Method and kit for detecting concentration of chiral amino acid

A chiral amino acid and amino acid technology, applied in the field of analytical chemistry, can solve the problem of inability to analyze the content of chiral amino acids, and achieve the effects of low emission background, high catalytic effect, and high detection sensitivity

Inactive Publication Date: 2012-09-26
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a chemiluminescence method with simple operation and high sensitivity, which can be used to detect enantiomer chiral amino acids in the body

Method used

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  • Method and kit for detecting concentration of chiral amino acid
  • Method and kit for detecting concentration of chiral amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Sample pretreatment

[0035] 1. Solution preparation:

[0036] ①PBS buffer solution: Weigh a certain amount of disodium hydrogen phosphate and sodium dihydrogen phosphate, prepare a phosphate buffer solution with a total concentration of 10mM, and adjust the pH of the solution to 5.0.

[0037] ②Amino acid solution: weigh D-alanine, dissolve it in 10mM PBS (pH 5.0), and prepare the concentration to be 1.0×10 -3 mol / L solution.

[0038] ③Copper ion solution: Weigh a certain amount of copper sulfate, dissolve it in 10mM PBS (pH 5.0), and prepare 1.0×10 -2 mol / L copper ion solution.

[0039] ④ Amino acid solution containing copper ions: Add solid copper sulfate to the mixed amino acid solution prepared in ② to make the final concentration 1.0×10 -2 mol / L.

[0040] 2. Purification of samples by cation exchange solid phase extraction

[0041] (1) Activate SCX SPE cartridge (model CNWBOND SCX, 500mg, 3ml, CNW company).

[0042] Wash the SPE cartridge three times with 3m...

Embodiment 2

[0046] D-type amino acid oxidase converts D-alanine (D-Ala) to hydrogen peroxide

[0047] 1. Solution preparation:

[0048] ① D-amino acid oxidase solution: Weigh a certain amount of D-amino acid oxidase and dissolve it in 10mM PBS (pH 8.3) at a concentration of 43U / ml.

[0049] ②D-alanine solution: Weigh D-alanine, dissolve it in 10mM PBS (pH 8.3), and prepare a concentration of 5.0×10 -6 , 1.0×10 -5 , 5.0×10 -5 , 1.0×10 -4 , 5.0×10 -4 , 1.0×10 -3 , 2.0×10 -3 A series of solutions in mol / L.

[0050] ③L-alanine solution: Weigh L-alanine, dissolve it in 10mM PBS (pH 8.3), and prepare a concentration of 2.0×10 -3 mol / L solution.

[0051] ④ Mixed solution of D-alanine and L-alanine: Weigh D-alanine and L-alanine, dissolve them in 10mMPBS (pH 8.3), and make final concentrations of 2.0×10 -3 mol / L mixed solution.

[0052] 2. D-amino acid conversion enzyme reaction

[0053] Take 3 μl of the above-mentioned D-amino acid oxidase solution (i.e. solution ①), 5 μl of the abov...

Embodiment 3

[0055] Chemiluminescent detection of D-alanine

[0056] 1. Solution preparation:

[0057] (1) Horseradish peroxidase solution: Weigh a certain amount of horseradish peroxidase and dissolve it in 100mMPBS (pH 8.0) with a concentration of 60U / ml.

[0058] (2) Luminescence reaction buffer: prepare 0.1mol / L phosphate buffer solution, and adjust the pH value of the solution to 10.

[0059] (3) Luminol solution: Weigh a certain amount of luminol, dissolve it in water and prepare a concentration of 1.0×10 -3 mol / L luminol solution. The concentration of luminol should not be too high, otherwise it is not easy to dissolve, and more alkali needs to be added, which is not conducive to the control of the pH of the reaction system.

[0060] 2. Horseradish peroxidase catalyzes luminol luminescence reaction

[0061] Take 50 μl of D-alanine conversion reaction solution by D-type amino acid oxidase, and add in sequence 397 μl of the above-mentioned luminescent reaction buffer, 50 μl of lum...

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Abstract

The invention discloses a method and a kit for detecting concentration of chiral amino acid. The method comprises the following steps of: 1) adding D-amino acid oxidase or L-amino acid oxidase into a sample to be tested for carrying out an enzyme catalysis reaction; 2) sequentially adding buffer solution, luminol and horse radish peroxidase into the obtained reaction solution in the step 1) for carrying out a chemiluminiscence reaction; and 3) measuring the luminous intensity of the reaction solution in the step 2). The method can be used for measuring chiral amino acid in an enantiomer. The method is not interfered by another chiral amino acid in the enantiomer or is not interfered by common metal ions. The method is easy to operate and high in sensitivity, and the content of the chiral amino acid in the enantiomer can be detected.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, in particular to a method for detecting chiral amino acids by chemiluminescence and a kit thereof. Background technique [0002] The level of amino acids in organisms reflects many life phenomena in organisms, and is closely related to many diseases. Therefore, especially the detection of D-type amino acids is of great significance in clinical diagnosis and clinical basic research. However, due to the lack of ultraviolet absorption, fluorescence activity and electrochemical activity of these compounds, most analytical methods require derivatization of such compounds; more importantly, the analysis of enantiomers often requires chiral derivatization reagents or other chiral Resolution techniques can be realized, such as separation by chiral chromatographic columns. Chemiluminescence (CL) detection has the characteristics of high sensitivity, simple method, and does not require any external li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
Inventor 曹旭妮倪志尧夏琨
Owner EAST CHINA UNIV OF SCI & TECH
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