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A highly sensitive detection method for various lung cancer markers based on nano-gold enhancement

A detection method, nano-gold technology, applied in high-sensitivity detection, nano-biocomposite probes and nano-gold enhancement, based on the microarray field, can solve the problems of powerlessness and low sensitivity in the detection of trace biomolecules, and achieve high sensitivity and operation steps Less time-consuming effect

Active Publication Date: 2016-03-30
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gold nanoparticles have the characteristics of high electron density. Brown particles can be seen under the microscope at the junction of gold nanoparticles. When these markers are agglomerated in large quantities at the corresponding ligands, red or pink spots can be seen with the naked eye, so they are widely used in Among the qualitative or semi-quantitative rapid detection methods, the biggest disadvantage of this type of detection technology is low sensitivity, the sensitivity of detecting protein is only ng / ml, and it is powerless for the detection of trace biomolecules (the progress of immunolabeling technology and the role of nanotechnology in it) Applications, Jia Chunping, Life Sciences, Volume 20, Issue 5, Pages 749-753)
In addition, most of these detection methods are suitable for detection systems with test strips or membranes as solid phase carriers. For micron or even nanoscale detection points on microarrays with glass as solid phase carriers, no further steps are required. The signal amplification of the gold nanometer is not detectable by the general microscope, especially the naked eye, and further amplification of the nano-gold signal is required.

Method used

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  • A highly sensitive detection method for various lung cancer markers based on nano-gold enhancement
  • A highly sensitive detection method for various lung cancer markers based on nano-gold enhancement
  • A highly sensitive detection method for various lung cancer markers based on nano-gold enhancement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: A highly sensitive detection method that can be used for standard CEA and P53 proteins

[0034] 1 Fabrication of the protein chip:

[0035] The monoclonal antibodies of carcinoembryonic antigen (CEA) and P53 protein were spotted on the aldehyde-modified glass substrate. Store overnight at room temperature and store at 4°C for later use.

[0036] Preparation of 2 nanometer gold biocomposite probes

[0037] Under the condition of pH 6~9, according to the colloidal gold agglutination experiment, determine the consumption of the detection antibody of CEA and P53 protein, and mix the antibodies of various concentrations with the nano gold solution at room temperature for 1-5 hours, and then add 0.1-20% ( Quality) of polyethylene glycol, overnight at 4°C, centrifuged, washed off unlabeled antibodies, resuspended with gold nanoparticles (bovine serum albumin BSA0.25-10%, polyvinylpyrrolidone PVP0.1-1%) , sucrose 1.5-10%, polyethylene glycol PEG 0.01-1%, Tween 200...

Embodiment 2

[0045] Embodiment 2: The assay method that can be used for the early detection of lung cancer

[0046] Carcinoembryonic antigen (CEA) is an acidic glycoprotein. The dynamic change of serum CEA level can not only be used for the early diagnosis of malignant tumors, but also reflect the patient's response to treatment and prognosis. Those whose measured value increases progressively have a poor prognosis. . Cytokeratin (Cytokeratin) 19 is of great value in the diagnosis of lung cancer and is an important marker of non-small cell lung cancer. The level of serum CK19 gradually increases with the increase of tumor stage, which can also predict the prognosis of lung cancer and help to determine the curative effect of surgery. Combined application of cytokeratin 19 and CEA, the coincidence rate of diagnosis of non-small cell lung cancer can reach 78%. Neuron-specific enolase (Neuron-Specific Enolase NSE), a glycolytic enzyme ubiquitous in mammalian tissues, is an important marker o...

Embodiment 3

[0056] Example 3: Determination method that can be used for detection of lung cancer micrometastasis

[0057] Mucin 1 (MucinsMUC1) is a large molecular weight glycoprotein, which is often abnormally expressed in tumor tissues and is associated with tumor invasion, metastasis and prognosis. The combined detection of MUC1, CEA and CK19 can be used to predict the metastasis and prognosis of lung cancer.

[0058] 1 Fabrication of protein chips

[0059] Carcinoembryonic antigen (CEA), cytokeratin (CK-19), mucin (MUC1), and quality control points (anti-mouse IgG) were spotted on the aldehyde-modified glass substrate. Store overnight at room temperature and store at 4°C for later use.

[0060] Preparation of 2 nanometer gold biocomposite probes

[0061] In the case of pH 6-9, according to the colloidal gold agglutination experiment, determine the amount of detection antibodies for CEA, CK-19, and MUC1 proteins, and mix the antibodies of each concentration with the nano-gold soluti...

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Abstract

The invention provides a nano-gold enhanced highly sensitive detection method for a plurality of lung cancer markers. The detection method comprises the following steps: 1) applying samples of capture antibodies of all the proteins to be detected on an aldehyde group-modified substrate; (2) marking polyclonal or monoclonal detection antibodies of the proteins to be detected on nano-gold; (3) mixing protein chips on which capture antibodies of the proteins to be detected are marked and nano-gold biological composite probes with protein samples to be detected, incubating obtained mixture for a period of time at a temperature of 37 DEG C, and eluting nano-gold probes which have not undergone reaction; 4) adding a gold enhanced reaction solution, observing the solution with naked eyes or microscope, or scanning and taking pictures of the solution with CCD, and determining corresponding protein concentration according to gray values. The method provided in the invention can be applied in a wide variety of fields like clinical diagnosis, monitoring of tumor metastasis, detection of antigens, antibodies and nucleic acids, health quarantine, environment detection, and sensitivity of the method in detecting proteins reaches pg / mL level.

Description

technical field [0001] The present invention relates to a highly sensitive detection method for various lung cancer markers based on nano-gold enhancement, especially a highly sensitive detection method based on microarray, nano-biocomposite probe and nano-gold enhancement, which can simultaneously detect multiple A lung cancer-related marker can be used for early detection, recurrence and metastasis monitoring of lung cancer. The invention belongs to the technical field of nanometer biological detection. Background technique [0002] Being able to detect biomolecules simply, quickly, and with high sensitivity has always been the direction of people's efforts, and it is of great significance in the fields of early diagnosis of diseases, rapid diagnosis, sanitation detection, and environmental monitoring. Lung cancer is the malignant tumor with the highest incidence rate and the largest number of deaths in the world. Lung cancer is also one of the malignant tumors with the w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/574
Inventor 贾春平景奉香赵辉金庆辉赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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