Cyanide detection using a substrate-regenerating peroxidase-based biosensor

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Abstract
An enzyme-based, dual working electrode system is described for the sensing of cyanide. Horseradish peroxidase (HRP) is incorporated as the sensing element. A continuous monitoring of oxidative activity by the enzyme results through the generation and regeneration of substrates at the electrode surfaces. Thus, HRP is oxidized by hydrogen peroxide generated from dissolved oxygen, at the primary electrode, and then reduced through the secondary electrode by mediated electron transfer using ferrocene as a carrier. Ferrocene regeneration at this electrode is proportional to the intrinisic activity of HRP. The dynamics of the system are investigated by using a rotating ring-disk electrode. The enzyme is immobilized to provide better control over its catalytic activity and to increase the lifetime of the biosensor. Cyanide inhibition can be modeled by reversible binding kinetics. Detection of cyanide is possible in submicromolar (ppb) concentrations, with a half maximal response at 2 .mu.M. The response time for detection of introduced cyanide is within 1 s. The sensor can be operated between 5 and 40.degree. C, and cyanide inhibition is unaffected by pH changes between 5 and 8. The sensor is reproducible for cyanide determination and is stable for over 6 months.