Optical techniques fluorometry

Fluorometry is a superior optical technique in terms of sensitivity and specificity. Merits of fluoroimmunoassays (FIAs) and fluoroimmuno-like assays (FILAs) include the stability and freedom from hazards of fluorescent labels compared to radioactive tracers, the moderate cost of analysis, the wide availability of the equipment needed, and the potential high sensitivity. In general, the sensitivity of fluorescence measurements is 10- to 1,000-fold higher than the absorption counterparts. 

This volume presents a cross section of recent advances in the development of novel chemical and biochemical sensors for on-line monitoring and control applications in the environmental, clinical, and bioprocess areas. These chapters illustrate how many of the key challenges for continuous monitoring are being addressed. The methods discussed include optical techniques ranging from near-infrared spectroscopy to lifetime-based phase fluorometry biosensors ranging from optical immu-nosensors to enzyme-electrodes as well as electrochemical, acoustic, and plasmon resonance techniques. 

In the previous sections, it has been shown how powerful the time-resolved fluorescence techniques are in real time probing of photoinduced processes and in allowing the determination of reaction rates from fluorescence lifetimes. The present section is devoted to the method of UV/vis transient absorption spectroscopy, which is a key method in probing non emissive species and is thus crucial to detect photoreaction products or intermediates following optical excitation of molecules in their electronic excited states. When carried out on short time scales, i.e. with femtosecond to subnanosecond excitation sources, fluorescent species can also be detected by their stimulated emission. Combining time-resolved fluorometry and transient absorption spectroscopy is ideal for the study of photochemical and photophysical molecular processes. 

Phase fluorometry is another useful fluorometric technique for the determination of substances with overlapping fluorescence spectra. In phase fluorometry the phase angle between the lamp pulse and the emission of fluorescent light allows discrimination between fluorescences of different origins. Phase-sensitive optics and electronics are rather complicated and, as in the case of time-resolved fluorometry, will be mentioned here only in passing. For further information on these areas, the reader is referred to the bibliography. 

Fluorescence measurements and detection can either be made under steady-state or time-resolved conditions. Some of the commonly used measurement techniques focus on changes in optical properties such as fluorescence intensity quenching, phase fluorometry (lifetime), polarization, surface plasmon resonance (SPR), and evanescent waves. Here, we will present detector systems based for (a) fluorescence intensity quenching and (b) phase fluorometry in detail. A few example references of integrated optical sensor systems based on the various optical measurement techniques are given in Table 1 and the reader is encourage to review those papers if more details are desired. 

Phase fluorometry, on the other hand, is based on the principle that the excited state lifetime of the fiuorophors vary with analyte concentrations. This technique provides a rugged, accurate and sensitive platform for the development of optical sensors as described below. 

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