Test paper for detecting carbamate and organophosphorus pesticides as well as preparation method and application of test paper

Abstract

The invention relates to the technical field of test paper, and particularly discloses test paper for detecting carbamate and organophosphorus pesticides as well as a preparation method and application of the test paper. The test paper comprises a PVC substrate, a sample pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the nitrocellulose membrane and the water absorption pad are sequentially stacked, and the nitrocellulose membrane is coated with a detection line formed by manganese dioxide nanoneedles wrapped by bovine serum albumin. The test paper provided by the invention can realize semi-quantitative detection of carbamate and organophosphorus pesticides by counting the disappearing number of the detection line, has the advantages of simplicity and convenience in operation, low cost, convenience in carrying, intuitive result interpretation, strong anti-interference capability and the like, and is particularly suitable for on-site rapid screening.

Description

Technical Field

[0001] This invention relates to the field of test strip technology, specifically to a test strip for detecting carbamates and organophosphorus pesticides, its preparation method, and its application. Background Technology

[0002] Carbamates and organophosphates are commonly used pesticides in agriculture, accounting for over 90% of their application. Therefore, efficient detection of these two pesticides is crucial for food quality monitoring; however, existing technologies have significant limitations. Enzyme inhibition is a traditional rapid detection method for carbamates and organophosphates. This method utilizes acetylcholinesterase (AChE) to catalyze the reaction of iodothioacetylcholine (ATChI) to produce thioacetylcholine. The thioacetylcholine then reacts with 5,5'-dithiobis(2-nitrobenzoic acid) to form a yellow compound. When carbamates and organophosphates are present in the sample, acetylcholinesterase activity is specifically inhibited, resulting in a reduced content of the yellow compound. The inhibition rate of acetylcholinesterase activity is determined by spectrophotometry, thus indicating the presence of carbamates and organophosphates in the sample. While this method is simple to operate, it still requires a colorimetric reagent and a portable spectrophotometer, making it costly and lacking portability for on-site testing. Summary of the Invention

[0003] This invention provides a test strip for detecting carbamates and organophosphorus pesticides, its preparation method, and its application. When detecting carbamates and organophosphorus pesticides, the MnO2 in the detection line of the test strip provided by this invention is reduced to colorless Mn. 2+ Ions cause a visual change in the detection line from brown to colorless. By counting the number of disappearing detection lines, a semi-quantitative analysis of the target pesticide can be achieved. It has the advantages of simple operation, low cost, portability, intuitive result interpretation and strong anti-interference ability, and is particularly suitable for rapid on-site screening.

[0004] The present invention provides a test strip for detecting carbamates and organophosphorus pesticides, comprising a PVC substrate, wherein a sample pad, a nitrocellulose membrane and an absorbent pad are disposed on the PVC substrate from left to right; At least one detection line is provided on the nitrocellulose membrane; The detection line is formed by drawing a solution of manganese dioxide nanoneedles coated with bovine serum albumin; bovine serum albumin forms coordination bonds with manganese ions on the surface of manganese dioxide nanoneedles, so that bovine serum albumin is coated on the surface of manganese dioxide nanoneedles.

[0005] When the test strip provided by this invention detects carbamates and organophosphorus pesticides, the MnO2 in the detection line is reduced to colorless Mn. 2+Ions cause a visual change in the detection line from brown to colorless. By counting the number of disappearing detection lines, a semi-quantitative analysis of the target pesticide can be achieved. It has the advantages of simple operation, low cost, portability, intuitive result interpretation and strong anti-interference ability, and is particularly suitable for rapid on-site screening.

[0006] Furthermore, the manganese dioxide nanoneedles have a γ-MnO2 needle-like morphology and a length of 5 nm to 22 nm.

[0007] Furthermore, the content of bovine serum albumin-encapsulated manganese dioxide nanoneedles in each detection line is 3.0 µg to 3.4 µg.

[0008] Furthermore, the number of detection lines is 3 to 5, and the spacing between adjacent detection lines is 3.8 mm to 4.2 mm.

[0009] This invention also provides a method for preparing test strips for detecting carbamates and organophosphorus pesticides, comprising the following steps: Bovine serum albumin solution, manganese sulfate monohydrate solution, and sodium hydroxide solution were mixed and reacted to obtain a reaction solution; the reaction solution was then dialyzed and concentrated sequentially to obtain a concentrated solution. The concentrated solution was diluted to 4 mg / mL~10 mg / mL to obtain a bovine serum albumin-encapsulated manganese dioxide nanoneedle solution. This solution was then uniformly coated onto a nitrocellulose membrane to form at least one detection line. After drying, a nitrocellulose membrane with a detection line was obtained. A nitrocellulose membrane with detection lines is attached to the middle section of a PVC substrate. Then, a sample pad and an absorbent pad are attached to the PVC substrate and the two ends of the nitrocellulose membrane, respectively, to obtain a test strip for detecting carbamates and organophosphorus pesticides.

[0010] Further, the volume ratio of the bovine serum albumin solution, manganese sulfate monohydrate solution, and sodium hydroxide solution is 40~60:1.5~2.5:0.5~1; The concentration of the bovine serum albumin solution is 1.2 mg / mL to 1.8 mg / mL, the concentration of the manganese sulfate monohydrate solution is 90 mmol / L to 110 mmol / L, and the concentration of the sodium hydroxide solution is 0.5 mol / L to 1.5 mol / L.

[0011] Furthermore, the reaction time is 50 min to 70 min.

[0012] Furthermore, the dialysis is performed by dialysis in water for 1 to 2 days using a dialysis bag with a molecular weight cutoff of 80 kDa to 100 kDa; the concentration is performed using an ultrafiltration tube with a molecular weight cutoff of 25 kDa to 30 kDa.

[0013] The present invention also provides the application of the test paper described in any one of the claims in the detection of carbamates and organophosphorus pesticides, comprising the following steps: The sample to be tested was mixed with phosphate buffer and shaken to obtain the sample extract; The acetylcholinesterase solution was mixed with the sample extract and incubated for reaction. Iodothioacetylcholine was then added to continue the reaction. After the reaction was completed, iodine solution was added for further reaction, followed by ascorbic acid solution to obtain the sample solution. Insert the sample pad end of the test strip into the sample solution and react for 5 to 10 minutes. Observe the disappearance of the color of the detection line on the nitrocellulose membrane. If the color on the detection line does not disappear, it indicates that the sample solution contains carbamate and organophosphorus pesticides. The more detection lines that do not disappear, the higher the content of carbamate and organophosphorus pesticides in the sample solution.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention involves preparing a BSA@MnO2 solution and uniformly coating it onto an NC membrane to form a T-line. This T-line is then assembled with a PVC substrate, a sample pad, and an absorbent pad to create a visual semi-quantitative test strip for the detection of carbamates and organophosphorus pesticides. The test strip provided by this invention is simple to use, small in size, and easy to carry, making it ideal for on-site detection of carbamates and organophosphorus pesticides. Furthermore, the test strip provided by this invention does not have color vision dependence, does not rely on multi-level signal conversion for interpretation, is easily identifiable by people with color vision deficiencies, is unaffected by ambient light, and has high accuracy, providing a novel approach for rapid on-site detection of carbamates and organophosphorus pesticides.

[0015] The test strip provided by this invention has lower raw material costs, is easier to obtain, and has a lower cost per test compared to the traditional spectrophotometer-based enzyme inhibition method. The test strip has only 4 components, the preparation process is simple, it is easy to scale up production, and the batch quality is more stable during production.

[0016] The test strip provided by this invention uses BSA@MnO2 in its T line as a colorimetric indicator, which can be reduced by ascorbic acid, changing from dark brown to colorless. When reacting with a detection system containing the pesticide, the activity of acetylcholinesterase is inhibited, leading to a reduction in the production of its catalytic product, thiocholine. This reduces the reduction effect on iodine solution, resulting in a relative increase in the amount of iodine solution available to consume ascorbic acid. More ascorbic acid is consumed, thus maintaining the colorimetric intensity and quantity of the detection line. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram of the test strip structure.

[0019] Figure 2 This is a diagram illustrating how to use the test strip.

[0020] Figure 3 The image shows the UV absorption spectrum of BSA@MnO2; the smaller images in the image show the appearance of the BSA@MnO2 solution.

[0021] Figure 4 The figures show the morphology and length distribution of BSA@MnO2; in the figures, A is a scanning electron microscope image of BSA@MnO2, and B is a length distribution map of BSA@MnO2.

[0022] Figure 5 The results are the blind sample test results of this test strip; among them, (1) is cowpea; (2) is baby bok choy; (3) is cabbage; (4) is apple; and (5) is cucumber.

[0023] Figure labeling: 1-sample pad, 2-NC membrane, 3-absorbent pad, 201-detection line, 4-PVC substrate, 5-sample solution. Detailed Implementation

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1: A method for preparing a test strip for detecting carbamates and organophosphorus pesticides.

[0026] Preparation of BSA@MnO2: 75 mg of bovine serum albumin (BSA, CAS No. 9048-46-8, Shanghai Yuanye Biotechnology Co., Ltd.) was dissolved in 50 mL of water to obtain a BSA solution. 1.9 mL of 100 mmol / L manganese sulfate monohydrate solution (CAS No. 10034-96-5, Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to the BSA solution, and the mixture was stirred for 1 min to obtain a mixture. 0.75 mL of 1 mol / L sodium hydroxide solution (CAS No. 1310-73-2, Tianjin Damao Chemical Reagent Partnership) was added to the mixture, and the reaction was carried out for 60 min to obtain a reaction solution. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed in deionized water for 1 day to obtain a dialysate. The dialysate was concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to obtain a concentrated solution of bovine serum albumin-coated manganese dioxide nanoneedles (BSA@MnO2).

[0027] BSA forms coordination bonds with manganese ions (V) on the surface of MnO2 nanoneedles, causing BSA to coat the surface of MnO2 nanoneedles; the MnO2 nanoneedles have a γ-MnO2 needle-like morphology, and MnO2 in this needle-like morphology can form a stable inclusion state with BSA, which is beneficial for the production of test strips.

[0028] Preparation of nitrocellulose membrane (NC membrane) with detection lines (T lines): Prepare a 10 mg / mL BSA@MnO2 solution by adding water to the concentrated BSA@MnO2 solution. Using a membrane scribing apparatus, coat the NC membrane 2 with the BSA@MnO2 solution at 4 mm intervals using a chromatography speed of 120 s / 4 cm, forming 5 T lines 201. Dry the NC membrane 2 at 45 °C for 7 h to obtain the NC membrane with T lines, where each T line contains 3.2 µg of BSA@MnO2.

[0029] Preparation of the Visualized Semi-Quantitative Test Strip: A PVC substrate 4 is placed as the bottom layer. The PVC substrate 4 is divided into three adhesive sections: 1.5 cm, 2 cm, and 2.5 cm. First, the NC film with T-lines is adhered to the 2.5 cm adhesive section on the surface of the PVC substrate. Then, sample pad 1 and absorbent pad 3 are sequentially installed onto the 1.5 cm and 2 cm adhesive sections on the PVC surface, respectively, with sample pad 1 and absorbent pad 3 in contact with the NC film 2, thus obtaining the visualized semi-quantitative test strip (structure as shown). Figure 1 As shown, the lower end is the sample pad 1, the middle is the NC membrane 2, and the upper section is the absorbent pad 3; there are 5 T lines 201 distributed on the NC membrane, with a spacing of 4 mm between the T lines 201; the distance between the lower T line 201 and the upper end of the sample pad 1 is 4.5 mm, and the distance between the upper T line 201 and the lower end of the absorbent pad 3 is 4.5 mm, which are used to detect carbamates and organophosphorus pesticides.

[0030] Example 2: Application of a test strip for detecting carbamates and organophosphorus pesticides.

[0031] I. Instruments and Materials 1. Test materials BSA, manganese sulfate monohydrate, and sodium hydroxide.

[0032] 2. Experimental apparatus The film-drawing instrument (XYZ 3D film-drawing and gold-spraying instrument HM3030, Shanghai Jinbiao Biotechnology Co., Ltd.), and the ultraviolet spectrophotometer (Nanodrop2000 micro spectrophotometer, Thermo Fisher Scientific).

[0033] II. Test Plan 1. Ultraviolet absorption spectrum scan of BSA@MnO2 The BSA@MnO2 concentrate prepared in Example 1 was diluted with water to prepare a 1 mg / mL BSA@MnO2 solution. The solution was added to a 1 cm cuvette and scanned in the wavelength range of 200 nm to 800 nm using a UV spectrophotometer. The absorbance was recorded.

[0034] 2. Morphology and length distribution of BSA@MnO2 The BSA@MnO2 concentrate from Example 1 was prepared into a 0.5 mg / mL BSA@MnO2 solution using water. The morphology image of BSA@MnO2 was obtained using transmission electron microscopy. The length of BSA@MnO2 nanoneedles was measured using imageJ image processing software. Then, the length distribution of BSA@MnO2 was obtained by processing and analyzing the data using OriginPro2024 data analysis software.

[0035] 3. Blind Sample Test 3.1 Test strip method The test strips prepared in Example 1 were used for detection. Cowpea pods, baby bok choy leaves, Chinese cabbage leaves, apple fruits, and cucumber fruits were selected, chopped into 0.4 ± 0.1 cm pieces, and mixed thoroughly to obtain five samples. 1 g of each sample was taken, and 3 mL of 10 mmol / L pH 7.4 phosphate buffer was added. The mixture was shaken for 2 min to obtain the sample extract. 50 μL of 40 μg / mL silkworm acetylcholinesterase (AChE) solution (Guangzhou Wanlian Biotechnology Co., Ltd., catalog number: WLNC0203) and 100 μL of sample extraction solution were added to the wells of a 96-well plate and reacted for 10 min. Then, 25 μL of iodothioacetylcholine (ATChI) (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S30724) was added and reacted for 10 min. After the reaction, 25 μL of 3 mmol / L iodine solution was added and reacted for 5 min. Finally, 100 μL of pH 4.0 ascorbic acid solution (100 mmol / L) was added to obtain sample solution 5. Figure 2 Insert the test strip by inserting the sample pad 1 end of the test strip into the microwell containing the sample solution 5. After reacting for 10 minutes, observe the number of T lines 201 (the number of T lines is positively correlated with the pesticide content, so the concentration of carbamate and organophosphorus pesticides in the sample can be qualitatively determined based on the number of T lines). If T lines are observed, the result is positive (containing pesticides); if no T lines are observed, the result is negative (not containing pesticides).

[0036] 4.2 Enzyme Inhibition Method The specific operating procedures refer to the second method (enzyme inhibition rate method, spectrophotometric method) of the national standard GB 5009.199-2003 "Rapid Detection of Organophosphorus and Carbamate Pesticide Residues in Vegetables".

[0037] III. Test Results 1. Ultraviolet absorption spectrum scan of BSA@MnO2 The UV-Vis absorption spectrum of BSA@MnO2 is as follows: Figure 3 As shown, BSA@MnO2 exhibits significant absorption in the wavelength range of 200 nm to 400 nm, with a maximum absorption peak at 229 nm. This unique optical property gives BSA@MnO2 a dark brown appearance, making it easy to observe with the naked eye and thus highly suitable for the preparation of T-lines.

[0038] 2. Morphology and length distribution of BSA@MnO2 The morphology and length distribution of BSA@MnO2 are shown in the figure. Figure 4 As shown. Figure 4 Image A is a scanning electron microscope image of BSA@MnO2, which shows that BSA@MnO2 has a fine needle-like shape. Figure 4Figure B shows the length distribution of BSA@MnO2, which ranges from 5 nm to 22 nm, with the highest concentration around 12 nm. The nanoscale structure of BSA@MnO2 endows it with a high specific surface area, significantly increasing the contact sites with ascorbic acid. This allows for efficient reduction and fading by ascorbic acid (during sample detection, acetylcholinesterase solution reacts with thioacetylcholine iodide to produce the reducing product thiocholine, followed by the addition of iodine solution to react with thiocholine, and finally the addition of ascorbic acid. When pesticides are present in the sample, the activity of acetylcholinesterase is inhibited, leading to a reduction in the amount of its catalytic product thioacetylcholine, thus reducing the reduction effect on iodine solution. This results in a relative increase in the amount of iodine solution available to consume ascorbic acid, allowing more ascorbic acid to be consumed, thereby maintaining the color intensity and quantity of the T line during detection).

[0039] 3. Blind sample testing The results of the blind sample test are shown in Table 1 and Figure 5 As shown, the test strip prepared in Example 1 was compared with the second method of enzyme inhibition rate method (spectrophotometric method) of the national standard "GB5009.199-2003 Rapid Detection of Organophosphorus and Carbamate Pesticide Residues in Vegetables". The test strip's detection range met the national standard value, indicating that the method has accuracy and reliability.

[0040] Table 1 Blind Sample Test Results Note: The "+" in the table a "This indicates the number of T lines that remain undiminished after the test strip has reacted for 10 minutes; the relative standard deviation (RSD)" b =Standard deviation ÷ Mean × 100%. c "This indicates that all lines will disappear after 10 minutes of reaction on the test strip."

[0041] This invention utilizes the color reaction between BSA@MnO2 and carbamates and organophosphorus pesticides to achieve visual detection of carbamates and organophosphorus pesticides. The prepared test strips can be used for visual semi-quantitative detection of carbamates and organophosphorus pesticides. In this invention, a T-line is prepared using BSA@MnO2, where the MnO2 is reduced to colorless Mn by ascorbic acid. 2+The ions react with ascorbic acid, causing the T-line to change from dark brown to colorless, a very noticeable color change. When detecting carbamates and organophosphate pesticides, the activity of acetylcholinesterase is specifically inhibited by these pesticides, leading to a reduction in the formation of its catalytic product, thiocholine. This reduces the reducing effect on iodine solution, resulting in a relatively increased amount of iodine solution available to consume ascorbic acid. More ascorbic acid is consumed, maintaining the color intensity and number of detection lines. The number of T-lines is directly proportional to the concentration of carbamates and organophosphate pesticides, thus achieving visual semi-quantitative detection of carbamates and organophosphate pesticides. This invention provides a test strip for rapidly detecting the concentration of carbamates and organophosphate pesticides, featuring ease of use, low cost, simple operation, and portability, making it suitable for on-site detection of carbamates and organophosphate pesticides.

[0042] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0043] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A test strip for detecting carbamates and organophosphorus pesticides, characterized in that, Includes a PVC substrate (4), on which a sample pad (1), a nitrocellulose membrane (2) and an absorbent pad (3) are arranged sequentially from left to right. At least one detection line (201) is provided on the nitrocellulose membrane (2). The detection line (201) is formed by drawing a solution of manganese dioxide nanoneedles coated with bovine serum albumin; bovine serum albumin forms coordination bonds with manganese ions on the surface of manganese dioxide nanoneedles, so that bovine serum albumin is coated on the surface of manganese dioxide nanoneedles.

2. The test strip for detecting carbamates and organophosphorus pesticides according to claim 1, characterized in that, The manganese dioxide nanoneedles have a γ-MnO2 needle-like morphology and a length of 5 nm to 22 nm.

3. The test strip for detecting carbamates and organophosphorus pesticides according to claim 1, characterized in that, The content of bovine serum albumin-encapsulated manganese dioxide nanoneedles in each detection line (201) is 3.0 µg to 3.4 µg.

4. The test strip for detecting carbamates and organophosphorus pesticides according to claim 3, characterized in that, The number of detection lines (201) is 3 to 5, and the spacing between adjacent detection lines (201) is 3.8 mm to 4.2 mm.

5. A method for preparing a test strip for detecting carbamates and organophosphorus pesticides according to any one of claims 1 to 4, characterized in that, Includes the following steps: Bovine serum albumin solution, manganese sulfate monohydrate solution, and sodium hydroxide solution were mixed and reacted to obtain a reaction solution; the reaction solution was then dialyzed and concentrated sequentially to obtain a concentrated solution. The concentrate was diluted to 4 mg / mL to 10 mg / mL to obtain a solution of manganese dioxide nanoneedles coated with bovine serum albumin. The solution was then uniformly coated onto a nitrocellulose membrane (2) to form at least one detection line (201). After drying, a nitrocellulose membrane with a detection line was obtained. The nitrocellulose membrane with the detection line is pasted on the middle section of the PVC substrate (4), and the sample pad (1) and the absorbent pad (3) are pasted on the PVC substrate (4) and the two ends of the nitrocellulose membrane (2) respectively to obtain the test paper for detecting carbamate and organophosphorus pesticides.

6. The preparation method for detecting carbamates and organophosphorus pesticides according to claim 5, characterized in that, The volume ratio of the bovine serum albumin solution, manganese sulfate monohydrate solution, and sodium hydroxide solution is 40~60:1.5~2.5:0.5~1; The concentration of the bovine serum albumin solution is 1.2 mg / mL to 1.8 mg / mL, the concentration of the manganese sulfate monohydrate solution is 90 mmol / L to 110 mmol / L, and the concentration of the sodium hydroxide solution is 0.5 mol / L to 1.5 mol / L.

7. The preparation method for detecting carbamates and organophosphorus pesticides according to claim 5, characterized in that, The reaction time is 50 min to 70 min.

8. The preparation method for detecting carbamates and organophosphorus pesticides according to claim 5, characterized in that, The dialysis is performed by dialysis in water for 1 to 2 days using a dialysis bag with a molecular weight cutoff of 80 kDa to 100 kDa; the concentration is performed using an ultrafiltration tube with a molecular weight cutoff of 25 kDa to 30 kDa.

9. The application of the test paper according to any one of claims 1 to 4 in the detection of carbamates and organophosphorus pesticides, characterized in that, Includes the following steps: The sample to be tested was mixed with phosphate buffer and shaken to obtain the sample extract; The acetylcholinesterase solution was mixed with the sample extract and incubated for reaction. Then, iodothioacetylcholine was added to continue the reaction. After the reaction was completed, iodine solution was added for further reaction, followed by ascorbic acid solution to obtain sample solution (5); Insert the sample pad (1) end of the test paper into the sample solution (5) and react for 5 min to 10 min. Observe the disappearance of the color of the detection line (201) on the nitrocellulose membrane (2). If the color on the detection line (201) does not disappear, it indicates that the sample solution (5) contains carbamate and organophosphorus pesticides. Moreover, the more detection lines (201) that do not disappear, the higher the content of carbamate and organophosphorus pesticides in the sample solution (5).

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