A dual-modal detection method for microcystin-LR based on MIP-Cu@CDs laccase activity

Abstract

The present invention belongs to the field of environmental and food pollution detection, and relates to a dual-modal detection method for microcystin-LR (MC-LR) based on regulating the activity of MIP-Cu@CDs-like laccases. The present invention uses cupric chloride dihydrate, citric acid and ethylenediamine as synthetic substrates, synthesizes fluorescent nanozymes with laccase-like activity in one step by a hydrothermal method, and encapsulates them through a molecular imprinting method, so that they can specifically identify MC-LR and regulate the activity of MIP-Cu@CDs-like laccases, thereby establishing a colorimetric and fluorescence dual-modal rapid determination method for MC-LR. The method of the present invention has a short detection time, high sensitivity and low cost. The dual-modality provides a built-in cross-reference correction, which greatly improves the reliability and accuracy of MC-LR detection in complex environmental samples and avoids false positive output of the test results.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental and food pollution detection, and in particular relates to a MIP-Cu@CDs laccase activity microcystin-LR dual-modal detection method and application thereof. Background Art

[0002] Microcystin-LR (MC-LR), as one of the common derivative forms of microcystins (MCs), is a heptapeptide toxin containing alanine and glutamic acid, and its structure contains special groups such as linolenic acid (Adda) and methacryloyl (Mdha). MC-LR is highly toxic. Ingestion of MC-LR can cause acute toxicity symptoms, and long-term exposure can lead to chronic toxicity. In severe cases, liver damage, nerve damage and even carcinogenicity can occur. MC-LR can also have a negative impact on aquatic organisms and ecosystems, destroy water quality and ecological balance, and ultimately pose a potential risk to human food safety. In view of the potential threat of MC-LR to the ecological environment and the health of organisms, it is crucial to strengthen the monitoring and management of MC-LR.

[0003] In recent years, the traditional MC-LR detection methods mainly include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS), protein phosphatase inhibition (PPI) and enzyme-linked immunosorbent assay (ELISA). GC-MS can detect MC-LR quickly and accurately through cumbersome steps, but the sample pretreatment is complicated and the analysis results are greatly affected by environmental factors. LC-MS detection of MC-LR has high sensitivity and selectivity and can detect multiple targets at the same time. However, the instruments used are relatively expensive and have high operating requirements for laboratory personnel, resulting in high laboratory costs. PPI can produce highly sensitive reactions to MC and help detect low concentrations of toxins. However, PPI may require complex experimental operations and data analysis, which increases the complexity of the experiment. Some PPIs may require a long experimental time and are not suitable for situations where rapid detection is required. ELISA is a fast and simple method that can perform quantitative and qualitative analysis. It has been widely used for the detection of MC, but it requires complex measurement steps and a time-consuming detection process. This is not only time-consuming for simple on-site detection methods, but also incompatible with rapid, efficient and high-throughput screening methods. Although nanozymes have shown extraordinary potential and broad application prospects in many fields, their limitations in specific situations cannot be ignored, especially in terms of selectivity and specific binding to target molecules.

[0004] Therefore, the rapid and accurate detection of microcystin-LR (MC-LR) still needs further research and improvement. Summary of the invention

[0005] The purpose of the present invention is to comprehensively utilize the advantages of fluorescent nanozymes and molecular imprinting technology to develop a detection reagent and a detection method that can quickly and accurately detect microcystin-LR (MC-LR), which is of great significance for protecting the ecological environment, food safety and public health.

[0006] To achieve the above-mentioned object and other related objects, in one aspect of the present invention, the present invention provides a method for preparing MIP-Cu@CDs fluorescent nanozyme, the method comprising:

[0007] S1. Synthesis of Cu@CDs fluorescent nanozyme: citric acid and cupric chloride dihydrate are added to ultrapure water, and ethylenediamine is added dropwise to the solution, and ultrasonic treatment is performed. The mixture is quickly transferred to a high-temperature reactor lined with polytetrafluoroethylene, and the high-temperature treatment is performed for a period of time. After the high-temperature treatment, the temperature of the reactants is lowered to room temperature, and then the reactants are separated by high-speed centrifugation to obtain a precipitate, and the precipitate is washed with ethanol, and the precipitate is dried into powder to obtain Cu@CDs;

[0008] S2. Dissolve the Cu@CDs and MC-LR obtained in S1 in ethanol, mix them ultrasonically, then add the imprinted molecules, stir and mix, dry the solution, and extract to obtain MIP-Cu@CDs fluorescent nanozyme.

[0009] In one embodiment, the present invention provides a method for preparing MIP-Cu@CDs fluorescent nanozyme, the method comprising:

[0010] (1) Synthesis of Cu@CDs fluorescent nanozyme: 1.921 g citric acid and 3.0 g copper chloride dihydrate were added to 20.0 mL ultrapure water, and 1.0 mL ethylenediamine was added dropwise to the solution. After ultrasonication for 30 min, the mixture was quickly transferred to a polytetrafluoroethylene-lined high-temperature reactor and heated at 200.0 °C for 4.0 h. After the temperature of the reactant dropped to room temperature, it was centrifuged at 11000 rpm for 10 min, and then the solution was separated, and the precipitate was washed with ethanol 3 times, and finally dried in an oven to obtain Cu@CDs.

[0011] (2) Synthesis of MIP-Cu@CDs fluorescent nanozyme: 0.12g Cu@CDs nanozyme and 0.16g MC-LR were dissolved in 10.0ml ethanol and ultrasonicated for 30min to mix thoroughly. Next, 0.1568g carboxyethylsilanetriol sodium salt and 0.633g silicon tetraacetate were added to the above mixture, and stirring continued for 30.0min in the dark, followed by slow addition of 200.0μL ammonia water to the above reaction system, and stirring continued for 12.0h. After stirring, the system was collected and freeze-dried into powder using a freeze dryer. Finally, the freeze-dried powder was collected, and the MIP-Cu@CDs fluorescent nanozyme was extracted using methanol Soxhlet extraction, and dried into powder in an oven. The MIP-Cu@CDs fluorescent nanozyme can be stored in a refrigerator at 4 degrees.

[0012] In the present invention, the present invention utilizes a one-pot hydrothermal method to directly obtain MIP-Cu@CDs fluorescent nanozyme with laccase-like activity from simple and easily available raw materials such as citric acid, cupric chloride dihydrate, and ethylenediamine without separating intermediates.

[0013] In one aspect of the present invention, the present invention provides a MC-LR dual-modal detection method based on MIP-Cu@CDs fluorescent nanozyme, wherein the MIP-Cu@CDs fluorescent nanozyme is prepared by the aforementioned method of the present invention. Dual-modal detection can use both absorbance detection mode and fluorescence detection mode.

[0014] In one aspect of the present invention, a dual-modal detection method and application of MC-LR based on MIP-Cu@CDs fluorescent nanozyme is provided, wherein the method is used to detect pollutants in the environment and food; preferably, the MIP-Cu@CDs laccase of the present invention is particularly suitable for the detection of MC-LR residues.

[0015] In one aspect of the present invention, the present invention provides a MC-LR dual-modal detection method based on MIP-Cu@CDs fluorescent nanozyme, the specific steps are:

[0016] (1) Determine the absorbance value A of MC-LR standard of different concentrations at 510 nm s : (Note: The maximum UV absorption peak of 2,4-DP and 4-AP oxidized to red products is around 510nm);

[0017] Pipette the MIP-Cu@CDs fluorescent nanozyme solution into a centrifuge tube, add 2,4-DP, 4-AP solution and pH 7.5 buffer, then add the standard solution, mix well and react for a while, transfer to a cuvette, and measure the absorbance at 510 nm with a UV spectrophotometer, which is recorded as the absorbance value A. s ;

[0018] (2) Determine the fluorescence intensity value F of MC-LR standard of different concentrations at 420nm under 340nm excitation s : (Note: The MIP-Cu@CDs fluorescent nanozyme of the present invention has fluorescent properties, has an optimal emission wavelength under 340nm excitation, and has a maximum emission wavelength at 420nm);

[0019] Pipette the MIP-Cu@CDs fluorescent nanozyme solution into a centrifuge tube, add 2,4-DP, 4-AP solution and pH 7.5 buffer, then add the standard solution, mix well and react for a while, transfer to a cuvette, and measure the fluorescence intensity at 420nm using a fluorescence spectrometer, which is recorded as the fluorescence value F s ;

[0020] (3) Constructing the linear regression equation of the standard

[0021] The absorbance values ​​A measured using different concentrations of standard substances s The UV spectrophotometer linear regression equation was constructed based on the concentration values ​​of the standard;

[0022] The fluorescence intensity values ​​F measured using different concentrations of standard substances s The linear regression equation of fluorescence spectrometer was constructed based on the concentration value of the standard substance;

[0023] (4) Use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested by UV spectrophotometer n ; Use the sample to be tested instead of the standard to measure the fluorescence intensity value F of the sample to be tested by fluorescence spectrometer n ;

[0024] (5) The absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the MC-LR residual value; the fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the MC-LR residual value;

[0025] Preferably, the concentration of the MIP-Cu@CDs fluorescent nanozyme solution is 0.2 mg / ml; the concentration of the 2,4-DP solution is 1.0 mmol / L; the concentration of the 4-AP solution is 1.0 mmol / L; the buffer solution is phosphate buffer (1x), pH 7.5; the volume ratio between the MIP-Cu@CDs fluorescent nanozyme solution, 2,4-DP solution, 4-AP and phosphate buffer is: 10:10:10:150-170; the buffer solution can also be acetic acid-sodium acetate buffer.

[0026] Preferably, the reaction time after uniform mixing in step (1) is: reaction at 50° C. for 20.0 min after uniform mixing.

[0027] Preferably, the test sample is aquatic food or environmental water containing residual MC-LR. Before the test, the test sample is filtered with filter paper or filter membrane with a pore size of 0.22 μm, and the filtrate is collected.

[0028] Preferably, the absorbance linear equation of the present invention is A=0.00195C MC-LR +0.05208; the linear equation of fluorescence spectrum is F=0.1248C MC-LR +62.1044;

[0029] In one aspect of the present invention, the present invention provides a MC-LR dual-modal detection method based on MIP-Cu@CDs fluorescent nanozyme, the method comprising:

[0030] S11 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution, 10.0 μL of 1.0 mmol / L 2,4-DP and 10.0 μL of 1.0 mmol / L 4-AP were added to 150.0 μL of phosphate buffer with a pH value of 7.5, and then 20.0 μL of MC-LR standard solution of different concentrations were added to react at 50 °C for 20.0 min, and the absorbance value A at 510 nm was measured by UV spectrophotometer. s ; and / or using a fluorescence spectrometer to measure the fluorescence intensity value F at 420nm s ;

[0031] S12 Absorbance value A measured using different concentrations of standard substances s The linear regression equation of UV spectrophotometer was constructed by using the concentration value of the standard substance at different concentrations. s The linear regression equation of fluorescence spectrum was constructed based on the concentration value of the standard;

[0032] S13 Use the sample to be tested that has been pre-treated by 0.22 μm filter paper or filter membrane to replace the standard sample and measure the absorbance value A of the sample to be tested by UV spectrophotometer n ; Use the sample to be tested instead of the standard to measure the fluorescence spectrum to obtain the fluorescence intensity value F of the sample to be tested n .

[0033] S14 converts the absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the MC-LR residual value; the fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the MC-LR residual value.

[0034] In one aspect of the present invention, the present invention can use both colorimetric and fluorescence modes to detect MC-LR simultaneously, or can use only one of the colorimetric or fluorescence modes to detect MC-LR. Relatively speaking, the sensitivity of detecting MC-LR in the fluorescence mode is the highest.

[0035] In one aspect of the present invention, the present invention discloses a MC-LR residue detection kit, the kit comprising MIP-Cu@CDs fluorescent nanozyme, 2,4-DP, buffer, 4-AP. Preferably, the buffer is a phosphate buffer or an acetic acid-sodium acetate buffer.

[0036] In one aspect of the present invention, the present invention also discloses the use of relevant detection reagents in dual-mode detection of MC-LR residues in environmentally complex samples.

[0037] The method established by the present invention has a good linear relationship between the change value of the absorption intensity at 510nm and the MC-LR concentration in the range of 1.12-800.0μg / L, and the regression equation is A=0.00195C MC-LR +0.05208, the correlation coefficient was 0.9958, and the detection limit was 0.885μg / L; there was a good linear relationship between the change in fluorescence intensity at 420nm and the MC-LR concentration in the range of 2.23-800.0μg / L, and the regression equation was F=0.1248C MC-LR +62.1044, the correlation coefficient is 0.9970, and the detection limit is 0.0139μg / L; it is obtained using the formula for the minimum detection limit (3σ / k, σ is the standard deviation of 11 blank samples).

[0038] The MIP-Cu@CDs fluorescent nanozyme of the present invention has laccase-like activity, wherein 2,4-DP and 4-AP are substrates of laccase-like enzymes, 2,4-DP is oxidized to generate corresponding quinone compounds, and 4-AP reacts with these oxidized quinone compounds to form red complexes. However, the presence of MC-LR blocks the cavity of molecular imprinting, thereby preventing the entry of 2,4-DP and 4-AP, thereby blocking the generation of red products, showing a decrease in the colorimetric signal dependent on the concentration of MC-LR, and thus developing a MC-LR detection method based on the color development of red products. At the same time, the red product generated by oxidation can absorb the energy of the fluorescence emission of the MIP-Cu@CDs fluorescent nanozyme itself at 420nm, resulting in its fluorescence quenching, which is due to the presence of electron transfer. When MC-LR blocks the molecular imprinting cavity, the energy transfer between the nanozyme and the colored product is blocked, thereby resulting in an increase in fluorescence intensity, showing an MC-LR concentration-dependent fluorescence signal enhancement, thereby developing a MC-LR fluorescence detection method based on the fluorescence change of the MIP-Cu@CDs fluorescent nanozyme.

[0039] In the present invention, a colorimetric and fluorescence dual-modal simultaneous detection of MC-LR is constructed using a molecularly imprinted copper-doped carbon dot fluorescent nanozyme with laccase-like activity. Compared with traditional methods (LC-MS, GC-MS), the new fluorescence analysis method based on nanotechnology performs better in terms of detection limit and linearity. Fluorescence detection has the advantages of sensitivity, accuracy and visualization, and can be used in combination with other mature methods to greatly shorten the detection process and cycle of MC-LR. In the fluorescence analysis of MC-LR, the optical, electronic and chemical properties of fluorescent nanomaterials can improve the sensitivity and resolution of fluorescence analysis, thereby achieving highly selective analysis of MC-LR. Molecular imprinting (MIP) has attracted widespread attention with its unique "lock and key", and it provides a new method to solve the specificity challenges of nanozymes. In this method, the template molecule is equivalent to a lock, and the molecularly imprinted polymer is a key that can accurately match it. Through the polymerization process, the template molecule guides the functional monomers to be arranged in space to form a cavity with a specific shape, size and chemical function. Once the polymerization is completed, the template molecule is removed, and the remaining cavity can specifically recognize and bind to the template molecule. By combining the dual advantages of MIP and nanozyme, the recognition selectivity of nanozyme for target molecules is significantly enhanced.

[0040] Beneficial effects:

[0041] Due to the application of the above technical solution of the present invention, the present invention has the following beneficial effects compared with the prior art:

[0042] 1. The MIP-Cu@CDs fluorescent nanozyme synthesis method of the present invention has the advantages of wide raw material sources, low cost, and economical and practical. Compared with other existing methods for synthesizing nanozymes, the materials are simple, the method is fixed, the cost-effectiveness is low, and it can be synthesized in large quantities.

[0043] 2. Compared with the existing traditional methods for detecting MC-LR, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS), protein phosphatase inhibition (PPI) and other methods, the present invention does not require complicated experimental operations and data analysis as well as time-consuming detection processes, and has a shorter detection time, higher sensitivity and lower cost.

[0044] 3. The present invention proposes a dual-modality detection method for MC-LR based on MIP-Cu@CDs fluorescent nanozyme. According to the relationship between MC-LR concentration and red product color intensity, MC-LR concentration and purple fluorescence change of MIP-Cu@CDs fluorescent nanozyme, a method for rapid determination of MC-LR by colorimetry and fluorescence dual modality can be established. In addition, through mutual confirmation between different modalities, the dual modality provides built-in cross-reference correction, which greatly improves the reliability and accuracy of MC-LR detection in complex environmental samples and avoids false positive output of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a synthesis diagram of MIP-Cu@CDs of the present invention and a principle diagram of dual-modal detection MC-LR of MIP-Cu@CDs+2,4-DP+4-AP system.

[0046] Figure 2 This is a transmission electron microscopy image of the MIP-Cu@CDs fluorescent nanozyme prepared in the present invention.

[0047] Figure 3 These are the UV spectra of various components measured by UV spectrophotometer, including: (1) 4-AP; (2) 2,4-DP; (3) 2,4-DP+4-AP; (4) MIP-Cu@CDs+4-AP; (5) MIP-Cu@CDs+2,4-DP+4-AP; (6) MIP-Cu@CDs+2,4-DP+4-AP+MC-LR.

[0048] Figure 4 Fluorescence spectra of various components measured by fluorescence spectroscopy, including (1) 4-AP; (2) 2,4-DP; (3) 2,4-DP+4-AP; (4) MIP-Cu@CDs+4-AP; (5) MIP-Cu@CDs+2,4-DP+4-AP; (6) MIP-Cu@CDs+2,4-DP+4-AP+MC-LR.

[0049] Figure 5 The optimized conditions for detecting MC-LR in the MIP-Cu@CDs+2,4-DP+4-AP system measured by UV spectrophotometer. (A) in the figure shows the optimization of pH, with the optimal pH being 7.0-7.5; (B) in the figure shows the optimization of reaction temperature, with the optimal temperature being 50°C.

[0050] Figure 6 The time optimization conditions for the MIP-Cu@CDs+2,4-DP+4-AP system. (A) in the figure shows the time optimization conditions for the MIP-Cu@CDs+2,4-DP+4-AP system to catalyze the generation of red products, and the optimal reaction time is 20.0 min; (B) in the figure shows the time optimization conditions for the MIP-Cu@CDs+2,4-DP+4-AP system to detect MC-LR, and the optimal reaction time is 20.0 min.

[0051] Figure 7 Shows the absorbance of the standard sample with different concentrations of MC-LR and the standard curve.

[0052] Figure 8 Figure 3 shows the fluorescence spectra and standard curves of MC-LR standards with different concentrations.

[0053] Fig. 9 The bar graph is a dual-mode detection of interfering substances in a complex environment, proving that the colorimetric and fluorescence dual-mode system of MIP-Cu@CDs+2,4-DP+4-AP has good selectivity for MC-LR, where (1) blank; (2) Ca 2+ ; (3) K + ; (4) Fe 3 + ; (5) Co 2+ ; (6) NO 2- ; (7) Al 3+ ; (8) Mn 2+ ; (9) glutamic acid; (10) ascorbic acid; (11) arginine; (12) leucine; (13) glyphosate; (14) imidacloprid; (MC-LR). DETAILED DESCRIPTION

[0054] The following specific embodiments illustrate the embodiments of the present invention, and those familiar with the technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. It should be noted that the structure, ratio, size, etc. illustrated by the accompanying drawings of this specification are only used to match the content disclosed in the specification, so as to be understood and read by those familiar with the technology, and are not used to limit the restrictive conditions that the present invention can implement, so there is no technical substantive significance, any structural modification, change of ratio relationship or adjustment of size. The following examples are provided to better understand the present invention, but not to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0055] Related reagents:

[0056] The raw materials used in the present invention: copper chloride dihydrate (99.0%), 2,4-DP (2,4-dichlorophenol, >98.0%), 4-AP (4-aminoantipyrine, 98.0%), silicon tetraacetate (95.0%), anhydrous ethanol (99.5%) and carboxyethylsilanetriol sodium salt (25.0wt.% aqueous solution) were purchased from Shanghai McLean Biochemical Technology Co., Ltd.; ethylenediamine (98.0%) and citric acid (99.5%) were purchased from Shanghai Aladdin Reagent Company; ammonia water was purchased from Yantai Shuangshuang Chemical Co., Ltd.; microcystin-LR (20.0 mg / L) was purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.; phosphate buffer (20.0X, pH 7.2-7.6) was purchased from Shanghai Shenggong Biotechnology Co., Ltd. The water used in the experimental process was ultrapure water.

[0057] Phosphate buffer (1.0X, pH 7.5) is prepared by diluting 50.0 mL of 20.0X phosphate buffer by 20 times to obtain 1.0X phosphate buffer, and then adjusting the pH of the phosphate buffer (pH = 7.2-7.6) to 7.5 using a pH meter with the aid of a phosphoric acid solution and a sodium hydroxide solution.

[0058] Embodiment 1:

[0059] A method for preparing a microcystin-LR-regulated laccase activity MIP-Cu@CDs fluorescent nanozyme, the method comprising:

[0060] S1: Synthesis of Cu@CDs fluorescent nanozyme. The synthesis of Cu@CDs nanozyme was carried out by a one-pot hydrothermal method. In short, 1.921g of citric acid and 3.0g of copper chloride dihydrate were added to 20.0mL of ultrapure water, and then 1.0mL of ethylenediamine was added dropwise to the solution. After ultrasonication for 30.0min, the mixture was quickly transferred to a high-temperature reactor lined with polytetrafluoroethylene. And heated at 200.0℃ for 4.0h. After the temperature of the reactant dropped to room temperature, centrifuged at 11000.0rpm for 10.0min, then the solution was separated, and the precipitate was washed 3 times with ethanol, and finally dried into powder in an oven to obtain Cu@CDs.

[0061] S2: Synthesis of MIP-Cu@CDs fluorescent nanozyme. First, 0.12g Cu@CDs nanozyme and 0.16g MC-LR were dissolved in 10.0ml ethanol and ultrasonicated for 30.0min to mix thoroughly. Next, 0.1568g carboxyethylsilanetriol sodium salt and 0.633g silicon tetraacetate were added to the above mixture, and stirring continued for 30.0min in the dark, followed by slow addition of 200.0μL ammonia water to the above reaction system, and stirring continued for 12.0h. After stirring, the system was collected and freeze-dried into powder using a freeze dryer. Finally, the freeze-dried powder was collected, and the MIP-Cu@CDs fluorescent nanozyme was extracted by methanol Soxhlet extraction, dried into powder in an oven, and stored in a refrigerator at 4 degrees.

[0062] MIP-Cu@CDs fluorescent nanozyme has laccase-like activity and can catalyze 4-AP to oxidize 2,4-DP to generate red products, accompanied by enhanced absorbance at 510nm. When MC-LR is present, MC-LR can inhibit the laccase-like activity of MIP-Cu@CDs fluorescent nanozyme through specific recognition of molecular imprinting film, thereby inhibiting the formation of red products. Moreover, the red products generated by oxidation can absorb the energy of the fluorescence emission of MIP-Cu@CDs fluorescent nanozyme itself at 420nm, resulting in its fluorescence quenching.

[0063] Embodiment 2:

[0064] A MC-LR dual-modal detection method based on MIP-Cu@CDs fluorescent nanozyme

[0065] 2.1 Optimization of MC-LR method conditions for detecting catalytic activity of MIP-Cu@CDs fluorescent nanozyme based on laccase activity

[0066] Pipette 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution into a centrifuge tube, add 1.0 mmol / L 2,4-DP and 1.0 mmol / L 4-AP solution, then add a buffer solution adjusted to pH 7.5, and the sample to be tested (200.0 μL system), mix well, react at 50 °C for 20.0 min, transfer to a cuvette, measure the absorbance value A at 510 nm with an ultraviolet spectrophotometer, measure the fluorescence spectrum under excitation at a wavelength of 340 nm with a fluorescence spectrophotometer, and record the fluorescence intensity value F;

[0067] Figure 3 The UV spectrum measured by UV spectrophotometer is shown. Figure 3 It can be clearly seen that MIP-Cu@CDs+2,4-DP+4-AP obviously generated a red product, and the absorbance at 510nm was enhanced. The combination of MIP-Cu@CDs+2,4-DP+4-AP+MC-LR inhibited the generation of red products and reduced the absorbance. Figure 4 The fluorescence spectrum measured by a fluorescence spectrometer. It can be found that the single MIP-Cu@CDs fluorescent nanozyme has obvious purple fluorescence emission, and the generated red product can quench the fluorescence of the MIP-Cu@CDs fluorescent nanozyme (as shown in Group 5), but when MC-LR is present (as shown in Group 6), the MIP-Cu@CDs fluorescent nanozyme recovers fluorescence because the generation of the red product is inhibited.

[0068] Embodiment 3:

[0069] In order to study the optimal reaction conditions for the generation of red products catalyzed by MIP-Cu@CDs fluorescent nanozymes, the optimal conditions of the detection reagents were optimized using the control variable method in the ultraviolet absorption spectrum mode. Figure 5 , Figure 5 Middle (A) is the optimization of pH under the same conditions, with the optimum being pH 7.5; Figure 5 Middle (B) is the optimization of reaction temperature. As the temperature increases, the absorbance value gradually increases until the temperature reaches 50°C, when the absorbance no longer changes significantly. Therefore, the optimal reaction temperature is 50°C.

[0070] Embodiment 4:

[0071] After the above optimization conditions were completed, UV absorption spectroscopy was used to confirm the optimal reaction time for the MIP-Cu@CDs fluorescent nanozyme to catalyze the production of red products under the same conditions.

[0072] Specifically: 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution, 10.0 μL of 1.0 mmol / L 2,4-DP and 10.0 μL of 1.0 mmol / L 4-AP were added to 150.0 μL of phosphate buffer with a pH value of 7.5, and the change of absorbance at 510 nm was measured by UV absorption spectroscopy every 2.0 min. Figure 6 As shown in (A), as the reaction time increases, the absorbance value gradually increases until 20.0 min, when the reaction reaches the equilibrium point and the absorbance no longer changes. Therefore, 20.0 min is confirmed to be the optimal reaction time for this system. Figure 6 As shown in (B), as the reaction time increases, the absorption peak of oxidized 2,4-DP continues to increase until 20.0 min, when the reaction reaches an equilibrium point, at which time 2,4-DP is completely oxidized and the absorbance at 510 nm no longer changes. Therefore, the optimal reaction time for detecting MC-LR is 20.0 min.

[0073] Example 5: Standard curve establishment and sample detection method

[0074] (1) Establish the linear equation for detection

[0075] 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution, 10.0 μL of 1.0 mmol / L 2,4-DP and 10.0 μL of 1.0 mmol / L 4-AP were added to 150.0 μL of phosphate buffer with a pH value of 7.5, and then 20.0 μL of MC-LR standard solution of different concentrations were added to react at 50 °C for 20.0 min, and the absorbance value A at 510 nm was measured by UV spectrophotometer. s ; Use fluorescence spectrometer to measure the fluorescence intensity value F at 420nm s ;

[0076] The measured fluorescence intensity values, absorbance values ​​and temperature values ​​are as follows:

[0077]

[0078] See also Figure 7 and Figure 8 After fitting and settling with origin software, it was found that there was a good linear relationship between the change in absorption intensity at 510 nm and the MC-LR concentration in the range of 1.12-800.0 μg / L, and the regression equation was A=0.00195C MC-LR+0.05208, the correlation coefficient was 0.9958, and the detection limit was 0.885μg / L; there was a good linear relationship between the change in fluorescence intensity at 420nm and the MC-LR concentration in the range of 2.23-800.0μg / L, and the regression equation was F=0.1248C MC-LR +62.1044, correlation coefficient was 0.9970, and detection limit was 0.0139 μg / L;

[0079] (2) Pretreatment of the test samples

[0080] The detection object is algae powder or environmental water with residual MC-LR.

[0081] Complex environmental samples require simple pretreatment before testing to remove floating impurities and insoluble matter, filter with filter paper or filter membrane with a pore size of 0.22μm, collect the filtrate, and adjust the pH to 7.5 with phosphate buffer before testing.

[0082] (3) Testing of samples to be tested

[0083] Determine the absorbance value A of the sample to be tested at 510nm n

[0084] Pipette 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution into a centrifuge tube, add 1.0 mmol / L 2,4-DP and 1.0 mmol / L 4-AP solution, then add pH 7.5 buffer solution and the sample to be tested (200.0 μL system), mix well, react at 50 °C for 20.0 min, transfer to a cuvette, and measure the absorbance at 510 nm using a UV spectrophotometer, which is recorded as the absorbance value A. n ;

[0085] Measure the fluorescence spectrum of the sample to be tested under 340nm wavelength excitation

[0086] Pipette 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution into a centrifuge tube, add 1.0 mmol / L 2,4-DP and 1.0 mmol / L 4-AP solution, then add pH 7.5 buffer solution and the sample to be tested (200.0 μL system), mix well, react at 50 °C for 20.0 min, transfer to a cuvette, and measure the fluorescence spectrum under 340 nm excitation with a fluorescence spectrophotometer. Record the fluorescence intensity at 420 nm as F n ;

[0087] (4) Calculate the MC-LR concentration in the sample to be tested

[0088] The linear equation of absorbance is A = 0.00195C MC-LR+0.05208; the linear equation of fluorescence spectrum is F=0.1248C MC-LR +62.1044; Substitute into (3) to get A n and F n , we can get the MC-LR concentration C in the sample to be tested MC-LR The value is the MC-LR residual value.

[0089] Example 6: Effects of different environmental interfering substances on the MC-LR dual-modal detection method based on MIP-Cu@CDs fluorescent nanozyme

[0090] See also Fig. 9 , showing the use of different samples to test the selectivity of the colorimetric and fluorescence dual-modal detection of the present invention in different environments. 1blank; 2Ca 2+ ; 3K + ;4Fe 3+ ;5Co 2+ ; 6NO2 - ;7Al 3+ ;8Mn 2+ ; 9 Glutamic acid (Glu); 10 Ascorbic acid (AA); 11 Arginine (Arg); 12 Leucine (Leu); 13 Glyphosate; 14 Imidacloprid; 15 Microcystin-LR (MC-LR). The results show that the present invention can specifically detect MC-LR, while other interfering substances have no effect on the method of the present invention, showing the anti-interference and high sensitivity of the present invention.

[0091] Example 7: Algal powder and water samples were tested as real samples

[0092] 7.1 Algae powder sample test:

[0093] Algae powder pretreatment: 2.0 g of algae powder flakes were ground, and then the powder was dissolved in 20.0 mL of methanol. After ultrasonic treatment for 30.0 minutes, the supernatant was taken as the sample solution after centrifugal filtration.

[0094] Before the test, the pH of the prepared sample solution was adjusted to 7.5. Then, 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution, 10.0 μL, 1.0 mmol / L 2,4-DP and 10.0 μL, 1.0 mmol / L 4-AP were added to the above sample solution. At this time, the standard addition method was used to add different concentrations of MC-LR to the above system, and after mixing evenly, the mixture was reacted at 50 ° C for 20.0 min and moved to the cuvette, and the change in absorbance at 510 nm was measured by ultraviolet absorption spectroscopy. In the same case, the fluorescence spectrum under excitation at a wavelength of 340 nm was measured using a fluorescence spectrophotometer.

[0095] 7.2 Actual water sample testing:

[0096] Before the test, the water sample was filtered with a 0.22 μm filter paper or filter membrane, and the filtrate was collected. The prepared sample solution was adjusted to pH 7.5. Then, 10.0 μL of 0.2 mg / mL MIP-Cu@CDs fluorescent nanozyme solution, 10.0 μL, 1.0 mmol / L 2,4-DP and 10.0 μL, 1.0 mmol / L 4-AP were added to the above sample solution. At this time, the standard addition method was used to add different concentrations of MC-LR to the above system, and after mixing evenly, the mixture was reacted at 50 ° C for 20.0 min and moved to a cuvette, and the change in absorbance at 510 nm was measured by ultraviolet absorption spectroscopy. In the same case, the fluorescence spectrum under excitation at a wavelength of 340 nm was measured using a fluorescence spectrophotometer.

[0097] See Table 1, which shows the different situations of colorimetric and fluorescence synergistic detection of actual samples. The recovery of the colorimetric method ranged from 99.90% to 111.29%, and the relative standard deviation (RSD) was less than 5.00%. Similarly, the recovery in the fluorescence mode ranged from 99.69% to 116.28%, with an RSD of less than 5.00%. This shows that the MIP-Cu@CDs fluorescent nanozyme has good feasibility and reproducibility in detecting MC-LR in actual samples.

[0098] Table 1 Results of actual sample detection using colorimetry and fluorescence synergistic detection

[0099]

[0100]

[0101] In summary, the present invention has developed a MC-LR dual-modal detection method based on MIP-Cu@CDs fluorescent nanozyme, which utilizes the decrease of colorimetric signal and the enhancement of fluorescence signal. In the colorimetric (visible to the naked eye) mode, the higher the MC-LR concentration, the fewer colored products generated by oxidation, and the lighter the red color visible to the naked eye; in the fluorescence mode, the higher the MC-LR concentration, the stronger the fluorescence emission of MIP-Cu@CDs fluorescent nanozyme at 420nm. Finally, a colorimetric and fluorescence dual-modal detection method for detecting MC-LR based on MIP-Cu@CDs fluorescent nanozyme can be established, and the results obtained by the colorimetric and fluorescence modes are used to verify the accuracy of the MC-LR detection results, thereby avoiding the output of false positive results. In addition, in different environmental scenarios, the most convenient and appropriate method of the above two methods can be selected to detect the MC-LR concentration in different environments, which greatly improves its scope of application.

[0102] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. A method for preparing MIP-Cu@CDs fluorescent nanozyme, characterized in that: The method comprises: S1. Synthesis of Cu@CDs fluorescent nanozyme: citric acid and cupric chloride dihydrate are added to ultrapure water, and ethylenediamine is added dropwise to the solution, and ultrasonic treatment is performed. The mixture is quickly transferred to a high-temperature reactor lined with polytetrafluoroethylene, and the high-temperature treatment is performed for a period of time. After the high-temperature treatment, the temperature of the reactants is lowered to room temperature, and then the reactants are separated by high-speed centrifugation to obtain a precipitate, and the precipitate is washed with ethanol, and the precipitate is dried into powder to obtain Cu@CDs; S2. Dissolve the Cu@CDs and MC-LR obtained in S1 together with ethanol, mix them ultrasonically, then add carboxyethylsilanetriol sodium salt, silicon tetraacetate and ammonia water, stir and mix, dry the solution, and extract MIP-Cu@CDs fluorescent nanozyme by methanol Soxhlet extraction; the MC-LR is microcystin-LR.

2. A method for preparing MIP-Cu@CDs fluorescent nanozyme, characterized in that: The method comprises: (1) Synthesis of Cu@CDs fluorescent nanozyme: 1.921 g citric acid and 3.0 g cupric chloride dihydrate were added to 20.0 mL ultrapure water, and 1.0 mL ethylenediamine was added dropwise to the solution. After ultrasonication for 30 min, the mixture was quickly transferred to a polytetrafluoroethylene-lined high-temperature reactor and heated at 200.0 °C for 4.0 h. After the temperature of the reactant dropped to room temperature, it was centrifuged at 11000 rpm for 10 min, and then the solution was separated and the precipitate was washed with ethanol three times. Finally, it was dried in an oven into powder to obtain Cu@CDs. (2) Synthesis of MIP-Cu@CDs fluorescent nanozyme: 0.12 g Cu@CDs nanozyme and 0.16 g microcystin-LR were dissolved in 10.0 ml ethanol and ultrasonicated for 30 min to mix thoroughly. Next, 0.1568 g carboxyethylsilanetriol sodium salt and 0.633 g silicon tetraacetate were added to the mixture and stirred in the dark for 30.0 min. Then, 200.0 mL ammonia water was slowly added to the reaction system and stirred for 12.0 h. After stirring, the system was collected and freeze-dried into powder using a freeze dryer. Finally, the freeze-dried powder was collected and the MIP-Cu@CDs fluorescent nanozyme was extracted using methanol Soxhlet extraction and dried in an oven into powder.

3. A MIP-Cu@CDs fluorescent nanozyme, characterized in that: The fluorescent nanozyme is prepared by the method described in claim 1 or claim 2.

4. A MC-LR dual-modal detection method based on the MIP-Cu@CDs fluorescent nanozyme prepared according to claim 1, characterized in that: The method comprises the following steps: (1) Determine the absorbance value A of MC-LR standard of different concentrations at 510 nm s : Pipette the MIP-Cu@CDs fluorescent nanozyme solution into a centrifuge tube, add 2,4-DP, 4-AP solution and pH 7.5 buffer, then add the standard solution, mix well and react for a while, transfer to a cuvette, and measure the absorbance at 510 nm with a UV spectrophotometer, which is recorded as the absorbance value A s ; And / or, (2) measuring the fluorescence intensity value F at 420 nm of MC-LR standard with different concentrations under 340 nm excitation s : Pipette the MIP-Cu@CDs fluorescent nanozyme solution into a centrifuge tube, add 2,4-DP, 4-AP solution and pH 7.5 buffer, then add the standard solution, mix well and react for a while, transfer to a cuvette, and measure the fluorescence intensity at 420 nm using a fluorescence spectrometer, which is recorded as the fluorescence value F s ; (3) Constructing the linear regression equation of the standard The absorbance values ​​A measured using different concentrations of standard substances s and the concentration value of the standard to construct a UV spectrophotometer linear regression equation; and / or, using the fluorescence intensity values ​​F measured by the standard at different concentrations s The linear regression equation of fluorescence spectrometer was constructed based on the concentration value of the standard substance; (4) Use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested using a UV spectrophotometer. n ; and / or, using the sample to be tested instead of the standard to perform fluorescence spectrometer measurement to obtain the fluorescence intensity value F of the sample to be tested n ; (5) The absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the MC-LR residual value; and / or, the fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the MC-LR residual value; The MC-LR is microcystin-LR, 2,4-DP is 2,4-dichlorophenol, and 4-AP is 4-aminoantipyrine.

5. The dual-modal detection method according to claim 4, characterized in that: The concentration of MIP-Cu@CDs fluorescent nanozyme solution is 0.2 mg / ml; the concentration of 2,4-DP solution is 1.0 mmol / L; the concentration of 4-AP solution is 1.0 mmol / L; the buffer solution is phosphate buffer, pH 7.5; wherein, the volume ratio between MIP-Cu@CDs fluorescent nanozyme solution, 2,4-DP solution, 4-AP and phosphate buffer is: 10: 10: 10: 150-170.

6. The dual-modal detection method according to claim 4, characterized in that: In the step (1), the reaction time after uniform mixing is: reacting at 50° C. for 20.0 min after uniform mixing.

7. The dual-modal detection method according to claim 4, characterized in that: The test samples are aquatic food or environmental water bodies with MC-LR residues. Before testing, the test samples are filtered with filter paper or filter membrane with a pore size of 0.22 mm and the filtrate is collected.

8. The dual-modal detection method according to claim 4, characterized in that: The linear regression equation of the UV spectrophotometer is A = 0.00195C MC-LR + 0.05208; the linear regression equation of fluorescence spectrometer is F = 0.1248C MC-LR +62.1044.

9. A MC-LR residue detection kit, characterized in that: The kit comprises the MIP-Cu@CDs fluorescent nanozyme prepared according to claim 1, 2,4-DP, buffer, and 4-AP, wherein the 2,4-DP is 2,4-dichlorophenol, and the 4-AP is 4-aminoantipyrine.

10. Use of the MC-LR residue detection kit according to claim 9, characterized in that: The kit is used to detect MC-LR contamination in environmental and food samples.

Images