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.
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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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