Quencher displacement

Fig. 10. Covalent and ionic potential curves for some NO-quencher pairs. Equation (26) was used to calculate the ionic curves, using the electron affinities of Table III. The covalent curves are Leonard-Jones potential of NO and the respective quencher, displaced by the electronic excitation energy of the appropriate Rydberg state. See text for further details.

Wang and co-workers [53] have succeeded in constructing a novel sensor based on the displacement of a small, structurally unrelated quencher from a MIP comprising a fluorescent functional monomer (Fig. 20.20). The principle of detection relies upon the enhanced fluorescence observed upon displacement of the quencher, p-nitrobenzaldehyde, from the L-tryptophan (r-Trp) binding sites within the polymer. 

Mixed Mechanism Probes. Several probe systems appear to function by both hydrolysis and hybridization mechanisms. These include hairpin probes, self-probing amplicon primers, and displacement probes. A hairpin probe functions similarly to a hairpin primer in that it is designed to increase in fluorescence when the distance between the quencher and the reporter increases upon target hybridization (see Figure 37-24, row five). Similarly, primers that... 

Figure 5. Electron transfer induced photochemical dehalogenation, dimerization and displacement. "Q" indicates a triplet quencher like cyclohexadiene.

The enhanced fluorescence of the ethidium-DNA complex can be quenched partially by the addition of a second ligand. Two mechanisms have been proposed to account for quenching, namely displacement of ethidium bromide by melting of the helix or electron transfer from external quencher molecules. The most efficient quenching occurs of course when both the quencher and ethidium bromide molecules are bound to DNA. This nondisplacement quenching is correlated with DNA-enhanced electron transfer, either from excited ethidium to an acceptor (methylviologen, copper (II) counterions) or from an donor to an excited ethidium acceptor. The DNA double helix works as a well-defined matrix for an organized electron transfer. It enhances its yield by a factor K. (DNA) K, (H,0), which is often in the order of 5 x 10. ... 

Fig. 1. Probes are indicated with quencher molecules in diamonds and reporter fluorophores as either red or blue symbols. Probe differences are indicated by striped boxes and are allele specific. The differences can be as small as a single nucleotide polymorphism. During annealing phase, probes hybridize to their specific sequences, while during the extension phase, the Taq polymerase displaces the probe and digests it releasing the reporter. In this example, samples of DNA that are homozygous for allele 1 (Al-1) would yield blue fluorescence only. DNA samples that are homozygous for allele 2 (Al-2) would yield red fluorescence only while heterozygotes would yield both blue and red fluorescence.

Several concepts (categories of action) that could result in cation enhanced fluorescence can be envisioned. Five examples are summarized here (a) the ion could cause subtle change(s) in energy levels or electron densities that lead to enhanced fluorescence, (b) the cation of interest could displace a quencher complexed by the crown, (c) the complexation of a cation could interrupt a quenching mechanism operable in the free crown, (d) complexation could adjust the conformation so that a new fluorescent excited state might form, (e) a crown ether used in an extraction method could promote the solubility of a fluorescent ion in a phase that is monitored for fluorescence. The literature of crown ethers contains examples employing each of these concepts (i-2i) with the possible exception of case (b). Seve concq)ts we have attempted to employ follow. 

The first concept to be considered is cation displacement of a complexed quencher of fluorescence. Scheme I illustrates the concq>t. To be operable the scheme requires a quencher that is complexed by a crown ether, a metal ion of interest that is not a quencher but is complexed effectively, and a crown ether ring that orients the complexed quencher so it will effectively quench the chromophore fluorescence. Scheme II shows a relatively simple crown ether system that we hoped would fulfill these requirements(22,2i). The l,5-naphtho-22-crown-6 compound was selected because of the ability of its crown ether band to hold a quencher against the face of the pi system of the naphthalene chromophore. The heavy atom ion Cs+ was selected as a quencher based on its propensity to increase inter-system crossing from the fluorescent Sj state to the nonfluorescent T state(i,2). It was likely, based on previous results(2), that potassium ion would be complexed by the crown, but not quench naphthalene fluorescence appreciably. 

Imagine that the volume containing the excited states and quenchers, initially randomly distributed, is subdivided into thin slabs of thickness a, assumed to be a few nanometers, and surrounded by impenetrable walls (see Fig. 2). Now the quenching reaction can occur only between excited states and quenchers within the same slab. The initial part of the decay will not be affected by this confinement, since at times shorter than c/ /2Z), where D is the diffusion coefficient, the displacements of the reactants are smaller than the width of the slab. At longer times, however, there will be less quenching than in the 3-D system, since only quenchers within the same slab as the excited state may take part. The frequency of encounters, and hence the rate of quenching, then depends on the diffusion in the two unlimited dimensions. 

---