Signal-enhanced test strip biosensor for detecting histone methylation

A histone methylation and biosensor technology, applied in biological testing, material inspection products, etc., can solve the problems of high toxicity of reagents, long operation time, cumbersome steps, etc., and achieve improved sensitivity, high specificity, and avoid operating steps complex effects

Active Publication Date: 2013-11-13
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the operation time of this method is long (about one day), the steps are cumbersome, a variety of expensive instruments and equipment and skilled operators are required, and the reagents used are highly toxic

Method used

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  • Signal-enhanced test strip biosensor for detecting histone methylation
  • Signal-enhanced test strip biosensor for detecting histone methylation
  • Signal-enhanced test strip biosensor for detecting histone methylation

Examples

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Embodiment 1

[0026] Embodiment 1: Preparation of the signal-enhanced test strip biosensor for detecting histone methylation according to the present invention.

[0027] The present invention will take the detection of H3K9 in HeLa cells as an example, and construct a sensor that utilizes the signal enhancement probe of AuNP-DNA to amplify the signal of the detection result of the test strip (see figure 1 ).

[0028] The preparation method of the test strip biosensor for detecting the signal enhancement of histone methylation according to the present invention comprises the following steps:

[0029] 1) Design a pair of complementary oligonucleotide sequences, denoted as DNA and c-DNA respectively.

[0030] The 5' end of DNA is marked with a sulfhydryl group whose sequence is:

[0031] 5'-SH-atcataagctcatacaatcactaa-3' (SEQ ID NO. 1).

[0032] The 5' end of c-DNA is labeled with biotin, and its sequence is:

[0033] 5'-biotin-ttagtgattgtatgagcttatgat-3'.

[0034] 2) The monoclonal a...

specific Embodiment

[0043] 1. Preparation of gold nanometer (colloidal gold) solution:

[0044] Add 100 ml of 0.01% HAuCl to a 500 ml round bottom flask 4 solution, magnetic stirring and heating to boiling; then add 4 ml 1% sodium citrate to the above solution, after the solution turns wine red, continue to boil for 10 minutes, stop heating and continue stirring for 15 minutes to obtain gold nano (colloidal gold) solution .

[0045] The colloidal gold solution was stored in the dark at 4°C, and the gold nanoparticles were identified by the maximum absorbance value at 520 nm.

[0046] 2. Preparation of AuNP-DNA signal enhancement probe:

[0047] Dissolve 1OD DNA in 100 μl deionized water, add to 5 times volume concentrated colloidal gold (15nm) solution, shake at low speed, 4°C overnight; after blocking with 1% bovine serum albumin for 30 minutes, add Na 3 PO 4and 1% SDS, respectively, to a final concentration of 0.01M and 0.01%, and gradually increase the NaCl concentration to 0.15 M in t...

Embodiment 2

[0058] Embodiment 2: The detection method of the test strip biosensor for detecting the signal enhancement of histone methylation according to the present invention.

[0059] In this example, the test strip biosensor prepared in Example 1 was used to detect the trimethylation of histone H3K9 in HeLa cells.

[0060] Taking the detection of H3K9me3 as an example, the detection method is as follows:

[0061] 1) HeLa cells were lysed, and histone crude extract was obtained by acid extraction.

[0062] 2) Add histone extract solution dropwise to the sample pad, after 10 minutes, add AuNP-DNA dropwise, and observe the experimental results after 5 minutes.

[0063] The following is a specific example:

[0064] 1. Obtaining crude histone extracts from HeLa cells:

[0065] a) HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum.

[0066] b) When the cells grow to a confluence of 90% in a 6cm culture dish, aspirate the culture medium, wash it twice wit...

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Abstract

The invention relates to a signal-enhanced test strip biosensor for detecting histone methylation. The test strip biosensor comprises a bottom plate with viscidity, a nitrocellulose membrane, a colloidal gold pad, an absorbent paper, and a sample pad, wherein the nitrocellulose membrane is attached to the middle part of the bottom plate; a monoclonal antibody of anti-histone H3 and a goat-anti-rat IgG antibody are sprayed on the nitrocellulose membrane; a detection line and a quality control line are respectively formed; the colloidal gold pad is attached to the bottom plate at one side of the nitrocellulose membrane; gold nanoparticle solution doubly marked by c-DNA (deoxyribonucleic acid) and the monoclonal antibody of anti-histone H3 is sprayed on the colloidal gold pad; the absorbent paper is attached to the bottom plate at the other side of the nitrocellulose membrane; the sample pad is attached to the bottom plate; histone extract is firstly dropwise added to the sample pad in detection; then an enhanced probe formed by DNA and gold nanoparticles in a coupling manner is dropwise added. By adopting the test strip biosensor provided by the invention, the histone methylation condition can be simply and rapidly detected; a detection signal is further amplified by the enhanced probe.

Description

technical field [0001] The present invention relates to a test strip biosensor for detecting histone methylation, in particular to a test strip biosensor for detecting signal enhancement of histone H3K9 trimethylation (H3K9me3) in HeLa cells . Background technique [0002] Histone is a general term for basic proteins that exist in combination with DNA in the nucleus of all eukaryotes. There are five types of histones, including H1, H2A, H2B, H3, and H4, which can interact with negatively charged phosphate groups in DNA. In eukaryotic cells, chromosomes are composed of repeating units called nucleosomes. Each nucleosome consists of a core histone 8-mer (consisting of two monomers each of H2A, H2B, H3, and H4), deoxyribonucleic acid (DNA) approximately 200 base pairs in length, and a monomer Histone H1. Nucleosomes carry epigenetic information through DNA or histone modifications, such as DNA methylation, histone methylation, phosphorylation, acetylation, sumoylation, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 曾令文葛晨晨余路新方志远
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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