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Preparation method of nanocluster mimic enzyme with visible-light activity and use of nanocluster mimic enzyme in colourimetry detection of trypsin

A trypsin and photoactive technology, which is applied in the field of nanotechnology and biological analysis and detection, and can solve problems such as the reduction of catalytic activity

Active Publication Date: 2015-03-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein template on the surface of the nanocluster is decomposed by trypsin, resulting in a change in the surface state of the nanocluster, causing aggregation and a decrease in catalytic activity

Method used

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  • Preparation method of nanocluster mimic enzyme with visible-light activity and use of nanocluster mimic enzyme in colourimetry detection of trypsin
  • Preparation method of nanocluster mimic enzyme with visible-light activity and use of nanocluster mimic enzyme in colourimetry detection of trypsin
  • Preparation method of nanocluster mimic enzyme with visible-light activity and use of nanocluster mimic enzyme in colourimetry detection of trypsin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] a. 5mL 50mg / mL bovine serum albumin, 1mL 1.25×10 -3 After mixing mol / L mercaptosuccinic acid with 5ml 0.01mol / L chloroauric (III) acid, add 1mL 5.0×10 -2 mol / L hydrogen peroxide solution and use NaOH to adjust the pH to pH=9; after stirring at 37°C for 24 hours, a gold nanocluster material is obtained;

[0017] b. Dialyze the obtained gold nanocluster material for 48 hours (change the water every four hours) to remove reaction impurities; take 100 μL of gold nanocluster material, add different concentrations of trypsin, and place it at 37° C. for 2 hours, then Add 0.3mL 5.0×10 -3 mol / L characteristic substrate 3,3',5,5'-tetramethylbenzidine and 2mL of 0.2mol / L acetate buffer solution with pH=4.0, set the volume to 5mL, and place it under visible light for 10min to show color. The characteristic absorption (λ max =652nm) to measure the absorbance of the system.

Embodiment 2

[0019] a. 5mL of 50mg / mL bovine serum albumin, 1mL of 1.75×10 -3 After mixing mol / L mercaptosuccinic acid with 5ml 0.01mol / L chloroauric (III) acid, add 500μL 1.0×10 -3 mol / L ascorbic acid, and use NaOH to adjust the pH to 11; after stirring at 37°C for 12 hours, the gold nanocluster material was obtained;

[0020] b. Dialyze the obtained gold nanocluster material for 48 hours (change the water every four hours) to remove reaction impurities; take 100 μL of gold nanocluster material, add different concentrations of trypsin, and place it at 37° C. for 2 hours, then Add 0.5mL 5.0×10 -3 mol / L characteristic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 0.2mol / L acetate buffer solution of 2mL pH=4.0, set Make up to 5mL, and place it under visible light for 10min to develop color. The characteristic absorption (λ max =417nm) to measure the absorbance of the system.

Embodiment 3

[0022] a. 5mL 50mg / mL bovine serum albumin, 1mL 4.25×10 -3 After mixing mol / L mercaptosuccinic acid with 5ml 0.01mol / L silver nitrate, add 1mL 5.0×10 dropwise -2 mol / L formaldehyde, and use NaOH to adjust the pH to 11; after stirring at 37°C for 12 hours, the silver nanocluster material was obtained;

[0023] b. Dialyze the obtained silver nanocluster material for 48 hours (change the water every four hours) to remove reaction impurities; take 100 μL of silver nanocluster material, add different concentrations of trypsin, place it at 37°C for 2 hours, add 0.5mL 5.0×10 -3 mol / L characteristic substrate 3,3',5,5'-tetramethylbenzidine and 2mL of 0.2mol / L acetate buffer solution with pH=4.0, set the volume to 5mL, and place it under visible light for 10min to show color. The characteristic absorption (λ max=652nm) to measure the absorbance of the system.

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Abstract

The invention provides a preparation method of nanocluster mimic enzyme with visible-light activity and a use of the nanocluster mimic enzyme in colourimetry detection of trypsin. Gold / silver nanoclusters modified by bovine serum albumin and mercaptosuccinic acid as surface modification agents has peroxidase-like catalytic characteristics under visible light irradiation and can catalyze chromogenic substrate oxidation. Compared with the peroxidase, the nanocluster mimic enzyme with visible-light activity is free of a high-concentration oxidizing agent and has high catalytic activity, good stability and eco-friendly catalytic conditions. Trypsin decomposes a protein template on the surface of the nanocluster so that the nanocluster surface state is changed and causes aggregation thereby reducing catalytic activity. The preparation method has a trypsin detection linear range of 9.0*10<-7> to 1.0*10<-3>g / mL and has a detection limit of 0.6 micrograms per milliliter far lower than trypsin content of urine and blood of patients.

Description

Technical field: [0001] The invention relates to the field of nanotechnology and the field of biological analysis and detection, in particular to the preparation of a novel visible light-induced nano-cluster imitation enzyme and its application in the detection of trypsin. Background technique: [0002] Natural enzymes can catalyze chemical reactions, and compared with chemical catalysis, natural enzymes have higher catalytic activity. Therefore, natural enzymes are widely used in various fields such as pesticide production, pharmaceutical process and food industry [James B. Chem. Soc. Rev. 2009, 38, 185-196]. However, natural enzymes are easily inactivated by external influences and are generally unstable to acids, alkalis, and heat, and are expensive, which limit their wide application. Therefore, the study of mimetic enzymes has aroused widespread interest. [0003] Recent studies have shown that the rapid development of nanotechnology provides a broader space for the s...

Claims

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Application Information

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IPC IPC(8): B01J23/52B01J23/50B22F9/24G01N21/31B82Y30/00B82Y40/00
Inventor 王光丽金璐怡吴秀明陶慧杨璇
Owner JIANGNAN UNIV
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