title: Reactive semiconductor nanocrystals for chemoselective biolabeling and multiplexed analysis.
abstract: Effective biological application of nanocrystalline semiconductor quantum dots continues to be hampered by the lack of easily implemented and widely applicable labeling chemistries. Here, we introduce two new orthogonal nanocrystal bioconjugation chemistries that overcome many of the labeling issues associated with currently utilized approaches. These chemistries specifically target either (1) the ubiquitous amines found on proteins or (2) thiols present in either antibody hinge regions or recombinantly introduced into other proteins to facilitate site-specific labeling. The amine chemistry incorporates aniline-catalyzed hydrazone bond formation, while the sulfhydryl chemistry utilizes nanocrystals displaying surface activated maleimide groups. Both reactive chemistries are rapidly implemented, yielding purified nanocrystal-protein bioconjugates in as little as 3 h. Following initial characterization of the nanocrystal materials, the wide applicability and strong multiplexing potential of these chemistries are demonstrated in an array of applications including immunoassays, immunolabeling in both cellular and tissue samples, in vivo cellular uptake, and flow cytometry. Side-by-side comparison of the immunolabeled cells suggested a functional equivalence between results generated with the amine and thiol-labeled antibody-nanocrystal bioconjugates in that format. Three-color labeling was achieved in the cellular uptake format, with no significant toxicity observed while simultaneous five-color labeling of different epitopes was demonstrated for the immunolabeled tissue sample. Novel labeling applications are also facilitated by these chemistries, as highlighted by the ability to directly label cellular membranes in adherent cell cultures with the thiol-reactive chemistry.
authors.affiliation: eBioscience, Inc., 10255 Science Center Drive, San Diego, California 92121, USA. travis.jennings@ebioscience.com
authors.forename: Travis L
authors.initials: TL
authors.lastname: Jennings
authors.forename: Sara G
authors.initials: SG
authors.lastname: Becker-Catania
authors.forename: Robert C
authors.initials: RC
authors.lastname: Triulzi
authors.forename: Guoliang
authors.initials: G
authors.lastname: Tao
authors.forename: Bradley
authors.initials: B
authors.lastname: Scott
authors.forename: Kim E
authors.initials: KE
authors.lastname: Sapsford
authors.forename: Samantha
authors.initials: S
authors.lastname: Spindel
authors.forename: Eunkeu
authors.initials: E
authors.lastname: Oh
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Jain
authors.forename: James B
authors.initials: JB
authors.lastname: Delehanty
authors.forename: Duane E
authors.initials: DE
authors.lastname: Prasuhn
authors.forename: Kelly
authors.initials: K
authors.lastname: Boeneman
authors.forename: W Russ
authors.initials: WR
authors.lastname: Algar
authors.forename: Igor L
authors.initials: IL
authors.lastname: Medintz
mesh_terms: D000588:Amines / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D003116:Color; D004768:Enterotoxins / Q000032:analysis; D005434:Flow Cytometry; D006801:Humans; D007118:Immunoassay; D007150:Immunohistochemistry; D045663:Quantum Dots; D012666:Semiconductors; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D000588:Amines; D004768:Enterotoxins; D013438:Sulfhydryl Compounds; C031818:enterotoxin B, staphylococcal