDC solution and NHS solution into the colloidal gold solution in the step (6); three antibodies OTa-Ab, DON-Ab, AFB1-Ab were added.
Further, in the step (4), three antigens (OTA-BSA, DON-BSA and AFB 1-BSA) are added dropwise to each SERS substrate (a porous silicon sputtered with nano silver is called a SERS substrate, the size of the SERS substrate is about 1.5cm x1.5cm, the adding mode is shown in a schematic diagram 1), three antigens are added dropwise to each antigen (OTA-BSA, DON-BSA and AFB 1-BSA), 0.3 mu L of each antigen is added dropwise, and the concentration is 60 mu g/mL, 80 mu g/mL and 80 mu g/mL in sequence.
Further, 0.25mL of chloroauric acid solution with a mass concentration of 2% and 0.35mL of trisodium citrate solution with a mass concentration of 1% are sequentially added to each 50mL of boiling ultrapure water in the step (5). The concentrations of the added three antibodies OTa-Ab, DON-Ab and AFB1-Ab were 80. Mu.g/mL, 40. Mu.g/mL and 60. Mu.g/mL.
Further, in the step (6), 2.5mL of NBA solution having a concentration of 50. Mu.M was added to each 22.5mL of the colloidal gold, 0.75mL of SH-PEG-SH solution having a concentration of 10. Mu.M was added, 5mL of SH-PEG-COOH solution having a concentration of 50. Mu.M was added, 6. Mu.L of EDC solution having a concentration of 40mg/mL was added, and 6. Mu.L of NHS solution having a concentration of 110mg/mL was added; the concentrations of the added three antibodies OTa-Ab, DON-Ab and AFB1-Ab were 80. Mu.g/mL, 40. Mu.g/mL and 60. Mu.g/mL.
Preferably, the preparation method of the sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS comprises the following steps:
(1) The P-type monocrystalline silicon wafer is sequentially ultrasonically cleaned by ultrapure water, acetone, ethanol and ultrapure water, and then dried by nitrogen for standby.
(2) And (3) assembling the silicon wafer treated in the step (1) into an electrochemical etching instrument, dripping electrolyte, performing electrochemical anodic oxidation to prepare porous silicon, cleaning with ethanol, and drying with nitrogen for later use.
(3) Fixing the porous silicon prepared in the step (2) in a reaction magnetron sputtering device, loading a silver target, and vacuumizing to 5 multiplied by 10 -4 Sputtering silver nano particles on the surface of porous silicon after Pa; cooling to room temperature and storing in nitrogen.
(4) And (3) physically adsorbing three mycotoxin artificial antigens (OTA-BSA, DON-BSA and AFB 1-BSA) on the surfaces of the porous silicon silver and silver nano particles prepared in the step (3) to prepare the SERS substrate.
(5) Sequentially adding chloroauric acid solution and trisodium citrate aqueous solution into boiling water, stirring and heating at a constant temperature of 100 ℃ until the mixture is mauve and does not change color, and cooling to room temperature for later use;
(6) Adding NBA 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.
The biochip sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS is application to simultaneous quantitative detection and analysis of multiple mycotoxins.
Preferably, the nano-silver-porous silicon-based simultaneous SERS detection multiple mycotoxins biochip sensor is applied to quantitative detection and analysis of three mycotoxins (OTA, AFB1 and DON); specifically, the SERS sensor based on nano silver-porous silicon is mainly used for synchronously detecting three mycotoxins (OTA, AFB1 and DON) in food and medicines.
The specific process of quantitative detection and analysis is as follows: buffer solutions of mycotoxin (OTA, DON, AFB 1) samples with different concentrations are added into SERS label solutions, so that three mycotoxins (OTA, DON, AFB) and three mycotoxin artificial antigens (OTA-BSA, DON-BSA and AFB 1-BSA) on a SERS substrate compete for binding to three mycotoxin antibodies (OTa-Ab, DON-Ab and AFB 1-Ab) on the SERS label, and quantitative analysis of the three mycotoxins is realized by detecting the NBA characteristic peak signal intensity bound on the SERS label.
Further, buffer solutions of mycotoxin (OTA, DON, AFB 1) samples with different concentrations are added into SERS label solutions, so that three mycotoxins (OTA, DON, AFB 1) and three mycotoxin artificial antigens (OTA-BSA, DON-BSA, AFB 1-BSA) on a SERS substrate compete for binding to three mycotoxin antibodies (OTa-Ab, DON-Ab, AFB 1-Ab) on the SERS label, and quantitative analysis of the three mycotoxins is realized by detecting the signal intensity of the NBA characteristic peaks bound on the SERS label, wherein the competition reaction time is 1h, the competition reaction temperature is 37 ℃, and the Raman shift of the Raman characteristic peaks of the NBA is detected to be 591cm -1 。
According to the invention, in the preparation process of SERS labels, a Nile Blue (NBA) solution for signal detection is added into a colloidal gold solution, under the oscillation condition, enrichment of NBA on the surfaces of gold nanoparticles is realized through physical adsorption, meanwhile, methoxy polyethylene glycol sulfhydryl solution and sulfhydryl polyethylene glycol carboxyl solution, specifically CH3O-PEG5000-SH with the molecular weight of 5000 and SH-PEG5000-COOH are added in the reaction process, au-S bonds are formed between sulfhydryl and gold atoms so as to fix the Au-S bonds on the surfaces of the gold nanoparticles, PEG polyethylene glycol is a macromolecular polymer, the molecular chain length and the function of spreading apart each other after connection are realized, the molecular chain length is used for preventing coagulation, and simultaneously, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to activate carbonyl groups positioned at the outer ends on SH-PEG5000-COOH and are combined with amino groups on three mycotoxin antibody chains through covalent bonds, so that gold nanoparticles with NBA and three mycotoxin antibodies are simultaneously modified.
In the preparation process of the SERS substrate, a completely cleaned silicon wafer is cut and placed on an anode, electrochemical etching is carried out in electrolyte after electrification, a layer of porous silicon can be formed on the surface of the silicon wafer, the process is a 4-electron oxidation process, two silicon atoms come from the electrode and two silicon atoms come from SiF2, si atoms are separated from the silicon wafer in a SiF4 form under F-nucleophilic attack to form a pore channel, siF4 and F-in solution react to form SiF62-, and finally a porous structure is formed. Placing porous silicon in a magnetron sputtering instrument, filling a silver target, and sputtering a layer of silver on the surface of the porous silicon, wherein the process is as follows: under the action of an electric field E, electrons collide with argon atoms in the process of flying to the porous silicon, so that Ar positive ions and new electrons are generated by ionization of the electrons, the new electrons fly to the porous silicon, ar ions are accelerated to fly to a cathode target under the action of the electric field, the surface of the silver target is bombarded with high energy, the silver target is sputtered in sputtered particles, and neutral silver atoms are deposited on the porous silicon to form silver nano particles. Three mycotoxin antigens (OTA-BSA, DON-BSA and AFB 1-BSA) are dripped on the surface of the silver layer and adsorbed on the surface of nano silver through covalent bonds.
The principle of the invention is shown in fig. 1, in which the immune competition reaction is designed: three antibodies (OTa-Ab, DON-Ab, AFB 1-Ab) of mycotoxin connected on the SERS label and corresponding mycotoxin artificial antigens (OTA-BSA, DON-BSA, AFB 1-BSA) on the SERS substrate are specifically combined, and the other part can be combined with the corresponding mycotoxin. If mycotoxin is not added, the mycotoxin antibody on the SERS label is combined with the mycotoxin antigen on the SERS substrate, and a higher NBA characteristic peak Raman signal is shown; if mycotoxins are added to the system, the mycotoxin antibodies attached to the SERS tag will bind to the mycotoxins first, and the remaining mycotoxin antibodies on the SERS tag bind to the mycotoxin antigen on the SERS substrate, and the raman signal is reduced. Therefore, the finally detected NBA characteristic peak Raman signal value can reflect the concentration of the added mycotoxins, so that three mycotoxins (OTA, AFB1, DON) in substances such as traditional Chinese medicines can be synchronously detected by establishing a standard curve detection curve of each toxin due to specificity of competitive binding. Specific measurement As shown in schematic diagram 1, different antigens are dripped at different positions of the SERS substrate, and a certain mycotoxin is needed to drip the SERS label liquid and the corresponding sample liquid at the corresponding antigen positions. 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 by detecting Raman signals of NBA on the surface of the SERS label, simultaneous quantitative analysis of three mycotoxins of OTA, DON and AFB1 is realized, 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%.
The invention prepares a brand-new biochip sensor for detecting various mycotoxins based on nano silver-porous silicon and SERS at the same time, wherein the process for preparing the SERS substrate improves the overall stability, particularly the stability of the process by adopting magnetron sputtering compared with spin coating and deposition is effectively improved, the error is reduced, the time cost is reduced when three mycotoxins are detected, and the dosage of antigen and antibody used by the ELISA kit is obviously reduced compared with that of the antigen and antibody used by the ELISA kit. The sensor prepared by the invention realizes simultaneous detection through the specificity of antigen-antibody combination by competition reaction. The competition reaction only needs 1h, the sample measurement only needs 0.3 seconds, three mycotoxins can be detected simultaneously, and the ELISA kit only can detect one mycotoxin. At present, 50uL of antibody and 50uL of artificial antigen are required to be dripped into a 96-well plate in each measurement of an ELISA kit, the dosage of 50-500ug of antigen-antibody required to be detected is far more than that of the ELISA kit, and the price of the mycotoxin artificial antigen and the mycotoxin artificial antigen with the market price of 1mg is about 2000-4000 yuan.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) The porous silicon is formed by electrochemical etching, the specific surface area is increased by the reticular structure, and more hot spots can be provided; (2) Silver nano particles are fixed on the surface of porous silicon by utilizing a magnetron sputtering technology, so that the stability is high, the error is low, the preparation is simple, more than 30 SERS substrates can be prepared by single magnetron sputtering, the SERS substrates can be produced in batches, can be stored in nitrogen for a long time, and have the condition of long-time storage and circulation in the market; (3) The determination sample only needs 0.024 mug OTA-BSA or 0.018 mug AFB1-BSA or 0.024 mug DON-BSA, and 0.024 mug OTa-Ab, 0.012 mug AFB1-Ab and 0.018 mug DON-Ab connected on SERS label, compared with the traditional enzyme-linked immunosorbent assay (ELISA), the amount of required artificial antigen and antibody is less, and the cost of single detection sample is reduced; (4) Compared with the traditional chromatographic technique or enzyme-linked immunosorbent assay (ELISA), the detection method using the sensor has the advantages of high sensitivity, good specificity, no damage to samples, capability of detecting various toxins simultaneously, and the like; (5) By using the sensor and the detection method provided by the invention, three mycotoxins (OTA, AFB1 and DON) can be detected simultaneously, the three mycotoxins can be detected simultaneously and quantitatively analyzed, each sample is detected only by 0.3S (only by 0.3S through Raman instrument laser), and the market demand of rapid and efficient detection is met. (6) The SERS sensor prepared by the invention can be produced in batch, has large specific surface area, high stability and long storage time, and can detect three mycotoxins simultaneously.
Drawings
FIG. 1 is a schematic diagram of simultaneous detection of three mycotoxins (OTA, AFB1, DON) based on nano-silver-porous silicon surface enhanced Raman spectroscopy;
FIG. 2 is an electron microscope image of a SERS substrate characterization;
FIG. 3 is a graph showing the SERS signal enhancement effect of NBA as a probe molecule;
FIG. 4 is a chart of SERS signals corresponding to different targets sputtered on the surface of porous silicon;
FIG. 5 is a graph of Raman intensity versus mycotoxin concentration;
FIG. 6 is a linear range diagram of OTA detection;
FIG. 7 is a graph of the linear range of AFB1 detection;
FIG. 8 is a linear range plot of DON detection;
FIG. 9 is a graph of OTA specificity analysis;
FIG. 10 is a diagram of AFB1 specificity analysis;
FIG. 11 is a DON specificity analysis chart;
FIG. 12 shows the labeled recovery rate of the method of the present invention in Pueraria lobata and Poria cocos malt;
FIG. 13 shows the labeled recovery of ELISA method in Pueraria lobata and Poria cocos malt.
Detailed Description
The invention is further described below with reference to specific embodiments and figures.
The experimental methods described in the examples, unless otherwise specified, are all conventional; the reagents and materials, unless otherwise specified, are commercially available.
Configuration of PB buffer: weigh 27.6g NaH 2 PO 4 Dissolving in water, metering to 1000mL to obtain solution A, weighing 71.6g Na 2 HPO 4 ﹒12H 2 O is dissolved by adding water, the volume is fixed to 1000mL, liquid B is prepared, 23mL of liquid A and 77mL of liquid B are taken and mixed evenly, 200mL of distilled water is added, and 0.1mol/mL PB buffer with pH being 7.4 can be obtained.
The main instruments used in the invention are as follows: the model of the laser confocal Raman microscope is OEPRO, and Hangzhou spectrum laser photoelectric technology Co., ltd; the reactive magnetron sputtering instrument is of the model SKY, and is manufactured by Shenyang scientific instruments Co., ltd; the model of the electrochemical etching instrument is NJ320B, and Nanj Hanlong experimental equipment limited company is manufactured by manufacturers; the model of the desk type low-speed automatic balancing centrifuge is TDZ5-WS, and the manufacturer is Changshaxiang intelligent centrifuge instrument limited company; the model of the ultrasonic cleaner is KQ-300B, and the manufacturer is Kunshan ultrasonic instrument Co., ltd; the model of the multifunctional enzyme-labeled instrument is Infinite200, which is available from Shanghai trade company; the shake incubator is of the model ZQTY-79, and manufacturers know Chu instruments limited company in Shanghai; the vortex mixer is model QL-861, manufactured by the Linbel instruments, inc. of Haimen, inc.; the model of the traditional Chinese medicine pulverizer is FW177, and the traditional Chinese medicine pulverizer is sold in the market Chang Hong pharmaceutical mechanical equipment factory.
Mycotoxin artificial antigens (OTA-BSA, DON-BSA, AFB 1-BSA) were purchased from Shandong blue Biotech Co., ltd; mycotoxin antibodies (OTa-Ab, DON-Ab, AFB 1-Ab) were purchased from Shandong blue Biotech Co.
Silver target material is purchased from advanced materials limited company, and has the purity of 99.99 percent.
The P-type monocrystalline silicon wafer is purchased from Lijing electronic Co., ltd, and has a resistivity of 0.0008-0.00012 omega and a thickness of 500-550 μm.
Example 1
Preparation of nano gold-porous silicon SERS substrate
(1) Cleaning a monocrystalline silicon wafer: cutting a P-type monocrystalline silicon wafer with resistivity of 0.0008-0.00012 omega into 1.5cm multiplied by 1.5cm, sequentially ultrasonically cleaning with ultrapure water for 10min, ultrasonically cleaning with acetone for 10min, ultrasonically cleaning with absolute ethyl alcohol for 10min, repeatedly ultrasonically cleaning with ultrapure water for 10min for 3 times, and drying with nitrogen for later use.
(2) Preparation of porous silicon: placing the treated silicon wafer into an electrochemical etching device, preparing electrolyte by using hydrofluoric acid with the mass fraction of 49% and absolute ethyl alcohol in a ratio of 3:1, dripping 1mL of electrolyte into a groove of the electrochemical etching device, respectively selecting square waves by using waveforms of an electrochemical etching instrument, selecting 400mA by using current, selecting 20S for a long time, selecting no jump of the jump times, starting the instrument, preparing porous silicon by electrochemical etching, repeatedly cleaning by using ethyl alcohol, and drying by using nitrogen for standby.
The scanning electron microscope image of the prepared porous silicon is shown as a graph a in fig. 2, the prepared porous silicon has a net structure, and the pore size of the porous silicon prepared by 400mA is 70-85nm; the network is regular, the specific surface area is increased by the network structure, and good hot spots and signal strength can be provided.
(3) And (3) a magnetron sputtering process: fixing the porous silicon sample prepared in the step (2) on a sample target, fixing the sample target in SKY reaction magnetron sputtering equipment, and filling a silver target (purity is 99.99%); setting a pre-sputtering voltage of 50W and a sputtering time of 30S, and setting the sputtering voltage of 50W and the sputtering time of 24min; vacuumizing to 5×10 -4 After Pa, starting the instrument, sputtering silver nano particles on the surface of the porous silicon, cooling for 15-20min, taking down the sample, and putting the sample into nitrogen for preservation, wherein the thickness of the silver layer of the nano silver-porous silicon is 200nm.
The electron microscope image after magnetron sputtering is shown in the graph b in fig. 2, compared with the graph a, the surface image shows that the porous silicon surface is sputtered with the silver particles of the millboard, the substrate provides good hot spots, and the SERS signal of the nano silver-porous silicon is obviously enhanced compared with that of the porous silicon.
(4) Dropping artificial antigen: respectively dripping three drops of 80 mug/mL OTA-BSA, 60 mug/mL AFB1-BSA and 80 mug/mL DON-BSA on the surface of the nano silver-porous silicon, wherein the specific situation of each drop is the same as that of the schematic diagram 1, and standing for 20min at 37 ℃; after completion, 0.3. Mu.L of PB solution was added dropwise to each spot to wash off the excess antigen, and the washing was repeated three times.
The surface diagrams of the SERS substrate electron microscope are shown in the diagram b and the diagram c in fig. 2, the front and the back of the nano silver-porous silicon immobilized antigen are not obvious from the surface diagram, further the cross section diagrams are shown in the diagram d and the diagram e in fig. 2, and compared with the diagram b, the diagram e can be used for showing that the mycotoxin artificial antigen is firmly combined on the surface of the nano silver-porous silicon; in summary, the SERS substrate prepared by the present invention is illustrated to have feasibility.
As can be seen from fig. 4, when the SERS substrates prepared from different materials are sputtered on the surface of the porous silicon, the SERS signal intensity of the SERS signal is higher than that of the sputtering of the silver target on the surface of the porous silicon (the sputtering method is the same as step 3) and the sputtering of the titanium target is performed before the sputtering of the silver target, so the nano silver-porous silicon is selected as the SERS substrate.
Example 2
Preparation of SERS tags
(1) Gold nanoparticle preparation: 50mL of double distilled water is boiled, 0.25mL of chloroauric acid solution (2%, w/w) and 0.375mL of trisodium citrate aqueous solution (1%, w/w) are sequentially added under vigorous stirring, stirring and heating are continued at 100 ℃, after the solution changes from black blue to mauve for about 10-15min, the color of the solution is not changed, heating is stopped, and the solution is cooled to room temperature under continuous stirring to obtain a colloidal gold solution.
(2) 22.5mL of colloidal gold is added with 2.5mL of NBA solution with the concentration of 50 mu M, after shaking and mixing at room temperature, 0.75mL of SH-PEG5000-COOH solution with the concentration of 10 mu mol/L is added, shaking (150 r/min) at room temperature for 20min, and then 5mL of SH-PEG5000-SH solution with the concentration of 50 mu mol/L is added, and shaking at room temperature for 3h. Excess PEG was removed by repeated centrifugation (10000 r/min,15 min) 3 times and the precipitate was dissolved in 1mL of PB solution to give Au@NBA@PEG nanosolution. To the resulting Au@NBA@PEG nanosolvent was added simultaneously 6. Mu.L of 40mg/mL EDC and 6. Mu.L of 110mg/mL NHS to activate the carboxyl groups to couple carboxylic acid functionalities on the nanoparticle surface to the antibody. After 20 minutes of vigorous reaction at room temperature, excess EDC and NHS were removed by centrifugation and the pellet was resuspended in 1mL PB and left to stand at 4 ℃.
(3) Immobilization of antibodies: adding the activated colloidal gold solution obtained in the step (2) into a centrifuge tube, wherein each tube contains 450 mu L of the activated colloidal gold solution, and 100 mu L of each of the OTa-Ab solution with the concentration of 80 mu g/mL, the AFB1-Ab solution with the concentration of 40 mu g/mL and the DON-Ab solution with the concentration of 60 mu g/mL and the colloidal gold solution with the concentration of 60 mu g/mL are simultaneously added: antibody (v/v) =1.5:1, shake-reacted overnight in a shaker at 4℃at 150r/min. After the reaction was completed, the excess antibody was removed by washing with centrifugation 3-4 times, and the pellet was resuspended in 450. Mu.L PB solution.
(4) And (3) closing colloidal gold: adding 300 mu L of 1% BSA solution into the colloidal gold solution with the antibody fixed in the step (3), reacting for 1h at 37 ℃, centrifugally washing for 3-4 times after the reaction is finished, and re-suspending the precipitate in 450 mu LPB solution to obtain SERS label solution.
Example 3
Establishment of OTA standard curve
(1) Preparing a series of concentration gradients of OTA solution: 9 concentration gradients of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000ng/mL of OTA solution with each concentration gradient of 50 mu L are configured.
(2) Competing reaction: at the position of OTA-BSA on 9 nano silver-porous silicon (prepared in example 1), 0.3. Mu.L of 9 concentration gradient OTA solution and 0.3. Mu.L of SERS label solution prepared in example 2 were sequentially dropped, reacted at 37℃for 1 hour, after the reaction was completed, 0.3. Mu.L of LPB solution was dropped at the reaction position for washing, and the operation was repeated 3-4 times.
(3) Determination of surface-enhanced raman spectroscopy: measuring the Raman spectrum intensity of the NBA characteristic peak; and thus the concentration of the optimal antigen. The laser confocal Raman microscope is used for detecting the intensity of the NBA Raman characteristic peak of each point, and the specific conditions are as follows: 785nm,550mA, integration time 3S, peak time 0.3S.
As can be seen from fig. 5, as the concentration of OTA increases, the raman signal gradually decreases, and a linear relationship is formed, and an OTA detection curve is sequentially established; as can be seen from FIG. 6, the linear detection range of OTA is 0.01-100 ng/mL, and the minimum detection limit is 2.87pg/mL.
Example 4
AFB 1 Establishment of a Standard Curve
Specific implementation stepsAs in example 3, it is not possible to switch the OTA in step (1) to AFB 1 The OTA-BSA in step (2) is changed into AFB 1 -BSA。
As can be seen from FIG. 5, with AFB 1 The concentration is increased, the Raman signal is gradually reduced, the linear relation is formed, and an OTA detection curve is sequentially established; as can be seen from FIG. 7, the linear detection range of OTA is 0.01-100 ng/mL, and the lowest detection limit is 0.39pg/mL.
Example 5
Establishment of DON standard curve
The procedure is as in example 3, except that the OTA in step (1) is replaced with DON and the OTA-BSA in step (2) is replaced with DON-BSA.
As can be seen from fig. 5, as the DON concentration increases, the raman signal gradually decreases, and the linear relationship is established, and an OTA detection curve is sequentially established; as can be seen from FIG. 8, the linear detection range of DON is 0.001-10 ng/mL, and the minimum detection limit is 1.86pg/mL.
Example 6
OTA specificity detection
The procedure is as in example 3, except that in step (1) a series of concentration gradients of the OTA solution are provided: three mixed mycotoxin solutions were prepared separately: OTA-AFB 1 -DON、OTB-AFB 1 -DON、AFB 1 DON, 10ng/mL of each toxin, the three mixed toxins were used instead of the series of concentration gradient OTAs, the remaining steps being identical.
As can be seen from FIG. 9, the Raman intensity of the NBA characteristic peak was significantly enhanced when the mixed mycotoxin was not containing OTA, compared with that containing OTA, indicating AFB 1 DON does not react with OTA-Ab in the system, and the specificity is good; when OTA is changed into a structural analogue OTB, the Raman intensity of the NBA characteristic peak has no obvious change, which indicates that the detection specificity of OTA is good.
Example 7
AFB1 specific assay
As in example 4, except that step (1) was conducted with a series of concentration gradients of AFB 1 Solution: four mixed mycotoxin solutions were prepared separately: AFB (alpha-fetoprotein) 1 -OTA-DON、AFG 1 -OTA-DON、AFG 2 OTA-DON, 10ng/mL of each toxin, replacing the serial concentration gradient AFB with these four mixed toxins 1 The remaining steps are the same.
As can be seen from FIG. 10, the mixed mycotoxin contains AFB 1 In contrast to the above, when the mixed mycotoxin does not contain AFB 1 When the system is used, the Raman intensity of the NBA characteristic peak is obviously enhanced, which indicates that OTA and DON are not compared with AFB in the system 1 Ab reacts with good specificity; AFB is carried out 1 AFG substituted by structural analogue 1 And AFG 2 When the Raman intensity of NBA characteristic peak is not changed obviously, the characteristic peak shows that for AFB 1 Has good detection specificity.
Example 8
DON-specific detection
The difference from example 5 is that step (1) is to configure a series of concentration gradients of DON solution: three mixed mycotoxin solutions were prepared separately: DON-AFB 1 -OTA、ZEN-AFB 1 -OTA、AFB 1 OTA, 10ng/mL of each toxin, replaces a series of concentration gradients DON with the three mixed toxins, the remaining steps being identical.
As can be seen from FIG. 11, the Raman intensity of the NBA characteristic peak was significantly enhanced when the mixed mycotoxin was free of DON, compared with that of DON contained in the mixed mycotoxin, indicating AFB 1 And OTA does not react with DON-Ab in the system, and the specificity is good; when DON is changed into structural analogue ZEN, the Raman intensity of the NBA characteristic peak has no obvious change, which shows that the detection specificity of OTA is good.
Example 9
Detection of labeled recovery rate of three mycotoxins in traditional Chinese medicine
(1) Sample treatment: pulverizing fructus Hordei Germinatus, radix Puerariae and Poria with Chinese medicinal pulverizer, sieving with 20 mesh sieve, weighing 15g of each sample, dividing into three parts, placing 5g of each part into 100mL triangular flask, diluting with 100% methanol solution to OTA-AFB of 0, 0.1, 1, 10ng/mL 1 Mixing DON mixed toxin solution (three toxin mixed solutions with four concentrations, each toxin being the above concentration), taking 2500 μL of each mixed toxin solution, adding into 5g of three Chinese medicinal samples, mixing thoroughly, placing into ventilation place until solvent evaporation is complete. 15mL of an extractant (methanol: water=7:3) was added thereto, extraction was performed in a shaker for 2 hours at 150r/min, filtration was performed with Whatman filter paper, and filtration was performed again with a 0.45 μm filter membrane, and the filtrate was the sample to be tested.
(2) Competitive immunoassay: combining the extracted traditional Chinese medicine sample to be tested with the SERS label in the embodiment 2 in equal proportion, reacting for 20min at 37 ℃, dripping the mixture on the SERS substrate prepared in the corresponding embodiment 1 (for example, dripping 0.3 mu L of the mixture on the position of OTA-BSA on the SERS substrate when the recovery rate of OTA is to be measured), reacting for 1h at 37 ℃, detecting the NBA Raman characteristic peak intensity of each point by using a laser confocal Raman microscope, and carrying out 550mA, integration time 3S and peak emergence time 0.3S under the condition of 785 nm. And calculating the recovery rate according to the NBA Raman characteristic peak intensity value, wherein the recovery rate is calculated according to the following formula:
as can be seen from FIG. 12, the OTA and AFB were measured by the present method 1 The recovery rate of DON in the poria cocos, the radix puerariae and the malt is 76.59+/-7.24% -91.02 +/-5.38%; the nano silver-porous silicon SERS detection system prepared by the invention can effectively detect three mycotoxins in grains, and has low detection limit and good detection effect.
Example 10
ELISA method for detecting labeled recovery rate of three mycotoxins in traditional Chinese medicine
(1) Sample treatment: weighing 20g of crushed and sieved malt, radix puerariae and poria cocos samples, and respectively adding 1ng/mL, 5ng/mL and OTA-AFB of 10ng/mL into a 1mLELISA kit 1 DON mixed toxin solution (namely, three ELISA kits (OTA, DON, AFB 1) were taken for each concentration, the toxin solutions were mixed), placed in a 250mL plug conical flask, 30mL petroleum ether and 100mL methanol water (7:3) solution were added, shaking was performed for 30min, filtration and separation funnel were performed with quick qualitative filter paper, and after the lower methanol water solution was separated, the methanol water solution was discharged into another conical flask. Placing 20mL of methanol aqueous solution (corresponding to 4g sample) in another 125mL separating funnel, adding 20mL of chloroform, shaking for 2min, standing for delamination, and standing for appearanceThe emulsification phenomenon can be realized by dripping methanol to promote layering. Discharging chloroform layer, filtering with quantitative slow filter paper containing about 10g anhydrous sodium sulfate pre-moistened with chloroform in 50mL evaporation dish, adding 5mL chloroform in separating funnel, repeatedly shaking and extracting, filtering to dry evaporation dish, washing filter with small amount of chloroform, washing liquid and evaporation dish. The evaporating dish was ventilated and volatilized in a 65 ℃ water bath under a fume hood. After cooling to room temperature, the sample solution is fully dissolved by 40mL of methanol water (35:65) to obtain the sample solution to be tested.
(2) OTA, DON and AFB are applied before use 1 Taking out the ELISA quantitative detection kit from the refrigerator, balancing for 15-20min in room temperature environment, and carefully reading the specification to operate according to the specification. Detecting absorbance at 492nm with multifunctional enzyme-labeled instrument, and calculating OTA and AFB 1 And the labeling recovery rate of DON in the Chinese medicine poria cocos, radix puerariae and malt.
As shown in FIG. 13, the recovery rate of Poria cocos, radix puerariae and malt by ELISA method is 70.78 + -6.13% -89.05+ -3.21%, which is lower than the method of the present invention, and only one mycotoxin can be detected by one ELISA kit; compared with the method, the SERS sensor based on the nano silver-porous silicon, which is prepared by the method, can effectively detect OTA, DON, AFB in grains 1 The invention further proves that the invention meets the market demand of rapidly, efficiently and accurately detecting a plurality of mycotoxins, and has wide market prospect.

Claims (10)

1. The sensor is characterized by comprising a SERS substrate and a SERS label, wherein the SERS substrate consists of silver nanoparticles and three mycotoxins connected with the surfaces of the silver nanoparticles by magnetron sputtering on the surfaces of the porous silicon, and 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 A artificial antigen and yellowAflatoxin B 1 The corresponding mycotoxin antibodies are ochratoxin A antibody, vomitoxin antibody and aflatoxin B 1 An antibody.
2. The SERS sensor based on nano silver-porous silicon according to claim 1, wherein the SERS label is formed by physically adsorbing NBA on the upper surface of gold nanoparticles, respectively modifying methoxy polyethylene glycol mercapto and mercapto polyethylene glycol carboxyl, and finally connecting mycotoxin antibody through covalent bond under the activation of EDC and NHS; the SERS substrate is prepared by preparing porous silicon through electrochemical etching after monocrystalline silicon is cleaned, and sputtering nano silver particles on the surface through magnetron sputtering, wherein the surface of the nano silver particles is covalently bonded with mycotoxin artificial antigens.
3. A method for preparing a sensor for simultaneous SERS detection of multiple mycotoxins based on nano silver-porous silicon according to claim 1, comprising the steps of:
(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 prepared in the step (2) after vacuumizing the porous silicon; cooling to room temperature to obtain porous Si-Ag inert gas and maintaining;
(4) The porous silicon silver prepared in the step (3) and the surface of silver nano particles are physically adsorbed with three mycotoxin artificial antigens (OTA-BSA, DON-BSA and AFB 1-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.
4. The process according to claim 3, wherein the resistivity of the P-type monocrystalline silicon piece in step (1) is preferably 0.0008 to 0.00012 Ω.
5. The preparation method of claim 3, wherein in the step (2), the processed silicon wafer is assembled into an electrochemical etcher by tabletting, electrolyte is dripped into the tablet, the porous silicon is prepared by electrochemical anodic oxidation, and the tablet is dried for standby after being cleaned; the electrolyte is prepared from hydrofluoric acid and absolute ethyl alcohol, porous silicon is prepared by electrochemical etching, the electrochemical etching current is 300-500mA, and the etching time is 10-30S.
6. The method of preparing a porous silicon according to claim 3, wherein the porous silicon prepared in step (3) is fixed in a reactive magnetron sputtering apparatus, a silver target is charged, and vacuum is applied to 5X 10 -4 Sputtering silver nano particles on the surface of porous silicon after Pa; cooling to room temperature and storing in nitrogen.
7. The preparation method according to claim 3, wherein the magnetron sputtering conditions are voltage 45-55W, sputtering time 22-26min, annealing 15-20min, and silver layer thickness is controlled by controlling sputtering conditions.
8. The preparation method of claim 4, wherein in the step (4), artificial antigens are dripped on a substrate, and three artificial antigens, namely OTA-BSA, DON-BSA and AFB1-BSA, are dripped on the substrate; adding a Nile blue solution, an SH-PEG-SH solution, an SH-PEG-COOH solution, an EDC solution and an NHS solution into the colloidal gold solution in the step (6); three antibodies OTa-Ab, DON-Ab, AFB1-Ab were added.
9. A nano-silver-porous silicon based simultaneous SERS detection of multiple mycotoxins as described in claim 1 for three mycotoxins (OTA, AFB 1 DON) in simultaneous quantitative detection analysis.
10. The use according to claim 9, wherein the quantitative detection analysis is performed by: buffer solutions of different mycotoxin (OTA, DON, AFB 1) samples with different concentrations are added into the SERS label solution, so that three mycotoxins and three mycotoxin artificial antigens on a SERS substrate compete for binding to three corresponding mycotoxin antibodies on the SERS label, and quantitative analysis of the three mycotoxins is realized simultaneously by detecting the NBA characteristic peak signal intensity bound on the SERS label.
CN202310524882.0A 2023-05-10 2023-05-10 Biochip sensor for simultaneously detecting multiple mycotoxins based on nano silver-porous silicon SERS and preparation method and application thereof Pending CN116660513A (en)

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