Method for detecting enzyme intermediates in real time by use of electrochemical biosensor

An intermediate and enzyme detection technology, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., can solve the problems of immature sensor applications

Inactive Publication Date: 2012-08-15
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, biosensors have been developed to the third generation, that is, biosensors without electronic mediators, but the application of such sensors is still immature

Method used

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  • Method for detecting enzyme intermediates in real time by use of electrochemical biosensor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Before the modification, the surface of the glassy carbon electrode was polished and polished with 0.05 mm of alumina paste covered on the emery cloth, and then the glassy carbon electrode was ultrasonically cleaned with acetone, 50% NaOH solution, 50% nitric acid, and double distilled water, respectively. Dry at room temperature.

[0026] Disperse 1 mg of activated multi-walled carbon nanotubes in 10 mL of N,N-dimethylformamide and vibrate ultrasonically to obtain a black suspension. 10 mL of the suspension was dropped on the surface of the glassy carbon electrode, and the solvent was evaporated under an infrared lamp to prepare an HRP-modified electrode.

[0027] Dissolve 2.5mg 300U / mL HRP and 4mg BSA in 0.2mL of KH 2 PO 4 -Na 2 HPO4 A mixed solution was formed in phosphate buffer (0.05mol / L, pH 7.0). Then 20 μL of the mixture was dropped onto the surface of the carbon nanotube-modified glassy carbon electrode. The electrodes were then placed in a closed containe...

Embodiment 2

[0029] Cyclic voltammetry experiments were performed with an electrochemical workstation and a conventional three-electrode system. Among them, modified glassy carbon electrode was used as working electrode; platinum wire was used as counter electrode; saturated calomel electrode (SCE) was used as reference electrode; PBS (0.05mol / L, pH 7.0) was used as supporting electrolyte. The electrolyte was degassed with nitrogen for 30 min before the test, and the electrochemical reactor was protected with nitrogen during the experiment.

[0030] The potential scanning range (relative to SCE) of the cyclic voltammetry experiment is -0.4~0.2V, and the scanning rate is 100mV / s. Experiments were performed at room temperature.

Embodiment 3

[0032] Experiments investigating the effect of substrates were performed in a reaction cell filled with 20 mL of supporting electrolyte. Under stirring, 1 mmol / L hydroquinone was added, and the baseline of the initial current was recorded at -0.25 V. Then, add 0.1 mL of 0.1 mol / L H at intervals of about 50 s 2 o 2 solution, a current-time curve showing a step-up trend was obtained.

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Abstract

The invention discloses a process method for detecting enzyme intermediates in real time by use of an electrochemical biosensor. The process method is realized by continuous measurement of a reduction current on sensor poles relevant to the intermediates. In an H2O2 redox reaction system catalyzed by an immobilized horseradish peroxidase, quantitative relationship is present between electron transfer and quantity of enzyme intermediates during the reaction process. Through examination of influence on enzymatic activity by a substrate, an inhibitor and both the substrate and the inhibitor, the method is proved to track the change of the enzymatic activity and the relative amount of the intermediates in real time. Compared with the traditional intermediate detection method, the method provided by the invention has the characteristics of high sensitivity and low cost, can be used as the standard for transient dynamics detection and can be further applied in laboratories, medical tests and industries.

Description

technical field [0001] The invention relates to a method for detecting enzyme intermediates. Specifically, it involves the use of electrochemical biosensors for real-time measurement of the relative amounts of enzyme-substrate complex intermediates and their variation with substrate and inhibitor concentrations. Background technique [0002] Enzyme activity detection is a basic skill in biochemistry and the foundation of medical diagnosis, drug design, food engineering, environmental monitoring and other fields. Traditional detection methods include liquid chromatography, ninhydrin chromogenic method, Nessler ammonia determination method, etc. These detections are a kind of final state measurement, and cannot continuously track the changes of substances during the reaction. Although methods such as ultraviolet spectroscopy and kinetic analysis can be used for continuous tracking, and the monitoring object is either the consumption of the substrate or the accumulation of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327
Inventor 黄积涛黄薇王缇缇邢达杰
Owner NANKAI UNIV
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