Fluorescence biosensor for detecting UDG (Uracil-DNA Glycosylase) and preparation method thereof
A biosensor and glycosylase technology, applied in the field of biosensors, can solve the problems of long detection period, low specificity and sensitivity, etc., and achieve the effect of fast detection speed, rapid detection and high specificity
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Embodiment 1
[0064] Example 1 Preparation of Circular Template and Composite Probe
[0065] Prepared with 50 mM Tris-HCl, 10 mM MgCl 2 , T4 DNA Ligase Reaction Buffer with 10 mM DTT and 1 mM ATP. Formulated with 10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , 140 mM NaCl, 1 mM KCl, 1 mM MgCl 2 , 1mM CaCl 2 , PBS buffer solution of pH=7.4.
[0066] (1) Mix 42 μL sterile water, 6 μL linear template (10 μM), 6 μL ligation probe (10 μM) and 6 μL 10× T4 DNA ligase buffer, denature at 95°C for 5 min, then Slowly cool down to room temperature to complete the hybridization, then add 3 μL of T4 DNA ligase (60 U / μL) to the reaction system, and react at 16°C for 20 hours; after that, the reaction system is placed in a water bath at 65°C for 15 minutes , inactivate the T4 DNA ligase in the system.
[0067] (2) Add 3 μL of exonuclease Ⅰ (20 U / μL) and 3 μL of exonuclease Ⅲ (100 U / μL) to the above reaction system and react at 37°C for 2 h; then put the reaction system at 85°C Heated in a water bath for 1...
Embodiment 2
[0069] Embodiment 2 Fluorescence intensity changes with the concentration of composite probe I
[0070] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:
[0071] (1) Mix 2 μL compound probe I (concentrations are 50 nM, 100 nM, 500 nM, 1 μM, 5 μM), 2 μL dNTP (1 mM), 2 μL phi29 DNA polymerase (1 U / μL), 2 μL endonuclease IV (1 U / μL) in 2 μL buffer (50 mMTris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5), add 2 μLUDG enzyme solution (1 U / mL) respectively, mix well and react at 37°C for 60 min;
[0072] (2) Add 2 μL of Composite Probe II (1 μM) to the solution in step (1), mix well and react at a constant temperature of 37°C for 60 min;
[0073] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 486 nm, the emission wavelength is 518 nm, and the detection range is 450 nm-530 nm, and the fluorescence signal changes a...
Embodiment 3
[0077] Embodiment 3 Fluorescence intensity changes with the concentration of compound probe II
[0078] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:
[0079] (1) Mix 2 μL compound probe I (1 μM), 2 μL dNTP (1 mM), 2 μL phi29 DNA polymerase (1 U / μL), 2 μL endonuclease IV (1 U / μL) in 2 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5), add 2 μL UDG enzyme solution (1 U / mL) respectively, mix well and react at a constant temperature of 37°C for 60 min;
[0080] (2) Add 2 μL of compound probe II (concentrations are 50 nM, 100 nM, 500 nM, 1 μM, 5 μM) to the solution in step (1), mix well and react at 37°C for 60 min;
[0081] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 486 nm, the emission wavelength is 518 nm, and the detection range is 450 nm-530 nm, and the fluorescence signal changes ...
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