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. 1998 Dec 15;70(24):5115-21.
doi: 10.1021/ac980876c.

Low-frequency modulation sensors using nanosecond fluorophores

J R Lakowicz  1 , F N Castellano, J D Dattelbaum, L Tolosa, G Rao, I Gryczynski
Affiliations

Low-frequency modulation sensors using nanosecond fluorophores

J R Lakowicz et al. Anal Chem. .

Abstract

We describe a new approach to fluorescence sensing based on a mixture of fluorophores, one of which is sensitive to the desired analyte. If a long-lifetime analyte-insensitive fluorophore is mixed with a short-lifetime analyte-sensitive fluorophore, the modulation of the emission at conveniently low frequencies becomes equal to the fractional fluorescence intensity of the sensing fluorophore. Under these conditions, the modulation can be used to determine the analyte concentration. This can be used with any fluorophore that changes intensity in response to analyte and does not require the sensing fluorophore to display a change in lifetime. The feasibility of modulation-based sensing was demonstrated using mixtures of 6-carboxyfluorescein and [Ru 2,2'-(bipyridyl)3]2+ as a pH sensor and of the calcium probe Fluo-3 and [Ru 2,2'-(bipyridyl)3]2+ as a calcium sensor.

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Figures

Figure 1.
Figure 1.
Emission spectra of the model sensor containing fluorescein in the internal aqueous phase and [Ru(bpy)3]2+ in an external PVA film.
Figure 2.
Figure 2.
Frequency-domain intensity decay of the fluorescein-[Ru(bpy)3]2+ model sensor.
Figure 3.
Figure 3.
Emission spectra of a pH sensor based on a mixture of 6-CF and [Ru(bpy)3]2+.
Figure 4.
Figure 4.
(Top) Frequency responses of the pH assay. (Bottom) Low-frequency modulation of the pH sensor.
Figure 5.
Figure 5.
Calculation curve of the pH sensor.
Figure 6.
Figure 6.
Emission spectra of a modulated calcium sensor based on Fluo-3 and [Ru(bpy)3]2+.
Figure 7.
Figure 7.
(Top) Modulation frequency response of the calcium assay. (Bottom) Phase angle frequency response of the calcium assay.
Figure 8.
Figure 8.
Phase and modulation calibration curves of the calcium sensor.
Chart 1.
Chart 1.
Schematic of the Sensors with a Nanosecond Fluorophore and [Ru(bpy)3]2+
See this image and copyright information in PMC

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