Fluorescence Quenching

The fluorescence of a compound can be quenched - i.e. reduced or completely switched off - by a series of processes without permanently destroying the fluorophore. The effect of fluorescence quenching thus reduces the intensity of fluorescence. As soon as the quencher is removed or the quenching mechanism is "switched off", the original fluorescence intensity reappears.

Either the excitation to the fluorescent state is already prevented by the quenching agent or the electronically excited state is deactivated to the ground state without radiation, i.e. the emission of a photon, in the presence of the quencher.

In practice, fluorescence quenching is used in many applications because it is an easily observed or measurable phenomenon. It can therefore be used as an indicator for processes taking place at the molecular level. The presence or absence of an analyte, for example, causes a change in the distance between fluorophore and quencher and thus a change in fluorescence. In addition to the fluorescence intensity, the lifetime can also be used as a measure for this change in some cases.

Molecular Beacon
A well-known example of this is the so-called "genepin", "hairpin" or "molecular beacon" technique. Here, a fluorescent dye and a quencher are located at the ends of a DNA single strand. A hybridization takes place by several complementary bases at the end pieces, i.e. a partial DNA double strand is formed. Thus, the middle part of the DNA forms a loop, the resulting structure resembles a hairpin. The fluorescence of the marker is quenched by the spatial proximity of the quencher. If a DNA strand is added to the solution, which is complementary to the base sequence of the first strand, these two strands hybridize by a stronger interaction of all the bases, so that dye and quencher are spatially separated. This causes fluorescence to reoccur. With this principle many different experiments can be conceived and conclusions can be drawn about molecular processes.
In addition to using a dye and a quencher, two fluorescent dyes can be used for this purpose, whose fluorescence behaves as described under Förster resonance energy transfer.

Y. Cheng, T. Stakenborg, P. van Dorpe, L. Lagae, M. Wang, H. Chen, G. Borghs, Fluorescence Near Gold Nanoparticles for DNA Sensing, Analytical Chemistry 83, 1307 (2011). → ATTO 550
A. Gianetti, S. Tombelli, Intracellular delivery of molecular beacons by PMMA nanoparticles and carbon nanotubes for mRNA sensing, Proceedings of SPIE - The International Society for Optical Engineering 8596, 85960 (2013). → ATTO 647N
R. Tsukanov, T. Tomov, Y. Berger, M. Liber, E. Nir, Conformational Dynamics of DNA Hairpins at Millisecond Resolution Obtained from Analysis of Single-Molecule FRET Histograms, The Journal of Physical Chemistry B 117, 16105 (2013). → ATTO 550, ATTO 647N
A. Hartmann, G. Krainer, M. Schlierf, Different Fluorophore Labeling Strategies and Designs Affect Millisecond Kinetics of DNA Hairpins, Molecules 19, 13735 (2014). → ATTO 532, ATTO 647N
S. Ebrahimi, Y. Akhlaghi, M. Kompany-Zareh, Å. Rinnan, Nucleic Acid Based Fluorescent Nanothermometers, ACS Nano 8, 10372 (2014). → ATTO 425
V. Weiss, C. Bliem, I. Gösler, S. Fedosyuk, M. Kratzmeier, D. Blaas, G. Allmaier, In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis, Analytical and Bioanalytical Chemistry, 1 (2016). → ATTO 488, ATTO 495, ATTO 633
S. Ochmann, C. Vietz, K. Trofymchuk, G. Acuna, B. Lalkens, P. Tinnefeld, Optical Nanoantenna for Single Molecule-Based Detection of Zika Virus Nucleic Acids without Molecular Multiplication, Analytical chemistry 89, 13000 (2017). → ATTO 542, ATTO 647N
L. Zhang, A. Bluhm, K.-J. Chen, N. Larkey, S. Burrows, Performance of nano-assembly logic gates with a DNA multi-hairpin motif, Nanoscale 9, 1709 (2017). → ATTO 633

Polymerase Chain Reaction
In addition, similar methods are used in the application of polymerase chain reaction (PCR) for the amplification of DNA. This allows the progress or end of the reaction to be indicated.

K. Stöhr, B. Hafner, O. Nolte, J. Wolfrum, M. Sauer, D.‐P. Herten, Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes, Analytical Chemistry 77, 7195 (2005). → ATTO 655
L. Alberti, S. Renaud, L. Losi, S. Leyvraz, J. Benhattar, G. Saretzki, High Expression of hTERT and Stemness Genes in BORIS/CTCFL Positive Cells Isolated from Embryonic Cancer Cells, PLOS ONE 9, e109921 (2014). → ATTO 647
Z. Fidan, A. Wende, U. Resch-Genger, Visible and red emissive molecular beacons for optical temperature measurements and quality control in diagnostic assays utilizing temperature-dependent amplification reactions, Analytical and Bioanalytical Chemistry 409, 1519 (2017). → ATTO 647N

Several phenomena can lead to fluorescence quenching. There are basically two different types, but in reality they often occur as a mixed form:

Dynamic quenching
The energy is transferred to the quencher molecule by the collision of the excited fluorophore with a quencher molecule and finally converted into heat. This type of diffusion related process is known as "collision quenching". Dynamic fluorescence quenching is described by the "Stern-Vollmer equation".

Static quenching
Fluorophore and quencher form a "stable" complex, which permanently reduces the concentration of fluorescent molecules.

Concentration quenching
An apparently trivial phenomenon is the so-called concentration quenching, i.e. the quenching of the fluorescence of similar molecules in a concentrated solution. However, the observed experimental findings are inconsistent and even contradictory, depending on the conditions.
We will only briefly discuss the case of organic dyes, such as rhodamines, in aqueous solution. This is mainly a static association of several dye molecules whose electron orbitals interact with each other. This changes the absorption spectrum and quenches the fluorescence. A more detailed description is given in the section "Dye-Aggregation".

Electron transfer
A direct contact of the excited molecule with the quencher is also necessary during the quenching by electron transfer, i.e. the transfer of an electron from the donor to the acceptor. In this way, the fluorescence of the oxazine dyes ATTO 655, ATTO 680 and ATTO 700 is quenched very effectively by guanosine, tryptophan and similar compounds.

N. Marmé, J.-P. Knemeyer, M. Sauer, J. Wolfrum, Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan, Bioconjugate Chemistry 14, 1133 (2003). → ATTO 590, ATTO 655, ATTO 680
S. Doose, H. Neuweiler, M. Sauer, A close look at fluorescence quenching of organic dyes by tryptophan, ChemPhysChem : a European journal of chemical physics and physical chemistry 6, 2277 (2005). → ATTO 655
R. Zhu, X. Li, X. Zhao, A. Yu, Photophysical Properties of Atto655
Dye in the Presence of Guanosine and Tryptophan in Aqueous Solution
, The Journal of Physical Chemistry B 115, 5001 (2011). → ATTO 655
Q. Sun, R. Lu, A. Yu, Structural Heterogeneity in the Collision Complex between Organic Dyes and Tryptophan in Aqueous Solution, The Journal of Physical Chemistry B 116, 660 (2012). → ATTO 655
Y. Zhang, S. Yuan, R. Lu, A. Yu, Ultrafast Fluorescence Quenching Dynamics of Atto655 in the Presence of N -Acetyltyrosine and N -Acetyltryptophan in Aqueous Solution, The Journal of Physical Chemistry B 117, 7308 (2013). → ATTO 655
S. Nanguneri, B. Flottmann, F. Herrmannsdörfer, K. Thomas, M. Heilemann, Single-molecule super-resolution imaging by tryptophan-quenching-induced photoswitching of phalloidin-fluorophore conjugates, Microscopy Research and Technique 77, 510 (2014). → ATTO 488
A. Sharma, J. Enderlein, M. Kumbhakar, Photon Antibunching Reveals Static and Dynamic Quenching Interaction of Tryptophan with Atto-655, The journal of physical chemistry letters 8, 5821 (2017). → ATTO 655

Resonance energy transfer
In the so-called Förster-Resonance-Energy-Transfer (FRET) the energy of an excited donor is transferred without radiation to a nearby acceptor. This reduces the fluorescence of the donor. If the acceptor is fluorescent, its fluorescence can appear instead, as if it had been directly excited. If the acceptor itself does not fluoresce, it only takes over the energy of the donor and therefore acts as a fluorescence quencher or quencher dye.

ATTO Quencher
Our quenchers ATTO 540Q, ATTO 575Q, ATTO 580Q and ATTO 612Q are designed to quench exclusively via the FRET mechanism, i.e. no fluorescence quenching takes place without overlapping of the emission spectrum of the fluorophore with the absorption spectrum of the quencher in question.
D. Betbeder, E. Lipka, M. Howsam, R. Carpentier, Evolution of availability of curcumin inside poly-lactic-co-glycolic acid nanoparticles: impact on antioxidant and antinitrosant properties, International journal of nanomedicine 10, 5355 (2015). → ATTO 540Q
R. Chulluncuy, C. Espiche, J. Nakamoto, A. Fabbretti, P. Milón, Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin, Antibiotics 5, 38 (2016). → ATTO 540Q
T. Kokko, L. Kokko, T. Soukka, T. Lövgren, Homogeneous non-competitive bioaffinity assay base on fluorescence resonance energy transfer, Anal. Chim. Acta 585, 120 (2007). → ATTO 612Q