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A kind of amino acid dehydrogenase electrode and preparation method and application thereof

An amino acid dehydrogenase and amino acid technology are applied in the field of amino acid dehydrogenase electrodes and their preparation, and achieve the effects of low cost, not easy to fall off, good stability and repeatability

Active Publication Date: 2021-09-17
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 polydopamine is used to prepare amino acid dehydrogenase electrodes

Method used

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  • A kind of amino acid dehydrogenase electrode and preparation method and application thereof
  • A kind of amino acid dehydrogenase electrode and preparation method and application thereof
  • A kind of amino acid dehydrogenase electrode and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (1) amino acid dehydrogenase crude enzyme preparation: according to the original sequence of amino acid dehydrogenase (AaDH) as shown in SEQ ID No.1, design the primers shown in SEQ ID No.2 and SEQ ID No.3, by A commercial company (Shanghai Bioengineering Co., Ltd.) used PCR to synthesize the gene sequence of amino acid dehydrogenase, which was used to connect with the vector pET28a to construct a plasmid.

[0050] SEQ ID No. name Sequence (NdeI and XhoI restriction sites are underlined) SEQ ID No.2 AaDH-F 5'-GGAATTC CATATG TTTGAAAAAATATCACAGCATG-3'

SEQ ID No.3 AaDH-R 5'-CCG CTCGAG ATGTCTAACCTGCCATTTTG-3'

[0051] The connection of the PCR product (gene sequence of amino acid 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 T4 DNA Ligase to obtain the transformation plasmid. The Compe...

Embodiment 2

[0063] (1)-(3) The experimental procedures are as the steps (1)-(3) of Example 1.

[0064] (4) Preparation of enzyme electrode rGO-PDA-AuNPs-AaDH: graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 h. Add 0.5g ascorbic acid to 10mL 1mg / mL GO for reduction for 12h, centrifuge at 12000rpm, remove the supernatant, add 10mL ultrapure water to wash once, centrifuge again, and add 20mL of 0.1M phosphate buffer solution (pH 7.0) to prepare 0.5mg / mL mL of rGO solution. Add 25mg dopamine (DA) and 5mL 2mg / mL AaDH to 10mL 0.5mg / mL rGO solution, add 3.5mM NaAuCl under slow stirring at room temperature 4 Carry out chemical oxidative polymerization and immobilize amino acid dehydrogenase. After 5 hours of reaction, the resulting dispersion is centrifuged at 10,000 rpm, the supernatant is discarded, and the precipitate (rGO-PDA-AuNPs-AaDH) is dispersed into 10 mL of phosphate buffer.

[0065] (5) Take 10 μL...

Embodiment 3

[0069] (1)-(3) The experimental procedures are as the steps (1)-(3) of Example 1.

[0070] (4) Preparation of enzyme electrode rGO-L-DOPA-AuNPs-AaDH: graphene oxide was prepared by the Hummer method, and a uniformly dispersed GO solution was obtained after ultrasonic dispersion for 3 h. Add 0.5 g of ascorbic acid to 1 mg / mL GO for reduction for 12 h, centrifuge at 12,000 rpm, remove the supernatant, add 10 mL of ultrapure water to wash once, and centrifuge again, then add 10 mL of 0.1M Tris-HCl buffer solution (p H 7.5) to prepare 1 mg / mL rGO solution. Add 25 mg levodopa (L-DOPA) + 5 mL 3 mg / mL AaDH to 10 mL of 1 mg / mL rGO solution, add 2.0 mM NaAuCl under slow stirring at room temperature 4 Carry out chemical oxidation polymerization and immobilize amino acid dehydrogenase. After 7 hours of reaction, the obtained dispersion is centrifuged at 10,000 rpm, the supernatant is discarded, and the precipitate (rGO-L-DOPA-AuNPs-AaDH) is dispersed into 10 mL of phosphate buffer mid...

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Abstract

The invention discloses an amino acid dehydrogenase electrode, its preparation method and application. The invention relates to the technical field of biosensors, in particular to an enzyme electrode, an enzyme biosensor and a preparation method and application thereof. The amino acid dehydrogenase electrode includes a base electrode, an electron conductor, an electron mediator, an ionic coenzyme and an amino acid dehydrogenase, which can realize effective immobilization of enzyme molecules and electron transfer between the enzyme and the electrode, and improve the catalytic activity and Sensitivity of the sensor. The amino acid dehydrogenase electrode of the present invention uses conductive polymer polydopamine modified reduced graphene oxide or carbon nanotubes with good biocompatibility as the electrode material, and has the advantages of simple manufacturing method, high detection sensitivity, fast and accurate, stable and repeatable It has the advantage of good performance and has a good application prospect.

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 amino acid 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 analy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/327G01N27/48C12Q1/00
CPCC12Q1/004C12Q1/005G01N27/3271G01N27/48
Inventor 王世珍姚光晓
Owner XIAMEN UNIV
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