FLUORESCENCE LIFETIME-BASED SENSING OF PH, CA-2(+), K+ AND GLUCOSE

Optical sensing of analytes is often accomplished using fluorescence detection. Quantitation is usually performed by measuring the fluorescence of a probe whose intensity changes in response to the analyte of interest. In the present report we describe sensing based on changes in lifetime or decay time of the probe. Such a sensing scheme is advantageous because, in contrast to intensity measurements, the decay times are mostly independent of intensity changes due to light losses, photobleaching or probe washout. Present technology allows nanosecond (ns) decay times to be measured by the phase-modulation method with inexpensive and robust instrumentation. In particular, we describe lifetime-based pH sensing using a green 543 nm He Ne laser, sensing of Ca2+ using 514 nm excitation and a Ca2+ competitive assay for glucose using an energy-transfer mechanism. We also discuss emerging applications of phase-modulation method sensing to cellular imaging and flow cytometry.