On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1401204
- author
- Laiwattanapaisal, W. ; Yakovleva, J. ; Bengtsson, Martin LU ; Laurell, Thomas LU ; Wiyakrutta, S. ; Meevootisom, V. ; Chailapakul, O. and Emneus, J.
- organization
- publishing date
- 2009
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- lab-on-a-chip, biosensors, enzymes, microfluidics
- in
- Biomicrofluidics
- volume
- 3
- issue
- 1
- article number
- 014104
- publisher
- American Institute of Physics (AIP)
- external identifiers
-
- wos:000264781300018
- scopus:64149126159
- pmid:19693397
- 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
- 2016-04-01 14:25:00
- date last changed
- 2022-03-29 20:51:45
@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.}},
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}},
keywords = {{lab-on-a-chip; biosensors; enzymes; microfluidics}},
language = {{eng}},
number = {{1}},
publisher = {{American Institute of Physics (AIP)}},
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}},
doi = {{10.1063/1.3098319}},
volume = {{3}},
year = {{2009}},
}