Natural Fluorescence Techniques

Fluorescence emission is a radiation de-exdtation process of an excited molecule, corresponding to the transition from its lowest singlet excited electronic state to the ground state. The ability of an excited molecule to emit fluorescence light when decaying to its ground state is defined by its fluorescence quantum yield, 

The fluorescence quantum yield corresponds to the fraction of absorbed energy that is lost in the form of fluorescence light. 

Fluorescence is a very sensitive technique capable of providing information on the structural status of the fluorophore or the molecules they are inserted in, making possible the monitoring of specific processes. In well defined and controled circumstances, it also allows for a quantitative determination of the fluorescence compounds, with detection limits within the parts per billion (ppb) range. 

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The use of these natural fluorescence techniques offers not only the possibility of studying the interaction of proteins with membranes, under convective and diffusive conditions, but also they may be easily extended to studies involving proteins and other porous materials such as chromatography media. The areas of application of these techniques will range from polypeptide and protein fractionation to the monitoring of systems where protein-surface interactions are relevant. 

Natural Fluorescence Techniques for Monitoring the Membrane Processing of Biological Molecules... 

The case studies presented in this chapter are very much based on the personal experience and research interests of the authors and they should illustrate, not limit, the high potential of using natural fluorescence techniques for the monitoring of membrane processes. The examples selected reflect, however, the authors perception about the areas of membrane research where natural fluorescence techniques may contribute to a better monitoring of membrane processes and, ultimately, to a better comprehension of the phenomena taking place when complex media interact with membranes. 

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

In this chapter, we present the theory and results of measurements on humic acid fractions using fluorescence techniques. The fluorescence techniques are attractive for this application because of the natural fluorescence of humic materials, the hi sensitivity of fluorescence detection, and the ability to directly observe the morphology of the molecule in aqueous solutions without the need for drying or applying harsh chemical conditions. Several interesting types of information are obtained from fluorescence measurements ... 

By their nature, many UV absorbers are amenable to analysis by fluorimetric analysis. In many instances visible fluorescence techniques are less subject to interference by other polymer additives in a polymer extract than are UV methods of analysis. In fluorescence analysis (ex at 367 nm, em at 400-440 nm) of a PS/Uvitex OB chloroform dissolution AOs such as Ionol CP, Ionox 330, Polygard and Wingstay T/W do not interfere detection limit of 10 ppm [41]. 

Mattson S, Christoffersson JO, Jonson R, et al. 1987. X-ray fluorescence technique for in vivo analysis of "natural" and administered trace elements. In Elis, Yasumuru, Morgan, eds. In vivo body composition studies. New York, NY Brookhaven National Laboratory, The Institute of Physical Sciences in Medicine. 

Hedeishi and McLaughlin [457] have reported the application of the Zeeman effect for the determination of mercury. Atomic absorption and atomic fluorescence techniques using closed system reduction-aeration have been applied widely to determine mercury concentrations in natural samples [458-472]. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

Whatever the technique used, it is important to note that (i) only an equivalent viscosity can be determined, (ii) the response of a probe may be different in solvents of the same viscosity but of different chemical nature and structure, (iii) the measured equivalent viscosity often depends on the probe and on the fluorescence technique. Nevertheless, the relative variations of the diffusion coefficient resulting from an external perturbation are generally much less dependent on the technique and on the nature of the probe. Therefore, the fluorescence techniques are very valuable in monitoring changes in fluidity upon an external perturbation such as temperature, pressure and addition of compounds (e.g. cholesterol added to lipid vesicles alcohols and oil added to micellar systems). 

Fluorescence detection is widely employed in electromigration techniques for samples that naturally fluoresce or are chemically modified to produce molecules containing a fluorescent tag. Indirect detection incorporating a fluorescent probe into the electrolyte solution is also employed. One of the most common fluorophores used for this purpose is fluorescein, which is a water soluble, stable, and relatively cheap compound. 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

To study the excited state one may use transient absorption or time-resolved fluorescence techniques. In both cases, DNA poses many problems. Its steady-state spectra are situated in the near ultraviolet spectral region which is not easily accessible by standard spectroscopic methods. Moreover, DNA and its constituents are characterised by extremely low fluorescence quantum yields (<10 4) which renders fluorescence studies particularly difficult. Based on steady-state measurements, it was estimated that the excited state lifetimes of the monomeric constituents are very short, about a picosecond [1]. Indeed, such an ultrafast deactivation of their excited states may reduce their reactivity something which has been referred to as a "natural protection against photodamage. To what extent the situation is the same for the polymeric DNA molecule is not clear, but longer excited state lifetimes on the nanosecond time scale, possibly of excimer like origin, have been reported [2-4],... 

It appears that fluorescence techniques are poised to receive more serious consideration for accelerated development efforts. Key obstacles remaining include stability of receptors and fluorophores, challenges that will possibly be met partially by results of the intense efforts of molecular biology, polymer science, and nanotechnology. Advances in nanomaterials such as quantum dots will likely enable improvements in optical stability and choice of excitation/emission wavelengths for various transduction methods. Stabilization of natural and artificial enzymes and rendering immunogenic protein receptors stealthy may also aid the pursuit. 

Fluorescence spectroscopy is characterized by a greater selectivity when compared with other spectro-photometric techniques because there is an excitation and an emission spectra, with maxima usually quite characteristic of a particular compound. It is also selective because of the limited number of organic compounds that fluoresce. It has greater sensitivity than spectrophotometric methods in solution 10 9-10 12 M can usually be measured while on a thin-layer chromatogram some naturally fluorescent compounds may be detected instrumentally in sub-nanogram amounts. 

The hydrophilic groups on mucoadhesive polymers and the large amount of water associated with mucin present two possible adhesion mechanisms (i) hydrogen bonding and (ii) interpenetration of a swollen gel network with hydrated mucin. Many methods have been used for the assessment of bioadhesive properties, including fluorescent techniques and tensile tests. By using these methods, a number of natural and synthetic polymers have been discovered possessing mucoadhesive properties. 

Multiple parameters can be measured for fluorescence photons count rate (or intensity), wavelength (X), polarization (p), arrival time (to), time delay after excitation (td), and location x,y on an imaging detector). These parameters carry information about the fluorophore that includes the nature of its environment, its interactions with other molecules, and its motions. Fluorescence techniques that exploit each of these parameters are described below. 

The electronic excited state is inherently unstable and can decay back to the ground state in various ways, some of which involve (re-)emission of a photon, which leads to luminescence phenomena (fluorescence, phosphorescence, and chemiluminescence) (22). Some biologic molecules are naturally fluorescent, and phosphorescence is a common property of many marine and other organisms. (Fluorescence is photon emission caused by an electronic transition to ground state from an excited singlet state and is usually quite rapid. Phosphorescence is a much longer-lived process that involves formally forbidden transitions from electronic triplet states of a molecule.) Fluorescence measurement techniques can be extremely sensitive, and the use of fluorescent probes or dyes is now widespread in biomolecular analysis. For example, the large increase in fluorescence... 

Opiate narcotics are thought to act at specific receptors in the brain since they exhibit stereospecific binding (1) and have been shown by fluorescence techniques to be localized at discrete regions in the central nervous system (2). While much effort has been made to isolate and characterize the opiate receptor C3), relatively little detailed information exists about the nature of the opiate binding site. Some investigators describe the receptor as a membrane bound protein or proteo-lipid (4) while others have used nerve cell components such as cerebroside sulfate or phosphatidyl inositol as models for the opiate receptor (5). 

Sensitive Optical Detectors. More sensitive optical techniques that have been used with CE include fluorescence, refractive index, chemiluminescence, Raman spectrophotometry, and circular dichroism. The most sensitive optical detection method used in CE is laser-induced fluorescence (LIE), which is capable of detection limits in the 10 to 10" mol (or better) range. This detection mode is easily accomplished with analytes that are either easily labeled with a fluorescent substrate (e.g., intercalators for double-stranded DNA) or are naturally fluorescent (e.g., proteins or peptides containing tryptophan). CE systems have also been interfaced with mass spectrometers, and electrochemical detection methods have been developed, although such detectors must be isolated electrically from the electrophoretic voltages. 

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