Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

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• 作者：Duane E. Prasuhn ; Anne Feltz ; Juan B. Blanco-Canosa ; Kimihiro Susumu ; Michael H. Stewart ; Bing C. Mei ; Aleksey V. Yakovlev ; Christina Loukou ; Jean-Maurice Mallet ; Martin Oheim ; Philip E. Dawson ; Igor L. Medintz
• 刊名：ACS Nano
• 出版年：2010
• 出版时间：September 28, 2010
• 年：2010
• 卷：4
• 期：9
• 页码：5487-5497
• 全文大小：619K
• 年卷期：0
• ISSN：1936-086X

文摘

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.