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Malate dehydrogenase electrode as well as preparation method and application thereof

A malate dehydrogenase and electrode technology, applied in the field of detection and analysis, can solve the problems of slow electron transfer rate, difficult regeneration, unsatisfactory kinetic characteristics, etc.

Active Publication Date: 2020-11-06
BIOLOGY INST OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the inventors have found that the oxidation overpotential of NADH is high, the electron transfer rate is too slow, and the kinetic characteristics are not ideal. During the electrochemical oxidation process, dimers will be deposited on the electrode surface to passivate the electrode, thereby making the NADH Direct measurement and regeneration of NAD+ become very difficult, redox regeneration of NAD+ based on electron mediator is an effective regeneration method, which can reduce the oxidation potential of NADH, Improve the electron transfer rate on the electrode surface and realize NAD+ regeneration during the use of the dehydrogenase electrode

Method used

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  • Malate dehydrogenase electrode as well as preparation method and application thereof
  • Malate dehydrogenase electrode as well as preparation method and application thereof
  • Malate dehydrogenase electrode as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0175] Embodiment 1 Chitosan-NAD + Preparation of complex

[0176] Take NAD + Add 1 mL of iodoacetic acid (1 mg / mL) to 5 mL (1 mg / mL) of the aqueous solution, and react in a water bath at 70 °C for 1 h. In the above NAD + Add 1 mL (1.3 mM) of sodium thiosulfate solution to the aqueous solution, adjust the pH to 11 with 1 M NaOH, and react in a water bath at 70°C for 1 h. After that, 1 mL of formaldehyde was added to react in a 70°C water bath for 1 h. Use 1M HCl to adjust the pH of the reaction system to be neutral, add NHS (10mg), EDC (10mg), add 0.1% chitosan solution 10mL [chitosan is medium viscosity chitosan (200-400mPa.s)], React in a water bath at 70°C for 1h. Carry out UV absorption full-wavelength scanning test for key steps.

[0177] Chitosan-NAD + The UV absorption wavelength results during the preparation of the composite are as follows figure 1 As shown, we mainly use NAD + Covalent linkage with chitosan with good water solubility and good film-forming pr...

Embodiment 2

[0177] Chitosan-NAD + The UV absorption wavelength results during the preparation of the composite are as follows figure 1 As shown, we mainly use NAD + Covalent linkage with chitosan with good water solubility and good film-forming properties to realize NAD + Immobilized. NAD + Starting with NAD + It exhibits the largest UV absorption peak at 260nm ( figure 1 -I). Firstly, under acidic environment, iodoacetic acid was used as alkylating agent for NAD + Alkylation of the 1-position nitrogen atom of adenine to give N1-carboxymethyl-NAD + . with NAD + Compared to N1-carboxymethyl-NAD + There is a blue-shifted peak, showing the maximum UV absorption peak at 250nm ( figure 1 -II). Using sodium thiosulfate as reducing agent for N1-carboxymethyl-NAD + Reduction is carried out to obtain N1-carboxymethyl-NADH which is more stable under alkaline conditions. Then carry out Dimroth rearrangement under the condition of strong base to obtain the C6-carboxymethyl NADH whose am...

Embodiment 3

[0191] Embodiment 3 The preparation method of glucose dehydrogenase electrode

[0192] (1) Preparation of glucose dehydrogenase electrode

[0193] All electrochemical measurements were performed in a typical three-electrode system (CHI 760D, CH Instruments). The composition of the three-electrode system includes: a platinum electrode as a counter electrode, an Ag / AgCl electrode as a reference electrode, and a glassy carbon electrode as a working electrode. During the experiment, the glassy carbon electrode with a diameter of 3mm was sequentially filled with Al with a certain particle size 2 o 3 The slurry was polished to a mirror surface on a polishing cloth. After each polishing, the surface dirt was washed off first, and then moved into an ultrasonic water bath for cleaning, each time for 1 min, and repeated three times. Finally, 1:1 ethanol, 1:1 NHO 3 Ultrasonic cleaning. After thorough washing, in 0.20mol / L KNO 3 Medium record 1×10 -3 mol / L K 3 Fe(CN) 6 The cyclic ...

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Abstract

The invention provides a malate dehydrogenase electrode as well as a preparation method and application thereof. The method comprises the following steps: chemically modifying a coenzyme factor smallmolecule NAD < + > non-active part through a chemical means, covalently connecting the chemically modified NAD < + > with a chitosan carrier to obtain an NAD < + >- chitosan compound, modifying a carbon nanotube on the surface of a substrate electrode, and detecting NADH by using the carbon nanotube as a substrate material; electrically depositing ABTS on the electrode, and achieving NAD <+> in-situ regeneration by using the ABTS as an electron mediator; dropwise adding an NAD < + >- chitosan compound to realize fixation of NAD < + > on the surface of the electrode; connecting dehydrogenase tothe chitosan carrier through glutaraldehyde cross-linking action to obtain a dehydrogenase electrode; the immobilization and regeneration method of NAD < + > can be combined with different types of dehydrogenases to prepare various dehydrogenase electrodes / biosensors. The method has wide practical application value in the field of biosensor preparation.

Description

[0001] This application is a divisional application with application number 2020101521251, application date of March 6, 2020, and the title of the invention "a coenzyme factor complex, enzyme electrode, enzyme sensor and its preparation method and application". technical field [0002] The invention belongs to the technical field of detection and analysis, and in particular relates to a malate dehydrogenase electrode and its preparation method and application. Background technique [0003] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0004] Oxidoreductases are the most abundant types of enzymes in nature. Among the six types of enzymes, 30% to 35% are oxidoreductases. Oxidase-dependent biosensors are matu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/30C12Q1/32C12Q1/00
CPCG01N27/3272G01N27/3278G01N27/308C12Q1/32C12Q1/001C12Q1/005Y02E60/50
Inventor 马耀宏郑岚孟庆军马恒张云娟杨艳杨俊慧
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI
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