Fabrication of a Planar-Form Screen-Printed Solid Electrolyte Modified

Anal. Chem. 2006, 78, 4219-4223

Technical Notes

Fabrication of a Planar-Form Screen-Printed Solid Electrolyte Modified Ag/AgCl Reference Electrode for Application in a Potentiometric Biosensor Wei-Yin Liao and Tse-Chuan Chou*

Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan

This study features the fabrication of a planar-form, solid electrolyte modified, (PSEM) Ag/AgCl reference electrode using a screen-printing method. The PSEM Ag/AgCl reference electrode uses agar gel as the inner electrolyte and chloroprene rubber for the liquid junction and insulator. These common low-cost materials and the simple fabrication processes involved render the proposed reference electrode an ideal candidate for cost-efficient mass production. It is shown that the developed reference electrode is insensitive to most of the physiologically important ionic species, including Na+, K+, Li+, Ca2+, NH4+, and Cl-, under continuous measurement conditions. Moreover, as with conventional commercial reference electrodes, the proposed reference electrode exhibits a reversible response, which is maintained until the agar gel dries out. The PSEM Ag/AgCl reference electrode is integrated with an iridium oxide modified Pt-based pH indicator electrode to form a chip-type pH biosensor. The performance of this biosensor is consistent with that obtained from a pH meter based on a macroscopic commercial Ag/AgCl reference electrode. The experimental results confirm that the proposed biosensor is capable of providing precise pH measurements of various real samples. Accordingly, the PSEM Ag/AgCl reference electrode presented in this study provides a viable alternative to the macroscopic Ag/AgCl reference electrode used in many conventional chip-based pH sensors. Recently, intensive research activity has focused on developing miniaturized electrochemical sensors for micro- or even submicroscale devices intended for extracelluar fluid measurement,1-3 clinical diagnosis,4,5 implantation,6,7 and micro total analysis * Corresponding author. Tel: 886-6-2757575 ext 62639. Fax: 886-6-2366836. E-mail: [email protected]. (1) Bakker, E. Anal. Chem. 2004, 76, 3285-3298. (2) Chen, J. R.; Miao, Y. Q.; He, N. Y.; Wu, X. H.; Li, S. J. Biotechnol. Adv. 2004, 22, 505-518. (3) Erickson, D.; Li, D. Q. Anal. Chim. Acta 2004, 507, 11-26. (4) Cunningham, D. D. Anal. Chim. Acta 2001, 429, 1-18. (5) Tudos, A. J.; Besselink G. A. J.; Schasfoort, B. M. Lab Chip 2001, 1, 8395. (6) Wilson, G. S.; Gifford, R. Biosens. Bioelectron. 2005, 20, 2388-2403. 10.1021/ac051562+ CCC: $33.50 Published on Web 05/16/2006

© 2006 American Chemical Society

system8,9 applications. Such sensors are generally fabricated using screen-printing or semiconductor-compatible fabrication techniques since these methods provide a precise control over the product dimensions, excellent uniformity, high reproducibility, and the potential for mass production. However, in developing these miniaturized electrochemical sensors, reducing the scale of the macroscopic commercial reference electrode with an internal filling solution remains a major challenge. Over the years, various fabrication methods have been proposed for producing miniaturized reference electrodes of different types. For example, solid-state reference electrodes have been fabricated by the following procedures: (1) coating a metallic surface with an electrolyte-doped polymeric material such as poly(vinyl acetate)/KCl,10 quaternized poly(chloromethylstrene)/Cl-,11 PVC/NaCl12, low melting point glass paste/KCl and silicon polymer/KCl,13 or commercial paste/KCl;14 (2) protecting the Ag/ AgCl electrode directly with Nafion;15,16 (3) depositing ion-selective membranes on parallel Ag/AgCl electrodes;17,18 (4) doping polyurethane with lipophilic salts and ionophores to form a liquid junction-free solvent-processible polymer membrane;19,20 and (5) (7) Newman, J. D.; Turner, A. P. F. Biosens. Bioelectron. 2005, 20, 24352453. (8) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (9) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (10) Diamond, D.; McEnroe, E.; McCarrick, M.; Lewenstam, A. Electroanalysis 1994, 6, 962-971. (11) Kinlen, P. J.; Heider, J. E.; Hubbard, D. E. Sens. Actuators, B 1994, 22, 13-25. (12) Nolan, M. A.; Tan, S. H.; Kounaves, S. P. Anal. Chem. 1997, 69, 12441247. (13) Cranny, A. W. J.; Atkinson, J. K. Meas. Sci. Techol. 1998, 9, 1557-1565. (14) Tymecki, L.; Zwierkowska, E.; Koncki, R. Anal. Chim. Acta 2004, 526, 3-11. (15) Moussy, F., Harrison, D. J. Anal. Chem. 1994, 66, 674-679. (16) Nolan, M. A.; Tan, S. H.; Kounaves, S. P. Anal. Chem. 1997, 69, 12441247. (17) Nagy, K.; Eine, K.; Syverud, K.; Aune, O. J. Electrochem. Soc. 1997, 144, L1-L2. (18) Eine, K.; Kjelstrup, S.; Nagy, K.; Syverud, K. Sens. Actuators, B 1997, 44, 381-388. (19) Lee, H. J.; Hong, U. S.; Lee D. K.; Shin J. H.; Nam, H.; Cha, G. S. Anal. Chem. 1998, 70, 3377-3383. (20) Yoon, H. J.; Shin J. H.; Lee D. K.; Nam, H.; Cha, G. S.; Strong T. D.; Brown, R. B. Sens. Actuators, B 2000, 24, 8-14.

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using a polyion-responsive membrane.21,22 In an alternative approach, the Suzuki groups employed microelectromechanical systems technology23-25 to fabricate micromachined liquid junction Ag/AgCl reference electrodes26-29 for chip-based potentiometric sensors. However, their fabrication process is complicated. This study develops a novel planar-form, solid electrolyte modified (PSEM) Ag/AgCl reference electrode fabricated from common low-cost materials using a simple screen-printing method. The electrochemical characteristics of the proposed reference electrode are examined by potentiometry in a two-electrode system and by cyclic voltammery in a three-electrode system. The stability of the proposed reference electrode was investigated by continuously measuring the potential shift relative to the signal obtained from a commercial reference electrode in various physiologically important ionic solutions. Finally, the proposed reference electrode was integrated with a pH indicator to form a chip-based pH sensor. This sensor was then used to perform in vitro measurements of various samples, including human serum, human whole blood, Coca Cola, tap water, orange juice, and low fat milk. EXPERIMENTAL SECTION Materials and Reagents. The chemicals used to fabricate the PSEM Ag/AgCl reference electrode were acquired from the following sources: agar (Lancaster), potassium chloride (Aldrich), iron(III) chloride (Showa), silver paste (Heraeus Inc.), chloroprene rubber (Kuo Sen enterprise, Taiwan), and alumina substrate (Laser Tek Taiwan Co.). The test sample solutions of lithium chloride, potassium chloride, sodium chloride, calcium chloride, magnesium chloride, ammonium chloride, calcium nitrate, sodium nitrate, and sodium hydrogen carbonate were all analytical grades and were purchased from Aldrich. The iridium chloride (IrCl4‚ xH2O), hydrogen peroxide, potassium oxalate, and potassium carbonate used in the fabrication of the pH indicator were purchased from Alfa Aesar and Showa. All of the solutions in the present study were made using deionized water (Millipore Water System) with a resistance of 18.3 MΩ. Structure and Fabrication of PSEM Ag/AgCl Reference Electrode and pH Indicator Electrodes. The structure of PSEM Ag/AgCl reference electrode is shown on the left-hand side of Figure 1 and was fabricated using a screen-printing technique. The alumina substrate of the proposed reference electrode measured approximately 3 cm × 0.5 cm (length × width). Initially, a silver paste was printed onto the substrate and dried at 150 °C for 30 min. The substrate was then calcined at 850 °C for 20 min. The area III (1 cm × 0.2 cm) of the resulting silver electrode was then immersed in a 1 M FeCl3 solution for 1 min to form a AgCl layer, which was then cleaned with deionized water. Agar solution (21) Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 2250-2259. (22) Meyerhoff, M. E.; Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C. Anal. Chem. 1996, 68, 168A-175A. (23) Lindner, E.; Buck, R. P. Anal. Chem. 2000, 72, 336A-345A. (24) Madaras, M. B.; Buck, R. P. Anal. Chem. 1996, 68, 3832-3839. (25) Cosofret, V. V.; Erdosy, M.; Johnson, T. A.; Buck, R. P. Anal. Chem. 1995, 67, 1647-1653. (26) Suzuki, H.; Hiratsuka A., T.; Sasaki, S.; Karube, I. Sens. Actuators, B 1998, 46, 104-113. (27) Suzuki, H.; Hirakawa, T.; Sasaki, S.; Karube, I. Sens. Actuators, B 1998, 46, 146-154. (28) Suzuki, H.; Shiroishi, H. Anal. Chem. 1999, 71, 5069-5075. (29) Suzuki, H.; Taura, T. J. Electrochem. Soc. 2001, 148, E468-E474.

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Figure 1. Schematic illustration of the PSEM Ag/AgCl reference electrode (left side) and pH indicator (right side): I, alumina substrate. II, Ag electrode. III, Ag/AgCl. IV, agar gel. V, chloroprene rubber. VI, Pt electrode. VII, iridium oxide.

(30 µL) at a temperature of 65 °C (0.8 g agar dissolved in 50 mL of 1 M KCl boiling solution) was smeared over the AgCl area and then left to cool at room temperature to form a KCl gel layer. Then, the KCl agar gel and silver electrode were covered with a protective chloroprene rubber, leaving just a binding pad area (for contacting the potentiostat) uncovered. Finally, the resulting multilayer electrode was then left for 2 days at room temperature to stabilize. The structure of the pH indicator electrode is shown on the right-hand side of Figure 1. The fabrication procedure for the pH indicator was the same as the development process of the PSEM Ag/AgCl reference electrode. But, the Pt was used as a conductive layer for the selective electrical deposition of iridium oxide. After the planar Pt electrode was formed, the electrode was immersed in a deposition solution prepared in accordance with the method presented by Yamanaka.30 The iridium oxide was deposited on the platinum electrode using cyclic voltammery between 0.0 and 0.6 V versus a commercial reference electrode at 20 mV s-1 for 300 cycles. Evaluation of the PSEM Ag/AgCl Reference Electrode. The potential shifts of the proposed reference electrode were established by performing potentiometric measurements using a two-electrode system in which the working electrode was the PSEM Ag/AgCl electrode and the reference electrode was a commercial Ag/AgCl electrode with an internal saturated KCl solution. The potential signal outputs of the two electrodes were measured in test solutions with chloride ion concentrations ranging from 10-6 to 0.3 M. The stability of the PSEM Ag/AgCl reference electrode was investigated by adding different salts, i.e., nitrate, carbonate, phosphate, acetate, and sulfate, to the test solutions in concentrations ranging from 10-6 to 0.3 or 10-1 M. Finally, cyclic voltammery with a scanning rate of 10 mV/s was employed to investigate the reversibility of the proposed electrode in a solution of 0.1 M KNO3. All of the measurement results were recorded using a potentiostat (CHI Instruments, model 1000). Application of the PSEM Ag/AgCl Reference Electrode to Potentiometric pH Biosensor for in Vitro Measurements. The PSEM Ag/AgCl reference electrode was integrated with the iridium oxide modified platinum pH indicator electrode by bonding the nonfunctional sides of the two electrodes with epoxy. To examine the characteristics of the pH indicator and the effect of (30) Yamanaka, K. Jpn. J. Appl. Phys. 1989, 28, 632-637.

Figure 2. Dynamic emf response curves of the PSEM Ag/AgCl electrode and unmodified Ag/AgCl electrode vs commercial Ag/AgCl reference electrode in solutions with various chloride ion concentrations.

pH on the PSEM Ag/AgCl reference electrode, the following combinations were coupled and tested: (1) PSEM Ag/AgCl reference electrode versus the commercial reference electrode, (2) pH indicator versus the commercial reference electrode, and (3) pH indicator electrode integrated with the PSEM Ag/AgCl reference electrode device. The pH dependence of the potential signal generated by these various combinations was measured by using potentiometric measurements in a buffer solution of 0.1 M KNO3, additionally containing H3PO4, H3BO3, and CH3COOH (all 0.01 M)31 together with arbitrary amount of 1 M KOH or 1M HNO3, added in a stepwise manner, to change the pH of the buffer solution. The pH of the buffer solution was continuously monitored using a commercial pH meter (model CyberScan pH/Ion 510, Eutech Instruments Pte Ltd., Singapore). To investigate the suitability of the developed pH biosensor for practical sensing applications, the device was used to perform in vitro measurements of various real samples, including human serum, human whole blood, standard buffer, Coca Cola, tap water, orange juice, and low fat milk. RESULTS AND DISCUSSION Electrochemical Characterization of the PSEM Ag/AgCl Reference Electrode. When a silver-silver chloride electrode is placed in an aqueous solution, the silver chloride achieves a reversible equilibrium with the solid silver and the chloride ions. At steady state, the equilibrium potential (E) associated with this reversible reaction can be expressed by the Nernst equation.32 Under the assumptions of an ideal solution behavior and a specific temperature (e.g., 25 °C), the Nernst equation of the silver-silver chloride electrode can be simplified such that the electrode potential is related to the chloride ion concentration via a slope of -59.16 mV/pCl-. Consequently, the Ag/AgCl electrode will be predicted to have potential shifts in the test solutions containing variable chloride ion concentrations. As shown by the dashed line in Figure 2, the unmodified Ag/AgCl electrode versus the (31) Fog, A. Sens. Actuators 1984, 5, 137-146. (32) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York, 2002.

commercial reference electrode has significant potential shifts of ∼275 mV as the chloride ion concentration in the test solutions is stepwise increased from 10-6 to 0.3 M. Hence, it cannot be a standard electrode under the solution of chloride ion concentration changing dramatically. However, the potential shift between the proposed reference electrode and the commercial reference electrode (solid lines in Figure 2) is very small, i.e., less than 10 mV, over the same chloride ion concentration range. In other words, a 1 order of magnitude change of the chloride ion concentration of the test solutions results in a potential shift of just 1.5-2 mV. From Figure 2, it can be seen that the proposed reference electrode shows only a small potential change in the test solutions containing different cations, e.g., potassium, sodium, lithium, calcium, magnesium, and ammonium. Therefore, it is apparent that the proposed reference electrode is not influenced by changes in the chloride ion concentration or by the presence of common alkaline-earth metal ions. This is likely because the agar gel layer of the electrode provides chloride ions, which enable the Ag/AgCl electrode to establish an equilibrium and therefore to generate a constant potential, a fundamental requirement for any commercial reference electrode. Moreover, the chloroprene rubber used as the outer layer of the proposed reference electrode prevents serious leakage of KCl from agar gel into the test solution and prevents the interference ions diffusing into the agar gel. An important characteristic of any reference electrode is a reversible and nonpolarizable32 property. In the current study, cyclic voltammery was performed using a three-electrode system comprising the PSEM Ag/AgCl electrode as the working electrode, a 4-cm2 Pt foil as the counter electrode, and a commercial reference electrode. The result shows a purely resistive load. According to the literature,32 the i-E curve of a cell with two ideal nonpolarizable electrodes as working and reference electrodes would like a pure resistance. Therefore, the result told us that the proposed reference electrode is nonpolarizable. In other words, the PSEM Ag/AgCl electrode has good reversibility and provides a viable alternative to established commercial reference electrodes. Stability of the PSEM Ag/AgCl Reference Electrode. In practical applications, it is important that the potential of the proposed reference electrode is not affected by the presence of other substances in the solution of interest. Accordingly, the sensitivity of the proposed electrode was evaluated by measuring its potential signal versus the commercial reference electrode continuously as the concentration of various salts in the testing solution was stepwise increased. Figure 3 shows that the potential shifts of the proposed electrode versus the commercial reference electrode vary by no more than 10 mV for different kinds of salt concentrations. In other words, the proposed reference electrode is insensitive to the salts typically used in the preparation of buffer solutions. From the results of Figure 3, it can be concluded that the stability characteristic of the proposed electrode rivals that of commercial reference electrodes. Therefore, the proposed reference electrode can be regarded as a potential standard for potentiometric sensors in aqueous solutions. Analytical Applications of the PSEM Ag/AgCl Reference Electrode. The PSEM Ag/AgCl reference electrode was integrated with an iridium oxide modified platinum electrode to form a chip-type pH biosensor. The corresponding response curves of the chip-type pH biosensor and individual electrode are presented Analytical Chemistry, Vol. 78, No. 12, June 15, 2006

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Figure 3. Dynamic emf response curves of the PSEM Ag/AgCl electrode vs commercial reference electrode in solutions of nitrate, acetate, carbonate, phosphate, and sulfate with varying concentrations.

Figure 5. Potential response curves of pH indicator with commercial reference electrode (solid line) and developed pH biosensor (dashed line). (I) standard buffer pH 4.01, (II) standard buffer pH 7.01, (III) standard buffer pH 10.01, (IV) Coca Cola, (V) tap water, (VI) orange juice, (VII) milk (low fat), (VIII) human serum, and (IX) human whole blood. Table 1. In Vitro pH Measurement Results Obtained Using Developed pH Biosensor and Commercial pH Meter samples standard bufferb pH 4.01 standard bufferb pH 7.01 standard bufferb pH 10.01 Coca Cola tap waterc orange juiced

Figure 4. Stability test: (a) the PSEM Ag/AgCl electrode vs commercial reference electrode in different pH solutions, (b) iridium oxide modified Pt pH indicator electrode vs commercial reference electrode and (c) chip-type pH biosensor. Curves obtained by stepwise titrating 1 M KOH solutions against 0.1 M KNO3, additionally containing H3PO4, H3BO3, and CH3COOH (all 0.01 M) within pH range of 2.00-10.11.

in Figure 4. The line a indicates the potential shift of the PSEM Ag/AgCl electrode versus a commercial reference electrode, the line b indicates the potential shift relative to the pH indicator and commercial reference electrode, and the line c refers to the potential shifts of chip-type pH biosensor. There are two important facts apparent from Figure 4. First, examining line a, it is found that potential shift of the PSEM Ag/AgCl reference electrode remains within 8-9 mV of the potential shift relative to the commercial reference electrode over a pH range of 2.00-10.11. In other words, the proposed reference electrode is insensitive to the pH change of the test solution. Second, comparing line b and line c in Figure 4, it is observed that the two response curves exhibit slopes equal to -74.40 mV/pH for the pH indicator relative to the commercial reference electrode and -74.35 mV/pH for the pH indicator relative to the PSEM Ag/AgCl reference electrode. The pH indicator provides the same response irrespective of 4222 Analytical Chemistry, Vol. 78, No. 12, June 15, 2006

milke(low

fat)

human serumf human

bloodf

found Ia IIa I II I II I II I II I II I II I II I II

4.01 4.05 7.02 6.97 9.95 10.03 2.47 2.47 7.56 7.51 3.82 3.96 6.47 6.46 7.44 7.32 7.35 7.34

3.98 4.07 7.00 6.99 9.97 9.96 2.45 2.49 7.51 7.48 3.92 3.80 6.49 6.45 7.33 7.30 7.38 7.37

3.97 4.05 7.01 7.03 9.99 9.97 2.48 2.44 7.55 7.55 3.94 3.97 6.51 6.43 7.32 7.46 7.44 7.43

average value

pH meter

3.99 4.07 7.01 7.00 9.97 9.99 2.47 2.47 7.54 7.51 3.89 3.91 6.49 6.47 7.36 7.36 7.39 7.38

4.01 7.01 10.01 2.47 7.53 3.86 6.54 7.38 7.39

a I: calculated from the calibration curve of the pH indicator versus commercial reference electrode (potential (mV) ) 763.45 mV - 74.4 pH). II: calculated from the calibration curve of the chip-type pH biosensor (potential (mV) ) 754.45 mV - 74.35 pH). b From Hanna Instruments. c From Taiwan Water Supply Co., Tainan, Taiwan. d From Wei Chuan Corp., Taiwan. e From Uni-President Corp., Tainan, Taiwan. f From health woman blood, Tainan, Taiwan.

whether the reference electrode is a commercial reference electrode or the solid electrolyte modified Ag/AgCl electrode. In other words, the PSEM Ag/AgCl reference electrode is a suitable standard electrode for pH biosensors. In Vitro Measurement Results of Developed Chip-Type pH Biosensor. Chip-type pH biosensors provide a convenient tool for performing the in vitro measurement of biosamples such as human serum and human whole blood. In this study, the developed pH biosensor was used to investigate the pH of various samples, including standard buffers, human serum, human whole blood, Coca Cola, tap water, orange juice, and milk (low fat). The corresponding results are presented in Figure 5 and Table 1.

Figure 5 shows the real-time potential as measured by the pH biosensor and pH indicator versus a commercial reference electrode. It can be seen that the pH biosensor rapidly attains a steady potential in all of the samples as well as the pH indicator using a commercial reference electrode. The steady potential measurements presented in Figure 5 can be converted to a corresponding pH value by means of the calibration curves in Figure 4. The equivalent pH values are summarized in Table 1 together with the average pH value. For comparison purposes, the pH of each sample was also measured using a commercial pH meter. It is clear that that the average pH values of the different samples obtained using the developed pH biosensor are in good agreement with the results obtained from the commercial pH meter, even in complex samples such as serum and blood. CONCLUSIONS Using inexpensive and commercially available materials (agar and chloroprene rubber), this study has successfully fabricated a planar-form solid electrolyte modified Ag/AgCl reference electrode using a straightforward screen-printing method. The electrochemical characterization results have shown that the proposed refer-

ence electrode is insensitive to the following: (1) chloride ions, (2) common alkaline-earth metal ions, (3) pH changes, and (4) nitrate, acetate, carbonate, phosphate, and sulfate salt concentrations in the solution of interest. The proposed reference electrode has been successfully integrated with a pH indicator to form a chip-type pH biosensor for in vitro measurements. The experimental results have shown that the developed pH biosensor can precisely identify the pH of various real samples, including human serum, human whole blood, Coca Cola, tap water, orange juice, and low fat milk. ACKNOWLEDGMENT The support provided to this study by the Ministry of Education of the Republic of China (Grant EX-94-E-FA09-5-4) and by National Cheng Kung University is gratefully acknowledged.

Received for review August 31, 2005. Accepted March 1, 2006. AC051562+

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