The properties of the final sol-gel material depend on, among other factors, the water concentration, and pH. Changes in the
water:TMOS ratio (R value) and/or pH during fabrication affects the porosity of the final sol-gel. The aim was to
investigate if changing sol-gel properties could alter the pH sensitivity of immobilised resorufin. We also modified the pH
response of the resorufin doped sol-gels by the addition of Triton X-100 surfactant (S3/S5). Addition of surfactants to solgels has been shown to shift the ground state pKa of entrapped indicators with a resulting shift in the absorption based pH
response curves.
11,12 A similar effect would be expected for the excited state pKa (pKa*), allowing the tuning of the
fluorescence lifetime pH response curves.
Resorufin doped PVA:TMOS copolymers (TP1 and TP2) were synthesised by slightly modifying the method of Cajlakovic
et al.
10 TP1 and TP2 were identical to the L2 and L3 schemes outlined in Ref. 10. The only significant difference in the
method is that resorufin was added in 1 cm
3 aliquots (0.01M resorufin sodium salt in methanol) to the
PVA/TMOS/EtOH/HCl mixtures. Once the copolymers had been dip-coated (~0.25 µm thick) onto the activated glass
slides they were conditioned in pH 3 phosphate buffer for several days. No leaching of resorufin from the copolymers was
observed at this pH. The final concentration of resorufin in the films was not calculated but is probably less than 10
-3 M.
We also attempted to produce a sol-gel with a lower PVA content (identical to L1 in Ref. 10) but this was found to gel too
quickly for dipcoating. This effect may be due to the presence of resorufin itself.
15
3. RESULTS AND DISCUSSION
0
0.2
0.4
0.6
0.8
1.0
350
450
550
650
B
Wavelength (nm)
Absorbance
0
0.2
0.4
0.6
0.8
1.0
350
450
550
650
A
Wavelength (nm)
Absorbance
Figure 1: Absorption spectra of resorufin in solution (
___
), sol-gel scheme S2 (
_ _ _
) and scheme S3 (-----), at low and high pH. (A) Low
pH case: resorufin in 0.9M HNO 3 solution, and in S2 and S3 at pH 3.1 and 3.6 respectively (0.1M phosphate buffer). (B) High pH case:
resorufin in 0.9M NaOH solution; in S2 (pH 9.0, 0.1 M phosphate buffer); and S3 (pH 9.0, 0.1 M phosphate buffer). All spectra were
normalised at their wavelength of maximum absorbance.
water:TMOS ratio (R value) and/or pH during fabrication affects the porosity of the final sol-gel. The aim was to
investigate if changing sol-gel properties could alter the pH sensitivity of immobilised resorufin. We also modified the pH
response of the resorufin doped sol-gels by the addition of Triton X-100 surfactant (S3/S5). Addition of surfactants to solgels has been shown to shift the ground state pKa of entrapped indicators with a resulting shift in the absorption based pH
response curves.
11,12 A similar effect would be expected for the excited state pKa (pKa*), allowing the tuning of the
fluorescence lifetime pH response curves.
Resorufin doped PVA:TMOS copolymers (TP1 and TP2) were synthesised by slightly modifying the method of Cajlakovic
et al.
10 TP1 and TP2 were identical to the L2 and L3 schemes outlined in Ref. 10. The only significant difference in the
method is that resorufin was added in 1 cm
3 aliquots (0.01M resorufin sodium salt in methanol) to the
PVA/TMOS/EtOH/HCl mixtures. Once the copolymers had been dip-coated (~0.25 µm thick) onto the activated glass
slides they were conditioned in pH 3 phosphate buffer for several days. No leaching of resorufin from the copolymers was
observed at this pH. The final concentration of resorufin in the films was not calculated but is probably less than 10
-3 M.
We also attempted to produce a sol-gel with a lower PVA content (identical to L1 in Ref. 10) but this was found to gel too
quickly for dipcoating. This effect may be due to the presence of resorufin itself.
15
3. RESULTS AND DISCUSSION
0
0.2
0.4
0.6
0.8
1.0
350
450
550
650
B
Wavelength (nm)
Absorbance
0
0.2
0.4
0.6
0.8
1.0
350
450
550
650
A
Wavelength (nm)
Absorbance
Figure 1: Absorption spectra of resorufin in solution (
___
), sol-gel scheme S2 (
_ _ _
) and scheme S3 (-----), at low and high pH. (A) Low
pH case: resorufin in 0.9M HNO 3 solution, and in S2 and S3 at pH 3.1 and 3.6 respectively (0.1M phosphate buffer). (B) High pH case:
resorufin in 0.9M NaOH solution; in S2 (pH 9.0, 0.1 M phosphate buffer); and S3 (pH 9.0, 0.1 M phosphate buffer). All spectra were
normalised at their wavelength of maximum absorbance.