A microneedle patch for detecting pesticide residues, a preparation method thereof, and an application method thereof

Abstract

The present invention provides a microneedle patch for detecting pesticide residues, a preparation method thereof and an application method thereof, relating to the technical field of pesticide residue detection. The microneedle patch proposed by the present invention is prepared from surface-modified multi-walled carbon nanotubes, polyethylene glycol diacrylate, triethylamine and acryloyl chloride. The prepared microneedle patch has excellent swelling ability and can quickly and effectively absorb pesticide residues in agricultural products. By pressing the microneedle patch on the surface of agricultural products, the pesticide residues can be collected by entering the interior of vegetable cells through the needle tips. After obtaining the eluate through the elution process, it is detected with an immunochromatographic test strip. It can pierce the cutin layer of plants or the human body in the least invasive way to detect internal pesticides, is suitable for on-site sampling and detection of real samples, and prevents the samples from being damaged during the detection process.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide residue detection, and particularly relates to a microneedle patch for detecting pesticide residues, a preparation method thereof, and an application method thereof. Background Art

[0002] Pesticide residues are commonly present in various agricultural products, including tea, fruits, and vegetables, and have become one of the most widely discussed issues in the world. Some pesticide residues, especially endogenous pesticides, are difficult to remove and can cause cancer, hormonal disorders, asthma, allergies, and other diseases. Therefore, it is crucial to develop reliable detection techniques to monitor the levels of pesticide residues in agricultural products. Traditional methods, such as gas chromatography and high-performance liquid chromatography, often require time-consuming sample processing and are difficult to detect pesticide residues in situ. In response to the above problems, a variety of detection methods have been reported, including electrochemical methods, surface-enhanced Raman spectroscopy, and colorimetry. Lateral flow immunoassay has the advantages of simple pretreatment, high sensitivity, fast reaction speed, and strong specificity, and is a promising method for detecting pesticide residues.

[0003] Common techniques for the pretreatment of general organophosphorus pesticides: liquid-liquid extraction, solid-phase extraction technology, QuEChERS method. These detection methods have problems such as long time consumption, poor anti-interference ability, cumbersome operation, and large consumption of organic solvents. Although the pretreatment methods of enzyme-linked immunosorbent assay and colloidal gold test strips are simpler than instrumental detection methods, the pretreatment methods will use homogenization or chopping, etc. Such pretreatment methods will increase matrix effect interference and can only detect pesticide residues on the surface of agricultural products, but not those inside. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a microneedle patch for detecting pesticide residues, a preparation method thereof, and an application method thereof, which can detect pesticide residues on the surface and inside of agricultural products in a convenient and minimally invasive manner simultaneously.

[0005] To achieve the above objectives, the technical solution of the present invention is realized through the following technical solutions:

[0006] In the first aspect, the present invention provides a preparation method of a microneedle patch for detecting pesticide residues, comprising the following steps:

[0007] S1: Mix polyethylene glycol diacrylate, triethylamine, and acryloyl chloride in a mass ratio of 99:0.5:0.5 to obtain a mixed solution;

[0008] S2: Inject the mixed solution into a silica gel microneedle mold, place it under vacuum, and irradiate the mixed solution with ultraviolet light to obtain a PEGDA patch;

[0009] S3: Immerse the PEGDA patch into a suspension of surface-modified multi-walled carbon nanotubes with a concentration of 50 mmol / L for ultrasonic soaking. After standing at room temperature, lift the upper transparent film to obtain a microneedle patch.

[0010] Further, the irradiation conditions of ultraviolet light in step S2 are: the irradiation intensity is 8 - 12 mW / cm 2 , for example, it can be 8, 9, 10, 11, 12 mW / cm 2 ; the wavelength is 365 nm, and the irradiation time is 40 - 50 min, for example, it can be 40, 41, 42, 45, 47, 50 min;

[0011] The vacuum standing time in step S2 is 12 - 18 min, for example, it can be 12, 14, 15, 18 min.

[0012] Further, the surface-modified multi-walled carbon nanotubes in step S3 include hydroxyl-modified multi-walled carbon nanotubes, carboxylated multi-walled carbon nanotubes, and aminated multi-walled carbon nanotubes, preferably hydroxyl-modified multi-walled carbon nanotubes.

[0013] Further, the ultrasonic soaking time in step S3 is 25 - 35 min, for example, it can be 25, 27, 30, 32, 35 min.

[0014] Further, the standing time at room temperature in step S3 is 10 - 15 h, for example, it can be 10, 11, 12, 13, 15 h.

[0015] In the second aspect, the present invention proposes a microneedle patch for detecting pesticide residues prepared by the above method.

[0016] In the third aspect, the present invention proposes the application of the microneedle patch for detecting pesticide residues prepared by the said method in detecting chlorpyrifos and / or methyl parathion in agricultural products.

[0017] In the fourth aspect, the present invention proposes an application method of a microneedle patch for detecting pesticide residues, and the application method includes:

[0018] Press the microneedle patch prepared by the above method on different parts of the object to be measured;

[0019] After pressing, soak the microneedle patch with PBS buffer to obtain an eluate;

[0020] Absorb the eluate, mix it evenly with the gold-labeled mixed antibody, and after mixing and standing, transfer the liquid to the sample pad of the colloidal gold test strip, and read the test result through a colloidal gold reader.

[0021] Further, the concentration of the PBS buffer is 0.02 mol / L, and the pH is 7.4.

[0022] Furthermore, after each press of the microneedle patch, a pause of 6 - 12 s is made.

[0023] Furthermore, the colloidal gold test strip is prepared according to the following steps:

[0024] Preparation of colloidal gold: Heat the chloroauric acid solution with a mass fraction of 1% until boiling, then add the trisodium citrate solution with a mass fraction of 1%, and continue heating until the color of the solution tends to be stable to obtain a colloidal gold solution;

[0025] Preparation of the colloidal gold test strip: Dilute the chlorpyrifos hapten - bovine serum albumin conjugate with PBS buffer to a concentration of 0.3 mg / mL and spray it on the NC membrane as the T1 line; Spray the methyl parathion hapten - bovine serum albumin conjugate at a position 5 mm above the T1 detection line and dilute it with PBS buffer to a concentration of 0.5 mg / mL as the T2 line; Spray goat anti - mouse secondary antibody at a position 5 mm above the T2 line as the C line;

[0026] Assembly of the colloidal gold test strip: Stick the NC membrane, sample pad, and absorbent pad on the PVC bottom plate, making the sample pad coincide with the NC membrane by 0.3 cm in the length direction and the NC membrane coincide with the absorbent pad by 0.3 cm in the length direction, and then cut it into test strips with a width of 3 mm using a strip cutter to obtain the colloidal gold test strip.

[0027] Furthermore, the gold - labeled mixed antibody is prepared according to the following steps:

[0028] Add 9 μL / mL of chlorpyrifos and methyl parathion antibodies to the colloidal gold solution, shake it on a shaker at 37 °C for 30 min, add 10% PEG - 20000 with a concentration of 10% and shake for 10 min, and then add 5% BSA with a mass - to - volume ratio and shake for 10 min;

[0029] After the reaction, place it in a high - speed refrigerated centrifuge and centrifuge at 12000 r / min for 5 min, discard the supernatant, and dissolve the lower - layer precipitate with 100 μL / mL of resuspension solution to obtain the gold - labeled mixed antibody.

[0030] The advantages of the microneedle patch for detecting pesticide residues, its preparation method, and application method provided by the present invention compared with the prior art are as follows:

[0031] The microneedle patch proposed by the present invention is prepared from surface-modified multi-walled carbon nanotubes, polyethylene glycol diacrylate, triethylamine, and acryloyl chloride. Among them, PEGDA has the advantages of non-toxicity, good biocompatibility, low immunogenicity, and convenient use. Under ultraviolet irradiation, the acrylate double bonds at the ends of PEGDA molecular chains break and combine with other PEGDA molecules to form crosslinking points and networks, and stable chemical bonds are formed between PEGDA molecules, thus forming an integral hydrogel structure. The addition of multi-walled carbon nanotubes can improve the adsorption rate and reduce the interference of matrix effects. The microneedle patch prepared by the present invention has excellent swelling ability and can quickly and effectively absorb pesticide residues in agricultural products. Press the microneedle patch on the surface of agricultural products, collect pesticide residues by entering the interior of vegetable cells through the needle tips, and then use a colloidal gold test strip to detect after obtaining the eluate through the elution process. It can pierce plants in the least invasive way to detect internal pesticides, is suitable for on-site sampling and detection of real samples, and prevents samples from being damaged during the detection process.

[0032] By performing surface treatment on the surface of MWNTs in the present invention, the problem of poor dispersion of MWNTs in organic solvents or aqueous solvents can be improved to enhance their dispersion in solvents. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for describing the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 Shows the tensile compressor test results of the microneedle patch prepared in the embodiment of the present invention;

[0035] Figure 2 Shows the water absorption test results of the microneedle patch prepared in the embodiment of the present invention;

[0036] Figure 3 Shows the specificity test results of the colloidal gold test strip prepared in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0038] The following examples will describe in detail the preparation process of the microneedle patch provided by the present invention, the preparation process of the colloidal gold test strip, the preparation process of the gold-labeled mixed antibody, the application methods and conditions of the microneedle patch, and the detection performance. Unless otherwise specified, the raw materials used in the examples are all general commercially available raw materials. Example 1:

[0039] Prepare the MN patch according to the following different raw materials and methods:

[0040] Combination 1: Mix PVA (polyvinyl alcohol) and HAMA (methacrylated hyaluronic acid) in a mass ratio of 3:6. Pour the obtained mixed solution onto the microneedle mold, and then place it in a vacuum for 30 min to allow the mixed solution to fill the cavity. After the microneedle mold is filled with the mixed solution, it is left at room temperature for 12 h to obtain a dried MN patch, which is then frozen at -20 °C for 12 h and then stored at 4 °C for 4 h. After repeating the above process three times, a hydrogel HAMA / PVA MN patch is obtained. HAMA is an environmentally friendly and pollution-free material with excellent formability and satisfactory mechanical properties, while ensuring the manufacturing integrity and excellent puncture performance of the patch.

[0041] Combination 2: Mix PEGDA (polyethylene glycol diacrylate), triethylamine, and acryloyl chloride in a mass ratio of 99:0.5:0.5 to obtain a mixed solution. Add the mixed solution to the silicone microneedle mold, place it in a vacuum for 15 min to remove the air bubbles in the mixed solution and ensure the uniform distribution of the mixture in the microneedle mold. Irradiate the mixture with an intensity of 10 mW / cm 2 and an ultraviolet wavelength of 365 nm for 45 min to obtain a hydrogel PEGDA patch.

[0042] Combination 3: Mix glycidyl methacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate, 1-hydroxycyclohexyl phenyl ketone, and 2-methoxyethanol in a mass ratio of 10:6.0:15.7:0.01:0.01 to obtain a mixed solution. Add the mixed solution to the silicone negative microneedle mold, place it in a vacuum for 15 min to ensure complete filling of the microneedle mold. Subsequently, place the microneedle mold under a UV lamp (365 nm) for 30 min to polymerize it. Then take out the MNs from the microneedle mold, wash them with distilled water and dry them to obtain a composite microneedle patch.

[0043] The microneedle patches prepared according to the above methods were subjected to tensile compressor testing, and the results are as Figure 1As shown, the results show that all three combinations can penetrate the fruit and vegetable epidermis. However, the micro-needle patch prepared according to Combination 1 has a much lower bearing capacity than the micro-needle patches prepared according to Combination 2 and Combination 3, with some swelling and deformation. The micro-needle patches prepared by the other two combinations are not deformed, and the micro-needle patch needs to have sufficient insertion force.

[0044] The water absorption rate of the micro-needle patch prepared according to the above method was tested, and the test results are as Figure 2 shown. It can be seen that the absorption rates are in the order of Combination 2 > Combination 1 > Combination 3, and the micro-needle patch needs to absorb the same liquid in a shorter time.

[0045] Based on the results of the above two tests, the micro-needle patch was prepared using the formula of Combination 2. The hydrogel PEGDA patches of Combination 2 were respectively immersed in suspensions of MWCNT-OH (hydroxyl-modified multi-walled carbon nanotubes), MWCNT-COOH (carboxylated multi-walled carbon nanotubes), and MWCNT-NH2 (aminated multi-walled carbon nanotubes) with a concentration of 50 mmol / L, soaked for 30 min under ultrasonic conditions, and left at room temperature for 12 h to obtain MWCNT / PEGDA / MN patches with different functional groups. The corresponding MN patches were named MNOH-50, MNCOOH-50, and MNNH2-50 (i.e., the micro-needle patches proposed in the present invention).

[0046] It should be noted that the modification method of multi-walled carbon nanotubes in the present invention is not specifically limited. For example, MWCNT-OH can be obtained by treating MWCNTs with a mixture of concentrated nitric acid and concentrated sulfuric acid or by its alkaline oxidation treatment to introduce hydroxyl functional groups on the surface of multi-walled carbon nanotubes. Example 2:

[0047] Prepare a colloidal gold test strip, including the following steps:

[0048] Prepare colloidal gold solution: First, add 100 mL of 1% chloroauric acid solution by mass to a 250 mL flask and heat to boiling. Then, add 1 mL of freshly prepared 1% trisodium citrate solution by mass under magnetic stirring. The color of the solution becomes colorless, and the solution is continuously heated until the color slowly turns blue and finally dark red. When the color of the solution tends to be stable, continue heating for 10 min, then stop heating and cool it at room temperature to obtain the colloidal gold solution. Seal it with a sealing film and store it at 4 °C for later use. Characterize the colloidal gold by ultraviolet spectrophotometer and transmission electron microscope (TEM).

[0049] Preparation of the test line and the quality control line: The following T line and C line were both sprayed on the NC membrane with a spraying volume of 1.0 μL / cm using a film spraying instrument. The chlorpyrifos and methyl parathion hapten-bovine serum albumin conjugates were used as the test lines (T1 line, T2 line) on the NC membrane, and the goat anti-mouse secondary antibody was used as the quality control line (C line). The specific process is as follows:

[0050] Dilute the chlorpyrifos hapten-bovine serum albumin conjugate with PBS buffer (pH 7.4, concentration 0.02 mol / L, containing 1% methanol by volume fraction) to a concentration of 0.3 mg / mL, and spray it on the NC membrane as the T1 line;

[0051] Spray the methyl parathion hapten-bovine serum albumin conjugate 5 mm above the T1 test line, and dilute it with PBS buffer (pH 7.4, concentration 0.02 mol / L, containing 1% methanol by volume fraction) to a concentration of 0.5 mg / mL as the T2 line;

[0052] Spray the goat anti-mouse secondary antibody (1 mg / mL, PBS buffer pH 7.4, 0.02 mol / L) 5 mm above the T2 line as the C line (quality control line). Place the coated NC membrane in an oven at 37 °C for 2 h for standby. Soak the sample pad in PBS buffer containing 1% BSA (1% mass / volume, that is, 1 g BSA in 100 mL), 2% sucrose (2% mass / volume, that is, 2 g sucrose in 100 mL), pH 7.4, and 0.02 mol / L for 2 h, and dry it at 37 °C for 2 h for standby.

[0053] Assembly of the colloidal gold test strip: Stick the NC membrane (nitrocellulose membrane), the sample pad, and the absorbent pad on the PVC bottom plate, making the sample pad overlap with the NC membrane by 0.3 cm (in the length direction), and the NC membrane overlap with the absorbent pad by 0.3 cm (in the length direction). Then use a strip cutting machine to cut it into test strips with a width of 3 mm, which are the colloidal gold test strips. Example 3:

[0054] Preparation of the gold-labeled mixed antibody: Add an appropriate amount of potassium carbonate solution to the colloidal gold solution prepared in Example 2 to adjust the pH, then add 9 μL / mL of chlorpyrifos and methyl parathion antibodies, shake on a shaker at 37 °C for 30 min, add 10% PEG-20000 by concentration, shake for 10 min, and then add 5% BSA (5% mass / volume, that is, 5 g BSA in 100 mL), shake for 10 min to block the vacant sites on the surface of the gold particles. After the reaction, place it in a high-speed refrigerated centrifuge and centrifuge at 12000 r / min for 5 min. Discard the supernatant, and dissolve the lower precipitate with 100 μL / mL of resuspension solution to obtain the gold-labeled mixed antibody for standby. Example 4:

[0055] The micro-needle patch prepared in Example 1 was used to detect chlorpyrifos and methyl parathion:

[0056] Chlorpyrifos was dissolved in methanol and then diluted to a series of concentrations (1, 5, 10, 50, 100, 500, 1000 ng / mL) with deionized water. The chlorpyrifos solutions with different dilution concentrations were sprayed onto the surface of Chinese cabbage leaves. At the same time, methyl parathion was dissolved in methanol and diluted to a series of concentrations (1, 5, 10, 50, 100, 500, 1000 ng / mL) with deionized water. The methyl parathion solutions with different dilution concentrations were sprayed onto the surface of Chinese cabbage leaves. Finally, MNOH-50, MNCOOH-50, and MNNH2-50 were pressed on different parts of the Chinese cabbage 5 - 8 times, with a 10-s pause for each press. Then, the patch was soaked in 1 mL of PBS buffer (pH 7.4, 0.02 mol / L) for 1 min. 100 μL of the eluate was taken and mixed well with the gold-labeled mixed antibody by pipetting, and left to stand for 2 min. The liquid was transferred to the sample pad of the colloidal gold test strip and waited for 8 min. The result was read by a colloidal gold reader. This method can detect chlorpyrifos and methyl parathion simultaneously.

[0057] The results of the recovery experiment of adding samples of MNOH-50, MNCOOH-50, and MNNH2-50 are shown in Table 1:

[0058] Table 1

[0059]

[0060] The MN patch was selected based on the recovery rate of the spiked results. The results showed that the result of the liquid extracted by MNOH-50 on the colloidal gold test strip was more obvious and had stronger anti-interference ability. Therefore, MNOH-50 was selected in the present invention for the subsequent pesticide adsorption experiment.

[0061] Example 5:

[0062] Specificity test of the micro-needle patch: The micro-needle patch MNOH-50 prepared in Example 1 was used to extract pesticide residues, and the specificity of this extraction method was determined using the colloidal gold test strip prepared in Example 2. The results are as Figure 3 shown, where A is phorate, B is profenofos, C is methamidophos, D is isocarbophos, E is acetamiprid, F is carbofuran, G is isoprocarb, H is propiconazole, I is omethoate, J is chlorpyrifos, K is methyl parathion, L is chlorpyrifos + methyl parathion, and T / T0 represents the ratio of the response signal intensity after adding different types of pesticides to the initial response signal intensity. It can be seen that the colloidal gold test strip prepared in the present invention did not show obvious cross-reaction phenomena with the above other types of compounds, indicating that this extraction method has good specificity.

[0063] Example 6:

[0064] Colloidal gold strip analysis of chlorpyrifos and methyl parathion in fruits and vegetables: The colloidal gold strip of MNOH-50 was used to detect chlorpyrifos and methyl parathion in spinach, cucumber, strawberry and apple, and its detection ability for pesticides in fruits and vegetables was evaluated.

[0065] The colloidal gold strip based on MNOH-50 can detect chlorpyrifos with a concentration of 5 ng / mL in spinach, cucumber, strawberry and apple respectively. The colloidal gold strip based on MNOH-50 can detect methyl parathion with a concentration of 8.5 ng / mL in spinach, cucumber, strawberry and apple respectively. The time for the patch to extract pesticides is faster than the pretreatment time of the general colloidal gold strip, and the operation is simple, without the need for scissors, knives, chopping boards, etc.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the microneedle patch in detecting chlorpyrifos and / or methyl parathion in agricultural products, characterized in that: The preparation method of the microneedle patch comprises the following steps: S1: polyethylene glycol diacrylate, triethylamine and acryloyl chloride are mixed in a mass ratio of 99:0.5:0.5 to obtain a mixed solution; S2: injecting the mixed solution into the silicone microneedle mold, placing it in vacuum for 12-18 minutes, and then irradiating the mixed solution with ultraviolet light to obtain a PEGDA patch; the ultraviolet irradiation conditions are: the irradiation intensity is 8-12mW / cm 2 , wavelength is 365nm, irradiation time is 40-50min; S3: Immerse the PEGDA patch in a 50 mmol / L surface-modified multi-walled carbon nanotube suspension for ultrasonic immersion, and after standing at room temperature, peel off the upper transparent film to obtain a microneedle patch; the surface-modified multi-walled carbon nanotubes include hydroxyl-modified multi-walled carbon nanotubes, carboxyl-modified multi-walled carbon nanotubes and amino-modified multi-walled carbon nanotubes.

2. The use of the microneedle patch according to claim 1 in detecting chlorpyrifos and / or methyl parathion in agricultural products, characterized in that: The ultrasonic immersion time in step S3 is 25-35 minutes.

3. The use of the microneedle patch according to claim 1 in detecting chlorpyrifos and / or methyl parathion in agricultural products, characterized in that: The time of standing at room temperature in step S3 is 10-15 hours.

4. The use of the microneedle patch according to claim 1 in detecting chlorpyrifos and / or methyl parathion in agricultural products, characterized in that: The applications include: Press the microneedle patch onto different parts of the object to be tested; After pressing, the microneedle patch was soaked in PBS buffer to obtain an eluent; Pipette the eluate and mix with the gold-labeled mixed antibody. After the mixture is allowed to stand, transfer the liquid to the sample pad of the colloidal gold test strip and read the test result using a colloidal gold card reader.

5. Use of the microneedle patch according to claim 4 in detecting chlorpyrifos and / or methyl parathion in agricultural products, characterized in that: The concentration of the PBS buffer is 0.02 mol / L, and the pH is 7.

4.

6. Use of the microneedle patch according to claim 4 in detecting chlorpyrifos and / or methyl parathion in agricultural products, characterized in that: Pause for 6-12 seconds after each press of the microneedle patch.

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