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Protease expression, activity, and inhibition play crucial roles in a multitude of biological processes; however, these three aspects of their function are difficult for any one bioanalytical probe to measure. To help address this challenge, we report a multifunctional concentric Förster resonance energy transfer (FRET) configuration that combines two modes of biorecognition using aptamers and peptide substrates coassembled to a central semiconductor quantum dot (QD). The aptamer is sensitive to the concentration of protease and the peptide is sensitive to its hydrolytic activity. The role of the QD is to serve as a nanoscale scaffold and initial donor for energy transfer with both Cyanine 3 (Cy3) and Alexa Fluor 647 (A647) fluorescent dyes associated with the aptamer and peptide, respectively. Using thrombin as a model protease, we show that a ratiometric analysis of the emission from the QD, Cy3, and A647 permits discrimination between thrombin and thrombin-like activity, and distinguishes between active, reversibly inhibited, and irreversibly inhibited thrombin. Reliable quantitative results were obtained from a kinetic analysis of the changes in FRET. This concentric FRET format, which capitalizes on both the physical and optical properties of QDs, should be adaptable to other protease targets for which both peptide substrates and binding aptamers are known. It is thus expected to become valuable a tool for the real-time analysis of protease activity and regulation.

Quantum Dot-Based Concentric FRET Configuration for the Parallel Detection of Protease Activity and Concentration