Preparation method and application of fluorescent sensor for detecting microRNA

A fluorescent sensor and microRNA technology, which is applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of autofluorescence interference, bleaching, etc., and achieve the effect of simple preparation method, low cost and safe operation

Inactive Publication Date: 2015-12-16
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the photobleaching of organic small molecule fluorescent donors commonly used in FRET technology is severe; moreover, in the traditional FRET analysis based on steady-state fluorescence, when excited by ultraviolet light, the autofluorescence interference of biological samples is very serious

Method used

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  • Preparation method and application of fluorescent sensor for detecting microRNA
  • Preparation method and application of fluorescent sensor for detecting microRNA
  • Preparation method and application of fluorescent sensor for detecting microRNA

Examples

Experimental program
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Embodiment 1

[0034] A method for preparing a fluorescent sensor for detecting microRNA, comprising the following steps:

[0035] 1) Preparation of polyacrylic acid (PAA)-modified GdF by solvothermal method 3 :Tb 3+ Nanoparticles

[0036] In a 50 mL round bottom flask, add 0.1 mmol GdCl to 12 mL, 17-18.2 MΩcm ultrapure water and 4.0 mL ethylene glycol mixed solvent 3 ·6H 2 O, 80 mg polyacrylic acid and 2.0 μmol TbCl 3 ·6H 2 O, stir until uniform and transparent, then heat to 50°C to obtain solution a; add 0.3mmol NH to 5.0mL ultrapure water and 2.0mL ethylene glycol mixed solvent in a 15mL test tube 4 F, stir at room temperature until uniform and transparent, then heat to 50°C to obtain solution b, while hot, add solution b dropwise to solution a at a rate of 1 drop per second, react for 30 minutes, centrifuge at 2000rpm for 5 minutes to collect solid products, and use Wash twice with ultrapure water and ethanol, and dry in vacuum for 10 hours to obtain polyacrylic acid-modified GdF ...

Embodiment 2

[0053] A method for preparing a fluorescent sensor for detecting microRNA, comprising the following steps:

[0054] 1) In a 100mL round bottom flask, add 0.2mmolGdCl to 17-18.2MΩcm, 24mL ultrapure water and 8.0mL ethylene glycol mixed solvent 3 ·6H 2 O, 160 mg polyacrylic acid and 4.0 μmol TbCl 3 ·6H 2 O, stir until uniform and transparent, then heat to 50°C to obtain solution a; add 0.6mmol NH to 10mL ultrapure water and 4.0mL ethylene glycol mixed solvent in a 50mL test tube 4 F, stir at room temperature until uniform and transparent, then heat to 50°C to obtain solution b, while hot, add solution b dropwise to solution a at a rate of 1 drop per second, react for 30 minutes, centrifuge at 2000rpm for 5 minutes to collect solid products, and use Wash twice with ultrapure water and ethanol, and dry in vacuum for 10 hours to obtain polyacrylic acid-modified GdF 3 :Tb 3+ Nanoparticle white powder product;

[0055] 2) Using 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC)...

Embodiment 3

[0066] A method for preparing a fluorescent sensor for detecting microRNA, comprising the following steps:

[0067] 1) In a 100mL round bottom flask, add 0.15mmolGdCl to 17-18.2MΩcm, 18mL ultrapure water and 6.0mL ethylene glycol mixed solvent 3 ·6H 2 O, 120 mg polyacrylic acid and 3.0 μmol TbCl 3 ·6H 2 O, stir until uniform and transparent, then heat to 50°C to obtain solution a; add 0.45mmol NH to 7.5mL ultrapure water and 3.0mL ethylene glycol mixed solvent in a 15mL test tube 4 F, stir at room temperature until uniform and transparent, then heat to 50°C to obtain solution b, while hot, add solution b dropwise to solution a at a rate of 1 drop per second, react for 30 minutes, centrifuge at 2000rpm for 5 minutes to collect solid products, and use Wash twice with ultrapure water and ethanol, and dry in vacuum for 10 hours to obtain polyacrylic acid-modified GdF 3 :Tb 3+ Nanoparticle white powder product;

[0068] 2) Using 1-ethyl-(3-dimethylaminopropyl) carbodiimide (E...

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Abstract

Disclosed is a preparation method of a fluorescent sensor for detecting microRNA. The preparation method comprises the following steps: preparing polyacrylic acid modified GdF3:Tb3+ nanoparticles through a solvothermal method; preparing a solution of GdF3:Tb3+ nanoparticles coupled to a single oligonucleotide chain probe I through catalytic condensation of EDC and NHS; preparing a fresh solution of colloidal gold nanoparticles through reducing chloroauric acid with sodium citrate; coupling a single oligonucleotide chain probe II to the gold nanoparticles by utilizing combination of sulfydryl groups and gold; and preparing a PBS solution of the fluorescent sensor for detecting microRNA. The invention has advantages that: the preparation method of the fluorescent sensor is simple, and is safe to operate, mild in condition, and low in cost; artificially synthesized hsa-miR-122-5p in a buffer solution can be sensitively and specifically recognized; and the fluorescent sensor has a linear range of 1fM-100pM, and a detection limit of 0.5fM.

Description

technical field [0001] The invention relates to the fields of chemical synthesis technology and biochemical analysis and detection technology, in particular to a preparation method and application of a fluorescent sensor for detecting microRNA. Background technique [0002] MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of about 22 nucleotides. play a very important regulatory role. Studies have shown that certain miRNAs can mediate the expansion and spread of tumors, are closely related to multidrug resistance, and can regulate the expression of multiple genes in the process of cancer metastasis. These miRNAs are likely to become important indicators for predicting cancer, such as : hsa-miR-122 plays an important role in the occurrence and development of liver cancer. At present, the detection methods of miRNAs mainly include reverse transcription polymerase chain reaction, microarray, electrochemistry,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C12Q1/68
Inventor 张毅段宏泉
Owner TIANJIN MEDICAL UNIV
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