Anisotropy-based sensing with reference fluorophores

Joseph R. Lakowicz, Ignacy Gryczynski, Zygmunt Gryczynski, Jonathan D. Dattelbaum

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.

Original languageEnglish
Pages (from-to)397-405
Number of pages9
JournalAnalytical Biochemistry
Volume267
Issue number2
DOIs
StatePublished - 15 Feb 1999

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