Quenched dynamics

Later on, such S-layer-based sensing layers were also used in the development of optical biosensors (optodes), where the electrochemical transduction principle was replaced by an optical one [97] (Fig. 10c). In this approach an oxygen-sensitive fluorescent dye (ruthenium(II) complex) was immobilized on the S-layer in close proximity to the glucose oxidase-sensing layer [97]. The fluorescence of the Ru(II) complex is dynamically quenched by molecular oxygen. Thus, a decrease in the local oxygen pressure as a result of... 

The data for sodium 9-anthroate in benzonitrile do not fit the pattern of the other derivatives since in this case kD + fccq > kMThis effect cannot be due to kD since this value is less than those of the other derivatives. Therefore cq must be greatly increased for the salt. This effect is thought to arise from both dynamic quenching and static quenching due to ion pairs. 

When anisotropy increases due to fluorescence lifetime decrease being coupled to any effect of dynamic quenching. 

Tomin VI, Oncul S, Smolarczyk G, Demchenko AP (2007) Dynamic quenching as a simple test for the mechanism of excited-state reaction. Chem Phys 342 126—134... 

Besides the quenching in the excited state dynamic quenching), there exists another type of quenching (static), which takes place in the ground state and occurs due to the formation of nonemitting complexes. 

To study the dynamic quenching in steady state approach, the Stem-Volmer relations are commonly used ... 

As it was shown recently [16, 17], dynamic quenching is also a powerful method for determining the kinetic or thermodynamic characteristics of photoreactions in the excited sate, and for changing the rates [18, 19] of such reactions as, for example, ESIPT. 

Tomin VI, Smolarczyk G (2008) Dynamic quenching of the multiband fluorescence of 3-hydroxyflavone. Opt Spectros 104 919-925... 

Tomin VI (2009) Effect of temperature and dynamic quenching on proton transfer in 3-hydroxyflavone. Opt Spectros 107 92-100... 

The lifetime, therefore, depends not only on the intrinsic properties of the fluorophore but also the characteristics of the environment. For example, any agent that removes energy from the excited state (i.e., dynamic quenching by oxygen) shortens the lifetime of the fluorophore. This general process of increasing the nonradiative decay rates is referred to as quenching. 

Kautsky 1931 first oxygen sensor (non-fiber optic) based on dynamic quenching of the phosphorescence of adsorbed dyes... 

Fluorescence quenching may be dynamic, if the photochemical process is the result of a collision between the photoexcited indicator dye and the quencher species, or static, when the luminophore and the quencher are preassociated before photoexcitation of the former20. It may be easily demonstrated that dynamic quenching in isotropic 3-D medium obeys the so-called Stem-Volmer equation (2)21 ... 

It may also happen that an association equilibrium exists between the luminescent indicator and the quencher. Non-associated indicator molecules will be quenched by a dynamic process however, the paired indicator dye will be instantaneously deactivated after absorption of light (static quenching). Equation 2 still holds provided static quenching is the only luminescence deactivation mechanism (i.e. no simultaneous dynamic quenching occurs) but, in this case, Ksv equals their association constant (Kas). However, if both mechanisms operate simultaneously (a common situation), the Stem-Volmer equation adopts more complicated forms, depending on the stoichiometry of the fluorophore quencher adduct, the occurrence of different complexes, and their different association constants. For instance, if the adduct has a 1 1 composition (the simplest case), the Stem-Volmer equation is given by equation 3 ... 

Huber C., Krause C., Werner T., Wolfbeis O.S., Serum chloride optical sensors based on dynamic quenching of the fluorescence of photo-immobilized lucigenin, Microchimica Acta 2003 142 245-253. 

In some cases it is possible to obtain a measure of the association constant for intercalation directly from fluorescence quenching data. This method is applicable when the dynamic quenching of the hydrocarbon fluorescence by DNA is small and when the intercalated hydrocarbon has a negligible fluorescence quantum yield compared to that of the free hydrocarbon. If these conditions are met, the association constant for intercalation, Kq, is equal to the Stern-Volmer quenching constant Kgy (76) and is given by Equation 1. 

In dynamic quenching (or diffusional quenching) the quenching species and the potentially fluorescent molecule react during the lifetime of the excited state of the latter. The efficiency of dynamic quenching depends upon the viscosity of the solution, the lifetime of the excited state (x ) of the luminescent species, and the concentration of the quencher [Q], This is summarized in the Stern-Volmer equation ... 

Fluorescence quenching is described in terms of two mechanisms that show different dependencies on quencher concentration. In dynamic quenching, the quencher can diffuse at least a few nanometers on the time scale of the excited state lifetime (nanoseconds). In static quenching, mass diffusion is suppressed. Only those dye molecules which are accidentally close to a quencher will be affected. Those far from a quencher will fluoresce normally, unaware of the presence of quenchers in the system. These processes are described below for the specific case of PMMA-Phe quenched by MEK. 

At an MEK concentration greater than 1 M, both the dynamic and the static quenching mechanisms have to be taken into account. Therefore, Frank and Vavilov s model of combined static and dynamic quenching model (28),... 

Figure 6. Calculated PMMA-Phe Fluorescence Intensity from Static and Dynamic Quenching Theory as a Function of MEK Concentration.

Similarly to bulk oxygen sensors, optical nanosensors rely on dynamic quenching of luminescence. Numerous indicators and polymeric materials were found suitable... 

Following an external perturbation, the fluorescence quantum yield can remain proportional to the lifetime of the excited state (e.g. in the case of dynamic quenching (see Chapter 4), variation in temperature, etc.). However, such a proportionality may not be valid if de-excitation pathways - different from those described above - result from interactions with other molecules. A typical case where the fluorescence quantum yield is affected without any change in excited-state lifetime is the formation of a ground-state complex that is non-fluorescent (static quenching see Chapter 4). 

Case C Q is not in large excess and mutual approach of M and Q is possible during the excited-state lifetime. The bimolecular excited-state process is then diffusion-controlled. This type of quenching is called dynamic quenching (see Section 4.2.2). At high concentrations of Q, static quenching may occur in addition to dynamic quenching (see Section 4.2.4). 

The excited-state lifetime of the uncomplexed fluorophore M is unaffected, in contrast to dynamic quenching. The fluorescence intensity of the solution decreases upon addition of Q, but the fluorescence decay after pulse excitation is unaffected. Quinones, hydroquinones, purines and pyrimidines are well-known examples of molecules responsible for static quenching. 

A linear relationship is thus obtained, as in the case of the Stern-Volmer plot (Eq. 4.10), but there is no change in excited-state lifetime for static quenching, whereas in the case of dynamic quenching the ratio I0/I is proportional to the ratio to/t of the lifetimes. 

Static and dynamic quenching may occur simultaneously, resulting in a deviation of the plot of Io/I against [Q] from linearity. 

Let us consider first the case of static quenching by formation of a non-fluorescent complex. The ratio I0/I obtained for dynamic quenching must be multiplied by the fraction of fluorescent molecules (i.e. uncomplexed)... 

Method II Dynamic quenching by totally micellized immobile quenchers It is assumed that the probability of quenching of a fluorescent probe in a given micelle is proportional to the number of quenchers residing in this micelle. The rate constant for de-excitation of a probe in a micelle containing n quencher molecules is given by... 

When a system contains a fluorophore in different environments (e.g. a fluo-rophore embedded in microheterogeneous materials such as sol-gel matrices, polymers, etc.) or more than one fluorophore (e.g. different tryptophanyl residues of a protein), the preceding relations must be modified. If dynamic quenching is predominant, the Stem-Volmer relation should be rewritten as... 

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 ... 

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