Fluorophore distance dependence

One of the most characteristic features of FRET is its sensitive dependency on the fluorophore distance. This is advantageously used to evaluate structures and conformational changes of peptides, glycopeptides, and proteins among other molecules [164-166], The conformational change of the lipopeptide antibiotic daptomycin from an inactive linear form to a biological active cyclic form... 

Figure 10.9 Sample architecture for the distance dependence of metal-enhanced Superoxide generation (Top), and graphical representation of the interpretation of metal-enhanced superoxide generation with an enhanced and distance dependent excitation rate (Bottom). F - Fluorophore, MEF - Metal-Enhanced Fluorescence, MEP — Metal-enhanced Phosphorescence, SiFs — Silver Island Films. EF- Enhancement factor = I Silver /I Glass. Adopted from ref [31 ].

At very short metal nanoparticle-fluorophore distances ( 1 to 3 nm), a large decrease in fluorescence, known as quenching, is expected [8,19,20]. At greater distances however, the fluorescence can undergo enhancement or continue to experience a degree of quenching. The examples outlined below will illustrate that whether enhancement or quenching is observed depends on nanoparticle size and shape, the distance between the fluorophore and the metal nanoparticle surface, and on the overlap between the SPR and the excitation and/or emission transitions in the fluorophore. 

Many experiments have been carried out where the distance between the fluorophore and the metal nanoparticle surface is varied yet only quenching is observed. In these cases the nanoparticles are usually small spherical metal nanoparticles. The following examples demonstrate distance-dependent quenching in a couple of nanoparticle-fluorophore systems. 

Both Geddes and the Lakowicz group s have investigated the metal-enhanced fluorescence of fluorophores on silver island films (SIFs) [11,26,27] and variously aggregated silver nanoparticles in solution [28,29]. One example of enhancement on SIFs is discussed below [26]. In this work the distance-dependent MEF of a monolayer of sulforfiodamine B (SRB) on SIFs was studied. A SRB monolayer was electrostatically incorporated into the Langmuir-Blodgett (LB) layers of octadecylamine (ODA) deposited... 

This technique is used to sense the proximity of two fluorescently labeled molecules. The fluorophore couple is chosen to have a donor that in the excited state transfers its energy by dipole-dipole coupling to an acceptor, that is, the second fluorophore, which re-emits the light at a longer wavelength [117]. Among others, FRET pairs with appropriate spectral overlaps are BFP-GFP or CFP-YFP. Because the transfer efficiency correlates with the inverse sixth power of the distance between the fluorophores and depends on their spatial orientation, the experimental detection of FRET is possible only for pairs separated in space by 1-10 nm. If position and orientation of the fluorophore pair are favorable and ERET occurs, the fluorescent emission of the donor is quenched whereas the acceptor begins to fluoresce. The transfer efficiency can be derived by the ratio of the two emissions [118]. 

Other popular techniques used in biological studies include FRET, which may be applied in these systems as well. The transfer of energy from a donor to an acceptor fluorophore is also distance dependent and can be used to explore the dynamics of the desorbed layer in terms of diffusion and exchange processes. 

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