Method for simultaneous luminescence sensing of two species using optical probes of different decay time, and its application to an enzymatic reaction at varying temperature

Chemical sensing, imaging and microscopy based on the use of fluorescent probes has so far been limited almost exclusively to the detection of a single parameter at a time. We present a scheme that can overcome this limitation by enabling optical sensing of two parameter simultaneously and even at identical excitation and emission wavelengths of two probes provided (a) their decay times are different enough to enable two time windows to be recorded, and (b) the emission of the shorter-lived probe decays to below the detectable limit while that of the other still can be measured. We refer to this new scheme as the dual lifetime determination (DLD) method and show that it can be widely varied by appropriate choice of probes and experimental settings. DLD is demonstrated to work by sensing oxygen and temperature independently from each other by making use of two probes, one for oxygen (a platinum porphyrin dissolved in polystyrene), and one for temperature [a europium complex dissolved in poly(vinyl methylketone)]. DLD was applied to monitor the consumption of oxygen in the glucose oxidase-catalyzed oxidation of glucose at varying temperatures. The scheme is expected to have further applications in cellular assays and biophysical imaging. Figure Principle behind the dual lifetime determination (DLD) method