Intrinsically Referenced Fluorimetric Sensing and Detection Schemes: Methods, Advantages and Applications

Abstract

The precision of analytical methods using fluorescent probes or biomolecular labels often is compromisedof a variety of conceivable interferences that may originate from the instrumental system, the sample,or the underlying sensor chemistry. Instrumental drifts of the optoelectronic system, photobleaching ofluminophores, or high intrinsic color and background fluorescence of the sample cannot be eliminated evenby extensive calibration procedures. As a result, intrinsically referenced methods are preferred toimprove optical chemo- or biosensor technology, and fluorescent bioimaging. Intrinsic referencing is oftenaccomplished by ratiometric techniques. These include (1) dual wavelength probes, (2) dual luminophoresensors, (3) lifetime-based assays, (4) dual lifetime referencing (in the time and frequency-domain), and(5) fluorescence anisotropy. Applications and advantages of the various approaches are outlined in thisreview, with a focus on widely used sensing methods for oxygen, pH, carbon dioxide, calcium, glucose,or temperature, and on biomolecular screening. In addition to ratiometric methods, fluorescence correlationspectroscopy represents another attractive tool to determine analyte concentrations via fluorescent probes.Many of these ratiometric approaches have the potential to pave the way for the development of calibration-freesensor and imaging schemes.