PROCESSIBLE POLYANILINE AS AN ADVANCED ... - ACS Publications

Publication Date (Web): May 19, 1999 ... Abstract. An advanced potentiometric pH transducer based on processible polyaniline (PCPAn) is reported on. ...
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Anal. Chem. 1999, 71, 2534-2540

PROCESSIBLE POLYANILINE AS AN ADVANCED POTENTIOMETRIC pH TRANSDUCER. APPLICATION TO BIOSENSORS Arkady A. Karyakin,*,†,‡ Maika Vuki,‡,§ Lylia V. Lukachova,† Elena E. Karyakina,†,‡ Andrey V. Orlov,| Galina P. Karpachova,| and Joseph Wang*,‡

Faculty of Chemistry of M.V. Lomonosov Moscow State University, 119899 Moscow, Russia, Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003, and A.V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Science, Leninskii prospect 29, 117912, Moscow, Russia

An advanced potentiometric pH transducer based on processible polyaniline (PCPAn) is reported on. Both glassy carbon and screen-printed carbon electrodes modified with PCPAn by dip-coating exhibited a fully reversible potentiometric response of approximately 90 mV/pH unit over the range from pH 3 to 9. Such a significantly higher potentiometric response of PCPAn-modified electrodes as compared with those of existing devices is explained on the basis of the thermodynamics of polyaniline redox reactions. The PCPAn-based pH transducers exhibit both good operational stability and prolonged shelf life and display a negligible response toward singly charged cations. The new thick-film pH transducer was employed for designing a potentiometric biosensor for urea. In the model solution which mimics blood serum, the ureasensitive electrode has a detection limit of 10-5 M urea and a maximum response of ∼120 mV. The attractive performance characteristics are advantageous over those of existing pH sensors and offer great promise for sensing and biosensing applications. The measurement of the solution pH is an important task required in clinical diagnostics, environmental and industrial control, and various branches of modern science and technology. The most widely used, glass-membrane, electrodes possess the highest stability in pH sensing, but the applications of them may suffer from the disadvantages of high impedance and fragility. A particular case of application of the pH transducers is the development of potentiometric biosensors. Since the pioneering work of Guilbault and Montalvo,1 potentiometric biosensors have been developed for a variety of important substances including urea, glucose, penicillin and organic phosphates. This interest in potentiometric biosensors relies on a variety of the enzymes, which catalyze the conversion of the target analyte while consuming or liberating protons. Some of these enzymes are already * Corresponding authors: RUSSIA (tel.) (7-095) 939 2804, (fax) 7-095-939 2742, (e-mail) [email protected]; USA (e-mail) [email protected]. † Moscow State University. ‡ New Mexico State University. § Permanent address: University of the South Pacific, Suva, Fiji. | Russian Academy of Science. (1) Guilbault, G. G.; Montalvo, J. J. Am. Chem. Soc. 1969, 91, 2164-2165.

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included in analytical kits as terminal ones, which recognize the specific analyte. In these cases, the potentiometric electrodes allow the direct, reagentless detection of analytes, whereas the amperometric or spectrophotometric methods require several sequential reactions. The main disadvantage of potentiometric biosensors is the dependence of their response on buffer capacitance. However, for the variety of systems in clinical diagnostics, the food industry, and environmental control, with either stable or low buffer capacitance, the potentiometric biosensors seem to be very useful. The fundamental sensitivity limit of potentiometric enzyme electrodes is the sensitivity of the pH transducer. New pH-sensitive transducers, offering high sensitivity, are highly desired for enhancing the performance of potentiometric biosensors. The commonly used glass pH electrode contains the internal solution with the reference. The response of the electrode is provided by the difference in the electrochemical potentials of hydrogen ions inside and those outside the H+-permeable glass membrane. The Nernst equation predicts 59 mV per pH unit for the theoretical limit of sensitivity of such electrodes. The more advanced technology of pH sensing based on Ion Selective Field Effect Transistors (ISFETs) allows both the miniaturization of the transducer and the improved precision of measurements. The actual sensitivity of silicone nitride based ISFETs to proton activity is 45-54 mV/pH unit,2 which is lower than that of glass electrodes (55-58 mV/pH unit). The sensitivity of the ISFETs can be improved by aminating the silicon surface. The pH response of the resulting membranes was reported to be Nernstian.3 However, silicon-based ISFETs seem to operate almost as ideally polarizable electrodes, which change their stationary potential upon proton absorption. Thus, the limit of their sensitivity is also a Nernstian slope of 59 mV per pH unit. This fundamental sensitivity limit of potentiometric pH transducers can be addressed using chemically modified electrodes. Indeed, when the electroactive material undergoes a redox reaction involving more than one proton per electron, the slope of the equilibrium potential of the corresponding modified electrode should exceed the value of 59 mV/pH unit. Certainly, (2) Caras, S. D.; Janata, J.; Saupe, D.; Schmitt, K. Anal. Chem. 1985, 57, 19171928. (3) Baccar, Z. M.; Jaffrezicrenault, N.; Lemiti, M. J. Electrochem. Soc. 1997, 144, 3989-3992. 10.1021/ac981337a CCC: $18.00

© 1999 American Chemical Society Published on Web 05/19/1999

the stationary potential of such electrodes is dependent on the presence of electroactive species in solution. However, for biosensor application this disadvantage is not significant. Indeed, the response can be measured as the difference in potentials of two similar electrodes, one of which contains the enzyme and the other of which does not. Such a differential circuit practically eliminates the influence of interferences such as pH, electroactive species, cations, etc. Among the various materials tested as possible pH transducers, the metal oxides were most intensively investigated. Fog and Buck4 investigated a number of metal oxides including RuO2, PtO2, TiO2, OsO2, IrO2, RhO2, Ta2O5, and SnO2. Their results indicated that IrO2 and RuO2 held the most promise, yielding a near Nernstian response over a wide pH range. There are also more recent reports on the use of platinum, palladium, ruthenium, and iridium oxides for pH sensing.5-8 An super Nernstian response of metal oxide pH transducers was reported in the case of iridium and tungsten oxides. The electrochemically deposited iridium oxides reached a slope of 83 mV/pH unit above pH 6, with an overall average of 74 mV/pH unit over a wide pH region.9 Tungsten oxide bronze exhibited a Nernstian behavior,10 but also was shown to exhibit a slope of 76 mV/pH unit in the pH range of 5-9.11 The highest potentiometric response, of 90-100 mV/pH unit in the range of 3-14, was reported for alkaline pretreated aluminum oxide electrodes.12 However the response was totally irreversible and was observed only in the acidic direction after keeping the electrode in a strong basic medium (pH > 10.5). Obviously, such electrodes are of no use for routine sensing and especially for biosensing applications. A number of organic materials were also tested as potential pH transducers. In particular, electrodes modified with anthraquinone,13 phthalocyanine,14 Co-porphirine,15 calixarenes,16 and polyaniline derivatives17,18 exhibited a near Nernstian response. The present paper describes a new, highly sensitive polyanilinebased pH sensor with many attractive performance characteristics. Polyaniline represents one of the most promising materials for pH sensing. As we already reported, the potentiometric response of ‘self-doped’ polyaniline was approximately 70 mV/pH unit in (4) Fog, A.; Buck, R. P. Sens. Actuators 1984, 5, 137-146. (5) Kreider, K. G.; Tarlov, M. J.; Cline, J. P. Sens. Actuators, B 1995, B28, 167-172. (6) Tarlov, M. R.; Semancik, S.; Kreider, K. J. Sensors and Actuators, B 1990, B1, 293-297. (7) Mihell, J. A.; Atkinson, J. K. Sens. Actuators, B 1998, B48, 505-511. (8) McMurray, H. N.; Douglas, P.; Abbot, D. Sens. Actuators, B 1995, B28, 9-15. (9) Baur, J. E.; Spaine, T. W. J. Electroanal. Chem. 1998, 443, 208-216. (10) Kriksunov, L. B.; Macdonald, D. D.; Millett, P. J. J. Electrochem. Soc. 1994, 141, 3002-3005. (11) Shuk, P.; Ramanujachary, K. V.; Greenblatt, M. Electrochim. Acta 1996, 41, 2055-2058. (12) Zhou, T. A.; Ottova, A.; Tien, H. T. J. Electrochem. Soc. 1994, 141, 11421146. (13) Shiu, K. K.; Song, F. Y.; Dai, H. P. Electroanalysis 1996, 8, 1160-1164. (14) Kang, T. F.; Xie, Z. Y.; Tang, H.; Shen, G. L.; Yu, R. Q. Talanta 1997, 45, 291-296. (15) Blair, T. L.; Allen, J. R.; Daunert, S.; Bachas, L. G. Anal. Chem. 1993, 65, 2155-2158. (16) Mlika, R.; Benouada, H.; Jaffrezicrenault, N.; Dumazet, I.; Lamartine, R.; Gamoudi, M.; Guillaud, G. Sens. Actuators, B 1998, B47, 43-47. (17) Wan, Q. J.; Zhang, X. J.; Zhang, C. G.; Zhou, X. Y. Chemical J. Chin. Univ.Chinese 1997, 18, 226-228. (18) Lindino, C. A.; Bulhoes, L. O. S. Anal. Chim. Acta 1996, 334, 317-322.

the range of pH 4-9.19 The use of polyaniline for the development of potentiometric biosensors with improved sensitivity was first reported by our group.20 Polyaniline (PAn) is the electroactive polymer obtained by either chemical21 or electrochemical22 oxidation of aniline. The term ‘polyaniline’ as commonly employed today, refers to a class of polymers consisting of up to 1000 or more p-phenyleneimine repeated units.23 Oxidation of this leucoemeraldine form leads to formation of iminoquinones in a polymer structure. In acidic aqueous solutions, the partially oxidized (emeraldine) form of PAn occurs. Emeraldine was found to be the most conductive among the various PAn oxidation states. A completely oxidized form, polyp-phenylene iminoquinone, is referred to usually as pernigraniline. Polyaniline has already found wide use in the development of chemical sensors. In particular, PAn-based conductometric sensors were used for analysis of NH3,24 H2,25 HCl,26 NO, NO2, and H2S27 in the gas phase. In aqueous media, PAn-based sensors were used for detection of NH4+ 28 and for determination of solution pH. In addition, PAn has been successfully used in the development of conductometric29 and potentiometric19,20,30 biosensors. As a sensing element PAn was used in different pH transducers including electrochemical, optical,31,32 and gravimetric ones.33 Nevertheless, the potentiometric devices first reported by our group20,30 seem to be the most versatile, providing signalprocessing simplicity. For regular polyaniline, the slopes of electrode potential vs pH were reported to be near Nernstian.17,18 An improved potentiometric response of apparently 70 mV/pH unit was reported for self-doped polyaniline.19 Recent investigations on the processing of polyaniline (PCPAn)34,35 have provided new opportunities to simplify the procedure of preparing PAn-modified electrodes. Regular chemically synthesized PAn can be solubilized in organic media by redoping it with organic acid. Casting of such processible polyaniline onto any solid support results in polymer films with (19) Karyakin, A. A.; Bobrova, O. A.; Lukachova, L. V.; Karyakina, E. E. Sens. Actuators, B 1996, 33, 34-38. (20) Karyakina, E. E.; Neftyakova, L. V.; Karyakin, A. A. Anal. Lett. 1994, 27, 2871-2882. (21) Willstater, R.; Dorogi, S. Chem. Ber. 1909, 42, 2143. (22) Diaz, A. F.; Logan, J. A. J. Electroanal. Chem. 1980, 111, 111-114. (23) MacDiarmid, A. G.; Epstein, A. J. Faraday Discuss. Chem. Soc. 1989, 317332. (24) Krutovertsev, S. A.; Sorokin, S. I.; Zorin, A. V.; Letuchy, Y. A.; Antonova, O. Y. Sens. Actuators, B 1992, B7, 492-495. (25) Domansky, K.; Baldwin, D. L.; Grate, J. W.; Hall, T. B.; Li, J.; Josowicz, M.; Janata, J. Anal. Chem. 1998, 70, 473-481. (26) Koul, S.; Dhawan, S. K.; Chandra, S.; Chandra, R. Indian J. Chem., Sect. A 1997, 36, 901-904. (27) Agbor, N. E.; Petty, M. C.; Monkman, A. P. Sens. Actuators, B 1995, B28, 173-179. (28) Laranjeira, J. M. G.; Deazevedo, W. M.; Dearaujo, M. C. U. Anal. Lett. 1997, 30, 2189-2209. (29) Hoa, D. T.; Kumar, T. N. S.; Punekar, N. S.; Srinivasa, R. S.; Lal, R.; Contractor, A. Q. Anal. Chem. 1992, 64, 2645-2646. (30) Karyakina, E. E.; Shkurko, O. A.; Varfolomeyev, S. D.; Karyakin, A. A. In Modern Enzymology: Problems and Trends; Kurganov, B. I., Kochetkov, S. N., Tishkov, V. I., Eds.; Nova Science Publishers: New York, 1994; pp 809813. (31) Grummt, U. W.; Pron, A.; Zagorska, M.; Lefrant, S. Anal. Chim. Acta 1997, 357, 253-259. (32) Pringsheim, E.; Terpetschnig, E.; Wolfbeis, O. S. Anal. Chim. Acta 1997, 357, 247-252. (33) Zhou, X. Y.; Cha, H. Y.; Yang, C.; Zhang, W. M. Anal. Chim. Acta 1996, 329, 105-109. (34) Cao, Y.; Smith, P.; Heeger, A. J. Synthetic Metals 1993, 57, 3514-3519. (35) Cao, Y.; Qiu, J. J.; Smith, P. Synth. Met. 1995, 69, 187-190.

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high electronic conductivity. The highest conductivity of cast films (300 S cm-1) was obtained for PAn dissolved in m-cresol with camphorsulfonic acid (CSA) as a doping agent. However, for sensor applications, the use of chloroform seems to be preferred. The conductivity of the PCPAn cast from chloroform is also rather high (>1 S cm-1). In the sections that follow we illustrate the use of processible polyaniline for the development of highly sensitive potentiometric pH transducers. The modified electrodes were prepared according to a very simple procedure involving casting of polyaniline solution onto the electrode surface and then allowing the volatile chloroform to evaporate. Different carbon-electrode supports were tested. Several attractive properties of PCPAn carbon sensors and their advantages over existing pH electrodes are demonstrated and discussed below. EXPERIMENTAL SECTION Reagents. Experiments were carried out with solutions prepared with Millipore (Milli-Q) water. Aniline was distilled under vacuum prior to use. Chloroform (HPLC grade) was purchased from Fisher Chemical Co. Ethanol was purchased from Equistar Chemicals in dehydrated form. D-Glucose, urea, camphorsulfonic acid, phenol, dodecylbenzenesulfonic acid sodium salt, Nafion (5% solution in light alcohols), and all inorganic salts were purchased from Aldrich and used as received. Urease (from jack beans (type VI)) was purchased from Sigma. Electrochemical Setup. Electrochemical measurements were carried out using either the Solartron 1286 electrochemical interface or the Autolab PGST 10 analyzer. The cyclic voltammograms were recorded using a three-compartment electrochemical cell with a platinum gauze counter electrode and a Ag|AgCl|1 M KCl electrode as reference. Glassy carbon disk electrodes were made by pressing glassy carbon (L1.5-mm) rods into Teflon tubes with outer diameters of 10 mm. Prior to use the glassy carbon electrodes were polished with 1-µm alumina to a mirror finish. Potentiometric measurements were carried out in the open-circuit mode in a simple glass vessel upon stirring. Visible Spectrum. A visible spectrum of PCPAn solution in chloroform was taken using a Hewlett-Packard 8452A UV-vis spectrophotometer. Screen-Printing Fabrication. A semiautomatic screen printer (model TF 100, MPM, Franklin, MA) was used for printing the planar working electrodes. Two types of commercial carbon inks were compared. These include Acheson Ink 49AB90 (Acheson Colloids, Ontario, CA) and Ercon Ink G-448-I (Ercon, Waltham, MA). The printing procedure was as described in ref 36.36 The typical area of a carbon screen-printed electrode was 2 × 4 mm. Preparation of Processible PAn (PCPAn). Chemical synthesis of PAn was carried out through low-temperature aniline oxidation with ammonium persulfate in 1 M HCl as described elsewhere.23 The emeraldine base was solubilized according to the procedure proposed in refs 34 and 35.34,35 Emeraldine base was mixed with camphor sulfonic acid in the ratio of 1 pphenyleneimine unit per 0.5-1.0 doping molecule. The mixture was then dissolved in chloroform to give a final concentration of 0.5% (weight). To improve the processibility of PAn, the phenol was added in a ratio of 0.5-1.0 per p-phenyleneimine unit. (36) Wang, J.; Tian, B.; Nascimento, V. B.; Angnes, L. Electrochim. Acta 1998, 43, 3459-3465.

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PCPAn-Modified Electrodes. These were prepared by syringing 1-5 µL of polyaniline solution onto the electrode surface and allowing chloroform to evaporate. Urea-Sensitive Biosensors. These were made by immobilization of urease onto the top of PCPAn-modified screen-printed electrodes. The immobilization procedure included casting 1 µL of urease water solution (50 mg/mL) onto the top of PCPAnmodified screen-printed electrodes. After the drop dried the electrode was covered by Nafion membrane by syringing 1 µL of the polyelectrolyte salt solution. The latter was prepared by dilution of the Nafion stock solution (5%) with ethanol and alkaline alcohol. The resulting enzyme electrodes were used immediately or stored in the refrigerator when not in use. The Potentiometric pH Response. This was investigated in open-circuit mode by immersing the PCPAn-modified electrodes in different buffer solutions containing 0.1 M buffer and 0.1 M KCl as the supporting electrolyte. The buffers used were: phosphate (pH 6-8), acetate (pH 4, 5), borate (pH 9), and 1 mM HCl (pH 3). The Potentiometric PCPAn-Based Biosensor. This was tested in batch mode, controlling the changing in open-circuit potential after addition of urea samples. The supporting electrolyte, 0.001 M phosphate + 0.1 M NaCl pH 7.0, was chosen as a rough model of blood serum. The stock solution of urea was prepared in the same buffer. The pH of the urea stock solution was controlled. RESULTS AND DISCUSSION Processible PAn for Modification of Electrodes. The main advantage of processible polyaniline is the high conductivity of casted polymer films. Initially PCPAn was designed for polymer blends.34,35 However, its application in chemical and biological sensors also seems to be very attractive. Indeed, the modified electrodes could be prepared by a simple dip-coating rather than by electrochemical polymerization. This obviously decreases the cost of the PAn-modified electrodes particularly when mass production is involved. Best results in polyaniline processing (considering conductivity of the resulting films) were obtained when emeraldine base was solubilized in cresols with the use of camphorsulfonic acid for redoping. However, such use of cresols as solvents for modification of electrodes seems not to be convenient because of their low volatility. Moreover, both ortho- and para-cresols are solid at room temperature, and m-cresol is highly viscous having a melting point of 18 °C. As a Follow up to the conclusion in ref 3535 that the phenolic structure of the solvent induces the processibility of polyaniline, we investigated solutions of phenol in different water-miscible solvents. Indeed the solution of phenol in i-propanol was a suitable medium for solubilization of PAn, but only in the case of high phenol concentration. Unfortunately, at such concentration the phenol solution also became less volatile. After a search for a suitable solvent for the processing of polyaniline, we chose chloroform. The complex of PAn-CSA was reported to be solubilized in CHCl3 up to a concentration of 0.5%.34,35 The commonly used ratio of CSA to p-phenyleneimine unit is 0.5, which is in a good agreement with the fact that in the fully doped PAn state only half of the nitrogen atoms of PAn are

Figure 1. Visible spectrum of 0.005% PCPAn in chloroform.

Figure 2. Cyclic voltammogram of PCPAn-modified glassy carbon electrode; 0.05 M sulfuric acid, sweep rate 50 mV/s.

protonated.23 To stabilize the PAn solution in chloroform we also added phenol in the same ratio to p-phenyleneimine unit. A typical visible spectrum of processible PAn in chloroform is shown in Figure 1. The color of PCPAn is mostly due to the absorption around 750 nm. In addition, an absorption band in the near UV region exists. The electroactivity of the PCPAn films was investigated. For this purpose, the polished glassy carbon electrodes were covered by PCPAn by dip-coating from its solution in chloroform. Figure 2 presents the cyclic voltammogram of the PCPAn-modified glassy carbon electrode. Two well-defined sets of peaks are seen, which correspond to the polyaniline redox reactions in acidic media. They are leucoemeraldine/emeraldine and emeraldine/pernigraniline conversions. The shape of the peaks is typical for electropolymerized polyaniline. We point to the absence of the third (or middle) seat of peaks which commonly appeared in cyclic voltammograms of electropolymerized PAn and corresponds to defects in its linear structure.37 Thus, the number of such defects in PCPAn is low. The latter conclusion was confirmed by continuous cycling of a PCPAn-modified electrode in 0.05 M sulfuric acid in the potential range from -0.1 to 0.8 V. At high anodic potentials, around 0.8 V, the destruction of polyaniline occurs, which causes the appearance of defects in its structure. Indeed, during continuous cycling in the given potential region the third set of peaks appeared in cyclic voltammograms of PCPAn-modified electrodes. In conclusion of this section, we point out that polyanilinemodified electrodes can be made using processible PAn. The procedure of modification is a simple dip-coating or casting of PCPAn solution onto the electrode support and followed by a few seconds of drying while chloroform is evaporated.

Figure 3. pH Dependence on pH of the stationary potential of PAnmodified electrodes: (a) four PCPAn-modified glassy carbon electrodes; (b) screen-printed electrodes modified: (n) with electropolymerized regular PAn, (B,9,2) with PCPAn.

Thermodynamics of PCPAn-Modified Electrodes. As we reported earlier,19,20 the open-circuit potential of the polyanilinemodified electrodes is pH dependent. In cyclic voltammograms of PAn-modified electrodes recorded in supporting electrolyte solutions (for example, Figure 2), no diffusion-controlled reactions were observed. Thus, the observed open-circuit potential is an equilibrium potential of the polyaniline redox reactions. Since the oxidation of leucoemeraldine to pernigraniline is accomplished by the evolution of protons, the equilibrium potential exhibits pH dependence. In neutral solutions the two protons per two electrons are transferred, to give according to the Nernstian equation ∂ E/∂ pH ) 59 mV/pH unit.38,39 The open-circuit potential of PCPAn-modified glassy carbon electrodes was investigated in buffer solutions of different pH. The corresponding pH dependence for four different PCPAnmodified electrodes is presented in Figure 3a. These data indicate that the reproducibility of the open-circuit potential values from electrode to electrode upon acid-to-base and base-to-acid titrations is quite good. The pH dependence of the open-circuit potential was fit to a linear function (Figure 3a). It can be seen, that with the exception of pH 9, the fitting is quite high. The slope of the potential vs pH plot was found to be 90 mV/pH unit. The greatly improved potentiometric pH response of the PCPAn-modified electrodes (in comparison to those based on regular polyaniline) can be explained in terms of the pH depen(37) Lapkowski, M. Synth. Met. 1990, 35, 169-182. (38) Huang, W.; Humphrey, B. D.; MacDiarmid, A. G. J. Chem. Soc., Faraday Trans. 1 1986, 82, 2385-2400. (39) Focke, W. W.; Wnek, G. E.; Wei, Y. J. Phys. Chem. 1987, 91, 5813-5818.

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dence of the equilibrium potential of PCPAn. When polyaniline is fully protonated, half of the nitrogen atoms in the leucoemeraldine state are associated with an extra proton. In contrast, in the pH region 0 < pH < 1, this extra proton is evolved when leucoemeraldine is oxidized to emeraldine. In weakly acidic media (pH > 4) this reaction is pH independent.38,39 The total leucoemeraldine/pernigraniline redox reaction in strong acidic media involves the transfer of three protons per two electrons,38,39 which gives an equilibrium potential vs pH plot with the slope of -89 mV/pH. We believe that in PCPAn the protonation with camphorsulfonic acid causes the association of half of its imino groups with the ‘extra’ proton in the leucoemeraldine state. Since camphorsulfonic acid is poorly soluble in water it is retained in the PCPAn upon open-circuit investigations. As a result, even in neutral solutions the equilibrium potential of PCPAn possesses the slope of 89 mV/pH unit, similar to that of regular polyaniline in strong acidic media. To confirm the above conclusion it was necessary to remove camphorsulfonic acid from PCPAn and to test the pH response of the resulting films. Several procedures were used for this purpose which consisted of continuous cycling in 0.05 M sulfuric acid and washing either with ethanol or with deionized water. Indeed, after such treatment the slope of the plot of the opencircuit potential of the PCPAn-modified electrodes vs the solution pH decreased to the lower limit of 60 mV/pH unit. Thus, through the protonation with camphorsulfonic acid, the redox reactions of PCPAn even in neutral media, are similar to those of regular PAn in strong acidic solutions. This makes the equilibrium potential of PCPAn modified electrodes pH dependent with a slope of 89 mV/pH unit. The higher slope of the plot of the equilibrium potential vs pH provides the 1.5-fold improved potentiometric response, making PCPAn extremely attractive for the development of pH transducers and potentiometric biosensors. PCPAn-Based pH Transducers. The screen-printing fabrication technology represents an attractive route for mass production of highly reproducible and yet low-cost (disposable) electrochemical transducers. We have chosen screen-printed carbon electrodes as support for PCPAn-based pH transducers. Preparation of the transducer involves syringing 1 µL of PCPAn solution onto the carbon strip surface and allowing the solvent to evaporate. The resulting electrodes showed potentiometric signals in response to changes in pH similar to those of the PCPAn-modified glassy carbon electrodes discussed above. We investigated how the method of fabricating screen-printed carbon electrodes affects the properties of the resulting transducers. The highest pH response of the PCPAn-based pH transducer was found for electrode strips fabricated from Acheson carbon ink cured at 120 °C. Lowering the curing temperature down to 90 °C did not significantly affect the response. In contrast, when the electrodes were made from Ercon carbon ink, the response of the corresponding transducer was 5-7% lower. Further work relied on the use of Acheson carbon ink for preparing the pH strip electrodes. The stationary potential of screen-printed electrodes is shown in Figure 3b. It can be seen that the results obtained for three different PCPAn-based strips were nearly identical, which illustrates the reproducibility of the transducer development. In the pH range from 4 to 8, the dependence of the stationary potential 2538 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999

Figure 4. Slope of the stationary potential of the plot of pH vs PCPAn concentration in its chloroform solution taken for deposition onto the (n) screen-printed and (9) glassy carbon electrodes.

on the pH is linear with a slope of approximately 90 mV/pH unit. Compared with the PCPAn-based glassy carbon electrodes, the screen-printed transducers seem to have a reduced response in the strongly acidic region. To demonstrate the advantage of PCPAn for the development of potentiometric transducers we compared them to the screenprinted electrodes, modified with regular electropolymerized PAn. The dependence of the steady-state potential of the latter electrode on the pH is shown in Figure 3b. Over the entire pH range investigated, the slope of Est vs pH was approximately 60 mV/pH unit, which agrees with the values reported for regular polyaniline.17,18 The dramatic sensitivity improvement offered by PCPAn film over that of the electropolymerized PAn is obvious from Figure 3b. For further practical applications it is essential to estimate the amount of PCPAn, which should be deposited onto the electrode support to provide the improved potentiometric response of the resulting transducers. For this, we investigated how the concentration of PCPAn in the depositing solution affected the properties of the electrodes. It can be seen from Figure 4 that when the concentration of PCPAn is higher than 0.2%, the corresponding electrodes exhibit the highest slope for the plot of the stationary potential vs pH. However, the decrease of concentration down to 0.05% causes less than 5% decrease in the pH response. Subsequent work was carried out with the pH transducers made with 0.5% PCPAn solution. By now we have considered the thermodynamic properties of PCPAn-modified electrodes. For analytical applications it is essential to know the actual pH response of the reporting transducers as well as their operational and storage stability. Figure 5 illustrates acid-to-base and base-to-acid titrations of PCPAnmodified screen-printed electrodes. It is seen that after the first scan exhibiting the slope of 97% of its maximum value, the subsequent two acid-to-base and three base-to-acid runs gave almost similar results with slopes of approximately 90 mV/pH units. Thus, the response of the PCPAn-based pH transducer is highly reproducible and rather stable. We investigated the storage stability of the PCPAn-based pH electrodes made from 0.5% PCPAn between the measurements. The electrodes were kept either in dry state at room temperature in open air or were kept in water. After 4-5 h of being in the dry state, the pH response of the PCPAn-based pH transducer was almost the same as its maximum value. However, after 24 h of

Figure 5. pH response of PCPAn-modified screen-printed electrode: (n) first acid-to-base titration, (0) two subsequent acid-tobase scans and three base-to-acid scans.

Figure 6. Calibration plot of PCPAn-based urea-sensitive potentiometric biosensor: (n) urea-sensitive electrode, (9) ‘enzyme-free’ electrode; 1 mM phosphate buffer, pH 7 + 0.1 M NaCl.

Table 1. Interference Effect of Cations on PCPAn-Based pH Electrode 0.01 M phosphate buffer pH 6 + (0.1 M cation)

∆E/mV

NaCl KCl LiCl NH4Cl CaCl2

0 6 -4 -1 -40

storage the response decreased by 14%. In the wet state the transducers were found to be more stable: after 24 h the pH response was 98% of its initial value. The short-term shelf life of the PCPAn-based transducers was estimated. When the electrodes were kept in open air at room temperature prior to measurements one could observe only a minor decrease in the pH response. Even after keeping the transducer for two weeks, the dependence of the open-circuit potential on the pH gave the slope of 90 mV/pH unit. Taking into account that the two-week storage stability is not a long period, we note that the shelf life of PCPAn-based transducers can be further drastically improved by keeping them in an inert atmosphere or under vacuum. The effect of potential interferences on the response of PCPAnbased pH transducers was also investigated. The open-circuit electrode potentials in 0.01 M phosphate (pH 6.0) containing different supporting electrolytes in a concentration of 0.1 M were compared. The results in Table 1 indicate that only a minor response to the singly charged cations was observed. Surprisingly, even the response to ammonium ion was negligible. On the contrary, cations with high complexing ability were expected to cause the potentiometric response because of the shifting of the PAn equilibrium potential. Indeed the response of PCPAn-modified electrodes to Ca2+ was detected (Table 1). Thus, the advanced pH transducers were developed on the basis of PCPAn. In addition to the simplicity of their preparation, the PCPAn-based transducers showed an improved response toward the pH, of 90 mV/pH, which was completely reversible and rather stable. Such attractive properties of the PCPAn pH strip were subsequently exploited for the benefit of improved potentiometric biosensors. PCPAn-Based Urea Biosensor. To confirm the possibility of the biosensor development on the basis of the PCPAn-modified

electrodes, a urea-sensitive potentiometric biosensor was prepared. Measurements of blood urea are very important to diagnose the kidney functions. Urease, being one of the oldest enzymes investigated, is known to decompose the urea molecule to two ammonia ions and one bicarbonate ion. In neutral media these products cause a rise in solution pH. The new biosensor was prepared by subsequent casting of the urease and Nafion layers onto the top of PCPAn-modified screenprinted electrodes. The biosensor was tested in a 1 mM phosphate buffer solution (pH 7.0) containing 0.1 M NaCl as supporting electrolyte, chosen as a rough model of blood serum. Figure 6 illustrates the typical calibration plot of the ureasensitive electrodes. The response time was approximately 50 s. The detection limit was 10-5 M, which was satisfactory for potentiometric devices tested in 1 mM neutral buffer solutions. The maximal value of the response calculated from the calibration plot was ∼120 mV. The ‘enzyme-free’ electrode strip gave a negligible response toward urea in similar conditions (Figure 6). The effect of the content of Nafion, in the polyelectrolyte solution used for coating the enzyme electrode, upon the behavior of the urea biosensor was assessed. When the Nafion content was higher than 0.25%, the maximum response value of the resulting potentiometric electrode was decreased. The electrode prepared from 0.1% Nafion exhibited almost a similar response, but possessed lower operational stability. Thus, Nafion content of 0.25% was found to be optimal for coating the enzyme electrodes. The operational stability of the optimal urease biosensor was investigated. Three subsequent calibration plots taken for the same electrode exhibited almost similar response values. When the electrode was kept overnight in the wet state at 4 °C, only a minor decrease in the response was observed. This particular calibration plot (recorded on the second day of using of this urease electrode) is shown in Figure 6. Hence the resulting urease biosensor exhibited rather high operational stability, suitable for analytical use. CONCLUSION We have described the preparation and attractive performance characteristics of a potentiometric pH transducer based on PCPAn. The advantages of such a transducer are the following: First, PCPAn-modified electrodes were prepared according to a very simple procedure involving the casting of the polyaniline solution Analytical Chemistry, Vol. 71, No. 13, July 1, 1999

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onto the desired electrode surface either by dip-coating or by syringing and allowing the volatile chloroform to evaporate. Second, PCPAn-based electrodes exhibited completely reversible, improved response toward pH changes of 90 mV/pH unit. Third, the PCPAn-modified transducers showed both good operational stability and prolonged shelf life. Fourth, only a minor interference of singly charged cations was detected. We are not sure that PCPAn-based pH transducers can compete with glass-membrane electrodes for use in pH meters; however, they seem to be very promising for biosensor application. Indeed the PCPAn-based pH transducer is expected to be very inexpensive and suitable for single-use sensors. In addition, it has higher sensitivity toward pH, which provides for a higher sensitivity of the resulting biosensor. To confirm the possibility of biosensor development on the basis of PCPAn, we made a urea-sensitive potentiometric elec-

trode. It displayed both high sensitivity and good operational stability in routine analytical applications. The problem with the application of unsubstituted polyanilines in bioelectrochemistry is the lack of their electroactivity in neutral solutions. Even the potentiometric response of PCPAn decreased in basic media starting from pH 9.0. As we reported earlier, the electroactivity of polyaniline can be prolonged in the basic solutions by using self-doped PAn instead of a regular polymer.40,41 Thus, for future improvement of the reported pH transducers, use of the self-doped processible polyaniline has to be undertaken.

(40) Karyakin, A. A.; Strakhova, A. K.; Yatsimirsky, A. K. J. Electroanal. Chem. 1994, 371, 259-265. (41) Karyakin, A. A.; Maltsev, I. A.; Lukachova, L. V. J. Electroanal. Chem. 1996, 402, 217-219.

Received for review December 1, 1998. Accepted March 24, 1999.

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ACKNOWLEDGMENT The authors thank the NATO-Linkage Grant SA. 12-3-02(HTECH.LG972789) and NIH grant R01RR14173-01 for financial support of this work. Maika Vuki acknowledges the financial support from University of the South Pacific.

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