Ultrasensitive multiplexed biosensors using FRET in
spectral and temporal resolution
Niko Hildebrandt
NanoBioPhotonics, Institut d’Electronique Fondamentale, Université Paris-Sud, Orsay, France
Förster Resonance Energy Transfer (FRET) is a distance dependent non-radiative
energy transfer between an excited donor and a ground state acceptor molecule or particle.
As FRET occurs at distances between ca. 1 and 20 nm, it is ideally suited for the
investigation of biological interactions, concentrations and/or distances. The combination of
two unique FRET fluorophores, namely terbium complexes (Tbs) and semiconductor
quantum dots (QDs), in time-resolved FRET offers many advantages for multiplexed
biosensing [1-3]. Tbs provide long excited state lifetimes (up to several milliseconds), which
allow efficient FRET to QD acceptors and nearly complete suppression of fluorescence
background by time-gated detection. Spectral and temporal multiplexing becomes possible
using different QD colors and the long Tb luminescence decay times, respectively. Moreover,
so-called FRET relays (multistep FRET from Tb-to-QD-to-dye) allow for an even higher
flexibility in the development of sophisticated biosensors.
This lecture will present various Tb- and QD-based homogeneous multiplexed FRET
biosensors using a variety of biological recognition molecules, such as antibodies [4,5],
nanobodies [6], peptides and oligonucleotides [7,8], for multiplexed detection of tumor
markers, proteases, DNA and RNA. The application of time-resolved FRET in imaging using
optically multifunctional antibodies, FRET investigation of nanomedicine crossing the bloodbrain-barrier and FRET-based molecular logic devices will also be discussed.
The FRET toolbox contains a myriad of different fluorophores and a large variety of
steady-state and time-resolved spectroscopy and microscopy detection technologies. The
efficient combination with in-vitro or in-vivo biological systems provides a highly sensitive
method for multiplexed detection of biomolecular interactions. Therefore, FRET is a powerful
tool for clinical prognostics and diagnostics and for a better understanding of biomolecular
structures and dynamics.
REFERENCES
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