Fluorophore properties fluorescence detection

Barone PW, Parker RS, Strano MS. In vivo fluorescence detection of glucose using a single-walled carbon nanotube optical sensor design, fluorophore properties, advantages, and disadvantages. Analytical Chemistry 2005, 77, 7556-7562. 

The three aromatic amino acids, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), are the only native amino acids with useful fluorescence properties. Figure la shows their absorption and fluorescence spectra. Their fluorescence properties are summarized in Table 1. Note that the relative absorption coefficients increase in the order Phe < Tyr < Trp. The fluorescence quantum yields increase in the same order. The product of absorption coefficient and fluorescence quantum yield can be taken as a measure of the brightness of the fluorophore. By the standards of fluorescent dyes (see below), the brightness of aU three amino acids is poor. Trp is the brightest of the three, and for proteins with a small number of Trp residues it may be possible to assign fluorescence decays to specific Trp residues. As a result, of the three fluorescent amino acids, Trp is by far the most widely exploited for its fluorescent properties. Fluorescence from Tyr is also detectable but may be masked by Trp fluorescence. Proteins often contain many Tyr residues, so it is often not possible to isolate the fluorescence from individual Tyr residues. Fluorescence from Phe is weak and not often used in fluorescence studies. 

Since tryptophan has a larger molar extinction coefficient, a redder absorbance, and is found in most proteins, this fluorophore is the one most frequently employed in fluorescence studies. The sensitivity of fluorescence detection is such that concentrations as low as 10" m can be studied with commercial fluori-meters, and, by selecting smaller cell pathlengths or longer excitation wavelengths, concentrations as high as lO " m can be studied. A very important property of tiyptophan fluorescence is that it is very dependent on the environment of the indole side chain, making tiyptophan fluorescence responsive to the structure of a protein. 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Fast librational motions of the fluorophore within the solvation shell should also be consideredd). The estimated characteristic time for perylene in paraffin is about 1 ps, which is not detectable by time-resolved anisotropy decay measurement. An apparent value of the emission anisotropy is thus measured, which is smaller than in the absence of libration. Such an explanation is consistent with the fact that fluorescein bound to a large molecule (e.g. polyacrylamide or monoglucoronide) exhibits a larger limiting anisotropy than free fluorescein in aqueous glycerolic solutions. However, the absorption and fluorescence spectra are different for free and bound fluorescein the question then arises as to whether r0 could be an intrinsic property of the fluorophore. 

---