Multiplexed Tracking of
Protease Activity Using a
Single Color of Quantum Dot Vector and a Time-Gated Förster
Resonance Energy Transfer Relay
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Abstract
Semiconductor quantum dots (QDs) are attractive probes
for optical
sensing and imaging due to their unique photophysical attributes and
nanoscale size. In particular, the development of assays and biosensors
based on QDs and Förster resonance energy transfer (FRET) continues
to be a prominent focus of research. Here, we demonstrate the application
of QDs as simultaneous donors and acceptors in a time-gated FRET relay
for the multiplexed detection of protease activity. In contrast to
the current state-of-the-art, which uses multiple colors of QDs, multiplexing
was achieved using only a single color of QD. The other constituents
of the FRET relay, a luminescent terbium complex and fluorescent dye,
were assembled to QDs via peptides that were selected as substrates
for the model proteases trypsin and chymotrypsin. Loss of prompt FRET
between the QD and dye signaled the activity of chymotrypsin; loss
of time-gated FRET between the terbium and QD signaled the activity
of trypsin. We applied the FRET relay in a series of quantitative,
real-time kinetic assays of increasing biochemical complexity, including
multiplexed sensing, measuring inhibition in a multiplexed format,
and tracking the proteolytic activation of an inactive pro-protease
to its active form in a coupled, multienzyme system. These capabilities
were derived from a ratiometric analysis of the two FRET pathways
in the relay and permitted extraction of initial reaction rates, enzyme
specificity constants, and apparent inhibition constants. This work
adds to the growing body of research on multifunctional nanoparticles
and introduces multiplexed sensing as a novel capability for a single
nanoparticle vector. Furthermore, the ability to track both enzymes
within a coupled biological system using one vector represents a significant
advancement for nanoparticle-based biosensing. Prospective applications
in biochemical research, applied diagnostics, and drug discovery are
discussed