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LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein of carcinogene C-myc

A sensor chip and recombinant protein technology, which is applied in the field of protein content detection in tumor cells, can solve the problems of application limitations, cumbersome operation, and low precision, and achieve the effects of good selectivity and reproducibility, simple assembly, and rapid quantification

Active Publication Date: 2012-11-28
CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, the detection methods for protein, antigen and antibody include Bradford method and traditional biological enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA), and there is no specific detection method for C-myc recombinant protein
However, the Bradford method has poor anti-interference and selectivity; the traditional ELISA method can only be qualitative or semi-quantitative, and the above-mentioned method is cumbersome to operate and has low precision, which limits its application

Method used

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  • LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein of carcinogene C-myc
  • LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein of carcinogene C-myc
  • LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein of carcinogene C-myc

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Assembly preparation of LSPR sensor chip:

[0018] 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;

[0019] 2) Add Piranha solution (Note: Piranha solution is a solution of concentrated sulfuric acid:hydrogen peroxide = 7:3) dropwise on the surface of the above-mentioned gold film for 15 seconds, take it out and rinse it repeatedly with twice distilled water for 3 times;

[0020] 3) Soak the gold film treated in the above steps in absolute ethanol and double distilled water for 60 seconds;

[0021] 4) Immerse the gold film treated in the above steps in a 50mmol / L DTT solution, place it for 10h to form a DTT monolayer, rinse it with absolute ethanol three times to remove the free DTT on the surface, and then rinse it repeatedly...

Embodiment 2

[0027] Determination of C-myc recombinant protein standard curve:

[0028] 1) The reagents used (double distilled water, PBS buffer solution) were sterilized and stored at 4°C for later use;

[0029] 2) Take 1μLC-myc recombinant protein (antigen) and place it in a 1.5mL small test tube, use PBS as the buffer solution, dilute it by 1-100000 times respectively, and prepare a series of solutions of 0-6.5μg / mL for later use;

[0030] 3) The LSPR sensor chip modified with the above-mentioned C-myc monoclonal antibody was used to test the C-myc recombinant protein samples of various concentrations, and the combination of the C-myc monoclonal antibody and the C-myc recombinant protein immune reaction caused the gold on the chip to The absorption peak of the localized surface plasmon resonance spectrum of nanoparticles shifts red, and the peak shift has a linear response relationship with the content of C-myc recombinant protein; The displacement value Δλ (nm) is the ordinate, and th...

Embodiment 3

[0033] Determination of C-myc recombinant protein content in liver cancer tissue:

[0034] Extraction of protein in tissue: take 0.1g tissue sample, rinse with pre-cooled PBS at 4°C to remove tissue foreign matter. Cut to 0.5mm with scissors 3 Small pieces were added to the prepared 1.0mL lysate, and then diluted with PBS for later use.

[0035] Determination of C-myc recombinant protein content: place the LSPR sensor chip prepared above in PBS buffer solution, first record the reading λ of the absorption peak wavelength position of PBS buffer solution 1 = 353.47nm. Take the above-mentioned diluted sample solution for measurement, and record the reading λ of the absorption peak wavelength position 2 = 354.68nm. The absorption peak displacement value (from, nm) of this sample can be obtained by following formula: Δλ=λ 2 -λ 1 , Substituting Δλ into the fitted standard curve equation, the concentration of C-myc recombinant protein in the liver cancer tissue sample can be calc...

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Abstract

The invention provides an LSPR (Localized Surface Plasmon Resonance) sensing chip for detecting recombinant protein (6) of carcinogene C-myc based on an LSPR spectrum, wherein a DTT (Dithiothreitol) monomolecular layer (2) is assembled on the surface of a gold film (1) of the LSPR sensing chip; a gold nano particle layer (3) is connected to the DTT monomolecular layer (2); a 3-sulfydryl propionicacid monomolecular layer (4) is modified on the surface of the gold nano particle layer (3); the 3-sulfydryl propionic acid monomolecular layer (4) and a monoclonal antibody (5) of the C-myc are combined through DMAP (Dimethylamino Pyridine)-EDC (Dichloroethane) activation; and the surface of gold nano particles is caused to generate the displacement of an LSPR absorption peak through the immunoreaction combination of the monoclonal antibody (5) of the C-myc and a recombinant protein (6) of the C-myc, thereby detecting the content of the recombinant protein (6) of the C-myc in cancerous tissues. The LSPR sensing chip provided by the invention has the beneficial effects of simplicity and convenience in assembly, rapidness in quantitation, high sensitivity and good selectivity and repeatability, can be used for realizing multi-channel detection and is prior to the traditional enzyme-linked immunosorbent assay (ELISA).

Description

technical field [0001] The invention belongs to the technical field of protein content detection in tumor cells, and relates to a sensor chip for detecting oncogene C-myc recombinant protein and a method thereof. 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 protein, antigen and antibody include Bradford method and traditional biological enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA), and ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/55G01N33/543G01N21/552
Inventor 曹忠王明星黄茜茜戴云林何婧琳张玲曾巨澜孙立贤
Owner CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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