Ion-Partitioning Membrane-Based Electrochemical Sensors

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Anal. Chem. 2000, 72, 1835-1842

Ion-Partitioning Membrane-Based Electrochemical Sensors Elizabeth A. Moschou and Nikolas A. Chaniotakis*

Laboratory of Analytical Chemistry, Department of Chemistry, University of Crete, 71 409 Iraklion, Crete, Greece

The application of ion-partitioning membranes on proton transducers for the development of electrochemical sensors is presented. The ion-partitioning membrane incorporates two different ionophores, one selective to protons and the other to analyte cations, as well as the necessary anionic lipophilic sites. As dictated by the electroneutrality principle, when the concentration of the analyte cations in the sample increases, the analyte cations are extracted into the membrane, displacing protons of equal charge out of the membrane. The pH-sensitive gate of a CHEMFET or the surface of a glass pH electrode is used as an internal transducer for the monitoring of the membrane proton flux. The resulting signal of the pH transducer is related to the concentration of the analyte cation present in the sample. The experiments presented here indicate that the observed CHEMFET’s signal is affected by the interaction of the pH-sensitive sensor element with protons released by the polymeric membrane. Polymeric membranes incorporating ion-selective carriers have been extensively used for the construction of ion-selective electrodes (ISEs), ion-selective field effect transistors (ISFETs), and optodes. The transduction mechanisms of the sensors based on these doped polymeric membranes can be either the potential difference generated at the membrane/aqueous solution interface or the changes of its optical properties. In the case of ISFETs, the membrane is used as the chemical recognition element, while transduction is obtained using a transistor. Since the introduction by Bergveld1 in 1970 of ion-selective field effect transistors, many theoretical and experimental studies have described the performance of these chemically sensitive electronic devices.2-7 When an electroactive membrane, solid or polymeric, is deposited on the top of a native gate oxide, a chemically modified field effect transistor (CHEMFET) is produced with selectivity depending on the electroactive membrane used. The deposition of pH-sensitive * Corresponding author. E-mail: [email protected]. Fax: +30-81-393601. (1) Bergveld, P. IEEE Trans. Biomed. Eng. 1970, 17, 70-71. (2) Janata, J.; Huber, R. J. Ion-Sel. Electrode Rev. 1979, 1, 31-79. (3) Sibbald, A. IEE Proc. 1983, 130, 233-44. (4) Van Den Berg, A.; Bergveld, P.; Reinhoudt, D. N.; Sudho ¨lter, E. J. R. Sens. Actuators 1985, 8, 129-148. (5) Van Hal, R. E. G.; Eijkel, J. C. T.; Bergveld, P. Adv. Colloid Interface Sci. 1996, 69, 31-62. (6) Van Hal, R. E. G.; Eijkel, J. C. T.; Bergveld, P. Sens. Actuators, B 1995, 24-25, 201-205. (7) Ho ¨gg, G.; Lutze, O.; Cammann, K. Anal. Chim. Acta 1996, 335, 103-109. 10.1021/ac991313j CCC: $19.00 Published on Web 03/10/2000

© 2000 American Chemical Society

metal oxides,8,9 such as Al2O3, SiO2, or Ta2O5, results in the construction of pH-ISFETs. The commonly accepted model to describe the sensitivity of a pH-ISFET as a proton transducer is the site-dissociation model.4-6 pH-ISFETs are the most common proton transducers that are being covered with polymeric membranes. The selectivity of a resulting CHEMFET depends on the ionophore incorporated into the polymeric membrane.1,2,5,10-15 The origin of the CHEMFET’s signal is the potential difference arising at the membrane/aqueous solution interface due to the complexation of the ion carrier with the analyte ion at the outer-phase boundary of the membrane. No significant changes are reported to occur at the inner membrane boundary and the internal interface between the polymeric membrane and the FET’s gate. Thus the pH-sensitive FET’s gate does not participate in the mechanism of the signal generation of the CHEMFETs presented so far.2,16 On the other hand, it has been reported that polymeric membranes are generally permeable to neutral species, such as NH3, CO2, and organic molecules, including weak acids and bases. These species can permeate the polymeric membrane and interact with the FET’s gate, affecting the CHEMFET’s signal.17-21 This was the first report of an FET gate interaction with species partitioned within the polymeric membrane, although considered as interferences to the CHEMFET’s potentiometric response. In some cases,19-21 a negative interference from carbon dioxide has been presented, while, with similar systems, a positive potential (8) Abe, H.; Esashi, M.; Matsuo, T. IEEE Trans. Electron Devices 1979, ED26, 1939-1344. (9) Akiyama, T.; Ujihira, Y.; Okabe, Y.; Sugano, T.; Niki, E. IEEE Trans. Electron Devices 1982, ED-29, 1936-1941. (10) Van der Wal, P. D.; van den Berg, A.; de Rooij, N. F. Sens. Actuators, B 1994, 18-19, 200-207. (11) Cobben, P. L. H. M.; Egberink, R. J. M.; Bomer, J. G.; Bergveld, P.; Verboom, W.; Reinhoudt, D. N. J. Am. Chem. Soc. 1992, 114, 10573-10582. (12) Stauthamer, W. P. R. V.; Engbersen, J. F. J.; Verboom, W.; Reinhoudt, D. N. Sens. Actuators, B 1994, 17, 197-201. (13) Jime´nez, C.; Marque´s, I.; Bartoli, J. Anal. Chem. 1996, 68, 3801-3807. (14) Lugtenberg, R. J. W.; Egberink, R. J. M.; van den Berg, A.; Egbersen, J. F. J.; Reinhoudt, D. N. J. Electroanal. Chem. Interfacial Electrochem. 1998, 452, 69-86. (15) Barbaro, A.; Colapicchioni, C.; Davini, E.; Mazzamurro, G.; Piotto, A.; Porcelli, F. Adv. Mater. 1992, 6, 402-408. (16) Moss, S. D.; Janata, J.; Johnson, C. C. Anal. Chem. 1975, 47, 2238-2243. (17) Li, X.; Verpoorte, E. M. J.; Harrison, D. J. Anal. Chem. 1988, 60, 493-498. (18) Shaw, J. Sens. Actuators, B 1993, 15-16, 81-85. (19) Van den Vlekkert, H.; Francis, C.; Grisel, A.; de Rooij, N. Analyst 1988, 113, 1029-1033. (20) Haak, J. R.; van der Wal, P. D.; Reinhoudt, D. N. Sens. Actuators, B 1992, 8, 211-219. (21) Fogt, E. J.; Untereker, D. F.; Norenberg, M. S.; Meyerhoff, M. E. Anal. Chem. 1985, 57, 1998-2002.

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response to acidic species has been shown.17,18 Furthermore, a CHEMFET constructed by the application of a positively charged membrane with adsorbed lysozyme was introduced.22 The CHEMFET presented a transient potential response caused by an intramembrane pH change (a short-term proton release by the FET’s gate toward the inner membrane boundary) during an ion step in the sample solution. In this paper, an electrochemical sensing system based on ionpartitioning membranes doped with one proton- and one cationselective carrier (similar to those used for the construction of optodes23,24) and a proton transducer is presented. The signal of the CHEMFET is affected by the potential difference developed at the internal pH-sensing element. The proton transducer employed can be any pH-sensing solid-state sensor such as a pHISFET or a glass pH electrode. As dictated by the electroneutrality principle, when the concentration of the analyte cation in the sample increases, the analyte cations are extracted into the membrane, displacing protons of equal charge out of the membrane. In such cases, a pH-sensitive sensor can monitor the proton release, and the signal generated can be directly related to the analyte concentration present in the sample. It should also be pointed out that, in the case of multicharged cations partitioning in the membrane (such as calcium, magnesium, etc.), the amount of protons escaping the membrane per cation partitioning will be larger, thus exhibiting higher sensitivities. Here, the experimental results supporting the proposed response mechanism of the electrochemical sensing system are presented. The proton transducer employed is a pH-ISFET owing to its simple membrane coating and adhesion over the FET’s gate. A glass pH electrode is also used for comparison. The ionpartitioning membranes used for the construction of the CHEMFETs incorporate valinomycin as the potassium-selective carrier. Initially, the effect of basic gaseous NH3 on the sensor signals is investigated. Studies comparing the response of a sensor having a direct deposition of the membrane over the sensor element to that of a sensor with a dextran layer between are undertaken. Such experiments can provide direct evidence of the interactions taking place at the internal interfaces of the sensor covered with an ion-partitioning membrane. The information obtained from the experiments involving the pH-sensitive sensors (the pH-ISFET and glass pH electrode) covered with ion-partitioning membranes of varying thicknesses and also the optical pH measurements performed can lead to the development of a valid model able to explain the observed signal signs, stabilities, and sensitivities. EXPERIMENTAL SECTION Reagents and Membranes. All reagents used were of puriss p.a. grade and were obtained from Fluka, while for all solution preparations deionized water (Barnstead NAN-O-Pure) was used. All the polymeric membrane components were Selectophores and were obtained from Fluka. The proton carriers used were ETH 5294, 9-(diethylamino)-5-(octadecanoylimino)-5H-benzo[a]phenoxazine, and ETH 1907, 4-(nonadecyl)pyridine. Valinomycin was incorporated as the K+ carrier, and potassium tetrakis(4-chlorophenyl)borate served as the anionic additive. Bis(2-ethylhexyl) (22) Eijkel, J. Ph.D. Thesis, University of Twente, Enschede, The Netherlands, 1995; ISBN 90-9008615-3. (23) Seiler, K. Ph.D. Thesis, ETH, Zu ¨ rich, 1990; No. 9221. (24) Bakker, E.; Bu ¨ hlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083-3132.

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Table 1. Compositions of the Ion-Partitioning Membrane Cocktails Used

membrane

H+ carrier

concn, mmol/kg H+ K+ carrier carrierc

I II III IV V VI VII VIII IX

ETH 5294 ETH 5294 ETH 5294 ETH 1907 ETH 1907 ETH 1907 ETH 1907 3-nitropa phenolb

26.03 6.10 4.62 23.15 22.57 21.70 20.53 111.4 63.77

9.08 9.63 9.36 2.16 10.71 19.80 63.75 90.34 58.76

molar ratiod

concn of additive,e mol/kg

0.35 1.58 2.00 0.09 0.48 0.93 3.10 0.81 0.92

9.26 9.89 10.28 11.09 10.68 11.09 10.47 128.2 66.92

a 3-Nitrophenol. b Phenolphthalein. c Valinomycin. d K+ carrier:H+ carrier. e Potassium tetrakis(4-chlorophenyl)borate.

sebacate (DOS) was the plasticizer and high molecular weight poly(vinyl chloride) (PVC) was the polymeric matrix used in all ion-partitioning membranes (2:1 by weight, respectively). The specific membrane compositions are presented in Table 1. The dextran solution used for the construction of the CHEMFET/ dextran sensor was prepared by the dilution of 10 mg of ((diethylamino)ethyl)dextran chloride (Sigma, D-9885) in 0.5 mL of 0.1 M KCl solution buffered with Tris-HCl to pH 7.4. Sensor Construction. The ISEs were prepared as described previously.25 A 0.01 M KCl solution buffered to pH 7.5 with Tris (tris(hydroxymethyl)aminomethane)-HCl served as the internal filling solution. The ISE electrodes were allowed to soak overnight in a solution of composition identical to that of the internal filling solution. Aluminum oxide pH-ISFETs (with internal Ag/AgCl reference electrodes) were obtained from Orion Research Inc. (Catalog No. 615700). CHEMFETs were prepared by casting 8 µL portions of the ion-partitioning membrane cocktail onto the pH-ISFET gates and were allowed to dry at room temperature for at least 20 min. The estimated membrane thicknesses were less than 10 µm. For the construction of a CHEMFET incorporating a dextran layer, a small amount of the dextran solution was cast over the top of the pH-ISFET’s gate. After 2 h, 8 µL of the blank membrane cocktail containing 66 wt % DOS and 34 wt % PVC was deposited over the dextran layer, and the ion-partitioning membrane was subsequently cast on top. The blank membrane was deposited between the dextran and the ion-partitioning membrane to postpone ion (H+ or K+) transport from the dextran layer toward the ionpartitioning membrane during the experiment. The CHEMFETs with and without dextran layers were left to soak in the testing solution for 30 min. For the pH-response studies, the solutions used were 10-4-10-1 M KCl with initial low pH values adjusted with 1 M HCl. The pH was titrated with either 1 M Tris or 2.5 M NaOH from low to high pH values. The calibration curves were established by standard additions of 1 M KCl to 10-5 M KCl solutions buffered with either Tris-HCl to pH 8.1 or citric acidTris to pH 5.1. Electrochemical Measurements. Potentiometric measurements of the ISEs were performed using an Ag/AgCl double(25) Chaniotakis, N. A.; Chasser, A. M.; Meyerhoff, M. E.; Groves, J. T. Anal. Chem. 1988, 60, 185-188.

Figure 1. Performances of the K+-selective CHEMFETs covered with membranes I-III (A-C, respectively) at different H+ and K+ concentrations [(9) 10-4 M, (b) 10-3 M, (2) 10-2 M, and (1) 10-1 M KCl].

junction reference electrode (Orion Research Inc., 900200). The CHEMFET’s signal was transduced to potential changes by a model 605 preamplifier (Orion Research Inc.). The glass bulb of a pH triode (Orion Research Inc., 9107 BN) was used as the matrix that was covered with the ion-partitioning membrane. An Orion ROSS pH electrode was used to monitor the sample pH during all experiments while the laboratory temperature was set to 20 ( 0.5 °C. The data were collected using a personal computer with software written in Basic. Simultaneous Optical and Potentiometric Measurements. For simultaneous monitoring of the optical and potentiometric responses, circular disk membranes VIII, IX, and VI with 0.66 cm diameters and 100 µm thicknesses were mounted in series on an optical glass slide. The same series of thick (100 µm) membranes prepared from the same membrane cocktails were cast over the tops of a CHEMFET and a CHEMFET/dextran sensor. After the membranes were stacked on the tops of the sensors and on the glass slide, they were allowed to soak in a 10-2 M KCl solution, pH 7.8, for 30 min. Finally, they were all immersed in the same stirred KCl-containing solution where the concentrations of both protons and potassium ions were changed to the desired values. The absorbance changes of the chromoionophores were monitored at a wavelength 435 nm for the 3-nitrophenol chromoionophore and at 476 nm for the phenolphthalein chromoionophore using a fiber-optic spectrometer (Ocean Optics Inc., SD 1000) run with SpectraScope version 2.55 (1996) software. A blank membrane with no chromoionophore added served as a reference. Simultaneously with the optical measurements, the potential responses of the electrodes were monitored using a personal computer. RESULTS AND DISCUSSION The experiments presented in this paper were designed to provide the required information for the elucidation of the response mechanism of a solid-state pH-sensor covered with an ion-partitioning membrane. Initially, the effect of the composition of the ion-partitioning membrane on the CHEMFET’s signal was investigated. For the membrane construction, the potassium ionophore valinomycin and the highly selective proton carrier ETH 5294 were used as initial carrier models. Three different ion-partitioning membranes (I-

III) containing different molar ratios of the two ionophores (Table 1) were applied on pH-ISFET gates for the construction of CHEMFETs. The performances of all three CHEMFETs with varying potassium and the proton concentrations are presented in Figure 1. As can be seen from Figure 1B, the CHEMFET covered with membrane II can respond to both protons and potassium ions, with sensitivity depending on the concentrations of the ions present in the sample. In the acidic region, the CHEMFET responds with greater sensitivity to protons than to potassium ions, while, in the basic region, the sensor exhibits higher sensitivity to potassium at the expense of proton sensitivity. In the regions between, the sensor’s signal is governed by the concentrations of both protons and potassium ions present in the sample. When the concentration of the proton carrier ETH 5294 is increased within the ion-partitioning membrane (membrane I), the range of the proton response is increased while the range of the potassium response is decreased (Figure 1A). In contrast, when the concentration of the proton carrier is dramatically decreased (membrane III), the range of the potassium response of the CHEMFET is increased enormously at the expense of the range of the proton response (Figure 1C). Furthermore, the potassium response of the CHEMFET covered with membrane III is due to the high concentration of valinomycin with respect to the H+ carrier and not to the anionic sites used, since the sensor does not respond to high concentrations of other alkali metals such as sodium. It can thus be concluded that the performance of a CHEMFET covered with an ion-partitioning membrane is dependent on the composition of the ion-partitioning membrane used as well as the relative concentration of the hydrogen and the potassium ions present in the sample. Furthermore, as expected, the performance of the CHEMFET is affected by the stability constants of the ion carriers used. From experimental results, it has been observed that when chromoionophores with higher stability constants (for example 107.7 vs 106.1) are used, the pH response of the CHEMFET is shifted toward lower pH values (1.96 pH units for above example) when all other membrane component and cation concentrations are kept constant. Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

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Figure 2. Potential responses of a CHEMFET and an ISE electrode (A and B, respectively) incorporating membranes IV (+), V (O), VI (×) and VII (-) to changes in the proton concentration of a 10-4 M KCl sample solution.

A series of experiments were then performed to prove that the observed signal of the sensor does not arise from the potential difference generated at the membrane/solution interface.26 Initially, possible signal generation at the pH-ISFET’s gate by the interaction with protons exiting the membrane was investigated. Such a signal can be generated solely by the hydrogen ion flux to or from the pH-sensitive ISFET’s gate. For this reason, two different sensors were constructed. The first one was produced by the application of the ion-partitioning membrane VI over the bare FET’s gate, and the second, by the application of a pacifying dextran layer between membrane VI and the gate (CHEMFET/ dextran sensor). By the use of such a buffering layer of the amino derivative, the pH-sensitive FET’s gate is always in contact with a medium of constant pH. No pH response is thus expected due to any hydrogen flux to or from the internal membrane interface, as the pH-sensitive FET’s gate is being “inactivated”. According to Van den Vlekkert et al.,19 when such a passive layer is applied between the FET’s gate and the polymeric membrane, the CHEMFET’s response is dictated only by the potential difference arising at the polymeric membrane/aqueous solution interface. Possible signal generation by the interaction of the FET’s gate with the protons exiting the membrane was investigated by the addition of gaseous ammonia to the sample solution. The addition of NH4Cl in a 10-2 M KCl solution adjusted to pH 12.7 using NaOH resulted in the formation of gaseous NH3 in the sample. While the effect of the addition of gaseous ammonia to the sample on the signal of the CHEMFET without the pH-buffered layer is large (-250 mV), this is not the case when the pH-buffered dextran layer is used (Figure 1S, Supporting Information). The CHEMFET/dextran sensor presented no potential response during the addition of 10-2 M NH3 to the sample solution. Thus, the CHEMFET’s pH-sensitive gate can interact with species diffusing into the ion-partitioning membrane, affecting the CHEMFET’s signal. To investigate whether a CHEMFET’s signal is due to the potential difference developed at the membrane/aqueous solution interface, CHEMFETs and conventional ion-selective electrodes were constructed using ion-partitioning membranes of identical composition. The proton carrier ETH 1907 and the potassium carrier valinomycin were the two competitive ionophores used (26) Bakker, E.; Pretsch, E. Anal. Chem. 1998, 70, 295-302.

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for the construction of the ion-partitioning membranes. The compositions of membranes IV-VII used for this set of experiments are presented in Table 1. Membrane IV contains the lowest molar ratio of potassium carrier to hydrogen carrier (0.09), while membranes V-VII contain gradually increasing molar ratios of the potassium ionophore to the proton ionophore. In Figure 2 are shown the pH responses of the CHEMFETs and the ISEs based on membranes IV-VII. From Figure 2A it can be seen that all four CHEMFETs show a large negative potential response to the increase of the sample pH. On the other hand, the ISEs based on membranes V-VII do not respond significantly to the pH changes of the sample solution (Figure 2B). The membrane IV-based ISE is the only ISE electrode that does respond to the sample pH changes in the range 3.3-8.9, while, for the pH range 8.9-12.4, the sensor does not respond to protons because of sodium interference. In contrast, the membrane IV-based CHEMFET responds to pH in the range 3.3-12.4, and thus it shows no alkali metal interference. It is therefore evident that the signal obtained by the CHEMFET does not primarily originate from the potential difference developed at the membrane/aqueous solution interface. Additionally, the magnitude of the observed pH response indicates that the signal of the CHEMFET arises from the interactions of the pH-sensitive FET’s gate with the hydrogen ions partitioned within the polymeric membrane. Subsequently, the response of the above sensors toward the potassium ions was examined. Solutions of pH 5.1 and 8.1 were used for the additions of potassium ions as shown in Figure 3A,B. The CHEMFETs present a transient positive potential response to the increase of the potassium ion concentration in the sample with increasing intensity as the concentration of valinomycin in the ion-partitioning membrane increases. This transient potential response of the CHEMFETs may be explained by the initial response, which is fast for the potassium ions at the outer interface, followed by a slower pH response at the inner interface of the sensor. Furthermore, the overall response of the CHEMFETs to potassium ions at steady state increases with an increasing valinomycin to ETH 1907 molar ratio. Additionally, this overall response at steady-state becomes larger as the sample pH increases from 5.1 to 8.1. Compared to the CHEMFETs, the membrane IV- to VII-based ISEs show a fast and stable response to the potassium ions with

Figure 3. Potential responses to the potassium ion concentration for (A, B) CHEMFETs (A, pH 5.1; B, pH 8.1) and (C, D) ISE electrodes (C, pH 5.1; D, pH 8.1) covered with membranes IV (+), V (O), VI (×) and VII (-).

theoretical sensitivity (Figure 3C,D for the samples at pH 5.1 and 8.1). The potential response of the ISEs is expected to be fast and stable with theoretical sensitivity since the origin of an ISE signal is the potential difference generated at the membrane/ aqueous solution interface, which reaches steady state very fast. The next set of experiments was designed to investigate whether there is a critical transport process that determines a CHEMFET’s behavior. Four different CHEMFETs were constructed by the application of the thick (100 µm) membranes IVVII. The potassium response of the CHEMFETs at a sample pH of 5.1 was recorded. A slowly increasing response (0.1 up to 0.3 mV/min) for more than 75 min after each increase of the analyte ion concentration in the sample was observed (Figure 2S, Supporting Information). This slowly increasing potential response of the CHEMFETs indicates that the origin of a CHEMFET’s signal is a time-dependent transport process. The mean sensitivity of the membrane V- to VII-based CHEMFETs is 63 mV/decade (vs 50 mV/decade sensitivity to potassium ions for the conventional ISEs, Figure 3C). It is also worth mentioning that the membrane IV-based CHEMFET presents a positive potential response after the addition of 0.1 M potassium ions, while the ISE counterpart shows negligible potassium response under identical conditions. It is thus concluded that the origin of a CHEMFET’s signal is due not only to the potential difference arising at the ionpartitioning membrane/aqueous solution interface, which is responsible for the short-term response of the sensor, but also to the potential difference generated by the interaction of the pHISFET’s gate with the protons partitioned within the polymeric

membrane, which dominates the long-term response of the CHEMFET. It has been reported27 that the time required for a polymeric membrane to reach the steady state when transport processes occur depends on the thickness of the polymeric membrane used. To verify more clearly that the origin of a CHEMFET’s signal is a time-dependent transport process, simultaneous optical and electrochemical measurements were performed on thick ionpartitioning membranes containing proton chromoionophores. Membranes VIII and IX contain chromoionophores with different absorbance maxima, allowing for the simultaneous measurements of the uncomplexed forms of the two indicators. Membrane VI was used to prevent leaching of the chromoionophores out of the first two membranes. Membrane VIII contains the pH indicator 3-nitrophenol as the H+ carrier and was the first layer applied on the pH-ISFET’s gate for the construction of a CHEMFET. Membrane IX, which contains the pH indicator phenolphthalein, was applied over membrane VIII. Finally, membrane VI was placed over membrane IX for protection. The thickness of each membrane was on the order of 100 µm. The layout of the CHEMFET constructed is the following:

pH-ISFET’s gate/membrane VIII/membrane IX/ membrane VI

A CHEMFET/dextran sensor and a glass plate incorporating the (27) Schneider, B.; Zwickl, T.; Federer, B.; Pretsch, E.; Lindner, E. Anal. Chem. 1996, 68, 4342-4350.

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Figure 4. Potential responses of a CHEMFET and a CHEMFET/ dextran sensor covered with the thick (100 µm) membranes VIII, IX, and VI in series during the protonation and the deprotonation of the proton carriers: (A) 10-2 M KCl (pH 7.8) to 10-5 M KCl (pH 5.0); (B) 10-2 M KCl (pH 5.0); (C) 10-1 M KCl (pH 5.0).

same series of 100 µm thick membranes were also constructed. All three systems, the CHEMFET, the CHEMFET/dextran sensor, and the glass plate, were immersed in the same test solution. The potentiometric responses of the two CHEMFETs as well as the absorbance changes of the two pH-indicators in the membranes mounted on the glass plate were monitored simultaneously. After soaking in a 10-2 M KCl, pH 7.8 solution for 1.5 h, all three systems were immersed in a 10-5 M KCl, pH 5.0 solution for protonation, and after a short equilibration time, KCl was added up to the final concentration 10-2 M KCl, pH 5.0 (deprotonation). The time-dependent absorbance changes of the two membranes during protonation and deprotonation were monitored optically. After an initial equilibration time (3-10 min), there is a continuous change in the absorbance of both membranes (Figure 3S, Supporting Information). The absorbance change of the inner, 3-nitrophenol-based, membrane during protonation was -0.0069 ( 0.0006 min-1 while that of the outer, phenolphthalein-based, membrane was -0.0032 ( 0.0005 min-1 for the period of at least 25 min. Analogous gradual absorbance changes of both membranes were monitored during deprotonation. For the inner membrane, the rate of absorbance increase of the deprotonated form of the chromoionophore was 0.216 ( 0.02 min-1 while that of the outer membrane was 0.14 ( 0.02 min-1 for the period of at least 25 min. It was evident that the absorbance changes occurring were gradual, indicating a transport dependency. From the optical measurements performed, it can be concluded that during the protonation there is a slow proton transport from the aqueous solution through membrane IX to the inner membrane VIII, while during the addition of potassium ions to the sample gradual deprotonation of both chromoionophores in both membranes takes place. The effect of the addition of potassium ions to the sample on the sensors’ signals is shown in Figure 4. The CHEMFET/dextran sensor presents a fast and stable signal, indicating the generation of the potential difference at the membrane/aqueous solution interface. On the other hand, more than 20 min is required for the dextran-free CHEMFET to produce a steady signal. The response behavior of the CHEMFET is similar to the one observed in the optical partitioning experiment presented above. Addition1840 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

ally, the overall response of the CHEMFET is much larger than that observed for the CHEMFET/dextran sensor (41.6 mV compared to 19.4 mV, respectively, for the change of the potassium ion concentration from 10-5 to 10-2 M). These results indicate that the slowly increasing large potential response of the CHEMFET is based not only on the potential difference arising at the membrane/aqueous solution interface but also on the response generated by the interaction of the FET’s gate with the species transported to the internal membrane boundary. Assuming that the electroneutrality principle holds within the bulk of the membrane, the extraction of potassium ions into the membrane phase must be accompanied by the release of protons of equal charge out of the membrane. Any extraction of potassium ions into the ion-partitioning membrane will thus initiate a slow proton transport toward the membrane’s boundaries. In the case of the dextran-free CHEMFET system, the protons displaced by the potassium ions that reach the internal interface between membrane VIII and the FET’s gate can be measured by the pHsensitive FET’s gate, resulting in a slow positive response. The same response should also be observed in the case of any pHsensitive sensor used as the internal pH-sensing element, such as a glass pH electrode. To verify the measurement of the proton flux to the membrane’s internal boundary by any pH-sensitive transducer, ionpartitioning membranes were mounted on a glass pH electrode’s surface. The thick, 100 µm, ion-partitioning membranes VIII/IX/ VI were applied on a glass pH electrode and a CHEMFET, as well as on a CHEMFET/dextran sensor for comparison. The responses of all three systems were monitored simultaneously as the potassium ion concentration increased in a pH 5.0 buffered aqueous solution. While the CHEMFET/dextran sensor does not respond to the increase of the potassium ion concentration (to 10-4 and 10-3 M), the CHEMFET and the pH electrode present a slow positive response. Both the pH-ISFET and the glass pH electrode are capable of measuring the protons being gradually displaced by the K+ ions and diffusing through the ion-partitioning membranes toward the pH-sensitive electrode surfaces. As in the case of the CHEMFET, both the molar ratio of the two ionophores present in the membrane and the concentrations of the two competing cations in the sample solution should affect the response of the pH electrode covered with an ion-partitioning membrane. Ion-partitioning membranes V and VI with valinomycin:ETH 1907 molar ratios of 0.50 and 0.98, respectively, were used to evaluate the influence of the carrier and cation ratios on the sensors’ responses. Comparing the performance of the pH electrode to that of a CHEMFET covered with the same membranes, we conclude that both sensors can measure the proton flux to the internal membrane’s boundary. Increasing the sample pH increases the sensitivity to potassium ions while lowering the potassium ion concentration increases the sensitivity to protons for both sensors (Figure 4S, Supporting Information). Additionally, it is shown that an increase in the molar ratio of one of the ionophores (valinomycin in the case of membrane VI) increases the sensitivity of a sensor to the specific ion (potassium). The potassium sensitivities of the glass pH electrode, for example, are 32.5 ( 0.9 and 18.1 ( 0.8 mV/decade while those of the CHEMFET are 55 ( 1 and 45 ( 1 mV/decade for membranes VI and V, respectively, at a sample pH of 7.5. From the experimental

Figure 5. Schematic representation of the mechanism for the response of a CHEMFET covered with an ion-partitioning membrane to the protons and analyte cations: Mz+, analyte cation; L and MLz+, free and complexed ionophores; C and CH+, unprotonated and protonated carrier; R-, lipophilic anionic sites; y and n, molar ratios of protons exiting the membrane phase toward the sample and the aqueous layer, respectively, when potassium ions are partitioned in the membrane phase.

evidence presented so far, it can be concluded that the signal of a CHEMFET covered with an ion-partitioning membrane is due to the potential difference at the membrane/aqueous solution interface as well as the proton flux at the inner membrane’s boundary. It is well established that water diffuses through PVC membranes,28-32 forming thin aqueous layers between the membranes and the hydrated surfaces of a pH electrode and a pHISFET.21,33,34 The proton flux at the inner boundary of a membrane affects the proton activity of the thin interfacial aqueous layer between the membrane and the surface of the proton transducer. The pH-sensitive transducer, either a pH-ISFET or a glass pH electrode, monitors the pH changes occurring at the thin interfacial aqueous layer, producing the observed signal. In Figure 5 is shown the schematic diagram of the proposed response mechanism of a CHEMFET covered with an ionpartitioning membrane. When an ion-partitioning membrane is mounted on the surface of a pH-sensitive transducer, either a pHISFET or a glass pH electrode, the observed sensor’s signal is due to the hydrogen ions transported to and from the pH-sensing element. The proton transport can only take place by cation exchange (hydrogen exchanging with another cation), as is the case of the present study using ion-exchange membranes, or by ion cotransport (hydrogen ion cotransported with an anion). Any hydrogen flux in or out of the bulk membrane phase will result in a pH change of the surrounding environment of the sensor. This environment can be either the thin aqueous layer between the membrane and the surface of the proton transducer or the sample solution contacting the ion-partitioning membrane. Therefore, the hydrogen flux in or out of the bulk of the ion-partitioning membrane will affect the activity of the hydrogen ions at the thin (28) Chan, A. D. C.; Li, X.; Harrison, D. J. Anal. Chem. 1992, 64, 2512-2517. (29) Chan, A. D. C.; Harrison, D. J. Anal. Chem. 1993, 65, 32-36. (30) Li, X.; Petrovic, S.; Harrison, D. J. Anal. Chem. 1996, 68, 1717-1725. (31) Li, X.; Rothmaier, M.; Harrison, D. J. Anal. Chem. 1996, 68, 1726-1734. (32) Zwickl, T.; Schneider, B.; Lindner, E.; Sokalski, T.; Schaller, U.; Pretsch, E. Anal. Sci. 1998, 14, 57-61. (33) Sandifer, J. R. Anal. Chem. 1988, 60, 1553-1562. (34) Miyahara, Y.; Yamashita, K.; Ozawa, S.; Watanabe, Y. Anal. Chim. Acta 1996, 331, 85-95.

aqueous layer between the internal membrane boundary and the surface of the proton transducer. The pH-sensitive transducer detects the pH changes occurring at the thin aqueous layer resulting in the observed sensor’s signal. The pH changes of the thin aqueous layer and also the sign of the sensor’s signal are controlled by the direction of the hydrogen ion flux. Also presented in Figure 5 is the proposed mechanism for the response of the CHEMFET to an increase in the potassium ion concentration of the sample. Increasing the potassium ion concentration of the sample will result in the extraction of K+ ions into the ion-partitioning membrane and the release of an equal charge of protons out of the membrane. The hydrogen ions can migrate toward the membrane/sample interface or diffuse through the bulk to the inner membrane’s boundary and finally to the thin aqueous layer. The decrease of the aqueous layer’s pH is measured by the pH-sensitive FET’s gate developing the positive potential response of the CHEMFET covered with an ionpartitioning membrane. Furthermore, decrease of the sample pH results in a positive proton flux (from the solution toward the bulk of the membrane and finally to the thin aqueous layer), generating a positive response of the sensor. In addition, due to the electroneutrality principle, the decrease of the sample pH will result in a negative potassium flux (from the membrane bulk phase to the aqueous solution). Details of the theory and its fit to experimental data will be presented in a paper currently in preparation. CONCLUSIONS A pH-sensing element (pH-ISFET or glass electrode) covered with an ion-partitioning membrane that contains, besides lipophilic anionic sites, two different ionophores, one selective to protons and the other selective to analyte cations, is introduced. The origin of the sensor’s signal is not only the potential difference arising at the membrane/aqueous solution interface but also the interaction of the pH sensor element with protons at the internal membrane interface. As analyte cations are extracted into the membrane, the displacement of an equal charge of protons can take place toward both sides of the membrane. This displacement will affect the proton activity of the interface between the pHsensitive FET gate and the membrane, which is most probably a thin aqueous layer. According to the presented response mechanism, under strict experimental conditions, the observed sensor signal is a quantitative measurement of the concentration of analyte cations present in the sample, similar to the case of optodes. Furthermore, due to the direct measurement of the pH, which is translated as an indirect measurement of the analyte cations, the sensitivity of the presented sensors toward multiply charged analyte ions will be greater than that obtained for singly charged counterparts. ACKNOWLEDGMENT We thank Erno¨ Pretch for helpful discussions and Orion Research Inc. for providing the pH-ISFETs, the pH electrodes, and the fiber-optic system. This work was supported by the Greek General Secretariat of Research. SUPPORTING INFORMATION AVAILABLE Effects of gaseous NH3 on the potential responses of a CHEMFET and a CHEMFET/dextran sensor incorporating the Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

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ion-partitioning membrane VI, shown for the addition of 10-2 M NH3 in a 10-2 M KCl, pH 12.7 sample solution (Figure 1S), the potential responses of CHEMFETs covered with the thick (100 µm) membranes IV-VII to the change of the potassium ion concentration in a pH 5.1 sample solution (Figure 2S), the timedependent absorbance changes of 3-nitrophenol (100 µm membrane VIII) and phenolphthalein (100 µm membrane IX) during protonation and deprotonation (Figure 3S), and the potential responses of (A, B) pH electrode and (C, D) a CHEMFET covered

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with membranes VI and V, respectively, to pH upon varying the potassium ion concentration (Figure 4S). This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review November 15, 1999. Accepted January 10, 2000. AC991313J