Lifetime-based optical sensing of pH using resonance energy transfer in sol-gel sensors

We describe the fabrication and testing of an optical pH sensor based on fluorescence lifetime measurements and sol-gel technology. The sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET), from a pH-insensitive donor to a pH-sensitive acceptor. The pH-dependent increase in the bromothymol blue acceptor absorbance results in increased energy transfer, reducing the lifetime of the Texas red hydrazide donor. The lifetimes were measured by the phase and modulation of the emission, relative to the modulated incident light, and were found to be insensitive to the total signal level and fluctuations in light intensity. However, the present sensors are sensitive to salt concentration and/or ionic strength. Importantly, this sol-gel sensor is not fragile, providing stable readings for days and can be repeatedly autoclaved without loss of sensitivity to pH. The use of FRET as the pH transduction mechanism can be reliably extended to longer wavelengths, and allows the future use of laser diode excitation sources.