Colorimetric detection of quercetin based on nh2-mil-101-cucco nanoszyme

Abstract

This invention provides a colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozymes. The method includes: preparing a first mixture by mixing NH2-MIL-101-CuCo nanozymes, TMB solution, and hydrogen peroxide solution with the sample to be tested, and a second mixture by mixing with a quercetin standard; reacting the mixture under the same reaction conditions to obtain absorbance values ​​A1 and A0 at predetermined wavelengths; determining the quercetin content in the sample based on A1 and linear regression curves of different concentrations of quercetin; the nanozyme is synthesized in one step via a hydrothermal method using copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid, and sodium hydroxide. The method utilizes hydrogen bonds formed by the active hydrogen atoms in the nanozyme and the hydroxyl groups in quercetin to specifically recognize and adsorb quercetin, thus enabling the detection of quercetin.

Description

Technical Field

[0001] This invention belongs to the field of food contamination detection technology, and particularly relates to a colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozyme. Background Technology

[0002] Quercetin is a natural flavonol compound widely found in various plants, with the molecular formula C. 15 H 10 O7 possesses abundant biological activity and pharmacological effects. Quercetin, due to its unique chemical structure, shows broad application prospects in antioxidation, anti-inflammation, and antitumor activities. However, the safety of quercetin also requires attention. High doses or excessive intake of quercetin can cause inflammation, DNA damage, hypertension, and cancer (Ultra-sensitive amperometric determination of quercetin by using a glassy carbon electron demodified with a nanocomposite prepared from aminated graphene quantum dots, thiolated β-cyclodextrin and gold nanoparticles, DOI:10.1007 / s00604-019-4106-1). Furthermore, quercetin can interact with some drugs, affecting their metabolism and thus their therapeutic efficacy (One-step solvothermal synthesis of nanoflake-nanorod WS2 hybrid for non-enzymatic detection of uric acid and quercetin 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 while effectively avoiding its harms.

[0003] Given the complexity of quercetin, its detection methods must be precise 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), and liquid chromatography-mass spectrometry (LC-MS) (Pharmacokinetic comparison of quercetin, isoquercitrin, and quercetin-3-O-β-D-glucuronide in rats by Methods such as HPLC-MS (DOI:10.7717 / peerj.6665) can accurately determine the quercetin content in samples, providing a scientific basis for quercetin research and application. HPLC has advantages such as high separation efficiency and good repeatability, and is widely used in quercetin content determination. GC-MS can be used to determine quercetin derivatives, but derivatization is required. LC-MS combines the separation performance of HPLC with the high sensitivity of mass spectrometry, providing a powerful tool for the analysis of quercetin in complex samples. However, these traditional detection methods also have certain limitations, such as complex sample pretreatment, high detection costs, long processing times, and demanding equipment requirements, making them unsuitable 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] This invention aims to at least partially solve one of the technical problems in related technologies. This invention provides a colorimetric detection method for quercetin based on NH2-MIL-101-CuCo nanozymes. The provided NH2-MIL-101-CuCo nanozymes have peroxidase-like activity, capable of catalyzing the generation of hydroxyl radicals from H2O2, which then oxidize TMB to blue ox-TMB. The hydroxyl groups in quercetin form hydrogen bonds with the active hydrogen atoms in the NH2-MIL-101-CuCo nanozymes. That is, the NH2-MIL-101-CuCo nanozymes can utilize the π-coordination effect of its metal active sites and the bonding effect of hydrogen bonds to complete the adsorption of quercetin, thereby affecting the enzyme activity of the NH2-MIL-101-CuCo nanozymes and exhibiting a concentration-dependent decrease in the colorimetric signal of quercetin. Therefore, a TMB-based colorimetric method for quercetin detection can be developed. In colorimetric (visible to the naked eye) mode, the higher the concentration of quercetin, the less the blue ox-TMB product generated by oxidation, and the paler the visible blue color. Based on this, the present invention provides a colorimetric modal detection method for quercetin based on NH2-MIL-101-CuCo nanozyme. This method allows for rapid on-site detection, making the detection process more convenient and possessing significant application value in the field of food contamination detection.

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

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

[0007] A first mixture was prepared, consisting of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution, and the sample to be tested. A second mixture was prepared, consisting of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution, and quercetin standards at different concentration gradients.

[0008] The first mixture and the second mixture were reacted under the same reaction conditions, and the absorbance of the reaction products at a predetermined wavelength was measured, corresponding to absorbance values ​​A1 and A0, respectively.

[0009] A linear regression curve was obtained based on the absorbance value A0 and quercetin standards at different concentration gradients. Based on the linear regression curve and the absorbance value A1, the content of quercetin in the sample to be tested was determined.

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

[0011] The NH2-MIL-101-CuCo nanozyme of this invention possesses peroxidase-like activity, catalyzing the generation of hydroxyl radicals from H2O2, which then oxidize TMB to blue ox-TMB. The hydroxyl groups in quercetin form hydrogen bonds with the active hydrogen atoms in the NH2-MIL-101-CuCo nanozyme. This means the NH2-MIL-101-CuCo nanozyme can utilize the π-coordination effect of its metal active site and the bonding effect of hydrogen bonds to adsorb quercetin, thus affecting the enzyme activity of the NH2-MIL-101-CuCo nanozyme and exhibiting a concentration-dependent decrease in the quercetin concentration-based colorimetric signal. This allows for the development of a quercetin detection method based on TMB colorimetry. In colorimetric (visible to the naked eye) mode, higher quercetin concentrations result in less ox-TMB produced by oxidation, and a paler, more visible blue color. Finally, a colorimetric modal detection method for quercetin based on NH2-MIL-101-CuCo nanozymes can be established.

[0012] The method provided by this invention, compared with traditional methods (LC-MS, GC-MS), exhibits better performance in terms of detection limit and linearity. Colorimetric detection offers advantages such as sensitivity, accuracy, and visualization, and can be combined with other mature methods to achieve rapid on-site detection of quercetin.

[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 a reaction at pH 3.0–4.0 and a temperature of 40–50°C for 10–20 minutes. According to a preferred embodiment of the present invention, the reaction conditions are a reaction at pH 3.5 and a temperature of 40°C for 15 minutes.

[0015] According to some embodiments of the present invention, the reaction conditions of pH 3.0 to 4.0 are obtained by adding an acetate-sodium acetate buffer solution with a pH of 3.0 to 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; and the volume ratio of the NH2-MIL-101-CuCo nanozyme solution, TMB solution, hydrogen peroxide solution and acetate-sodium acetate buffer solution is 15:10:10:145-165.

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

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

[0018] The mixed solution was reacted at pH 3.0–4.0 and 40°C–50°C, and the absorbance of the product after the reaction was measured at a wavelength of 640–660 nm.

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

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

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

[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 pretreated using a 0.22 μm filter membrane before detection.

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

[0025] Copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide were mixed in a predetermined molar ratio and reacted at a predetermined temperature. The mixture was then cooled to obtain the mixed product.

[0026] The mixture was centrifuged, the precipitate was washed, and dried to obtain the NH2-MIL-101-CuCo nanozyme.

[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 to 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] A third aspect of the present invention provides a method for colorimetric modal detection of quercetin based on NH2-MIL-101-CuCo nanozymes, comprising:

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

[0032] The first mixture and the second mixture were reacted at pH 3.0–4.0 and 40–50°C for 10–20 minutes, respectively. The absorbance of the products after the reaction at a wavelength of 652 nm was measured, corresponding to absorbance values ​​A1 and A0, respectively.

[0033] A linear regression curve was obtained based on the absorbance value A0 and quercetin standards at different concentration gradients. Based on the linear regression curve and the absorbance value A1, the content of quercetin in the sample to be tested was determined.

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

[0035] According to a fourth aspect of the present invention, an NH2-MIL-101-CuCo nanozyme for colorimetric detection of quercetin is provided, which is obtained by a one-pot hydrothermal method using copper 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 were mixed in a molar ratio of 1:1:0.005-0.1:0.5-1 and reacted at 150-160℃ for 8-15 hours. The mixture was then cooled to obtain the mixed product.

[0037] The mixture was centrifuged, the resulting precipitate was 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 solution, TMB solution and hydrogen peroxide solution;

[0039] The kit mentioned also includes an acetate-sodium acetate buffer solution with a pH of 3.0–4.0 or a phosphate buffer solution with a pH of 3.0–4.0.

[0040] The beneficial effects achieved by this invention are at least as follows:

[0041] (1) The NH2-MIL-101-CuCo nanozyme used in this invention has a high specific surface area, porous structure, and excellent chemical stability. These advantages enable the NH2-MIL-101-CuCo nanozyme to have more active sites, which can improve its adsorption capacity for quercetin. Moreover, compared with other existing methods for synthesizing nanozymes, the materials are simple, the method is fixed, the cost is low, and it can be synthesized in large quantities.

[0042] (2) Compared with existing traditional methods for detecting quercetin, such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), the method provided by this invention does not require complex instruments or professional skills and can be used for rapid on-site detection. Furthermore, this method has high sensitivity and a low detection limit, meaning that quercetin can be effectively detected even at trace levels. In particular, the NH2-MIL-101-CuCo nanozyme used in this invention has high peroxidase-like activity and can catalyze the generation of hydroxyl radicals from H2O2, thereby oxidizing TMB to blue ox-TMB. However, the hydroxyl groups in quercetin form hydrogen bonds with the active hydrogen atoms in the NH2-MIL-101-CuCo nanozyme. This means the NH2-MIL-101-CuCo nanozyme can utilize the π-coordination effect of its metal active site and the bonding effect of hydrogen bonds to adsorb quercetin, thus affecting the enzyme activity of the NH2-MIL-101-CuCo nanozyme and exhibiting a concentration-dependent decrease in the colorimetric signal. Therefore, a TMB-based colorimetric method for quercetin detection can be developed. In summary, this invention develops a colorimetric modal detection method for quercetin based on the NH2-MIL-101-CuCo nanozyme, utilizing the decrease in colorimetric signal.

[0043] (3) This invention proposes a colorimetric modal detection method for quercetin based on NH2-MIL-101-CuCo nanozyme. Based on the relationship between quercetin concentration and TMB color development, a rapid colorimetric method for quercetin determination can be established. Furthermore, this method not only simplifies the detection process and improves efficiency but also significantly reduces reagent consumption, achieving a green and environmentally friendly detection goal. It provides new possibilities for rapid on-site detection of quercetin, making the detection process more convenient and possessing significant application value in the field of food contamination detection. Attached Figure Description

[0044] Figure 1The synthesis diagram of NH2-MIL-101-CuCo and the schematic diagram of the colorimetric modal detection of quercetin in the NH2-MIL-101-CuCo+TMB+H2O2 system are provided according to embodiments of the present invention.

[0045] Figure 2 Transmission electron microscopy image and elemental distribution diagram of NH2-MIL-101-CuCo nanozyme provided according to an embodiment of the present invention.

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

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

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

[0049] Figure 6 The ultraviolet spectra of the combinations (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, measured by an ultraviolet spectrophotometer according to an embodiment of the present invention.

[0050] Figure 7 Optimized conditions for the detection of quercetin in the NH2-MIL-101-CuCo+TMB+H2O2 system, measured by a UV spectrophotometer according to embodiments of the present invention. Figure (A) shows the optimized pH result, with an optimal pH of 3.5; Figure (B) shows the optimized reaction temperature result, with an optimal temperature of 40℃. Figure (C) shows the optimized time conditions for the catalytic formation of oxTMB in the NH2-MIL-101-CuCo+TMB+H2O2 system, with an optimal reaction time of 15.0 min; Figure (D) shows the optimized time conditions for the detection of quercetin in the NH2-MIL-101-CuCo+TMB+H2O2 system, with an optimal reaction time of 15.0 min.

[0051] Figure 8 Absorbance and standard curves of quercetin at different concentrations provided according to embodiments of the present invention.

[0052] Figure 9The histograms for detecting interfering substances in complex environments using the colorimetric mode provided by the embodiments of the present invention demonstrate that the colorimetric mode system of NH2-MIL-101-CuCo+TMB+H2O2 has good selectivity for quercetin and similar flavonoids. Detailed Implementation

[0053] The 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 intended to explain the present invention, but should not be construed as limiting the present invention.

[0054] Metal-organic frameworks (MOFs) possess advantages such as high porosity, large specific surface area, and good chemical and thermal stability, showing broad application prospects in adsorption, separation, and catalysis. Utilizing MOF materials for quercetin detection offers rapid, highly sensitive, highly selective, and highly stable results, enabling highly sensitive and selective detection of quercetin. Therefore, MOF-based detection technologies demonstrate significant application potential in quercetin detection, providing new approaches for rapid and accurate detection. In this regard, this paper develops a novel colorimetric method for detecting quercetin using MOFs. This method leverages the high sensitivity and selectivity of MOF materials, achieving rapid identification of quercetin through color changes, thereby simplifying the analytical process, improving analytical efficiency, reducing reagent consumption, and achieving a green and environmentally friendly detection goal. This provides new possibilities for rapid on-site detection of quercetin.

[0055] Specifically, this invention utilizes the NH2-MIL-101-CuCo nanozyme to detect quercetin content. During research, it was found that compared to other nanozymes, such as the NH2-MIL-101-Fe nanozyme, the NH2-MIL-101-CuCo nanozyme provided by this invention has a very low detection limit of only 0.030 μM. Compared to other methods, it exhibits higher accuracy, greater precision, and higher sensitivity. Furthermore, the colorimetric detection of quercetin using the nanozyme provided by this invention can be directly observed with the naked eye, without requiring complex operations or specialized personnel. In addition, it is resistant to interference from 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 was obtained directly from readily available raw materials using a one-pot hydrothermal method, without intermediate separation, and possessing peroxidase-like activity.

[0057] The NH2-MIL-101-CuCo nanozyme is synthesized in one step via a hydrothermal method using copper 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 specific embodiments, the predetermined molar ratio is 1:1:0.005-0.1:0.5-1. During the research, it was found that when the molar ratio of copper chloride dihydrate to cobalt chloride hexahydrate was 1:1, the prepared nanozyme exhibited very high activity. Adding 2-aminoterephthalic acid during the preparation process increased the amount of modified NH2, thereby increasing the number of hydrogen atoms in the prepared nanozyme and enhancing its ability to adsorb quercetin. The research also found that changes in the raw material ratio affected the activity of the prepared nanozyme. For example, when only one metal element is present, such as only copper chloride dihydrate or cobalt chloride hexahydrate, the prepared nanozyme exhibited poor activity.

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

[0060] According to the specific implementation method, 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 (tablets) were dissolved in 30.0 mL of ultrapure water. After sonication 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 reactants cooled to room temperature, they were centrifuged at 12000.0 rpm for 10.0 min, the solution was separated, and the precipitate was washed with ethanol. Finally, the precipitate was dried into powder in an oven to obtain NH2-MIL-101-CuCo nanozyme.

[0061] This NH2-MIL-101-CuCo nanozyme can be used for colorimetric detection of quercetin. It can be used to detect quercetin residues in various samples, such as protein powder. Detecting quercetin residues in food can ensure food safety and quality, preventing food safety issues caused by excessive addition or improper handling. Furthermore, detecting quercetin residues in proteins can be used to assess its effects on drug metabolism, biological activity, and potential health impacts, thus providing a scientific basis for drug development and clinical applications.

[0062] The provided NH2-MIL-101-CuCo nanozyme can be used for the colorimetric detection of quercetin, specifically including:

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

[0064] The first mixture and the second mixture were reacted under the same reaction conditions, and the absorbance of the product after the reaction was measured at a predetermined wavelength, corresponding to absorbance values ​​A1 and A0, respectively.

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

[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 aforementioned reaction conditions were carried out at pH 3.0–4.0 and temperature 40°C–50°C for 10–20 minutes. According to a preferred embodiment of the invention, the same reaction conditions were carried out at pH 3.5 and temperature 40°C for 15 minutes. After the reaction was completed, the sample was 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 acetate-sodium acetate buffer solution with a pH of 3.5; the volume ratio of the NH2-MIL-101-CuCo nanozyme solution, TMB solution, H2O2 solution and acetate-sodium acetate buffer solution is 15:10:10:145-165; in addition, the buffer solution used can also be a phosphate buffer solution.

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

[0070] The method established in this invention shows a good linear relationship between the change in absorption intensity at 652 nm and the quercetin concentration in the range of 0.88-80.84 μM (indicating a very wide range of quercetin concentrations), with the regression equation being ΔA = 0.0284C. 槲皮素 -0.0327, correlation coefficient 0.9955, detection limit 0.030 μM; the minimum detection limit was obtained using the formula (3σ / k, where σ is the standard deviation of 11 blank samples). According to a specific embodiment, the absorbance linear equation of this invention is ΔA = 0.0284C. 槲皮素 -0.0327.

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

[0072] Relevant reagents:

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

[0074] The preparation method of acetate-sodium acetate buffer (pH 3.5) is as follows: Take 50.0 mL of acetate-sodium acetate buffer with pH = 5.5, and adjust the pH of the acetate-sodium acetate buffer with pH = 5.5 to 3.5 using a pH meter and with the help of hydrochloric acid solution and sodium hydroxide solution.

[0075] Example 1

[0076] Example 1 prepared NH2-MIL-101-CuCo nanozymes with peroxidase-like activity by the following method, specifically including:

[0077] S1: Synthesis of NH2-MIL-101-CuCo nanozymes: NH2-MIL-101-CuCo nanozymes were synthesized using a one-pot hydrothermal method. Specifically, this included:

[0078] For reference 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 (tablets) were dissolved in 30.0 mL of ultrapure water. After sonication for 30.0 min, the mixture was rapidly transferred to a high-temperature reactor lined with polytetrafluoroethylene (PTFE). The reactor was heated at 150.0 °C for 10.0 h. After the reactants cooled to room temperature, they were centrifuged at 12000.0 rpm for 10.0 min. The solution was then separated, and the precipitate was washed three times with ethanol. Finally, the precipitate was dried into powder in an oven to obtain the NH2-MIL-101-CuCo nanozyme.

[0079] The synthesized NH2-MIL-101-CuCo nanozyme was then analyzed. TEM images showed that the synthesized NH2-MIL-101-CuCo was cubic in shape, indicating successful synthesis of the NH2-MIL-101-CuCo nanozyme. Simultaneously, EDS spectra confirmed the uniform distribution of C, N, O, Cu, and Co in NH2-MIL-101-CuCo (e.g., ...). Figure 2 As shown), this is consistent with the measurement results of X-ray photoelectron spectroscopy (XPS). Figure 3 (As shown), this provides strong evidence for the successful synthesis of NH2-MIL-101-CuCo.

[0080] In addition, such as Figure 4 As shown, in the Fourier transform infrared spectrum, the NH2-MIL-101-CuCo nanozyme exhibits activity in the range of 1000–500 cm⁻¹. -1 and 3500~3300cm -1 Characteristic peaks appeared at the locations, corresponding to the skeletal stretching vibration absorption peak of terephthalic acid and the stretching vibration absorption peak of N-H, respectively, which also indicates that NH2-MIL-101-CuCo nanozyme was successfully prepared.

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

[0082] Example 2

[0083] Example 2 explored the feasibility of using the NH2-MIL-101-CuCo nanozyme prepared in Example 1 for the detection of quercetin, specifically including:

[0084] In 0.5 mL centrifuge tubes, add the following solutions respectively: 1.0 mmol / L TMB (labeled 1); 1.0 mmol / L H2O2 solution (labeled 2); 1.0 mmol / L TMB and 1.0 mmol / L H2O2 solution (labeled 3); 0.15 mg / mL NH2-MIL-101-CuCo nanozyme and 1.0 mmol / L TMB (labeled 4); 0.15 mg / mL NH2-MIL-101-CuCo nanozyme, 1.0 mmol / L TMB and 1.0 mmol / L H2O2 solution (labeled 5); 0.15 mg / mL NH2-MIL-101-CuCo nanozyme and 1.0 mmol / L TMB. TMB, 1.0 mmol / L H2O2 solution, and quercetin (labeled 6) were added, and then a 200.0 μL system of acetate-sodium acetate buffer (pH=3.5) was prepared. After mixing thoroughly, the mixture was reacted at 40 °C for 15.0 min. The mixture was then transferred to a cuvette, and the absorbance at 652 nm was measured using a UV spectrophotometer and recorded as absorbance value A1.

[0085] Figure 6 The ultraviolet spectra of each label are shown, measured using an ultraviolet spectrophotometer. Figure 6 The numbers 1 through 6 represent mixtures 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 label 5, NH2-MIL-101-CuCo+TMB+H2O2, clearly generated oxTMB, and the absorbance at 652nm was enhanced; the experimental group corresponding to label 6, NH2-MIL-101-CuCo+TMB+H2O2+quercetin combination, inhibited the generation of oxTMB and reduced the absorbance.

[0088] The results indicate that the prepared NH2-MIL-101-CuCo nanozyme possesses high peroxidase-like activity, capable of catalyzing the generation of hydroxyl radicals from H2O2, which in turn oxidize TMB to blue ox-TMB. However, the hydroxyl groups in quercetin form hydrogen bonds with the active hydrogen atoms in the NH2-MIL-101-CuCo nanozyme. This means that the NH2-MIL-101-CuCo nanozyme can utilize the π-coordination effect of its metal active sites and the bonding effect of hydrogen bonds to adsorb quercetin, thus affecting the enzyme activity of the NH2-MIL-101-CuCo nanozyme and exhibiting a concentration-dependent decrease in the quercetin-based colorimetric signal. Therefore, a TMB-based colorimetric method for quercetin detection can be developed.

[0089] Example 3

[0090] To further investigate the optimal reaction conditions for the formation of the blue product oxTMB catalyzed by NH2-MIL-101-CuCo nanozyme, Example 3 optimized the optimal conditions for the detection reagent using the controlled variable method in UV absorption spectroscopy mode.

[0091] See Figure 7 As shown, A through C represent the optimization of multiple conditions, such as pH, reaction temperature, and reaction time, using a system without the analyte quercetin (NH2-MIL-101-CuCo nanozyme + TMB + hydrogen peroxide solution system). Figure 7 (A) shows the absorbance results under different pH conditions, with all other conditions being equal. The results indicate that pH 3.5 is optimal.

[0092] Figure 7 (B) shows the absorbance results at different reaction temperatures under the same conditions. As the temperature increases, the absorbance value gradually increases until it no longer changes significantly at 40℃. Therefore, the optimal reaction temperature is 40℃.

[0093] Figure 7 (C) Absorbance results at different reaction times under the same conditions. 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 has been confirmed as the optimal reaction time for this system.

[0094] The detection substance quercetin was added to the optimized system described above, such as... Figure 7 As shown in (D), the absorption peak of oxTMB increases continuously with the increase of reaction time until the reaction reaches the equilibrium point at 15.0 min. At this time, TMB is completely oxidized and the absorbance at 652 nm no longer changes. Therefore, the optimal reaction time for detecting quercetin is 15.0 min.

[0095] Example 4

[0096] Referring to the optimal reaction conditions explored in the above embodiments, a standard curve was established in Example 4, and the samples were tested, including:

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

[0098] 15.0 μL of NH2-MIL-101-CuCo nanozyme solution (0.15 mg / mL), 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 acetate-sodium acetate buffer solution (pH 3.5). Then, 20.0 μL of quercetin standard solutions of different concentrations were added. The mixture was reacted at 40 °C for 15.0 min, and 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 Figure 8 As shown, Origin software fitting analysis revealed a strong linear relationship between the change in absorption intensity at 652 nm and the quercetin concentration in the range of 0.88–80.84 μM, with the regression equation being ΔA = 0.0284C. 槲皮素 -0.0327, correlation coefficient was 0.9955, and detection limit was 0.030 μM.

[0103] (2) Sample pretreatment

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

[0105] (3) Detection of the sample to be tested

[0106] Transfer 15.0 μL of NH2-MIL-101-CuCo nanozyme solution with a concentration of 0.15 mg / mL into a centrifuge tube, add 1.0 mmol / L TMB and 1.0 mmol / L H2O2 solution, and then add the pretreated sample (200.0 μL of the prepared system) prepared in step (2) with the pH adjusted to 3.5. After mixing evenly, react at 40℃ for 15.0 min, transfer to a cuvette, and measure the absorbance value at 652 nm using a UV spectrophotometer. Record the absorbance value as A1.

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

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

[0109] Example 5

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

[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 acetate-sodium acetate buffer solution with a pH of 3.5. Then, 20.0 μL of 80 μM of each substance was added, and the mixture was reacted at 40 °C for 15.0 min. The absorbance at 652 nm was then measured using a UV spectrophotometer.

[0112] See Figure 9 This demonstrates the environmental selectivity of the method provided by the present invention, tested using different samples. Figure 9 The substances added, as indicated by each label, are: 1. Blank (i.e., acetate-sodium acetate buffer solution 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 uses the method provided by this invention to select protein powder as a real sample for testing. The protein powder used was randomly purchased and sampled from Hefei, China.

[0116] According to the references, the protein powder was first pretreated: 1.0g of protein powder (pea protein powder, chickpea protein powder, soy protein powder and microbial protein powder) was dissolved in 30.0mL of ultrapure water, sonicated for 30.0 minutes, centrifuged and filtered, and the supernatant was used as the sample solution.

[0117] Before testing, 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 point, using the standard addition method, different concentrations of quercetin were added to the system, mixed thoroughly, and reacted at 40 °C for 15.0 min. The mixture was then transferred to a cuvette, and the absorbance at 652 nm was measured using UV absorption spectroscopy.

[0118] Table 2 illustrates different scenarios for colorimetric detection of real samples. The recoveries of the colorimetric method ranged from 100.35% to 121.65%, with relative standard deviations (RSDs) less than 5.00%. This demonstrates the feasibility and reproducibility of using the NH2-MIL-101-CuCo nanozyme for detecting quercetin in real samples.

[0119] Table 2 Detection Results

[0120]

[0121]

[0122] In summary, this invention has developed a colorimetric modal detection method for quercetin based on the NH2-MIL-101-CuCo nanozyme, utilizing the decrease in colorimetric signal. In the colorimetric (visible to the naked eye) mode, the higher the concentration of quercetin, the less the blue product oxTMB generated by oxidation, and the paler the visible blue color. Finally, a colorimetric modal detection method for quercetin based on the NH2-MIL-101-CuCo nanozyme can be established. Furthermore, this method allows for rapid on-site detection, making the detection process more convenient and possessing significant application value in the field of food contamination detection.

[0123] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," and "detailed implementation" refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0124] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to 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 was prepared, consisting of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution, and the sample to be tested. A second mixture was prepared, consisting of NH2-MIL-101-CuCo nanozyme, TMB solution, hydrogen peroxide solution, and quercetin standards at different concentration gradients. The first mixture and the second mixture were reacted under the same reaction conditions, and the absorbance of the reaction products at a predetermined wavelength was measured, corresponding to absorbance values ​​A1 and A0, respectively; the reaction conditions were pH 3.0~4.0 and 40℃~50℃ for 10~20 minutes. A linear regression curve was obtained based on the absorbance value A0 and quercetin standards at different concentration gradients. Based on the linear regression curve and the absorbance value A1, the content of quercetin in the sample to be tested was determined. The NH2-MIL-101-CuCo nanozyme is synthesized in one step via a hydrothermal method using copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid, and sodium hydroxide; the molar ratio of copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid, and sodium hydroxide is 1:1:0.005-0.01:0.5-1.

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

3. The method according to claim 2, characterized in that, The predetermined wavelength is 652 nanometers.

4. The method according to claim 1, characterized in that, The reaction conditions were pH 3.5 and 40°C for 15 minutes.

5. The method according to claim 1, characterized in that, The reaction conditions of pH 3.0-4.0 were obtained by adding an acetate-sodium acetate buffer solution with a pH of 3.0-4.

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

6. The method according to claim 1, characterized in that, The sample to be tested was a protein powder containing quercetin.

7. The method according to claim 6, characterized in that, The protein powder containing quercetin was pretreated using a 0.22-micron filter membrane before detection.

8. 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 were mixed in a predetermined molar ratio and reacted at a predetermined temperature. The mixture was then cooled to obtain the mixed product. The precipitate obtained by centrifugation and washing of the mixed product was dried to obtain the NH2-MIL-101-CuCo nanozyme.

9. The method according to claim 8, characterized in that, The predetermined temperature is 150~160 degrees Celsius.

10. The method according to claim 8, characterized in that, The reaction is carried out at the predetermined temperature for 8 to 15 hours.

11. A quercetin detection kit, characterized in that, The kit includes NH2-MIL-101-CuCo nanozyme, quercetin standard, buffer solution, TMB solution and hydrogen peroxide solution; The NH2-MIL-101-CuCo nanozyme is obtained via a one-pot hydrothermal process using copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid, and sodium hydroxide, comprising: Copper chloride dihydrate, cobalt chloride hexahydrate, 2-aminoterephthalic acid and sodium hydroxide were mixed in a molar ratio of 1:1:0.005-0.01:0.5-1 and reacted at 150-160 degrees Celsius for 8-15 hours. After cooling, a mixed product was obtained. The precipitate obtained by centrifugation and washing of the mixed product was dried to obtain the NH2-MIL-101-CuCo nanozyme.

12. The kit according to claim 11, characterized in that, The kit includes an acetate-sodium acetate buffer or a phosphate buffer at pH 3-4.

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