Functionalized melamine sensing sponge, preparation method thereof and thiram colorimetric-fluorescence visual dual-mode detection method

Abstract

The invention provides a functionalized melamine sensing sponge, a preparation method thereof and a thiram colorimetric-fluorescence visual dual-mode detection method, and belongs to the technical field of food safety detection. According to the invention, thiram is specifically combined with PDA-coated CuO2 in the sensing sponge, so that the nano-enzyme catalytic activity of the sensing sponge is excited, colorless TMB is catalyzed to be oxidized into blue oxTMB, and a characteristic absorption peak exists at 650 nm; meanwhile, the generated oxTMB can quench red fluorescence of RhB at HOF at 580 nm through an inner filtering effect; the color and fluorescence intensity change have a linear relationship with the concentration of the thiram, and the method can be used for quantitative detection of the thiram. Along with the increase of the concentration of thiram, the color of the sensing sponge is changed from pink to blue-green due to the generation of oxTMB under sunlight, and the red fluorescence is gradually weakened due to the fluorescence quenching effect under ultraviolet light. Therefore, dual-mode visual detection of colorimetric and fluorescence signals can be realized.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a functionalized melamine-sensing sponge, its preparation method, and a dual-mode detection method for thiram (a type of melamine) using colorimetric and fluorescence visualization. Background Technology

[0002] Tetramethylthiuram disulfide (Thiram), chemically known as tetramethylthiuram disulfide, is a fungicide with advantages such as simple structure, broad antibacterial spectrum, strong antibacterial activity, and low price. It is widely used in modern agriculture to protect crops, vegetables, and fruits from pests. However, due to improper use, thiram residues are easily accumulated and enter the human body through the food chain, potentially causing reproductive and neurotoxic effects. Currently, many countries have strictly regulated the residue limits of thiram in food. Therefore, establishing accurate and sensitive methods for detecting thiram residues is of great significance for ensuring food quality and safety and promoting the healthy development of agriculture.

[0003] In recent years, colorimetric and fluorescence-based sensing technologies have attracted widespread attention due to their rapid response and ease of operation. However, existing methods often rely on expensive and sophisticated laboratory equipment, such as ultraviolet spectrophotometers or fluorescence spectrophotometers, which are insufficient to meet the practical needs of rapid on-site screening. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a functionalized melamine-sensing sponge, its preparation method, and a dual-mode detection method for thiram residue using colorimetric-fluorescence visualization. The functionalized melamine-sensing sponge provided by this invention possesses excellent selectivity and anti-interference capabilities, enabling dual-mode visualization detection and quantitative analysis of thiram residue under both sunlight and ultraviolet light, with high sensitivity and accuracy.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a functionalized melamine sensing sponge, comprising a melamine sponge, PDA@CuO2 loaded in the melamine sponge, and a fluorescent reference signal material; The PDA@CuO2 comprises a PDA carrier and CuO2 nanoparticles distributed in the PDA carrier; The fluorescent reference signal material is RhB@HOF, which includes a hydrogen-bonded organic framework and Rhodamine B encapsulated in the hydrogen-bonded organic framework. The hydrogen-bonded organic framework is formed from melamine and cyanuric acid.

[0006] Preferably, the preparation method of the PDA@CuO2 includes the following steps: Dopamine hydrochloride solution is added to an alkaline solution to carry out a polymerization reaction, thus obtaining PDA; After mixing PDA dispersion, copper chloride solution and alkaline buffer, hydrogen peroxide solution was added to carry out the oxidation reaction to obtain PDA@CuO2.

[0007] Preferably, the preparation method of the RhB@HOF includes the following steps: Melamine, rhodamine B, cyanuric acid, and an organic solvent were mixed and encapsulated to obtain RhB@HOF.

[0008] Preferably, the PDA@CuO2 content in the functionalized melamine sensing sponge is 0.15~0.3 mg / cm³. 3 The mass content of the fluorescent reference signal material is 8~10 mg / cm³. 3 .

[0009] This invention provides a method for preparing the above-mentioned functionalized melamine sensing sponge, comprising the following steps: PDA@CuO2, RhB@HOF and water were mixed to obtain a dispersion; The dispersion was added to a melamine sponge and then filtered to obtain a functionalized melamine sensing sponge.

[0010] This invention provides the application of the above-mentioned functionalized melamine sensing sponge in the detection of thiram.

[0011] This invention provides a thiram-based detection kit, comprising a functionalized melamine-sensing sponge, a TMB solution, and an acidic buffer solution.

[0012] Preferably, the concentration of the TMB solution is 4-6 mM; and the pH value of the acidic buffer solution is 4.

[0013] This invention provides a colorimetric-fluorescence visualization dual-mode detection method for thiram, comprising the following steps: The test solution is mixed with an acidic buffer solution to obtain a pretreatment solution; The pretreatment solution was added to the functionalized melamine sensing sponge to carry out a coordination reaction, and the functionalized melamine sensing sponge after coordination reaction was obtained. TMB solution was added to the functionalized melamine sensing sponge after the coordination reaction was completed, and an oxidation reaction was carried out to obtain the melamine sensing sponge to be tested. The melamine-sensing sponge to be tested was exposed to sunlight and photographed. The RGB values ​​of the color information in the photograph were extracted, and the (G+B) / R value was calculated. The concentration of thiram in the test solution was obtained according to the pre-established first standard curve. The first standard curve is the linear relationship between the concentration of thiram and the (G+B) / R value under sunlight irradiation. The melamine-sensing sponge to be tested was irradiated with ultraviolet light and a fluorescence photograph was taken. The R value in the RGB values ​​of the photograph color information was extracted, and the concentration of thiram in the test solution was obtained according to the pre-established second standard curve. The second standard curve is the linear relationship between the concentration of thiram and the R value under ultraviolet irradiation conditions.

[0014] Preferably, the volume ratio of the test solution to the acidic buffer solution is 1:9~11.

[0015] The coordination reaction takes 8-12 minutes; The oxidation reaction takes 3-5 minutes. The wavelength of the ultraviolet irradiation is 350~370nm.

[0016] This invention provides a functionalized melamine sensing sponge, comprising a melamine sponge, a PDA@CuO2 nanozyme and an RhB@HOF fluorescent reference signal material loaded in the melamine sponge. In this invention, the RhB@HOF is pink under sunlight and emits red fluorescence at 580 nm upon excitation at a wavelength of 365 nm. In the presence of thiram, thiram reacts with PDA@CuO2 in the sensing sponge, exciting the latter to catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxTMB, which has a characteristic absorption peak at 650 nm. Simultaneously, the generated oxTMB quenches the red fluorescence of RhB@HOF at 580 nm through an internal filtration effect. Both color and fluorescence intensity changes are linearly related to the thiram concentration, thus enabling quantitative detection. With increasing thiram concentration, the color of the sensing sponge under sunlight changes from pink (the original color of RhB@HOF) to blue-green (a composite color of oxTMB and the residual original color), and the red fluorescence gradually weakens under ultraviolet light, thereby achieving dual-mode visual detection of thiram using both colorimetric and fluorescence methods. The sensing sponge of this invention possesses excellent selectivity and anti-interference capabilities, enabling highly sensitive and accurate visual quantitative analysis of thiram residues. The results of the examples show that the detection limit of the functionalized melamine sensing sponge of the present invention is 0.0087 µg / mL under sunlight and 0.0065 µg / mL under ultraviolet light. Moreover, the color and fluorescence changes can be identified by the naked eye without the assistance of precision instruments. This indicates that the functionalized melamine sensing sponge constructed in this invention has broad application prospects in rapid visual analysis and detection in Thiram. Attached Figure Description

[0017] Figure 1 Here is a scanning electron microscope image of PDA@CuO2; Figure 2 Here is a scanning electron microscope image of RhB@HOF; Figure 3 This is a color comparison image of the functionalized melamine sensing sponge in Example 1 under sunlight and ultraviolet light; Figure 4 Photographs and corresponding standard curves showing the color changes of functionalized melamine-sensing sponge under sunlight and fluorescence as the concentration of Thiram increases; Figure 5 Photographs and corresponding standard curves of the color change of melamine sensing sponge loaded with DT-Cu and RhB@HOF under sunlight and fluorescence as the Thiram concentration increases; Figure 6 Photographs showing the color changes of a melamine-sensing sponge loaded with copper chloride dihydrate and RhB@HOF under sunlight and fluorescence as the Thiram concentration increases. Detailed Implementation

[0018] This invention provides a functionalized melamine sensing sponge, comprising a melamine sponge, and a PDA@CuO2 nanozyme and a fluorescent reference signal material loaded in the melamine sponge; The PDA@CuO2 comprises a polydopamine (PDA) carrier and CuO2 nanoparticles distributed in the PDA carrier; The fluorescent reference signal material is RhB@HOF, which comprises a hydrogen-bonded organic framework (HOF) formed by the self-assembly of melamine and cyanuric acid through hydrogen bonds and rhodamine B (RhB) encapsulated in the hydrogen-bonded organic framework.

[0019] In this invention, the melamine sponge can be a commercially available product, with a porosity preferably of 45-55%, more preferably 45-50%. This invention does not impose any special limitations on the size of the melamine sponge; it can be designed according to actual testing requirements. As a specific embodiment of this invention, the melamine sponge has a size of 1 cm × 1 cm and a thickness of 5 mm.

[0020] In this invention, the method for preparing PDA@CuO2 preferably includes the following steps: Dopamine hydrochloride solution is added to an alkaline solution to carry out a polymerization reaction, thus obtaining PDA; After mixing PDA dispersion, copper chloride solution and alkaline buffer, hydrogen peroxide solution was added to carry out the oxidation reaction to obtain PDA@CuO2.

[0021] This invention involves adding a dopamine hydrochloride solution to an alkaline solution to carry out a polymerization reaction to obtain PDA. In this invention, the pH value of the alkaline solution is preferably 8-10. The alkaline solution is preferably a mixture of ammonia, ethanol, and water, with a volume ratio preferably of 0.4:12:27-30. The concentration of the dopamine hydrochloride solution is preferably 0.10-0.20 g / mL, more preferably 0.15 g / mL. The volume ratio of the dopamine hydrochloride solution to the alkaline solution is preferably 1:38-42, more preferably 1:40. In this invention, the polymerization reaction is preferably carried out under stirring conditions, the polymerization temperature is preferably room temperature, and the reaction time is preferably 24 hours.

[0022] This invention involves mixing a PDA dispersion, a copper chloride solution, and an alkaline buffer solution, then adding hydrogen peroxide solution to induce an oxidation reaction, yielding PDA@CuO2. In this invention, the concentration of the PDA dispersion is preferably 0.2–0.4 g / mL, more preferably 0.3 g / mL. The concentration of the copper chloride solution is preferably 0.1–0.2 M; the alkaline buffer solution is preferably a sodium bicarbonate-carbonate buffer solution with a concentration of 0.1 M and a pH of 9.1. In this invention, the volume ratio of the PDA dispersion, copper chloride solution, and alkaline buffer solution is preferably 1:5:4–6. In this invention, the mixing method is preferably stirring, and the mixing time is preferably 4 hours.

[0023] In this invention, the mass concentration of the hydrogen peroxide solution is preferably 25-30%, more preferably 28-30%; the volume ratio of the PDA dispersion to the hydrogen peroxide solution is preferably 8-10:1. In this invention, the oxidation reaction is preferably carried out under stirring conditions, the oxidation reaction temperature is preferably room temperature, and the reaction time is preferably 30 min. In this invention, the synthesis of PDA@CuO2 is based on the self-polymerization of dopamine hydrochloride under alkaline conditions to generate PDA; then Cu... 2+ The PDA chelates with PDA and is oxidized by H2O2 to form PDA@CuO2. After the reaction is complete, the reaction solution is centrifuged, washed, and dried to obtain PDA@CuO2.

[0024] In this invention, the preparation method of RhB@HOF preferably includes the following steps: Melamine, rhodamine B, cyanuric acid, and an organic solvent were mixed and encapsulated to obtain RhB@HOF.

[0025] In this invention, the mass ratio of melamine to cyanuric acid is preferably 1:0.9~1.1, more preferably 1:1; the molar ratio of melamine to rhodamine B is preferably 100 mg:0.13~0.26 mmol, more preferably 100 mg:0.18~0.22 mmol. In this invention, the organic solvent is preferably one or more of dimethyl sulfoxide, N,N-dimethylacetamide, and ethanol. This invention does not have special requirements on the amount of the organic solvent used, as long as it is sufficient to dissolve the above components.

[0026] In this invention, the encapsulation reaction is preferably carried out under stirring conditions, the temperature of the encapsulation reaction is preferably room temperature, and the time is preferably 16-20 h, more preferably 18 h. After the encapsulation reaction, the resulting encapsulation reaction solution is preferably centrifuged, washed with alcohol, and dried to obtain RhB@HOF.

[0027] In this invention, the PDA@CuO2 content in the functionalized melamine sensing sponge is preferably 0.15~0.3 mg / cm³. 3 More preferably, it is 0.2~0.25 mg / cm³. 3 The preferred mass content of the fluorescent reference signal material is 8~10 mg / cm³. 3 More preferably 9 mg / cm 3 .

[0028] This invention provides a method for preparing the above-mentioned functionalized melamine sensing sponge, comprising the following steps: PDA@CuO2, RhB@HOF and water were mixed to obtain a dispersion; The dispersion was added to a melamine sponge and filtered to obtain a functionalized melamine sensing sponge.

[0029] This invention involves mixing PDA@CuO2, RhB@HOF, and water to obtain a dispersion. Preferably, an aqueous solution of PDA@CuO2 and an aqueous solution of RhB@HOF are prepared first, and then the two are mixed. In this invention, the concentration of the PDA@CuO2 aqueous solution is preferably 0.3~0.6 mg / mL, more preferably 0.4~0.5 mg / mL; the concentration of the RhB@HOF aqueous solution is preferably 16~20 mg / mL, more preferably 16~18 mg / mL. The volume ratio of the PDA@CuO2 aqueous solution to the RhB@HOF aqueous solution is preferably 1:0.9~1.1, more preferably 1:1.

[0030] The present invention preferably pre-treats the melamine sponge, the pre-treatment preferably including sequential alcohol washing, water washing and drying; the alcohol washing is preferably ethanol washing, preferably once, the water washing is preferably ultrapure water washing, preferably twice; the drying is preferably vacuum drying, the drying temperature is preferably 60°C.

[0031] In this invention, the filtration is preferably performed by allowing the solution to be filtered by gravity for 30 seconds. After filtration, the resulting functionalized melamine sensing sponge is preferably cut and dried. The drying is preferably performed under vacuum, and the drying temperature is preferably 60°C.

[0032] This invention provides the application of the above-mentioned functionalized melamine sensing sponge in the detection of thiram.

[0033] This invention provides a thiram-based detection kit, comprising a functionalized melamine-sensing sponge, a TMB solution, and an acidic buffer solution.

[0034] In this invention, the concentration of the TMB solution is preferably 4-6 mM, more preferably 5 mM; the acidic buffer solution is preferably an acetate-sodium acetate buffer solution; in this invention, the concentration of the buffer solution is preferably 0.1M, and the pH value is preferably 4.

[0035] This invention provides a colorimetric fluorescence visualization dual-mode detection method for thiamethoxam, comprising the following steps: The test solution is mixed with an acidic buffer solution to obtain a pretreatment solution; The pretreatment solution was added to the functionalized melamine sensing sponge to carry out a coordination reaction, and the functionalized melamine sensing sponge after coordination reaction was obtained. TMB solution was added to the sensing sponge that had completed the coordination reaction to carry out an oxidation reaction, and the melamine sensing sponge to be tested was obtained. The melamine-sensing sponge to be tested is exposed to sunlight and photographed. The RGB values ​​of the color information in the photograph are extracted, and the (G+B) / R value is calculated. The concentration of thiram in the test solution is obtained according to the pre-established first standard curve. The first standard curve is the linear relationship between the concentration of thiram and the (G+B) / R value under sunlight irradiation. The sensor sponge to be tested is irradiated with ultraviolet light and a fluorescence photograph is taken. The R value in the RGB value of the photograph color information is extracted, and the concentration of thiram in the test solution is obtained according to the pre-established second standard curve. The second standard curve is the linear relationship between the concentration of thiram and the R value under ultraviolet irradiation conditions.

[0036] This invention involves mixing the test solution with an acidic buffer solution to obtain a pretreatment solution. In this invention, when the analyte is a solid, the preparation method of the test solution preferably includes the following steps: Add 10 mL of 10% acetonitrile solution (pH=4) to 1 g of sample and soak for 10 min. Filter using a 0.22 µm filter membrane and collect the supernatant. Add an appropriate amount of polyvinylpyrrolidone to the supernatant, filter again through a 0.22 µm filter membrane, and dilute the resulting extract 10 times with 10% acetonitrile solution (pH=4) to obtain the test solution.

[0037] In this invention, the volume ratio of the test solution to the acidic buffer solution is preferably 1:9 to 11, more preferably 1:10. The pH value of the pretreatment solution is preferably 4.

[0038] In this invention, the pretreatment solution is added to a functionalized melamine sensing sponge to perform a coordination reaction, resulting in a functionalized melamine sensing sponge after the coordination reaction. In this invention, the preferred volume of the pretreatment solution is 490-510 μL, more preferably 500 μL. In this invention, the preferred temperature for the coordination reaction is room temperature, and the preferred time is 8-12 min, more preferably 10 min.

[0039] In this invention, a TMB solution is added to the functionalized melamine sensing sponge after the coordination reaction to carry out an oxidation reaction, thereby obtaining the melamine sensing sponge to be tested. In this invention, the volume of the added TMB solution is preferably 40-60 μL, more preferably 50 μL. In this invention, the oxidation reaction is preferably carried out at room temperature for 3-5 minutes, more preferably 4 minutes.

[0040] This invention involves exposing a melamine-sensing sponge to be tested to sunlight and taking photographs. The RGB values ​​of the colors in the photographs are extracted, and the (G+B) / R value is calculated. The concentration of thiram in the test solution is then obtained based on a pre-established first standard curve. The first standard curve represents the linear relationship between the thiram concentration and the (G+B) / R value under sunlight exposure. Preferably, the photographs are taken using a mobile phone's built-in camera, and the process for converting the color information in the sunlight-exposed photographs into RGB data is preferably color recognition. In this invention, the method for obtaining the first standard curve preferably includes the following steps: A series of thiram standard solutions with gradient concentrations were provided as test solutions. The melamine sensing sponge was used to conduct the tests and take sunlight photos according to the above method to obtain the (G+B) / R values ​​corresponding to each concentration of thiram standard solution. A standard curve was plotted with the thiram concentration as the x-axis and the (G+B) / R value as the y-axis.

[0041] In this invention, the solvent for the gradient concentration thiram standard solution is preferably ethanol with a mass fraction of 10%, and the gradient concentrations are 0 µg / mL, 0.01 µg / mL, 0.05 µg / mL, 0.1 µg / mL, 0.5 µg / mL, 1 µg / mL, 2 µg / mL, 3 µg / mL, 4 µg / mL, 5 µg / mL, 7 µg / mL, and 10 µg / mL.

[0042] In one specific embodiment of the present invention, the linear regression equation of the first standard curve is y = 0.190x + 1.791 (R² + π / 2). 2 =0.993), the detection limit is 0.0087 µg / mL, and the detection range is 0.01~7 µg / mL.

[0043] This invention involves irradiating a melamine-sensing sponge under test with ultraviolet light and capturing a fluorescence photograph. The R value is extracted from the RGB values ​​of the photograph, and the concentration of thiram in the test solution is obtained based on a pre-established second standard curve. The second standard curve represents the linear relationship between the thiram concentration and the R value under ultraviolet irradiation. In this invention, the wavelength of the ultraviolet irradiation is preferably 350-370 nm, more preferably 365 nm. The method for obtaining the second standard curve preferably includes the following steps: A series of thiram standard solutions with gradient concentrations were provided as test solutions. The tests were conducted using a sensing sponge according to the method described above, and fluorescence images were captured. The R values ​​corresponding to each concentration of thiram standard solution were obtained. A standard curve was plotted with thiram concentration on the x-axis and R values ​​on the y-axis. The gradient concentration thiram standard solution system is as described above.

[0044] In one specific embodiment of the present invention, the linear regression equation of the second standard curve is y = -18.356x + 129.642 (R² + π / 2). 2 =0.993), the detection limit is 0.0065 µg / mL, and the detection range is 0.01~7 µg / mL.

[0045] In this invention, after the melamine sensing sponge is irradiated with sunlight to test the concentration of thiram in the test solution, it can be used to continue to irradiate with ultraviolet light to test the concentration of thiram in the test solution.

[0046] The following detailed description, in conjunction with embodiments, illustrates the functionalized melamine-sensing sponge provided by the present invention, its preparation method, and the dual-mode detection method of thiram-colorimetric-fluorescence visualization. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1 (1) Synthesis of PDA@CuO2 First, a PDA carrier was prepared by mixing 400 µL of ammonia, 12 mL of anhydrous ethanol, and 27 mL of ultrapure water, and stirring at room temperature for 30 min. Then, 1 mL of dopamine hydrochloride solution (0.15 g / mL) was added, and stirring continued for 24 h. After the reaction was complete, the reaction solution was centrifuged at 10000 rpm for 10 min to collect the precipitate, washed three times with ultrapure water, and redispersed in 40 mL of ultrapure water to obtain a PDA dispersion (concentration 0.04 g / mL) for later use. 40 mL of the above PDA dispersion was mixed with 200 mL of copper chloride dihydrate solution (0.1 M), and 160 mL of sodium bicarbonate-carbonate buffer solution (0.1 M, pH=9.1) was added, and stirring continued for 4 h. Then, 4 mL of hydrogen peroxide solution (30% by mass) was added, and the reaction continued for 30 min. The final product was centrifuged at 10000 rpm for 10 min, washed three times with ultrapure water, and vacuum dried at 60 °C to obtain the PDA@CuO2 composite material, which was stored at room temperature. The scanning electron microscope image of the obtained PDA@CuO2 is shown below. Figure 1 As shown, by Figure 1 It can be seen that PDA@CuO2 is a uniform sphere with a rough surface and visible burr-like structure at the edges, with an average diameter of about 950 nm.

[0048] (2) Synthesis of RhB@HOF fluorescence reference signal 100 mg of melamine and 0.26 mmol of rhodamine B were dissolved together in 6 mL of DMSO, and 100 mg of cyanuric acid was dissolved in 4 mL of DMSO. The two solutions were mixed and stirred at room temperature for 16–20 h. After the reaction was complete, the precipitate was collected by centrifugation at 8000 rpm for 10 min, washed three times with ethanol, and finally dried under vacuum at 60 °C to obtain the RhB@HOF fluorescent reference material. The scanning electron microscope image of RhB@HOF is shown below. Figure 2 As shown, by Figure 2 As can be seen, RhB@HOF has a regular spherical morphology with irregular sheet-like structures attached to its surface, and an average size of about 3.88 µm.

[0049] (3) Construction of functionalized melamine sensing sponge Prepare 10 mL of 0.4 mg / mL PDA@CuO2 aqueous solution and 10 mL of 16 mg / mL RhB@HOF aqueous solution, mix them well and set aside; take a 5 mm thick melamine sponge, cut it into 6 cm × 6 cm pieces, wash it once with ethanol and twice with ultrapure water, and dry it under vacuum at 60 °C for later use. Using a vacuum filtration device, the above mixed solution was uniformly loaded onto a pretreated melamine sponge; the sponge loaded with PDA@CuO2 and RhB@HOF was cut into 1 cm × 1 cm pieces and dried under vacuum at 60°C to obtain a functionalized melamine sensing sponge, wherein the mass content of PDA@CuO2 was 0.22 mg / cm³. 3 The mass content of the fluorescent reference signal material is 8.8 mg / cm³. 3 .

[0050] Figure 3 This is a color comparison image of melamine sponge before and after loading with PDA@CuO2 and RhB@HOF under sunlight and ultraviolet light. Figure 3 As shown, the loaded sensing sponge appears pink under sunlight and exhibits red fluorescence under ultraviolet light.

[0051] Example 2: Method for detecting Thiram using functionalized melamine-sensing sponge A series of Thiram standard solutions of different concentrations (0 µg / mL, 0.01 µg / mL, 0.05 µg / mL, 0.1 µg / mL, 0.5 µg / mL, 1 µg / mL, 2 µg / mL, 3 µg / mL, 4 µg / mL, 5 µg / mL, 7 µg / mL, 10 µg / mL) were prepared using a 10% ethanol solution. 500 µL of the above Thiram solutions were added to the functionalized melamine sensing sponge obtained in Example 1, and a coordination reaction was carried out at room temperature for 10 min. Then, 50 µL of a 5 mM TMB solution was added, and the reaction was continued at room temperature for another 4 min. After the reaction was complete, the samples were placed under sunlight and a 365 nm UV lamp, and photographs were taken using a smartphone (iPhone 15). Color information from photos was extracted using a WeChat mini-program (color recognition) and converted into RGB data. Standard curves were then established for Thiram concentration versus (G+B) / R value under sunlight and Thiram concentration versus R value under fluorescence, thereby enabling quantitative analysis of Thiram. Results are as follows: Figure 4 As shown.

[0052] Figure 4 In the image, A is a photograph of the sensor sponge under sunlight as the concentration of Thiram changes; B is the corresponding fluorescence photograph under a 365 nm UV lamp; C is the standard curve of Thiram concentration versus (G+B) / R value under sunlight; and D is the standard curve of Thiram concentration versus R value under fluorescence.

[0053] like Figure 4As shown in Figure A, with the increase of Thiram concentration, the color of the sensing sponge under sunlight gradually changes from pink to blue-green. This is mainly because Thiram coordinates with PDA@CuO2, activating its enzyme-like activity and catalyzing the oxidation of colorless TMB to blue oxTMB; while RhB@HOF is pink under sunlight, the superposition of the two causes the color to transition from pink to blue-green. Figure 4 The value of (G+B) / R in the figure indicates that the (G+B) / R value increases with increasing Thiram concentration, showing a good linear relationship with concentration in the range of 0.01~7 µg / mL. The linear regression equation is y=0.190x+1.791(R). 2 =0.993), and the detection limit is 0.0087 µg / mL.

[0054] like Figure 4 As shown in Figure B, the red fluorescence of the sensing sponge gradually quenches as the Thiram concentration increases. Figure 4 The D-value in the data shows that the corresponding R-value decreases with increasing concentration, and good linearity is also observed in the range of 0.01~7 µg / mL. The linear regression equation is y=-18.356x+129.642(R). 2 =0.993), and the detection limit is 0.0065 µg / mL. In summary, the color changes from pink to blue-green under sunlight, and the red fluorescence gradually weakens under fluorescence. These changes can be identified by the naked eye without the aid of sophisticated instruments. This indicates that the functionalized melamine-sensing sponge probe constructed in this invention can achieve sensitive visual analysis of thiram and has broad application prospects in rapid on-site detection.

[0055] Example 3 Using commercially available green tea leaves as the actual sample, the practical application potential of the constructed sensing sponge for the detection of thiram was evaluated using the standard addition method. The specific steps are as follows: Different concentrations of thiram standard (0, 0.05, 0.1, and 1 μg / g) were added to 1 g of tea sample, and the mixture was allowed to stand at room temperature for 2 h to ensure sufficient absorption of thiram. Subsequently, 10 mL of 10% acetonitrile solution (pH=4) was added to the sample, and the mixture was soaked for 10 min. The solution was filtered using a 0.22 µm filter membrane, and the supernatant was collected. An appropriate amount of cross-linked polyvinylpyrrolidone (PVP-P) was added to the supernatant, and the mixture was filtered again. The resulting extract was diluted 10-fold with 10% acetonitrile (pH=4) as the test solution.

[0056] The functionalized melamine sensing sponge constructed in Example 1 was analyzed according to the steps in Example 2. The determination results of Thiram in the tea samples are shown in Table 1.

[0057] Table 1. Results of Thiram determination in tea samples

[0058] The results showed that the recovery rate of thiram in tea samples was 95.4%–103.4%, with a relative standard deviation of 1.05%–2.47%, indicating good recovery and high precision. This demonstrates that the functionalized melamine sensing sponge and its colorimetric-fluorescence dual-mode detection method provided by this invention have good accuracy and reliability in the detection of thiram, and possess good application potential in practical sample analysis.

[0059] Comparative Example 1 To more clearly demonstrate the superiority of the PDA@CuO2 functionalized sensing sponge of this invention, the PDA@CuO2 nanozyme in the functionalized melamine sensing sponge of Example 1 was replaced with either diaminotriazole-modified copper nanozyme (DT-Cu) or copper chloride dihydrate (2H2O·CuCl2) nanozyme, while the nanozyme loading remained unchanged. Thiram was detected according to the method described in Example 2.

[0060] Photographs of the color changes of melamine sensing sponges loaded with DT-Cu and RhB@HOF under sunlight and fluorescence with increasing Thiram concentration, and the corresponding standard curves are shown below. Figure 5 As shown, Figure 5 In the image, A is a photograph of the sensing sponge under sunlight as the concentration of Thiram changes; B is the corresponding fluorescence photograph under a 365 nm UV lamp; C is the standard curve of Thiram concentration versus (G+B) / R value under sunlight; and D is the standard curve of Thiram concentration versus R value under fluorescence. Figure 5 As shown in A and B, under the same experimental conditions, the sponge modified with diaminotriazole-modified copper nanozyme (DT-Cu) exhibits color and fluorescence responses depending on the concentration of thiram. Specifically, the DT-Cu-modified sponge changes color from pink to pinkish-purple under sunlight, and the corresponding (G+B) / R value is linear in the range of 0.01~7 µg / mL, with the linear equation being y=0.03x+1.728(R). 2 =0.895), and the detection limit was 0.4783 µg / mL; under 365 nm UV light, the linear equation for the fluorescence R value was y = -11.46x + 128.625 (R = 0.895), and the detection limit was 0.4783 µg / mL; under 365 nm UV light, the linear equation for the fluorescence R value was y = -11.46x + 128.625 (R 2 =0.905), and the limit of detection is 0.3743 µg / mL (see [reference]). Figure 5 (C and D in the original text). Compared with the PDA@CuO2 system of this invention, its linear correlation coefficient is lower and the detection limit is several times higher, indicating that the DT-Cu nanozyme has limited sensing performance due to its weak enzyme activity, different mechanism of action with the target, and poor signal stability.

[0061] The image shows the color change of a melamine-sensing sponge loaded with copper chloride dihydrate and RhB@HOF under sunlight and fluorescence as the Thiram concentration increases. Figure 6 As shown. Figure 6 In the image, A shows a photograph of the sensing sponge under sunlight as the concentration of Thiram changes, and B shows the corresponding fluorescence photograph under a 365 nm UV lamp. It can be seen that while the sponge modified with copper chloride dihydrate also exhibits color and fluorescence changes, the large amount of active ingredient lost during the modification process due to the high water solubility of copper chloride dihydrate makes it difficult to stably immobilize in the sponge, resulting in weak signal response, poor reproducibility, and difficulty in achieving sensitive and reliable detection.

[0062] In summary, while the comparative materials could induce changes in visual signals, none could match the high sensitivity, excellent linear correlation, and stable and reliable detection performance of the PDA@CuO2 functionalized sensing sponge of this invention. This fully demonstrates the crucial role of the PDA@CuO2 composite structure in enhancing sensing performance, highlighting the innovation and technical advantages of this invention.

[0063] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A functionalized melamine sensing sponge, characterized in that, Includes melamine sponge, PDA@CuO2 loaded in the melamine sponge, and fluorescent reference signal material; The PDA@CuO2 comprises a PDA carrier and CuO2 nanoparticles distributed in the PDA carrier; The fluorescent reference signal material is RhB@HOF, which includes a hydrogen-bonded organic framework and Rhodamine B encapsulated in the hydrogen-bonded organic framework. The hydrogen-bonded organic framework is formed from melamine and cyanuric acid.

2. The functionalized melamine sensing sponge according to claim 1, characterized in that, The preparation method of the PDA@CuO2 includes the following steps: Dopamine hydrochloride solution is added to an alkaline solution to carry out a polymerization reaction, thus obtaining PDA; After mixing PDA dispersion, copper chloride solution and alkaline buffer, hydrogen peroxide solution was added to carry out the oxidation reaction to obtain PDA@CuO2.

3. The functionalized melamine sensing sponge according to claim 1, characterized in that, The preparation method of the RhB@HOF includes the following steps: Melamine, rhodamine B, cyanuric acid, and an organic solvent were mixed and encapsulated to obtain RhB@HOF.

4. The functionalized melamine sensing sponge according to claim 1, characterized in that, The functionalized melamine sensing sponge contains PDA@CuO2 at a mass content of 0.15~0.3 mg / cm³. 3 The mass content of the fluorescent reference signal material is 8~10 mg / cm³. 3 .

5. The method for preparing the functionalized melamine sensing sponge according to any one of claims 1 to 4, characterized in that, Includes the following steps: PDA@CuO2, RhB@HOF and water were mixed to obtain a dispersion; The dispersion was added to a melamine sponge and then filtered to obtain a functionalized melamine sensing sponge.

6. The application of the functionalized melamine sensing sponge according to any one of claims 1 to 4 or the functionalized melamine sensing sponge prepared by the preparation method according to claim 5 in the detection of thiram.

7. A thiamethoxam detection kit, characterized in that, Includes functionalized melamine sensing sponge, TMB solution, and acidic buffer solution; The functionalized melamine sensing sponge is the functionalized melamine sensing sponge according to any one of claims 1 to 4 or the functionalized melamine sensing sponge prepared by the preparation method according to claim 5.

8. The thiamethoxam detection kit according to claim 7, characterized in that, The concentration of the TMB solution is 4-6 mM; the pH value of the acidic buffer solution is 4.

9. A colorimetric-fluorescence visualization dual-mode detection method for thiram, characterized in that, Includes the following steps: The test solution is mixed with an acidic buffer solution to obtain a pretreatment solution; The pretreatment solution was added to the functionalized melamine sensing sponge to carry out a coordination reaction, and the functionalized melamine sensing sponge after the coordination reaction was obtained. TMB solution was added to the functionalized melamine sensing sponge after the coordination reaction was completed, and an oxidation reaction was carried out to obtain the melamine sensing sponge to be tested. The melamine-sensing sponge to be tested was exposed to sunlight and photographed. The RGB values ​​of the color information in the photograph were extracted, and the (G+B) / R value was calculated. The concentration of thiram in the test solution was obtained according to the pre-established first standard curve. The first standard curve is the linear relationship between the concentration of thiram and the (G+B) / R value under sunlight irradiation. The melamine-sensing sponge to be tested was irradiated with ultraviolet light and a fluorescence photograph was taken. The R value in the RGB values ​​of the photograph color information was extracted, and the concentration of thiram in the test solution was obtained according to the pre-established second standard curve. The second standard curve is the linear relationship between the concentration of thiram and the R value under ultraviolet irradiation conditions.

10. The detection method according to claim 9, characterized in that, The volume ratio of the test solution to the acidic buffer solution is 1:9~11. The coordination reaction takes 8-12 minutes; The oxidation reaction takes 3-5 minutes. The wavelength of the ultraviolet irradiation is 350~370nm.

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