Detection method of substance with specific action with nucleic acid beacon

A specificity and beacon technology, applied in the direction of material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of long preparation cycle, low cost, and reduced material biocompatibility, and achieve simplified preparation difficulty, low-cost detection, biological Effect of Compatibility Improvement

Inactive Publication Date: 2013-06-26
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned carbon materials are difficult to prepare, and usually need to purchase special preparation raw materials or use more complicated experimental conditions, the preparation cycle is long, and the cost is not low enough; and organic solvents are used in the preparation process or dispersion process, which will greatly reduce the cost of materials. biocompatibility

Method used

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  • Detection method of substance with specific action with nucleic acid beacon
  • Detection method of substance with specific action with nucleic acid beacon
  • Detection method of substance with specific action with nucleic acid beacon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Take 6 μL of 3 μM tetramethylrhodamine-single-stranded DNA (TAMRA-ssDNA) Tris-HCl (10 mM, pH 7.4, containing 0.15 M NaCl, the same below) solution, add different volumes of 0.1 mg / mL Sodium dodecylbenzenesulfonate stabilized carbon nanoparticle solution (SDBS-CNPs)), add Tris-HCl to make the volume to 600 μL, so that the SDBS-CNPs concentration is 0, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055 mg / mL, after incubation at 37°C for 1 h, the down-conversion fluorescence was detected. Prepare a series of Tris-HCl solutions containing different concentrations of target DNA (0, 0.5, 1, 5, 10, 30, 50 and 100 nM), incubate at 42°C for 2 h, then add SDBS-CNPs solution to make the concentration 0.055 mg / mL. Incubate at 37°C for 40 min, detect the down-converted fluorescence, and plot the concentration of target DNA and the ratio of the fluorescence intensity of the sample to the fluorescence intensity of the sample without target DNA. For samples of unknown ...

Embodiment 2

[0030] (1) Preparation of up-converting fluorescent nanoparticles (UCPs): Take 2 ml of 0.25 mol / L rare earth nitrate solution (the molar ratio of rare earth ions is yttrium ion: ytterbium ion: erbium ion = 80:18:2, add Add 18 ml of absolute ethanol, then add 8 ml of an aqueous solution containing 900 mg of polyacrylic acid, and stir for 10 min; add 8 ml of an aqueous solution containing 0.210 g of sodium fluoride to the above mixed solution, continue stirring for 20 min, and place it under high pressure for reaction In the kettle, under stirring conditions, hydrothermal reaction was carried out at 200 °C for 10 h; stop heating and keep stirring to cool to room temperature, centrifuge to separate the solid product, wash with absolute ethanol and ultrapure water three times, and vacuum dry at room temperature for 12 h , to obtain up-converting fluorescent nanoparticles with carboxyl groups on the surface.

[0031] (2) Surface labeling of UCPs: Dissolve 5 mg of the upconvertin...

Embodiment 3

[0036] Take 30 μL of 0.6 mg / mL UCPs Tris-HCl solution labeled with beacon DNA (the synthesis and labeling methods of UCPs are the same as in Example 2), add different amounts of carbon dioxide nanoparticles (CNPs oxide) aqueous solution, and constant volume to 600 μL. After incubation at 30°C for 90 min, upconversion fluorescence was detected using 980 nm excitation light. Add different amounts of adenosine triphosphate (ATP) to the Tris-HCl solution containing 0.03 mg / mL beacon DNA-UCPs and 0.04 mg / mL CNPs oxide, incubate at 30°C for 90 min, and measure the upconversion fluorescence with 980 nm excitation light. Calculate the ratio of the fluorescence intensity F of the sample added with ATP to the fluorescence intensity F0 of the sample without ATP, and plot the ratio of ATP concentration to F / F0 to obtain a standard curve. For samples with unknown concentrations, add beacon DNA-UCPs solution and CNPs oxide aqueous solution and add Tris-HCl to make up to 600 μL, so that...

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Abstract

The invention discloses a detection method of a substance with specific action with a nucleic acid beacon. The detection method comprises the following steps: adding carbon nano-particle solution with different concentration in a nucleic acid beacon solution provided with a certain concentration and annotated with a fluorophore at one end, incubating, measuring the fluorescence intensity at room temperature to obtain a quenching curve, adding target object with different concentration in a plurality of groups of nucleic acid solutions provided with a certain concentration and annotated with fluorophore at one end, incubating, adding the carbon nano-particle solution with a certain volume, incubating, measuring the fluorescence intensity, obtaining a standard curve, and computing the concentration of the target object. The method disclosed by the invention is simple for operation, good in biocompatibility, and can be used for effectively, flexibly and fast detecting the biomolecule in low cost, and the raw material is easily available.

Description

technical field [0001] The invention relates to a detection method for a substance capable of specifically interacting with a nucleic acid beacon, and belongs to the technical field of molecular beacon detection. Background technique [0002] The traditional molecular beacon is to label small molecules at both ends of the hairpin structure beacon molecule, such as Texas Red, Fluoresein, etc. as fluorescent groups, 4-(4'-dimethyl Amino azophenyl) benzoic acid (DABCYL) etc. are used as quenching groups. In the free state, the two types of molecules are close (about 7-10 nm), and fluorescence resonance energy transfer occurs, and the fluorescence emitted by the fluorophore is absorbed by the quencher molecule. When the test sample (complementary single-stranded DNA / RNA, molecules that interact specifically with the beacon ring aptamer region, etc.) that interacts with the base of the beacon molecule is added to the detection system, a hybrid is formed, and the beacon stem is c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 刘志洪曾令瑜袁云霞沈佩
Owner WUHAN UNIV
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