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Electrochemical sensor detection method of thrombin based on aptamer-gold nanoparticle-enzyme composite

A thrombin and gold nanoparticle technology, applied in the field of analytical chemistry, can solve the problem that the sensitivity of the protein detection method does not reach the nanomolar and picomolar level, and achieve the effect of large specific surface area, good separation effect and high activity

Inactive Publication Date: 2012-02-15
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sensitivity of the protein detection methods introduced above has not yet reached the nanomolar and picomolar levels.

Method used

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  • Electrochemical sensor detection method of thrombin based on aptamer-gold nanoparticle-enzyme composite
  • Electrochemical sensor detection method of thrombin based on aptamer-gold nanoparticle-enzyme composite
  • Electrochemical sensor detection method of thrombin based on aptamer-gold nanoparticle-enzyme composite

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Experimental program
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Effect test

Embodiment 1

[0025] The steps for detecting thrombin using an electrochemical method based on the signal amplification of the aptamer-gold nanoparticles-horseradish peroxidase complex are as follows:

[0026] (1) Synthesize gold-coated magnetic nanoparticles and immobilize the thrombin aptamer I capture probe (the sequence of this aptamer I is 5′SH-(CH 2 ) 6 -GGTTGGTGT GGTTGG). Take 50 μL of the gold-coated magnetic nanoparticle suspension and wash it with tris-hydrochloric acid buffer solution (pH 7.4, containing 100 mmol L -1 Sodium chloride, 5mmol·L -1 Potassium chloride, 1mmol L -1 Magnesium chloride, 5mmol·L -1 Calcium chloride) was washed three times, and then re-dispersed into 495 μL tris-hydrochloric acid buffer solution and sonicated for 30 min. 5 μL sulfhydryl-modified aptamer I (100 μmol L -1 ) was added thereto, and stirred gently at room temperature for 2h. Magnetic separation, followed by washing three times with tris-hydrochloric acid buffer solution, to remove unreac...

Embodiment 2

[0031] Under the same experimental conditions, four other proteins (human serum albumin, lysozyme, fibrin, immunoglobulin G) were used to replace thrombin to measure its electrochemical response, and the four proteins when they coexisted with thrombin respectively. electrochemical response.

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Abstract

The invention relates to a method for detecting thrombin with an electrochemical aptamer sensor based on aptamer-gold nanoparticle-horseradish peroxidase signal amplification, belonging to the technical field of analytical chemistry, characterized in that: a thrombin aptamer I is fixed on the gold coated magnetic nanoparticles as a capture probe, an aptamer II is double-labeling by gold nanoparticle and horseradish peroxidase to form an aptamer-gold nanoparticle-horseradish peroxidase composite as a detection probe; the capture probe and the detection probe are combined with thrombin to form a [gold coated magnetic nanoparticle-aptamer I] / thrombin / [ aptamer II-gold nanoparticle-horseradish peroxidase] sandwich structure; and the sandwich structure is fixed on the surface of the electrode, and thrombin is detected through enzyme catalytic reaction. The method has the advantages of simple operation, rapid response, high sensitivity, and strong specificity. The detection limit of thrombin is 30 fmol.L<-1>, and the linear range is 0.1-60 pmol.L<-1>.

Description

technical field [0001] The invention relates to a method for measuring thrombin based on an aptamer-gold nanoparticle-horseradish peroxidase signal amplification electrochemical technique and belongs to the field of analytical chemistry. Background technique [0002] In recent years, protein detection has attracted widespread attention in many biological fields such as disease diagnosis and basic research. In many protein detections, traditional techniques use antibodies to identify proteins. However, new recognition technologies using aptamers as recognition elements are constantly being developed and researched. Aptamers are a class of oligonucleotide fragments with a special sequence of 3-D structure screened by exponential enrichment ligand system technology, which can be RNA or DNA. At present, the aptamers screened by this technology can specifically recognize different targets, mainly including: ions, small organic molecules, peptides, proteins and complete cells. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/34C12Q1/68C12Q1/56C12Q1/28G01N27/26
Inventor 张友玉赵洁曾跃刘美玲
Owner HUNAN NORMAL UNIVERSITY
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