Construction and application of terminal transferase activity dynamic analysis method based on alternating-current impedance technology
A technology of terminal transferase and AC impedance, which is applied in the field of functional biomaterials and biosensing, can solve undiscovered problems and achieve high sensitivity
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Embodiment 1
[0030] Embodiment 1 sensor preparation
[0031] Electrode 1: 0.05 aluminum oxide (Al 2 o 3 ) was polished and polished on the suede, followed by ultrasonic cleaning in absolute ethanol and ultrapure water for 5min, in 0.5M H 2 SO 4 The cyclic voltammetry scan was performed at -0.4~1.5V until a stable and repeatable cyclic voltammetry curve was obtained, and the electrode was washed slowly with ultrapure water, which was marked as Electrode 1.
[0032] Electrode 2: Add 2.5 μL of thiol DNA solution with a concentration of 1 μM to the surface of Electrode 1 dropwise, place in a refrigerator at 4°C for 12 hours, and then treat with 1.0 mM mercaptoethanol (MCH) for 30 minutes (replacing the non-Au-S bond-immobilized capture probe on the surface of the electrode). Needle), ultrapure water buffer wash electrode, labeled Electrode 2.
[0033] Electrode 3: Prepare a TdT reaction solution with a total volume of 5 μL, which contains 3 μL of ultrapure water, 0.5 μL of 5×TdT buffer, 0....
Embodiment 2
[0036] Embodiment 2 Feasibility experiment
[0037] In order to prove that the electrochemical sensor in the present invention can realize the dynamic analysis of TdT detection and its related microenvironment, based on the sensor prepared in Example 1, alternately use the 10mM PB solution of pH 7.0 and pH 5.8 to incubate, and in 5mM [Fe ( EN) 6 ] 3- / 4- EIS measurements were performed in solution. See figure 2 , compared with the absence of TdT, the electrochemical impedance value measured after the sensor was incubated in 10mM PB (pH 7.0) and 10mM PB (pH 5.8) changed significantly, and showed a certain rule (off-on-off). It is proved that the sensor can be used for TdT activity detection and its related microenvironmental dynamic analysis.
Embodiment 3
[0038] Embodiment 3 sensor is used for TdT activity detection
[0039] Based on the sensor prepared in Example 1, in the step of preparing Electrode 3, the concentration of TdT was changed, and the final concentration of TdT was controlled to be: 0, 5×10 -5 , 1×10 -4 , 2×10 -4 , 5×10 -4 , 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 U / mL, a series of electrochemical biosensors were prepared as in Example 1. Experimental results such as image 3 Shown, after 10mMPB solution (pH 7.0) is processed, the greater the TdT concentration, the greater the electrochemical impedance value, the linear correlation equation of the electrochemical impedance value to the logarithmic value of the TdT concentration is Ret=2029lg TdT +10247, R 2 =0.9991, the linear range is 5×10 -5 ~0.1U / mL, the linear correlation equation shows that the detection limit is 2.1×10 -5 U / mL, after 10mM PB solution (pH 5.8) treatment, the greater the TdT concentration, the electrochemical impedance val...
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