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- Collins, A., et al.
(författare)
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Interference elimination in glutamate monitoring with chip integrated enzyme microreactors
- 2001
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Ingår i: Electroanalysis. - 1040-0397. ; 13:6, s. 425-431
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Tidskriftsartikel (refereegranskat)abstract
- On-chip enzyme reactors are often used in medical/pharmaceutical analysis due to their inherent advantages, such as high sample throughput, low reagent consumption, stability, reproducibility and low cost. The present work describes a different application of such microreactors, namely, elimination of interfering ascorbate signals in glutamate monitoring using ascorbate oxidase modified silicon chip microreactors of different sizes (5.3 and 0.95 muL). Glutamate was monitored with a previously developed redox hydrogel integrated bienzyme electrode, based on coupled glutamate oxidase and horseradish peroxidase, inserted in a miniaturized flow cell operated at -50 mV (vs. Ag/AgCl). The developed on-line analysis system was characterized with regard to dilution effects, detection limit, response time and interference ability using model solutions and real samples. Off-line in vivo glutamate measurements could be made by injecting rat brain microdialysate samples collected before and after KCl stimulation without any interference of ascorbate, I Within the studied flow rate range (2-25 muL/min), 1 mM and 200 muM ascorbate could he totally eliminated using the larger and the mailer microreactor, respectively.
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- Mikeladze, E, et al.
(författare)
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Characterization of a glutamate biosensor based on a novel glutamate oxidase integrated into a redox hydrogel
- 2002
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Ingår i: Electroanalysis. - 1040-0397. ; 14:15-16, s. 1052-1059
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Tidskriftsartikel (refereegranskat)abstract
- A newly isolated and purified glutamate oxidase was used to construct amperometric biosensors for glutamate monitoring. The enzyme-producing microorganism belongs to the genus of Streptomyces - Streptomyces sp. Z-11-6 - and is capable of producing extracellular L-glutamate oxidase. The microorganism was obtained by mutagenesis with HNo2 from a parent strain isolated from soil of Lypetskaya region, Russia, followed by conventional screening methods. The developed biosensor is based on a coupled enzyme system (glutamate oxidase and horseradish peroxidase), the enzymes being crosslinked to a redox polymer (PVI19-dmeOs) using poly(ethylene glycol) diglycidyl ether as crosslinker. ne characteristics of the obtained biosensors were compared with those obtained for similarly constructed electrodes based on a commercially available glutamate oxidase from Yamasa Corp., Japan. The biosensors were operated at - 50 mV (vs. Ag/AgCl 0.1 M KCl) in a flow injection system. Special attention has been focused on the selectivity of the biosensors, evaluating their responses in the presence of several potentially interfering substances, possibly present in brain microdialysates (ascorbic acid, aspartate, cysteine, dopamine, DOPAC, GABA, glutamine, glycine, 5-HIAA, HMPG, HVA, serotonin, tryptophan, tyrosine and uric acid). The biosensors based on the two enzymes displayed similar bioanalytical characteristics but different selectivity patterns.
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- Mikeladze, E, et al.
(författare)
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Redox hydrogel-based bienzyme microelectrodes for amperometric monitoring of L-glutamate
- 2002
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Ingår i: Electroanalysis. - 1040-0397. ; 14:6, s. 393-399
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Tidskriftsartikel (refereegranskat)abstract
- Fabrication and characterization of amperometric bienzyme L-glutamate sensitive microelectrodes are the prerequisite for monitoring changes Of L-glutamate concentration at glutamate-secreting cell cultures. The design of the glutamate microelectrodes is based on incorporating L-glutamate oxidase and horseradish peroxidase into a redox-hydrogel containing PVI19-dmeOs as the redox mediator and immobilizing this system onto the surface of platinum microdisk electrodes using, a dip-coating procedure. For amperometric measurements Of L-glutamate, these redox hydrogel-based bienzyme microelectrodes can be operated at low working potentials (-50 mV vs. Ag/AgCl) decreasing the influence of electroactive interferants possibly present in biological samples. The L-glutamate microsensors are characterized by a good operation stability and sensitivity (0.038+/-0.005 mAM(-1)), a low detection limit (0.5 muM in a conventional amperometric set-up and 0.03 muM in a Faraday cage, defined as three times the signal-to-noise ratio), a linear range up to 50 muM and a response time of about 35 s. The glutamate biosensors have been applied for the direct measurement Of L-glutamate release (upon chemical stimulation) from a population of immortalized hippocampal neurons (HN10 cells) demonstrating the possibility to amperometrically monitor in-Situ L-glutamate secretion from these cells.
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