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AC Impedance-Type DNA Electrochemical Sensor Based on Probe DNA Controlled Assembly Interface

A technology of AC impedance and controlled assembly, applied in the field of biosensing, can solve the problems of high density of DNA probes, limited steric hindrance and charge, and insignificant changes in the AC impedance value of hybridization reactions, etc.

Inactive Publication Date: 2011-12-21
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the high density of DNA probes on the electrode surface, the traditional AC impedance DNA electrochemical sensor has obvious steric hindrance and charge effects before DNA hybridization. When the concentration of the detected target DNA is low, the increased The steric hindrance and charge are very limited, and the AC impedance value of the hybridization reaction has increased, but the change is not obvious, which makes the sensitivity of the method not high

Method used

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  • AC Impedance-Type DNA Electrochemical Sensor Based on Probe DNA Controlled Assembly Interface
  • AC Impedance-Type DNA Electrochemical Sensor Based on Probe DNA Controlled Assembly Interface
  • AC Impedance-Type DNA Electrochemical Sensor Based on Probe DNA Controlled Assembly Interface

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Experimental program
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Effect test

Embodiment 1

[0031] The preparation steps of the AC impedance type DNA electrochemical sensor based on the probe DNA controlled assembly interface are as follows:

[0032] (1) Piranha solution (30% H 2 o 2 Concentrated H with a concentration of 98% 2 SO 4 , mixed at a volume ratio of 1:3) ultrasonicated for 5 min, ultrasonically cleaned with deionized water twice, each time for 5 min, and then washed with 0.3 μm and 0.05 μm Al 2 o 3 The mixture of water and water is polished to a mirror surface, followed by ultrasonic cleaning with ethanol and distilled water. Place the sonicated electrode at 0.5 M H 2 SO 4 The cyclic voltammetry scans to stability, washes with double distilled water, N 2 Blow dry for later use;

[0033] (2) Take 4 μl of probe DNA (oligonucleotides synthesized by Baosheng Bioengineering Co., Ltd.) solution, drop-coat it on the surface of the pretreated bare gold electrode, leave it at room temperature for 2 h, wash with PBS washing solution, double distilled water ...

Embodiment 2

[0035] The detection steps of the target DNA by the AC impedance type DNA electrochemical sensor based on the probe DNA controlled assembly interface are as follows:

[0036] (1) The capture probe immobilized on the surface of the electrode obtained in Example 1 and the target DNA (oligonucleotides synthesized by Baosheng Bioengineering Co., Ltd.) were hybridized in a hybridization buffer solution at 37 °C for 40 min in a water bath to form double-stranded DNA. Rinse the surface of the electrode with 10 mM PBS washing solution (pH 7.4) to remove unhybridized DNA strands, and then rinse with double distilled water before testing;

[0037] (2) Immerse the electrode prepared in step (1) in 4 mM K containing 0.1 M KCl 3 [Fe(CN) 6 ] / K 4 [Fe(CN) 6 ] (1:1) solution, the open circuit potential is the initial potential, and the frequency range is 10 5 Hz~1.0Hz, record the AC impedance curve. AC impedance diagram see Figure 3A The curve a.

Embodiment 3

[0039] The preparation of AC impedance type DNA electrochemical sensor and the detection steps of PML / RARα fusion gene are as follows:

[0040] (1) Piranha solution (30% H 2 o 2 Concentrated H with a concentration of 98% 2 SO 4 , mixed at a volume ratio of 1:3) ultrasonicated for 5 min, ultrasonically cleaned with deionized water twice, each time for 5 min, and then washed with 0.3 μm and 0.05 μm Al 2 o 3 The mixture of water and water is polished to a mirror surface, followed by ultrasonic cleaning with ethanol and distilled water. Place the sonicated electrode at 0.5 M H 2 SO 4 The cyclic voltammetry scans to stability, washes with double distilled water, N 2 Blow dry for later use;

[0041] (2) The sequence of the DNA capture probe labeled with thiol at the 5' end is: 5'-SH-(CH 2 ) 6 -T 10 CTTCA GAACT GCTGC TCTGG GTCTC AATGG-3', prepared as a 1 μM solution;

[0042] (3) Take 4 μl of the capture probe DNA solution in step (2), drop-coat it onto the surface of th...

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Abstract

The invention discloses an alternating current impedance type deoxyribonucleic acid (DNA) electrochemical sensor based on a probe DNA control assembly interface, which comprises an electrode and capture probe DNA, wherein the electrode adopts a gold electrode, and the capture probe DNA adopts sulfydryl modified DNA. The alternating current impedance type DNA electrochemical sensor is characterized in that the sulfydryl modified DNA is modified onto the surface of the gold electrode through the chemical bonding effect of Au-S bonds with the gold electrode at the room temperature and the absorption effect of the probe basic group parts and the gold so that the capture probe DNA flatly lies on the surface of the gold electrode for forming a capture probe DNA assembly layer, and bovine serum albumin exists on the surface of the capture probe DNA assembly layer and is used as sealing agents and protection agents. The impedance change before and after the hybridization is used as indicationsignals, the method adopts the surface assembly chemical technology for building the flat lying type DNA probe identification interface, simultaneously, the form of the probe maintains the flat lyingstate on the surface of the electrode without being influenced by the blank hybrid condition, and the method has the characteristics of high sensitivity, good specificity and the like.

Description

technical field [0001] The invention relates to a preparation method of an AC impedance type DNA electrochemical sensor based on a DNA-controlled assembly interface, and belongs to the technical field of biosensing. Background technique [0002] The AC impedance method is the process of using the change of the electrode surface resistance to reflect the change of the electrode surface morphology. The modified electrode is studied by electrochemical impedance spectroscopy, and the Randle circuit is used for simulation. The obtained Nyquist diagram includes two parts: semicircle and oblique line, of which the semicircle The part is the high-frequency region, which is controlled by the electron transfer process; the linear part is the low-frequency region, which represents the diffusion-controlled step. The change of the Nyquist diagram can reflect the magnitude of the electron transfer rate on the electrode surface, so as to understand the information about the dynamics of DNA...

Claims

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Application Information

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IPC IPC(8): C12M1/34C12Q1/68G01N27/327
Inventor 陈伟刘银环林新华刘爱林陈元仲
Owner FUJIAN MEDICAL UNIV
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