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Amperometric organic- and aqueous-phase peroxide biosensors were prepared with native and N-hydroxysuccinimide ester (NHS)-modified horseradish peroxidase (HRP). The ε-amino functions of the free lysine residues of HRP were selectively modified with homobifunctional suberic acid bis(N-hydroxysuccinimide ester) (SA-NHS) or ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) (EG-NHS). The biosensors were based on electrostatic complexation of the HRP variants with an osmium bis(bipyridyl)poly(4-vinylpyridine) polymer. The enzyme electrodes were operated in both aqueous (0.1 M phosphate buffer, pH 7.0) and organic (90% CH3CN) phases, for the detection of hydrogen peroxide and methyl isothiocyanate. Kinetic analysis of the steady-state amperometry data showed that chemical modification of HRP brought up to a ∼5-fold enhancement of the biosensor sensitivity for H2O2 and an improvement in both sensor stability and organotolerance. Also, NHS modification of HRP results in a 3-fold extension of the range of linear response of the peroxide sensor in both aqueous and nonaqueous media. When used as a methyl isothiocyanate sensor in the organic phase, the amperometric responses of the biosensors were doubled on changing from native HRP to NHS-HRP electrodes.

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