Sensitivity of fluorescence measurements

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. 

There is one domain, however, in which EWIF is probably unsurpassable. Due to the high sensitivity of fluorescence measurements, significant data can be obtained in much less than one minute. Therefore, the kinetics of formation of the adsorbed layer can be monitored almost in real time. Schemes in which the incident beam is kept fixed but where the detection is performed at various angles should further speed up the measurements especially if the single channel photomultiplier is replaced by a linear position sensitive detector. 

The sensitivities of particular spectroscopic teclmiques to specific chemical features are described more fully in tire next section. Perhaps tire most common and versatile probes of reaction dynamics are time-resolved UV-vis absorjDtion and fluorescence measurements. Wlren molecules contain cliromophores which change tlieir stmcture directly or experience a change of environment during a reaction, changes in absorjDtion or fluorescence spectra can be expected and may be used to monitor tire reaction dynamics. Altliough absorjDtion measurements are less sensitive tlian fluorescence measurements, tliey are more versatile in tliat one need not rely on a substantial fluorescence yield for tire reactants, products or intennediates to be studied. 

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 ... 

After equilibration, the amount of bound and free-labeled antigen can be measured, and a calibration curve can be used to determine the analyte. Radioactive labels have been extensively used because of the sensitivity of the measurement however, they have several disadvantages, such as the waste disposal problem and the unstable nature of reagents. CL tags were therefore considered attractive alternatives due to their low (excellent) detectability, which was not fully provided by most fluorescent labels. 

The main advantage of fluorescence techniques is their sensitivity and measurements of nanogram (10—9 g) quantities are often possible. The reason for the increased sensitivity of fluorimetry over that of molecular absorption spectrophotometry lies in the fact that fluorescence measurements use a non-fluorescent blank solution, which gives a zero or minimal signal from the detector. Absorbance measurements, on the other hand, demand a blank solution which transmits most of the incident radiation and results in a large response from the detector. The sensitivity of fluorimetric measurements can be increased by using a detector that will accurately measure very small amounts of radiation. 

In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

If no fluorophore exists in a given area of the thin-layer plate, then no emission signal can be obtained. The fluorescence of the sample is then an absolute quantity relative to this zero signal and proportional to the number of emitting species present in the sample. However, in practice, the adsorbent does contain trace amounts of fluorescent impurities and thus background noise is observed, but usually at a lower level than experienced in absorption measurements. The low background noise is an important factor in the high sensitivity of fluorescence. 

Surface pressure distribution measurement is of fundamental importance in the experimental study of aerodynamic problems in the fields of avionics, car, rocket, aerospace, and aircraft design [1]. The conventional methods based on pressure taps or transducers have a number of limitations. The most serious problem is that their very nature limits them to providing information only at discrete points on the surface of a substrate. A new approach to surface pressure distribution measurement, the use of pressure-sensitive paint (PSP), has recently developed that offers the potential of revolutionizing the nature of such measurements in the field of aerodynamics. This method employs the oxygen sensitivity of fluorescent materials in the form of a paint, in conjunction with image processing techniques, to map the pressure field over... 

FDCD measurements, and a basic theoretical formalism for this technique, were first reported by Turner, Tinoco and Maestre in 1974 [5]. In this experiment one uses the selectivity and sensitivity of luminescence measurements to probe the local chiral environment of fluorescent chromophores. The ultimate goal in many applications of FDCD is to relate the observed differential fluorescence signal to the conventional CD measurement. In certain multi-component absorbing systems this procedure may be difficult. This technique is sometimes applied to systems for which CD measurements are impossible or very difficult. FDCD, like CPL and other polarization sensitive techniques, is not immune to troublesome background and noise problems, and these will be discussed in Section 3. The only detailed discussion of the applicability of FDCD measurements, and other characteristics of the technique has been presented by Turner in 1978 [6]. In this chapter we will also list some of the more recent applications of FDCD. 

The great sensitivity of fluorescence spectral, intensity, decay and anisotropy measurements has led to their widespread use in synthetic polymer systems, where interpretations of results are based upon order, molecular motion, and electronic energy migration (1). Time-resolved methods down to picosecond time-resolution using a variety of detection methods but principally that of time-correlated single photon counting, can in principle, probe these processes in much finer detail than steady-state techniques, but the complexity of most synthetic polymers poses severe problems in interpretation of results. 

Chemical and biological microstructures have been probed by means of arrays of excitable donor and acceptors whose spacing are measured by means of energy transfer . The structures can be determined from the measured spread of separation distances. Intracellular sensitization of fluorescence has been applied to biological systems which are studied by a combination of microfluorimetry and fluorescence spectroscopy . ... 

The classic text by Udenfriend (50) initiated the surge in interest in the use of fluorescence by analytical and clinical chemists. This approach was of interest because of its potential for significant increases in analytical sensitivity. In fact, the fluorescence signal by the chromophore is 100 to 1000 times greater than the sensitivity of absorbance measurements. Because of this level of analytical sensitivity, fluorescence has been applied almost exclusively to the measurement of compounds at low concentrations. 

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