Miniature Sodium-Selective Ion-Exchange Optode with Fluorescent

sodium-selective, crown ether-capped calix[4]arene iono- phore, capable of ... 5th ed.; Molecular Probes, Inc.: Eugene, OR, 1992. (4) Munkholm, C.; Wa...
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Anal. Chem. 1996, 68, 2656-2662

Miniature Sodium-Selective Ion-Exchange Optode with Fluorescent pH Chromoionophores and Tunable Dynamic Range Michael Shortreed, Eric Bakker,† and Raoul Kopelman*

Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055

An extension into the fluorescence mode of ion-exchange optodes is described, allowing miniaturization and its concomitant benefits. A micrometer-size, fluorescent fiber-optic sodium sensor is described, based on a highly sodium-selective, crown ether-capped calix[4]arene ionophore, capable of ratiometric operation. Three sensor configurations are given, employing different lipophilic, fluorescent pH chromoionophores (Nile Blue derivatives), demonstrating the ability to improve the detection limit and tune the dynamic range to the desired region of interest. Two of the sensors are of special interest in that their working ranges lie within those desired for measuring intracellular cytosolic or blood levels of sodium at the respective physiological pH. These optodes have excellent sodium selectivity, with other physiologically relevant cations (e.g., potassium, calcium, and magnesium) being highly discriminated. Three simple mathematical relationships are given for the three experimentally used fluorescent signal mechanisms (intensity, intensity ratios, and inner-filter or energy transfer effects), permitting visualization on a single graph and enabling direct comparison of the different sensors’ optical responses on a common platform. Finally, these optodes measure the sample’s sodium activity, rather than the concentration, provided that the sample’s pH is measured simultaneously by another sensor, such as a glass electrode. There has been recent interest in developing fiber-optic chemical sensors (optodes) that would reliably measure electrolytes in restricted volumes and under physiological conditions without some of the drawbacks often encountered in imaging applications, which include dye sequestration in intracellular organelles, effects of toxicity of the fluorescent dye, quenching of fluorescence by heavy metals, dye loss through leakage, ion buffering, cellular autofluorescence, alteration of fluorescence by sample viscosity, and poor resolution at the edge of cells.1,2 Recently, indicator molecules have been synthesized that can be excited in the visible, greatly reducing the problem of cellular autofluorescence.3 In principle, optodes could alleviate most of these problems. The sensitivity of fluorescence-based measurements coupled with covalent attachment of complexing agents prompted the development of poly(acrylamide) hydrogel-type

optodes.4-6 A small number of water-soluble fluorescent chelators, such as Calcium Green, were developed originally for imaging applications and recently were used in making hydrogel-based ion sensors. An optode for calcium was successfully developed using Calcium Green as the fluorescent chelator.7 Unfortunately, highly selective water-soluble chelators are not available for sodium or potassium. Also, complex derivatization schemes are often required in order to attach the chelator to the polymer matrix. The derivatization and covalent immobilization might affect the final selectivity, and the binding constant of the chelator is fixed, which prevents fine tuning of the dynamic range and detection limit, in contrast to the sensors described in this manuscript. Ion-selective microelectrodes have been developed that can reliably measure several of the most interesting electrolytes (e.g., potassium, calcium, magnesium, and hydrogen ions8,9) in single biological cells. These devices tend to be somewhat noisier and less selective than larger, more traditionally sized ion-selective electrodes and require a reference electrode also to be inserted into the cell. An interesting alternative is fluorescent optical microsensors. However, the measurement of intracellular levels of sodium (5-18 mM Na+) has remained a challenge for both devices, considering the high level of potassium (80-160 mM K+) and the absence of an ionophore with sufficient selectivity.8 The Nicolsky coefficient, a measure of selectivity, required for intracellular assays of sodium in such a background of potassium, is log K ) -3.5. Ion-selective electrode membranes that contain commercially available sodium ionophores, ETH 227,10 ETH 157, ETH 2120, and ETH 4120,11 as well as bis[(12-crown-4)methyl]dodecyl methyl malonate,12 have the following respective selectivities: log K ) -2.3, -0.4, -1.5, -1.4, and -2.0. While these ionophores may have acceptable selectivity for work in blood serum, they are not quite adequate for intracellular assays. Yamamoto and co-workers13,14 recently reported the synthesis of

† Current address: Department of Chemistry, Auburn University, Auburn, AL 36849. (1) Roe, J. N.; Szoka, F. C.; Verkman, A. S. Analyst 1990, 115, 353-358. (2) Poenie, M. Cell Calcium 1990, 11, 85-91. (3) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 5th ed.; Molecular Probes, Inc.: Eugene, OR, 1992.

(4) Munkholm, C.; Walt, D. R.; Milanovich, F. P.; Klainer, S. M. Anal. Chem. 1986, 58, 1427-1430. (5) Tan, W.; Shi, Z.-Y.; Kopelman, R. Anal. Chem. 1992, 64, 2985-2990. (6) Tan, W.; Shi, Z.-Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (7) Shortreed, M.; Kuhn, M.; Hoyland, B.; Kopelman, R. Anal. Chem. 1996, 68, 1414-1418. (8) Ammann, D. Ion-Selective Micro-electrodes; Springer-Verlag: Berlin-Heidelberg, 1986. (9) Schaller, U.; Spichiger, U. E.; Simon, W. Pflugers Arch. 1993, 423, 338342. (10) Steiner, R. A.; Oehme, M.; Ammann, D.; Simon, W. Anal. Chem. 1979, 51, 351-353. (11) Huser, M.; Gehrig, P. M.; Morf, W. E.; Simon, W.; Lindner, E.; Jeney, J.; To´th, K.; Pungor, E. Anal. Chem. 1991, 63, 1380-1386. (12) Shono, T.; Okahara, M.; Ikeda, I.; Kimura, K.; Tamura, H. J. Electroanal. Chem. 1982, 132, 99-105.

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S0003-2700(96)00035-2 CCC: $12.00

© 1996 American Chemical Society

an ionopore for sodium, with a selectivity against potassium given by log K ) -5.0. We have repeated the synthesis and report here on this ionophore’s performance in an optical microsensor. The response mechanism of all the poly(vinyl chloride) (PVC) bulk optodes employed here was first described by Simon and co-workers15-18 and expatiated on extensively by Suzuki et al.19-21 and Wolfbeis et al.22 The nature and concentration, within the membrane phase, of the lipophilic anionic charge sites, the chromoionophore, and the ionophore exactly define the optode response. The effect on the response produced by substituting a given chromoionophore by another one whose pKa differs is known a priori, and it has been shown23 that the range of sensitivity can be tuned and the limit of detection can be extended, while retaining the selectivity, by such chromoionophore substitution. This leads to a highly versatile sensing methodology. This knowledge has been used here to develop an optical sodium sensor with a dynamic range tuned to the desired regions of interestsin this case, for intracellular and extracvellular measurements at the respective physiological pH values. These features are not available with ion-selective electrodes (ISEs). Though ISEs typically have a large dynamic range, this range cannot be extended below the typical detection limits, which are of the order of 1 µM in unbuffered ion solutions. These optode fundamentals have been used to fabricate heavy-metal ion-slective optodes24,25 with detection limits below that of any ISE. A majority of plasticized PVC membrane-based optodes has relied upon absorbance changes as the signal transduction mechanism. While absorbance is sufficiently sensitive for many routine sensing applications, it is not sensitive enough for microscopic measurements. Analyte ions are extracted from the sample into the membrane, effectively lowering the sample concentration and introducing a small error.17 The higher sensitivity of fluorescence over absorbance is well known, and its use in optical sensors provides a means to significantly reduce the required membrane volume while retaining the same level of signal-to-noise ratio. Furthermore, the equilibrium response time of these optodes is limited, in part, by diffusion of the analyte within the liquid polymer membrane; therefore, reduction in the total membrane volume shortens the response time. In the present work, we have extended the absorbance-based approach of Simon and co-workers to one based on fluorescence, using several ratiometric modes, with the aim of achieving optode miniaturization and its concomitant advantages. We have inves(13) Yamamoto, H.; Shinkai, S. Chem. Lett. 1994, 1115-1118. (14) Yamamoto, H.; Sakaki, T.; Shinkai, S. Chem. Lett. 1994, 469-472. (15) Morf, W. E.; Seiler, K.; Lehmann, B.; Behringer, C.; Tan, S. S. S.; Hartman, K.; Sorensen, P. R.; Simon, W. Ion Selective Electrodes; Akade´miai Kiado´: Budapest, 1989; pp 115-131. (16) Morf, W. E.; Seiler, K.; Sorensen, P. R.; Simon, W. Ion Selective Electrodes; Akade´miai Kiado´: Budapest, 1989; pp 141-152. (17) Seiler, K.; Simon, W. Anal. Chim. Acta 1992, 266, 73-87. (18) Bakker, E.; Simon, W. Anal. Chem. 1992, 64, 1805-1812. (19) Suzuki, K.; Ohzora, H.; Tohda, K.; Miyazaki, K.; Watanabe, K.; Inoue, H.; Shirai, T. Anal. Chim. Acta 1990, 237, 155-164. (20) Watanabe, K.; Nakagawa, E.; Yamada, H.; Hisamoto, H.; Suzuki, K. Anal. Chem. 1993, 65, 2704-2710. (21) Hisamoto, H.; Watanabe, K.; Nakagawa, E.; Siswanta, D.; Shichi, Y.; Suzuki, K. Anal. Chim. Acta 1994, 299, 179-187. (22) He, H.; Li, H.; Mohr, G.; Kova´cs, B.; Werner, T.; Wolfbeis, O. S. Anal. Chem. 1993, 65, 123-127. (23) Lerchi, M.; Bakker, E.; Rusterholz, B.; Simon, W. Anal. Chem. 1992, 64, 1534-1540. (24) Lerchi, M.; Reitter, E.; Simon, W.; Pretsch, E.; Chowdhury, D. A.; Kamata, S. Anal. Chem. 1994, 66, 1713-1717. (25) Bakker, E.; Willer, M.; Pretsch, E. Anal. Chim. Acta 1993, 282, 265-271.

tigated the fluorescent properties of a number of fluorescent lipophilic chromoionophores which show sufficient photostability and brightness. Simon and co-workers have used these chromoionophores26 successfully in the construction of absorbancebased optodes. The efficacy of these chromoionophores as fluorescent indicators is reported here. In addition, we have derived three simple mathematical relationships that can be applied to the commonly used fluorescent signal mechanisms (intensity, intensity ratios, and inner-filter or energy transfer effects) so that they can be visualized on a single graph, thus enabling direct comparison of the different sensors’ optical responses on a common platform. This article also describes the development of the first optode for sodium that should be capable of making intracellular measurements without interference by potassium, calcium, or magnesium. In addition, it demonstrates the first simultaneous measurement of pH-buffered sodium solutions with ISEs and optodes, where both sensors employ the same selective sodium ionophore, making it possible to perform direct comparisons of the optical and electrochemical sensors’ apparent selectivities. Response time, detection limit, lifetime, and reversibility data are also presented. EXPERIMENTAL SECTION Reagents. Poly(vinyl chloride) (PVC), potassium tetrakis[3,5bis(trifluoromethyl)phenyl]borate (KTFPB), chromoionophore I (ETH 5294), chromoionophore II (ETH 2439), chromoionophore III (ETH 5350), potassium ionophore I (valinomycin), potassium ionophore III (BME-44), 2-nitrophenyl octyl ether (o-NPOE), and bis(2-ethylhexyl) sebacate (DOS) were obtained from Fluka Chemical Corp. (Ronkonkoma, NY). The fluorescent label 1,1′dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18) was obtained from Molecular Probes, Inc. (Eugene, OR). The tert-butylated calix[4]arene tetramethyl ester ionophore was a gift from S. Harris (School of Chemical Sciences, Dublin City University, Dublin, Ireland).27 The 1,3-bridged calix[4]crown sodium ionophore was synthesized according to the procedure described by Yamamoto et al.14 The product was purified with alumina chromatography and fully characterized by 1H and 13C NMR. All solutions were prepared with salts of the highest available quality. Buffer solutions were prepared from potassium citrate (Aldrich, Milwaukee, WI), HEPES [4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid], the HEPES sodium salt (Sigma, St. Louis, MO), and the HEPES potassium salt (Fluka Chemical Corp.). Standard solutions were prepared from NaCl, KCl, and MgCl2‚6H2O (Aldrich) and CaCl2‚6H2O (Fluka Chemical Corp.). Standardized solutions of 1.0 N HCl were obtained from Aldrich. All solutions were prepared in 18 MΩ water, Barnstead I Thermolyne Nanopure II system (Dubuque, IA). Membrane Preparation. Both the optode and ISE membranes were prepared to contain 30 mmol/kg ionophore, 15 mmol/kg lipophilic anionic sites (KTFPB), and 15 mmol/kg chromoionophore (optode only). Membranes contained 33% PVC and 66% DOS by weight, which is quite common.17,18,23,28-30 All components were dissolved in freshly distilled tetrahydrofuran (26) Bakker, E.; Lerchi, M.; Rosatzin, T.; Rusterholz, B.; Simon, W. Anal. Chim. Acta 1993, 278, 211-225. (27) O’Conner, K. M.; Cherry, M.; Svehla, G.; Harris, S. J.; McKervey, M. A. Talanta 1994, 41, 1207-1217. (28) Pioda, L. A. R.; Simon, W.; Bosshard, H.-R.; Curtius, H. C. Clin. Chim. Acta 1970, 29, 289-293.

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(THF). Optodes were prepared by dip-coating silanized optical fibers. The silanization procedure is that of Mosbach.31 Optodes were used immediately after preparation and then discarded. Exact membrane compositions are as follows: the first potassium sensor contained 2.8 mg of valinomycin, 1.6 mg of KTFPB, 1.2 mg of ETH 5350, 77.6 mg of DOS, and 37.9 mg of PVC; the second potassium sensor cotnained 2.4 mg of BME-44, 1.7 mg of KTFPB, 1.1 mg of ETH 5350, 77.3 mg of o-NPOE, and 38.5 mg of PVC; the first sodium sensor contained 3.2 mg of tert-butylated calix[4]arene tetramethyl ester, 1.5 mg of KTFPB, 1.0 mg of ETH 5350, 76.4 mg of DOS, and 38.0 mg of PVC; the microsensor contained 3.6 mg of 1,3-bridged calix[4]crown, 2.0 mg of KTFPB, 1.2 mg of ETH 5350, 97.2 mg of DOS, and 48.6 mg of PVC; macrosensor 1 contained 2.8 mg of 1,3-bridged calix[4]crown, 1.5 mg of KTFPB, 1.2 mg of ETH 2439, 71.0 mg of DOS, and 35.5 mg of PVC (1000:1 chromoionophore/DiIC18); macrosensor 2 contained 2.9 mg of 1,3-bridged calix[4]crown, 1.9 mg of KTFPB, 1.1 mg ETH 5294, 76.1 mg DOS and 38.1 mg PVC; macro-sensor 3 contained 3.6 mg 1,3-bridged calix[4]crown, 2.0 mg of KTFPB, 1.2 mg of ETH 5350, 97.2 mg of DOS, and 48.6 mg of PVC; and the ISE contained 6.5 mg of 1,3-bridged calix[4]crown, 3.3 mg of KTFPB, 159.9 mg of DOS, and 77.7 mg of PVC. The electrodes were conditioned in 0.01 M NaCl overnight, and the emf measurements were performed with the following cell:

Ag; AgCl, 1 M KCl|sample|| membrane||0.01 M NaCl, AgCl; Ag Optodes. Macrosensors were fabricated with all-silica, multimode optical fiber with a 105 µm core and a 240 µm cladding diameter (General Fiber Optics, Inc., Cedar Grove, NJ). Microsensors were fabricated with all-silica, single-mode optical fiber with a 3 µm core and an 80 µm cladding diameter (Thor Labs, Newton, NJ). The thickness of the membranes on both the macro- and microsensors was monitored visually with an inverted optical microscope using a 100× oil immersion objective with a numerical aperture of 1.3. The reticule of an optical microscope was calibrated using a standard sample, and the smallest division of the reticule was determined to be slightly less than 1 µm (∼950 nm) at the magnification used. The fibers were measured both end-on and side-on, before and after application of the membranes. The thickness of the membranes was determined to be in the range of 1.5-2.5 µm. Optics. The complete optical path (Figure 1) included an Ion Laser Technology (Salt Lake City, UT) argon ion laser, a 514.5 nm laser band-pass filter (Newport Corp., Irvine, CA), neutral density filters (Melles Griot), a Uniblitz shutter controller (Rochester, NY), a fiber coupler (Newport Corp.), a sample stage (see below), an Olympus inverted fluorescence microscope, IMT-II (Lake Success, NY), Nikon 50 mm f/1.8 camera lenses, an Acton 150 mm spectrograph (Acton, MA), and a Princeton Instruments 1024 × 256 liquid nitrogen-cooled CCD array (Trenton, NJ). Sample Stages. Optode measurements were completed in an in-house-built glass chamber (Figure 1b) constructed of microscope slides with a 50 mL capacity. The sample was (29) Dinten, O.; Spichiger, U. E.; Chaniotakis, N.; Gehrig, P.; Rusterholz, B.; Morf, W. E.; Simon, W. Anal. Chem. 1991, 63, 596-603. (30) Morf, W. E.; Seiler, K.; Rusterholz, B.; Simon, W. Anal. Chem. 1990, 62, 738-742. (31) Mosbach, K. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press, Inc.: New York, 1976; Vol. XLIV.

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Figure 1. (A) Configuration of instruments for the optode measurements. (B) Reaction vessel and sample stage. (C) Flow cell.

continuously agitated by bubbling of nitrogen gas, and either the hydrogen ion activity or the metal ion activity was measured simultaneously with an ISE. A Rabbit (Woburn, MA) peristaltic pump was used in conjunction with a custom-built flow cell (Figure 1c), constructed of square quartz tubing (2 mm i.d.) to measure the reproducibility and response time of the optodes. The flow rate was 8 mL/min. In addition, a Wavetek function generator (San Diego, CA) was employed to initiate consecutive integrations by the CCD detector. Data Acquisition. Potentiometric signals were recorded with a Fisher pH meter and with a Macintosh IIcx computer with an NB-MIO-16X A/D I/O board (National Instruments, Austin, TX) and two high-impedance input, four-channel electrode interface modules (World Precison Instruments), controlled by LabView 2 software (National Instruments). An Ingold HA glass pH electrode (Mettler-Toledo Process Inc., Wilmington, MA) was used in the optode measurements. All optode measurements were made by titration with aqueous solutions of hydrochloric acid or the metal chloride salt, with 2 min allowed to reach steady state. The hydrogen ion activity was measured simultaneously with the optical signal. Calculations. Liquid junction potentials were corrected according to the Henderson formalism. Activity coefficients were calculated according to the two-parameter Debye-Huckel formalism of Meier.32 RESULTS AND DISCUSSION Principle of Operation. A complete description of the optode response mechanisms used here can be found in the references of Simon and co-workers.15,16,18 The metal ion activity aI+ (eq 1) is a function of the hydrogen ion activity aH+ and a number of constants, including [Ltot], the total ionophore concentration, [Ctot], the total chromoionophore concentration, and [Rtot ], the total lipophilic charge site concentration. The parameter R has been

aI+ )

1

( )(

Kexch

)

RaH+ [Rtot] - (1 - R)[Ctot] 1 - R [L ] - {[R- ] - (1 - R)[C ]} tot tot tot

(1)

defined as the relative portion of unprotonated chromoionophore.18 Replacing the chromoionophore with another whose pKa differs (32) Meier, P. C. Anal. Chim. Acta 1982, 136, 363-368.

Figure 2. Sodium optode responses with three different optode configurations (see text): 0, approach II (ETH 5350); O, approach I (ETH 5294); and b, approach III (ETH 2439/DiIC18). The curves are theoretical predictions (see eq 2) of the optical response with no adjustable parameters. The vertical dashed lines delimit the typical activity ratios found in whole blood, whereas the solid vertical lines delimit the typical activity ratios found in intracellular media.

directly changes the overall membrane exchange constant. The effect is to shift both the range of sensitivity and the detection limit while retaining the selectivity. This is elegantly shown the work of Lerchi et al.23 Equation 1 was used here to help predict which chromoionophore would yield an optical sensor with a dynamic range covering the range of values typically found in intracellular environments, with the added requirements of operating at physiological pH and exploring fluorescence-based ratiometric optodes. Optode Development. Ionophore Selection. Poly(vinyl chloride) membrane optodes for sodium and potassium were constructed by employing many commercially available ionophores, including valinomycin, BME-44, and a tert-butylated calix[4]arene tetramethyl ester, which have been previously used in the construction of both ion-selective electrodes33 and bulk optodes.28,34,35 Valinomycin proved to be far too polar an ionophore and was rapidly leached from the membrane phase into the aqueous solution.29 The optodes prepared by us with BME-44 had lifetimes longer than those of the valinomycin-based optodes. A description of a fluorescent BME-44-based potassium optode is the subject of another article.36 The tert-butylated calix[4]arene tetramethyl ester ionophore proved to be quite stable in the membrane phase, and the response was reproducible. Unfortunately, the tert-butylated calix[4]arene tetramethyl ester lacks sufficient selectivity against potassium to perform intracellular measurements. Only the 1,3-bridged calix[4]crown ionophore satisfied both the stability and sodium-selectivity requirements. Optimization of the Optical Signal. An important function of the chromoionophore is its ability to vary, in a controlled fashion, the detection limit and the dynamic range of the optode. Figure 2 is a plot of the response functions of the three optodes developed for use here, each with the 1,3-bridged calix[4]crown derivative but with a different chromoionophore. These shifts are predicted from theory and demonstrate the versatility of such optodes as (33) Umezawa, Y. CRC Hardbook of Ion-Selective Electrodes: Selectivity Coefficients; CRC Press: Boca Raton, FL, 1990. (34) Lindner, E.; To´th, K.; Jeney, J.; Horvath, M.; Pungor, E.; Bitter, I.; Agai, B.; Toke, L. Microchim. Acta 1990, 1, 157-168. (35) Cunningham, K.; Svehla, G.; Harris, S. J.; McKervey, M. A. Analyst 1993, 118, 341-345. (36) Shortreed, M. R.; Dourado, S.; Kopelman, R. Sens. Actuators B, in press.

sensors (Figure 2). These results are discussed in greater detail below. It is important to point out that these detection limit and dynamic range extension mechanisms are unique to ion-selective optodes. It is also important to note the codependence of the response on pH. Hydrogen ion activity measurements must be carried out in buffered solutions, or the pH must be simultaneously monitored in order to achieve accurate determinations. The challenge lies not only in adapting each chromoionophore for use in ratiometric fluorescence-based measurements, but also in deriving suitable mathematical relationships so that the fluorescent signal of the optical sensor can be plotted in a conventional manner. The degree of protonation of the chromoionophore, (1 - R), as measured by absorbance, is a commonly used representation of the optode response. The purpose of the three following mathematical relationships is to provide a common platform whereby sensors with different response mechanisms can be directly compared. The indicators chosen for this study were originally intended to be used in absorbance-based measurements, without consideration of their fluorescence signals. Three Nile Blue derivatives26 were chosen for use in this work: ETH 5350, ETH 5294, and ETH 2439. The chromoionophore ETH 5350 displays a significant emission peak shift in its fluorescence spectrum as the pH is altered. The chromoionophore ETH 5294 also shows such a shift, though a portion of its fluorescence spectrum remains invariant. Fluorescence spectra of both chromoionophores are thus amenable to ratiometric measurements, which compensate for both photobleaching and excitation power fluctuations. For the purpose of demonstration, the ETH 5294 response is described using intensity only (approach I below), and the ETH 5350 response is described using intensity ratios (approach II below), though either system is adequate for either dye. Finally, the inner-filter effect (approach III below) was used with the chromoionophore ETH 2439 and an unreactive fluorescer, DiIC18. The theoretical response is derived below, showing that (1 - R), the degree of protonation of the chromoionophore, is precisely and consistently defined for all three systems. The fluorescence spectra of the fully protonated and deprotonated membranes are shown in Figure 3. All three systems proved to have sufficient fluorescence quantum efficiency and photostability for both the macro- and microsensors, with judicious choices of excitation wavelength, excitation power (∼10-6-10-3 W), and illumination time (0.2 s). Approach I: Development of a Simple Fluorescence IntensityBased Response Mechanism (ETH 5294). Perhaps the most common representation of optical sensor response is the fluorescence intensity at a single emission wavelength. According to Skoog and Leary,37 at low concentrations (∼0.05 M), the fluorescent radiation from the system is proportional to the concentration C of the fluorescer (eq 2) and therefore is proportional to the overall absorbance at constant excitation power, where F is the fluorescence and A is the absorbance. The term k includes the

F ) k′A ) kC

(2)

molar absorptivity, the path length, the quantum efficiency, and the excitation power. This relationship can be applied to formulate a new expression for (1 - R), the degree of protonation of a chromoionophore. The term R can be determined experimentally (37) Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis, 4th ed.; Saunders College Publishing: Fort Worth, TX, 1992.

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Figure 3. Fluorescence spectra of the three optical systems under the conditions of complete protonation and deprotonation: (A) approach I (ETH 5294), (B) approach II (ETH 5350), and (C) approach III (ETH 2439/DiIC18). O, Chromoionophore in the basic form; b, chromoionophore in the acidic form.

(eq 3) by measurement of the absorbance of the system compared with the absorbances of the totally protonated indicator, AP, and the totally deprotonated indicator, AD. Our new expression for R

R)

AP - A [C] ) [Ctot] AP - AD

(3)

(eq 4) is formulated in terms of the fluorescence of the chromoionophore when it is fully protonated, FP, when it is fully deprotonated, FD, and when it is in some intermediate state, F.

R)

FP - F FP - FD

(4)

Approach II: Development of the Fluorescence Intensity Ratio (ETH 5350). When dealing with an indicator whose fluorescence emission maximum (λmax) changes wavelength upon protonation or deprotonation, we wish to formulate an expression for R that will allow the experimentalist to compensate for fluctuations in excitation power, collection geometry, and photobleaching. Starting with the fluorescence intensity of the protonated chromoionophore FP (λmax1) and the deprotonated chromoionophore FD (λmax2) gives eq 5. The constant K can be evaluated under the intermedi-

FP kPCP kP(1 - R)[Ctot] (1 - R) ) ) )K FD kDCD R kDR[Ctot] 2660

Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

(5)

ate condition where the concentrations of protonated and unprotonated indicator are equal; that is, where R ) 0.5. In this article, the contribution of the basal level of fluorescence is eliminated from the ratio prior to plotting. Approach III: Development of the Inner-filter Effect (ETH 2439/ DiIC18). Finally, it is important to formulate an expression for (1 - R) that applies to systems employing energy transfer or innerfilter effects. The use of energy transfer and inner-filter effects in chemical sensors is well documented,1,22,38-41 though, until now, they have not been adapted for use in thermodynamically describable optode membrane systems. The inner-filter effect involves modulation of a fluorescing species by a second absorber which is chemically different than the fluorescer. This technique requires good overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. The necessary concentration of acceptor for typical systems is in the low millimolar range.39-41 There are no lower limits on the concentration of the emitter (donor); however, at high emitter concentrations, selfabsorption or quenching may occur. As it is used in this article, the fluorescence of an inert molecule, DiIC18, is modulated by the changing absorbance of a pH-sensitive indicator (chromoionophore). The basic form of the chromoionophore ETH 2439 is highly absorbing in the region where DiIC18 is fluorescent, but the acidic form is not. As the chromoionophore gets deprotonated in response to changing sodium activities, the absorbance of the basic form of ETH 2439 increases dramatically. Since the absorbance spectrum of the basic form of the chromoionophore has excellent overlap with the emission spectrum of DiIC18, the emission of the DiIC18 decreases concomitantly. The concentration of absorber used in these measurements was ∼15 mM, which is quite sufficient and should compensate at least partially for the limited thickness of the membrane. In such a small volume, near-field or direct, shortrange energy transfer effects may also play a role.42 Care must always be taken in choosing appropriate emitter/acceptor pairs, as differing rates of photobleaching and membrane solubility may introduce artifacts into the measurement. In addition, the basic form of the chromoionophore is also fluorescent, so that as the DiIC18 emission intensity decreases, the chromoionophore intensity increases. We are able to eliminate the excitation power and collection geometry dependence of the signal by taking the ratio of the emission intensities of the two fluorescent species. To formulate an expression for (1 - R), we begin with a ratio of the standard equations of fluorescence for the protonated chromoionophore, FP, and the inner-filter, FI, which is modulated by the absorbance of the deprotonated form of the indicator. The fluorescence from both species is measured simultaneously with the aid of an array detector; therefore, the ratio (eq 6) clearly has no excitation power dependence. Rewriting

FP kPCP ) FI k C e-DCD

(6)

I I

eq 6 in terms of the degree of protonation of the chromoionophore (38) Jordan, D. M.; Walt, D. R.; Milanovich, F. P. Anal. Chem. 1987, 59, 437439. (39) Yuan, P.; Walt, D. R. Anal. Chem. 1987, 59, 2391-2394. (40) Gabor, G.; Chadha, S.; Walt, D. R. Anal. Chim. Acta 1995, 313, 131-137. (41) Gabor, G.; Walt, D. R. Anal. Chem. 1991, 63, 793-796. (42) Kopelman, R.; Lieberman, K.; Lewis, A.; Tan, W. J. Lumin. 1991, 48, 871875.

Table 1. Selectivity Values of the Optodes Tested in Comparison with the Electrode and the Necessary Selectivities for Measurement of Sodium in Intracellular Media pot log KNaJ

ISE J

required8

FIM13

SSM

buffered

optode

-3.5 -1.4 -3.1

-5.0 -4.4 -4.5

-3.8 -3.8 -5.1