CHA amplification reaction system of HCV core protein and ultra-sensitive visual detection method
A reaction system, core protein technology, applied in the field of analytical chemistry
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[0033] In the CHA amplified reaction system of the present invention, the preparation method of the B-H2 functionalized nanofiber membrane comprises the following steps: (1) dissolving in N,N-dibutylammonium bromide after mixing polystyrene and tetrabutylammonium bromide In methyl formamide, obtain PS / TBAB / DMF solution; The mixture quality of described polystyrene and tetrabutylammonium bromide is 15~25% of described PS / TBAB / DMF solution quality;
[0034] (2) Electrospinning the PS / TBAB / DMF solution, and drying to obtain a PS nanofiber membrane; the voltage during the electrospinning is 10-20kV, and the injection speed is 0.15-0.45mL / h, The receiving distance is 7~15cm, and the collection time is 1.5~3h;
[0035] (3) Utilize the atmospheric pressure air plasma that dielectric barrier discharge produces to process described PS nanofiber membrane, obtain the PS nanofiber membrane after processing; The voltage of described treatment is 40~50V, and electric current is 1.2~2.5 A, d...
Embodiment 1
[0056]Using DMF as a solvent, prepare a PS / TBAB / DMF solution with a mass fraction of 20%, in which TBAB is 0.3% (w / v), stir at room temperature for 24 hours, and perform electrospinning after mixing evenly. Spinning conditions: voltage 15kV, sample injection speed 0.3mL / h, receiving distance 10cm, collection time 2h, ambient humidity 45%-50%. The nanofiber film received on the aluminum foil was placed in an oven at 80° C. for 4 hours, and then the nanofiber film was cut into discs with a diameter of d=5 mm for later use.
[0057] The hydrophilicity of the PS nanofiber membrane surface was improved by atmospheric pressure air plasma generated by dielectric barrier discharge (DBD). The voltage was set to 45V, the current was 2.2A, and the treatment time was 2min. Immediately soak the plasma-treated PS nanofiber membrane in 100 μL 3 μM avidin solution for 1 h, take it out and wash it three times with ultrapure water, then soak it in 100 μL 1.6 μM B-H2 solution for 1 h, take it ou...
Embodiment 2
[0059] 100 μL of HCV core protein was reacted with annealed C7-I (0.4 μM) for 3 h, and the B-H2 functionalized nanofiber membrane prepared in Example 1 was soaked in the reaction solution for 16 h to carry out CHA amplification reaction. After the reaction, take it out and wash it three times with ultrapure water, and finally soak it in 100 μL of 4 μM hemin solution for 2 hours, take it out and wash it three times with ultrapure water. The DNAzyme / nanofiber membrane was obtained and stored in a refrigerator at 4°C.
[0060] The DNAzyme / nanofiber membrane was washed, blotted dry with filter paper and placed at the bottom of a 96-well plate, and 100 μL containing 240 μM H was added to each well. 2 o 2 MES buffer. After reacting for 30 minutes, add 100 μL containing 0.4mM HAuCl 4 MES buffer solution, 50 μL of 100 μM glutathione was added after 20 min of reaction. Then take a picture with a digital camera and detect it with a UV-Vis spectrophotometer. The wavelength test range...
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