On-line enzymatic amplification by substrate cycling in a dual bioreactor with rotation and amperometric detection.
The amplification approach centered on the cycling of two reversibly interconvertible chemical species sequentially participating in two different enzyme-catalyzed reactions (enzymatic amplification by substrate cycling) has been implemented on-line into a continuous-flow/stopped-flow/continuous-flow operation. The implementation is illustrated with the determination of L-lactate in a dual enzyme reactor containing immobilized lactate oxidase (LOD, EC 1.1.3.x) to catalyze the oxidation of L-lactate by dissolved oxygen. The immobilized LOD was affixed to a rotating disk in the lower part of the flow-through cell. Immobilized lactate dehydrogenase (EC 1.1.1.27), affixed to the top part of the cell regenerates L-lactate with the mediation of beta-NADH as the hydrogen donor. The substrate cycling permits the generation of H2O2 beyond the stoichiometric limitation, and this is detected at a stationary Pt-ring electrode located at the bottom part of the cell. The stationary Pt-ring electrode is positioned concentrically to the rotating disk containing the immobilized LOD. The resulting amplified response permits, in a simple manner, achievement of detection limits as low as 0.3 fmol.liter-1 and allows the processing of 30 samples per hour.
Department of Chemistry, Oklahoma State University, Stillwater 74078-0447.
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Raba
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Mottola
D000818:Animals; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004798:Enzymes / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010376:Pediococcus / Q000201:enzymology; D011817:Rabbits
D004798:Enzymes; D004800:Enzymes, Immobilized; D007773:Lactates; D009243:NAD; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D007770:L-Lactate Dehydrogenase; C021497:lactate 2-monooxygenase