Continuous-Flow/Stopped-Flow and Rotating Bioreactors in the Determination of Glucose

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Abstract
The high sensitivity that can be attained by enzymatic amplification via substrate cycling has been verified by on-line interfacing of a rotating bioreactor and continuous-flow/stopped-flow/continuous-flow processing [Raba, J.; Mottola, H. A. Anal. Biochem.1994, 220, 297−302]. The determination of glucose levels was possible with a limit of detection of 0.2 fmol·L-1 in the processing of as many as 30 samples per hour. Determination at such low levels is of interest in several situations encountered in fermentation biotechnology and clinical chemistry, and this determination in culture broths illustrates the capabilities of the proposed approach. The glucose oxidase/glucose dehydrogenase coupled system was used by immobilizing glucose oxidase (EC 1.1.3.4) on the top of a rotating disk while glucose dehydrogenase (EC 1.1.1.47) was immobilized on the top part of the flow-through cell. Substrate cycling was realized via NADH/NAD+ that, in conjunction with glucose dehydrogenase, regenerates glucose, the substrate in the glucose oxidase-catalyzed reaction. This cycling permits generation of H2O2 (detected at Pt ring electrode concentric to the rotating disk) beyond stoichiometric limitations. This permits a 100-fold increase in the sensitivity for glucose determination when compared with the determination involving no substrate cycling.