Microneedle patch for detecting pesticide residues as well as preparation method and application method of microneedle patch

Abstract

The invention provides a microneedle patch for detecting pesticide residues as well as a preparation method and an application method of the microneedle patch, and relates to the technical field of pesticide residue detection. The microneedle patch provided by the invention is prepared from the multi-walled carbon nanotubes subjected to surface modification, polyethylene glycol diacrylate, triethylamine and acryloyl chloride, the prepared microneedle patch has excellent expansion capacity, pesticide residues in agricultural products can be quickly and effectively absorbed, the microneedle patch is pressed on the surfaces of the agricultural products, and the pesticide residues in the agricultural products can be effectively absorbed. The needle tip enters the interior of vegetable cells to collect pesticide residues, eluent is obtained through an elution process, then the immunochromatography test strip is used for detection, the cuticle of a plant or a human body can be punctured in a minimum invasion mode, internal pesticide is detected, and the device is suitable for on-site sampling and detection of a real sample, and has a wide application prospect. And the sample is prevented from being damaged in the detection process.

Description

Technical Field

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

[0002] Pesticide residues are commonly found 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, hormone disorders, asthma, allergies, allergies, and other diseases. Therefore, it is crucial to develop reliable detection technologies 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 immunochromatography has the advantages of simple pretreatment, high sensitivity, fast reaction speed, and strong specificity, and is a promising method for pesticide residue detection.

[0003] The commonly used techniques for pretreatment of organophosphorus pesticides are: liquid-liquid extraction, solid phase extraction, QuEChERS method. These detection methods have the problems of long time consumption, poor anti-interference, cumbersome operation, large amount of organic solvents, etc. Although the pretreatment methods of enzyme-linked immunosorbent assay and colloidal gold test strips are simpler to operate than instrument 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 inside agricultural products. Summary of the invention

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

[0005] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions: In a first aspect, the present invention provides a method for preparing a microneedle patch for detecting pesticide residues, comprising 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 a silicone microneedle mold, placing it in a vacuum, and irradiating the mixed solution with ultraviolet light to obtain a PEGDA patch; S3: immersing the PEGDA patch in a 50 mmol / L surface-modified multi-walled carbon nanotube suspension for ultrasonic immersion, leaving it at room temperature and then peeling off the upper transparent film to obtain a microneedle patch.

[0006] Furthermore, the ultraviolet irradiation conditions in step S2 are: the irradiation intensity is 8-12mW / cm 2 , for example, 8, 9, 10, 11, 12 mW / cm 2 ; The wavelength is 365nm, and the irradiation time is 40-50min, for example, it can be 40, 41, 42, 45, 47, 50min; The vacuum placement time in step S2 is 12-18 minutes, for example, 12, 14, 15, or 18 minutes.

[0007] Furthermore, the surface-modified multi-walled carbon nanotubes in step S3 include hydroxyl-modified multi-walled carbon nanotubes, carboxyl-modified multi-walled carbon nanotubes and amino-modified multi-walled carbon nanotubes, preferably hydroxyl-modified multi-walled carbon nanotubes.

[0008] Furthermore, the ultrasonic immersion time in step S3 is 25-35 min, for example, 25, 27, 30, 32, or 35 min.

[0009] Furthermore, the time of standing at room temperature in step S3 is 10-15 hours, for example, it can be 10, 11, 12, 13, or 15 hours.

[0010] In a second aspect, the present invention proposes a microneedle patch for detecting pesticide residues prepared by the above method.

[0011] In a third aspect, the present invention proposes the use of the microneedle patch for detecting pesticide residues prepared by the method described above in detecting chlorpyrifos and / or methyl parathion in agricultural products.

[0012] In a fourth aspect, the present invention provides an application method of a microneedle patch for detecting pesticide residues, the application method comprising: Pressing the microneedle patch prepared according to the above method on 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.

[0013] Furthermore, the concentration of the PBS buffer is 0.02 mol / L and the pH is 7.4.

[0014] Furthermore, the microneedle patch pauses for 6-12 seconds after each pressing.

[0015] Furthermore, the colloidal gold test strip is prepared according to the following steps: Preparation of colloidal gold: Heat a 1% chloroauric acid solution to boiling, then add a 1% trisodium citrate solution, and continue heating until the color of the solution becomes stable to obtain a colloidal gold solution; Preparation of colloidal gold test strips: dilute the chlorpyrifos hapten-bovine serum protein conjugate with PBS buffer to a concentration of 0.3 mg / mL, and spray it on the NC membrane to form the T1 line; spray methyl parathion hapten-bovine serum protein conjugate 5 mm above the T1 detection line, and dilute it with PBS buffer to a concentration of 0.5 mg / mL to form the T2 line; spray goat anti-mouse secondary antibody 5 mm above the T2 line to form the C line; Assemble colloidal gold test strips: stick the NC membrane, sample pad, and absorbent pad on the PVC base plate, so that the sample pad and the NC membrane overlap by 0.3 cm in the length direction, and the NC membrane and the absorbent pad overlap by 0.3 cm in the length direction, and then use a strip cutter to cut into test strips with a width of 3 mm to obtain colloidal gold test strips.

[0016] Furthermore, the gold-labeled mixed antibody is prepared according to the following steps: Add 9 μL / mL of chlorpyrifos and methyl parathion antibody to the colloidal gold solution, shake on a shaker at 37°C for 30 min, add 10% PEG-20000 and shake for 10 min, then add 5% BSA by mass volume ratio and shake for 10 min; After the reaction, the mixture was placed in a high-speed refrigerated centrifuge at 12000 r / min and centrifuged for 5 min. The supernatant was discarded and the lower precipitate was dissolved with 100 μL / mL resuspension solution to obtain the gold-labeled mixed antibody.

[0017] The microneedle patch for detecting pesticide residues provided by the present invention and its preparation method and application method have the following advantages compared with the prior art: The microneedle patch proposed by the present invention is prepared from multi-walled carbon nanotubes, polyethylene glycol diacrylate, triethylamine and acryloyl chloride with surface modification, wherein PEGDA has the advantages of non-toxicity, good biocompatibility, low immunogenicity, and convenience in use. Under ultraviolet irradiation, the acrylate double bond at the end of the PEGDA molecular chain breaks and combines with other PEGDA molecules to form crosslinking points and networks, and stable chemical bonds are formed between PEGDA molecules to form an overall hydrogel structure. The addition of multi-walled carbon nanotubes can improve the adsorption rate and reduce the interference of the matrix effect. The microneedle patch prepared by the present invention has excellent expansion ability, can quickly and effectively absorb pesticide residues in agricultural products, press the microneedle patch on the surface of agricultural products, enter the vegetable cells through the needle tip to collect pesticide residues, and then use colloidal gold test strips to detect after obtaining the eluate through the elution process, which can pierce the plant in a minimally invasive manner to detect internal pesticides, is suitable for on-site sampling and detection of real samples, and prevents the samples from being destroyed during the detection process.

[0018] The present invention can improve the problem of poor dispersibility of MWNTs in organic solvents or water solvents by performing surface treatment on the surfaces of MWNTs, so as to improve their dispersibility in the solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The tensile compressor test results of the microneedle patch prepared in the embodiment of the present invention are shown; Figure 2 The water absorption test results of the microneedle patch prepared in the embodiment of the present invention are shown; Figure 3 The specificity test results of the colloidal gold test strip prepared in the examples of the present invention are shown. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The following examples respectively describe in detail the preparation process of the microneedle patch, the preparation process of the colloidal gold test strip, the preparation process of the gold-labeled mixed antibody, the application method and conditions of the microneedle patch, and the detection performance provided by the present invention. Unless otherwise specified, the raw materials used in the examples are all general commercially available raw materials. Embodiment 1:

[0023] MN patches were prepared using the following different materials and methods: Combination 1: PVA (polyvinyl alcohol) and HAMA (methacryloyl hyaluronic acid) were mixed in a mass ratio of 3:6, and the obtained mixed solution was cast on the microneedle mold, and then placed in vacuum for 30 minutes to allow the mixed solution to fill the cavity. After the microneedle mold was filled with the mixed solution, it was placed at room temperature for 12 hours to obtain a dry MN patch, and then frozen at −20°C for 12 hours, and then stored at 4°C for 4 hours. After the above process was repeated three times, a hydrogel HAMA / PVAMN patch was 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.

[0024] Combination 2: PEGDA (polyethylene glycol diacrylate), triethylamine and acryloyl chloride were mixed in a mass ratio of 99:0.5:0.5 to obtain a mixed solution, which was added to the silicone microneedle mold and placed in a vacuum for 15 minutes to remove bubbles in the mixed solution and ensure that the mixture was evenly distributed in the microneedle mold. 2 The mixture was irradiated with an ultraviolet wavelength of 365 nm and an intensity of 450 nm for 45 min to obtain a hydrogel PEGDA patch.

[0025] Combination 3: Glycidyl methacrylate, trimethylolpropane trimethacrylate, trimethylol glycol dimethacrylate, 1-hydroxycyclohexyl phenyl ketone and 2-methoxyethanol were mixed in a mass ratio of 10:6.0:15.7:0.01:0.01 to obtain a mixed solution, which was added to the silica gel negative electrode microneedle mold and placed in a vacuum for 15 minutes to ensure that the microneedle mold was completely filled. Subsequently, the microneedle mold was placed under a UV lamp (365nm) for 30 minutes to polymerize it. The MNs were then removed from the microneedle mold, washed with distilled water and dried to obtain a composite microneedle patch.

[0026] The microneedle patch prepared according to the above method was subjected to tensile compression test, and the results were as follows: Figure 1 As shown, the results show that all three combinations can penetrate the epidermis of fruits and vegetables, but the microneedle patch prepared according to combination one has a much lower bearing capacity than the microneedle patches prepared according to combinations two and three, with some swelling and deformation, while the microneedle patches prepared according to the other two combinations are not deformed, and the microneedle patch needs to have sufficient insertion force.

[0027] The microneedle patch prepared according to the above method was tested for water absorption. The test results are as follows: Figure 2 As shown, it can be seen that the absorption rate is combination 2 > combination 1 > combination 3, and the microneedle patch absorbs the same liquid in a shorter time.

[0028] Based on the results of the above two tests, combination formula No. 2 was selected to prepare microneedle patches. The hydrogel PEGDA patches of combination 2 were immersed in MWCNT-OH (hydroxyl-modified multi-walled carbon nanotubes), MWCNT-COOH (carboxyl-modified multi-walled carbon nanotubes), and MWCNT-NH2 (amino-modified multi-walled carbon nanotubes) suspensions with a concentration of 50 mmol / L, respectively, and immersed for 30 minutes under ultrasonic conditions and placed at room temperature for 12 hours 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 microneedle patches proposed in the present invention).

[0029] It should be noted that the modification method of multi-walled carbon nanotubes is not specifically limited in the present invention. For example, MWCNT-OH can be prepared by treating MWCNTs with a mixture of concentrated nitric acid and concentrated sulfuric acid or alkaline oxidation treatment to introduce hydroxyl functional groups on the surface of multi-walled carbon nanotubes. Embodiment 2:

[0030] The preparation of colloidal gold test strips comprises the following steps: Preparation of colloidal gold solution: First, add 100 mL of 1% chloroauric acid solution to a 250 mL flask and heat to boiling. Then add 1 mL of freshly prepared 1% trisodium citrate solution under magnetic stirring. The color of the solution changes to colorless. Continue to heat the solution until the color slowly changes to blue and finally to dark red. When the color of the solution tends to be stable, continue heating for 10 minutes, then stop heating and cool it at room temperature to obtain a colloidal gold solution. Seal it with a sealing film and store it at 4°C for later use. Colloidal gold was characterized by UV spectrophotometer and transmission electron microscopy (TEM).

[0031] Preparation of test lines and quality control lines: The following T lines and C lines were sprayed with a film sprayer at a spray volume of 1.0 μL / cm. Chlorpyrifos, methyl parathion hapten-bovine serum protein conjugates were used as test lines (T1 line, T2 line) on the NC membrane, and goat anti-mouse secondary antibody was used as the quality control line (C line). The specific process is as follows: The chlorpyrifos hapten-bovine serum protein conjugate was diluted to a concentration of 0.3 mg / mL with PBS buffer (pH 7.4, concentration 0.02 mol / L, containing 1% methanol by volume) and sprayed on the NC membrane to form the T1 line; Spray methyl parathion hapten-bovine serum protein conjugate 5 mm above the T1 detection line and dilute it with PBS buffer (pH 7.4, concentration 0.02 mol / L, containing 1% methanol by volume) to a concentration of 0.5 mg / mL, which is the T2 line; Spray goat anti-mouse secondary antibody (1 mg / mL, PBS buffer pH 7.4, 0.02 mol / L) at 5 mm on the T2 line, which is the C line (quality control line). Dry the coated NC membrane at 37 ° C for 2 hours and set aside. Soak the sample pad in PBS buffer containing 1% BSA (mass volume ratio is 1%, that is, 100 ml contains 1g BSA), 2% sucrose (mass volume ratio is 2%, that is, 100 ml contains 2g sucrose), pH 7.4, 0.02 mol / L for 2 hours, and dry it at 37 ° C for 2 hours.

[0032] Assemble colloidal gold test strips: stick the NC membrane (nitrocellulose membrane), sample pad, and absorbent pad on the PVC base plate, so that the sample pad overlaps with the NC membrane by 0.3 cm (in the length direction), and the NC membrane overlaps with the absorbent pad by 0.3 cm (in the length direction), and then use a strip cutter to cut it into test strips with a width of 3 mm, which are colloidal gold test strips. Embodiment 3:

[0033] Preparation of gold-labeled mixed antibodies: 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 minutes, add 10% PEG-20000, shake for 10 minutes, then add 5% BSA (mass volume ratio is 5%, that is, 100 ml contains 5g BSA), shake for 10 minutes, and block the free sites on the surface of the gold particles. After the reaction, put it into a high-speed refrigerated centrifuge at 12000r / min, centrifuge for 5 minutes, discard the supernatant, dissolve the lower precipitate with 100 μL / mL resuspension solution, and obtain the gold-labeled mixed antibody for use. Embodiment 4:

[0034] The microneedle patch prepared in Example 1 was used to detect chlorpyrifos and methyl parathion: Chlorpyrifos was dissolved in methanol and then diluted with deionized water to a series of concentrations (1, 5, 10, 50, 100, 500, 1000 ng / mL), and the chlorpyrifos solutions of different dilution concentrations were sprayed on the surface of cabbage leaves. At the same time, methyl parathion was dissolved in methanol and diluted with deionized water to a series of concentrations (1, 5, 10, 50, 100, 500, 1000 ng / mL), and the methyl parathion solutions of different dilution concentrations were sprayed on the surface of cabbage leaves. Finally, MNOH-50, MNCOOH-50, and MNNH2-50 were pressed on different parts of the cabbage for 5 to 8 times, and each pressing paused for 10 seconds. Then, the patch was soaked in 1 mL of PBS buffer (pH 7.4, 0.02 mol / L) for 1 minute, 100 uL of the eluate was pipetted and mixed with the gold-labeled mixed antibody, and then allowed to stand for 2 minutes. The liquid was transferred to the sample pad of the colloidal gold test strip, and the results were read by the colloidal gold card reader after waiting for 8 minutes. This method can detect chlorpyrifos and methyl parathion at the same time.

[0035] The results of the sample recovery experiments of MNOH-50, MNCOOH-50 and MNNH2-50 are shown in Table 1: Table 1

[0036] The MN patch was selected based on the recovery rate of the spiked results. The results showed that the liquid extracted by MNOH-50 had a more obvious result on the colloidal gold test strip and had stronger anti-interference. Therefore, the present invention selected MNOH-50 for subsequent pesticide adsorption experiments.

[0037] Embodiment 5: Specificity test of microneedle patch: The microneedle patch MNOH-50 prepared in Example 1 was used to extract pesticide residues, and the colloidal gold test strip prepared in Example 2 was used to determine the specificity of this extraction method. The results are shown in FIG. Figure 3 As shown, 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, and 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 by the present invention does not show obvious cross-reaction with the above other types of compounds, indicating that the extraction method has good specificity.

[0038] Embodiment 6: Colloidal gold test strip analysis of chlorpyrifos and methyl parathion in fruits and vegetables: MNOH-50 colloidal gold test strips were used to detect chlorpyrifos and methyl parathion in spinach, cucumber, strawberry, and apple to evaluate its ability to detect pesticides in fruits and vegetables.

[0039] The colloidal gold test strip based on MNOH-50 can detect chlorpyrifos with a pesticide concentration of 5 ng / mL in spinach, cucumber, strawberry, and apple. The colloidal gold test strip based on MNOH-50 can detect methyl parathion with a pesticide concentration of 8.5 ng / mL in spinach, cucumber, strawberry, and apple. The time for extracting pesticides from the patch is faster than the pre-treatment time of general colloidal gold test strips, and the operation is simple, without the need for scissors, knives, cutting boards, etc.

[0040] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a microneedle patch for detecting pesticide residues, characterized in that: The following steps are involved: 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 a silicone microneedle mold, placing it in a vacuum, and irradiating the mixed solution with ultraviolet light to obtain a PEGDA patch; S3: immersing the PEGDA patch in a 50 mmol / L surface-modified multi-walled carbon nanotube suspension for ultrasonic immersion, leaving it at room temperature and then peeling off the upper transparent film to obtain a microneedle patch.

2. The method for preparing a microneedle patch for detecting pesticide residues according to claim 1, characterized in that: The irradiation conditions of ultraviolet light in step S2 are: irradiation intensity is 8-12mW / cm 2 , wavelength is 365nm, irradiation time is 40-50min; The vacuum placement time in step S2 is 12-18 minutes.

3. The method for preparing a microneedle patch for detecting pesticide residues according to claim 1, characterized in that: The surface-modified multi-walled carbon nanotubes in step S3 include hydroxyl-modified multi-walled carbon nanotubes, carboxyl-modified multi-walled carbon nanotubes and amino-modified multi-walled carbon nanotubes.

4. The method for preparing a microneedle patch for detecting pesticide residues according to claim 1, characterized in that: The ultrasonic immersion time in step S3 is 25-35 minutes.

5. The method for preparing a microneedle patch for detecting pesticide residues according to claim 1, characterized in that: The time of standing at room temperature in step S3 is 10-15 hours.

6. A microneedle patch for detecting pesticide residues, characterized in that: The method is prepared by any one of claims 1 to 5.

7. Use of the microneedle patch for detecting pesticide residues prepared by the method according to any one of claims 1 to 5 in detecting chlorpyrifos and / or methyl parathion in agricultural products.

8. An application method of a microneedle patch for detecting pesticide residues, characterized in that: The application method comprises: Pressing the microneedle patch prepared by the method according to any one of claims 1 to 5 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.

9. The application method of the microneedle patch for detecting pesticide residues according to claim 8, characterized in that: The concentration of the PBS buffer is 0.02 mol / L, and the pH is 7.

4.

10. The application method of the microneedle patch for detecting pesticide residues according to claim 8, characterized in that: Pause for 6-12 seconds after each press of the microneedle patch.

Images