Quenching (fluorescence)

The effect of fluorescence quenching (English quenching) denotes operations that result in a decrease in the intensity of fluorescence of the fluorophore without the fluorophore is destroyed.

There are a number of effects that can lead to fluorescence quenching, for example:

• Complex formation
• Internal conversion
• Energy transfer to other molecules, so-called quencher

Indistinguishable from the decrease in the fluorescence quenching due to high excitation intensity, or (usually unwanted ) of chemical changes of the dye, for example by oxidation in the presence of oxygen. This type of fluorescence decrease is called the fading of the dye or photobleaching. The fluorophore is thereby irreversibly destroyed. In contrast, the fluorescence quenching of a fluorophore is reversible by a quencher: Fluorescence increases again when the quencher is removed.

Quenching effects

The quenching effects include all the operations that either transfer the excited state of the fluorophore -radiatively to the ground state or prevent the fluorophore can transition to the excited state.

The dynamic quenching of the excited fluorophore, the energy is transmitted through the collision with a quencher molecule at this quencher, wherein the energy ultimately turns into heat. This type of quenching is also referred to as collisional quenching. The decrease in fluorescence by dynamic quenching can be described by the Stern-Volmer equation, especially with the Stern-Volmer equation for dynamic quenching.

In static quenching fluorophore and quencher form a complex whose fluorescence is reduced or absent. Due to the complex formation, the concentration of fluorophores capable of fluorescence is reduced. The decrease in fluorescence by static quenching can with a modification of the Stern-Volmer equation, the Stern-Volmer equation for static quenching, are described.

The resonant energy transfer the energy of the excited state of the fluorophore D (donor) nonradiatively due to resonance effects of a second molecule A (acceptor) is transmitted. Thus, the fluorescence of the fluorophore D. decreases the resonance energy transfer can be described by the Förster resonance energy transfer ( FRET).

Often a quencher species is able to quench the fluorophore with more than one effect. It then arise mixing effects. Often dynamic quenching and static quenching occur together. The reduction of the fluorescence by simultaneous static and dynamic quenching can be described in extension of the Stern-Volmer equation for Mischquenching.

When the excitation photon energy is captured. When quenching, this energy is distributed in the material. This leads to a temperature increase. Due to the thermal motion of the absorber are also encouraged and can radiate thereby. This is a consequence of Kirchhoff 's law of radiation. The radiation escapes the material energy, which becomes cooler. Mostly, these side effects can be ignored, but in special operations these may be significant.

Applications

Since the quenching of fluorescence is a readily observable and measurable phenomenon, it offers itself as an indicator of which takes place at the molecular level processes. A fundamental principle is, that are brought home by the presence or absence of a target substance in solution a fluorophore and quencher be each other or away from each other. In the basic research while the fluorescence lifetime is often measured. The simpler alternative to measure the fluorescence intensity is more common in optical sensors. Examples:

As fluorophores ruthenium (II ) complexes used with α - Dimin ligand ( perylene, Decacylen, pyrene butyric acid ).

A proof of potassium ions work with a short DNA fragment ( telomeric sequence), at the ends of the dye and the quencher are covalently bound. In solution, they are separated from each other and the fluorescent dye. The DNA fragment Diaper However, a potassium ion, so they touch and the fluorescence is quenched.

Another detection exploits the fact that DNA once it hybridizes to its complementary strand, assuming a rigid, linear shape. In this application, the deletion is completed when, are separated from each other in the presence of a correct base-pairing complementary strand, the fluorophore and quencher, which are attached to the ends of the strand.

Swell

• Quenching. In: Rompp online. Georg Thieme Verlag, accessed on 21 March 2014.

• Spectroscopy
• Solid State Physics