Biotin/avidin system for the generation of fully renewable DNA sensor based on biotinylated polypyrrole film

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.

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, environment, medical-legal and food industry. Biosensors have been prepared by various immobilization strategies such as adsorption, direct covalent binding, entrapment in a polymer 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 electropolymerization in one step of the ECP matrix, opening the possibility for miniaturization and (ii) versatile functionalization, either by grafting or doping, of desired biological entities. In most cases, polypyrrole has been used for the immobilization of biomolecules because its electropolymerization is feasible in aqueous solutions at a low potential, which is compatible with most of the molecules of biological 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 copolymerization of pyrrole and oligonucleotide-substituted pyrrole [5]. This process leads in one step to the irreversible immobilization 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 polypyrrole-biotin film through an intermediate avidin layer. The procedure involves three steps: (i) the electrosynthesis of the polypyrrole-biotin film at an electrode surface; (ii) the immobilization of the avidin layer on this polymer via the biotin entities linked to the polypyrrole network; (iii) the anchoring of the biomolecules bearing biotin entities on the polypyrrole-biotin/avidin scaffolding to elaborate a bioactive surface. The successive immobilization of avidin and of biotinylated biomolecules, was made possible due to the strong avidin–biotin interaction (binding constant, 10¹⁵ M⁻¹) [9]. This strategy presents the advantages of (i) avoiding the use of chemical reagents that may damage more fragile biological species; (ii) anchoring biomolecules only at the surface of the film, which induces a high sensitivity; (iii) being versatile through the wide variety of commercially available biotin conjugates.

In previous works, we have studied the immobilization of avidin on polypyrrole-biotin by the use of a quartz crystal microbalance (QCM) [10], which is an extremely sensitive mass sensor able to monitor mass variations at the sub-nanogram level. By comparison of the gravimetric measurements achieved on biotinylated and unbiotinylated polypyrrole films, it has been possible to estimate the amount of avidin anchored specifically by biotin/avidin interaction on the biotinylated polymer. This quantity corresponds to little more than one monolayer calculated for a monomolecular layer of closely packed avidin in a flat orientation. This apparent excess could be explained by water entrapped within the avidin layer, as shown by Caruso et al. [11]. Moreover, we have demonstrated that the frequency changes observed were not induced by a viscoelastic effect and thus, the gravimetric detection by QCM constitutes a suitable method to study the immobilization of biomolecules on a polypyrrole film [12]. We have also studied, by QCM, the elaboration of a DNA sensor constructed by immobilization of biotinylated-specific nucleotides (probe molecules) on an avidin layer previously anchored on a biotinylated polypyrrole homopolymer electrosynthesized from monomer 1 (Fig. 1) [7]. The bioassembly, polypyrrole-biotin/avidin/ODN probe obtained in this way was used as biosensor for DNA and showed some regeneration ability by solubilization of the avidin layer.

In order to round off this preliminar study, the work presented herein described an extended study of the regeneration processes offered by such a sensor. Dissociation of the biological assembly can take place at two different levels and is selectively activated to regenerate (Fig. 2) either the underlying polypyrrole-biotin layer by solubilization of the avidin layer (strategy 1) or the polypyrrole-biotin/avidin/ODN probe by denaturation of the DNA duplex (strategy 2). Both dissociation processes operate under mild conditions, and with a minimum loss of bioactivity upon recycling. Two detection approaches have been employed QCM and fluorescence microscopy, to monitor the efficiency of the sensor assembly and to give control over hybridization event. Before this study, we had determined the optimal conditions to elaborate this DNA sensor. To do that, copolymers containing different amounts of biotin were electrosynthesized in order to evaluate the influence of the biotin content in polypyrrole matrix on the DNA hybridization capacity. Due to the relatively large size of avidin, the optimization of the recognition capacity of these biosensors should not require the synthesis of a polypyrrole film with a high surface density of biotin units, i.e. pyrrole-biotin homopolymers. Thus, biotinylated copolymers were electrochemically synthesized from biotinylated and non-biotinylated monomers 1 and 2 (Fig. 1) in various ratios. The amounts of avidin units and ODN probes immobilized onto these copolymers have been evaluated from microgravimetry and fluorescence measurements, respectively.