Fluorescence detection method for hydrolase activity

Abstract

The invention discloses a fluorescence detection method for hydrolase activity. The method comprises the following steps: hatching detection liquid containing a substrate and a Cu<2+>-bathocuproine disulfonic acid complex with a to-be-detected hydrolase solution, taking the Cu<2+>-bathocuproine disulfonic acid complex as a fluorescent probe, and reducing the fluorescent probe by a reducing product obtained by hydrolyzing the substrate by virtue of hydrolase, so as to generate the Cu<2+>-bathocuproine disulfonic acid complex and weaken a fluorescent signal of the detection liquid. The higher is the activity of the hydrolase in a sample, the lower fluorescence emission intensity of the detection liquid is. The fluorescence detection method disclosed by the invention has high detection sensitivity on hydrolase activity, good selectivity, strong interference resistance, good reproducibility, simple operation and short detection time and can be used for quick and high-sensitivity detection of the hydrolase activity in a clinical sample, and the detection cost is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of biological analysis and relates to a fluorescence detection method for hydrolase activity. Background technique [0002] Hydrolase is a general term for a class of enzymes that catalyze hydrolysis reactions, and widely exists in various animals, plants and microorganisms. Simple, rapid and sensitive detection of hydrolase activity is of great significance for early diagnosis of diseases, observation of curative effect and monitoring of prognosis. [0003] At present, fluorescence analysis and colorimetric analysis are mostly used for the determination of hydrolytic enzyme activity. Among them, the fluorescence analysis method is based on the change of the fluorescence intensity of the substance in the solution to quantitatively analyze the content of the substance, which has excellent characteristics such as low detection cost, simple equipment, easy operation, and high accuracy. Taking alkaline phospha...

AI Technical Summary

Problems solved by technology

Taking alkaline phosphatase as an example, there are two types of fluorescence analysis methods currently available for its activity determination: (1) Based on the principle of polymerization-induced luminescence, alkaline phosphatase hydrolyzes water-soluble phosphoric acid-tetraphenylethylene to form insoluble Tetraphenylethylene in water, tetraphenylethylene will produce fluorescence after polymerization, so as to realize the fluorescence analysis of alkaline phosphatase, but this method has defects such as poor selectivity, low sensitivity and insufficient anti-interference ability (1.Gu X, et al. (2013).A new fluorometric turn-on assay for alkaline phosphatase and inhibitor screening based on aggregation and deaggregation oftetraphenylethylene molecules[J].Analyst,138(8):2427-2431; 2.Liang J,et al.(2013). Fluorescent light-up probe with aggregation-induced emission characteristics for alkaline phosphatase sensing and activity study[J].ACS Applied Materials&Interfaces,5(17):8784-8789.); (2) Using carbon quantum dots as fluorescent probes, carbon quantum dots When the carboxyl groups enriched on the surface are triggered to aggregate, the fluorescence can be quenched, thereby realizing the fluorescence analysis of alkaline phosphatase. However, this method has defects such as high detection cost, poor anti-interference ability and possible toxicity (1. Qian Z, etal.(2015).Carbon quantum dots-based recyclable real-time fluorescence assayfor alkaline phosphatase with adenosine triphosphate as substrate[J].Analytical Chemistry,87(5):2966-2973; 2.Qian Z S,et al .(2015).A real-time fluorescent assay for the detection of alkaline phosphatase activity based on carbon quantum dots[J].Biosensors and Bioelectronics,68:675-680.)

Images