Method for detecting enzyme kinetics in capillary

A technology of enzyme kinetics and capillary tubes, which is applied in the field of detection of enzyme kinetics in capillaries, can solve the problems of no detection of enzyme kinetics, etc., and achieve the effects of expanded application, high repeatability and simple operation

Inactive Publication Date: 2015-12-09
CHANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for detecting enzyme kinetics in capillary
  • Method for detecting enzyme kinetics in capillary

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Embodiment 1

[0020] ATTO590-H6-QD detection of thrombin dynamics

[0021] The quantum dot bioprobe consists of fluorescent dye "ATTO590", quantum dot "CdSe / ZnSQDs565nm", polypeptide "DDDLVPRGSGP 9 G 2 h 6 "composition.

[0022] 1. Fat-soluble QDs are converted into water-soluble QDs by GSH

[0023] Mix 18mgGSH, 5mgKOH, and 250μL of methanol, take 40μL of the mixed solution and add it to 200μL of fat-soluble quantum dots, and shake for 30min. After shaking, 200 μL of 1 mM NaOH was added, and the fat-soluble quantum dots were transferred to the water phase. Take out the quantum dots in the upper layer, add 1 mL of methanol and 30 μL of NaCl (30 mg / mL) to precipitate, and dissolve in boric acid buffer (pH 7.4, 10 mM). The precipitation was repeated twice, and finally dissolved in 200 μL of boron buffer (pH 7.4, 10 mM).

[0024] 2. Peptide synthesis and labeling

[0025] Peptide DDDLVPRGSGP 9 G 2 h 6 It was synthesized by Fmoc solid-phase synthesis. Activate the Fmoc-protected amino...

Embodiment 2

[0033] Cy5-H6-QDs detection of thrombin kinetic changes

[0034] The quantum dot bioprobe consists of fluorescent dye "Cy5", quantum dot "CdSe / ZnSQDs605nm", polypeptide "DDDLVPRGSGP 9 G 2 h 6 "composition.

[0035] Other steps are with embodiment 1.

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Abstract

The invention relates to the nano-biotechnology field, and provides a method for detecting enzyme kinetics in a capillary. The method is characterized in that the method comprises the following steps: firstly, quantum dots and polypeptides with fluorescent labels are mixed and reacted outside a capillary, and a quantum dot biological probe is obtained; secondly, fluorescence capillary electrophoresis detection is carried out, namely, enzymes and quantum dot biological probes interact with each other in a capillary, a relation of a ratio of peak value areas of an acceptor detection channel and a donor detection channel and reaction time is determined through fluorescence detection, and a peak area ratio-time standard curve is plotted. The beneficial effects of the method are as follows: operation is simple, the repeatability is high, and applications of quantum dot probes in the bioanalysis field are developed further.

Description

technical field [0001] The invention relates to the field of nanobiology technology, in particular to a method for detecting enzyme kinetics in a capillary. Background technique [0002] Common methods for detecting enzyme kinetics include spectrophotometry, fluorescence and radionuclide methods, etc., but these methods are cumbersome to operate and consume a lot of samples, and the enzyme content in organisms is often trace amounts, so the traditional method has no effect on enzyme kinetics. The detection of science and other aspects has great limitations. [0003] Quantum dots (QDs), as a novel fluorescent material, have the advantages of broad excitation spectrum, narrow and symmetrical emission spectrum, tunable color, anti-photobleaching and long fluorescence lifetime. Combining quantum dots with fluorescent peptides, when the distance between them is less than When the radius is large, non-radiative energy transfer occurs, that is, fluorescence resonance energy tran...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 王建浩顾亚琴滕一万陈瑶蒋鹏举邱琳李进晨王车礼杨丽樊杰刘菲菲柳丽
Owner CHANGZHOU UNIV
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