A general methodology toward drug/dye incorporated living copolymer-protein hybrids: (NIRF dye-glucose) copolymer-avidin/BSA conjugates as prototypes.

Azide-terminated poly(tert-butyl acrylate) was synthesized via atom transfer radical polymerization (ATRP). Subsequent deprotection was performed to yield poly(acrylic acid) (PAA) possessing a reactive chain-end. A one-pot sequential amidation of the PAA with the amine derivatives of a near-infrared fluorescent dye (ADS832WS) and glucose produced NIRF dye-incorporated water-soluble copolymers. End-group modifications were performed to produce alkyne/biotin-terminated copolymers which were further employed to generate dye-incorporated polymer-protein hybrids via the biotin-avidin interaction with avidin or "click" bioconjugation with azide-modified BSA. We have overcome two fundamental limitations in the synthesis of bioconjugates: (a) the basic restriction in the diversity of copolymers which can be synthesized for producing bioconjugates, (b) the limitation in the number of dyes/drug molecules that can be attached per protein molecule. The copolymers possessed enhanced optical properties compared to the dye due to increased solubility in water. Potential utility of these copolymers and conjugates in multiwell plate based assays, cell surface imaging and in vivo animal imaging were explored.