Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Quenching (fluorescence)

Quenching of quinine fluorescence (right) by chloride ions (left)

Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on pressure and temperature. Molecular oxygen and the iodide ion are common chemical quenchers. Quenching poses a problem for non-instant spectroscopic methods, such as laser-induced fluorescence.

Quenching is made use of in optode sensors; for instance the quenching effect of oxygen on certain ruthenium complexes allows the measurement of oxygen saturation in solution. Quenching is the basis for fluorescence resonance energy transfer (FRET) assays. Quenching and dequenching upon interaction with a specific molecular biological target is the basis for activatable optical contrast agents for molecular imaging.

Quenching mechanisms

There are a few distinct mechanisms by which energy can be transferred nonradiatively (without absorption or emission of photons) between two dyes, a donor and an acceptor. Förster resonance energy transfer (FRET or FET) is a dynamic quenching mechanism because energy transfer occurs while the donor is in the excited state. FRET is based on classical dipole-dipole interactions between the transition dipoles of the donor and acceptor and is extremely dependent on the donor – acceptor distance (R), falling off at a rate of 1/R⁶. FRET also depends on the donor-acceptor spectral overlap (see figure below) and the relative orientation of the donor and acceptor transition dipole moments. FRET can typically occur over distances up to 100 Å.

Dexter (also known as exchange or collisional energy transfer) is another dynamic quenching mechanism. Dexter energy transfer is a short-range phenomenon that decreases with e^-R and depends on spatial overlap of donor and quencher molecular orbitals. In most donor fluorophore – quencher acceptor situations, the Förster mechanism is more important than the Dexter mechanism. With both Förster and Dexter energy transfer, the shapes of the absorption and fluorescence spectra of the dyes are unchanged.

Exciplex (excited state complex) formation is a third dynamic quenching mechanism.

The remaining energy transfer mechanism is static quenching (also referred to as contact quenching). Static quenching can be a dominant mechanism for some reporter-quencher probes. Unlike dynamic quenching, static quenching occurs when the donor and acceptor molecules are in the ground state. The donor and acceptor molecules bind together to form a ground state complex, an intramolecular dimer with its own unique properties, such as being nonfluorescent and having a unique absorption spectrum. Dye aggregation is often due to hydrophobic effects – the dye molecules stack together to minimize contact with water. Planar aromatic dyes that are matched for association through hydrophobic, electrostatic and steric forces can enhance static quenching. High temperatures and addition of surfactants tend to disrupt ground state complex formation.

Comparison of static and dynamic quenching mechanisms