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 cellsurface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.To perform single-molecule imaging in cells using dyes or fluorescent proteins, one must constantly contend with a weak signal, which typically bleaches in <10 s. Gold particles or latex beads allow stable single-particle tracking via their scattering, but are generally very large (30-500 nm) 1 . QDs are an alternative probe as they exhibit fluorescence so bright that single molecules can be imaged on an epifluorescence microscope, and their photostability allows hours of illumination without bleaching 2 . However, the full potential of QDs for cellular imaging has not yet been realized because of problems with large QD size (typically 20-30 nm for biocompatible red-emitting QDs 2 ), the difficulty of delivering QDs into the cytosol, the instability of antibody-mediated targeting of QDs and QD multivalency. Previously we addressed the problem of binding instability by targeting streptavidin-functionalized QDs to cellular proteins that were site-specifically biotinylated by E. coli biotin ligase (BirA) 3 . In this work, we addressed QD multi-valency and minimized the size of biomolecule-conjugated QDs (Fig. 1a).QDs that are 20-30 nm can impair trafficking of proteins to which they are attached and restrict access to crowded cellular locations such as synapses 4 . A large fraction of QD size comes from the passivating layer, often a polyacrylic acid polymer or phospholipid micelle 2 . It is challenging to reduce the passivating layer without also increasing nonspecific interactions between QDs and cells, increasing QD self-aggregation and degrading quantum yield 5 . We 1 online) based on a dihydrolipoic acid (DHLA) head group to chelate the ZnCdS shell, 8 ethylene glycol (PEG) units to protect from nonspecific interactions, and a carboxylic acid tail to allow covalent coupling to biomolecules and to confer electrophoretic mobility 6,7 . When we used this DHLA-PEG 8 -CO 2 H ligand to coat 605 nm-emitting CdSe-ZnCdS QD cores (Supplementary Methods online), the resulting small QDs (sQDs) had a hydrodynamic diameter of 11.1 ± 0.1 nm, not much larger than an immunoglobulin gamma antibody (9.7 ± 0.1 nm; Supplementary Fig. 2a online). The quantum yield remained high (~40%), and the QDs were stable and monodispersed in PBS ( Supplementary Fig. 2).We tested whether the reduction in QD size improved access of the QDs to neuronal synapses. We fused the rat GluR2 subunit of the AMPA-type glutamate receptor, which localizes to postsynaptic membranes, to a 15-amino-acid `acceptor ...

We present a new class of polymeric ligands for quantum dot (QD) water solubilization to yield biocompatible and derivatizable QDs with compact size (~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 (RAFT)-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 non-specific 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.

The spectral linewidth of an ensemble of fluorescent emitters is dictated by a combination of the single emitter linewidths and sample inhomogeneities. For semiconductor nanocrystals, efforts to tune ensemble linewidths for optical applications have focused primarily on eliminating sample inhomogeneities because conventional single-molecule methods cannot reliably build accurate ensemble-level statistics for single-particle linewidths. Photon-correlation Fourier spectroscopy in solution (S-PCFS) offers a unique approach to investigating single-nanocrystal spectra with large sample statistics, without user selection bias, with high signal-to-noise ratios, and at fast timescales. With S-PCFS, we directly and quantitatively deconstruct the ensemble linewidth into contributions from the average single-particle linewidth and from sample inhomogeneity. We demonstrate that single-particle linewidths vary significantly from batch to batch and can be synthetically controlled. These findings crystallize our understanding of the synthetic challenges facing underdeveloped nanomaterials such as InP and InAs core/shell particles and introduce new avenues for the synthetic optimization of fluorescent nanoparticles.