Quercetin colorimetric detection method based on NH2-MIL-101-CuCo nano-enzyme

Abstract

The invention provides a colorimetric detection method for quercetin on the basis of NH2-MIL-101-CuCo nano enzyme and a preparation method of the quercetin. The method comprises the following steps: preparing a first mixture formed by mixing NH2-MIL-101-CuCo nano-enzyme, a TMB (Tetramethylbenzidine) solution and a hydrogen peroxide solution with a sample to be detected respectively, and preparing a second mixture formed by mixing with a quercetin standard substance; reacting under the same reaction condition after the reaction to obtain absorbance values A1 and A0 under a preset wavelength; determining the content of the quercetin in the sample to be detected based on the A1 and a linear regression curve of the quercetin with different concentrations; the nano-enzyme is obtained by one-step synthesis of copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide through a hydrothermal method. According to the method provided by the invention, hydrogen bonds are formed by combining active hydrogen atoms in the nano-enzyme with hydroxyl groups in the quercetin, so that the quercetin can be specifically recognized and adsorbed, and the detection of the quercetin is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of food contamination detection, and in particular relates to a quercetin colorimetric detection method based on NH2-MIL-101-CuCo nanozyme. Background Art

[0002] Quercetin is a natural flavonol compound widely found in many plants. Its molecular formula is C 15 H 10 O7, has rich biological activities and pharmacological effects. Due to its special chemical structure, quercetin has broad application prospects in anti-oxidation, anti-inflammatory, and anti-tumor. However, the safety of quercetin also needs to be taken seriously. In the case of high doses or excessive intake, quercetin can cause inflammation, DNA damage, hypertension, and cancer (Ultra-sensitive amperometric determination of quercetin by using a glassy carbonelectrodemodified with a nanocomposite prepared from aminated graphenequantum dots,thiolatedβ-cyclodextrin and gold nanoparticles,DOI:10.1007 / s00604-019-4106-1). In addition, quercetin can also interact with some drugs, thereby affecting their metabolism in the body and thus affecting the efficacy (One-step solvothermal synthesis of nanoflake-nanorod WS2 hybrid for non-enzymatic detection of uric acid andquercetin in blood serum, DOI: 10.1016 / j.msec.2019.110217). Therefore, strengthening the monitoring and management of quercetin is of great significance for guiding its rational use, achieving health benefits and effectively avoiding its hazards.

[0003] Given the complexity of quercetin, its detection method also needs to be accurate and sensitive. Commonly used detection techniques include high performance liquid chromatography (HPLC) (Antioxidant study of quercetin and their metal complex and determination of stability constant by spectrophotometry method, DOI: 10.1016 / j.foodchem.2013.09.080), gas chromatography-mass spectrometry (GC-MS) (Efficient extraction methods and callus culture of quercetin from Indian white radish: yield analysis via HPLC quantification and assessment of cytotoxic activity, DOI: 10.1111 / ijfs.17043), liquid chromatography-mass spectrometry (LC-MS) (Pharmacokinetic comparison of quercetin, isoquercitrin, and quercetin-3-O-β-D-glucuronide in rats by HPLC-MS, DOI:10.7717 / peerj.6665), etc. These methods can accurately determine the quercetin content in the sample, providing a scientific basis for the research and application of quercetin. HPLC has the advantages of high separation efficiency and good repeatability, and has been widely used in the determination of quercetin content; GC-MS can be used for the determination of quercetin derivatives, but they need to be derivatized; LC-MS combines the separation performance of HPLC with the high sensitivity of mass spectrometry, providing a powerful means for the analysis of quercetin in complex samples. However, these traditional detection methods also have certain limitations, such as complex sample pretreatment, high detection cost, long time consumption, and demanding equipment requirements, making them difficult to use for large sample screening and real-time monitoring. Therefore, effective quercetin detection methods should be developed to protect public health. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. The present invention provides a quercetin colorimetric detection method based on NH2-MIL-101-CuCo nanozyme. The provided NH2-MIL-101-CuCo nanozyme has a peroxidase-like activity, can catalyze H2O2 to produce hydroxyl radicals, and then oxidize TMB to blue ox-TMB. The hydroxyl groups in quercetin will generate hydrogen bonds with the active hydrogen atoms in the NH2-MIL-101-CuCo nanozyme, that is, the NH2-MIL-101-CuCo nanozyme can play the π coordination effect of its metal active site and use the bond and effect of hydrogen bonds to complete the adsorption of quercetin, thereby affecting the enzyme activity of the NH2-MIL-101-CuCo nanozyme, showing a decrease in the colorimetric signal dependent on the concentration of quercetin, so that a quercetin detection method based on TMB color development can be developed. In the colorimetric (visible to the naked eye) mode, the higher the quercetin concentration, the less blue product ox-TMB generated by oxidation, and the lighter the blue color visible to the naked eye. Based on this, the present invention provides a colorimetric modal detection method for detecting quercetin based on NH2-MIL-101-CuCo nanozyme. This method can quickly perform on-site detection, making the detection process more convenient, and has important application value in the field of food contamination detection.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozyme, comprising:

[0007] A first mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and a sample to be tested, and a second mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and quercetin standards with different concentration gradients are prepared respectively;

[0008] The first mixture and the second mixture are reacted under the same reaction conditions, and the absorbance of the reaction products at a predetermined wavelength is measured, which correspond to absorbance values ​​A1 and A0, respectively;

[0009] Obtaining a linear regression curve based on the absorbance value A0 and quercetin standards with different concentration gradients, and determining the content of quercetin in the sample to be tested based on the linear regression curve and the absorbance value A1;

[0010] The NH2-MIL-101-CuCo nanozyme is synthesized in one step by a hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide.

[0011] The NH2-MIL-101-CuCo nanozyme in the present invention has a peroxidase-like activity, can catalyze H2O2 to produce hydroxyl radicals, and then oxidize TMB to blue ox-TMB. The hydroxyl in quercetin can generate hydrogen bonds with the active hydrogen atoms in NH2-MIL-101-CuCo nanozyme, that is, NH2-MIL-101-CuCo nanozyme can play the π coordination effect of its metal active site and use the bond and effect of hydrogen bond to complete the adsorption of quercetin, thereby affecting the enzyme activity of NH2-MIL-101-CuCo nanozyme, showing that the colorimetric signal of quercetin concentration dependence decreases, so that a quercetin detection method based on TMB color development can be developed. In colorimetric (visible to the naked eye) mode, the higher the quercetin concentration, the less the blue product ox-TMB generated by oxidation, and the lighter the blue visible to the naked eye. Finally, a colorimetric detection method based on NH2-MIL-101-CuCo nanozyme for detecting quercetin was established.

[0012] Compared with traditional methods (LC-MS, GC-MS), the method provided by the present invention is a new nanotechnology-based method that performs better in terms of detection limit and linearity. Colorimetric detection has the advantages of sensitivity, accuracy and visualization, and can be used in combination with other mature methods to achieve rapid detection of quercetin on site.

[0013] According to an embodiment of the present invention, the predetermined wavelength is 640-660 nanometers. According to a preferred embodiment of the present invention, the predetermined wavelength is 652 nanometers.

[0014] According to an embodiment of the present invention, the reaction conditions are pH 3.0-4.0, 40-50°C for 10-20 minutes. According to a preferred embodiment of the present invention, the reaction conditions are pH 3.5, 40°C for 15 minutes.

[0015] According to some embodiments of the present invention, the reaction condition of pH 3.0-4.0 is obtained by adding acetic acid-sodium acetate buffer with a pH value of 3.0-4.0; the concentration of the NH2-MIL-101-CuCo nanozyme solution is 0.15 mg / mL; the concentration of the TMB solution is 1.0 mmol / L; the concentration of the hydrogen peroxide solution is 1.0 mmol / L; the volume ratio between the NH2-MIL-101-CuCo nanozyme solution, TMB solution, hydrogen peroxide solution and acetic acid-sodium acetate buffer is 15:10:10:145-165.

[0016] The second aspect of the present invention provides a colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozyme, comprising:

[0017] The NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and the sample to be tested are mixed, and then mixed with quercetin standards of different concentration gradients to obtain a mixed solution;

[0018] The mixed solution is reacted at a pH of 3.0 to 4.0 and a temperature of 40° C. to 50° C., and the absorbance of the product after the reaction is measured at a wavelength of 640 to 660 nanometers;

[0019] Based on the absorbance value, determining the content of quercetin in the sample to be tested;

[0020] The NH2-MIL-101-CuCo nanozyme is synthesized in one step by a hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide.

[0021] Using NH2-MIL-101-CuCo nanozyme and the standard addition method for colorimetric detection of quercetin can reduce the interference of sample matrix effects and improve the accuracy and reliability of analytical results. This method calculates the concentration of the substance to be tested in the sample by adding a known amount of standard solution to the sample to be tested and measuring its absorbance change; it can compensate for the signal enhancement or inhibition effect caused by the sample matrix, thereby improving the accuracy of the analysis.

[0022] According to an embodiment of the present invention, the sample to be tested is a protein powder containing quercetin.

[0023] According to an embodiment of the present invention, the protein powder containing quercetin is pre-treated using a 0.22 μm filter membrane before detection.

[0024] According to an embodiment of the present invention, the NH2-MIL-101-CuCo nanozyme is prepared by the following method:

[0025] Copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide are mixed in a predetermined molar ratio, reacted at a predetermined temperature, and cooled to obtain a mixed product;

[0026] The mixed product is centrifuged, the precipitate is washed, and the NH2-MIL-101-CuCo nanozyme is obtained by drying;

[0027] According to an embodiment of the present invention, the predetermined molar ratio is 1:1:0.005-0.01:0.5-1. According to a preferred embodiment of the present invention, the predetermined molar ratio is 1:1:0.008:0.5.

[0028] According to an embodiment of the present invention, the predetermined temperature is 150-160 degrees Celsius.

[0029] According to an embodiment of the present invention, the reaction is carried out at the predetermined temperature for 8 to 15 hours.

[0030] The third aspect of the present invention provides a colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozyme, comprising:

[0031] A first mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and a sample to be tested, and a second mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and quercetin standards with different concentration gradients were prepared respectively;

[0032] The first mixture and the second mixture are reacted at pH 3.0-4.0 and 40-50° C. for 10-20 minutes, respectively, and the absorbance of the reaction products at a wavelength of 652 nanometers is measured, which correspond to absorbance values ​​A1 and A0, respectively;

[0033] Obtaining a linear regression curve based on the absorbance value A0 and quercetin standards with different concentration gradients, and determining the content of quercetin in the sample to be tested based on the linear regression curve and the absorbance value A1;

[0034] The NH2-MIL-101-CuCo nanozyme is synthesized in one step by a hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide.

[0035] According to a fourth aspect of the present invention, there is provided an NH2-MIL-101-CuCo nanozyme for colorimetric detection of quercetin, which is obtained by a one-pot hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide, comprising:

[0036] Copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide are mixed in a molar ratio of 1:1:0.005-0.1:0.5-1, reacted at 150-160° C. for 8-15 hours, and cooled to obtain a mixed product;

[0037] The mixed product is centrifuged, the obtained precipitate is washed, and then dried to obtain the NH2-MIL-101-CuCo nanozyme.

[0038] According to a fifth aspect of the present invention, a quercetin detection kit is provided, the kit comprising NH2-MIL-101-CuCo nanozyme, buffer, TMB solution and hydrogen peroxide solution;

[0039] The kit mentioned also includes an acetic acid-sodium acetate buffer at pH 3.0-4.0 or a phosphate buffer at pH 3.0-4.0.

[0040] The beneficial effects achieved by the present invention are at least:

[0041] (1) The NH2-MIL-101-CuCo nanozyme used in the present invention has a high specific surface area, a porous structure and excellent chemical stability. These advantages enable the NH2-MIL-101-CuCo nanozyme to have more active sites, which can improve the adsorption capacity of quercetin. 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.

[0042] (2) Compared with the existing traditional methods for detecting quercetin, such as high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), etc., the method provided by the present invention does not require complex instruments or professional skills and can be quickly detected on site. In addition, the method has high sensitivity and low detection limit, which means that quercetin can be effectively detected even at trace levels. In particular, the NH2-MIL-101-CuCo nanozyme used in the present invention has a high peroxidase-like activity, which can catalyze H2O2 to produce hydroxyl radicals, and then oxidize TMB to blue ox-TMB. However, the hydroxyl group in quercetin can generate hydrogen bonds with the active hydrogen atoms in NH2-MIL-101-CuCo nanozyme, that is, NH2-MIL-101-CuCo nanozyme can play the π coordination effect of its metal active site and use the bond and effect of hydrogen bonds to complete the adsorption of quercetin, thereby affecting the enzyme activity of NH2-MIL-101-CuCo nanozyme, showing a decrease in the colorimetric signal dependent on quercetin concentration, so that a quercetin detection method based on TMB color development can be developed. In summary, the present invention has developed a quercetin colorimetric modal detection method based on NH2-MIL-101-CuCo nanozyme using a decrease in colorimetric signal.

[0043] (3) The present invention proposes a colorimetric modality detection method for quercetin based on NH2-MIL-101-CuCo nanozyme. According to the relationship between quercetin concentration and TMB color development, a colorimetric modality rapid determination method for quercetin can be established. In addition, this method not only simplifies the detection process and improves efficiency, but also greatly reduces reagent consumption, achieves the goal of green and environmentally friendly detection, provides new possibilities for on-site rapid detection of quercetin, makes the detection process more convenient, and has important application value in the field of food contamination detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A synthesis diagram of NH2-MIL-101-CuCo provided according to an embodiment of the present invention and a principle diagram of colorimetric detection of quercetin using the NH2-MIL-101-CuCo+TMB+H2O2 system.

[0045] Figure 2 This is a transmission electron microscope image and element distribution map of the NH2-MIL-101-CuCo nanozyme provided according to an embodiment of the present invention.

[0046] Figure 3 This is an X-ray photoelectron energy diagram of the NH2-MIL-101-CuCo nanozyme provided according to an embodiment of the present invention.

[0047] Figure 4 This is an infrared image of NH2-MIL-101-CuCo nanozyme provided according to an embodiment of the present invention.

[0048] Figure 5 This is a diagram of the hydrated particle size and potential of the NH2-MIL-101-CuCo nanozyme provided according to an embodiment of the present invention.

[0049] Figure 6 Ultraviolet spectra of (1) TMB, (2) H2O2 (3) TMB+H2O2, (4) NH2-MIL-101-CuCo+TMB, (5) NH2-MIL-101-CuCo+TMB+H2O2, and (6) NH2-MIL-101-CuCo+TMB+H2O2+quercetin combination measured by ultraviolet spectrophotometer according to an embodiment of the present invention.

[0050] Figure 7 The optimized conditions for detecting quercetin by the NH2-MIL-101-CuCo+TMB+H2O2 system measured by an ultraviolet spectrophotometer according to an embodiment of the present invention. Figure (A) shows the optimization result of pH, with the optimal value being pH 3.5; Figure (B) shows the optimization result of reaction temperature, with the optimal value being 40°C. Figure (C) shows the optimized time conditions for the catalytic generation of oxTMB by the NH2-MIL-101-CuCo+TMB+H2O2 system, with the optimal reaction time being 15.0min; Figure (D) shows the optimized time conditions for detecting quercetin by the NH2-MIL-101-CuCo+TMB+H2O2 system, with the optimal reaction time being 15.0min.

[0051] Figure 8 The absorbance and standard curve of quercetin at different concentrations provided according to an embodiment of the present invention.

[0052] Fig. 9This is a bar graph of the colorimetric mode provided in an embodiment of the present invention for detecting interfering substances in a complex environment, proving that the colorimetric mode system of NH2-MIL-101-CuCo+TMB+H2O2 has good selectivity for quercetin and similar flavonoids. DETAILED DESCRIPTION

[0053] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0054] Metal-Organic Frameworks (MOFs) have the advantages of high porosity, large specific surface area, good chemical and thermal stability, and have broad application prospects in adsorption, separation, catalysis, etc. The MOFs material can be used to detect quercetin quickly, with high sensitivity, high selectivity, and high stability, and can achieve high sensitivity and high selectivity detection of quercetin. Therefore, the detection technology based on metal frameworks shows great application prospects in the detection of quercetin, providing new ideas for the rapid and accurate detection of quercetin. In view of this, this paper developed a new method for detecting quercetin based on colorimetric metal frameworks. This method uses the high sensitivity and selectivity of MOFs materials to achieve rapid identification of quercetin through color changes, thereby simplifying the analysis process, improving analysis efficiency, reducing reagent consumption, and achieving green and environmentally friendly detection goals, providing new possibilities for on-site rapid detection of quercetin.

[0055] Specifically, the present invention uses NH2-MIL-101-CuCo nanozyme to detect the content of quercetin. During the research process, it was found that compared with other nanozymes, such as NH2-MIL-101-Fe nanozyme, the NH2-MIL-101-CuCo nanozyme provided by the present invention has a very low detection limit of only 0.030 μM. Compared with other methods, its detection accuracy is higher, more precise, and highly sensitive. Moreover, the colorimetric detection of quercetin using the nanozyme provided by the present invention can be directly observed by the naked eye without the need for complicated operations and professionals. In addition, it can also resist the interference of various substances, including but not limited to Cd 2+ , Fe 3 + Mg 2+ , Cu 2+ , Hg 2+ 、CO3 2- 、Al 3+ 、Co 2+ , Ca 2+ 、NO2 - , ascorbic acid, serine, glutamic acid, arginine, glycine, glucose, rutin, etc.

[0056] The NH2-MIL-101-CuCo nanozyme used is prepared by a one-pot hydrothermal method from simple and readily available raw materials without separation of intermediates, and the NH2-MIL-101-CuCo nanozyme with peroxidase-like activity is directly obtained.

[0057] The NH2-MIL-101-CuCo nanozyme is synthesized in one step by a hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide. These substances are mixed in a predetermined molar ratio and reacted at a predetermined temperature to obtain the NH2-MIL-101-CuCo nanozyme.

[0058] According to a specific embodiment, the predetermined molar ratio is 1:1:0.005-0.1:0.5-1. During the study, it was found that when the molar ratio of cupric chloride dihydrate and cobalt chloride hexahydrate is 1:1, the activity of the prepared nanozyme is very high. When 2-aminoterephthalic acid is added during the preparation process, the modified NH2 will increase, thereby increasing the hydrogen atoms of the prepared nanozyme and enhancing the ability to adsorb quercetin. During the study, it was found that when the ratio of raw materials changes, it will affect the activity of the prepared nanozyme. For example, when it contains only one metal element, such as only cupric chloride dihydrate or cobalt chloride hexahydrate, the activity of the prepared nanozyme is poor.

[0059] According to a specific embodiment, the predetermined temperature is 150 to 160 degrees Celsius. According to a specific embodiment, the reaction is carried out at the predetermined temperature for 8 to 15 hours.

[0060] According to a specific embodiment, 0.5g of copper chloride dihydrate, 0.5g of cobalt chloride hexahydrate, 0.004g of 2-aminoterephthalic acid, and 0.25g of sodium hydroxide (flaky) are dissolved in 30.0mL of ultrapure water, and after ultrasonication for 30.0min, the mixture is quickly transferred to a high-temperature reactor lined with polytetrafluoroethylene and heated at 150.0°C for 10.0h. After the temperature of the reactant drops to room temperature, centrifuge at a speed of 12000.0rpm for 10.0min, separate the solution, wash the precipitate with ethanol, and finally dry it in an oven into powder to obtain NH2-MIL-101-CuCo nanozyme.

[0061] The NH2-MIL-101-CuCo nanozyme can be used for colorimetric detection of quercetin. It can be used for the detection of a variety of samples, for example, it can be used to detect quercetin residues in protein powder. By detecting quercetin residues in food, the safety and quality of food can be ensured, and food safety problems caused by excessive addition or improper handling can be avoided. In addition, the detection of quercetin residues in protein can also be used to evaluate its effects on drug metabolism, biological activity and potential health effects, thereby providing a scientific basis for drug development and clinical applications.

[0062] The provided NH2-MIL-101-CuCo nanozyme can be used to perform a colorimetric detection method for quercetin, specifically including:

[0063] preparing a first mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and a sample to be tested, and preparing a second mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and quercetin standards with different concentration gradients;

[0064] The first mixture and the second mixture are reacted under the same reaction conditions, and the absorbance of the reaction products at a predetermined wavelength is measured, which correspond to absorbance values ​​A1 and A0, respectively;

[0065] A linear regression curve is obtained based on the absorbance value A0 and quercetin standards with different concentration gradients, and the content of quercetin in the sample to be tested is determined based on the linear regression curve and the absorbance value A1.

[0066] The predetermined wavelength mentioned is 640-660 nanometers. According to a preferred embodiment of the present invention, the predetermined wavelength is 652 nanometers.

[0067] The same reaction conditions mentioned above are 10 to 20 minutes at pH 3.0 to 4.0 and 40 to 50 degrees Celsius. According to a preferred embodiment of the present invention, the same reaction conditions are 15 minutes at pH 3.5 and 40 degrees Celsius. After the reaction is completed, the mixture is transferred to a cuvette for detection.

[0068] According to a specific embodiment, the concentration of the NH2-MIL-101-CuCo nanozyme solution is 0.15 mg / mL; the concentration of the TMB solution is 1.0 mmol / L; the concentration of the H2O2 solution is 1.0 mmol / L; the buffer solution is an acetic acid-sodium acetate buffer with a pH of 3.5; the volume ratio between the NH2-MIL-101-CuCo nanozyme solution, TMB solution, H2O2 solution and acetic acid-sodium acetate buffer is: 15:10:10:145-165; in addition, the buffer used can also be a phosphate buffer.

[0069] Preferably, the test sample is a plant protein powder containing residual quercetin. Before the test, the test sample is filtered with a filter paper or filter membrane with a pore size of 0.22 μm, and the filtrate is collected.

[0070] The method established by the present invention has a good linear relationship between the change value of the absorption intensity at 652nm and the quercetin concentration in the range of 0.88-80.84μM (it can be seen that the quercetin concentration range spans a very large range), and the regression equation is ΔA=0.0284C 槲皮素 -0.0327, the correlation coefficient is 0.9955, and the detection limit is 0.030 μM; the formula for the minimum detection limit (3σ / k, σ is the standard deviation of 11 blank samples) is used to obtain the absorbance linear equation of the present invention is ΔA=0.0284C 槲皮素 -0.0327.

[0071] The following specific embodiments of the present invention are described. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. It should be noted that the following embodiments are provided to better understand the present invention, but are not intended to limit the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

[0072] Related reagents:

[0073] The raw materials used in the present invention: 2-aminoterephthalic acid (>98.0%), quercetin (>95.0%) were purchased from Shanghai Aladdin Reagent Company; copper chloride dihydrate (99.0%), cobalt chloride hexahydrate (99.9%) and TMB (3,3',5,5'-tetramethylbenzidine, 99.5%) were purchased from Shanghai McLean Biochemical Technology Co., Ltd.; H2O2, i.e. hydrogen peroxide solution and sodium hydroxide were purchased from Sinopharm Group Pharmaceutical Co., Ltd.; acetic acid-sodium acetate buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd. All chemicals were purchased directly from suppliers without further purification; the water used in the experimental process was ultrapure water.

[0074] Acetic acid-sodium acetate buffer (pH 3.5) is prepared by taking 50.0 mL of acetic acid-sodium acetate buffer with a pH of 5.5, and adjusting the pH of the acetic acid-sodium acetate buffer with a pH of 5.5 to 3.5 using a pH meter and with the aid of a hydrochloric acid solution and a sodium hydroxide solution.

[0075] Example 1

[0076] Example 1: NH2-MIL-101-CuCo nanozyme with peroxidase-like activity was prepared by the following method, which specifically includes:

[0077] S1: Synthesis of NH2-MIL-101-CuCo nanozyme: NH2-MIL-101-CuCo nanozyme was synthesized by a one-pot hydrothermal method. Specifically, it includes:

[0078] References Figure 1 As shown, 0.5 g of copper chloride dihydrate, 0.5 g of cobalt chloride hexahydrate, 0.004 g of 2-aminoterephthalic acid, and 0.25 g of sodium hydroxide (flaky) were dissolved in 30.0 mL of ultrapure water. After ultrasonication for 30.0 min, the mixture was quickly transferred to a high-temperature reactor lined with polytetrafluoroethylene. And heated at 150.0 ° C for 10.0 h. After the temperature of the reactant dropped to room temperature, it was centrifuged at 12000.0 rpm for 10.0 min, and then the solution was separated, and the precipitate was washed with ethanol 3 times, and finally dried in an oven to powder to obtain NH2-MIL-101-CuCo nanozyme.

[0079] Then the synthesized NH2-MIL-101-CuCo nanozyme was analyzed. From the TEM image, it can be seen that the synthesized NH2-MIL-101-CuCo is cubic in shape, indicating that the NH2-MIL-101-CuCo nanozyme was successfully synthesized. At the same time, the EDS spectrum results confirmed the uniform distribution of C, N, O, Cu and Co in NH2-MIL-101-CuCo (such as Figure 2 ), which is consistent with the results of X-ray photoelectron spectroscopy (XPS) (as shown in Figure 3 This provides strong evidence for the successful synthesis of NH2-MIL-101-CuCo.

[0080] In addition, if Figure 4 As shown in the Fourier transform infrared spectrum, NH2-MIL-101-CuCo nanozyme has a wavelength of 1000-500 cm -1 and 3500~3300cm -1 Characteristic peaks appeared at the positions, corresponding to the skeleton stretching vibration absorption peak of terephthalic acid and the stretching vibration absorption peak of N—H, which also indicated that NH2-MIL-101-CuCo nanozyme was successfully prepared.

[0081] like Figure 5 As shown, the average diameter of NH2-MIL-101-CuCo nanozyme is about 300 nm, and the zeta potential is 8.27±0.1 mV, which also confirms that the present invention successfully prepared NH2-MIL-101-CuCo nanozyme.

[0082] Example 2

[0083] Example 2 Using the NH2-MIL-101-CuCo nanozyme prepared in Example 1, whether it can be used for the detection of quercetin was developed, specifically including:

[0084] In a 0.5 mL centrifuge tube, 1.0 mmol / L TMB (label 1) was added; 1.0 mmol / L H2O2 solution (label 2); 1.0 mmol / L TMB and 1.0 mmol / L H2O2 solution (label 3); 0.15 mg / mL NH2-MIL-101-CuCo nanozyme and 1.0 mmol / L TMB (label 4); 0.15 mg / mL NH2-MIL-101-CuCo nanozyme, 1.0 mmol / L TMB and 1.0 mmol / L H2O2 solution (label 5); 0.15 mg / mL NH2-MIL-101-CuCo nanozyme, 1.0 mmol / L TMB, 1.0mmol / LH2O2 solution and quercetin (label 6), then add acetic acid-sodium acetate buffer (prepare 200.0μL system) with pH=3.5, mix well, react at 40℃ for 15.0min, transfer to a cuvette, and measure the absorbance at 652nm with a UV spectrophotometer, which is recorded as absorbance value A1.

[0085] Figure 6 The UV spectrum of each number measured by a UV spectrophotometer is shown. Figure 6 The numbers 1 to 6 represent mixed systems of different substances:

[0086] 1: TMB; 2: H2O2; 3: TMB+H2O2; 4: NH2-MIL-101-CuCo+TMB; 5: NH2-MIL-101-CuCo+TMB+H2O2; 6: NH2-MIL-101-CuCo+TMB+H2O2+Quercetin.

[0087] from Figure 6 We can clearly see that the experimental group corresponding to number 5, NH2-MIL-101-CuCo+TMB+H2O2, obviously produced oxTMB, and the absorbance at 652nm was enhanced; the experimental group corresponding to number 6, NH2-MIL-101-CuCo+TMB+H2O2+quercetin combination inhibited the production of oxTMB, and the absorbance decreased.

[0088] The results show that the prepared NH2-MIL-101-CuCo nanozyme has a high peroxidase-like activity, which can catalyze H2O2 to produce hydroxyl radicals, and then oxidize TMB to blue ox-TMB. However, the hydroxyl groups in quercetin will form hydrogen bonds with the active hydrogen atoms in the NH2-MIL-101-CuCo nanozyme, that is, the NH2-MIL-101-CuCo nanozyme can play the role of π coordination of its metal active site and use the bonding and action of hydrogen bonds to complete the adsorption of quercetin, thereby affecting the enzyme activity of the NH2-MIL-101-CuCo nanozyme, showing a decrease in the colorimetric signal dependent on the concentration of quercetin, so that a quercetin detection method based on TMB color development can be developed.

[0089] Example 3

[0090] In order to further study the optimal reaction conditions for the generation of the blue product oxTMB catalyzed by NH2-MIL-101-CuCo nanozyme, Example 3 optimized the optimal conditions of the detection reagent under the ultraviolet absorption spectroscopy mode using the controlled variable method.

[0091] See also Figure 7 As shown, A to C are the optimization of multiple conditions such as pH, reaction temperature and reaction time using a system without adding the test substance quercetin (NH2-MIL-101-CuCo nanozyme + TMB + hydrogen peroxide solution system). Figure 7 (A) shows the absorbance results under different pH conditions when other conditions are the same. The results show that the optimal pH is 3.5.

[0092] Figure 7 (B) shows the absorbance results at different reaction temperatures when other conditions are the same. As the temperature increases, the absorbance value gradually increases until the temperature reaches 40°C, when the absorbance no longer changes significantly, so the optimal reaction temperature is 40°C.

[0093] Figure 7 (C) Absorbance results at different reaction times when other conditions are the same. As the reaction time increases, the absorbance value gradually increases until 15.0 min, when the reaction reaches the equilibrium point and the absorbance no longer changes. Therefore, 15.0 min is confirmed to be the optimal reaction time for this system.

[0094] The test substance quercetin was added to the above optimized system, such as Figure 7 As shown in (D), with the increase of reaction time, the absorption peak of oxTMB continued to increase until 15.0 min, when the reaction reached the equilibrium point, at which time TMB was completely oxidized and the absorbance at 652 nm no longer changed. Therefore, the optimal reaction time for detecting quercetin was 15.0 min.

[0095] Example 4

[0096] Referring to the optimal reaction conditions found in the above examples, Example 4 established a standard curve and tested the samples, including:

[0097] (1) Establish the linear equation for detection

[0098] 15.0 μL of 0.15 mg / mL NH2-MIL-101-CuCo nanozyme solution, 10.0 μL of 1.0 mmol / L TMB, and 10.0 μL of 1.0 mmol / L H2O2 were added to 145.0 μL of acetic acid-sodium acetate buffer with a pH value of 3.5, followed by the addition of 20.0 μL of quercetin standard solution of different concentrations, and the mixture was reacted at 40 °C for 15.0 min. The absorbance value A0 at 652 nm was measured using a UV spectrophotometer.

[0099] The measured absorbance values ​​are shown in Table 1 below:

[0100] Table 1 Absorbance values ​​corresponding to different quercetin concentrations

[0101]

[0102] See also Figure 8 As shown, after fitting and settling with origin software, it was found that there was a good linear relationship between the change in absorption intensity at 652nm and the quercetin concentration in the range of 0.88-80.84μM, and the regression equation was ΔA=0.0284C 槲皮素 -0.0327, correlation coefficient was 0.9955, and detection limit was 0.030 μM.

[0103] (2) Pretreatment of samples to be tested

[0104] The sample to be tested is a protein powder with residual quercetin. The protein powder is centrifuged to obtain the supernatant, and then the sample to be tested is filtered using a filter paper or filter membrane with a pore size of 0.22 μm, the filtrate is collected, and the pH is adjusted to 3.5 with an acetic acid-sodium acetate buffer for testing.

[0105] (3) Testing of samples to be tested

[0106] Pipette 15.0 μL of 0.15 mg / mL NH2-MIL-101-CuCo nanozyme solution into a centrifuge tube, add 1.0 mmol / L TMB and 1.0 mmol / L H2O2 solution, and then add the pretreated sample prepared in step (2) with the pH value adjusted to 3.5 (the prepared system is 200.0 μL), mix well, react at 40°C for 15.0 min, transfer to a cuvette, and measure the absorbance at 652 nm using a UV spectrophotometer, which is recorded as absorbance A1;

[0107] (4) Calculate the concentration of quercetin in the sample to be tested.

[0108] The linear equation of the combined absorbance is ΔA = 0.0284C 槲皮素 -0.0327; and the absorbance value A1 measured in step (3), the quercetin concentration C in the sample to be tested is calculated 槲皮素 The value of is the quercetin residue value.

[0109] Example 5

[0110] Example 5 further studied the effects of different environmental interfering substances on the quercetin colorimetric detection method based on NH2-MIL-101-CuCo nanozyme.

[0111] 15.0 μL of 0.15 mg / mL NH2-MIL-101-CuCo nanozyme solution, 10.0 μL of 1.0 mmol / L TMB and 10.0 μL of 1.0 mmol / L H2O2 were added to 145.0 μL of acetic acid-sodium acetate buffer with a pH value of 3.5, and then 20.0 μL of 80 μM of each substance was added and reacted at 40°C for 15.0 min, and the absorbance at 652 nm was measured using a UV spectrophotometer.

[0112] See also Fig. 9 , demonstrating the environmental selectivity of the method provided by the present invention tested using different samples. Fig. 9 The numbers in the table correspond to the substances added: 1 blank (i.e., acetic acid-sodium acetate buffer at pH 3.5); 2 Cd 2 + ;3Fe 3+ ; 4Mg 2+ ; 5Cu 2+ ;6Hg 2+ ;7CO3 2- ;8Al 3+ ;9Co 2+ ;10Ca 2+ ;11NO2 -; 12 Ascorbic acid; 13 Serine; 14 Glutamic acid; 15 Arginine; 16 Glycine; 17 Glucose; 18 Rutin; 19 Quercetin.

[0113] The results show that the present invention can specifically detect quercetin and similar flavonoids, while other interfering substances have no effect on the method of the present invention, demonstrating the anti-interference and high sensitivity of the present invention.

[0114] Example 6

[0115] Example 6 Using the method provided by the present invention, protein powder was selected as a real sample for testing. The protein powder used was randomly purchased and sampled from Hefei, China.

[0116] According to the literature, the protein powder was first pretreated: 1.0 g of protein powder (pea protein powder, chickpea protein powder, soy protein powder and microbial protein powder) was dissolved in 30.0 mL of ultrapure water, and after ultrasonic treatment for 30.0 minutes, the supernatant was taken as the sample solution after centrifugal filtration.

[0117] Before the test, the pH of the prepared sample solution was adjusted to 3.5. Then, 15.0 μL of 0.15 mg / mL NH2-MIL-101-CuCo nanozyme solution, 10.0 μL of 1.0 mmol / L TMB, and 10.0 μL of 1.0 mmol / L H2O2 were added to the sample solution. At this time, different concentrations of quercetin were added to the above system by the standard addition method, mixed evenly, reacted at 40 ° C for 15.0 min, moved to a cuvette, and measured the change in absorbance at 652 nm by ultraviolet absorption spectroscopy.

[0118] As shown in Table 2, different situations of colorimetric detection of actual samples are shown. The recovery of the colorimetric method ranged from 100.35% to 121.65%, and the relative standard deviation (RSD) was less than 5.00%. This shows that the NH2-MIL-101-CuCo nanozyme has good feasibility and reproducibility in detecting quercetin in actual samples.

[0119] Table 2 Test results

[0120]

[0121]

[0122] In summary, the present invention has developed a colorimetric modal detection method for quercetin based on NH2-MIL-101-CuCo nanozyme using a colorimetric signal drop. In the colorimetric (visible to the naked eye) mode, the higher the concentration of quercetin, the less blue product oxTMB generated by oxidation, and the lighter the blue color visible to the naked eye. Finally, a colorimetric modal detection method for detecting quercetin based on NH2-MIL-101-CuCo nanozyme can be established. In addition, the method can quickly carry out on-site detection, making the detection process more convenient, and has important application value in the field of food contamination detection.

[0123] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementation methods", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0124] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozyme, characterized in that: include: A first mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and a sample to be tested, and a second mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and quercetin standards with different concentration gradients are prepared respectively; The first mixture and the second mixture are reacted under the same reaction conditions, and the absorbance of the reaction products at a predetermined wavelength is measured, which correspond to absorbance values ​​A1 and A0, respectively; Obtaining a linear regression curve based on the absorbance value A0 and quercetin standards with different concentration gradients, and determining the content of quercetin in the sample to be tested based on the linear regression curve and the absorbance value A1; The NH2-MIL-101-CuCo nanozyme is synthesized in one step by a hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide.

2. The method according to claim 1, characterized in that The predetermined wavelength is 640-660 nanometers, preferably 652 nanometers.

3. The method according to claim 1, characterized in that The reaction conditions are pH 3.0-4.0, 40°C-50°C for 10-20 minutes; Optionally, the reaction is carried out at pH 3.5 and 40° C. for 15 minutes.

4. The method according to claim 3, characterized in that The reaction conditions of pH 3.0-4.0 are obtained by adding acetic acid-sodium acetate buffer with a pH value of 3.0-4.

0. The concentration of the NH2-MIL-101-CuCo nanozyme solution is 0.15 mg / mL; the concentration of the TMB solution is 1.0 mmol / L; the concentration of the hydrogen peroxide solution is 1.0 mmol / L; the volume ratio of the NH2-MIL-101-CuCo nanozyme solution, TMB solution, hydrogen peroxide solution and acetic acid-sodium acetate buffer is 15:10:10:145-165.

5. A colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozyme, characterized in that: include: The NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and the sample to be tested are mixed, and then mixed with quercetin standards of different concentration gradients to obtain a mixed solution; The mixed solution is reacted at a pH of 3.0 to 4.0 and a temperature of 40° C. to 50° C., and the absorbance of the reaction product at a wavelength of 640 to 660 nanometers is measured; Based on the absorbance measurement result, determining the content of quercetin in the sample to be tested; The NH2-MIL-101-CuCo nanozyme is synthesized in one step by a hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide.

6. The method according to claim 1, characterized in that The sample to be tested is a protein powder containing quercetin; Optionally, the quercetin-containing protein powder is pre-treated using a 0.22 μm filter membrane before detection.

7. The method according to claim 1, characterized in that The NH2-MIL-101-CuCo nanozyme was prepared by the following method: Copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide are mixed in a predetermined molar ratio, reacted at a predetermined temperature, and cooled to obtain a mixed product; The mixed product is centrifuged, the precipitate is washed, and the precipitate is dried to obtain the NH2-MIL-101-CuCo nanozyme; Optionally, the predetermined molar ratio is 1:1:0.005-0.01:0.5-1; Optionally, the predetermined temperature is 150 to 160 degrees Celsius; Optionally, the reaction is carried out at the predetermined temperature for 8 to 15 hours.

8. A colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozyme, characterized in that: include: A first mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and a sample to be tested, and a second mixture of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution and quercetin standards with different concentration gradients are prepared respectively; The first mixture and the second mixture are reacted at pH 3.0-4.0 and 40-50° C. for 10-20 minutes, and the absorbance of the reaction product at a wavelength of 652 nanometers is measured, which correspond to absorbance values ​​A1 and A0, respectively; Obtaining a linear regression curve based on the absorbance value A0 and quercetin standards with different concentration gradients, and determining the content of quercetin in the sample to be tested based on the linear regression curve and the absorbance value A1; The NH2-MIL-101-CuCo nanozyme is synthesized in one step by a hydrothermal method using cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide.

9. An NH2-MIL-101-CuCo nanozyme for colorimetric detection of quercetin, characterized in that: Obtained by a one-pot hydrothermal process from cupric chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide, including: Copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide are mixed in a molar ratio of 1:1:0.005-0.01:0.5-1, reacted at 150-160 degrees Celsius for 8-15 hours, and then cooled to obtain a mixed product; The mixed product is centrifuged, the precipitate is washed, and the NH2-MIL-101-CuCo nanozyme is obtained by drying.

10. A quercetin detection kit, characterized in that: The kit includes NH2-MIL-101-CuCo nanozyme, buffer, TMB solution and hydrogen peroxide solution; Optionally, the kit includes acetic acid-sodium acetate buffer or phosphate buffer at pH 3-4.

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