A ratiometric fluorometer for reduced sensitivity against solvent artifacts

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] When measuring fluorescence emission intensity, both the dye concentration and the optical absorption properties of the solvent influence the measured intensity. As a consequence, intensity-based assays (for example quenching studies or viscosity measurements using molecular rotors) lack precision. We present a ratiometric fluorophotometer capable of determining fluorescence emission simultaneously with fluid absorption at the dye's excitation and absorption wavelengths. Using suitable models, dye turbidity, emission re-absorption and dye concentration can be determined, and the measured emission intensity corrected. Validation of the prototype showed excellent correlation of measured values with commercial fluorometer (R2=0.99) and spectrometer (R2=0.97). In addition, only instrument constants remain to link emission intensity to quantum yield, a metric which is generally hard to determine. Using the same instrument with only minor modifications, fluorescence measurement through an optical fiber is possible. This allows measurements in remote areas, very small samples, and closed fluid loops. This instrument is characterized by inexpensive design, robust technology based on light-emitting diodes, its flexibility, the reduced footprint due to the integration of two standard instruments, and its increased measurement precision.