A fluorescence ratiometric detection scheme for ammonium ions based on the solvent sensitive dye MC 540

Principles of wavelength ratiometric evaluation of fluorescence have been extensively used to cancel out and reference sensor response not associated with the analyte. Main limitations of this technique include wavelength ratiometric properties inherent only to a restricted number of solute specific fluorescent dyes. This study presents a dual-excitation/dual-emission ratiometric detection scheme applied on a two-phase coextraction based fluorosensor. Rather than utilizing spectral shifts associated with various complexation and acid–base equilibria of solute specific indicators, we made use of the fluorescence shift induced by the phase transfer of the solvent sensitive dye merocyanine 540 (MC 540) upon ammonium–nonactin coextraction in a hydrogel–ether emulsion. The two-phase ratiometric fluorosensor was exposed to conditions frequently reported to alter performance of fluorescence based optical sensors. Sensor response was unaffected by O2 concentrations, and signals caused by variations in excitation light intensity and pH were found more or less completely eliminated by the ratiometric procedure. Selectivity towards NH4+ and possible interferences from K+ were more accurately evaluated using the dual-excitation/dual-emission ratio, compared to a single-excitation/single-emission fluorescence scheme. It was not possible to eliminate artifacts associated with fluctuations in temperature, effective MC 540 concentrations and sample ionic strength by the ratiometric reference scheme, most likely due to monomer–dimer complexation of the indicator dye and the formation of larger aggregates at concentrations exceeding the critical salt concentration of MC 540. Principles described for a two-phase ratiometric detection procedure associated with solvent sensitive dyes rather than solute specific fluorescent dyes may provide a powerful tool for direct solute detection. Thus, the limited versatility of wavelength ratiometric fluorescence detection to specially designed compounds with wavelength ratiometric properties directly linked to interactions with the analyte, may be overcome.