Bioconjugation of hydroxylated semiconductor nanocrystals and background-free biomolecule detection

Semiconductor nanocrystals emerge as fluorescent bioprobes for long-term imaging and multiplexed assays; however, there is a challenge of making nanocrystals biocompatible without nonspecific bindings to background molecules. Here, we report the bioconjugation of small-sized, hydroxylated nanocrystals, enabling highly sensitive detection of various biomolecules with little or no nonspecific binding. Zinc-blende CdSe/ZnS nanocrystals were passivated with 3-mercapto-1-propanol (MPO) and activated to amine-reactive succinimidyl carbonate derivatives and then covalently linked to amine-functionalized biomolecules, such as biotin, DNA, and hemagglutinin peptide, by forming a carbamate linkage. Tris(3-hydroxypropyl)phosphine was added to stabilize the zinc-thiolate linkage on nanocrystals. For comparison, CdSe/ZnS nanocrystals were passivated with 3-mercaptopropionic acid (MPA) and conjugated with aminated biomolecules. Photoluminescence properties of organic, water-soluble, and bioconjugated nanocrystals were characterized. Significantly, the bioconjugates of hydroxylated (CdSe/ZnS-MPO) nanocrystals exhibited brighter photoluminescence with longer lifetimes than those of carboxylated (CdSe/ZnS-MPA) nanocrystals. Specific and nonspecific interactions between nanocrystals and biomolecules were examined by incubating nanocrystal-bioconjugates with avidin-agarose beads, anti-hemagglutinin affinity matrix, DNA glass slide, or avidin glass slide. CdSe/ZnS-MPO nanocrystals showed little or no nonspecific binding to both agarose beads and glass slides, whereas CdSe/ZnS-MPA nanocrystals exhibited significant nonspecific binding due to the carboxyl-amine interactions. Notably, CdSe/ZnS-MPO bioconjugates yield about 20 times brighter images than CdSe/ZnS-MPA bioconjugates in both DNA hybridization and biotin-streptavidin binding. Hydroxylated nanocrystals stabilized by hydroxyphosphine are small, bright, and photostable in physiological conditions, and their bioconjugates afford background-free detection of specific biomolecular interactions, positioning them for an ideal fluorescent probe to biological settings.