ECL detection method of unlabeled DNA based on porphyrin and DNA double helix groove mosaicism action
A detection method and double helix technology, applied in the field of DNA biosensors, can solve the problems of unstable reagents, uneven solution mixing, difficulty in realizing time and space control, etc., and achieve the effect of improving sensitivity and signal-to-noise ratio.
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Embodiment 1
[0028] Preparation and detection of sensors
[0029] Glassy carbon electrodes (GCE) with a diameter of 5 mm were polished on the suede of 0.3 nm and 0.05 nm alumina for 3 min, respectively. The alumina adsorbed on the electrode surface was rinsed with ultrapure water each time, and then acetone, ethanol, deionized Sonicate with water and dry with nitrogen. Hairpin probes H1 and H2 were first incubated at 95°C for 5 min and then lowered to room temperature for 1 h for activation. SH-cDNA was prepared into a 1 μM solution with 10 mM PBS, and TCEP was added to prevent the formation of disulfide bonds between sulfhydryl groups. The concentration of TCEP in the solution was 100 μM.
[0030] 25 μL of the nano-gold modified electrodes synthesized in the above steps were dried at room temperature, washed with ultrapure water, and then blown dry with nitrogen. 20 μL of SH-cDNA was drop-coated on the surface of the gold nanoparticles-modified electrode, and the water vapor was saturat...
Embodiment 2
[0034] 1. HCR reaction nucleic acid molecular weight observation, the steps are as follows:
[0035] 2.5% agarose gel was prepared with TBE buffer, 10 μL of EB / 100 mL solution was added and mixed, heated in a microwave oven for 5 min, and poured into the template to cool to gel when the temperature dropped to about 50 °C. After the samples of each lane were stained with bromophenol blue, 6 μM of each was added dropwise. The voltage was set to 80mV and the time was 90min. Lane 1 is the 50-1000bp DNALadder-H2 purchased from Shanghai Shenggong; Lane 2 is the solution of 10μM H1, 10μM H2, 0.1μM tDNA in 10mM pH7.4PBS after accelerated reaction for 2h at 37℃; 3 is the PBS solution of 10μM H1; 4 is 10 μM H2 in PBS; 5 and 6 are blank. like image 3 As shown, after a period of time, the bands of H1 and H2 moved the farthest distance, while the solution after HCR reaction showed discrete bands aggregated at the beginning of the band, indicating that the molecular weight of DNA in the...
Embodiment 3
[0041] 25 μL of gold nanoparticles modified GCE, after drying, 20 μL of SH-cDNA was drop-coated on the surface of the nano-gold modified electrode, overnight at room temperature; after washing with PBS buffer, blocked with 10 μL of 1 mM MCH for 2 h, and modified with 20 μL of tDNA of different concentrations. Concentration gradient of 10 -11 M~10 -7 M, incubate at 37°C for 3h; wash off the tDNA that is not bound to cDNA, mix 10μL H1 (1μM) and 10μL H2 (1μM) dropwise on the electrode surface, and incubate at 37°C for 3h; finally modify the sensing surface with 50μM ZnPPIX in PBS solution , and incubated at room temperature for 4 h. The whole process was saturated with water vapor, and each step was gently rinsed with PBS buffer.
[0042] The sensors were constructed following the same steps as above, replacing the tDNA with single-, di- and tri-base mismatched DNA.
[0043] The glassy carbon electrode constructed with tDNA and mismatched DNA was used as the working electrode,...
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