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A preparation method and application of an electrochemical biosensor for simultaneous detection of exo I and TdT

A biosensor and electrochemical technology, applied in the field of functional biomaterials and biosensing, can solve the problems of inability to form AgNCs, signal drop, and influence on electrochemical signal output, and achieve good application prospects, easy operation, and fast detection speed Effect

Active Publication Date: 2021-02-09
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, before TdT plays the role of tool enzyme, introducing Exo I into the system can hydrolyze the sulfhydryl DNA immobilized on the surface of the gold electrode, but cannot form AgNCs, resulting in a decrease in signal, and the change of Exo I concentration affects the output of electrochemical signals

Method used

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  • A preparation method and application of an electrochemical biosensor for simultaneous detection of exo I and TdT
  • A preparation method and application of an electrochemical biosensor for simultaneous detection of exo I and TdT
  • A preparation method and application of an electrochemical biosensor for simultaneous detection of exo I and TdT

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Preparation of sensor

[0039] (1) The specific preparation steps of the electrochemical biosensor are as follows:

[0040] Preparation of Electrode 1:

[0041] First, the gold electrode (2 mm in diameter) was polished on the suede with Al2O3 powder for 5 min, and after polishing, the electrode was placed in an ultrasonic cleaner and ultrasonically cleaned with twice distilled water for 5 min, and then washed with N 2 Blow dry, labeled Electrode 1.

[0042] Preparation of Electrode 2:

[0043] Take mercapto DNA solution (2.5 μL, 10 μM), drop-coat it on the surface of Electrode 1, incubate overnight in a refrigerator at 4°C, wash the electrode slowly with distilled water, treat it with 1.0 mM mercaptohexanol (MCH) for 30 min, and replace the non-Au-S on the surface of the electrode. Bond immobilized thiol DNA, wash the electrode slowly with distilled water, labeled as Electrode 2.

[0044] Preparation of Electrode 3:

[0045] On the electrode surface of...

Embodiment 2

[0049] Embodiment 2 Feasibility experiment

[0050] In the preparation process of Electrode 3 in Example 1, before using TdT extension, introduce Exo I solution (final concentration is 500U / mL), incubate at 37°C for 30min for sensor preparation, and then prepare the sensor as in step (1), Used to detect the electrochemical response of ExoI. At the same time, the electrochemical response of TdT was detected by using the sensor prepared in Example 1.

[0051] Using square wave voltammetry, set the potential range to 0-0.3V and the amplitude to 25mV. The result is as figure 2 , 3 Shown: When there is no Exo I, the electrochemical biosensor has an obvious response signal, but in the presence of Exo I, the electrochemical biosensor has almost no response signal in PBS (0.1M, pH 7.0) (such as figure 2 ). It is proved that the sensor can be used for Exo I activity detection. On the contrary, when there is TdT, the electrochemical biosensor has obvious electrochemical response...

Embodiment 3

[0052] Example 3 Detection of different concentrations of Exo I activity

[0053] In the process of preparing the Exo I electrochemical sensor, change the Exo I concentration (the control final concentration is respectively: 0, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, 1000U / mL). Experimental results such as Figure 4 As shown, the linear range of the current response of the sensor to the logarithmic value of Exo I concentration is 0.1~500U / mL, and the linear correlation equation is y=-7.12lgC Exo I +20.4, R 2 =0.9951, the detection limit is 0.05U / mL, indicating that the sensor can realize highly sensitive detection of Exo I activity.

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Abstract

The invention discloses a preparation method of an electrochemical biosensor for simultaneously detecting Exo I and TdT. Sulfhydryl DNA and a gold electrode surface form an Au-S covalent bond spontaneously by a mercapto part and the sulfhydryl DNA is fixed on the gold electrode surface. Polymerization of the 3'-OH tail of the TdT catalytic sulfydryl DNA and dCTP is carried out and a generated rich-C DNA long chain can used for forming AgNCs; and the TdT activity is detected by a dissolved voltammetric signal of Ag. Before TdT polymerization, Exo I is introduced and the sulfhydryl DNA can be hydrolyzed, so that the formation of AgNCs is affected finally; and thus the ExoI activity is detected. The electrochemical signal intensity can be affected by changing the ExoI or TdT concentration; and on the basis of the principle, a high-efficiency electrochemical biosensor for simultaneously detecting Exo I and TdT is prepared.

Description

technical field [0001] The invention relates to an electrochemical biosensor, in particular to a preparation method and application of an electrochemical biosensor capable of simultaneously detecting Exo I and TdT, and belongs to the technical field of functional biomaterials and biosensing. Background technique [0002] Biosensor is a high-tech that has grown through the interpenetration of various disciplines such as biology, chemistry, physics, medicine, and electronic technology. Electrochemical biosensing technology is a kind of biosensor. It is a new type of detection technology developed by combining the specific recognition of biomolecules with high-sensitivity sensing technology. It has the characteristics of fast, sensitive, and easy operation. It has been widely used in various clinical fields. Compared with traditional detection technologies, electrochemical biosensing technology has the following advantages: high sensitivity and high precision, which can realiz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/327
Inventor 胡宇芳胡丹丹张青青马少华王邃郭智勇
Owner NINGBO UNIV
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