Chemistry Reference
In-Depth Information
2.2
Influencing Factors for FRET
Amplification of C* emission upon excitation of CPE, relative to that upon direct
excitation of C* is an important advantage of CPE-based FRET sensors, which
benefits from the rapid intrachain and interchain energy migration from CPE to C*
via FRET. The detection sensitivity of CCP-based DNA sensor thus is enhanced to
an extent dependent on the signal amplification of C* emission.
Amplification
factor
is defined as the intensity ratio of the saturated CCP-sensitized C* emission
to the intrinsic C* emission in the absence of CCP. To acquire large signal
amplification, it is necessary to review the factors affecting the FRET process
from CCP to C*. Equation (
1
) describes the calculation of FRET rate (
K
FRET
)[
67
]:
6
1
t
D
R
0
r
DA
K
FRET
ð
r
DA
Þ/
(1)
R
0
6
4
d
2
J
9000
ð
In
10
Þ
Q
D
k
ð
l
Þ
F
D
ð
l
Þ
e
A
ð
l
Þ
l
l
R
0
R
0
¼
;
J
ð
l
Þ¼
128
p
5
Nn
4
F
D
ð
l
Þ
d
l
where
r
DA
is the donor-acceptor distance,
t
D
is the lifetime of donor in the absence
of acceptor, and
R
0
is known as the F¨rster distance.
Q
D
is the quantum yield of the
donor in the absence of acceptor,
N
is Avogadro's number,
n
is the refractive index
of the medium, and
k
is the orientation factor. The overlap integral,
J
(
l
), expresses
the degree of spectral overlap between the donor emission
F
D
(
l
), and the absorption
of acceptor
).
The F¨ rster equation is valid for ideal FRET processes when the donor and
acceptor have well-matched energy levels. Otherwise, a competing process to
FRET, photoinduced electron transfer (PET), is likely to occur. Although PET is a
favorite mechanism for fluorescence quenching assays, it constitutes an undesir-
able energy-wasting channel in FRET assays that significantly reduces the inten-
sity of signal output and in turn the overall sensitivity. Scheme
7
represents two
e
A
(
l
Situation A: FRET
Situation B: PET
Acceptor
h
γ
h
γ
Donor
Donor
Quencher
Scheme 7 Effect of relative orbital energy levels preferred for FRET and PET
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