Electrogenerated trisbipyridyl Ru(II)-/nitrilotriacetic-polypyrene copolymer for the easy fabrication of label-free photoelectrochemical immunosensor and aptasensor: Application to the determination of thrombin and anti-cholera toxin antibody Yao Wenjuan a,b , Alan Le Goff a , Nicolas Spinelli a , Michael Holzinger a , Guo-Wang Diao b , Dan Shan b , Eric Defrancq a , Serge Cosnier a,n a Universite´ Joseph Fourier, De´partement de Chimie Mole´culaire, UMR-5250, ICMG FR-2607, CNRS, Grenoble, France b School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China article info Article history: Received 17 September 2012 Received in revised form 10 November 2012 Accepted 12 November 2012 Available online 22 November 2012 Keywords: Electropolymerization Photoelectrochemistry Aptasensor Immunosensor Pyrene Metallopolymers abstract A bifunctional copolymer was electrogenerated, which allows efficient bioreceptor immobilization and transduction of the biorecognition event. This copolymer was formed using pyrenebutyric acid Na 0 ,Na- bis(carboxymethyl)-L-lysine amide (NTA–pyrene) and [tris-(2,2 0 -bipyridine) (4,4 0 -bis(4-pyrenyl-1- ylbutyloxy)-2,2 0 -bipyridine] ruthenium(II) hexafluorophosphate (Ru(II)–pyrene) complex. The pyrene groups, present in both compounds, undergo oxidative electropolymerization on platinum electrodes. The resulting copolymer contains NTA moieties, which were used as a versatile immobilization system for biotin- and histidine-tagged biomolecules, while Ru(II)–pyrene was employed as a photoelec- trochemical transducing molecule. The efficiency of this copolymer for biomolecule anchoring was investigated with biotin- and histidine- tagged glucose oxidases, biotin-tagged cholera toxin and a histidine-tagged thrombin aptamer. The constructed enzyme electrodes exhibited an amperometric response toward glucose at 0.6 V vs SCE, demonstrating the anchoring of this enzyme via two coordination systems. An immunosensor configuration based on the immobilization of biotin-tagged cholera toxin was applied to the detection of anti-cholera antibody while the aptasensor based on the immobilization of histidine-tagged thrombin aptamer was tested for thrombin determination. The biorecognition events were monitored via the evolution of the photocurrent intensity generated by the polymerized Ru(II)–pyrene in the presence of visible light and a sacrificial donor (ascorbate). The binding of the targets hinders the diffusion of the sacrificial donor, inducing thus a photocurrent decrease. The constructed immunosensor presents a specific label-free photoelectrochemical response to anti-cholera antibody without labeling step, the detection limit being 0.2 mg mL 1 . The label-free photoelectrochemical response of the aptasensor varies linearly with thrombin concentrations up to 10 pmol L 1 , the detection limit being 1  10 13 mol L 1 . & 2012 Elsevier B.V. All rights reserved. 1. Introduction DNA sensors and immunosensors constitute a valuable alter- native to centralized analytical techniques, since they are easy, inexpensive, fast, and selective tools in many applications includ- ing medical diagnostic, genetic screening, environmental control as well as food and agricultural analyses. These affinity sensors require one binding partner (the probe element) to be immobilized on a surface where both the immu- noreaction or hybridization (i.e., the recognition event), and the signal generation take place. To acquire a high sensitivity, the immobilization method of immunologic materials or single- stranded DNA is extremely important. The latter should maintain the biological activity close to the transducer surface without loss of accessibility with the probe. Furthermore, the ‘ideal’ immobiliza- tion method can provide specific properties to the biological deposit propitious to a label-free transduction of the recognition event. One elegant method to immobilize bioreceptors on the transducer is the combination of electrogenerated polymers with supramolecular or coordination complexes, called affinity interactions (Cosnier, 2005, 2007; Ronkainen et al., 2010). Electrogenerated polymers have shown their great benefit in biosensor setups due to the possibility of the reproducible and stable modification of conductive surfaces (Cosnier and Holzinger, 2011). Common electropolymerized affinity Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.11.013 n Corresponding author. Tel.: þ33 456 520810; fax: þ33 476 514267. E-mail address: serge.cosnier@ujf-grenoble.fr (S. Cosnier). Biosensors and Bioelectronics 42 (2013) 556–562