CN114134138A

CN114134138A - Ionic liquid polymer-based electrochemical modification material for pesticide detection and preparation method and application thereof

Info

Publication number: CN114134138A
Application number: CN202111514368.6A
Authority: CN
Inventors: 张谦; 宛菀; 夏立新; 王慧婷; 陈雅贤; 李顺
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2021-12-13
Filing date: 2021-12-13
Publication date: 2022-03-04
Anticipated expiration: 2041-12-13
Also published as: CN114134138B

Abstract

The invention discloses an ionic liquid polymer-based electrochemical modification material for pesticide detection and a preparation method and application thereof. A dispersed water phase is formed by ionic liquid, acetylcholinesterase, a cross-linking agent and an initiator; an oil phase consists of dodecane and span 80; dropwise adding the dispersed water phase into the oil phase, adding TEMED into the obtained emulsion, and carrying out polymerization reaction to obtain AChE @ PIL; adding AChE @ PIL into the gold nano solution to obtain the ionic liquid polymer-based electrochemical modification material AChE @ PIL/AuNPs. The electrochemical modified material prepared by the invention has excellent conductivity and biocompatibility, and the material is applied to biosensors, so that the application of the material in the fields of electrochemical analysis, biosensing and the like is expanded.

Description

Ionic liquid polymer-based electrochemical modification material for pesticide detection and preparation method and application thereof

Technical Field

The invention belongs to the field of bioelectrochemistry, relates to a novel biosensor, and particularly relates to an ionic liquid polymer-based electrochemical modification material and application thereof in pesticide detection.

Background

In the past few years, agricultural chemicals have been widely used as herbicides, insecticides, and fungicides in the fields of agriculture, industry, medicine, and the like, because of their advantages such as high efficiency, convenient use, small amount, short half-life, and the like. Generally, they pose a great threat to both humans and animals due to their high toxicity. Therefore, there is a need to develop a rapid and reliable pesticide detection method for public food safety and environmental detection. Traditional pesticide residue detection methods include Gas Chromatography (GC), Liquid Chromatography (LC), Mass Spectrometry (MS), Capillary Electrophoresis (CE), and the like. Although the analysis techniques have the characteristics of sensitivity and effectiveness in pesticide analysis, the defects of complex instruments, complex techniques, complex and time-consuming sample pretreatment and the like still exist, and the determination cost is high. Amperometric acetylcholinesterase (AChE) biosensors have proven to be a suitable alternative due to their advantages of fast response, simplicity, convenience, and low assay cost.

A biosensor is an instrument that senses a biological substance and converts the concentration of the biological substance into an electrical signal for detection, and is composed of an immobilized biological substance, a transducer, and a signal amplification device, and has attracted much attention in recent years.

Various techniques have been used to introduce enzymes into immobilization matrices (e.g., many natural and synthetic materials), such as entrapment, covalent adsorption and adsorption. In these methods, embedding the enzyme into an immobilization matrix is relatively simple and inexpensive, and causes relatively little interference with the structure and function of the native enzyme. Therefore, this method is also often used for mass production. Methods employing polymer-encapsulated enzymes that entrap the enzyme in a Polymeric Ionic Liquid (PIL) formed by microemulsion polymerization are gaining importance.

The PIL provides a biocompatible microenvironment for the enzyme, so that the enzyme activity is maintained and the enzyme loss is effectively reduced by the embedding method, and the high stability of the electrode material is guaranteed. However, since the established method is composed of only substances having poor conductivity, such as PIL and AChE, the introduction of a highly conductive substance is necessary. The PIL can also be combined with gold nanoparticles, so that the stability of the electrochemical biosensor is ensured, and the conductivity is further enhanced.

Therefore, based on the above analysis, if AChE @ PIL and gold nanoparticles can be combined to form a complex, the complex will have the advantages of both.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation method and application of an ionic liquid polymer-based electrochemical modification material for pesticide detection. The composite material AChE @ PIL/AuNPs provided by the invention has excellent conductivity and biocompatibility, can ensure that the immobilized enzyme keeps bioactivity, and can promote direct electron transfer between the enzyme and the surface of an electrode. Can be applied to the fields of electrochemical analysis, biosensing, pesticide detection and the like.

The technical scheme adopted by the invention is as follows: an ionic liquid polymer-based electrochemical modification material for pesticide detection is AChE @ PIL/AuNPs, and the preparation method comprises the following steps:

1) preparation of dispersed water phase: dissolving the ionic liquid, acetylcholinesterase (AChE), a cross-linking agent and an initiator in a Tris-HCl dispersing agent solution, and uniformly stirring to obtain a dispersed water phase;

2) preparing an oil phase: taking dodecane and span 80, and uniformly stirring to obtain an oil phase;

3) synthesis of AChE @ PIL: dropwise adding the dispersed water phase into the oil phase in a dropwise adding manner to obtain emulsion; adding Tetramethylethylenediamine (TEMED) into the emulsion, carrying out polymerization reaction at 25 ℃ for 60min, washing the obtained precipitate with acetone and PBS, carrying out centrifugal separation, and carrying out freeze drying to obtain AChE @ PIL;

4) synthesis of AChE @ PIL/AuNPs: and adding the AChE @ PIL into a centrifugal tube, adding a gold nano solution (AuNPs), shaking for 10min at room temperature, and centrifuging to obtain a solid substance to obtain the ionic liquid polymer-based electrochemical modification material AChE @ PIL/AuNPs.

Further, in the step 1), the ionic liquid is 1-vinyl-3-ethylimidazole bromine salt (ViEtim)⁺Br^-) (ii) a The cross-linking agent is N, N' -methylene bisacrylamide; the initiator is ammonium persulfate.

Furthermore, the mass ratio of the 1-vinyl-3-ethylimidazole bromine salt to the N, N' -methylene bisacrylamide to the ammonium persulfate is 100:5: 2.

Further, in the step 2), dodecane and span 80 are mixed in a volume ratio of 3: 1.

Further, in step 3), the volume ratio of the oil phase to the water phase is 1: 5.

Further, in the step 4), the volume ratio of the gold nanoparticle solution AChE @ PIL is 200: 1.

The invention provides an application of an ionic liquid polymer-based electrochemical modification material in electrochemical detection of pesticides.

Further, the method is as follows: coating an ionic liquid polymer-based electrochemical modified material AChE @ PIL/AuNPs on a glassy carbon electrode GCE to prepare a GCE/AChE @ PIL/AuNPs modified electrode; a GCE/AChE @ PIL/AuNPs modified electrode is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as an auxiliary electrode to form a three-electrode system, and the three-electrode system is placed in a solution containing pesticide for electrochemical detection.

Further, the pesticide is dichlorvos.

The invention has the following beneficial effects:

1. the ionic liquid polymer-based electrochemical modification material AChE @ PIL/AuNPs provided by the invention has good repeatability, anti-interference capability and ultrahigh thermal stability and storage stability, and has higher accuracy in analysis of practical samples such as peach, tap water and the like.

2. The ionic liquid polymer-based electrochemical modification material AChE @ PIL/AuNPs provided by the invention is prepared into a biosensor, shows excellent electrochemical activity, and can realize high sensitivity, low detection limit and high selectivity detection on pesticides.

3. According to the invention, the ionic liquid polymer can provide a biocompatible microenvironment for the enzyme, and is beneficial to the maintenance of the enzyme activity, and the embedding method effectively reduces the loss of the enzyme and ensures the high stability of the electrode material.

Drawings

FIG. 1 is a scanning electron micrograph of PIL (a), AChE @ PIL (b), and AChE @ PIL/AuNPs (c).

FIG. 2 is a histogram of zeta potentials of AChE, ILs, AChE @ PIL and AuNPs.

FIG. 3 shows the UV-Vis absorption spectra (UV-vis) of the supernatants from AuNPs (b) and AChE @ PIL/AuNPs (a).

FIG. 4 is an x-ray diffraction spectrum (XRD) of AChE @ PIL/AuNPs (a) and AChE @ PIL (b).

FIG. 5 is an x-ray photoelectron spectroscopy (XPS) of AChE @ PIL/AuNPs (a) and AChE @ PIL (b).

FIG. 6 is a Fourier transform infrared (FT-IR) spectrum of AChE (c), AChE @ PIL/AuNPs (b), and AChE @ PIL (a).

FIG. 7 is the Raman spectra of AChE @ PIL/AuNPs (a) and AChE @ PIL (b).

FIG. 8 is a Raman difference spectrum of AChE @ PIL/AuNPs and AChE @ PIL.

FIG. 9 shows a graph comprising 5X 10 of GCE (a), GCE/PIL (b), GCE/AChE @ PIL (c) and GCE/AChE @ PIL/AuNPs (d)^-3M Fe(CN)₆ ^3-/4-Cyclic Voltammograms (CVs) in 0.5M KCl solution at a scan rate of 0.2Vs^-1。

FIG. 10 shows a graph of GCE/PIL (a), GCE/AChE @ PIL (b) and GCE/AChE @ PIL/AuNPs (c) in a sample containing 5X 10^-3M Fe(CN)₆ ^3-/4-Impedance plot in 0.5M KCl solution.

FIG. 11 is Differential Pulse Voltammograms (DPVs) of GCE/PIL (c), GCE/AChE @ PIL (b), and GCE/AChE @ PIL/AuNPs (a) in 0.1M PBS, pH 7.5 in 8.0mM ATCl.

FIG. 12 is a graph of anodic peak current (a) versus scan rate (b) for GCE/AChE @ PIL/AuNPs in 8.0mM ATCl solution in 0.1M PBS, pH 7.5.

FIG. 13 is a Differential Pulse Voltammogram (DPVs) (a) and double reciprocal linear dependence of peak current on ATCl solution concentration (b) for GCE/AChE @ PIL/AuNPs in 0.1M PBS, pH 7.5 at various concentrations of ATCl solution.

FIG. 14 shows the storage stability (a) and high temperature stability (b) of GCE/AChE @ PIL/AuNPs.

FIG. 15 shows the presence of 8mM glucose (a), 8mM PO, respectively, in GCE/AChE @ PIL/AuNPs in 0.1M PBS containing 8mM ATCl₄ ^3-(b) Citric acid (c), Mg²⁺(d)，SO₄ ^2-(e)，NO₃ ^-(f)，Fe³⁺(g)，Cu²⁺(h) Remaining signals from 10min incubations with interferents such as carbaryl (i), methyl parathion (j) and malathion (k).

FIG. 16 is a calibration graph (b) of DPV response (a) and determination of dichlorvos concentration for GCE/AChE @ PIL/AuNPs after 10min inhibition by dichlorvos at various concentrations in 0.1M PBS (pH 7.5) and 10min after inhibition.

Detailed Description

For better understanding of the technical solution of the present invention, specific examples are described in further detail, but the solution is not limited thereto.

Example 1 an ionic liquid polymer-based electrochemically modified material AChE @ PIL/AuNPs

The preparation method comprises the following steps:

1. preparation of ionic liquids

In a 250mL round bottom flask, 30.0mL of 1-vinylimidazole and 42.8mL of ethyl bromide were added. The mixed solution was heated under reflux at a reaction temperature of 70 ℃ for 10 hours. After the reaction is stopped, cooling to room temperature, transferring the reaction product to a beaker, recrystallizing with acetonitrile-ethyl acetate, filtering, and drying in vacuum to obtain the ionic liquid 1-vinyl-3-ethylimidazole bromine salt (ViEtim)⁺Br^-)。

2. Synthesis of AChE @ PIL

Preparation of dispersed water phase: dissolving 0.5mL of ionic liquid, 0.5mg of acetylcholinesterase AChE, 0.01g of cross-linking agent N, N' -methylene bisacrylamide and 0.0040g of initiating agent ammonium persulfate in 1mL of dispersing agent Tris-HCl solution with the concentration of 20mM, and uniformly stirring to obtain a dispersed water phase.

Preparing an oil phase: and (2) taking the dodecane and the span 80 according to the volume ratio of 3:1, and uniformly stirring to obtain an oil phase.

According to the volume ratio, the oil phase and the water phase are 1:5, and the dispersed water phase is dripped into the oil phase in a dripping manner to obtain emulsion; adding TEMED into the emulsion, performing polymerization reaction at 25 ℃ for 60min, washing the obtained precipitate with acetone and PBS, performing centrifugal separation (4000rpm, 10min), and performing freeze drying to obtain AChE @ PIL.

3. Synthesis of AChE @ PIL/AuNPs

Adding 50 mu L of AChE @ PIL into a centrifuge tube, adding 10mL of gold nano solution (AuNPs), shaking at room temperature for 10min, observing that the solution is changed from white to purple, and centrifuging (4000rpm for 10min) to separate a purple solid, namely AChE @ PIL/AuNPs.

(II) Synthesis of comparative example PIL

Preparation of dispersed water phase: 0.5mL of ionic liquid, 0.01g of cross-linking agent N, N' -methylene bisacrylamide and 0.0040g of initiator ammonium persulfate are dissolved in 1mL of dispersant Tris-HCl solution with the concentration of 20mM and stirred uniformly to obtain a dispersed water phase.

Preparing an oil phase: according to the volume ratio, dodecane and span 80 are mixed to be 3:1, and the dodecane and the span 80 are uniformly stirred to obtain an oil phase;

according to the volume ratio, the oil phase and the water phase are 1:5, and the dispersed water phase is dripped into the oil phase in a dripping manner to obtain emulsion; adding TEMED into the emulsion, performing polymerization reaction at 25 deg.C for 60min, washing the obtained precipitate with acetone and PBS, centrifuging (4000rpm, 10min), and freeze drying to obtain PIL.

(III) characterization of materials and electrodes

FIG. 1 is a typical SEM image of PIL (a), AChE @ PIL (b), and AChE @ PIL/AuNPs (c). All three are spherical structures generated by a concentrated emulsion polymerization reaction, and c in fig. 1 shows that the morphology of the binary components is not changed by further assembling the AuNPs, and it is noted that no scattered AuNPs are observed, which indicates that the AuNPs are completely loaded on the surface of AChE @ PIL in an assembled form.

FIG. 2 is a Zeta potential histogram of AChE, ILs, AChE @ PIL and AuNPs. The Zeta potentials of AChE and ILs are-6.02 mV and 6.36mV respectively, the negatively charged AChE and the positively charged ILs generate stronger interaction, and the Zeta potentials of a binary system formed by polymerization and AuNPs are 24.9mV and-5.48 mV respectively, so that the positively charged binary system generated by embedding can be used for further assembling the negatively charged AuNPs under the electrostatic action.

FIG. 3 shows the UV-Vis absorption spectra (UV-vis) of the supernatants from AuNPs (b) and AChE @ PIL/AuNPs (a). As can be seen from curve b in fig. 3, the characteristic absorption peak of AuNPs is at 519nm, which cannot be observed in curve a, because the electrostatic assembly of AChE @ PIL and AuNPs causes AuNPs to appear in the precipitate, while AuNPs alone cannot be completely precipitated at 4000rpm, so that the characteristic absorption peak of AuNPs at 519nm is observed, and by monitoring this assembly process, it is apparent that AuNPs are effectively enriched to the surface of the former binary structure, and the AChE @ PIL/AuNPs ternary composite structure is formed.

FIG. 4 is an x-ray diffraction spectrum (XRD) of AChE @ PIL/AuNPs (a) and AChE @ PIL (b). Four strong diffraction peaks corresponding to the (111), (200), (220), and (311) crystal planes of Au were clearly observed in the X-ray diffraction pattern of AChE @ PIL/AuNPs at 38.184 °, 44.392 °, 64.576 °, and 77.547 °, indicating the presence of AuNPs. The simultaneous presence of amorphous peaks in the 20-30 ° range of the X-ray diffraction patterns of AChE @ PIL/AuNPs and AChE @ PIL indicates that both contain organic components and that amorphous structures are present, indicating that both AuNPs and amorphous polymer structures are present in the AChE @ PIL/AuNPs complex of the invention.

FIG. 5 is an x-ray photoelectron spectroscopy (XPS) of AChE @ PIL/AuNPs (a) and AChE @ PIL (b). Compared with AChE @ PIL (curve b), the XPS spectrum of AChE @ PIL/AuNPs (curve a) shows characteristic peaks of Au at 82.96eV and 87.79eV, which is consistent with the conclusions of FIGS. 3 and 4.

FIG. 6 is a Fourier transform infrared (FT-IR) spectrum of AChE (c), AChE @ PIL/AuNPs (b), and AChE @ PIL (a). As shown in FIG. 6, the curve b has 1700-1500cm more^-1The in-range peak, attributed to C ═ N characteristic stretching vibration, confirms the presence of PIL in AChE @ PIL. While curve b is at 3360cm compared to curve a^-1，3100cm^-1Peaks at (B) were assigned to the C-H stretching vibration of alkynyl group and the C-H stretching vibration of benzene ring, respectively, and were located at 1735cm^-1The peak at (a) is due to the stretching vibration of the C ═ O functional group, and the above evidence confirms the simultaneous presence of AChE and PIL in AChE @ PIL, which is essentially consistent with the results obtained from XPS analysis.

FIG. 7 is the Raman spectra of AChE @ PIL/AuNPs (a) and AChE @ PIL (b). As shown in FIG. 7, comparing the curve a and the curve b, it is obvious that the Raman signal intensity is increased by adding AuNPs, and the AChE @ PIL/AuNPs can realize Raman enhancement (SERs), which proves that the AuNPs and the AChE @ PIL are tightly combined, and the AuNPs are positioned on the surface of the AChE @ PIL and not in the environment.

FIG. 8 is a Raman difference spectrum of AChE @ PIL/AuNPs and AChE @ PIL. The difference spectrum is very similar to the Raman signature of AChE, confirming the availability of an active site of enzyme on the electrode surface, 1600cm^-1Left and right and 1265cm^-1The peaks at the left and right belong to the secondary structures amine i and amine iii from AChE, 1620cm^-1To 1700cm^-1And 1200cm^-1To 1300cm^-1The region in between is related to C-O stretching vibration, regulated by secondary structure (alpha helix and beta sheet), 1550cm^-1The band at (A) is characteristic of a tryptophan residue, 1400cm^-1To 1500cm^-1The bands of (A) are predominantly associated with the side chain CH₂Is related to shear vibration of 1362cm^-1The occurrence of banding is due to CH₂Flexural vibration, 800cm^-1To 1150cm^-1The bands in between are considered tensile oscillations of the AChE fatty side chains (C-C and C-N). 921cm^-1，1072cm^-1The band at (B) is related to the presence of alanine, and is 946cm^-1The bands in (A) are related to lysine, glutamic acid and serine. Thus, AChE embedded in PIL/AuNPs still has native enzymatic activity.

Example 2 application of Ionic liquid Polymer-based electrochemically modified Material AChE @ PIL/AuNPs (one) preparation of GCE/AChE @ PIL/AuNPs modified electrode

1. Pretreatment of glassy carbon electrodes

The experiment adopts a glassy carbon electrode with the diameter of 3mm, and uses Al with the diameters of 1.0, 0.3 and 0.05 mu m respectively₂O₃To glassy carbonPolishing, and ultrasonically cleaning with ultrapure water for 1 min. A glassy carbon electrode (GC) is used as a working electrode, a platinum wire is used as a counter electrode, and an Ag/AgCl electrode is used as a reference electrode to form a three-electrode system. At 1mM K₃Fe(CN)₆The electrochemical Cyclic Voltammetry (CV) test was performed in 1M KCl solution. When the peak position difference of the oxidation peak and the reduction peak of the electrode is less than 70.0mV, the electrode meets the requirement of activation cleaning. Taking out the glassy carbon electrode, cleaning with ultrapure water, and purifying with high-purity nitrogen (N)₂) And drying for later use.

2. Preparation of GCE/AChE @ PIL/AuNPs modified electrode

5mg of AChE @ PIL/AuNPs prepared in example 1 and 10.0. mu.L of nafion solution with a concentration of 0.5 wt% were taken and mixed well. And (3) dropwise coating the obtained AChE @ PIL/AuNPs solution on the surface of a pretreated Glassy Carbon Electrode (GCE), drying at room temperature, continuously dropwise coating 5 mu L of nafion (0.5 wt%), and completely drying at 4 ℃ to obtain the GCE/AChE @ PIL/AuNPs modified electrode.

(II) characterization of electrochemical properties of GCE/AChE @ PIL/AuNPs modified electrode

In the electrochemical characterization test, the experiment was performed in 0.1M PBS buffer solution with pH 7.5, and at a sweep rate of 200 mV/s. A three-electrode system is adopted, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as an auxiliary electrode, and a GCE/AChE @ PIL/AuNPs modified electrode is used as a working electrode. Under the same experimental conditions, the GCE/AChE @ PIL/AuNPs modified electrode was scanned for 50 consecutive turns before each test.

FIG. 9 shows a graph comprising 5X 10 of GCE (a), GCE/PIL (b), GCE/AChE @ PIL (c) and GCE/AChE @ PIL/AuNPs (d)^-3M Fe(CN)₆ ^3-/4-Cyclic Voltammograms (CVs) in 0.5M KCl solution at a scan rate of 0.2Vs^-1. All modified electrodes showed a good pair of reversible redox peaks with a zeta potential (Ep) of 0.23V, which is Fe (CN)₆ ^3-/4-The characteristic potential of the electrical pair. The different potential difference and peak current show the step-by-step assembly process of the material, as shown by curve b and curve c, the potential difference of GCE/PIL and GCE/AChE @ PIL is 93mV and 114mV respectively, the peak current is 123 muA and 63 muA respectively, AChE is used as a protein molecule, and the poor conductivity of the protein molecule obstructs the transfer of electrons, so that complex AChE-containing materials are causedThe composite material modified electrode has larger peak potential difference and lower conductivity. As shown by a curve a and a curve b, the potential difference of the GCE/AChE @ PIL/AuNPs and the potential difference of the GCE/AChE @ PIL are both 114mV, and the peak currents are 155 muA and 63 muA respectively, which indicates that the AuNPs can obviously improve the conductivity of the electrode material.

FIG. 10 shows a graph of GCE/PIL (a), GCE/AChE @ PIL (b) and GCE/AChE @ PIL/AuNPs (c) in a sample containing 5X 10^-3M Fe(CN)₆ ^3-/4-Impedance plot in 0.5M KCl solution. The resistance value of GCE/PIL electron transfer is 27.53 omega, the resistance value of GCE/AChE @ PIL is changed to 49.21 omega after enzyme embedding, and the resistance value is obviously increased after AChE embedding, thereby proving the successful immobilization of the enzyme. It is noted that the electron transfer resistance of the enzyme electrode added with AuNPs is obviously reduced to 26.30 omega compared with GCE/AChE @ PIL under the same enzyme dosage, which is consistent with the CV test result obtained before, and the GCE/AChE @ PIL/AuNPs are proved to have the lowest electron transfer resistance value and the highest conductivity.

FIG. 11 is Differential Pulse Voltammograms (DPVs) of GCE/PIL (c), GCE/AChE @ PIL (b), and GCE/AChE @ PIL/AuNPs (a) in 0.1M PBS, pH 7.5 in 8.0mM ATCl. The oxidation peak currents of GCE/AChE @ PIL/AuNPs (a) and GCE/AChE @ PIL (b) are 6.1 muA and 2.4 muA respectively, and the former is 2.54 times that of the latter. It can be obviously seen that the peak current of the modified electrode containing enzyme added by AuNPs is obviously higher than that of GCE/AChE @ PIL, because the AuNPs obviously improve the electron transfer capability of the modified electrode.

FIG. 12 is a graph of anodic peak current (a) versus scan rate (b) for GCE/AChE @ PIL/AuNPs in 8.0mM ATCl solution in 0.1M PBS, pH 7.5. When the sweep rate is 40mV. s, as shown by a in FIG. 12^-1-220mV.s^-1When the range is increased, the oxidation peak current increases and the oxidation peak shifts to a positive potential. As shown in fig. 12 b, the peak current is proportional to the square root of the sweep rate, which represents the diffusion control process for this electrode.

(III) characterization of electrocatalytic performance of GCE/AChE @ PIL/AuNPs modified electrode

The experiment was carried out in 0.1M pH 7.5 PBS buffer containing ATCl (Acetylthiocholine chloride) at a sweep rate of 200 mV/s. A three-electrode system is adopted, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as an auxiliary electrode, and a GCE/AChE @ PIL/AuNPs modified electrode is used as a working electrode. Under the same experimental conditions, the GCE/AChE @ PIL/AuNPs modified electrode was scanned for 50 consecutive turns before each test.

FIG. 13 is a Differential Pulse Voltammogram (DPVs) (a) and double reciprocal linear dependence of peak current on ATCl solution concentration (b) for GCE/AChE @ PIL/AuNPs in 0.1M PBS, pH 7.5 at various concentrations of ATCl solution. In the range of 0-10mM ATCl concentration, the peak current signal increases with the increase of the ATCl concentration, and then the ATCl is continuously added, so that the peak current has no obvious change. Michaelis Menten constant (Km) was measured with a Lineweaver Burke eq. (1/Iss-Km/Imax.1/c +1/Imax) where Iss is the steady state current after injection of enzyme substrate, Imax is the maximum current calculated at saturating concentrations, and c is the substrate concentration. The Km obtained is 9.66 mM.

FIG. 14 shows the storage stability (a) and high temperature stability (b) of GCE/AChE @ PIL/AuNPs. The biosensor current remained at 95% of its initial response after 30 days. After heat treatment of the electrodes at 35, 45 and 55 ℃ for 20 minutes, their residual activity showed a tendency to increase first and then decrease. When the embedded AChE is heated at 55 ℃ for 20 minutes, the embedded AChE retains 73% of the initial activity, while the unprotected AChE is easily inactivated at high temperature, and when the temperature is in the range of 42-48 ℃, the activity of the AChE is reduced by 60%. It is clear that AChE's thermal stability is significantly improved by its entrapment.

(IV) Selectivity study of GCE/AChE @ PIL/AuNPs

FIG. 15 shows the presence of 8mM glucose (a), 8mM PO, respectively, in GCE/AChE @ PIL/AuNPs in 0.1M PBS containing 8mM ATCl₄ ^3-(b) Citric acid (c), Mg²⁺(d)，SO₄ ^2-(e)，NO₃ ^-(f)，Fe³⁺(g)，Cu²⁺(h) Remaining signals from 10min incubations with interferents such as carbaryl (i), methyl parathion (j) and malathion (k). In glucose, PO₄ ^3-Citric acid, Mg²⁺，SO₄ ^2-，NO₃ ^-，Fe³⁺And Cu²⁺In the presence of (c), no significant interference is obtained. In addition, the test samples were also tested for possible co-presence of methyl parathion and horsePhothion was investigated. Methyl parathion and malathion were found to affect the detection of carbaryl. This indication may be due to the fact that these pesticides compete for the binding site of acetylcholinesterase to malathion. The results show that the constructed biosensor based on enzyme inhibition has universality and acceptable selectivity for pesticide detection.

(V) pesticide detection research of GCE/AChE @ PIL/AuNPs

The experiment was carried out at a sweep rate of 200mV/s in 0.1M pH 7.5 PBS buffer containing varying concentrations of dichlorvos (DDVP). A three-electrode system is adopted, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as an auxiliary electrode, and a GCE/AChE @ PIL/AuNPs modified electrode is used as a working electrode. Under the same experimental conditions, the GCE/AChE @ PIL/AuNPs modified electrode was scanned for 50 consecutive turns before each test.

FIG. 16 is a calibration graph (b) of DPV response (a) and determination of dichlorvos concentration for GCE/AChE @ PIL/AuNPs after 10min inhibition by dichlorvos at various concentrations in 0.1M PBS (pH 7.5) and 10min after inhibition. When the concentration of DDVP is 1375 ng.mL^-1When the enzyme activity is high, the enzyme inhibition rate reaches 70 percent, and the linear range is 0.125-1375 ng.mL^-1Linear fit regression with correlation coefficients of 0.90 and 0.96 for Inhibition (%) 6.22log c (DDVP) +12.45 and Inhibition (%) 27.56log c (DDVP) -17.32. LOD was 0.038 (calculated according to the 3 σ rule).

Claims

1. An ionic liquid polymer-based electrochemical modification material for pesticide detection is characterized in that the ionic liquid polymer-based electrochemical modification material is AChE @ PIL/AuNPs, and the preparation method comprises the following steps:

1) preparation of dispersed water phase: dissolving the ionic liquid, acetylcholinesterase AChE, a cross-linking agent and an initiator in a Tris-HCl dispersing agent solution, and uniformly stirring to obtain a dispersed water phase;

3) synthesis of AChE @ PIL: dropwise adding the dispersed water phase into the oil phase in a dropwise adding manner to obtain emulsion; adding TEMED into the emulsion, carrying out polymerization reaction at 25 ℃ for 60min, washing the obtained precipitate with acetone and PBS, carrying out centrifugal separation, and carrying out freeze drying to obtain AChE @ PIL;

4) synthesis of AChE @ PIL/AuNPs: and adding the AChE @ PIL into a centrifugal tube, adding a gold nano solution, shaking at room temperature for 10min, and centrifuging to obtain a solid substance to obtain the ionic liquid polymer-based electrochemical modification material AChE @ PIL/AuNPs.

2. The ionic liquid polymer-based electrochemical modification material for pesticide detection as claimed in claim 1, wherein in step 1), the ionic liquid is 1-vinyl-3-ethylimidazole bromine salt; the cross-linking agent is N, N' -methylene bisacrylamide; the initiator is ammonium persulfate.

3. The ionic liquid polymer-based electrochemical modification material for pesticide detection according to claim 2, characterized in that the mass ratio of 1-vinyl-3-ethylimidazole bromide to N, N' -methylene bisacrylamide to ammonium persulfate is 100:5: 2.

4. The ionic liquid polymer-based electrochemical modification material for pesticide detection as claimed in claim 1, wherein in step 2), dodecane is 80 ═ 3:1 by volume.

5. The ionic liquid polymer-based electrochemical modification material for pesticide detection according to claim 1, wherein in the step 3), the oil phase and the water phase are 1:5 in volume ratio.

6. The ionic liquid polymer-based electrochemical modification material for pesticide detection as claimed in claim 1, wherein in step 4), the volume ratio of the gold nanoparticle solution AChE @ PIL is 200: 1.

7. The use of the ionic liquid polymer-based electrochemically modified material of claim 1 in the electrochemical detection of pesticides.

8. Use according to claim 7, characterized in that the method is as follows: coating an ionic liquid polymer-based electrochemical modified material AChE @ PIL/AuNPs on a glassy carbon electrode GCE to prepare a GCE/AChE @ PIL/AuNPs modified electrode; a GCE/AChE @ PIL/AuNPs modified electrode is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as an auxiliary electrode to form a three-electrode system, and the three-electrode system is placed in a solution containing pesticide for electrochemical detection.

9. Use according to claim 7 or 8, characterized in that the pesticide is dichlorvos.