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Intracellular micro-RNA non-enzymatic amplification detection method based on electrostatic affinity nano-transporter and cell imaging

A non-enzymatic amplification and cell imaging technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as sensitivity limitations

Inactive Publication Date: 2019-03-19
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, sensitivity is still limited by a single fluorescent emitter

Method used

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  • Intracellular micro-RNA non-enzymatic amplification detection method based on electrostatic affinity nano-transporter and cell imaging
  • Intracellular micro-RNA non-enzymatic amplification detection method based on electrostatic affinity nano-transporter and cell imaging
  • Intracellular micro-RNA non-enzymatic amplification detection method based on electrostatic affinity nano-transporter and cell imaging

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Synthesis of Embodiment 1 Nano Gold Particles (AuNPs)

[0095] When synthesizing AuNPs, positively charged poly-L-lysine (PLL) was used as a capping agent and a reducing agent, and PLL reduced gold salt ions (AuCl 4 -1 ), the metal ions induce the oxidation of the amine to nitrite, a direct redox reaction occurs, and AuNPs are spontaneously formed, and the PLL is encapsulated on the surface of the AuNPs to stabilize the formation of nanoparticles. The stabilization of the resulting AuNPs is ensured by a combination of steric and electrostatic interactions of charged peptides. First, dissolve 30mg of PLL in 3mL of water, shake and mix for 5 minutes to dissolve it completely, and prepare a PLL solution with a concentration of 10mg / mL; take 10uL of chloroauric acid solution with an original concentration of 1.52mol / L, add it to 142uL of water, and mix well. Mix well, avoid light, and prepare a chloroauric acid solution with a concentration of 0.1mol / L; finally add nuclea...

Embodiment 2

[0096] Example 2 Establishment of microRNA non-enzymatic amplification platform based on electrostatic affinity nanotransporter and its sensitivity experiment

[0097]According to the principle of the microRNA non-enzymatic amplification platform based on the electrostatic affinity nanotransporter, the hairpin probes H1 and H2 required for the non-enzymatic amplification system were designed for the microRNA21 sequence, and their sequences are shown in Table 1. The hairpin probe H1 adopts a double-headed labeling strategy, and its 5' end and 3' end are respectively labeled with a fluorescent group FAM and a quencher group BHQ-1; the 3' end of H2 is labeled with a fluorescent group FAM, and the 5' end The 8 base T marks BHQ-1. The hairpin probes were all synthesized by Huada Gene Technology Co., Ltd., and H1 and H2 were dissolved in TE buffer (final concentration 100 μM, stored in -20 refrigerator).

[0098] Used sequence in the embodiment of table 1

[0099]

[0100] In o...

Embodiment 3

[0103] Example 3 Specific verification of the intracellular microRNA non-enzymatic amplification platform based on electrostatic affinity nanotransporter

[0104] In order to verify the specificity of the intracellular microRNA non-enzymatic amplification platform based on the electrostatic affinity nanotransporter, microRNA210 and microRNA214, which have a base sequence similar to microRNA21, were added to the platform respectively, and the amount added was 10 pmol respectively, and the fluorescent signal was detected. Experimental results such as Figure 4 As shown, the Control group is a blank control. The fluorescence intensity values ​​of the microRNA210, microRNA214 and Control control groups at the emission wavelength of 518nm are 801.775a.u., 788.7a.u., and 739.5a.u., respectively, and the fluorescence intensities of these three groups are basically the same; The fluorescence intensity value at the wavelength of 518nm was 3763a.u., and a strong fluorescence signal was ...

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Abstract

The invention discloses an intracellular micro-RNA non-enzymatic amplification detection method based on an electrostatic affinity nano-transporter and cell imaging. According to the method, sequencesof specific DNA (deoxyribonucleic acid) hairpin probes H1 and H2 with fluorescent labels are designed by the aid of a non-enzymatic amplification system according to a detection target micro-RNA sequence, the H1, the H2 and gold nano-particles are assembled into compounds to be transferred to tumor cells, the amplification system and the H1 can be combined when the amplification system contains micro-RNA to be detected, so that the neck of the H1 is opened, parts of generated single-chain structures and the H2 act, the neck of the H2 is opened, the distance between a fluorescent group FAM andBHQ-1 quenching group is increased, fluorescence strength is increased and detected by a fluorescence spectrometer, and intracellular imaging conditions are recorded by a confocal laser scanning microscope. The method is simple in principle, low in detection cost and has the advantages that constant-temperature non-enzymatic amplification is achieved, and the method is high in sensitivity, simpleto operate, easy to popularize and the like.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to an intracellular microRNA non-enzymatic amplification detection method based on an electrostatic affinity nanotransporter and its application in cell imaging. Background technique [0002] microRNA (miRNA) is a highly conserved small single-stranded RNA with a length of 18-25nt and encoded by non-coding endogenous genes. So far, the functional mechanism of miRNA in the regulation of gene expression has received great attention. Studies have shown that miRNA is involved in many important cellular activities, including early development, virus defense, organ development and formation, cell proliferation, and apoptosis. In addition, their expression levels are directly related to various cancers and can reflect the expression of oncogenes or tumor suppressor genes. Therefore, miRNAs can serve as valuable biomarkers for cell-level studies and related disea...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2525/301
Inventor 黄曦廖玉辉赵钊艳
Owner THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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