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
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  • Abstract
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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 a

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  • Detection method for activity of telomerase
  • Detection method for activity of telomerase
  • Detection method for activity of telomerase

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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...

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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

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Application Information

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IPC IPC(8): G01N21/78G01N21/65C12Q1/68C12Q1/48
Inventor 王著元宗慎飞钟嫄崔一平
Owner SOUTHEAST UNIV
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