RESUMO
We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.
Assuntos
Células/citologia , Células/metabolismo , Pontos Quânticos , Receptores de Superfície Celular/análise , Animais , Antígeno Carcinoembrionário , Sobrevivência Celular , Células Cultivadas , Eletroforese em Gel de Ágar , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Ligantes , Nanotecnologia/métodos , Polietilenoglicóis , Ratos , Receptores de Glutamato/metabolismo , Coloração e Rotulagem/métodos , Estreptavidina , Sinapses/metabolismoRESUMO
We present a new class of polymeric ligands for quantum dot (QD) water solubilization to yield biocompatible and derivatizable QDs with compact size (approximately 10-12 nm diameter), high quantum yields (>50%), excellent stability across a large pH range (pH 5-10.5), and low nonspecific binding. To address the fundamental problem of thiol instability in traditional ligand exchange systems, the polymers here employ a stable multidentate imidazole binding motif to the QD surface. The polymers are synthesized via reversible addition-fragmentation chain transfer-mediated polymerization to produce molecular weight controlled monodisperse random copolymers from three types of monomers that feature imidazole groups for QD binding, polyethylene glycol (PEG) groups for water solubilization, and either primary amines or biotin groups for derivatization. The polymer architecture can be tuned by the monomer ratios to yield aqueous QDs with targeted surface functionalities. By incorporating amino-PEG monomers, we demonstrate covalent conjugation of a dye to form a highly efficient QD-dye energy transfer pair as well as covalent conjugation to streptavidin for high-affinity single molecule imaging of biotinylated receptors on live cells with minimal nonspecific binding. The small size and low serum binding of these polymer-coated QDs also allow us to demonstrate their utility for in vivo imaging of the tumor microenvironment in live mice.