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Sensors 2016, 16(10), 1565; doi:10.3390/s16101565

Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters

UCD School of Chemistry, University College Dublin, Belfield, Dublin D04 N2E5, Ireland
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Academic Editor: Alexander Star
Received: 28 July 2016 / Revised: 7 September 2016 / Accepted: 18 September 2016 / Published: 23 September 2016
(This article belongs to the Section Biosensors)
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Abstract

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/PEI2/GluOx5/PEGDE, displayed good sensitivity (LOD < 0.2 μM), response time (t90% < 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. View Full-Text
Keywords: glutamate biosensor stability; biomedical applications; amperometry; surface enzyme loading and affinity; permselective polymer; poly(ortho-phenylenediamine) glutamate biosensor stability; biomedical applications; amperometry; surface enzyme loading and affinity; permselective polymer; poly(ortho-phenylenediamine)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Ford, R.; Quinn, S.J.; O’Neill, R.D. Characterization of Biosensors Based on Recombinant Glutamate Oxidase: Comparison of Crosslinking Agents in Terms of Enzyme Loading and Efficiency Parameters. Sensors 2016, 16, 1565.

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