Fluorescence quenching

Introduction
Quenching of fluorescence is a physiochemical process that describes the decreasing of the fluorescent intensity of fluorphores. There are two different ways of quenching: static and dynamic quenching. It has to be mentioned that there is a difference between quenching and the decrease of fluorescence because of an high state of molecular excitement or chemical changes of dye (like oxydation).

Static Quenching
Static quenching (or contact quenching) of fluorescence is, when a Fluorphor (F) and a Quencher (Q) are creating a complex (FQ) which is not fluorescent. The chemical equation therefore is: F + Q ⇌ FQ

The chemical equilibrium (Ks) between the Fluorphor, the Quencher and the complex FQ is formed by the law of mass action and equal to the Stern-Volmer-constant (Ksv). There [FQ] stands for the concentration of the complex FQ, [F] for the concentration of the loose Fluorfor and [Q] for the loose Quencher.

The Stern-Volmer-equation describes the dependence of the fluorescent intensity of a fluorescent dye on the concentration of an quenching material (Quencher). It was created by the two physical chemists Otto Stern and Max Volmer in 1919. There F0 is the fluorescent intensity of the fluorescent dye without the quencher, F is the fluorescent intensity of fluorescent dye with the quencher. Ks stands for the Stern-Volmer-constant and [Q] for the concentration of the quencher.

Dynamic Quenching
are non-radioactive, short-range quenching methods that occur between two light-sensitive molecules (called donor and acceptor fluorophores). Exchange of electrons between donor and acceptor fluorophore happens non-radioactively – without releasing of photons during the process.

Förster Energy Transfer
is a non-radioactive phenomenon in which donor fluorophore transmits electron(s) to acceptor fluorophore without coming in direct contact. Fluorescence Resonance Energy Transfer (FRET) is a technique for measuring distance between two light sensitive molecules; it relies on Forster energy transfer principle. Since this process is so distance-dependent, using of it has become almost exclusively biology-driven. The most useful application is measuring the size of large molecules. A donor-acceptor pair of fluorophores is attached to a big molecule and length is measured over the time that passes while donor transmits energy to the acceptor.

Dexter Energy Transfer
similarly to Forster Energy Transfer, is a non-radioactive and short range process; though by principle and range they are different. Dexter Energy transfer happens only when two chemical groups (by fluorophores- intermolecular) or two parts (fluorophores-intramolecular) of a chemical group collide and exchange electrons bilaterally. Ground-State group (G) might come into contact with Excited, fluorescent group (E) and take away the electrons that account for fluorescence. Dexter energy transfer stands out as it requires a so-called “Wavefunction Overlap” between two fluorophores, in other terms; - atoms on molecules or chemical groups need to overlap their electron clouds. The energy transferring electron will travel from donor to acceptor and occupy acceptor’s leas occupied orbital. Energy Transfer results in donor’s returning from excited to ground state and acceptor’s maintaining the ground state due to greater electronegativity it has. Now that the energy is transferred there are no fluorophores that are excited and emit light, and we can say that the quenching process (decreasing of light emission) has happened.

Applications
Quenching is a really good visible physical phenomenon, that's why it is used as an indicator for molecular processes.

The ends of short DNA fragments (telomer sequence), connected with a fluorescent dye and quencher through a covalent bond, are divided in a solution. The dye is producing light. If the there are any potassium-ions present, the DNA fragment is wrapping around the potassium-ion and the endings are touching each other. The fluorescence is quenched (lightning stops).
 * 1) Indicator for Potassium-ions

If DNA strand is hybridising with an opposite strand it gets quite linear and stiff. Fluorphor and Quencher are attached to the ends. If the bases are paring correctly the Fluorphor and Quencher disconnect and the quenching stops.
 * 1) Indicator for DNA hybridisation

On a Rubidium complex u can show the level of oxygen saturation depending on the level of quenching.
 * 1) Indicator of loose Oxygen