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DNA detection method based on rapid nanogold aggregation induced by nucleic acid dye

A nucleic acid dye, nano-gold technology, applied in the field of biosensing, can solve problems such as difficulty in quantitative determination, and achieve the effects of high sensitivity, fast polymerization speed and stable color

Pending Publication Date: 2019-12-13
CHENGDU UNIVERSITY OF TECHNOLOGY
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the salt-induced aggregation of AuNPs is a relatively long kinetic process, and the color changes continuously after aggregation, making quantitative determination difficult.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] In a 96-well plate, add different concentrations of p53 gene (5'-TTC CTC TGT GCG CCG GTC TCT CCT-3') and its probe strand (CS-p53: 5'-AGG AGAGAC CGG CGC ACAGAG GAA-3' ) (10nM, prepared in pH 7.4 phosphate buffer), incubate for 1min, then add nucleic acid dye SYBR Green I (final concentration 0.94μM) and AuNPs (4nM) into the solution in turn, stir well, and measure the absorbance of AuNPs at 680nm Under the same conditions, measure the blank signal; realize the visual semi-quantitative analysis of the target DNA by comparing the color change of AuNPs with or without the target; through the absorbance value of AuNPs at 680nm, realize the accurate quantification of the target DNA .

Embodiment 2

[0015] In a 96-well plate, add different concentrations of p53 gene (5'-TTC CTC TGT GCG CCG GTC TCT CCT-3') and its probe strand (CS-p53: 5'-AGG AGAGAC CGG CGC ACAGAG GAA-3' ) (10nM, prepared in pH 7.4 phosphate buffer), incubate for 2min, then add the nucleic acid dye Pico Green (final concentration 0.096μM) and AuNPs (4nM) into the solution in turn, stir well, and measure the absorbance value of AuNPs at 680nm ;Under the same conditions, measure the blank signal; realize the visual semi-quantitative analysis of the target DNA by comparing the color change of AuNPs with or without the target; through the absorbance value of AuNPs at 680nm, realize the accurate quantification of the target DNA.

[0016] Using Examples 1 and 2 to visually detect p53, the detection limit is 0.1nM; using ultraviolet spectrophotometry, the detection limit is 3pM. By changing the sequence of the recognition probe chain, the system can be applied to the detection of any target nucleic acid.

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Abstract

The invention relates to a DNA detection method based on rapid nanogold aggregation induced by a nucleic acid dye. A charge neutralization effect between the nucleic acid dye with positive charges andnanogold with negative charges is used to induce aggregation of nanogold rapidly. The nucleic acid dye with positive charges can be embedded into the dsDNA and is prevented from interacting with AuNPs, so that the aggregation process is easily regulated and controlled by the dsDNA and is further used for DNA detection. After that the nucleic acid dye induces the aggregation of the nanogold, darkblue is generated and is in contrast with original red of the nanogold, so that the detection sensitivity is high, and the target DNA of 0.1nM can be detected; a spectrophotometric method is adopted,and the detection limit of the target DNA can reach 3pM and is 50 times lower than that of a traditional salt-induced aggregation method. Meanwhile, the method is simple and convenient to operate, does not need the steps of separation, marking and the like, can be expanded to detection of DNA of any other sequence, and has a good application prospect.

Description

technical field [0001] The invention relates to a method for detecting DNA based on nucleic acid dye-induced rapid aggregation of gold nanometers, and establishes a gold nanometer-free colorimetric sensing platform for visual detection of DNA, belonging to the technical field of biosensing. Background technique [0002] DNA (deoxyribonucleic acid) affects life activities such as growth and development of organisms, aging and disease, and is the basis of gene expression. Nucleic acid molecules have become important research objects in biology, molecular genetics, medicine and chemistry because they carry genetic information and are related to life activities such as growth, development, reproduction and canceration of organisms (Science., 2002 297:1536- 1540). There are many methods for nucleic acid detection, mainly including fluorescence method (Anal. Chem. Commun., 2015, 51:14465-14468) and chemiluminescence (Anal. Chem., 2013, 82:5511-5517). In contrast, the colorimetr...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/543G01N21/82G01N21/78
CPCG01N33/5308G01N33/54393G01N21/82G01N21/78G01N2021/825
Inventor 张信凤李琳范晓娅雷凤洁
Owner CHENGDU UNIVERSITY OF TECHNOLOGY
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