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DNA detection method based on electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and enzyme

A gold nanoparticle and terminal extension technology, which is applied in the field of electrochemical biosensor research, can solve problems such as increased cost, high cost, and increased complexity of the reaction system, and achieves the effects of easy simplification, strong applicability, and easy miniaturization

Active Publication Date: 2016-08-10
NANJING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the polymerase chain reaction technology requires strict control of temperature changes, and requires relatively complex reaction instruments, which are costly; and the rolling circle replication amplification process also requires the addition of reaction templates, which increases the complexity of the reaction system and increases the cost. cost

Method used

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  • DNA detection method based on electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and enzyme
  • DNA detection method based on electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and enzyme
  • DNA detection method based on electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and enzyme

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Preparation of Au-DNA and its results

[0038] Using the method for detecting DNA based on terminal extension, gold nanoparticles and enzyme three-stage amplified electrochemical sensor of the present invention, the operation of preparing Au-DNA is as follows: centrifuge and concentrate 1000mL of gold nanoparticle solution to 300μL, and its final concentration is about 1 nmol / L; slowly add 20 μL of 5'-terminal sulfhydryl-labeled signal probe solution with a concentration of 50 μmol / L, and the final concentration is 3.3 μmol / L; shake and react at 350 rpm for 16 hours, then add an equal volume of 0.02 mol / L PBS ( PH=7, 0.2mol / L NaCl), aged for 40 hours; centrifuged at 10000rpm for 15 minutes, added 0.01mol / L PBS (PH=7, 0.1mol / L NaCl), centrifuged at 10000rpm for 15 minutes, respectively PBS (0.01M, 0.25M NaCl, PH=7), PBST (0.01M, 0.25M NaCl, 0.1% Tween, PH=7), PBSB (0.01M, 0.25M NaCl, 1% bovine serum albumin, PH=7) to prepare Au-DNA solution. The prepared Au-...

Embodiment 2

[0039] Example 2: Effects of different assembly densities on electrochemical signal detection results.

[0040] The method for detecting DNA based on terminal extension, gold nanoparticles and enzyme three-stage amplified electrochemical sensor of the present invention is used, and the completely complementary DNA sequence is used as the target fragment. All the operating steps are as described above, wherein the blocking solution HS-(CH 2 ) 11 -The concentration of EG2-OH is 0.5mmol / L, the concentration of target DNA is 1μmol / L, and the assembly density is 10, 5, 1, 0.5, 0.2, 0.1μmol / L in turn, and the influence of different assembly densities on the detection of electrochemical signals is analyzed The impact of the analysis results are attached image 3 As shown in (A), before 0.5 μmol / L, the signal-to-noise ratio increases with the decrease of the assembly density, and after 0.5 μmol / L, the signal-to-noise ratio decreases with the decrease of the assembly density, so the o...

Embodiment 3

[0041] Example 3: Different HS-(CH 2 ) 11 - Effect of blocking concentration of EG2-OH on electrochemical detection results.

[0042] Using the method for detecting DNA based on terminal extension, gold nanoparticles and enzyme three-stage amplified electrochemical sensor described in the present invention, using a fully complementary DNA sequence as the target fragment, all the operating steps are as described in Example 2 above, wherein the assembly The density is 0.5 μmol / L, the concentration of target DNA is 1 μmol / L, and the blocking solution HS-(CH 2 ) 11 The concentration of -EG2-OH was 2, 1.5, 1, 0.5, 0.1, 0mmol / L in order to analyze different HS-(CH 2 ) 11 The influence of -EG2-OH concentration on the detection of electrochemical signals, the analysis results are as attached image 3 As shown in (B), before 0.5mmol / L, with HS-(CH 2 ) 11 -The signal-to-noise ratio increases with the decrease of EG2-OH concentration, after 0.5mmol / L, along with HS-(CH 2 ) 11 -T...

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Abstract

The invention discloses a DNA detection method based on an electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and oxido-reductase. The method comprises: modifying the surface of a gold electrode with a layer of sulfydryl-modified capturing probe molecules for capturing an objective DNA molecule; after the objective DNA molecule is captured by the capturing probe, modifying the surface of the electrode with complementary pairing of signal-probe-modified gold nanoparticles and basic groups on the other end of the objective DNA molecule; in the catalytic action of a terminal extension enzyme, extending biotin-labeled deoxyribonucleotide to 3' terminal of the signal probe; during the extension process, introducing biotin for specific binding to avidin-labeled horse radish peroxidase; and modifying the surface of the electrode with horse radish peroxidase. High-sensitivity and high-specificity detection of the objective DNA molecule is realized through detection of change of an electrochemical signal generated from an electrolyte catalyzed by horse radish peroxidase. The method is used for detection of a DNA sample in the fields, such as molecular recognition, clinical diagnosis, environmental monitoring, and food security.

Description

technical field [0001] The invention belongs to the research field of electrochemical biosensors, and relates to a method for detecting DNA based on terminal extension, gold nanoparticles and enzyme-catalyzed three-stage amplified electrochemical sensors. Background technique [0002] DNA detection has been widely used in molecular identification, clinical diagnosis, environmental monitoring, food safety and so on. However, the concentration of DNA to be detected in actual samples is often very low, so designing and researching sensors with high sensitivity and selectivity has become the focus of research. In order to improve the sensitivity of DNA detection, various signal amplification strategies have emerged, including nucleic acid amplification signal amplification strategies, nanomaterial signal amplification strategies, and enzyme-catalyzed signal amplification strategies. [0003] Currently commonly used nucleic acid amplification techniques include polymerase chain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 万莹王鹏娟苏岩杨树林
Owner NANJING UNIV OF SCI & TECH
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