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Antibody detection method for combining three-chain DNA molecular beacons with nanopore technology for combination

A technology of DNA molecules and antibodies, which is applied in recombinant DNA technology, DNA/RNA fragments, microbial measurement/inspection, etc. It can solve the problems of low signal characteristics, blocked signal unfavorable high-sensitivity detection, strong background interference, etc.

Inactive Publication Date: 2018-08-31
INST OF CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, clogged signals are very unfavorable for high-sensitivity detection because of their low signal characteristics and strong background interference

Method used

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  • Antibody detection method for combining three-chain DNA molecular beacons with nanopore technology for combination
  • Antibody detection method for combining three-chain DNA molecular beacons with nanopore technology for combination
  • Antibody detection method for combining three-chain DNA molecular beacons with nanopore technology for combination

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, the preparation of DNA molecular beacon

[0081] 1. Preparation of antibody capture elements

[0082] 1. Preparation of single-stranded DNA molecules II

[0083] Each single-stranded DNA molecule II was artificially synthesized separately.

[0084] A total of 7 types of single-stranded DNA molecule II were designed, as follows:

[0085] (1)

[0086] (2)

[0087] (3)

[0088] (4)

[0089] (5)

[0090] (6)

[0091] (7)

[0092] The single underline marks the binding region B, the double underline marks the binding region C, and the middle section is between the binding region B and the binding region C.

[0093] 2. An antigen molecule is connected to the 5' end and the 3' end of the DNA molecule synthesized in step (1) (the antigen molecule is directly synthesized by Sangon Biotech and the antigen molecule is connected to the end of the DNA molecule through a covalent bond), to obtain Antibody capture elements numbered (1)-(7).

[00...

Embodiment 2

[0122] Embodiment 2, the construction of sample cell system

[0123] The sample cell system consists of two compartments (cis and trans), both of which have a volume of 1.5 mL, and are separated by a 20-μm thick polytetrafluoroethylene film (the middle of the film has a diameter of about 100- 150μM small hole), use a glass capillary to drop a mixed solution of 1 volume part of n-hexadecane and 10 volume parts of n-pentane into the small hole of the polytetrafluoroethylene film (the dropping amount is about 2 to 3 microliters ), then electrolyte buffer (3M KCl, 10mM Tris, pH 5.0) and 1,2-diphytanyl lecithin (lipids) (1,2-diphytanyl lecithin with Add in the form of phospholipid stock solution, the concentration of phospholipid stock solution is 10 mg / ml, and the addition amount is about 15 microliters; the addition amount of electrolyte buffer solution is 1 mL), and mix with a pipette to make it self-assemble to form a phospholipid bilayer . Then 1 μl of α-hemolysin heptamer p...

Embodiment 3

[0127] Embodiment 3, the detection of antibody

[0128] Antibodies to be tested: Dig antibody (Abcam Company, product number: ab6212), 5BrdU antibody (Abcam Company, product number: ab8152), HIVp17 antibody (Abcam Company, product number: ab9067), streptavidin (Sigma-Aldrich Company, product number: 85878) and dig fab fragement (Roche company, catalog number: 11093274910).

[0129] 1. Preparation of three-chain molecular beacon and antibody detection

[0130] 1. The DNA molecule shown in sequence 1 is artificially synthesized, and the 5' and 3' ends of the DNA molecule shown in sequence 1 are simultaneously modified with the antigen molecule (digoxigenin molecule) corresponding to the Dig antibody, and the Dig antibody captures the element; The Dig antibody capture element and the probe numbered (a) in Example 1 were mixed in equimolar amounts, and incubated together at 4° C. for 2 h to form a Dig antibody DNA molecular beacon.

[0131] The Dig antibody capture element is sh...

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Abstract

The invention discloses an antibody detection method for combining a three-chain DNA molecular beacon with a nanopore technology for combination. The method comprises the steps that the three-chain DNA molecular beacon used for detecting an antibody is designed and synthesized, when the antibody is combined with the three-chain DNA molecular beacon, the molecular beacon structure is destroyed, anda probe is released; an antibody-antibody capture DAN molecular complex cannot penetrate through a nanopore due to too large volume, the probe passes through the nanopore and can generate a characteristic current signal, the signal specificity is extremely high, molecular recognition can be accurately achieved, and the background interference problem is avoided. In addition, the unique thermodynamics and kinetic properties of the molecular beacon are combined with the nanopore single molecular technique, and high sensitivity detection on the variable antibody is achieved. In addition, the method has the advantages that implementation is easy, reagent consumption is extremely low, and large-scale equipment is not needed.

Description

technical field [0001] The invention relates to an antibody detection method combined with triple-strand DNA molecular beacon and nanopore technology. Background technique [0002] Antibody is an important biochemical indicator, and its rapid and sensitive detection is not only of great significance for disease diagnosis, but also an important means of curative effect observation and disease prevention. At present, there are mainly two kinds of antibody detection techniques commonly used, enzyme-linked immunosorbent assay and Western blot. However, these two methods are not only complicated to operate and consume a large amount of reagents, but also the results obtained are usually semi-quantitative results. In recent years, with the rapid development of nanotechnology, various methods based on nanomaterials to detect antibodies with high sensitivity and selectivity have been developed. However, large instruments are still required and the process is cumbersome. Therefore...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804C12N15/11
CPCC12Q1/6804C12Q2525/10C12Q2565/50
Inventor 吴海臣刘蕾郭秉元
Owner INST OF CHEM CHINESE ACAD OF SCI
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