A Fluorescence Ratiometric Protein Assay Using Light‐Harvesting Conjugated Polymers

Abstract

AbstractSummary: A highly selective protein assay was created which combines the fluorescent ratiometric technique based on FRET with the light‐harvesting properties of conjugated polymers. The cationic poly[(9,9‐bis(6′‐N,N,N‐trimethylammonium)‐hexyl)‐fluorene phenylene] bromide (PFP‐NMe) and the negatively charged biotinylated fluorescein probe (Fl‐B) were used to detect the target protein streptavidin optically. The strong electrostatic interactions between PFP‐NMe and fluorescein result in efficient FRET from PFP‐NMe to fluorescein. In the presence of streptavidin, however, the biotin moiety of Fl‐B specifically associates with streptavidin and the fluorescein molecule is buried deeply in the adjacent vacant binding sites. This separates the fluorescein spatially from the PFP‐NMe moiety, resulting in inefficient FRET from PFP‐NMe to fluorescein. Although a nonspecific protein, such as BSA, shows nonspecific interactions with PFP‐NMe, it does not affect the fluorescent ratio value of PFP‐NMe to fluorescein. Hence, the charged neutral complex of two oppositely charged conjugated polymers can eliminate the nonspecific interactions, and thus optimize their application in protein assays.A schematic representation of the protein assay operation.magnified imageA schematic representation of the protein assay operation.