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Preparation method of electrochemical biosensor for simultaneously detecting Exo I and TdT and application thereof

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

Active Publication Date: 2019-06-07
NINGBO UNIV
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  • Abstract
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  • 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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  • Preparation method of electrochemical biosensor for simultaneously detecting Exo I and TdT and application thereof
  • Preparation method of electrochemical biosensor for simultaneously detecting Exo I and TdT and application thereof
  • Preparation method of electrochemical biosensor for simultaneously detecting Exo I and TdT and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of sensor

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

[0040] Preparation of Electrode 1:

[0041] First, polish the gold electrode (diameter 2mm) on the suede with aluminum oxide powder for 5 minutes. After polishing, place the electrode in an ultrasonic cleaner and ultrasonically clean it with double distilled water for 5 minutes, and then use N 2 Blow dry and label Electrode 1.

[0042] Preparation of Electrode 2:

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

[0044] Preparation of Electrode 3:

[0045] On the surface of Electrode 2 electrode, add 5.0μL TdT reaction solution (c...

Embodiment 2

[0049] Example 2 Feasibility experiment

[0050] In the preparation process of Electrode 3 in Example 1, before using TdT for extension, introduce Exo I solution (final concentration of 500U / mL), incubate at 37°C for 30 minutes 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 sensor prepared as in Example 1 was used to detect the electrochemical response of TdT.

[0051] Using square wave voltammetry, set the potential range from 0 to 0.3V and the amplitude to 25mV. The result is figure 2 , 3 Shown: when there is no Exo I, the electrochemical biosensor has an obvious response signal, and when Exo I is present, 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 an obvious electrochemical response...

Embodiment 3

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

[0053] In the process of preparing Exo I electrochemical sensor, change the Exo I concentration (control the final concentration respectively: 0, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, 1000U / mL). The experimental results are as Figure 4 As shown, the linear range of the sensor's current response to the log 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 achieve 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 for detecting Exo I and TdT simultaneously, and belongs to the technical field of functional biomaterials and biosensing. Background technique [0002] Biosensor is a high-tech developed by the mutual penetration of biology, chemistry, physics, medicine, electronic technology and other disciplines. Electrochemical biosensing technology is a type of biosensor. It is a new type of detection technology developed by combining the specific recognition of biomolecules with highly sensitive sensing technology. It has the characteristics of fast, sensitive, and easy to operate. It has been widely used in various clinical fields. Compared with traditional detection technology, electrochemical biosensing technology has the following advantages: high sensitivity, high precision, can realize trace analysis of biological target mo...

Claims

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Application Information

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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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