Test method for trypsin

A detection method and trypsin technology, applied in the field of protein detection, can solve the problems of complex preparation process, easy to be interfered by other substances, low sensitivity, etc., and achieve the effects of simple synthesis method, good fluorescence performance and high quantum yield.

Inactive Publication Date: 2012-07-18
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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Problems solved by technology

However, the preparation process of this fluorescent polymer is complicated, the cost is high, and the synthesis is difficult, which limits the development of protein detection by fluorescence photometry.
[0005] In order to promote the application of fluorescence spectrometry in the field of protein detection, the existing technology is committed to the development of a simple method for the synthesis of fluorescent probes, such as Xu Jianping of Zhejiang University who synthesized a quaternized tetraphenyl salt and bovine serum albumin (BSA) complex, and using this complex as a fluorescent probe, the contents of trypsin and antitrypsin were detected simultaneously by fluorescence spectrophotometry (Jian-Ping Xu, Yuan Fang, Zhe-Gang Song, Ju Mei , Lan Jia, An-Jun Qin, Jing-Zhi Sun, Jian Ji, Ben-Zhong Tang.Analyst, 2011, 136(11), 2315-2321.), the synthesis of this fluorescent probe mainly relies on quaternized ammonium The electrostatic force between the ammonium cation of the tetraphenyl salt and the carboxyl anion of BSA is weak, but this electrostatic force is easily interfered by other substances, which makes the detected content of trypsin inaccurate and low in sensitivity

Method used

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  • Test method for trypsin
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Examples

Experimental program
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Effect test

Embodiment 1

[0058] 5mL molar concentration of 10mmol / L chloroauric acid solution mixed with 5mL mass concentration of 50mg / mL bovine serum albumin solution, reacted at 37°C for 10 minutes, and then added a mass concentration of 30% sodium hydroxide solution , the pH value of the solution was adjusted to 12, and then the obtained mixed solution was stirred and reacted at 37° C. for 12 hours to obtain bovine serum albumin-gold (BSA-Au) nanoclusters.

[0059] The present invention detects the fluorescence spectrum properties of BSA-Au nanoclusters, and obtains that the luminescent color of the BSA-Au nanoclusters prepared in this embodiment is red light; the fluorescence intensity of the metal nanoclusters is measured, and the results show that BSA with a molar concentration of 5 μmol / L - The fluorescence intensity of Au is 171.

Embodiment 2

[0061] The BSA-Au metal nanocluster solution prepared in Example 1 with a molar concentration of 5 μmol / L was placed in a 37°C water bath for 2 hours, and the fluorescence excitation wavelength was 385nm, and the excitation slit and emission slit were 10nm and 20nm, respectively. The fluorescence intensity was obtained as 171. Metal nanoclusters containing 5 μmol / L BSA-Au were mixed with a mass concentration of 1×10 -8 g / mL, 1×10 -7 g / mL, 1×10 -6 g / mL, 1×10 -5 g / mL or 1×10 -4 The g / mL trypsin solution was reacted in a water bath at 37°C for 2 hours, and its fluorescence intensity was detected.

[0062] The fluorescence intensity values ​​after the reaction obtained in this example are 155, 121, 105, 86 and 62 respectively, and the calculation is 1×10 -8 g / mL, 1×10 -7 g / mL, 1×10 -6 g / mL, 1×10 -5 g / mL or 1×10 -4 The difference of the fluorescence intensity corresponding to the trypsin solution of g / mL is 16, 50, 66, 85 and 109 respectively, the logarithm of the mass con...

Embodiment 3

[0064] The BSA-Au metal nanocluster solution prepared in Example 1 with a molar concentration of 5 μmol / L was placed in a 37°C water bath for 2 hours, and the fluorescence excitation wavelength was 385nm, and the excitation slit and emission slit were 10nm and 20nm, respectively. The fluorescence intensity was obtained as 171. Will contain a mass concentration of 5 × 10 -8 A mixed solution of g / mL trypsin and BSA-Au metal nanoclusters with a molar concentration of 5 μmol / L was reacted in a water bath at 37 °C for 2 hours, and the fluorescence intensity of the reaction product was detected in parallel three times.

[0065] The differences between the fluorescence intensity of the reaction product obtained in this example and the fluorescence intensity of 5 μmol / L BSA-Au metal nanoclusters are 36.0, 36.2, and 36.6, respectively, which are calculated according to the standard curve obtained in Example 1. The mass concentration of trypsin in the medium was 4.8×10 -8 g / mL, 4.9×10...

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Abstract

The invention provides a test method for trypsin. The test method comprises the following steps that: the fluorescence intensity of a metal nanocluster is tested; the trypsin reacts with the metal nanocluster, so that a reaction product is obtained, and the fluorescence intensity of the reaction product is tested; the difference between the fluorescence intensity of the metal nanocluster and the fluorescence intensity of the reaction product is calculated, and according to the difference and a predetermined standard curve, the content of the trypsin is obtained. The test method provided by the invention adopts the metal nanocluster as a fluorescence probe, the metal nanocluster comprises protein and metal, the property of the trypsin in fluorescently quenching the metal nanocluster is utilized, moreover, the degree of fluorescence quenching is positively correlated to the concentration of the trypsin, and the fluorospectrophotometry is adopted to determine the content of the trypsin. The test method provided by the invention has the advantages of high accuracy, high sensitivity and high selectivity, and an experimental result shows that the sensitivity of the test method provided by the invention on the test of the trypsin is 1.0 multiplied by 10 negative 9g / mL.

Description

technical field [0001] The invention relates to the technical field of protein detection, in particular to a method for detecting trypsin. Background technique [0002] Trypsin is a hydrolytic enzyme secreted by the pancreas, which can hydrolyze amino acid compounds linked by peptide bonds. As an important protein-digesting enzyme, trypsin is widely used in medicine, food and industrial production. It is clinically used to treat empyema, hemothorax, surgical inflammation, ulcer, traumatic injury, etc. It can also be sprayed The form of inhalation is used to dissolve mucus; in addition, trypsin is closely related to the secretory function of the pancreas, and the content of trypsin is an important basis for the clinical diagnosis of pancreas-related diseases such as acute pancreatitis, chronic pancreatitis and pancreatic cancer. Therefore, the detection of trypsin content plays an important role in the quality control of human health, medicine and food, and the quality detec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 徐国宝胡连哲
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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