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Temperature-controllable electrochemical DNA biosensor and preparation method thereof

A biosensor and electrochemical technology, applied in the field of nucleic acid detection and bioanalysis, can solve the problems of enzyme molecular structure denaturation, difficult operation, complicated device, etc., and achieve the effect of high sensitive operation, fast detection and low cost

Inactive Publication Date: 2017-04-26
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Exonuclease III is a biological macromolecule that is very sensitive to temperature changes. When the temperature is lower than the optimum temperature, the activity of the enzyme is poor, but if the temperature is too high, the molecular structure of the enzyme will be irreversibly denatured and the enzyme will be inactivated.
Most of the amperometric biosensors reported in the past control the overall temperature change of the experimental system, and the required devices are complicated and difficult to operate.

Method used

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  • Temperature-controllable electrochemical DNA biosensor and preparation method thereof
  • Temperature-controllable electrochemical DNA biosensor and preparation method thereof
  • Temperature-controllable electrochemical DNA biosensor and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A temperature-controlled exonuclease-based The preparation method of the electrochemical DNA biosensor of target cycle signal amplification, such as figure 1 shown, including the following steps:

[0034] (1) Design a capture probe DNA strand CP, the DNA strand CP is complementary to the target DNA strand TP, and can form a double-stranded structure with a 3' blunt end, inducing exonuclease III from the 3' of the double-stranded structure The blunt end digests the capture probe from the 3' end to the 5' end, thereby releasing the target DNA; the 5' end of the CP chain is thiolated, so that the CP is modified to the surface of the gold wire thermode through the gold-sulfur bond; among them, the capture probe CP The DNA sequence of the target DNA strand TP is: 5'-SH-(CH2)6-TTTTC TGTGC GCCGG TCTCT CCCA-3', the DNA sequence of the target DNA strand TP is: 5'-TGGGA GAGAC CGGCG CACAG AGGAAG-3';

[0035] (2) The gold wire thermal electrode is used on the suede with 0.05mm A...

Embodiment 2

[0039] To investigate the influence of temperature on the stability of gold wire thermode modified with capture probe CP, such as figure 2 As shown, the specific steps are as follows:

[0040] (1) Soak the thiolated capture probe-modified gold wire thermal electrode obtained in step (3) of Example 1 in a detection solution containing 10 mM Tris-HCl and 5 mM hexaammine ruthenium trichloride at a pH of 7.4, Then form a three-electrode system with silver silver chloride participating electrode and platinum wire contrast electrode, and perform SWV detection to obtain the initial SWV curve of ruthenium;

[0041] (2) Put the gold wire thermode modified with the thiol capture probe that has undergone the initial detection in the buffer solution containing 10mM Tris-HCl with a temperature of 30°C, 35°C, 40°C, 45°C, and 50°C and a pH of 7.4. Soak in the middle for 1h, after soaking, use SWV to detect in the ruthenium trichloride hexaammonium ruthenium detection solution, and obtain t...

Embodiment 3

[0043] To optimize the experimental temperature.

[0044] Only change the reaction temperature in step (4) of Example 1 from 0 to 40°C, and carry out experiments in turn, and other reaction conditions are the same as in Example 1; modify the thiol capture probe obtained in step (3) of Example 1 with CP The gold wire thermal electrode of the three chloride hexaammonium ruthenium detection solution carries out SWV detection, obtains the peak current I of ruthenium 0(RuHex) ; The electrochemical biosensor finally obtained in the embodiment 1 step (4) is detected in the detection solution of hexaammonium ruthenium trichloride with SWV, and the peak current of the obtained ruthenium is compared with the obtained I 0(RuHex) Take the absolute value of the difference: |ΔI RuHex |=|I RuHex -I 0(RuHex) | with |ΔI RuHex |Plotted against temperature, such as image 3 As shown, it can be seen that with the increase of temperature |ΔI RuHex | becomes larger, indicating that the increa...

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Abstract

The invention provides a temperature-controllable electrochemical DNA biosensor and a preparation method thereof. The preparation method comprises fixing a sulfhydrylated single-chain DNA complementary with a target DNA sequence segment to the surface of a gold wire thermode so that a capture probe is obtained, carrying out complementation with the target DNA to obtain a double-chain structure with a flush end 3', and inducing an excision enzyme III to digest the capture probe from the flush end 3' to 5' of the double-chain structure so that the target DNA is released, carries out novel hybridization digestion cycling with other capture probes so that finally, the amount of the capture probes on the surface of the electrode is significantly reduced. Before and after digestion cycling, a reduction degree of electrode surface capture probe electrochemical adsorption signals to hexaamimineruthenium trichloride in the detection liquid is in a linear relationship with the logarithm of a DNA concentration so that the detection of the target DNA can be achieved. The detection method provided by the invention has the characteristics of short detection time, high sensitivity, low detection line and good selectivity to the detection of the target DNA.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, in particular to a temperature-controllable electrochemical DNA biosensor and a preparation method thereof, which are applied in the field of nucleic acid detection. Background technique [0002] Rapid, accurate, and sensitive detection of specific DNA sequences is of great significance in the fields of preventive diagnosis of gene-related diseases, environmental monitoring, and food monitoring. Usually, there are only trace amounts of target substances in actual biochemical analysis samples, so high sensitivity and high accuracy have always been the improvement goals of analytical detection methods. In response to this demand, in addition to the direct development of highly sensitive methods, the amplification detection method can reduce the design difficulty of analytical methods or sensors and the dependence on professional large-scale instruments. [0003] According to different ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327
CPCG01N27/26G01N27/3275
Inventor 吴韶华王芳芳孙建军张标米真真
Owner FUZHOU UNIV
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