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LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting plasmid DNA (deoxyribonucleic acid) containing oncogene C-myc antigen fragment

A sensor chip and oncogene technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of anti-interference, insufficient selectivity, application limitations, cumbersome operation, etc., and achieve good selectivity and reproducibility, High sensitivity, rapid quantitative effect

Inactive Publication Date: 2011-06-01
CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method is cumbersome to operate, low in precision, can only be qualitative or semi-quantitative, and its anti-interference and selectivity are insufficient, so its application is limited

Method used

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  • LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting plasmid DNA (deoxyribonucleic acid) containing oncogene C-myc antigen fragment
  • LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting plasmid DNA (deoxyribonucleic acid) containing oncogene C-myc antigen fragment
  • LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting plasmid DNA (deoxyribonucleic acid) containing oncogene C-myc antigen fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Assembly preparation of LSPR sensor chip:

[0020] 1) A layer of 100nm gold film was coated on the surface of polystyrene plastic and silicon oxide glass substrate by magnetron sputtering coating method, and the vacuum degree of the coating film was controlled to be 1.0×10 -4 Pa, the coating speed is Make the surface of the gold film smooth and smooth to form a gold plate electrode;

[0021] 2) Add Piranha solution (Note: Piranha solution is a solution of concentrated sulfuric acid: hydrogen peroxide = 7:3) dropwise on the surface of the gold plate electrode to soak for 15s, take it out and rinse it with twice distilled water repeatedly for 3 times;

[0022] 3) Soak the above electrodes in absolute ethanol and double distilled water for 60s;

[0023] 4) Immerse the above electrode in 50mmol / L DTT solution, place it for 10h to form a DTT monolayer, rinse it with absolute ethanol for 3 times to remove the free DTT on the surface, and then rinse it with twice distilled ...

Embodiment 2

[0029] Determination of standard curve of plasmid DNA containing C-myc antigen fragment:

[0030] 1) The reagents used (double distilled water, PBS buffer solution) were sterilized and stored at 4°C for later use;

[0031] 2) Take 1 μL of plasmid DNA in a 1.5mL small test tube, use PBS as a buffer solution, dilute 1-100000 times respectively, and prepare a series of solutions of 0-1.5 μg / mL for later use;

[0032] 3) The LSPR sensor chip modified with the above-mentioned C-myc monoclonal antibody was used to test the plasmid DNA samples of various concentrations respectively, and the immunoreaction of the C-myc monoclonal antibody with the plasmid DNA containing the C-myc antigen fragment was combined to cause a reaction on the chip. The absorption peak of local surface plasmon resonance spectrum of gold nanoparticles is red-shifted, and the peak displacement has a linear response relationship with the content of plasmid DNA; with the concentration C (μg / mL) of plasmid DNA as ...

Embodiment 3

[0035] Determination of the concentration of plasmid DNA containing the C-myc antigen fragment in Escherichia coli:

[0036] In this embodiment, the alkaline lysis method is used to extract the plasmid DNA containing the C-myc antigen fragment in Escherichia coli, and the specific operation method is as follows:

[0037] 1. Take 1% Escherichia coli cells containing the plasmid in 2ml LB medium, shake and culture overnight at 37°C, take 1.5ml cells into an Ep tube, centrifuge at 4000rpm for 3min, and discard the supernatant.

[0038] 2. Add 0.1ml solution I (1% glucose, 50mM / L EDTA pH8.0, 25mM / L Tris-HCl pH8.0) and mix well. Add 0.2ml solution II (0.2mM / L NaOH, 1% SDS), gently invert to mix, and place in ice bath for 5min. Add 0.15ml precooling solution III (5mol / L KAc, pH4.8), gently invert and mix well, and place in ice bath for 5min.

[0039] 3. Centrifuge at 1000rpm for 20min, take the supernatant into another new Ep tube, add an equal volume of isoamyl alcohol, mix well,...

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Abstract

The invention provides an LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting plasmid DNA (deoxyribonucleic acid) containing an oncogene C-myc antigen fragment on the basis of LSPR. The LSPR sensing chip is characterized in that one DTT (Dithiothreitol) unimolecule layer (2) is assembled on the surface of a gold film (1) of the LSPR sensing chip; a gold nanoparticle layer (3) is accessed; the surface of the gold nanoparticle layer (3) is furnished with a 3-mercaptopropionic acid unimolecule layer (4); then DMAP-EDC [4-Dimethylamino pyridine-1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide] activation ensures that the 3-mercaptopropionic acid unimolecule layer (4) is combined with a C-myc monoclonal antibody (5); and the immune response combination of the C-myc monoclonal antibody (5) and the plasmid DNA (6) containing the C-myc antigen fragment causes that the surface of the gold nanoparticle generates the displacement of a local surface plasma resonance absorption peak so as to detect the content of the plasmid DNA (6) containing the C-myc antigen fragment in canceration tissues. The invention has the beneficial effects that the LSPR sensing chip has the advantages of multiple-path detection, high sensitivity and good selectivity and repeatability, is simple and convenient to assemble and is quick to quantify. The LSPR sensing chip is superior to the LSPR sensing chip manufactured with the traditional chromatin immunoprecipitation (ChIP) method.

Description

technical field [0001] The invention belongs to the technical field of detecting plasmid DNA content in tumor cells, and relates to a sensor chip and a method for detecting plasmid DNA containing an oncogene C-myc antigen fragment. Background technique [0002] C-myc is a proto-oncogene that plays an important role in the regulation of DNA synthesis, apoptosis, differentiation and cell cycle. The C-myc recombinant protein encoded by the C-myc gene not only plays an extremely important role in cell life activities such as cell proliferation, cell differentiation and cell cycle, but also closely participates in the transformation of cell tumors. The expression of C-myc recombinant protein is closely related to the initiation and cancerous degree of many cancer tissues and cell tumors. [0003] Currently, the detection methods for plasmid DNA include horizontal agarose gel electrophoresis and the traditional chromatin immunoprecipitation (Chromatin immunoprecipitation, ChIP) m...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/574
Inventor 曹忠黄茜茜戴云林王明星何婧琳张玲曾巨澜孙立贤
Owner CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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