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Signal labeled molecule for DNA oxidative damage product 8-hydroxydeoxyguanosine and labeling method

A technology of hydroxydeoxyguanosine and signal labeling, which is applied in biological testing, material analysis, material analysis through optical means, etc. It can solve the problems of higher detection value than the real value, difficulty in antibody preparation, and cross-reaction. High labeling rate, good specificity, and easy preparation

Inactive Publication Date: 2015-07-22
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the experimental operation is cumbersome, and the incomplete enzymatic digestion of DNA may lead to a result that the detection value is higher than the true value
When using GC-MS to detect 8-hydroxydeoxyguanosine, it is necessary to derivatize 8-hydroxydeoxyguanosine in advance, and the derivatization reaction can easily lead to the generation of by-products, resulting in false positive results (Ravanat, JL. et al. Chem. Res. Toxicol. 1995, 8:1039-1045.)
Monoclonal antibody and ELISA method have high sensitivity and good repeatability when detecting 8-hydroxydeoxyguanosine, but there is cross reaction, and the preparation of antibody is not easy (Osawa T.et al.Oxidative Stress and Aging.Birkhauser Verlag,Basel, Switzerland [C], 1995, pp367-374.)

Method used

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  • Signal labeled molecule for DNA oxidative damage product 8-hydroxydeoxyguanosine and labeling method
  • Signal labeled molecule for DNA oxidative damage product 8-hydroxydeoxyguanosine and labeling method
  • Signal labeled molecule for DNA oxidative damage product 8-hydroxydeoxyguanosine and labeling method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1, Synthesis of Signal Marker Molecules

[0042] 1. Take a 50ml two-necked round bottom flask, one of which is protected by a nitrogen balloon, and add 1.28g of 4-methyl-4'-(3-hydroxypropyl)-2,2'-bipyridine, DCC1. 545g, NHS0.575g, DMF15ml, stirred at room temperature for 5h.

[0043] 2. Take another 50ml two-necked round bottom flask, one of which is protected by nitrogen gas, and the other is equipped with a 50ml constant pressure dropping funnel, and 10.1g of spermine (diaminopropyltetramethylenediamine) and 10ml of DMF are added to the bottle. Transfer the reaction solution obtained after the reaction in Step 1 into a constant pressure dropping funnel under nitrogen protection, and slowly add it dropwise to the reaction flask under vigorous stirring. After the dropwise addition, continue to react for 1 h, remove DMF under reduced pressure, and add Add 2M hydrochloric acid and dichloromethane to the compound, separate liquid, wash twice with saturated sodium ...

Embodiment 2

[0045] Embodiment 2, gold electrode surface DNA incubation time optimization

[0046] The DNA modified with sulfhydryl groups at the end was dissolved in the buffer containing fresh TCEP-Tris, 4 μl was dropped onto the surface of the treated gold electrode, covered with the electrode cap, and incubated for 8h, 16h and 40h, respectively. After the end, wash with deionized water, blow dry with nitrogen, soak in 200ul of mercaptohexanol and incubate for a period of time, wash with deionized nitrogen and dry with nitrogen. First place the modified electrode in a Tris solution containing 3.5 μM RuHex to scan CV at a scan rate of 50 mV / s. Then washed with water and placed in a Tris solution containing 50 μM RuHex to scan CV at a scan rate of 50 mV / s. Comparing the CV diagrams under different incubation times, it was found that the DNA assembly on the electrode surface was saturated and relatively stable after 40 hours of incubation.

Embodiment 3

[0047] Embodiment 3, gold electrode surface DNA concentration optimization

[0048] Five different concentrations of 0.1, 0.5, 1, 2, and 5 μM DNA solutions with end-modified sulfhydryl groups were selected and added dropwise to the surface of the gold electrode, and incubated at room temperature. Wash with deionized water, dry with nitrogen, soak in mercaptohexanol for a period of time, wash with deionized water and dry with nitrogen. The modified electrode was placed in a Tris solution containing 50 μM RuHex to scan CV at a scan rate of 50 mV / s. Comparing the CV plots of different concentrations of DNA, it was found that the concentration of DNA reached saturation when the concentration was 2 μM.

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Abstract

The invention discloses a signal labeled molecule for specific detection of a DNA oxidative damage product 8-hydroxydeoxyguanosine and a labeling method. The signal labeled molecule at least contains two functional groups: one functional group can be connected to 8-hydroxydeoxyguanosine through a covalent bond; and the other functional group can generate a detection signal. The signal labeled molecule specifically reacts with 8-hydroxydeoxyguanosine in the presence of a certain concentration of an oxidizing agent so as to form covalent labeling. According to the signal generated by the labeled molecule, a corresponding detection method can be adopted. By measuring a signal such as an optical signal, an electric signal, an electrochemiluminescence signal or a photo-electrochemical signal and the like which is generated by the labeled molecule, quantitative detection of 8-hydroxydeoxyguanosine is detected. The labeled molecule finishes bonding and signal labeling of 8-hydroxydeoxyguanosine through a one-step reaction, and reaction conditions are mild. The signal labeled molecule has good specificity and high labeling rate, and is very suitable for quick and quantitative detection of 8-hydroxydeoxyguanosine.

Description

technical field [0001] The invention relates to a signal marker molecule and a marker method that can be used to detect 8-hydroxydeoxyguanosine, a product of oxidative damage to DNA. The method is simple to operate and has high specificity, and can be used for the quantitative analysis of 8-hydroxydeoxyguanosine. Background technique [0002] 8-hydroxydeoxyguanosine (8-hydroxydeoxguanosine, 8-oxodG) is recognized as the most important marker of DNA oxidative damage products. The production of 8-OxodG mainly comes from the attack of a large number of reactive oxygen species (reactive oxygen species, ROS) produced by the metabolic activation of chemical carcinogens or ionizing radiation on the C8 position of DNA deoxyguanosine. Since 8-hydroxydeoxyguanosine can pair not only with base A, but also with base C, this damage often leads to G→T mutations during DNA replication. If this damage is not repaired in time, it will cause gene instability, and then produce gene or genoto...

Claims

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Application Information

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IPC IPC(8): G01N33/50
CPCG01N21/763
Inventor 郭良宏吴一萍
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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