CN114660151B - Preparation method and application of double-electric-field driving sensor - Google Patents
Preparation method and application of double-electric-field driving sensor Download PDFInfo
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Abstract
The invention belongs to the field of detecting pesticide residues by using electrochemical sensors, and particularly relates to a preparation method and application of a double-electric-field driving sensor. Firstly, preparing a flexible printing substrate, and effectively adjusting the double-electric-field distribution according to a plurality of electrode arrays on a flexible printing electrode; the sensor is endowed with good biocompatibility, catalytic performance and conductivity by modifying RGO and gold nanoparticles on the surface of the electrode. Meanwhile, pesticide antibodies are introduced to selectively identify pesticide molecules, double electric field driving is applied, micro-flow and particles in the solution can move directionally, and analytes can flow to the surface of the biosensor quickly, so that the combination efficiency of the analytes and a sensor surface probe is improved, and the reaction time is shortened. The complementary double electric field mode can effectively prevent the bubbles from being generated on the surface of the sensor due to overlarge voltage, so that a more effective electric field enhancement strategy is established, and the sensitivity and the detection efficiency of the sensor are improved. The rapid and selective detection of pesticide residues in natural samples is realized.
Description
Technical Field
The invention belongs to the field of detecting pesticide residues by using electrochemical sensors, and particularly relates to a preparation method and application of a double-electric-field driving sensor.
Background
Pesticides are substances or mixtures for preventing, controlling and reducing harmful organisms, and with the development of modern agriculture, the application of pesticides has become one of essential links in crop production. However, excessive use of pesticides can cause groundwater and food contamination, with serious consequences for humans and animals. Prolonged exposure to pesticides in humans can lead to deleterious effects on health, such as neurotoxicity, genotoxicity, mutagenicity and carcinogenicity. Although various laboratory-based analytical methods such as gas chromatography, high performance liquid chromatography, mass spectrometry and enzyme-linked immunosorbent assay have been widely used, they all suffer from their respective disadvantages such as the use of expensive instruments, time-consuming procedures and the need for trained personnel. Electrochemical immunosensors have the advantages of strong specificity, high sensitivity and the like, and are often used as effective detection methods. However, the above techniques rely on the reaction of the receptor with the target molecule, which occurs through diffusion-dominated transport kinetics. In practical samples where the concentration of the target analyte in the complex matrix is low, the immune reaction may take several hours or even days to proceed, and in extreme cases may not even occur, which is not conducive to rapid and sensitive detection in practical samples.
At present, no forming device for realizing rapid detection of pesticide residues by using a double-electric-field driven electrochemical sensor exists in the market. In the existing technology for detecting pesticide residues by using an electrochemical immunosensor, a patent ' a preparation method of a methyl parathion immunosensor based on combination of two electrochemical methods and an application ' CN105806922A ' discloses a preparation method of a biological immunosensor based on a functional nano material and a method for detecting methyl parathion by using the sensor. The sensor obtained by the method is convenient to carry and low in cost. However, the sensor is complex in manufacturing process, long in sensing time and not suitable for rapid detection.
Disclosure of Invention
Aiming at the problems of complex manufacture, low sensitivity, slow response speed and the like of the traditional technology for detecting pesticide residues by using an electrochemical immunosensor, the invention discloses a preparation method of a double-electric-field driving sensor and application thereof.
The preparation of the double electric field driving sensor comprises the steps of manufacturing a flexible printing substrate, constructing a double electric field driving module and modifying a working electrode. The invention adopts the flexible printed circuit technology to manufacture the flexible printed substrate, thereby reducing the manufacturing cost of the sensor. The double-electric-field distribution can be effectively adjusted according to the plurality of electrode arrays on the flexible printed electrode. The sensor is endowed with good biocompatibility, excellent catalytic performance and conductivity by modifying Reduced Graphene Oxide (RGO) and gold nanoparticles (AuNPs) on the surface of the electrode. Meanwhile, pesticide antibodies are introduced to selectively identify pesticide molecules. Under the condition of external double-electric-field driving, micro-flow and particles in the solution can move directionally, and analytes can flow to the surface of the biosensor quickly, so that the binding efficiency of the analytes and the antibody on the surface of the sensor is improved, and the reaction time of the immunosensor is greatly shortened. Especially, the complementary double electric field mode can effectively prevent the bubbles from being generated on the surface of the sensor due to overlarge voltage, so that a more effective electric field enhancement strategy is established, and the sensitivity and the detection efficiency of the sensor are improved. The double-electric-field driving sensor designed according to the principle realizes selective detection of pesticide residues in natural samples, and has the advantages of simple manufacture, low cost, easy carrying, short detection time and the like.
The invention is realized by the following technical scheme:
a preparation method of a double electric field driving sensor comprises the following steps: the method comprises the steps of manufacturing a flexible printing substrate, constructing a double electric field driving module and modifying a working electrode.
Step one, manufacturing a flexible printing substrate:
designing the shape of the circuit by using computer drawing software; cutting the flexible polyimide substrate; one end of the flexible polyimide substrate is provided with N conductive holes which are connected by circuits and marked as N 1 、N 2 、N 3 ……N n-1 、N n N is a positive integer not less than 5; spraying a layer of uniform copper film on the surface of the flexible polyimide substrate; coating a photoresist on the copper film according to a pre-designed circuit shape by utilizing a photoetching technology; placing the flexible polyimide substrate coated with the photoresist under ultraviolet light for exposure; developing the exposed flexible polyimide substrate by using a chemical developer; after developing, dissolving the unexposed area, and leaving the exposed area to form a copper foil circuit; will be provided withThe developed flexible polyimide substrate was immersed in potassium aurous cyanide (KAu (CN) 2 ) In the solution, an unclosed annular gold film is formed at the other end opposite to the conductive hole by electroplating technology to serve as a counter electrode, and the open end of the annular gold film of the counter electrode is connected with the conductive hole N 1 Electrically connecting; a working electrode and a conductive hole N are arranged in the inner region of the counter electrode 2 Are electrically connected with each other; coating arc Ag/AgCl slurry on the blank annular region of the unclosed annular ring of the counter electrode, drying, and using as a reference electrode, the reference electrode and the conductive hole N 3 Electrically connecting; the counter electrode, the working electrode and the reference electrode formed at this time are called a three-electrode system; a hollow reaction cavity is arranged on the surface of the flexible polyimide substrate, and the three-electrode system is arranged in the hollow area of the hollow reaction cavity; and is not contacted with the hollow reaction cavity; finally obtaining a flexible printing substrate;
step two, constructing a double electric field driving module: electroplating two groups of independent copper foil areas on the back surface of the three-electrode system of the flexible printing substrate prepared in the step one, wherein each copper foil area consists of M independent copper foils, and M is a positive integer; the area distribution of each group of copper foil area can be adjusted through the copper foil; the two groups of copper foil areas pass through the conductive holes N of the flexible polyimide substrate respectively 4 、N 5 The double-channel signal generator is connected to provide a bias sine electric field; setting a dynamic bias voltage and an inter-peak voltage; each copper foil area is distributed with a group of electric fields; in the operation process, a dual-channel signal generator is started to provide a group of complementary electric field environments for the hollow area of the reaction cavity of the three-electrode system;
step three, modification of the working electrode:
firstly, preparing a Graphene Oxide (GO) solution; then dripping GO solution on the surface of a three-electrode system in a reaction cavity, carrying out electrodeposition in an electrochemical workstation, and washing a working electrode with distilled water after the electrodeposition; second, preparation of chloroauric acid (HAuCl) 4 ) Solution of HAuCl 4 Dropwise adding the solution on the surface of a three-electrode system in the reaction cavity, performing electrodeposition in an electrochemical workstation, and washing the working electrode with distilled water after the electrodeposition; then sulfuric acid (H) 2 SO 4 ) Three electrodes dripped into reaction cavityActivating the working electrode on the surface of the system; washing the working electrode with distilled water after activation, dripping pesticide antibody on the surface of the three-electrode system in the reaction cavity for incubation after washing, and washing the working electrode with distilled water after the pesticide antibody incubation is finished; continuously dripping Bovine Serum Albumin (BSA) on the surface of the three-electrode system in the reaction cavity for incubation after washing, and washing the working electrode with distilled water after the BSA incubation is finished; and finally obtaining the double electric field driving sensor.
Further, the thickness of the copper film in the first step is 15-25 μm; the photoresist is one of linear phenolic resin and polymethyl methacrylate; the chemical developer is one of sodium carbonate or potassium carbonate, and the concentration is 0.1-0.4 g/mL; the concentration of the potassium aurous cyanide solution is 1-5 g/L.
Further, the thickness of the annular gold film in the first step is 0.2-0.7 μm; the working electrode is in the shape of a tridentate and consists of ten electrically connected circular gold films; each edge of the trident is respectively provided with three circular gold films, the remaining circular gold film is arranged at the junction of the three edges, and the circular gold film at the junction and the conductive hole N are arranged 2 Are electrically connected with each other;
the thickness of the arc Ag/AgCl is 0.1-0.5 mm; the drying temperature is 70-90 ℃, and the drying time is 30-40 min.
Furthermore, in the second step, the number M of the copper foils is an even number between 10 and 20, the copper foils are square, and the side length is 1 to 1.5mm.
Furthermore, the dual-channel signal generator in the second step is any type of device with a dual-channel electric field, the dynamic bias voltage is set to be 40-60% of the peak-to-peak voltage, and the peak-to-peak voltage is set to be 2-6V.
Further, in the third step, the concentration of the graphene oxide solution is 2-6 mg/mL, the dropping amount is 10-15 mu L, and the electrodeposition time is 5-8 min; the concentration of the chloroauric acid solution is 1-3 mM, the dripping amount is 10-15 mu L, and the electrodeposition time is 3-5 min; said H 2 SO 4 The concentration is 0.3-0.6M, the dripping amount is 20-30 mu L, and the activation time is 150-300s.
Further, the concentration of the pesticide antibody in the third step is 5-20 mug/mL, the dripping amount is 10-15 mug L, and the incubation time is 40-70 min; the concentration of the bovine serum albumin is 0.5-2%, the dropping amount is 10-15 mu L, and the incubation time is 30-60 min.
The double-electric-field driving sensor is applied to detection of pesticide residues, and comprises the following steps:
step one, establishing a current/concentration (I/C) standard curve:
preparing a series of pesticide standard liquid drops with different concentrations, adding the pesticide standard liquid drops to the surface of a three-electrode system in a reaction cavity of the double electric field driving sensor for incubation, simultaneously starting a double-channel electric field generating device in the incubation process, and using the incubated sensor for a Differential Pulse Voltammetry (DPV) test to generate a current peak value; establishing a standard curve according to the corresponding pesticide concentration and the peak value;
step two, calculating the concentration of the pesticide to be detected according to the established I/C standard curve:
under the condition of double electric field driving, pesticide sample liquid is dripped on the surface of a three-electrode system in a reaction cavity for incubation; and performing DPV test after incubation, generating a certain current intensity in an electrochemical window, and calculating the concentration of the pesticide to be tested according to the established I/C standard curve.
Further, in the first step, the pesticide is any one of chlorpyrifos, methyl parathion, isocarbophos, imidacloprid, paraoxon, atrazine, triazophos, carbendazim, thiacloprid or carbarfan, the concentration of the standard pesticide solution is 0.01-550 mu g/L, the dropping amount is 10-15 mu L, and the incubation time is 1-5 min;
the dynamic bias voltage of the dual-channel electric field generating device is set to be 40-60% of peak-to-peak voltage, and the peak-to-peak voltage is set to be 2-6V.
Further, the dripping amount of the pesticide sample liquid in the step two is 10-15 mu L, and the incubation time is 1-5 min; the dynamic bias voltage of the dual-channel electric field generating device is set to be 40-60% of peak-to-peak voltage, and the peak-to-peak voltage is set to be 2-6V.
The invention also provides application of the double-electric-field driving sensor in rapid detection of pesticide residues in actual samples.
Compared with the prior art, the invention has the beneficial effects that:
the flexible printing substrate is light in weight, can be freely bent and folded, is convenient to carry, and can realize electronic element assembly and circuit connection according to flexible design.
The combination of the RGO and AuNPs materials of the present invention exhibits excellent conductivity, high specific surface area and excellent biocompatibility.
The immunosensor designed by the invention has good selectivity, and effectively overcomes the interference of other components in actual samples.
The double-electric-field driving module provides two complementary electric fields for the reaction cavity, induces solution convection, effectively accelerates pesticide molecules to move to the surface of the electrode, and enables immune reaction to be completed in a short time.
Drawings
FIG. 1 is a schematic diagram of electric field distribution of a dual electric field driving module;
FIG. 2 is a front (A) and back (B) diagram of the dual electric field driven sensor of the present invention;
FIG. 3 is a modification process of a working electrode;
FIG. 4A is a diagram of the two electric field driving module for accelerating the immune response; b is a single electric field driving module accelerated immunoreaction diagram; c is an immune response map driven by no electric field;
FIG. 5 is a standard graph of a methyl parathion assay;
in the figure, 1-flexible polyimide substrate; 2-a conductive via; 3-copper film; 4-copper foil circuit; 5-a pair of electrodes; 6-a working electrode; 7-a reference electrode; 8-a reaction chamber; 9-copper foil.
Detailed Description
For the purpose of facilitating an understanding of the invention, reference will now be made in detail to embodiments of the invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. The reaction chamber 8 used in the invention is made of transparent plastic material;
a control test of the incubation time of methyl parathion under different electric field driving conditions comprises the following steps:
the method comprises the following steps of firstly, manufacturing a flexible printing substrate: designing the shape of the circuit by using computer drawing software; cutting the flexible polyimide substrate 1 (length: 3cm, width: 1.5 cm); one end of a flexible polyimide substrate 1 is provided with 5 conductive holes 2 connected by lines and marked as N 1 、N 2 、N 3 、N 4 、N 5 (ii) a Spraying a copper film 3 with the uniform thickness of 20 mu m on the surface of the flexible polyimide substrate 1; coating photoresist linear phenolic resin on the copper film 3 according to a pre-designed circuit shape by utilizing a photoetching technology; placing the flexible polyimide substrate 1 coated with the photoresist under ultraviolet light for exposure; developing the exposed flexible polyimide substrate 1 by using 0.2g/mL sodium carbonate; after development, the unexposed area is dissolved, and the exposed area is left to form a copper foil circuit 4; the flexible polyimide substrate 1 after development was immersed in 4g/L of potassium aurous cyanide (KAu (CN) 2 ) In the solution, an unclosed annular gold film with a thickness of 0.3 μm is formed at the other end of the conductive hole 2 by electroplating technology as a counter electrode 5, and the open end of the annular gold film of the counter electrode 5 and the conductive hole N are connected 1 Electrically connecting; the working electrode 6 composed of ten circular gold films is arranged in the inner area of the counter electrode 5 and is in the shape of a trident, each edge of the trident is provided with three circular gold films, the remaining circular gold film is arranged at the junction of the three edges, each gold film is electrically connected, and the junction of the gold films and the conductive hole N are arranged 2 Are electrically connected with each other; coating arc Ag/AgCl slurry with thickness of 0.5mm on the blank annular region of the unclosed annular ring of the counter electrode 5, drying at 75 deg.C for 40min in a vacuum drying oven to obtain a reference electrode 7, the reference electrode 7 and the conductive hole N 3 Electrically connecting; the counter electrode 5, the working electrode 6, and the reference electrode 7 formed at this time are referred to as a three-electrode system; in the three electrode bodiesA transparent hollow plastic reaction cavity 8 is arranged on the surface of the area formed by the system, and the three-electrode system is arranged in the hollow area of the hollow plastic reaction cavity 8 and is not contacted with the hollow plastic reaction cavity 8; finally obtaining a flexible printing substrate;
step two, constructing a double electric field driving module: electroplating two groups of independent copper foil areas on the back surface of the three-electrode system of the flexible printing substrate prepared in the step one, wherein each copper foil area consists of 12 independent copper foil sheets 9; the area distribution of each group of copper foil area can be adjusted by mutually welding the independent copper foils 9; two groups of copper foil areas respectively pass through N of the conductive holes 2 of the flexible polyimide substrate 1 4 、N 5 Connecting a double-channel signal generator (SDG 6052X, beijing Boyu communication Ming technology Co., ltd.) to provide a bias sinusoidal electric field, setting the dynamic bias voltage to be 3V, and setting the peak-to-peak voltage to be 6V; when a double-channel signal generator is started by adopting a double-electric-field module, each copper foil area is distributed with a group of electric fields, and a group of complementary electric field environments are provided for the hollow area of the reaction cavity 8 of the three-electrode system; starting a single-channel mode of the signal generator when a single electric field module is adopted;
FIG. 1 is a schematic diagram of electric field distribution of a dual electric field driving module; FIG. 2 is a front and back view of the dual electric field driven sensor of the present invention. It can be seen from fig. 1 that there are 2 sets of electric fields in the reaction chamber, and the electric field 1 and the electric field 2 act on two independent sets of copper foil regions respectively.
Step three, modification of the working electrode: firstly, preparing a 4mg/mL Graphene Oxide (GO) solution; then dripping 15 mu LGO solution on the surface of a three-electrode system in the reaction chamber 8, carrying out electrodeposition for 7min in an electrochemical workstation, and washing the working electrode 6 with distilled water after the electrodeposition; next, 1mM chloroauric acid (HAuCl) was prepared 4 ) Solution, 12. Mu.L of HAuCl 4 Dropwise adding the solution on the surface of a three-electrode system in the reaction cavity 8, performing electrodeposition for 250s in an electrochemical workstation, and washing the working electrode 6 with distilled water after the electrodeposition; then 25. Mu.L of 0.5M sulfuric acid (H) 2 SO 4 ) The surface of a three-electrode system dripped in the reaction cavity 8 activates the working electrode; washing the working electrode 6 with distilled water after activation, and dripping 12 mu L and 15 mu g/mL methyl-p-sulfur on the surface of the three-electrode system in the reaction cavity 8 after washingIncubating the phosphorus antibody for 50min, and washing the working electrode 6 with distilled water after the incubation of the methyl parathion antibody is finished; after washing, continuously dripping 15 mu L of 1% Bovine Serum Albumin (BSA) on the surface of the three-electrode system in the reaction cavity 8 for incubation for 40min, and washing the working electrode 6 with distilled water after the BSA incubation is finished; FIG. 3 shows a functional modification process of a working electrode of a sensor, and a double electric field driving sensor is obtained after the final modification.
Step four, carrying out comparison test on the parathion-methyl incubation time under different electric field driving conditions: dripping 100 mu g/L and 12 mu L of methyl parathion on the surface of a three-electrode system in the double-electric-field driving sensor reaction cavity 8 for incubation; the methyl parathion incubation process is divided into three groups, wherein the first group starts a double electric field module, the second group starts a single electric field module, and the third group disables the electric field module; in the first group, the current intensity was measured every 15 s; in the second group, the current intensity was measured every 30 s; in the third group, the current intensity was measured every 15 min; the incubation time was recorded when no significant change in current intensity occurred anymore.
In FIG. 4, A-C are the incubation times of methyl parathion under the conditions of double electric field driving, single electric field driving and no electric field driving, which are 60s, 180s and 90min in sequence. As can be seen from FIG. 4, the time of the immune reaction is greatly shortened under the driving condition of the electric field. In particular, the dual electric field driving module exhibits a shorter reaction time than the single electric field module, which is benefited from the fact that the reaction rate between methyl parathion molecules and antibodies is accelerated by two complementary electric field environments in the reaction system.
Example 1:
taking methyl parathion in an actual sample as an example, a preparation method and application of a double electric field driving sensor comprise the following steps:
step one, manufacturing a flexible printing substrate: designing the shape of the circuit by using computer drawing software; cutting the flexible polyimide substrate 1 (length: 3cm, width: 1.5 cm); one end of a flexible polyimide substrate 1 is provided with 5 conductive holes 2 which are connected by circuits and marked as N 1 、N 2 、N 3 、N 4 、N 5 (ii) a Spraying a layer on the surface of a flexible polyimide substrate 1A copper film 3 with a uniform layer thickness of 15 μm; coating photoresist linear phenolic resin on the copper film 3 according to a pre-designed circuit shape by utilizing a photoetching technology; placing the flexible polyimide substrate 1 coated with the photoresist under ultraviolet light for exposure; developing the exposed flexible polyimide substrate 1 by using 0.1g/mL sodium carbonate; after development, the unexposed area is dissolved, and the exposed area is left to form a copper foil circuit 4; the developed flexible polyimide substrate 1 was immersed in 2g/L of potassium aurous cyanide (KAu (CN) 2 ) In the solution, an unclosed annular gold film with a thickness of 0.5 μm is formed at the other end of the conductive hole 2 by electroplating technology as a counter electrode 5, and the open end of the annular gold film of the counter electrode 5 and the conductive hole N are connected 1 Electrically connecting; the working electrode 6 composed of ten circular gold films is arranged in the inner area of the counter electrode 5 and is in the shape of a trident, each edge of the trident is provided with three circular gold films, the remaining circular gold film is arranged at the junction of the three edges, each gold film is electrically connected, and the junction of the gold films and the conductive hole N are arranged 2 Are electrically connected with each other; coating arc Ag/AgCl slurry with thickness of 0.4mm on the blank annular region of the unclosed annular ring of the counter electrode 5, drying at 80 deg.C for 30min in a vacuum drying oven to obtain a reference electrode 7, the reference electrode 7 and the conductive hole N 3 Electrically connecting; the counter electrode 5, the working electrode 6, and the reference electrode 7 formed at this time are referred to as a three-electrode system; a transparent hollow plastic reaction cavity 8 is arranged in the region formed by the three-electrode system, and the three-electrode system is arranged in the hollow region of the hollow plastic reaction cavity 8 and is not in contact with the hollow plastic reaction cavity 8; finally obtaining a flexible printing substrate;
step two, constructing a double electric field driving module: electroplating two groups of independent copper foil areas on the back surface of the three-electrode system of the flexible printing substrate prepared in the step one, wherein each copper foil area consists of 10 independent copper foil sheets 9; the area distribution of each group of copper foil area can be adjusted by mutually welding the independent copper foils 9; two groups of copper foil areas respectively pass through N of the conductive holes 2 of the flexible polyimide substrate 1 4 、N 5 A two-channel signal generator (EDU 33212A, keysight Inc. USA) is connected to provide a bias sinusoidal electric field, the dynamic bias voltage is set to be 2V,the peak-to-peak voltage was set to 5V; each copper foil area is distributed with a group of electric fields; in the operation process, a dual-channel signal generator is started to provide a group of complementary electric field environments for the hollow area of the reaction cavity 8 of the three-electrode system;
step three, modification of the working electrode: firstly, preparing a 3mg/mL Graphene Oxide (GO) solution; then dripping 10 mu LGO solution on the surface of a three-electrode system in the reaction chamber 8, carrying out electro-deposition for 5min in an electrochemical workstation, and washing the working electrode 6 with distilled water after deposition; next, 2mM chloroauric acid (HAuCl) was prepared 4 ) Solution, 10. Mu.L of HAuCl 4 Dropwise adding the solution on the surface of a three-electrode system in the reaction cavity 8, performing electrodeposition for 200s in an electrochemical workstation, and washing the working electrode 6 with distilled water after the electrodeposition; then 20. Mu.L of 0.6M sulfuric acid (H) 2 SO 4 ) Dropwise adding the surface of a three-electrode system in the reaction cavity 8 to activate the working electrode; washing the working electrode 6 with distilled water after activation, dripping 10 mu L and 20 mu g/mL of methyl parathion antibody on the surface of the three-electrode system in the reaction cavity 8 after washing for incubation for 40min, and washing the working electrode 6 with distilled water after the incubation of the methyl parathion antibody is finished; after washing, continuously dripping 15 mu L of 2% Bovine Serum Albumin (BSA) on the surface of the three-electrode system in the reaction cavity 8, incubating for 30min, and washing the working electrode 6 with distilled water after the BSA incubation is finished; and finally finishing modification to obtain the double-electric-field driving sensor.
Step four, establishing a current/concentration (I/C) standard curve: dripping a series of methyl parathion (0.01-550 mu g/L) with different concentrations on the surface of a three-electrode system in a reaction cavity 8 of the double-electric-field driving sensor for incubation for 1min, and simultaneously starting a double-channel electric field generating device; under the action of electric field force, methyl parathion molecules reach the surface of the electrode in a short time and are rapidly captured by methyl parathion antibodies; using the incubated sensor for DPV test to generate a current peak value; and establishing a standard curve according to the corresponding concentration and peak value of the methyl parathion. FIG. 5 is a standard curve chart of the methyl parathion measurement, and the I/C standard curve is as follows: i =0.013C +0.303 2 And the detection range is 0.1-300 mu g/L, wherein the detection range is 0.993.
Step five, detecting an actual sample:
(1) Pretreating a cabbage sample: taking 25g of cabbage samples, washing the cabbage samples with deionized water, and then cutting the cabbage samples into pieces; adding 50mL of acetonitrile into the minced sample, and placing the mixture in an ultrasonic environment for 30min for extraction; filtering the supernatant with filter paper, adding into a 100mL volumetric flask, and diluting to a constant volume with methanol to a scale mark; and obtaining the pesticide sample liquid.
(2) And (3) calculating the concentration of the methyl parathion to be detected according to the established I/C standard curve: under the condition of double electric field driving, 12 mu L of pesticide sample liquid is dripped on the surface of a three-electrode system in the reaction cavity 8 for incubation for 1min, then DPV test is carried out, certain current intensity is generated in an electrochemical window, and the concentration of the methyl parathion to be tested is calculated according to the established I/C standard curve.
To further verify the accuracy and sensitivity of a dual electric field driven immunosensor of the present invention, the system of the present invention was compared to standard High Performance Liquid Chromatography (HPLC). The test solution was filtered through a 0.45 μm filter before HPLC analysis. In the DPV test and HPLC assay, three measurements per sample were averaged to reduce random errors.
The result is shown in table 1, the error between the detection result of the method and the HPLC result is within 5.2%, and the established method is suitable for detecting actual samples and has better accuracy. In addition, the detection time of the double electric field driving module is extremely short (1 min), and the method can be suitable for rapid detection of actual samples.
TABLE 1 comparison of the results of the test of cabbage samples by the method and the standard method
In conclusion, the invention is a system for detecting pesticide residues by combining the electrochemical immunosensor with the double-electric-field driving module for the first time. The self-made flexible printed electrode can form a specific circuit through external design, and is light in weight, bendable and convenient to carry; the RGO and AuNPs modified on the surface of the working electrode effectively increase the conductivity of the surface of the working electrode of the sensor; the introduction of the pesticide antibody enables the sensor to show excellent selectivity in a complex sample environment; the complementary double electric fields are introduced in the pesticide incubation process, so that the adsorption efficiency is effectively improved, and the immunoreaction is accelerated. The double-electric-field-driven electrochemical immunosensor has the advantages of being simple to manufacture, high in sensitivity, strong in selectivity and the like, and provides a new prospect for detecting pesticide residues in complex samples such as natural environments and agricultural products.
Description of the invention: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the various embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (10)
1. A preparation method of a double electric field driving sensor is characterized by comprising the following steps:
designing a circuit shape by using computer drawing software; cutting a flexible polyimide substrate (1), and arranging N conductive holes (2) connected by a circuit at one end of the flexible polyimide substrate (1) and marking the conductive holes as N 1 、N 2 、N 3 ……N n-1 、N n N is a positive integer not less than 5; spraying a layer of uniform copper film (3) on the surface of the flexible polyimide substrate (1); coating photoresist on the copper film (3) according to a pre-designed circuit shape by utilizing a photoetching technology; placing the flexible polyimide substrate (1) coated with the photoresist under ultraviolet light for exposure; developing the exposed flexible polyimide substrate (1) by using a chemical developer; after development, the unexposed area is dissolved, and the exposed area is left to form a copper foil circuit (4); dipping the developed flexible polyimide substrate (1) in a potassium aurous cyanide solution, forming an unclosed annular gold film at the other end opposite to the conductive hole (2) by utilizing an electroplating technology to serve as a counter electrode (5), wherein one end of the annular gold film of the counter electrode (5) is opened and the conductive hole N 1 Electrically connecting; at the counter electrode(5) Is provided with a working electrode (6) and a conductive hole N 2 Are electrically connected with each other; coating arc-shaped Ag/AgCl slurry on a blank annular region at the position of an unclosed annular ring of the counter electrode (5), drying to obtain a reference electrode (7), and using the reference electrode (7) and the conductive hole N 3 Electrically connecting; the counter electrode (5), the working electrode (6) and the reference electrode (7) formed at this time are called a three-electrode system; a hollow reaction cavity (8) is arranged on the surface of the flexible polyimide substrate (1), and three electrode systems are all arranged in the hollow area of the hollow reaction cavity (8); and is not in contact with the hollow reaction chamber (8); finally obtaining a flexible printing substrate;
step two, electroplating two groups of independent copper foil areas on the back surface of the three-electrode system of the flexible printing substrate prepared in the step one, wherein each copper foil area consists of M independent copper foil sheets (9), and M is a positive integer; the area distribution of each group of copper foil area can be adjusted through a copper foil (9); the two groups of copper foil areas pass through the conductive holes (2) N of the flexible polyimide substrate (1) respectively 4 、N 5 The double-channel signal generator is connected to provide a bias sine electric field; setting a dynamic bias voltage and an inter-peak voltage; each copper foil area is distributed with a group of electric fields; in the operation process, a dual-channel signal generator is started to provide a group of complementary electric field environments for the hollow area of the reaction cavity (8) of the three-electrode system;
step three, preparing a graphene oxide solution; then, dropwise adding a graphene oxide solution on the surface of a three-electrode system in the reaction cavity (8), carrying out electrodeposition in an electrochemical workstation, and washing the working electrode (6) with distilled water after the electrodeposition; secondly, preparing a chloroauric acid solution, dropwise adding the chloroauric acid solution on the surface of a three-electrode system in the reaction cavity (8), performing electrodeposition in an electrochemical workstation, and washing the working electrode (6) with distilled water after the electrodeposition; then dropwise adding sulfuric acid on the surface of a three-electrode system in the reaction cavity (8) to activate the working electrode (6); washing the working electrode (6) with distilled water after activation, dripping pesticide antibody on the surface of the three-electrode system in the reaction cavity (8) after washing for incubation, and washing the working electrode (6) with distilled water after the pesticide antibody incubation is finished; continuously dripping bovine serum albumin on the surface of the three-electrode system in the reaction cavity (8) for incubation after washing, and washing the working electrode (6) with distilled water after incubation is finished; and finally obtaining the double electric field driving sensor.
2. The method for manufacturing a dual electric field driven sensor according to claim 1, wherein the thickness of the copper film in the first step is 15 to 25 μm; the photoresist is one of linear phenolic resin and polymethyl methacrylate; the chemical developer is one of sodium carbonate or potassium carbonate, and the concentration is 0.1-0.4 g/mL; the concentration of the potassium aurous cyanide solution is 1-5 g/L.
3. The method for preparing a dual electric field driven sensor according to claim 1, wherein the thickness of the circular ring gold film in the first step is 0.2-0.7 μm; the working electrode (6) is in a trident shape and consists of ten electrically connected circular gold films; each edge of the trident is respectively provided with three circular gold films, the remaining circular gold film is arranged at the junction of the three edges, and the circular gold film at the junction and the conductive hole N are arranged 2 Are electrically connected with each other;
the thickness of the arc Ag/AgCl is 0.1-0.5 mm; the drying temperature is 70-90 ℃, and the drying time is 30-40 min.
4. The method for preparing a dual electric field driven sensor according to claim 1, wherein the number M of the copper foils in the second step is an even number between 10 and 20, and the copper foils are square with a side length of 1 to 1.5mm.
5. The method for preparing a dual electric field driven sensor according to claim 1, wherein the dual channel signal generator in step two is any type of device having a dual channel electric field generator, 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.
6. The method for preparing the dual electric field driven sensor according to claim 1, wherein the concentration of the graphene oxide solution in step three is 2-6 mg/mL, the dropping amount is 10-15 μ 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 mu L, and the electrodeposition time is 3-5 min; the concentration of the sulfuric acid is 0.3-0.6M, the dropping amount is 20-30 mu L, and the activation time is 150-300s.
7. The method for preparing the double electric field driven sensor according to claim 1, wherein the concentration of the pesticide antibody in step three is 5-20 μ g/mL, the dropping amount is 10-15 μ L, and the incubation time is 40-70 min; the concentration of the bovine serum albumin is 0.5-2%, the dripping amount is 10-15 mu L, and the incubation time is 30-60 min.
8. Use of a dual electric field driven sensor prepared according to the method of any one of claims 1 to 7 for detecting pesticide residues, characterized by the following steps:
step one, establishing a current/concentration standard curve:
preparing a series of pesticide standard liquid drops with different concentrations, adding the pesticide standard liquid drops to the surface of a three-electrode system in a reaction cavity (8) of the double-electric-field driving sensor for incubation, simultaneously starting a double-channel electric field generating device in the incubation process, and using the incubated sensor for a differential pulse voltammetry test to generate a current peak value; establishing a standard curve according to the corresponding pesticide concentration and the peak value;
step two, under the condition of double electric field driving, pesticide sample liquid is dripped on the surface of a three-electrode system in a reaction cavity (8) for incubation; and performing DPV test after incubation, generating a certain current intensity in an electrochemical window, and calculating the concentration of the pesticide to be tested according to the established I/C standard curve.
9. The use according to claim 8, wherein the pesticide in the first step is any one of chlorpyrifos, methyl parathion, isocarbophos, imidacloprid, paraoxon, atrazine, triazophos, carbendazim, thiacloprid or carbarfan, the concentration of the pesticide standard solution is 0.01-550 μ g/L, the dropping amount is 10-15 μ L, and the incubation time is 1-5 min;
the dynamic bias voltage of the dual-channel electric field generating device is set to be 40-60% of peak-to-peak voltage, and the peak-to-peak voltage is set to be 2-6V.
10. The use according to claim 8, wherein the pesticide sample liquid is dripped in the second step in an amount of 10-15 μ L, and the incubation time is 1-5 min; the dynamic bias voltage of the dual-channel electric field generating device is set to be 40-60% of peak-to-peak voltage, and the peak-to-peak voltage is set to be 2-6V.
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| US11402375B2 (en) * | 2010-08-05 | 2022-08-02 | Abbott Point Of Care Inc. | Magnetic immunosensor with trench configuration and method of use |
| CA3120967A1 (en) * | 2018-11-30 | 2020-06-04 | Perkinelmer Health Sciences, Inc. | Biological sample preparation using electric fields |
| CN113970585A (en) * | 2021-09-03 | 2022-01-25 | 江苏大学 | Enhanced adsorption electrochemical immunosensor and preparation method and detection method thereof |
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| CN103558374A (en) * | 2013-11-19 | 2014-02-05 | 山东理工大学 | Quick pesticide residue detector with current-mode immunosensor |
| CN109632905A (en) * | 2019-01-14 | 2019-04-16 | 南京邮电大学 | A kind of flexible non-enzymatic glucose sensor of graphene-supported copper nano particles and preparation method thereof |
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