Fluoresence detection

Quantification. High Pressure Liquid Chromatography. In plasma, blood or urine sensitivity 1 ng/ml, fluorescence detection—E. T. Lin et al., J. Chromat., 1980, 183, Biomed. AppL, 9, 361-311. In plasma sensitivity 500 pg/ml, fluoresence detection—P. A. Reece, J. Chromat., 1980, 227 Biomed. AppL, 10, 188-192. 

When fluoresence detection is employed, the most important consideration... 

Advanced Ion-trap mass spectrometry Bioaerosol Detector System based on Aerogel UV Fluoresence Detection of BW Agents on Surfaces Parallel Micro Separations-based Detection (PMSD) Taqman PCR-based BW assays Automated Nucleic Acid Extractor Deployable diagnostic kit for biowarfare agents... 

Product solutions were passed over a size exclusion diromatograpl column to remove 1-ArOH formed from reaction of l-ArN2 with water. Hiese solutions containing tagged polyelectrolyte were hydrolyzed in 1 M NaOH at ambient temperature for 30 hours to ensure complete hydrolysis of the ester linkages. The half-life for the hydrolysis of the model compound 1-ArOAc is only about 5 minutes under these conditions based on its measured second order rate constant of 2.8 X 10 M sec After the hydrolyses were complete, the pH of each solution was adjusted to 7.0 by titrating wiA 6 M HCl and monitoring with a pH meter. The yields of 1-ArOH were analyzed by HPLC by fluoresence detection in triplicate 100 pi injections. 

Comparison between stable isotope dilution assay (SIDA) and Liquid ehromatography-fluoreseence detection (LC-FD) for food folates. Folate contents are calculated as folic acid in tg/100g. SIDA generally yields higher folate contents as 5-formyl-H4folate was not detectable in LC-FD due to weak fluorescence. 

Elemental Precision. For the determination of element concentrations in a multi-component phase, it is assumed that, for suitably thin samples absorption and fluoresence of X-rays are negligible. Thus, the relative intensities of characteristic X-ray peaks can be related to the concentration ratio of elements by a factor, k [4]. This k-factor accounts for the relative efficiencies of X-ray production and detection during the analysis. This relationship can be expressed by the equation... 

L., and Sewing, K.-Fr. (1994) High performance liquid chromatographic analysis of phospholipids from different sources with combined fluoresence and ultraviolet detection, Anal. Biochem. 220, 172-180. 

Co combines with calcein to form a chelate that does not have fluoresence in the neutral pH range. However, in the presence of EDTA, a stronger Co chelator, calcein is released and can be readily detected at very low concentrations. Therefore, the inclusion of EDTA or other strong chelators in these measurements will cause interfere to the quenching effect of Co. ... 

Another promising type of optical affinity sensor has recently been described by IVettnak and Wolfbeis [221, 222]. The fluoresence of flavoproteins glucose oxidase, lactate monooxygenase) changes with the interaction between enzyme and substrate, and this can be used for substrate detection. As the reduced prosthetic group is reoxidized by Oj, the process is reversible, so in this case the affinant is self-regenerating. 

Burst-recording in the continuous-flow mode can be eombined with multidetector operation. The detectors can be used to record the fluoreseence in different wavelength intervals or under different angles of polarisation. This multiparame-ter detection technique delivers the lifetime, the angle of polarisation, the fluorescence anisotropy, and the emission wavelength within the individual bursts [442, 500]. 

The optical setup is similar to that shown in Fig. 5.129, page 198. A frequeney-doubled YAG laser is used for excitation. The wavelength is 632 nm, the pulse width 150 ps, the repetition rate 76 MHz. An NA =1.3 oil immersion lens foeuses the laser into the sample. The sample is mounted on a piezo-driven sean stage. The fluoreseence is collected back through the microscope lens and separated from the exeitation by a dichroic mirror. A second dichroic mirror splits the fluoreseenee into two wavelength intervals, which are detected by two single-photon APD... 

Use of polarized light to excite fluorescence, and measurement of the state of polarization of the emitted light introduce another set of measurable parameters that can characterize structures and dynamics of molecules. The anisotropy of the polarization of fluoresence after excitation by linearly polarized light provides the rotational diffusion coefficient, or rotational correlation time, of the fluorophore. When there is fluorescence energy transfer, analysis of the anisotropy of both donor and acceptor can reveal the relative orientation, and the relative motion. Measurement of fluorescence after excitation by circularly polarized light provides the fluorescence-detected circular dichroism. This measurement characterizes the chiral environment of the ground state of the fluorophore. If the circular polarization of the fluorescence is measured, the circularly polarized luminescence is obtained. This measurement characterizes the chirality of the excited state. 

Quantitation may be done crudely on the spots separated by planar chromatographic techniques such as TLC or slab gel electrophoresis (see Chapter 13). One might compare the optical density, the fluoresence, or the degree of stationary phase fluoresence suppression by the unknown spot to a series of standards of known concentration. In contrast, the electrical signals from the variety of detectors used in various column chromatography instruments can be precisely, reproducibly, and linearly related to the amount of analyte passing through the detector cell. If all parameters of injection, separation, and detection are carefully controlled from run to run, and especially if appropriate quantitative internal standards are incorporated in the procedure, accuracy and precision better than +1 % may be attained. 

Fig. 2. A protein-detecting microarray. Each square in the grid represents a different feature of the array that would be impregnated with a particular protein ligand (blue shapes). When the sample is applied to the chip, each ligand will capture its target protein (orange and red coils in blow-up). The amount of target protein bound to each feature of the array would be quantitated with probes such as fluorescently labeled antibodies against the captured proteins. A fluoresence scanner would then measure the intensity of fluorescence (differently shaded green squares) at each spot, which would reflect the level of captured protein. (See Color Insert.)...

Claimed advantages of this hybrid device compared to traditional mieroehips inelude lower eost in terms of materials and preparation, UV-detection with no need for fluoreseent labels, the abihty to... 

Similar to the labeling of enzymes with redox mediators, genosensors were developed for sequence-specific DNA detection by making use of the electrochemieal properties of organometallic compounds, mainly ferrocene and its derivatives. " Eleetroehemical genosensors are partieularly attractive because they are highly sensitive and robust, cheap compared to other detection modes sueh as fluoreseence, and they can be easily miniaturized. The potential in this technique has attraeted commereial interest. ... 

XRF technique is suitable for bulk chemical analysis of major elements e.g. Si, Ti, Al, Fe, Mn, Mg, Ca, Na, K and P For trace element analysis with abundances >1 ppm can also be done for Ba, Ce, Co, Cr, Cu, Ga, La, Nb, Ni, Rb, Sc, Sr, Rh, U, V, Y, Zr and Zn with a detection limit of few ppm. For materials whieh are eompositionally similar, suitable standards are available. Samples containing high abundances of elements eorreetions are to be made for absorption and fluoreseence effeets. XRE is eommonly used to identify traees and for quantitative estimation, good resolution of the peaks are neeessary. It ean also analyze thin films. 

Photoassociation of like atoms to form homonuclear molecules, emphasizing the widely studied alkali metals, is treated first. The simple one-color experiments are described in detail, including the variety of techniques used for detection trap loss (decrease in atomic fluoresence), direct detection of excited molecule ionization, detection of fragments by resonance-enhanced multiphoton ionization, and detection of ground or metastable molecules (formed by decay of the upper photoassociation level) by resonance-enhanced multiphoton ionization. 

Fluoreseence indicators and materials as reagents for detection in general ... 

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