CN109253999B - Preparation and application of pesticide quick test card - Google Patents
Preparation and application of pesticide quick test card Download PDFInfo
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- CN109253999B CN109253999B CN201811092703.6A CN201811092703A CN109253999B CN 109253999 B CN109253999 B CN 109253999B CN 201811092703 A CN201811092703 A CN 201811092703A CN 109253999 B CN109253999 B CN 109253999B
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Abstract
The invention relates to the fields of nano materials, catalysis and analytical chemistry, and particularly relates to preparation and application of a pesticide rapid test card. The method utilizes cobaltosic oxide nanospheres with peroxidase activity, acetylcholinesterase, choline oxidase and a color-developing agent to modify filter paper to obtain a rapid detection card, and realizes simple and rapid detection of acetylcholine and organophosphorus pesticides. The novel colorimetric pesticide detection method has strong practicability, so the method has wide application prospect in the fields of food detection, analytical chemistry, environmental engineering and agriculture.
Description
Technical Field
The invention relates to the fields of nano materials, catalysis and analytical chemistry, and particularly relates to preparation and application of a pesticide rapid test card.
Technical Field
With the development of agricultural technology, pesticides are widely applied. Pesticide residues become an important hidden danger threatening the safety of human food, so that the detection of pesticides becomes more important and is greatly concerned by people. There are many papers on the detection of pesticides, for example, Jung et al in M13 bacteriophages/silver nanowire surface-enhanced Raman scattering sensor for sensitive and selective detection of pesticides by Raman using M13 phage functionalized silver nanowires (ACS Applied Materials & Interfaces2018,10, 10388-10397). Pumera et al, in Two-dimensional 1T-phase transition metal dichalcogenides as nanocarriers to enhance enzyme activity in electrochemical detection of pesticide(s) ((ACS Nano2017, 11, 5774-5784). Colorimetric detection of organophosphorous pesticides by immobilizing acetylcholinesterase onto silver nanoparticles as described in Acetylcholinesterase (AChE) -mediated immobilization of silver nanoparticles for the detection of organophosphorous pesticides by Mukherjee et alRSC Advances2016, 6, 64769-64777). Although the reports all realize the detection of the pesticide, the detection process is complex, and a new method needs to be developed to realize the rapid detection of the pesticide.
The test strip has been widely used in life as a portable rapid detection method, such as a blood glucose test strip, a urine analysis test strip, and the like. Therefore, many documents have also conducted some research on test strips. Yan et al in Nanozyme-strip for Rapid local diagnosis of Ebola, the colloidal gold in the traditional test strip is replaced by magnetic nano-enzyme, the ebola virus is detected by a color development method, and the sensitivity of the test strip is improved by 100 times (Biosensors and Bioelectronics2015,74, 134-141). Cai et al, in A simple and rapid colloidal gold-based immunochloromatographic strip for detection of FMDV derivative A, combined immunoassay and test strip (ACS Applied Materials & Interfaces2014,6, 21624-21631). Although the test strip has been applied to a certain extent, the existing method has a complex manufacturing process and high cost, and the action mechanism of the test strip is essentially different from that of pesticide small molecule detection. Therefore, it is necessary to develop a new method for realizing simple and rapid preparation and detection of small molecules of pesticides.
Disclosure of Invention
The invention aims to provide preparation and application of a pesticide quick test card.
In order to achieve the purpose, the invention adopts the technical scheme that:
the preparation method of the pesticide quick test card is characterized in that the preparation process of the pesticide quick test card is as follows:
(1) dropping a Bovine Serum Albumin (BSA) solution with the mass concentration of 1% on a cellulose membrane, standing for 1 hour and drying at room temperature;
(2) respectively dripping 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with the mass concentration of 2% on the cellulose membrane modified with BSA in the step (1), standing for 5 hours, washing with water and drying at room temperature;
(3) uniformly dripping the solution of the composite catalytic material on the cellulose membrane obtained in the step (2), standing overnight at 4 ℃, washing with water and drying;
(4) and (4) dropwise coating the organic color developing agent solution on the cellulose membrane modified with the composite catalytic material in the step (3), and drying at 4 ℃ to obtain the rapid test card.
The preparation process of the composite catalytic material is characterized by comprising the following steps:
(1) adding a certain amount of sodium borohydride into a mixed solution containing BSA and divalent cobalt ions, reacting, centrifuging and drying to obtain Co3O4 NPs；
(2) Mixing a certain amount of cobaltosic oxide nanospheres (Co)3O4NPs) is added into a 0.2mM tert-butyl carbamate (BOC) solution, and the mixture is washed and dried after being kept stand for 2 hours;
(3) the Co obtained in the step (2) is mixed3O4Adding NPs into a mixed solution of EDC and NHS with the mass concentration of 2%, standing for 5 hours, washing with water and drying;
(4) the Co obtained in the step (3) is put into3O4NPs are added into a mixed solution of acetylcholinesterase (AchE) and choline oxidase (ChOx), reacted overnight at 4 ℃, washed and dried to prepare the composite catalytic material.
The preparation and application of the pesticide quick test card are provided, and the composite catalytic material can be used for detecting pesticides.
Preferably, the pesticide tach consists of a substrate, a composite catalytic material and a color developing agent, wherein the composite catalytic material is Co modified with AchE and Chox3O4NPs, composite catalytic material and color developing agent are fixed on the substrate.
More preferably, the substrate can be a nitrocellulose membrane, a cellulose acetate membrane, polyester fiber, glass fiber, non-woven fabric, and the like; the organic color developing agent is 2,2 ' -dinitro-bis (3-ethylbenzthiazoline-6-sulfonic acid) diamine salt, 3 ', 5,5 ' -tetramethyl benzidine, dopamine or phenol.
Further preferably, the detection application of the pesticide tach is characterized in that the application process is as follows:
(1) taking two rapid test cards, adding a certain amount of organophosphorus or carbamate pesticide solution on one rapid test card, and leaving the other rapid test card without pesticide for 5 minutes to ensure that the organophosphorus or carbamate pesticide and the composite catalytic material fully act;
(2) then adding a certain amount of acetylcholine on the rapid-determination card, standing for 5 minutes to enable the acetylcholine to fully react with the composite catalytic material;
(3) observing the color change of the rapid test card to realize the qualitative detection of the organophosphorus or carbamate pesticide, and if the color of the rapid test card is lighter than that of the rapid test card without the organophosphorus or carbamate pesticide, indicating that the organophosphorus or carbamate pesticide exists in the system;
(4) the intelligent mobile phone is used for photographing, and the photographing can be carried out under the irradiation of the LED lamp during photographing so as to prevent the pictures from being fuzzy. Reading the specific RGB value of the speed measuring card in the photo by using color recognition software of the mobile phone, and obtaining the RGB value by BNCalculating to obtain B/(R + G + B)NSubstituted into BNAnd calculating the concentration of the pesticide by using a concentration working curve equation to realize quantitative detection of the organophosphorus or carbamate pesticide.
More preferably, the organophosphorus pesticide can be organophosphorus pesticide such as paraoxon, parathion, phosphorus dibromide, diazinon and the like; the carbamate pesticide can be carbamate pesticide such as metolcarb, carbaryl, aldicarb, carbofuran, cicada powder, pirimicarb and the like.
The invention has the following effects:
1. the invention utilizes the series reaction of cobaltosic oxide nanospheres with the activity of mimic peroxidase, acetylcholinesterase and choline oxidase to construct a sensor for rapidly detecting organophosphorus pesticides.
2. The composite catalytic material synthesized by the invention and the prepared test strip realize one-pot pesticide detection and avoid the step reaction.
3. The invention has certain practical value in actual life and can be expanded to quantitative analysis, catalysts and other aspects in the fields of food fermentation, biomedicine, chemical industry, environment, biotechnology and the like.
Drawings
Fig. 1 is a flowchart of a manufacturing process of a speed measuring card according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of a detection of a speed measurement card according to an embodiment of the present invention.
Fig. 3 illustrates the feasibility of the speed measuring card detection provided by the embodiment of the present invention.
Fig. 4 is a one-pot pH optimization of the composite catalytic material provided in the embodiments of the present invention.
Fig. 5 is a photograph of a qualitative test of the speed test card according to the embodiment of the present invention.
Fig. 6 is a standard working curve of quantitative determination of pesticide provided by the embodiment of the invention.
Detailed Description
In order to further illustrate the contents of the present invention, some examples will be further illustrated below, but the present invention is not limited to the illustrated examples. The specific experimental conditions or methods in the following examples, if not noted, were carried out according to conventional conditions or methods in the art.
Example 1
The preparation process of the composite catalytic material is characterized by comprising the following steps:
(1) adding a certain amount of sodium borohydride into a mixed solution containing BSA and divalent cobalt ions, and obtaining Co through the reduction of the sodium borohydride and the oxidation of oxygen3O4NPs are centrifuged and dried to obtain solid;
(2) a certain amount of Co3O4NPs were added to a 0.2mM BOC solution to protect Co3O4Standing amino groups on the NPs for 2 hours, washing with water, and drying;
(3) the Co obtained in the step (2) is mixed3O4NPs were added to a mixed solution of EDC and NHS at a mass concentration of 2% to activate Co3O4Standing carboxyl on NPs for 5 hours, washing with water and drying;
(4) the Co after the carboxyl activation obtained in the step (3) is treated3O4NPs are added into the mixture of AchE and ChOx, and the amino groups on the AchE and ChOx react with Co3O4The carboxyl groups on the NPs are subjected to an amide reaction to be fixed to Co3O4And (4) carrying out an overnight reaction on the NPs at the temperature of 4 ℃, washing with water, and drying to obtain the composite catalytic material.
Example 2
Preparation process of the speed measuring card (fig. 1):
(1) dropping a BSA solution with the mass concentration of 1% on a cellulose membrane, standing for 1 hour and drying at room temperature;
(2) respectively dripping EDC and NHS with the mass concentration of 2% on the cellulose membrane (selecting the cellulose nitrate membrane with good water absorbability) modified with BSA in the step (1) to activate the carboxyl of the BSA, standing for 5 hours, washing with water and drying at room temperature;
(3) uniformly dripping the solution of the composite catalytic material on the cellulose membrane obtained in the step (2), enabling amino on the composite catalytic material to react with carboxyl on BSA (bovine serum albumin) to enable the composite catalytic material to be fixed on the cellulose membrane, standing overnight at 4 ℃, washing with water and drying;
(4) and (4) dropwise coating the organic color developing agent solution on the cellulose membrane modified with the composite catalytic material in the step (3), and drying at 4 ℃ to obtain the rapid test card.
Detection principle of the tach card (fig. 2):
the pesticide quick test card consists of a substrate, enzyme and a color developing agent, wherein the substrate adopts nitrocellulose filter paper, and Co connected with AchE and ChOx3O4NPs and a color developing agent are fixed on a substrate, and the detection basic principle is as follows: when acetylcholine is added, acetylcholine first generates choline under the catalytic action of AchE, and the generated choline continuously reacts with Chox to generate H2O2Then Co with peroxidase-mimetic activity3O4NPs catalyze H2O2The ABTS is changed in color, and the speed measuring card is changed from colorless to green; organophosphorus or carbamate pesticides can inhibit the activity of AchE, so that a series of catalytic and oxidation reactions cannot be normally carried out, the color change of the rapid-determination card is small or unchanged, and the quantitative analysis of the pesticides can be realized according to the color change condition of the rapid-determination card.
Example 3
Feasibility of rapid test card detection:
catalytic reaction system (a) the colorimetric card was cut into pieces and placed in a solution of acetylcholine (1 mmol/L) (pH = 5.0), and after 5 minutes of reaction, the reaction solution was scanned at full wavelength. For another set of control experiments (b) the cards were cut into pieces and placed in buffer (pH = 5.0) and after 5 minutes of reaction, the reaction solution was scanned at full wavelength.
As shown in fig. 3, the catalytic reaction system (a) has a distinct uv absorption peak, and the catalytic reaction system (b) has no uv absorption peak, demonstrating that the catalytic reaction can only proceed in the presence of acetylcholine.
Example 4
One-pot pH optimization of the composite catalytic material:
the catalytic reaction system comprises a composite catalytic material (30 mu g/mL), acetylcholine (1 mmol/L) and buffers with different pH values (pH 1.0-2.0, glycine-hydrochloric acid buffer solution, pH 3.0-6.0, acetic acid-sodium acetate buffer solution, pH 7.0-8.0, phosphate buffer solution, pH 9.0, Tris-hydrochloric acid buffer solution). After 5 minutes of reaction at room temperature (25 ℃), the absorbance of the reaction solution at 650 nm was measured. As shown in FIG. 4 (a), the optimum pH was about 5.0.
Another set of control experiments was performed, in which the catalytic reaction system included Co3O4NPs (30. mu.g/mL), AchE (3.75. mu.g/mL), ChOx (1.25. mu.g/mL), acetylcholine (1 mmol/L) and buffers of different pH (pH 1.0-2.0, glycine-hydrochloric acid buffer; pH 3.0-6.0, acetic acid-sodium acetate buffer; pH 7.0-8.0, phosphate buffer; pH 9.0-10.0, Tris-hydrochloric acid buffer). After 5 minutes of reaction at room temperature (25 ℃), the absorbance of the reaction solution at 650 nm was measured. As shown in FIG. 4 (b), the activity was higher at a pH of about 3.0 to 6.0, but the activity was lower than that of 3 (a).
Example 5
Qualitative detection of the speed test card:
(1) taking three speed measuring cards, wherein pesticide is not added in the speed measuring card (a), 10 ng/L of organophosphorus or carbamate pesticide is added in the speed measuring card (b), and 100 ng/L of organophosphorus or carbamate pesticide is added in the speed measuring card (c), and placing for 5 minutes to enable the organophosphorus or carbamate pesticide to fully act with the composite catalytic material;
(2) then adding 1 mmol/L acetylcholine on the rapid-determination card, standing for 5 minutes to enable the acetylcholine to fully react with the composite catalytic material;
(3) the intelligent mobile phone is used for photographing, and the photographing can be carried out under the irradiation of the LED lamp during photographing so as to prevent the pictures from being fuzzy. As shown in fig. 5, the color of the rapid measurement card (a) without pesticide is changed to green, the color of the rapid measurement card (b) with pesticide of smaller concentration is changed to lighter than that of the rapid measurement card (a), and the color of the rapid measurement card (c) with pesticide of larger concentration is not changed, which indicates that the pesticide concentration dropped on the rapid measurement card (c) is the largest, and the rapid measurement card (b) is the next time, and the rapid measurement card (a) is not added with pesticide, which proves the feasibility of pesticide detection by the rapid measurement card, therefore, the rapid measurement card can realize pesticide detection.
Example 6
Standard working curve for pesticide quantitative determination:
(1) taking eight rapid test cards, dripping parathion pesticides (0-50 ng/mL) with different concentrations on the rapid test cards, and standing for 5 minutes to ensure that the parathion pesticides and the composite catalytic material have full action;
(2) then 10 mul 10 mM acetylcholine is added on the rapid-determination card, and the card is kept stand for 5 minutes to ensure that the card and the composite catalytic material fully react;
(3) the intelligent mobile phone is used for photographing, and the photographing can be carried out under the irradiation of the LED lamp during photographing so as to prevent the pictures from being fuzzy. Reading the specific RGB value of the speed measuring card in the photo by using color recognition software of the mobile phone, and obtaining the RGB value by BNCalculating to obtain B/(R + G + B)NObtaining a standard curve, wherein the specific working curve is shown in FIG. 6, the linear range is 0-20ng/L, and the equation is y = -0.0194x +0.7975 (R)2=0.9981）。
Claims (6)
1. A preparation method of a pesticide quick test card is characterized by comprising the following steps: the pesticide quick test card consists of a substrate, a composite catalytic material and a color developing agent, wherein the composite catalytic material is cobaltosic oxide nanospheres (Co) modified with acetylcholinesterase (AchE) and choline oxidase (ChOx)3O4NPs), the composite catalytic material and the color developing agent are fixed on the substrate; the preparation method of the pesticide quick test card comprises the following steps:
(1) dropping a Bovine Serum Albumin (BSA) solution with the mass concentration of 1% on a cellulose membrane, standing for 1 hour and drying at room temperature;
(2) respectively dripping 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with the mass concentration of 2% on the cellulose membrane modified with BSA in the step (1), standing for 5 hours, washing with water and drying at room temperature;
(3) uniformly dripping the solution of the composite catalytic material on the cellulose membrane obtained in the step (2), standing overnight at 4 ℃, drying and washing with water;
(4) and (4) dropwise coating the organic color developing agent solution on the cellulose membrane modified with the composite catalytic material in the step (3), and drying at 4 ℃ to obtain the rapid test card.
2. The method for preparing the pesticide tach according to claim 1, characterized in that the preparation process of the composite catalytic material is as follows:
(1) adding sodium borohydride into a mixed solution containing BSA and divalent cobalt ions, reacting, centrifuging and drying to obtain Co3O4NPs；
(2) Mixing Co3O4NPs are added into a 0.2mM tert-butyl carbamate (BOC) solution, kept stand for 2 hours, washed with water and dried;
(3) the Co obtained in the step (2) is mixed3O4Adding NPs into a mixed solution of EDC and NHS with the mass concentration of 2%, standing for 5 hours, washing with water and drying;
(4) the Co obtained in the step (3) is put into3O4And adding NPs into the mixed solution of the AchE and the ChOx, reacting at 4 ℃ overnight, washing with water, and drying to obtain the composite catalytic material.
3. The method for preparing the pesticide tach according to claim 1, characterized in that: the substrate is a nitrocellulose membrane, a cellulose acetate membrane, polyester fiber, glass fiber, non-woven fabric and the like; the organic color developing agent is 2,2 ' -dinitro-bis (3-ethylbenzthiazoline-6-sulfonic acid) diamine salt, 3 ', 5,5 ' -tetramethyl benzidine, dopamine or phenol.
4. The preparation method of the pesticide tach according to claim 1, characterized in that the tach is used for pesticide detection, and the steps are as follows:
(1) taking two rapid test cards, adding an organophosphorus or carbamate pesticide solution on one rapid test card, and leaving the other rapid test card without pesticide for 5 minutes to ensure that the organophosphorus or carbamate pesticide and the composite catalytic material fully act;
(2) then adding acetylcholine on the rapid-determination card, and standing for 5 minutes to enable the acetylcholine to fully react with the composite catalytic material;
(3) observing the color change of the rapid test card to realize the qualitative detection of the organophosphorus or carbamate pesticide, and if the color of the rapid test card is lighter than that of the rapid test card without the organophosphorus or carbamate pesticide, indicating that the organophosphorus or carbamate pesticide exists in the system;
(4) the intelligent mobile phone is used for photographing, and the photographing is carried out under the irradiation of the LED lamp during photographing so as to prevent the pictures from being fuzzy.
5. The detection application of the pesticide tach of claim 4, characterized in that: reading the specific RGB value of the speed measuring card in the photo by using color recognition software of the mobile phone, and obtaining the RGB value by BNB is calculated as B/(R + G + B)NSubstituted into BNAnd calculating the concentration of the pesticide by using a concentration working curve equation to realize quantitative detection of the organophosphorus or carbamate pesticide.
6. The detection application of the pesticide tach of claim 4, characterized in that: the organophosphorus pesticide is organophosphorus pesticide such as paraoxon, parathion, phosphorus dibromide, diazinon and the like; the carbamate pesticide is carbamate pesticide such as metolcarb, carbaryl, aldicarb, carbofuran, cicada powder, pirimicarb and the like.
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| CN115356336B (en) * | 2022-10-21 | 2022-12-13 | 广东江门中医药职业学院 | Method for rapidly detecting trace dibromophosphate residue in agricultural products |
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| EP1175509A2 (en) * | 1999-04-26 | 2002-01-30 | U.S. Army Medical Research and Materiel Command | Immobilized enzymes as biosensors for chemical toxins |
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