CN111007137A - Method and equipment for detecting organic phosphorus - Google Patents
Method and equipment for detecting organic phosphorus Download PDFInfo
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- CN111007137A CN111007137A CN201911415861.5A CN201911415861A CN111007137A CN 111007137 A CN111007137 A CN 111007137A CN 201911415861 A CN201911415861 A CN 201911415861A CN 111007137 A CN111007137 A CN 111007137A
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Abstract
The invention belongs to the field of pollutant detection, and particularly relates to an organophosphorus detection method and organophosphorus detection equipment. The method is based on the electrostatic attraction effect of thiocholine and ferricyanide ions, and an electrochemical sensing system is constructed for detecting the content of organic phosphorus. Firstly, depositing nanogold on the surface of a treated glassy carbon electrode, and then utilizing the action of an Au-S covalent bond to enable thiocholine generated after Acetylthiocholine (ATCH) is hydrolyzed under the action of acetylcholinesterase (AChE) to be assembled on the surface of the electrode. The assembled electrode was subjected to electrochemical measurements in a potassium ferricyanide solution containing a lower ion concentration. The method can realize rapid detection of organophosphorus pesticide residues, and has the advantages of high sensitivity, strong specificity and the like. The detection method can be applied to practical operation of field detection of agricultural and sideline products.
Description
Technical Field
The invention belongs to the field of pollutant detection, and particularly relates to an organophosphorus detection method and organophosphorus detection equipment.
Background
China is rich in land resources and is a big country for agricultural production. With the continuous development of modern economy and the continuous progress of science and technology, the use of pesticides is more and more common in order to reduce the labor of farmers on working. Among them, Organophosphorus Pesticides (OPs for short) are one of the most widely used varieties of Pesticides due to their high effectiveness and durability. However, since the agricultural chemicals are used in excess, a large amount of OPs that are not effectively used remain in agricultural products, soil and water, causing a serious problem of environmental pollution. Meanwhile, OPs, as a neurotoxin, can inhibit the activity of Acetylcholinesterase (AChE) in the central nervous system, block the catalytic hydrolysis pathway of Acetylcholine (ACh), cause the continuous accumulation of Acetylcholine content, further block neurotransmitter transmission, and further cause great harm to human bodies. Therefore, besides preventing the abuse of pesticides, establishing an effective method for detecting the OPs residues is of great significance to the environmental protection and the human health maintenance.
In the prior art, the detection method of OPs residues mainly comprises gas chromatography, high performance liquid chromatography, gas chromatography/mass spectrometry combined technology, enzyme-linked immunosorbent assay and the like. Although the modern instrument analysis method for detecting OPs has higher sensitivity and accuracy, the defects of complicated sample pre-enrichment and pretreatment processes, long analysis time, high cost, expensive and complicated instruments and the like exist, and the requirements of rapid analysis and the like cannot be met. Although the enzyme-linked immunosorbent assay is rapid and efficient, the detection cost is high, and the real-time field detection of batch products cannot be realized. There is a need to develop a method for detecting OPs, which has high sensitivity, rapid detection, strong specificity and no need of special pretreatment on samples, so as to meet the requirement of detecting OPs residues.
Disclosure of Invention
The invention aims to provide a method for detecting organophosphorus, which utilizes target molecule acetylcholinesterase of organophosphorus pesticide to prepare an enzyme sensor, can realize rapid detection of organophosphorus pesticide residue, and has the advantages of high sensitivity, strong specificity and the like.
In order to solve the technical problems, the technical scheme of the invention is as follows:
an organophosphorus detection method comprises the following steps:
the step (1) comprises the preparation of the assembly liquid to be tested: treating an acetylcholinesterase solution by using a sample to be detected, then adding an acetylthiocholine solution, and carrying out enzymatic reaction to obtain an assembly liquid to be detected;
preparing a nano gold electrode with the surface covered with nano gold particles;
and (3) preparing an assembled electrode to be tested: treating the nano-gold electrode by using an assembly liquid to be tested to enable the nano-gold particles to adsorb thiocholine, so as to obtain an assembly electrode to be tested;
the step (4) comprises the detection of electrochemical response signals: and detecting an electrochemical response signal of the assembled electrode to be detected by using cyclic voltammetry or differential pulse voltammetry, and using a solution containing ferricyanide ions as a detection base solution for soaking the assembled electrode to be detected.
By adopting the technical scheme, the technical principle is as follows:
thioacetyl choline is capable of being hydrolyzed in the presence of acetylcholinesterase to produce thiocholine. In the presence of an organophosphorus pesticide (or organophosphorus), the organophosphorus pesticide is capable of inhibiting the activity of acetylcholinesterase, thereby preventing the production of thiocholine. And placing the nano-gold electrode in standard assembly liquid or assembly liquid to be tested for a period of time, and assembling the thiocholine generated by hydrolysis on the surface of the nano-gold electrode through Au-S bonds. When the organophosphorus pesticide exists, the assembling amount of the thiocholine is reduced along with the increase of the concentration of the organophosphorus pesticide. And (3) placing the electrode (standard assembly electrode or assembly electrode to be tested) assembled with the thiocholine into a ferricyanide ion solution with lower ionic strength for electrochemical determination, wherein cations on the thiocholine can make the ferricyanide ions approach the surface of the electrode through electrostatic attraction, and the potential gradient of the surface of the electrode is kept unchanged, so that a larger current value is generated under low ion concentration. Therefore, the greater the amount of thiocholine assembled on the electrode surface, the greater the electrochemical signal measured. When the organophosphorus pesticide is detected, the higher the concentration of the organophosphorus pesticide, the lower the amount of the thiocholine assembled on the surface of the electrode, and the smaller the detected electrochemical signal, so that the electrochemical sensing system with reduced signal is constructed for detecting the organophosphorus.
The method comprises the steps of firstly enabling the organophosphorus pesticide and acetylcholinesterase to fully act, inhibiting the activity of the acetylcholinesterase (the inhibition degree is related to the concentration of the organophosphorus pesticide), and then enabling the acetylcholinesterase and the chlorinated acetylthiocholine to fully reflect to generate thiocholine (the generation amount of the thiocholine is related to the activity degree of the acetylcholinesterase). And assembling the generated thiocholine on the nanogold electrode to form an assembled electrode to be detected, and detecting the change of an electrochemical response signal on the assembled electrode to be detected to obtain the concentration information of the organic phosphorus in the sample to be detected so as to realize the rapid detection of the organic phosphorus.
Has the advantages that:
(1) the detection method combines an enzyme inhibition method and a biosensor method, combines a key enzyme AChE for detecting OPs with an electrochemical detection method, and realizes the detection of OPs. The inventor finds and skillfully utilizes the electrostatic attraction effect of the thiocholine and the ferricyanide ions, successfully constructs simple, efficient, sensitive, convenient, economical and practical enzyme inhibition electrochemical biosensing equipment and a detection method, and realizes the high-sensitivity determination of OPs pesticides.
(2) The method has the advantages of good linear range, low detection limit, high accuracy, good specificity and the like. The electrochemical sensing technology provides a new method for detecting the residual quantity of OPs pesticides by using a relatively simple detection program and convenient experimental conditions, and has potential application prospect in the field detection of agricultural and sideline products. The concentration range of methyl parathion (an OPs) detected by the method is 0.1 mu g/L-100 mu g/L, the logarithm value of the concentration of the OPs of the electrochemical response signal is in a linear relation in the range, and the detection limit of the method is 0.046 mu g/L. Therefore, the detection method and the corresponding equipment have the characteristics of wide linear range and low detection limit, and can be used for field detection of various scenes.
(3) The method is used for detecting OPs, special pretreatment is not needed to be carried out on a sample, special equipment is not needed, and the sample preparation method is simple.
(4) The detection method has stronger specificity and selectivity to the organophosphorus pesticides, and experiments prove that the interference of non-organophosphorus substances to the detection method is smaller under the condition of not carrying out any pretreatment.
(5) In the prior art, AChE is usually fixed on a detection electrode when the OPs are detected by using AChE, but after AChE is fixed on the electrode as a macromolecular protein, the protein conformation of AChE is affected, so that the catalytic activity of AChE is further changed and unstable, and the detection result is inaccurate. According to the technical scheme, AChE is not fixed on the electrode, but the catalytic product thiocholine is adsorbed by the electrode, so that the detection is realized. The thiocholine is a stable small molecular substance, so that the detection result is more accurate.
Further, the step (1) also comprises the preparation of a standard assembly liquid series; the standard assembling liquid series comprises a plurality of standard assembling liquids, and the preparation method of the standard assembling liquids comprises the following steps: treating an acetylcholinesterase solution by using a standard solution containing an organophosphorus pesticide, adding an acetylthiocholine solution, and carrying out enzymatic reaction to obtain a standard assembly solution; the concentration of the organophosphorus pesticide in the standard solution is known, the number of the standard solutions is a plurality, and the concentrations of the organophosphorus pesticide in the standard solutions are different;
step (3) also includes the preparation of standard assembly electrodes: processing the nano-gold electrodes by using standard assembly liquid, wherein one nano-gold electrode corresponds to one standard assembly liquid, so that the nano-gold particles adsorb thiocholine to obtain a plurality of standard assembly electrodes;
the step (4) also comprises the acquisition of the concentration of the organophosphorus pesticide: respectively detecting electrochemical response signals of a plurality of standard assembly electrodes by using a cyclic voltammetry method or a differential pulse voltammetry method, and using a solution containing ferricyanide ions as a detection base solution for soaking the standard assembly electrodes; establishing a standard curve of the electrochemical response signal and the concentration of the organophosphorus pesticide in the standard solution; and obtaining the concentration of the organophosphorus pesticide in the solution of the sample to be detected according to the electrochemical response signal and the standard curve of the assembled electrode to be detected.
By adopting the technical scheme, the relation between the concentration of OPs and the electrochemical signal is found by utilizing the difference of the concentration of OPs in the standard assembling liquid series, a standard curve for reacting the relation between the electrochemical response signal and the concentration of the organophosphorus pesticide in the standard solution is drawn and established, the specific concentration value of OPs in the sample to be detected can be obtained through the standard curve, and the quantitative detection is realized.
Further, in the step (3), the time for treating the nano-gold electrode by using the standard assembly liquid or the assembly liquid to be tested is more than or equal to 30min at the temperature of 35-40 ℃.
By adopting the technical scheme and the assembly time and temperature, the gold nanoparticles and the thiocholine can spontaneously form Au-S covalent bonds, so that the thiocholine molecules can be fully assembled on the surface of the electrode. Too short a time, the adsorption of thiocholine is insufficient.
Further, in the step (4), the ferricyanide ions in the detection base solution are provided by potassium ferricyanide, and the detection base solution further contains potassium chloride.
By adopting the technical scheme, the potassium ferricyanide is common ferricyanide.
Further, in the detection base solution, the concentration of potassium ferricyanide was 0.5mmol/L and the concentration of potassium chloride was 1 mmol/L.
By adopting the technical scheme, the potassium chloride with the concentration can ensure that the response signals before and after modification of the thiocholine are more obviously compared when the electrochemical determination is carried out on the electrode modified by the thiocholine; potassium ferricyanide at the above concentrations can provide a sufficient amount of ferricyanide ion for thiocholine binding.
Further, in the step (1), the acetylthiocholine solution is one of a chlorinated acetylthiocholine solution, an iodinated acetylthiocholine solution and a brominated acetylthiocholine solution.
By adopting the technical scheme, the chlorinated acetylthiocholine, the iodized acetylthiocholine and the brominated acetylthiocholine are all conventional acetylthiocholine, are easy to obtain and have clear physicochemical properties.
Further, in the step (4), when cyclic voltammetry is used, the scanning potential is-0.1-0.5V, and the potential scanning speed is 50 mV/s; when the differential pulse voltammetry is used, the scanning potential is-0.2-0.5V, the pulse period is 0.2s, the pulse amplitude is 0.025V, and the pulse width is 0.05 s.
By adopting the technical scheme, the electrochemical response signal of the assembled electrode to be detected or the standard assembled electrode can be obtained, the strength of the electrochemical response signal is moderate, and the OPs can be accurately detected.
Further, in the step (2), the preparation method of the nano gold electrode comprises the following steps: grinding the glassy carbon electrode on polishing flannelette with the assistance of aluminum oxide polishing powder with the particle size of 0.05 mu m and 0.3 mu m, and then cleaning the glassy carbon electrode by using ultrapure water; and putting the polished and cleaned glassy carbon electrode into a chloroauric acid solution, reducing the chloroauric acid into nano-gold particles by using a potentiostatic method, and depositing the nano-gold particles on the surface of the glassy carbon electrode to obtain the nano-gold electrode.
By adopting the technical scheme, the nano gold electrode can be obtained, the preparation method is simple, and the preparation efficiency is high.
Further, in the step (1), the preparation method of the standard assembling liquid comprises the following steps: adding 10 mu g/mL acetylcholinesterase solution into standard solution with known concentration of organophosphorus pesticide, incubating for at least 30min at 37 ℃, adding 10mmol/L acetylthiocholine chloride solution and phosphate buffer solution with pH value of 8.4, and incubating for at least 20min at 37 ℃ to obtain standard assembly solution;
the preparation method of the assembly liquid to be tested comprises the following steps: adding 10 mu g/mL acetylcholinesterase solution into a sample to be detected, incubating for 30min at 37 ℃, adding 10mmol/L acetylthiocholine chloride solution and phosphate buffer solution with the pH value of 8.4, and incubating for 20min at 37 ℃ to obtain an assembly liquid to be detected.
By adopting the technical scheme, the organophosphorus pesticide and acetylcholinesterase can fully act in the first incubation process, and the acetylcholinesterase and acetylthiocholine chloride can fully react to generate thiocholine in the second incubation process.
Further, the device for detecting the organic phosphorus comprises an electrochemical workstation, wherein a working electrode of the electrochemical workstation is a glassy carbon electrode with a nano-gold particle layer deposited on the surface, and thiocholine is assembled on the outer side of the nano-gold particle layer through an Au-S bond.
By adopting the technical scheme, the thiocholine is assembled on the surface of the glassy carbon electrode with the surface deposited with the nano-gold particle layer through the Au-S bond, and the OPs can be accurately detected through the strength of an electrochemical response signal.
Drawings
FIG. 1 is a graph of DPV response for different OPs concentrations for example 1.
FIG. 2 is a standard curve of the relationship between the electrochemical response value and the concentration of OPs in example 1.
FIG. 3 is a graph showing the selectivity analysis of the detection method of comparative example 1.
FIG. 4 is a comparison of the electrodes of Experimental example 1 before and after assembly without the target.
FIG. 5 is a comparison of the electrodes of Experimental example 1 before and after assembly with a target.
Fig. 6 is a signal comparison graph of the nanogold electrode of experimental example 2 at different ion concentrations.
Fig. 7 is a graph comparing signals of the gold nano-electrode assembled with thiocholine of experimental example 2 at different ion concentrations.
FIG. 8 is a DPV response graph of the nano-gold electrode of Experimental example 3 in the assembling liquid without OPs under different soaking time.
Fig. 9 is a graph of the trend between different assembly times and DPV response signals for experimental example 3.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1:
1. electrochemical measurement conditions:
secondary ultra-pure water is used in the whole experiment process, and the mixture is added with KCl containing 1mmol/L and 0.5mmol/L K3[Fe(CN)6]The mixed solution of (a) is used as a detection base solution; the whole measurement process is carried out in an electrolytic cell formed by a traditional three-electrode system, wherein the working electrode is a glassy carbon electrode (GCE, phi is 3mm) or a modified glassy carbon electrode (the specific modification mode is shown in the following of the embodiment), the counter electrode is a platinum wire electrode, and a silver-silver chloride electrode (AgCl/Ag) is a reference electrodeAnd a specific electrode. Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) were measured using an electrochemical workstation with the following parameters: the scanning potential of CV is-0.1V-0.5V, and the potential scanning speed is 50 mV/s; the DPV has a scanning potential of-0.2V to 0.5V, a pulse period of 0.2s, a pulse amplitude of 0.025V and a pulse width of 0.05 s.
2. Establishment of a Standard Curve
All solutions in this step were prepared with ultrapure water, as follows: adding 25 mu L of a series of OPs solutions with different concentrations (the concentration sequence is 0 mu g/L,0.1 mu g/L,0.3 mu g/L,0.6 mu g/L,1 mu g/L,3 mu g/L,6 mu g/L,10 mu g/mL and 100 mu g/L, the OPs specifically uses methyl parathion standard) and 25 mu L of 10 mu g/mL AChE solution into a 1.5mL centrifuge tube, and incubating for 30min at 37 ℃ (in the scheme, the reaction between OPs and AChE can be ensured to be sufficient by incubating for 30min and more than 30 min); then 50 μ L of 10mmol/L thiocholine chloride solution and 50 μ L of PBS solution (pH 8.4) were added to the centrifuge tube and incubated at 37 ℃ for 20min (defined as standard assembly solution) (in this case, incubation for 20min and more than 20min ensures that AChE can sufficiently catalyze the formation of thiocholine chloride from thiocholine chloride). In the scheme, the chlorinated acetylthiocholine solution can be replaced by other acetylthiocholine solutions, such as an acetylthiocholine iodide solution, an acetylthiocholine bromide solution and the like.
At 0.3 and 0.05 μm of aluminum oxide (Al)2O3) Grinding a glassy carbon electrode on polishing flannelette with the aid of polishing powder, cleaning the glassy carbon electrode with ultrapure water, and immersing the electrode into 10mL of 1% chloroauric acid (HAuCl)4) Depositing for 30s in the solution under a potential of-0.2V to deposit a layer of bright and uniform nano-gold particles (AuNPs) on the surface of the glassy carbon electrode to obtain the nano-gold electrode.
Immersing the glassy carbon electrode (nano gold electrode) subjected to gold deposition treatment in a standard assembly liquid, assembling for 30min at 37 ℃ (37 ℃ is the optimal temperature, and the smooth assembly can be guaranteed within the temperature range of 35-40 ℃), then cleaning with ultrapure water, and wiping excess water with filter paper to obtain the standard assembly electrode. Finally, the assembled electrode is arrangedIn 5mL of 1mmol/L KCl and 0.5mmol/L K3[Fe(CN)6]The CV or DPV measurement was carried out in the solution (detection base solution) of (1), and the DPV measurement was specifically used in this example.
According to the detection result (figure 1, DPV response graphs corresponding to different OPs concentrations), a standard curve (figure 2) is established, and the relation between the reaction electrochemical response signal and the concentration of the organophosphorus pesticide in the standard solution is fitted to obtain a linear equation: i ═ 0.705lgc-2.95(r ═ 0.9912), where c denotes the concentration of OPs (μ g/L) and I denotes the detected current value (μ a). As is clear from FIGS. 1 and 2, in the concentration range of 0.1. mu.g/L to 100. mu.g/L, the response value of the reduction peak current gradually decreased as the concentration of OPs gradually increased. That is, the higher the concentration of OPs, the stronger the inhibition degree of the activity of OPs on AChE, and the less thiocholine assembled on the electrode, so that the electrochemical signal is smaller. The electrochemical response signal and the logarithm value of the OPs concentration are in a linear relation, the detection limit obtained by calculation is 0.046 mu g/L, and the experimental result shows that the measured electrochemical signal is in inverse proportion to the concentration of the target Substance (OPs), so that the method can realize high-sensitivity determination of the organic phosphorus.
Example 2
This example uses the detection system established in example 1 to perform the detection of a sample. In order to verify the effectiveness of the scheme in the application of the practical sample analysis, in the embodiment, the white cabbage juice and the apple juice are used for replacing ultrapure water to prepare methyl parathion into solutions with different concentrations (0.1 mug/L, 1 mug/L and 10 mug/L), namely, the sample to be detected is prepared from the white cabbage juice and the apple juice instead of the ultrapure water, and the liquid environment where OPs are actually located in the practical detection process is simulated. The organic phosphorus detection of the scheme was performed on the 6 samples, and the detection concentration of OPs was quantitatively calculated. The detection process comprises the following steps: and treating the AChE solution by using the sample solution to be detected, and then reacting the treated AChE solution with the acetylthiocholine chloride solution to form the assembly solution to be detected. Adding 25 mu L of sample solution to be detected and 25 mu L of AChE solution with the concentration of 10 mu g/mL into a 1.5mL centrifuge tube, and incubating for 30min at 37 ℃ (in the scheme, the reaction between OPs and AChE can be ensured to be sufficient by incubating for 30min and more than 30 min); then 50 mu L of 10mmol/L acetyl chloride is added into the centrifuge tubeThe thiocholine solution and 50 μ L PBS solution (pH 8.4) were incubated at 37 ℃ for 20min before use (defined as the test assembly) (in this case, incubation for 20min and 20min or more ensured that AChE catalyzed sufficient chlorination of acetylthiocholine to produce thiocholine). In the scheme, the chlorinated acetylthiocholine solution can be replaced by other acetylthiocholine solutions, such as an acetylthiocholine iodide solution, an acetylthiocholine bromide solution and the like. In the same manner as in example 1, the method for preparing the gold nano-electrode in this embodiment includes immersing a glassy carbon electrode (gold nano-electrode) subjected to gold deposition treatment in an assembly liquid to be tested, assembling at 37 ℃ for 30min (37 ℃ is an optimal temperature, and smooth assembly can be guaranteed within a temperature range of 35-40 ℃), then cleaning with ultrapure water, and wiping excess water with filter paper to obtain an assembly electrode to be tested. Finally, the assembled electrode is placed in 5mL of KCl with the concentration of 1mmol/L and K with the concentration of 0.5mmol/L3[Fe(CN)6]The present example specifically adopts DPV measurement to obtain current value (μ a) data, and then calculates the concentration of OPs in the sample solution to be measured according to the linear equation in example 1. In this example, the electrochemical measurement conditions were the same as in example 1.
The results are shown in table 1, and the recovery rate of the detection results of the actual samples (simulation) is between 93.30% and 107.0%, so that the constructed enzyme inhibition electrochemical sensing detection method has high anti-interference capability and accuracy for detecting the residual quantity of organic phosphorus in agricultural products.
Table 1: detection result of organophosphorus residues in sample
Comparative example 1:
to examine the selectivity of the constructed sensor to OPs, an interference experiment was performed using propyl hydroxybenzoate (PPB), p-phthalone (DBP), Atrazine (ATZ), Ascorbic Acid (AA), endotoxin (LPS), and target OPs according to the experimental procedure under the same conditions (using the standard curve and sample detection method of example 1), respectively. As shown in FIG. 3, the electrical signals of the 5 interfering substances used were all high and all were closer to the electrical signal response value of the OPs blank group; the small electric signal of methyl parathion (OPs is 1 mug/L) containing target substance indicates that the experimental method has specificity for detecting the organic phosphorus. The letter symbols in fig. 3 respectively represent: a is 1. mu.g/L OPs, b is 50. mu.g/L PPB, c is 50. mu.g/L DBP, d is 50. mu.g/LATZ, e is 50. mu.g/L AA, f is 50. mu.g/L LPS, and g is blank. The detection base solution of this comparative example was 1mmol/LKCl and 0.5mmol/L K3[Fe(CN)6]The mixed solution of (1).
Experimental example 1: principle verification experiment of detection method
To verify the detection principle of the present invention, the gold-deposited electrode (nanogold electrode) was loaded with thiocholine (to obtain an assembled electrode) according to example 1 (establishment of standard curve), and the assembled electrode was placed in a detection base solution of 1mmol/L KCl and 0.5mmol/L K3[Fe(CN)6]CV measurement was performed on the mixed solution of (1). In fig. 4, curve a is the nanogold electrode, curve b is the nanogold electrode (assembled electrode) after being assembled in the assembly solution without the target object (POs), and the electric signal of curve b in the detection base solution is obviously higher than that of curve a. In the absence of OPs, AChE catalyzes the hydrolysis of acetylthiocholine chloride to generate a large amount of thiocholine, and the thiocholine is assembled on the surface of the electrode through Au-S bonds and can promote electron transfer, so that the electric signal of the assembled electrode is higher than that of the nanogold electrode. In fig. 5, curve a is a gold deposition processing electrode (nano gold electrode), curve b is an assembled electrode after adding the target substance OPs, and electrochemical signals of curve a and curve b are approximate in the detection base solution; this is because OPs inhibit AChE activity in the presence of OPs, thereby preventing the production of thiocholine. Therefore, the surface of the electrode is not assembled with the thiocholine, and the electrochemical signal value of the electrode is close to that of the nanogold electrode. The analysis of combining fig. 4 and fig. 5 shows that: under the condition without target OPs, the electrochemical signal of the assembled electrode is higher than that of a nano gold electrode because the thiocholine can promote electron transfer; under the condition of target OPs, the electrochemical signal value of the assembled electrode is close to that of the electrode modified by the nano-gold. Thus, it can be proved that OPs can inhibit the activity of AChE, reduce the generation of thiocholine,thereby reducing the promotion of electron transfer.
Experimental example 2: optimization of ion concentration in detection base solution
In order to improve the performance of the constructed sensor, the signal comparison before and after the assembly of the thiocholine is more obvious, and the ion concentration of the detection base solution is optimized. As shown in fig. 6, as the KCl concentration in the detection base solution decreased, the redox current of the AuNPs-modified electrode (nanogold electrode) was observed to decrease, and when the KCl concentration in the detection base solution was 1mmol/L, almost no current was observed; however, as can be observed in fig. 7, when the KCl concentration of the base solution is detected to be 1mmol/L, the redox current of the electrode assembled with thiocholine is significantly higher than that of the AuNPs modified electrode (nanogold electrode). And in the solution for detecting the KCl concentration of the base solution as 10mmol/L and 100mmol/L, the redox current response values before and after electrode assembly are not obviously changed. The experimental result shows that the electrochemical determination of the electrode modified by the thiocholine under the condition of detecting the KCl concentration of the base solution to be 1mmol/L can make the response signal comparison before and after the modification of the thiocholine more obvious. Thus, the optimal detection base solution adopted in the invention is 5mL of KCl with the concentration of 1mmol/L and 0.5mmol/L K3[Fe(CN)6]The mixed solution of (1).
Experimental example 3: optimization of thiocholine Assembly time
The gold nanoparticles and the thiocholine can spontaneously form Au-S covalent bonds, so that the thiocholine can be assembled on the gold nanoparticle modified glassy carbon electrode. To examine the optimal conditions of the assembly process, the electrode treated by gold deposition is soaked in the assembly solution without targets (POs) for different time periods, and the detection base solution is 5mL of KCl with the concentration of 1mmol/L and 0.5mmol/L K3[Fe(CN)6]The DPV measurement was performed in the mixed solution (see fig. 8); a trend plot of the time to assembly of thiocholine was then made from the measured values of DPV (see figure 9). The graph shows that the current response value is gradually increased along with the increase of the assembly time of the thiocholine, and finally, the current response value is stable around 30 min. Therefore, in the present invention, the assembly time of the thiocholine is 30min to ensure that the thiocholine molecules can be fully assembled on the surface of the electrode.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. The method for detecting the organic phosphorus is characterized by comprising the following steps of:
the step (1) comprises the preparation of the assembly liquid to be tested: treating an acetylcholinesterase solution by using a sample to be detected, then adding an acetylthiocholine solution, and carrying out enzymatic reaction to obtain an assembly liquid to be detected;
preparing a nano gold electrode with the surface covered with nano gold particles;
and (3) preparing an assembled electrode to be tested: treating the nano-gold electrode by using an assembly liquid to be tested to enable the nano-gold particles to adsorb thiocholine, so as to obtain an assembly electrode to be tested;
the step (4) comprises the detection of electrochemical response signals: and detecting an electrochemical response signal of the assembled electrode to be detected by using cyclic voltammetry or differential pulse voltammetry, and using a solution containing ferricyanide ions as a detection base solution for soaking the assembled electrode to be detected.
2. The method for detecting organophosphorus according to claim 1, wherein step (1) further comprises the steps of preparing a standard assembly liquid series; the standard assembling liquid series comprises a plurality of standard assembling liquids, and the preparation method of the standard assembling liquids comprises the following steps: treating an acetylcholinesterase solution by using a standard solution containing an organophosphorus pesticide, adding an acetylthiocholine solution, and carrying out enzymatic reaction to obtain a standard assembly solution; the concentration of the organophosphorus pesticide in the standard solution is known, the number of the standard solutions is a plurality, and the concentrations of the organophosphorus pesticide in the standard solutions are different;
step (3) also includes the preparation of standard assembly electrodes: processing the nano-gold electrodes by using standard assembly liquid, wherein one nano-gold electrode corresponds to one standard assembly liquid, so that the nano-gold particles adsorb thiocholine to obtain a plurality of standard assembly electrodes;
the step (4) also comprises the acquisition of the concentration of the organophosphorus pesticide: respectively detecting electrochemical response signals of a plurality of standard assembly electrodes by using a cyclic voltammetry method or a differential pulse voltammetry method, and using a solution containing ferricyanide ions as a detection base solution for soaking the standard assembly electrodes; establishing a standard curve of the electrochemical response signal and the concentration of the organophosphorus pesticide in the standard solution; and obtaining the concentration of the organophosphorus pesticide in the solution of the sample to be detected according to the electrochemical response signal and the standard curve of the assembled electrode to be detected.
3. The method for detecting organophosphorus according to claim 2, wherein in the step (3), the time for treating the nanogold electrode with the standard assembly liquid or the assembly liquid to be detected at 35-40 ℃ is greater than or equal to 30 min.
4. The method of claim 2, wherein in step (4), the ferricyanide ions in the detection base solution are provided by potassium ferricyanide, and the detection base solution further contains potassium chloride.
5. The method for detecting organophosphorus according to claim 4, wherein the concentration of potassium ferricyanide in the detection base solution is 0.5mmol/L, and the concentration of potassium chloride in the detection base solution is 1 mmol/L.
6. The method for detecting organophosphorus according to any one of claims 2 to 5, wherein in step (1), the acetylthiocholine solution is one of a chlorinated acetylthiocholine solution, an iodinated acetylthiocholine solution and a brominated acetylthiocholine solution.
7. The method for detecting organophosphorus according to claim 6, wherein in the step (4), when cyclic voltammetry is used, the scanning potential is-0.1 to 0.5V, and the potential scanning speed is 50 mV/s; when the differential pulse voltammetry is used, the scanning potential is-0.2-0.5V, the pulse period is 0.2s, the pulse amplitude is 0.025V, and the pulse width is 0.05 s.
8. The method for detecting organophosphorus according to any one of claims 1 to 5, wherein in the step (2), the nanogold electrode is prepared by: grinding the glassy carbon electrode on polishing flannelette with the assistance of aluminum oxide polishing powder with the particle size of 0.05 mu m and 0.3 mu m, and then cleaning the glassy carbon electrode by using ultrapure water; and putting the polished and cleaned glassy carbon electrode into a chloroauric acid solution, reducing the chloroauric acid into nano-gold particles by using a potentiostatic method, and depositing the nano-gold particles on the surface of the glassy carbon electrode to obtain the nano-gold electrode.
9. The method for detecting organophosphorus according to claim 6, wherein in step (1), the standard assembling solution is prepared by: adding 10 mu g/mL acetylcholinesterase solution into standard solution with known concentration of organophosphorus pesticide, incubating for at least 30min at 37 ℃, adding 10mmol/L acetylthiocholine chloride solution and phosphate buffer solution with pH value of 8.4, and incubating for at least 20min at 37 ℃ to obtain standard assembly solution;
the preparation method of the assembly liquid to be tested comprises the following steps: adding 10 mu g/mL acetylcholinesterase solution into a sample to be detected, incubating for 30min at 37 ℃, adding 10mmol/L acetylthiocholine chloride solution and phosphate buffer solution with the pH value of 8.4, and incubating for 20min at 37 ℃ to obtain an assembly liquid to be detected.
10. The organophosphorus detection equipment comprises an electrochemical workstation, and is characterized in that a working electrode of the electrochemical workstation is a glassy carbon electrode with a nanogold particle layer deposited on the surface, and thiocholine is assembled on the outer side of the nanogold particle layer through Au-S bonds.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111766226A (en) * | 2020-06-28 | 2020-10-13 | 中国人民解放军军事科学院军事医学研究院 | Kit and method for detecting organophosphorus toxicant |
| CN112114016A (en) * | 2020-09-22 | 2020-12-22 | 广西壮族自治区农业科学院农产品质量安全与检测技术研究所 | Electrochemical method for detecting 3-nitropropionic acid |
| CN115389584A (en) * | 2022-08-01 | 2022-11-25 | 中国人民解放军国防科技大学 | Portable electrochemical acetylcholinesterase sensor for organophosphorus field detection and application thereof |
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2019
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111766226A (en) * | 2020-06-28 | 2020-10-13 | 中国人民解放军军事科学院军事医学研究院 | Kit and method for detecting organophosphorus toxicant |
| CN112114016A (en) * | 2020-09-22 | 2020-12-22 | 广西壮族自治区农业科学院农产品质量安全与检测技术研究所 | Electrochemical method for detecting 3-nitropropionic acid |
| CN112114016B (en) * | 2020-09-22 | 2022-09-23 | 广西壮族自治区农业科学院农产品质量安全与检测技术研究所 | Electrochemical method for detecting 3-nitropropionic acid |
| CN115389584A (en) * | 2022-08-01 | 2022-11-25 | 中国人民解放军国防科技大学 | Portable electrochemical acetylcholinesterase sensor for organophosphorus field detection and application thereof |
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