WO2023212991A1 - 一种双电场驱动传感器的制备方法及其应用 - Google Patents

一种双电场驱动传感器的制备方法及其应用 Download PDF

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WO2023212991A1
WO2023212991A1 PCT/CN2022/095898 CN2022095898W WO2023212991A1 WO 2023212991 A1 WO2023212991 A1 WO 2023212991A1 CN 2022095898 W CN2022095898 W CN 2022095898W WO 2023212991 A1 WO2023212991 A1 WO 2023212991A1
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electric field
electrode
dual
pesticide
reaction chamber
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French (fr)
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张文
苏小雨
袁磊
周璇
胡矩涛
邹小波
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江苏大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

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  • the invention belongs to the field of electrochemical sensors for detecting pesticide residues, and specifically relates to a preparation method and application of a dual electric field driven sensor.
  • Pesticides are substances or mixtures used to prevent, control and reduce harmful organisms. With the development of modern agriculture, the application of pesticides has become an essential link in crop production. However, excessive use of pesticides can lead to groundwater and food contamination, causing serious consequences for humans and animals. Long-term human exposure to pesticides can cause harmful effects on health, such as neurotoxicity, genotoxicity, mutagenicity and carcinogenesis. Although various laboratory-based analytical methods, such as gas chromatography, high-performance liquid chromatography, mass spectrometry, and enzyme-linked immunosorbent assay methods have been widely used, they all have their own shortcomings, such as the use of expensive instruments, time-consuming process and requires professionally trained personnel.
  • Electrochemical immunosensors have the advantages of strong specificity and high sensitivity, and are often used as effective detection methods.
  • the above techniques rely on receptor reactions with target molecules that occur via diffusion-dominated transport kinetics.
  • the concentration of target analytes in complex matrices is very low, and the immune reaction may take several hours or even days to proceed. In extreme cases, it may not even occur, which is not conducive to rapid detection in actual samples. , sensitive detection.
  • the present invention discloses a preparation method and application of a dual electric field driven sensor, which combines external complementary dual electric field driving and antigen-antibody reaction Specificity for rapid determination of pesticide residues in natural samples.
  • the preparation of the dual electric field driven sensor includes the production of a flexible printing substrate, the construction of a dual electric field driven module and the modification of the working electrode.
  • the present invention uses flexible printed circuit technology to manufacture flexible printed substrates, thereby reducing sensor production costs. Multiple electrode arrays on flexible printed electrodes can effectively adjust the dual electric field distribution.
  • RGO reduced graphene oxide
  • AuNPs gold nanoparticles
  • the microflow and particles in the solution can move in a directional manner, and the analyte can quickly flow to the surface of the biosensor, thereby improving the binding efficiency of the analyte and the antibody on the sensor surface, and greatly shortening the reaction time of the immunosensor.
  • the complementary dual electric field mode can effectively prevent excessive voltage from generating bubbles on the sensor surface, thereby establishing a more effective electric field enhancement strategy and improving sensor sensitivity and detection efficiency.
  • the dual electric field driven sensor designed by the present invention based on the above principles has realized the selective detection of pesticide residues in natural samples, and has the advantages of simple production, low cost, easy portability, and short detection time.
  • a method for preparing a dual electric field driving sensor includes the following steps: making a flexible printing substrate, constructing a dual electric field driving module, and modifying a working electrode.
  • Step 1 Production of flexible printing substrate:
  • K gold cyanide K gold cyanide
  • An unclosed ring gold film is formed as a counter electrode.
  • One end of the opening of the counter electrode ring gold film is electrically connected to the conductive hole N 1 ; a working electrode is provided in the internal area of the counter electrode and is electrically connected to the conductive hole N 2 . Electrically connected; apply arc-shaped Ag/AgCl slurry on the blank annular area of the unclosed ring of the counter electrode, and use it as a reference electrode after drying.
  • the reference electrode is electrically connected to the conductive hole N 3 ; at this time, the The counter electrode, working electrode, and reference electrode are called a three-electrode system; a hollow reaction chamber is provided on the surface of the flexible polyimide substrate, and the three-electrode system is in the hollow area of the hollow reaction chamber; and is not in contact with the hollow reaction chamber ; Finally, a flexible printing substrate is obtained;
  • Step 2 Construction of a dual electric field drive module: Two sets of independent copper foil areas are electroplated on the back of the three-electrode system of the flexible printing substrate prepared in step 1.
  • Each copper foil area is composed of M independent copper foil sheets. M is a positive integer; the area distribution of each group of copper foil areas can be adjusted through copper foil sheets; the two groups of copper foil areas are connected to the dual-channel signal generator through the conductive holes N 4 and N 5 of the flexible polyimide substrate respectively, providing Bias the sinusoidal electric field; set the dynamic bias voltage and peak-to-peak voltage; each copper foil area is assigned a set of electric fields; turn on the dual-channel signal generator during operation to provide a signal to the hollow area of the three-electrode system reaction cavity A set of complementary electric field environments;
  • the pesticide antibody is dripped onto the surface of the three-electrode system in the reaction chamber for incubation. After the pesticide antibody incubation is completed, the working electrode is rinsed with distilled water; after rinsing Continue to drop bovine serum albumin (BSA) onto the surface of the three-electrode system in the reaction chamber for incubation. After the BSA incubation is completed, rinse the working electrode with distilled water; finally, a dual electric field driven sensor is obtained.
  • BSA bovine serum albumin
  • the thickness of the copper film in step 1 is 15-25 ⁇ m; the photoresist is one of novolac resin and polymethylmethacrylate; the chemical developer is sodium carbonate or potassium carbonate.
  • the concentration is 0.1 ⁇ 0.4g/mL; the concentration of the potassium gold cyanide solution is 1 ⁇ 5g/L.
  • the thickness of the circular gold film in step 1 is 0.2-0.7 ⁇ m;
  • the working electrode is in the shape of a trident and consists of ten electrically connected circular gold films; each side of the trident has three There is a circular gold film, and the remaining circular gold film is at the intersection of the three sides.
  • the circular gold film at the intersection is electrically connected to the conductive hole N 2 ;
  • the arc-shaped Ag/AgCl thickness is 0.1-0.5 mm; the drying temperature is 70-90°C, and the drying time is 30-40 minutes.
  • the number M of copper foil sheets in step 2 is an even number between 10 and 20, and the shape of the copper foil sheets is square with a side length of 1 to 1.5 mm.
  • the dual-channel signal generator described in step 2 is any type of dual-channel electric field generating device, the dynamic bias voltage is set to 40-60% of the peak-to-peak voltage, and the peak-to-peak voltage is set to 2-6V.
  • the concentration of the graphene oxide solution in step 3 is 2-6 mg/mL, the dripping amount is 10-15 ⁇ L, and the electrodeposition time is 5-8 min; the concentration of the chloroauric acid solution is 1-3 mM, The dropping amount is 10-15 ⁇ L, and the electrodeposition time is 3 to 5 minutes; the H 2 SO 4 concentration is 0.3 to 0.6 M, the dropping amount is 20-30 ⁇ L, and the activation time is 150-300 s.
  • the concentration of the pesticide antibody described in step 3 is 5-20 ⁇ g/mL, the dripping amount is 10-15 ⁇ L, and the incubation time is 40-70 min; the concentration of bovine serum albumin is 0.5%-2%, and the dropwise addition The volume is 10-15 ⁇ L, and the incubation time is 30-60min.
  • a series of pesticide standard liquids of different concentrations are prepared and added dropwise to the surface of the three-electrode system in the reaction chamber of the dual electric field driven sensor for incubation.
  • the dual-channel electric field generating device is turned on during the incubation process, and the incubated sensor is used for Differential pulse voltammetry (DPV) test produces a current peak; establishes a standard curve based on the corresponding pesticide concentration and peak size;
  • DUV Differential pulse voltammetry
  • Step 2 Calculate the concentration of the pesticide to be tested based on the established I/C standard curve:
  • the pesticide sample liquid is added dropwise to the surface of the three-electrode system in the reaction chamber for incubation; after incubation, a DPV test is performed to generate a certain current intensity in the electrochemical window. According to the established I/C standard curve Calculate the concentration of the pesticide to be tested.
  • the pesticide described in step one is any one of chlorpyrifos, parathion-methyl, acetozoate, imidacloprid, paraoxon, atrazine, triazophos, carbendazim, thiacloprid or carbofuran. species, the concentration of the pesticide standard solution is 0.01-550 ⁇ g/L, the dripping amount is 10-15 ⁇ L, and the incubation time is 1-5 min;
  • the dynamic bias voltage of the dual-channel electric field generating device is set to 40-60% of the peak-to-peak voltage, and the peak-to-peak voltage is set to 2-6V.
  • the dropping amount of the pesticide sample liquid in step 2 is 10-15 ⁇ L, and the incubation time is 1-5 min; the dynamic bias voltage of the dual-channel electric field generating device is set to 40-60% of the peak-to-peak voltage.
  • the peak-to-peak voltage is set to 2 ⁇ 6V.
  • the invention also provides the application of the above-mentioned dual electric field driven sensor in rapid detection of pesticide residues in actual samples.
  • the flexible printing substrate of the present invention is light in weight, can be bent and folded freely, is easy to carry, and can realize electronic component assembly and circuit connection according to flexible design.
  • RGO and AuNPs materials described in the present invention exhibits excellent conductivity, high specific surface area and superior biocompatibility.
  • the immunosensor designed in the present invention has good selectivity and effectively overcomes the interference of other components in the actual sample.
  • the dual electric field driving module of the present invention provides two complementary electric fields into the reaction chamber, inducing solution convection, effectively accelerating the movement of pesticide molecules to the electrode surface, and completing the immune reaction in a short time.
  • Figure 1 is a schematic diagram of the electric field distribution of the dual electric field drive module
  • Figure 2 is a front (A) and reverse (B) physical view of the dual electric field drive sensor of the present invention
  • Figure 3 shows the modification process of the working electrode
  • A is a diagram of the accelerated immune response of the dual electric field drive module
  • B is a diagram of the accelerated immune response of the single electric field drive module
  • C is a diagram of the immune response of no electric field drive
  • Figure 5 is a standard curve diagram for the determination of methyl parathion
  • 1-flexible polyimide substrate 2-conductive hole; 3-copper film; 4-copper foil circuit; 5-counter electrode; 6-working electrode; 7-reference electrode; 8-reaction chamber; 9 -Copper foil sheets.
  • Step 1 Production of flexible printing substrate: Use computer drawing software to design the circuit shape; cut the flexible polyimide substrate 1 (length: 3cm, width: 1.5cm); set a circuit on one end of the flexible polyimide substrate 1 The five connected conductive holes 2 are marked N 1 , N 2 , N 3 , N 4 , and N 5 ; spray a layer of copper film 3 with a uniform thickness of 20 ⁇ m on the surface of the flexible polyimide substrate 1; use photolithography Technology: Coat the photoresist novolac resin on the copper film 3 according to the pre-designed circuit shape; place the flexible polyimide substrate 1 coated with the photoresist under UV light for exposure; exposure The final flexible polyimide substrate 1 is developed using 0.2g/mL sodium carbonate; after development, the unexposed area is dissolved, leaving the exposed area to form a copper foil circuit 4; the developed flexible polyimide substrate 1 is immersed In 4g/L potassium gold cyanide (KAu(CN) 2 ) solution, use electroplating technology to form an un
  • One end of the opening of the circular gold film is electrically connected to the conductive hole N1 ; in the internal area of the counter electrode 5, there is a working electrode 6 composed of ten circular gold films, which is in the shape of a trident, and each of the tridents There are three circular gold films on each side of the strip, and the remaining circular gold film is at the intersection of the three sides. Each gold film is electrically connected to each other, and the intersection of the gold films is electrically connected to the conductive hole N 2 ; Coat the blank annular area of the unclosed ring of the counter electrode 5 with a 0.5mm arc-shaped Ag/AgCl slurry, place it in a vacuum drying oven at 75°C and dry for 40 minutes as the reference electrode 7.
  • the reference electrode 7 and The conductive hole N 3 is electrically connected; the counter electrode 5, the working electrode 6, and the reference electrode 7 formed at this time are called a three-electrode system; a transparent hollow plastic reaction chamber 8 is provided on the surface of the area formed by the three-electrode system.
  • the three-electrode system is all within the hollow area of the hollow plastic reaction chamber 8 and is not in contact with the hollow plastic reaction chamber 8; a flexible printing substrate is finally obtained;
  • Step 2 Construction of a dual electric field driving module: Two sets of independent copper foil areas are electroplated on the back of the three-electrode system of the flexible printing substrate prepared in step 1. Each copper foil area is composed of 12 independent copper foil sheets 9 ; The area distribution of each group of copper foil areas can be adjusted by welding independent copper foil sheets 9 to each other; the two groups of copper foil areas are respectively connected to dual-channel signals through N 4 and N 5 of the conductive holes 2 of the flexible polyimide substrate 1
  • the generator (SDG6052X, Beijing Boyu Xunming Technology Co., Ltd.) provides a biased sinusoidal electric field, sets the dynamic bias voltage to 3V, and sets the peak-to-peak voltage to 6V; when using a dual electric field module, turn on the dual-channel signal generator, each The copper foil area is assigned a set of electric fields to provide a set of complementary electric field environments for the hollow area of the three-electrode system reaction chamber 8; when using a single electric field module, turn on the single-channel mode of the signal generator;
  • Figure 1 is a schematic diagram of the electric field distribution of the dual electric field drive module;
  • Figure 2 is a front and back view of the dual electric field drive sensor of the present invention. It can be seen from Figure 1 that there are two sets of electric fields in the reaction chamber. Electric field 1 and electric field 2 act on two independent sets of copper foil areas respectively.
  • Step 3 Modification of the working electrode: First prepare a 4 mg/mL graphene oxide (GO) solution; then drop 15 ⁇ L GO solution on the surface of the three-electrode system in reaction chamber 8, conduct electrodeposition on the electrochemical workstation for 7 minutes, and deposit Then rinse the working electrode 6 with distilled water; secondly, prepare a 1mM chloroauric acid (HAuCl 4 ) solution, drop 12 ⁇ L HAuCl 4 solution on the surface of the three-electrode system in the reaction chamber 8, and perform electrodeposition for 250s on the electrochemical workstation.
  • GO graphene oxide
  • FIG. 3 shows the functional modification process of the sensor working electrode. The final modification is completed to obtain a dual electric field driven sensor.
  • Step 4 Control test of methyl parathion incubation time under different electric field driving conditions: drop 100 ⁇ g/L, 12 ⁇ L methyl parathion into the surface of the three-electrode system in the reaction chamber 8 of the dual electric field driving sensor for incubation; A The parathion-based incubation process is divided into three groups. The first group turns on the dual electric field module, the second group turns on the single electric field module, and the third group disables the electric field module. In the first group, the current intensity is measured every 15 seconds; in the second group In the first group, the current intensity was measured every 30 s; in the third group, the current intensity was measured every 15 min; when the current intensity no longer changed significantly, the incubation time was recorded.
  • a to C in Figure 4 are the incubation times of methyl parathion under dual electric field driving, single electric field driving, and no electric field driving conditions, respectively, which are 60s, 180s, and 90min respectively. It can be seen from Figure 4 that under electric field driving conditions, the time of immune reaction is greatly shortened. In particular, the dual electric field drive module shows a shorter reaction time than the single electric field module, which is due to the two complementary electric field environments in the reaction system accelerating the reaction rate between the methyl parathion molecules and the antibody.
  • a preparation method and application of a dual electric field driven sensor includes the following steps:
  • Step 1 Production of flexible printing substrate: Use computer drawing software to design the circuit shape; cut the flexible polyimide substrate 1 (length: 3cm, width: 1.5cm); set a circuit on one end of the flexible polyimide substrate 1 The five connected conductive holes 2 are marked N 1 , N 2 , N 3 , N 4 , and N 5 ; spray a layer of copper film 3 with a uniform thickness of 15 ⁇ m on the surface of the flexible polyimide substrate 1; use photolithography Technology: Coat the photoresist novolac resin on the copper film 3 according to the pre-designed circuit shape; place the flexible polyimide substrate 1 coated with the photoresist under UV light for exposure; exposure The final flexible polyimide substrate 1 is developed using 0.1g/mL sodium carbonate; after development, the unexposed area is dissolved, leaving the exposed area to form a copper foil circuit 4; the developed flexible polyimide substrate 1 is immersed In 2g/L potassium gold cyanide (KAu(CN) 2 ) solution, use electroplating technology to form an un
  • One end of the opening of the circular gold film is electrically connected to the conductive hole N1 ; in the internal area of the counter electrode 5, there is a working electrode 6 composed of ten circular gold films, which is in the shape of a trident, and each of the tridents There are three circular gold films on each side of the strip, and the remaining circular gold film is at the intersection of the three sides. Each gold film is electrically connected to each other, and the intersection of the gold films is electrically connected to the conductive hole N 2 ; Coat the blank annular area of the unclosed ring of the counter electrode 5 with a 0.4mm arc-shaped Ag/AgCl slurry, place it in a vacuum drying oven at 80°C and dry for 30 minutes as the reference electrode 7.
  • the reference electrode 7 and The conductive hole N 3 is electrically connected; the counter electrode 5, the working electrode 6, and the reference electrode 7 formed at this time are called a three-electrode system; a transparent hollow plastic reaction chamber 8 is provided in the area formed by the three-electrode system, and The three-electrode system is in the hollow area of the hollow plastic reaction chamber 8 and is not in contact with the hollow plastic reaction chamber 8; a flexible printing substrate is finally obtained;
  • Step 2 Construction of a dual electric field driving module: Two sets of independent copper foil areas are electroplated on the back of the three-electrode system of the flexible printing substrate prepared in step 1. Each copper foil area is composed of 10 independent copper foil sheets 9 ; The area distribution of each group of copper foil areas can be adjusted by welding independent copper foil sheets 9 to each other; the two groups of copper foil areas are respectively connected to dual-channel signals through N 4 and N 5 of the conductive holes 2 of the flexible polyimide substrate 1
  • the generator (EDU33212A, American Keysight Company) provides a biased sinusoidal electric field, sets the dynamic bias voltage to 2V, and sets the peak-to-peak voltage to 5V; each copper foil area is assigned a set of electric fields; dual-channel signals are turned on during operation.
  • a generator provides a set of complementary electric field environments to the hollow area of the three-electrode system reaction chamber 8;
  • Step 3 Modification of the working electrode: First prepare a 3 mg/mL graphene oxide (GO) solution; then add 10 ⁇ L GO solution dropwise on the surface of the three-electrode system in reaction chamber 8, conduct electrodeposition on the electrochemical workstation for 5 minutes, and deposit Then rinse the working electrode 6 with distilled water; secondly, prepare a 2mM chloroauric acid (HAuCl 4 ) solution, drop 10 ⁇ L HAuCl 4 solution on the surface of the three-electrode system in the reaction chamber 8, and perform electrodeposition for 200s on the electrochemical workstation.
  • GO graphene oxide
  • BSA bovine serum albumin
  • Step 4 Establish a current/concentration (I/C) standard curve: add a series of different concentrations of methyl parathion (0.01 to 550 ⁇ g/L) dropwise to three of the reaction chambers 8 of the dual electric field drive sensor.
  • the surface of the electrode system is incubated for 1 minute, and the dual-channel electric field generating device is turned on at the same time; under the action of the electric field force, the methyl parathion molecules reach the electrode surface in a short time and are quickly captured by the methyl parathion antibody; the incubated molecules are
  • the sensor is used for DPV testing to generate a current peak; a standard curve is established based on the corresponding methyl parathion concentration and peak size.
  • Figure 5 is a standard curve diagram for the determination of methyl parathion according to the present invention.
  • the system of the present invention was compared with standard high performance liquid chromatography (HPLC). Before HPLC detection, the liquid to be tested was filtered with a 0.45 ⁇ m filter membrane. In DPV test and HPLC determination, each sample was measured three times and averaged to reduce random errors.
  • HPLC high performance liquid chromatography
  • the present invention is the first system that uses an electrochemical immunosensor combined with a dual electric field drive module to detect pesticide residues.
  • the self-made flexible printed electrode can form a specific circuit through external design, and is lightweight, bendable, and easy to carry; the RGO and AuNPs modified on the surface of the working electrode effectively increase the conductivity of the sensor working electrode surface; the pesticide antibody
  • the introduction enables the sensor to show excellent selectivity in complex sample environments; the introduction of complementary double electric fields during the pesticide incubation process effectively improves the adsorption efficiency and accelerates the immune reaction.
  • This dual electric field-driven electrochemical immunosensor has the advantages of simple fabrication, high sensitivity, and strong selectivity, and provides new prospects for the detection of pesticide residues in complex samples such as natural environments and agricultural products.

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Abstract

一种双电场驱动传感器的制备方法及其应用。采用柔性印刷电路技术制造柔性印刷基底，利用柔性印刷电极上多个电极阵列有效调节双电场分布；通过在电极表面修饰RGO和金纳米粒子赋予传感器良好的生物相容性、催化性能和导电性能。同时引入农药抗体来选择性的识别农药分子，在外加双电场驱动的条件下，溶液中微流和颗粒可以定向运动，分析物能快速流向生物传感器表面，从而提高了分析物与传感器表面探针的结合效率，缩短反应时间。互补的双电场模式能有效的防止电压过大而在传感器表面产生气泡，从而建立了更有效的电场增强策略，提高了传感器灵敏度和检测效率。

Description

一种双电场驱动传感器的制备方法及其应用 技术领域
本发明属于电化学传感器检测农药残留领域，具体涉及一种双电场驱动传感器的制备方法及其应用。
背景技术
农药是用来预防、控制和减少有害生物的物质或混合物，随着现代农业的发展，农药的应用已成为作物生产中必不可少的环节之一。然而，农药的过度使用会导致地下水和食物污染，对人类和动物造成严重影响。人类长期接触农药对健康可导致有害影响，如神经毒性、遗传毒性、致突变性和致癌性。尽管各种基于实验室的分析方法，如气相色谱、高效液相色谱、质谱和酶联免疫吸附测定方法已被广泛应用，但它们都存在着各自的缺点，如使用昂贵的仪器、耗时的过程和需要受过专业培训的人员。电化学免疫传感器具有特异性强、灵敏度高等优点，常被用作有效的检测方法。然而，上述技术依赖于受体与目标分子的反应，这些反应通过扩散主导的运输动力学发生。在实际样品中，目标分析物在复杂的基质中浓度很低，免疫反应的进行可能需要几个小时甚至几天的时间，在极端情况下，甚至可能不会发生，这不利于实际样品中快速、灵敏地检测。
目前市场上并无成型的利用双电场驱动电化学传感器实现农药残留快速检测的设备。在现有的应用电化学免疫传感器检测农药残留技术中，专利“一种基于两种电化学方法联用的甲基对硫磷免疫传感器的制备方法及应用”CN105806922A公开了一种基于功能纳米材料的生物免疫传感器的制备方法，以及利用所述传感器对甲基对硫磷进行检测的方法。该方法得到的传感器携带方便，成本低廉。但该传感器制作的过程复杂，传感时间长，不适合快速检测。
发明内容
针对传统电化学免疫传感器检测农药残留技术存在制作复杂，灵敏度低、响应速度慢等问题，本发明公开一种双电场驱动传感器的制备方法及其应用，结合外部互补的双电场驱动和抗原抗体反应的特异性，快速测定自然样品中的农药残留。
所述双电场驱动传感器的制备，包括柔性印刷基底的制作、双电场驱动模块的构建和工作电极的修饰。本发明采用柔性印刷电路技术制造柔性印刷基底，降低了传感器制作成本。根据柔性印刷电极上多个电极阵列能够有效的调节双电场分布。通过在电极表面修饰还原性氧化石墨烯(RGO)和金纳米粒子(AuNPs)赋予传感器良好的生物相容性、优良的催化性能和导电性能。同时引入农药抗体来选择性的识别农药分子。在外加双电场驱动的条件下，溶液中微流和颗粒的可以定向运动，分析物能快速流向生物传感器表面，从而提高了分析物与传感器表面抗体的结合效率，大大缩短了免疫传感器的反应时间。尤其是互补的双电场模 式能有效的防止电压过大而在传感器表面产生气泡，从而建立了更有效的电场增强策略，提高了传感器灵敏度和检测效率。本发明根据以上原理设计出的双电场驱动传感器已经实现对自然样本中农药残留的选择性检测，同时具有制作简单，成本低，易携带，检测时间短等优点。
本发明通过以下技术方案实现：
一种双电场驱动传感器的制备方法，包括以下步骤：柔性印刷基底的制作、双电场驱动模块的构建和工作电极的修饰。
步骤一、柔性印刷基底的制作：
利用计算机绘图软件设计电路形状；对柔性聚酰亚胺基板进行裁剪；在柔性聚酰亚胺基板的一端设置线路连接的N个导电孔并标记为N 1、N 2、N 3……N n-1、N n，N为不小于5的正整数；在柔性聚酰亚胺基板表面喷涂上一层均匀的铜膜；利用光刻技术将光致抗蚀剂按照预先设计的电路形状涂覆在铜膜上；将已涂覆光致抗蚀剂的柔性聚酰亚胺基板放置紫外在灯下进行曝光；曝光后的柔性聚酰亚胺基板利用化学显影剂进行显影；显影后，未曝光区域被溶解，留下曝光区域形成铜箔线路；将显影后的柔性聚酰亚胺基板浸渍在氰化亚金钾(KAu(CN) 2)溶液中，利用电镀技术在相对导电孔的另一端形成一个未闭合的圆环金膜作为对电极，对电极圆环金膜开口的一端与导电孔N 1电性连接；在对电极的内部区域设有工作电极，与导电孔N 2之间电性连接；在对电极未闭合圆环处的空白环形区域处涂覆圆弧型Ag/AgCl浆料，干燥后作为参比电极，参比电极与导电孔N 3电性连接；此时形成的对电极、工作电极、参比电极称为三电极体系；在柔性聚酰亚胺基板表面上设有中空反应腔，三电极体系均在中空反应腔的中空区域内；且与中空反应腔不接触；最终得到柔性印刷基底；
步骤二、双电场驱动模块的构建：在步骤一所制备的柔性印刷基底的三电极体系的背面电镀出两组独立的铜箔区，每个铜箔区域由M个独立的铜箔片组成，M为正整数；每组铜箔区的面积分布可以通过铜箔片来调整；两组铜箔区分别通过柔性聚酰亚胺基板的导电孔N 4、N 5连接双通道信号发生器，提供偏置正弦电场；设定动态偏置电压和峰间电压；每个铜箔区被分配一组电场；在运行过程中开启双通道信号发生器，对该三电极体系反应腔的中空区域提供一组互补的电场环境；
步骤三、工作电极的修饰：
首先制备氧化石墨烯(GO)溶液；然后将GO溶液滴加在反应腔中的三电极体系表面，在电化学工作站进行电沉积，沉积后用蒸馏水冲洗工作电极；其次制备氯金酸(HAuCl 4)溶液，将HAuCl 4溶液滴加在反应腔中的三电极体系表面，在电化学工作站进行电沉积，沉积后用蒸馏水冲洗工作电极；然后将硫酸(H 2SO 4)滴加在反应腔中的三电极体系表面对工作电极进行活化；活化后用蒸馏水冲洗工作电极，冲洗后向反应腔中的三电极体系表面滴加农药 抗体进行孵育，农药抗体孵育完成后用蒸馏水冲洗工作电极；冲洗后继续向反应腔中的三电极体系表面滴加牛血清蛋白(BSA)进行孵育，BSA孵育完成后用蒸馏水冲洗工作电极；最终得到双电场驱动传感器。
进一步的，步骤一中所述铜膜厚度为15～25μm；所述光致抗蚀剂为线性酚醛树脂、聚甲基丙烯酸甲酯中的一种；所述化学显影剂为碳酸钠或碳酸钾中的一种，浓度为0.1～0.4g/mL；所述氰化亚金钾溶液的浓度为1～5g/L。
进一步的，步骤一中所述圆环金膜厚度为0.2～0.7μm；所述工作电极呈三叉戟的形状，由十个电性连接的圆形金膜组成；三叉戟的每条边分别有三个圆形金膜，剩下的一个圆形金膜在三条边的交汇处，交汇处的圆形金膜与导电孔N 2之间电性连接；
所述弧型Ag/AgCl厚度为0.1～0.5mm；所述干燥的温度为70～90℃，干燥的时间为30～40min。
进一步的，步骤二中所述铜箔片数量M为10～20之间的偶数，铜箔片形状为正方形，边长为1～1.5mm。
进一步的，步骤二中所述双通道信号发生器为任意型号的具有双通道电场发生装置，动态偏置电压设置为峰间电压的40～60％，峰间电压设置为2～6V。
进一步的，步骤三中所述氧化石墨烯溶液的浓度为2～6mg/mL，滴加量为10-15μL,电沉积时间为5～8min；所述氯金酸溶液的浓度为1～3mM，滴加量为10-15μL，电沉积时间为3～5min；所述H 2SO 4浓度为0.3～0.6M，滴加量为20-30μL，活化时间为150-300s。
进一步的，步骤三中所述农药抗体的浓度为5～20μg/mL，滴加量为10-15μL，孵育时间为40～70min；所述牛血清蛋白的浓度为0.5％～2％，滴加量为10-15μL，孵育时间为30～60min。
一种所述双电场驱动传感器应用于农残的检测，包括以下步骤：
步骤一、建立电流/浓度(I/C)标准曲线：
配制一系列不同浓度的农药标准液滴加到所述的双电场驱动传感器的反应腔中的三电极体系表面进行孵育，同时在孵育过程中开启双通道电场发生装置，将孵育完成的传感器用于差分脉冲伏安法(DPV)测试，产生电流峰值；根据对应的农药浓度和峰值大小建立标准曲线；
步骤二、根据建立的I/C标准曲线计算出待测农药浓度：
在双电场驱动条件下，取农药样品液滴加在反应腔中的三电极体系表面进行孵育；孵育后进行DPV测试，在电化学窗口中产生一定的电流强度，根据建立的I/C标准曲线计算出待测农药浓度。
进一步的，步骤一中所述农药为毒死蜱、甲基对硫磷、水胺硫磷、吡虫啉、对氧磷、莠去津、三唑磷、多菌灵、噻虫啉或卡巴呋喃的任意一种，所述农药标准液的浓度为0.01～550μg/L， 滴加量为10-15μL，孵育时间为1～5min；
所述双通道电场发生装置的动态偏置电压设置为峰间电压的40～60％，峰间电压设置为2～6V。
进一步的，步骤二中所述农药样品液的滴加量为10-15μL，孵育时间为1～5min；所述双通道电场发生装置的动态偏置电压设置为峰间电压的40～60％，峰间电压设置为2～6V。
本发明还提供了上述双电场驱动传感器在实际样品农药残留快速检测中的应用。
与现有技术相比，本发明的有益效果是：
本发明所述的柔性印刷基底重量轻，可自由弯曲和折叠，便于携带，可根据灵活的设计实现电子元件组装和电路连接。
本发明所述的RGO和AuNPs材料的结合呈现出优良的导电性、高比表面积和优越的生物相容性。
本发明所设计的免疫传感器具有良好的选择性，有效克服了实际样品中其他组分的干扰。
本发明所述的双电场驱动模块向反应腔中提供了两个互补的电场，诱导溶液对流，有效的加速了农药分子向电极表面运动，使免疫反应在短时间内完成。
附图说明
图1为双电场驱动模块的电场分布示意图；
图2为本发明的双电场驱动传感器正(A)、反(B)实物图；
图3为工作电极的修饰过程；
图4中A为双电场驱动模块加速免疫反应图；B为单电场驱动模块加速免疫反应图；C为无电场驱动免疫反应图；
图5为甲基对硫磷测定的标准曲线图；
图中，1-柔性聚酰亚胺基板；2-导电孔；3-铜膜；4-铜箔线路；5-对电极；6-工作电极；7-参比电极；8-反应腔；9-铜箔片。
具体实施方式
为了方便理解本发明，下面结合实例详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。在本发明中除非另有限制，本说明书使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。一旦存在矛盾，可以根据具体情况理解本说明书中的定义。本发明所使用的反应腔8为透明塑料材质；
在不同电场驱动条件下甲基对硫磷孵育时间对照试验，包括以下步骤：
步骤一、柔性印刷基底的制作：利用计算机绘图软件设计电路形状；对柔性聚酰亚胺基板1进行裁剪(长：3cm，宽：1.5cm)；在柔性聚酰亚胺基板1的一端设置线路连接的5个 导电孔2并标记为N 1、N 2、N 3、N 4、N 5；在柔性聚酰亚胺基板1表面喷涂上一层均匀的厚度为20μm铜膜3；利用光刻技术将光致抗蚀剂线性酚醛树脂按照预先设计的电路形状涂覆在铜膜3上；将已涂覆光致抗蚀剂的柔性聚酰亚胺基板1放置紫外在灯下进行曝光；曝光后的柔性聚酰亚胺基板1利用0.2g/mL碳酸钠进行显影；显影后，未曝光区域被溶解，留下曝光区域形成铜箔线路4；将显影后的柔性聚酰亚胺基板1浸渍在4g/L氰化亚金钾(KAu(CN) 2)溶液中，利用电镀技术在相对导电孔2的另一端形成一个未闭合的厚度为0.3μm圆环金膜作为对电极5，对电极5圆环金膜开口的一端与导电孔N 1电性连接；在对电极5的内部区域设有由十个圆形金膜组成的工作电极6，其呈三叉戟的形状，三叉戟的每条边分别有三个圆形金膜，剩下的一个圆形金膜在三条边的交汇处，每个金膜之间电性连接，金膜交汇处与导电孔N 2之间电性连接；在对电极5未闭合圆环处的空白环形区域处涂覆厚度为0.5mm圆弧型Ag/AgCl浆料，放置在真空干燥箱75℃干燥40min后作为参比电极7，参比电极7与导电孔N 3电性连接；此时形成的对电极5、工作电极6、参比电极7称为三电极体系；在三电极体系所形成的区域表面上设有透明的中空塑料反应腔8，三电极体系均在中空塑料反应腔8的中空区域内，且与中空塑料反应腔8不接触；最终得到柔性印刷基底；
步骤二、双电场驱动模块的构建：在步骤一所制备的柔性印刷基底的三电极体系的背面电镀出两组独立的铜箔区，每个铜箔区域由12个独立的铜箔片9组成；每组铜箔区的面积分布可以通过相互焊接独立的铜箔片9来调整；两组铜箔区分别通过柔性聚酰亚胺基板1的导电孔2的N 4、N 5连接双通道信号发生器(SDG6052X,北京博宇讯铭科技有限公司)提供偏置正弦电场，设定动态偏置电压为3V，峰间电压设置为6V；采用双电场模块时开启双通道信号发生器，每个铜箔区被分配一组电场，对该三电极体系反应腔8的中空区域提供一组互补的电场环境；采用单电场模块时开启信号发生器单通道模式；
图1为双电场驱动模块的电场分布示意图；图2为本发明的双电场驱动传感器正反实物图。从图1可以看出反应腔中存在2组电场，电场1和电场2分别作用于两组独立的铜箔区。
步骤三、工作电极的修饰：首先制备4mg/mL的氧化石墨烯(GO)溶液；然后将15μL GO溶液滴加在反应腔8中的三电极体系表面，在电化学工作站进行电沉积7min，沉积后用蒸馏水冲洗工作电极6；其次制备1mM氯金酸(HAuCl 4)溶液，将12μL HAuCl 4溶液滴加在反应腔8中的三电极体系表面，在电化学工作站进行电沉积250s，沉积后用蒸馏水冲洗工作电极6；然后将25μL、0.5M硫酸(H 2SO 4)滴加在反应腔8中的三电极体系表面对工作电极进行活化；活化后用蒸馏水冲洗工作电极6，冲洗后向反应腔8中的三电极体系表面滴加12μL、15μg/mL甲基对硫磷抗体进行孵育50min，甲基对硫磷抗体孵育完成后用蒸馏水冲洗工作电极6；冲洗后继续向反应腔8中的三电极体系表面滴加15μL、1％的牛血清蛋白(BSA)孵育40min，BSA孵育完成后用蒸馏水冲洗工作电极6；图3为传感器工作电极功 能修饰过程，最终修饰完成得到双电场驱动传感器。
步骤四、不同电场驱动条件下甲基对硫磷孵育时间对照试验：将100μg/L、12μL甲基对硫磷滴入所述的双电场驱动传感器反应腔8中的三电极体系表面孵育；甲基对硫磷孵育过程分为三组，第一组开启双电场模块，第二组开启单电场模块，第三组禁用电场模块；在第一组中，每15s测定一次电流强度；在第二组中，每30s测定一次电流强度；在第三组中，每15min测定一次电流强度；当电流强度不再发生明显变化时记录孵育时间。
图4中A～C为分别采用双电场驱动、单电场驱动、无电场驱动条件下的甲基对硫磷孵育时间，依次为60s、180s、90min。从图4可以看出，在有电场驱动条件下，免疫反应的时间大大缩短。特别是所述双电场驱动模块呈现出比单电场模块更短的反应时间，这得益于反应体系中两个互补的电场环境加速了甲基对硫磷分子与抗体之间的反应速率。
实施例1：
以检测实际样品中的甲基对硫磷为例，一种双电场驱动传感器的制备方法和应用，包括以下步骤：
步骤一、柔性印刷基底的制作：利用计算机绘图软件设计电路形状；对柔性聚酰亚胺基板1进行裁剪(长：3cm，宽：1.5cm)；在柔性聚酰亚胺基板1的一端设置线路连接的5个导电孔2并标记为N 1、N 2、N 3、N 4、N 5；在柔性聚酰亚胺基板1表面喷涂上一层均匀的厚度为15μm铜膜3；利用光刻技术将光致抗蚀剂线性酚醛树脂按照预先设计的电路形状涂覆在铜膜3上；将已涂覆光致抗蚀剂的柔性聚酰亚胺基板1放置紫外在灯下进行曝光；曝光后的柔性聚酰亚胺基板1利用0.1g/mL碳酸钠进行显影；显影后，未曝光区域被溶解，留下曝光区域形成铜箔线路4；将显影后的柔性聚酰亚胺基板1浸渍在2g/L氰化亚金钾(KAu(CN) 2)溶液中，利用电镀技术在相对导电孔2的另一端形成一个未闭合的厚度为0.5μm圆环金膜作为对电极5，对电极5圆环金膜开口的一端与导电孔N 1电性连接；在对电极5的内部区域设有由十个圆形金膜组成的工作电极6，其呈三叉戟的形状，三叉戟的每条边分别有三个圆形金膜，剩下的一个圆形金膜在三条边的交汇处，每个金膜之间电性连接，金膜交汇处与导电孔N 2之间电性连接；在对电极5未闭合圆环处的空白环形区域处涂覆厚度为0.4mm圆弧型Ag/AgCl浆料，放置在真空干燥箱80℃干燥30min后作为参比电极7，参比电极7与导电孔N 3电性连接；此时形成的对电极5、工作电极6、参比电极7称为三电极体系；在三电极体系所形成的区域上设有透明的中空塑料反应腔8，且三电极体系在中空塑料反应腔8的中空区域内，且与中空塑料反应腔8不接触；最终得到柔性印刷基底；
步骤二、双电场驱动模块的构建：在步骤一所制备的柔性印刷基底的三电极体系的背面电镀出两组独立的铜箔区，每个铜箔区域由10个独立的铜箔片9组成；每组铜箔区的面积分布可以通过相互焊接独立的铜箔片9来调整；两组铜箔区分别通过柔性聚酰亚胺基板1的导 电孔2的N 4、N 5连接双通道信号发生器(EDU33212A,美国Keysight公司)提供偏置正弦电场，设定动态偏置电压为2V，峰间电压设置为5V；每个铜箔区被分配一组电场；在运行过程中开启双通道信号发生器，对该三电极体系反应腔8的中空区域提供一组互补的电场环境；
步骤三、工作电极的修饰：首先制备3mg/mL的氧化石墨烯(GO)溶液；然后将10μL GO溶液滴加在反应腔8中的三电极体系表面，在电化学工作站进行电沉积5min，沉积后用蒸馏水冲洗工作电极6；其次制备2mM氯金酸(HAuCl 4)溶液，将10μL HAuCl 4溶液滴加在反应腔8中的三电极体系表面，在电化学工作站进行电沉积200s，沉积后用蒸馏水冲洗工作电极6；然后将20μL、0.6M硫酸(H 2SO 4)滴加在反应腔8中的三电极体系表面对工作电极进行活化；活化后用蒸馏水冲洗工作电极6，冲洗后向反应腔8中的三电极体系表面滴加10μL、20μg/mL甲基对硫磷抗体进行孵育40min，甲基对硫磷抗体孵育完成后用蒸馏水冲洗工作电极6；冲洗后继续向反应腔8中的三电极体系表面滴加15μL、2％的牛血清蛋白(BSA)孵育30min、BSA孵育完成后用蒸馏水冲洗工作电极6；最终修饰完成得到双电场驱动传感器。
步骤四、建立电流/浓度(I/C)标准曲线：将一系列不同浓度的甲基对硫磷(0.01～550μg/L)滴加到所述的双电场驱动传感器的反应腔8中的三电极体系表面进行孵育1min，同时开启双通道电场发生装置；在电场力的作用下，甲基对硫磷分子在短时间内达到电极表面，被甲基对硫磷抗体快速捕获；将孵育完成的传感器用于DPV测试，产生电流峰值；根据对应的甲基对硫磷浓度和峰值大小建立标准曲线。图5为本发明所述甲基对硫磷测定的标准曲线图，所述I/C标准曲线为：I＝0.013C+0.303,R 2＝0.993，检测范围为0.1～300μg/L。
步骤五、实际样品的检测：
(1)白菜样品的预处理：取25g白菜样品，用去离子水清洗后切碎；切碎后的样品加入50mL乙腈放置在超声环境中30min进行萃取；将上清液用滤纸过滤后加入100mL容量瓶中，用甲醇定容至刻度线；得到农药样品液。
(2)根据建立的I/C标准曲线计算出待测甲基对硫磷浓度：在双电场驱动条件下，滴加12μL农药样品液在反应腔8中的三电极体系表面孵育1min，随后进行DPV测试，在电化学窗口中产生一定的电流强度，根据建立的I/C标准曲线计算出待测甲基对硫磷浓度。
为了进一步验证本发明所述一种双电场驱动免疫传感器的准确度和灵敏度，将本发明的所述体系与标准高效液相色谱(HPLC)进行对照。在进行HPLC检测前，将待测液用0.45μm滤膜过滤。在DPV试验和HPLC测定中，每个样品测量三次取平均值以减小随机误差。
结果如表1所示，本方法检测结果与HPLC结果的误差在5.2％以内，表明所建立的方法适用于实际样品的检测，并具有较好的准确性。另外，本方法的双电场驱动模块检测时间极 短(1min)，能够适用于实际样品的快速检测。
表1 本方法检测白菜样品与标准方法检测结果对比
Figure PCTCN2022095898-appb-000001
综上所述，本发明是首次使用电化学免疫传感器结合双电场驱动模块来检测农药残留的体系。自制的柔性印刷电极能够通过外部的设计形成特定的电路，且重量轻，可弯折，便于携带；在工作电极表面修饰的RGO和AuNPs有效的增加了传感器工作电极表面的导电性；农药抗体的引入使传感器在复杂的样品环境中表现出优良的选择性；农药孵育过程中引入互补的双电场有效的有效改善了吸附效率，加速免疫反应的进行。该双电场驱动电化学免疫传感器具有制作简单，灵敏度高，选择性强等优势，为自然环境，农产品等复杂样品中农药残留检测提供了新前景。
说明：以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案；因此，尽管本说明书参照上述的各个实施例对本发明已进行了详细的说明，但是本领域的普通技术人员应当理解，仍然可以对本发明进行修改或等同替换；而一切不脱离本发明的精神和范围的技术方案及其改进，其均应涵盖在本发明的权利要求范围内。

Claims (10)

  1. 一种双电场驱动传感器的制备方法，其特征在于，步骤如下：
    步骤一、利用计算机绘图软件设计电路形状；对柔性聚酰亚胺基板(1)进行裁剪，然后在柔性聚酰亚胺基板(1)的一端设置线路连接的N个导电孔(2)并标记为N 1、N 2、N 3……N n-1、N n，N为不小于5的正整数；在柔性聚酰亚胺基板(1)表面喷涂上一层均匀的铜膜(3)；利用光刻技术将光致抗蚀剂按照预先设计的电路形状涂覆在铜膜(3)上；将已涂覆光致抗蚀剂的柔性聚酰亚胺基板(1)放置紫外在灯下进行曝光；曝光后的柔性聚酰亚胺基板(1)利用化学显影剂进行显影；显影后，未曝光区域被溶解，留下曝光区域形成铜箔线路(4)；将显影后的柔性聚酰亚胺基板(1)浸渍在氰化亚金钾溶液中，利用电镀技术在相对导电孔(2)的另一端形成一个未闭合的圆环金膜作为对电极(5)，对电极(5)圆环金膜开口的一端与导电孔N 1电性连接；在对电极(5)的内部区域设有工作电极(6)，与导电孔N 2之间电性连接；在对电极(5)未闭合圆环处的空白环形区域处涂覆圆弧型Ag/AgCl浆料，干燥后作为参比电极(7)，参比电极(7)与导电孔N 3电性连接；此时形成的对电极(5)、工作电极(6)、参比电极(7)称为三电极体系；在柔性聚酰亚胺基板(1)表面上设有中空反应腔(8)，三电极体系均在中空反应腔(8)的中空区域内；且与中空反应腔(8)不接触；最终得到柔性印刷基底；
    步骤二、在步骤一所制备的柔性印刷基底的三电极体系的背面电镀出两组独立的铜箔区，每个铜箔区域由M个独立的铜箔片(9)组成，M为正整数；；每组铜箔区的面积分布可以通过铜箔片(9)来调整；两组铜箔区分别通过柔性聚酰亚胺基板(1)的导电孔(2)N 4、N 5连接双通道信号发生器，提供偏置正弦电场；设定动态偏置电压和峰间电压；每个铜箔区被分配一组电场；在运行过程中开启双通道信号发生器，对该三电极体系反应腔(8)的中空区域提供一组互补的电场环境；
    步骤三、首先制备氧化石墨烯溶液；然后将氧化石墨烯溶液滴加在反应腔(8)中的三电极体系表面，在电化学工作站进行电沉积，沉积后用蒸馏水冲洗工作电极(6)；其次制备氯金酸溶液，将氯金酸溶液滴加在反应腔(8)中的三电极体系表面，在电化学工作站进行电沉积，沉积后用蒸馏水冲洗工作电极(6)；然后将硫酸滴加在反应腔(8)中的三电极体系表面对工作电极(6)进行活化；活化后用蒸馏水冲洗工作电极(6)，冲洗后向反应腔(8)中的三电极体系表面滴加农药抗体进行孵育，农药抗体孵育完成后用蒸馏水冲洗工作电极(6)；冲洗后继续向反应腔(8)中的三电极体系表面滴加牛血清蛋白进行孵育，孵育完成后用蒸馏水冲洗工作电极(6)；最终得到双电场驱动传感器。
  2. 根据权利要求1所述的双电场驱动传感器的制备方法，其特征在于，步骤一中所述铜膜厚度为15～25μm；所述光致抗蚀剂为线性酚醛树脂、聚甲基丙烯酸甲酯中的一种；所述化学显影剂为碳酸钠或碳酸钾中的一种，浓度为0.1～0.4g/mL；所氰化亚金钾溶液的浓度为1～5g/L。
  3. 根据权利要求1所述的双电场驱动传感器的制备方法，其特征在于，步骤一中所述圆环金膜厚度为0.2～0.7μm；所述工作电极(6)呈三叉戟的形状，由十个电性连接的圆形金膜组成；三叉戟的每条边分别有三个圆形金膜，剩下的一个圆形金膜在三条边的交汇处，交汇处的圆形金膜与导电孔N 2之间电性连接；
    所述弧型Ag/AgCl厚度为0.1～0.5mm；所述干燥的温度为70～90℃，干燥的时间为30～40min。
  4. 根据权利要求1所述的双电场驱动传感器的制备方法，其特征在于，步骤二中所述铜箔片数量M为10～20之间的偶数，铜箔片形状为正方形，边长为1～1.5mm。
  5. 根据权利要求1所述的双电场驱动传感器的制备方法，其特征在于，步骤二中所述双通道信号发生器为任意型号的具有双通道电场发生装置，动态偏置电压设置为峰间电压的40～60％，峰间电压设置为2～6V。
  6. 根据权利要求1所述的双电场驱动传感器的制备方法，其特征在于，步骤三中所述氧化石墨烯溶液的浓度为2～6mg/mL，滴加量为10-15μL，电沉积时间为5～8min；所述氯金酸溶液的浓度为1～3mM，滴加量为10-15μL，电沉积时间为3～5min；所述硫酸的浓度为0.3～0.6M，滴加量为20-30μL，活化时间为150-300s。
  7. 根据权利要求1所述的双电场驱动传感器的制备方法，其特征在于，步骤三中所述农药抗体的浓度为5～20μg/mL，滴加量为10-15μL，孵育时间为40～70min；所述牛血清蛋白的浓度为0.5％～2％，滴加量为10-15μL，孵育时间为30～60min。
  8. 根据权利要求1-7任一项所述方法制备的双电场驱动传感器用于检测农药残留的用途，其特征在于，步骤如下：
    步骤一、建立电流/浓度标准曲线：
    配制一系列不同浓度的农药标准液滴加到所述的双电场驱动传感器的反应腔(8)中的三电极体系表面进行孵育，同时在孵育过程中开启双通道电场发生装置，将孵育完成的传感器用于差分脉冲伏安法测试，产生电流峰值；根据对应的农药浓度和峰值大小建立标准曲线；
    步骤二、在双电场驱动条件下，取农药样品液滴加在反应腔(8)中的三电极体系表面进行孵育；孵育后进行DPV测试，在电化学窗口中产生一定的电流强度，根据建立的I/C标准曲线计算出待测农药浓度。
  9. 根据权利要求8所述的用途，其特征在于，步骤一中所述农药为毒死蜱、甲基对硫磷、水胺硫磷、吡虫啉、对氧磷、莠去津、三唑磷、多菌灵、噻虫啉或卡巴呋喃的任意一种，所述农药标准液的浓度为0.01～550μg/L，滴加量为10-15μL，孵育时间为1～5min；
    所述双通道电场发生装置的动态偏置电压设置为峰间电压的40～60％，峰间电压设置为2～6V。
  10. 根据权利要求8所述的用途，其特征在于，步骤二中所述农药样品液的滴加量为10-15 μL，孵育时间为1～5min；所述双通道电场发生装置的动态偏置电压设置为峰间电压的40～60％，峰间电压设置为2～6V。
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CN117088985A (zh) * 2023-05-31 2023-11-21 南京大学 一种基于bret的甲基对硫磷生物传感器、构建方法及其应用
CN117088985B (zh) * 2023-05-31 2024-05-10 南京大学 一种基于bret的甲基对硫磷生物传感器、构建方法及其应用

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