Chemistry Reference
In-Depth Information
fluorescent stains for nucleic acids. When nucleic acids such as DNA are added to a
quenched solution of the Eu
III
helicate containing EB or AO, the organic stain intercalates
between the DNA strands and the initial Eu
III
luminescence is restored.
The quenching of a luminophore is collisional and/or static in nature and is modelled
by Stern-Volmer theory. For collisional quenching resulting from particle diffusion in
solution (Equation 6.9):
I
0
I
¼
1
þ
k
q
t
0
Q
½¼
1
þ
½
ð
6
:
9
Þ
K
D
Q
in which
I
0
and
I
are the emission intensities in absence and in presence of the quencher
Q, respectively,
k
q
is the bimolecular rate constant,
t
0
the observed lifetime in absence of
quencher and
K
D
the dynamic quenching constant. Intuitively, 1/
K
D
is the quencher
concentration for which 50% of the luminescence intensity is lost. When collisional
quenching occurs, the lifetime decreases in parallel to the luminescence intensity
(Equation 6.10):
I
0
I
¼
t
0
ð
6
:
10
Þ
t
Static quenching results when the luminophore and the quencher form a nonlumines-
cent ground-state complex with association constant
K
S
(Equation 6.11):
I
0
I
¼
1
þ
K
S
Q
½
ð
6
:
11
Þ
In this case, the lifetime of the emitting species remains unchanged since the effect of
the process is simply to remove a fraction of luminescent molecules from observation.
Therefore
1. When both dynamic and static quenching occurs, the equations com-
bine into Equation 6.12:
t
0
/
t
¼
I
0
I
2
1
¼
ð
K
D
þ
K
S
Þ
½þ
Q
K
D
K
S
Q
½
ð
6
:
12
Þ
In this case, lifetime dependence follows Equation 6.9.
Two situations are illustrated in Figure 6.12. On the left side, both intensities and life-
timesvaryinthesamewaywithincreasingconcentrationofAO,pointingtoapure
dynamic quenching, with
K
D
¼
10
8
M
1
s
1
.On
the right side,
I
0
/
I
clearly deviates from linearity with an upward curvature while the
10
5
M
1
6.7(1)
and
k
q
¼
2.7(1)
t
0
/
t
plot is linear; this is diagnostic for EB inducing simultaneous dynamic and static quench-
ing, with associated constants
K
D
¼
10
4
M
1
,
k
q
¼
10
7
M
1
s
1
and
3.03(5)
1.23(4)
10
3
M
1
.
The analytical method has been tested for five different types of DNA and limits of
detection are reported in Table 6.9. The method is rugged in that it is insensitive to pH in
the range 3-10 and results are affected by less than
K
S
¼
2.0(1)
5% upon the addition of a 1000-fold
excess of potentially interfering substances such as glucose, sodium hydrogenophosphate,
edta, Ca
II
,Mg
II
,Zn
II
,Fe
II
,Fe
III
, citrate and urate. In contrast, the addition of bovine serum
Search WWH ::
Custom Search