Fluorescence quenching
The effect of fluorescence quenching ( English quenching ) indicates operations that a decrease in the intensity of fluorescence of a fluorophore effect without this that is destroyed.
A number of effects can cause fluorescence quenching, for example:
- Complex education
- internal conversion
- Energy transfer to other molecules , the quencher . This type of fluorescence quenching is reversible : the fluorescence increases again as soon as the quencher is removed.
A distinction must be made between quenching and the decrease in fluorescence due to high excitation intensities or (mostly unwanted) chemical changes in the dye , e.g. B. by oxidation in the presence of oxygen. This type of decrease in fluorescence is referred to as dye fading or photobleaching ; the fluorophore is irreversibly destroyed.
Quenching effects
The quenching effects include all processes that either transform the excited state of the fluorophore into the ground state without radiation or else prevent the fluorophore from being able to transition into the excited state.
- Dynamic quenching
In dynamic quenching, the energy of the excited fluorophore is transferred to this quencher molecule through the collision with a quencher molecule, with the energy ultimately being converted into heat . This type of quenching is also known as collision quenching . The reduction in fluorescence due to dynamic quenching can be described with the Stern-Volmer equation , especially with the Stern-Volmer equation for dynamic quenching .
- Static quenching
In static quenching, the fluorophore and quenching molecule form a complex , the fluorescence of which is reduced or completely absent. The complex formation reduces the concentration of fluorescent fluorophores. The reduction in fluorescence due to static quenching can be described with a modification of the Stern-Volmer equation, the Stern-Volmer equation for static quenching .
- Resonance energy transfer
In the case of resonance energy transfer, the energy of the excited state of the fluorophore D (donor) is transferred without radiation to a second molecule A (acceptor) through resonance effects. This reduces the fluorescence of the fluorophore. The resonance energy transfer can be described using the Förster resonance energy transfer (FRET).
- Blending effects
Often a quenching molecule species can quench the fluorophore via more than one effect, which results in mixing effects. Dynamic and static quenching often occur together, which can be described by expanding the Stern-Volmer equation for mixed quenching .
- Side effects
When excited, photon energy is captured; when quenched, it is distributed in the material. This results in an increase in temperature. The absorbers are also stimulated by the heat movement and can therefore radiate. This is a consequence of Kirchhoff's law of radiation . The radiation removes energy from the material, which makes it cooler. Most of the time, these side effects can be neglected, but they can be significant for specific processes.
Applications
Since the quenching of fluorescence is a phenomenon that is easy to observe and measure, it can be used as an indicator for processes taking place at the molecular level. A fundamental principle is that the presence or absence of a target substance in solution brings a fluorophore and its quencher closer to one another (no fluorescence) or distant from one another (fluorescence). In basic research , the fluorescence lifetime is often measured. The simpler alternative of measuring the fluorescence intensity is found more often with optical sensors. Examples:
- Oxygen measurement with the pO 2 - Optrode
Ruthenium (II) complexes with α- dimine ligands ( perylene , decacylene , pyrenebutyric acid ) are used as fluorophores .
- Potassium ion indicator
Evidence for potassium ions operates with a short DNA fragment ( telomere - sequence ), at the ends of dye and quencher covalently are. In solution they are separated from each other and the dye fluoresces. However, if the DNA fragment wraps around a potassium ion, they touch and the fluorescence is quenched. No fluorescence occurs when the target substance is present .
- DNA hybridization indicator
Another evidence uses the fact that as soon as DNA hybridizes with its counterstrand, it adopts a more rigid, linear shape. In this application, the deletion is ended as soon as - in the presence of a correctly base-pairing counter-strand - the fluorophore and quencher, which are attached to the ends of the strand, are separated from one another. If the target substance is present, fluorescence takes place.
- Optimization of hyperpolarization processes
In the hyperpolarization of gases, quenching molecules are inserted in order to reduce the rate of spontaneous emission of the gas molecules in the excited state and thus to protect other gas molecules from re- absorption of unpolarized light .
Web links
- Entry on quenching. In: Römpp Online . Georg Thieme Verlag, accessed on March 21, 2014.
Individual evidence
- ^ W. Happer, WA Van Wijngaarden: An optical pumping primer . In: Hyperfine Interactions . tape 38 , no. 1-4 , December 1987, ISSN 0304-3843 , pp. 435-470 , doi : 10.1007 / BF02394855 ( springer.com [accessed February 15, 2020]).