DIRECT ELECTROCHEMISTRY OF MEMBRANE-ENTRAPPED HORSERADISH PEROXIDASE: AMPEROMETRIC DETECTION OF HYDROGEN PEROXIDE. 

T. Ferri¹, A. Poscia², R. Santucci³

¹Dipartimento di Chimica, Università di Roma ‘La Sapienza’ and Dipartimento di Medicina Sperimentale e Scienze Biochimiche, 

²Menarini Industrie Farmaceutiche Riunite SpA, Firenze

³Università di Roma ‘Tor Vergata’, Rome, Italy.

In the last years, electrochemical biosensors (based on enzymes which catalize redox reactions) have found wide application for detection of a large body of biological substrates. The most recent amperometric biosensors, indicated as ‘third-generation biosensors’, do not make use of mediators; they monitor the presence of small molecules forming or disappearing during the reaction between an enzyme and the substrate (as the hydrogen peroxide formation or the oxygen disappearance) through the measurement of the current generated.

The recent successfull employment of multienzymatic systems is mainly due to the ability to change the analyte in a form electrochemically detectable through a sequence of reactions, or eliminate intereferring substances by their convertion into inactive electrochemical compounds.

We describe here an amperometric biosensor based on horseradish peroxidase (HRP) entrapped within a tributylmethyl phosphonium chloride polymer bound (polystyrene crosslinked with 1% divynil benzene) anionic exchange resin at a pyrolytic graphite (PG) electrode. Data obtained indicate that the immobilized protein is electrochemically active within a wide pH range (pH 3.0-12.0) even in the absence of mediators, and shows catalytic activity in the hydrogen peroxide electroreduction. The ability of the system to acts as a biosensor has been tested by entrapping choline oxidase (or glucose oxidase) in the membrane together with HRP, and detecting the presence of choline (or glucose), respectively, in solution. The system revealed to act efficiently: to a good stability, it coupled rapid electron-transfer at the electrode and good biological selectivity. This system may represent a promising example of simple ‘solid-state’ sensor to be employed in electrochemical analysis of biomolecules in solution. 

Keywords: Electrochemical biosensor, horseradish peroxidase, hydrogen peroxide.