Electrogeneration of a biotinylated poly(pyrrole–ruthenium(II)) film for the construction of photoelectrochemical immunosensor{ Naoufel Haddour, Serge Cosnier* and Chantal Gondran Laboratoire d’Electrochimie Organique et de Photochimie Re ´dox(CNRS UMR 5630) Institut de Chimie Mole ´culaire de Grenoble FR CNRS 2607, Universite ´ Joseph Fourier, BP 53, 38041 Grenoble cedex9, France. E-mail: serge.cosnier@ujf-grenoble.fr; Fax: 133 4 76 51 42 67; Tel: 133 4 76 51 49 98 Received (in Cambridge, UK) 15th July 2004, Accepted 6th August 2004 First published as an Advance Article on the web 17th September 2004 A biotinylated photosensitive polymer was electrogenerated from on a ruthenium complex bearing biotin and pyrrole groups; the resulting polypyrrolic film allowed the bioaffine immobilisation of avidin and biotinylated cholera toxin and the photoelectrochemical detection of the corresponding antibody. The development of non-manual immobilization methods of biomolecules on surface attracted a continuous substantial attention due to the exponential emergence of immunosensors and biochips as valuable tools in diagnostic laboratories and medical treatment. Among the conventional immobilization procedures, the strong affinity interactions between the glycopro- tein avidin and four biotins, a vitamin (association constant K a ~ 10 15 M 21 ) 1 have been extensively used for binding biological species to surfaces in various fields such as immunohistochemistry, 2 enzyme-linked immunoassay (ELISA) 3 and DNA hybridization. 4 The anchoring of the protein or oligonucleotide monolayer was performed by the formation of avidin–biotin bridges between biotinylated surfaces and avidin-conjugated enzymes, or biotiny- lated enzymes, antibodies, bacteria or oligonucleotides. 5 The avidin–biotin technique constitutes one of the few immobilization procedures involving solely a single attachment point of the biomolecule facilitating thus recognition phenomenon such as immunoreaction, hybridization or protein-ligand system. This affinity-driven immobilization method was mainly coupled with sophisticated fluorescent detection technologies. 6 These transduc- tions, however, require an additional labeling step of the target. With the aim of developing alternative transduction approach free of label, we report here, the electrogeneration of the first example of a biotinylated redox polypyrrole film allowing both the immobilization of biomolecules and the detection of their biological interactions via the change of its photoelectrochemical properties. For this purpose, to our best knowledge, the synthesis of the first electropolymerizable biotinylated metal complex possessing elec- trochemical and photochemical activities is described. The electrogeneration of polymer films indeed is one of the few procedures of surface functionalization with molecular reagents that allows the reproducible functionalization of conductive surfaces of complex geometry with a precise spatial resolution. In this study a novel biotin-labeled ruthenium (II) tris(bipyridyl) complex functionalized by four pyrrole groups (Fig. 1), was prepared by reaction of dichloro bis[4,4’ -bis(4-pyrrole-1-butyl)2,2’ - bipyridyl] ruthenium(II) with 4,4’ -bis(biotin)2,2’ -bipyridine and characterized by H 1 H NMR and UV-visible absorption spectra as well as by EI mass spectrometry. The dichloro bis[4,4’ -bis(4- pyrrole-1-butyl)2,2’ -bipyridyl] ruthenium(II) was synthesized according to the published procedure 7 by reaction of the 4,4’ -bis(4-pyrrole-1-butyl)2,2’ -bipyridine with Ru(III)Cl 3 and characterized by 1 H NMR. As previously reported, the 4,4’ -bis(biotin)-2,2’ -bipyridine ligand was prepared by esterification of 4,4’ -bis(hydroxymethyl)-2,2’ -bipyridine with biotin, using the carbodiimide method and characterized by 1 H NMR and FAB mass spectrometry. 8 The electrochemical behavior of the ruthenium complex (1 mM) was investigated in CH 3 CN 1 0.1 M nBu 4 NClO 4 . Upon reductive scanning, the monomer exhibits three successive reversible peak systems at 21.72 V (DE p ~ 60 mV), 21.97 V (DE p ~ 70 mV) and 22.24 V (DE p ~ 70 mV) corresponding to the successive one- electron reduction of the three bipyridyl ligands. These values are similar to those previously reported for tris(bipyridyl)ruthenium(II) complex containing pyrrole groups. 9 Upon oxidative scanning, the cyclic voltammogram displays a weakly reversible anodic peak at 0.92 V on the one-electron oxidation wave of the complex. This behavior clearly indicates the irreversible oxidation of the pyrrole groups via the one-electron oxidation of the metal center (Ru II / Ru III ) since N-alkylpyrroles are oxidized around 1.0 V (Fig. 2A). 10 Since the electrogeneration of the pyrrole cation radical is followed by its coupling while protons are released, the electropolymerisa- tion properties of the ruthenium complex were investigated by repeated potential cycling over the range 0–1.1 V. The continuous growth of reversible peak systems due to the Ru II/III couple clearly { Electronic supplementary information (ESI) available: Synthetic proce- dures and characterization data. See http://www.rsc.org/suppdata/cc/b4/ b410727f/ DOI: 10.1039/b410727f Fig. 1 Structure of tris(bipyridyl)ruthenium(II) complex. Fig. 2 A. Cyclic voltammogram recorded at a platinum electrode (diameter 5 mm) of the tris(bipyridyl)ruthenium(II) complex (1 mM) in CH 3 CN 1 0.1 M nBu 4 NClO 4 . Scan rate 0.1 V s 21 . B. Cyclic voltammograms of polypyrrole–ruthenium–platinum electrode (C ~ 1.34 6 10 29 mol cm 22 ) in CH 3 CN 1 0.1 M LiClO 4 . Scan rate 0.1 V s 21 . 2472 Chem. Commun. , 2004, 2472–2473 This journal is ß The Royal Society of Chemistry 2004 Published on 17 September 2004. Downloaded by UNIVERSITY OF ALABAMA AT BIRMINGHAM on 26/10/2014 01:56:36. View Article Online / Journal Homepage / Table of Contents for this issue