A method for the simultaneous detection of two miRNAs based on the FRET effect

A satellite-shaped, gold rod technology, applied in the field of material chemistry, can solve the problems of strong background signal, short life, unstable optical signal of organic dyes, etc., and achieve the effects of good selectivity, low detection limit, good selectivity and sensitivity

Active Publication Date: 2019-09-03
JIANGNAN UNIV +1
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  • Abstract
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Problems solved by technology

In addition, many FRET-based detection methods have been developed to detect metal particles, cancer markers, etc., which have become a hot topic and have attracted widespread attention. However, they mainly focus on the detection of a miRNA, and the optical signal of organic dyes is not The disadvantages of stability, short lifespan, and strong background signal limit the application of this technology in the simultaneous detection of multiple miRNAs in cells.

Method used

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  • A method for the simultaneous detection of two miRNAs based on the FRET effect
  • A method for the simultaneous detection of two miRNAs based on the FRET effect
  • A method for the simultaneous detection of two miRNAs based on the FRET effect

Examples

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Effect test

Embodiment 1

[0025] Example 1 Preparation of Gold Rod Core-Upconversion Satellite Nano-Assembly

[0026] All glass instruments were soaked in aqua regia, rinsed with double distilled water, and dried for later use. The water used in the experiment is 18.2MΩ Milli-Q ultrapure water.

[0027] (1) Synthesis of gold nanorods: the gold nanorods with lateral plasmon absorption peak controlled at 780nm were synthesized by seed crystal growth method;

[0028] a. Synthesis of seed crystals: at room temperature, add 0.05mL of tetrachloroauric acid trihydrate with a concentration of 10mM to 1mL of 0.2M cetyltrimethylammonium olfactory ammonium solution, and the color of the solution changes from colorless to yellowish brown, then add 0.12mL of newly prepared sodium borohydride solution, stir rapidly for 2min, the color of the solution changes from yellowish brown to light brown;

[0029] b. Growth of gold nanorods: 5mL of 1mM tetrachloroauric acid trihydrate was added to 5mL, 0.2M hexadecyltrimethyla...

Embodiment 2

[0039] Example 2 Detection of Intracellular Cancer Markers

[0040] (1) Gold rod core-upconversion satellite nano-assembly to modify membrane-penetrating peptide: the gold rod core-upconversion satellite structure assembly obtained in step (4) of Example 1: PEG5000: membrane-penetrating peptide TAT at a molar concentration of 1:1000 : 100, mixed evenly, incubated at room temperature for 12h, centrifuged at 7000rpm for 10min, removed the supernatant, and the pellet was resuspended in the cell culture medium;

[0041] (2) Gold rod nucleus-upconversion satellite nano-assembly for intracellular detection of two miRNAs: the surface-modified gold rod nucleus-upconversion satellite-like structure assembly directly enters the cell for detection. When the analyte exists in the cell miR-21, which binds to sequence DNA 1 The combination of the complementary fragments in the gold rods leads to the dissolution of the upconversion nanoparticles on the end face of the gold rod. Under the ex...

Embodiment 3

[0044] Example 3 Detection of Extracellular Cancer Markers

[0045] In vitro, different concentrations of miR-21 and miR-200b were added to gold rod core-upconversion satellite nanoassemblies. The result is as image 3 As shown, with the increase of the concentration of miR-21 and miR-200b, the degree of dissociation of the up-conversion nanoparticles on the end face and the side surface increases, and FRET is generated under 980nm excitation, which transfers energy to the fluorescent molecules TAMRA and Cy5.5, respectively, lead to increased fluorescence. At the same time, the concentration of miR-21 and miR-200b showed a good linear relationship with the fluorescence intensity of TAMRA and Cy5.5, respectively.

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Abstract

A method for simultaneously detecting two types of miRNAs on the basis of FRET effects, relating to the field of material chemistry. According to the method, a gold rod nucleus-upconversion satellite-shaped structure assembly is synthesized, a standard curve is built, a golden rod nucleus-upconversion satellite-shaped nanocrystal assembly with a uniform structure and good biocompatibility is finally prepared, and a platform capable of implementing quantitative detection of two types of intracellular miRNAs on the basis of FRET effects is provided.

Description

technical field [0001] The invention relates to a method for simultaneously detecting two miRNAs based on the FRET effect, in particular to a miRNA ultrasensitive detection platform based on a FRET effect-based gold rod-upconversion satellite assembly, which belongs to the field of material chemistry. Background technique [0002] MicroRNA (miRNA) is a small molecule RNA regulatory factor that widely exists in eukaryotic cells. It usually has a length of about 21 bases and plays an important role in the regulation of development. Studies have found that the abnormal expression of miRNA is related to a variety of cancers, which also makes miRNA a new biological marker for cancer diagnosis and provides a new means for the treatment of human diseases. However, due to its low content in cells, small size and easy degradation, it is very difficult to quantitatively detect miRNAs in cells. miRNA microarray hybridization and real-time quantitative polymerase chain reaction (PCR) a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/6841
Inventor 匡华瞿爱华胥传来徐丽广刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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