A ratio fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables

Abstract

The application relates to the technical field of material science, analytical chemistry and food safety detection technology, in particular to a ratio fluorescent carbon dot microneedle patch for detecting pesticide residues of fruits and vegetables, and a preparation method thereof. The preparation method comprises the following steps: Fe-CDs and MnO2 are prepared into Fe-CDs / MnO2 nanosheet hybrid materials; the Fe-CDs / MnO2 nanosheet hybrid materials, acetylcholine, acetylcholine esterase and o-phenylenediamine are added into a PVA solution under vacuum conditions; a solvent and a crosslinking agent are further added; after the reaction is completed, the Fe-CDs / MnO2 / PVA microneedle patch is obtained by pouring into a microneedle patch mold and drying. The microneedle patch can realize rapid, accurate, on-site operable and low-cost detection of pesticide residues of fruits and vegetables in combination with RGB color recognition software.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of material science, analytical chemistry and food safety detection technology, and particularly relates to a ratio fluorescent carbon dot microneedle patch for detecting pesticide residues of fruits and vegetables. BACKGROUND

[0002] The development of agricultural modernization cannot be separated from the use of various pesticides, but due to the misuse of pesticides and the lack of efficient detection means 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 divided into chromatography, biosensor method and immunoassay method.

[0003] Chromatography utilizes the slight difference in distribution coefficient between different substances in the stationary phase and the mobile phase, which leads to different moving speeds in different phases, so as to realize the separation, analysis and detection of target substances, and has a very wide application in the fields of analytical chemistry and organic chemistry. At present, chromatographic analysis technology is widely used in the field of pesticide residue detection, mainly including gas chromatography, liquid chromatography and the like. Chromatographic analysis has the advantages of high separation efficiency, wide application range and high sensitivity, but this method needs to be operated by professional personnel and has high precision requirements for instruments.

[0004] The biosensor method uses molecules (enzymes, aptamers, etc.) of biological origin as recognition elements, and combines with corresponding physical or chemical signal output to achieve the purpose of high-performance detection of target substances. The biosensor method is often combined with various optical and electrical analysis methods, and has the characteristics of good sensitivity and selectivity.

[0005] The immunoassay method uses labeled antibodies (or antigens) to compete with the detection object for binding, which can be used for screening tests of some toxic substances. The immunoassay method has the advantage of high selectivity and has been developed for high-performance detection of pesticide residues. According to the difference of signal substances, the immunoassay method can be divided into various types. Among them, the common ones include enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), immunochromatography (ICA) and the like. The immunoassay method has the advantages of good specificity and high sensitivity, but it needs to be combined with the development technology of pesticide antibodies, and the development cost is high. At the same time, the pesticide category needs to be determined before the immunoassay, which is difficult to meet the demand of simultaneous detection of multiple types of pesticide residues.

[0006] It can be seen that the existing pesticide detection technology has at least the following problems: 1. Lack of rapid and on-site detection methods. Most of the current detection technologies require complex equipment and professional operation, which is difficult to perform instant detection in fruit and vegetable picking sites, agricultural markets and other places, and cannot meet the demand of consumers and producers for rapid acquisition of detection results. 2. The accuracy of some detection methods is greatly affected by environmental factors. For example, enzyme inhibition method and some detection methods based on biosensor, small changes in environmental temperature, humidity, pH value and other factors may cause detection result deviation, reducing the reliability of detection. 3. High detection cost. The high-precision detection method such as chromatography has high equipment and operation cost, and the antibody preparation cost of immune analysis method is also high, which limits its large-scale application. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art and provide a ratio fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables, which realizes rapid, accurate, on-site operable and low-cost detection of pesticide residues in fruits and vegetables.

[0008] The purpose of the present application is achieved by 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 with a mass ratio of 100-300:1 in water, stirring at a speed of 800-1000 r / min for 5-10 min, drying to obtain Fe-CDs / MnO2 nanosheet hybrid material, adding the Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase and o-phenylenediamine to a 10%-15% PVA solution under vacuum conditions according to a mass ratio of 3-3.2:0.8-1.2:0.5-0.7:0.4-0.8, then adding a solvent, and adding a crosslinking agent with a total mass of 10%-20% of the Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase and o-phenylenediamine, stirring at 500-1000 rpm for 1-2 h, pouring into a microneedle patch mold and drying to obtain a Fe-CDs / MnO2 / PVA microneedle patch.

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

[0010] The dried pomelo peel powder is added to the acetone solution and mixed uniformly, then FeCl3·6H2O is added, and the reaction is continued at 100-120℃ for 5-8h, then the supernatant is obtained by centrifugation, and the Fe-CDs are obtained by drying after filtration, wherein the mass ratio of the dried pomelo peel powder, acetone solution and FeCl3·6H2O is in the range of 4-4.2:50-100:0.8-1.2.

[0011] Preferably, the preparation method of the dried pomelo peel powder comprises the following steps: washing the pomelo peel, cutting the pomelo peel into pieces, drying the pomelo peel at 50 DEG C for 12 hours, and finally crushing the pomelo peel into powder.

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

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

[0014] Further, the preparation method of the MnO2 nanosheet comprises the following steps:

[0015] The MnCl2.4H2O is dissolved in water, then tetramethylammonium hydroxide pentahydrate and 30% hydrogen peroxide are added, and the mixture is stirred for 12 hours; the product is centrifuged and washed to obtain the MnO2 nanosheet; and the mass ratio of the MnCl2.4H2O, the tetramethylammonium hydroxide pentahydrate and the 30% hydrogen peroxide ranges from 0.5 to 0.8: 2.1 to 2.4: 1.8 to 2.2.

[0016] Further, the types of the cross-linking agent include one or more of glutaraldehyde, glycerol, diphenyl methane diisocyanate and disuccinimidyl succinate, and the types of the solvent include one or more of chloroform, carbon disulfide, carbon tetrachloride and toluene.

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

[0018] The application further provides a method for detecting pesticide residues in fruits and vegetables, which comprises using the ratio fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables.

[0019] The Fe-CDs / MnO2 / PVA microneedle patch is used to prick the surface of the fruits and vegetables to be detected, and the juice of the fruits and vegetables is absorbed; after 5 minutes, the reaction chamber provided with a 365 nm ultraviolet lamp is irradiated, a deep learning model is learned, and a photo is collected by using a smart phone; and an App with an RGB color recognition function is used for analysis.

[0020] The principle of the application is that acetylcholinesterase (AChE) catalyzes the conversion of thioacetylcholine (ATCh) into reduced thiocholine (TCh), and further reduces MnO2 nanosheets into Mn 2+ Due to the Mn 2+Without oxidase-like activity, ortho-phenylenediamine (OPD) cannot be oxidized. In the presence of organophosphorus, the fluorescence signal of the system is weakened by dynamic quenching of the fluorescence of Fe-CDs. In addition, the presence of organophosphorus reduces the activity of AChE, inhibiting the production of TCh, thereby reducing the decomposition of MnO2 nanosheets. The residual MnO2 nanosheets further catalyze the oxidation of colorless ortho-phenylenediamine (OPD) to generate colored oxOPD using oxidase-like activity, increasing the fluorescence signal of the system. Therefore, the fluorescence intensity ratio of Fe-CDs and oxOPD is measured to quantitatively detect organophosphorus.

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

[0022] 1. Rapid detection: The microneedle patch prepared by the present application can be directly inserted into the surface of fruits and vegetables to quickly contact and adsorb pesticide residues. The detection process is simple and fast, and the detection result can be obtained in a short time (such as a few minutes to tens of minutes), meeting the demand of on-site rapid detection.

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

[0024] 3. Simple operation. No complex sample pretreatment and professional equipment operation are required. Consumers or detection personnel only need to press the microneedle patch and use a portable fluorescence detector to perform detection, which is easy to popularize and apply.

[0025] 4. Low cost. The raw materials for preparing ratio fluorescent carbon dots are cheap and easy to obtain, and the preparation process of microneedle patches is relatively simple, without the need for expensive large-scale instruments and equipment, which reduces the detection cost and is conducive to large-scale production and application.

[0026] 5. In-situ detection and non-destructive detection. The microneedle patch only penetrates the surface of fruits and vegetables to a very shallow depth during detection, causing minimal damage to the whole fruit or vegetable, allowing in-situ detection without affecting the subsequent sale and consumption of fruits and vegetables. At the same time, it also avoids the errors caused by sampling in the traditional sampling detection method. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The surface morphology of Fe-CDs was characterized. (a) Transmission electron micrograph of Fe-CDs; (b) X-ray diffraction pattern of Fe-CDs;

[0028] Figure 2Characterization of the performance of Fe-CDs. (a) UV-Vis 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) X-ray electron energy spectrum full spectrum of Fe-CDs; (b) X-ray electron energy spectrum C1s spectrum of Fe-CDs; (c) X-ray electron energy spectrum N1s spectrum of Fe-CDs; (d) X-ray electron energy spectrum O1s spectrum of Fe-CDs;

[0030] Figure 4 Stereo microscope image of Fe-CDs / MnO2 / PVA microneedle;

[0031] Figure 5 Ratio fluorescence detection of Fe-CDs / MnO2 / PVA microneedle;

[0032] Figure 6 Fitting standard curve. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be described in further detail below in conjunction with the accompanying drawings, but the protection scope of the present application is not limited to the following description.

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

[0035] (1) Preparation of Fe-CDs

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

[0037] (2) Preparation of MnO2 nanosheets

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

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

[0040] After mixing Fe-CDs and MnO2 nanosheets with a mass ratio of 300:1 in 2 mL ultrapure water, a mixed solution was obtained by stirring at a speed of 800 r / min for 5 min. Finally, Fe-CDs / MnO2 nanosheet powder was obtained by freeze-drying.

[0041] (4) Preparation of microneedle mold

[0042] Using 3D printing technology, materials such as polydimethylsiloxane (PDMS) were used to design the shape (such as conical) and size (needle length 500 μm, needle base diameter 200 μm, needle spacing 400 μm) of the microneedle, and the microneedle mold was printed.

[0043] (5) Preparation of microneedle patch

[0044] A PVA solution with a mass percentage concentration of 15% was prepared, and a mixed solution of Fe-CDs / MnO2, acetylcholine, acetylcholinesterase, and o-phenylenediamine with a mass ratio of 3:1:0.6:0.5 was directly added under vacuum conditions. Crosslinking agent (glutaraldehyde, amount of 15% of the mass of the polymer) and solvent (chloroform) were added, and the materials were uniformly mixed under stirring conditions (such as 500-1000 rpm stirring for 2 h). The mixed materials were filled into the microneedle mold, and the microneedle mold was placed in an oven at 60°C for 4 h to solidify, and then naturally dried to obtain the Fe-CDs / MnO2 / PVA microneedle patch.

[0045] Experimental Example 1 Characterization of Fe-CDs carbon dots

[0046] The morphology, lattice spacing, and other microstructures of Fe-CDs were characterized by scanning transmission electron microscopy and X-ray diffractometry. As shown in Figure 1 (a), the transmission electron microscopy image of Fe-CDs shows that Fe-CDs are uniformly dispersed near-spherical particles and do not significantly aggregate in water. To further study the structural information of Fe-CDs, high-resolution transmission electron microscopy was used to observe the lattice spacing of a single Fe-CD. Figure 1(a) shows that the lattice structure of Fe-CDs is clear, and the lattice spacing is 0.21 nm. In addition, Fe-CDs are further characterized by X-ray diffractometer, and Figure 1 (b) shows that a typical crystal diffraction peak can be observed, and the diffraction angle 2θ = 25.6° is the characteristic diffraction peak of carbon, corresponding to the (002) crystal plane of graphite, which represents the stacking mode of aromatic layers, indicating that the synthesized Fe-CDs have the lattice structure of graphite and the degree of graphitization is high. The above results show that Fe-CDs are successfully prepared and can be used in subsequent experiments.

[0047] The formation of Fe-CDs is further proved by UV-vis spectrum and fluorescence properties. As shown in Figure 2 (a), the UV-vis spectrum of Fe-CDs shows two shoulder peaks at 210 nm and 285 nm, which are respectively attributed to the π-π* transition of aromatic sp 2 2 domain and n-π* transition of C=N / C=O bond, which are the characteristic absorption peaks of Fe-CDs. In the inset of Figure 2 (a), the Fe-CDs solution is white under sunlight, and blue fluorescent under 365 nm UV lamp, showing the unique fluorescence properties of Fe-CDs. In addition, the fluorescence quenching ability of Fe-CDs is also investigated. As shown in Figure 2 (b), the synthesized Fe-CDs show UV absorption in the range of 350-560 nm, which is largely overlapped with its fluorescence excitation peak (Ex: 430 nm) and emission peak (Em: 510 nm). The PL properties of Fe-CDs are characterized in the excitation wavelength range of 340-480 nm. As shown in Figure 2 (c), the fluorescence emission peak of Fe-CDs shows red shift with the increase of excitation wavelength. At the excitation wavelength of 510 nm, the fluorescence emission wavelength of Fe-CDs reaches the maximum value of 430 nm, showing a more pure blue fluorescence, which is consistent with the results in the inset of Figure 2 (a). At the same time, the PL properties of Fe-CDs are characterized in the emission wavelength range of 460-550 nm. As shown in Figure 2 (d), the fluorescence excitation peak of Fe-CDs shows blue shift with the decrease of emission wavelength. At the emission wavelength of 430 nm, the fluorescence excitation wavelength of Fe-CDs reaches the maximum value of 510 nm, which is consistent with the results of Figure 2 (b).

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

[0049] The surface element analysis of Fe-CDs is further carried out by X-ray electron spectrometer, as shown in Figure 3(a) The results show that 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%). Figure 3 (b)-(d) are the high-resolution X-ray electron spectrograms corresponding to C1s, N1s and O1s, respectively, and the fitting peaks appear at 285.6, 399.1 and 533.9 eV, respectively, corresponding to C—C / C=C, C—N, C=O and C—O bonds.

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

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

[0052] Experimental Example 4 Fe-CDs / MnO2 / PVA microneedles for detection of pesticide residues in actual fruit and vegetable samples and grading standards

[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. Specifically, Figure 5 reflects the change in fluorescence color of Fe-CDs / MnO2 / PVA microneedles in the presence or absence of organophosphorus. By pressing the microneedle patch to the surface and inside of the fruit and vegetable, the fruit and vegetable juice was absorbed, and after 5 min, the picture was taken under the irradiation of 365 nm ultraviolet lamp and analyzed by App with RGB color recognition function. (This experimental example uses a mobile phone App color recognition colorimeter, Xi'an Banli Ge Ban Software Co., Ltd.). Then the pesticide residue content was calculated according to the fitting standard curve ( Figure 6 ), and HPLC was used to detect the test sample to obtain the reference value of pesticide residues 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 the spiked experiment, and the recovery rate was from 96.2% to 110.8%, which showed that it had high reliability and stability in practical application. In addition, according to the current national pesticide maximum residue standards GB2763.1-2022 "Food safety national standard maximum residue limits of 112 kinds of pesticides in food such as 2,4-D sodium butyrate" and GB2763-2021 "Food safety national standard maximum residue limits of pesticides in food", Fe-CDs / MnO2 / PVA microneedles are based on the maximum residue limit to formulate three rating standards of unqualified, qualified and excellent, which can provide fruit and vegetable pesticide residue information for consumers, market supervision departments, catering enterprises and others in real time, and ensure food quality and safety and human health.

[0058] The above only describes the preferred embodiments of the present application, and it should be understood that the present application is not limited to the forms disclosed herein, and should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concepts described herein by the above teachings or related technical or knowledge. Any modification and change made by those skilled in the art without departing from the spirit and scope of the present application shall be within the protection scope of the claims of the present application.

Claims

1. A ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables, characterized in that, The preparation method includes the following steps: Fe-CDs and MnO2 nanosheets with a mass ratio of 100-300:1 are mixed in water and stirred at 800-1000 r / min for 5-10 min. After drying, Fe-CDs / MnO2 nanosheet hybrid material is obtained. The Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase, and o-phenylenediamine are added to a 10%-15% PVA solution under vacuum conditions at a mass ratio of 3-3.2:0.8-1.2:0.5-0.7:0.4-0.

8. The solvent and a crosslinking agent of 10%-20% of the total mass of the Fe-CDs / MnO2 nanosheet hybrid material, acetylcholine, acetylcholinesterase, and o-phenylenediamine are added. The mixture is stirred at 500-1000 rpm for 1-2 h and then poured into a microneedle patch mold and dried to obtain 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 includes the following steps: The dried grapefruit peel powder was added to an acetone solution and mixed evenly. Then FeCl3·6H2O was added, and the reaction was continued at 100-120℃ for 5-8 hours. After centrifugation, the supernatant was obtained, filtered, and dried to obtain the Fe-CDs. The mass ratio of the dried grapefruit peel powder, acetone solution, and FeCl3·6H2O ranged from 4 to 4.2: 50 to 100: 0.8 to 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 preparation method of the MnO2 nanosheets includes the following steps: Manganese chloride tetrahydrate was dissolved in water, and then tetramethylammonium hydroxide pentahydrate and 30% hydrogen peroxide were added. The mixture was stirred for 12 hours, and the product was centrifuged and washed to obtain the MnO2 nanosheets. The mass ratio of manganese chloride tetrahydrate, tetramethylammonium hydroxide pentahydrate and 30% hydrogen peroxide ranged from 0.5 to 0.8: 2.1 to 2.4: 1.8 to 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 crosslinking agent includes one or more of glutaraldehyde, glycerol, diphenylmethane diisocyanate, and disuccinimide succinic acid, and the solvent includes 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 includes 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 includes using a ratiometric fluorescent carbon dot microneedle patch for detecting pesticide residues in fruits and vegetables as described in any one of claims 1-5, and further includes the following steps: The Fe-CDs / MnO2 / PVA microneedle patch was inserted into the surface of the fruits and vegetables to be tested, and the juice of the fruits and vegetables was absorbed. After 5 minutes, the vegetables were irradiated with a 365nm ultraviolet lamp. The deep learning model was used to learn the data and the smartphone was used to collect photos. The data was then analyzed using an app with RGB color recognition function.

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