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Fluorescence Quenching
The fluorescence of a compound can be quenched, meaning it is reduced or completely switched off by processes that do not destroy the fluorophore permanently. Fluorescence quenching reduces the intensity of fluorescence, but the original intensity reappears as soon as the quencher is removed or the quenching mechanism is switched off.
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Fluorescence quenching occurs when the quenching agent either prevents excitation to the fluorescent state or deactivates the electronically excited state to the ground state without emission of a photon in the presence of the quencher.
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This easily observable and measurable phenomenon is frequently employed in various applications as an indicator for molecular-level processes. ?The existence or nonexistence of an analyte triggers a modification in the spacing between the fluorophore and quencher, thus resulting in an alteration in fluorescence. Additionally, in certain circumstances, the lifetime can serve as a measure for this alteration aside from the fluorescence intensity.
Molecular Beacon
The genepin, hairpin or molecular beacon technique is a well-known example used in molecular biology. Fluorescent dye and a quencher are positioned at the ends of a single strand DNA. The ends then go through a process of hybridization, forming a partial double-stranded DNA, due to several complementary bases present at the end pieces. ?This arrangement will cause the middle DNA portion to form a loop, resulting in a hairpin-like structure.The fluorescence of the marker is suppressed by the proximity of the quencher. When a DNA strand, complementary to the base sequence of the first strand, is added to the solution, hybridization occurs between the two strands through a stronger interaction of all the bases. As a result, the positioning of dye and quencher becomes separated, leading to fluorescence reoccurring. ?This principle can be applied to conceive various experiments and draw conclusions about molecular processes.Two fluorescent dyes, whose fluorescence behaves as described under F?rster resonance energy transfer, may be employed for this purpose alongside a dye and a quencher.
Polymerase Chain Reaction
Furthermore, comparable techniques are utilised in the implementation of polymerase chain reaction (PCR) for the enhancement of DNA. This enables the detection of the advancement or conclusion of the reaction.
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Fluorescence can be quenched by various phenomena. In reality, these phenomena are often a mixture of two different types. However, only two types can be distinguished.
Dynamic quenching
The energy is transferred to the quencher molecule when the excited fluorophore collides with it, and the energy is ultimately converted into heat. This diffusion process is referred to as "collision quenching". The Stern-Vollmer equation describes dynamic fluorescence quenching.
Static quenching
The fluorophore and quencher combine to create a "stable" complex that decreases the concentration of fluorescent molecules permanently.
Concentration quenching
Concentration quenching is a seemingly insignificant phenomenon where the fluorescence of similar molecules in a concentrated solution is suppressed. However, experimental observations have shown inconsistencies and contradictions under different conditions.
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This discussion will focus on organic dyes like rhodamines in aqueous solutions, where several dye molecules form a static association and their electron orbitals interact with each other. This alters the spectrum of absorption and extinguishes the fluorescence. A more detailed account is provided in the "Dye-Aggregation" section.
Electron transfer
Direct contact between the excited molecule and the quencher is necessary for quenching by electron transfer, which involves an electron transferring from the donor to the acceptor. This effectively quenches the fluorescence of oxazine dyes ATTO 655, ATTO 680, and ATTO 700 by guanosine, tryptophan, and similar compounds.
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