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Amine dehydrogenase electrode and preparation method and application thereof

An amine dehydrogenase and electrode technology, applied in the field of biosensors, to achieve superior electronic performance, good stability and repeatability, and good chemical modification performance

Active Publication Date: 2019-05-28
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But up to now, there is no report that polyethyleneimine is used to prepare amine dehydrogenase electrodes.

Method used

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  • Amine dehydrogenase electrode and preparation method and application thereof
  • Amine dehydrogenase electrode and preparation method and application thereof
  • Amine dehydrogenase electrode and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031](1) Preparation of crude enzyme of amine dehydrogenase: according to the original sequence of amine dehydrogenase shown in SEQ ID NO.1, primers shown in SEQ ID NO.2 and SEQ ID NO.3 were designed, provided by a commercial company ( Shanghai Bioengineering Co., Ltd.) used the PCR method to synthesize the gene sequence of amine dehydrogenase, which was used to connect with the vector pET28a to construct a plasmid.

[0032] Ligation of the PCR product (the gene sequence of amine dehydrogenase) and the vector pET28a: the PCR product and the vector pET28a were double digested with NdeI and XhoI, after purification, they were reacted overnight at 4°C under the action of T4DNA ligase to obtain the transformation plasmid. Competent cells were prepared using the Competent Cell Preparation Kit. Add 10ng of transformation plasmid to the competent cells, mix gently, place on ice for 30 minutes, place in a water bath at 42°C for 45 seconds, and immediately place on ice for 1 to 2 minu...

Embodiment 2

[0043] (1)-(3) The experimental procedures are as the steps (1)-(3) of Example 1.

[0044] (4) Preparation of enzyme electrode modification material rGO-PEI: graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 h. Under stirring, 100 mL of water, 200 mg of KOH and 300 mg of 187KDa branched PEI were sequentially added, stirred evenly, and 4.6 mL of 4.9 mg / mL GO solution was slowly added. After heating (80°C) to reflux for 24 hours, add 8 mL of 2mol / L sodium borohydride for reduction, and continue the reaction for 2 hours. After the reaction was completed, rGO-PEI was obtained by centrifugation and drying, and a 0.1 mg / mL rGO-PEI solution was prepared.

[0045] (5) Preparation of rGO-PEI-AmDH: Add the amine dehydrogenase solution obtained in step (2) with an enzyme concentration of 10 mg / ml to the amine dehydrogenase solution obtained in step 4) at a volume ratio of 1:1 at 4°C 0.1 mg / mL rGO-PEI s...

Embodiment 3

[0050] (1)-(3) The experimental procedures are as the steps (1)-(3) of Example 1.

[0051] (4) Preparation of enzyme electrode modification material rGO-PEI: graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 hours. Under stirring, 7 mL of water, 100 mg of KOH and 200 mg of PEI were sequentially added, stirred evenly, and 4.6 mL of 4.9 mg / mL GO solution was slowly added. After heating (80°C) to reflux for 24 hours, add 8 mL of 2mol / L sodium borohydride for reduction, and continue the reaction for 2 hours. After the reaction was completed, rGO-PEI was obtained by centrifugation and drying, and a 1 mg / ml rGO-PEI solution was prepared. The polyethyleneimine includes linear PEI with a molecular weight range of 217KDa, and its concentration is 50mg / ml;

[0052] (5) Preparation of rGO-PEI-AmDH: Add the amine dehydrogenase solution with an enzyme concentration of 5 mg / mL obtained in step (2) to the ...

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Abstract

The invention discloses an amine dehydrogenase electrode and a preparation method and application thereof, and relates to the technical field of biosensors, in particular to an enzyme electrode, an enzyme biosensor and a preparation method and application of the electrode. The electrode comprises a substrate electrode, an electron conducting layer and an amine dehydrogenase layer, the effective immobilization of enzyme molecules and the electron transfer between an enzyme and the electrode can be achieved, and the catalytic activity of the enzyme molecules and the sensitivity of the sensor areimproved. With conductive polymer polyethyleneimine-modified graphene and carbon nanotubes with good biocompatibility as an electrode material, the enzyme electrode has the advantages of simple manufacturing method, high detection sensitivity, quickness, accuracy and good stability and repeatability, and has good application prospects.

Description

technical field [0001] The invention relates to the technical field of biosensors, and relates to an enzyme electrode, an enzyme biosensor and its preparation method and application, in particular to an amine dehydrogenase electrode and its preparation method and application. Background technique [0002] Biosensors have broad application prospects in the fields of biomedical testing, disease diagnosis and treatment, food analysis, environmental monitoring, industrial process detection and control, poison detection, and war biochemical early warning. The bottleneck of the industrialization of biosensing technology is the manufacturing technology of key devices and analysis systems. Focusing on enzyme molecular devices, innovative design in molecular design, device assembly, analysis model construction, etc., and application demonstration in key fields such as medical care, environment, and industry are the research hotspots of biosensors. [0003] At present, the analysis m...

Claims

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Application Information

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IPC IPC(8): G01N27/30G01N27/327G01N27/48C12Q1/00B82Y30/00B82Y5/00B82Y15/00
Inventor 王世珍姚光晓
Owner XIAMEN UNIV
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