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On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate

Laiwattanapaisal, W.; Yakovleva, J.; Bengtsson, Martin LU ; Laurell, Thomas LU ; Wiyakrutta, S.; Meevootisom, V.; Chailapakul, O. and Emneus, J. (2009) In Biomicrofluidics 3(1).
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 mu m depth and 25 mu m width and (ii) polystyrene Poros (TM) beads with a particle size of 20 mu m. 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... (More)
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 mu m depth and 25 mu m width and (ii) polystyrene Poros (TM) beads with a particle size of 20 mu m. 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 (epsilon(ex)=340 nm, epsilon(em)=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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
lab-on-a-chip, biosensors, enzymes, microfluidics
in
Biomicrofluidics
volume
3
issue
1
publisher
American Institute of Physics
external identifiers
  • wos:000264781300018
  • scopus:64149126159
ISSN
1932-1058
DOI
10.1063/1.3098319
language
English
LU publication?
yes
id
304d35b6-8a59-4b4b-99fe-e93a2882ecb5 (old id 1401204)
date added to LUP
2009-06-12 08:50:29
date last changed
2017-06-11 04:15:18
@article{304d35b6-8a59-4b4b-99fe-e93a2882ecb5,
  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 mu m depth and 25 mu m width and (ii) polystyrene Poros (TM) beads with a particle size of 20 mu m. 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 (epsilon(ex)=340 nm, epsilon(em)=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.},
  articleno    = {014104},
  author       = {Laiwattanapaisal, W. and Yakovleva, J. and Bengtsson, Martin and Laurell, Thomas and Wiyakrutta, S. and Meevootisom, V. and Chailapakul, O. and Emneus, J.},
  issn         = {1932-1058},
  keyword      = {lab-on-a-chip,biosensors,enzymes,microfluidics},
  language     = {eng},
  number       = {1},
  publisher    = {American Institute of Physics},
  series       = {Biomicrofluidics},
  title        = {On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate},
  url          = {http://dx.doi.org/10.1063/1.3098319},
  volume       = {3},
  year         = {2009},
}