title: Detecting kallikrein proteolytic activity with peptide-quantum dot nanosensors.
abstract: Contamination and adulterants in both naturally derived and synthetic drugs pose a serious threat to the worldwide medical community. Developing rapid and sensitive sensors/devices to detect these hazards is thus a continuing need. We describe a hydrophilic semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) nanosensor for monitoring the activity of kallikrein, a key proteolytic enzyme functioning at the initiation of the blood clotting cascade. Kallikrein is also activated by the presence of an oversulfated contaminant recently found in preparations of the drug heparin. Quantitatively monitoring the activity of this enzyme within a nanosensor format has proven challenging because of inherent steric and kinetic considerations. Our sensor is designed around a central QD donor platform which displays controlled ratios of a modular peptidyl substrate. This peptide, in turn, sequentially expresses a terminal oligohistidine motif that mediates the rapid self-assembly of peptides to the QD surface, a linker-spacer sequence to extend the peptide away from the QD surface, a kallikrein recognized-cleavage site, and terminates in an acceptor dye-labeling site. Hydrophilic QDs prepared with compact, zwitterionic surface coatings were first evaluated for their ability to self-assemble the dye-labeled peptide substrates. An optimized two-step protocol was then utilized where high concentrations of peptide were initially digested with purified human kallikrein and samples collected at distinct time points were subsequently diluted into QD-containing solutions for assaying. This sensor provided a quantitative FRET-based readout for monitoring kallikrein activity and comparison to a calibration curve allowed estimation of the relevant Michaelis-Menten kinetic descriptors. The results further suggest that almost any protease should be amenable to a QD-based FRET assay format with appropriate design considerations.
authors.affiliation: Center for Bio/Molecular Science and Engineering, Code 6900, and ‡Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory , Washington, DC, 20375 United States.
authors.forename: Joyce C
authors.initials: JC
authors.lastname: Breger
authors.forename: Kim E
authors.initials: KE
authors.lastname: Sapsford
authors.forename: Jessica
authors.initials: J
authors.lastname: Ganek
authors.forename: Kimihiro
authors.initials: K
authors.lastname: Susumu
authors.forename: Michael H
authors.initials: MH
authors.lastname: Stewart
authors.forename: Igor L
authors.initials: IL
authors.lastname: Medintz
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007610:Kallikreins / Q000032:analysis*; D010455:Peptides / Q000737:chemistry*; D059748:Proteolysis; D045663:Quantum Dots / Q000737:chemistry*; D012666:Semiconductors
chemical_list: D010455:Peptides; D007610:Kallikreins