Introduction to Fluorescence Sensing

pp 65-118

Fluorescence Detection Techniques

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Fluorescence is the phenomenon of the emission of a light quanta by a molecule or material (fluorophore) after initial electronic excitation in a light-absorption process. After excitation, a molecule resides for some time in the so-called excited state and its fluorescence emission can be observed usually with a lower energy (longer wavelength) than the excitation. The time range of fluorescence emission (fluorescence lifetime) depends on both the fluorophore and its interactions with the local environment. Thus, for organic dyes it is in the picosecond (ps) to nanosecond (ns) time range, typically 10-8–10-11 s. Fluorescence is a part of a more general phenomenon, luminescence. The latter includes the emission of species excited in the course of chemical reactions (chemiluminescence), biochemical reactions (bioluminescence) or upon oxidation/reduction at an electrode (electrochemiluminescence). Important for sensing is also emission with a long lifetime from triplet state (phosphorescence). The duration of these types of luminescence can be much longer than the fluorescence. For semiconductor nanocrystals it can be tens of nanoseconds; for organometallic compounds and lanthanide complexes — hundreds of nanoseconds, up to milliseconds (ms).

Several parameters of fluorescence emission can be recorded and all of them can be used in sensing (Fig. 3.1). Fluorescence intensity F can be measured at the given wavelengths of excitation and emission (usually, band maxima). Its dependence on emission wavelength, Fem) gives the fluorescence emission spectrum. If this intensity is measured over the excitation wavelength, one can obtain the fluorescence excitation spectrum Fex). Emission anisotropy, r (or the similar parameter, polarization, P) is a function of the fluorescence intensities obtained at two different polarizations, vertical and horizontal. Finally, emission can be characterized by the fluorescence lifetime τF, fluorescence-detected excited-state lifetime what is often called. All of these parameters can be determined as a function of excitation and emission wavelengths. They can be used for reporting on sensor-target interactions and a variety of possibilities exist for their employment in sensor constructs.