A Simple Hybrid Circuit for Direct Determination of Fluorescence Lifetimes

Abstract

A new circuit for the measurement of fluorescence lifetime is described. The technology may be used in sensors which rely on fluorescence quenching in the presence of chemical species such as O2 or H+. Fluorescence is excited by pulses of light from an LED modulated with a square wave. The same square wave is used to gate the resulting luminescence signal from the fluorophore in order to eliminate non-specific luminescence detected during the LED pulse, and to extract the first harmonic of the resulting train of fluorescence decays. The first harmonic is then compared to the excitation signal using simple digital logic, resulting in a pulse width modulated signal. This signal has direct trigonometric relationships to the fluorescence lifetime and to the concentration of the quenching molecule. Evaluation of a prototype of this circuit was successful; the relationships between the sensor output and quenching concentration were as predicted by theory. Observed lifetimes of the fluorophore Ruthenium (4,7-diphenyl-1,10-phenanthroline)32+ immobilized in a sol-gel (~3 to 13 µs) exceeded the lifetimes reported for the same fluorophore dissolved in liquid solvents, indicating a stabilizing effect of the sol-gel and comparatively low values of oxygen permeability in this material. Rate constants for emission and quenching of the fluorophore by oxygen in the sol-gel were determined to be 7.6 x 104 s-1 and 2.8 x 105 (atm·s)-1, respectively. The simplicity of the circuit design compared to alternative technologies is particularly compelling where rapid multiplexing to many parallel sensors is required.