Fluorescence quenching refers to the phenomenon that the fluorescent signal emitted by fluorescent dyes is weakened due to the aggregation of molecules or the mutual interference between them. Fluorescence quenching is a physicochemical process that reduces the intensity of light emitted by fluorescent molecules. Fluorescence quenching is a reversible process, which means that if the distance between molecules is increased, their fluorescence will become stronger. This point is that it is different from fluorescent bleaching. Fluorescence bleaching is an irreversible process. After the dye molecules are bleached, the molecular structure has been destroyed, and the molecules can no longer emit fluorescence. Fluorescence quenching mainly occurs in these situations, such as when the dye molecules are insoluble, the concentration of the dye molecules is too high, or the density of the dye molecules labeled on the surface of the macromolecule is too high.
The interaction between fluorescent molecules and solvents or other molecules, causing the reduction of fluorescence intensity is called quenchers, such as halide ions, heavy metal ions, oxygen molecules, nitro compounds, diazo compounds, carboxyl, and carbonyl compounds are common fluorescence quenchers. When the concentration of a fluorescent substance is too large, a self-quenching phenomenon will occur.
The fluorescence quenching phenomenon needs to be considered in the fluorescent dye labeling experiments of macromolecules. When labeling, the ratio of macromolecule/dye needs to be controlled. Do not over-label. Too much dye on the surface of the macromolecule will make the distance between the dye molecules too close, which will cause fluorescence quenching, and the fluorescence of the final over-labeled product will not be strong. In addition, it should be noted that fluorescence quenching does not mean that the fluorescence signal is gone, but lower than expected, so the dye molecules under the quenching condition can still emit fluorescence, but it is weaker. Using the phenomenon of fluorescence quenching, many fluorescent probes can be developed. For example, the probe shown in the figure below deforms after detecting the target product, the distance between the dye molecules at both ends increases, the quenching phenomenon disappears, and the fluorescence is enhanced.
Fluorescence quenching is divided into static quenching and collisional quenching
Static quenching is caused by the formation of complexes between fluorescent and quenching molecules. Once the complex is formed, it is non-fluorescent. The formation of the complex occurs before any electronic excitation takes place.
Collisional quenching occurs when the excited fluorophore experiences contact with an atom or molecule that can facilitate non-radiative transitions to the ground state. Excited-state molecule collides with the quencher molecule and returns to the ground state non-radiatively.
There are many reasons for fluorescence quenching, and the mechanism is also very complicated, including:
1. Loss of energy due to the collision between the molecules of the fluorescent substance and the molecules of the quencher;
2. The molecule of the fluorescent substance interacts with the molecule of the quencher to form a coordination compound that does not emit light by itself;
3. The existence of dissolved oxygen makes the fluorescent substance oxidize, or because of the paramagnetism of the oxygen molecule, it promotes the crossing between the systems, so that the excited singlet state of the fluorescent molecule changes to the triplet state;
4. When the concentration of a fluorescent substance is too large, a self-quenching phenomenon will occur.
Applications of Fluorescence Quenching
Fluorescence quenching can be used as an indicator of DNA hybridization, where a fluorophore and a quenching molecule attach to the ends of single-stranded DNA and come close to each other, forming a loop. When the DNA hybridizes and pairs with another single-stranded DNA strand, the fluorophore-quencher complex is pulled apart, allowing the fluorophore to generate light.
The Förster mechanism of fluorescence quenching can be used to infer the distance between donor and acceptor molecules, depending on the strength of the quenching. This determines the size or conformation of the protein and detects any interactions between the proteins.