Biomedical Engineering Reference
In-Depth Information
pulse oximeter cannot discriminate between O
2
and CO binding, as the changes in
hemoglobin are similar.
9.4.2.2 Fluorescent-Based Techniques
Fluorescent (described in Chapter 8) labels render a biomolecule (or a biological
system) fluorescent so to make it amenable to fluorescence spectroscopy. Labeling
proteins with fluorophores is an approach in developing a number of immunoas-
says. The human genome project is based on the fluorescent labeling of nucleic
acids, which enables a faster sequencing than other methods. Labels are attached to
the species of interest by covalent binding via a reactive group that forms a chemi-
cal bond with other groups such as amino, hydroxy, sulfhydryl, or carboxy. Labels
are expected to be inert to other chemical species present in the environment, for
example, to pH. Labels preferably have long-wave excitation and emission to re-
duce background luminescence of biological matter, and/or long decay times so that
background luminescence decays much faster than the luminescence of the label.
As a result, there is a substantial interest in the design of long-wave and long-decay
luminescent labels.
A subtechnique within fluorescence is based on the
fluorescence resonance en-
ergy transfer
(FRET), which is a process in which the energy from one fluorophore
(the energy donor, D) in an excited state is transferred to another chromophore (the
energy acceptor, A). The donor and acceptor are selected such that the absorption
spectrum of the acceptor is in the same wavelength region as the emission spectrum
of the donor. FRET
biosensors are used to study molecular events from single cells
to whole organisms. They are unique because of their spontaneous fluorescence
and targeting specificity to both organelles and tissues. German physicist Theoder
Förster, in whose honor some refer to FRET
as the Forster resonance energy trans-
fer, developed a quantitative theory based on the assumption that the transfer of
energy occurs through dipole-dipole interactions of the donor and the acceptor.
The efficiency of the energy transfer (
η
tr
), which can be experimentally measured
by the fluorescence intensities or by the lifetime of the fluorophore, is expressed in
terms of a Forster distance constant
R
0
as
R
6
0
η
=
(9.8)
(
)
tr
6
6
R
+
δ
0
where
is the distance between the acceptor and the donor. The advantage of FRET
is the dependence (and thus sensitivity) of the energy transfer efficiency on the sixth
power of the distance (
δ
) between the chromophores.
Hence, FRET is also used
to measure changes in distances in the molecular interactions.
R
0
depends on the
degree of overlap of the donor emission and the acceptor absorption spectrum and
calculated using the relation
δ
1/6
2
⎛
Jq
κ
⎞
d
RA
[
]
=
9,772.42
(9.9)
⎜
⎟
0
4
⎝
⎠
