Quenching, of fluorescence

Quenching of Fluorescence As described in Section 1.4.A, a ide vari of small molecules or ions can act as quenchas of fluorescence  

Collisional quenching of fluorescence requires contact between the fluorophore and the quencher. For quenching to occur the quencher must diffuse to and collide with the fluorophore in the excited state. If this occurs the fluorophore returns to the ground state without emission of a photon (Fig. 4). Many small molecules act as collisional quenchers of fluorescence [6,12]. These include iodide, acrylamide, halogenated hydrocarbons and occasionally amines and metal ions. The excited state lifetimes provide ample opportunity for quenching. For instance, acrylamide is known to be an efficient quencher of tryptophan fluorescence [12,13]. Suppose its 

Once again melittin illustrates the effect of protein structure on the fluorescence emission. Acrylamide quenching data for melittin monomer and tetramer are shown in Fig. 8. Stem-Volmer plots are often used to present quenching data. The Stem-Volmer equation is 

If every collisional event results in quenching, the bimolecular rate constant can be estimated using the diffusion constants of the fluorophore (Dp) quencher (Dq) and the radius expected for contact (i ), 

The quenching data for both the monomeric and tetrameric forms of melittin indicate the tryptophan residues are accessible to acrylamide with the accessibility being greater in the monomeric state. This conclusion is reached by comparison with acrylamide quenching data for NATA. At 25 °C in water the acrylamide 

---

In addition to the processes that can compete with fluorescence within the molecule itself, external actions can rob the molecule of excitation energy. Such an action or process is referred to as quenching. Quenching of fluorescence can occur because the dye system is too warm, which is a very common phenomenon. Solvents, particularly those that contain heavy atoms such as bromine or groups that ate detrimental to fluorescence in a dye molecule, eg, the nitro group, ate often capable of quenching fluorescence as ate nonfluorescent dye molecules. 

In order to clear up the mechanism of inactivation of excited states, we examined the processes of quenching of fluorescence and phosphorescence in PCSs by the additives of the donor and acceptor type253,2S5,2S6 Within the concentration range of 1 x 1CT4 — 1 x 10"3 mol/1, a linear relationship between the efficiency of fluorescence quenching [(/0//) — 1] and the quencher concentration was found. For the determination of quenching constants, the Stem-Volmer equation was used, viz. 

Fig. 14. Quenching of fluorescence of PP solution in benzene. Quenchers (Q) (1) tetracyanoethylene, (2) chioranil, (3) trinitrobenzene / fluorescence in relative units in the absence of a quencher, / in the presence of a quencher...

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. 

Mooradian (1993) has studied the antioxidant properties of 14 steroids in a non-membranous system in which the fluorescence of the protein phycoerythrin was measured in the presence of a lipid peroxyl radical generator (ABAP). Oxidation of the protein produces a fluorescent species. Quenching of fluorescence by a test compound indicates antioxidant activity. Oestrone, testosterone, progesterone, androstenedione, dehydroepian-drosterone, cortisol, tetrahydrocortisone, deoxycorti-... 

Karapetyan, N. V. (2007). Non-photochemical quenching of fluorescence in cyanobacteria. Biochemistry (Moscow) 72(10) 1127-1135. 

Bimolecular reactions with paramagnetic species, heavy atoms, some molecules, compounds, or quantum dots refer to the first group (1). The second group (2) includes electron transfer reactions, exciplex and excimer formations, and proton transfer. To the last group (3), we ascribe the reactions, in which quenching of fluorescence occurs due to radiative and nonradiative transfer of excitation energy from the fluorescent donor to another particle - energy acceptor. 

Optical sensors for oxygen measurement are attractive since they can be fast, do not consume oxygen and are not easily poisoned. The most common method adopted in construction is based on quenching of fluorescence from appropriate chemical species. The variation in fluorescence signal (I), or fluorescence decay time (x) with oxygen concentration [O2] is described by Stem-Volmer equation91 ... 

In 1888, Walter studied the quenching of fluorescence, by the concentration effect, of fluorescein solutions. Nicols and Merrit observed in 1907, in solutions of eosine and resoruflne, the symmetry existing between their absorption and fluorescence spectra. In 1910, Ley and Engelhardt determined the fluorescence quantum yield of various benzene derivatives, values that were still referred to until recent years [18], The works by Lehmann and Wood, around 1910, marked the beginning of analysis based on fluorescence [4],... 

Fluorescence may be decreased or completely eliminated by interactions with other chemical species. This phenomenon is called quenching of fluorescence. Obviously, if the fluorescence of a fluorophore generated in a CL reaction is quenched the observation of chemiluminescence will be precluded. 

While there are only a few examples that can be used for direct detection of desired analytes, many simple molecules and ions do not have optical activity under regular conditions, a chemical reaction is needed to generate an optically active species. The reactions can be acid-base, ion pairing, complexation reactions, or quenching of fluorescence by 02, paramagnetic molecules, etc. Optical sensors for a few analyte or group analytes are summarized below. 

Considerable effort has been devoted to the determination of cross-sections for quenching of fluorescence from some of the lower states, but apart from the first two excited states zz IIj and A2h+, little attention has been paid to their photochemistry. 

Lakowicz JR (2006) Quenching of fluorescence, Chapter 8. In Principles of fluorescence spectroscopy, 3rd edn. Springer, New York... 

Dynamic quenching of fluorescence is described in Section 4.2.2. This translational diffusion process is viscosity-dependent and is thus expected to provide information on the fluidity of a microenvironment, but it must occur in a time-scale comparable to the excited-state lifetime of the fluorophore (experimental time window). When transient effects are negligible, the rate constant kq for quenching can be easily determined by measuring the fluorescence intensity or lifetime as a function of the quencher concentration the results can be analyzed using the Stern-Volmer relation ... 

In calix[4]arenes bearing four anthracene moieties on the lower rim (30),(62) changes in fluorescence intensity are observed on binding of alkali metal ions quenching of fluorescence by Na+ may arise from interaction of four anthracene residues brought in closer proximity to one another enhancement of fluorescence by K+ is difficult to explain. 

W. R. Ware, Oxygen quenching of fluorescence in solution An experimental study of diffusion process, /. Phys. Chem. 66, 455—458 (1962). 

J. R. Lakowicz and G. Weber, Quenching of fluorescence by oxygen A probe for structural fluctuation in macromolecules, Biochemistry 12, 4161 1170(1973). 

In summary, the use of fluorescence lifetime monitoring for temperature sensing at high temperatures is based on the phenomenon of thermal quenching of fluorescence, while this phenomenon is j u st the very obstacle that blocks the extending of the measurement further into higher temperatures. Therefore, fluorescence thermometry is intrinsically more effective for measurement within moderate temperature regions, due to this fundamental nature of the fluorescence emission itself. 

Quenching of fluorescence of tryptophan residues, coenzyme fluoro-phores, or extrinsic probes buried in the interior of proteins by colli-sional quencher molecules diffusing through the protein matrix/7,25 27)... 

---