Single molecule studies of multiple-fluorophore labeled antibodies. Effect of homo-FRET on the number of photons available before photobleaching

Rafal Lushowski, Evgenia G. Matveeva, Ignacy Gryczynski, Ewald A. Terpetschnig, Leoniel Patsenker, Gabor Laczko, Julian Borejdo, Zygmunt Gryczynski

Research output: Contribution to journalReview article

38 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)411-420
Number of pages10
JournalCurrent Pharmaceutical Biotechnology
Volume9
Issue number5
DOIs
StatePublished - 1 Dec 2008

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Photobleaching
Energy Transfer
Photons
Fluorescence
Antibodies
Chemical Phenomena
Observation
Physical Phenomena
Biological Phenomena
Computer Systems
Immunoassay
Proteins
Immunoglobulin G

Cite this

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title = "Single molecule studies of multiple-fluorophore labeled antibodies. Effect of homo-FRET on the number of photons available before photobleaching",
abstract = "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 F{\"o}rster-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.",
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Single molecule studies of multiple-fluorophore labeled antibodies. Effect of homo-FRET on the number of photons available before photobleaching. / Lushowski, Rafal; Matveeva, Evgenia G.; Gryczynski, Ignacy; Terpetschnig, Ewald A.; Patsenker, Leoniel; Laczko, Gabor; Borejdo, Julian; Gryczynski, Zygmunt.

In: Current Pharmaceutical Biotechnology, Vol. 9, No. 5, 01.12.2008, p. 411-420.

Research output: Contribution to journalReview article

TY - JOUR

T1 - Single molecule studies of multiple-fluorophore labeled antibodies. Effect of homo-FRET on the number of photons available before photobleaching

AU - Lushowski, Rafal

AU - Matveeva, Evgenia G.

AU - Gryczynski, Ignacy

AU - Terpetschnig, Ewald A.

AU - Patsenker, Leoniel

AU - Laczko, Gabor

AU - Borejdo, Julian

AU - Gryczynski, Zygmunt

PY - 2008/12/1

Y1 - 2008/12/1

N2 - 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.

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M3 - Review article

C2 - 18855695

AN - SCOPUS:58149279552

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EP - 420

JO - Current Pharmaceutical Biotechnology

JF - Current Pharmaceutical Biotechnology

SN - 1389-2010

IS - 5

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