Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip

A thrombin inhibitor and peptide microarray technology, applied in the field of peptide microarray chips, can solve the problems of unsuitable thrombin screening and achieve accurate judgment

Active Publication Date: 2015-05-20
中科应化(长春)科技有限公司
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Problems solved by technology

[0005] In order to solve the technical problem that the existing method for detecting thrombin in analytical chemistry is not suitable for the detection of thrombin in the blood coagulation environment and the screening of thrombin inhibitors, the present invention provides a polypeptide microarray chip for detecting thrombin activity in plasma and method for screening thrombin inhibitors

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  • Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip
  • Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip
  • Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip

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preparation example Construction

[0052] The preparation method of the polypeptide microarray chip of the present invention is a well-known technology for those skilled in the art, such as the contact method. This embodiment provides a preparation process thereof, but the present invention is not limited thereto. The specific steps are:

[0053] (1) Use the product produced by Beijing Boao Biological Co., Ltd. The SmartArrayer136 spotting system spots the peptide solution on Polymer three-dimensional substrate D surface;

[0054] (2) Place the above-mentioned chip in a vacuum drying oven to evacuate, and react overnight at 30° C. to obtain the reacted chip;

[0055] (3) Block the reacted chip with 1% (w / v) bovine serum albumin to make a polypeptide microarray chip.

[0056] In the present invention, the polypeptide lattice concentration of the polypeptide microarray chip is generally 0.1-2mg / mL, but not limited thereto, the greater the concentration of the polypeptide lattice in the polypeptide microarray ...

Embodiment 1

[0108] combine figure 2 and Figure 10 Illustrative Example 1

[0109] Peptide microarray chip detection of thrombin activity:

[0110] (1) The polypeptide microarray chip with 8 kinds of polypeptide substrates, the concentration of the polypeptide array is 0.5mg / mL, and the 8 kinds of polypeptide substrates are respectively:

[0111] Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys (biotin);

[0112] Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys(biotin);

[0113] Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);

[0114] Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);

[0115] Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys (biotin);

[0116] Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys (biotin);

[0117] Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);

[0118] The polypeptide microarray chip of Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys (biotin)...

Embodiment 2

[0123] combine image 3 and Figure 10 Illustrative Example 2

[0124] Peptide microarray chip detection of thrombin activity:

[0125] (1) The concentration of the peptide matrix is ​​0.2, 0.3, 0.35, 0.5, 1, 1.5 mg / mL, and the peptide substrate is Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe -Arg-Val-Val-Lys (biotin) polypeptide microarray chip was reacted with 810nM thrombin for 1h to obtain the reacted polypeptide microarray chip;

[0126] (2) respectively reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;

[0127] (3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.

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Abstract

The invention discloses a method for detecting thrombin activity and screening a thrombin inhibitor in plasma by using a polypeptide microarray chip, which solves the technical problem that the thrombin detection method in the existing analytical chemistry is inapplicable to thrombin detection and thrombin inhibitor screening in a blood clotting environment. The method comprises the following steps: reacting a polypeptide microarray chip with activated plasma or activated plasma containing a thrombin inhibitor at 36-38 DEG C for more than 40 minutes, reacting with avidin for more than 1 hour, reacting with polypeptide modified gold nanoparticles for more than 1 hours, and detecting resonant light scattering signals of the gold nanoparticles on the polypeptide microarray chip, thus realizing the detection of the thrombin activity and the screening of the thrombin inhibitor. According to the invention, the thrombin activity can be directly detected in plasma, and common plasma, hypercoagulable plasma and hypocoagulable plasma can be preliminarily distinguished; and in a blood clotting environment, the thrombin inhibitor can be screened, thus providing a foundation for the development and application of thrombin inhibitor medicaments.

Description

technical field [0001] The invention relates to a polypeptide microarray chip for detecting thrombin activity in blood plasma and a method for screening thrombin inhibitors, belonging to the technical field of polypeptide microarray chips. Background technique [0002] Thrombin is a serine protease. In the blood coagulation pathway, on the one hand, it converts fibrinogen into fibrin, promotes platelet aggregation, and accelerates blood coagulation. On the other hand, it activates the anticoagulant mechanism to prevent thrombus formation. (A Brief Historical Review of the Waterfall / Cascade of Blood CoaguLation, J. Biol. Chem., 2003, 278, 50819-50832). [0003] Blood coagulation is a complex network process regulated by multiple factors, and changes in the concentration of a certain factor will change the concentration of other factors in the system. Thrombin activity detection can more accurately reflect the combined effect of each factor in the coagulation pathway (In Vivo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/56G01N33/68
Inventor 王振新苏敏
Owner 中科应化(长春)科技有限公司
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