An ache@au np immobilized enzyme paper chip, a preparation method and application thereof

Abstract

The application discloses an AChE@AuNPs immobilized enzyme paper chip and a preparation method and application thereof, and belongs to the technical field of pesticide detection. The preparation method of the AChE@AuNPs immobilized enzyme paper chip comprises the following steps: adding HAuCl4 solution dropwise on a paper sheet, standing, then adding a reducing agent solution to react, then adding a surfactant solution and a buffer solution, and obtaining a paper chip with in-situ generated nanogold after reaction; adding a concanavalin A solution dropwise on the paper chip with in-situ generated nanogold, reacting, then adding a bovine serum albumin solution to react, finally adding an AChE solution, and obtaining an AChE@AuNPs immobilized enzyme system after reaction; and the AChE@AuNPs immobilized enzyme system and a detection area constitute the AChE@AuNPs immobilized enzyme paper chip. The AChE@AuNPs immobilized enzyme paper chip has the advantages of low preparation cost, portability, rapid detection, clear results and the like.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of pesticide detection, in particular to an AChE@AuNPs immobilized enzyme paper chip and a preparation method and application thereof. BACKGROUND

[0002] Pesticide residue detection as an important part of food safety has been widely concerned in recent years, and high-efficiency and rapid detection of pesticide residues has become a research hotspot of many scholars. There are various detection methods for pesticide residues, and currently, chromatography, enzyme-linked immunoassay, biosensor method and enzyme inhibition method are commonly used. These methods play a significant role in pesticide qualitative and quantitative detection. Chromatography is a detection method with wide application, high separation efficiency, fast analysis speed, good sensitivity and easy purification, but it has problems such as complex sample pretreatment, expensive equipment, time-consuming analysis and high professional level of technical personnel, and cannot meet the requirements of on-site rapid detection of samples. The enzyme-linked immunoassay requires simple equipment, can detect liquid food without pretreatment, can realize multi-batch sample detection of solid samples in a short time, and is very suitable for on-site detection of pesticide residues. However, this method has the following shortcomings: large instrument size, large matrix interference of the sample itself, and low precision when detecting small molecules. The biosensor method has high selectivity, simple operation and fast response speed, but this method requires high equipment, has high detection cost and poor selectivity. The enzyme inhibition method is simple, fast and accurate and reliable, but this method is not ideal in qualitative and quantitative detection, has few detectable pesticide types, poor adaptability, narrow coverage and is prone to false negative and false positive phenomena. It can be seen that the current detection methods can meet the actual pesticide detection requirements to a certain extent, but are limited by factors such as site, equipment, technology and cost, and are difficult to meet the actual demand of on-site and rapid screening of a large number of samples. SUMMARY

[0003] The application aims to provide an AChE@AuNPs immobilized enzyme paper chip and a preparation method and application thereof, so as to solve the problems in the prior art.

[0004] To achieve the above-mentioned purpose, the application provides the following solutions.

[0005] One of the technical solutions of the application is a preparation method of an AChE@AuNPs immobilized enzyme paper chip, which comprises the following steps:

[0006] The HAuCl4 solution is dropped on the paper sheet, and after standing, the reducing agent solution is dropped for reaction, and then the surfactant solution and the buffer solution are dropped for reaction, so that the paper chip with in-situ generated nanogold is obtained;

[0007] The paper chip for generating gold nanoparticles in situ is added with a concanavalin A solution, and after reaction, a bovine serum albumin solution is added for reaction, and finally an AChE solution is added for reaction, so that an AChE@AuNPs immobilized enzyme system is obtained after reaction.

[0008] The AChE@AuNPs immobilized enzyme system and the detection area constitute the AChE@AuNPs immobilized enzyme paper chip.

[0009] Further, the reducing agent solution comprises a sodium citrate solution; and the surfactant solution comprises a CTAB solution.

[0010] The buffer is a PBS buffer with pH = 8 and 10 mmol / L.

[0011] The molar ratio of HAuCl4 in the HAuCl4 solution, the reducing agent in the reducing agent solution and the surfactant in the surfactant solution is 6:1:0.3.

[0012] Further, the reaction time after adding the reducing agent solution is 5 min; and the reaction time after adding the surfactant solution and the buffer is 15 min.

[0013] Further, the amount ratio of HAuCl4 in the HAuCl4 solution and AChE in the AChE solution is 6 mmol:50000 U.

[0014] The amount ratio of concanavalin in the concanavalin A solution, bovine serum albumin in the bovine serum albumin solution and AChE in the AChE solution is 1.1 mg:5 mg:10 U.

[0015] Further, the reaction time after adding the concanavalin A solution is 60-120 min; the reaction time after adding the bovine serum albumin solution is 15 min; and the reaction time after adding the AChE solution is 30-150 min.

[0016] Further, the reaction time after adding the concanavalin A solution is 110 min; the reaction time after adding the bovine serum albumin solution is 15 min; and the reaction time after adding the AChE solution is 90 min.

[0017] The technical solution two of the application is an AChE@AuNPs immobilized enzyme paper chip prepared by the preparation method.

[0018] The technical solution three of the application is an application of the AChE@AuNPs immobilized enzyme paper chip in detecting pesticides inhibiting AChE activity.

[0019] The fourth technical solution of the present application is a detection method of a pesticide inhibiting AChE activity, comprising the following steps:

[0020] The color developing reaction substrate is ATCh, and the color developing agent is 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0021] The color developing reaction substrate is ATCh, and the color developing agent is 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0022] Further, the pesticide inhibiting AChE activity includes organophosphorus pesticides.

[0023] Further, the pesticide inhibiting AChE activity includes malathion, dimethoate and other small molecule substances.

[0024] Further, when the concentration of the AChE solution used for preparing the AChE@AuNPs immobilized enzyme paper chip is 10 U / mL, and the dosage is 6.0 μL, the concentration of the color developing reaction substrate solution is 8.65 mmol / L, and the dosage is 3-8 μL.

[0025] Further, when the concentration of the AChE solution used for preparing the AChE@AuNPs immobilized enzyme paper chip is 10 U / mL, and the dosage is 5.0 μL, the dosage of the color developing reaction substrate solution is 6 μL, and the concentration is 5.19-12.10 mmol / L.

[0026] The principle of the AChE@AuNPs immobilized enzyme paper chip for detecting organophosphorus pesticides is as follows:

[0027] Under certain conditions, organophosphorus pesticides have an inhibitory effect on the activity of AChE, and the inhibition degree is positively correlated with the pesticide concentration. The present application takes AChE and AuNPs immobilized enzyme as the reaction system, takes ATCh as the enzyme color developing reaction substrate, and takes DTNB as the color developing agent for color developing reaction. When the sample does not contain the target pesticide, AChE normally catalyzes the hydrolysis of ATCh (iodized acetylthiocholine) to generate TCh (thiocholine), and TCh reacts with the color developing agent DTNB to generate color developing reaction, and the color changes from colorless to yellow (the substrate ATCh color developing principle diagram is shown in Figure 1 ); otherwise, if the sample contains the target pesticide, the activity of AChE is inhibited, and the amount of product is reduced or even no product is generated. The RGB value after reaction is read, the enzyme inhibition rate is calculated, and the pesticide residue in the sample is quantitatively analyzed.

[0028] The application discloses the following technical effects:

[0029] (1) The AChE@AuNPs immobilized enzyme paper chip has the advantages of low preparation cost, portability, rapid detection, clear results, no need to use high-end equipment, and low requirement for technical personnel.

[0030] (2) The AChE@AuNPs immobilized enzyme paper chip has the dual characteristics of enzyme-like activity and nanomaterial performance, can improve the stability of enzyme molecules, and realizes more sensitive and accurate detection of pesticides.

[0031] (3) The AChE@AuNPs immobilized enzyme paper chip has the characteristics of good detection accuracy, strong specificity and good stability, and can be used for detection of organophosphorus pesticide residues in vegetables and fruits. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0033] Figure 1 It is a color development principle diagram for substrate ATCh;

[0034] Figure 2 It is a structural schematic diagram of the AChE@AuNPs immobilized enzyme paper chip;

[0035] Figure 3 It is a preparation process schematic diagram of the AChE@AuNPs immobilized enzyme system;

[0036] Figure 4 It is an influence of color development reaction substrate concentration on color development results in Example 2;

[0037] Figure 5 It is an influence of enzyme bonding time on color development results in Example 3;

[0038] Figure 6 It is an influence of ConA reaction time on color development results in Example 4;

[0039] Figure 7 It is an influence of color development reaction substrate dosage on color development results in Example 5;

[0040] Figure 8 It is an enzyme reaction kinetics curve prepared in Example 6;

[0041] Figure 9Enzyme inhibition reaction kinetics curve prepared for Example 7;

[0042] Figure 10 Intra-day stability of AChE@AuNPs immobilized enzyme paper chip determined in Example 8;

[0043] Figure 11 Standard curve plot prepared after malathion was determined using AChE@AuNPs immobilized enzyme paper chip in Example 9;

[0044] Figure 12 Standard curve plot prepared after malathion was determined using AChE@AuNPs immobilized enzyme paper chip in Example 9;

[0045] Figure 13 Standard curve plot prepared after malathion was determined using AChE@AuNPs immobilized enzyme paper chip in Example 9;

[0046] Figure 14 Standard curve plot prepared after malathion was determined using AChE@AuNPs immobilized enzyme paper chip in Example 9;

[0047] Figure 15 Plot of enzyme activity inhibition rate vs. concentration of malathion determined using AChE@AuNPs immobilized enzyme paper chip in Example 10;

[0048] Figure 16 Plot of enzyme activity inhibition rate vs. concentration of malathion determined using AChE@AuNPs immobilized enzyme paper chip in Example 10;

[0049] Figure 17 Results plot of actual sample detection using AChE@AuNPs immobilized enzyme paper chip in Example 10, wherein a is Shanghai green, b is tomato, and c is yellow sprout;

[0050] Figure 18 Residual activity of enzyme after repeated inhibition / revival of AChE@AuNPs immobilized enzyme paper chip in Example 11;

[0051] Figure 19 Residual activity of immobilized enzyme after repeated use of AChE@AuNPs immobilized enzyme paper chip in Example 12. DETAILED DESCRIPTION

[0052] Various illustrative embodiments of the present application are now described in detail below. The following description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In other instances, well-known structures and functions have not been described in detail in order to avoid obscuring the understanding of the present application.

[0053] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included in the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the smaller ranges.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.

[0055] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative only.

[0056] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0057] I. Instruments used in the present application are as follows:

[0058] (1) pH meter (PB-10, Sartorius);

[0059] (2) Laser particle size potential instrument (Zen-3600, Malvern, UK);

[0060] (3) UV-Vis spectrophotometer (UV-2102PCS, Unico (Shanghai) Instruments Co., Ltd.);

[0061] (4) Magnetic semi-automatic hot press (G311, Fujian Aplis Electrical Co., Ltd.);

[0062] (5) Single-hole puncher (0.6 cm, Deli Group Co., Ltd.);

[0063] (6) Wax jet printer (Xerox Phaser 8570, Fuji Xerox).

[0064] II. Reagents used in the present application are as follows:

[0065] Chloroauric acid trihydrate (≥99.9%, Shanghai Aldrin Biotech Co., Ltd.); Iodine solution of pralidoxime (2-PAM, Shanghai Aldrin Biotech Co., Ltd.); Sodium citrate (analytical pure, Bikeman Biotech); Acetylcholinesterase (Shanghai Yuan Ye Biotech Co., Ltd.); Iodoacetylthiocholine (98%, Shanghai Aldrin Biotech Co., Ltd.); Tris-hydroxymethyl aminomethane (Tianjin Kemio Reagent Co., Ltd.); Concanavalin A (≥98%, Baishabiotech Co., Ltd.); Phosphate buffered saline PBS (pH 8, Yida Technology Co., Ltd.); Bovine serum albumin (≥98%, Zhongmei Biotech Co., Ltd.); 5, 5'-dithiobis (2-nitrobenzoic acid) (≥98%, Shanghai Aldrin Biotech Co., Ltd.); Marathion (analytical standard 1000 μg / mL, Tianjin Alta Technology Co., Ltd.); Dimethoate (analytical standard, Shanghai Aldrin Biotech Co., Ltd.); Deionized water (Xianyang Xiyuan Pure Water Co., Ltd.); Gastrodin (Shanghai Yuan Ye Biotech Co., Ltd., ≥98.0%).

[0066] Thirdly, the solution preparation method adopted in the present application is as follows:

[0067] (1) 2.96 mg / mL tetrachloroauric acid solution: 29.60 mg of tetrachloroauric acid was accurately weighed, dissolved with deionized water and made up to 10 mL in a volumetric flask to obtain a tetrachloroauric acid solution with a concentration of 2.96 mg / mL.

[0068] (2) Sodium citrate solution: 500.00 mg of sodium citrate dihydrate was accurately weighed, dissolved with deionized water and made up to 10 mL in a volumetric flask to obtain a sodium citrate solution with a concentration of 0.1700 mol / L.

[0069] (3) CTAB solution: 36.45 mg of cetyltrimethylammonium bromide (CTAB) was accurately weighed, dissolved with deionized water and made up to 10 mL in a volumetric flask to obtain a CTAB solution with a concentration of 0.01 mol / L.

[0070] (4) Iodine solution of pralidoxime: 100.00 mg of iodine solution of pralidoxime was accurately weighed, dissolved with deionized water and made up to 10 mL in a volumetric flask, shaken well and stored in the refrigerator under light protection.

[0071] (5) Tris-HCl buffer solution: 60.57 mg of Tris solid was accurately weighed, dissolved with deionized water and made up to 25 mL in a volumetric flask, and 1 mol / L HCl solution was added to adjust the pH to 7.5 to obtain a Tris-HCl buffer solution with a concentration of 20 mmol / L.

[0072] (6) 10 U / mL acetylcholinesterase solution (AChE): acetylcholinesterase 50 mg was weighed and dissolved in 1 mL of Tris-HCl buffer solution (20 mmol / L, pH 7.5) to prepare an AChE solution with a concentration of 10 U / mL, which was then aliquoted into centrifuge tubes and stored frozen.

[0073] (7) Iodized thioacetylcholine solution (ATCh): ATCh 25.00 mg was precisely weighed, dissolved in deionized water, and made up to 10 mL in a volumetric flask to obtain an iodized thioacetylcholine solution with a concentration of 8.65 mmol / L, which was stored frozen.

[0074] (8) DTNB solution: 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solid 4.00 mg was precisely weighed, dissolved in 1.00 mL of PBS solution (10 mmol / L, pH 7.4) to obtain a DTNB solution with a concentration of 4 mg / mL, which was stored refrigerated and protected from light.

[0075] (9) Concanavalin A solution: Concanavalin A (ConA) 0.02750 g was precisely weighed, dissolved in 25 mL of PBS solution (10 mmol / L, pH 7.4) to obtain a concanavalin A solution with a concentration of 1.1 mg / mL, which was stored frozen.

[0076] (10) Preparation of bovine serum albumin solution: bovine serum albumin (BSA) 0.050 g was weighed and dissolved in 10 mL of PBS solution (10 mmol / L, pH 7.4) to obtain a bovine serum albumin solution with a concentration of 5 mg / mL.

[0077] (11) Gastrodin solution: Gastrodin 0.0110 g was precisely weighed, dissolved in deionized water, and made up to 2 mL in a volumetric flask to obtain a gastrodin stock solution.

[0078] Example 1

[0079] A method for preparing an AChE@AuNPs immobilized enzyme paper chip:

[0080] The AChE@AuNPs immobilized enzyme paper chip is composed of two parts, the first part (Part I) is a 6 mm circular paper sheet containing the AChE@AuNPs immobilized enzyme reaction system (the circular paper sheet is made of Whatman No. 1 qualitative filter paper using a 6 mm punch), and the second part (Part II) is a detection zone with a channel that can be connected to Part I. The structural diagram is shown in Figure 2 .

[0081] (1) On a 6mm diameter paper disc, 2μL of 3mmol / L HAuCl4solution was dropped, 2min later, 4μL of 0.25mmol / L sodium citrate solution was dropped, after 5min, 3μL of 0.1mmol / L CTAB and 2μL of PBS (pH 8, 10mmol / L) solution was dropped, and the reaction was allowed to proceed for 15min to obtain the paper disc with in situ generated AuNPs.

[0082] (2) Part I: 5.0μL of 1.1mg / mL ConA solution was dropped on the paper disc with in situ generated AuNPs prepared in step (1), and the reaction was allowed to proceed for 1h, then 5.0μL of 5mg / mL BSA solution was dropped on the paper disc to block the non-specific binding sites, and the reaction was allowed to proceed for 15min at room temperature. After the paper disc was washed with PBS (pH 7.4) for 3 times and dried with filter paper, 5.0μL of 10U / mL AChE solution was dropped and the reaction was allowed to proceed for 30min at room temperature. After the reaction was completed, the paper disc was washed with PBS (pH 7.4) for 3 times in the same way to obtain the immobilized enzyme reactor. The preparation process of AChE@AuNPs immobilized enzyme system (immobilized enzyme reactor) is shown in Figure 2. Figure 3 .

[0083] (3) Part II: detection zone

[0084] Whatman 1 qualitative filter paper was cut into a detection zone with a channel, the width of the channel was 1.5mm, the length was 4mm, and the diameter of the detection zone was 4mm.

[0085] (4) Part I and Part II constitute AChE@AuNPs immobilized enzyme paper chip.

[0086] Example 2

[0087] Effect of color reaction substrate concentration on color development results:

[0088] The AChE@AuNPs immobilized enzyme system (Part I) of the AChE@AuNPs immobilized enzyme paper chip prepared in Example 1 was added with 6 μL of ATCh solution with a concentration of 5.19, 6.92, 8.65, 10.37, 12.10 mmol / L, respectively, and then connected with the channel of the detection zone after reaction (enzyme and substrate) for 5 min. Under the capillary action of the paper, the enzyme solution filled the detection zone through the channel, and Part I was separated from Part II. After the paper chip was naturally dried for 3 min, 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added to the non-channel area of the detection zone (Part II), and then the result was recorded by taking a photo after reaction for 1 min. The RGB (red, yellow, and blue three channels superimposed channel) value of the color development result was read by using Photoshop software, and the result was repeated for 3 times. The results are shown in Table 1 and Figure 4 (mean).

[0089] Table 1 Influence of color development substrate concentration on color intensity

[0090]

[0091] ATCh is used as the substrate of the enzyme inhibition color development reaction, and the concentration of ATCh is closely related to the product generation amount. As can be seen from Table 1 and Figure 4 , within the range of 5.19 mmol / L to 8.65 mmol / L, the RGB value increases with the increase of the substrate concentration, and the measured RGB value gradually decreases when the concentration exceeds this range. According to the enzyme reaction kinetics equation, when the substrate concentration is small, the product generation speed increases with the increase of the substrate concentration. However, when the substrate concentration increases to a certain amount, the enzyme is gradually saturated with the substrate, and at this time, the increase of the reaction speed is not proportional to the substrate concentration. Therefore, in order to ensure the stability and high signal intensity in the substrate detection process, 8.65 mmol / L should be selected as the optimal concentration in the reaction process.

[0092] Example 3

[0093] Influence of enzyme bonding time on color development result:

[0094] The preparation method of the AChE@AuNPs immobilized enzyme paper chip is the same as that in Example 1, except that the reaction time after adding the AChE solution in step (2) is 30, 50, 70, 90, 110, 130, and 150 min, respectively, and seven different AChE@AuNPs immobilized enzyme paper chips are prepared.

[0095] 6μL of 8.65mmol / L thioacetylcholine iodide solution (ATCh solution) was added dropwise to the AChE@AuNPs immobilized enzyme system (Part I) of the prepared seven AChE@AuNPs immobilized enzyme paper chips, respectively. After 5min of reaction (enzyme and substrate), the detection zone was connected with the channel, and under the action of capillary force, the enzyme solution filled the detection zone through the channel, and Part I was separated from Part II. After the paper chip was naturally dried for 3min, 1.2μL of 4mg / mL DTNB solution was added dropwise to the non-channel area of the detection zone (Part II), and after 1min of reaction, the result was recorded by taking a photo. The RGB value of the color result was read by Photoshop software, and the result was repeated for three times. The results are shown in Table 2 and Figure 5 (mean).

[0096] Table 2 Influence of enzyme bonding time on color intensity

[0097]

[0098]

[0099] It can be seen from Table 2 and Figure 5 that in the range of 30-90min, the RGB value of the color reaction increases continuously with the increase of time, and the RGB value read at 110min is the largest, indicating that at this time, the enzyme (AChE) is fully bonded with the concanavalin A, and after 110min, the enzyme and ConA are bonded to saturation, and the amount of bonding no longer increases.

[0100] Example 4

[0101] Influence of ConA reaction time on color result:

[0102] The preparation method of the AChE@AuNPs immobilized enzyme paper chip is the same as that of Example 1, except that in step (2), the reaction time after adding the AChE solution is 110min, and the reaction time after adding the concanavalin A solution (ConA) is 60, 75, 90, 105 and 120min, respectively, and five kinds of immobilized enzyme reactors are prepared.

[0103] 6 μL of 8.65 mmol / L thioacetylcholine iodide solution (ATCh solution) was added dropwise to the AChE@AuNPs immobilized enzyme system (Part I) of the five prepared AChE@AuNPs immobilized enzyme paper chips, respectively. After 5 min of reaction (enzyme and substrate), the detection zone was connected with the channel, and under the action of capillary force, the enzyme solution filled the detection zone through the channel, and Part I was separated from Part II. After the paper chip was naturally dried for 3 min, 1.2 μL of 4 mg / mL DTNB solution was added dropwise to the non-channel area of the detection zone (Part II), and after 1 min of reaction, the result was recorded by taking a photo. The RGB (red, yellow, and blue three channels superimposed channel) value of the color development result was read by Photoshop software, and the result was repeated for 3 times. The results are shown in Table 3 and Figure 6 (mean).

[0104] Table 3 Influence of ConA reaction time on color intensity

[0105]

[0106] It can be seen from Table 3 and Figure 6 that the optimal reaction time is 90 min. ConA forms a covalent bond with AuNPs, thereby realizing the connection of ConA to the surface of the paper. The influence of the reaction time of 60-120 min on the RGB value of color development was determined, and the color intensity reached the maximum after 90 min of reaction time, indicating that the ConA immobilization reached a saturated state at this time. In a longer time, the color intensity decreased, because the ConA molecules became too crowded and interfered with each other in space when combined with AchE. Therefore, the reaction time of 90 min was selected for the enzyme inhibition method reaction.

[0107] Example 5

[0108] Influence of loading amount on color development:

[0109] The preparation method of the AChE@AuNPs immobilized enzyme paper chip was the same as that of Example 1, except that in step (2), the reaction time after adding the AChE solution was 110 min, and the reaction time after adding the concanavalin A solution (ConA) was 90 min.

[0110] The AChE@AuNPs immobilized enzyme system (Part I) of the AChE@AuNPs immobilized enzyme paper chip prepared in this example was added with 3, 4, 5, 6, 7, 8 μL of ATCh solution, and the ATCh concentration was the optimal concentration of 8.65 mmol / L. After 5 min of reaction (enzyme and substrate), the detection zone was connected with the channel, and under the action of the capillary of the paper, the enzymatic solution filled the detection zone through the channel, and the Part I and Part II were separated. After the paper chip was naturally dried for 3 min, 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added to the non-channel area of the detection zone (Part II), and after 1 min of reaction, the result was recorded by taking a photo. The RGB value of the color development result was read by using Photoshop software, and the operation was repeated for 3 times. The results are shown in Table 4 and Figure 7 (average value).

[0111] Table 4 Influence of the amount of color development substrate on color intensity

[0112]

[0113] It can be seen from Table 4 and Figure 7 that the optimal amount of ATCh is 6 μL. When the amount of substrate is sufficient, AChE will continue to catalyze the generation of TCH and DTNB color development. Therefore, within a certain period of time, the amount of substrate affects the RGB value of the color development reaction. When the amount of substrate is 3-6 μL, the color development reaction increases with the increase of the amount of substrate. When the amount of substrate is more than 6 μL, the color intensity value gradually decreases with the increase of the amount of substrate, which indicates that when the amount of sample is 6 μL, the substrate and enzyme have been fully reacted.

[0114] Example 6

[0115] Enzymatic kinetics:

[0116] The AChE@AuNPs immobilized enzyme paper chip was prepared according to the method of Example 1 with the optimal conditions of the related factors (the reaction time after adding AChE solution was 110 min, and the reaction time after adding ConA was 90 min), and the detection was performed using the paper chip. Specifically, 6 μL of different concentrations (0.69, 1.04, 2.07, 3.46, 4.84, 6.22 and 7.61 mmol / L) of ATCh solution (substrate) was added to the AChE@AuNPs immobilized enzyme system (Part I) of the AChE@AuNPs immobilized enzyme paper chip, and after 5 min of reaction (enzyme and substrate), the detection zone was connected with the channel, and under the capillary action of the paper, the enzyme solution filled the detection zone through the channel, and Part I was separated from Part II. After the paper chip was naturally dried for 3 min, 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added to the non-channel area of the detection zone (Part II), and after 1 min of reaction, the result was recorded by taking a photo, the RGB value of the color development result was read by using Photoshop software, and the operation was repeated for 3 times. The results are shown in Table 5.

[0117] Table 5 Color intensity at different substrate concentrations

[0118]

[0119] The enzymatic reaction kinetics equation is the Michaelis equation, and the equation is shown in formula (1):

[0120]

[0121] where V max is the maximum reaction rate, [S] is the substrate concentration, V is the reaction rate, Michaelis-Menten binding constant (K m ) is the substrate concentration when the enzymatic reaction rate is half of the maximum reaction rate, and reflects the affinity of the enzyme for the substrate. The smaller K m , the greater the affinity of the enzyme for the substrate.

[0122] The RGB value was used instead of the reaction rate V, the substrate concentration was used as the horizontal coordinate, and the RGB value was used as the vertical coordinate. The Michaelis-Menten curve fitting Michaelis equation was performed in origin software to obtain K m and V max. , and the results are shown in Figure 8 .

[0123] As can be seen from Table 5 and Figure 8 , when the ATCh concentration increased from 0.69 mmol / L to 7.61 mmol / L, the color intensity increased.

[0124] The Michaelis-Menten graph was used for non-linear regression to determine the K m and V max of the immobilized AChE m =(0.15±0.0048)mmol / L, V max =(76.24±0.11)mmol / L / min. Compared with the previously reported K m =(0.21±0.02)mmol / L, V max =(24.52±0.07)mmol / L / min, the K m was significantly reduced, and the V max was significantly increased, indicating that the enzyme and substrate on the AChE@AuNPs immobilized enzyme paper chip prepared in the application had higher affinity.

[0125] Example 7

[0126] Enzyme inhibition kinetics:

[0127] (1) The concentration of gastrodin was 0 μmol / L: The AChE@AuNPs immobilized enzyme paper chip was prepared according to the method of Example 1 using the optimal conditions of the related factors (the reaction time after adding AChE solution was 110 min, and the reaction time after adding ConA was 90 min), and the paper chip was used for detection, specifically: 6 μL of different concentrations (0.2, 0.4, 0.6, 1.0, 1.5 mg / mL) of iodinated thioacetylcholine solution (ATCh solution, substrate) was added to the AChE@AuNPs immobilized enzyme system (Part I) of the AChE@AuNPs immobilized enzyme paper chip, and after 5 min of reaction (enzyme and substrate), the detection zone was connected with the channel, and under the action of paper capillary, the enzyme solution filled the detection zone through the channel, and the first part was separated from the second part. After the paper chip was naturally dried for 3 min, 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added to the non-channel area of the detection zone (Part II), and after 1 min of reaction, the result was recorded by taking a photo, and the RGB value of the color development result was read by Photoshop software, repeated 3 times, and the average value of RGB under different concentrations was calculated.

[0128] (2) The concentration of Gastrodin is 3.5 μmol / L: According to the method of step (1), 6 μL of different concentrations (0.2, 0.4, 0.6, 1.0, 1.5 mg / mL) of iodized thioacetylcholine solution (and the iodized thioacetylcholine solution contains Gastrodin with a concentration of 3.5 μmol / L) is added to the AChE@AuNPs immobilized enzyme paper chip system (Part I) prepared under the optimized optimal conditions, and then the reaction (enzyme and substrate) is carried out for 5 min, and then connected with the channel of the detection area, under the capillary action of the paper, the enzyme solution fills the detection area through the channel, separates Part I from Part II, and then 1.2 μL of DTNB solution with a concentration of 4 mg / mL is added to the non-channel area of the detection area (Part II) after the paper chip is naturally dried for 3 min, and then the reaction is carried out for 1 min, and then the result is recorded by taking a photo with a mobile phone, and then the RGB value of the color development result is read by using Photoshop software, and then the average value of RGB under different concentrations is calculated by repeating 3 times.

[0129] (3) The concentration of Gastrodin is 7.0 μmol / L: According to the method of step (1), 6 μL of different concentrations (0.2, 0.4, 0.6, 1.0, 1.5 mg / mL) of iodized thioacetylcholine solution (and the iodized thioacetylcholine solution contains Gastrodin with a concentration of 7.0 μmol / L) is added to the AChE@AuNPs immobilized enzyme paper chip system (Part I) prepared under the optimized optimal conditions, and then the reaction (enzyme and substrate) is carried out for 5 min, and then connected with the channel of the detection area, under the capillary action of the paper, the enzyme solution fills the detection area through the channel, separates Part I from Part II, and then 1.2 μL of DTNB solution with a concentration of 4 mg / mL is added to the non-channel area of the detection area (Part II) after the paper chip is naturally dried for 3 min, and then the reaction is carried out for 1 min, and then the result is recorded by taking a photo with a mobile phone, and then the RGB value of the color development result is read by using Photoshop software, and then the average value of RGB under different concentrations is calculated by repeating 3 times.

[0130] The enzyme inhibitor double-reciprocal curve formula is as formula (2)

[0131]

[0132] wherein V, K m , [S] have the same meaning as in Example 6, and Gastrodin is an AChE inhibitor. Taking Gastrodin as an example, the paper chip immobilized AChE inhibition kinetics is investigated.

[0133] Taking 1 / ATCh (i.e. 1 / [S]) as the horizontal coordinate and 1 / RGB as the horizontal coordinate, the double-reciprocal curve is constructed by fitting in Origin software, and the results are shown in Figure 9 .

[0134] From Figure 9As can be seen from the figure, the intercept of the three straight lines increases with the increase of the concentration of the inhibitor (Gastrodin), and intersects in the second quadrant, which proves that Gastrodin is a non-competitive inhibitor, and the AChE@AuNPs immobilized enzyme paper chip prepared in the application can be used for the determination of the inhibition rate of AChE.

[0135] Example 8

[0136] Reproducibility test:

[0137] (1) Inter-day reproducibility: according to the method in Example 1, the enzyme bonding time (110 min) and ConA reaction time (90 min) were set as the optimal conditions to prepare the AChE@AuNPs immobilized enzyme paper chip, then 6 μL of iodized thioacetylcholine solution (ATCh solution) with a concentration of 8.65 mmol / L was added dropwise, and after 5 min of reaction (enzyme and substrate), the channel of the detection zone was connected, under the capillary action of the paper, the enzyme solution filled the detection zone through the channel, and the first part and the second part were separated, after the paper chip was naturally dried for 3 min, 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added dropwise in the non-channel area of the detection zone (the second part), and after 1 min of reaction, the result was recorded by taking a photo with a mobile phone, and the RGB value of the color development result was read by Photoshop software, the same AChE@AuNPs immobilized enzyme paper chip was detected for 5 consecutive days, once a day, and after each detection, the buffer was used for cleaning, and the results are shown in Table 6.

[0138] Table 6 Inter-day reproducibility test results

[0139]

[0140] As can be seen from Table 6, the inter-day precision is 1.04%, which is within the range of 2%, indicating that the method has good inter-day reproducibility.

[0141] (2) Intra-day reproducibility: according to the method in Example 1, the enzyme bonding time (110 min) and ConA reaction time (90 min) were set as the optimal conditions to prepare the AChE@AuNPs immobilized enzyme paper chip, then 6 μL of iodized thioacetylcholine solution (ATCh solution) with a concentration of 8.65 mmol / L was added dropwise, and after 5 min of reaction (enzyme and substrate), the channel of the detection zone was connected, under the capillary action of the paper, the enzyme solution filled the detection zone through the channel, and the first part and the second part were separated, after the paper chip was naturally dried for 3 min, 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added dropwise in the non-channel area of the detection zone (the second part), and after 1 min of reaction, the result was recorded by taking a photo with a mobile phone, and the RGB value of the color development result was read by Photoshop software, the same AChE@AuNPs immobilized enzyme paper chip was detected for 5 times within 1 day, and after each detection, the buffer was used for cleaning, and the results are shown in Table 7.

[0142] Table 7 Intra-day repeatability experiment results

[0143]

[0144] As can be seen from Table 7, the RSD% value is 0.76%, indicating that the method has good intra-day repeatability.

[0145] (3) Intra-day stability: 40 consecutive tests were performed using the same prepared enzyme reactor to determine the degree of enzyme activity reduction. Under the same conditions, free enzyme was also compared, and the enzyme was directly titrated on the blank paper chip, which was the process of making free enzyme. The results are shown in Figure 10 .

[0146] As can be seen from Figure 10 , the AChE activity of the AChE@AuNPs immobilized enzyme paper chip showed a downward trend after 40 consecutive tests, but still retained more than 85% of the initial enzyme activity.

[0147] Example 9

[0148] Pesticide standard solution detection:

[0149] (1) Two different organophosphorus pesticides (malathion, dimethoate) were selected, and the pesticides were prepared into a series of standard working solutions (concentrations were 1.5, 3.0, 6.0, 12.0, 24.0 μg / mL) respectively using 10% acetonitrile aqueous solution, and stored in a freezer away from light.

[0150] (2) According to the method of Example 1, combined with the single factor experiment investigated above, the optimal reaction conditions were determined (the reaction time after adding AChE solution was 110 min, and the reaction time after adding concanavalin A (ConA) solution was 90 min), and the AChE@AuNPs immobilized enzyme paper chip was prepared. The pesticide standard working solution with different concentrations was mixed with ATCh solution (8.65 mmol / L) in a volume ratio of 6:3, and 6 μL of the obtained mixed solution was added to the AChE@AuNPs immobilized enzyme paper chip system (Part I) for reaction for 5 min; the enzyme solution was absorbed by the detection zone (Part II), and under the action of paper capillary, the enzyme solution filled the detection zone through the channel, separating Part I from Part II. After the paper chip was naturally dried for 3 min, 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added to the non-channel area of the detection zone (Part II), and after reaction for 1 min, the result was recorded by taking a photo, the RGB value was measured, the enzyme inhibition rate was calculated by the measured RGB value, and the logarithmic value of the measured pesticide concentration was taken as the abscissa, and the enzyme inhibition rate was taken as the ordinate, to establish the corresponding standard curve graph, the results are shown in Tables 8-9 and Figures 11-12 .

[0151] Control test: using UV-spectrophotometry, 30 μL of AChE solution with a concentration of 10 U / mL, 100 μL of ATCh solution with a concentration of 10 mmol / L, 100 μL of PBS buffer (10 mmol / L, pH 7.4) were added into a beaker, then 25 μL of pesticide standard working solution with different concentrations was added respectively, the reaction was carried out for 5 min, 1.0 mL of SDS (enzyme reaction terminator, sodium dodecyl sulfate) solution with a concentration of 4% was added to terminate the reaction, the solution was diluted to 10 mL with PBS buffer (10 mmol / L, pH 7.4), finally 1.2 μL of DTNB solution with a concentration of 4 mg / mL was added, after the reaction, the absorbance at 412 nm was determined by using a UV-vis spectrometer, the enzyme inhibition rate was calculated according to the determined absorbance, the standard curve was established by taking the concentration of the measured pesticide as the horizontal coordinate and the enzyme inhibition rate as the vertical coordinate, and the results are shown in Figures 13-14 .

[0152] When the AChE@AuNPs immobilized enzyme paper chip was used for detection, the calculation formula of the enzyme inhibition rate (IR) was as follows:

[0153]

[0154] wherein, CI0 and CI1 represent the RGB values of the blank sample (without pesticide) and the measured sample (with pesticide) after analysis respectively.

[0155] When the UV-spectrophotometry was used for detection, the calculation formula of the enzyme inhibition rate (IR) was as follows:

[0156]

[0157] wherein, A0 and A1 represent the absorbance of the blank sample (without pesticide, the RGB value is 78.84) and the measured sample (with pesticide) respectively.

[0158] Table 8: results of the determination of dimethoate by using the AChE@AuNPs immobilized enzyme paper chip

[0159]

[0160] Table 9: results of the determination of malathion by using the AChE@AuNPs immobilized enzyme paper chip

[0161]

[0162]

[0163] As can be seen from Figures 11-12 , the fitting curves R 2The values ​​of 0.9997 and 0.9991, respectively, indicate that the standard curve has a good linear relationship; the IC50 of pesticides against AChE is usually used to calculate the effective value of pesticides against AChE. 50 The value is defined as the limit of detection (LOD) for pesticides by the experimental method. The LOD (μg / mL) for dimethoate and malathion were calculated to be 1.035±0.1024 and 2.045±0.78518, respectively. The LOD values ​​reported by this method are close to those of similar research methods, indicating that this method is feasible.

[0164] Example 10

[0165] Pesticide residue determination of actual samples

[0166] Weigh 2.5g each of Chinese cabbage, Shanghai bok choy, and tomato into a mortar and grind them. Add 10mL of 10% acetonitrile aqueous solution to each and homogenize. Sonicate the homogenized solution for 30min, centrifuge for 3min, first coarsely filter it once with medium-speed qualitative filter paper, and then filter it twice with a 0.22μm filter membrane to obtain the vegetable sample extract. Measure and read the RGB values ​​according to the method in Example 9.

[0167] The relationship between the inhibition rate of dimethoate on enzyme activity and its concentration, as determined using AChE@AuNPs immobilized enzyme paper chips (fitted curve equation), is shown in the figure below. Figure 15 The relationship between the inhibition rate of dimethoate on enzyme activity and its concentration, determined by ultraviolet-spectrophotometry, is shown in the figure (fitted curve equation). Figure 16 The results of detecting actual samples using AChE@AuNPs immobilized enzyme paper chips are shown in the figure. Figure 17 , Figure 17 In the diagram, a represents Shanghai bok choy, b represents Chinese cabbage, and c represents tomatoes.

[0168] Table 10. Standard solution curve data determined using AChE@AuNPs immobilized enzyme paper chip.

[0169]

[0170] Table 11 shows the detection of actual samples using AChE@AuNPs immobilized enzyme paper chips.

[0171]

[0172]

[0173] from Figures 15-16 As can be seen, from 0.30 μg / mL to 1.50 μg / mL, the AChE inhibition rate increased linearly with the increase of organophosphorus pesticide concentration, and the R value of the fitted curve was [value missing]. 2All are greater than 0.99. According to the method in Example 9, the RGB values of Shanghai green, yellow sprout and tomato are 68.43, 67.57 and 65.55 respectively. According to the fitting curve equation, the concentrations of organophosphorus pesticides (calculated by taking Rogor as a standard to calculate the total concentration of organophosphorus pesticides) in Shanghai green, yellow sprout and tomato are 1.21 mg / kg, 1.33 mg / kg and 1.60 mg / kg respectively. The organophosphorus pesticide residues in the three kinds of vegetables measured by UV-spectrophotometry are 1.29 mg / kg, 1.41 mg / kg and 1.70 mg / kg respectively. The results of the two methods are similar, and the pesticide residues of the three samples all meet the requirements of the national standard for the limit of organophosphorus pesticide residues in vegetables.

[0174] Example 11

[0175] Investigation of the activity of AChE@AuNPs immobilized enzyme paper chip:

[0176] Organophosphorus pesticides are very firm in binding with the -OH of serine site in AChE molecules, so they can irreversibly inhibit enzyme activity. The inhibited enzyme molecules cannot recover by themselves, but it is very important to restore the activity of the inhibited enzyme for realizing continuous multiple sample detection of the immobilized enzyme reactor.

[0177] In the present application, pralidoxime iodide (2-PAM, concentration of 10 mg / mL) is used as a reactivator, 5 mg / mL of Rogor is used as an inhibitor to carry out color reaction with substrate ATCh (10 mg / mL), and 10 μL of 2-PAM (10 mg / mL) is added on the paper chip after the use of the inhibitor for inhibition for 10 min, and the immobilized enzyme reactor is repeatedly determined for 13 times in this way. The results are shown in Figure 18 .

[0178] As can be seen from Figure 18 , after adding 2-PAM on the inhibited enzyme reactor, the relative activity of the enzyme decreases less, thereby indicating that the reactivation of the immobilized enzyme reactor can be realized, and after 13 times of repeated inhibition and reactivation of the enzyme using 2-PAM, the activity of AChE can still be maintained at about 75%, thereby indicating that the reactivation of the AChE@AuNPs immobilized enzyme paper chip can realize the repeated detection of organophosphorus pesticides.

[0179] The application successfully combines nano gold and acetylcholinesterase together, and constructs a directional immobilized enzyme reactor (AChE@AuNPs immobilized enzyme paper chip). The AChE@AuNPs immobilized enzyme paper chip is used to determine two kinds of pesticide standard solutions of malathion and dimethoate, a standard curve is established according to the pesticide concentration and the corresponding RGB value, the curve is linear well; the organic phosphorus pesticide content of several vegetables is determined, the results are similar to the results of related published literatures, and meet the national standards. The method has good accuracy, qualified relative error, good repeatability, low cost, easy to use and fast analysis characteristics, and is expected to be used for the detection of pesticide residues in actual vegetable samples.

[0180] Example 12

[0181] Investigation of the storage activity of AChE@AuNPs immobilized enzyme paper chip:

[0182] The day-to-day retention activity of free enzyme and immobilized enzyme is investigated, and the results are shown in Table 1. Figure 19 As shown in Table 1, after continuously determining for 7 days on the same enzyme reactor, the activity of the immobilized enzyme (AChE@AuNPs immobilized enzyme paper chip) is 78.06% of the initial activity, while the activity of the free enzyme is only 3.63%, which indicates that the immobilized enzyme reactor (AChE@AuNPs immobilized enzyme paper chip) prepared by the method of the application has good stability and repeatability, and it can also prove that the method of immobilizing AChE greatly improves the activity of the enzyme.

[0183] The above-described examples only describe the preferred modes of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements of the technical solutions of the application made by those skilled in the art shall fall within the protection scope determined by the claims of the application.

Claims

1. A preparation method of an AChE@AuNPs immobilized enzyme paper chip for detecting organophosphorus pesticides, characterized in that, The method comprises the following steps: dropping HAuCl4 solution on the paper sheet, standing, then dropping reductant solution to react, then dropping surfactant solution and buffer solution, and obtaining the paper chip with in-situ generated gold nanoparticles after reaction; dropping concanavalin A solution on the paper chip with in-situ generated gold nanoparticles, reacting, then dropping bovine serum albumin solution to react, finally dropping AChE solution, and obtaining AChE@AuNPs immobilized enzyme system after reaction; the AChE@AuNPs immobilized enzyme system and the detection area constitute the AChE@AuNPs immobilized enzyme paper chip; the reductant solution comprises sodium citrate solution; the surfactant solution comprises CTAB solution; the buffer solution is PBS buffer solution with pH=8 and 10 mmol / L; the molar ratio of HAuCl4 in the HAuCl4 solution, reductant in the reductant solution and surfactant in the surfactant solution is 6:1:0.3; the reaction time after dropping the reductant solution is 5 min; and the reaction time after dropping the surfactant solution and the buffer solution is 15 min; the dosage ratio of HAuCl4 in the HAuCl4 solution and AChE in the AChE solution is 6 mmol:50000 U; the dosage ratio of concanavalin A in the concanavalin A solution, bovine serum albumin in the bovine serum albumin solution and AChE in the AChE solution is 1.1 mg:5 mg:10 U; the reaction time after dropping the concanavalin A solution is 60-120 min; the reaction time after dropping the bovine serum albumin solution is 15 min; and the reaction time after dropping the AChE solution is 30-150 min. 2.An AChE@AuNPs immobilized enzyme paper chip prepared by the preparation method in claim 1. 3.Use of the AChE@AuNPs immobilized enzyme paper chip in claim 2 in detection of pesticides inhibiting AChE activity.

4. A method for detecting a pesticide which inhibits AChE activity, characterized by, The method comprises the following steps: dropping color reaction substrate and pesticides inhibiting AChE activity on the AChE@AuNPs immobilized enzyme system of the AChE@AuNPs immobilized enzyme paper chip in claim 2, connecting with the detection area after reaction, then dropping color developing agent on the detection area to develop color, finally determining RGB value according to the color developing degree, and calculating the content of the pesticides inhibiting AChE activity according to the RGB value; the color reaction substrate is ATCh; and the color developing agent is 5,5'-dithiobis(2-nitrobenzoic acid).

5. The detection method according to claim 4, characterized in that, the pesticides inhibiting AChE activity comprise organophosphorus pesticides.

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