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A Fluorescence Ratiometric Protein Assay Using Light‐Harvesting Conjugated Polymers
Author(s) -
An Lingling,
Tang Yanli,
Wang Shu,
Li Yuliang,
Zhu Daoben
Publication year - 2006
Publication title -
macromolecular rapid communications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.348
H-Index - 154
eISSN - 1521-3927
pISSN - 1022-1336
DOI - 10.1002/marc.200600214
Subject(s) - fluorescein , chemistry , streptavidin , förster resonance energy transfer , conjugated system , moiety , fluorescence , fluorophore , biotinylation , polymer , photochemistry , biotin , stereochemistry , organic chemistry , biochemistry , physics , quantum mechanics
Summary: 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 3 + ) 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 3 +and fluorescein result in efficient FRET from PFP‐NMe 3 +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 3 +moiety, resulting in inefficient FRET from PFP‐NMe 3 +to fluorescein. Although a nonspecific protein, such as BSA, shows nonspecific interactions with PFP‐NMe 3 + , it does not affect the fluorescent ratio value of PFP‐NMe 3 +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.