Biomedical Engineering Reference
In-Depth Information
sixth degree of the distance separating the donor and acceptor dipoles, allowing for
a sensitive gauge of intermolecular distances in the range 10 to 100 A.
Utilization of fluorophores with long lifetimes allows for time-gated fluorescence
data acquisition, employed in time-resolved fluorescence (TRF) and FRET (TR-
FRET). The underlying phenomenon, sometimes called photoluminescence , bridges
the fluorescence and luminescence domains. The most frequently utilized slow-
decaying fluorophores are two lanthanides, europium and terbium, whose caged
forms could be attached to one of two binding partners, directly or through a labeled
antibody. Long-lived fluorescence of lanthanides provides sustained light output
spanning several hundreds microseconds; for comparison, the lifespan of common
fluorophores is measured on the pico- and nanosecond scale requiring simultaneous
excitation and emission for their detection. Therefore, time-gated data acquisition
allows for a significant increase in assay sensitivity through selective elimination
of short-lived background light sources, such as nonspecific fluorescence and light
reflection and scattering.
The sensitivity provided by the third main spectrophotometric detection tech-
nique, luminescence, could easily surpass that of the TRF approaches. This is due
to the extremely low (near-zero) background inherent in luminescence. This advan-
tage of luminescence has long been appreciated and made use of in the blotting
protein detection techniques. Luminescent approaches could be grouped into biolu-
minescent and chemiluminescent techniques, depending on whether a participation
and catalytic reaction of a special enzyme from a class of luciferases is critical for
photon generation.
Two additional approaches blend some of the features of fluorescence and lumi-
nescence phenomena. Similar to FRET, bioluminescence resonance energy transfer
(BRET) relies on nonradiative energy transfer though dipole-dipole coupling; how-
ever, it employs a luminescent entity as the energy donor, as opposed to the fluores-
cence moiety employed in FRET approaches. Strong inverse correlation dependence
of the efficiency of energy transfer on the distance between donor and acceptor
molecules, inherent in all resonance energy transfer applications, makes it suitable
for studying biomolecular interactions in vitro and within live cells. Variations in
luciferases, their substrates, and fluorescent acceptor moieties resulted in several
BRET applications developed over the years.
The second approach is called amplified luminescent proximity homogeneous
assay (AlphaScreen). This assay approach is aimed at detecting biomolecular inter-
actions through specialized bead chemistry. In these assays, two types of beads,
donor and acceptor, are present. The donor bead, which contains a photosensitizer,
phthalocyanine, generates singlet oxygen molecules storing extra energy in a single
excited electron upon excitation at 680 nm. Singlet oxygen is capable of transferring
its energy to thioxene moieties of the acceptor beads, resulting in light emission in the
range 520 to 620 nm. The singlet oxygen's 4-
s half-life defines the limited range of
its diffusion equal to about 200 nm away from the donor bead. Therefore, emission
light is generated only when donor and acceptor beads are present in close proximity.
Each detection technique has inherent strengths and weaknesses. The main weak-
ness of any particular detection approach is a potential compound interference. Most
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