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Method for quantitatively detecting activity of 8-OhdG (8-hydroxy-2'-deoxyguanosine) based on aniline deposited electrochemical sensing electrode

A sensing electrode and quantitative detection technology, applied in the field of biosensing, can solve the problem that the detected value is higher than the real value, achieve good environmental stability, avoid mutual entanglement, and reduce the effect of detection limit

Active Publication Date: 2017-02-22
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is cross-reaction in this method, which may cause the detected value to be higher than the true value

Method used

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  • Method for quantitatively detecting activity of 8-OhdG (8-hydroxy-2'-deoxyguanosine) based on aniline deposited electrochemical sensing electrode
  • Method for quantitatively detecting activity of 8-OhdG (8-hydroxy-2'-deoxyguanosine) based on aniline deposited electrochemical sensing electrode
  • Method for quantitatively detecting activity of 8-OhdG (8-hydroxy-2'-deoxyguanosine) based on aniline deposited electrochemical sensing electrode

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The electrochemical sensing electrode based on polyaniline deposition quantitatively detects the activity of 8-hydroxydeoxyguanosine, and the detection steps are:

[0042] 1) Mix 0.5 μL each of the three single strands A, B, and C with sulfhydryl groups modified at the 5’ end and D chain with 8-hydroxydeoxyguanosine aptamer at the 5’ end in 40 μL TM buffer and 0.5 μL TCEP in equal proportions In the method, prepare a sample with a final concentration of 0.5 μM, then put the prepared sample in a 90°C hot water bath for 10 minutes, then quickly cool down to 1°C, and keep it in 1°C for more than 40 minutes, you can get the DNA tetrahedral structure with sulfhydryl groups;

[0043] 2) The modification of the DNA tetrahedral structure with the sulfhydryl group connected to the 8-OHdG aptamer on the gold electrode to obtain the electrode with the DNA tetrahedral structure connected to the 8-OHdG aptamer at the top: first soak the gold electrode in piranha Lotion (H 2 o 2 a...

Embodiment 2

[0047] Embodiment 2: The electrochemical sensing electrode based on polyaniline deposition quantitatively detects the activity of 8-hydroxydeoxyguanosine, and the detection steps are:

[0048] 1) Mix 2 μL each of the three single strands A, B, and C with sulfhydryl groups modified at the 5’ end and D strand with 8-hydroxydeoxyguanosine aptamer at the 5’ end in 50 μL TM buffer and 2 μL TCEP in equal proportions, Prepare a sample with a final concentration of 2 μM, then place the prepared sample in a hot water bath at 100°C for 3 minutes, then quickly cool down to 5°C, and keep at 5°C for more than 20 minutes to obtain the thiol-containing DNA tetrahedral structure; the TM buffer solution is 20mM Tris-HCl solution containing pH=8.0 and 50mM MgCl 2 solution, CH 3 COOH-CH 3 COONa buffer solution containing 100mM CH 3 COOH and 100mM CH 3 The buffer solution of pH=4.3 of COONa;

[0049] 2) The modification of the DNA tetrahedral structure with the sulfhydryl group connected to ...

Embodiment 3

[0054] The electrochemical sensing electrode based on polyaniline deposition quantitatively detects the activity of 8-hydroxydeoxyguanosine, and the detection steps are:

[0055] 1) Mix 1.25 μL each of the three single chains A, B, and C with sulfhydryl groups modified at the 5’ end and D chain with 8-hydroxydeoxyguanosine aptamer at the 5’ end in 45 μL TM buffer and 1.25 μL TCEP in equal proportions In the method, prepare a sample with a final concentration of 1.25 μM, then place the prepared sample in a 95°C hot water bath for 6 minutes, then quickly cool down to 3°C, and keep it in 3°C for more than 30 minutes, and you can get the DNA tetrahedral structure with sulfhydryl groups;

[0056] 2) The modification of the DNA tetrahedral structure with the sulfhydryl group connected to the 8-OHdG aptamer on the gold electrode to obtain the electrode with the DNA tetrahedral structure connected to the 8-OHdG aptamer at the top: first soak the gold electrode in piranha Lotion (H 2...

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Abstract

The invention discloses a method for quantitatively detecting the activity of 8-OhdG (8-hydroxy-2'-deoxyguanosine) based on an aniline deposited electrochemical sensing electrode. The method comprises the following steps of preparing a DNA (Deoxyribonucleic Acid) tetrahedral structure with sulfhydryl; modifying, on a gold electrode, the DNA tetrahedral structure with the sulfhydryl to obtain an electrode of which the top end is connected with an 8-OhdG aptamer and which has the DNA tetrahedral structure with the sulfhydryl; forming a G-quadplex electrode; forming a polyaniline deposited electrochemical sensing electrode; detecting a generated current signal of polyaniline by utilizing an electrochemical method, so as to detect the activity of the 8-OhdG. By using the method for quantitatively detecting the activity of the 8-OhdG based on the aniline deposited electrochemical sensing electrode, the detection sensitivity on the 8-OhdG is greatly improved. In compassion with a conventional electrochemical detection method using a reduction peak of the 8-OhdG as a signal, the detection limit is decreased by two orders of magnitude. By using the method for quantitatively detecting the activity of the 8-OhdG based on the aniline deposited electrochemical sensing electrode, the preparation of a complicated material and a DNA labeling probe is not needed; the defects that the preparation of the material and the DNA labeling probe causes that the detection cost is high, the operation is fussy and the reproducibility is poor can be avoided. The method for quantitatively detecting the activity of the 8-OhdG based on the aniline deposited electrochemical sensing electrode has the advantages of being low in cost, being quick, simple and convenient, and being high in sensitivity.

Description

technical field [0001] The invention belongs to the biosensing technology for quantitatively detecting the activity of 8-hydroxydeoxyguanosine, in particular to a method for quantitatively detecting the activity of 8-OhdG with an electrochemical sensing electrode based on aniline deposition. Background technique [0002] Oxidative damage caused by oxidative stress is an important factor that causes various diseases and injuries in humans. 8-hydroxydeoxyguanosine (8-hydroxy-2'-deoxyguanosine, 8-OHdG) is one of the DNA oxidative damage modification products caused by reactive oxygen species (ROS). There are many reasons for its formation, mainly ionizing radiation, chemical carcinogens Metabolic activation and normal cell metabolism produce a large amount of ROS to directly attack guanine (dG) in DNA, and oxidize deoxyguanosine to 8-OHdG. 8-OHdG can be cleaved and cleared by the body's specific DNA repair enzymes and excreted through the kidneys with urine. Once it escapes t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26
CPCG01N27/26
Inventor 卫伟范佳慧张雪红徐晓林陈昌慧刘松琴
Owner SOUTHEAST UNIV
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