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Thrombin detection method and kit thereof

A detection kit and detection method technology, applied in the field of chemical and biological sensing, can solve the problems of lower detection efficiency, higher detection cost, and different scope of application, and achieve the effect of good specificity and low detection limit

Active Publication Date: 2018-11-06
CIXI INST OF BIOMEDICAL ENG NINGBO INST OF MATERIALS TECH & ENG CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the methods for detecting thrombin mainly include electrochemical methods, nano-gold chromogenic method, surface-enhanced Raman method (SERS), fluorescent signal detection method, etc., but all of them have certain shortcomings, such as different scopes of application, and need to rely on Large-scale detection instruments or the synthesis of probes that require signal labeling, etc., greatly increase the detection cost and reduce the detection efficiency

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  • Thrombin detection method and kit thereof
  • Thrombin detection method and kit thereof
  • Thrombin detection method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] (1) Design and synthesis of nucleic acid for detection: Unmodified thrombin aptamer DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3', can be used without further purification; Synthesis method Manually synthesize a complementary PNA chain with a length of 10 bases: AAC CTG CCT C, cleave the crude PNA in the peptide synthesis tube and wash it with anhydrous ether three times, centrifuge and discard the supernatant, dry the sample with nitrogen and use Dissolved in ultrapure water. The sample was purified on HPLC, and the target peak was taken for mass spectrometry identification. The product of the target peak was collected after multiple purifications, and the product was lyophilized and dissolved in ultrapure water, and the concentration was quantified with an ultraviolet-visible absorption spectrometer.

[0039] (2) Detection of thrombin and exploration of its detection limit: PNA and DNA (2 μM) at the same concentration were added to Tris-HCl buffer as subst...

Embodiment 2

[0043](1) Design and synthesis of nucleic acid for detection: Unmodified thrombin aptamer DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3', can be used without further purification; Synthesis method Manually synthesize a complementary PNA chain with a length of 10 bases: AAC CTG CCT C, cleave the crude PNA in the peptide synthesis tube and wash it with anhydrous ether three times, centrifuge and discard the supernatant, dry the sample with nitrogen and use Dissolved in ultrapure water. The sample was purified on HPLC, and the target peak was taken for mass spectrometry identification. The product of the target peak was collected after multiple purifications, and the product was lyophilized and dissolved in ultrapure water, and the concentration was quantified with an ultraviolet-visible absorption spectrometer.

[0044] (2) Detection of thrombin and exploration of its detection limit: PNA and DNA (1.5 μM) at the same concentration were added to Tris-HCl buffer as subs...

Embodiment 3

[0046] (1) Design and synthesis of nucleic acid for detection: Unmodified thrombin aptamer DNA sequence: 5'-AGT CCGTGG TAG GGC AGG TTG GGG TGA CT-3', can be used without further purification; Synthesis method Manually synthesize a complementary PNA chain with a length of 10 bases: AAC CTG CCT C, cleave the crude PNA in the peptide synthesis tube and wash it with anhydrous ether three times, centrifuge and discard the supernatant, dry the sample with nitrogen and use Dissolved in ultrapure water. The sample was purified on HPLC, and the target peak was taken for mass spectrometry identification. The product of the target peak was collected after multiple purifications, and the product was lyophilized and dissolved in ultrapure water, and the concentration was quantified with an ultraviolet-visible absorption spectrometer.

[0047] (2) Detection of thrombin and exploration of its detection limit: PNA and DNA (2.5 μM) at the same concentration were added to Tris-HCl buffer as sub...

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Abstract

The invention discloses a thrombin detection method and a kit thereof. The method comprises the following steps: 1) synthesizing a complementary PNA chain according to a DNA sequence of a thrombin aptamer; 2) mixing DNA in the thrombin aptamer with the synthesized PNA chain in the step 1) to be prepared into a substrate solution; 3) adding a color developing agent into the substrate solution and observing solution color; 4) adding a sample to be tested, observing solution color and judging a concentration of thrombin in the sample to be tested. The detection method disclosed by the invention has the advantages of quickness and convenience in detection, extremely-low requirement for instruments and equipment, high sensitivity and good specificity.

Description

technical field [0001] The application relates to a thrombin detection method and a kit thereof, belonging to the technical field of chemical and biological sensing. Background technique [0002] Thrombin, as a hemostatic drug, can catalyze fibrinogen into fibrin to promote blood coagulation. It is often used for local hemostasis of capillary bleeding and tissue healing after surgery. The lack of thrombin or the abnormal production mechanism will lead to a variety of hemorrhagic diseases, such as cerebral hemorrhage, gastrointestinal bleeding, etc., and even cause cancer in severe cases. Therefore, thrombin is often used as a biomarker for the diagnosis of related diseases. At present, the methods for detecting thrombin mainly include electrochemical methods, nano-gold chromogenic method, surface-enhanced Raman method (SERS), fluorescent signal detection method, etc., but all of them have certain shortcomings, such as different scopes of application, and need to rely on Lar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N21/78
CPCG01N21/3103G01N21/78
Inventor 徐梦佳赵超邢淑付盼徐小军徐皖星
Owner CIXI INST OF BIOMEDICAL ENG NINGBO INST OF MATERIALS TECH & ENG CHINESE ACAD OF SCI
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