Fluorescence ratiometric assays of hydrogen peroxide and glucose in serum using conjugated polyelectrolytes

Abstract

A new water-soluble cationic polyfluorene with boronate-protected fluorescein (peroxyfluor-1) covalently linking to the polymer backbone (PF-FB) was synthesized as a fluorescence probe to optically detect hydrogen peroxide (H₂O₂) and glucose in serum. The peroxyfluor-1 exists as a lactone form which is colorless and non-fluorescent. Without addition of H₂O₂, the fluorescence resonance energy transfer (FRET) from fluorene units (donor) to peroxyfluor-1 (acceptor) is absent and only blue donor emission is observed upon excitation of the fluorene units. In the presence of H₂O₂, the peroxyfluor-1 can specifically react with H₂O₂ to deprotect the boronate protecting groups and to generate green fluorescent fluorescein. The absorption of fluorescein overlaps the emission of polyfluorene, which encourages efficient FRET from the fluorene units to the fluorescein. By triggering the shift in emission color and the ratio change of blue to green emission intensities, it is possible to assay H₂O₂ and its concentration change in buffer solution and in serum. The PF-FB probe can detect H₂O₂ in the range from 4.4 to 530 µM. Since glucose oxidases (GOx) can specifically catalyze the oxidation of β-D-(+)-glucose to generate H₂O₂, glucose detection is also realized with the PF-FB probe as the signal transducer. Thus, an optical assay for hydrogen peroxide (H₂O₂) and glucose in serum was created which combines a fluorescent ratiometric technique based on FRET with the light-harvesting properties of conjugated polymers.