Anal. Chem. 1999, 71, 1540-1543
Probing the Dissociation State of Acid-Base Indicators by Time-Resolved Lanthanide Luminescence: A Convenient Transduction Scheme for Optical Chemical Sensors Manfred A. Kessler
Analytical Division, Institute of Organic Chemistry, Karl-Franzens University, 8010 Graz, Austria
The paper describes a new method for the optical measurement of pH based on time-resolved luminescence. The intense line emission of a europium terpyridine chelate is strongly quenched by bromothymol blue (BTB). Since only the blue base form acts as a quencher, the decay time of the luminescence directly reflects the protonation state of BTB and, hence, the pH of the solution. The experimental results are in good agreement with a theoretical model that accommodates both the Henderson-Hasselbalch equation and the Stern-Volmer law. Decay time-based response curves have the same sigmoidal shape as conventional titration curves, but their apparent pKa is shifted to lower pH, depending on the total concentration of BTB used. All luminescence decay curves featured single-exponential decay. With 8 µmol/L BTB, decay times decreased from 1172 µs at pH 2.60 to 120 µs at pH 8.94, and the apparent pKa shifted from 7.0 to 6.1. A Stern-Volmer quenching constant of 9.55 × 105 was obtained for the base form of BTB. The method appears particularly useful as a transduction scheme for optical chemical sensors, since it makes conventional indicator dyes available to decay time-based sensing. The optical measurement of pH is based on indicator dyes that are either protonated or deprotonated, depending on their pKa and the actual pH. The dependence of pH obeys the mass action law and is described by the Henderson-Hasselbalch equation (eq 2). Regardless of individual indicator properties, titration curves always have the same sigmoidal shape, with the maximum slope at pH ) pKa. In addition to the visual estimation of pH with indicator test strips, instrumental techniques give more precise and accurate results.1 Depending on the optical properties of the indicator, the actual protonation state is transduced into an optical signal by measurement of absorption,2 reflection,3 or fluorescence emission.4 However, in contrast to the pH electrode, the measurement is usually limited to a narrow range of typically 2-3 pH (1) Jordan, D. M.; Walt, D. R.; Milanovich, F. P. Anal. Chem. 1987, 59, 437. (2) Kirkbright, G. F.; Narayanaswami, R.; Welti, N. A. Analyst 1984, 109, 1025. (3) Holobar, A.; Weigl, B. H.; Trettnak, W.; Benes, R.; Lehmann, H.; Rodriguez, N. V.; Wollschlager, A.; Oleary, P.; Raspor, P.; Wolfbeis, O. S. Sens. Actuators B 1993, 11, 425. (4) Zhujun, Zh.; Seitz, W. R. Anal. Chim. Acta 1984, 160, 47.
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units, centered at the pKa of the indicator used.5 Therefore, indicators with tailored pKa have been used for special applications.6-9 Recently, new approaches to optical sensing of pH have been introduced, exploiting the photoinduced electron transfer (PET)10-12 and the fluorescence resonance energy transfer (FRET).13 The pH response based on PET is caused by intramolecular quenching of the excited fluorophor by the free electron pair of an amino group that is covalently attached by a spacer. Since only free amino groups quench, the measured fluorescence intensity directly reflects the protonation state and hence the pH. FRET is based on deactivation of a fluorescent donor/acceptor couple.13 Since both PET and FRET result in dynamic quenching, the lifetime of the excited state and, at the same time, the decay time of the fluorescence emission are decreased. In fact, this has already been exploited for optical sensing of pH.12-14 Time-resolved fluorometry is preferable to conventional fluorometry, since the fluorescence decay time is independent of the concentration of the indicator, even if most of it has been photobleached, leached out, or decomposed.15 Second, there are no problems with turbid samples, inner filter effect, and cuvette geometry. However, the decay times of common fluorescent indicators are typically of a few nanoseconds,16 which makes suitable instrumentation sophisticated and expensive. In contrast, time-resolved measurements of the long-lived luminescence of lanthanides can be performed by rather simple electronics and (5) Wolfbeis, O. S. In Molecular Luminescence Spectroscopy: Methods and Applications. Part II; Schulman, S. G., Ed.; Wiley: New York, 1988; pp 202220. (6) Russell, D. A.; Pottier, R. H.; Valenzeno, D. P. J. Photochem. Photobiol. B 1995, 29, 17. (7) Pomeroy, R. S.; Baker, M. E.; Denton, M. B.; Dickson, A. G. Appl. Spectrosc. 1995, 49, 1729. (8) Hamm, L. L.; Heringsmith, K. S.; Weiner, I. D. Int. Rev. Exp. Pathol. 1996, 36, 161. (9) Baldini, F.; Bracci, S.; Cosi, F.; Bechi, P.; Pucciani, F. Appl. Spectrosc. 1994, 48, 549. (10) Draxler, S.; Lippitsch, M. E. Appl. Opt. 1996, 35, 4117. (11) Desilva, A. P.; Gunaratne, H. Q. N.; Lynch, P. L. M. J. Chem. Soc., Perkin Trans. 2 1995, 685. (12) Murtaza, Z.; Chang, Q.; Rao, G.; Lin, H.; Lakowicz, J. R. Anal. Biochem. 1997, 247, 216. (13) Lakowicz, J. R.; Szmacinski, H.; Karakelle, M. Anal. Chim. Acta 1993, 272, 179. (14) Draxler, S.; Lippitsch, M. E. Sens. Actuators B 1995, 29, 199. (15) Szmacinski, H.; Lakowicz, J. R. Sens. Actuators B 1995, 29, 16. (16) Guilbault, G. G. Practical Fluorescence, Theory, Methods, and Techniques; Dekker: New York, 1973; pp 16-17. 10.1021/ac971313o CCC: $18.00
© 1999 American Chemical Society Published on Web 03/19/1999
instrumentation. Lanthanide labels are known for their unique luminescence properties with large Stokes shifts, sharp emission lines, and decay times of milliseconds.17 This is a major advantage for discrimination of short-lived background fluorescence and scattered excitation light and is exploited in various immunoassays18,19 and for ultrasensitive detection in liquid chromatography.20 Some terbium chelates exhibit pH-sensitive luminescence, which has also been suggested for use in optical sensors.21 Furthermore, although not experimentally verified, a variety of “gedanken” sensors have been designed, including a europium chelate that is covalently linked to Congo red.13 In a previous work, it was shown that europium chelates are susceptible to quenching by various species, including heavy metals and organic dyes.22 Among the latter, the base form of bromothymol blue (BTB) strongly quenched the luminescence of an europium terpyridine chelate. Since the acid form did not quench, this appeared to be a novel approach to decay time-based optical sensing of pH that may be applicable to other conventional pH indicators as well. Although the pH dependence of such systems looks complex at the first sight, it is well-described by theory and governed by a combination of the HendersonHasselbalch equation and the Stern-Volmer law for dynamic quenching. In this work, the response of such systems is theoretically described and experimentally proven for BTB. The combination of conventional acid-base indicators with timeresolved lanthanide luminescence represents a new transduction scheme for the optical measurement of pH that is theoretically described and experimentally proven here for the first time.
a repetition rate of 15 Hz and a pulse duration of 500 ps. Emission was collected by a lens in right-angle geometry, passed an OG 550-nm long-pass filter (Schott, Mainz, Germany), and measured by a red-sensitive photomultiplier tube (R 928, Hamamatsu, Herrsching, Germany) with a built-in voltage supply. The output signal was recorded by an oscilloscope (LeCroi 9410 dual 150MHz oscilloscope; Le Croi, Heidelberg, Germany) triggered by the excitation pulse. Low-noise decay curves were obtained by averaging 1000 single curves and transmitted to a computer. All experimental decay curves obeyed single-exponential decay. The offset (A0), amplitude (A1), and decay time (τ) were extracted from nonlinear least-squares fits with the function I(t) ) A0 + A1e-(t/τ). In Figure 2, offset values (A0) were subtracted from the decay curves in order to obtain linear plots. All measurements were performed at 22 °C. Theoretical Background. The relation between the luminescence intensity and the concentration of a quencher is described by the Stern-Volmer equation
MATERIALS AND METHODS Reagents. Bromothymol blue (standard for microscopy) was from Fluka (Buchs, Switzerland) and used without further purification. [2,2′,2′′,2′′′-[(4′-Phenyl-2,2′:6′,2′′-terpyridine-6,6′′-diyl)bis(methylenenitrilo)]tetrakis(acetato)]europium(III) (EuTerpy) was synthesized according to a published procedure.23 All measurements were performed in aqueous buffer solutions containing 150 µmol/L EuTerpy, 10 mmol/L phosphate, and 150 mmol/L NaCl. Nanopure deionized water was used for all buffer preparations. Instrumentation and Data Processing. Uncorrected luminescence spectra and intensity-based measurements were performed on a Shimadzu RF-5001 PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) with band-passes of 3 nm for both excitation and emission. Time-resolved measurements were performed in an optical black box using 1-cm quartz cuvettes. Europium luminescence was excited at 337 nm by a nitrogen laser (PNL 200, Laser Technik, Berlin, Germany) with an average output power of 12 mW. The laser operated in pulsed mode with
pH ) pKa + log([A]/[HA])
(17) Choppin, G. R.; Bu ¨ nzli, J. C. G. Lanthanide Probes in Life, Medical and Environmental Sciences; Elsevier: Amsterdam, 1989; Chapter 7. (18) Dickson, E. F. G.; Pollak, A.; Diamandis, E. P. J. Photochem. Photobiol. B 1995, 27, 3. (19) Dickson, E. F. G.; Pollak, A.; Diamandis, E. P. Pharmacol. Ther. 1995, 66, 207. (20) Okabayashi, Y.; Kitagawa, T. Anal. Chem. 1994, 66, 1448. (21) deSilva, A. P.; Gunaratne, H. Q. N.; Rice, T. E. Angew. Chem. 1996, 108, 2253. (22) Kessler, M. A. Anal. Chim. Acta 1998, 364, 125. (23) Mukkala, V. M.; Helenius, M.; Hemmila¨, I.; Kankare, J.; Takalo, H. Helv. Chim. Acta 1993, 76, 1361.
I0/I ) τ0/τ ) 1 + KSV[Q]
(1)
where I0 and I are the luminescence intensities in the absence and presence, respectively, of a quencher Q, present at the concentration [Q], and KSV is the Stern-Volmer quenching constant. Instead of intensities, the decay times give an equivalent expression with τ0 and τ standing for the luminescence decay times in the absence and presence, respectively, of a quencher.5 The relationship between the protonation state of an indicator and the pH is governed by the Henderson-Hasselbalch equation
(2)
with [A] and [HA] denoting the equilibrium concentrations of the base and acid forms, respectively, and pKa being the acid-base constant of the indicator. In systems where only one form of the indicator (e.g., the blue anion of BTB) is a dynamic quencher, the pH dependence of the luminescence decay time is derived from eqs 1 and 2 and given by
pH ) pKa + log{(τ0/τ - 1)/(AtKSV - (τ0/τ - 1))}
(3)
with At denoting the total concentration of indicator. If both the acid and base forms are quenching, a more complex relation results from the Stern-Volmer equation for multiple quenching,5
I0/I ) τ0/τ ) 1 + KSV1[Q1] + KSV2[Q2]
(4)
with individual Stern-Volmer constants for each quenching species. In our case, indexes 1 and 2 refer to the acid and base forms of the indicator, respectively. Derived from (2) and (4), the resulting equations
pH ) pKa + log{(KSV1At - τ0/τ + 1)/(τ0/τ - KSV2At - 1)} (5) and Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
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τ) τ0(10(pH-pKa) + 1)/(KSV1At + 10(pH-pKa)(KSV2At + 1) + 1) (6)
theoretically describe the decay time-based optical measurement of pH. RESULTS AND DISCUSSION Luminescence Properties of EuTerpy. The excitation and emission spectra of EuTerpy along with its chemical structure are shown in Figure 1. The sharp emission lines at 586, 595, 614, and 693 nm originate from F7J r D50 transitions of the Eu3+ ion.24 Single-exponential decay was observed throughout all experiments, and decay times in the absence of BTB were independent of pH (between 2 and 13) and the type of buffer used and almost independent of temperature (within statistical fluctuations) between 20 and 80 °C. The precision of the decay time in the absence of quenchers was determined at 1270 ( 6.0 µs (n ) 10). Response to BTB. Even low concentrations of BTB strongly quenched the luminescence of EuTerpy by decreasing its decay time (τ). Since the amplitude (A1) was almost not affected, quenching is considered entirely dynamic. This is also supported by a linear relationship between I0/I - 1 and the concentration of BTB, as predicted by the Stern-Volmer law.5 From decay time measurements and eq 1, the Stern-Volmer constant was determined at 9.55 × 105 L/mol. In order to find out the photophysical mechanism that is responsible for the strong quenching action of BTB, the quenching ability of several other compounds was investigated. Thus, ammonia and aliphatic amines did not quench, whereas weak static and dynamic quenching was observed with colorless to pale yellow aromatic amines and aromatic nitro compounds. Based on the implication that the Stern-Volmer constant for neither of the compounds tested was above 5 × 103 L/mol, it is assumed that the blue color of BTB is ultimately connected with the strong dynamic quenching action. In fact, the absorption maximum of BTB at 615 nm25 almost exactly matches the wavelength of the main emission line of europium at 614 nm, which fulfills the requirement of spectral overlap between the donor (i.e., the europium ion) and the acceptor (i.e., BTB) in long-range energy transfer.26 The latter is therefore assumed to provide the main photophysical pathway for quenching of EuTerpy by BTB. Optical Measurement of pH. Figure 2 shows luminescence decay curves of EuTerpy in presence of 8.0 µmol/L BTB at three different pH values. Depending on the actual protonation state of BTB, the luminescence of EuTerpy was strongly quenched by the blue base form, whereas quenching by the pale yellow acid form was not significant. The almost linear curves on a logarithmic scale indicate the single-exponential nature of the luminescence decay. Figure 3 shows the pH dependence of the decay time at two different concentrations of BTB. Although the response obeys eq 3 rather than the Henderson-Hasselbalch law, the curves have exactly the same sigmoidal shape as conventional (24) Li, M.; Selvin, P. R. J. Am. Chem. Soc. 1995, 117, 8132. (25) Green, F. J. The Sigma-Aldrich Handbook of Stains, Dyes and Indicators; Aldrich Chemical Co.: Milwaukee, WI, 1990; p 180. (26) Parker, C. A. Photoluminescence of Solutions; Elsevier: Amsterdam, 1968; p 77.
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Figure 1. Excitation (λem ) 614 nm) and emission (λex ) 337 nm) spectra together with the chemical structure of EuTerpy. The sharp emission lines originate from F7J r D50 transitions of Eu3+.
Figure 2. Single-exponential decay curves of the luminescence of EuTerpy in the presence of 8.0 µmol/L BTB: (A) pH ) 6.95, τ ) 244 µs; (B) pH ) 6.15; τ ) 591 µs; (C) pH ) 5.47, τ ) 936 µs. Decay times τ are reflected by the slope.
Figure 3. Experimental (symbols) and calculated (solid lines) responses to pH of EuTerpy in the presence of BTB at concentrations of 8.0 (A) and 24.0 µmol/L (B). Note the difference between the apparent pKa and its literature value of 7.0.
titration curves. As the main difference, the position of the point of inflection (i.e., the apparent pKa) depends on the amount of indicator used and is distinct from the thermodynamic pKa by a shift to lower values with increasing total concentration of BTB. This is caused by the Stern-Volmer law, which results in higher sensitivity of the decay time at low quencher concentrations. Consequently, the shift of the apparent pKa should be reversed with indicators exhibiting a blue acid form and a yellow base form. Apart from the shifted apparent pKa, the curves are characterized by the minimum decay time, τmin. Since almost the total amount of BTB is converted into its base form at strongly alkaline pH, τmin is calculated from eq 1 with [Q] being equal to At. Calculated curves of Figure 3 agree well with experimental results and are based on the following parameters: KSV ) 9.55 × 105 L/mol; pKa ) 7.0;25 At ) 8.0 µmol/L (curve A); and At ) 24.0 µmol/L (curve B). Surprisingly, use of the value of 1270 µs for τ0, determined in absence of BTB, did not give satisfactory fits,
even if weak quenching by the acid form of BTB had been assumed. Therefore, τ0 was varied in a curve fit and determined at 1170 and 1080 µs, for curves A and B, respectively. The discrepancy in τ0 is possibly due to impurities that accompany BTB and retain their deep color at low pH. Interference by Heavy Metals. Some heavy metals interfere, since they also strongly quench the luminescence of EuTerpy. Both static- and dynamic-type quenching have been observed with heavy metals.22 Static quenching is predominant with Hg2+, Cd2+, Zn2+, and Pb2+, whereas Cu2+, Ni2+, Co2+, Cr3+, Fe3+, and Mn2+ quench via both mechanisms. A special case is encountered with Cu2+, as it is by far the strongest dynamic quencher among heavy metals, with a Stern-Volmer constant of 1.39 × 105L/mol.22 As far as dynamic quenching is concerned, heavy metals compete with BTB and, therefore, strongly interfere. In contrast, static quenching may only be a problem if the emission intensity becomes too weak to allow an accurate determination of the decay time. Potential Applications. The dependence of the working function on the indicator concentration has to be taken into account when optical sensors based on this principle are being designed. As with absorption-based optical sensors, leaching or decomposition of the indicator would affect the working function and hence would limit the long-term stability of the sensor. On the other hand, the shift in the apparent pKa provides an elegant way for tuning the sensitivity and the working range of sensors by simply adjusting the concentration of the indicator. As a big advantage, there is no need of new tailor-made indicators, since various commercially available indicators may be used. Moreover, this principle may also be applied for improving sensors for other analytes that are based on color changes of appropriate indicators. In particular, tuning the response of sensors based on plasticized
membranes with incorporated sensor chemistry27-30 may be a promising application and a novel approach to decay time-based sensing of a variety of analytes. The use of lipophilic membranes will probably solve the problem of interference by heavy metals. CONCLUSION The method provides a convenient transduction principle for the optical measurement of pH. It may be applied to a variety of conventional pH indicators both in solution and in sensor membranes. Apart from the fact that the response depends on the indicator concentration, this approach provides all the advantages of decay time-based sensing, including insensitivity to turbidity, cuvette geometry, refractive index of the sample, scattered light, and short-lived background fluorescence. Apart from sensors for pH, the method appears suitable for application to other optical chemical sensors and may provide new ways for tuning the sensitivity and working range of these sensors. ACKNOWLEDGMENT This project was supported by the Austrian Academy of Sciences within the Austrian Programme for Advanced Research and Technology (APART). Technical support by the Institute for Chemical and Optical Sensors, Joanneum Research, Graz, Austria, is acknowledged. Received for review December 4, 1997. Accepted July 8, 1998. AC971313O (27) Mills, A.; Monaf, L. Analyst 1996, 121, 535. (28) Mills, A.; Chang, Q. Anal. Chim. Acta 1994, 285, 113. (29) Trinkel, M.; Trettnak, W.; Reininger, F.; Benes, R.; Oleary, P.; Wolfbeis, O. S. Anal. Chim. Acta 1996, 320, 235. (30) Mills, A.; Wild, L.; Chang, Q. Mikrochim. Acta 1995, 121, 225.
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