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 toxinantibody

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−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.

► A polypyrene copolymer bearing NTA and trisbypyridyl Ru(II) groups was formed.
► The film shows photoelectrochemical and affinity binding properties vs biomolecules.
► A photoelectrochemical immunosensor for anti-cholera toxin antibody was achieved.
► An extremely sensitive photoelectrochemical aptasensor for thrombin was elaborated.