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Glutamine detection method based on double enzyme coupling

A technology of glutamine and detection method, applied in the field of glutamine determination, can solve problems such as damage to plant samples, lack of a specific detection method for glutamine, etc., and achieve the effects of convenient operation, low price and little damage

Active Publication Date: 2019-08-30
SUZHOU CHIEN SHIUNG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also studies that apply fluorescence analysis to the detection of amino acids, but there is a lack of specific detection methods for glutamine. In addition, such methods require a certain amount of sample and have certain damage to plant samples.

Method used

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  • Glutamine detection method based on double enzyme coupling
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038](1) Prepare the TE buffer solution (10mM tris-hydrochloric acid, 1mM sodium edetate, pH 7.4) for dissolving the required DNA, respectively, the sequences are 5'-TTTGGGTAGGGCGGGTTGGG-3', 5 The DNA of the two segments of DNA of '-CGAACTCTGCAACATAAAAAA-3' was dissolved in the above-mentioned TE buffer solution to obtain DNA solution A and DNA solution B with a concentration of 10 μM, and stored at 4°C;

[0039] (2) prepare potassium phosphate buffer solution (200mM K 2 HPO 4 -KH 2 PO 4 , 400mM NaCl, pH 7.4), using a small amount of dimethyl sulfoxide to dissolve hemin, and then prepare with the potassium phosphate buffer to obtain a heme solution with a concentration of 10 μM, and store it at 4°C;

[0040] (3) Mix the above DNA solution A, DNA solution B and heme solution at a volume ratio of 1:1:2, let stand at room temperature for 30-60 minutes to obtain a DNA mimic enzyme solution, and store at 4°C;

[0041] (4) Prepare glutamine synthetase working solution, the reac...

Embodiment 2

[0047] (1) Prepare the TE buffer solution (10mM tris-hydrochloric acid, 1mM sodium edetate, pH 7.4) for dissolving the required DNA, respectively, the sequences are 5'-TTTGGGTAGGGCGGGTTGGG-3', 5 The DNA of the two segments of DNA of '-CGAACTCTGCAACATAAAAAA-3' was dissolved in the above-mentioned TE buffer solution to obtain DNA solution A and DNA solution B with a concentration of 10 μM, and stored at 4°C;

[0048] (2) prepare potassium phosphate buffer solution (200mM K 2 HPO 4 -KH 2 PO 4 , 400mM NaCl, pH 7.4), using a small amount of dimethyl sulfoxide to dissolve hemin, and then prepare with the potassium phosphate buffer to obtain a heme solution with a concentration of 10 μM, and store it at 4°C;

[0049] (3) Mix the above DNA solution A, DNA solution B and heme solution at a volume ratio of 1:1:2, let stand at room temperature for 30-60 minutes to obtain a DNA mimic enzyme solution, and store at 4°C;

[0050] (4) prepare glutamine synthetase working solution, this re...

Embodiment 3

[0056] (1) Prepare the TE buffer solution (10mM tris-hydrochloric acid, 1mM sodium edetate, pH 7.4) for dissolving the required DNA, respectively, the sequences are 5'-TTTGGGTAGGGCGGGTTGGG-3', 5 The DNA of the two segments of DNA of '-CGAACTCTGCAACATAAAAAA-3' was dissolved in the above-mentioned TE buffer solution to obtain DNA solution A and DNA solution B with a concentration of 10 μM, and stored at 4°C;

[0057] (2) prepare potassium phosphate buffer solution (200mM K 2 HPO 4 -KH 2 PO 4 , 400mM NaCl, pH 7.4), using a small amount of dimethyl sulfoxide to dissolve hemin, and then prepare with the potassium phosphate buffer to obtain a heme solution with a concentration of 10 μM, and store it at 4°C;

[0058] (3) Mix the above DNA solution A, DNA solution B and heme solution at a volume ratio of 1:1:2, let stand at room temperature for 30-60 minutes to obtain a DNA mimic enzyme solution, and store at 4°C;

[0059] (4) Prepare glutamine synthetase working solution, the rea...

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Abstract

The invention adopts a double enzyme coupling system of a G-quadruplet-Hemin peroxide mimic enzyme and a glutamine synthetase; the selectivity of the glutamine synthetase is used for specifically recognizing glutamine; the glutamine is quantitatively converted into a fluorescence signal; the content of glutamine in a sample is detected via a standard curve method. The detection method is convenient to operate, low in cost, quick, accurate, high in sensitivity, and relatively small in damage to the sample, and can be widely applied to the field of biology and agriculture.

Description

technical field [0001] The invention relates to a method for measuring glutamine, more specifically, to a dual-enzyme coupling system capable of rapidly measuring glutamine in leaves, and the application of the method in the fields of biology and agriculture. [0002] technical background [0003] Glutamine is an important intermediate product of plant nitrogen metabolism. The content of glutamine in plant leaves can reflect the information of plant nitrogen utilization. Through the analysis of glutamine, the nitrogen demand of plants can be obtained. The development of fine detection of glutamine is of great significance for the precise management of nitrogen in crops. At present, the quantitative analysis of glutamine is mainly determined by high performance liquid chromatography. High performance liquid chromatography has good reproducibility and accuracy, but there are limiting factors such as relatively high instrument cost and daily analysis maintenance cost, long ana...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/533G01N33/535C12Q1/28C12Q1/26
CPCC12Q1/26C12Q1/28G01N21/64G01N21/6428G01N33/533G01N33/535G01N2021/6417G01N2021/6441
Inventor 朱志强金晨
Owner SUZHOU CHIEN SHIUNG INST OF TECH
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