Various flow-injection analysis arrangements employing immobilized oxidase and peroxidase enzymes in conjunction with fluoride ion-selective electrode detection are described. Selective measurement of substrates is based on the oxidase-catalyzed production of hydrogen peroxide. The liberated peroxide is detected via a coupled peroxidase reaction involving a fluoroaromatic reagent added to the carrier stream to yield free fluoride ions. Three specific arrangements are evaluated: (1) both enzymes coimmobilized on a single membrane covering the fluoride electrode detector, (2) both enzymes immobilized on controlled-pore glass within a flow-through reactor place before the potentiometric detector, and ( 3 ) two separate enzyme reactors placed in series upstream from the electrode. Optimum sensitivity toward model analyte glucose using glucose oxidase is achieved with arrangements based on the immobilized enzyme reactors. With these systems, detection of glucose at concentrations ranging from 0.1 to 10 mM is possible at throughputs of more than 30 samples/h.
UVTRODUCTIONThe peroxidase-catalyzed reaction of parahalogenated aromatic amine substrates with hydrogen peroxide was first reported by Hughs and Sanders [ 11. In a reaction involving transient formation of free radicals, halide ions are liberated. Some 30 years later, Siddiqi [ 2 ] demonstrated that hydrogen peroxide generated from certain highly selective oxidase enzymes could be quantitated by employing such peroxidase-catalyzed reactions in conjunction with direct potentiometric detection of the liberated fluoride ions with a solid-state fluoride ion-selective electrode. For determining various oxidase substrates, the following generic peroxidase coupled enzymatic sequence can be used; o x r h e substrate + 0, -products + H,O, (1)The final products o f the peroxidation reactions (reaction 2) vary considerably depending on the specific fluorocompound used as the cosubstrate for the reaction (e.g., 4-fluorophenol, tetrafluorophenol, 4-fluoroaniline, etc.).