Self-Referencing Intensity Measurements Based on Square-Wave Gated Phase-Modulation Fluorimetry

Abstract

An adaptation of square-wave gated phase-modulation (GPM) fluorimetry allows for self-referenced intensity measurements without the complexity of dual excitation or dual emission wavelengths. This AC technique utilizes square-wave excitation, gated detection, a reference emitter, and a sensor molecule. The theory and experimental data demonstrating the effectiveness and advantages of the adapted GPM scheme are presented. One component must have an extremely short lifetime relative to the other. Both components are affected identically by changes in intensity of the excitation source, but the sensor intensity also depends on the concentration of the analyte. The fluctuations of the excitation source and any optical transmission changes are eliminated by ratioing the sensor emission to the reference emission. As the concentration of the analyte changes, the corresponding sensor intensity changes can be quantified through several schemes including digitization of the signal and digital integration or AC methods. To measure pH, digital methods are used with Na3[Tb(dpa)3] (dpa = 2,6-pyridinedicarboxylic acid) as the long-lived reference molecule and fluorescein as the short-lived sensor molecule. Measurements from the adapted GPM scheme are directly compared to conventional ratiometric measurements. Good agreement between the data collection methods is demonstrated through the apparent pKa. For the adapted GPM measurements, conventional measurements, and a global fit the apparent pKa values agree within less than 2%. A key element of the adapted GPM method is its insensitivity to fluctuations in the source intensity. For a roughly 8-fold change in the excitation intensity, the signal ratio changes by less than 3%.

pH-Dependent Photophysical Behavior of Rhenium Complexes Containing Hydroxypyridine Ligands

“…2 The large intensity change may also make them useful as intensity-based pH probes in a variety of self-referencing sensing schemes. 12…”

Optical Chemical Sensors

“…Additionally, Borisov et al recently reported the use of a modified DLR (m-DLR) technique that facilitates the simultaneous detection of two analytes. 268 A related technique is that of gated phase-modulation (GPM) fluorometry, 269 which is similar to DLR in that it makes use of two luminophores with long and short lifetimes, but unlike DLR, square-wave modulation is employed in conjunction with gated detection in order to develop sensors for analytes such as pH.…”