Self-referencing intensity measurements based on square-wave gated phase-modulation fluorimetry.

Area of Science:

• Analytical Chemistry
• Spectroscopy
• Fluorescence Techniques

Background:

• Conventional ratiometric measurements often require dual wavelengths, increasing complexity.
• Intensity fluctuations from excitation sources can compromise measurement accuracy.
• Self-referenced methods are desirable for robust analytical measurements.

Purpose of the Study:

• To adapt square-wave gated phase-modulation (GPM) fluorimetry for self-referenced intensity measurements.
• To demonstrate the effectiveness and advantages of the adapted GPM scheme.
• To quantify analyte concentration by eliminating excitation source intensity variations.

Main Methods:

• Utilized square-wave excitation, gated detection, a reference emitter, and a sensor molecule.
• Employed a ratiometric approach comparing sensor emission to reference emission.
• Applied digital methods with Na3[Tb(dpa)3] as a long-lived reference and fluorescein as a short-lived sensor for pH measurement.

Main Results:

• The adapted GPM scheme provides self-referenced intensity measurements, eliminating the need for dual wavelengths.
• Measurements showed high accuracy, with apparent pKa values agreeing within 2% compared to conventional methods.
• The method demonstrated excellent insensitivity to excitation source intensity fluctuations (less than 3% signal change for an 8-fold intensity change).

Conclusions:

• The adapted GPM fluorimetry offers a robust and accurate method for self-referenced intensity measurements.
• This technique simplifies measurements by avoiding dual excitation or emission wavelengths.
• The GPM method's insensitivity to source intensity fluctuations makes it highly reliable for quantitative analysis.