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.
Kim
Yongwook
Y
Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Korea.
Kim
Wonjung
W
Yoon
Hye-Joo
HJ
Shin
Seung Koo
SK
D001710:Biotin / Q000737:chemistry*; D019187:Cadmium Compounds / Q000737:chemistry; D002254:Carbonates / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006900:Hydroxylation; D015394:Molecular Structure; D049329:Nanostructures / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D020005:Propanols / Q000737:chemistry; D018036:Selenium Compounds / Q000737:chemistry; D012666:Semiconductors; D012995:Solubility; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry; D015032:Zinc / Q000737:chemistry; D017967:Zinc Compounds / Q000737:chemistry
C518434:3-mercaptopropanol; D019187:Cadmium Compounds; D002254:Carbonates; D005456:Fluorescent Dyes; D010455:Peptides; D020005:Propanols; D018036:Selenium Compounds; D013388:Succinimides; D013438:Sulfhydryl Compounds; D013440:Sulfides; D017967:Zinc Compounds; C085786:succinimidyl carbonate; D001710:Biotin; D004247:DNA; C058667:cadmium selenide; D015032:Zinc; C031238:zinc sulfide