Fluorescence based methods

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

Ghiselli A, Serafini M, Maiani G, Azzini E and Ferro-Luzzi A. 1995. A fluorescence-based method for measuring total plasma antioxidant capability. Free Radic Biol Med 18(1) 29—36. 

The various fluorescence-based methods for the determination of fluidity and molecular mobility are summarised in Table 8.1. Attention should be paid to the comments indicated in the last column. 

Progress in instrumentation has considerably improved the sensitivity of fluorescence detection. Advanced fluorescence microscopy techniques allow detection at single molecule level, which opens up new opportunities for the development of fluorescence-based methods or assays in material sciences, biotechnology and in the pharmaceutical industry. 

Other fluorescence-based methods to investigate target-ligand interactions use fluorescence correlation spectroscopy (FCS) or fluorescence resonance energy transfer (FRET) [8, 12, 46]. 

A number of different detection technologies have been established, including label-based and label-free detection systems (Fig. 2). Fluorescence-based methods are the most commonly... 

Fig. 7 Selected 2PA spectra obtained by absolute fluorescence-based methods, a Spectra for fluorescein, rhodamine B, coumarin 307, and , -p-bis(o-methylstyryl)benzene (the solvent is indicated in the legend) obtained by Xu and Webb [78]. In the case of coumarin 307, the ordinate displays the quantity rjSy where rj is the fluorescence quantum yield, b Spectrum for , -p-bis(o-methylstyryl)benzene (this spectrum is obtained from the tabulated values for the band shape reported by Kennedy and Lytle [90] and the cross section at 585 nm reported by Fisher et al. [80], to correct for a typographical error in the 1986 paper). Part (a) reproduced with permission from [78]. 1996, Optical Society of America...

Currently, there is a need for high-throughput determination of nucleic acid sequences. At present, detection systems most commonly employ fluorescence-based methods. However, wide spread applications of such methods are limited by low speed, high cost, size, and number of incubations steps, among other factors. Application of electrochemical methods in affinity DNA sensors presents likely a promising alternative, allowing miniaturization and cost reduction, and potentially allowing application in point-of-care assays. 

Another fluorescence-based method for assaying activity and enantioselectivity of synthetic catalysts, specifically in the acylation of chiral alcohols, was recently reported [27]. The idea is to use a molecular sensor that fluoresces upon formation of an acidic product (acetic acid). Adaptation to high-throughput evaluation of enantioselective lipases or esterases needs to be demonstrated. 

With peptide and oligonucleotide libraries prepared by split synthesis, the structure of a bound ligand on an individual bead can be determined by microsequencing [ 17,18]. This approach has been applied to testing libraries where the bead bound compounds are evaluated for their ability to bind to monoclonal antibodies or other recognition macromolecules. In one such approach, fluorescence-based methods are used to select the most... 

For numerical chromosomal alterations, it is possible to analyze interphase cells using FISH with whole chromosome paints, or chromosomal specific probes. Thus, it is feasible to assess nondisjunctional events in any cell type, including target cells for tumorigenesis. Other fluorescence-based methods described in the section on in vitro methods (Section 25.3.4b) can be used in vivo, but they are of rather limited value for basic mutagenicity assays. 

Letsinger R. Elghanian G. Viswanadham, a fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles. Analytical Chemistry 2000, 72, 5535-5541. 

Compared to fluorescence, SERRS has some significant advantages. Unlike fluorescence-based methods, SERRS measurements are unaffected by quenching by oxygen or other species, are less sensitive to photobleaching, and are inherently... 

It is noteworthy to add that the enantiomeric composition determination method based on chiral IL is not specific to near-infrared spectroscopic technique but is rather general as it can be used with other spectroscopic techniques as well. In fact, we have successfully demonstrated that fluorescence technique can also be used with this method. Furthermore, the fluorescence based method has relatively higher sensitivity and lower limit... 

Kenworthy AK (2006) Fluorescence-based methods to image palmitoylated proteins. Methods 40 198-205... 

Since a fluorescence based method is now used in automated DNA sequencing procedure photophysics is set to play a significant part... 

Once you have pinned your molecule down, you need some way of observing what it gets up to. Fluorescence-based methods are usually the most sensitive here, because photomultipliers and CCD devices (see Chapter 2) can readily detect the single photons emanating from individual fluorescent groups. Moreover, the intrinsic fluorescence of proteins or other fluorescence probes is very sensitive to conformational changes and is well understood for bulk samples. 

A fluorescence-based method was successfully tested for iodine determination in iodine-supplemented food, such as table salt and milk powder. It is based on the fluorescent quenching capability of iodine/triodide using a highly fluorescent compound consisting of a synthetic metal ion receptor coupled with a signaling element. The detection limit for triodide down to a concentration of 10 mol was reported (Zhao et al., 2003). 

Interactions between the HIV-1 regulatory protein. Rev, and the Rev responsive element (RRE) RNA, as well as the effect of inhibitors on these interactions, were monitored using a single-particle fluorescence-based method [203]. This biosensing method combined FRET and the colocalization of fluorophores as the means of detection. In the presence of an analyte, the dye-labeled recognition complex was assembled on the fluorescent QDs, and these were tunneled, under flow, into two detector channels that simultaneously analyzed the fluorescence of the QDs and the FRET emission of the dye. The two emissions were observed simultaneously only if... 

These criteria limit the applicahility of fluorescent dye-based sensors to only a handful of analytes. If a fluorescence-based method does not exist or is Incompatible with an optical sensor, an absorption sensor is the only alternative. There exist a large number of different absorbing dyes that are sensitive, selective and absorb in convenient regions of the spectrum. Several absorbance-based fiber-optic sensors have been described (8,25.26). Unfortunately, a major drawback with absorption spectroscopy is its inherent insensitivity. We decided to explore the possibility of using other optical techniques that can be used with indirect methods. A successful example has been presented which combines the sensitivity of the fluorescence measurement with the specificity of an absorbing dye and is based on an energy transfer mechanism (15). 

In spite of such advances, all these extremely powerful fluorescence-based methods of analysis were necessarily restricted... 

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