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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

Active Publication Date: 2020-10-16
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no related reports on the dynamic monitoring of TdT activity and the screening of small molecule inhibitors in different microenvironments based on i-motif impedance switches at home and abroad

Method used

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  • Construction and application of terminal transferase activity dynamic analysis method based on alternating-current impedance technology
  • Construction and application of terminal transferase activity dynamic analysis method based on alternating-current impedance technology
  • Construction and application of terminal transferase activity dynamic analysis method based on alternating-current impedance technology

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses construction and application of a terminal transferase activity analysis method based on an alternating-current impedance technology, which comprises the following steps of: introducing cytidine triphosphate (dCTP) into the surface of a gold electrode modified by 5 '-sulfydryl DNA by using TdT to form a C-rich DNA long chain; the DNA long chain is kept in an irregular single-chain state under the condition that the pH value is 7.0; a relatively large electrochemical impedance value is obtained; on the contrary, under the condition that the pH value is 5.8, due to the fact that C is partially protonated, a stable i-motif structure can be formed; a small electrochemical impedance value (semi-protonated C-rich DNA in i-motif is beneficial to electron conduction) is obtained, a very meaningful electrochemical impedance switch is formed, the electrochemical impedance value depends on the activity of TdT, and based on this, dynamic analysis of the activity of TdT in different microenvironments and screening of small-molecule inhibitors of TdT are realized.

Description

technical field [0001] The invention relates to the dynamic analysis of terminal transferase activity, in particular to the construction of an electrochemical analysis method for terminal transferase activity based on AC impedance technology, and belongs to the technical fields of functional biological materials and biosensing. Background technique [0002] As we all know, terminal deoxynucleotidyl transferase (TdT) is a special DNA polymerase that can repeatedly add deoxyribonucleotides (dNTP) at the 3'-OH end of DNA containing three or more nucleotides , forming a special DNA long chain. TdT expression is suppressed in most human cells, and it is only expressed on immature lymphocytes and acute lymphoblastic leukemia cells, so TdT can be used as a clinical diagnostic marker for such diseases. It can be seen that the level of TdT activity is of great significance for exploring TdT-related pathological functions and disease diagnosis and treatment. Conventional TdT activit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/48G01N27/04
CPCG01N27/3277G01N27/48G01N27/041
Inventor 胡宇芳胡丹丹郭智勇王邃
Owner NINGBO UNIV
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