Rhodamine energy transfer

Blomberg, K., Hurskainen, P. and Hemmila, I. (1999). Terbium and rhodamine as labels in a homogeneous time-resolved fluorometric energy transfer assay of the b-subunit of human chorionic gonadotropin in serum. Clin. Chem. 45, 855-61. 

The second label also may be a fluorescent compound, but doesn t necessarily have to be. As long as the second label can absorb the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce fluorescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe, N-(4-aminobutyl)-N-ethylisoluminol, and reactive fluorescent derivatives of fluorescein, rhodamine, and the cyanine dyes (Chapter 9). For a review of these techniques, see Morrison (1992). 

Fig. 16 Addition of 0.017-nmol aliquots of a rhodamine B-labeled streptavidin and b Texas Red-X-labeled streptavidin to 1.51 nmol of 43. Energy transfer observed in both cases with amplified emission of the dyes to the light-harvesting conjugated polymers. Direct excitation of the dyes at 575 and 585 nm correspond to 0.100 nmol of streptavidin.

The interactions of the dye acceptors to polymers could also be witnessed in the case of solid films. In this case, the sterically more restrictive cavities of the polymeric film 46 allowed better orbital interaction with the smaller and more flexible rhodamine B dye, and accordingly higher energy transfer with rhodamine B-labeled streptavidin was observed compared to Texas Red-... 

Kaplan and Jortner [164] have observed dipole—dipole energy transfer between the second excited state of rhodamine 6G and 2,5-bis(5 -f-butyl-2-benzoxazolyl)thiophene in ethanol. The donor excited state lifetime was estimated to be 0.19 ps based on energy transfer by Forster kinetics. 

In another picosecond laser study of Forster energy transfer, Sato et al. [165] have studied systems of rhodamine 6G (R6G) and/or 3,3 -diethyl-thiacarbocyanine iodide (DODCI). Satisfactory agreement between the experimentally observed decay of R6G fluorescence and that based on the Forster kinetics [eqn. (85) with a calculated R0 — 5.9 nm and r = 0] was noted. However, from eqn. (85), Uiim 10-9 m2 s-1, so that Forster... 

Fluorescence depolarisation by energy transfer (rather than rotational relaxation) between donor molecules of the same type can occur. Eisenthal [174] excited solutions of rhodamine 6G (9 mmol dm-3) in glycerol with 530 nm light from a frequency-doubled neodymium laser. The polarisation... 

Triplet—triplet energy transfer from benzophenone to phenanthrene in polymethylmethacrylate at 77 and 298 K was studied by steady-state phosphorescence depolarisation techniques [182], They were unable to see any clear evidence for the orientational dependence of the transfer probability [eqn. (92)]. This may be due to the relative magnitude of the phosphorescence lifetime of benzophenone ( 5 ms) and the much shorter rotational relaxation time of benzophenone implied by the observation by Rice and Kenney-Wallace [250] that coumarin-2 and pyrene have rotational times of < 1 ns, and rhodamine 6G of 5.7 ns in polymethyl methacrylate at room temperature. Indeed, the latter system of rhodamine 6G in polymethyl methacrylate could provide an interesting donor (to rose bengal or some such acceptor) where the rotational time is comparable with the fluorescence time and hence to the dipole—dipole energy transfer time. In this case, the definition of R0 in eqn. (77) is incorrect, since k cannot now be averaged over all orientations. 

Fluorescein is an energy acceptor for chromophores such as naphthalene and anthracene and acts as energy donor toward Eosin and Rhodamine, so derivatives have been used for singlet-singlet energy transfer studies. According to Forster s theory [68] the rate constant for energy transfer increases... 

Membranes fusion can be studied using the energy-transfer mechanism. In fact, membrane vesicles labeled with both NBD and rhodamine probes are fused with unlabeled vesicles. In the labeled vesicles, upon excitation of NBD at 470 nm, emission from rhodamine is observed at 585 nm as a result of energy transfer from NBD to rhodamine. The average distance separating the donor from the acceptor molecules increases with fusion of the vesicules, thereby decreasing the energy-transfer efficiency (Struck et al. 1981). 

Another example of improved sensitivity due to modulation of lanthanide photophysics by ancillary ligands can be found in the europium and terbiiun chelates used in time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays (100,101). Due to their line-type emissions and long decay times, the lanthanide chelate is used as a donor, with some visible-absorbing dye such as Alexa 647 or a rhodamine derivative as the acceptor. Without the helper ligand, the lanthanides would be unprotected from solvent and have much shorter decay times, making them unsuitable for such an assay. 

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