Ratio fluorescent carbon dot microneedle patch for detecting pesticide residues on fruits and vegetables

Abstract

The invention relates to the technical field of material science, analytical chemistry and food safety detection technologies, in particular to a ratiometric fluorescent carbon dot microneedle patch for detecting fruit and vegetable pesticide residues, and a preparation method comprises the following steps: preparing Fe-CDs and MnO2 into a Fe-CDs / MnO2 nanosheet hybrid material; and then adding the Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholin esterase and o-phenylenediamine into a PVA solution under a vacuum condition, then adding a solvent and a cross-linking agent, after the reaction is completed, pouring the mixture into a microneedle patch mold, and drying to obtain the Fe-CDs / MnO2 / PVA microneedle patch. The microneedle patch is combined with RGB color identification software, so that rapid, accurate, on-site operable and low-cost detection on pesticide residues of fruits and vegetables can be realized.

Description

Technical Field

[0001] The invention relates to the technical fields of material science, analytical chemistry and food safety detection technology, and in particular to a ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables. Background Art

[0002] The development of agricultural modernization is inseparable from the use of various pesticides. However, due to the abuse of pesticides and the lack of efficient detection methods by relevant departments, public safety incidents caused by pesticide residues are common. At present, a variety of methods have been developed for the detection of pesticide residues, which can be subdivided into chromatography, biosensor method and immunoassay.

[0003] Chromatography uses the slight difference in the distribution coefficients of different substances between the stationary phase and the mobile phase, resulting in different movement speeds in different phases, to achieve separation, analysis and detection of target substances. It has very wide applications in analytical chemistry, organic chemistry and other fields. At present, chromatographic analysis technology is widely used in the field of pesticide residue detection, mainly including gas chromatography, liquid chromatography, etc. Chromatographic analysis has the advantages of high separation efficiency, wide application range and high sensitivity, but this method requires professional personnel to operate and has high precision requirements for instruments.

[0004] The biosensor method uses biological molecules (enzymes, aptamers, etc.) as recognition elements, coupled with corresponding physical or chemical signal outputs, to achieve high-performance detection of target objects. The biosensor method is often combined with various optical and electrical analysis methods, and is characterized by good sensitivity and selectivity.

[0005] Immunoassay uses labeled antibodies (or antigens) to competitively bind to the test object, and can be used for screening tests of certain toxic drugs. Immunoassay has the advantage of high selectivity and has been developed for high-performance detection of pesticide residues. Depending on the signal, immunoassay can be divided into many types. Among them, the common ones include enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), immunochromatography (ICA), etc. Immunoassay has the advantages of good specificity and high sensitivity, but it needs to be combined with the corresponding pesticide antibody research and development technology, and the development cost is relatively high. At the same time, the pesticide category needs to be clarified before conducting immunoassay, which is difficult to meet the needs of simultaneous detection of multiple types of pesticide residues.

[0006] It can be seen that the existing problems with pesticide detection technology are at least: 1. Lack of rapid, field-applicable detection methods. Most of the current detection technologies require complex equipment and professional operations, making it difficult to conduct instant detection at fruit and vegetable picking sites, farmers' markets and other places, and cannot meet the needs of consumers and producers for rapid acquisition of test results. 2. The accuracy of some detection methods is greatly affected by environmental factors. For example, for enzyme inhibition methods and some biosensor-based detection methods, slight changes in ambient temperature, humidity, pH value, etc. may lead to deviations in test results, reducing the reliability of the test. 3. The detection cost is high. High-precision detection methods such as chromatography have high equipment and operating costs, and the cost of antibody preparation for immunoassays is also high, which limits large-scale applications. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables, so as to achieve rapid, accurate, on-site operable and low-cost detection of pesticide residues in fruits and vegetables.

[0008] The object of the present invention is achieved through the following technical scheme: a ratio fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables, the preparation method comprising the following steps: mixing Fe-CDs and MnO2 nanosheets in a mass ratio of 100 to 300:1 in water, stirring at a speed of 800 to 1000 r / min for 5 to 10 minutes, and drying to obtain a Fe-CDs / MnO2 nanosheet hybrid material in a mass ratio of 3 to 3.2: 0.8 to 1.2: 0.5 to 0.7: 0.4 to 0.8 The Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase, and o-phenylenediamine are added to a 10%-15% PVA solution under vacuum conditions, and then a solvent and a cross-linking agent accounting for 10%-20% of the total mass of the Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase, and o-phenylenediamine are added, stirred at 500-1000rpm for 1-2h, poured into a microneedle patch mold and dried to obtain a Fe-CDs / MnO2 / PVA microneedle patch.

[0009] Furthermore, the preparation method of the Fe-CDs comprises the following steps:

[0010] The grapefruit peel dry powder is added to the acetone solution and mixed evenly, and then FeCl3·6H2O is added, and the reaction is continued at 100-120°C for 5-8 hours, and then centrifuged to obtain the supernatant, which is filtered and dried to obtain the Fe-CDs, wherein the mass ratio of the grapefruit peel dry powder, the acetone solution and the FeCl3·6H2O is in the range of 4-4.2:50-100:0.8-1.2.

[0011] Preferably, the preparation method of the grapefruit peel dry powder is: wash the grapefruit peel, cut it into pieces, dry it at 50° C. for 12 hours, and finally grind it into powder.

[0012] Preferably, the supernatant is filtered using a polyethersulfone membrane (0.22 μm).

[0013] Preferably, the drying method is freeze-drying.

[0014] Furthermore, the method for preparing the MnO2 nanosheets comprises the following steps:

[0015] Manganese chloride tetrahydrate is dissolved in water, and then tetramethylammonium hydroxide pentahydrate and 30% hydrogen peroxide are added, stirred for 12 hours, and the product is centrifuged and washed to obtain the MnO2 nanosheets, wherein the mass ratio of the manganese chloride tetrahydrate, tetramethylammonium hydroxide pentahydrate and 30% hydrogen peroxide is in the range of 0.5-0.8:2.1-2.4:1.8-2.2.

[0016] Furthermore, the types of the cross-linking agent include one or more of glutaraldehyde, glycerol, diphenylmethane diisocyanate and disuccinimidyl succinic acid, and the types of the solvent include one or more of chloroform, carbon disulfide, carbon tetrachloride and toluene.

[0017] Furthermore, the preparation method of the microneedle patch mold includes the following steps: using 3D printing technology or photolithography technology to make a microneedle patch mold, the material of the microneedle patch mold is polydimethylsiloxane, the size of the microneedle patch mold is 300-800μm, the needle bottom diameter is 100-300μm, and the needle spacing is 200-500μm.

[0018] The present invention also provides a method for detecting pesticide residues in fruits and vegetables, comprising using the above-mentioned ratio fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables, and further comprising the following steps:

[0019] The Fe-CDs / MnO2 / PVA microneedle patch was inserted into the surface of the fruits and vegetables to be tested to absorb the fruit and vegetable juice. After 5 minutes, it was irradiated in a reaction chamber equipped with a 365nm ultraviolet lamp. The deep learning model was used to learn and the photos were collected by a smartphone, and the App with RGB color recognition function was used for analysis.

[0020] The principle of the present invention is that acetylcholinesterase (AChE) catalyzes the conversion of thioacetylcholine (ATCh) into reduced thiocholine (TCh), which further reduces the MnO2 nanosheets to Mn 2+ . Due to Mn 2+It does not have oxidase-like activity, so o-phenylenediamine (OPD) cannot be oxidized. In the presence of organophosphate, the fluorescence signal of the system is weakened by dynamically quenching the fluorescence of Fe-CDs. In addition, the presence of organophosphate reduces the activity of AChE, inhibits the production of TCh, and thus reduces the decomposition of MnO2 nanosheets. The residual MnO2 nanosheets further catalyze the oxidation of colorless o-phenylenediamine (OPD) to generate colored oxidized OPD (oxOPD) using oxidase-like activity, increasing the fluorescence signal of the system. Therefore, organophosphate is quantitatively detected by measuring the fluorescence intensity ratio of Fe-CDs and oxOPD.

[0021] The beneficial effects of the present invention are:

[0022] 1. Rapid detection: The microneedle patch prepared by the present invention can directly penetrate the surface of fruits and vegetables, quickly contact and adsorb pesticide residues. The detection process is simple and fast, and the test results can be obtained in a short time (such as a few minutes to more than ten minutes), meeting the needs of on-site rapid detection.

[0023] 2. High sensitivity and accuracy. The ratio fluorescence detection method detects pesticide residues by changing the ratio of the fluorescence intensity of two emission wavelengths. It can effectively reduce the interference of environmental factors (such as temperature, pH value, etc.) on the test results, improve the sensitivity (the detection limit can be as low as ppb level) and accuracy of the test, and has higher reliability compared with traditional detection methods.

[0024] 3. Easy to operate. No complicated sample pretreatment and professional equipment operation are required. Consumers or testers only need to press the microneedle patch and use a portable fluorescence detector for testing, which is easy to promote and apply.

[0025] 4. Low cost. The raw materials for preparing ratiometric fluorescent carbon dots are cheap and easily available. The preparation process of microneedle patches is relatively simple and does not require expensive large-scale instruments and equipment, which reduces the detection cost and is conducive to large-scale production and application.

[0026] 5. In-situ testing and non-destructive testing. The microneedle patch only penetrates the surface of fruits and vegetables to a very shallow depth during testing, causing minimal damage to the fruits and vegetables as a whole. In-situ testing can be achieved without affecting the subsequent sales and consumption of fruits and vegetables. At the same time, it also avoids the errors caused by sampling in traditional sampling and testing methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Characterization of the surface morphology of Fe-CDs. (a) Transmission electron microscopy image of Fe-CDs; (b) X-ray diffraction image of Fe-CDs;

[0028] Figure 2Characterization of the performance of Fe-CDs. (a) UV-visible absorption spectrum of Fe-CDs; (b) Fluorescence excitation and emission spectra of Fe-CDs; (c) Fluorescence emission spectra of Fe-CDs at different excitation wavelengths; (d) Fluorescence excitation spectra of Fe-CDs at different emission wavelengths;

[0029] Figure 3 Characterization of the performance of Fe-CDs. (a) Full spectrum of X-ray electron energy spectrum of Fe-CDs; (b) C1s spectrum of X-ray electron energy spectrum of Fe-CDs; (c) N1s spectrum of X-ray electron energy spectrum of Fe-CDs; (d) O1s spectrum of X-ray electron energy spectrum of Fe-CDs;

[0030] Figure 4 It is a stereomicroscope image of Fe-CDs / MnO2 / PVA microneedle;

[0031] Figure 5 for the ratiometric fluorescence detection of Fe-CDs / MnO2 / PVA microneedles;

[0032] Figure 6 Fitting the standard curve. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0034] Example 1 Preparation of Fe-CDs / MnO2 / PVA microneedle patch

[0035] (1) Preparation of Fe-CDs

[0036] Fe-CDs were prepared by solvothermal method. The grapefruit peel was washed, cut into pieces, and dried at 50°C for 12h. After the grapefruit peel was crushed into powder, 4.0g of powder was weighed and added to 80mL of acetone solution, and stirred thoroughly at 25°C for 3min. It was transferred to a reactor (100mL), and 1.0g of FeCl3·6H2O was added thereto and the reaction was continued at 120°C for 5h. After natural cooling, the resulting mixture was centrifuged at 8000r / min for 10min, and the supernatant was filtered using a polyethersulfone membrane (0.22μm). Finally, the Fe-CDs were obtained by freeze drying.

[0037] (2) Preparation of MnO2 nanosheets

[0038] Manganese chloride tetrahydrate (0.593 g) was dissolved in 10 mL of aqueous solution and transferred to a 100 mL round-bottom culture bottle. Then, 20 mL of tetramethylammonium hydroxide pentahydrate (2.1748 g) and H2O2 (30%, 2 mL) were added to the water, quickly added and vigorously stirred at room temperature for 12 h. Subsequently, the product was centrifuged at 10,000 r / min for 15 min and washed several times with ethanol before freeze drying to obtain MnO2 nanosheets.

[0039] (3) Preparation of Fe-CDs / MnO2 nanosheet hybrid materials

[0040] After mixing Fe-CDs and MnO2 nanosheets at a mass ratio of 300:1 in 2 mL of ultrapure water, the mixture was stirred at 800 r / min for 5 min to obtain a mixed solution, and finally freeze-dried to obtain Fe-CDs / MnO2 nanosheet powder.

[0041] (4) Microneedle mold production

[0042] Using 3D printing technology and materials such as polydimethylsiloxane (PDMS), the shape (such as cone) and size (needle length 500μm, needle base diameter 200μm, and needle spacing 400μm) of the microneedle are designed and the microneedle mold is printed.

[0043] (5) Preparation of microneedle patches

[0044] Prepare a PVA solution with a mass percentage concentration of 15%, directly add a mixed solution of Fe-CDs / MnO2, acetylcholine, acetylcholinesterase, and o-phenylenediamine with a mass ratio of 3:1:0.6:0.5 under vacuum conditions, add a crosslinker (glutaraldehyde, 15% of the polymer mass) and a solvent (chloroform), and mix the materials evenly under stirring conditions (such as stirring at 500-1000rpm for 2h). Fill the mixed material into the microneedle mold, place the microneedle mold in an oven at 60°C for curing for 4h, and dry it naturally to obtain a Fe-CDs / MnO2 / PVA microneedle patch.

[0045] Experimental Example 1 Characterization of Fe-CDs Carbon Dots

[0046] Scanning transmission electron microscopy and X-ray diffractometer were used to characterize the morphology, lattice spacing and other microstructures of Fe-CDs. Figure 1 As shown in (a), the transmission electron microscopy image of Fe-CDs shows that Fe-CDs are uniformly dispersed nearly spherical particles, and there is no obvious aggregation when dispersed in water. To further study the structural information of Fe-CDs, high-resolution transmission electron microscopy was used to observe the lattice spacing of single Fe-CDs. Figure 1(a) shows that the lattice structure of Fe-CDs is clearly visible, and the lattice spacing is 0.21nm. In addition, Fe-CDs were further characterized by X-ray diffractometer. Figure 1 A typical crystal diffraction peak can be observed in (b), with a diffraction angle of 2θ = 25.6°, which is a characteristic diffraction peak of carbon and corresponds to the (002) crystal plane of graphite. This crystal plane represents the stacking mode of aromatic layers, indicating that the synthesized Fe-CDs have the lattice structure of graphite and a high degree of graphitization. The above results show that Fe-CDs have been successfully prepared and can be used for subsequent experiments.

[0047] The formation of Fe-CDs was further confirmed by UV-vis spectroscopy and fluorescence properties. Figure 2 As shown in (a), the UV-vis spectrum of Fe-CDs showed two shoulder peaks at 210 nm and 285 nm, which were respectively attributed to the aromatic sp 2 The π-π* transition of the structural domain and the n-π* transition of the C=N / C=O bond are the characteristic absorption peaks of Fe-CDs. Figure 2 In the inset of (a), the Fe-CDs solution appears white under sunlight, but shows blue fluorescence under 365nm UV light, showing the unique fluorescence properties of Fe-CDs. In addition, the fluorescence quenching ability of Fe-CDs was also investigated. Figure 2 As shown in (b), the synthesized Fe-CDs exhibit UV absorption in the range of 350-560nm, which overlaps with its fluorescence excitation peak (Ex: 430nm) and emission peak (Em: 510nm) in a large area. The PL characteristics of Fe-CDs are characterized in the excitation wavelength range of 340-480nm. Figure 2 As shown in (c), the fluorescence emission peak of Fe-CDs red-shifts with the increase of excitation wavelength. At an excitation wavelength of 510 nm, the fluorescence emission wavelength of Fe-CDs reaches a maximum of 430 nm, showing a purer blue fluorescence, which is consistent with Figure 2 (a) The results are consistent with those in the illustration. At the same time, the PL characteristics of Fe-CDs were characterized in the emission wavelength range of 460-550 nm. Figure 2 As shown in (d), the fluorescence excitation peak of Fe-CDs blue-shifts as the emission wavelength decreases. At an emission wavelength of 430 nm, the fluorescence excitation wavelength of Fe-CDs reaches a maximum of 510 nm, which is consistent with Figure 2 (b) Results.

[0048] Experimental Example 2 Composition and structure of Fe-CDs carbon dots

[0049] The surface elements of Fe-CDs were further analyzed by X-ray electron spectrometer, such as Figure 3As shown in (a), Fe-CDs are mainly composed of C (mass fraction 38.9%), O (mass fraction 39.8%) and a small amount of N (mass fraction 12.6%) elements. Figure 3 (b) to (d) are the high-resolution X-ray electron energy spectra corresponding to C1s, N 1s and O1s, respectively. The fitting peaks appear at 285.6, 399.1 and 533.9 eV, corresponding to C—C / C=C, C—N, C=O and C—O bonds, respectively.

[0050] Experimental Example 3 Characterization of Fe-CDs / MnO2 / PVA microneedles

[0051] The Fe-CDss / MnO2 / PVA microneedles were characterized by a stereo microscope. The results showed that Fe-CDs and MnO2 were successfully loaded in the PVA microneedle cavity and the color was yellow. This was because Fe-CDs and MnO2 were cross-linked inside the mold to form more hydrogen bonds, which provided a basis for the detection of pesticide residues in actual fruit and vegetable samples.

[0052] Experimental Example 4: Application of Fe-CDs / MnO2 / PVA microneedles in the detection and classification of pesticide residues in actual fruit and vegetable samples

[0053] The sensor was further applied to the recovery experiment of fruit and vegetable samples to evaluate its reliability and applicability in detecting pesticides in actual samples. Figure 5 It reflects the fluorescence color change of Fe-CDs / MnO2 / PVA microneedles in the presence or absence of organic phosphorus. The microneedle patch is pressed onto the surface and inside of fruits and vegetables to absorb the fruit and vegetable juice. After 5 minutes, it is irradiated under 365 nm ultraviolet light, and pictures are taken and analyzed using an App with RGB color recognition function. (This experimental example uses a mobile phone App color recognition color picker, Xi'an Banligeban Software Co., Ltd.). Then, according to the fitting standard curve ( Figure 6 ) to calculate the pesticide residue content, and use HPLC to test the sample to obtain the reference value of the pesticide residue in the sample. The test results are as follows:

[0054] Table 1 Determination of pesticides in spiked samples

[0055]

[0056] ND: Not detected.

[0057] The pesticide residues on the surface of fruits were determined by spiked experiments, with recovery rates ranging from 96.2% to 110.8%. The results showed that it has high reliability and stability in practical applications. In addition, according to the current national maximum pesticide residue standards GB2763.1-2022 "National Food Safety Standard Maximum Residue Limits of 112 Pesticides including 2,4-D Butyrate Sodium Salt in Food" and GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food", Fe-CDs / MnO2 / PVA microneedles use the maximum residue as the benchmark to formulate three rating standards of unqualified, qualified and excellent, providing consumers, market supervision departments, catering companies, etc. with real-time information on fruit and vegetable pesticide residues to ensure food quality safety and human health.

[0058] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables, characterized in that: The preparation method comprises the following steps: mixing Fe-CDs and MnO2 nanosheets in a mass ratio of 100 to 300:1 in water, stirring at a speed of 800 to 1000 r / min for 5 to 10 minutes, drying to obtain a Fe-CDs / MnO2 nanosheet hybrid material, adding the Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase and o-phenylenediamine to a 10% to 15% PVA solution under vacuum conditions at a mass ratio of 3 to 3.2: 0.8 to 1.2: 0.5 to 0.7: 0.4 to 0.8, then adding a solvent and a cross-linking agent accounting for 10% to 20% of the total mass of the Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase and o-phenylenediamine, stirring at 500 to 1000 rpm for 1 to 2 hours, pouring the mixture into a microneedle patch mold and drying to obtain a Fe-CDs / MnO2 / PVA microneedle patch.

2. The ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables according to claim 1, characterized in that: The preparation method of the Fe-CDs comprises the following steps: The grapefruit peel dry powder is added to the acetone solution and mixed evenly, and then FeCl3·6H2O is added, and the reaction is continued at 100-120°C for 5-8 hours, and then centrifuged to obtain the supernatant, which is filtered and dried to obtain the Fe-CDs, wherein the mass ratio of the grapefruit peel dry powder, the acetone solution and the FeCl3·6H2O is in the range of 4-4.2:50-100:0.8-1.

2.

3. The ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables according to claim 1, characterized in that: The method for preparing the MnO2 nanosheets comprises the following steps: Manganese chloride tetrahydrate is dissolved in water, and then tetramethylammonium hydroxide pentahydrate and 30% hydrogen peroxide are added, stirred for 12 hours, and the product is centrifuged and washed to obtain the MnO2 nanosheets, wherein the mass ratio of the manganese chloride tetrahydrate, tetramethylammonium hydroxide pentahydrate and 30% hydrogen peroxide is in the range of 0.5-0.8:2.1-2.4:1.8-2.

2.

4. The ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables according to claim 1, characterized in that: The types of the cross-linking agent include one or more of glutaraldehyde, glycerol, diphenylmethane diisocyanate and disuccinimidyl succinic acid, and the types of the solvent include one or more of chloroform, carbon disulfide, carbon tetrachloride and toluene.

5. The ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables according to claim 1, characterized in that: The preparation method of the microneedle patch mold comprises the following steps: using 3D printing technology or photolithography technology to make the microneedle patch mold, the material of the microneedle patch mold is polydimethylsiloxane, the size of the microneedle patch mold is 300-800μm, the needle bottom diameter is 100-300μm, and the needle spacing is 200-500μm.

6. A method for detecting pesticide residues in fruits and vegetables, characterized in that: The method comprises using the ratio fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables as claimed in any one of claims 1 to 5, and further comprising the following steps: The Fe-CDs / MnO2 / PVA microneedle patch was inserted into the surface of the fruits and vegetables to be tested to absorb the fruit and vegetable juice. After 5 minutes, it was irradiated with 365nm ultraviolet light, and the deep learning model was used to learn and the photos were collected by a smartphone, and the App with RGB color recognition function was used for analysis.

Images