Anal. Chem. 1998, 70, 3892-3897
Strategies To Design pH Optodes with Luminescence Decay Times in the Microsecond Time Regime Ute Kosch, Ingo Klimant,* Tobias Werner, and Otto S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany
We present the first optical pH sensor with luminescence decay times longer than 1 µs. It is based on radiationless energy transfer from a luminescent ruthenium(II) complex as the donor to a colored pH indicator as the acceptor. The metal-ligand complex ruthenium(II) tris4,4′-diphenyl-2,2′-bipyridyl was selected as the donor, and bromothymol blue and a reactive azo dye were selected as pH-sensitive acceptors. Strategies for coimmobilization of transition metal complexes and pH indicators into a hydrogel matrix were evaluated and discussed. pH transition intervals between 7 and 9 allow the measurement in the physiological range and also under marine conditions. Signal changes of up to 50% over 2.5 pH units are observed, depending on the respective acceptor content in the sensing film. Luminescence lifetime measurements of the sensing films were performed in the frequency domain with a blue LED as the light source. The effect of molecular oxygen (acting both as a quenching and a bleaching agent, thereby limiting the accuracy and the long-term stability of such sensors) was found to be crucial for practical applications. Since leaching of the acceptor also limits the stability of the sensing films, several strategies to reduce this effect were evaluated. Optical sensors based on the measurement of luminescence intensity frequently suffer from interferences by changes of turbidity, refractive index, or color of the sample. Changes in the optoelectronic system such as drifts of the light source and the photodetector, bending of optical fibers, and displacement or even delamination of the sensing layer may also cause signal changes to occur. Furthermore, degradation of the indicator caused by photobleaching and leaching are critical. Extensive referencing and recalibration procedures are, therefore, necessary to overcome these problems. The measurement of the luminescence decay time as a parameter which is almost independent of the absolute signal height can solve such problems and, therefore, has substantial advantages in practice. Decay time-based sensing schemes have been described for optical sensing of oxygen, pH, and CO2.1-7 However, only oxygen optodes have been found to be useful for (1) Bacon, J. R.; Demas, J. N. Anal. Chem. 1987, 59, 2780. (2) Vanderkoi, J. M.; Maniara, G.; Green, T. J.; Wilson, T. F. J. Biol. Chem. 1987, 262, 5476.
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practical applications so far. The indicators applied are characterized by luminescence lifetimes ranging from a few hundred nanoseconds to a few milliseconds, which are measurable with instruments with rather inexpensive optoelectronical components. Lifetime-based sensing of parameters such as pH and pCO2 is of great interest but has not been realized in practice. Nevertheless, there are different schemes described in the literature to design such sensors. One approach for lifetime-based pH sensing uses the pH-dependent photoinduced electron transfer (PET) of diethylaminoethylpyrene.8 This indicator shows a lifetime in the range of 100 ns but needs to be excited in the UV, where no inexpensive solid-state light sources are available. The use of the pH-dependent fluorescence decay time of seminaphthofluorescein (SNAFL) and seminaphthorhodafluorescein (SNARF) was suggested by Szmacinski and Lakowicz9 and Thompson et al.10 Both indicators are fluorescent in the protonated and the deprotonated forms, with lifetimes ranging from 0.5 to 5 ns. Such short lifetimes require modulation frequencies of >100 MHz.11,12 This, in turn, may compromise resolution, which, in biomedical applications, is expected to be (0.01 pH unit. A luminescent pH indicator based on a ruthenium(II) complex was described by Murtaza et al. recently.13 This pH probe shows different lifetimes in the protonated and the deprotonated forms in aqueous solution (ranging from 200 to 300 ns), but a pH sensor was not realized. A promising way to realize lifetime-based pH optodes is the use of fluorescence (“Fo¨rster”) energy transfer (FET) from a pHinsensitive luminescent donor to a pH-sensitive colored acceptor.14,15 The excited state of the donor is deactivated by the (3) Papkovsky, D. B.; Ponomarev, G. V.; Trettnak, W.; O’Leary, P. Anal. Chem. 1995, 67, 4112. (4) McEvoy, A. K.; McDonagh, C. M.; MacCraith, B. D. Analyst 1996, 121, 785. (5) Wolfbeis, O. S. Fiber Optic Chemical Sensors and Biosensors; CRC Press: Boca Raton, FL, 1991. (6) Wolfbeis, O. S.; Leiner, M. J. P.; Posch, H. E. Mikrochim. Acta 1986, 3, 359. (7) Klimant, I.; Wolfbeis, O. S. Anal. Chem. 1995, 67, 3160. (8) Draxler, S.; Lippitsch, M. E. Sens. Actuators B 1995, 29, 199. (9) Szmacinski, H.; Lakowicz, J. R. Anal. Chem. 1993, 65, 1668. (10) Thompson, R. B.; Frisoli, J. K.; Lakowicz, J. R. Anal. Chem. 1992, 64, 2075. (11) Demas, J. N. Excited-State Lifetime Measurements; Academic Press: New York, 1983. (12) O’Connor, D. V.; Phillips, D. Time-Correlated Single Photon Counting; Academic Press: New York, 1984. (13) Murtaza, Z.; Chang Q.; Rao G.; Lin, H.; Lakowicz, J. R. Anal. Biochem. 1997, 247, 216. (14) Lakowicz, J. R. Anal. Chim. Acta 1993, 272, 179. (15) Jordan, D. M.; Walt, D. R.; Milanovich, F. P. Anal. Chem. 1987, 59, 437. S0003-2700(97)01282-1 CCC: $15.00
© 1998 American Chemical Society Published on Web 08/08/1998
accepor, thereby causing a decrease in both the quantum yield and the decay time of the donor. FET-based optodes have been realized by several groups by measuring either intensity or decay time as the analyte-dependent parameter but only in the range of a few nanoseconds.16,17 The use of transition metal complexes as donor opens a promising way to obtain LED-compatible pH sensors with lifetimes in the microsecond range and was first suggested by Lakowicz.14 Here we demonstrate that co-immobilization of such luminophores with colored pH indicators results in pH optodes with decay times of a few microseconds, which paves the way to frequency domain pH sensing at low modulation frequencies. EXPERIMENTAL SECTION Materials. 4,4′-Diphenyl-2,2′-bipyridyl (dph-bpy), bromothymol blue sodium salt (Aldrich), the reactive pH indicator N-9 (Merck), and the polyurethane hydrogel D4 (Tyndall-PlainsHunter Ltd.) were obtained from the indicated suppliers and were used without further purification. The polyester support, with a thickness of 125 µm, was from Goodfellow (Cambridge, UK). Phosphate and borate buffer solutions were prepared from distilled water containing 3.5% (w/w) sodium chloride (GR standard, Merck) to simulate the salinity of seawater and deoxygenated by bubbling nitrogen through the solutions for at least 1 h. Synthesis of the Ruthenium Complexes. Ruthenium(II) Tris(4,4′-diphenyl-2,2′-bipyridyl) Chloride Solution. A total of 225.9 mg (864 µmol) of RuCl3‚3H2O and 800 mg (2.59 mmol) of 4,4′diphenyl-2,2′-bipyridyl were dissolved in 5 mL of ethylene glycol and 0.2 mL of water and refluxed for 45 min, during which time the color of the solution turned from orange to red. After the solution was cooled to room temperature, 50 mL of acetone was added to obtain the crude solution. Ruthenium(II) Tris(4,4′-diphenyl-2,2′-bipyridyl) Chloride Heptahydrate, Ru(dph-bpy)Cl (1). Ten milliliters of the crude solution was treated with 50 mL of a 1 M solution of NaCl, upon which a dark-red precipitate was formed. It was filtered, washed with 20 mL of water and 40 mL of ether, and dried in a desiccator over solid NaOH. Yield 85%, mp >245 °C. Elemental analysis (calcd/ found) for C66H62N6O7RuCl2 (1223.25): C, 64.80/64.73; H, 5.11/ 4.99; N, 6.87/6.56. Ruthenium(II) Tris(4,4′-diphenyl-2,2′-bipyridyl) Perchlorate Bishydrate, Ru(dph-bpy)ClO4 (2). Twenty milliliters of the crude solution was treated with 100 mL of a 1 M solution of perchloric acid and 100 mL of water. The orange-brown precipitate was filtered by suction and washed with water and ether. For purification, the precipitate was dissolved in acetone. Water was added in small quantities until a precipitate started to form. The solvent was allowed to evaporate slowly, and the resulting crystals were dried in a desiccator, affording dye 2 in 92% yield, mp >245 °C. Elemental analysis (calcd/found) for C66H52N6O10RuCl2 (1261.17): C, 62.86/64.16; H, 4.16/4.18; N, 6.66/6.66. Synthesis of Ion Pairs. Ion pairs of anionic bromothymol blue with cetyltrimethylammonium (BTB-CTA, 3) and tridodecylmethylammonium (BTB-TDMA, 4) were prepared by analogy to a protocol from Werner et al.,18 except that the ion pairs were (16) Gabor, G.; Chadha, S.; Walt, D. R. Anal. Chim. Acta 1995, 313, 131. (17) Roe, J. N.; Szoka, F. C.; Verkman, A. S. Analyst 1990, 115, 353. (18) Werner, T.; Klimant, I.; Wolfbeis, O. S. Analyst 1995, 120, 1627.
Figure 1. Chemical structure of ion pair Ru(dph-bpy)BTB. Table 1. Composition of Sensing Films with Immobilized Ru(dph-bpy)BTB (5) as Donor/Acceptor Ion Paira membrane M1 M2 a
5 (mmol/kg) 0.5 1
membrane M3 M4
5 (mmol/kg) 2 5
All membranes are 12.5 µm thick.
extracted with CH2Cl2. The ion pair with Ru(dph-bpy)3 as the cationic counterion was prepared by the following procedure. Ru(II) Tris(4,4′-diphenyl-2,2′-bipyridyl)bisbromothymol Blue Hexahydrate, Ru(dph-bpy)BTB (5). A solution of 104 mg (162 µmol) of bromothymol blue sodium salt in 3 mL in 0.1 M NaOH was treated with 0.1 M HCl until the indicator was completely converted into its acidic yellow form. This solution was dropped into 93 mg of the ruthenium complex 1 dissolved in 500 mL of water. After 3 days, a red-brown precipitate was formed and was filtered by suction. The crude material was washed with water and dried in a desiccator with NaOH as a drying agent to yield 52 mg (21.9 mmol, 27%) of red-brown ion pair 5, whose chemical structure is given in Figure 1 (mp >245 °C). Elemental analysis (calcd/found) for C120H114Br4N6O16S2Ru (2379.13): C, 60.58/60.38; H, 4.83/4.61; N, 3.53/3.87; Preparation of Sensor Membranes. A 5% (w/w) solution of hydrogel was prepared by dissolving 4 g of polymer D4 in 72 g of ethanol and 8 g of water and vigorously stirring for 5 h. Preparation of Sensor Membranes M1-M4. The membranes were obtained by dissolving the ion pair 5 in the polymer solution. Indicator concentrations are given in Table 1 and refer to the mass of hydrogel employed. The mixture was spread onto the polyester support in a wet thickness of 250 µm using a home-made device. The layers were dried at room temperature for at least 1 day to remove all traces of the solvent. The thickness of the dried membranes was estimated to be 12 µm. Preparation of Sensor Membranes M5-M11. For preparation of membranes M5-M11, the ruthenium complex 1 and ion pairs 3 or 4 were dissolved in the polymer solution simultaneously in the concentrations given in Table 2. The further process is analogous to the protocol for sensor membranes M1-M4. Membranes M5-M8 were spread in a wet thickness of 250 µm, Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Table 2. Composition of Sensing Films with Ru(dph-bpy) and BTB Immobilized Separately Ru(dph-bpy)Cl ion pair BTB thickness of membrane (1) (mmol/kg) of BTB (mmol/kg) membrane (µm) M5 M6 M7 M8 M9 M10 M11
3 3 3 3 5 5 10
3 3 3 3 3 4 3
1 2 5 10 10 10 10
12.5 12.5 12.5 12.5 3 3 3
Figure 3. Experimental setup for measuring decay times.
Figure 2. Chemical structure of the dye-hydrogel conjugate. The azo dye is covalently immobilized via a vinyl sulfonyl group. One of the substituents R is a hydroxyl group which, on dissociation, causes the color change to occur.
whereas membranes M9-M11 had a wet thickness of 60 µm. This results in sensing films with a dry thickness of ∼12.5 and 3 µm, respectively. Preparation of Sensor Membrane M12. This sensor was obtained by a two-step procedure. First, a cocktail composed of 5 g of a hydrogel solution in ethanol/water (see above) and 1.58 mg of Ru(dph-bpy)ClO4 (2), was spread on the polyester support in a wet thickness of 120 µm. After drying, the membrane had a thickness of 6 µm. The pH indicator N-9 was covalently immobilized onto the hydrogel layer according to a procedure described previously,19 except that hydrogel was used as the polymeric support instead of cellulose acetate (Figure 2). In essence, 1.0 mg of N-9 was ground with 1 drop of concentrated sulfuric acid in a mortar with a pestle and allowed to stand for 30 min in a desiccator over solid NaOH. Then, 500 mL of water was added, and the solution was neutralized with a 1 N sodium hydroxide solution until the color turned to green. Three hydrogel films (3 × 5 cm) were placed in this solution with stirring. After 5 min, 250 mg of sodium carbonate and, after another 5 min, 0.5 mL of a 32% sodium hydroxide solution were added, upon which the color of the solution turned to deep blue. After 75 min, the membrane was removed from the reaction solution and washed with copious quantities of water. The content of coupled pH indicator and, therefore, the pH sensitivity can be tuned by varying the reaction time during the coupling procedure. All membranes were conditioned in a buffer solution of pH 7 for 1 h before measurements. Apparatus. All measurements were taken at 21 °C. pH reference measurements were performed with a microprocessor (19) Weigl, B. H.; Holobar, A.; Rodriguez, N. V.; Wolfbeis, O. S. Anal. Chim. Acta 1993, 282, 335.
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pH meter (WTW, Weilheim) calibrated with Aldrich pH standards of pH 4.00 and 7.00 at 21 °C. Fluorescence spectra were acquired using a Perkin-Elmer LS 50 B spectrofluorometer with a pulsed xenon discharge lamp as a light source. Absorption spectra were measured using a Perkin-Elmer Lambda 14 P spectrophotometer. Frequency domain lifetime measurements were performed with a fiber-optic setup schematically shown in Figure 3. The optical system consists of a blue LED as light source (λmax 470 nm, NSPB 500, from Nichia), a blue glass filter (BG 12, Schott Mainz), a bifurcated glass fiber bundle (2 mm diameter), a red-sensitive photomultiplier tube (PMT, H5701-02, Hamamatsu), and an emission filter (OG 570, Schott Mainz). A dual-phase lock-in amplifier (DSP 830, Stanford Research Inc.) was used for sine wave modulation of the LED at a frequency of typically 75 kHz and for the detection of the phase shift of the luminescence signal. The average decay time τ was calculated by using the following relationship:
τ)
tan φ 2πf
where φ is the measured phase angle and f the modulation frequency of the LED. The accuracy and the reproducibility of the measurement setup were 0.05°. Frequency spectra were measured on the multifrequency phase fluorometer K2 from ISS, with an argon ion laser as light source operated at the 488-nm line. Experimental Procedure. Sensor membrane spots of 5 mm were punched and placed on the bottom of the wells of a microplate using silicone high-vacuum grease (product no. 7922, Merck). The sensor spots were illuminated via a fiber bundle which was placed below the bottom of a microplate, as shown in Figure 3. Buffer solutions were pipetted into the wells and renewed immediately before measurement to ensure that no oxygen diffuses from the microplate into the solution. For each variation of the pH, the spots in the microplates were first washed twice with water and buffer solution and then adjusted to the new pH for 3 min. The cross-sensitivity to oxygen was measured in oxygen-saturated buffer solutions. For all other measurements, the buffers were deoxygenated by bubbling nitrogen through the solutions before pipetting them into the wells.
Figure 5. pH response of membranes M5-M8 and relation between decay time and concentration of BTB in the membrane.
Figure 4. Absorption spectra of BTB in the acidic (1) and basic (2) forms, respectively, and excitation (3) and emission spectra (4) of Ru(dph-bpy)Cl; overlap integral is hatched.
RESULTS AND DISCUSSION Choice of Materials. To obtain a useful two-indicator system whose fluorescence intensity and decay time are modulated by the pH-dependent efficiency of Fo¨rster energy transfer (FET), a luminescent donor and a colored pH-sensitive acceptor are required which are in close spatial proximity.14 In the described sensors, both were co-immobilized in hydrogel at high concentrations. Depending on the respective pH, the acceptor changes its color and deactivates the excited state of the donor. This effect changes both the luminescence intensity and the decay time of the sensor. To obtain a sufficiently high signal change in either intensity or decay time, a strong overlap between the absorption of the deprotonated or the protonated form of the pH indicator with the emission spectrum of the donor is essential. We apply the hydrophobic ruthenium(II) tris(4,4′-diphenyl-2,2′bipyridyl) complex 1 as a promising candidate for the donor due to its high quantum yield of ∼0.3 and a luminescence decay time of 1.95 µs, measured in ethanol-methanol (4:1 v/v) solution at 20 °C under vacuum-deaerated conditions.20 Its absorption spectrum perfectly overlaps the emission of bright blue LEDs, and its broad red emission spectrum overlaps the absorption of blue pH indicators such as the sulfophthalein dyes. Compared to the widely used oxygen probe ruthenium(II) tris(4,7-diphenyl1,10-phenanthroline), it is 4 times less susceptible to quenching by oxygen. Bromothymol blue (BTB) was selected as the acceptor due to its pK of ∼7.2 and its absorption spectrum, which, in the deprotonated state, perfectly overlaps the emission spectrum of the ruthenium complex 1, as shown in Figure 4. Since BTB is available as the water-soluble sodium salt only, it was made lipophilic by ion pair formation with long-chain quarternary ammonium ions such as cetyltrimethylammonium (CTA) and tridodecylmethylammonium (TDMA). In addition, a special donor/acceptor ion pair between the anionic BTB and the cationic luminescent ruthenium complex was prepared. This ion pair (5) can be prepared easily, is well retained in the polymer matrix, and is almost insoluble in water. In an alternative approach, a reactive, pH-dependent azo dye (N-9) was used as the pH indicator. Unlike in the previous cases, it was covalently coupled onto the hydrogel matrix to avoid any (20) Cook, M. J.; Lewis, A. P.; McAuliffe, G. S. G.; Skarda, V.; Thompson, A. J.; Glasper, J. L.; Robbins, D. J. J. Chem. Soc., Perkin Trans. 2 1984, 1293.
leaching. Its use in absorption-based optical pH sensing has been described previously.19 N-9 and BTB show similar spectral properties in that N-9 also turns from yellow to blue on changing from acidic to basic conditions, and its pK of ∼7.4 is similar to that of BTB. The polyurethane hydrogel D4 was chosen as the polymer matrix because of its hydrophilicity, which ensures a homogeneous distribution of the indicators and a fast pH response. Furthermore, the presence of a high fraction of hydroxyl groups in this matrix allows the covalent coupling of the reactive N-9. Sensor Design. We have investigated three strategies for coimmobilization of donor and acceptor. In the first, donor and acceptor were dissolved separately (M5-M11) as single species. In the second, donor and acceptor formed a special ion pair, 5 (M1-M4). In the third, the acceptor was covalently linked to the hydroxyl groups of the matrix, while the donor was dissolved in the hydrogel. Response to pH. Response times (t90) of sensors M1-M11 are ∼10 s in the pH transition interval between 6.5 and 9. t99 values are within 15 s on going from pH 6.5 to 9 and within 20 s on changing from acidic to basic conditions. Membranes M12M14 respond (t90, t99) within 7 s on going from pH 6.5 to 9. On changing from basic to acidic conditions, t90 increases to 25 s and t99 to 50s. To further minimize the response times, the thickness of the membranes may be reduced. The pH-dependent work functions of membranes M5-M8 were established at different concentrations of the acceptor (Figure 5). As expected, the rate of FET strongly depends on the concentration of BTB in the membrane. Membrane M8, which possesses the highest concentration of BTB (10 mmol/ kg), undergoes a decrease in the decay time (∆τ) from 1.42 to 0.72 µs on going from pH 6.5 to 9. This corresponds to a phase shift of 15° at a modulation frequency of 75 kHz. As predicted by Fo¨rster’s law (with its R6 dependency of the efficiency of FET), the rate of FET becomes rather small in the case of decreasing (1-5 mmol/kg) concentrations of the pH indicator (acceptor), resulting in small (0.11-0.26 µs) changes in decay time over the same pH range (Table 3). To obtain sensing materials with a better defined distance between donor and acceptor membranes, M1-M4 were prepared, where donor and acceptor form an ion pair (5). In this case, we expected intramolecular FET to occur preferably between the two partners of a single ion pair. The shape of the pH calibration curves was expected to be nearly independent of the total concentration of the indicators. Surprisingly, these membranes behave similarly to M5-M8, where both indicators are dissolved Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Table 3. Average Decay Time τ (in µs) at pH 6.5 and 9, Decrease of the Decay Time ∆τ (µs) of Membranes M1-M11 on Going from pH 6.5 to 9, and the Respective pSh Values τ (µs) membrane
pH 6.5
pH 9
∆τ (µs)
pSha
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11
1.59 1.58 1.52 1.32 1.57 1.54 1.46 1.42 1.26 1.27 1.20
1.53 1.48 1.34 0.75 1.46 1.40 1.20 0.72 0.89 0.76 0.60
0.06 0.10 0.18 0.57 0.11 0.14 0.26 0.70 0.37 0.51 0.60
8.1 8 8.1 8.3 8 8 8.2 8.4 8 8.2 8
Figure 6. pH calibration curve of membrane M12 with covalently coupled pH indicator N-9; average over five sensor membranes with mean standard deviation. Inset: Calibration curve of M12 measured in deaerated and in air-saturated buffers.
a Defined as the pH at which the signal has decreased to one-half on going from pH 6.5 to 9.
separately. No significant improvement of the sensing properties was obtained in terms of the efficiency of energy transfer (and hence the change in decay time). Again, the rate of FET strongly depends on the concentration of the acceptor. It is evident that no efficient energy transfer occurs between the partners of a donor/acceptor ion pair, probably due to a shielding effect of the water molecules around the charged groups of donor and acceptor, or a wrong orientation of the dipole moments of both partners. The maximum decrease of ∆τ of M4 (with its ion pair concentration of 5 mmol/kg) is 0.57 µs. With decreasing indicator concentration, the rate of FET drops, and hence ∆τ is as small as 0.06-0.18 µs (Table 3). Because there is no linear relation between the measured phase angles (as well as the respective decay times) and the concentration of the deprotonated indicator, the calibration curves do not obey the Henderson-Hasselbalch equation. It, therefore, is impossible to define a pKa value for such optodes. Rather, a so-called pSh value is introduced here which describes the pH transition interval and is defined as the pH at which 50% of the relative signal change has occurred in the relevant pH interval between pH 6.5 and 9. Similar to the pKa, the pH sensitivity is highest around the pSh. At pH values higher pH 9, a significant swelling of the films was observed, causing a decrease in the rate of energy transfer and an increase in the apparent lifetime. Membranes M1-M11 show pSh values of around 8 ( 0.2 at any indicator concentrations, whereas the transition point of membranes containing N-9 (M12) is pH 7.4 (Figure 6). From the frequency spectra analysis (Figure 7), it was found that there was no single-exponential decay of the luminescence signal. This is not unexpected, since the immobilization of ruthenium complexes in polymeric matrixes frequently results in a heterogeneous site distribution. It is also evident that the selected modulation frequency of 75 kHz is not the optimal choice. At higher modulation frequencies (in the range between 150 and 200 kHz), the lifetime change results in a greater phase shift and, therefore, a better pH resolution. Unfortunately, with our fiberoptic setup, we had an upper limit of 100 kHz. At very low pH values, a decrease in the luminescence decay time with increasing acceptor concentration was observed. This 3896 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
Figure 7. Frequency response of membrane M12. Phase values are indicated with ×, modulation values with 9. A double-exponential fit gives the following parameters. pH 5: τ1 ) 1.44 µs (0.96), τ2 ) 0.25 µs (0.04). pH 7: τ1 ) 1.06 µs (0.87), τ2 ) 0.20 µs (0.13). pH 9: τ1 ) 0.76 µs (0.62), τ2 ) 0.16 µs (0.38).
effect was rather unexpected, since there is no spectral overlap between the absorption spectrum of the deprotonated BTB and the emission of Ru(dph-bpy). The decay times of membranes M1-M4 decreased from 1.59 to 1.32 µs at pH 6.5. Similarly, they dropped from 1.57 to 1.42 µs in the case of membranes M5-M8. We conclude that BTB itself may acts as a quencher of luminescence, due to the presence of its bromo substituents. In line with this, we find dyes containing even more bromo substituents (such as bromophenol blue and bromocresol purple) to almost completely quench the luminescence of Ru(dph-bpy) in hydrogel. On the other hand, sulfophthaleins such as m-cresol purple, thymol blue, which lack bromo atoms, show no significant quenching. Indicator Leaching. The calibration curves of membranes M1-M9 were not stable over time. This is caused by leaching of the pH indicator in its deprotonated form. As the result, a continuous increase in decay times was observed at pH’s >7. Three different strategies were pursued to reduce this effect. (1) The counterion of BTB was exchanged by using the more lipophilic TDMA instead of CTA. As the result, leaching was reduced by a factor of 2 (membrane M10). (2) Ru(dph-bpy), being cationic, can act as a counterion for the negative BTB. When preparing membranes with an excess of the ruthenium complex (M11), we found a significant improvement (by a factor of 4) of signal stability at uncompromised pH sensitivity. A beneficial side effect results from the higher concentration of luminophore in the membrane, which results in
a higher luminescence signal and, therefore, an improved signalto-noise ratio. (3) The best way to avoid leaching is to covalently couple the acceptor onto the matrix. This was realized by linking the reactive pH indicator N-9 to the hydroxyl groups of the hydrogel. The calibration curve of the resulting membrane M12 is perfectly stable for at least a few weeks of continuous immersion in water, and no dye leaching was observed at all. The pH transition interval of M12 is in the range between pH 6.5 and 9.5. Photostability. The photobleaching of membrane M12 was studied at pH 6 (where the pH indicator is almost completely present in the protonated form) and at pH 9, where the deprotonated form prevails. M12 was illuminated with the focused light of a bright blue LED (0.5 mW/cm2) over a period of 30 min. No photodecomposition of the ruthenium complex was found, but the pH indicator bleached, particularly in its deprotonated form. In air-saturated buffer at pH 6, a signal drift of 14 ns and, at pH 9, of 55 ns was observed. Under anaerobic conditions, no significant drift was measurable at pH 6, whereas at pH 9 a signal drift of 17.5 ns was found, indicating a photostability almost 4 times better than that in the presence of oxygen. It is obvious, as well, that any degradation of the acceptor, caused by leaching or photobleaching, will result in a decrease of the rate of FET and, therefore, a change of the calibration curve. Hence, one of the classical advantages of decay time-based optical sensors is lost. Cross-Sensitivity to Oxygen. The luminescence of ruthenium-ligand complexes is prone to quenching by oxygen. This is a critical factor in terms of accuracy and photobleaching, and the effects of oxygen, therefore, were studied for membrane M12, which is the most suitable one for practical use. Calibration curves were measured in both air-saturated and oxygen-free buffers. Quenching by oxygen was found to be modest and is more expressed at low pH, where the decay time of the ruthenium complex is longest. The decay time is reduced from 1.38 to 1.31 µs at pH 6 on changing from oxygen-free to air-saturated solution. At pH 8, the change is -0.04 µs. Quenching by oxygen at air levels causes an apparent pH shift of -0.17 pH unit at pSh, as can be seen in the inset of Figure 6. (21) Gruber, W. R.; O’Leary, P.; Wolfbeis, O. S. Proc. SPIE-Soc. Photo-Instrum. Eng. 1995, 2388, 148.
Storage Stability. For sensing films of type M12, no effect on the calibration curves was found after at least 6 months when the films were kept at room temperature in the dark and in the protonated form of the indicator. Comparison to Existing pH Sensors. The sensors described here are the first pH optodes with lifetimes in the microsecond range. They can be efficiently excited with the blue LED and are highly luminescent. Modulation frequencies in the range of 75-200 kHz allow lifetime measurements with the same solid-state instruments which already exist for optical oxygen sensing.21 This is a distinct advantage over lifetime sensors with decay times the nanosecond range13 and over others which need UV light sources.9 The phase signal is not influenced by fluctuations of the LED or the sensitivity of the PMT. In addition, bending effects and changes of the optical properties of the sample do not interfere with the measurement. The pH range of the sensor can easily be varied by proper selection of the pH indicator, e.g., from the class of sulfophthalein dyes. It is obvious that such sensors open a promising method of self-referenced optical pH sensing with low-cost instrumentation. Outlook. The sensing scheme presented here for optical pH sensing is also useful for designing optodes for basic or acidic gases. Soaking membrane M12 with a hydrogen carbonate buffer and covering it with a gas-permeable silicone membrane results in a lifetime-based pCO2 optode. Conceivably, sensors for ammonia or sulfur dioxide may be obtained as well. The development of a pCO2 sensor along the lines presented here is in progress. Furthermore, there is a great interest for other luminescent transition metal complexes which are less quenchable by oxygen to overcome the major drawbacks of the sensors described and to make them more suitable for practical use. ACKNOWLEDGMENT This work was supported by the Commission of the European Community under the Marine Science & Technology (MAST III) program, project no. 950029. Received for review November 24, 1997. Accepted April 27, 1998. AC971282X
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