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

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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; Laurell, Thomas; Wiyakrutta, S.; Meevootisom, V.; Chailapakul, O.; Emneus, J.

I: Biomicrofluidics, Vol. 3, Nr. 1, 014104, 2009.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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Laiwattanapaisal, W, Yakovleva, J, Bengtsson, M, Laurell, T, Wiyakrutta, S, Meevootisom, V, Chailapakul, O & Emneus, J 2009, 'On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate', Biomicrofluidics, vol. 3, nr. 1, 014104. https://doi.org/10.1063/1.3098319

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Laiwattanapaisal, W. ; Yakovleva, J. ; Bengtsson, Martin ; Laurell, Thomas ; Wiyakrutta, S. ; Meevootisom, V. ; Chailapakul, O. ; Emneus, J. / On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate. I: Biomicrofluidics. 2009 ; Vol. 3, Nr. 1.

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TY - JOUR

T1 - On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate

AU - Laiwattanapaisal, W.

AU - Yakovleva, J.

AU - Bengtsson, Martin

AU - Laurell, Thomas

AU - Wiyakrutta, S.

AU - Meevootisom, V.

AU - Chailapakul, O.

AU - Emneus, J.

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

KW - lab-on-a-chip

KW - biosensors

KW - enzymes

KW - microfluidics

U2 - 10.1063/1.3098319

DO - 10.1063/1.3098319

M3 - Article

VL - 3

JO - Biomicrofluidics

JF - Biomicrofluidics

SN - 1932-1058

IS - 1

M1 - 014104

ER -