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Fluorescence detection method of cysteine

A technology for fluorescence detection and cysteine, which is applied in the field of fluorescence detection of cysteine, can solve complex and high-cost problems, achieve high sensitivity and specificity, save detection costs, and simplify operation steps

Inactive Publication Date: 2011-09-14
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

) developed a fluorescent "on" molecular beacon probe for the detection of cysteine, which is also based on Hg 2+ Competitive coordination by cysteine ​​and T-T mismatches has high sensitivity and selectivity, but it not only requires double fluorescent labeling of molecular beacons but also requires heating of the solution, so this method is not only costly but also also more complicated

Method used

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  • Fluorescence detection method of cysteine
  • Fluorescence detection method of cysteine
  • Fluorescence detection method of cysteine

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Add 2mL buffer (10mM MOPS, containing 0.1M NaNO 3 , pH 7.50), add 2 μL Hg 2+ (70 μM) and mercury ion-specific DNA 10 μL (2 μM), mix evenly for 2 minutes, add 5 μL Sybr Green I (25×), mix evenly, and perform fluorescence test after 2 minutes. On this basis, 4 μL (70 μM) of cysteine ​​was added, mixed evenly, and the fluorescence test was performed immediately. At this moment, the final concentration of each substance is: [MSD]=10nM, [Hg 2+ ]=70nM, [Sybr Green I]=1.225×10 -7 M, [cysteine] = 140 nM.

[0037] Fluorescence spectra before and after addition of cysteine figure 2 As shown, where a is the fluorescence spectrum before the addition of cysteine, the fluorescence intensity at the maximum emission wavelength is 116.7, b is the fluorescence spectrum after the addition of cysteine, and the fluorescence intensity at the maximum emission wavelength is 15. Therefore, the present invention can quantitatively detect cysteine.

[0038] Selection of the concentration o...

Embodiment 2

[0043] Add 2mL buffer (10mM MOPS, containing 0.1M NaNO 3 , pH 7.50), add 2 μL Hg 2+ (70 μM) and mercury ion-specific DNA 10 μL (2 μM), mix evenly for 2 minutes, add 5 μL Sybr Green I (25×), mix evenly, and perform fluorescence test after 2 minutes. On this basis, different concentrations of cysteine ​​were added, mixed evenly, and the fluorescence test was performed immediately. The result is as Figure 4 As shown, it shows that the present invention can quantitatively detect different concentrations of cysteine, and the concentration of cysteine ​​at 7-84nM is directly proportional to the fluorescence intensity.

Embodiment 3

[0045] Replace cysteine ​​with other amino acids and repeat the steps of Example 1, the results are as follows Figure 5 shown. It shows that the present invention is not interfered by other amino acids and has good selectivity.

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Abstract

The invention discloses a fluorescence detection method of cysteine. In the method, mercury ion specific DNA (mismatching of 7 pieces of thymine-thymine) is in a random curled state without the presence of mercury ions; after the mercury ions are added, a thymine-Hg2<+>-thymine (T-Hg2<+>-T) complex is formed, so that the mercury ion specific DNA has a hairpin-shaped structure; fluorescent dye Sybr Green I, which can be combined with double-chain DNA to emit light and has high fluorescence intensity, is added; and amino acids to be tested are added, when the cysteine is detected, the cysteine and the mercury ions can form a more stable complex, so that the mercury ions can be extracted from the T-Hg2<+>-T complex, the hairpin-shaped structure unwinds, the fluorescence intensity is reduced, and other amino acids cannot cause the change of the fluorescence intensity. The method is high in sensitivity and specificity, simple and quick; and the whole process can be finished within 5 minutes.

Description

technical field [0001] The invention relates to the field of cysteine ​​detection, in particular to a cysteine ​​fluorescence detection method. Background technique [0002] Cysteine ​​belongs to protein amino acid and is a very important sulfhydryl substance in organisms. It participates in the reduction process of cells in organisms, and has physiological functions such as regulating the metabolism of phospholipids in the liver and protecting liver cells from poison damage. Changes in the content of cysteine ​​in organisms or metabolic disorders can lead to the occurrence of some diseases. By measuring the content of cysteine ​​in organisms, the diagnosis of certain metabolic diseases can be realized, so the rapid and sensitive detection of cysteine ​​can be realized. is of great significance. There are many reported cysteine ​​determination methods, mainly spectrophotometry (O.Rusin, N.N.S.Luce,; R.A.Agbaria, J.O.Escobedo, S.Jiang, I.M.Warner, F.B.Dawan, K.Lian and R.M.S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 徐慧陈建农高淑丽柳全文
Owner LUDONG UNIVERSITY
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