CN114134138B - Ionic liquid polymer-based electrochemical modification material for pesticide detection and preparation method and application thereof - Google Patents
Ionic liquid polymer-based electrochemical modification material for pesticide detection and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an ionic liquid polymer-based electrochemical modification material for pesticide detection, and a preparation method and application thereof. The dispersion water phase is composed of ionic liquid, acetylcholinesterase, a cross-linking agent and an initiator; an oil phase is composed of dodecane and span 80; dropwise adding a dispersed water phase into the oil phase, adding TEMED into the obtained emulsion, and performing polymerization reaction to obtain AChE@PIL; adding AChE@PIL into the gold nano solution to obtain an ionic liquid polymer-based electrochemical modification material AChE@PIL/AuNPs. The electrochemical modification material prepared by the invention has excellent conductivity and biocompatibility, and the application of the material in the fields of electrochemical analysis, biological sensing and the like is expanded by applying the material to biological sensors.
Description
Technical Field
The invention belongs to the field of bioelectrochemistry, relates to a novel biosensor, and in particular relates to an ionic liquid polymer-based electrochemical modification material and application thereof in pesticide detection.
Background
In the past few years, pesticides have been widely used as herbicides, insecticides and bactericides in the fields of agriculture, industry, medicine and the like because of their advantages of high efficiency, convenient use, small dosage, short half-life and the like. In general, there is a great threat to both humans and animals due to their high toxicity. Therefore, there is a need to develop a rapid, reliable pesticide detection method for public food safety and environmental detection. The conventional pesticide residue detection methods include Gas Chromatography (GC), liquid Chromatography (LC), mass Spectrometry (MS), capillary Electrophoresis (CE), and the like. Although these analysis techniques have the characteristics of sensitivity and effectiveness in pesticide analysis, there are disadvantages of complex instruments, complex techniques, complex pretreatment of samples, time-consuming and the like, resulting in high measurement cost. Amperometric acetylcholinesterase (AChE) biosensors have proven to be a suitable alternative because of their advantages of fast response, simplicity, convenience, and low assay cost.
A biosensor is an instrument that is sensitive to a biological substance and converts its concentration into an electric signal for detection, and is composed of an immobilized biological substance, a transducer, and a signal amplification device, and has recently received much attention.
A wide variety of techniques have been used to introduce enzymes into an immobilization matrix (e.g., many natural and synthetic materials), such as entrapment, covalent adsorption and adsorption. In these methods, embedding enzymes in an immobilization matrix is relatively simple and inexpensive and produces relatively little interference with the structure and function of the native enzyme. Therefore, this method is also often used for mass production. The use of polymer-encapsulated enzymes has been gaining attention, which entraps enzymes in Polymeric Ionic Liquids (PILs) formed by microemulsion polymerization.
PIL provides a biocompatible microenvironment for the enzyme, is beneficial to maintaining the enzyme activity, and effectively reduces the loss of the enzyme by an embedding method, so that the high stability of the electrode material is ensured. However, since the established method is composed of only substances of poor conductivity such as PIL and AChE, the introduction of highly conductive substances is necessary. The PIL can also bind to gold nanoparticles, which further enhances conductivity while ensuring electrochemical biosensor stability.
Thus, 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 immobilized enzyme maintains bioactivity, and can promote direct electron transfer between the enzyme and the electrode surface. Can be applied to the fields of electrochemical analysis, biological sensing, pesticide detection and the like.
The technical scheme adopted by the invention is as follows: the ionic liquid polymer-based electrochemical modification material for pesticide detection is AChE@PIL/AuNPs, and the preparation method comprises the following steps:
1) Preparing a dispersed aqueous phase: dissolving ionic liquid, acetylcholinesterase (AChE), a cross-linking agent and an initiator in a dispersant Tris-HCl solution, and uniformly stirring to obtain a dispersed water phase;
2) Preparing an oil phase: mixing dodecane and span 80, and stirring to obtain oil phase;
3) Synthesis of ache@pil: dropwise adding the dispersed water phase into the oil phase in a dropwise adding mode to obtain emulsion; adding tetramethyl ethylenediamine (TEMED) into the emulsion, performing polymerization reaction at 25deg.C for 60min, washing the obtained precipitate with acetone and PBS, centrifuging, and lyophilizing to obtain AChE@PIL;
4) Synthesis of ache@pil/AuNPs: adding AChE@PIL into a centrifuge tube, adding gold nano-solution (AuNPs), vibrating for 10min at room temperature, centrifuging, and taking solid to obtain the ionic liquid polymer-based electrochemical modification material AChE@PIL/AuNPs.
Further, in step 1), the ionic liquid is 1-vinyl-3-ethylimidazole bromide (Vietim + Br - ) The method comprises the steps of carrying out a first treatment on the surface of the The cross-linking agent is N, N' -methylene bisacrylamide; the initiator is ammonium persulfate.
Further, 1-vinyl-3-ethylimidazole bromide in mass ratio, N' methylenebisacrylamide: ammonium persulfate=100:5:2.
Further, in step 2), dodecyl/span 80=3:1 by volume ratio.
Further, in step 3), the oil phase is water phase=1:5 according to the volume ratio.
Further, in the step 4), according to the volume ratio, the gold nano-solution is AChE@PIL=200:1.
The invention provides an application of an ionic liquid polymer-based electrochemical modification material in electrochemical detection of pesticides.
Further, the method comprises the following steps: coating an ionic liquid polymer-based electrochemical modification material AChE@PIL/AuNPs on a glassy carbon electrode GCE to prepare a GCE/AChE@PIL/AuNPs modification electrode; the GCE/AChE@PIL/AuNPs modified electrode is used as a working electrode, the Ag/AgCl electrode is used as a reference electrode, the platinum electrode is used as an auxiliary electrode, a three-electrode system is formed, and the three-electrode system is placed in a solution containing pesticides 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 is prepared into a biosensor, has good repeatability, anti-interference capability, ultrahigh thermal stability and storage stability, and has higher accuracy in analysis of actual 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 of pesticides.
3. In the invention, the ionic liquid polymer not only can provide a biocompatible microenvironment for the enzyme, but also 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 microscope image of PIL (a), AChE@PIL (b), and AChE@PIL/AuNPs (c).
Fig. 2 is a zeta potential bar graph of AChE, ILs, AChE@PIL and AuNPs.
FIG. 3 is an ultraviolet-visible absorption spectrum (UV-vis) of AuNPs (b) and AChE@PIL/AuNPs (a) supernatants.
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 spectrum (FT-IR) of AChE (c), AChE@PIL/AuNPs (b) and AChE@PIL (a).
FIG. 7 is a Raman spectrum of AChE@PIL/AuNPs (a) and AChE@PIL (b).
FIG. 8 is a Raman spectrum of AChE@PIL/AuNPs and AChE@PIL.
FIG. 9 is a schematic diagram of GCE (a), GCE/PIL (b), GCE/AChE@PIL (c) and GCE/AChE@PIL/AuNPs (d) in a composition containing 5X 10 -3 M Fe(CN) 6 3-/4- Cyclic Voltammogram (CVs) in 0.5M KCl solution at a scan rate of 0.2Vs -1 。
FIG. 10 is a graph of GCE/PIL (a), GCE/AChE@PIL (b) and GCE/AChE@PIL/AuNPs (c) in the presence of 5X 10 -3 M Fe(CN) 6 3-/4- Impedance plot in 0.5M KCl solution.
FIG. 11 is a Differential Pulse Voltammogram (DPVs) of GCE/PIL (c), GCE/AChE@PIL (b) and GCE/AChE@PIL/AuNPs (a) in 8.0mM ATCl solution in 0.1M PBS, pH 7.5.
FIG. 12 is a graph (b) of anodic peak current (a) versus scan rate for GCE/AChE@PIL/AuNPs in 8.0mM ATCl solution at 7.5 pH in 0.1M PBS.
FIG. 13 is a graph (a) of Differential Pulse Voltammograms (DPVs) and a graph (b) of peak current versus double reciprocal linearity of ATCl solution concentration for GCE/AChE@PIL/AuNPs in 0.1M PBS, pH 7.5, different concentrations of ATCl solution.
FIG. 14 is the storage stability (a) and high temperature stability (b) of GCE/AChE@PIL/AuNPs.
FIG. 15 is a graph of GCE/AChE@PIL/AuNPs in 0.1M PBS containing 8mM ATCl, 8mM glucose (a), 8mM PO, respectively 4 3- (b) Citric acid (c), mg 2+ (d)，SO 4 2- (e)，NO 3 - (f)，Fe 3+ (g)，Cu 2+ (h) Hatching of interferents such as carbaryl (i), methyl parathion (j) and malathion (k)Remaining signal in 10 minutes.
FIG. 16 is a graph (b) showing the DPV response (a) and the dichlorvos concentration measurements of GCE/AChE@PIL/AuNPs after 10min of dichlorvos inhibition and without 10min of dichlorvos inhibition at different concentrations of 0.1M PBS (pH 7.5).
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 electrochemical modification 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 bromoethane were added. The mixed solution was heated under reflux at a reaction temperature of 70℃for 10 hours. After the reaction was stopped, the reaction product was cooled to room temperature, transferred to a beaker, recrystallized from acetonitrile-ethyl acetate, filtered and dried under vacuum to give the product as an ionic liquid 1-vinyl-3-ethylimidazole bromide (ViEtIm + Br - )。
2. Synthesis of AChE@PIL
Preparing a dispersed aqueous phase: 0.5mL of ionic liquid, 0.5mg of acetylcholinesterase AChE,0.01g of cross-linking agent N, N' -methylene bisacrylamide and 0.0040g of initiator ammonium persulfate are taken and dissolved in 1mL of dispersant Tris-HCl solution with the concentration of 20mM, and the mixture is stirred uniformly to obtain a dispersed water phase.
Preparing an oil phase: according to the volume ratio, dodecyl/span 80=3:1, and the dodecyl and span 80 are taken and stirred uniformly to obtain an oil phase.
According to the volume ratio, the oil phase is water phase=1:5, and the dispersed water phase is dropwise added into the oil phase in a dropwise adding mode to obtain emulsion; adding TEMED into the emulsion, polymerizing at 25deg.C for 60min, washing the obtained precipitate with acetone and PBS, centrifuging (4000 rpm,10 min), and lyophilizing to obtain AChE@PIL.
3. Synthesis of AChE@PIL/AuNPs
50 mu L of AChE@PIL is taken in a centrifuge tube, 10mL of gold nano-solution (AuNPs) is added, the solution is observed to turn from white to purple after shaking for 10min at room temperature, and a purple solid is obtained by centrifugation (4000 rpm,10 min) for separation, and is AChE@PIL/AuNPs.
(II) Synthesis of comparative example PIL
Preparing a dispersed aqueous phase: 0.5mL of ionic liquid, 0.01g of cross-linking agent N, N' -methylene bisacrylamide and 0.0040g of initiator ammonium persulfate are taken and dissolved in 1mL of dispersant Tris-HCl solution with the concentration of 20mM, and the mixture is stirred uniformly to obtain a dispersed water phase.
Preparing an oil phase: according to the volume ratio, dodecyl/span 80=3:1, and the dodecyl and span 80 are taken and stirred uniformly to obtain an oil phase;
according to the volume ratio, the oil phase is water phase=1:5, and the dispersed water phase is dropwise added into the oil phase in a dropwise adding mode to obtain emulsion; adding TEMED into the emulsion, polymerizing at 25deg.C for 60min, washing the precipitate with acetone and PBS, centrifuging (4000 rpm,10 min), and lyophilizing to obtain PIL.
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 were spherical structures generated by concentrated emulsion polymerization, and fig. 1c shows that further assembly of AuNPs did not change morphology of the binary component, notably, no scattered AuNPs were observed, indicating that the AuNPs were fully loaded on the ache@pil surface in 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 positively charged ILs generate stronger interaction, the Zeta potentials of a binary system formed by polymerization and AuNPs are 24.9mV and-5.48 mV respectively, and the positively charged binary system generated by embedding can further assemble the negatively charged AuNPs under the electrostatic effect.
FIG. 3 is an ultraviolet-visible absorption spectrum (UV-vis) of AuNPs (b) and AChE@PIL/AuNPs (a) supernatants. As can be seen from curve b in fig. 3, the characteristic absorption peak of AuNPs at 519nm was not observed in curve a, because the electrostatic assembly of ache@pil with AuNPs caused AuNPs to appear in the precipitate, whereas AuNPs alone could not completely precipitate at 4000rpm, so that the characteristic absorption peak of AuNPs at 519nm was observed, and by monitoring this assembly process, it was clearly observed that AuNPs were effectively enriched to the previous binary structure surface, and the ache@pil AuNPs ternary composite structure was formed.
FIG. 4 is an x-ray diffraction spectrum (XRD) of AChE@PIL/AuNPs (a) and AChE@PIL (b). In the X-ray diffraction pattern of AChE@PIL/AuNPs, four strong diffraction peaks at 38.184 degrees, 44.392 degrees, 64.576 degrees and 77.547 degrees are obviously observed, and the diffraction peaks respectively correspond to the (111), (200), (220) and (311) crystal faces of Au, which indicate the existence of AuNPs. The presence of both amorphous peaks in the 20-30 ° range of the X-ray diffraction patterns of ache@pil/AuNPs and ache@pil indicates the presence of both organic components and amorphous structures, indicating the presence of both AuNPs and amorphous polymer structures in the ache@pil/AuNPs complexes of the invention.
FIG. 5 is an x-ray photoelectron spectroscopy (XPS) of AChE@PIL/AuNPs (a) and AChE@PIL (b). Compared to ache@pil (curve b), the XPS spectra of ache@pil/AuNPs (curve a) showed characteristic peaks of Au at 82.96eV and 87.79eV, which is consistent with the conclusions drawn in fig. 3 and 4.
FIG. 6 is a Fourier transform infrared spectrum (FT-IR) of AChE (c), AChE@PIL/AuNPs (b) and AChE@PIL (a). As shown in FIG. 6, there is 1700-1500cm more in curve b than in curve c -1 The presence of PIL in ache@pil was confirmed by the in-range peak, attributed to the c=n characteristic stretching vibration. At the same time, compared with curve a, at 3360cm in curve b -1 ，3100cm -1 Peaks respectively belonging to C-H stretching vibration of alkynyl and C-H stretching vibration of benzene ring, and further located at 1735cm -1 The peak at is due to the stretching vibration of the c=o functionality, the evidence above demonstrates the simultaneous presence of AChE and PIL in ache@pil, which is substantially consistent with the conclusions reached by XPS analysis.
FIG. 7 is a Raman spectrum of AChE@PIL/AuNPs (a) and AChE@PIL (b). As shown in fig. 7, comparing curve a and curve b, it is evident that the addition of AuNPs can increase the raman signal intensity, ache@pil/AuNPs can achieve raman enhancement (SERs), demonstrating tight binding between AuNPs and ache@pil, with AuNPs located on the ache@pil surface rather than in the environment.
FIG. 8 is a schematic diagram of a preferred embodiment of the present inventionRaman difference spectra of ache@pil/AuNPs and ache@pil. The difference spectrum is very similar to the raman features of AChE, confirming the availability of enzyme active sites on the electrode surface, 1600cm -1 Left and right 1265cm -1 The peaks at the left and right are respectively of secondary structure amines i and iii from AChE, 1620cm -1 Up to 1700cm -1 And 1200cm -1 To 1300cm -1 The region between is related to C-O stretching vibration, regulated by secondary structure (alpha helix and beta sheet), 1550cm -1 The band at this position is characteristic of tryptophan residues, 1400cm -1 Up to 1500cm -1 The bands of (2) being predominantly with CH of side chains 2 Is related to the shear vibration of 1362cm -1 The banding occurs due to CH 2 Bending vibration of 800cm -1 To 1150cm -1 The bands in between are considered as the stretching vibrations of the AChE fatty side chains (C-C and C-N). 921cm -1 ，1072cm -1 The band at this point is related to the presence of alanine, at the same time 946cm -1 The bands at this point are related to lysine, glutamic acid and serine. Thus, AChE embedded by PIL/AuNPs still has native enzymatic activity.
Example 2 preparation of Ionic liquid Polymer-based electrochemical modification Material AChE@PIL/AuNPs application (one) GCE/AChE@PIL/AuNPs modification electrode
1. Pretreatment of glassy carbon electrode
The experiment uses glassy carbon electrodes with diameters of 3mm, al with diameters of 1.0, 0.3 and 0.05 μm 2 O 3 Polishing the glassy carbon electrode, and ultrasonically cleaning the glassy carbon electrode for 1min by using ultrapure water. The three-electrode system is formed by taking a glassy carbon electrode (GC) as a working electrode, a platinum wire as a counter electrode and an Ag/AgCl electrode as a reference electrode. In 1mM K 3 Fe(CN) 6 Electrochemical Cyclic Voltammetry (CV) tests were performed in 1M KCl solution. When the peak position difference between the oxidation peak and the reduction peak of the electrode is less than 70.0mV, the electrode is proved to meet 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 standby.
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 0.5wt% were taken and mixed well. And (3) dripping the AChE@PIL/AuNPs solution on the surface of the pretreated Glassy Carbon Electrode (GCE), continuously dripping 5 mu L nafion (0.5 wt%) after drying at room temperature, and completely drying at 4 ℃ to obtain the GCE/AChE@PIL/AuNPs modified electrode.
Characterization of electrochemical Properties of (II) GCE/AChE@PIL/AuNPs modified electrode
In the electrochemical characterization test, the experiment was performed in a PBS buffer solution at a pH of 0.1 M=7.5, 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 cycles before each test.
FIG. 9 is a schematic diagram of GCE (a), GCE/PIL (b), GCE/AChE@PIL (c) and GCE/AChE@PIL/AuNPs (d) in a composition containing 5X 10 -3 M Fe(CN) 6 3-/4- Cyclic Voltammogram (CVs) in 0.5M KCl solution at a scan rate of 0.2Vs -1 . All modified electrodes showed a pair of good reversible redox peaks with a formula potential (Ep) of 0.23V, which is Fe (CN) 6 3-/4- Characteristic potential of the pair. The different potential differences and peak currents show the stepwise assembly process of the material, as shown in curve b and curve c, the potential differences of GCE/PIL and GCE/AChE@PIL are 93mV and 114mV respectively, the peak currents are 123 mu A and 63 mu A respectively, AChE is used as a protein molecule, and the conductivity of AChE is poor, so that the electron transfer is blocked, and therefore, the AChE-containing composite material modified electrode has larger peak potential difference and lower conductivity. As shown in curve a and curve b, the potential difference of GCE/AChE@PIL/AuNPs and GCE/AChE@PIL is 114mV, and peak currents are 155 mu A and 63 mu A respectively, which shows that AuNPs can obviously improve the conductivity of an electrode material.
FIG. 10 is a graph of GCE/PIL (a), GCE/AChE@PIL (b) and GCE/AChE@PIL/AuNPs (c) in the presence of 5X 10 -3 M Fe(CN) 6 3-/4- Impedance plot in 0.5M KCl solution. The GCE/PIL electron transfer resistance value is 27.53 omega, the GCE/AChE@PIL resistance value becomes 49.21 omega after enzyme is embedded, and the resistance value is obviously increased after AChE is embedded, so that successful immobilization of the enzyme is proved. Notably, auNP was added at the same enzyme levelThe electron transfer resistance of the enzyme electrode of s is obviously reduced to 26.30 omega compared with that of GCE/AChE@PIL, which is consistent with the CV test result obtained before, and the GCE/AChE@PIL/AuNPs has the lowest electron transfer resistance value and the highest conductivity.
FIG. 11 is a Differential Pulse Voltammogram (DPVs) of GCE/PIL (c), GCE/AChE@PIL (b) and GCE/AChE@PIL/AuNPs (a) in 8.0mM ATCl solution in 0.1M PBS, pH 7.5. The oxidation peak currents of GCE/AChE@PIL/AuNPs (a) and GCE/AChE@PIL (b) were 6.1. Mu.A and 2.4. Mu.A, respectively, the former being 2.54 times that of the latter. It is evident that the peak current of the enzyme-containing modified electrode added by AuNPs is significantly higher than GCE/AChE@PIL, since AuNPs significantly improves the electron transfer capability of the modified electrode.
FIG. 12 is a graph (b) of anodic peak current (a) versus scan rate for GCE/AChE@PIL/AuNPs in 8.0mM ATCl solution at 7.5 pH in 0.1M PBS. As shown in FIG. 12 a, when the sweep rate is 40mV.s -1 -220mV.s -1 When the range increases, 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.
Characterization of the electrocatalytic Properties of the GCE/AChE@PIL/AuNPs modified electrode
The experiment was performed in a PBS buffer solution containing ATCl (acetylthiocholine chloride) at a pH of 0.1 M=7.5 and 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 cycles before each test.
FIG. 13 is a graph (a) of Differential Pulse Voltammograms (DPVs) and a graph (b) of peak current versus double reciprocal linearity of ATCl solution concentration for GCE/AChE@PIL/AuNPs in 0.1M PBS, pH 7.5, different concentrations of ATCl solution. In the concentration range of 0-10mM ATCl, as the concentration of ATCl increases, the peak current signal increases, and then ATCl is continuously added, so that the peak current has no obvious change. Michaelis Menten constant (Km) was used as Lineweaver Burke eq. (1/Iss = Km/Imax 1/c + 1/Imax) to evaluate the activity of AChE in interaction with ATCl, where Iss is steady state current after injection of enzyme substrate, imax is maximum current calculated at saturation concentration, c is substrate concentration. The Km was found to be 9.66mM.
FIG. 14 is 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 treating the electrodes at 35, 45 and 55 ℃ for 20 minutes, their residual activity showed a tendency to rise before fall. Heating at 55deg.C for 20 min, the embedded AChE retained 73% of the initial activity, whereas unprotected AChE was susceptible to deactivation at high temperatures, with a 60% decrease in AChE activity when the temperature was in the range of 42-48deg.C. It is evident that the thermal stability of AChE is significantly improved by its embedding.
(IV) Selectivity study of GCE/AChE@PIL/AuNPs
FIG. 15 is a graph of GCE/AChE@PIL/AuNPs in 0.1M PBS containing 8mM ATCl, 8mM glucose (a), 8mM PO, respectively 4 3- (b) Citric acid (c), mg 2+ (d)，SO 4 2- (e)，NO 3 - (f)，Fe 3+ (g)，Cu 2+ (h) The remaining signal in 10 minutes of incubation of interferents such as carbaryl (i), methyl parathion (j) and malathion (k). In glucose, PO 4 3- Citric acid, mg 2+ ，SO 4 2- ，NO 3 - ，Fe 3+ And Cu 2+ No significant interference is obtained in the presence of (c). In addition, methyl parathion and malathion, which may coexist in the test sample, were also investigated. Methyl parathion and malathion were found to affect the detection of carbaryl. This evidence may be due to the fact that these pesticides compete for the binding sites of acetylcholinesterase and malathion. The results show that the constructed biosensor based on enzyme inhibition has universality and acceptable selectivity for pesticide detection.
Pesticide detection study of GCE/AChE@PIL/AuNPs
The experiment was performed in PBS buffer solution with different concentrations of dichlorvos (DDVP) at a sweep rate of 200mV/s at 0.1M pH=7.5. 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 cycles before each test.
FIG. 16 is a graph (b) showing the DPV response (a) and the dichlorvos concentration measurements of GCE/AChE@PIL/AuNPs after 10min of dichlorvos inhibition and without 10min of dichlorvos inhibition at different concentrations of 0.1M PBS (pH 7.5). When the DDVP concentration is 1375 ng.mL -1 When the enzyme inhibition rate reaches 70%, the linear range is 0.125-1375 ng.mL -1 Linear fit regression of Inhibition (%) =6.22 log c (DDVP) +12.45 and Inhibition (%) =27.56 log c (DDVP) -17.32, correlation coefficients of 0.90 and 0.96.LOD is 0.038 (calculated by 3 sigma rule).
Claims (8)
1. The ionic liquid polymer-based electrochemical modification material for pesticide detection is characterized by being AChE@PIL/AuNPs, and the preparation method comprises the following steps:
1) Preparing a dispersed aqueous phase: dissolving an ionic liquid, acetylcholinesterase AChE, a cross-linking agent and an initiator in a dispersant Tris-HCl solution, and uniformly stirring to obtain a dispersed water phase; the ionic liquid is 1-vinyl-3-ethylimidazole bromide; the cross-linking agent is N, N' -methylene bisacrylamide; the initiator is ammonium persulfate;
2) Preparing an oil phase: mixing dodecane and span 80, and stirring to obtain oil phase;
3) Synthesis of ache@pil: dropwise adding the dispersed water phase into the oil phase in a dropwise adding mode to obtain emulsion; adding TEMED into the emulsion, performing polymerization reaction at 25 ℃ for 60min, washing the obtained precipitate with acetone and PBS, centrifuging, and freeze-drying to obtain AChE@PIL;
4) Synthesis of ache@pil/AuNPs: and (3) adding AChE@PIL into a centrifuge tube, adding a gold nano solution, vibrating for 10min at room temperature, and centrifuging to obtain a solid substance, thereby obtaining the ionic liquid polymer-based electrochemical modification material AChE@PIL/AuNPs.
2. The ionic liquid polymer-based electrochemical modification material for pesticide detection according to claim 1, wherein the mass ratio of 1-vinyl-3-ethylimidazole bromide to N, N' -methylenebisacrylamide to ammonium persulfate=100:5:2.
3. An ionic liquid polymer-based electrochemical modification material for pesticide detection as claimed in claim 1, wherein in step 2), dodecyl/span 80=3:1 is used in terms of volume ratio.
4. An ionic liquid polymer-based electrochemical modification material for pesticide detection as claimed in claim 1, wherein in step 3), the oil phase is water phase=1:5 in terms of volume ratio.
5. The ionic liquid polymer-based electrochemical modification material for pesticide detection as set forth in claim 1, wherein in the step 4), the gold nano-solution is ache@pil=200:1 in terms of volume ratio.
6. Use of the ionic liquid polymer-based electrochemical modification material according to claim 1 for electrochemical detection of pesticides.
7. The use according to claim 6, characterized in that the method is as follows: coating an ionic liquid polymer-based electrochemical modification material AChE@PIL/AuNPs on a glassy carbon electrode GCE to prepare a GCE/AChE@PIL/AuNPs modification electrode; the GCE/AChE@PIL/AuNPs modified electrode is used as a working electrode, the Ag/AgCl electrode is used as a reference electrode, the platinum electrode is used as an auxiliary electrode, a three-electrode system is formed, and the three-electrode system is placed in a solution containing pesticides for electrochemical detection.
8. The use according to claim 6 or 7, wherein the pesticide is dichlorvos.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106248748A (en) * | 2016-07-22 | 2016-12-21 | 云南省产品质量监督检验研究院 | A kind of acetylcholinesterasebiosensor biosensor and application thereof |
| CN106770564A (en) * | 2016-11-10 | 2017-05-31 | 河南工业大学 | Acetylcholinesterasebiosensor biosensor for detection of organic phosphorus pesticide and preparation method thereof |
| CN110031521A (en) * | 2019-05-15 | 2019-07-19 | 贵州大学 | The preparation of acetylcholinesterasebiosensor biosensor and to organic phosphorus detection application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009073927A1 (en) * | 2007-12-13 | 2009-06-18 | Monash University | Electrochemical nanocomposite biosensor system |
| CN106248748A (en) * | 2016-07-22 | 2016-12-21 | 云南省产品质量监督检验研究院 | A kind of acetylcholinesterasebiosensor biosensor and application thereof |
| CN106770564A (en) * | 2016-11-10 | 2017-05-31 | 河南工业大学 | Acetylcholinesterasebiosensor biosensor for detection of organic phosphorus pesticide and preparation method thereof |
| CN110031521A (en) * | 2019-05-15 | 2019-07-19 | 贵州大学 | The preparation of acetylcholinesterasebiosensor biosensor and to organic phosphorus detection application |
Non-Patent Citations (2)
| Title |
|---|
| Enzyme Immobilization in Polymerized Ionic Liquids-based Hydrogels for Active and Reusable Biocatalysts;A. Grollmisch et al.;Syn Open;第2卷;192-199 * |
| Progressive development in biosensors for detection of dichlorvos pesticide: A review;Archana Mishra;Journal of Environmental Chemical Engineering;第9卷;105067 * |
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