Biosensors & Bioelectronics 10 (1995) 735-742 The design of enzyme sensors based on the enzyme structure M. Alvarez-lcaza*, H.M. Kalisz, H.J. Hecht, K.-D. Aumann, D. Schomburg & R.D. Schmid GBF-Gesellschaft fiir Biotechnoiogische Forschung mbH, Mascheroder Weg 1, D-38124 Braunscheweig, Germany. Tel: [49] (0)531 61810 Fax: [49] (0)531 6181515. (Received 4 August 1994; revised 13 February 1995; accepted 14 February 1995) Abstract: The size of some of the reported electron mediators for glucose oxidase compared with the available space to penetrate the active site, implies that electrons have to move along the protein structure. Theoretical and experimental evidence predicts that it is possible to have electron transfer at the required rate used in biosensors ~200 to 1840 electrons s -~) for the distances in glucose oxidase (20 to 25 A). Use of the program "Pathways" (together with the knowledge of the enzyme structure) allowed us to find an electron pathway within the enzyme. This pathway has a maximum electron coupling between the active site and the surface of the enzyme. The pathway reaches the surface near functional groups that can be used for oriented immobilization of the enzyme. Experimental confirmation of this particular pathway has been attempted but it is still elusive. Keywords: glucose oxidase, direct electron transfer, electron tunnelling, electron pathway, oriented enzyme immobilization. INTRODUCTION Glucose oxidase is by far the most widely used enzyme in biosensors (Wilson & Turner, 1992). Knowledge of its structure (Hecht et al., 1993) has to have profound consequences in the way biosensors are designed and manufactured. With knowledge of the enzyme structure, in addition to elucidation of the enzyme reaction mechanism, it should be possible to engineer the interaction * Present Address: Cranfield Biotechnology Ltd., 74 Sunderland Rd., Sandy, Bedfordshire, SG19 1QY, UK. Tel" [44] (0)1767 682282. Fax: [44] (0)1767 691864. with other systems and to base the enzyme sensor design on a more scientific approach. The enzyme reoxidation process is a fundamen- tal interaction of glucose oxidase with the rest of the measuring system of a electrochemical biosensor. Mediation by low molecular weight compounds such as oxygen or ferrocene (Cass et al., 1984) has been the most widely used and reported form of shuttling electrons from the enzyme active site to the electrode. However, reoxidation of the enzyme can also be achieved by the use of electron relays attached to the enzyme structure (Degani & Heller, 1987). In addition, electron transfer using polysiloxane (Hale et al., 1989) or polypyridine polymers (Gregg & Heller, 1990) as well as high molecular 0956-5663/95/$09.50 © 1995 Elsevier Science Ltd. 735