Quantum dot-fluorescent protein pairs as fluorescence resonance energy transfer pairs

Abstract

Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide. The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.