Lifetime-Based
ApplicationsUsing Square and SETA dyes
Lifetime-Based
ApplicationsIn the last twenty years, fluorescence has become a powerful tool for the study of biochemical and biological processes. Some of the parameters that are utilized are fluorescence intensity, fluorescence polarization, fluorescence energy transfer and fluorescence lifetime. The fluorescence lifetime of a florescent molecule is defined as the average time the molecule spends in the excited state before it returns to the ground state by emitting a photon. Typical fluorescence lifetimes for common fluorophores such as fluoresceins and rhodamines are in the order of a few ns.
The fluorescence lifetimes of several Square and SETA Dyes have been measured. After covalent binding to a protein, the mean fluorescence decay time of the Square and SETA Dyes increases on average about five times (e.g. from 0.5 to 2.5 ns). In contrast, the mean lifetime of Cy5 increases only from 1 to 1.33 ns upon covalent binding to protein (Table 1). The intensity decays of Cy5 are less distinct and indicate that Cy5 is less sensitive to the environment than the related Square and SETA Dyes.
Table 1. Fluorescence Lifetime
|
Compound |
Environment |
Average Lifetime, τ [ns] |
|
Square-635 |
Water |
0.5 |
|
Square-635 |
Protein |
2.5 |
|
Cy5 |
Water |
1.0 |
|
Cy5 |
Protein |
1.3 |
Because the fluorescence lifetime of some fluorescent molecules is highly dependent on their environment it can function as a parameter to observe a variety of interesting molecular binding events such as receptor-ligand binding or antigen-antibody interactions.
The lifetime-change of Square and SETA Dyes upon binding to proteins can be utilized for homogeneous Lifetime-based Binding Assays (LBA). In these assays Square- and SETA-labeled and unlabeled antigens would compete for the binding sites on the antibodies and fluorescence lifetime can be used as a sensitive parameter to trace the binding event. Like in Fluorescence Polarization Assays LBAs require only a single label. Unlike Florescence Resonance Energy Transfer (FRET)-type assays, LBAs are not spatially restricted. The assay format would work on large biomolecules or cells as well as on smaller molecules given the binding event is accompanied by a change in fluorescence lifetime.