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Method and sensor for detecting nucleic acid on-site damage by photoelectrochemistry

A photoelectrochemical and sensor technology, applied in the field of detection, can solve the problems of long time, no parallelism, complex optical detection instruments, etc., and achieve the effect of simple production

Inactive Publication Date: 2009-03-25
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most of the above are cell tests. Although the cycle has been shortened from several months to a few days for animal tests, the time is still too long
Moreover, the test involves multiple steps, and the operation needs to be carried out by professionals in the cell laboratory. There is no parallelism, and it is not suitable for large-scale screening of environmental compounds.
Gene sensor technology has the advantages of high throughput and high parallelism, but it involves complex sample pretreatment (nucleic acid extraction and labeling), probe design and strict hybridization conditions, coupled with complex and expensive optical detection instruments, which is not conducive to Popularization and promotion (refer to E.F. Nuwaysir et al., Molecular Carcinogenesis, 24: 153 (1999))

Method used

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  • Method and sensor for detecting nucleic acid on-site damage by photoelectrochemistry
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  • Method and sensor for detecting nucleic acid on-site damage by photoelectrochemistry

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Catalytic photocurrents of the signaling molecule bipyridylruthenium.

[0058] The succinamide (NHS)-activated bipyridyl ruthenium signal molecule was covalently labeled on avidin to form avidin-Ru. Spread 10 μL of avidin-Ru solution evenly on the prepared nano-SnO at room temperature 2 Electrode surface, static adsorption for 1 hour, take out water and shake for 5 minutes, blow dry with nitrogen, get avidin-Ru modified semiconductor electrode, SnO 2 / avidin -Ru. Use the same method to modify different nucleotides on the electrode surface, such as: double-stranded DNA (ds-DNA), single-stranded DNA (ss-DNA), polyguanine nucleotide (polyG), polythymidine acid (polyA), polycytosine nucleotide (polyC), polyuracil nucleotide (polyU). Then various nucleotide-modified semiconductor electrode sensors are placed in the electrolytic cell, and the photocurrent is detected in the phosphate buffer solution. The electrochemical workstation used is CHI 630A, and the detection is ba...

Embodiment 2

[0060] Modified electrode SnO 2 / avidin-Ru / ds-DNA / PDDA / GOx detection of metal ion Fe 2+ genotoxicity.

[0061] Avidin-Ru, ds-DNA, polydiallyldimethylamine hydrochloride (PDDA), and glucose oxidase (GOx) were sequentially assembled on the surface of the semiconductor electrode according to the method described in Example 1 to obtain a modified sensor Electrode: SnO 2 / avidin-Ru / ds-DNA / PDDA / GOx, see figure 2 . Place the modified sensor in 1mM FeSO 4 / 50mM glucose solution, react at 37°C for 1h. The glucose in the solution is used here to react with the glucose oxidase assembled on the electrode to produce hydrogen peroxide, which can react with Fe 2+ The Fenton reaction produces highly active oxygen and damages nucleic acids, which is the main way for metal ions to exhibit genotoxicity in the body. After the reaction, the electrode was taken out, rinsed with water, dried with nitrogen gas, and placed in 20mM phosphate buffer to detect the photocurrent. Separate FeSO 4 A...

Embodiment 3

[0063] Modified electrode SnO 2 / avidin-Ru / ds-DNA / Hb was used to detect the potential genotoxicity of styrene.

[0064] Avidin-Ru, ds-DNA, and hemoglobin (hemoglobin, Hb) were sequentially assembled on the surface of the semiconductor electrode according to the method described in Example 1 to obtain a modified sensor electrode SnO 2 / avidin-Ru / ds-DNA / Hb, see Figure 4 . Here, hemoglobin can convert styrene into genotoxic styrene oxide under the action of hydrogen peroxide, causing nucleic acid damage, similar to the metabolism of styrene in the human liver, which is also the nucleic acid damage pathway of most organic substances in the body. That is, in the living body, it is first catalyzed by the cytochrome P450 enzyme in the liver and converted into an active metabolite, and then covalently combined with the base of the nucleic acid to form an adduct, causing damage to the nucleic acid. The prepared sensor electrode SnO 2 / avidin-Ru / ds-DNA / Hb placed in 2mM H 2 o 2 / 2...

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Abstract

The invention relates to a method and a sensor for detecting on-site damage of nucleic acid in photo-electrochemistry, and provides a method for detecting gene toxicity of chemical substances simply, quickly and sensitively. The method comprises the following steps: (a) an analyte possibly having gene toxicity is contacted with the nucleic acid; (b) if the analyte to be detected expresses the gene toxicity after being activated, a corresponding enzyme is used as an activating agent; (c) photo-electrochemical active molecules are used as a signal indicator; and (d) the combination and / or reaction between the analyte and the nucleic acid is elevated, that is, in the presence of electrodes, current generated by electron transfer between the photoelectrical signal molecules and the electrodes under the action of light is evaluated, and qualitative analysis or quantitative analysis for the gene toxicity of the analyte is carried out according to the difference of photocurrent response before and after the nucleic acid contacts with the analyte. The method and the sensor are applied in the fields of toxicity screening for industrial chemicals, toxicity detecting for environmental pollutants, toxicity testing for synthetic drugs, environmental toxicology research, and the like.

Description

technical field [0001] The invention relates to the technical field of detection, and relates to a method and a sensor for photoelectrochemical detection of on-site damage to nucleic acid for qualitative and quantitative detection of genotoxicity of chemical substances through a photoelectrochemical method. technical background [0002] With the improvement of our country's industrialization level, a large number of chemical substances have entered the ecological environment. According to statistics, nearly 100,000 chemical substances have been synthesized, and 1,000 new compounds are synthesized every year. These substances are potentially harmful to the human body, and some of these compounds have been proven to be mutagenic, carcinogenic, mutagenic and other genotoxic. At present, the evaluation of the toxicity of compounds on the body is mainly carried out through cell and animal experiments. The experiment cycle is long, manpower and material resources are expensive, a...

Claims

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Application Information

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IPC IPC(8): G01N23/227C12Q1/68
Inventor 郭良宏梁敏敏
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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