3.3
Resorufin in TMOS-PVA copolymers.
TP1 shows a ~0.8 ns change in
τ
over the pH 5.8 to 8.0 range (Fig. 5) with the decay being bi-exponential in the range pH
5.8 – 7 and, above pH 8, the decay becomes mono-exponential with only the anion being present. Lifetimes could not be
measured below pH ~5.5 because the fluorescence emission from the ~0.25 µm thick films was too weak.
In TP2, the fluorescence decay is mono-exponential with a constant lifetime of ~2.7 ns over the pH 5.8 to 8 at both emission
wavelengths. We surmise that, as the PVA content increases, the matrix becomes more lipophilic and hydrophobic,
resulting in the pKa* being shifted to lower pH. For both TP1 and TP2 it was not possible to make accurate lifetime
measurements above ~pH 8.0 because of extensive leaching.
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
6.0
6.5
7.0
7.5
8.0
pH
Average
τ (ns)
Figure 5: Average lifetime versus pH for resorufin doped TP1 at 600 nm (o) and 650 nm (∇), and TP2 at 660 nm (∆) and 650 nm (□).
Lifetimes were recorded in 0.1M phosphate buffer with 460 nm excitation.
4. CONCLUSIONS:
Resorufin in aqueous solution shows potential for use as a lifetime based pH probe since it is has a relatively large lifetime
change of ~3.5 ns (
τ
at 600 nm) over pH 2 to 10, is not quenched by oxygen or chloride, and is unaffected by changes in
buffer concentration. Furthermore, it can be excited using inexpensive 460 nm LED light sources. The pH sensing range of
resorufin can be shifted slightly to different pH ranges by measuring the lifetime at different emission wavelengths.
Emission wavelength 550 nm gave the best response over the desired 6 – 8 pH range, although the optimum sensing range
at this emission wavelength is between pH 5 - 6. At longer emission wavelengths, 600 and 650 nm, the best sensing range
with the largest average lifetime change is between pH 2 – 6.
Resorufin can be introduced into sol-gels and retains its lifetime based pH sensitivity. However, there is a decrease in the
absolute change in
τ
with pH, and a shift in the apparent pKa* which results in the flattening of the pH response curve,
reducing its effectiveness as a pH sensor. The apparent pKa* and pH sensing range can be shifted by addition of detergent
TX-100 to the sol-gel, and therefore the pH response can be tuned to a particular pH range. Unfortunately, all the sol-gel
and TMOS-PVA copolymers supports investigated here were all susceptible to extensive leaching particularly at alkaline
Resorufin in TMOS-PVA copolymers.
TP1 shows a ~0.8 ns change in
τ
over the pH 5.8 to 8.0 range (Fig. 5) with the decay being bi-exponential in the range pH
5.8 – 7 and, above pH 8, the decay becomes mono-exponential with only the anion being present. Lifetimes could not be
measured below pH ~5.5 because the fluorescence emission from the ~0.25 µm thick films was too weak.
In TP2, the fluorescence decay is mono-exponential with a constant lifetime of ~2.7 ns over the pH 5.8 to 8 at both emission
wavelengths. We surmise that, as the PVA content increases, the matrix becomes more lipophilic and hydrophobic,
resulting in the pKa* being shifted to lower pH. For both TP1 and TP2 it was not possible to make accurate lifetime
measurements above ~pH 8.0 because of extensive leaching.
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
6.0
6.5
7.0
7.5
8.0
pH
Average
τ (ns)
Figure 5: Average lifetime versus pH for resorufin doped TP1 at 600 nm (o) and 650 nm (∇), and TP2 at 660 nm (∆) and 650 nm (□).
Lifetimes were recorded in 0.1M phosphate buffer with 460 nm excitation.
4. CONCLUSIONS:
Resorufin in aqueous solution shows potential for use as a lifetime based pH probe since it is has a relatively large lifetime
change of ~3.5 ns (
τ
at 600 nm) over pH 2 to 10, is not quenched by oxygen or chloride, and is unaffected by changes in
buffer concentration. Furthermore, it can be excited using inexpensive 460 nm LED light sources. The pH sensing range of
resorufin can be shifted slightly to different pH ranges by measuring the lifetime at different emission wavelengths.
Emission wavelength 550 nm gave the best response over the desired 6 – 8 pH range, although the optimum sensing range
at this emission wavelength is between pH 5 - 6. At longer emission wavelengths, 600 and 650 nm, the best sensing range
with the largest average lifetime change is between pH 2 – 6.
Resorufin can be introduced into sol-gels and retains its lifetime based pH sensitivity. However, there is a decrease in the
absolute change in
τ
with pH, and a shift in the apparent pKa* which results in the flattening of the pH response curve,
reducing its effectiveness as a pH sensor. The apparent pKa* and pH sensing range can be shifted by addition of detergent
TX-100 to the sol-gel, and therefore the pH response can be tuned to a particular pH range. Unfortunately, all the sol-gel
and TMOS-PVA copolymers supports investigated here were all susceptible to extensive leaching particularly at alkaline