Micromachined Severinghaus-Type Carbon Dioxide Electrode

Anal. Chem. 1999, 71, 1737-1743

Micromachined Severinghaus-Type Carbon Dioxide Electrode Hiroaki Suzuki* and Hiroaki Arakawa

Institute of Materials Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba Science City, 305-8573, Japan Satoshi Sasaki and Isao Karube

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan

The Severinghaus-type pCO2 electrode was miniaturized and batch-fabricated using semiconductor and micromachining techniques. Anodically grown iridium oxide film (AIROF) was employed as the pH sensing element to detect a local pH change caused by the infusion of CO2. The AIROF showed a super-Nernstian response with a slope of approximately -80 mV/pH at 25 °C. A novel thinfilm Ag/AgCl structure was also used. It features a hydrophobic membrane which covers the entire silver layer and the AgCl layer grown from the periphery of the silver pattern. The open-circuit potential of the Ag/AgCl element drifted to the negative side at -0.1 to -0.2 mV h-1. A microcavity in which the electrolyte solution was filled was anisotropically etched in a silicon substrate, and a silicone rubber gas-permeable membrane was formed on the sensitive area. The miniature pCO2 electrode showed a distinct response to the variation in concentration of dissolved CO2. The inherent characteristics of the Severinghaus electrode were confirmed in terms of its response and calibration curve. The selectivity of the electrode was satisfactory in view of its application to clinical analysis. The partial pressure of CO2 (pCO2) as one of the blood gases is a critical index in assessing the rapidly changing status of patients who are critically ill or undergo a surgical operation. The measurements are usually accomplished on discrete blood samples using a very expensive instrument placed remote from the patient. Although the acquired data are reliable, they do not reflect the present status due to the time-delay between sampling and measurement.1 Therefore, a supplemental portable device which monitors rapidly changing pCO2 level is desired. An innovative alternative will be the µTAS (Micro Total Analysis System)2 or an implantable device connected to an extracorporeal in-line or on-line sensing system.1 Once such a technology is realized, it will revolutionize modern clinical analysis. In order to construct such sophisticated devices a reliable miniature pCO2 sensing * Corresponding author: (tel.) 81-298-53-5598, (fax) 81-298-55-7440, (e-mail) [email protected]. (1) Collison, M. E.; Meyerhoff, M. E. Anal. Chem. 1990, 62, 425A-437A. (2) Kopp, M. U.; Mello, A. J.; Manz, A. Science 1998, 280, 1046-1048. 10.1021/ac9811468 CCC: $18.00 Published on Web 03/27/1999

© 1999 American Chemical Society

device is becoming more and more important. The Severinghaustype pCO2 electrode is a potential candidate. 3,4 Its original structure consists of a pH glass electrode, a reference electrode, an electrolyte solution, and a hydrophobic gas-permeable membrane. The membrane minimizes the interference by nonvolatile materials resulting in the excellent selectivity of the device.5 Several miniaturized versions of the electrode have also been proposed employing the configuration and basic operational principle of the Severinghaus electrode. These include the application of the liquid-membrane electrode,6,7,8 the optode,9,10 and the ISFET.11,12 However, each has its inherent problems: fabrication, cost, stability, reliability, and the necessity of an expensive detection system (in the case of the optode). In this sense there exist no totally successful micro pCO2 sensors at present, and it is desired that more and more possibilities will be proposed. As illustrated with the ISFET, microfabrication technologies can provide inexpensive miniature devices. However, simplification of both structure and fabrication will be more and more critical in the coming decade if several sensors are integrated on a micro system, and further cost reduction is required. In view of its formation and membrane impedance, iridium oxide is an appropriate pH sensing element. As shown later, a whole structure of the ISFET can be replaced with a simple pattern of the iridium oxide depending upon the size of the device. Several methods of formation of and peculiar electrochemical behaviors of the corresponding oxides have been reported.12-23 From a technological point of view we chose the electrochemically grown oxide (AIROF: Anodic Iridium Oxide Film)16-23 because no thermal (3) Stow, R. W.; Baer, R. F.; Randall, B. F. Arch. Phys. Med. Rehabil. 1957, 38, 646-650. (4) Severinghaus, J. W.; Bradley, A. F. J. Appl. Physiol. 1958, 13, 515-520. (5) Guilbault, G. G.; Shu, F. R. Anal. Chem. 1972, 44, 2161-2166. (6) Opdycke, W. N.; Meyerhoff, M. E. Anal. Chem. 1986, 58, 950-956. (7) Shin, J. H.; Sakong, D. S.; Nam, H.; Cha, G. S. Anal. Chem. 1996, 68, 221225. (8) Zhao, P.; Cai, W. J. Anal. Chem. 1997, 69, 5052-5058. (9) Zhujun, Z.; Seitz, W. R. Anal. Chim. Acta 1984, 160, 305-309. (10) He, X.; Rechnitz, G. A. Anal. Chem. 1995, 67, 2264-2268. (11) Shimada, K.; Yano, M.; Shibatani, K.; Komoto, Y.; Esashi, M.; Matsuo, T. Med. Biol. Eng. Comput. 1980, 18, 741-745. (12) Tsukada, K.; Miyahara, Y.; Shibata, Y.; Miyagi, H. Sens. Actuators, B 1990, B2, 291-295. (13) Katsube, T.; Lauks, I.; Zemel, J. N. Sens. Actuators 1982, 2, 399-410.

Analytical Chemistry, Vol. 71, No. 9, May 1, 1999 1737

treatment which is detrimental to other elements in the device is necessary, and the control of the growth rate or membrane thickness is easy. Unlike previous studies of micro chemical sensors, our present study has also focused on the elements other than the indicator electrode. The Ag/AgCl reference electrode is one such element, and the applicability of our novel thin-film Ag/ AgCl structure24 was tested. Including the characteristics of these discrete elements, the fabrication and performance characteristics of the miniature pCO2 electrode will be described. EXPERIMENTAL SECTION Materials and Reagents. Glass wafers (3′′, 500 µm thick) were purchased from Corning Japan, Tokyo. (100)-oriented silicon wafers (3′′, 400 µm thick) were purchased from Japan Silicon Ltd. A photosensitive polyimide prepolymer (Photoneece 3100) was a gift from Toray. A positive photoresist (S1400-31) used in fabrication was purchased from Shipley Far East, Tokyo. A negative photoresist, OMR-83, used in fabrication and the electrode structure was purchased from Tokyo Ohka Kogyo, Kawasaki, Japan. One component, RTV silicone rubber, KE347T, used for the gas-permeable membrane was purchased from Shin-Etsu Chemical, Tokyo. The reagents used in fabricating the electrode were of semiconductor grade and were purchased from Kanto Chemicals, Tokyo and Wako Pure Chemicals Industries, Osaka. A photocurable adhesive (BENEFIX PC) was purchased from Adell, Tokyo. All reagents used to examine the electrode performance were purchased from Wako Pure Chemicals Industries except for acetoacetic acid obtained from Sigma Chemical Co., St. Louis, MO. They were of analytical reagent grade and were used without further purification. Distilled water was used throughout the experiments. Overall Electrode Structure and Fabrication. Figure 1 shows the structure of the miniature pCO2 electrode. The basic structure and details of its fabrication have been described in our previous papers.24,25 The dimensions of the chip were approximately 1.5 mm wide, 13 mm long, and 0.9 mm thick. Electrode patterns were formed on a 7740 glass substrate. All metal layers were sputter-deposited. The base layers consisted of 200-nm-thick gold patterns with a 40-nm-thick chromium adhesive layer. Threehundred-nanometer-thick iridium and 300-nm-thick silver patterns were formed on the areas of the base layer shown in Figure 1. (14) Ianniello, R. M.; Yacynych, A. M. Anal. Chim. Acta 1983, 146, 249-253. (15) Sato, Y.; Ono, K.; Kobayashi, T.; Wakabayashi, H.; Yamanaka, H. J. Electrochem. Soc. 1987, 134, 570-575. (16) Olthuis, W.; Robben, M. A. M.; Bergveld, P.; Bos, M.; van der Linden, W. E. Sens. Actuators, B 1990, B2, 247-256. (17) Kinoshita, E.; Ingman, F.; Edwall, G.; Thulin, S.; Glab, S. Talanta 1986, 33, 125-134. (18) Hitchman, M. L.; Ramanathan, S. Analyst (Cambridge, U.K.) 1988, 113, 35-39. (19) Conway, B. E.; Mozota, J. Electrochim. Acta 1983, 28, 9-16. (20) Buckley, D. N.; Burke, L. D.; Mulcahy, J. K. J. Chem. Soc. Faraday Trans. 1 1976, 72, 1896-1902. (21) Pickup, P. G.; Birss, V. I. J. Electrochem. Soc. 1988, 135, 126-133. (22) Anderson, D. J.; Najafi, K.; Tanghe, S. J.; Evans, D. A.; Levy, K. L.; Hetke, J. F.; Xue, X.; Zappia, J. J.; Wise, K. D. IEEE Trans. Biomed. Eng. 1989, 36, 693-704. (23) Burke, L. D.; Mulcahy, J. K.; Whelan D. P. J. Electroanal. Chem. 1984, 163, 117-128. (24) Suzuki, H.; Hiratsuka, A.; Sasaki, S.; Karube, I., Sens. Actuators 1998, B46, 104-113. (25) Suzuki, H.; Sugama, A.; Kojima, N. Sens. Actuators, B 1993, B10, 91-98.

1738 Analytical Chemistry, Vol. 71, No. 9, May 1, 1999

Figure 1. Structure of the miniature pCO2 electrode. (a) Silicon container (outside). (b) The other side of (a) on which the container for the electrolyte solution and a liquid junction was formed. (c) Glass substrate on which the detecting electrodes are formed. (d) Completed miniature pCO2 electrode. (e) Cross-section of the Ag/AgCl element used in the pCO2 electrode along the line x-x′. (f) Cross-section of the currently used Ag/AgCl element for comparison. In (a) and (d) the gas-permeable membrane is formed on the entire area indicated by the arrows. In (c) the dashed lines and the black areas in the Ag/ AgCl element indicate the underlying gold backbone layer and the portions where the AgCl layers are formed. In (e) and (f) the arrows indicate the direction of AgCl formation.

The area of the iridium pattern was 500 µm × 500 µm. The structure of the thin-film Ag/AgCl element was basically the same as reported elsewhere,24 and details will be described below. The container which accommodates the internal filling solution was made by anisotropically etching a (100) silicon substrate in a 35% KOH solution at 80 °C using an SiO2 mask. The surface of the etched silicon substrate was thermally oxidized to obtain an SiO2 layer (1.0-µm-thick) to ease introduction of the electrolyte solution. A through-hole was formed in the sensitive area, and the distance between the AIROF and the gas-permeable membrane was reduced to 200 µm by etching the silicon substrate from both sides. A recess for the electrolyte solution was formed around the area corresponding to the iridium pattern and the Ag/ AgCl reference electrode. To reduce the influence of CO2 influx from the reference-electrode compartment, the two recesses were connected with a narrow groove (50 µm × 1.35 mm at the silicon surface). Another through-hole was formed near the pad to

introduce the electrolyte solution. The volume of the internal cavity thus made was approximately 1.6 µL. The through-hole in the sensitive area was covered with a gaspermeable membrane. In forming the membrane, the positive photoresist was filled in the through-hole from the other side and was dried. Then the silicone-rubber prepolymer was coated on the sensitive area. After the silicone rubber was cured, the photoresist was removed in acetone, and the silicon substrate was thoroughly rinsed in fresh acetone. The obtained membrane had a thickness of 100 ( 10 µm. The glass and silicon substrates were aligned under an optical microscope and fixed with a clip. Then the photocurable adhesive was permeated from the edges of the substrates using capillary action and was cured under a UV light (Toshiba FL-20). Ninety miniature reference electrodes were batch-fabricated on a 3′′ wafer and were diced into chips. To examine the difference in the response characteristics, three types of internal-filling solution, 0.1 M KCl + 10 mM NaHCO3, 0.1 M KCl + 20 mM NaHCO3, and 0.1 M KCl + 50 mM NaHCO3, were prepared. AgCl was saturated in the solutions. Filling the microcavity with the electrolyte solution was accomplished by immersing the chip in the electrolyte solution in a beaker, placing it in a vacuum chamber, and evacuating the chamber.25 Because air bubbles often prevented optimal filling, they were removed by centrifuging the chip. Detailed Structure and Fabrication of the Thin-Film Ag/ AgCl Element. As discussed later, the currently used thin-film Ag/AgCl element (Figure 1f) is damaged by HCO3- ion which is an essential component in the Severinghaus-type pCO2 electrode. In our previous study a novel thin-film Ag/AgCl structure was proposed (Figure 1e)24 mainly to obtain a durable thin-film Ag/ AgCl element which can be used in a liquid-junction reference electrode with an internal electrolyte solution saturated with KCl. It was also verified that the novel element was markedly durable against interfering ions including I-, Br-, S2-, and HCO3- which form sparingly soluble salts with Ag+. The Ag/AgCl element used in the miniature pCO2 electrode has a gold/chromium backbone layer (200-nm-thick) at the center. A silver layer (300-nm-thick) was then formed over the area shown, which made partial contact with the gold layer. The entire silver layer was covered with a hydrophobic negative photoresist layer (OMR-83, 2.8 µm). AgCl was grown galvanostatically from the edges of the silver pattern (Figure 1c,e) by applying a constant current of 2 µA for 33 min in a 0.1 M KCl-HCl buffer solution (pH 2.2). Approximately one-third of the silver layer was converted to AgCl. To perform a comparative study, the conventional thin-film Ag/ AgCl sample without the upper photoresist layer was prepared. The gold layer was formed under the entire silver layer to obtain enough adhesion. The same amount of AgCl layer was grown. Operational Principle. The basic operational principle of the Severinghaus electrode has been given theoretically in some previous literature.5,26-29 Its response is governed by a series of equilibrium processes. When a sample solution containing CO2 (26) Ross, J. W., Riseman, J. H.; Kruger, J. A. Pure Appl. Chem. 1973, 36, 473487. (27) Fielder, U.; Hansen, E. H.; Ru˚zˇicˇka, J. Anal. Chim. Acta 1975, 74, 423435. (28) Lopez, M. E. Anal. Chem. 1984, 56, 2360-2366. (29) Janata, J. Principles of Chemical Sensors; Plenum Press: New York, 1989; pp 148-152.

is in contact with the sensing area, the dissolved CO2 diffuses through the gas-permeable membrane and the following equilibria are sequentially established

CO2 (aq) + H2O a H2CO3 (Kh ) 2.6 × 10-3)

(1)

H2CO3 a H+ + HCO3- (K1 ) 1.72 × 10-4)

(2)

HCO3- a H+ + CO32- (K2 ) 5.59 × 10-11)

(3)

where Kh is the Henry’s law constant, and K1 and K2 are the first and second dissociation constants of H2CO3. By taking the dissociation of water and the balance of charge into account, the relation between [H+] and pCO2 is given as follows,

[H+]3 + [NaHCO3][H+]2 - (K1Kh pCO2 + KW)[H+] 2K1K2Kh pCO2 ) 0 (4) where [NaHCO3] is the concentration of NaHCO3 in the internal electrolyte, and KW is the dissociation constant of water. Within an appropriate pCO2 range, an approximation reduces eq 4 to the well-known Henderson-Hasselbalch equation.29 The pH given by eq 4 is related to the potential of the AIROF through the Nernst equation. The relation between the shape of the curve and the NaHCO3 concentration in the internal electrolyte has been given by rigorous numerical analysis using eq 4.26,27 As discussed later, the response of the pCO2 electrode is influenced by the existence of various interfering acids. A similar formulation has been given by taking them into account.28 Preparation of Buffer Solutions and Standard Solutions. Buffer solutions were prepared using a pH measuring instrumentation consisted of a TOA Electronics HM-20S pH meter, with a combination pH glass electrode previously calibrated in precision buffers of pH 4.01 and 6.86 (TOA Electronics). Solutions of NaHCO3, 1.0 and 0.1 M in concentration, were prepared as the standard solutions to generate CO2 in the external solution. In evaluating the electrode’s selectivity, standard 1.0 M aqueous solutions for respective compounds were prepared. Sparingly soluble compounds were added as powder. The standard solutions were stored in a refrigerator when not in use. Formation and Evaluation of the AIROF and the Miniature pCO2 Electrode. Electrochemical growth and characterization of the AIROF was performed with a Hokuto Denko HA-151 potentiostat/galvanostat. In forming the AIROF the periphery of the iridium pattern and portions other than the pads were protected with the positive photoresist. Otherwise, the underlying chromium adhesive layer was completely lost after the potential cycling. The iridium pattern was immersed in a 0.1 M H2SO4 solution with an Ag/AgCl reference electrode (Horiba 2080A-06T, internal solution: saturated with KCl and AgCl) and a platinum plate counter electrode. The iridium site on the chip was used as the working electrode, and a triangular voltage waveform was applied there between -0.3 and +1.4 V (vs Ag/AgCl) at 0.5 V/s using a Hokuto Denko HB-111 function generator. The current and potential were recorded on a Graphtec WX1100 X-Y recorder. In evaluating the pH response of the AIROF, the AIROF pattern and the macroscopic reference electrode above were immersed Analytical Chemistry, Vol. 71, No. 9, May 1, 1999

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in a series of stirred buffer solution and the potential of the AIROF was measured with a Hokuto Denko HE-106 electrometer. The level of resolution achieved by the electrometer was (0.1 mV. The buffer solutions used were 20 mM citrate-NaOH (pH 3.95, 4,96, and 5.98), 20 mM KH2PO4-NaOH (pH 5.95, 6.93, and 7.72), and 20 mM borate-NaOH (pH 7.72, 8.69, and 9.50). The stability of the thin-film Ag/AgCl element was examined by immersing the Ag/AgCl element on the chip and a macroscopic Ag/AgCl reference electrode (Horiba 2535A-06T, internal solution: saturated with KCl and AgCl) and measuring the open-circuit potential with the electrometer. The macroscopic electrode has a double junction, and its outer compartment was filled with the external solution used for the measurement to minimize the errors caused by the contamination of effusing KCl and AgCl. The stability was examined in the same solutions used for the internalfilling solution of the pCO2 electrode. A 0.1 M KCl solution containing no NaHCO3 was also used for the experiment. In evaluating the completed miniature pCO2 electrode, its sensitive area was immersed in a stirred 1 mM H2SO4 solution filled to the top of a 50 mL Erlenmeyer flask. The flask was tightly sealed with Parafilm (American National Can, WI), and two small holes were formed to insert the electrode chip and to inject the sample solution. After the sensitive area of the miniature pCO2 electrode was immersed in the H2SO4 solution and the electrode was allowed to stabilize, CO2 was produced by injecting and acidifying a predetermined amount of the NaHCO3 stock solution. For higher concentrations of NaHCO3, an appropriate amount of 2.0 M H2SO4 solution was added to effectively generate CO2. In examining the response to the reverse direction, 1.0 M NaOH solution was added to change CO2 into ions after the response to CO2 was allowed to reach a stabilized level. The potential variation against the step change of the CO2 concentration was recorded with the electrometer. The data of the series of experiments were recorded on a TOA Electronics PRR-5011 strip-chart recorder. Measurements were all made at 25.0 ( 0.1 °C. RESULTS AND DISCUSSION Growth and pH Response of the AIROF. A thick oxide film can be grown on the surface of iridium by progressively cycling its potential. The successful growth of the oxide depends on both the anodic and cathodic limits of the cycling.20 Our cathodic limit (-0.3 V (vs Ag/AgCl)) was set near the onset of H2 evolution, while the anodic limit (+1.4 V (vs Ag/AgCl)) was set near the onset of O2 evolution. Cyclic voltammograms obtained during the oxide growth are shown in Figure 2. Progressively cycling the potential to the potential limits expanded the curves outward indicating the increase in charge capacity. The oxide is a hydrated oxyhydroxide, and the major anodic and cathodic peaks have been attributed to an Ir(III)/Ir(IV) transition given by the following equation:18

Figure 2. Cyclic voltammograms during the growth of the AIROF in 0.1 M H2SO4 at 25 °C. The numerals in the figure indicate the repetition of potential cycling. Scan rate: 0.5 V/s.

The Nernst plot of the AIROF formed by 2000 cyclings is shown in Figure 3. The obtained plot was so-called superNernstian with a slope of approximately -80 mV/pH at 25 °C. The obtained value of the slope was fairly close to that of Hitchman et al. using an iridium wire.18 Dependence of the slope on the number of cyclings is plotted in the inset of Figure 3. The slope leveled off after a relatively small number of potential cyclings, which coincides with the results given by others in the literature.17,23 As with the anion sensitivity of the AIROF,18,23 no substantial effect was confirmed. Owing to the low impedance of the AIROF, no preamplification was necessary, and a stable output was obtained for the dimensions of the pattern used here. Stability of the Thin-Film Ag/AgCl Element. The stability of the thin-film Ag/AgCl element is affected by the presence of ions other than Cl- which form an insoluble salt with Ag+. Although the influence might be trivial for macroscopic Ag/AgCl reference electrodes, distinct abnormalities have been confirmed for the thin-film Ag/AgCl elements.24 With the solubility product Ksp(AgzX) of an interfering anion Xz-, the thin-film Ag/AgCl layer will undergo metathesis under the following condition:30,31

Ksp(AgCl)/Ksp(AgzX)1/z > a(Cl-) / a(Xz-)1/z

(6)

2[IrO2(OH)2-x(2 + x)H2O](2-x)- + (3 - 2x)H+ + 2e- a [Ir2O3(OH)33H2O]3- + 3H2O (5) The peculiar behavior of the voltammogram, the mechanism of the oxide film growth, the nature of the film, and its pH response have been understood well16-23 and are not described in detail here. 1740 Analytical Chemistry, Vol. 71, No. 9, May 1, 1999

The interference from anions which form sparingly soluble salts (e.g., I- and S-) is explained with the above criterion. However, this is not the case for Ag2CO3 considering the pKa1 and pKa2 of carbonic acid and the solubility product of Ag2CO3 (30) Janata, J. Principles of Chemical Sensors; Plenum Press: New York, 1989; pp 110-111. (31) Morf, W. E.; Kahr, G.; Simon, W. Anal. Chem. 1974, 46, 1538-1543.

Table 1. Variation of the Potential and Lifetime of the Thin-Film Ag/AgCl Elements currently used type

novel type

electrolyte solution

variation (mV h-1)

lifetime (h)

variation (mV h-1)

lifetime (h)

1a 2b 3c

-0.20 -0.30 -0.44

>48 23 26

-0.12 -0.22 -0.13

>48 >48 >48

a 0.1 M KCl (AgCl not saturated). b 0.1 M KCl + 20 mM NaHCO 3 (AgCl not saturated). c 0.1 M KCl + 20 mM NaHCO3 (AgCl saturated). Three measurements were conducted for each case, and the best data are presented. For both types of electrode AgCl was formed at 2 µA for 33 min in 0.1 M KCl-HCl buffer (pH 2.2, 25 °C). The lifetime means the elapsed time until the catastrophic potential shift is observed. The measurements were stopped at 48 h.

Figure 3. Dependence of the potential of the AIROF on pH at 25 °C. The potential of the AIROF was measured in: 20 mM citrateNaOH (n), 20 mM KH2PO4-NaOH (9), and 20 mM borate-NaOH buffers (4). The oxide was grown by 2000 potential cyclings. The inset shows the dependence of the slope on the repetition of cycling.

(8.45 × 10-12 at 25 °C), although a noticeable potential shift has also been observed in the presence of bicarbonate.24 The dissolution of AgCl would proceed until sparingly soluble Ag2CO3 is formed sufficiently to reach an equilibrium. Even in a solution saturated with Ag2CO3, the dissolution from the Ag/AgCl element would proceed microscopically to maintain the equilibrium as a whole. In this respect, the degradation of the thin-film Ag/AgCl element starts once the element makes contact with the electrolyte solution. The durability of the thin-film Ag/AgCl element used in the pCO2 electrode (Figure 1e) was examined in comparison with the currently used thin-film Ag/AgCl element (Figure 1f). The results are summarized in Table 1. For all the cases examined, the potential gradually drifted to the negative side. A distinct difference was observed between the solutions with and those without NaHCO3. Durability and potential drift were superior in the solution containing no NaHCO3. In the presence of NaHCO3, however, the lifetime of the currently used Ag/AgCl element became markedly short. When it broke, a substantial catastrophic potential shift exceeding 200 mV was observed as experienced in our previous study.24 The lifetime of the two types of element was elongated to some extent in the solution saturated with AgCl, although there was still a distinct difference compared with the bicarbonate-free solution. The effect of NaHCO3 concentration was not clearly distinguished. The durability and stability of the novel structure depend significantly on the adhesion between respective layers and the glass substrate. The critical portion of the present Ag/AgCl element was like this: glass/(Ag or AgCl)/negative photoresist. The adhesion between the substrate and the silver layer in particular was poorer than in our previous study,24 which

could not effectively suppress the dissolution of AgCl. The novel structure was more influenced by the equilibrium relations other than Ag/AgCl. The underlying gold support area would be a possible cause to make a mixed potential. Several ISFET type miniature pCO2 sensors have been reported using a thin-film Ag/AgCl reference electrode. Considering the results above, it is possible that the thin-film Ag/AgCl reference electrode used in these devices might not have given a stable potential depending on the thickness of AgCl, the concentration of NaHCO3, and aging. Response Time of the Completed pCO2 Electrode. The response time of the Severinghaus electrode depends on the concentration of the internal NaHCO3 and the variation and direction of the step change of pCO2. Such a behavior has already been reported by Severinghaus and Bradley.4 The response characteristics of the pCO2 electrode are usually described by the theoretical model by Ross et al.,26 where l ) the thickness of the

t)

[

]

dCB lm ∆C 1+ ln Dk dC C2

(7)

internal electrolyte layer, m ) the thickness of the gas-permeable membrane, D ) the diffusion coefficient of CO2 in the gaspermeable membrane, k ) the partition coefficient of CO2 between the aqueous solutions and the gas-permeable membrane, C ) the concentration of H2CO3 in the internal electrolyte layer, dCB/dC ) (d[HCO3-] + d[CO32-])/d[H2CO3], ∆C ) difference in the initial and final H2CO3 concentrations,  ) |(C2 - C)/C2|, C2 ) the final concentration of H2CO3 in the aqueous solutions, and t ) the time required to achieve the concentration C in the internal electrolyte layer. Figure 4 shows typical response curves of the miniature pCO2 electrode. The output potential was very stable, and no noise or fluctuation was observed. However, the response was relatively slow. The volume of the sensitive area between the gas-permeable membrane and the AIROF was relatively large in this pCO2 electrode. Along with the diffusion coefficient of the gas-permeable membrane, the product lm in eq 7 is considered to be large compared with ordinary macroscopic electrodes and to be a major cause for the slow response. Furthermore, the solution-gas equilibrium of CO2 is slow26 and might have partially contributed to the slow response. The application of a heterogeneous air-gap Analytical Chemistry, Vol. 71, No. 9, May 1, 1999

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Figure 4. Typical response curves of the miniature pCO2 electrode retraced from a real chart. The concentrations in the figure indicate the final concentrations of the added NaHCO3. Two-hundred microliters of 1 M NaOH was added to examine the response to the reverse change. The internal electrolyte: 0.1 M KCl + 20 mM NaHCO3.

membrane instead of the present homogeneous silicone-rubber membrane has been proven effective because the former gives a “kD” value 104 times larger.26 Although there needs to be a technical breakthrough to apply such a membrane to microsensors, it can be a good solution in fabricating a fast-responding pCO2 electrode. Hydration of CO2 can also be a rate-determining step, and carbonic anhydrase has been used in some previous pCO2 electrodes to accelerate the reaction.8 However, with reference to the obtained values of the response time, this effect is not dominating in our miniature pCO2 electrode. The response time depends on pCO2 and the direction of its change.4,5 The larger the variation of pCO2, the faster the response. Furthermore, the response to an increasing step change is faster than that to the corresponding decreasing change, which is peculiar to systems in which diffusion is dominating.5 The expected tendency was clearly confirmed. In the examined pCO2 range, the 90% response time for the increasing change was shortest (3.0 min) for a 5-mM step change of injected NaHCO3 and longest (23 min) for 0.2-mM step change. The same tendency was also confirmed for the decreasing step change, and the response time was 2-6 times longer than that for the increasing change of pCO2. The influx of CO2 from the reference-electrode compartment can also influence the response time.32 Although the contribution from this factor is difficult to estimate, the effect is considered to be small even if it exists, considering the dimensions of the liquid-junction. The internal NaHCO3 concentration also affects the response time. It has been pointed out both experimentally4-6 and theoretically26 that the response becomes faster when more dilute internal NaHCO3 is used. The effect is enhanced at low levels of pCO2, while the response is virtually independent of the internal NaHCO3 concentration at higher pCO2.32 However, the dependency was hardly distinguished because the control of the gas-permeable (32) Jensen, M. A.; Rechnitz, G. A. Anal. Chem. 1972, 51, 1972-1977.

1742 Analytical Chemistry, Vol. 71, No. 9, May 1, 1999

Figure 5. Calibration curves for the miniature pCO2 electrode at 25 °C. The shaded area in the figure indicates an approximately linear region which follows the Henderson-Hasselbalch equation. The slope of the area is 80 mV/decade. The internal electrolyte solution contains : 50 mM (n), 20 mM (9), and 10 mM (4) of NaHCO3.

membrane thickness was not rigorous enough with the present level of technology. Calibration Curve for the Completed pCO2 Electrode. The internal NaHCO3 concentration also affects sensitivity, linearity of the calibration curve, and detection limit.6,26,29 The effect has been explained clearly on the basis of a theoretical analysis,27 which was in line with the experimental result using a macroscopic pCO2 electrode.5 Typical calibration curves for the completed miniature pCO2 electrode are shown in Figure 5, in which the steady-state potential values vs the logarithm of the sample NaHCO3 concentration are plotted. As a general tendency, all curves showed an approximately linear region denoted by the shaded area in the figure, and their slope decreased below this range. The dependence of the slope of the linear region on the internal NaHCO3 concentration was not clear for the present device because of the scatter of the data points. On the other hand, a clear dependency of the detection limit was observed. The lowest concentrations shown in the figure should be considered to be the lowest detection limits for this device. With 10 mM internal NaHCO3, the detection limit reached 5 µM. The detection limit is usually determined quantitatively using the S/N ratio. However, its accurate determination was actually difficult for this pCO2 electrode because of its increasingly slower response around the low pCO2 region and possible drift of the baseline caused by some factors (e.g., escape of the gaseous CO2 from the flask) during the long time required for the measurement. Although the experiment was conducted in a solution purged with nitrogen, this is not the case in real situations. Because the dissolved CO2 concentration in equilibrium with air is considered to be around 10-30 µM, the detection limit in a real sample without N2 purging should be considered to be around 100 µM as pointed out elsewhere in the literature.5,27 The upper limit of the linear region is determined by the fact that the solubility of CO2 is limited and HCO3- produced by its hydrolysis approaches the original HCO3concentration in the electrolyte.5,27 Influence by Interfering Materials. Owing to the hydrophobic gas-permeable membrane, only gaseous acidic or basic species can cause a pH shift in the sensing area.5,6 Interference by volatile

Table 2. Response of the Miniature pCO2 Electrode to Organic and Inorganic Acids compound CO2 acetoacetic oxalacetic H2S

pKa 6.35 (pK1) 10.33 (pK2) 3.58 2.15 (pK1) 4.06 (pK2) 7.00 (pK1) 12.92 (pK2)

relative selectivitya

90% response (min)

1.00

5

0.93 0.37

∼145 ∼75

0.64

1

aThe relative selectivity was defined as the relative response ∆E / 2 ∆E1, where subscripts 1 and 2 refer to the variation of the potential with CO2 (NaHCO3) and respective interfering acids in separate solutions. The concentrations of the sample solutions were 1 mM. H2S was produced from Na2S. The other examined compounds include L-ascorbic acid, DL-lactic acid, L-malic acid, oxalic acid, pyruvic acid, succinic acid, acetic acid, benzoic acid, formic acid, propionic acid, and SO2 (added as Na2SO3). The response to these acids was not detected.

inorganic and organic acids has been examined.28 Because a major application of the miniature pCO2 electrode will be clinical analysis, its selectivity against clinically important acids which exist in human blood,28 including nonvolatile acids, was examined. Table 2 summarizes the relative selectivity to the acids. Among examined organic acids the electrode responded to oxalacetic acid and acetoacetic acid and their finally stabilized potential levels were comparable to that of NaHCO3. However, the response to these acids was very slow. The interference by these two acids has also been reported elsewhere in the literature and has been ascribed to the production of CO2 as a result of their slow decomposition in acidic media.28 In view of the response time to CO2 the influence by these acids will be much smaller in practical situations. No response was observed to the other organic acids (L-ascorbic acid, DL-lactic acid, L-malic acid, oxalic acid, pyruvic acid, and succinic acid). Interference by some other organic and inorganic acids was also examined. Among examined acids, only H2S responded. Not only substantial interference was observed but also the response was faster than that to NaHCO3, a result that was also confirmed by Lopez.28 Furthermore, an accidental potential shift to the negative (or reverse) side was often observed before the response reached a stabilized level. This phenomenon was observed (33) Pucacco, L. R.; Carter, N. W. Anal. Biochem. 1978, 90, 427-434.

especially for aged devices and would be ascribed to the damage to the Ag/AgCl element by S2- ion. Although this interference was negligible in our previous study,24 improvements in the thinfilm Ag/AgCl structure will be critical if the electrode is used in an environment in which H2S exists. Response to the other acids (acetic acid, benzoic acid, formic acid, propionic acid, and SO2) was not detected. Because the interference by the three compounds mentioned above was supposed to originate from the experimental methods which use acidification, the effect was also examined in 0.1 M Tris-HCl buffer solution at pH 7.4. No response was confirmed in this medium. As with the metal oxide pH sensing element, interference by redox pairs has been pointed out,33 the most common species being oxygen. To examine this effect, the potential of the miniature pCO2 electrode was stabilized in the air-saturated 0.1 M Tris-HCl buffer solution, and dissolved oxygen was removed by adding Na2SO3. However, no change was detected. CONCLUSIONS The Severinghaus-type pCO2 electrode was miniaturized using micromachining techniques. It was confirmed that its characteristics were in line with the theoretical prediction and those of the macroscopic Severinghaus electrode. The AIROF would probably be the simplest and the most inexpensive pH sensing element formed by the thin-film process. Preamplification of the signal was not necessary; therefore, it would be suitable for a disposable blood-gas sensor. The incorporation of the electrolyte solution will be difficult for such use, and further simplification would be desirable. The original design of the Severinghaus-type pCO2 electrode is so general that it can be applied to other gas-sensing electrodes which work on the basis of similar operational principles. In applying the device to blood-gas analysis the adsorption of proteins on the sensitive area might affect the sensor response. Such practical problems will be investigated in our next stage using real blood samples. ACKNOWLEDGMENT This research was supported by the Grant from Casio Science Promotion Foundation. Received for review October 20, 1998. Accepted February 5, 1999. AC9811468

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