Method for detecting trypsin and inhibitor of trypsin on basis of phosphorescence copper nano-cluster

A trypsin inhibition, copper nanocluster technology, applied in the field of biosensing, can solve the problems of high price, low detection limit, complicated operation, etc., and achieve the effects of high sensitivity, fast response, and simple synthesis

Active Publication Date: 2018-11-06
NANJING UNIV OF TECH
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Problems solved by technology

The above detection methods generally have the disadvantage...

Method used

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  • Method for detecting trypsin and inhibitor of trypsin on basis of phosphorescence copper nano-cluster
  • Method for detecting trypsin and inhibitor of trypsin on basis of phosphorescence copper nano-cluster
  • Method for detecting trypsin and inhibitor of trypsin on basis of phosphorescence copper nano-cluster

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Effect test

Embodiment 1

[0040]Example 1: At room temperature, take DMF and deionized water to prepare a mixed solution, and the volume ratio of DMF / water is 2:1. Add 20 μL of glutathione (0.2M) to 480 μL of mixed solvent and shake gently until uniform. Add the copper sulfate solution into the glass bottle, so that the concentration of copper ions in the mixed solution is 500 μM, put it into a 20° C. shaking box, stir and shake at a slow speed for 1 minute, and form phosphorescent copper nanoclusters. The prepared phosphorescent copper nanoclusters were measured by ultraviolet-visible spectrophotometer and fluorescence spectrophotometer to draw absorption spectrum curve and fluorescence spectrum curve, and the phosphorescence lifetime and fitting curve of copper nanoclusters were drawn by lifetime detection. Such as figure 1 , figure 2 with image 3 .

Embodiment 2

[0041] Example 2: At room temperature, take DMF and deionized water to prepare a mixed solution, and the volume ratio of DMF / water is 2:1. Add 20 μL of glutathione (0.2M) to 480 μL of mixed solvent and shake gently until uniform. Take the copper sulfate solution and add it into a glass bottle, so that the copper ion concentration in the mixed solution is 500 μM, put it into a 20° C. shaking box, stir and shake at a slow speed for 1 minute, and form phosphorescent copper nanoclusters. A certain amount of copper nanoclusters was freeze-dried, and characterized by transmission electron microscopy (TEM) and X-ray photoelectric spectroscopy (XPS). Such as Figure 4 , Figure 5 with Image 6 .

Embodiment 3

[0042] Example 3: Cytochrome C solution (2 mg / mL) was prepared with prepared Tris-HCl (50 mM, pH 8.9) buffer. The absorption spectrum curve is drawn by UV-Vis spectrophotometer measurement, such as Figure 7 .

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Abstract

The invention relates to a method for detecting trypsin and an inhibitor of the trypsin on the basis of a phosphorescence copper nano-cluster, and belongs to the technical field of nanometer biosensing. Glutathione (GSH) and copper sulfate (CuSO4) are added in a mixed solvent of DMF (Dimethyl Formamide) and deionized water to prepare a copper nano-cluster having a phosphorescent property. Throughthe electrostatic interaction of cytochrome C (Cyt c), the fluorescence intensity of Cu NCs is reduced. The Cyt c incubated trypsin is added in the prepared Cu NCs. The Cyt c is hydrolyzed by the trypsin, the existence of the trypsin effectively inhibits the quenching effect of Cyt c on Cu NCs. Therefore, the trypsin is quantitatively detected through the change of the fluorescence intensity. A phosphorescence copper nano-cluster probe has the characteristics that the reaction is simple and fast, the detection of the trypsin is highly sensitive, and the chemical stability and the biological compatibility are good.

Description

technical field [0001] The invention relates to a method for detecting trypsin and its inhibitor based on phosphorescent copper nanoclusters, belonging to the technical field of biosensing. Background technique [0002] Trypsin is a serine proteolytic enzyme extracted from the pancreas. Changes in the content of trypsin in the organism directly reflect the function of the pancreas and the changes in the disease. It is a very important protease in the vertebrate organism. Therefore, the development of simple, rapid and accurate detection of trypsin activity is of great significance for disease diagnosis and treatment. Currently, there are various methods for detecting trypsin such as: enzyme-linked immunosorbent assay, gel electrophoresis, mass spectrometry, colorimetry, and fluorescence analysis. The above detection methods generally have the disadvantages of low detection limit, high price and complicated operation. Therefore, the fluorescence analysis method based on th...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/64G01N21/6428
Inventor 刘金华慈乔乔张倩晨张承武李林
Owner NANJING UNIV OF TECH
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