Bioconjugation of Hydroxylated Semiconductor Nanocrystals and Background-Free Biomolecule Detection
journal contributionposted on 2010-07-21, 00:00 authored by Yongwook Kim, Wonjung Kim, Hye-Joo Yoon, Seung Koo Shin
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.
DNA hybridizationglass slidesCdSeDNA glass slidecarbamate linkageagarose beadsPhotoluminescence propertiesavidin glass slidenanocrystals biocompatiblebackground moleculesMPAMPObiomolecular interactionsHydroxylated Semiconductor NanocrystalsHydroxylated nanocrystalsmultiplexed assaysbioconjugated nanocrystalshemagglutinin peptide20 timesbindinghydroxylated nanocrystalsaminated biomolecules