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Fluorescence sensor for detecting pyrophosphatase and preparation method thereof

A fluorescence sensor and pyrophosphatase technology, applied in the field of analytical chemistry, can solve the problems of inconvenient operation, complicated operation, high price and the like, and achieve the effects of strong practicability, simple operation steps and low cost

Inactive Publication Date: 2014-12-24
FUZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the general disadvantage of these methods is that either large-scale instruments and equipment are required, which are not only expensive, complicated to operate, but also inconvenient to operate, or require special reagents, which are expensive and some will cause harm to the human body.

Method used

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  • Fluorescence sensor for detecting pyrophosphatase and preparation method thereof
  • Fluorescence sensor for detecting pyrophosphatase and preparation method thereof
  • Fluorescence sensor for detecting pyrophosphatase and preparation method thereof

Examples

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Embodiment 1

[0029] The following examples illustrate the application of the method of the present invention to prepare a fluorescent pyrophosphatase sensor and apply it to the detection of the pyrophosphatase inhibitor sodium fluoride concentration in conjunction with the accompanying drawings:

[0030] 1. Pyrophosphatase hydrolyzes pyrophosphate. Specific steps are as follows:

[0031] A. Weigh the enzyme lyophilized powder to make a mother solution with a certain weight of 1 U / μL, and dilute it to the required concentration according to the gradient;

[0032] B. Add 2 μL of pyrophosphatase with different gradients to 4-hydroxyethylpiperazineethanesulfonic acid buffer solution (10mM HEPES, pH=7.2) containing 125 μM pyrophosphate, mix well, and place in a 37°C water bath for 60 minutes at room temperature ;

[0033] C. Add 2 μL of copper sulfate solution with a concentration of 10 mM to the mixed solution, and continue the reaction in a 37° C. water bath for 20 minutes.

[0034] 2. Cli...

Embodiment 2

[0038] 1. Detection of pyrophosphatase inhibitor sodium fluoride concentration, the specific steps are as follows:

[0039] Add 0 μM, 2.0 μM, 1.6, 1.2 μM, 0.8 μM and 0.4 μM enzyme inhibitor sodium fluoride to the buffer containing 4.0 mU pyrophosphatase respectively, and after standing at room temperature for 30 minutes, add 125 μM pyrophosphoric acid in a water bath at 37°C for 60 Minutes, then add 100 μM copper sulfate, continue to keep warm in water bath for 20 minutes, finally add the click reagent and keep it in the dark for 15 minutes, measure the fluorescence with a fluorescence spectrophotometer, and record the data.

[0040] 2. The method of the present invention prepares the specificity of pyrophosphatase detection of the fluorescent pyrophosphatase sensor, and the specific steps are as follows:

[0041]In order to detect the specificity of the fluorescent pyrophosphatase sensor of the present invention, the pyrophosphatase used in Example 1-1 was replaced with other...

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Abstract

The invention discloses a fluorescence sensor for detecting pyrophosphatase and preparation method thereof, with pyrophosphoric acid only before adding the pyrophosphatase, the pyrophosphoric acid and the cupric compound react for producing the complex, the sodium ascorbate cannot reduce the complex cupric compound into the cuprous compound as the catalyst, the azide coumarin and the propargyl alcohol cannot have the 1.3-dipole cycloaddition, so that the fluorescence intensity of the system is low; after adding the pyrophosphatase, which can hydrolyze the pyrophosphoric acid for inhibiting the formation of cupric compound complex, the click chemistry reaction continues, the fluorescence intensity of the system is greatly improved, the relationship between the fluorescence intensity and the amount of the pyrophosphatase is linear relationship. The sensor is successfully used for detecting pyrophosphatase inhibitor sodium fluoride, the fluorescence sensor for detecting pyrophosphatase and the preparation method thereof are simple in operation, low in cost, high in sensitivity and good in specificity.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, in particular to a method for detecting pyrophosphatase with a fluorescent chemical sensor based on click chemistry. Background technique [0002] Pyrophosphatase (EC 3.6.1.1) or inorganic pyrophosphatase is a class of hydrolytic enzymes that specifically convert pyrophosphate (P 2 o 7 4? , PPi) hydrolyzed to inorganic phosphate. Hydrolysis of pyrophosphate by pyrophosphatase is a high-energy releasing reaction that can be coupled to several thermodynamically unfavorable transformations in order to drive them (Biswas et al., 2013. Nucleic acids research 41, e56.; Cestari et al ., 2013. Journal of biomolecular screening 18, 490-497.). Early studies have shown that pyrophosphatase plays an important role in lipid metabolism (including lipid synthesis and breakdown), calcium absorption, and bone formation and deoxyribonucleic acid (DNA) synthesis (Ilias et al., 2006. Biochimica et biophysica ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 林振宇徐可丰郭隆华邱彬陈国南
Owner FUZHOU UNIVERSITY
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