Azadioxatriangulenium: A long fluorescence lifetime fluorophore for large biomolecule binding assay

Thomas Just Sørensen, Erling Thyrhaug, Mariusz Szabelski, Rafal Luchowski, Ignacy Gryczynski, Zygmunt Gryczynski, Bo W. Laursen

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

Original languageEnglish
Article number025001
JournalMethods and Applications in Fluorescence
Volume1
Issue number2
DOIs
StatePublished - 1 Jun 2013

Fingerprint

Fluorophores
Biomolecules
Assays
Anisotropy
Fluorescence
Coloring Agents
life (durability)
fluorescence
Dyes
anisotropy
dyes
Labels
rabbits
molecular weight
Immunoglobulin G
Molecular weight
Barreling
bioassay
immunoassay
Bioassay

Cite this

Sørensen, Thomas Just ; Thyrhaug, Erling ; Szabelski, Mariusz ; Luchowski, Rafal ; Gryczynski, Ignacy ; Gryczynski, Zygmunt ; Laursen, Bo W. / Azadioxatriangulenium : A long fluorescence lifetime fluorophore for large biomolecule binding assay. In: Methods and Applications in Fluorescence. 2013 ; Vol. 1, No. 2.
@article{06df3fd259954a09857b500e042d1ed5,
title = "Azadioxatriangulenium: A long fluorescence lifetime fluorophore for large biomolecule binding assay",
abstract = "Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75{\%}, and an increase in the steady-state anisotropy of 18{\%}. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.",
author = "S{\o}rensen, {Thomas Just} and Erling Thyrhaug and Mariusz Szabelski and Rafal Luchowski and Ignacy Gryczynski and Zygmunt Gryczynski and Laursen, {Bo W.}",
year = "2013",
month = "6",
day = "1",
doi = "10.1088/2050-6120/1/2/025001",
language = "English",
volume = "1",
journal = "Methods and applications in fluorescence",
issn = "2050-6120",
publisher = "IOP Publishing Ltd.",
number = "2",

}

Azadioxatriangulenium : A long fluorescence lifetime fluorophore for large biomolecule binding assay. / Sørensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

In: Methods and Applications in Fluorescence, Vol. 1, No. 2, 025001, 01.06.2013.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Azadioxatriangulenium

T2 - A long fluorescence lifetime fluorophore for large biomolecule binding assay

AU - Sørensen, Thomas Just

AU - Thyrhaug, Erling

AU - Szabelski, Mariusz

AU - Luchowski, Rafal

AU - Gryczynski, Ignacy

AU - Gryczynski, Zygmunt

AU - Laursen, Bo W.

PY - 2013/6/1

Y1 - 2013/6/1

N2 - Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

AB - Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

UR - http://www.scopus.com/inward/record.url?scp=84875196200&partnerID=8YFLogxK

U2 - 10.1088/2050-6120/1/2/025001

DO - 10.1088/2050-6120/1/2/025001

M3 - Article

AN - SCOPUS:84875196200

VL - 1

JO - Methods and applications in fluorescence

JF - Methods and applications in fluorescence

SN - 2050-6120

IS - 2

M1 - 025001

ER -