Search

CN-119915755-B - Based on NH2Quercetin colorimetric detection method of MIL-101-CuCo nano enzyme

CN119915755BCN 119915755 BCN119915755 BCN 119915755BCN-119915755-B

Abstract

The invention provides a quercetin colorimetric detection method based on NH 2 -MIL-101-CuCo nano enzyme. The method comprises the steps of preparing a first mixture of NH 2 -MIL-101-CuCo nano enzyme, TMB solution and hydrogen peroxide solution which are respectively mixed with a sample to be tested and a second mixture of the nano enzyme, reacting the mixture with a quercetin standard substance under the same reaction condition to obtain absorbance values A 1 and A 0 under the preset wavelength, and determining the content of quercetin in the sample to be tested based on A 1 and linear regression curves of different concentrations of quercetin, wherein the nano enzyme is obtained by one-step synthesis of copper chloride dihydrate, cobalt chloride hexahydrate, 2-amino terephthalic acid and sodium hydroxide through a hydrothermal method. The provided method can specifically identify and adsorb the quercetin by combining active hydrogen atoms in the nano enzyme and hydroxyl groups in the quercetin to form hydrogen bonds, so that the detection of the quercetin is realized.

Inventors

  • ZHANG SHUO
  • HE FENGJIAO
  • ZHOU SHUANG
  • SHEN YIZHONG
  • LUO PENGJIE
  • LI JINGGUANG
  • WU YONGNING

Assignees

  • 国家食品安全风险评估中心
  • 合肥工业大学

Dates

Publication Date
20260505
Application Date
20241129

Claims (12)

  1. 1. A quercetin colorimetric detection method based on NH 2 -MIL-101-CuCo nano enzyme is characterized by comprising the following steps: Respectively preparing a first mixture of NH 2 -MIL-101-CuCo nano enzyme, TMB solution, hydrogen peroxide solution and sample to be tested, and a second mixture of NH 2 -MIL-101-CuCo nano enzyme, TMB solution, hydrogen peroxide solution and quercetin standard substances with different concentration gradients; The first mixture and the second mixture are reacted under the same reaction condition, the absorbance of a reaction product under the preset wavelength is measured, and the absorbance value A 1 and the absorbance value A 0 are respectively corresponding, wherein the reaction condition is that the reaction is carried out for 10-20 minutes at the temperature of pH 3.0-4.0 and 40-50 ℃; Obtaining a linear regression curve based on the absorbance value A 0 and quercetin standard substances with different concentration gradients, and determining the content of quercetin in the sample to be detected based on the linear regression curve and the absorbance value A 1 ; the NH 2 -MIL-101-CuCo nano enzyme is obtained by one-step synthesis of copper chloride dihydrate, cobalt chloride hexahydrate, 2-amino terephthalic acid and sodium hydroxide through a hydrothermal method, wherein the molar ratio of the copper chloride dihydrate to the cobalt chloride hexahydrate to the 2-amino terephthalic acid to the sodium hydroxide is 1:1:0.005-0.01:0.5-1.
  2. 2. The method of claim 1, wherein the predetermined wavelength is 640-660 nanometers.
  3. 3. The method of claim 2, wherein the predetermined wavelength is 652 nanometers.
  4. 4. The method according to claim 1, wherein the reaction conditions are a reaction at a temperature of ph3.5 at 40 ℃ for 15 minutes.
  5. 5. The method according to claim 1, wherein the reaction condition of pH 3.0-4.0 is obtained by adding acetic acid-sodium acetate buffer with pH 3.0-4.0, wherein the concentration of the NH 2 -MIL-101-CuCo nano enzyme 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 NH 2 -MIL-101-CuCo nano enzyme solution, the TMB solution, the hydrogen peroxide solution and the acetic acid-sodium acetate buffer is 15:10:10:145-165.
  6. 6. The method of claim 1, wherein the sample to be tested is a protein powder containing quercetin.
  7. 7. The method of claim 6, wherein the quercetin-containing protein powder is pre-treated with a 0.22 micron filter membrane prior to detection.
  8. 8. The method according to claim 1, wherein the NH 2 -MILs-101-CuCo nanoenzyme is prepared by: mixing copper chloride dihydrate, cobalt chloride hexahydrate, 2-amino terephthalic acid and sodium hydroxide according to a predetermined molar ratio, reacting at a predetermined temperature, and cooling to obtain a mixed product; And centrifuging and washing the obtained precipitate of the mixed product, and drying to obtain the NH 2 -MIL-101-CuCo nano enzyme.
  9. 9. The method of claim 8, wherein the predetermined temperature is 150-160 degrees celsius.
  10. 10. The method of claim 8, wherein the reaction is carried out at the predetermined temperature for 8-15 hours.
  11. 11. A quercetin detection kit is characterized by comprising NH 2 -MIL-101-CuCo nano enzyme, a quercetin standard substance, a buffer solution, a TMB solution and a hydrogen peroxide solution; Wherein the NH 2 -MIL-101-CuCo nano enzyme is obtained by a one-pot hydrothermal method through copper chloride dihydrate, cobalt chloride hexahydrate, 2-amino terephthalic acid and sodium hydroxide, and comprises the following steps: Mixing copper chloride dihydrate, cobalt chloride hexahydrate, 2-amino terephthalic acid and sodium hydroxide according to the molar ratio of 1:1:0.005-0.01:0.5-1, reacting for 8-15 hours at 150-160 ℃, and cooling to obtain a mixed product; And centrifuging and washing the obtained precipitate of the mixed product, and drying to obtain the NH 2 -MIL-101-CuCo nano enzyme.
  12. 12. The kit of claim 11, wherein the kit comprises an acetic acid-sodium acetate buffer or a phosphate buffer at a pH of 3-4.

Description

Quercetin colorimetric detection method based on NH 2 -MIL-101-CuCo nano enzyme Technical Field The invention belongs to the technical field of food pollution detection, and particularly relates to a quercetin colorimetric detection method based on NH 2 -MIL-101-CuCo nano enzyme. Background Quercetin (Quercetin) is a natural flavonol compound widely existing in various plants, has a molecular formula of C 15H10O7, and has abundant bioactivity and pharmacological action. The quercetin has wide application prospects in the aspects of antioxidation, anti-inflammation, anti-tumor and the like due to the special chemical structure thereof. However, the safety problem of quercetin is also important. Under the condition of high dosage or excessive intake, the quercetin can cause inflammation, DNA damage, hypertension, cancer and the like (Ultra-sensitive amperometric determination of quercetin by using a glassy carbon electrodemodified with a nanocomposite prepared from aminated graphene quantum dots,thiolatedβ-cyclodextrin and gold nanoparticles,DOI:10.1007/s00604-019-4106-1)., in addition, the quercetin can also interact with some medicines to influence the metabolism in the body, so that the curative effect (One-step solvothermalsynthesis of nanoflake-nanorod WS2 hybrid for non-enzymatic detection of uric acid andquercetin in blood serum,DOI:10.1016/j.msec.2019.110217). is influenced, the monitoring and the management of the quercetin are enhanced, and the method has very important significance for guiding the reasonable use of the quercetin, not only realizing the health benefit, but also effectively avoiding the harm of the quercetin. In view of the complexity of quercetin, the detection method is also required to be accurate and sensitive. Common detection technologies include high performance liquid chromatography (HPLC)(Antioxidant study of quercetin and their metal complex and determination ofstability 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 Indianwhite 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) and the like, and the methods can accurately measure the content of the quercetin in the sample and provide scientific basis for research and application of the quercetin. The HPLC method has the advantages of high separation efficiency, good repeatability and the like, is widely applied to quercetin content measurement, can be used for measuring quercetin derivatives, but needs to be derived, and the LC-MS combines the separation performance of HPLC with the high sensitivity of mass spectrum, thereby providing a powerful means for analyzing quercetin in complex samples. However, these conventional detection methods also have certain limitations, such as complex sample pretreatment, high detection cost, long time consumption, and severe requirements on equipment, and are difficult to be used for large sample screening and real-time monitoring. Therefore, an effective quercetin detection method should be developed to protect public health. Disclosure of Invention The present invention aims to solve at least one of the technical problems in the related art to some extent. The invention provides a quercetin colorimetric detection method based on NH 2 -MIL-101-CuCo nano enzyme. The provided NH 2 -MIL-101-CuCo nano enzyme has peroxidase-like activity, can catalyze H 2O2 to generate hydroxyl free radicals, and further oxidizes TMB into blue ox-TMB. The hydroxyl in the quercetin can generate hydrogen bonds with active hydrogen atoms in NH 2 -MIL-101-CuCo nano enzyme, namely the NH 2 -MIL-101-CuCo nano enzyme can play pi coordination action of metal active sites of the quercetin and can adsorb the quercetin by utilizing the bond and action of the hydrogen bonds, so that the enzyme activity of the NH 2 -MIL-101-CuCo nano enzyme is influenced, the color comparison signal of the quercetin is reduced in a concentration-dependent manner, and a quercetin detection method based on TMB color development can be developed. In colorimetric (macroscopic) mode, the higher the quercetin concentration, the less the oxidation to form the blue product ox-TMB, and the lighter the macroscopic blue. Based on the method, the invention provides a colorimetric mode detection method for detecting quercetin based on NH 2 -MIL-101-CuCo nano enzyme. The method can be used for rapidly carrying out on-site detection, so that the detection process is more convenient and has important application value in the field of food pollution detection. Specifically, the invention provides the following technical scheme: The first aspect of the invention provides a quercetin color