Quenching fluorescence enhancement

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

Samples for studies of CDx effects on fluorescence enhancement in organic solution were prepared using pyrene, because pyrene possesses a long lifetime and is very susceptible to quenching and enhancement in solution (23). An aliquot of pyrene stock solution in cyclohexane was placed under a nitrogen purge to evaporate the cyclohexane. Samples were redissolved in a 1 A mixture of Isopropyl ether and 1-butanol, which was saturated with aqueous CDx solution. Pyrene samples were also prepared in which the organic solvent was not saturated with CDx solution. The mixed solvent was used in order to minimize the effects of ether evaporation and thus allow more accurate quantitation. Fluorescence measurements were made on diluted samples of these solutions. The solvent used to make up the... 

Contrary to the observations with TNP-ATP, however, Mg enhanced eosin fluorescence, whereas ions decreased the fluorescence enhancement of Mg and caused a fluorescence quench in the absence of added ions [99]. The fluorescence enhancement caused by Mg was explained by an increase in the number of eosin-binding sites. Fuller [98] on the other hand, has challenged this explanation and argued that only an increase in enhancement factor (i.e., movement of the fluorophore to a more hydrophobic region of the protein) can explain the Mg -induced fluorescence increase. 

The extent of fluorescence quenching often depends on the sorbent medium and is generally more severe for silica gel than for chemically bonded sorbents [183]. In many cases the emission signal can be enhanced by application of a viscous liquid to the layer before scanning the plate. Common fluorescence enhancing... 

Fig. 3 Typical ICT probes (left) and representative spectroscopic responses toward selected metal ions (right). Color code (left) coordinating atoms in blue, bridgehead atom of the fluorophore that takes part in complexation in orange, formal donor fragment in red, formal acceptor fragment in green (right) hypsochromic shifts in red, bathochromic shifts in green, fluorescence enhancement in violet, fluorescence quenching in blue. Symbols in table Aabs, 7em,

Figure 5 shows two typical core-shell structures (a) contains a metal core and a dye doped silica shell [30, 32, 33, 78-85] and (b) has a dye doped silica core and a metal shell [31, 34]. There is a spacer between the core and the shell to maintain the distance between the fluorophores and the metal to avoid fluorescence quenching [30, 32, 33, 78-80, 83]. Usually, the spacer is a silica layer in this type of nanostructures. Various Ag and Au nanomaterials in different shapes have been used for fluorescence enhancement. Occasionally, Pt and Au-Ag alloys are selected as the metal. A few fluorophores have been studied in these two core-shell structures including Cy3 [30], cascade yellow [78], carboxyfluorescein [78], Ru(bpy)32+ [31, 34], R6G [34], fluorescein isothiocyanate [79], Rhodamine 800 [32, 33], Alexa Fluor 647 [32], NIR 797 [82], dansylamide [84], oxazin 725 [85], and Eu3+ complexes [33, 83]. 

Class 2 fluorophores that can reversibly bind an analyte. If the analyte is a proton, the term fluorescent pH indicator is often used. If the analyte is an ion, the term fluorescent chelating agent is appropriate. Fluorescence can be either quenched upon binding (CEQ type Chelation Enhancement of Quenching), or enhanced (CEF type Chelation Enhancement of Fluorescence). In the latter case, the compound is said to be fluorogenic [e.g. 8-hydroxyquinoline (oxine)]. 

Calixarene containing a dioxotetraaza unit, PET-18, is responsive to transition metal ions like Zn2+ and Ni2+. Interaction of Zn2+ with the amino groups induces a fluorescence enhancement according to the PET principle. In contrast, some fluorescence quenching is observed in the case of Ni2+. PET from the fluorophore to the metal ion is a reasonable explanation but energy transfer by electron exchange (Dexter mechanism) cannot be excluded. 

Fabbrizzi L., Lichelli M., Paliavicini P., Sacchi D., Taglietti A. (1996) Sensing of Transition Metals Through Fluorescence Quenching or Enhancement, Analyst 121, 1763-8. 

Case 1 Complexation with fluorescence enhancement. If the metal ion itself is not quenching, binds to the azamacrocyle, and does not form a complex capable of absorbing the emitted light, a large CHEF effect (25- to 190-fold, depending on the... 

The fluorescence emission maximum, quantum yield, and lifetime of a fluorophore are very sensitive to its immediate environment. A blue shift in the emission maximum and an increase in the fluorescence quantum yield or lifetime is generally observed when a fluorophore is transferred form a polar solvent to a nonpolar one or when it binds to a hydro-phobic protein site. Furthermore, fluorescence quenching or enhancement may result from interactions of the fluorophore with various structural elements in its vicinity. 

This behavior facihtates two-channel fluorescence imaging, resulting in contrast due to fluorescence quenching at the shorter wavelengths (Xem of 3 from 425 to 620 nm) and fluorescence enhancement at longer wavelengths (A,em of 3a from 520 to 700 nm) as presented in Fig. 32. 

Ferrocene has been widely investigated as an electron donor and its electron donating ability can be tuned by redox reactions. As anticipated, when a ferrocene unit is covalently connected to an electron acceptor moiety that shows intrinsic fluorescence, the fluorescence of the acceptor moiety would be largely quenched because of the photoinduced electron transfer between ferrocene and the fluorescent acceptor. For instance, triad 15 that contains perylene diimide flanked by two ferrocene moieties, shows rather weak fluorescence due to the photoinduced electron transfer between perylene diimide and ferrocene units. Either chemical or electrochemical oxidation of ferrocene unit lead to fluorescence enhancement. This is simply because the electron donating ability of ferrocene is reduced after oxidation and accordingly the photoinduced electron transfer is prohibited. In this way, the fluorescence intensity of 15 can be reversibly modulated by sequential electrochemical oxidation and reduction. Therefore, a new redox fluorescence switch can be established with triad 15.25... 

Indicates quenching, - indicates fluorescence enhancement Indicator concentrations 1-5 ixmolL-1, ion concentration 100 pjnolL-1... 

Just as the sulfur-based signaling systems have been successful at achieving clear fluorescence enhancements with intrinsically quenching transition metal ions since 1993, there is a growing body of literature on a parallel development with aliphatic amine-based compounds. Bharadwaj and coworkers... 

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