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Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor

A hybridization chain reaction and single-molecule counting technology, applied in the field of immunosensors, can solve the problems of affecting the authenticity of detection, prone to false positives, and poor repeatability, so as to reduce the generation of false positive signals and reduce the interference to the background , the effect of high detection sensitivity

Inactive Publication Date: 2014-07-23
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, enzyme-linked immunosorbent assay is mostly used for the detection of TNF-α, but this method has problems such as poor repeatability, low sensitivity, high detection limit, etc., and it is prone to false positives, which affects the authenticity of the detection

Method used

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  • Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor
  • Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor
  • Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor

Examples

Experimental program
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Effect test

Embodiment 1

[0056] An immunosensor based on hybridization chain reaction and single molecule counting, composed of tumor necrosis factor-α capture antibody (anti-TNF-α), monoclonal rabbit anti-human tumor necrosis factor-α detection antibody (Bio-anti-TNF-α ), streptavidin, biotinylated primers and hairpin probes;

[0057] The base sequence of the biotinylated primer (Bio-1) is 5'-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3';

[0058] The hairpin probe consists of a hairpin probe H1 and a hairpin probe H2, wherein the base sequence of the hairpin probe H1 is 5'-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3';

[0059] The base sequence of hairpin probe H2 is 5'-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3'.

[0060] The preparation method of the immunosensor comprises the following steps:

[0061] (1) Construction of the sandwich immune reaction process: 50 μL of tumor necrosis factor-α capture antibody (anti-TNF-α) (20pM) was dropped ont...

Embodiment 2

[0072] An immunosensor based on hybridization chain reaction and single molecule counting, in its preparation method, the concentration of anti-TNF-α is 10pM; the blocking agent is bovine serum albumin, and the concentration is 60mM; the concentration of Bio-anti-TNF-α is 20pM, the concentration of streptavidin is 10pM, the concentration of Bio-I is 0.5nM, in the mixture of hairpin probes H1 and H2, the concentrations of H1 and H2 are the same, both are 5nM;

[0073] The volume ratio of the mixture of anti-TNF-α, blocking agent, Bio-anti-TNF-α, streptavidin, Bio-I, hairpin probes H1, H2 is 1:2:1:2:1 ; All the other are with embodiment 1.

Embodiment 3

[0075] An immunosensor based on hybridization chain reaction and single molecule counting, in its preparation method, the concentration of anti-TNF-α is 30pM; the blocking agent is bovine serum albumin, and the concentration is 20mM; the concentration of Bio-anti-TNF-α is 40pM, the concentration of streptavidin is 80pM, the concentration of Bio-I is 0.1nM, in the mixture of hairpin probes H1 and H2, the concentrations of H1 and H2 are the same, both are 15nM;

[0076] The volume ratio of the mixture of anti-TNF-α, blocking agent, Bio-anti-TNF-α, streptavidin, Bio-I, hairpin probes H1, H2 is 2:1:2:1:2 ; All the other are with embodiment 1.

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Abstract

The invention discloses an immunosensor based on hybrid chain reaction and single molecule counting. The immunosensor consists of a capturing antibody, a detection antibody, streptavidin, a biotinylated primer and a hairpin probe, wherein the capturing antibody is anti-TNF-alpha; the detection antibody is Bio-anti-TNF-alpha; a base sequence of the biotinylated primer (Bio-I) is 5'-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3'; the hairpin probe consists of a hairpin probe H1 and a hairpin probe H2; a base sequence of the hairpin probe H1 is 5'-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3'; a base sequence of the hairpin probe H2 is 5'-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3'. The invention also provides a preparation method of the immunosensor and application of the immunosensor in detection of the TNF-alpha. The immunosensor disclosed by the invention has the advantages of high detection sensitivity, small sample use amount, no need of enzyme amplification and the like, and quantitative detection on low-concentration TNF-alpha can be realized.

Description

technical field [0001] The invention relates to an immunosensor based on hybridization chain reaction and single molecule counting and its application. Background technique [0002] Single-molecule detection (SMD), a method built on the basis of the analysis of single molecules, has so far received much attention in life science research. This method is widely used in cell imaging, studies of protein interactions, and quantitative detection of DNA and proteins. In the above-mentioned applications, quantitative detection is achieved by counting target molecules one by one, and this quantitative detection means represents the ultimate limit of detection. Traditional methods of averaging assays quantify the relationship between signal intensity and target concentration. The higher the signal intensity, the higher the concentration of the measured target. Different from the average determination method, in the SMD quantitative method, the signal intensity is no longer so impo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/552G01N33/533G01N21/64
CPCG01N33/552G01N2333/525G01N2333/7151
Inventor 王磊姜玮代爽
Owner SHANDONG UNIV
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