A method for preparing a microfluidic paper-based chip for rapid pesticide detection, and the microfluidic paper-based chip for rapid pesticide detection and its application.

Abstract

This application discloses a method for preparing a microfluidic paper-based chip for rapid pesticide detection, as well as the chip itself and its applications. The preparation method includes sequentially constructing irregular U-shaped hydrophilic-hydrophobic channels using paper fibers as a substrate; modifying the endpoints of the constructed irregular U-shaped hydrophilic-hydrophobic channels with a modifier, then immobilizing a chromogenic reagent at the endpoints of the modified irregular U-shaped hydrophilic-hydrophobic channels to obtain immobilized U-shaped hydrophilic-hydrophobic channels; drop-coating an auxiliary reaction reagent into the immobilized U-shaped hydrophilic-hydrophobic channels and aging them to obtain aged U-shaped hydrophilic-hydrophobic channels; and attaching the unmodified hydrophilic channel portions of the aged U-shaped hydrophilic-hydrophobic channels with water-blocking tape to obtain the microfluidic paper-based chip for rapid pesticide detection. This invention achieves rapid detection of various pesticide residues based on colorimetric principles and can be applied to the detection of actual samples. The method is simple to operate, highly sensitive, and low in cost, showing promising commercial application prospects.

Description

Technical Field

[0001] This application relates to a method for preparing a microfluidic paper-based chip for rapid pesticide detection, as well as the microfluidic paper-based chip for rapid pesticide detection and its application, belonging to the field of rapid pesticide detection technology. Background Technology

[0002] Pesticide residues seriously threaten human health. Long-term consumption of agricultural products containing pesticide residues can lead to their accumulation in the digestive system, causing gastrointestinal diseases. Simultaneously, the body's efforts to eliminate these toxins increase the burden on the liver, potentially leading to conditions such as cirrhosis. Furthermore, numerous studies have demonstrated that many pesticides have teratogenic, carcinogenic, and mutagenic effects. Therefore, rapid detection of pesticide residues in edible agricultural products and timely disposal of products with excessive pesticide residues are of great significance.

[0003] There is a need to develop a rapid detection method that can simultaneously determine multiple types of pesticide residues, is inexpensive, and is suitable for routine pesticide residue monitoring of agricultural products. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-functional microfluidic paper-based chip for rapid pesticide detection based on colorimetric principles. This paper-based chip can simultaneously and rapidly determine multiple types of pesticides, and has good stability and high sensitivity, which can meet the needs of rapid on-site early warning detection of pesticide residues in fruit and vegetable samples.

[0005] According to one aspect of this application, a method for preparing a microfluidic paper-based chip for rapid pesticide detection is provided, the method comprising the following steps:

[0006] (1) Irregular U-shaped hydrophilic and hydrophobic channels were constructed sequentially using paper fibers as a substrate;

[0007] (2) After modifying the endpoints of the constructed irregular U-shaped hydrophilic-hydrophobic channel with a modifier, the chromogenic reagent is then immobilized at the endpoints of the modified irregular U-shaped hydrophilic-hydrophobic channel to obtain the immobilized U-shaped hydrophilic-hydrophobic channel.

[0008] (3) The auxiliary reaction reagent is drop-coated into the immobilized U-shaped hydrophilic-hydrophobic channel and aged to obtain an aged U-shaped hydrophilic-hydrophobic channel.

[0009] (4) The hydrophilic channel portion of the aged U-shaped hydrophilic-hydrophobic channel that has not been modified by the modifier is pasted with water-blocking tape to obtain a microfluidic paper-based chip for rapid pesticide detection.

[0010] Optionally, two sets of irregular U-shaped designs are printed on the paper substrate using wax spraying technology. The wax is melted by high-temperature heating and then penetrates into the paper fibers to construct U-shaped hydrophilic and hydrophobic channels. The paper substrate surface at the four ends of the two sets of U-shaped hydrophilic channels is modified, and then colorimetric reagents that react with different pesticides are drop-coated on them for effective immobilization. Subsequently, auxiliary reaction reagents are drop-coated at appropriate positions in the channels. After aging at room temperature, water-blocking tape is attached to the hydrophilic channels other than the immobilized colorimetric reagents, thereby preparing a multi-functional microfluidic paper-based chip for rapid detection of pesticide residues. This chip is then applied to the rapid early warning detection of multiple types of pesticide residues in actual fruit and vegetable samples.

[0011] The paper substrate surface is modified by drop-coating a cyclodextrin-bonded silica gel solution to form a porous structure. In this method, a silane precursor, cyclodextrin, dimethyl sulfoxide, hydrochloric acid, water, and surfactant are mixed in a mass ratio of 1:(1-3):(5-8):(0.2-0.5):(0.5-1):(0.1-0.2) and stirred at 500-800 rpm for 4 hours at room temperature.

[0012] In this application, the design of the irregular U-shaped hydrophilic channel shape is determined by the reaction amount and the amount of auxiliary reaction reagents. That is, the hydrophilic channel is thickened for reactions that require a large amount of test liquid, and the hydrophilic channel is lengthened for reactions that require a large amount of auxiliary reaction reagents.

[0013] Optionally, the method for preparing the modifier includes:

[0014] The modifier is obtained by stirring a mixture containing a silane precursor, cyclodextrin, dimethyl sulfoxide, hydrochloric acid, water, and a surfactant at a speed of 500–800 rpm for 4–6 h.

[0015] Optionally, the mass ratio of the silane precursor, cyclodextrin, dimethyl sulfoxide, hydrochloric acid, water, and surfactant is 1:(1-3):(5-8):(0.2-0.5):(0.5-1):(0.1-0.2).

[0016] Optionally, the silane precursor is selected from methyltriethoxysilane and / or n-octyltriethoxysilane.

[0017] Optionally, the cyclodextrin is selected from β-cyclodextrin and / or γ-cyclodextrin.

[0018] Optionally, the surfactant is selected from Tween-20 and / or Triton.

[0019] Optionally, the concentration of the hydrochloric acid is 2 to 4 g / L.

[0020] Optionally, the colorimetric reagent includes p-nitrobenzaldehyde, 1-(2-pyridinium azo)-2-naphthol, palladium chloride, and a new cuprous reagent.

[0021] Optionally, the concentrations of p-nitrobenzaldehyde, 1-(2-pyridiniazo)-2-naphthol, palladium chloride, and neocubic reagent are respectively 4-8 g / L, 1-4 g / L, 5-10 g / L, and 4-10 g / L.

[0022] Optionally, the auxiliary reaction reagents include o-dinitrobenzene, sodium hydroxide, and copper chloride.

[0023] Optionally, the concentrations of o-dinitrobenzene, sodium hydroxide, and copper chloride are 10–20 g / L, 80–100 g / L, and 8–12 g / L, respectively.

[0024] The colorimetric reagents are selected from commercial indicators based on the characteristic groups of the pesticides to be tested, including p-nitrobenzaldehyde (4-8 g / L solvent ethanol), 1-(2-pyridiniazo)-2-naphthol (1-4 g / L solvent ethanol), palladium chloride (5-10 g / L solvent 10% acetic acid aqueous solution), and neocopper(4-10 g / L solvent ethanol).

[0025] Auxiliary reaction reagents are essential reagents for exposing pesticide characteristic groups or for use in conjunction with colorimetric reagents, including o-dinitrobenzene (10-20 g / L solvent ethanol), sodium hydroxide (80-100 g / L solvent water), and copper chloride (8-12 g / L solvent water).

[0026] According to another aspect of this application, a method described above is provided for preparing a microfluidic paper-based chip for rapid pesticide detection. The pesticides in the microfluidic paper-based chip for rapid pesticide detection include dimethoate, malathion, dazomet, fenitrothion, zineb, thiram, mancozeb, cypermethrin, lambda-cyhalothrin, quinoline copper, Bordeaux mixture, and zinc thiamethoxam.

[0027] According to another aspect of this application, a method for detecting pesticide residues using a microfluidic paper-based chip for rapid pesticide detection, as described above, is provided, the method comprising the following steps:

[0028] (1) Extract the fruit and vegetable samples to be tested with phosphate buffer solution to obtain pesticide test solution;

[0029] (2) Drop the pesticide test solution into the microfluidic paper-based chip for rapid detection of pesticides in all-in-one. Observe the endpoint of the U-shaped hydrophilic and hydrophobic channel of the microfluidic paper-based chip for rapid detection of pesticides in all-in-one. If the colorimetric reagent changes color significantly, it is determined that there are pesticide residues in the fruit and vegetable samples.

[0030] If the colorimetric reagent shows no significant change, the fruit and vegetable samples are deemed to have no pesticide residues.

[0031] Optionally, in step (1), the pH value of the phosphate buffer solution is 7.5 to 8.5.

[0032] In this application, the specific detection steps for pesticide residues in fruit and vegetable samples are as follows:

[0033] (1) Pretreatment: For fruit and vegetable samples with no pigment or little pigment, cut the fruit and vegetable samples into pieces of about 1 cm with scissors, take 20g of sample and put it into a test tube, add 10mL of phosphate buffer solution (pH 7.5) to extract the pesticide in the sample by shaking to obtain the test solution; For fruit and vegetable samples with more pigment, the whole plant extraction method is adopted. After weighing the sample, the pesticide in the sample is extracted by shaking with an equal weight of phosphate buffer solution (pH 7.5) to obtain the test solution.

[0034] (2) Color reaction: Place absorbent filter paper under the microfluidic paper-based chip for rapid detection of pesticides. Add 1 mL of test solution to the bottom of each of the two sets of U-shaped hydrophilic channels of the paper-based chip. The solution dissolves the auxiliary reaction reagent in the channel and reacts due to the capillary action of the paper base. Finally, it reaches the end of the immobilized colorimetric reagent and is continuously enriched. After the solution is completely absorbed by the absorbent filter paper, let it stand for 3 min.

[0035] (3) Result determination: Observe the color of the colorimetric reagent. If the color of the colorimetric reagent changes significantly, it can be determined that there is pesticide residue in the sample. If the colorimetric reagent does not change or changes slightly, it can be determined that there is no pesticide residue in the sample.

[0036] The beneficial effects that this application can produce include:

[0037] 1) The pesticide rapid detection microfluidic paper-based chip provided in this application has the ability to simultaneously and rapidly detect multiple types of pesticides on one card through hydrophilic and hydrophobic channel design, which greatly reduces the detection cost of unknown pesticide residues in samples;

[0038] 2) The pesticide rapid detection microfluidic paper-based chip provided in this application has the ability to modify the paper surface by bonding silica gel solution with cyclodextrin, thereby obtaining a porous structure on the paper surface and achieving efficient capture of the analyte. In addition, the hydrophobic cavity of cyclodextrin can effectively embed and immobilize the hydrophobic indicator (color developer), so that the indicator is not washed away as the analyte liquid is continuously enriched during the detection process, thereby increasing the detection sensitivity.

[0039] 3) This application provides a method for preparing a multi-functional pesticide rapid detection microfluidic paper-based chip, which realizes rapid detection of various pesticide residues based on the colorimetric principle and is applied to the detection of actual samples. The method is simple to operate, highly sensitive, and low in cost, and has commercial application prospects. Attached Figure Description

[0040] Figure 1This refers to the multi-functional pesticide rapid detection microfluidic paper-based chip prepared in Examples 1 and 2 of this application.

[0041] Figure 2 The detection effect of the multi-functional pesticide rapid detection microfluidic paper-based chip in Example 3 of this application on the pesticide mixture test solution. Detailed Implementation

[0042] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0043] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased through commercial channels.

[0044] Example 1

[0045] Fabrication of a microfluidic paper-based chip for rapid detection of all pesticides:

[0046] Two sets of irregular U-shaped design patterns (such as...) are printed on the paper surface using wax spray printing technology. Figure 1 As shown in the figure, wax was melted by high-temperature heating and then penetrated into paper fibers to construct U-shaped hydrophilic-hydrophobic channels. The paper substrate surface at the four endpoints of the two sets of U-shaped hydrophilic channels was modified as follows: methyltriethoxysilane, β-cyclodextrin, dimethyl sulfoxide, hydrochloric acid (2 g / L), water, and Tween-20 were mixed in a mass ratio of 1:2:5:0.3:1:0.2 and stirred at 800 rpm for 4 hours at room temperature to obtain modified solution I. 2 μL of this modified solution was then taken... The above solution was drop-coated onto the paper substrate surface at the two ends of a group of U-shaped hydrophilic channels; methyltriethoxysilane, γ-cyclodextrin, dimethyl sulfoxide, hydrochloric acid (2.5 g / L), water, and Triton X-100 were mixed in a mass ratio of 1:2:7:0.3:0.6:0.1 and stirred at 600 rpm for 4 hours at room temperature to obtain modified solution II. 2 μL of the above solution was drop-coated onto the paper substrate surface at the two ends of another group of U-shaped hydrophilic channels. Next, colorimetric reagents that react with different pesticides were drop-coated at the four endpoints for effective immobilization. The colorimetric reagents, from left to right, were p-nitrobenzaldehyde (8 g / L solvent ethanol), neocubic reagent (5 g / L solvent ethanol), palladium chloride (10 g / L solvent 10% acetic acid aqueous solution), and 1-(2-pyridiniazo)-2-naphthol (2 g / L solvent ethanol). Subsequently, auxiliary reaction reagents were drop-coated in the channels. In the middle of the first group of U-shaped hydrophilic channels, from left to right, were o-dinitrobenzene (15 g / L solvent ethanol), sodium hydroxide (85 g / L solvent water), and copper chloride (10 g / L solvent water). After aging at room temperature, water-blocking tape was attached to the hydrophilic channels other than those immobilized with colorimetric reagents, thus preparing a multi-functional microfluidic paper-based chip for rapid detection of pesticide residues.

[0047] Example 2

[0048] Fabrication of a microfluidic paper-based chip for rapid detection of all pesticides:

[0049] Two sets of irregular U-shaped design patterns (such as...) are printed on the paper surface using wax spray printing technology. Figure 1 As shown), wax was melted by high-temperature heating and then infiltrated into paper fibers to construct U-shaped hydrophilic and hydrophobic channels. The paper substrate surface at the four endpoints of the two sets of U-shaped hydrophilic channels was modified as follows: methyltriethoxysilane, β-cyclodextrin, dimethyl sulfoxide, hydrochloric acid (4 g / L), water, and Tween-20 were mixed in a mass ratio of 1:3:5:0.4:1:0.15 and stirred at 500 rpm for 4 hours at room temperature to obtain modified solution I. 2... The above solution was drop-coated onto the paper substrate surface at the two ends of a group of U-shaped hydrophilic channels. Octyltriethoxysilane, γ-cyclodextrin, dimethyl sulfoxide, hydrochloric acid (3 g / L), water, and Tween-20 were mixed in a mass ratio of 1:2:8:0.5:0.8:0.2 and stirred at 700 rpm for 4 hours at room temperature to obtain modified solution II. 2 μL of the above solution was then drop-coated onto the paper substrate surface at the two ends of another group of U-shaped hydrophilic channels. Next, colorimetric reagents that react with different pesticides were drop-coated at the four endpoints for effective immobilization. The colorimetric reagents, from left to right, were p-nitrobenzaldehyde (4 g / L solvent ethanol), neocubic reagent (4 g / L solvent ethanol), palladium chloride (5 g / L solvent 10% acetic acid aqueous solution), and 1-(2-pyridiniazo)-2-naphthol (2 g / L solvent ethanol). Subsequently, auxiliary reaction reagents were drop-coated in the channels. In the middle of the first group of U-shaped hydrophilic channels, from left to right, were o-dinitrobenzene (15 g / L solvent ethanol), sodium hydroxide (100 g / L solvent water), and copper chloride (12 g / L solvent water). After aging at room temperature, water-blocking tape was attached to the hydrophilic channels other than those immobilized with colorimetric reagents, thus preparing a multi-functional microfluidic paper-based chip for rapid detection of pesticide residues.

[0050] Example 3

[0051] The effectiveness of a multi-functional microfluidic paper-based chip for rapid pesticide detection in detecting pesticide mixtures:

[0052] Place absorbent filter paper under the all-in-one pesticide rapid detection microfluidic paper-based chip and secure the chip and filter paper with staples. Prepare mixed solutions of pesticides (1 mg / L each) including lambda-cyhalothrin, thiram, dazomet, and dimethoate. Add 1 mL of each pesticide mixture to the bottom of the two U-shaped channels of the all-in-one pesticide rapid detection microfluidic paper-based chip. The solution moves into the U-shaped hydrophilic channels under capillary action, and finally accumulates and develops color at the endpoint of the U-shaped channel containing the detection reagent. After all the test solution is absorbed, let it stand for 3 minutes. Figure 2It can be seen that the color of the indicator at the four endpoints of the paper-based chip changed significantly, indicating that the prepared paper-based chip can detect mixed pesticides and has high detection sensitivity.

[0053] Example 4

[0054] A multi-functional microfluidic paper-based chip for rapid pesticide detection was used to detect pesticides in commercially available cabbage samples.

[0055] (1) Pretreatment: Cut the cabbage sample into pieces about 1cm in size with scissors, put 20g of sample into a test tube, add 10mL of phosphate buffer solution (pH 7.5) to extract the pesticide in the sample by shaking to obtain the test solution;

[0056] (2) Color reaction: Place absorbent filter paper under the microfluidic paper-based chip for rapid detection of pesticides. Add 1 mL of test solution to the bottom of each of the two sets of U-shaped hydrophilic channels of the paper-based chip. The solution dissolves the auxiliary reaction reagent in the channel and reacts due to the capillary action of the paper base. Finally, it reaches the end of the immobilized colorimetric reagent and is continuously enriched. After the solution is completely absorbed by the absorbent filter paper, let it stand for 3 min.

[0057] (3) Result determination: Observing the color of the colorimetric reagent, it was found that the color of the colorimetric reagent did not change, so it was determined that there was no pesticide residue in the sample.

[0058] Example 5

[0059] A multi-functional microfluidic paper-based chip for rapid pesticide detection was used to detect pesticides in commercially available leek samples.

[0060] (1) Pretreatment: The whole plant extraction method was adopted. After weighing the chives, the pesticides in the chives were extracted by shaking with an equal weight of phosphate buffer solution (pH 7.5) to obtain the test solution;

[0061] (2) Color reaction: Place absorbent filter paper under the microfluidic paper-based chip for rapid detection of pesticides. Add 1 mL of test solution to the bottom of each of the two sets of U-shaped hydrophilic channels of the paper-based chip. The solution dissolves the auxiliary reaction reagent in the channel and reacts due to the capillary action of the paper base. Finally, it reaches the end of the immobilized colorimetric reagent and is continuously enriched. After the solution is completely absorbed by the absorbent filter paper, let it stand for 3 min.

[0062] (3) Result determination: Observing the color of the colorimetric reagent, it was found that the color of the colorimetric reagent at the left end of the first U-shaped channel turned pink, so it was determined that there was pesticide residue in the sample.

[0063] The leek sample was pretreated using the national standard method and then measured using gas chromatography. The results showed that the leek sample contained 2 mg / kg of cypermethrin, which was consistent with the detection results of the microfluidic paper-based chip for rapid detection of all pesticides, indicating that the paper-based chip detection results are reliable.

[0064] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for preparing a microfluidic paper-based chip for rapid pesticide detection, characterized in that, The preparation method includes the following steps: (1) Irregular U-shaped hydrophilic and hydrophobic channels were constructed sequentially using paper fibers as a substrate; (2) After modifying the endpoints of the constructed irregular U-shaped hydrophilic-hydrophobic channel with a modifier, the chromogenic reagent is then immobilized at the endpoints of the modified irregular U-shaped hydrophilic-hydrophobic channel to obtain the immobilized U-shaped hydrophilic-hydrophobic channel. (3) The auxiliary reaction reagent is drop-coated into the immobilized U-shaped hydrophilic-hydrophobic channel and aged to obtain an aged U-shaped hydrophilic-hydrophobic channel; (4) The hydrophilic channel portion of the aged U-shaped hydrophilic-hydrophobic channel that has not been modified by the modifier is pasted with water-blocking tape to obtain a microfluidic paper-based chip for rapid pesticide detection.

2. The preparation method according to claim 1, characterized in that, The method for preparing the modifier includes: The modifier is obtained by stirring a mixture containing a silane precursor, cyclodextrin, dimethyl sulfoxide, hydrochloric acid, water, and a surfactant at a speed of 500-800 rpm for 4-6 h.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the silane precursor, cyclodextrin, dimethyl sulfoxide, hydrochloric acid, water, and surfactant is 1:(1~3):(5~8):(0.2~0.5):(0.5~1):(0.1~0.2).

4. The preparation method according to claim 2, characterized in that, The silane precursor is selected from methyltriethoxysilane and / or n-octyltriethoxysilane; The cyclodextrin is selected from β-cyclodextrin and / or γ-cyclodextrin; The surfactant is selected from Tween-20 and / or Triton; The concentration of the hydrochloric acid is 2~4 g / L.

5. The preparation method according to claim 1, characterized in that, The colorimetric reagents include p-nitrobenzaldehyde, 1-(2-pyridiniazo)-2-naphthol, palladium chloride, and a new cuprous reagent; The concentrations of p-nitrobenzaldehyde, 1-(2-pyridiniazo)-2-naphthol, palladium chloride, and neocubic reagent are respectively 4~8 g / L, 1~4 g / L, 5~10 g / L, and 4~10 g / L.

6. The preparation method according to claim 1, characterized in that, The auxiliary reaction reagents include o-dinitrobenzene, sodium hydroxide, and copper chloride.

7. The preparation method according to claim 6, characterized in that, The concentrations of o-dinitrobenzene, sodium hydroxide, and copper chloride are 10~20 g / L, 80~100 g / L, and 8~12 g / L, respectively.

8. The preparation method according to any one of claims 1 to 7 for preparing a microfluidic paper-based chip for rapid pesticide detection, characterized in that, The pesticides in the microfluidic paper-based chip for rapid pesticide detection include dimethoate, malathion, dazomet, fenitrothion, zineb, thiram, mancozeb, cypermethrin, lambda-cyhalothrin, quinoline copper, Bordeaux mixture, and zinc thiamethoxam.

9. The method for detecting pesticide residues using a microfluidic paper-based chip for rapid pesticide detection as described in claim 8, characterized in that, The method includes the following steps: (1) Extract the fruit and vegetable samples to be tested with phosphate buffer solution to obtain the pesticide test solution; (2) Drop the pesticide test solution into the microfluidic paper-based chip for rapid detection of pesticides, and observe the endpoint of the U-shaped hydrophilic and hydrophobic channel of the microfluidic paper-based chip for rapid detection of pesticides. If the colorimetric reagent changes color significantly, it is determined that there are pesticide residues in the fruit and vegetable samples. If the colorimetric reagent shows no significant change, the fruit and vegetable samples are deemed to have no pesticide residues.

10. The method according to claim 9, characterized in that, In step (1), the pH value of the phosphate buffer solution is 7.5~8.5.

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