Biosensors and Bioelectronics 22 (2006) 116–123 Characterization of an organic phase peroxide biosensor based on horseradish peroxidase immobilized in Eastman AQ Anastassija Konash, Edmond Magner ∗ Material and Surface Science Institute and Department of Chemical and Environmental Science, University of Limerick, Limerick, Ireland Received 7 October 2005; received in revised form 2 December 2005; accepted 7 December 2005 Available online 15 February 2006 Abstract Due to their frequent occurrence in food, cosmetics and pharmaceutical products, and their poor solubility in water, the detection of peroxides in organic solvents has aroused significant interest. For diagnostics or on-site testing, a fast and specific experimental approach is required. Although aqueous peroxide biosensors are well known, they are usually not suitable for nonaqueous applications due to their instability. Here we describe an organic phase biosensor for hydrogen peroxide based on horseradish peroxidase immobilized in an Eastman AQ 55 polymer matrix. Rotating disc amperometry was used to examine the effect of the solvent properties, the amount and pH of added buffer, the concentration of peroxide and ferrocene dimethanol, and the amount of Eastman AQ 55 and of enzyme on the response of the biosensor to hydrogen peroxide. The response of the biosensor was limited by diffusion. Linear responses (with detection limits to hydrogen peroxide given in parentheses) were obtained in methanol (1.2 M), ethanol (0.6 M), 1-propanol (2.8 M), acetone (1.4 M), acetonitrile (2.6 M), and ethylene glycol (13.6 M). The rate of diffusion of ferrocene dimethanol was more constrained than the rate of diffusion of hydrogen peroxide, resulting in a comparatively narrow linear range. The main advantages of the sensor are its ease of use and a high degree of reproducibility, together with good operational and storage stability. © 2005 Elsevier B.V. All rights reserved. Keywords: Horseradish peroxidase; Eastman AQ; Hydrogen peroxide; Nonaqueous solvents 1. Introduction The detection of peroxides in organic solvents is of interest in a number of areas including the food industry (Chan, 1987), polymer synthesis (Ayyagari et al., 2002), and for the detection of environmental pollutants (Adeyoju et al., 1994a,b). In food products, organic peroxides are the products of the autooxida- tion of unsaturated lipids in oil and fat (Chan, 1987). They can significantly reduce the quality of food by affecting its flavor and appearance. For on-site testing, fast and reliable measurements are required. Traditional techniques such as spectrophotome- try or chromatography usually employ laboratory-based instru- mentation and time-consuming sample preparation procedures, which complicates their use in fast-response mode. Enzyme electrodes are a potential alternative, offering a fast-response time, high sensitivity, ease of use and transportation. Such biosensors usually operate at low applied potentials, rendering ∗ Corresponding author. Tel.: +353 61 202629; fax: +353 61 213529. E-mail address: edmond.magner@ul.ie (E. Magner). them much less subject to interfering reactions when compared to the direct detection of peroxide at e.g. platinum electrodes where high overpotentials are required (Clark and Lyons, 1962). Horseradish peroxidase (HRP) is the most widely used enzyme in the detection of hydrogen peroxide (Ruzgas et al., 1996). HRP catalyses the oxidation of a wide variety of organic compounds in the presence of hydrogen peroxide in aqueous and organic solutions. Hydrogen peroxide reacts with HRP to generate the potent oxidant Compound I. The latter is then reduced by an elec- tron donor (M) in subsequent steps. Although peroxidase-based biosensors for aqueous media are well known (Ruzgas et al., 1996), they are usually not suitable for nonaqueous samples due to the instability of the enzyme and/or the immobilization matrix. Interest in the development of organic phase biosensors arises in the detection of hydrophobic analytes which are only spar- ingly soluble in water. They and their matrices can be readily solubilized in organic solvents. Additional advantages of organic phase biosensors include elimination of microbial contamina- tion, reduction of interference from water-soluble compounds, an extended linear range, and enhanced thermostability and sen- sitivity (Li et al., 1998; Wang et al., 1991). Organic solvents 0956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2005.12.010