CN110455898B - Preparation of electrochemical sensor with high-specific-surface nanogold as signal amplification carrier and application of electrochemical sensor in pesticide combined toxicity evaluation - Google Patents
Preparation of electrochemical sensor with high-specific-surface nanogold as signal amplification carrier and application of electrochemical sensor in pesticide combined toxicity evaluation Download PDFInfo
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Abstract
The invention belongs to the technical field of biosensing, and particularly relates to preparation of an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier and application of the electrochemical sensor in pesticide combined toxicity evaluation, which comprises the following steps: growing Au nano seeds, preparing high-specific-surface-area nano-gold materials, preparing an aptamer sensor and generating an electrochemical signal. The invention ensures the reliability and stability of the sensor; the capture efficiency of the target cells is improved; the electron transfer rate is improved; the electric signal recognition capability of the chlorpyrifos shows a higher level and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of biosensing, and particularly relates to preparation of an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier and application of the electrochemical sensor in pesticide combined toxicity evaluation.
Background
Organic phosphorus compounds (OPs) are a class of pesticides widely used in agriculture, have high insecticidal activity and are widely applied to agricultural production. It is estimated that more than 200 million tons of this insecticide are used worldwide each year to control and kill insects and pests in crop fields. The chlorpyrifos is mainly used for preventing and controlling different kinds of pests in crop fields, including termites, mosquitoes, roundworms, cutworms, corn rootworm, flea beetles, flies, fire ants, lice and the like. Chlorpyrifos is commonly introduced into the environment by different routes, such as rain discharge into surface waters, spray drift or soil erosion, and is therefore often found in air, food and water. The fish death events are reported to be related to the chlorpyrifos in the water body, thereby proving that the content of the chlorpyrifos in the water body can reach hundreds of parts per million. The toxicity of Chlorpyrifos (CP) poses many potential hazards to human health. In addition, it can enter the human body by inhaling the air containing chlorpyrifos. Eating contaminated food or direct contact with chlorpyrifos can lead to severe poisoning. It is reported that chlorpyrifos can be absorbed by the human body through the respiratory tract, resulting in the occurrence of respiratory symptoms such as cough, wheeze, airway inflammation, etc. In addition, other symptoms may occur, such as headache, nausea, numbness, coordination problems, dizziness, tremor, abdominal cramps, blurred vision, bradycardia, and the like. High doses of CP may lead to convulsions and even death from cardiovascular failure.
Carbofuran is a carbamate insecticide used as a broad-spectrum insecticide for killing insects in crops and plants, and is widely applied to agricultural production. Mites and nematodes. The residual carbofuran in agricultural products and human living environment poses potential threats to human health and animal husbandry. This is because carbofuran changes the oxidative balance and stability of the cell membrane of human erythrocytes. Due to its high toxicity, China sets the Maximum Residual Limit (MRL) of grains and oilseeds to 0.1 or 0.2 mg-kg-1The Environmental Protection Agency (EPA) sets the MRL of its selected agricultural products (including mung beans, bananas, coffee and rice) to 0.1 mg-kg-1. Some countries in the united states, canada and the european union have banned the use of carbofuran in vegetables, fruits, herbs and tea. It affects different organs of the human body, such as the brain, liver, muscles and heart. Thus, carbofuran can inhibit the function of acetylcholinesterase, thereby effectively preventingStopping nerve transmission. The detection technology of carbofuran is various, such as chromatography, electrochemical method, aptamer sensor and the like. However, the chromatographic method and the electrochemical method have the defects of complex pretreatment requirement, poor repeatability, poor selectivity and the like. In addition, adapter sensors typically require a label and an indicator signal.
The general detection method of the pesticides is realized by connecting Gas Chromatography (GC) or high performance liquid chromatography with mass spectrometry (HPLC-MS), the method has the characteristics of high stability and high sensitivity, and the national standard of pesticide detection in many countries is still detected by large-scale instruments such as GC or HPLC. However, the detection of large instruments is difficult to realize in many places due to factors such as high components, complex pretreatment of samples, need of professionals and the like, so the development of a rapid and simple detection method is still the direction of pesticide research.
The electrochemical biosensor takes a bioactive substance as a sensitive element, takes an electrode as a signal converter, converts a chemical signal of a detected object into an electric signal, and detects the concentration of a target object according to the intensity of the electric signal. The metal nano material is widely applied to electrochemical sensors due to excellent electric conductivity, high specific surface area and excellent catalytic performance.
Disclosure of Invention
The invention aims to solve the problems and provides an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier, which is used for detecting and evaluating the combined toxicity of diebi and carbofuran on cells and has high sensitivity, high stability and easiness in use.
According to the technical scheme of the invention, the preparation method of the electrochemical sensor with the high specific surface nano-gold as the signal amplification carrier comprises the following steps,
(1) weighing hexadecyltrimethylammonium chloride (CTAC), adding into 1-50mL deionized water, adjusting CTAC concentration to 0.1-1.0wt%, adding 0.1-5.0mL, 1-30mM HAuCl4Water solution, keeping the reaction solution in water bath at 20-50 ℃; then adding KBr solution of 5-100 μ L and 0.1-5.0mM, standing for 10-30min, then adding KI solution of 5-100 μ L and 0.1-5.0mM, standing for 10-30min, and continuing to add 800 μ L and 5-Standing for 30-60min after 100mM of L-ascorbic acid solution to grow Au nano seeds;
(2) CTAC and 0.1-5.0mL of 1-30mM HAuCl4Adding the mixed solution into 1-50mL of deionized water to prepare a growth solution, and adjusting the concentration of CTAC to 0.1-1.0 wt%; adding 0.1-10.0 mu L of L-glutathione with the concentration of 0.1-50mM into the growth solution, inverting the test tube, oscillating and mixing uniformly, then adding 1.0-100.0 mu L of the Au nano seed solution obtained in the step (1), oscillating and mixing uniformly; placing the solution in a water bath at 20-50 ℃ for 30-60min, performing centrifugal enrichment, and washing for 1-3 times to obtain a high-specific-surface-area nanogold material which is dispersed in 3-5mL of ultrapure water;
(3) immersing the glassy carbon electrode in an etching solution for 5-20 minutes, polishing the glassy carbon electrode to a mirror surface by using alumina slurry, sequentially washing the glassy carbon electrode in water, ethanol and water by ultrasonic waves for 1-3 minutes, and cleaning the glassy carbon electrode at 0.1-2.0M H2SO4Further electrochemically activated in solution, rinsed with ultra pure water and washed with N2Drying;
(4) dripping 1-25 μ L of 0.1-10.0 μ M DNA aptamer on the surface of the glassy carbon electrode treated in the step (3), incubating overnight at 0-20 ℃, and then washing with deionized water to remove unbound DNA aptamer; dropwise adding 5-10 mu L of the high specific surface area nano-gold material solution obtained in the step (2), and fixing the high specific surface area nano-gold material on a glassy carbon electrode through an Au-S bond to form an aptamer sensor;
(5) the aptamer sensor is incubated with PBS solution of HepG-2 cells with pH of 6.0-8.0 and concentration of 0.05-0.25M at 20-50 ℃ for 1-5 hours to capture the HepG-2 cells to form a sandwich structure, and then washed with PBS for 1-3 times to prepare the electrochemical aptamer sensor.
Further, the etching solution in the step (3) adopts H2SO4:30％H2O2And = 3:1 etching solution.
Further, the aptamer sensor obtained in the step (4) is stored in a refrigerator at 0-8 ℃ before use.
Further, the electrochemical aptamer sensor obtained in the step (5) is placed in 0.1-10mL of a solution containing 0.1-10.0mM of hydroquinone and 0.1-10.0mM H2O2In PBS solution of (a).
The invention also provides an application of the electrochemical sensor with the nanogold with the high specific surface area as the signal amplification carrier in pesticide combined toxicity evaluation, which is used for detecting chlorpyrifos, wherein the electrochemical sensor is an electrochemical aptamer sensor prepared by the preparation method according to any one of claims 1 to 3, and the electrochemical aptamer sensor comprises the following steps: dripping 5 μ L of chlorpyrifos standard solution or sample solution onto the surface of the electrochemical sensor, incubating at 30-40 deg.C for 1-2 hr, washing with PBS and adding N2Drying; the electrochemical sensor was immersed in 10mM phosphate buffered saline at pH 7.4 and measured using differential pulse voltammetry, with a pulse amplitude of 50Mv set, and a differential pulse voltammetry curve was recorded between-0.3V and 0.5V.
The invention also provides an application of the electrochemical sensor with the high specific surface nano gold as the signal amplification carrier in the joint toxicity evaluation of pesticides, the electrochemical sensor is used for the joint toxicity evaluation of binary organophosphorus pesticides on a cell level, the electrochemical sensor is an electrochemical aptamer sensor prepared by the preparation method of any one of claims 1 to 3, and the electrochemical aptamer sensor comprises the following steps: taking the treated HepG-2 cells growing to the logarithmic phase, adding a mixed solution of chlorpyrifos and carbofuran prepared according to the concentration ratio of 1:1 into the HepG-2 cells, diluting the chlorpyrifos and the carbofuran with different concentrations by using acetone before use, adding a cell culture medium into the mixed solution to prepare the required concentration, keeping the volume concentration of the acetone to be lower than 0.5 percent, and taking the cell medium containing 0.5 percent of the volume concentration of the acetone as a control group of an experiment; and adjusting the concentration of the mixed solution of chlorpyrifos and carbofuran, and testing the differential pulse voltammetry response of the electrochemical sensor.
The invention has the beneficial effects that:
the nanogold can be used as a carrier for fixing the DNA aptamer due to good biocompatibility, so that the reliability and stability of the sensor are ensured;
because the synthesized nano-gold has large specific surface area and high surface free energy, the immobilized amount of the DNA aptamer is greatly improved, and the capture efficiency of the target cells is improved;
the gold nanoparticles have good electron mediating capability, can promote electron transfer between a reaction system and the surface of an electrode, and improve the electron transfer rate;
an aptamer-cell-DNA nano probe sandwich-shaped electrochemical sensing system is constructed on the surface of the electrode, the signal amplification capability is remarkably improved, the electric signal recognition capability of chlorpyrifos is shown to be higher, and the sensitivity is high.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example one
A method for preparing an electrochemical sensor with high specific surface area nano gold as a signal amplification carrier comprises the following steps,
(1) weighing CTAC, adding into 1mL deionized water, adjusting CTAC concentration to 0.5wt%, and adding 0.1mL 15mM HAuCl4Water solution, keeping the reaction solution in a water bath at 20 ℃; then adding 5 mu L of 2.5mM KBr solution, standing for 10min, then adding 5 mu L of 2.5mM KI solution, standing for 10min, continuing adding 100 mu L of 55mM L-ascorbic acid solution, and standing for 30min to grow Au nano seeds;
(2) CTAC and 0.1mL of 15mM HAuCl4Adding the mixed solution into 1mL of deionized water to prepare a growth solution, and adjusting the concentration of CTAC to be 0.5 wt%; adding 0.1.0 mu L of 25mM L-glutathione into the growth solution, inverting the test tube, oscillating and mixing uniformly, then adding 1.0 mu L of the Au nano-seed solution obtained in the step (1), oscillating and mixing uniformly; placing the solution in a water bath at 20 ℃ for 30min, performing centrifugal enrichment, and washing for 1 time to obtain a high-specific-surface-area nanogold material which is dispersed in 3mL of ultrapure water;
(3) immersing the glassy carbon electrode in an etching solution (H)2SO4:30％H2O2= 3: 1), then polishing the glassy carbon electrode to a mirror surface by using alumina slurry, washing the glassy carbon electrode for 1min by ultrasonic wave in water, ethanol and water in sequence, and cleaning the glassy carbon electrode at 0.1M H2SO4Further electrochemically activated in solution, rinsed with ultra pure water and washed with N2Drying;
(4) dripping 1 mu L of 5.0 mu M DNA aptamer on the surface of the glassy carbon electrode treated in the step (3), incubating overnight at 0 ℃, and then washing with deionized water to remove unbound DNA aptamer; dripping 5 mu L of the high specific surface area nano-gold material solution obtained in the step (2), and fixing the high specific surface area nano-gold material on a glassy carbon electrode through an Au-S bond to form an aptamer sensor; stored in a refrigerator at 0-8 ℃ before use;
(5) incubating the aptamer sensor with PBS solution with pH of 6.0 and containing HepG-2 cells with concentration of 0.15M for 3 hours at 20 ℃ to capture the HepG-2 cells (target cells) to form a sandwich-shaped structure, and then washing the sandwich-shaped structure for 1 time by PBS to prepare the electrochemical aptamer sensor; in electrochemical detection, 0.1-10mL of a reagent containing 0.1-10.0mM of hydroquinone and 0.1-10.0mM of H is placed2O2In PBS solution of (a).
Example two
1. A method for preparing an electrochemical sensor with high specific surface area nano gold as a signal amplification carrier comprises the following steps,
(1) weighing CTAC, adding into 25mL deionized water, adjusting CTAC concentration to 0.6wt%, adding 2.5mL, 30mM HAuCl4Aqueous solution, keeping the reaction solution in a water bath at 35 ℃; then adding 50 mu L of 5.0mM KBr solution, standing for 20min, then adding 55 mu L of 5.0mM KI solution, standing for 22min, continuing to add 450 mu L of 100mM L-ascorbic acid solution, and standing for 45min to grow Au nano seeds;
(2) CTAC and 2.5mL of 30mM HAuCl4Adding the mixed solution into 25mL of deionized water to prepare a growth solution, and adjusting the concentration of CTAC to be 0.6 wt%; adding 5.2 mu L of L-glutathione with the concentration of 50mM into the growth solution, inverting the test tube, oscillating and mixing uniformly, then adding 55 mu L of the Au nano-seed solution obtained in the step (1), oscillating and mixing uniformly; placing the solution in a water bath at 40 ℃ for 45min, then carrying out centrifugal enrichment, washing for 2 times, and dispersing the obtained high-specific-surface-area nanogold material in 4mL of ultrapure water;
(3) immersing the glassy carbon electrode in an etching solution (H)2SO4:30％H2O2= 3: 1) for 13 minutes, then polishing the glassy carbon electrode to a mirror surface by using alumina slurry, and cleaning the glassy carbon electrode by ultrasonic wave in water, ethanol and water in sequence1-3min, the cleaned glassy carbon electrode is at H of 1.2M2SO4Further electrochemically activated in solution, rinsed with ultra pure water and washed with N2Drying;
(4) dripping 15 mu L of DNA aptamer with the concentration of 10.0 mu M on the surface of the glassy carbon electrode treated in the step (3), incubating overnight at 10 ℃, and then washing with deionized water to remove unbound DNA aptamer; dripping 7 mu L of the high specific surface area nano-gold material solution obtained in the step (2), and fixing the high specific surface area nano-gold material on a glassy carbon electrode through an Au-S bond to form an aptamer sensor; stored in a refrigerator at 0-8 ℃ before use;
(5) incubating the aptamer sensor with PBS (phosphate buffer solution) with the pH of 7.0 and the concentration of 0.15M of HepG-2 cells at 38 ℃ for 5 hours to capture the HepG-2 cells to form a sandwich structure, and washing the sandwich structure for 2 times by using PBS to prepare the electrochemical aptamer sensor; in electrochemical detection, 0.1-10mL of a reagent containing 0.1-10.0mM of hydroquinone and 0.1-10.0mM of H is placed2O2In PBS solution of (a).
EXAMPLE III
1. A method for preparing an electrochemical sensor with high specific surface area nano gold as a signal amplification carrier comprises the following steps,
(1) weighing CTAC, adding into 50mL deionized water, adjusting CTAC concentration to 0.1wt%, adding 5.0mL 1mM HAuCl4Water solution, keeping the reaction solution in a water bath at 50 ℃; then adding 100 mu L of 0.1mM KBr solution, standing for 30min, then adding 50 mu L of 0.1mM KI solution, standing for 30min, continuing to add 800 mu L of 5mM L-ascorbic acid solution, and standing for 60min to grow Au nano seeds;
(2) CTAC and 5.0mL of 1mM HAuCl4Adding the mixed solution into 50mL of deionized water to prepare a growth solution, and adjusting the concentration of CTAC to be 0.1 wt%; adding 10.0 mu L of L-glutathione with the concentration of 0.1mM into the growth solution, inverting the test tube, oscillating and mixing uniformly, then adding 100.0 mu L of the Au nano-seed solution obtained in the step (1), oscillating and mixing uniformly; placing the solution in a water bath at 50 ℃ for-60 min, then carrying out centrifugal enrichment, and washing for 3 times to obtain a high specific surface area nanogold material which is dispersed in 5mL of ultrapure water;
(3) dipping and carving a glassy carbon electrodeEtching solution (H)2SO4:30％H2O2= 3: 1), then polishing the glassy carbon electrode to a mirror surface by using alumina slurry, washing the glassy carbon electrode for 3min by ultrasonic waves in water, ethanol and water in sequence, and cleaning the glassy carbon electrode at 2.0M H2SO4Further electrochemically activated in solution, rinsed with ultra pure water and washed with N2Drying;
(4) dripping 25 mu L of DNA aptamer with the concentration of 0.1 mu M on the surface of the glassy carbon electrode treated in the step (3), incubating overnight at 20 ℃, and then washing with deionized water to remove unbound DNA aptamer; dripping 10 mu L of the high specific surface area nano-gold material solution obtained in the step (2), and fixing the high specific surface area nano-gold material on a glassy carbon electrode through an Au-S bond to form an aptamer sensor; stored in a refrigerator at 0-8 ℃ before use;
(5) incubating the aptamer sensor with a PBS solution which has the pH of 8.0 and contains HepG-2 cells with the concentration of 0.25M at 50 ℃ for 1 hour to capture the HepG-2 cells to form a sandwich structure, and then washing the sandwich structure for 3 times by using PBS to prepare the electrochemical aptamer sensor; in electrochemical detection, 0.1-10mL of a reagent containing 0.1-10.0mM of hydroquinone and 0.1-10.0mM of H is placed2O2In PBS solution of (a).
The embodiment relates to preparation of a second-level ultrahigh specific surface area nanogold material which is an octahedron high specific surface area nanogold material and is used as a seed and preparation of an electrochemical sensor which is based on the nanogold material and is used as a signal amplification DNA carrier, and the preparation is used for evaluating the combined toxicity of chlorpyrifos and carbofuran on cells. The appropriate amount of CTAC was added to ultrapure water, followed by the addition of aqueous HAuCl4 solution, and the vial was held in a water bath. And then, sequentially adding a small amount of KBr and KI solution, standing for 30-60min, partially substituting chloride by the bromide and the iodide to form an Au precursor to adjust the morphology of the nano gold, and further growing into the needed octahedral Au nano seeds at a relatively quick growth rate after adding a reducing agent L-ascorbic acid due to the lower reduction potential of the bromide and the iodide relative to the chloride. The octahedral nanogold prepared by the invention has the average particle size of 204.5 nm, uniform size and regular and ordered shape. Similarly, in the step (2), a special growth direction in the secondary structure is constructed by adding L-glutathione with chiral conformation, and finally, the solution is centrifugally enriched and dispersed in a proper amount of ultrapure water. The shape grown on the basis of the octahedral nanogold has extremely rich thorn-shaped edges, and the property of the biocatalyst is greatly enhanced when the octahedral nanogold is used as a nano carrier. Reacting and combining with DNA aptamer under a certain condition, and incubating the product with peroxidase and hemin to prepare the DNA nano probe for signal amplification. And then fixing the thiolated specific aptamer through an Au-S bond to be used as a modified electrode to capture the target cell. As the concentration of chlorpyrifos increases, the differential pulse voltammetric signal linearly decreases.
Application embodiment 1
An application of an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier in pesticide combined toxicity evaluation is used for detecting chlorpyrifos, and the electrochemical sensor is an electrochemical aptamer sensor prepared by the preparation method of the embodiment and comprises the following steps: dripping 5 μ L of chlorpyrifos standard solution or sample solution onto the surface of the electrochemical sensor, incubating at 30-40 deg.C for 1-2 hr, washing with PBS and adding N2Drying; the electrochemical sensor was immersed in 10mM phosphate buffered saline at pH 7.4 and measured using differential pulse voltammetry, with a pulse amplitude of 50Mv set, and a differential pulse voltammetry curve was recorded between-0.3V and 0.5V. The DPV response of the prepared aptamer sensors to chlorpyrifos was tested by varying the concentration of chlorpyrifos. The reproducibility of the aptamer sensor was tested by detecting 100fM chlorpyrifos, which verified high reproducibility.
Application example two
An application of an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier in pesticide combined toxicity evaluation is used for combined toxicity evaluation of a binary organophosphorus pesticide on a cell level, and the electrochemical sensor is an electrochemical aptamer sensor prepared by the preparation method of the embodiment and comprises the following steps: taking the treated HepG-2 cells growing to the logarithmic phase, adding a mixed solution of chlorpyrifos and carbofuran prepared according to the concentration ratio of 1:1 into the HepG-2 cells, diluting the chlorpyrifos and the carbofuran with different concentrations by using acetone before use, adding a cell culture medium into the mixed solution to prepare the required concentration, keeping the volume concentration of the acetone to be lower than 0.5 percent, and taking the cell medium containing 0.5 percent of the volume concentration of the acetone as a control group of an experiment; and adjusting the concentration of the mixed solution of chlorpyrifos and carbofuran, and testing the differential pulse voltammetry response of the electrochemical sensor. Because the electrochemical sensor has specific response to chlorpyrifos, a second pesticide carbofuran is added on the basis of the detection of the application example I, and the response change of the sensor is tested to evaluate whether the effects of the binary pesticides are synergistic or antagonistic or additive. The method can also be used for the combined toxicity evaluation of chlorpyrifos and other organophosphorus pesticides on a cellular level.
The electrochemical biosensor of the present invention has two main components: a Glassy Carbon Electrode (GCE) interface modified by gold nanoparticles with high specific surface area and a multifunctional hybrid nano probe. Thiolated DNA aptamers can be attached to the electrode interface via Au-S bonds for HepG-2 cell capture. The designed nanoprobe integrates the specific recognition of target cells through high-affinity aptamer, and amplifies electrochemical signals based on high-specific-surface-area nanogold. Compared with other pesticide electrochemical aptamer sensors, the electrochemical sensor based on the nanogold material with the high specific surface area as the signal amplification DNA carrier has the advantages of ultrahigh sensitivity, high stability and easiness in use, and is also used for combined toxicity evaluation of chlorpyrifos and carbofuran in real cells. Wherein, the cells are cultured by Japanese premna herb and carbofuran, and then are dripped on a glassy carbon electrode decorated with functionalized nano gold for electrochemical measurement. The aptamer sensor can still be tested after being stored at 4 ℃ for two weeks, and the aptamer sensor is known to have high stability.
Claims (6)
1. A method for preparing an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier is characterized by comprising the following steps,
(1) weighing hexadecyl trimethyl ammonium chloride, adding into 1-50mL deionized water, adjusting the concentration of hexadecyl trimethyl ammonium chloride to 0.1-1.0wt%, and adding 0.1-5.0mL of 1-30mM HAuCl4Water solution, keeping the reaction solution in water bath at 20-50 ℃; then adding 5-100 parts ofMu L of 0.1-5.0mM KBr solution, standing for 10-30min, then adding 5-100 mu L of 0.1-5.0mM KI solution, standing for 10-30min, continuing to add 100-800 mu L of 5-100mM L-ascorbic acid solution, and standing for 30-60min to grow Au nano seeds;
(2) mixing hexadecyl trimethyl ammonium chloride with 0.1-5.0mL of 1-30mM HAuCl4Adding into 1-50mL deionized water to prepare a growth solution, and adjusting the concentration of hexadecyl trimethyl ammonium chloride to 0.1-1.0 wt%; adding 0.1-10.0 mu L of L-glutathione with the concentration of 0.1-50mM into the growth solution, inverting the test tube, oscillating and mixing uniformly, then adding 1.0-100.0 mu L of the Au nano seed solution obtained in the step (1), oscillating and mixing uniformly; placing the solution in a water bath at 20-50 ℃ for 30-60min, performing centrifugal enrichment, and washing for 1-3 times to obtain a high-specific-surface-area nanogold material which is dispersed in 3-5mL of ultrapure water;
(3) immersing the glassy carbon electrode in an etching solution for 5-20 minutes, polishing the glassy carbon electrode to a mirror surface by using alumina slurry, sequentially washing the glassy carbon electrode in water, ethanol and water by ultrasonic waves for 1-3 minutes, and cleaning the glassy carbon electrode at 0.1-2.0M H2SO4Further electrochemically activated in solution, rinsed with ultra pure water and washed with N2Drying;
(4) dripping 1-25 μ L of 0.1-10.0 μ M DNA aptamer on the surface of the glassy carbon electrode treated in the step (3), incubating overnight at 0-20 ℃, and then washing with deionized water to remove unbound DNA aptamer; dropwise adding 5-10 mu L of the high specific surface area nano-gold material solution obtained in the step (2), and fixing the high specific surface area nano-gold material on a glassy carbon electrode through an Au-S bond to form an aptamer sensor;
(5) the aptamer sensor is incubated with PBS solution of HepG-2 cells with pH of 6.0-8.0 and concentration of 0.05-0.25M at 20-50 ℃ for 1-5 hours to capture the HepG-2 cells to form a sandwich structure, and then washed with PBS for 1-3 times to prepare the electrochemical aptamer sensor.
2. The method for preparing the electrochemical sensor with the nanogold as the signal amplification carrier according to claim 1, wherein the etching solution in the step (3) adopts H2SO4:30％H2O2And = 3:1 etching solution.
3. The method for preparing an electrochemical sensor with nanogold as a signal amplification carrier according to claim 1, wherein the aptamer sensor obtained in the step (4) is stored in a refrigerator at 0-8 ℃ before use.
4. The method for preparing the electrochemical sensor with nanogold as a signal amplification carrier according to claim 1, wherein the electrochemical aptamer sensor obtained in the step (5) is placed in a 0.1-10mL container containing 0.1-10.0mM of hydroquinone and 0.1-10.0mM of H in electrochemical detection2O2In PBS solution of (a).
5. The application of an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier in pesticide combined toxicity evaluation is characterized in that the electrochemical sensor is used for detecting chlorpyrifos, is an electrochemical aptamer sensor prepared by the preparation method according to any one of claims 1 to 3, and comprises the following steps: dripping 5 μ L of chlorpyrifos standard solution or sample solution onto the surface of the electrochemical sensor, incubating at 30-40 deg.C for 1-2 hr, washing with PBS and adding N2Drying; the electrochemical sensor was immersed in 10mM phosphate buffered saline at pH 7.4 and measured using differential pulse voltammetry, with a pulse amplitude of 50Mv set, and a differential pulse voltammetry curve was recorded between-0.3V and 0.5V.
6. The application of an electrochemical sensor with high specific surface area nanogold as a signal amplification carrier in the joint toxicity evaluation of pesticides is characterized in that the electrochemical sensor is used for the joint toxicity evaluation of binary organophosphorus pesticides on a cell level, and the electrochemical sensor is an electrochemical aptamer sensor prepared by the preparation method according to any one of claims 1 to 3, and comprises the following steps: taking the treated HepG-2 cells growing to the logarithmic phase, adding a mixed solution of chlorpyrifos and carbofuran prepared according to the concentration ratio of 1:1 into the HepG-2 cells, diluting the chlorpyrifos and the carbofuran with different concentrations by using acetone before use, adding a cell culture medium into the mixed solution to prepare the required concentration, keeping the volume concentration of the acetone to be lower than 0.5 percent, and taking the cell medium containing 0.5 percent of the volume concentration of the acetone as a control group of an experiment; and adjusting the concentration of the mixed solution of chlorpyrifos and carbofuran, and testing the differential pulse voltammetry response of the electrochemical sensor.
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