Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Detection method for activity of telomerase

A technology for activity detection and telomerase, which is applied in biochemical equipment and methods, microbial determination/inspection, Raman scattering, etc. Telomerase activity detection steps, improving reliability, and avoiding the effect of PCR reaction process

Active Publication Date: 2014-01-22
SOUTHEAST UNIV
View PDF4 Cites 15 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for detecting telomerase activity, which can react telomerase activity through dual signal channels of colorimetry and SERS technology. On the one hand, it can quickly and qualitatively distinguish samples with different telomerase activities through colorimetry. On the other hand, the accurate quantitative analysis of the telomerase activity of the sample is carried out by SERS technology, which solves the problems of low sensitivity, complicated procedure and high cost in the detection of telomerase activity in the prior art

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Detection method for activity of telomerase
  • Detection method for activity of telomerase
  • Detection method for activity of telomerase

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0044] The preparation method of described capturing base comprises the following steps:

[0045] The first step is to prepare iron ferric oxide nanoparticles by solvothermal method;

[0046] In the second step, the improved The method wraps the silica shell on the surface of the iron ferric oxide nanoparticles prepared in the first step, and then further modifies the surface of the silica shell with amino groups;

[0047] The third step, adopting the seed growth method to wrap the gold shell layer on the surface of the surface amino-modified silica shell wrapped iron ferric oxide nanoparticles prepared in the second step;

[0048] In the fourth step, a telomerase substrate is connected to the surface of the iron ferric oxide nanoparticles coated with gold shells prepared in the third step to obtain a capture substrate.

[0049] The preparation method of the report label comprises the following steps:

[0050] The first step, adopting sodium citrate reduction method to pre...

Embodiment 1

[0054] Using 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) as the Raman molecule, human cervical cancer cells (HeLa), human breast cancer cells (SKBR3, MCF7) and normal human embryonic lung Fibroblasts (MRC5) are cells to be tested, and the method for detecting telomerase activity is carried out using the method of the present invention.

[0055] Step 1, preparation of iron ferric oxide nanoparticles

[0056] Ferric oxide nanoparticles were prepared by solvothermal method. Add 2.7g ferric chloride hexahydrate (FeCl 3 ·6H 2 O), 1g polyethylene glycol (PEG, molecular weight 200) and 3.6g sodium acetate, stirred for half an hour to make it fully mixed. Subsequently, the mixed solution was charged into a polytetrafluoroethylene reactor at 200° C. for 8 hours. The product is magnetically separated and washed repeatedly with deionized water to obtain ferric oxide nanoparticles. Disperse ferric iron tetroxide nanoparticles into 50 mL of deionized water, use argon gas to drive away t...

Embodiment 2

[0076] Using the 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) molecule as the Raman molecule and human breast cancer cells (SKBR3) as the cells to be tested, the method of the present invention is used to quantify the activity of telomerase Detection experiment.

[0077] Prepare the capture substrate and the reporter label according to steps 1 to 6 in Example 1. According to the CHAPS method described in step 7 of Example 1, telomerase in SKBR3 cells was extracted, and the telomerase extract was diluted with RNase inhibitor-treated iced CHAPS lysate to correspond to 1000 cells / mL, 100 cells / mL, 10 cells / mL, 1 cell / mL. For telomere extension reaction, take 20 μL capture substrate, mix with 19 μL TRAP buffer (same as Step 4 of Example 1), 5 μL 10 mM dNTPs, 1 μL RNase inhibitor, and telomerase extracts of different cell concentrations (telomere Enzyme extracts were replaced with pure CHAPS lysate). The mixture was shaken at 30° C. for 3 h for telomere extension reaction, and the...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a detection method for activity of telomerase. The method comprises the following steps: step 1, extracting telomerase in to-be-detected cells by using the CHAPS method; step 2, subjecting a captured substrate and the to-be-detected telomerase to extension and subjecting an extension product and a reporter label solution to a hybridization reaction, wherein the captured substrate is prepared by connecting a telomerase substrate to the surface of a gold shell-coated ferriferrous oxide nanoparticle, and the reporter label solution is prepared by connecting a telomere complementary sequence and a Raman molecule to the surface of a spherical gold nanoparticle; and step 3, observing the color of a hybridization reaction product by using colourimetry or carrying out dual signal path detection by using SERS technology so as to obtain SERS signals. According to the invention, colourimetry and SERS technology are integrated into a same telomerase activity detection system, colourimetry is used to realize rapid qualitative analysis of a sample, then the SERS technology is used to realize accurate quantitative analysis of the sample, and combination of the two methods enables rapid high-sensitivity telomerase activity detection to be realized.

Description

technical field [0001] The invention relates to the field of detection of telomerase activity, in particular to the detection of telomerase activity through dual signal channels of colorimetry and SERS technology. Background technique [0002] The ends of linear chromosomes in eukaryotic organisms are protected by DNA-protein complexes called "telomeres" to avoid degradation of chromosomal DNA, end fusion, abnormal recombination, etc. Due to the problem of DNA terminal replication, as cells divide, telomere DNA shortens continuously, and cells gradually age, lose their ability to proliferate and die. Telomerase is a ribonucleoprotein that can use its own RNA as a template to synthesize and extend the telomere sequence, thereby maintaining the stability of telomere length and structure. Telomerase is inhibited in normal somatic cells and is only expressed in stem and germ cells. The overexpression of telomerase is closely related to the immortalization and carcinogenesis of...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N21/78G01N21/65C12Q1/68C12Q1/48
Inventor 王著元宗慎飞钟嫄崔一平
Owner SOUTHEAST UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com