Multiple biological toxin detection chips and their preparation and detection methods

Abstract

Provided is a multi-biotoxin detection chip, comprising a PDMS chip layer and a microelectrode layer bonded to each other, wherein the PDMS chip layer is provided with an injection hole and at least two sample outlet holes, and its bonding surface is provided with pits and connection grooves between the injection hole and each sample outlet hole, which are connected in sequence; after bonding with the microelectrode layer, each connection groove forms a flow channel, and each pit forms a detection cavity; on the bonding surface of the microelectrode layer, each thin layer detection electrode unit respectively arranged in the bonding corresponding area of ​​each detection cavity comprises a working electrode and a counter electrode; wherein the working electrode is a BDD thin film electrode generated by boron-doped diamond, and each working electrode is respectively modified with a nucleic acid aptamer corresponding to the target biotoxin to be detected by the working electrode; the counter electrode is a platinum thin layer electrode formed by platinum 1000 through a sputtering process; the invention also provides a manufacturing method and a detection method of such a chip; the invention can realize high-sensitivity rapid detection of multiple indicators of biotoxins.

Description

Technical field:

[0001] The present invention relates to the technical field of biosensors and biotoxin detection, and in particular to a chip for detecting multiple biotoxins and a manufacturing and detection method thereof. Background technology:

[0002] Biotoxins are a class of toxic substances derived from animals, plants or microorganisms, and are mainly divided into animal toxins, plant toxins, microbial toxins and marine biotoxins. Among them, microbial toxins are generally secondary metabolites produced by microorganisms during their growth, such as aflatoxin M1 (AFM1), aflatoxin B1 (AFB1), vomitoxin (DON) and zearalenone toxin (ZEN). Biotoxins can participate in the metabolic process by specifically selectively acting on cell membranes, ion channels, receptors, ribosomal proteins, etc., and cause lethal and toxic effects on other organisms.

[0003] Biological toxins are closely related to people's health and life. Biological toxin contamination may occur in every link of food production, transportation, consumption and eating. People will be poisoned after eating food contaminated by biological toxins, and in more serious cases, their lives will be endangered. As one of the important factors affecting food safety, highly sensitive and specific detection of biological toxins is of utmost importance to ensure human health and food safety. At present, traditional methods for detecting biological toxins include immunological methods, real-time mass spectrometry, and gas-liquid chromatography. However, all of the above methods have the disadvantages of long detection process, large instruments and professional operators, making it difficult to conduct rapid on-site detection of food in the production process.

[0004] Biosensors are very suitable for real-time monitoring and on-site detection of biological toxins due to their high sensitivity and rapid detection. At present, most biosensors for biological toxin detection use aptamers as recognition elements to specifically detect different biological toxins. For example, patent document CN110632156A has developed an aptamer sensor for detecting aflatoxin B1. -13 ~1×10 -8 mol / L detection range has good linear relationship, and the detection limit can reach 5.5×10 -14mol / L, with extremely high detection sensitivity. However, this detection method can only detect a single biological toxin, with low detection efficiency, and cannot meet the needs of simultaneous detection of multiple components on site. Patent document CN114609209A discloses a dual-target aptamer sensor for two biological toxins, aflatoxin B1 (AFB1) and zearalenone toxin (ZEN). Differential pulse voltammetry (DPV) can be used to generate non-interfering current peaks at different potentials to achieve simultaneous detection of AFB1 and ZEN. Although this method realizes multi-component detection, the working electrode and counter electrode of the sensor are still discrete, and an integrated sensor is not formed, so it does not meet the needs of easy operation in on-site rapid detection scenarios. Summary of the invention:

[0005] To overcome the above problems, the present invention combines microfluidic chips with electrochemical detection technology to provide a microchip for the joint detection of multiple biological toxins based on boron-doped diamond film, providing a convenient and fast detection method for the field of food safety monitoring.

[0006] The technical problem to be solved by the present invention is to provide a multiple biological toxin detection chip, which can integrate the detection functions of multiple biological toxins, such as the detection functions of four biological toxins that normally need to be detected in the field of food safety monitoring technology: aflatoxin M1 (AFM1), aflatoxin B1 (AFB1), vomitoxin (DON) and zearalenone toxin (ZEN), so as to solve the problems of complex structure and cumbersome steps of existing biological toxin detection devices, and especially provide a variety of biological toxin detection accurate, fast and convenient detection means for the field of food safety monitoring. The present invention also provides a method for manufacturing such a multiple biological toxin detection chip, and a detection method using such a multiple biological toxin detection chip.

[0007] In order to solve the above technical problems, the technical solutions adopted by the various biotoxin detection chips of the present invention are:

[0008] A chip for detecting multiple biological toxins comprises a PDMS chip layer and a microelectrode layer which are bonded to each other, characterized in that the PDMS chip layer is provided with an injection hole and at least two sample outlet holes at intervals, and pits are respectively provided between the injection hole and each sample outlet hole on the bonding surface of the PDMS chip, and connecting grooves are respectively provided between the injection hole and each pit, and between each pit and its corresponding sample outlet hole, and are connected in sequence; after the PDMS chip is bonded to the microelectrode layer, each connecting groove forms a flow channel on the microelectrode layer, and each pit forms a detection cavity; on the bonding surface of the microelectrode layer, corresponding thin layer detection electrode units are respectively arranged in the bonding corresponding areas of each detection cavity, and each of the thin layer detection electrode units comprises a working electrode and a counter electrode; wherein the working electrode is a BDD thin film electrode generated by boron-doped diamond, and each of the working electrodes is respectively modified with a nucleic acid aptamer corresponding to the target biological toxin to be detected by the working electrode; and the counter electrode is a platinum thin layer electrode formed by a sputtering process of platinum 1000.

[0009] The following is a further solution of the various biotoxin detection chips of the present invention:

[0010] The PDMS chip layer is provided with an injection hole located at the center of the PDMS chip layer, two pits on both sides of the injection hole and two corresponding outlet holes, respectively forming two detection cavities; on the bonding surface of the microelectrode layer, in the corresponding areas of each detection cavity bonding, two corresponding thin layer detection electrode units are respectively arranged, the target biological toxins are two of the four biological toxins of aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin, and each working electrode is modified with nucleic acid aptamers corresponding to two of the above four biological toxins.

[0011] The PDMS chip layer is provided with an injection hole located at the center of the PDMS chip layer and three pits and corresponding three outlet holes are separated around the injection hole, respectively forming three detection cavities; on the bonding surface of the microelectrode layer, in the corresponding areas of each detection cavity bonding, three corresponding thin layer detection electrode units are respectively arranged, the target biological toxins are three of the four biological toxins of aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin, and each working electrode is modified with nucleic acid aptamers corresponding to three of the above four biological toxins.

[0012] The PDMS chip layer is provided with an injection hole located at the center of the PDMS chip layer, and four pits and four outlet holes are separated around the injection hole to form four detection cavities respectively; on the bonding surface of the microelectrode layer, in the corresponding areas of each detection cavity bonding, four corresponding thin layer detection electrode units are respectively arranged, and the target biological toxins are four biological toxins: aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin, and each working electrode is modified with nucleic acid aptamers corresponding to the above four biological toxins.

[0013] Each of the pits is provided with a plurality of flow dividing connecting grooves which are connected to the injection hole after merging.

[0014] On the bonding surface of the microelectrode layer (2), the end of the working electrode (7) is connected to its wiring welding block (11), and the working electrode (7) includes a square main block (9) and a long straight bar (10) extending outward to its wiring welding block (11); the end of the counter electrode (8) is also connected to its wiring welding block (11), and the counter electrode (8) is in a shape surrounding the outside of the square main block (9) of the working electrode (7) and extending outward to its wiring welding block (11); the wiring welding blocks (11) at the ends of the working electrode (7) and the counter electrode (8) are both arranged at the proximal side of the bonding surface of the microelectrode layer (2).

[0015] In order to solve the above technical problems, the technical solution adopted by the method for manufacturing a plurality of biological toxin detection chips of the present invention is:

[0016] The preparation method of the above-mentioned multiple biological toxin detection chips includes the preparation of a PDMS chip layer and a microelectrode layer which can be carried out separately in no particular order, and bonding the prepared PDMS chip layer and the microelectrode layer; the preparation of the PDMS chip layer includes: first preparing a silicon substrate mold as its molding mold, and then preparing the PDMS chip layer; it is characterized in that: before the prepared PDMS chip layer and the microelectrode layer are bonded, the microelectrode layer also needs to be modified with a nucleic acid aptamer for its working electrode to construct a specific detection unit.

[0017] The following is a further scheme of the detection method of the various biological toxin detection chips of the present invention:

[0018] The process of modifying the working electrode of the microelectrode layer with the nucleic acid aptamer is as follows: 10 μL of gold deposition solution is dripped on the surface of the working electrode and incubated at room temperature for 4 hours, after which a layer of nano-gold particles will be deposited on the surface of the working electrode, wherein the gold deposition solution comprises: 0.5% w / v chloroauric acid HAuCl4, 50 mg / mL potassium bicarbonate KHCO3, and 5 mg / mL glucose Glucose; then 5 μM AFM1, AFB1, DON, and ZEN aptamers are dripped on the working electrode in each electrode unit respectively, incubated at room temperature for 4 hours, rinsed with Tris-HCl three times, 20 μL of 1 μM MCH is dripped on the working electrode area, incubated in a constant temperature shaker at 25°C for 1 hour, and then rinsed with Tris-HCl to complete the modification.

[0019] The preparation of the silicon substrate mold uses a single-sided polished four-inch single crystal silicon wafer as the mold substrate, and specifically includes the following steps:

[0020] Step 1. Cleaning: Use a mixture of H2SO4 and H2O2 to clean the silicon wafer, with the volume ratio of the two being: v:v=7:1.

[0021] Step 2. Glue coating: Glue coating the substrate with negative resist SU8-2075, 500r*30s, 3000r*60s, 50μm thick.

[0022] Step 3: Pre-baking, hot plate temperature 95°C, time 45 min.

[0023] Step 4: Photolithography, exposure time is 28s.

[0024] Step 5: Post-baking, oven temperature 95°C, time 7 min.

[0025] Step 6: Development, development time 5 minutes, spin drying at 2100r*60s.

[0026] Step 7. Hard bake, 150℃, 15min.

[0027] Finally, a silicon substrate mold with a microstructure height of 50μm is obtained.

[0028] The preparation process of the PDMS chip layer is as follows:

[0029] Step 1. Place the prepared silicon substrate mold in a fluorosilane steam box and incubate for 4 hours to facilitate the subsequent peeling of the PDMS chip;

[0030] Step 2. Weigh the PDMS prepolymer and curing agent in a weight ratio of 10:1, mix them evenly, and place them in a vacuum desiccator at a vacuum degree of 13 psi for 30 minutes to remove most of the bubbles.

[0031] Step 3. Place the silicon substrate mold on a horizontal platform, pour the PDMS mixture, and let it stand for 30 minutes to allow the microstructure to be filled with PDMS.

[0032] Step 4. Heat in an oven at 80°C for 1 hour. After the PDMS is completely cured, carefully peel the PDMS off the silicon wafer.

[0033] Step 5. Use a syringe with a needle of a specific aperture to punch holes according to the positions of the inlet and outlet on the PDMS chip.

[0034] Step 6. Cut the chip along the cutting lines to obtain a single PDMS chip, and store it by applying white film on the upper and lower surfaces.

[0035] The preparation process of the microelectrode layer is as follows:

[0036] Step 1. Select a single-side polished four-inch silicon wafer as a substrate, and use thermal oxidation to prepare a 2 μm thick silicon dioxide (SiO2) layer as an insulating isolation layer.

[0037] Step 2. Choose to grow a 3-5 μm diamond film using hot wire chemical deposition technology.

[0038] Step 3. Use metal sputtering technology to prepare an Al mask layer with a thickness of 450 nm, and then spin-coat 2.4 μm thick LC100A4 photoresist to pattern the Al layer.

[0039] Step 4: Remove the Al layer by wet etching, and then use oxygen plasma to dry etch the BDD area to obtain the BDD working electrode structure.

[0040] Step 5. Use a metal sputtering process to prepare a 0.4 μm thick Pt1000 layer as a counter electrode structure; then use acetone to remove the photoresist to complete the preparation of the Pt electrode area.

[0041] Step 6. Slice and cut along the cutting lines to obtain individual electrode chips; use solder paste to solder the connecting wires to the solder joints of each electrode unit, either before or after cutting.

[0042] The bonding process of the prepared PDMS chip layer and the microelectrode layer is as follows:

[0043] The white film on the upper surface of the PDMS chip was torn off, and the chip and the microelectrode layer were placed in a plasma cleaner for cleaning for 60 seconds. Then, an alignment keyer was used to key the two parts together according to the alignment marks, thereby completing the preparation of the entire chip.

[0044] In order to solve the above technical problems, the technical solution adopted by the detection method of the multiple biological toxin detection chip of the present invention is:

[0045] A method for detecting multiple biological toxins, using the multiple biological toxin detection chip described above, firstly performing a simulation test process on the multiple biological toxin detection chip described above as follows, testing and calibrating the multiple biological toxin detection chip, adding a series of standard sample solutions of different concentrations containing at least one of four biological toxins, aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin and having known concentration values, measuring a series of initial impedance values ​​Rct0 and post-reaction impedance values ​​Rct of each working electrode unit with different values, performing a series of concentration gradient experiments, obtaining a series of data of relative impedance values ​​y of each biological toxin concentration value and the corresponding working electrode unit, substituting into the linear relationship between a certain biological toxin concentration and the relative impedance value y value: y=A+Blgx, wherein the relative impedance value y=(Rct-Rct0) / Rct0, A and B are constants, x represents a certain biological toxin concentration, and lgx represents the logarithm of x with base 10; after a series of calculations, the values ​​of constants A and B in the linear relationship between various biological toxin concentrations and their relative impedance values ​​y values ​​are obtained.

[0046] The specific test process is as follows: (1) First, add 10mM Fe(CN)6 3- / 4- (1) 0.1M KCl solution was added to fill each detection cavity, and then the initial impedance value Rct0 of each working electrode unit was measured; (2) Tris-HCl solution was injected to rinse the chip, and then the sample solution to be tested was added to fill each detection cavity, and incubated at room temperature for 40 minutes; (3) deionized water was injected to rinse the chip, and then 10mM Fe(CN)6 3- / 4- Fill each detection cavity with 0.1 M KCl solution and detect the post-reaction impedance value Rct of each electrode unit; (4) calculate the relative impedance value y = (Rct-Rct0) / Rct0 of each detection unit and substitute it into the linear relationship between the corresponding biotoxin concentration and the relative impedance value y: y = A + Blgx to obtain the biotoxin concentration x in the sample.

[0047] The present invention is a multi-biotoxin detection chip based on boron-doped diamond electrodes, which efficiently integrates a biotoxin detection electrode array with a microfluidic chip, thereby achieving multi-index combination and high-sensitivity rapid detection of biotoxins.

[0048] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0049] 1. The present invention has developed a microchip based on boron-doped diamond film, which integrates multiple detection cavities and electrode systems, and realizes the function of simultaneously detecting four biological toxins in one chip, greatly improving the detection efficiency of biological toxins. 2. The present invention utilizes semiconductor technology to prepare microchips, integrates the working electrode and the counter electrode into one, can achieve batch manufacturing, and uses chemical deposition methods to deposit nanogold particles on the surface of the BDD electrode to increase the fixed amount of the aptamer, which can greatly improve the detection sensitivity of biological toxins. 3. The various biological toxin detection chips of the present invention have a wide range of applications, and other types of recognition elements can be modified on the surface of the working electrode for other types of biological toxin detection. 4. The present invention has accurate, fast and convenient detection, and provides an accurate, fast and convenient detection method for a variety of biological toxins in the field of food safety monitoring. Description of the drawings:

[0050] Figure 1 It is a three-dimensional schematic diagram of the assembly state of the various biotoxin detection chips of the present invention.

[0051] Figure 2 This is a three-dimensional schematic diagram of the separation state of the PDMS chip layer and the microelectrode layer of the multiple biological toxin detection chip of the present invention.

[0052] Figure 3 Schematic diagram of the microelectrode layer.

[0053] Figure 4 It is a three-dimensional schematic diagram of the bonding surface of the PDMS chip layer.

[0054] Figure numerals: 1. PDMS chip layer, 2. microelectrode layer, 3. injection hole, 4. output hole, 5. pit, 6. connection groove, 7. working electrode, 8. counter electrode, 9. square main block, 10. long straight bar, 11. wiring welding block. Specific implementation method:

[0055] The specific implementation of the present invention is further described in detail below with reference to the accompanying drawings.

[0056] The present invention has a variety of biotoxin detection chips, such as Figure 1 , Figure 2 As shown, it includes a PDMS chip layer 1 and a microelectrode layer 2 that are bonded to each other, and the PDMS chip layer 1 is provided with a sample inlet 3 and at least two sample outlets 4 at intervals. Figure 2 , Figure 4 As shown, the bonding surface of the PDMS chip has pits 5 between the sample inlet 3 and each sample outlet 4, and connecting grooves 6 are respectively formed between the sample inlet 3 and each pit 5, and between each pit 5 and its corresponding sample outlet 4, and are connected in sequence. After the PDMS chip is bonded to the microelectrode layer 2, as shown in FIG. Figure 2 As shown, on the microelectrode layer 2, each connection groove 6 forms a flow channel, and each pit 5 forms a detection cavity. Figure 2 , Figure 3 As shown, on the bonding surface of the microelectrode layer 2, corresponding thin layer detection electrode units are respectively arranged in the corresponding areas of each detection cavity, and each thin layer detection electrode unit includes a working electrode 7 and a counter electrode 8. The working electrode 7 is a BDD thin film electrode generated by boron-doped diamond, and each working electrode 7 is respectively modified with a nucleic acid aptamer corresponding to the target biological toxin to be detected by the working electrode 7; the counter electrode 8 is a platinum thin layer electrode formed by platinum 1000 through a sputtering process.

[0057] like Figure 2 , Figure 4 As shown, each pit 5 is provided with a plurality of flow dividing connecting grooves 6 which are connected to the injection hole 3 after merging. The following three arrangements of the injection hole 3, the sample outlet hole 4 and the pit 5 on the PDMS chip layer 1 are recommended.

[0058] Arrangement method 1: The PDMS chip layer 1 is provided with an injection hole 3 located at the center of the PDMS chip layer 1, two pits 5 and two corresponding sample outlet holes 4 on both sides of the injection hole 3, respectively forming two detection cavities; on the bonding surface of the microelectrode layer 2, in the corresponding areas of each detection cavity bonding, two corresponding thin layer detection electrode units are respectively arranged, and the target biological toxins are two of the four biological toxins of aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin, and each working electrode 7 is modified with nucleic acid aptamers corresponding to two of the above four biological toxins.

[0059] Arrangement method 2: The PDMS chip layer 1 is provided with an injection hole 3 located at the center of the PDMS chip layer 1, and three pits 5 and corresponding three outlet holes 4 are separated around the injection hole 3, so as to form three detection cavities respectively; on the bonding surface of the microelectrode layer 2, in the corresponding areas of each detection cavity, three corresponding thin layer detection electrode units are respectively arranged, and the target biological toxins are three of the four biological toxins of aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin, and each working electrode 7 is modified with nucleic acid aptamers corresponding to three of the above four biological toxins.

[0060] Arrangement method three: the PDMS chip layer 1 is provided with an injection hole 3 located at the center of the PDMS chip layer 1 and four pits 5 and four outlet holes 4 separated around the injection hole 3, respectively forming four detection cavities; on the bonding surface of the microelectrode layer 2, in the corresponding areas of each detection cavity, four corresponding thin layer detection electrode units are respectively arranged, and the target biological toxins are aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin. Each working electrode 7 is modified with nucleic acid aptamers corresponding to the above four biological toxins.

[0061] like Figure 2, Figure 3 As shown, on the bonding surface of the microelectrode layer 2, the end of the working electrode 7 is connected to its wiring welding block 11, and the working electrode 7 includes a square main block 9 and a long straight bar 10 extending outward to its wiring welding block 11. The end of the counter electrode 8 is also connected to its wiring welding block 11, and the counter electrode 8 is in a form surrounding the outside of the square main block 9 of the working electrode 7 and extending outward to its wiring welding block 11; the wiring welding blocks 11 at the ends of the working electrode 7 and the counter electrode 8 are both arranged at the proximal side of the bonding surface of the microelectrode layer 2.

[0062] The preparation method of the multi-biotoxin detection chip of the present invention comprises the preparation of a PDMS chip layer 1 and a microelectrode layer 2 which can be carried out in any order, and bonding the prepared PDMS chip layer 1 and the microelectrode layer 2; the preparation of the PDMS chip layer 1 comprises: firstly preparing a silicon substrate mold as its molding mold, and then preparing the PDMS chip layer 1; it is characterized in that: before bonding the prepared PDMS chip layer 1 and the microelectrode layer 2, the microelectrode layer 2 also needs to be modified with a nucleic acid aptamer for its working electrode 7 to construct a specific detection unit.

[0063] The process of nucleic acid aptamer modification of the working electrode 7 of the microelectrode layer 2 is as follows: 10 μL of gold deposition solution is added to the surface of the working electrode 7 and incubated at room temperature for 4 hours. After that, a layer of nano-gold particles will be deposited on the surface of the working electrode 7. The gold deposition solution consists of: 0.5% w / v chloroauric acid HAuCl4, 50 mg / mL potassium bicarbonate KHCO3, and 5 mg / mL glucose Glucose; then 5 μM AFM1, AFB1, DON and ZEN aptamers are added to the working electrode 7 in each electrode unit respectively, incubated at room temperature for 4 hours, rinsed with Tris-HCl three times, and 20 μL of 1 μM MCH was added to the working electrode 7 area. It was incubated in a constant temperature shaker at 25°C for 1 hour, and then rinsed with Tris-HCl to complete the modification.

[0064] The preparation of the silicon substrate mold uses a single-sided polished four-inch single crystal silicon wafer as the mold substrate, which specifically includes the following steps:

[0065] Step 1. Cleaning: Use a mixture of H2SO4 and H2O2 to clean the silicon wafer, with the volume ratio of the two being: v:v=7:1.

[0066] Step 2. Glue coating: Glue coating the substrate with negative resist SU8-2075, 500r*30s, 3000r*60s, 50μm thick.

[0067] Step 3: Pre-baking, hot plate temperature 95°C, time 45 min.

[0068] Step 4: Photolithography, exposure time is 28s.

[0069] Step 5: Post-baking, oven temperature 95°C, time 7 min.

[0070] Step 6: Development, development time 5 minutes, spin drying at 2100r*60s.

[0071] Step 7. Hard bake, 150℃, 15min.

[0072] Finally, a silicon substrate mold with a microstructure height of 50μm is obtained.

[0073] The preparation process of PDMS chip layer 1 is as follows:

[0074] Step 1. Place the prepared silicon substrate mold in a fluorosilane steam box and incubate for 4 hours to facilitate the subsequent peeling of the PDMS chip.

[0075] Step 2. Weigh the PDMS prepolymer and curing agent in a weight ratio of 10:1, mix them evenly, and place them in a vacuum desiccator at a vacuum degree of 13 psi for 30 minutes to remove most of the bubbles.

[0076] Step 3. Place the silicon substrate mold on a horizontal platform, pour the PDMS mixture, and let it stand for 30 minutes to allow the microstructure to be filled with PDMS.

[0077] Step 4. Heat in an oven at 80°C for 1 hour. After the PDMS is completely cured, carefully peel the PDMS off the silicon wafer.

[0078] Step 5. Use a syringe with a needle of a specific aperture to punch holes according to the positions of the inlet and outlet on the PDMS chip.

[0079] Step 6. Cut the chip along the cutting lines to obtain a single PDMS chip, and store it by applying white film on the upper and lower surfaces.

[0080] The preparation process of the microelectrode layer 2 is as follows:

[0081] Step 1. Select a single-side polished four-inch silicon wafer as a substrate, and use thermal oxidation to prepare a 2 μm thick silicon dioxide (SiO2) layer as an insulating isolation layer.

[0082] Step 2. Choose to grow a 3-5 μm diamond film using hot wire chemical deposition technology.

[0083] Step 3. Use metal sputtering technology to prepare an Al mask layer with a thickness of 450 nm, and then spin-coat 2.4 μm thick LC100A4 photoresist to pattern the Al layer.

[0084] Step 4: Remove the Al layer by wet etching, and then use oxygen plasma to dry etch the BDD area to obtain the BDD working electrode 7 structure.

[0085] Step 5. Use a metal sputtering process to prepare a 0.4 μm thick Pt1000 layer as the counter electrode 8 structure; then use acetone to remove the photoresist to complete the preparation of the Pt electrode area.

[0086] Step 6. Slice and cut along the cutting lines to obtain individual electrode chips; use solder paste to solder the connecting wires to the solder joints of each electrode unit, either before or after cutting.

[0087] The bonding process of the prepared PDMS chip layer 1 and the microelectrode layer 2 is as follows:

[0088] The white film on the upper surface of the PDMS chip was torn off, and the chip was placed in a plasma cleaner together with the microelectrode layer 2 for cleaning for 60 seconds. Then, an alignment keyer was used to key the two parts together according to the alignment marks, thus completing the preparation of the entire chip.

[0089] The method for detecting multiple biological toxins of the present invention uses the multiple biological toxin detection chips mentioned above, first simulates the following specific test process for the prepared multiple biological toxin detection chips, tests and calibrates the multiple biological toxin detection chips, adds a series of standard sample solutions of different concentrations containing at least one of the four biological toxins, aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin, and the concentration values ​​of which are known, measures a series of initial impedance values ​​Rct0 and post-reaction impedance values ​​Rct of each working electrode 7 unit with different values, performs a series of concentration gradient experiments, obtains a series of data of the relative impedance values ​​y of each biological toxin concentration value and the corresponding working electrode 7 unit, substitutes into the linear relationship between the concentration of a certain biological toxin and the relative impedance value y value: y=A+Blgx, wherein the relative impedance value y=(Rct-Rct0) / Rct0, A and B are constants, x represents the concentration of a certain biological toxin, and lgx represents the logarithm of x with base 10; and obtains the values ​​of constants A and B in the linear relationship between the concentration of various biological toxins and their relative impedance values ​​y values ​​through a series of calculations.

[0090] The specific test process is as follows: (1) First, add 10mM Fe(CN)6 3- / 4- (1) 0.1M KCl solution was added to fill each detection cavity, and then the initial impedance value Rct0 of each working electrode 7 unit was measured; (2) Tris-HCl solution was injected to rinse the chip, and then the sample solution to be tested was added to fill each detection cavity, and incubated at room temperature for 40 minutes; (3) deionized water was injected to rinse the chip, and then 10mM Fe(CN)6 3- / 4-Fill each detection cavity with 0.1 M KCl solution and detect the post-reaction impedance value Rct of each electrode unit; (4) calculate the relative impedance value y = (Rct-Rct0) / Rct0 of each detection unit and substitute it into the linear relationship between the corresponding biotoxin concentration and the relative impedance value y: y = A + Blgx to obtain the biotoxin concentration x in the sample.

Claims

1. A chip for detecting multiple biological toxins, comprising a PDMS chip layer (1) and a microelectrode layer (2) bonded to each other, characterized in that: The PDMS chip layer (1) is provided with an injection hole (3) and at least two sample outlet holes (4) at intervals; a bonding surface of the PDMS chip is provided with pits (5) between the injection hole (3) and each sample outlet hole (4); a connection groove (6) is provided between the injection hole (3) and each pit (5), and between each pit (5) and its corresponding sample outlet hole (4), and they are connected in sequence; after the PDMS chip is bonded to the microelectrode layer (2), each connection groove (6) forms a flow channel on the microelectrode layer (2), and each pit (5) forms a detection cavity; on the bonding surface of the microelectrode layer (2), in the corresponding areas of each detection cavity bonding, corresponding thin layer detection electrode units are respectively arranged, and each of the thin layer detection electrode units includes a working electrode (7) and a counter electrode (8); wherein the working electrode (7) is a BDD thin film electrode generated by boron-doped diamond, and each of the working electrodes (7) is modified with a nucleic acid aptamer corresponding to the target biological toxin to be detected by the working electrode (7); and the counter electrode (8) is a platinum thin layer electrode formed by a sputtering process of platinum 1000.

2. The multiple biological toxin detection chip according to claim 1, characterized in that: The PDMS chip layer (1) is provided with an injection hole (3) located at the center of the PDMS chip layer (1), two recesses (5) and two corresponding sample outlet holes (4) surrounding the injection hole (3) on both sides, respectively forming two detection cavities; on the bonding surface of the microelectrode layer (2), two corresponding thin layer detection electrode units are respectively arranged in the bonding corresponding areas of each detection cavity; the target biological toxins are two of the four biological toxins of aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin; and each working electrode (7) is respectively modified with nucleic acid aptamers corresponding to two of the above four biological toxins.

3. The multiple biological toxin detection chip as claimed in claim 1, characterized in that: The PDMS chip layer (1) is provided with an injection hole (3) located at the center of the PDMS chip layer (1) and three recesses (5) and corresponding three output holes (4) are arranged around the injection hole (3), respectively forming three detection cavities; on the bonding surface of the microelectrode layer (2), corresponding three thin-layer detection electrode units are respectively arranged in the bonding corresponding areas of each detection cavity; the target biological toxins are three of the four biological toxins of aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin; and each of the working electrodes (7) is respectively modified with nucleic acid aptamers corresponding to three of the above four biological toxins.

4. The multiple biological toxin detection chip as claimed in claim 1, characterized in that: The PDMS chip layer (1) is provided with an injection hole (3) located at the center of the PDMS chip layer (1), and four recesses (5) and four sample outlet holes (4) are arranged around the injection hole (3), respectively forming four detection cavities; on the bonding surface of the microelectrode layer (2), in the corresponding areas of the bonding of each detection cavity, four corresponding thin-layer detection electrode units are respectively arranged; the target biological toxins are four biological toxins, namely, aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin; and the working electrodes (7) are respectively modified with nucleic acid aptamers corresponding to the above four biological toxins.

5. The multiple biological toxin detection chip as claimed in claim 1, characterized in that: Each of the recesses (5) is provided with a plurality of flow-dividing connection grooves (6) which are connected to the injection hole (3) after merging.

6. The multiple biological toxin detection chip according to claim 1, characterized in that: On the bonding surface of the microelectrode layer (2), the end of the working electrode (7) is connected to its wiring welding block (11), and the working electrode (7) includes a square main block (9) and a long straight bar (10) extending outward to its wiring welding block (11); the end of the counter electrode (8) is also connected to its wiring welding block (11), and the counter electrode (8) is in a shape surrounding the outside of the square main block (9) of the working electrode (7) and extending outward to its wiring welding block (11); the wiring welding blocks (11) at the ends of the working electrode (7) and the counter electrode (8) are both arranged at the proximal side of the bonding surface of the microelectrode layer (2).

7. The method for preparing a chip for detecting multiple biological toxins according to any one of claims 1 to 6, comprising preparing a PDMS chip layer (1) and a microelectrode layer (2) in no particular order, and bonding the prepared PDMS chip layer (1) and the microelectrode layer (2); the preparation of the PDMS chip layer (1) comprises: First, a silicon substrate mold is prepared as a molding mold, and then a PDMS chip layer (1) is prepared; the characteristic is that before the prepared PDMS chip layer (1) and the microelectrode layer (2) are bonded, the microelectrode layer (2) also needs to be modified with a nucleic acid aptamer for its working electrode (7) to construct a specific detection unit.

8. The method for preparing a chip for detecting multiple biological toxins according to claim 7, characterized in that: The process of modifying the working electrode (7) of the microelectrode layer (2) with the nucleic acid aptamer is as follows: 10 μL of gold deposition solution is dripped onto the surface of the working electrode (7), and incubated at room temperature for 4 hours. After that, a layer of nano-gold particles will be deposited on the surface of the working electrode (7). The gold deposition solution consists of: 0.5% w / v HAuCl4, 50 mg / mL potassium bicarbonate KHCO3, and 5 mg / mL glucose Glucose; then 5 μM AFM1, AFB1, DON and ZEN aptamers are dripped onto the working electrode (7) in each electrode unit, respectively, and incubated at room temperature for 4 hours. The aptamer is rinsed three times with Tris-HCl, and 20 μL of 1 μM MCH is dripped onto the working electrode (7) area. The aptamer is incubated in a constant temperature shaker at 25°C for 1 hour, and then rinsed with Tris-HCl to complete the modification.

9. The method for preparing a chip for detecting multiple biological toxins according to claim 7, characterized in that: The preparation of the silicon substrate mold uses a single-sided polished four-inch single crystal silicon wafer as the mold substrate, and specifically includes the following steps: Step 1. Cleaning: Use a mixture of H2SO4 and H2O2 to clean the silicon wafer, with the volume ratio of v:v=7:1; Step 2. Glue coating: Glue coating the substrate, negative resist SU8-2075, 500 r*30 s, 3000 r*60 s, 50 μm thick; Step 3. Pre-baking, hot plate temperature 95 °C, time 45 min; Step 4. Photolithography, exposure time is 28 s; Step 5. Post-baking, oven temperature 95°C, time 7 min; Step 6. Development, development time 5 min, spin drying at 2100 r*60 s; Step 7. Hard baking, 150 ℃, 15 min; Finally, a microstructured silicon substrate mold is obtained; The preparation process of the PDMS chip layer (1) is as follows: Step 1. Place the prepared silicon substrate mold in a fluorosilane vapor box and incubate for 4 h to facilitate the subsequent peeling of the PDMS chip; Step 2. Weigh the PDMS prepolymer and curing agent in a weight ratio of 10:1, mix them well, and place them in a vacuum desiccator at a vacuum degree of 13 psi for 30 min to remove most of the bubbles; Step 3. Place the silicon substrate mold on a horizontal platform, pour the PDMS mixture, and let it stand for 30 min to allow the microstructure to be filled with PDMS; Step 4. Heat in an oven at 80 °C for 1 h. After the PDMS is completely cured, carefully peel the PDMS off the silicon wafer. Step 5. Use a syringe with a needle of a specific aperture to punch holes according to the positions of the inlet and outlet on the PDMS chip; Step 6. Cut the chip along the cutting line to obtain a single PDMS chip, and paste white film on the upper and lower surfaces for preservation; The preparation process of the microelectrode layer (2) is as follows: Step 1. Select a single-side polished four-inch silicon wafer as the substrate, and use thermal oxidation to prepare a 2 μm thick silicon dioxide (SiO2) layer as an insulating isolation layer; Step 2. Choose to use hot wire chemical deposition technology to grow 3-5 μm diamond film; Step 3. Prepare an Al mask layer with a thickness of 450 nm using metal sputtering technology, and then spin-coat a 2.4 μm thick LC100A4 photoresist to pattern the Al layer; Step 4. Remove the Al layer by wet etching, and then use oxygen plasma to dry etch the BDD area to obtain a BDD working electrode (7) structure; Step 5. Using a metal sputtering process, prepare a 0.4 μm thick Pt1000 layer as a counter electrode (8) structure; then use acetone to remove the photoresist to complete the preparation of the Pt electrode area; Step 6. Cut the electrode chips along the cutting lines to obtain individual electrode chips; solder the connecting wires to the solder joints of each electrode unit using solder paste, either before or after cutting; The bonding process of the prepared PDMS chip layer (1) and the microelectrode layer (2) is as follows: The white film on the upper surface of the PDMS chip is torn off and placed in a plasma cleaning machine together with the microelectrode layer (2) for cleaning for 60 seconds. Then, an alignment keyer is used to key the two parts together according to the alignment marks, thereby completing the preparation of the entire chip.

10. A method for detecting multiple biological toxins, characterized in that: Using the multiple biotoxin detection chip as described in any one of claims 1 to 6; First, the multiple biological toxin detection chip prepared as claimed in claim 7 or 8 is simulated with the following specific test process, and the multiple biological toxin detection chip is tested and calibrated. By adding a series of standard sample solutions of different concentrations containing at least one of the four biological toxins, aflatoxin M1, aflatoxin B1, vomitoxin and zearalenone toxin, and the concentration values ​​of which are known, a series of initial impedance values ​​Rct0 and post-reaction impedance values ​​Rct of each working electrode (7) unit with different values ​​are measured, and a series of concentration gradient experiments are carried out to obtain a series of data of the relative impedance value y of each biological toxin concentration value and the corresponding working electrode (7) unit, and the data are substituted into the linear relationship between the concentration of a certain biological toxin and the relative impedance value y value: y=A+Blgx, wherein the relative impedance value y=(Rct-Rct0) / Rct0, A and B are constants, x represents the concentration of a certain biological toxin, and lgx represents the logarithm of x with base 10; after a series of calculations, the values ​​of constants A and B in the linear relationship between the concentration of various biological toxins and their relative impedance values ​​y values ​​are obtained; The specific test process is as follows: (1) First, add 10 mM Fe(CN)6 3- / 4- 0.1M KCl solution was added to fill each detection cavity, and then the initial impedance value Rct0 of each working electrode (7) unit was detected; (2) Tris-HCl solution was injected to rinse the chip, and then the sample solution to be tested was added to fill each detection cavity, and incubated at room temperature for 40 minutes; (3) Deionized water was injected to rinse the chip, and then 10 mM Fe(CN)6 3- / 4- Fill each detection cavity with 0.1 M KCl solution and detect the post-reaction impedance value Rct of each electrode unit; (4) calculate the relative impedance value y = (Rct-Rct0) / Rct0 of each detection unit and substitute it into the linear relationship between the corresponding biotoxin concentration and the relative impedance value y: y = A + Blgx to obtain the biotoxin concentration x in the sample.

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