Fluorescence concentration quenching

Chen, R.F., and Knutson, J.R. (1988) Mechanism of fluorescent concentration quenching of carboxyfluo-rescein in liposomes Energy transfer to nonfluorescent dimers. Anal. Biochem. 172, 61. 

Chen RF, Knutson JR (1988) Mechanism of fluorescence concentration quenching of car-boxyfluorescein in liposomes energy transfer to nonfluorescent dimers. Anal Biochem 172(l) 61-77... 

A high concentration of the fluorescent dye itself in a solvent or matrix causes concentration quenching. Rhodamine dyes exhibit appreciable concentration quenching above 1.0%. Yellow dyes, on the other hand, can be carried to 5 or even 10% in a suitable matrix before an excessive dulling effect, characteristic of this type of quenching, occurs. Dimerization of some dyes, particularly those with ionic charges on the molecules, can produce nonfluorescent species. 

Fluorescent small molecules are used as dopants in either electron- or hole-transporting binders. These emitters are selected for their high photoluminescent quantum efficiency and for the color of their emission. Typical examples include perylene and its derivatives 44], quinacridones [45, penlaphenylcyclopenlcne [46], dicyanomethylene pyrans [47, 48], and rubrene [3(3, 49]. The emissive dopant is chosen to have a lower excited state energy than the host, such that if an exciton forms on a host molecule it will spontaneously transfer to the dopant. Relatively small concentrations of dopant are used, typically in the order of 1%, in order to avoid concentration quenching of their luminescence. 

That oxetane formation results from a singlet state reaction follows from the following evidence (a) Acetone fluorescence is quenched by addition of the olefin, (b) oxetane formation is relatively insensitive to piperylene, and (c) cis-trans isomerization of the olefin is quenched at high olefin concentrations but oxetane formation is not affected. Since oxetane formation was... 

DASPE-TFPB), respectively. The obtained solid precipitates were brightly emissive whereas that of the native DASPE-I were almost nonemissive (Fig. 7a the photo is taken under normal illumination and UV-light irradiation). This indicates that, in the solid of the ion-pair species between DASPE+ and TPB (or TFPB ), concentration quenching is effectively suppressed, and more importantly, these ion-pair complexes can generate fluorescent... 

Figure 11.1. An example of concentration quenching. In ruby, the observed decay time of the fluorescence at 77K is independent of chromium concentration up to a concentration of around 03 weight% of CrjOj in the material/251...

Finally it is worth mentioning the relation between fluorescence properties and the concentration of activator content. The fluorescence intensity increases initially with increases in the concentration of activator content. However, the increase in the concentration above a certain critical value can lead to a reduction in fluorescence intensity. This is called concentration quenching. It can also be observed from the reduction in the lifetime with the increase in the concentration, as shown in Figure 11.1 in the case of ruby, where the activator is the Cr3+ ion. Therefore, it is advantageous to select an activator concentration short of the critical value to achieve a good level of fluorescence intensity in a practical sensor system. 

Figure 14.17. Liposome fluoroimmunoassay depiction, in which antigen-liposome conjugates containing fluorescent dye (concentration quenched) compete with analyte antigen for antibody binding sites, followed by wash and detergent lysis, to release the fluorophore for fluorescence measurement.

A protein induced after coliphage N4 infection has been studied. Although it has one or two tryptophans, its intrinsic fluorescence is dominated by the ten tyrosines/1111 Tryptophan fluorescence is seen after denaturing the protein. Upon binding to single-stranded DNA, the tyrosine fluorescence is quenched. This signal has been used to demonstrate that the binding affinity is very dependent on salt concentration and is also very sensitive to the nucleotide sequence. 

The dependence of and S2 fluorescence intensities on the sample concentration was investigated between 10 - 10 M, and a sample concentration of less than 10 M was chosen to minimize the effect of concentration quenching. Two-photon absorption measurements were carried out by the excitation by relatively low power density (<10 photons cm" pulse ) in order to avoid the saturation effect on the laser power. Under these experimental conditions, the following rate equations for the concentration of S2 molecule is derived ... 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

The SeaDog sensor utilized in this work is capable of near real-time detection of low concentrations of explosives in water. The sensor utilizes novel sensing materials originally developed by collaborators at MIT. These materials are fluorescent polymers that are highly emissive when deployed as solid-state thin films. When the polymers interact with nitroaromatic explosives such as TNT, the fluorescence is quenched [3-5], The response of these materials to target analytes... 

Because of their high sensitivity, fluorescence detectors are particularly useful in trace analysis when either tire sample size is small or the analyte concentration is extremely low. Although fluorescence detectors can become markedly nonlinear at concentrations where absorption detectors are still linear in response, their linear dynamic range is more than adequate for most trace analysis applications. Unfortunately, fluorometric detectors are often susceptible to background fluorescence and quenching effects that can plague all fluorescence measurements. 

The normal violet fluorescence band of pyrene solutions shows concentration-quenching which is accompanied by the appearance of a blue structureless emission band. Forster and Kasper40 showed that the blue band is due to emission from an excited dimer formed by the combination of an excited singlet molecule with a molecule in the ground state. Most of the light in both spectral bands has a relatively short lifetime but Stevens and Hutton87 observed a long-lived component of the dimer... 

Values of Ar5xlO-1o for anthracene fluorescence A for Oa quenching, B for concentration quenching, C calculated from equation (6.4). 

Excimer formation has been assumed to be a necessary step in cou centration quenching. It is not essential that the eczimer shall be alv-, fluorescent (step 7, vide infra). It may promote radiationless procc within the molecule (step 8, vide infra). We shall use the various s involved in concentration quenching to set up a rate expression as a lsv j exercise in a complex situation (Appendix III). The separate processtj involved in the photokinetic scheme are ... 

It is called trivial because it does not require any energetic interaction between the donor and the acceptor. It is merely reabsorption of fluorescence radiation in accordance with Beer s Law and shows r 2 dependence on donor-acceptor distance. Although called trivial, it causes radiation imprisonment and can be important factor to be considered in fluorescence measurements. It may introduce error and distort emission spectrum by absorbing only that portion which overlaps its absorption spectrum. It is specially troublesome in studies on concentration quenching. 

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