Flow‐injection analysis with immobilized oxidase/peroxidase enzymes and fluoride electrode detection

Abstract

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.).

Operation of ion-selective electrode detectors in the sub-Nernstian/linear response range: application to flow-injection/enzymic determination of L-glutamine in bioreactor media

“…The ammonium-ion-selective membrane electrode was prepared by incorporating nonaction into a plasticized poly(vinyl chloride) (PVC) membrane (18) and then mounting a piece of this membrane within a Phillips electrode body (ISE-561, Glasblaserei Moller, Zurich). The electrode was fitted with a special cap for use as a flow-through potentiometric detector in a large-volume wall-jet configuration (19). The membrane electrode, connected to the FIA manifold via a short length of narrow bore Teflon tubing, and a saturated calomel reference electrode (SCE) were placed in a large beaker of reagent buffer (see ref 19).…”

“…The electrode was fitted with a special cap for use as a flow-through potentiometric detector in a large-volume wall-jet configuration (19). The membrane electrode, connected to the FIA manifold via a short length of narrow bore Teflon tubing, and a saturated calomel reference electrode (SCE) were placed in a large beaker of reagent buffer (see ref 19). Potentiometric response of the working electrode relative to the reference was detected with an Accumet Model 910 pH/mV meter (Fisher Scientific, Romulus, MI) and recorded on a Fisher Recordall Series 5000 strip-chart recorder.…”