Hydrogen Peroxide Biosensors Based on Direct Electron Transfer from Plant Peroxidases Immobilized on Self-Assembled Thiol-Monolayer Modi®ed Gold Electrodes Szilveszter Gaspar, + Heiko Zimmermann, ++ Irina Gazaryan, +++ Elisabeth Cso Èregi, + and Wolfgang Schuhmann* ++ + Department of Biotechnology, University of Lund, S-22100 Lund, Sweden ++ Analytische Chemie±Elektroanalytik & Sensorik, Ruhr-Universita Èt Bochum, D-44780 Bochum, Germany; e-mail: woschu@anachem.ruhr-uni-bochum.de +++ Department of Chemistry, Moscow State University, Vorobyovy Gory, Ru-119899 Moscow, Russia Received: March 27, 2000 Final version: July 20, 2000 Abstract A recently characterized tobacco peroxidase (TOP) has been adsorbed on differently modi®ed mixed alkylthiol monolayers, and the direct electron transfer between the enzyme and the monolayer-modi®ed electrode has been investigated. The comparison of the electrocatalytic activities of adsorbed TOP and horseradish peroxidase (HRP) showed no ef®cient direct electron transfer for HRP, whereas a signi®cant electrocatalytic current could be observed for TOP in the presence of hydrogen peroxide. The use of differently charged monolayers suggests that the electron-transfer rate of the electrocatalytic reduction of hydrogen peroxide is dependent on the orientation of the adsorbed tobacco peroxidase, probably due to electrostatic interactions between charged head groups at the monolayer and the protein shell. Keywords: Biosensor, Self-assembled monolayer, Direct electron transfer, Peroxidase, Hydrogen peroxide 1. Introduction For an enzyme-based amperometric biosensor one possibility to transduce the complementary biological recognition event to a useful electrical signal is the direct electron transfer (DET) between the enzyme's redox center and the electrode surface. DET avoids the main drawback of a mediated electron transfer, mainly, that a free-diffusing redox mediator may leak out from the electrode surface into the bulk solution [1]. As predicted by the Marcus theory [2, 3], the electron-transfer rate is expo- nentially dependent on the distance between the redox centers which are involved in the redox process. However, due to the fact that in general the enzymes' redox center is deeply buried within the protein shell the electron-transfer distance is often considerable and hence, the electron-transfer rate is slow. Therefore, in order to shorten the electron-transfer distance a proper orientation of the enzyme on the electrode surface is required [4, 5]. In addition, glycosylation of the enzyme rep- resents another important factor, since it may increase the distance between an electrode and the cofactor and thus, slow down the DET [6]. One possibility to control the electron-transfer distance between the enzyme's redox center and the electrode is to develop an appropriate immobilization method. Recently, it has been demonstrated that self-assembled thiol monolayers (SAMs), which are chemisorbed on Au electrodes show versatile char- acteristics with respect to their utilization as anchor layers for enzymes [7±9]. This is due to the fact that, on the one hand both Faradaic and non-Faradaic background currents are dramatically reduced leading to improved sensitivity [10], and, on the other hand, SAMs can be adapted to suppress interfering compounds at the modi®ed electrode surface while preventing possible dena- turation of proteins at the electrode surface [11, 12]. In addition, the monolayer can be tailored with functional terminal groups allowing the covalent immobilization of the biocatalytically active proteins [13±15]. To avoid steric hindrance during the enzyme immobilization, mixed monolayers consisting of short unsubstituted alkylthiols and longer alkylthiols bearing a functional group have been used [9, 16, 17]. During the formation of mixed monolayers one has to take into account that SAM formation is slower for disul®des [18]. It is well known that two different alkyl thiols having a similar alkyl-chain length form completely miscible binary monolayers [19], while different chain lengths form preferentially distinctive domains [20]. Mediated electron transfer has been reported for a variety of proteins immobilized on SAMs [21±24]. Using the advantages of SAMs as anchor layers, DET for glucose oxidase [25], recon- stituted horseradish peroxidase [9], microperoxidase-11 [26, 27], cytochrome c peroxidase [28] and cytochrome c oxidase [29] have already been described. In this work, we report on the development of a H 2 O 2 -sensitive electrode based on DET between different plant peroxidases and thiol-modi®ed Au electrodes. In our approach the monolayer was tailored on the one hand to avoid the steric hindrance between the bulky enzyme molecules and on the other hand, to offer a proper orientation of the enzyme molecules, thus, shortening the elec- tron-transfer distance and hence, achieving a faster electron- transfer rate. 2. Experimental 2.1. Materials Di-(N-succinimidyl)-3,3 0 -dithiodipropionat (cat. No. 43789) was purchased from Fluka (Buchs, Switzerland). 3-Carboxy- propyldisul®de (3-CPDS; cat. No. 10844) was purchased from Acros Organics (Geel, Belgium). Horseradish peroxidase, (HRP; RZ  3.4; Biozyme, Gwent, UK) and tobacco peroxidase (TOP), isolated and puri®ed as previously described [30], were used as biorecognition elements. 1,6-Diaminohexane (cat. No. 804323), 284 Electroanalysis 2001, 13, No. 4 # WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2001 1040-0397/01/0403±0284 $17.50.50=0