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Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters
Amperometric l-glutamate (Glu) biosensors, based on both wild-type and a recombinant form of l-glutamate oxidase (GluOx), were designed and characterized in terms of enzyme-kinetic, sensitivity and stability parameters in attempts to fabricate a real-time Glu monitoring device suitable for future lo...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087354/ https://www.ncbi.nlm.nih.gov/pubmed/27669257 http://dx.doi.org/10.3390/s16101565 |
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author | Ford, Rochelle Quinn, Susan J. O’Neill, Robert D. |
author_facet | Ford, Rochelle Quinn, Susan J. O’Neill, Robert D. |
author_sort | Ford, Rochelle |
collection | PubMed |
description | Amperometric l-glutamate (Glu) biosensors, based on both wild-type and a recombinant form of l-glutamate oxidase (GluOx), were designed and characterized in terms of enzyme-kinetic, sensitivity and stability parameters in attempts to fabricate a real-time Glu monitoring device suitable for future long-term detection of this amino acid in biological and other complex media. A comparison of the enzyme from these two sources showed that they were similar in terms of biosensor performance. Optimization of the loading of the polycationic stabilization agent, polyethyleneimine (PEI), was established before investigating a range of crosslinking agents under different conditions: glutaraldehyde (GA), polyethylene glycol (PEG), and polyethylene glycol diglycidyl ether (PEGDE). Whereas PEI-free biosensor designs lost most of their meager Glu sensitivity after one or two days, configurations with a 2:5 ratio of dip-evaporation applications of PEI(1%):GluOx(400 U/mL) displayed a 20-fold increase in their initial sensitivity, and a decay half-life extended to 10 days. All the crosslinkers studied had no effect on initial Glu sensitivity, but enhanced biosensor stability, provided the crosslinking procedure was carried out under well-defined conditions. The resulting biosensor design based on the recombinant enzyme deposited on a permselective layer of poly-(ortho-phenylenediamine), PoPD/PEI(2)/GluOx(5)/PEGDE, displayed good sensitivity (LOD < 0.2 μM), response time (t(90%) < 1 s) and stability over a 90-day period, making it an attractive candidate for future long-term monitoring of Glu concentration dynamics in complex media. |
format | Online Article Text |
id | pubmed-5087354 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-50873542016-11-07 Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters Ford, Rochelle Quinn, Susan J. O’Neill, Robert D. Sensors (Basel) Article Amperometric l-glutamate (Glu) biosensors, based on both wild-type and a recombinant form of l-glutamate oxidase (GluOx), were designed and characterized in terms of enzyme-kinetic, sensitivity and stability parameters in attempts to fabricate a real-time Glu monitoring device suitable for future long-term detection of this amino acid in biological and other complex media. A comparison of the enzyme from these two sources showed that they were similar in terms of biosensor performance. Optimization of the loading of the polycationic stabilization agent, polyethyleneimine (PEI), was established before investigating a range of crosslinking agents under different conditions: glutaraldehyde (GA), polyethylene glycol (PEG), and polyethylene glycol diglycidyl ether (PEGDE). Whereas PEI-free biosensor designs lost most of their meager Glu sensitivity after one or two days, configurations with a 2:5 ratio of dip-evaporation applications of PEI(1%):GluOx(400 U/mL) displayed a 20-fold increase in their initial sensitivity, and a decay half-life extended to 10 days. All the crosslinkers studied had no effect on initial Glu sensitivity, but enhanced biosensor stability, provided the crosslinking procedure was carried out under well-defined conditions. The resulting biosensor design based on the recombinant enzyme deposited on a permselective layer of poly-(ortho-phenylenediamine), PoPD/PEI(2)/GluOx(5)/PEGDE, displayed good sensitivity (LOD < 0.2 μM), response time (t(90%) < 1 s) and stability over a 90-day period, making it an attractive candidate for future long-term monitoring of Glu concentration dynamics in complex media. MDPI 2016-09-23 /pmc/articles/PMC5087354/ /pubmed/27669257 http://dx.doi.org/10.3390/s16101565 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Ford, Rochelle Quinn, Susan J. O’Neill, Robert D. Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters |
title | Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters |
title_full | Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters |
title_fullStr | Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters |
title_full_unstemmed | Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters |
title_short | Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters |
title_sort | characterization of biosensors based on recombinant glutamate oxidase: comparison of crosslinking agents in terms of enzyme loading and efficiency parameters |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087354/ https://www.ncbi.nlm.nih.gov/pubmed/27669257 http://dx.doi.org/10.3390/s16101565 |
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