Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 87

important for experimental reasons (e.g., suppression of scattering and background)
arises from the fact that the experimentally relevant spectra are the absorption
spectrum of the donor and the emission spectrum of the acceptor ( 3 ), in contrast to
the mechanistically important spectra shown in Fig. 34 [ 206 ]. The RET process can
take place when the emission spectrum of the donor overlaps with the absorption
spectrum of the acceptor (Fig. 34 ). The optimum distance between the donor in the
excited state and the acceptor in the ground state for RET to occur lies between 1
and 10 nm. Moreover, it is important to mention that the energy is transferred
through a nonradiative pathway and no fluorescence (of the donor) is involved.
Today, RET is very popular and widely used in various fields of analytical, life, and
materials sciences.

1 1

virtual
Stokes

Dn ¼ n abs ð D RET Þn em ð A RET Þ

D RET Þ

(3)

l abs ð l em ð A RET Þ

R 0
R 0 þ

E RET ¼ (4)

r 6

According to F¨ rster's theory [ 207 ], the efficiency of the resonance energy
transfer ( E RET ) shows an inverse sixth-power dependence on the distance ( r )
between donor and acceptor ( 4 ), where R 0 is the so-called F¨ rster distance at
which the transfer efficiency is 50%. FRET is therefore a very sensitive process,
well-suited for translating small conformational changes into large intensity
modulations. R 0 represents a characteristic property for a given donor-acceptor
pair. Other important molecular parametersthathavetobeconsideredinRET
system design are the overlap integral between D RET emission and A RET absorp-
tion spectrum, the fluorescence quantum yield of D RET, and the mutual orienta-
tion of the two partners. Details will not be discussed here but the reader is
referred to [ 208 ] and relevant literature cited therein. Depending on the various
D RET that can principally be employed, RET can be divided into FRET (fluores-
cence resonance energy transfer), where the donor is a molecule intrinsically
able to fluoresce and Bioluminescence Resonance Energy Transfer (BRET)

Fig. 34 Spectral
representation of the RET
process, highlighting the
mechanistically important
area of overlap between
emission spectrum of the
donor and absorption
spectrum of the acceptor.
(Reprinted with permission
from [ 206 ]. Copyright 2009
Springer)