Preparation method of nanoscale enzyme detection reagent for metronidazole residue in apple

Abstract

This invention relates to a method for preparing a nanozyme detection reagent for carbofuran residues in apples, belonging to the field of pesticide residue and food safety technology. Specific steps: (1) Prepare mesoporous polydopamine powder using polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer, dopamine hydrochloride, etc.; (2) Prepare a copper-manganese bimetallic carbon dot solution using citric acid, copper chloride, manganese chloride, and ethylenediamine; (3) Prepare the nanozyme detection reagent using the mesoporous polydopamine solution and the copper-manganese bimetallic carbon dot solution. Mesoporous polydopamine has a large specific surface area, providing numerous sites for carbon dot loading and effectively adsorbing carbofuran molecules. The copper-manganese bimetallic carbon dots give the nanozyme detection reagent good enzyme-like activity and generate pesticide concentration-dependent colorimetric and photothermal signals. This invention achieves mutual verification of dual-mode output signals, significantly improving detection reliability; and completes detection within 1.5 h.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for preparing a nanozyme detection reagent for detecting carbofuran residues in apples. Background Technology

[0002] In apple cultivation, carbofuran is widely used due to its high efficacy in controlling pests such as aphids and thrips, as well as its short residual period. However, with increasingly stringent food safety regulations, the detection of carbofuran residues in apples has become a key research focus. Because carbofuran readily degrades in the environment through hydrolysis into its more toxic metabolite, carbofuran, and its residual period may be prolonged under acidic conditions, accurate detection of carbofuran and its metabolites in apples is particularly crucial.

[0003] In the detection of carbofuran residues in apples, traditional large-scale instrumental methods, such as gas chromatography-mass spectrometry (GC-MS), while possessing high accuracy and reliability, are complex and costly, making them unsuitable for the rapid screening of large numbers of samples during apple production. Rapid assays based on acetylcholinesterase inhibition, although simple to operate, struggle to specifically identify individual carbofuran components when dealing with various interfering substances present in apples, leading to frequent misdiagnosis. Mesoporous materials, with their high specific surface area, tunable pore structure, and customizable surface chemistry, exhibit significant application value in various fields. However, research on mesoporous materials in pesticide residue detection is relatively limited.

[0004] Currently, several sensing technologies have been explored for the detection of carbofuran residues in apples. Colorimetry has attracted attention due to its intuitive operation and rapid response; however, the rich pigment background of apples can easily interfere with color interpretation, leading to inaccurate quantitative analysis. While photothermal sensing technology can achieve extremely high sensitivity, it is sensitive to changes in ambient temperature and exhibits poor stability in uncontrolled environments such as orchards. Given the risks of weak anti-interference capabilities and false positive / false negative results when dealing with the complex matrix of apples using a single detection mode, constructing a multimodal detection system has become an important direction for improving detection reliability. In particular, a dual-modal strategy combining colorimetry and photothermal detection can effectively compensate for the shortcomings of a single technology through the mutual corroboration of different signals. Summary of the Invention

[0005] To address the issue of low specificity in current dual-modal detection technologies for carbofuran residues in apples, this invention provides a method for preparing a nanozyme detection reagent for carbofuran in apples.

[0006] The preparation steps of a nanozyme detection reagent for carbofuran residues in apples are as follows:

[0007] (1) Preparation of mesoporous polydopamine powder

[0008] (1.1) Add 500 mg of polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer (F127) to 50 mL of 50% ethanol aqueous solution and mix evenly by ultrasonication to obtain polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer solution.

[0009] The ethanol-water solution was prepared by mixing ethanol and ultrapure water at a volume ratio of 1:1.

[0010] (1.2) Add 13 mL of 10 mg / L tris(hydroxymethyl)aminomethane (Tris) solution and 0.8 mL of 1,3,5-trimethylbenzene solution to the polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer solution and sonicate until homogeneous; stir on a magnetic stirrer to obtain a milky white solution;

[0011] (1.3) Add 0.75 g of dopamine hydrochloride to the milky white solution and stir magnetically for 24 h in the dark; centrifuge to obtain mesoporous polydopamine precipitate;

[0012] (1.4) Dissolve the mesoporous polydopamine precipitate in 20 mL of ultrapure water and sonicate to obtain an ultrasonic mesoporous polydopamine solution.

[0013] (1.5) In a freeze dryer, the ultrasonic mesoporous polydopamine solution is freeze-dried into flocculent powder to obtain mesoporous polydopamine powder, which is then sealed and stored.

[0014] The mesoporous polydopamine powder has a particle size of 90–120 nm;

[0015] (2) Preparation of copper-manganese bimetallic carbon dot solution

[0016] Dissolve 0.05 g citric acid, 0.07 g copper chloride, 0.05 g manganese chloride and 100 μL ethylenediamine (≥98%) in 5 mL of ultrapure water and react with sonication.

[0017] The reaction was carried out in a sealed reactor at 180.0℃ for 6.0 h.

[0018] The copper-manganese bimetallic carbon point solution was separated by centrifugation and stored at 4°C.

[0019] (3) Preparation of nanozyme detection reagent

[0020] (3.1) Dissolve mesoporous polydopamine powder in ultrapure water to prepare a mesoporous polydopamine solution with a concentration of 0.6 mg / mL;

[0021] (3.2) Mix 5 mL of 0.6 mg / mL mesoporous polydopamine solution and 1 mL of copper-manganese bimetallic carbon dot solution, and stir on a magnetic stirrer at 180 rpm for 24 h to obtain a mesoporous polydopamine solution loaded with copper-manganese bimetallic carbon dots.

[0022] (3.3) The mesoporous polydopamine solution loaded with copper-manganese bimetallic carbon dots was centrifuged to obtain a precipitate; the supernatant was removed by centrifuging at 10,000 rpm for 13 min; the supernatant was removed by eluting twice with ultrapure water at 10,000 rpm for 13 min to obtain the precipitate.

[0023] (3.4) Dissolve the precipitate in 12 mL of ultrapure water, sonicate to dissolve, and prepare nanozyme detection reagent; store at 4℃;

[0024] The nanozyme detection reagent has a colorimetric linear detection range of 0-2 μM and 0-1200 μM, and a photothermal detection range of 0-1200 μM, wherein the goodness of fit R0 is [value missing]. 2 The values ​​were 0.97532, 0.99409, and 0.99025, respectively, with a detection limit of 0.89 μM; the recoveries ranged from 82% to 120%.

[0025] Further technical solutions are as follows:

[0026] In steps (1.1), (1.2), (1.4), (2), and (3.4), the ultrasonic treatment conditions are: power 300W and time 5min.

[0027] In step (1.2), the magnetic stirring conditions are: 180 rpm for 30 min.

[0028] In step (1.3), the stirring conditions are: 180 rpm for 24 h.

[0029] In step (1.3), the supernatant was removed by centrifuging at 10,000 rpm for 20 min in a centrifuge; the supernatant was then removed by eluting three times with a 50% ethanol aqueous solution at 10,000 rpm for 10 min; and then eluted once with ultrapure water at 10,000 rpm for 10 min to obtain mesoporous polydopamine precipitate.

[0030] In step (1.5), the freeze-drying conditions are: temperature -50℃ and time 24 h.

[0031] In step (2), the centrifugation conditions are as follows: centrifuge at 10,000 rpm for 15 min to separate the first supernatant; repeat the centrifugation for 15 min to separate the second supernatant, which is the copper-manganese bimetallic carbon point solution.

[0032] In step (3.3), the centrifugation conditions are as follows: centrifuge at 10,000 rpm for 13 min, remove the supernatant; repeat the centrifugation for 13 min, wash twice with ultrapure water, remove the supernatant, and obtain the precipitate.

[0033] The beneficial technical effects of this invention are reflected in the following aspects:

[0034] 1. The inventiveness of the preparation method of the nanozyme detection reagent of this invention

[0035] This invention uses low-cost citric acid, copper chloride, and manganese chloride as raw materials, and synthesizes copper and manganese co-doped into a carbon matrix through high-temperature synthesis. Utilizing the synergistic catalytic effect of the copper-manganese bimetallic compound, the reaction energy barrier is effectively lowered and the catalytic efficiency is improved, thereby enhancing the colorimetric signal. This allows the nanozyme detection reagent to achieve a detection limit of 0.89 μM for carbofuran.

[0036] Furthermore, addressing the issues of easy aggregation and limited stability of existing copper-manganese bimetallic carbon dot nanozymes, this invention innovatively proposes an in-situ loading strategy. This strategy is simple and environmentally friendly; specifically, a magnetic stirrer is used to uniformly anchor copper-manganese bimetallic carbon dots with excellent enzyme-like activity within a mesoporous polydopamine framework through π-π stacking and hydrogen bonding interactions. This design not only effectively solves the aggregation effect of carbon dots by utilizing the high specific surface area and abundant pore structure of mesoporous polydopamine, significantly enhancing its dispersibility and structural stability, but also, as a highly efficient adsorption and enrichment material, mesoporous polydopamine itself can actively capture and enrich carbofuran in the apple matrix. Compared to simple detection based on active sites, this dual enhancement mechanism of "loading-enrichment" makes it easier for target molecules to access the active sites of copper-manganese bimetallic carbon dots under the adsorption of mesoporous polydopamine, thereby significantly improving the catalytic signal output and detection sensitivity of the sensing platform, enabling trace detection of carbofuran in the concentration range of 0-2 μM. The design of this composite material ingeniously combines the catalytic properties of carbon dots with the enrichment capacity of mesoporous materials, providing a new technical approach for the precise detection of trace pesticides in apple matrices.

[0037] 2. The inventiveness of the nanozyme detection reagent of this invention

[0038] To address the shortcomings of traditional detection methods based on acetylcholinesterase inhibition in specifically identifying carbofuran in the presence of multiple inhibitors, this invention synthesizes copper- and manganese-doped carbon dots and successfully loads them onto mesoporous polydopamine to synthesize a nanocomposite material. Experimental results demonstrate that the nanoenzyme complex exhibits good peroxidase activity and can be used for the specific and rapid detection of carbofuran residues in apples.

[0039] The unique chemical property of thiocarbofuran is that it can hydrolyze under acidic conditions to generate carbofuran and a reduced sulfide. This reduced sulfide can both inhibit the peroxidase-like activity of nanomaterials and consume hydroxyl radicals generated by hydrogen peroxide. The designed nanoenzyme detection reagent exhibits good enzyme-like activity under acidic conditions, catalyzing the generation of hydroxyl radicals from hydrogen peroxide, thereby oxidizing 3,3',5,5'-tetramethylbenzidine solution to produce a blue product. The unique chemical property of thiocarbofuran reduces the amount of hydroxyl radicals in the system after its addition, thus inhibiting the formation of the blue product. Furthermore, by simultaneously selecting different pesticides used in apple cultivation that are closely related to carbofuran but have similar structures, the specificity of colorimetric and photothermal detection modes was evaluated. The dual-signal normalized detection values ​​for other pesticides were all higher than 0.85, indicating that the nanozyme detection reagent of this invention exhibits significant specificity for carbofuran in complex samples under both colorimetric and photothermal modes, significantly improving detection selectivity. Under both colorimetric and photothermal detection modes, based on the excellent enzyme-like activity of the nanozyme detection reagent and the adsorption effect of mesoporous polydopamine, a detection limit as low as 0.89 μM for carbofuran was achieved, meeting the needs of trace detection. The correlation coefficients for linear fitting in both colorimetric and photothermal modes were both higher than 0.97, demonstrating that the concentration and signal response of this reagent exhibit a good correlation when detecting carbofuran, achieving the goal of rapid and accurate detection in practical applications.

[0040] The linear regression equation established under standard conditions in this invention exhibits good environmental tolerance and can be directly applied to apple detection without the need to re-establish a standard curve, saving detection time. It is suitable for rapidly estimating the preliminary concentration of carbofuran residues on crops under emergency conditions. For example, experimental results show that, after calculation, the concentrations of carbofuran measured using both colorimetric and photothermal dual-channel methods in apples at concentrations of 300 μM, 600 μM, and 900 μM are almost identical to the actual concentrations, with spiked recoveries of 82%-120%. When the results from the two signals are mutually corroborated, the reliability of the results is significantly improved; any deviation indicates a potential anomaly requiring verification. This mechanism significantly improves the accuracy and reliability of the detection results. It can be seen that even without establishing a corresponding standard curve and linear regression equation under apple conditions, the standard curve and linear regression equation established under acetate-sodium acetate buffer conditions still have good versatility and can be rapidly applied to apple sample detection. Attached Figure Description

[0041] Figure 1 These are the property characterization diagrams of the mesoporous polydopamine and the Zeta potential diagrams of the nanomaterials of this invention;

[0042] Figure 2 The transmission electron microscope image and elemental mapping diagram of the mesoporous polydopamine loaded with copper-manganese bimetallic carbon dots in this invention are shown.

[0043] Figure 3 The graph shows the colorimetric-photothermal dual-modal linear detection equation of the nanozyme reagent in this invention, as well as the detection graph of the actual sample.

[0044] Figure 4 This is a diagram illustrating the specificity of the nanozyme reagent in this invention. Detailed Implementation

[0045] The present invention will now be described in detail with reference to specific embodiments.

[0046] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] Unless otherwise specified, the raw materials used in the following examples are all conventional biochemical reagents; unless otherwise specified, the experimental methods are all conventional methods; unless otherwise specified, the quantitative tests in the following examples are all repeated three times and the results are averaged; unless otherwise specified, the percentages in the following examples are all mass percentages.

[0048] Unless otherwise specified, all other raw materials used in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0049] It should be noted that the acetate-sodium acetate buffer solution used in this application has a pH of 3.

[0050] It should be noted that instruments, equipment, raw materials, reagents, or methods not mentioned in this application are conventional or well-known technical methods to those skilled in the art, and will not be described in detail in this application.

[0051] Example 1

[0052] The preparation steps of a nanozyme detection reagent for detecting carbofuran residues in apples are as follows:

[0053] (1) Preparation of mesoporous polydopamine powder

[0054] (1.1) 500 mg of polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer (F127) was added to 50 mL of 50% ethanol aqueous solution and ultrasonically mixed for 5 min at 300 W to obtain a polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer solution.

[0055] A 50% ethanol aqueous solution is prepared by mixing ethanol and ultrapure water at a volume ratio of 1:1.

[0056] (1.2) Add 13 mL of 10 mg / L tris(hydroxymethyl)aminomethane (Tris) solution and 0.8 mL of 1,3,5-trimethylbenzene solution to the polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer solution, and sonicate for 5 min at 300 W power; then stir for 30 min at 180 rpm in a magnetic stirrer to obtain a milky white solution.

[0057] (1.3) Add 0.75 g of dopamine hydrochloride to the milky white solution and stir magnetically at 180 rpm in the dark for 24 h. After the reaction is complete, centrifuge the reactants at 10,000 rpm for 20 min and remove the supernatant. Then, elute three times with 50% ethanol aqueous solution at 10,000 rpm for 10 min and finally elute once with ultrapure water at 10,000 rpm for 10 min to obtain mesoporous polydopamine precipitate.

[0058] (1.4) Dissolve the mesoporous polydopamine precipitate in 20 mL of ultrapure water and sonicate it for 5 min at 300 W to obtain an ultrasonic mesoporous polydopamine solution.

[0059] (1.5) The ultrasonic mesoporous polydopamine solution was freeze-dried for 24 h at a temperature of -50℃ in a freeze dryer to obtain mesoporous polydopamine powder, which was then sealed and stored.

[0060] See Figure 1 In Figure A, transmission electron microscopy revealed that the mesoporous polydopamine powder had a particle size of 90-120 nm and exhibited good morphology. (See also...) Figure 1 According to BET results, the pore size of the mesoporous polydopamine powder is 9.459 nm.

[0061] (2) Preparation of copper-manganese bimetallic carbon dot solution

[0062] Dissolve 0.05 g citric acid, 0.07 g copper chloride, 0.05 g manganese chloride and 100 μL ethylenediamine (≥98%) in 5 mL of ultrapure water and sonicate for 5 min at 300 W.

[0063] The reaction was carried out in a sealed reactor at 180.0℃ for 6.0 h.

[0064] Centrifuge at 10,000 rpm for 15 min to separate the first supernatant. Repeat centrifugation for 15 min to separate the second supernatant, which is the copper-manganese bimetallic carbon point solution; store at 4℃.

[0065] See Figure 1 The particle size and distribution of copper-manganese bimetallic carbon dots were observed using a transmission electron microscope. It was found that the particle size of the copper-manganese bimetallic carbon dots was 2-3 nm and they were evenly distributed.

[0066] (3) Preparation of nanozyme detection reagent

[0067] (3.1) Dissolve mesoporous polydopamine powder in ultrapure water to prepare a mesoporous polydopamine solution with a concentration of 0.6 mg / mL.

[0068] (3.2) Mix 5 mL of 0.6 mg / mL mesoporous polydopamine solution and 1 mL of copper-manganese bimetallic carbon dot solution, and stir on a magnetic stirrer at 180 rpm for 24 h to obtain a mesoporous polydopamine solution loaded with copper-manganese bimetallic carbon dots.

[0069] (3.3) The mesoporous polydopamine solution loaded with copper-manganese bimetallic carbon dots was centrifuged to obtain a precipitate; the supernatant was removed by centrifuging at 10,000 rpm for 13 min; the supernatant was removed by eluting twice with ultrapure water at 10,000 rpm for 13 min to obtain the precipitate.

[0070] (3.4) Dissolve the precipitate in 12 mL of water and sonicate for 5 min at 300 W to prepare nanozyme detection reagent; store at 4℃.

[0071] See Figure 1 In Figure D, the potentials of mesoporous polydopamine, copper- and manganese-doped bimetallic carbon dots, and mesoporous polydopamine loaded with copper- and manganese-doped bimetallic carbon dots were measured using a Malvern potentiometer. "1" represents copper- and manganese-doped bimetallic carbon dots, "2" represents mesoporous polydopamine, and "3" represents mesoporous polydopamine loaded with copper- and manganese-doped bimetallic carbon dots. Compared to mesoporous polydopamine, the potential of mesoporous polydopamine loaded with bimetallic carbon dots decreased significantly, proving the successful loading of bimetallic carbon dots. See also... Figure 2 In Figure A, the particle size and distribution of mesoporous polydopamine loaded with copper-manganese bimetallic carbon dots were observed using transmission electron microscopy. It was found that the particle size of the mesoporous polydopamine loaded with copper-manganese bimetallic carbon dots was 90-120 nm, and the distribution was uniform. (See also...) Figure 2 B in Figure 2 The elemental mapping results of F in the figure, for mesoporous polydopamine loaded with copper-manganese bimetallic carbon dots, further demonstrate that the bimetallic carbon dots and mesoporous polydopamine achieve a good and uniform bond. Figure 2 B in Figure 2 C in Figure 2 D in Figure 2 E and Figure 2 F in the diagram represents the elemental mappings for carbon, nitrogen, oxygen, copper, and manganese, respectively. The results show that each element is widely and uniformly distributed.

[0072] The nanozyme detection reagent prepared in Example 1 showed colorimetric linearity of 0-2 μM and 0-1200 μM for carbofuran residues on apples, and photothermal linearity of 0-1200 μM, with goodness of fits of 0.97532, 0.97344, and 0.99025, respectively. The limit of detection was as low as 0.89 μM; the recovery rate was 82%-120%.

[0073] Example 2

[0074] The detection linear equations for the colorimetric mode and the photothermal mode are established separately. The specific operation steps are as follows:

[0075] (1) Trace colorimetric detection

[0076] Add 300 μL of acetate-sodium acetate buffer to 50 μL of thiocarbamate solutions of different concentrations, and incubate at 37°C and 200 rpm for 20 min under acidification. Then add 50 μL of nanozyme detection reagent solution and 50 μL of 5 mM hydrogen peroxide solution, and perform an enzyme reaction at 37°C and 200 rpm for 10 min. Finally, add 50 μL of 4.8 g / L 3,3',5,5'-tetramethylbenzidine solution, and perform color development at 37°C and 200 rpm for 10 min to obtain a trace concentration reaction solution. The absorbance of the trace concentration reaction solution is read using a UV spectrophotometer.

[0077] See Figure 3 In section A, the linear relationship between different concentrations of carbofuran in the concentration range of 0-2 μM and absorbance was investigated. The linear regression equation was absorbance = -0.06903 × [carbofuran (μM)] + 1.39623. The detection limit for trace colorimetric signals in the dual-modal detection technology of carbofuran was 0.89 μM, where R... 2 =0.97532.

[0078] (2) Wide range of concentration colorimetric-photothermal detection

[0079] 100 μL of acetate-sodium acetate buffer was added to 250 μL of thiocarbofuran solutions of different concentrations. The mixture was incubated at 37°C and 200 rpm for 30 min under acidification. Then, 50 μL of nanozyme detection reagent solution and 50 μL of 50 mM hydrogen peroxide solution were added, and the enzyme reaction was carried out at 37°C and 200 rpm for 10 min. Finally, 50 μL of 4.8 g / L 3,3',5,5'-tetramethylbenzidine solution was added, and the mixture was incubated at 37°C and 200 rpm for color development for 10 min, yielding a wide range of concentration reaction solutions. The absorbance of the wide range of concentration reaction solutions was read using a UV spectrophotometer.

[0080] In addition, the reaction solution with a wide range of concentrations was irradiated with a 660 nm laser for 10 min, and the temperature change of the reaction solution with a wide range of concentrations was read using a temperature imager.

[0081] See Figure 3 In section B, the linear relationship between different concentrations of carbofuran in the concentration range of 0-1200 μM and absorbance was investigated. The linear regression equation was normalized absorbance = -0.0005336 × [carbofuran (μM)] + 0.90783. The detection limit for colorimetric signals with a wide concentration range in the dual-modal detection technology of carbofuran was 25.52 μM, where R... 2 =0.99409.

[0082] See Figure 3 The study investigated the linear relationship between different concentrations of carbofuran in the 0-1200 μM range and temperature changes. The linear regression equation was normalized temperature change = -0.0004359 × [carbofuran (μM)] + 0.9495. The detection limit for the wide-range concentration photothermal signal of carbofuran in the dual-modal detection technique was 185.92 μM, where R... 2 =0.99025.

[0083] Example 3

[0084] Testing of real samples

[0085] The nanozyme detection reagent prepared in Example 1 and the linear regression equation constructed in Example 2 were used to detect real food samples of apples.

[0086] In this embodiment 3, in order to verify whether the linear regression equation constructed according to embodiment 2 can be effectively applied to the determination of apples in an interfering environment, a quantitative spraying of pesticides was performed to explore the differences in the concentrations of thiamethoxam detected by nanozymes in apple samples, so as to know that even in real food samples, the nanozyme detection reagent established in this application can be effectively detected under the influence of the food matrix.

[0087] The apples used in Example 3 were purchased from a supermarket and were of the Fuji variety. The apples were stored at room temperature during the purchase.

[0088] Apples were rinsed thoroughly with water and dried. They were then evenly sprayed with three different concentrations of carbofuran: 300 μM, 600 μM, and 900 μM. After drying, the spiked samples were obtained. Finally, 5 mL of ultrapure water was repeatedly sprayed onto the sample surface, and the outflow was the test solution. Subsequently, in accordance with Example 2, a colorimetric-photothermal dual-modal signal was detected. The concentration of carbofuran in the apples was obtained by substituting the normalized ultraviolet absorption intensity value minus the normalized temperature change value into the obtained linear regression equation.

[0089] See Figure 3 In section D, the experimental results show that, after calculation, the concentration of carbofuran measured by colorimetry and photothermal dual channels in apples under artificial spraying conditions is almost close to its true concentration of 300 μM, 600 μM, and 900 μM. It can be seen that even without establishing the corresponding standard curve and linear regression equation under the apple sample environment, the standard curve and linear regression equation established under the acetate-sodium acetate buffer solution still have good versatility.

[0090] Therefore, the nanozyme detection reagent prepared by this invention has good versatility and can be quickly applied to the detection of real samples.

[0091] Example 4

[0092] The other operations in this embodiment 4 are the same as in embodiment 2, except that:

[0093] Replace thiamethoxam with etoxazole, acetamiprid, isoprocarb, dinotefuran, chlorothalonil, pirimicarb, and propoxur.

[0094] See Figure 4 A and Figure 4 B in the middle, where Figure 4 In this context, A represents the normalized ultraviolet absorbance value for different pesticides. Figure 4 In the figure, B represents the normalized temperature variation (RTD) value for different pesticides. Experimental results showed that the normalized UV absorption intensity and normalized temperature variation values ​​for etoxazole were 1.284 and 0.956, respectively; for acetamiprid, they were 0.861 and 1.007; for isoprocarb, they were 0.920 and 0.993; for dinotefuran, they were 0.922 and 0.995; for chlorothalonil, they were 1.465 and 1.017; for pirimicarb, they were 0.943 and 0.985; and for propoxur, they were 0.925 and 0.978. Meanwhile, under the same conditions, the normalized UV absorbance and normalized temperature change of the test solution containing carbofuran were 0.484 and 0.585, respectively. This demonstrates that the nanozyme prepared in this invention has good specificity for carbofuran.

[0095] This invention develops a specific detection reagent for carbofuran residues in apples, possessing the following technical features: significantly enhanced detection sensitivity for the target pesticide, achieving accurate identification even in trace samples, thus effectively avoiding false negative results. Utilizing the unique chemical property of carbofuran hydrolysis under acidic conditions to produce reducing sulfides, specific identification of carbofuran in complex samples is achieved, significantly improving detection selectivity. Compared with traditional methods, this nanozyme-based detection technology significantly shortens analysis time and improves detection efficiency, better meeting the need for rapid response to carbofuran residues in apples in food safety monitoring. Furthermore, the synthesis of mesoporous polydopamine and the doping of carbon dots are simple, reducing dependence on experimental materials and equipment, helping to control overall detection costs and improving the economic practicality of the technology. The detection process is simple to operate, reducing the need for complex instruments and lowering operational difficulty, which is beneficial to improving laboratory efficiency. Simultaneously, this method can simultaneously acquire colorimetric and photothermal dual-modal signals in a single detection, not only improving detection sensitivity but also reducing misjudgments caused by operational errors through mutual correction between signals.

[0096] Those skilled in the art will readily understand that the above Embodiment 1 is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a nanozyme detection reagent for carbofuran residues in apples, characterized in that, The operation steps are as follows: (1) Preparation of mesoporous polydopamine powder (1.1) Add 500 mg of polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer (F127) to 50 mL of 50% ethanol aqueous solution and mix evenly by ultrasonication to obtain polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer solution. The ethanol-water solution was prepared by mixing ethanol and ultrapure water at a volume ratio of 1:

1. (1.2) Add 13 mL of 10 mg / L tris(hydroxymethyl)aminomethane (Tris) solution and 0.8 mL of 1,3,5-trimethylbenzene solution to the polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer solution and sonicate until homogeneous; stir on a magnetic stirrer to obtain a milky white solution; (1.3) Add 0.75 g of dopamine hydrochloride to the milky white solution and stir magnetically for 24 h in the dark; centrifuge to obtain mesoporous polydopamine precipitate; (1.4) Dissolve the mesoporous polydopamine precipitate in 20 mL of ultrapure water and sonicate to obtain an ultrasonic mesoporous polydopamine solution. (1.5) In a freeze dryer, the ultrasonic mesoporous polydopamine solution is freeze-dried into flocculent powder to obtain mesoporous polydopamine powder, which is then sealed and stored. The mesoporous polydopamine powder has a particle size of 90–120 nm; (2) Preparation of copper-manganese bimetallic carbon dot solution Dissolve 0.05 g citric acid, 0.07 g copper chloride, 0.05 g manganese chloride and 100 μL ethylenediamine (≥98%) in 5 mL of ultrapure water and react with sonication. The reaction was carried out in a sealed reactor at 180.0℃ for 6.0 h. The copper-manganese bimetallic carbon point solution was separated by centrifugation and stored at 4°C. (3) Preparation of nanozyme detection reagent (3.1) Dissolve mesoporous polydopamine powder in ultrapure water to prepare a mesoporous polydopamine solution with a concentration of 0.6 mg / mL; (3.2) Mix 5 mL of 0.6 mg / mL mesoporous polydopamine solution and 1 mL of copper-manganese bimetallic carbon dot solution, and stir on a magnetic stirrer at 180 rpm for 24 h to obtain a mesoporous polydopamine solution loaded with copper-manganese bimetallic carbon dots. (3.3) Centrifuge the mesoporous polydopamine solution loaded with copper-manganese bimetallic carbon dots to obtain the precipitate; (3.4) Dissolve the precipitate in 12 mL of ultrapure water, sonicate to dissolve, and prepare nanozyme detection reagent; store at 4℃; The nanozyme detection reagent has a colorimetric linear detection range of 0-2 μM and 0-1200 μM, and a photothermal detection range of 0-1200 μM, wherein the goodness of fit R0 is [value missing]. 2 The values ​​were 0.97532, 0.99409, and 0.99025, respectively, with a detection limit of 0.89 μM; the recoveries ranged from 82% to 120%.

2. The preparation method according to claim 1, characterized in that: In steps (1.1), (1.2), (1.4), (2), and (3.4), the ultrasonic treatment conditions are: power 300W and time 5min.

3. The preparation method according to claim 1, characterized in that: In step (1.2), the magnetic stirring conditions are: 180 rpm for 30 min.

4. The preparation method according to claim 1, characterized in that: In step (1.3), the stirring conditions are: 180 rpm for 24 h.

5. The preparation method according to claim 1, characterized in that: In step (1.3), the supernatant was removed by centrifuging at 10,000 rpm for 20 min in a centrifuge; the supernatant was then removed by eluting three times with a 50% ethanol aqueous solution at 10,000 rpm for 10 min; and then eluted once with ultrapure water at 10,000 rpm for 10 min to obtain mesoporous polydopamine precipitate.

6. The preparation method according to claim 1, characterized in that: In step (1.5), the freeze-drying conditions are: temperature -50℃ and time 24 h.

7. The preparation method according to claim 1, characterized in that: In step (2), the centrifugation conditions are as follows: centrifuge at 10,000 rpm for 15 min to separate the first supernatant; repeat the centrifugation for 15 min to separate the second supernatant, which is the copper-manganese bimetallic carbon dot solution.

8. The preparation method according to claim 1, characterized in that: In step (3.3), the centrifugation conditions are as follows: centrifuge at 10,000 rpm for 13 min, remove the supernatant; repeat the centrifugation for 13 min, wash twice with ultrapure water, remove the supernatant, and obtain the precipitate.

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