Novel methods for fluorescence sensing

We describe a new method for fluorescence sensing based on measurements of the steady state polarization of an analyte-sensitive fluorophore in the presence of a reference fluorophore with known polarization. The basic concept is that the polarization of a mixture reflects a weighted average of the polarization of the emitting species. By use of reference fluorophores the starting values can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the sensing fluorophore due to the analyte result in changes in the polarization of the combined emission. A wide dynamic range is available because of the freedom to select high or low starting polarization values. Polarization-based sensing was demonstrated for pH using 6-carboxy fluorescein. We also show that polarization sensing can be used for measurements of oxygen and glucose. Polarization sensing can have numerous applications in clinical and analytical chemistry.