Analytica Chimica Acta 515 (2004) 271–277 Biotin/avidin system for the generation of fully renewable DNA sensor based on biotinylated polypyrrole film A. Dupont-Filliard, M. Billon ∗ , T. Livache, S. Guillerez Laboratoire d’Electrochimie Moléculaire et Structures des Interfaces, Département de Recherche Fondamentale sur la Matière Condensée, CNRS-CEA-Université J. Fourier, CEA-Grenoble, UMR 5819, 17 Avenue des Martyrs, F-38054 Grenoble Cedex 9, France Received 23 October 2003; received in revised form 22 March 2004; accepted 30 March 2004 Available online 18 May 2004 Abstract A DNA sensor based on electropolymerized biotinylated polypyrrole film was prepared. Biotinylated DNA probes were immobilized on the polypyrrole-biotin film via an intercalated layer of avidin creating the sensing layer, polypyrrole-biotin/avidin/DNA probe. From this sensor, gravimetric measurements performed with a quartz crystal microbalance (QCM) showed that the amount of DNA probe immobilized is controlled by the quantity of biotin units included within the polypyrrole-biotin film. The maximum coverage of DNA probes was achieved with a copolymer electrosynthesized from a pyrrole-biotin and un-biotinylated pyrrole monomer ratio of 1/5. Furthermore, the sensing layer can be re-generated several times with a minimum loss of activity. After the hybridization between immobilized DNA probes and complementary DNA strands, two ways of regeneration are possible. The complementary oligonucleotide (ODNc) strands can be selectively removed: (i) by solubilization of the avidin layer, which can be lifted off to give the polypyrrole-biotin film on which a new DNA sensor can be built (strategy 1) or (ii) by denaturation of the DNA duplex, leaving the polypyrrole-biotin/avidin/ODN probes sensor ready for use in a subsequent recognition processor (strategy 2). For both strategies, the first regeneration step lead to a loss of activity of 15–20%. Subsequent regenerations can be achieved without further loss of activity. © 2004 Elsevier B.V. All rights reserved. Keywords: Avidin; Biotin; Polypyrrole; Renewable; Sensor; DNA 1. Introduction Over the last few years, the immobilization of DNA strands on electrode surfaces of different types has been the subject of numerous studies within the framework of the development of DNA sensors or DNA chips [1]. Areas of application of these sensors include clinical, environ- ment, medical-legal and food industry. Biosensors have been prepared by various immobilization strategies such as adsorption, direct covalent binding, entrapment in a poly- mer matrix or indirect binding by the use of intermediate systems [2]. Electronic conducting polymers (ECPs) appear to be particularly suitable substrates for the construction of elaborate sensing layers at the surface of an electrode [3,4]. This is due to their key properties, i.e. (i) easy electropoly- merization in one step of the ECP matrix, opening the ∗ Corresponding author. Tel.: +33-4-38784833; fax: +33-4-38785097. E-mail address: billon@drfmc.ceng.cea.fr (M. Billon). possibility for miniaturization and (ii) versatile functional- ization, either by grafting or doping, of desired biological entities. In most cases, polypyrrole has been used for the immobilization of biomolecules because its electropolymer- ization is feasible in aqueous solutions at a low potential, which is compatible with most of the molecules of biolog- ical interest. Thus, many examples have shown it possible to include single-stranded DNA in the bulk or at the surface of polypyrrole films. The immobilization of single-stranded DNA has been realized by an electrochemical copolymer- ization of pyrrole and oligonucleotide-substituted pyrrole [5]. This process leads in one step to the irreversible im- mobilization of ODN units in a copolymer film. Another way to anchor biomolecules on polypyrrole films consists in post-functionalization. For example, DNA strands have been covalently attached by amide bond formation on a preformed ECP having active ester groups [6]. More recently, a new approach to post-functionalization, based an biotin/avidin affinity system, has been used [7,8]. The biomolecules are anchored on a modified 0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.03.072