Redox labelled avidin for enzyme sensor architectures Celestino Padeste a, *, Beat Steiger b , Andreas Grubelnik a,1 , Louis Tiefenauer a a Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland 2 b Electrochemistry Laboratory, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland Received 24 June 2002; received in revised form 4 November 2002; accepted 15 May 2003 Abstract Conjugates of avidin with ferrocene and with microperoxidase 8 have been used as electrochemically active molecular building blocks. Assemblies of the conjugates with biotinylated glucose oxidase or lactate oxidase on gold electrodes were tested as enzyme sensors for glucose and lactate. The electrochemical detection is based either on ferrocene-mediated oxidation of the substrate in oxygen-free solution, or on microperoxidase-catalysed reduction of H 2 O 2 which is enzymatically produced from the substrate and molecular oxygen. Glucose and lactate were detectable with both detection principles in concentrations down to 1 or 0.1 mM, respectively. The molecular architecture concept allows quick adaptation of the sensors to other analytes, and it provides a platform for arrays of sensors with different selectivity. # 2003 Elsevier B.V. All rights reserved. Keywords: Avidin; Ferrocene; Microperoxidase; Biosensor; Lactate; Glucose 1. Introduction The potential of the avidin-biotin system to tailor properties of bioanalytical devices has generally been recognised and documented since many years (Wilchek and Bayer, 1990). Avidin is used as a versatile and stable anchoring system for biotinylated functional molecules such as enzymes or antibodies. Furthermore, it repre- sents an ideal building block for three-dimensional architectures, because the two pairs of biotin-binding pockets are arranged on opposite sides of the molecule (Pugliese et al., 1993), leading to defined molecular networks. Avidin based sensing layers have also been described for various types of enzyme electrodes (Pantano and Kuhr, 1993; Hoshi et al., 1995; Liu et al., 1995; Anzai et al., 1998; Chen et al., 1998; Anicet et al., 1998a,b, 1999; Cosnier, 1999; Cosnier et al., 1999; De Lacey et al., 2000; Trojanowicz and Miernik, 2001; Yoon et al., 2001; Cosnier et al., 2002). By variation of the number of layers of avidin and biotinylated enzyme and of their molar ratio, it is possible to adapt the response of the sensors in a relatively wide range (Du et al., 1996). In electrochemical sensors, however, the non-conductive protein layers impose an electrical barrier. Concepts to establish an electrochemical contact between the enzyme centres and the electrode surface have recently been reviewed (Armstrong and Wilson, 2000; Habermu ¨ller et al., 2000; Willner and Katz, 2000). In avidin based systems, ferrocene derivatives were used as soluble mediators (Anicet et al., 1998a). In order to prevent the mediators from leaking out of the protein layers, they were attached via biotin to avidin (Anicet et al., 1998b). Alternatively to redox mediation, sensors based on consecutive reactions have been presented. The enzymatic reaction product generated in the avidin/ enzyme layer is diffusing to the electrode surface, where it is electrochemically detected, either in a direct reaction at the electrode surface (Hoshi et al., 1995) or catalysed by additionally immobilised enzymes or enzyme frag- ments (Wright et al., 1995; Vreeke and Rocca, 1996; Mousty et al., 2001). In this work two different redox labels were cova- lently attached to avidin in order to combine the biotin- binding capability either with redox mediation or with * Corresponding author. Tel.:  /41-56-310-2141; fax:  /41-56-310- 2646. E-mail address: celestino.padeste@psi.ch (C. Padeste). 1 Present address: EMPA, CH-900, St. Gallen, Switzerland. 2 http://www.psi.ch/lmn. Biosensors and Bioelectronics 19 (2003) 239 /247 www.elsevier.com/locate/bios 0956-5663/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0956-5663(03)00214-8