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Current Pharmaceutical Biotechnology


ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Single Molecule Studies of Multiple-Fluorophore Labeled Antibodies. Effect of Homo-FRET on the Number of Photons Available Before Photobleaching

Author(s): Rafal Luchowski, Evgenia G. Matveeva, Ignacy Gryczynski, Ewald A. Terpetschnig, Leonid Patsenker, Gabor Laczko, Julian Borejdo and Zygmunt Gryczynski

Volume 9, Issue 5, 2008

Page: [411 - 420] Pages: 10

DOI: 10.2174/138920108785915094

Price: $65


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 Forster-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.

Keywords: Multiple-Fluorophore, Antibodies, Photons, Photobleaching, single molecule detection (SMD), Forster-type energy-transfer (FRET), fluorescence

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