Anal. Chem. 1999, 71, 5069-5075
Microfabricated Liquid Junction Ag/AgCl Reference Electrode and Its Application to a One-Chip Potentiometric Sensor Hiroaki Suzuki* and Hisanori Shiroishi
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
A liquid-junction Ag/AgCl reference electrode was microfabricated. A silver thin-film pattern was covered with a polyimide protecting layer with a 50-µm-wide slit at the center of the pattern, and the AgCl layer was grown from there into the silver layer. A liquid junction was formed with a photocurable hydrophilic polymer. The electrolyte layer was formed by screen-printing a paste containing fine KCl powder. A silicone rubber passivation covered the entire area except for the pad and the end of the junction. The novel thin-film Ag/AgCl element could maintain its expected potential for longer than 30 h in a saturated KCl solution. The completed miniature liquidjunction reference electrode could maintain a stable potential level within (1 mV for longer than 100 h with the aid of a poly(vinylpyrrolidone) (PVP) matrix in the electrolyte layer. Fluctuation of the potential was less than 0.1 mV at each moment. Dependency on external KCl concentration and pH was shown to be insignificant. The miniature reference electrode was integrated with an iridium oxide pH indicator electrode. No significant difference was observed between the potential obtained with the miniature reference electrode and that obtained with a macroscopic commercial Ag/AgCl electrode, which confirmed that the miniature reference electrode was applicable to one-chip potentiometric sensors. Miniaturization of electrochemical sensors has advanced dramatically during the last two decades. However, a serious problem remaining has been the unavailability of a reliable miniature reference electrode. In employing a micro indicator electrode including the ISFET (Ion Sensitive Field Effect Transistor), an alternative has been to use either a conventional macroscopic reference electrode or an integrated pseudo-reference electrode making direct contact with a sample solution. However, the former nullifies the significance of miniaturizing the sensor, while the latter requires stringent control of the activity of the primary ion which contributes to the electrode potential (e.g., Cl- for the * Corresponding author: (tel.) 81-298-53-5598, (fax) 81-298-55-7440, (e-mail)
[email protected]. 10.1021/ac990437t CCC: $18.00 Published on Web 10/08/1999
© 1999 American Chemical Society
Ag/AgCl electrode). In either case, the number of applications in which the microsensor can be used will be limited. With the recent advent of µTAS (Micro Total Analysis System) technology,1 microfabrication of the reference electrode has become a critical issue, the realization of which is urgently desired. One approach commonly used for the miniaturization of electrodes is the so-called solid-state reference electrode. However, as miniaturization brings with it many problems, many different approaches have been cited in the literature. In some of the studies, Cl- ions were trapped in an internal hydrogel layer, and the entire layer was covered with a cation-exchange membrane like Nafion to construct a liquid-junction-free Ag/AgCl electrode.2 Electrodes in which KCl and AgCl were directly incorporated in the Nafion were also reported.3 The electrodes with permselective membranes become sensitive to ions other than those of interest.2-4 In another approach a membrane was employed which was insensitive to all the ions which existed in the anticipated environment.5 This approach has often been used for the construction of REFETs (Reference Field-Effect Transistors).6-8 In this case, however, the exchange current densities of the membrane9 to possible ion-exchange processes are minimized and the resulting potential is a mixed potential which is influenced significantly by factors such as protein adsorption.9,10 The error caused by polarization limits the applicability to general environments. The problems could be overcome by special membrane selection if the anticipated environment is well-known. This work (1) Kopp, M. U.; Mello, A. J.; Manz, A. Science (Washington, D.C.) 1998, 280, 1046-1048. (2) Nolan, M. A.; Tan, S. H.; Kounaves, S. P. Anal. Chem. 1997, 69, 12441247. (3) Kinlen, P. J.; Heider, J. E.; Hubbard, D. E. Sens. Actuators, B 1994, B22, 13-25. (4) Ito, S.; Kobayashi, F.; Baba, K.; Asano, Y.; Wada, H. Talanta 1996, 43, 135-142. (5) Lee, H. J.; Hong, U. S.; Lee, D. K.; Shin, J. H.; Nam, H.; Cha, G. S. Anal. Chem. 1998, 70, 3377-3383. (6) Tahara, S.; Yoshii, M.; Oka, S. Chem. Lett. 1982, 3, 307-310. (7) Oyama, N.; Ohsaka, T.; Ikeda, S.; Okuaki, K. Anal. Sci. 1989, 5, 729-734. (8) Skowronska-Ptasinska, M.; van der Wal, P. D.; van den Berg, A.; Bergveld, P.; Sudho ¨lter, E. J. R.; Reinhoudt, D. N. Anal. Chim. Acta 1990, 230, 6773. (9) Cammann, K. Anal. Chem. 1978, 50, 936-940. (10) Collins, S.; Janata, J. Anal. Chim. Acta 1982, 136, 93-99.
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was done in search of a system that could overcome the described limitations. A good reference electrode must ideally be reversible and nonpolarizable.11 In other words, the electrode potential must be dominated by a reaction with a high exchange current density so as not to be affected by any current flow across the interface. Therefore, it is quite natural to miniaturize a conventional liquidjunction reference electrode to achieve a high level of accuracy. In previous studies, liquid-junction electrodes made of a hydrophilic polymer layer for impregnating a KCl solution and a liquid junction formed as a small opening in the polymer layer were studied.12,13 Similarly, a glass-fiber filter was used to impregnate the electrolyte solution and for the liquid junction.14 In other cases, a microcontainer with a through-hole for a liquid junction was formed using anisotropic etching of silicon, and a restraining material was used for the junction. The used materials include a porous glass,15 porous silicon,16,17 and pHEMA (poly(hydroxyethyl methacrylate)).17 In constructing a miniature liquid-junction Ag/AgCl reference electrode, durability of the Ag/AgCl element in concentrated KCl solutions is the most critical determining factor for the electrode lifetime. However, no previous reference to this problem was found. As mentioned later, the lifetime of a thin-film Ag/AgCl element is very short. We have already proposed a novel thinfilm Ag/AgCl structure which showed a dramatic improvement in its durability.18 It consisted of a hydrophobic polymer layer which covered the entire surface of a silver thin-film pattern and the AgCl layer formed from the edges into the silver layer. Also, a micromachined liquid-junction Ag/AgCl reference electrode was fabricated employing the Ag/AgCl structure.19 The reference electrode could maintain a stable potential within (1 mV for several hours. A problem with the liquid-junction electrode was that the amount of KCl stored was insufficient because the KCl could not be stored in solid form. Also, the effusion of KCl from the liquid junction was observed to be too fast for practical applications. Therefore, in this study an electrolyte layer containing fine KCl powder was screen-printed to elongate the electrode’s lifetime and was activated just before use by injecting a solution saturated with KCl and AgCl. A photocurable hydrophilic polymer was used as a restraining material for the liquid junction. Furthermore, the AgCl layer was grown from a slit at the center into the silver layer. This structure fixed the silver layer more tightly to the glass substrate, which resulted in a more reproducible electrode lifetime. In this report, details of the fabrication and (11) Ives, D. J.; Janz, G. J. Reference Electrodes; Academic Press: New York, 1961; pp14-26. (12) Sinsabaugh, S. L.; Fu, C. W.; Fung, C. D. Proc. of the Electrochem. Soc. 1986, 86-14, 66-73. (13) Arquint, Ph.; van den Berg, A.; van der Schoot, B. H.; de Rooij, N. F.; Bu ¨ hler, H.; Morf, W. E.; Du ¨ rselen, L. F. J. Sens. Actuators, B 1993, B13-14, 340344. (14) Mroz, A.: Borchardt, M.; Diekmann, C.; Cammann, K.; Knoll, M.; Dumschat, C. Analyst (Cambridge, U.K.) 1998, 123, 1373-1376. (15) Yee, S.; Jin, H.; Lam, L. K. C. Sens. Actuators 1988, 15, 337-345. (16) Smith, R. L., Scott, D. C. IEEE Trans. Biomed. Eng. 1986, BME-33, 8390. (17) van den Berg, A.; Grisel, A.; van den Vlekkert, H. H.; de Rooij N. F. Sens. Actuators, B 1990, B1, 425-432. (18) Suzuki, H.; Hiratsuka, A.; Sasaki, S.; Karube, I. Sens. Actuators, B 1998, B46, 104-113. (19) Suzuki, H.; Hirakawa, T.; Sasaki, S.; Karube, I. Sens. Actuators, B 1998, B46, 146-154.
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performance characteristics of the novel liquid-junction reference electrode, along with its application to a one-chip potentiometric sensor, will be presented. EXPERIMENTAL SECTION Materials and Reagents. 7740 glass wafers (3′′, 500 µm thick) were purchased from Corning Japan, Tokyo, Japan. A photosensitive polyimide prepolymer (Photoneece 3140) was a gift from Toray, Tokyo. A positive photoresist (S1400-31) used in fabrication was purchased from Shipley Far East, Tokyo. An ENT-2000 resin was a gift from Kansai Paint, Osaka, Japan. One-component RTV silicone rubber, KE347T, used for passivation was purchased from Shin-Etsu Chemical, Tokyo. The reagents used in fabricating the electrode were of semiconductor grade and 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. They were of analytical reagent grade and used without further purification. Distilled water was used throughout the experiments. Structure and Fabrication of the Liquid-Junction Ag/AgCl Reference Electrode. The miniature liquid-junction Ag/AgCl reference electrode was fabricated following the ordinary microfabrication processes. Figure 1a shows its decomposed structure. The dimensions of the chip were approximately a 1.5-mm width and a 13-mm length. A U-shaped gold backbone pattern (200-nmthick) was formed on a glass substrate using a 40-nm-thick chromium adhesive layer. The line width of the pattern was 100 µm. Next, a 300-nm-thick silver layer was formed only on the area enclosed by the gold pattern (0.9 mm × 6.4 mm). The silver layer made contact only with the U-shaped portion of the gold layer, and the other area was directly formed on the glass substrate. Because the adhesion of the silver layer on the glass substrate was poor, six square through-holes (100 µm square) were formed in the silver layer to affix it with the following polyimide layer. Then, a 10-µm-thick polyimide layer was formed, which has a slit (50 µm × 6.2 mm) at the center of the silver pattern and a recess to form a liquid junction at one end of the chip. The slit was formed only on the silver pattern and was used to generate the AgCl layer and as an opening for the Ag/AgCl element to be in contact with the electrolyte solution. The recess was separated from the silver pattern. In curing the polyimide, the chip was subjected to bakings for 15 min at 150 °C, 15 min at 200 °C, and 30 min at 300 °C. For these experiments, 90 chips were batch-fabricated on a 3-in. wafer up to the formation of the electrode and polyimide patterns for both the reference electrode and the indicator electrode described next. The chips were cut out using a dicing saw, and discrete chip-level processes were followed after that. The AgCl layer was grown from the slit into the silver layer as described later. Note that no gold layer exists under the area where AgCl is formed. To form a 50-µm-wide liquid junction, the ENT-2000 resin was cast from a 0.7 mm square area in the recess of polyimide and was cured under a UV light. A major component of the resin is poly(ethylene glycol), and the cross-linked polymer gives a hydrophilic porous structure. The electrolyte layer was screen-printed through a 250-µm-thick mask, and the solvent of the paste, 2-propanol, was allowed to evaporate. In preparing the electrolyte paste, KCl was ground into powder using a mortar and
Figure 1. Structure of the miniature liquid-junction Ag/AgCl reference electrode (a) and the AIROF thin-film indicator electrode (b). The indicated layers are sequentially formed.
pestle. One and two-tenths grams of the powdered KCl was mixed with 0.15 g of PVP and 450 µL of 2-propanol to obtain a homogeneous paste. The electrolyte layer was 0.9-mm-wide and covered the entire area of the Ag/AgCl element and the square recess of the liquid junction. The entire structure was passivated with the silicone rubber except for the pad and the end of the liquid junction (see Figure 1a). The reference electrode was activated by injecting 10 µL of a solution saturated with KCl and AgCl from the vicinity of the pad through the passivation using a microsyringe. Although a microscopic through-hole was left after the injection, the area was not immersed in the external electrolyte solution when in use. For comparison, a currently used Ag/AgCl element was also fabricated using the same silver pattern. A silver layer was formed on a gold backbone layer and a AgCl layer was formed on the entire surface. Because the U-shaped gold pattern did not give a good result with the currently used structure, the gold layer was formed under the entire silver layer. The thicknesses of the chromium, gold, and silver layers were the same. The electrode was not subjected to bakings at temperatures higher than 80 °C because the element was fatally damaged by interdiffusion from the gold underlayer.18,20 The AgCl layer was grown on the basis of the same conditions as the novel element. Formation of the AgCl Layer. In growing AgCl, a constant current was applied to the silver thin-film pattern in an unstirred 1.0 M KCl/HCl buffer solution (pH 2.2) using a Hokuto-Denko HA-151 potentiostat/galvanostat and a platinum plate counter electrode. To check chronopotentiograms during the AgCl growth, a commercial macroscopic Ag/AgCl reference electrode (Horiba 2080A-06T with a ceramic-plug junction and an internal solution saturated with KCl and AgCl) was used. As mentioned later, the optimum current was determined to be 3 µA. The current density at the beginning of the growth was calculated to be 0.97 mA/cm2 from the dimensions of the slit. Structure and Fabrication of the pH Indicator Electrode. Figure 1b shows the structure of the anodic iridium oxide film (AIROF) micro pH indicator electrode used to test the miniature liquid-junction reference electrode. The dimensions of the chip (20) Belser, R. B. J. Appl. Phys. 1960, 31, 562-570.
were matched to those of the reference electrode. A 200-nm-thick gold backbone layer was formed on a glass substrate as before. After a lift-off pattern was formed with the positive photoresist, a 300-nm-thick iridium layer was sputter-deposited. The photoresist covered the area other than the large rectangular area in Figure 1b. The iridium thin film on the photoresist was lifted off, and the iridium pattern was formed on the area as shown in Figure 1b. Because the iridium thin film was very brittle, its pattern was formed only on the necessary area on the gold backbone layer. The periphery of the iridium pattern and the lead were protected with a 1.9-µm-thick polyimide layer. The area of the exposed iridium layer was 0.8 mm × 4.9 mm. Electrochemical growth and characterization of the iridium oxide was carried out in a 1.0 M LiClO4 solution.21 Although an H2SO4 solution was used in our previous study,22 the chromium adhesive layer was completely lost during the oxide formation unless appropriate protection was applied at the periphery of the iridium pattern. The dissolution was not observed in the LiClO4 solution. The above-mentioned potentiostat, commercial Ag/AgCl reference electrode, and platinum-plate counter electrode were used. The potential of the iridium pattern was cycled by applying a triangular voltage waveform between - 0.9 and + 1.1 V (vs Ag/ AgCl) at 0.5 V/s using a Hokuto-Denko HB-111 function generator. Cyclic voltammograms during the growth of the AIROF were recorded on a Graphtec WX1100 X-Y recorder. The cycling was stopped at the maximum of the main peak. The mechanism of the oxide growth and the characteristics of the AIROF as a pH indicator electrode have been described elsewhere23-25 and are not described in detail here. The glass substrates with the liquid-junction Ag/AgCl reference electrode and the pH indicator electrode were bonded backto-back. In performing this, the two substrates were fixed with a clip, and the photocurable adhesive was permeated into the (21) Pickup, P. G.; Birss, V. I. J. Electrochem. Soc. 1988, 135, 126-133. (22) Suzuki, H.; Arakawa, H.; Sasaki, S.; Karube, I. Anal. Chem. 1999, 71, 17371743. (23) Conway, B. E.; Mozota, J. Electrochim. Acta 1983, 28, 9-16. (24) Kinoshita, E.; Ingman, F.; Edwall, G.; Thulin, S.; Glab, S. Talanta 1986, 33, 125-134. (25) Hitchman, M. L.; Ramanathan, S. Analyst (Cambridge, U.K.) 1988, 113, 35-39.
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interface using capillary action. Then the chip was exposed to UV light for 10 min to cure the adhesive. Although we used two substrates to complete the one-chip device, it is a technique commonly used in micromachining. The method will be more effective in increasing the production yield of the device than forming all necessary elements on the same substrate. Other Procedures. The open-circuit potential of the thin-film Ag/AgCl element, the completed miniature liquid-junction electrode, or the AIROF indicator electrode was measured in an appropriate solution with a Hokuto Denko HE-106 electrometer having an input impedance of 1 × 1013 Ω. The level of resolution achieved by the electrometer was (0.1 mV. Durability of the thin-film Ag/AgCl element was examined in a solution saturated with KCl and AgCl. An excess amount of KCl and AgCl were added to the solution until these compounds precipitated, and the solution was stirred more than 1 h to reach saturation prior to the experiment. The experiment was conducted in the solution which was in contact with air and saturated with it. The above-mentioned commercial reference electrode was used as the potential standard. In examining the pH dependence of the potential of the miniature liquid-junction reference electrode and the AIROF indicator electrode, the following combinations were used: (1) the miniature liquid-junction reference electrode vs the commercial reference electrode, (2) the AIROF indicator electrode vs the on-chip miniature liquid-junction reference electrode (onechip pH sensor), (3) the AIROF indicator electrode vs the commercial reference electrode. One of these pairs was immersed in an unstirred buffer solution. The used buffer solutions were 20 mM citrate/NaOH, 20 mM KH2PO4/NaOH, and 20 mM borate/NaOH, each of which contained 0.1 M KCl. Separate solutions of various pHs were prepared. All pH measurements were conducted using 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). To examine the response of the one-chip pH sensor to pH step changes, the chip was immersed in 5 mM KH2PO4 solution containing 0.1 M KCl, and an arbitrary amount of NaOH was added to change the pH of the solution stepwise. The solution pH was simultaneously measured with the combination pH glass electrode mentioned above. It must be noted that the measured potential values contain the liquid-junction potential of the macroscopic electrode because we cannot separately measure the contributions from each respective electrode of the pair. Therefore, we assumed that the macroscopic commercial electrode functioned ideally. Actually, the liquid junction potential of the macroscopic electrode was found to be negligible as shown in Figure 2. The data of the series of experiments were recorded on a TOA Electronics PRR-5011 strip-chart recorder. The amount of the solutions used in the formation and evaluation of the electrode elements was 100 mL. All experiments were conducted at a constant temperature of 25.0 ( 0.1 °C. RESULTS AND DISCUSSION Formation of the AgCl Layer. An optimum growth condition for AgCl was determined with reference to the chronopotentiogram. Because of the existence of the polyimide layer which tightly fixes the silver layer, current density increases during the 5072 Analytical Chemistry, Vol. 71, No. 22, November 15, 1999
Figure 2. Durability of the bare thin-film Ag/AgCl elements in an unstirred solution saturated with KCl and AgCl. (a) novel structure, (b) currently used structure. The same silver patterns were used. The numerals in the figure indicate the length of time to form the AgCl layer at 3 µA. Approximately 80% of the silver layer was converted into AgCl for the elements noted “40 min”. The dashed lines indicate the ideal expected potential considering the geometry of the cell. Although this experiment was conducted in a solution containing no PVP, its existence influenced the lifetime of the Ag/AgCl element and was effective in elongating it further as shown in Figure 3.
growth of AgCl at a constant current. This is recognized by polarization in the corresponding chronopotentiogram. At sufficiently high current, the AgCl layer did not grow smoothly, and a substantial portion of the silver layer remained intact even after a significant polarization was observed. A high-quality AgCl layer could not be obtained under such a polarized state possibly due to oxygen evolution.26 Whenever the current was set below 5 µA, the chronopotentiogram was clear-cut, and all the silver layer was completely consumed within the transition time. The potential of the stable flat region in the chronopotentiogram settled in the vicinity of a value expected from the Nernst equation and Clconcentration. With reference to the obtained result and the length of time necessary for the growth of AgCl, the current was set at 3 µA. Durability of the Ag/AgCl Element in a Saturated KCl Solution. In concentrated KCl solutions, solubility of AgCl is higher than that in water by 2 orders of magnitude, and silver exists in the form of AgCln+1-n (n ) 1-3).27,28 Figure 2a shows the durability of the thin-film Ag/AgCl element used in the liquidjunction electrode shown in Figure 1a in an unstirred solution saturated with KCl and AgCl. As the AgCl layer was grown into the silver layer, its lifetime became longer and longer. The element (26) Harzdorf, C. Anal. Chim. Acta 1982, 136, 61-67. (27) Ito, S.; Hachiya, H.; Baba, K.; Asano, Y.; Wada, H. Talanta 1995, 42, 16851690. (28) Katan, T.; Szpak, S.; Bennion, D. N. J. Electrochem. Soc. 1973, 120, 883888.
whose AgCl layer was grown for 40 min had maintained an expected level of potential as an Ag/AgCl electrode for longer than 30 h until a catastrophic potential shift was observed. Fluctuation of the potential of a normally functioning element at each moment was less than 0.1 mV, which was below the detection limit of our instrument. For comparison, the same experiment was also conducted using the currently used Ag/AgCl structure (Figure 2b). Even if the AgCl layer was grown thicker, the lifetime of the electrode was only slightly elongated. The expected potential level could not be maintained even for 10 min. Note the significant difference in time scale between the two sets of data. The results indicate that the polyimide protecting layer in the novel thin-film Ag/AgCl structure worked quite effectively in suppressing the dissolution of AgCl. The growth of the AgCl layer proceeds in a peculiar manner through the growth of AgCl mounds.29 If the AgCl layer is grown from the surface, a homogeneous membrane cannot be obtained especially at the beginning of the process. Harzdorf reported that complete coverage could not be achieved for an AgCl layer thinner than 1 µm. This will permit permeation of the electrolyte solution into the Ag/AgCl interface and cause its destruction.26 Although quality of the AgCl layer also depends on the current density during its growth,26 this would have little effect considering its thickness. It is not easy to examine what is happening in the membrane microscopically when the electrode is breaking. We suppose that a substantial portion of the silver layer had become exposed directly to the electrolyte solution as a result of the dissolution or detachment of the AgCl layer from the underlying silver. Actually, the dissolution of the AgCl layer was often recognized by visual inspection. Although it has been found that an AgCl film spontaneously precipitates upon simply immersing silver in 1-2 M KCl solution, no films are formed in saturated KCl solution.28 The resulting potential will be a mixed potential, which is observed when two or more simultaneously proceeding oxidation and reduction reactions are involved in exhibiting a potential at the electrode/electrolyte interface.9,30,31 Because the current and potential of each contribution are related with the Butler-Volmer equation, the electrode potential settles at a potential under the zero total current condition. It is evident that it is not an equilibrium state. The response on the basis of the mixed potential mechanism is highly dependent on the stirring rate, pH, ionic strength, and ionic environment of the sample solution.9,30-32 The resulting potential is unstable, lacks selectivity and reproducibility, and is influenced by adsorption. In Figure 2 the value of the exhibited potential after the electrode had broken varied substantially, which is considered to be a typical behavior of a mixed potential. In this state the contribution from the Ag/AgCl equilibrium is considered to be small. Otherwise, the high-exchange current reaction of Ag/AgCl would draw the resulting potential closer to its equilibrium potential.33 (29) Katan, T.; Szpak, S.; Bennion, D. N. J. Electrochem. Soc. 1974, 121, 757764. (30) Meruva, R. K.; Meyerhoff, M. E. Anal. Chem. 1996, 68, 2022-2026. (31) Harn, P. B.; Johnson, D. C.; Wechter, M. A.; Voigt, A. F. Anal. Chem. 1974, 46, 553-558. (32) Janata, J. Principles of chemical sensors; Plenum Press: New York, 1989; pp 107-108. (33) Janata, J. Principles of chemical sensors; Plenum Press: New York, 1989; pp 100-102.
Figure 3. Variation of the open-circuit potential of the completed miniature liquid-junction reference electrode. The experiment was conducted in an unstirred 50 mM KH2PO4/NaOH buffer solution containing 0.1 M KCl (pH 7.0). The dashed line indicates the ideal expected potential considering the geometry of the cell. The potential increase at the end of the lifetime (indicated by the arrow) is the Nernst response to decreasing Cl- concentration.
Stability and Lifetime of the Completed Liquid-Junction Reference Electrode. Two main factors which determine the lifetime of a liquid-junction reference electrode are (1) the durability of the thin-film Ag/AgCl element and (2) the effusion and concurrent dilution of the internal KCl solution. If the former is dominant, Figure 2 indicates that the potential shifts to the negative side after the lifetime is up. In the latter case, the Nernst equation suggests that the potential of a normally functioning Ag/ AgCl element drifts to the positive side as the internal KCl is diluted. To establish a stable liquid-junction potential which responds quickly to the sample replacement, a reproducible and timeindependent interface is a prerequisite.34 A free-diffusion junction would be better in this context than those using restraining materials.34,35 However, the effusion of KCl from a miniature reference electrode with this kind of junction was too fast to suppress the dilution of the internal electrolyte.19 To obtain a longlived miniature liquid-junction electrode, a restraining material had to be used to suppress the effusion of KCl. In this study the ENT2000 resin was used as the restraining material. Because the polymer was photocurable, the formation of the liquid junction was easily made by casting it in the polyimide recess and curing it. Contrary to casting polymers dissolved in a solvent and evaporating it, no substantial change in volume was observed, and an expected thickness was obtained without repeating the procedure. Any hydrophilic polymers could be used for the restraining material unless they show pH responses when used in the junction. A major difference would be the rate of effusion of KCl. The faster the effusion, the smaller the liquid junction potential and, vice versa, the shorter the lifetime. The dimensions of the junction also affect the lifetime. Considering the results of our preliminary experiments, the width of the junction was made 50 µm. An appropriate length was chosen considering the ease of pattern formation. Figure 3 shows the variation of the open-circuit potential of the completed miniature liquid-junction reference electrode. The stability within (1.0 mV was maintained for longer than 100 h (34) Dohner, R. E.; Wegmann, D.; Morf, W. E.; Simon, W. Anal. Chem. 1986, 58, 2585-2589. (35) Covington, A. K.; Whalley, P. D.; Davison, W. Anal. Chim. Acta 1985, 169, 221-229.
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with sporadic variations of the potential exceeding this limit. A shift of 1 mV causes an error of approximately 0.02 pH in pH measurement and an approximately 4-8% error in concentration for the measurement of mono- and divalent ions around room temperature.4,27 The level of stability obtained with the present miniature reference electrode would be within an allowable range in ordinary ion-selective-electrode measurements.4 The potential increased to the positive side when the lifetime was up. This is the Nernst response to decreasing Cl- ions. The start of potential increase and the final dissolution of KCl, visible through the silicone rubber, were observed to coincide. Compared with the result shown in Figure 2, the durability of the thin-film Ag/AgCl element was improved further. We suppose this was because the PVP matrix in the internal electrolyte suppressed the dissolution of the AgCl layer more effectively. A similar effect has been observed with Nafion and polyurethane coatings.36 In some previous studies major concerns seem to have been only to decrease the rate of effusion using more effective blocking materials.16,17 The effusion rate of the internal solution of a conventional macroscopic electrode is 0.1 to several milliliters per day.4 Continuous effusion of KCl is essential in eliminating the liquid junction potential effectively.35 If the effusion is stopped, the potential of the reference electrode becomes unstable and fluctuating.4,27,35 Generally, a flow of salt bridge solution along illdefined paths cannot produce a stable and reproducible liquid junction.34,35 In evaluating the performance of miniature liquid-junction Ag/ AgCl reference electrodes, the value of the potential, the extent of potential drift, and fluctuation will be the points to be checked. When a macroscopic Ag/AgCl reference electrode with a saturated KCl internal solution is used as a standard, the potential of a normally functioning miniature Ag/AgCl electrode will settle in the vicinity of the dashed line in Figure 3. Otherwise, it is suspected that either the Ag/AgCl element did not work normally or the liquid junction potential was not eliminated sufficiently due to clogging or dilution of the internal solution. Note that the direction of potential change in Figures 2 and 3 is opposite. Therefore, it is possible that even a miniature liquid-junction reference electrode with a degrading thin-film Ag/AgCl element and diluted internal electrolyte solution exhibits a potential somewhere in the vicinity of the dashed line in Figure 3. We have to check carefully whether the potential is that of a normally functioning electrode or a spurious potential of a broken electrode. Dependence of the Potential of the Miniature LiquidJunction Reference Electrode on External KCl Concentration and pH. A feature which distinguishes a liquid-junction reference electrode from a pseudo-reference electrode is its independence from the potential on external KCl concentration. The potential of the pseudo-Ag/AgCl reference electrode varies on the basis of the Nernst equation, while the dependence must ideally be nullified with the liquid-junction reference electrode. To check this, the potential of the miniature liquid-junction reference electrode was measured in 50 mM KH2PO4/NaOH buffer solutions containing various concentrations of KCl (Figure 4). As a general tendency, variation of the potential was small compared with the Nernst response of the Ag/AgCl element to Cl-. In this sense, the electrode gave a potential expected as a liquid-junction (36) Moussy, F.; Harrison, D. J. Anal. Chem. 1994, 66, 674-679.
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Figure 4. Dependence of the open-circuit potential of the miniature liquid-junction reference electrode on external KCl concentration. The experiment was conducted in an unstirred 50 mM KH2PO4/NaOH buffer solution (pH 7.0) containing various concentrations of KCl.
reference electrode. However, careful examination of the result reveals 2.5 mV of potential difference exists between 1 mM and 1 M solutions, with the potential approaching to zero at higher KCl concentrations. This indicates that the liquid-junction potential was not eliminated completely in diluted KCl solutions as a result of the slow diffusion of KCl. The cause of this liquid junction potential will be the existence of ions other than K+ and Cl-. In view of Figure 4, the electrode potential would be affected by these ions when the external KCl concentration is low and would depend on ionic strength. Depending on the situations, the adjustment of the effusion rate of internal KCl will again be required. The potential dependence on pH was also examined in the three types of buffer solutions mentioned before (citrate/NaOH, KH2PO4/NaOH, and borate/NaOH) whose pHs ranged approximately between 4 and 10. The major origin of the pH dependency will be the liquid junction. However, no noticeable tendency was found, and variation of the measured potential values was within ( 0.2 mV. The result also showed that the effect of different types of buffer solutions was negligible. Applicability of the Miniature Liquid-Junction Reference Electrode to a One-Chip Potentiometric Sensor. The durability of the miniature reference electrode was tested from a different point of view. The one-chip sensor with the integrated miniature reference electrode was stored by immersing the sensitive area of the pH indicator electrode and the liquid junction of the reference electrode in an unstirred 50 mM KH2PO4/NaOH buffer solution containing 0.1 M KCl (pH 7.0, 25 °C). The pH dependence of the indicator electrode potential was examined at 1 and 25 h after the start of the experiment using either the on-chip miniature reference electrode or the commercial reference electrode (Figure 5). No substantial difference in the output potential was observed between the two sets of data taken with the miniature reference electrode and the commercial reference electrode. Note that this level of coincidence can be achieved only with a liquid-junction reference electrode. In Figure 5, a slight difference in the output potential is observed between the data taken at different times. This is clearly due to the drift of the potential of the indicator electrode, which is peculiar to the AIROF.25 Improvement in the indicator electrode potential was not pursued here because it was beyond the scope our present study.
Figure 5. Dependence of the AIROF indicator electrode potential on pH. The potential was measured against the miniature liquidjunction reference electrode (O) and the macroscopic reference electrode (b). The chip consisting of the indicator electrode and the miniature reference electrode was stored by immersion in 50 mM KH2PO4/NaOH buffer solution containing 0.1 M KCl (pH 7.0). The pH dependence was examined at (a) 1 h and (b) 25 h after the activation of the miniature reference electrode. The buffer solutions used were 20 mM citrate/NaOH (pH: approximately 4-6), 20 mM KH2PO4/NaOH (pH: approximately 6-8), and 20 mM borate/NaOH (pH: approximately 8-10).
Figure 6 shows a typical response curve of the one-chip pH sensor to stepwise pH changes. The solution pH was simultaneously measured with the pH combination glass electrode. The output potential was stable, and a distinct response was observed. The 90% response times were generally found to be less than 10 s, which seems reasonable, especially if the time required to change the solution pH was considered. The same experiment was conducted in some other buffer solutions whose pHs ranged approximately from 3 to 10. Similar clear-cut responses were observed. CONCLUSIONS A planar miniature liquid-junction Ag/AgCl reference electrode was fabricated using thin-film and thick-film processes. This type of electrode could be classified as one of the most long-lived and stable microfabricated liquid-junction reference electrodes. Although its lifetime is still limited, it will be sufficient for a shortterm disposable use. The novel technologies and ideas presented here are promising, and the fabrication of miniature reference electrodes with even longer lifetimes has become a technical
Figure 6. Response curve of the one-chip pH sensor to pH step changes. The numerals above the curve indicate the pH at the corresponding stabilized level which was simultaneously measured with the pH glass electrode. An arbitrary amount of NaOH was added to a 5 mM KH2PO4 solution containing 0.1 M KCl successively at the times indicated by the arrows.
problem. As already mentioned, the lifetime of the liquid-junction reference electrode is dominated by two major factors: (1) durability of the thin-film Ag/AgCl element and (2) dilution of the internal KCl. The obtained results suggested that the existence of a PVP layer on the Ag/AgCl element was effective in suppressing the dissolution of AgCl, which will be a critical point in realizing a more durable thin-film Ag/AgCl element. As with the second point, no further improvement could be expected because the total amount of stored KCl is a determining factor. The designing of a novel liquid-junction structure might be helpful in reducing the effusion of KCl further. However, it must be noted that it is a tradeoff between the elimination of the liquid-junction potential. The effective use of dead volume on the chip will also be an effective solution especially in the µTAS (Micro Total Analysis System), and even the lifetime of one month could be attained. Because the liquid-junction Ag/AgCl reference electrode is so basic and general, many applications can be considered including one-chip potentiometric sensors and electrochemical analyses in a minute sample solution. ACKNOWLEDGMENT This work was partially supported by the Research for the Future Program of JSPS. Received for review April 26, 1999. Accepted August 16, 1999. AC990437T
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