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

W. Laiwattanapaisal, J. Yakovleva, Martin Bengtsson, T. Laurell, S. Wiyakrutta, V. Meevootisom, O. Chailapakul, Jenny Emnéus

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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 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.
    Original languageEnglish
    JournalBiomicrofluidics
    Volume3
    Issue number1
    Pages (from-to)014104
    ISSN1932-1058
    DOIs
    Publication statusPublished - 2009

    Bibliographical note

    Copyright (2009) American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics.

    Keywords

    • biosensors
    • microfluidics
    • lab-on-a-chip
    • enzymes

    Cite this

    Laiwattanapaisal, W., Yakovleva, J., Bengtsson, M., Laurell, T., Wiyakrutta, S., Meevootisom, V., Chailapakul, O., & Emnéus, J. (2009). On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate. Biomicrofluidics, 3(1), 014104. https://doi.org/10.1063/1.3098319