Photophysics of “Floppy” Dyads as Potential Biomembrane Probes

Abstract

In the study a dyad (C6 probe), constructed of two dyes with highly different hydrophobicities, was investigated by steady-state and time-resolved spectroscopic techniques in chloroform, methanol, and in phospholipid vesicles, respectively. The dyad was built on two dyes: the lipophilic benzo[a]pyrene (BaP) and the hydrophilic sulforhodamine B (SRB). The dyes were linked via a short, but flexible alkyl chain (six C-atoms). Based on their spectroscopic properties, BaP and SRB showed a very efficient non-radiative resonance energy transfer in solution. Incorporation into a lipid bilayer limited the relative flexibility (degree of freedom) between donor and acceptor and was used for the investigation of fundamental photophysical aspects (especially of FRET) as well as to elucidate the potential of the dyad to probe the interface of vesicles (or cells). The location of the two dyes in vesicles and their respective accessibility for interactions with dye-specific antibodies was investigated. Based on the alteration of the anisotropy, on the rotational correlation time as well as on the diffusion coefficient the incorporation of the C6 probe into the vesicles was evaluated. Especially the limitation in the relative movements of the two dyes was considered and used to differentiate between potential parameters, that influence the energy transfer in the dyad. Transient absorption spectroscopy (TAS) and pulsed-interleave single molecule fluorescence experiments were performed to better understand the intramolecular interactions in the dyad. Finally, in a showcase for a biosensing application of the dyads, the binding of an SRB-specific antibody was investigated when the dyad was incorporated in vesicles. Graphical Abstract.

Antibody Binding at the Liposome–Water Interface: A FRET Investigation toward a Liposome-Based Assay

“…In the presence of liposomes, the rotation of mm239-DTT was distinctly slower (Figure , left). In the data analysis the “wobble-in-a-cone” model was used. − Based on this model the rotational correlation time calculated for the anisotropy decay kinetics increased to Φ = 1.42 ns, and a semicone angle of θ k = 48° was found. The increase of the steady-state anisotropy as well as the rotational correlation time of the dye in the presence of liposomes showed once more that the movement of the dye was restricted and underlined its incorporation into the lipid phase of the liposome.…”