Enzyme-Electropolymer-Based Amperometric Biosensors: An Innovative Platform for Time-Temperature Integrators JOSE Ä I. REYES-DE-CORCUERA, ², | RALPH P. CAVALIERI,* ,‡ JOSEPH R. POWERS, ² JUMING TANG, § AND DONG H. KANG ² Department of Food Science and Human Nutrition, Washington State University, Pullman, Washington 99164-6376, Agricultural Research Center, Washington State University, Pullman, Washington 99164-6240, and Department of Biological Systems Engineering, Washington State University, Pullman, Washington 99164-6120 A novel exogenous time-temperature integrator (TTI) based on an amperometric glucose oxidase biosensor is presented. The TTI consists of the enzyme entrapped within an electrochemically generated poly(o-phenylenediamine) (PoPD) thin film deposited on the interior wall of a platinum (Pt) or a platinized stainless steel (Pt-SS) capsule. After thermal treatment, the TTI is mounted in a continuous flow system and connected to a potentiostat for amperometric detection of residual enzyme activity. A measurement is completed within 10 min. Isothermal treatments were carried out between 70 and 79.7 °C. Thermal inactivation of the immobilized enzyme followed apparent first-order kinetics with z values of 6.2 ( 0.6 and 6.6 ( 0.8 °C for Pt and Pt-SS capsules, respectively. These z values suggest that the proposed TTIs have the potential to assess pasteurization processes that target microorganism such as Listeria monocytogenes and Escherichia coli O157:H7. KEYWORDS: Time-temperature integrator; glucose oxidase; amperometric biosensor; pasteurization INTRODUCTION In view of the need for assessing the efficacy of thermal processing of food in operations where traditional instrumenta- tion like thermocouples or resistance temperature detectors cannot be implemented, several research groups have been developing different strategies to evaluate the time-temperature history of foods (1, 2). One strategy is the use of so-called time- temperature integrators (TTIs). Exogenous and endogenous TTIs from different origins (microbiological, enzymatic, chemical, and physical) have been compared (3). Enzymatic TTIs offer the advantage of being relatively easy to assay, they can be inexpensive, and the dependency of the rate of heat inactivation of some enzymes is similar to that of several pathogens of interest to the food industry. A detailed review of intrinsic endogenous TTIs with potential for use in the heat treatment of milk has been published (4), and six of these potential TTIs have been recently investigated (5). However, oftentimes, intrinsic endogenous TTIs have the disadvantage of the intrinsic variability of the concentration of the TTI component and its thermal stability. For example, triose phosphate isomerase has been proposed to verify roast beef processing (6), but its activity and thermal stability varied with muscle type. Other components present in the food in variable concentrations can also affect the thermal stability of the TTI. In contrast, exogenous TTIs can operate under controlled composition. The stability of some enzymes used as TTIs can be adjusted by changing their pH (7, 8) or adding stabilizing agents (9). Amylase (10-14), horseradish peroxidase (15-18), and r-phycoerytrhin (19) have been used as exogenous TTIs for food pasteurization. Some drawbacks of exogenous TTIs include the following: (i) if the TTI volume is relatively large (i.e. gel cubes), temperature gradients may develop throughout its volume and their applica- tion may be limited to processes where the heating transient is negligible with respect to processing time; (ii) when the substance used as the indicator needs to be extracted from a capsule, the substance might adhere to the surface of the capsule, making its recovery difficult for rapid assay; and (iii) once the substance used as indicator is retrieved, analysis often requires long preparation, which sometimes includes grinding, centrifu- gation, incubation, and finally the assay time itself (13). Most of the recent research in TTIs has focused on the use of R-amylases. The group of Hendrickx has focused on TTIs for food sterilization (3, 14, 20) and has recently developed TTIs based on Bacillus lichenformis R-amylase, sucrose, and salts immobilized on glass beads and equilibrated at low moisture content to increase the stability of the enzyme (21). This research provided not only a thermostable TTI for sterilization processes but a rapid spectrophotometric reading of the TTI response that obviates the need for an expensive differential scanning calorimeter used in previous reports. A disadvantage of that TTI is that, as mentioned above, it still requires collecting and * Corresponding author. Telephone: 509-335-4563. Fax: 509-356751. E-mail: cavalieri@wsu.edu. ² Department of Food Science and Human Nutrition. ‡ Agricultural Research Center. § Department of Biological Systems Engineering. | Current address: University of Florida, 700 Experiment Station Rd., Lake Alfred, FL 33850-2299. 8866 J. Agric. Food Chem. 2005, 53, 8866-8873 10.1021/jf051103+ CCC: $30.25 © 2005 American Chemical Society Published on Web 10/22/2005