Publication: On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate
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Issued Date
2009-04-15
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ISSN
19321058
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2-s2.0-64149126159
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Mahidol University
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SCOPUS
Bibliographic Citation
Biomicrofluidics. Vol.3, No.1 (2009)
Suggested Citation
Wanida Laiwattanapaisal, J. Yakovleva, M. Bengtsson, T. Laurell, S. Wiyakrutta, V. Meevootisom, O. Chailapakul, J. Emnéus On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate. Biomicrofluidics. Vol.3, No.1 (2009). doi:10.1063/1.3098319 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/27244
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Title
On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate
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Abstract
Two microfluidic systems have been developed for specific analysis of L-glutamate in food based on substrate recycling fluorescence detection. L-glutamate dehydrogenase and a novel enzyme, D-phenylglycine aminotransferase, were covalently immobilized on (i) the surface of silicon microchips containing 32 porous flow channels of 235 μm depth and 25 μm width and (ii) polystyrene Poros™ beads with a particle size of 20 μm. The immobilized enzymes recycle L-glutamate by oxidation to 2-oxoglutarate followed by the transfer of an amino group from D-4-hydroxyphenylglycine to 2-oxoglutarate. The reaction was accompanied by reduction of nicotinamide adenine dinucleotide (NAD+) to NADH, which was monitored by fluorescence detection εex=340 nm, εem=460 nm). First, the microchip-based system, L-glutamate was detected within a range of 3.1-50.0 mM. Second, to be automatically determined, sequential injection analysis (SIA) with the bead-based system was investigated. The bead-based system was evaluated by both flow injection analysis and SIA modes, where good reproducibility for L-glutamate calibrations was obtained (relative standard deviation of 3.3% and 6.6%, respectively). In the case of SIA, the beads were introduced and removed from the microchip automatically. The immobilized beads could be stored in a 20% glycerol and 0.5 mM ethylenediaminetetraacetic acid solution maintained at a pH of 7.0 using a phosphate buffer for at least 15 days with 72% of the activity remaining. The bead-based system demonstrated high selectivity, where L-glutamate recoveries were between 91% and 108% in the presence of six other L-amino acids tested. © 2009 American Institute of Physics.