Biotin−Fluorophore Conjugates with Poly(ethylene glycol) Spacers Retain Intense Fluorescence after Binding to Avidin and Streptavidin

Conventional biotin−fluorophore conjugates with ∼14 atom spacers lose most of their fluorescence when binding to avidin or streptavidin, as is demonstrated in the present study. This explains the unusual fact that only biotinylated marker enzymes, but not fluorescent biotins, are regularly used in bioanalytic assays. Novel biotin−spacer−fluorophore conjugates are presented that retain intense fluorescence when binding to avidin or streptavidin. Preservation of fluorescence depends upon the use of poly(ethylene glycol) (PEG) spacers, which are shown not to interfere with biotin function. The observed absence of nonspecific interactions may also be attributed to the PEG chain. These novel fluorescent biotins are expected to be excellent new tools in fluorescence microscopy and related techniques.