Intensity-based assay

The various possible schemes for fluorescence sensing are summarized in Figure 1.1. At present, most fluorescence assays are based on the standard intensity-based methods, in which the intensity of the probe molecule changes in response to the analyte of interest. However, there has been the realization that lifetime-based methods possess intrinsic advantages for chemical sensing. (A more detailed description of... 

At present, most fluorescence sensors or assays are based on intensity measurements, i.e., intensity-based sensing, in which the intensity of the probe molecules change in response to the analyte of interest. Intensity-based methods are initially the easiest to implement because many fluorescent probes change intensity in response to analytes. These intensity changes can be due to changes in extinction coefficient due to probe ionization, changes in quantum yield of the probe on analyte binding, or due... 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

Membrane permeabilization activity of peptides is currently measured by the use of artificial membrane bilayers, such as liposomes or erythrocytes. The hposome leakage assay can be performed by using spectrofluorimetry with a concentration-dependent quenching of a dye (calcein, carboxyfluorescein) encapsulated in liposomes. Disruption of hposomes in the presence of peptide-inducing leakage will lead to an increase in the fluorescence intensity of the liposome solution. Erythrocyte lysis assay is based on the absorption of hemoglobin, which can be measured once released into the extracellular medium upon erythrocyte lysis in the presence of peptide. 

A method has been described for determination of camphor in dilute solutions and in plants (189). The assay is based on the formation of a yellow colour with phenylhydrazine (Camphor phenylhydra-zone). The colour produced after absorption on Whatman No.120 paper and the intensity of the yellow colour produced was compared with that produced from a sample of known concentration. 

FIGURE 2.3 Dose-response curves for protease inhibitor with autofluorescence characteristics, (a) Result of standard fluorescence intensity-based assay employing an AMC-labeled substrate at a concentration of 2 pM. Profiling data could not be obtained due to the interference of the compound s fluorescence with the readout, (b) Result obtained from assay employing a RhllO-based substrate at a concentration of 0.5 pM. The profiling data (IC50 value of 335 nM and Hill coefficient of 1.0) were obtained for the depicted data set. 

In principle, intensity measurement is no different from the well-based immunoblot or other homogeneous well-based assay format such as an in-cell Western. However, because individual nuclei are identified and used as the mask, precise normalization to cell number can be achieved. In addition, microscopy with high quality cameras offers much higher signal-to-noise ratios, thus making many more assays possible. Finally, if a threshold can be set and cells classified as responders or non-responders, an intensity-based assay can be turned into a cytometry assay. 

The assays are based on membrane potential sensitive fluorescent dyes, which are relocated from the inside of the cells (or vice versa) and cause an alteration in the fluorescence intensity or flip between the inner and the outer sides of the membrane bilayer and transfer fluorescence resonance energy to another dye located outside of the membrane [25, 26]. In this assay, cells, transfected with hERG potassium channel, contribute to the resting membrane potential and the inhibition of this channel by test compounds results in depolarization of the cell membrane, entry of fluorescent dye in... 

In comparison to intensity-based methods, techniques that rely on FRET provide large changes in emission profiles and open the opportunity for ratio-metric fluorescence measurements [59]. Assays of this type are less prone to false positives from nonspecific binding events. 

As mentioned above, assay readouts based on fluorescence are probably the most commonly used of all HTS detection techniques. A number of different flavors of fluorescence-based assays exist. The simplest one, called fluorescence intensity (FLINT) was used, for example, to assay for inhibitors of a proteolytic enzyme that releases a fluorescent component upon cleavage of a specially designed substrate, as shown in Figure 6.3. In this case the enzyme of interest is Cathepsin K, a cysteine protease critical to bone resorption, inhibitors of which are of interest to fight osteoporosis. ... 

The Ferric Reducing Antioxidant Power (FRAP) assay uses an electron transfer reaction to assess the reducing ability of a given antioxidant. This colorimetric assay is based on the reduction of an Fe(ni)-tripyridyltriazine (TPTZ) complex to its intensely blue coloured Fe(II) analogue (Schane 4.4) under acidic conditions [11]. 

Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio International) is an assay based on the proximity of a lanthanide cryptate donor and a fluorescent acceptor molecule whose excitation wavelength overlaps that of the cryptate s emission. The utility of this technique is based on the time resolved fluorescence properties of lanthanides. Lanthanides are unique in the increased lifetime of their fluorescence decay relative to other atoms, so a delay in collection of the emission intensity removes the background from other fluorescent molecules. An example of the HTRF assay is a generic protein-protein interaction assay shown in Fig. 2. 

Averell and Norris (3) have developed an analytical method adapted to the determination of parathion in spray or dust residues, which is sensitive to about 20 micrograms. It is based upon the reduction of parathion with zinc to the amino compound, diazotiza-tion, and coupling with Bratton and Marshall s amine, which gives an intense magenta color with an absorption peak at 555 millimicrons. Bowen and Edwards (6) have used the polarograph to assay technical grades of parathion and its formulations. 

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