Reduction of Multiexponential Background in Fluorescence with Phase-Sensitive Detection

Abstract

Fluorescence detection in a complex matrix often requires the reduction of background signals. Fluorescence lifetime-based methods have been an effective approach to suppressing the undesired background, especially when substantial spectral overlap precludes optical filtering. We introduce a methodology of suppressing a fluorescence background that contains multiple species for the quantification of a single fluorescent analyte. The method is based on the recognition that the multiexponential decay of the fluorescence background can be described by a single phase angle when the sample is excited by a sinusoidally modulated beam. The reduction of the total background is accomplished by setting the detector 90 degrees out of phase from the total background fluorescence. The background reduction is demonstrated with ternary samples containing pyrene (τ = 18.2 ns), anthracene (τ = 4.5 ns), and 1,4-bis(5-phenyl-2-oxazolyl)benzene (POPOP, τ = 1.35 ns). The component with the shortest lifetime (POPOP) is successfully analyzed after the background from longer-lived components (pyrene and anthracene) is reduced. The phase-sensitive detection enables simultaneous suppression of background fluorescence with lifetimes longer and shorter than the analyte, as demonstrated by the quantification of anthracene in a background composed of pyrene and POPOP. These samples present situations that cannot be treated by traditional methods of fluorescence background reduction. An effective fluorescence spectrum of the analyte is collected after the multiexponential background is eliminated. The method is especially useful when the total concentration of the background displays significant fluctuations. When the variations in the background fluorescence intensity are comparable or exceed the analyte fluorescence, the analyte cannot be quantified with steady state fluorescence measurements. The phase-sensitive method, however, effectively quantifies the analyte with excellent linearity in calibration.