Quenching donor-acceptor probes

Donor-acceptor probes are very useful as fluorescent lables in biochemical assay and sensing based on the fluorescence quenching mechanisms. In a closed state form where the donor and acceptor are close to each other, the fluorescence of the donor (reporter) is highly quenched. In an open state, where the donor and receptor are spaced away from each other due to biochemical reactions, the donor fluorescnece is restored. The changes of the fluorescence intensity of the reporter has been used for DNA detection, immunoassay, enzyme sensing and detection of many other bimolecules. 

The duplex probe configuration provides for a close proximity of the donor/acceptor pair and may lead to two mechanisms of quenching FRET and direct transfer (contact-mediated) quenching [78]. Placement of the fluorophore and quencher toward the centre of the duplex probe sequence may further add to the quenching efficiency as the ends of duplexes are known to breathe and are not as tightly bound as internal base pairs. Duplex probes are relatively easy to synthesize as the fluorophore and quencher moieties do not have to be incorporated into the same strand. 

RET occurs via long-range dipole-dipole interactions and does not require direct molecular contact between the analyte and the probe. For RET to occur, a donor -acceptor system with spectral overlap is required, such that the emission spectrum of donor (D) overlaps with the absorption spectrum of acceptor (A). The acceptor may or may not be a lumophore. In RET sensors the presence of the analyte perturbs the electronic transitions of either the donor or acceptor in some way, so that the efficiency of the RET process is affected. Experimentally, RET shares many similarities with collisional quenching, typically resulting in a decrease the luminescence intensity and lifetime of the probe. Mechanistically, however, the processes are quite different and differ in both their concentration and distance dependencies. 

Fig. 6.11. Two types of FRET probes. (A) Ratiometric probes are formed by two fluorescent molecules that allow determination of emission ratio. (B) Quenched probes feature a donor fluorophore and a quencher. The emission increase of the donor after release of the acceptor is detected. Both types are frequently used to build proteinases probes.

Fig. 6.21. Principle of detection of lipopolysaccharide (LPS) with the CD14-derived probe. It relies on the formation of a ground state complex between fluorescein and rhodamine in aqueous solution with quenching of donor and acceptor fluorescence. Spectrum A shows hypothetical fluorescence emission spectra of this complex. After LPS binding, the peptide sequence gets straightened prohibiting the close contact between the two fluorophores and leading to the recovery of red fluorescence (Spectra B).

Fig. 3 Typical ICT probes (left) and representative spectroscopic responses toward selected metal ions (right). Color code (left) coordinating atoms in blue, bridgehead atom of the fluorophore that takes part in complexation in orange, formal donor fragment in red, formal acceptor fragment in green (right) hypsochromic shifts in red, bathochromic shifts in green, fluorescence enhancement in violet, fluorescence quenching in blue. Symbols in table Aabs, 7em,

This type of probe, often called fluorescent photoinduced electron transfer (PET) sensors, has been extensively studied (for reviews, see Refs. 22 and 23). Figure 2.2 illustrates how a cation can control the photoinduced charge transfer in a fluoroiono-phore in which the cation receptor is an electron donor (e.g., amino group) and the fluorophore (e.g., anthracene) plays the role of an acceptor. On excitation of the fluorophore, an electron of the highest occupied molecular orbital (HOMO) is promoted to the lowest unoccupied molecular orbital (LUMO), which enables photoinduced electron transfer from the HOMO of the donor (belonging to the free cation receptor) to that of the fluorophore, causing fluorescence quenching of the latter. On... 

Another approach of energy transfer-based probe has been demonstrated for a-Amylase sensing/98 In this case amylose was doubly labeled, by fluorescein derivative (donor) and Procion Red MX8B (acceptor). As a-Amylase catalyzes the cleavage of the amylose into smaller units, the average distance between fluorescein and Procion Red increases, which reduces the degree of quenching. The rate of increase in fluorescence intensity is proportional to ee-amylase activity. 

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