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Electrochemical method for detecting activity of uracil-DNA glycosylase based on DNA NANOTREE

A glycosylase activity, electrochemical technology, applied in the field of functional biomaterials and biosensing, can solve the problems of electrode interface electron transfer barrier, electrochemical impedance signal amplification, etc., achieve good application prospects, easy operation, accurate results Effect

Active Publication Date: 2020-01-10
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the biocatalysis of G4 / hemin, 3,3-diaminobenzidine (DAB) was converted to hydrogen peroxide (H 2 o 2 ) oxidation to form a non-conductive insoluble precipitate (IP), which looks like a DNANANOTREE, at which point the electron transfer between the electrode interface and the redox probe is greatly hindered, resulting in a significant amplification of the electrochemical impedance signal
At present, there is no report on this kind of electrochemical method based on DNA NANOTREE to detect the activity of uracil-DNA glycosylase at home and abroad.

Method used

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  • Electrochemical method for detecting activity of uracil-DNA glycosylase based on DNA NANOTREE
  • Electrochemical method for detecting activity of uracil-DNA glycosylase based on DNA NANOTREE
  • Electrochemical method for detecting activity of uracil-DNA glycosylase based on DNA NANOTREE

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Experimental program
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Effect test

Embodiment 1

[0027] The preparation of embodiment 1 sensor

[0028] (1) Au: Put the bare gold electrode on the polishing cloth (containing Al with a particle size of 0.05 μm 2 o 3 suspension) and polished in water for 5 min, then buffered and washed three times with distilled water to obtain Au.

[0029] (2) Electrode 1:

[0030] ① Drop H0 (5 μL, 0.05 μM) onto the electrode surface, assemble overnight, and wash slowly with distilled water. Then soak the electrode in mercaptohexanol (MCH) (1mM) aqueous solution to assemble to seal the unassembled area on the surface of the gold electrode to reduce non-specific adsorption of other biomolecules, and wash slowly with distilled water; ② Prepare 2.5 μL UDG reaction system (UDG reaction buffer, 0.05μM DNA1 and 1U / mL UDG, incubate at 37°C for 1h), then modify the reaction solution on the above-mentioned ① electrode, incubate at 37°C for 30min, wash slowly with distilled water; ③Prepare HCR reaction solution: 1μL 10×Tris reaction buffer, hairpi...

Embodiment 2

[0036] Example 2 UDG activity detection

[0037] In the process of preparing the sensor, in order to study whether the sensor can be applied to the detection of UDG activity, the electrochemical impedance response of the sensor prepared with and without UDG was studied during the sensor preparation process. The results are as follows: figure 2 , proving that the sensor can be used to detect UDG activity.

[0038] Subsequently, during the sensor preparation process, different concentrations of UDG (0, 0.0001, 0.0004, 0.001, 0.004, 0.01, 0.04, 0.1, 0.4, 1, 4, 10 U / mL) were added to detect the electrochemical impedance of the prepared sensor response, the result is as image 3 , between 0.0001~4U / mL, there is a good linear relationship between the AC impedance intensity and the logarithm of the UDG concentration, and the linear equation is y=9366lgC UDG +38860,R 2 =0.9924, the detection limit is 0.00003U / mL, which shows that the sensor can realize highly sensitive detection o...

Embodiment 3

[0039] Example 3 Detection of UDG inhibitor UGI

[0040] In order to prove that the established method can be used for the screening of UDG inhibitors, the UGI model was selected, Figure 4 Described are the AC impedance response curves of the sensor with different UGI concentrations (0, 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2U / mL). As shown in the figure, as the concentration of UGI increases, the electrochemical signal gradually weakens. When the concentration of UGI (1U / mL) is equivalent to that of UDG (1U / mL), the electrochemical impedance signal reaches a plateau, which indicates that the activity of UDG is suppressed by UGI. Effective suppression. From the above experimental results, it can be speculated that the analytical method we established has potential application value for screening UDG inhibitors.

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Abstract

The invention discloses an electrochemical method for detecting the activity of uracil-DNA glycosylase based on a DNA NANOTREE. The method comprises the following specific steps of performing Au treatment, modifying Au with H0, opening a hairpin structure of chain initiation by utilizing the UDG, initiating HCR amplification along with the introduction of single-stranded DNA1, using TdT to catalyze extension of 3'-OH of H1 and H2, forming a G4 structure under the action of Pb<2+>, inserting hemin into the G4 structure, and under the biocatalytic effect of G4 / hemin, oxidizing 3,3-diaminobenzidine (DAB) by hydrogen peroxide (H2O2) to form non-conductive IP. As a result, electron transfer between an electrode interface and a redox probe is greatly impeded, thereby resulting in significant amplification of an electrochemical impedance signal. In a sensor preparation process, the UDG concentration and the inhibitor UGI concentration are changed, and the influences of a series of prepared sensors on the electrochemical impedance signal are explored. The method has the advantages of high sensitivity, high detection speed, accurate and reliable result and low cost.

Description

technical field [0001] The invention relates to an electrochemical impedance sensor and a detection method thereof, in particular to an electrochemical method for detecting uracil-DNA glycosylase activity based on DNANANOTREE, which belongs to the technical field of functional biological materials and biosensing. Background technique [0002] The integrity and accuracy of the genome is of paramount importance to all living things. However, many environmental factors in everyday life can cause irreversible damage to DNA, such as radiation and toxic chemicals. A common damage to deaminated cytosines can cause damage to dU, which is misincorporated during DNA replication. If not repaired, permanent genetic mutations may result. Uracil-DNA glycosylase (UDG) is an indispensable DNA damage repair enzyme in uracil-induced lesions, which can flip uracil by catalyzing the breaking of the N-glycosidic bond between uracil and deoxyribose, exposing apurinic / The apyrimidinic (AP) sit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/12
CPCG01N27/3276G01N27/125
Inventor 胡宇芳张青青胡丹丹詹甜玉王邃郭智勇
Owner NINGBO UNIV
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