Protein chip detection by colloidal gold and silver reinforcing process

A protein chip, protein technology, applied in the biological field, can solve problems such as inapplicability

Inactive Publication Date: 2005-07-06
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, another detection method of protein chips - enzyme-linked immunoreaction detection method - is not suitable for the detection of protein chips based on glass slides

Method used

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  • Protein chip detection by colloidal gold and silver reinforcing process
  • Protein chip detection by colloidal gold and silver reinforcing process
  • Protein chip detection by colloidal gold and silver reinforcing process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] General detection conditions for colloidal gold-silver enhanced method

[0054] (a) The protein chip and the sample to be detected were reacted at room temperature for 1 hour, then washed with PBST (PBS containing 0.1% Tween 100), PBS and deionized water successively, and dried by centrifugation at 200g; first complex;

[0055] (b) reacting the protein chip in step (a) with the recognition molecule that recognizes the first complex to form a second complex composed of the first complex and the recognition molecule, wherein the recognition molecule has the first coupling The counterpart; the reaction conditions and washing and drying methods are the same as step (a);

[0056] (c) mixing the protein chip of step (b) with the colloidal gold solution with the second coupling partner to form a third complex composed of the second complex and colloidal gold, wherein the second coupling partner and Coupling is formed between the first coupling partners; the reaction conditio...

Embodiment 2

[0060] Influence of the mixing time of silver enhanced solution and protein chip on the results of detection by colloidal gold-silver enhanced method

[0061] The method of Example 1 was repeated, except that the silver enhancement solutions A and B were then mixed and reacted with the protein chip, and the reaction was stopped at 5, 10, 15, 20, 25 and 30 minutes, respectively. Then measure the gray value.

[0062] The result is as figure 2 As shown, at different concentrations (the spotting concentration of human IgG on the protein chip was 250 μg / ml. The concentrations of biotin-conjugated anti-human IgG were 110 ng / ml (▲), 1.1 μg / ml (■), and 11 μg / ml(●)), when the silver enhancement solution is mixed with the protein chip, the mixing time should be 5-30 minutes, more preferably 5-25 minutes.

Embodiment 3

[0064] The effect of the number of spotting proteins bound to each spotting area in the protein chip on the results

[0065] The method of Example 1 was repeated, except that the quantity of the spotting protein bound to each spotting area in the protein chip was changed. Then measure the gray value.

[0066] The result is as image 3 As shown, the dependence of the gray value of the spots and the concentration of human IgG immobilized on the chip is shown. Wherein, the inner illustration is the colloidal gold-silver enhanced detection image of the microarray formed by different concentrations of human IgG. The spotting concentrations of human IgG in columns 1 to 12 are as follows; / ml, 7.8 μg / ml, 3.9 μg / ml, 1.95 μg / ml and 0.97 μg / ml. The concentration of biotin-conjugated anti-human IgG was 11 μg / ml; the silver enhancement time was 15 minutes. Data in the curves are taken as the mean of 7 replicate points in the inset.

[0067] The results showed that the lowest detecti...

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Abstract

This invention discloses a protein chip test method through colloidal gold and silver strengthening method, which comprises the following steps: a, making the protein chip react with the sample to form the first compound; b, making the chip react with the first identifying molecule of first compound to form second compound; c, mixing the chip with colloidal gold solution of the second coupled pair part to form the second compound and third compound composed of colloidal gold; d, mixing the chip with silver strengthening solution to make the silver ions form metal silver under function of gold catalyzing; e, stopping silver strengthening reaction and measuring the gray degrees of each point sample area.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting a protein chip through a colloidal gold-silver enhancement method. Background technique [0002] Protein chip is an important tool to study the dynamic changes of the proteome of organisms, and it can be used to simultaneously detect multiple protein marker molecules in clinical tests. Generally, a protein chip is a microarray formed on a substrate by various probes that can specifically recognize different protein marker molecules in a sample. Existing protein chips mostly adopt the detection method (MacBeath, G., and Schreiber, S.L. (2000) Science 289, 1760-1763.Robinson, W.H.et al (2002) Nature Medicine 8, 295-301. Zhu, H. et al (2001) Science 293, 2101-2105). However, the high prices of fluorescent probes and laser biochip scanners limit the wide application of biochips, especially in clinical testing. [0003] Colloidal gold labeling and silver-enhanced...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/68
Inventor 阮康成梁汝强谭翠燕
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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