Single molecule studies of multiple-fluorophore labeled antibodies. Effect of homo-FRET on the number of photons available before photobleaching.
Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the behavior of individual and complex molecular systems in real time. SMD enables the characterization of complex molecular interactions and reveals basic physical phenomena underlying chemical and biological processes. We present here a systematic study of the quenching efficiency of Förster-type energy-transfer (FRET) for multiple fluorophores immobilized on a single antibody. We simultaneously monitor the fluorescence intensity, fluorescence lifetime, and the number of available photons before photobleaching as a function of the number of identical emitters bound to a single IgG antibody. The detailed studies of FRET between individual fluorophores reveal complex through-space interactions. In general, even for two or three fluorophores immobilized on a single protein, homo-FRET interactions lead to an overall non-linear intensity increase and shortening of fluorescence lifetime. Over-labeling of protein in solution (ensemble) results in the loss of fluorescence signal due to the self-quenching of fluorophores making it useless for assays applications. However, in the single molecule regime, over-labeling may bring significant benefits in regards to the number of available photons and the overall survival time. Our investigation reveals possibilities to significantly increase the observation time for a single macromolecule allowing studies of macromolecular interactions that are not obscured by ensemble averaging. Extending the observation time will be crucial for developing immunoassays based on single-antibody.
Center for Commercialization of Fluorescence Technologies, Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76106, USA.
Rafal
R
Luchowski
Evgenia G
EG
Matveeva
Ignacy
I
Gryczynski
Ewald A
EA
Terpetschnig
Leonid
L
Patsenker
Gabor
G
Laczko
Julian
J
Borejdo
Zygmunt
Z
Gryczynski
D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015336:Molecular Probe Techniques
D000936:Antigen-Antibody Complex