Bioconjugate-based microfluidic platform for detection of various pesticides and application of bioconjugate-based microfluidic platform

Abstract

The invention belongs to the technical field of micro-fluidic platforms for detecting various pesticides, and discloses a micro-fluidic platform for detecting various pesticides based on a bioconjugate and application of the micro-fluidic platform. The bioconjugate-based microfluidic platform for detecting various pesticides comprises a bioconjugate electrode and a sensor, and the bioconjugate electrode is formed by connecting a bioconjugate to a snakelike laser-induced graphene electrode through an amido bond; the bioconjugate is formed by a mesoporous silica composite material aptamer / glucose / PMSN which modifies a pesticide aptamer and wraps glucose and an aminated ferrihydrite nano composite material NH2-Fh-ssDNA which modifies a DNA single chain through base complementary pairing, and the bioconjugate is marked as NH2-Fh-dsDNA / glucose / PMSN. The microfluidic platform provided by the invention is based on a microfluidic technology, integrates a series of functions such as solution transfer, target capture, reagent mixing and separation, biological / chemical reaction and detection (signal output) and the like into the core layer sensing module, and can realize simultaneous and accurate detection of various pesticides.

Description

Technical Field

[0001] The present application relates to a microfluidic platform for detecting various pesticides, and in particular to a microfluidic platform for detecting various pesticides based on bioconjugates and its application. Background Art

[0002] Pesticides play a key role in modern agriculture, especially in the prevention and control of pests and diseases of fruits such as apples. As a class of highly effective and broad-spectrum insecticides, neonicotinoid pesticides have become one of the fastest growing insecticide categories in the world due to their unique mechanism of action and good environmental compatibility. Imidacloprid, acetamiprid, clothianidin and thiamethoxam, as representatives of neonicotinoid pesticides, act on nicotinic acetylcholine receptors in the nervous system of insects, causing insect neurotoxicity and death, and are widely used in pest control of fruit trees such as apples. High-performance liquid chromatography-tandem mass spectrometry, gas chromatography-mass spectrometry and other technologies have become the mainstream methods for pesticide residue analysis. The above detection methods can provide high sensitivity and high selectivity detection, but they also have disadvantages such as complex sample pretreatment and high equipment cost. At the same time, they are also poorly portable and cannot achieve on-site rapid detection of pesticides.

[0003] Microfluidic technology integrates multiple steps of biochemical analysis into one platform, realizes automated operation, improves experimental repeatability and accuracy, and its high-throughput characteristics enable the simultaneous detection of multiple solutions, greatly improving efficiency, reducing the consumption of reagents and solutions, reducing costs and contamination risks, and due to its portability, it is suitable for rapid on-site detection. The microfluidic platform, through its highly integrated and automated characteristics, achieves trace consumption of samples and reagents, rapid analysis, and reduces contamination, and has high-throughput processing capabilities, making it suitable for multiple fields such as biomedical research and clinical diagnosis.

[0004] However, to date, research on constructing a bioconjugate-mediated microfluidic platform for the simultaneous detection of multiple pesticides has not been reported. Summary of the invention

[0005] In order to solve the technical gap of bioconjugate-mediated microfluidic platform in the prior art in the detection of multiple pesticides, the present application provides a microfluidic platform based on bioconjugate for the detection of multiple pesticides and its application.

[0006] In order to solve the above technical problems, this application adopts the following technical solutions:

[0007] A microfluidic platform for detecting multiple pesticides based on bioconjugates comprises a bioconjugate electrode and a sensor. The bioconjugate electrode is formed by connecting a bioconjugate to a serpentine laser-induced graphene electrode via an amide bond. The bioconjugate is formed by base complementary pairing of a mesoporous silica composite material aptamer / glucose / PMSN that modifies a pesticide aptamer and encapsulates glucose and an aminated ferrihydrite nanocomposite material NH2-Fh-ssDNA that modifies a single-stranded DNA, and is recorded as NH2-Fh-dsDNA / glucose / PMSN.

[0008] Preferably, the aptamer / glucose / PMSN and the NH2-Fh-ssDNA are placed in Tris-HCl buffer and mixed, and the temperature change is controlled appropriately, so that the pesticide aptamer and ssDNA are combined through hydrogen bonds and annealed to form a bioconjugate NH2-Fh-dsDNA / glucose / PMSN.

[0009] Preferably, the aptamer / glucose / PMSN is prepared by mixing glucose, pesticide aptamer and positively charged modified mesoporous silica microspheres PMSN.

[0010] Preferably, the pesticide DNA single-stranded ssDNA is mixed with the amination ferrihydrate nanoparticles NH2-Fh, so that the single-stranded ssDNA is modified on the surface of the amination ferrihydrate nanoparticles NH2-Fh to form an amination ferrihydrate nanocomposite material modified with single-stranded DNA, which is recorded as NH2-Fh-ssDNA.

[0011] The sensor described in the present application includes at least one aptamer / AuNPs-LIG anode and a PtNPs-LIG cathode.

[0012] Preferably, the preparation method of the aptamer / AuNPs-LIG anode is as follows: using a polyimide film as a substrate, laser scanning a carbon source polyimide to form laser-induced graphene LIG, then dripping a precursor chloroauric acid solution, and laser printing the laser-induced graphene LIG dripped with the precursor solution to form an AuNPs-laser-induced graphene electrode, recorded as AuNPs-LIG; immersing the AuNPs-LIG electrode in a slightly acidified phosphate buffer solution to activate the carboxyl group on the surface of AuNPs-LIG, and incubating the activated electrode with the aptamer solution to graft the amino group at the end of the aptamer to the carboxyl group on the surface of the electrode to form an aptamer / AuNPs-LIG anode.

[0013] Preferably, the preparation method of the PtNPs-LIG cathode is to use a polyimide film as a substrate, form laser-induced graphene LIG by laser scanning a carbon source polyimide, then drip a precursor chloroplatinic acid solution, and laser print the dried laser-induced graphene LIG dripped with the precursor solution to form a PtNPs-laser-induced graphene cathode, denoted as Pt NPs-LIG.

[0014] Preferably, the microfluidic platform is a multi-layer structure, the bioconjugate electrodes and sensors are located in a core layer, and further comprises a bottom layer, an interlayer and a top layer which are sequentially arranged on the core layer.

[0015] Finally, the present application also provides an application of the microfluidic platform, and a method for detecting a variety of pesticides using the microfluidic platform includes:

[0016] One or more pesticide solutions to be tested pass through the microfluidic platform, and different pesticides undergo chain displacement reactions with the corresponding dsDNA of the bioconjugate electrode NH2-Fh-dsDNA / glucose / PMSN, releasing NH2-Fh-ssDNA. At the same time, the coated glucose is released from PMSN and flows into the anode of the sensor. Glucose is used as fuel to prompt the sensor to output a voltage signal, thereby achieving simultaneous and accurate detection of pesticides.

[0017] Preferably, the detection method of multiple pesticides specifically includes:

[0018] One or more pesticide solutions to be tested flow through the bioconjugate electrode, and the bioconjugate modified on the serpentine laser-induced graphene electrode specifically captures a specific type of pesticide. The pesticide undergoes a chain displacement reaction with the dsDNA corresponding to NH2-Fh-dsDNA / glucose / PMSN, and the NH2-Fh-ssDNA is released. At the same time, the coated glucose is released from the PMSN, and the pesticide is retained in the serpentine mixing channel of the bioconjugate electrode. The released NH2-Fh-ssDNA and glucose flow out along the serpentine mixing channel with the solution.

[0019] The released NH2-Fh-ssDNA and glucose flow into the sensor anode, the NH2-Fh-ssDNA is complementary paired with the aptamer corresponding to the aptamer / AuNPs-LIG anode, the Au NPs with grape oxidase-like activity on the sensor anode catalyzes the oxidation of glucose into gluconic acid and H2O2, the Fh with peroxidase-like activity catalyzes the oxidation of H2O2 into H2O, and the electrons generated at the anode reach the cathode through the external circuit;

[0020] The PtNPs with laccase-mimicking activity on the cathode of the sensor PtNPs-LIG obtain electrons, catalyze oxygen reduction, form a circuit, and then simultaneously generate multiple voltage signals. The size of the voltage signal is positively correlated with the pesticide content, realizing the simultaneous and accurate detection of one or more pesticides.

[0021] This application provides a microfluidic platform and application based on bioconjugates for multiple pesticide detection. It fills the gap in bioconjugate-mediated microfluidic platforms in multiple pesticide detection technology, and also brings the following beneficial effects:

[0022] The present application provides a microfluidic platform for the detection of various pesticides. The pesticide to be detected passes through the bioconjugate electrode of its core layer, which can retain the pesticide in the serpentine mixing channel corresponding to the bioconjugate electrode, preventing the pesticide from continuing to flow into the anode to compete with NH2-Fh-ssDNA and the specific recognition of the aptamer, thereby generating interference signals that affect subsequent reactions. Therefore, the bioconjugate electrode-serpentine laser-induced graphene electrode can avoid the interference of pesticides on subsequent reactions at the anode. At the same time, the serpentine electrode and the serpentine mixing channel can set a longer path on a given surface area unit, thereby increasing the residence time of the pesticide solution, allowing the pesticide to have enough time to fully combine with its aptamer, which is conducive to improving the accuracy of pesticide detection.

[0023] The microfluidic platform sensor provided in the present application includes at least one aptamer / AuNPs-LIG anode and a PtNPs-LIG cathode. A variety of neonicotinoid pesticides undergo chain displacement reactions with the dsDNA in the bioconjugate electrode corresponding to NH2-Fh-dsDNA / glucose / PMSN, and NH2-Fh-ssDNA is released. At the same time, the coated glucose is released from PMSN and flows into the anode of the sensor. Glucose is used as a fuel to cause the sensor to output a voltage signal, thereby achieving simultaneous and accurate detection of a variety of neonicotinoid pesticides. Secondly, if the generated H2O2 accumulates too much, it will affect the service life and stability of the battery. The released NH2-Fh-ssDNA has Fh with peroxidase-like activity, which can catalyze the oxidation of H2O2 to H2O, avoiding excessive accumulation of H2O2, which is conducive to accelerating electron transfer and improving reaction efficiency, thereby improving the service life and stability of the battery.

[0024] The bioconjugate-mediated microfluidic platform for simultaneous detection of multiple pesticides provided by the present application integrates a series of functions such as solution transfer, target capture, reagent mixing and separation, biological / chemical reaction and detection (signal output) into the core layer sensor module based on microfluidic technology, which can realize the simultaneous detection of multiple pesticides. Based on the microfluidic technology of the present application, a "one-negative-to-multiple-positive" biofuel cell can be constructed. Taking the detection of imidacloprid, acetamiprid, clothianidin and thiamethoxam as examples, corresponding aptamers are selected, multiple bioconjugates are synthesized, and they are directly fixed to four serpentine mixing channels, respectively, to construct a bioconjugate-mediated microfluidic platform for simultaneous detection of multiple pesticides. Only one step of adding samples in the cathode area is required, and the solution can pass through the serpentine mixing channel, triggering the in-situ recognition conversion of four targets at the same time, and the glucose coated by the bioconjugate is released. Glucose is used as a fuel to prompt the sensor to output a voltage signal, realizing simultaneous and accurate detection of multiple pesticides.

[0025] It can be seen from the above technical solutions that compared with traditional pesticide detection methods, the technical solution provided by this application is based on a bioconjugate-mediated microfluidic platform for simultaneous detection of multiple pesticides, which breaks away from traditional immunological and electrochemical detection methods and realizes simultaneous and accurate detection of multiple pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The schematic diagram of the microfluidic platform provided in this application is (a), the schematic diagram of the assembly of the microfluidic platform (b), and the schematic diagram of the use state of the microfluidic platform (c);

[0027] Figure 2 A solution flow diagram of the microfluidic platform provided in this application;

[0028] Figure 3 A solution flow diagram of the microfluidic platform provided in this application;

[0029] Figure 4 The effect diagram of Au NPs-LIG enzyme-mimicking activity provided in the effect example of the present application;

[0030] Figure 5 This is a diagram showing the enzyme-mimicking activity of Pt NPs-LIG provided in the embodiment of the present application;

[0031] Figure 6 The Fh enzyme activity effect diagram provided by the effect example of this application;

[0032] Figure 7 Schematic diagram of the overall structure and detection method of the microfluidic platform provided in the embodiment of the present application. DETAILED DESCRIPTION

[0033] The present application discloses a microfluidic platform and application for multiple pesticide detection based on bioconjugates. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0034] In order to enable those skilled in the art to better understand the present application, the present application is further described in detail below in conjunction with specific implementation methods.

[0035] A microfluidic platform for multiple pesticide detection based on bioconjugates comprises a bioconjugate electrode and a sensor, wherein the bioconjugate electrode is formed by connecting the bioconjugate to a serpentine laser induced graphene electrode via an amide bond, wherein the bioconjugate is formed by base complementary pairing of a mesoporous silica composite material aptamer / glucose / PMSN that modifies a pesticide aptamer and encapsulates glucose and an aminated ferrihydrite nanocomposite material NH2-Fh-ssDNA that modifies a single strand of DNA, and is recorded as NH2-Fh-dsDNA / glucose / PMSN.

[0036] In a preferred embodiment, the aptamer / glucose / PMSN and the NH2-Fh-ssDNA are placed in Tris-HCl buffer and mixed, and the temperature is appropriately controlled so that the aptamer and ssDNA are bound by hydrogen bonds and annealed to form a bioconjugate NH2-Fh-dsDNA / glucose / PMSN;

[0037] In a specific embodiment, the following preparation method may be preferably used:

[0038] 0.2~0.3mmol / L aptamer / glucose / PMSN and 0.2~0.3mmol / L NH2-Fh-ssDNA were dissolved in 10~12mmol / L Tris-HCl, pH 7.5~8.0, 40~50mmol / L NaCl and 1-1.5mmol / L ethylenediaminetetraacetic acid buffer; then equal amounts of aptamer / glucose / PMSN and NH2-Fh-ssDNA in Tris-HCl solution were mixed in a microtube and annealed according to the following temperature curve: first incubated at 90-95°C for 5-10 minutes to destroy any pre-existing hydrogen bonds, then incubated at 60-65°C for 30-40 minutes, then incubated at 40-50°C for 30-40 minutes, then incubated at 30-37°C for 30-40 minutes, and finally incubated at 20-25°C for 30-40 minutes. After annealing, the microtube was stored at 3-4°C for 2-3 hours to stabilize the double-stranded DNA structure, and finally the bioconjugate NH2-Fh-dsDNA / glucose / PMSN was obtained.

[0039] The bioconjugate electrode described in the present application is formed by connecting the NH2-Fh-dsDNA / glucose / PMSN bioconjugate to a serpentine laser-induced graphene electrode via an amide bond.

[0040] The aptamer / glucose / PMSN described in the present application is prepared by mixing glucose, pesticide aptamer and positively charged modified mesoporous silica microspheres PMSN;

[0041] Specifically, it can be prepared by the following method:

[0042] 830-850 μL NaOH (2-2.2 mol / L) was added to a CTAB solution (0.2-0.3 wt%) and stirred at 70-80°C for 20-30 min. Under continuous stirring, 1-1.5 mL TEOS was added dropwise to the above solution and stirred vigorously for 2-3 h to obtain a white precipitate. The precipitate was then collected by centrifugation, washed, and then vacuum dried at 60-70°C. Then, the obtained solid product was refluxed for 10-12 h using a solution consisting of hydrochloric acid and methanol to remove the surfactant template to obtain MSN. 40-50 mg MSN was dispersed in 2-3 mL PDDA solution (0.1-0.2 wt%) to form positively charged mesoporous silica microspheres PMSN.

[0043] 1000-1100 μL of glucose (1-2 mol / L) and 10-15 μL of pesticide aptamer (1-2 nmol / L) were mixed with the PMSN formed above to obtain a composite material aptamer / glucose / PMSN. The pesticide aptamer described in this application was purchased from Sangon Biotech (Shanghai) Co., Ltd., wherein:

[0044] The aptamer of the pesticide imidacloprid includes a DNA fragment as shown in SEQ ID NO.1;

[0045] The aptamer of the pesticide acetamiprid includes a DNA fragment as shown in SEQ ID NO.2;

[0046] The aptamer of the pesticide clothianidin includes a DNA fragment as shown in SEQ ID NO.3;

[0047] The aptamer of the pesticide thiamethoxam includes a DNA fragment as shown in SEQ ID NO.4.

[0048] The NH2-Fh-ssDNA described in this application is prepared by mixing single-stranded ssDNA of different pesticides with amination ferrihydrate nanoparticles NH2-Fh, so that the single-stranded ssDNA is modified on the surface of the amination ferrihydrate nanoparticles NH2-Fh, thereby forming an amination ferrihydrate nanocomposite material of modified DNA single strand, which is recorded as NH2-Fh-ssDNA.

[0049] In the specific embodiment, the following method can be preferably prepared:

[0050] NaOH was added dropwise to the FeCl3 solution and stirred vigorously to prepare ferrihydrite nanoparticles Fh. Fh was mixed with 3-aminopropyltriethoxysilane (APTES) and ssDNA to modify the NH2-Fh surface with ssDNA to form an aminated ferrihydrite nanocomposite material NH2-Fh-ssDNA modified with a single-stranded DNA.

[0051] The single-stranded SSDNA of DAN described in this application was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the single-stranded SSDNA of DAN was modified with a carboxyl group at the 5′ end, wherein:

[0052] The single-stranded DNA (ssDNA) corresponding to the pesticide imidacloprid includes a DNA fragment as shown in SEQ ID NO.5;

[0053] The single-stranded DNA (ssDNA) corresponding to the pesticide acetamiprid includes a DNA fragment as shown in SEQ ID NO.6;

[0054] The single-stranded DNA (ssDNA) corresponding to the pesticide clothianidin includes the DNA fragment shown in SEQ ID NO.7

[0055] The single-stranded DNA (ssDNA) corresponding to the pesticide thiamethoxam includes a DNA fragment as shown in SEQ ID NO.8.

[0056] Secondly, the sensor described in the present application includes at least one aptamer / AuNPs-LIG anode and one PtNPs-LIG cathode. The number of aptamer / AuNPs-LIG anodes can be reasonably set according to the type and quantity of pesticides, and a "one-negative to multiple-positive" biofuel cell can be constructed. In a preferred embodiment, taking the detection of imidacloprid, acetamiprid, clothianidin and thiamethoxam as an example, four aptamer / AuNPs-LIG anodes are correspondingly set.

[0057] In a preferred embodiment, the preparation method of the aptamer / AuNPs-LIG anode is as follows: using a polyimide film as a substrate, laser scanning a carbon source polyimide to form a laser-induced graphene LIG, then dripping a precursor chloroauric acid solution, and laser printing the laser-induced graphene LIG dripped with the precursor solution to form an AuNPs-laser-induced graphene electrode, recorded as AuNPs-LIG; immersing the AuNPs-LIG electrode in a slightly acidified phosphate buffer solution to activate the carboxyl group on the surface of AuNPs-LIG, and incubating the activated electrode with the aptamer solution to graft the amino group at the end of the aptamer to the carboxyl group on the surface of the electrode to form an aptamer / AuNPs-LIG anode.

[0058] The preparation of the aptamer / Au NPs-LIG anode can be preferably carried out according to the following method:

[0059] Take a chloroauric acid aqueous solution, dilute it with a chitosan solution, and stir it at room temperature for 1 to 2 hours to obtain a 5 to 10 mmol / L chloroauric acid solution;

[0060] With polyimide film as substrate, laser scanning of carbon source polyimide is used to form laser-induced graphene LIG, and then a precursor solution is added dropwise and dried for use; the dried laser-induced graphene LIG with the precursor solution added dropwise is laser printed, and the high temperature and high pressure generated by the laser instantly reduce chloroauric acid on LIG in situ and synchronously to generate AuNPs, which is recorded as AuNPs-LIG;

[0061] The prepared Au NPs-LIG electrode was immersed in a slightly acidified phosphate buffer solution (PBS) containing 40-50 mg / mL EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) and 40-50 mg / mL N-hydroxysuccinimide (NHS), the pH of the phosphate buffer solution was 5.5-6.5, and the solution was allowed to stand for 30-40 minutes to activate the carboxyl groups on the surface of Au NPs-LIG. The unreacted EDC and NHS were removed by secondary water rinsing, and 1.5-2 μL of aptamer (2-3 μmol / L) solution was dropped on the activated AuNPs-LIG surface, and then incubated in a wet environment at 35-37°C for 4-5 hours. Through an amide-like reaction, the anode-aptamer / AuNPs-LIG anode of the sensor was obtained.

[0062] In a preferred embodiment, the preparation method of the PtNPs-LIG cathode is to use a polyimide film as a substrate, form laser-induced graphene LIG by laser scanning a carbon source polyimide, then drip a precursor chloroplatinic acid solution, and laser print the dried laser-induced graphene LIG dripped with the precursor solution to form a PtNPs-laser-induced graphene cathode, denoted as Pt NPs-LIG;

[0063] The preparation of PtNPs-LIG can be preferably carried out according to the following method:

[0064] Take 5 mL of 1% chloroplatinic acid aqueous solution, dilute it to 10 mL with chitosan solution, and stir at room temperature for 1 to 2 hours to obtain a 10 mmol / L chloroplatinic acid solution;

[0065] With polyimide film as substrate, laser scanning of carbon source polyimide is used to form laser-induced graphene LIG, and then a precursor solution is added and dried for use; the dried laser-induced graphene LIG with the precursor solution is laser printed, and the high temperature and high pressure generated instantly by the laser in situ and synchronously reduce chloroauric acid on LIG to generate Pt NPs, thereby obtaining the sensor cathode, which is recorded as Pt NPs-LIG.

[0066] like Figure 1 As shown, the microfluidic platform can be preferably a multilayer structure, the bioconjugate electrode 1c and the sensor are located in the core layer 1, the sensor includes at least one aptamer / AuNPs-LIG anode 1a and a PtNPs-LIG cathode 1b, and the bioconjugate electrode 1c, the aptamer / AuNPs-LIG anode 1a and the PtNPs-LIG cathode 1b are integrated in the core layer sensing module. A "one-negative-to-multi-positive" biofuel cell can be constructed, and taking the detection of imidacloprid, acetamiprid, clothianidin and thiamethoxam as an example, the anode can preferably be set to four.

[0067] In a specific application embodiment, the microfluidic platform includes, in addition to the core layer 1, a bottom layer 2, an interlayer 3 and a top layer 4 which are sequentially arranged on the core layer 1. For example, four anodes are preferably arranged. Figure 1 (a) as shown:

[0068] Bottom layer 2: correspondingly arranged above the core layer 1, including a cathode microchamber 2b, four anode microchambers 2a surrounding the cathode microchamber, and a serpentine mixing channel 2c connected to the cathode and anode microchambers. The cathode and anode of the sensor are fixed in the cathode microchamber and the four anode microchambers respectively, which can be used to store solutions. The bioconjugate electrodes 1c are fixed in the multiple serpentine mixing channels 2c respectively. The serpentine mixing channels and the bioconjugate electrodes are used to identify and capture pesticides.

[0069] Interlayer 3: Located above the bottom layer, it also has a sample outlet 3b, a sample inlet 3a and four serpentine mixing channels 2c connected thereto. Correspondingly, the bottom layer 2 is also provided with a sample inlet and a sample outlet connected to the serpentine channels;

[0070] Top layer 4: located at the top of the platform, correspondingly provided with one inlet and four outlets for introducing and discharging solutions.

[0071] like Figure 1 As shown in (c), when the microfluidic platform is used, the pesticide solution to be tested flows into the microfluidic platform through the injection port on the top layer, first flows into the cathode microchamber, and after the solution fills the cathode microchamber, the solution is divided into four and enters four serpentine mixing channels at the same time, and then the solution flows to the four anode microchambers, and finally, under the action of capillary force, the solution is discharged from the microfluidic system through the injection port.

[0072] In a specific design embodiment, software is used to design microfluidic graphics, and a laser cutting and engraving machine is used to cut PI tape, and the microfluidic platform is constructed from bottom to top and layer by layer. The parameters of the software graphic design can be specifically set to 1100-1300DPI, and the cutting power is 2-3W. Specifically: the PI tape is stuck on the glass plate and cut by a laser cutting and engraving machine. The specific cutting pattern is the bottom pattern designed by the software. After the cutting is completed, the PI tape is stuck on the bottom layer of the printed microfluidic system again, and the microfluidic interlayer is cut by a laser cutting and engraving machine. The specific pattern is the interlayer pattern designed by the software. The top layer is cut and prepared again in the same way to realize the design of the microfluidic platform.

[0073] Finally, the present application also provides an application of the microfluidic platform, wherein the microfluidic platform is used to detect one or more pesticides, and the detection method comprises the following steps:

[0074] One or more pesticide solutions to be detected pass through the microfluidic platform, and different pesticides undergo chain displacement reactions with the corresponding dsDNA of the bioconjugate electrode NH2-Fh-dsDNA / glucose / PMSN, releasing NH2-Fh-ssDNA. At the same time, the coated glucose is released from PMSN and flows into the anode of the sensor. Glucose is used as fuel to prompt the sensor to output a voltage signal, thereby realizing simultaneous and accurate detection of one or more pesticides.

[0075] In a preferred embodiment, the method for detecting multiple pesticides can be specifically performed according to the following steps:

[0076] One or more pesticide solutions to be tested flow through the bioconjugate electrode in the identification conversion zone, and the bioconjugate modified on the serpentine laser-induced graphene electrode specifically captures a specific type of pesticide. Different pesticides undergo chain displacement reactions with the corresponding dsDNA of NH2-Fh-dsDNA / glucose / PMSN, and NH2-Fh-ssDNA is released. At the same time, the coated glucose is released from PMSN, and the pesticides are retained in the serpentine mixing channel of the bioconjugate electrode. The released NH2-Fh-ssDNA and glucose flow out along the serpentine mixing channel with the solution.

[0077] The released NH2-Fh-ssDNA and glucose flow into the anode, and the NH2-Fh-ssDNA is complementary paired with the aptamer corresponding to the aptamer / AuNPs-LIG anode. The AuNPs with grape oxidase-like activity on the sensor anode catalyze the oxidation of glucose into gluconic acid and H2O2, and the Fh with peroxidase-like activity catalyzes the oxidation of H2O2 into H2O. The electrons generated at the anode reach the cathode through the external circuit.

[0078] The PtNPs with laccase-mimicking activity on the PtNPs-LIG cathode obtain electrons, catalyze oxygen reduction, form a circuit, and then simultaneously generate multiple voltage signals. The size of the voltage signal is positively correlated with the pesticide content, realizing the simultaneous and accurate detection of multiple neonicotinoid pesticides.

[0079] In a preferred embodiment, the voltage signal can be converted by the programmable single chip 5 module and transmitted wirelessly to achieve simultaneous and accurate detection of multiple pesticides.

[0080] Effect Example

[0081] 1.1 Verification of the glucose oxidase-mimicking activity of Au NPs-LIG at the sensor anode

[0082] Experimental system a: The catalytic reaction system includes 0.5 mmol / L TMB solution, 50 μg / mL HRP solution, 50 mmol / L glucose solution, HAC-NaAC buffer with a pH of 4.0 and Au NPs-LIG prepared in the present application, and reacts at 37°C for 30 minutes, and the absorbance value within 400-800 nm is detected by UV-visible spectrophotometer;

[0083] Three control experiments: In the control experiment b, the Au NPs-LIG was not added to the catalytic reaction, and the absorbance value was detected after 30 minutes of reaction under the same conditions as the experimental system a; in the control experiment c, glucose and HRP were not added to the catalytic system, and the absorbance value was detected after 30 minutes of reaction under the same conditions as the experimental system a; in the control experiment d, the catalytic system was TMB solution (0.5 mmol / L) in HAC-NaAC (pH 4.0) buffer, and the absorbance value was detected after 30 minutes of reaction under the same conditions as the above experimental system.

[0084] like Figure 4 As shown, experimental system a shows an ultraviolet absorption peak at 652 nm, indicating that the Au NPs-LIG prepared in the present application can catalyze the hydrolysis of glucose to generate H2O2 and gluconic acid in a system with a pH of 4.0, and the generated H2O2 is catalyzed and decomposed by horseradish peroxidase to generate active oxygen, so that the colorless reduced TMB is oxidized to blue oxidized TMB, forming an ultraviolet absorption peak at 652 nm. The Au NPs-LIG prepared in the present application has simulated glucose oxidase activity.

[0085] 1.2 Verification of the laccase-mimicking activity of Pt NPs-LIG as the sensor cathode

[0086] Experimental system e: The catalytic reaction system includes 0.5 mmol / L ABTS solution, PtNPs-LIG prepared by printing in this application, and HAC-NaAC buffer with a pH of 4.0, and reacts at 37°C for 30 minutes, and the absorbance value within 300-600 nm is detected by a UV-visible spectrophotometer;

[0087] A control experiment: In the control experiment f, the Pt NPs-LIG provided by the present application was not added to the catalytic reaction, and the absorbance value was detected after the reaction was carried out for 30 minutes under the same conditions as the experimental system d.

[0088] like Figure 5 As shown, experimental system e shows an ultraviolet absorption peak at 420 nm, indicating that the Pt NPs-LIG prepared in the present application can catalyze the decomposition of ABTS into ABTS free radicals in a pH 4.0 system, and an ultraviolet absorption peak appears at 420 nm. The Pt NPs-LIG prepared in the present application has simulated laccase activity.

[0089] 1.3 Verification of the ability of ferrihydrite nanoparticles Fh to simulate peroxidase activity

[0090] Experimental system f: The catalytic reaction system comprises 0.5 mmol / L TMB solution, HAC-NaAC buffer solution with a pH of 4.0 and the ferrihydrite nanoparticles Fh prepared in the present application, and the reaction is carried out at 37° C. for 30 minutes, and the absorbance value within 400 to 800 nm is detected by a UV-visible spectrophotometer;

[0091] Three control experiments: Among them, in the catalytic reaction of control experiment g, the ferrihydrite nanoparticles Fh prepared in the present application were not added, and the absorbance value was detected after 30 minutes of reaction under the same conditions as in experimental system f; in the catalytic system of control experiment h, H2O2 was not added, and the absorbance value was detected after 30 minutes of reaction under the same conditions as in experimental system f; the catalytic system of control experiment i was TMB solution (0.5 mmol / L) in HAC-NaAC (pH 4.0) buffer, and the absorbance value was detected after 30 minutes of reaction under the same conditions as in the above-mentioned experimental system.

[0092] like Figure 6 As shown, the experimental system f shows an ultraviolet absorption peak at 652nm, indicating that the ferrihydrite nanoparticles Fh of the present application can catalyze the decomposition of H2O2 to generate active oxygen in a system with a pH of 4.0, so that TMB is oxidized to oxTMB, and an ultraviolet absorption peak is formed at 652nm, proving that the Fh prepared in Example 2 has simulated peroxidase activity.

[0093] 1.3 Bioconjugate-mediated microfluidic platform for simultaneous detection of multiple pesticides

[0094] Taking the detection of four neonicotinoid pesticides, imidacloprid, acetamiprid, clothianidin and thiamethoxam, as an example, in this embodiment, four sensor anodes and anode microchambers are correspondingly provided;

[0095] like Figure 7 Schematic diagram of four neonicotinoid pesticide solutions, imidacloprid, acetamiprid, clothianidin, and thiamethoxam, passing through the microfluidic platform. The solutions flow into the microfluidic system through the inlet on the top layer, first flowing into the cathode microchamber. After the solution fills the cathode microchamber, the solution is divided into four and enters four serpentine mixing channels at the same time. Then the solution flows to the four anode microchambers. Finally, the solution is discharged from the microfluidic platform through the outlet under the action of capillary force.

[0096] During the flow detection process of four neonicotinoid pesticides, imidacloprid, acetamiprid, clothianidin and thiamethoxam undergo chain displacement reaction with the corresponding dsDNA in NH2-Fh-dsDNA / glucose / PMSN, NH2-Fh-ssDNA is released, and the coated glucose is released from PMSN at the same time and flows into the anode. The Au NPs with grape oxidase-like activity on the sensor anode catalyze the oxidation of glucose into gluconic acid and H2O2. The released NH2-Fh-ssDNAFh has peroxidase-like activity and can catalyze the oxidation of H2O2 into H2O. The electrons generated at the anode reach the cathode through the external circuit. The PtNPs with simulated laccase activity on the cathode of the sensor PtNPs-LIG obtain electrons, catalyze oxygen reduction, and form a loop, thereby generating multiple voltage signals at the same time. Glucose is used as fuel to prompt the sensor to output voltage signals. The voltage signals are converted by the programmable single-chip module and transmitted wirelessly to achieve simultaneous and accurate detection of the four neonicotinoid pesticides.

[0097] The present application constructs a "one-negative-to-multiple-positive" biofuel cell. Taking the detection of imidacloprid, acetamiprid, clothianidin and thiamethoxam as an example, the corresponding aptamers are selected, and multiple bioconjugates are fixed on four serpentine mixing channels. A bioconjugate-mediated microfluidic platform is constructed to simultaneously detect multiple pesticides. Only one step of sample addition is required in the cathode area, and the solution can pass through the serpentine mixing channel, triggering the in-situ recognition conversion of the four targets at the same time, and the glucose coated with the bioconjugate is released. Glucose is used as fuel to prompt the sensor to output a voltage signal, thereby realizing real-time monitoring of one or more pesticides.

[0098] The above is only a preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A microfluidic platform based on bioconjugates for multiple pesticide detection, characterized in that: The invention comprises a bioconjugate electrode and a sensor. The bioconjugate electrode is formed by connecting a bioconjugate to a serpentine laser-induced graphene electrode through an amide bond. The bioconjugate is formed by a mesoporous silica composite material aptamer / glucose / PMSN which modifies a pesticide aptamer and encapsulates glucose, and an aminated ferrihydrite nanocomposite material NH2-Fh-ssDNA which modifies a single-stranded DNA through base complementary pairing, which is recorded as NH2-Fh-dsDNA / glucose / PMSN.

2. The microfluidic platform according to claim 1, characterized in that: The aptamer / glucose / PMSN and the NH2-Fh-ssDNA are placed in Tris-HCl buffer and mixed, and the temperature is controlled appropriately so that the aptamer and ssDNA are bound by hydrogen bonds and annealed to form a bioconjugate NH2-Fh-dsDNA / glucose / PMSN.

3. The microfluidic platform according to claim 1, characterized in that: The aptamer / glucose / PMSN is prepared by mixing glucose, pesticide aptamer and positively charged modified mesoporous silica microspheres PMSN.

4. The microfluidic platform according to claim 1, characterized in that: The pesticide DNA single-stranded ssDNA is mixed with the aminated ferrihydrate nanoparticles NH2-Fh, so that the single-stranded ssDNA is modified on the surface of the aminated ferrihydrate nanoparticles NH2-Fh, forming an aminated ferrihydrate nanocomposite material modified with the DNA single strand, which is recorded as NH2-Fh-ssDNA.

5. The microfluidic platform according to claim 1, characterized in that: The sensor includes at least one aptamer / AuNPs-LIG anode and a PtNPs-LIG cathode.

6. The microfluidic platform according to claim 5, characterized in that: The preparation method of the aptamer / AuNPs-LIG anode is as follows: using a polyimide film as a substrate, laser scanning a carbon source polyimide to form a laser-induced graphene LIG, then dripping a precursor chloroauric acid solution, and laser printing the laser-induced graphene LIG dripped with the precursor solution to form an AuNPs-laser-induced graphene electrode, recorded as Au NPs-LIG; immersing the Au NPs-LIG electrode in a slightly acidified phosphate buffer solution to activate the carboxyl group on the surface of Au NPs-LIG, and incubating the activated electrode with a pesticide aptamer solution to graft the amino group at the end of the aptamer to the carboxyl group on the surface of the electrode to form an aptamer / AuNPs-LIG anode.

7. The microfluidic platform according to claim 5, characterized in that: The preparation method of the PtNPs-LIG cathode is as follows: using a polyimide film as a substrate, laser scanning a carbon source polyimide to form laser-induced graphene LIG, then dripping a precursor chloroplatinic acid solution, and laser printing the dried laser-induced graphene LIG dripped with the precursor solution to form a PtNPs-laser-induced graphene cathode, recorded as Pt NPs-LIG.

8. The microfluidic platform according to claim 1, characterized in that: The microfluidic platform is a multi-layer structure, the bioconjugate electrodes and sensors are located in the core layer, and the microfluidic platform also includes a bottom layer, an interlayer and a top layer which are sequentially arranged on the core layer.

9. A method for detecting multiple pesticides using the microfluidic platform according to any one of claims 1 to 8, characterized in that: include: One or more pesticide solutions to be detected pass through the microfluidic platform, and different pesticides undergo chain displacement reactions with the corresponding dsDNA of the bioconjugate electrode NH2-Fh-dsDNA / glucose / PMSN, releasing NH2-Fh-ssDNA. At the same time, the coated glucose is released from PMSN and flows into the anode of the sensor. Glucose is used as fuel to prompt the sensor to output a voltage signal, thereby achieving simultaneous and accurate detection of pesticides.

10. A detection method according to claim 9, characterized in that: include: One or more pesticide solutions to be tested flow through the bioconjugate electrode, and the bioconjugate modified on the serpentine laser-induced graphene electrode specifically captures a specific type of pesticide. The pesticide undergoes a chain displacement reaction with the corresponding dsDNA in NH2-Fh-dsDNA / glucose / PMSN, and the NH2-Fh-ssDNA is released. At the same time, the coated glucose is released from the PMSN, and the pesticide is retained in the serpentine mixing channel of the bioconjugate electrode. The released NH2-Fh-ssDNA and glucose flow out along the serpentine mixing channel with the solution. The released NH2-Fh-ssDNA and glucose flow into the anode of the sensor, and the NH2-Fh-ssDNA is complementary paired with different aptamers corresponding to the aptamer / AuNPs-LIG anode. The AuNPs with grape oxidase-like activity on the anode of the sensor catalyze the oxidation of glucose into gluconic acid and H2O2, and the Fh with peroxidase-like activity catalyzes the oxidation of H2O2 into H2O. The electrons generated at the anode reach the cathode through an external circuit. The PtNPs with laccase-mimicking activity on the sensor cathode PtNPs-LIG obtain electrons, catalyze oxygen reduction, form a circuit, and then simultaneously generate multiple voltage signals. The size of the voltage signal is positively correlated with the pesticide content, realizing the simultaneous and accurate detection of one or more pesticides.

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