TY  - JOUR
A1  - Hoang, Hoa T.
A1  - Haubitz, Toni
A1  - Kumke, Michael Uwe
T1  - Photophysics of "Floppy" Dyadsas Potential Biomembrane Probes
JF  - Journal of fluorescence
N2  - 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.
KW  - Forster resonance energy transfer(FRET)
KW  - Antibody binding
KW  - Vesicles
KW  - Anisotropy
KW  - Fluorescence correlation
KW  - Single molecule fluorescence
KW  - Transient absorption
KW  - Pulsed interleaved excitation
Y1  - 2018
U6  - https://doi.org/10.1007/s10895-018-2286-4
SN  - 1053-0509
SN  - 1573-4994
VL  - 28
IS  - 5
SP  - 1225
EP  - 1237
PB  - Springer
CY  - New York
ER  - 
TY  - JOUR
A1  - Hoang, Hoa T.
A1  - Mertens, Monique
A1  - Wessig, Pablo
A1  - Sellrie, Frank
A1  - Schenk, Jörg A.
A1  - Kumke, Michael Uwe
T1  - Antibody Binding at the Liposome-Water Interface
BT  - a FRET Investigation toward a Liposome-Based Assay
JF  - ACS Omega
N2  - Different signal amplification strategies to improve the detection sensitivity of immunoassays have been applied which utilize enzymatic reactions, nanomaterials, or liposomes. The latter are very attractive materials for signal amplification because liposomes can be loaded with a large amount of signaling molecules, leading to a high sensitivity. In addition, liposomes can be used as a cell-like "bioscaffold" to directly test recognition schemes aiming at cell-related processes. This study demonstrates an easy and fast approach to link the novel hydrophobic optical probe based on [1,3]dioxolo[4,5-f]-[1,3]benzodioxole (DBD dye mm239) with tunable optical properties to hydrophilic recognition elements (e.g., antibodies) using liposomes for signal amplification and as carrier of the hydrophobic dye. The fluorescence properties of mm239 (e.g., long fluorescence lifetime, large Stokes shift, high photostability, and high quantum yield), its high hydrophobicity for efficient anchoring in liposomes, and a maleimide bioreactive group were applied in a unique combination to build a concept for the coupling of antibodies or other protein markers to liposomes (coupling to membranes can be envisaged). The concept further allowed us to avoid multiple dye labeling of the antibody. Here, anti-TAMRA-antibody (DC7-Ab) was attached to the liposomes. In proof-of-concept, steady-state as well as time-resolved fluorescence measurements (e.g., fluorescence depolarization) in combination with single molecule detection (fluorescence correlation spectroscopy, FCS) were used to analyze the binding interaction between DC7-Ab and liposomes as well as the binding of the antigen rhodamine 6G (R6G) to the antibody. Here, the Forster resonance energy transfer (FRET) between mm239 and R6G was monitored. In addition to ensemble FRET data, single-molecule FRET (PIE-FRET) experiments using pulsed interleaved excitation were used to characterize in detail the binding on a single-molecule level to avoid averaging out effects.
KW  - energy-transfer
KW  - immunoassay
KW  - complexes
KW  - probes
Y1  - 2018
U6  - https://doi.org/10.1021/acsomega.8b03016
SN  - 2470-1343
VL  - 3
IS  - 12
SP  - 18109
EP  - 18116
PB  - American Chemical Society
CY  - Washington
ER  -