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Label-free electrochemical sensing detection method used for activity of protein tyrosine phosphatase

A technology of tyrosine phosphatase and tyrosine, which is applied in biochemical equipment and methods, determination/inspection of microorganisms, etc. Effect

Inactive Publication Date: 2017-07-18
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] 1) The existing fluorescence method involves complex chemical synthesis of enzyme substrate fluorescent group labeling; 2) Capillary electrophoresis needs to be combined with other methods, and the operation is cumbersome and time-consuming; 3) ELISA is subject to antibody preparation, purification and ease Obtained and other factors are constrained

Method used

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  • Label-free electrochemical sensing detection method used for activity of protein tyrosine phosphatase
  • Label-free electrochemical sensing detection method used for activity of protein tyrosine phosphatase

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Embodiment 1

[0033] Embodiment 1 The functional modification of the semiconductor electrode surface

[0034] Since we use a sensor detection method, it is necessary to functionally modify the surface of the indium tin oxide (ITO) electrode. The modifiers that can be used are generally organic compounds with poor conductivity, which may inhibit the electrochemical response of the electronic mediator. We have carried out different degrees of silanization modification on the surface of the electrode, and respectively placed the ITO electrode in a toluene solution containing 1% 3-glycidyl etheroxypropyltrimethoxysilane to react for different times (0.5, 1, 2, 4 , 8, 10, 12, 16, 20, 24 hours). After the reaction is completed, ultrasonically sonicate in toluene and absolute ethanol for 5 minutes respectively, then blow dry with nitrogen gas, and bake in a muffle furnace at 115° C. for 20 hours. X-ray photoelectron spectroscopy (XPS) showed that the surface content of the modifiers increased wit...

Embodiment 2

[0035] Example 2 Covalent immobilization of polypeptide molecular film on the surface of indium tin oxide

[0036] First cut the functionally modified ITO electrode into a size of 2.5cm×0.5cm, wash it in pure water, blow dry with nitrogen, and evenly cover and spread 10 μL of different concentrations of peptide solutions (2.0M phosphate buffer solution, pH 7.4) on the 0.5cm×0.5cm ITO electrode, placed in a humid container at 25°C for 24 hours. After the surface reaction, the electrode was shaken and washed in 20 mM phosphate buffer containing 0.1% Tween-20, 20 mM phosphate buffer containing 150 mM sodium chloride, and 20 mM phosphate buffer for 5 minutes, and dried with nitrogen. Finally, the ITO electrode was placed in a Tris-HCl solution to seal the remaining reaction sites on the electrode surface. Rinse with deionized water and dry with nitrogen gas. XPS and cyclic voltammetry were used to measure the fixed amount of polypeptide and the electrochemical activity of polype...

Embodiment 3

[0037] The detection of embodiment 3 protein tyrosine phosphatase activity

[0038] According to the optimized conditions, place the ITO electrode with the substrate polypeptide immobilized on the surface in the dephosphorylation reaction solution (containing 50mM NaCl, 1mM DTT, 0.05% Tween-20 and PTP or PTP and inhibitor), and react at 37°C for a period of time. time. The electrode was shaken and washed in 20mM phosphate buffer solution containing 0.1% Tween-20, 20mM phosphate buffer solution containing 150mM sodium chloride, and 20mM phosphate buffer solution for 5 minutes, and dried with nitrogen gas for electrochemical detection.

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Abstract

The invention discloses a simple, rapid and label-free electrochemical sensing detection method used for the activity of protein tyrosine phosphatase (PTP). A substrate (containing phosphotyrosine) polypeptide molecular membrane is constructed on a semiconductor electrode by utilizing a covalent immobilization technology; tyrosine in a polypeptide molecule is used as an electrochemical signal probe and is combined with a catalytic amplification function of a metal complex of an electron mediator osmium with a suitable electric potential, so as to establish an electrochemical biosensor for quantitatively detecting the activity of the PTP; the minimum detection limit is 10nM; and the method can also be applied to rapid screening of PTP inhibitors and medicine molecules. The electrochemical sensing method has the greatest advantage that antibodies and signal labeled molecule are not needed and operation steps are simple; and the electrochemical sensing method is very suitable for quantitative detection of the activity of the protein tyrosine phosphatase.

Description

[0001] Technical field: [0002] The invention relates to an electrochemical biosensing method for label-free detection of protein tyrosine phosphatase activity. The method is simple in operation and high in sensitivity, and can be used for quantitative analysis of protein tyrosine phosphatase activity. [0003] technical background: [0004] Reversible phosphorylation of protein tyrosine is an important way to regulate eukaryotic cell signaling pathways. Phosphorylation is co-regulated, and eukaryotic cells regulate intracellular and intercellular communication through this reversible balance (see literature Lim W.A. et al. Cell, 2010, 142(5):661-667.). Compared with PTK, the research on PTP started late, and it was not until nearly two decades that people gradually realized its importance in human health and diseases. There are many types of PTPs, which are widely expressed in various tissues and cell types. There are more than 100 confirmed PTP family members. PTPs can reg...

Claims

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Application Information

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IPC IPC(8): C12Q1/42
CPCC12Q1/005C12Q1/42
Inventor 杨郁郭良宏任肖敏
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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