RESUMO
A bifunctional copolymer was electrogenerated, which allows efficient bioreceptor immobilization and transduction of the biorecognition event. This copolymer was formed using pyrenebutyric acid Nα',Nα-bis(carboxymethyl)-L-lysine amide (NTA-pyrene) and [tris-(2,2'-bipyridine) (4,4'-bis(4-pyrenyl-1-ylbutyloxy)-2,2'-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 photoelectrochemical 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 µg mL⻹. The label-free photoelectrochemical response of the aptasensor varies linearly with thrombin concentrations up to 10 pmol L⻹, the detection limit being 1×10⻹³ mol L⻹.