title: Quantum dot peptide biosensors for monitoring caspase 3 proteolysis and calcium ions.
abstract: The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD's surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis-Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed [3 + 2] azide-alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor solution increased the dyes fluorescence, altering the donor-acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.
authors.affiliation: Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, 4555 Overlook Ave., S.W.,Washington, D.C. 20375, USA.
authors.forename: Duane E
authors.initials: DE
authors.lastname: Prasuhn
authors.forename: Anne
authors.initials: A
authors.lastname: Feltz
authors.forename: Juan B
authors.initials: JB
authors.lastname: Blanco-Canosa
authors.forename: Kimihiro
authors.initials: K
authors.lastname: Susumu
authors.forename: Michael H
authors.initials: MH
authors.lastname: Stewart
authors.forename: Bing C
authors.initials: BC
authors.lastname: Mei
authors.forename: Aleksey V
authors.initials: AV
authors.lastname: Yakovlev
authors.forename: Christina
authors.initials: C
authors.lastname: Loukov
authors.forename: Jean-Maurice
authors.initials: JM
authors.lastname: Mallet
authors.forename: Martin
authors.initials: M
authors.lastname: Oheim
authors.forename: Philip E
authors.initials: PE
authors.lastname: Dawson
authors.forename: Igor L
authors.initials: IL
authors.lastname: Medintz
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis*; D053148:Caspase 3 / Q000378:metabolism*; D004738:Engineering; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D045663:Quantum Dots
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D053148:Caspase 3; D002118:Calcium