Journal article
On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate
Chulalongkorn University1
Evolva SA2
Lund University3
Mahidol University4
Bioanalytics Group, Biomedical Micro Systems Section, Department of Micro- and Nanotechnology, Technical University of Denmark5
Biomedical Micro Systems Section, Department of Micro- and Nanotechnology, Technical University of Denmark6
Department of Micro- and Nanotechnology, Technical University of Denmark7
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.
Language: | English |
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Publisher: | American Institute of Physics |
Year: | 2009 |
Pages: | 14104 |
ISSN: | 19321058 |
Types: | Journal article |
DOI: | 10.1063/1.3098319 |
ORCIDs: | Emnéus, Jenny |