Fluorescence intensity/quenching assays

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. 

Recently, Czamik et al. have reported the use of the acyclic protonated amine host 9 as a chemosensor of pyrophosphate. Typical fluorescence sensing methods rely on the ability of a complexed anion to quench the fluorophore. The fluorescence intensity of host 9, however, is actually increased upon complexation of anions and its 2200-fold selectivity of pyrophosphate over phosphate allows for real-time assay of pyrophosphate hydrolysis by inorganic pyrophosphatase.18... 

Donor-acceptor probes are very useful as fluorescent lables in biochemical assay and sensing based on the fluorescence quenching mechanisms. In a closed state form where the donor and acceptor are close to each other, the fluorescence of the donor (reporter) is highly quenched. In an open state, where the donor and receptor are spaced away from each other due to biochemical reactions, the donor fluorescnece is restored. The changes of the fluorescence intensity of the reporter has been used for DNA detection, immunoassay, enzyme sensing and detection of many other bimolecules. 

Fluorescence intensity assays detect an increase or a decrease in the strength of a fluorescence emission. Based on the origin of this change in fluorescence, the two main classes are fluorogenic assays and fluorescence quench assays. 

Fig. 2.3. (a) Fluorescence spectroscopic changes observed in a proteolytic enzyme assay using PPE-SO3 (3) and K-pNA. Initial addition of KpNA quenches fluorescence, and then addition of peptidase gives rise to fluorescence recovery. Solid line Initial fluorescence, [PPE-SO3] = 1.0 pM, phosphate buffer solution, pH 7.1 Dotted line fluorescence after addition of 167 nM K-pNA. Fluorescence intensity as a function of time (5-200 min) after addition of porcine intestinal peptidase (3.3 pg mL ). (b) Mechanism of the turn-on CPE-based sensor... 

Although a number of quantitative assays are available for allele frequency estimation, most are difficult and expensive to develop or implement. In this chapter, we describe the most versatile and least expensive assays for allele frequency estimation, namely, the template-directed dye-terminator incorporation assay with fluorescence quenching detection (the FQ-TDI assay [3]). The FQ-TDI assay is a real-time homogeneous primer extension assay based on two principles, namely, that deoxyribonucleic acid (DNA) polymerase catalyzes the allele-specific incorporation of a dye-terminator at the polymorphic site and that the fluorescence intensities of a fluorescent dye decreases significantly when it is incorporated into primers. 

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