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

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%.

[1]  J. Demas,et al.  Oxygen sensors based on luminescence quenching of metal complexes:  osmium complexes suitable for laser diode excitation. , 1996, Analytical chemistry.

[2]  Joseph R. Lakowicz,et al.  Measurement of the Intensity of Long-Lifetime Luminophores in the Presence of Background Signals Using Phase-Modulation Fluorometry , 1999, Applied spectroscopy.

[3]  I Klimant,et al.  Fluorescent imaging of pH with optical sensors using time domain dual lifetime referencing. , 2001, Analytical chemistry.

[4]  I Klimant,et al.  Optical sensor for seawater salinity , 2000, Fresenius' journal of analytical chemistry.

[5]  O. Wolfbeis,et al.  Multi-ion imaging using fluorescent sensors in a microtiterplate array format , 2002 .

[6]  Fluoro reactants and dual luminophore referencing: a technique to optically measure amines. , 2001, Analytical chemistry.

[7]  I Klimant,et al.  Dual lifetime referencing as applied to a chloride optical sensor. , 2001, Analytical chemistry.

[8]  Ingo Klimant,et al.  Dual lifetime referenced optical sensor membrane for the determination of copper(II) ions , 2002 .

[9]  Jennifer W. Parker,et al.  Fiber-optic sensors for pH and carbon dioxide using a self-referencing dye , 1993 .

[10]  N. Mack,et al.  Multicomponent lifetime-based pH sensors utilizing constant-lifetime probes. , 2002, Analytical Chemistry.

[11]  Benjamin A. DeGraff,et al.  New Method of Rapid Luminescence Lifetime Determination Using Square-Wave Excitation , 2001 .

[12]  A. Persoons,et al.  Fluorescence lifetime resolution with phase fluorimetry , 1983 .

[13]  H Szmacinski,et al.  Optical measurements of pH using fluorescence lifetimes and phase-modulation fluorometry. , 1993, Analytical chemistry.

[14]  E. Gratton,et al.  The Measurement and Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluorometry , 1984 .

[15]  J. Demas,et al.  Elimination of fluorescence and scattering backgrounds in luminescence lifetime measurements using gated-phase fluorometry. , 2002, Analytical chemistry.

[16]  R. Kopelman,et al.  High-performance fiber-optic pH microsensors for practical physiological measurements using a dual-emission sensitive dye. , 1997, Analytical chemistry.

[17]  J N Demas,et al.  Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes. , 2001, Analytical chemistry.