CN101943703A - Nanotechnology-based trace protein detection method - Google Patents

Abstract

A micro-protein detection method based on nanotechnology combines immunoenzyme-linked adsorption technology, tyramide signal amplification technology, and aggregation of gold nanoparticles modified with different biomolecules to establish a method for detecting micro-prostate-specific Experimental methods for proteins such as sex antigen (PSA). In this method, an antibody against the protein to be tested (such as PSA) is immobilized on the surface of the substrate, and after capturing the protein to be tested in the sample, another antibody containing horseradish peroxidase is incubated with the protein to be tested (such as PSA). (HRP) active antibody. Under certain conditions, HRP catalyzes biotin-tyramide to produce biotin deposition. The phenomenon of aggregation of biotin-labeled DNA-modified gold nanoparticles and streptavidin-modified gold nanoparticles is used to further amplify the signal, followed by silver staining. Finally, use the software to analyze the data of the experimental results. The method has a very low detection limit and a wide detection range, and can realize the detection of antigens in rabbit serum with complex components, and has important application prospects.

Description

A micro-protein detection method based on nanotechnology

technical field

The invention relates to a method for detecting trace proteins based on nanotechnology, which belongs to the technical field of analytical biochemistry.

Background technique

Measuring tiny amounts of proteins is essential in disease diagnosis and biomedical research. However, the currently used assay methods have the disadvantages of poor sensitivity and linear range. For example, prostate-specific antigen (PSA) is a single-chain glycoprotein encoded by hKLK3 gene, secreted by prostate epithelial cells, has chymotrypsin activity, and has a molecular weight of about 34000 g/mol. As a biomarker, PSA is widely used in disease screening, diagnosis and post-treatment monitoring. However, the content of PSA in human serum is extremely low. Therefore, higher requirements are placed on the detection limit and sensitivity of the detection method.

Immunoenzyme-linked adsorbent assay is an immunoassay method widely used in clinical testing at present, with high specificity. The working principle of ELISA is based on the immobilization of antigen or antibody and enzymatic labeling of antigen or antibody. The antigen or antibody is adsorbed on the surface of the solid phase carrier, and the antigen or antibody bound to the surface of the solid phase carrier still maintains its immunological activity; the antigen or antibody can be linked with the enzyme through a covalent bond to form an enzyme conjugate, and the enzyme-labeled antigen Or the antibody retains both its immunological activity and enzymatic activity. Due to the high catalytic efficiency of the enzyme, the result of the immune reaction is indirectly amplified, so that the determination method can achieve high sensitivity.

At present, horseradish peroxidase is commonly used to label antibodies. Horseradish peroxidase can not only catalyze chemiluminescence, but also catalyze biotin-tyramide under certain conditions to generate a large number of biotin activation molecules that bind to proteins near the antigen-antibody binding site A large amount of biotin is deposited on the amino acid residues. The technique is called tyramide signal amplification. If the aggregation phenomenon of biotin-labeled DNA-modified gold nanoparticles and streptavidin-modified gold nanoparticles is used in this experiment, further amplification of the signal can be achieved.

This method realizes the specificity of the detection method through the immunoenzyme-linked adsorption experiment, realizes the signal amplification through the tyramide signal amplification technology and the aggregation phenomenon of gold nanoparticles, and achieves extremely low detection limit, high sensitivity and wide detection range in protein determination the goal of.

Contents of the invention

The purpose of the present invention is to provide a method based on immunoenzyme-linked adsorption technology, tyramide signal amplification technology and gold nanoparticle aggregation to detect proteins such as trace PSA. This method has a wide linear range, extremely low detection limit and high of precision.

Technical scheme of the present invention: a method for detecting trace PSA or other proteins based on immunoenzyme-linked adsorption technology, tyramide signal amplification technology and gold nanoparticle aggregation phenomenon, characterized in that the steps are: (1) using double Antibody sandwich method to capture the protein to be tested; (2) Using tyramide signal amplification technology to generate a large amount of biotin deposition at the antigen-antibody binding site; (3) Gold nanoparticles aggregated at the biotin deposition site and silver staining experiments; (4) Scanning and data analysis.

(1) Use the double-antibody sandwich method to capture proteins on the solid phase: use the immunoenzyme-linked adsorption method to capture proteins such as PSA and bind them to antibodies with horseradish peroxidase activity;

For example, dilute the PSA monoclonal antibody into a solution of a certain concentration, take 1 μL each to spot on the epoxy-based substrate, place it in an incubator, vacuumize at 37°C overnight, take out the substrate, and add 10% After blocking with BSA solution at room temperature for 5 h, wash with PBST solution. Then paste the fence on the substrate, dilute the PSA standard with rabbit serum, and dilute its concentration successively to 5fg/μL, 2.5fg/μL, 125ag/μL, 6.25ag/μL and 330.5zg/μL, each take 20μL and dilute a series of Concentrations of PSA dissolved in PBS were added to the pens and incubated overnight at 4°C. After overnight incubation, the substrate was taken out and washed with PBST solution. After that, PSA diluted in PBS and another monoclonal antibody with horseradish peroxidase activity were added. After incubation for a period of time, it was washed with PBST solution.

(2) Use tyramide signal amplification technology to generate a large amount of biotin deposition at the antigen-antibody binding site: add biotin-tyramide solution to generate a large amount of biotin deposition at the antigen-antibody binding site;

Add biotin-tyramide solution on the substrate, the control group does not add biotin-tyramide solution, and wash with PBST solution after reacting for a period of time.

(3) Gold nanoparticles aggregated at the biotin deposition site and silver staining experiment: gold nanoparticles modified with different biomolecules aggregated at the biotin deposition site and the signal was further amplified by silver staining;

Utilizing the interaction between sulfhydryl groups and gold nanoparticles, biotin-labeled DNA was modified on the surface of gold nanoparticles, and streptavidin was modified on the surface of gold nanoparticles by using the electrostatic adsorption between protein and gold nanoparticles, thus preparing two kinds of Gold nanoparticles modified with different biomolecules. Add a certain amount of streptavidin-modified gold nanoparticles (SA-AuNP) into the reaction pool where the substrate is placed, and incubate for a period of time. After washing with PBST, a certain amount of biotin-labeled DNA-modified gold nanoparticles (biotin-DNA-AuNP) was added to the reaction pool and incubated for a period of time. Afterwards, wash with PBST, repeat the above gold nanoparticle incubation process, and silver stain the substrate after the incubation. Mix silver staining reagent A and B, add it to the reaction tank with the substrate, soak for a period of time, take it out, after cleaning, add a certain concentration of sodium thiosulfate solution, soak for 2 minutes, wash and dry.

(4) Scanning and data analysis;

After the chips were dried, they were placed on a scanner and scanned. The gray value of the image results was analyzed using ImageJ, and then data analysis was performed with Origin software.

Description of drawings

Figure 1 Electron micrograph of gold nanoparticles.

Fig. 2 UV-Vis absorption spectrum of gold nanoparticles.

Fig. 3 UV-Vis absorption spectra of AuNP, biotin-DNA-AuNP and SA-AuNP.

Figure 4 Inverted image of the substrate.

Fig. 5 Standard curve of the linear relationship between the mass of the antigen on the substrate and the gray value.

Detailed ways

It is characterized in that the steps are: (1) using the double antibody sandwich method to capture PSA or other protein samples to be tested on the solid phase; (2) using tyramide signal amplification technology to generate a large amount of biotin deposition at the antigen-antibody binding site; (3) Aggregation of gold nanoparticles at biotin deposition sites and silver staining experiments; (4) Scanning and data analysis.

(1) Capture PSA or other protein samples to be tested by double-antibody sandwich method on the solid phase: use immunoenzyme-linked adsorption method to capture PSA and bind it to an antibody with horseradish peroxidase activity;

Dilute the PSA monoclonal antibody into a solution of a certain concentration, take 1 μL each and spot it on the epoxy-based substrate, place it in an incubator, vacuumize at 37°C overnight, take out the substrate, and add 10% BSA to the reaction pool The solution was blocked, and after blocking at room temperature for 5 h, it was washed with PBST solution. Then paste the fence on the substrate, dilute the PSA standard with rabbit serum, and dilute its concentration successively to 5fg/μL, 2.5fg/μL, 125ag/μL, 6.25ag/μL and 330.5zg/μL, each take 20μL and dilute a series of Concentrations of PSA dissolved in PBS were added to the pens and incubated overnight at 4°C. After overnight incubation, the substrate was taken out and washed with PBST solution. After that, PSA diluted in PBS and another monoclonal antibody with horseradish peroxidase activity were added. After incubation for a period of time, it was washed with PBST solution.

(2) Use tyramide signal amplification technology to generate a large amount of biotin deposition at the antigen-antibody binding site: add biotin-tyramide solution to generate a large amount of biotin deposition at the antigen-antibody binding site;

Add biotin-tyramide solution on the substrate, the control group does not add biotin-tyramide solution, and wash with PBST solution after reacting for a period of time.

(3) Gold nanoparticles aggregated at the biotin deposition site and silver staining experiment: gold nanoparticles modified with different biomolecules aggregated at the biotin deposition site and the signal was further amplified by silver staining;

Utilizing the interaction between sulfhydryl groups and gold nanoparticles, biotin-labeled DNA was modified on the surface of gold nanoparticles, and streptavidin was modified on the surface of gold nanoparticles by using the electrostatic adsorption between protein and gold nanoparticles, thus preparing two kinds of Gold nanoparticles modified with different biomolecules. Add a certain amount of streptavidin-modified gold nanoparticles (SA-AuNP) into the reaction pool where the substrate is placed, and incubate for a period of time. After washing with PBST, a certain amount of biotin-labeled DNA-modified gold nanoparticles (biotin-DNA-AuNP) was added to the reaction pool and incubated for a period of time. Afterwards, wash with PBST, repeat the above gold nanoparticle incubation process, and silver stain the substrate after the incubation. Mix silver staining reagent A and B, add it to the reaction tank with the substrate, soak for a period of time, take it out, after cleaning, add a certain concentration of sodium thiosulfate solution, soak for 2 minutes, wash and dry.

(4) Scanning and data analysis;

After the chips were dried, they were placed on a scanner and scanned. The gray value of the image results was analyzed using ImageJ, and then data analysis was performed with Origin software.

Claims (1)

1. trace protein detection method based on nanometer technology

A kind of trace protein detection method based on nanometer technology is characterized in that step is: (1) utilizes double antibody sandwich method to catch testing protein on solid phase; (2) utilize the tyrasamine signal amplification technique to produce the plain deposition of large number of biological at the antigen-antibody binding site; (3) gold nano grain is assembled and silver dyes experiment mutually in the biotin deposition site; (4) scanner uni data analysis.

(1) utilizing double antibody sandwich method to catch PSA or other target protein on the solid phase: utilize immuno-enzymatic connection adsorption method catch protein such as PSA and make it in conjunction with on the antibody of horseradish peroxidase activity is arranged;

For example, the PSA monoclonal antibody is diluted to certain density solution, respectively gets 1 μ L and select on the epoxy radicals substrate, place incubator, 37 ℃ vacuumize spend the night after, substrate is taken out, the BSA solution of adding 10% sealing in reaction tank behind the room temperature sealing 5h, is cleaned with PBST solution.On substrate, paste fence then, with rabbit anteserum dilution PSA standard items, its concentration is diluted the L for 5fg/ μ successively, 2.5fg/ μ L, 125ag/ μ L, 6.25ag/ μ L and 330.5zg/ μ L respectively get 20 μ L the PSA standard items of the PBS of a series of concentration dissolving are joined in the fence, in 4 ℃ of overnight incubation.After the overnight incubation, substrate is taken out, clean with PBST solution, afterwards, the PSA another kind that adds the PBS dilution has the monoclonal antibody of horseradish peroxidase activity, hatch a period of time after, clean with PBST solution.

(2) utilize the tyrasamine signal amplification technique to produce the plain deposition of large number of biological: to add biotin-tyramide solution, produce the plain deposition of large number of biological at the antigen-antibody binding site at the antigen-antibody binding site;

Add biotin-tyramide solution on substrate, control group does not add biotin-tyramide solution, cleans with PBST solution after reaction a period of time.

(3) gold nano grain is assembled and silver dyes experiment mutually in the biotin deposition site: the gold nano grain that different biological molecules is modified is assembled mutually and is used the further amplifying signal of silver staining method in the biotin deposition site;

Utilize the interaction of sulfydryl and gold nano grain, with the dna modification of biotin mark to the gold nano grain surface, utilize the electrostatic adsorption of protein and gold nano grain that Streptavidin is modified the gold nano grain surface, thereby prepare the gold nano grain that two kinds of different biological molecules are modified.In being placed with the reaction tank of substrate, add the gold nano grain (SA-AuNP) that a certain amount of Streptavidin is modified, hatch a period of time.After PBST cleans, in reaction tank, add the gold nano grain (biotin-DNA-AuNP) of the dna modification of a certain amount of biotin mark again, hatch a period of time.Clean with PBST afterwards, repeat above-mentioned gold nano grain and hatch process, hatch and finish the back and substrate is carried out silver dye.Silver-colored transfection reagent A liquid and B liquid are mixed, join in the reactive tank that is placed with substrate, soak a period of time, take out, after the cleaning, add certain density hypo solution, soak 2min, after the cleaning, oven dry.

(4) scanner uni data analysis;

After the chip oven dry, place on the scanner and scan, image result uses ImageJ to analyze its gray-scale value, carries out data analysis with Origin software then.

Images