0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
2
4
6
8
10 12
A
pH
τ (ns)
0
20
40
60
80
100
2
4
6
8
10 12
B
pH
% Amplitude
Figure 3: Resorufin in 0.1m phosphate buffer. (A) τ 1 (о), τ 2 (□), and
τ
(∇) plotted as a function of pH. Emission wavelength 600 nm.
(B) % Amplitude of τ 1 (о) and τ 2 (□) plotted as a function of pH.
Resorufin doped sol-gel matrices (S2/S3/S4) also show pH dependence (Fig. 4). At an emission wavelength of 600 nm, the
effect of pH on the fluorescence lifetime for S2 and S4 are very similar (Fig. 4A), indicating that increased pH and
decreased water content during sol-gel fabrication therefore has no significant effect on the pH response curve of sol-gel
immobilised resorufin. There is, however, a significant difference between S2/S4 and S3, indicating that the surfactant has
a significant influence on the pKa* of resorufin. This same effect is seen at emission wavelengths of 550 nm (Fig. 4C) and
650 nm (Fig. 4D). At 550 nm, however, the change in
τ
with pH is much reduced since at this emission wavelength the
longer lifetime R
- species only has a very weak fluorescence and as such a smaller contribution to the overall fluorescence.
Inclusion of resorufin into sol-gel matrices results in a number of changes to the average lifetime versus pH curves. The
first point of note is that the fluorescence lifetime is considerably reduced to ~2.3 to 2.5 ns as opposed to ~3.5 ns in solution
at pH 9 (Fig 4A). The difference in the lifetime vs. pH curves between the sol-gels and resorufin in solution is due to the
acidic microenvironment within the sol-gels. Immobilisation of indicator dyes in sol-gel affects both the excited state acidbase equilibrium and apparent excited state pKa (pKa*). In solution, the approximate pKa* (calculated from the point at
which the contribution of each lifetime component is equal) is ~5.7, while in S2/S4 it is ~ 6.8 and in S3 it occurs at ~ 6.4
(Fig 3B & 4B). More accurate values for pKa* cannot be obtained from the sol-gel materials because of extensive leaching
at high pH (>9.0). In S2, at pH 9.0, the contribution from RH is ~36%, while at similar pH in solution the dye exists solely
as R
− (Fig. 3 & 4). Due to the increased pKa*, and the relatively greater contribution from the shorter lifetime RH,
τ
is
much reduced in the sol-gel as opposed to solution. Entrapment also results in
τ
having a lower sensitivity to pH, as
evidenced by the smaller change of 1.48 ns compared to 2.6 ns over the pH range 3 – 9. This decreased pH sensitivity and
lengthening of the pH response curve has been reported elsewhere for other sol-gel immobilised indicators.
Addition of surfactants to sol-gels has been shown to shift the ground state pKa of entrapped indicators and thus provide a
convenient method for fine-tuning the response of optical sensors.
11,12 In the S3 case, the addition of non-ionic TX-100 to
0.5
1.0
1.5
2.0
2.5
3.0
3.5
2
4
6
8
10 12
A
pH
τ (ns)
0
20
40
60
80
100
2
4
6
8
10 12
B
pH
% Amplitude
Figure 3: Resorufin in 0.1m phosphate buffer. (A) τ 1 (о), τ 2 (□), and
τ
(∇) plotted as a function of pH. Emission wavelength 600 nm.
(B) % Amplitude of τ 1 (о) and τ 2 (□) plotted as a function of pH.
Resorufin doped sol-gel matrices (S2/S3/S4) also show pH dependence (Fig. 4). At an emission wavelength of 600 nm, the
effect of pH on the fluorescence lifetime for S2 and S4 are very similar (Fig. 4A), indicating that increased pH and
decreased water content during sol-gel fabrication therefore has no significant effect on the pH response curve of sol-gel
immobilised resorufin. There is, however, a significant difference between S2/S4 and S3, indicating that the surfactant has
a significant influence on the pKa* of resorufin. This same effect is seen at emission wavelengths of 550 nm (Fig. 4C) and
650 nm (Fig. 4D). At 550 nm, however, the change in
τ
with pH is much reduced since at this emission wavelength the
longer lifetime R
- species only has a very weak fluorescence and as such a smaller contribution to the overall fluorescence.
Inclusion of resorufin into sol-gel matrices results in a number of changes to the average lifetime versus pH curves. The
first point of note is that the fluorescence lifetime is considerably reduced to ~2.3 to 2.5 ns as opposed to ~3.5 ns in solution
at pH 9 (Fig 4A). The difference in the lifetime vs. pH curves between the sol-gels and resorufin in solution is due to the
acidic microenvironment within the sol-gels. Immobilisation of indicator dyes in sol-gel affects both the excited state acidbase equilibrium and apparent excited state pKa (pKa*). In solution, the approximate pKa* (calculated from the point at
which the contribution of each lifetime component is equal) is ~5.7, while in S2/S4 it is ~ 6.8 and in S3 it occurs at ~ 6.4
(Fig 3B & 4B). More accurate values for pKa* cannot be obtained from the sol-gel materials because of extensive leaching
at high pH (>9.0). In S2, at pH 9.0, the contribution from RH is ~36%, while at similar pH in solution the dye exists solely
as R
− (Fig. 3 & 4). Due to the increased pKa*, and the relatively greater contribution from the shorter lifetime RH,
τ
is
much reduced in the sol-gel as opposed to solution. Entrapment also results in
τ
having a lower sensitivity to pH, as
evidenced by the smaller change of 1.48 ns compared to 2.6 ns over the pH range 3 – 9. This decreased pH sensitivity and
lengthening of the pH response curve has been reported elsewhere for other sol-gel immobilised indicators.
Addition of surfactants to sol-gels has been shown to shift the ground state pKa of entrapped indicators and thus provide a
convenient method for fine-tuning the response of optical sensors.
11,12 In the S3 case, the addition of non-ionic TX-100 to