Sensing Caspase 3 Activity with Quantum Dot−Fluorescent Protein Assemblies

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Abstract
We demonstrate the use of a hybrid fluorescent protein semiconductor quantum dot (QD) sensor capable of specifically monitoring caspase 3 proteolytic activity. mCherry monomeric red fluorescent protein engineered to express an N-terminal caspase 3 cleavage site was ratiometrically self-assembled to the surface of QDs using metal-affinity coordination. The proximity of the fluorescent protein to the QD allows it to function as an efficient fluorescence resonance energy transfer acceptor. Addition of caspase 3 enzyme to the QD−mCherry conjugates specifically cleaved the engineered mCherry linker sequence, altering the energy transfer with the QD and allowing quantitative monitoring of proteolytic activity. Inherent advantages of this sensing approach include bacterial expression of the protease substrate in a fluorescently appended form, facile self-assembly to QDs, and the ability to recombinantly modify the substrate to target other proteases of interest.