1 research outputs found
Quantum Dots as FoĢrster Resonance Energy Transfer Acceptors of Lanthanides in Time-Resolved Bioassays
We
report a flexible and modular design for biosensors based on
exploiting semiconductor quantum dots (QDs) and their excellent FoĢrster
resonance energy transfer (FRET) acceptor properties along with the
long-lived fluorescent lifetimes of lanthanide donors. We demonstrate
the formatās wide application by developing a broad adenosine
diphosphate (ADP) sensor with quantitative and high-throughput capabilities
as a kinase/ATPase assay method. The sensor is based on a Terbium
(Tb)-labeled antibody (Ab) that selectively recognizes ADP versus
ATP. The Tb-labeled Ab (Ab-Tb) acts as a FRET donor to a QD, which
has an ADP modified His<sub>6</sub>-peptide conjugated to its surface
via metal-affinity coordination. This strategy of using self-assembly,
modified peptides to present antibody epitopes on QD surfaces is readily
transferable to other assays of interest. We utilize time-resolved
FRET (TR-FRET) to measure the amounts of Ab-Tb bound to the QD by
looking at the emission ratio of the QD and Tb in a time-gated manner,
minimizing background signal. With the addition of free ADP the antibody
is competitively separated from the QD and a change in the ratiometric
emission signal correlates with the free ADP concentration. The sensor
obtained a detection limit below 10 nM of free ADP and quantitation
limit of 35 nM ADP using 8 nM of sensor. Quantitative values were
obtained for a model enzyme (glucokinase) kinetics, as well as demonstrations
of the assays capability to distinguish enzyme inhibitors. We discuss
future outlooks and note areas for improvement in similar design strategies