Selective Permeation of Hydrogen Peroxide through Polyelectrolyte

A layer-by-layer deposition technique for constructing poly- electrolyte ..... (PAA/PVS)2PAA film-coated electrode may be used for determin- ing H2O2 ...
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Anal. Chem. 2001, 73, 5310-5315

Selective Permeation of Hydrogen Peroxide through Polyelectrolyte Multilayer Films and Its Use for Amperometric Biosensors Tomonori Hoshi,† Hidekazu Saiki,† Sachie Kuwazawa,† Chikako Tsuchiya,† Qiang Chen,‡ and Jun-ichi Anzai*,†

Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan, and Life Sciences College, Nankai University, Weijin Road 94, Tianjin 300071, China

A platinum electrode was coated with polyelectrolyte multilayer (PEM) films to prepare an amperometric hydrogen peroxide sensor which can be used in the presence of possible interferences such as ascorbic acid, uric acid, and acetaminophen. The PEM films were prepared on the surface of a Pt disk electrode by an alternate deposition of polycation and polyanion from the aqueous solutions through electrostatic force of attraction. The Pt electrodes coated with a poly(allylamine)/poly(vinyl sulfate) or poly(allylamine)/poly(styrenesulfonate) film were used successfully for detecting H2O2 selectively in the presence of the possible interfering agents. It was suggested that H2O2 can diffuse into the PEM film smoothly while the ascorbic acid, uric acid, and acetaminophen cannot penetrate the film by a size exclusion mechanism. On the other hand, the electrodes coated with PEM films containing poly(ethyleneimine) or poly(diallyldimethylammonium chloride) were not useful for the selective determination of H2O2. The results were rationalized based on the different permeability of the films due to the different molecular density or packing in the PEM films. The PEM film-coated electrode was useful for constructing glucose biosensors by coupling with glucose oxidase. A layer-by-layer deposition technique for constructing polyelectrolyte multilayer (PEM) films has attracted much attention because of its simplicity in procedure and wide choice of materials.1 This technique involves an alternate adsorption of anionic and cationic polyelectrolytes from solution onto a solid surface through electrostatic force of attraction. Recent studies revealed that anionic and cationic layers in PEM films do not form a well-defined layered structure as often depicted but significantly * Corresponding author: (e-mail) [email protected]. † Tohoku University. ‡ Nankai University. (1) (a) Decher, G.; Hong, J. D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (b) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210-211, 831. (c) Kaschak, D. M.; Mallouk, T. E. J. Am. Chem. Soc. 1996, 118, 4222. (d) Decher, G. Science 1997, 277, 1232. (e) Clark, S. L.; Montague, M. F.; Hammond, P. T. Macromolecules 1997, 30, 7237. (f) Lowack, K.; Helm, C. A. Macromolecules 1998, 31, 823. (g) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Colloids Surf. 1999, 146, 337. (h) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213.

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interpenetrate each other as a consequence of charge compensation within multilayers.2 Still, the thickness of PEM films depends precisely in the nanometer level on the number of layers. Another merit of PEM films is that the polarity of the surface charge can be controlled arbitrarily by changing the type of polyelectrolyte in the outermost layer. Polycations such as poly(diallyldimethylammoniun chloride), poly(allylamine), and poly(ethyleneimine) have been frequently used to prepare PEM films, while poly(styrenesulfonate), poly(vinyl sulfate), and poly(acrylic acid) are typical anionic counterparts. Recently, dyes,3 host-guest compounds,4 redox or conducting polymers,5 proteins,6 and nucleic acids7 have also been employed as components of PEM films. These functional PEM films find applications to separation, sensing, catalysis, etc. It was recently reported that PEM film-coated electrodes exhibit selective response to redox-active ions and molecules in (2) (a) Lo ¨sche, M.; Schmit, J.; Decher, G.; Bouwman, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893. (b) Schlenoff, J. B.; Ly, H.; Li, M. J. Am. Chem. Soc. 1998, 120, 7626. (3) Cooper, T. M.; Campbell, A. L.; Crane, R. L. Langmuir 1995, 11, 2713. (b) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224. (c) Ariga, K.; Onda, M.; Lvov, Y.; Kunitake, T. Chem. Lett. 1997, 25. (d) Tedeschi, C.; Caruso, F.; Mo¨hwald, H.; Kirstein, S. J. Am. Chem. Soc. 2000, 122, 5841. (4) (a) Yang, X.; Johnson, S.; Shi, J.; Holesinger, T.; Swanson, B. Sens. Actuators, B 1997, 45, 87. (b) Nabok, A. V.; Davis, F.; Hassan, A. K.; Ray, A. K.; Majeed, R.; Ghassemlooy, Z. Mater. Sci. Eng. C 1999, 8-9, 123. (5) (a) Hodak, J.; Etchenique, R.; Calvo, E. J. Langmuir 1997, 13, 2708. (b) Laurent, D.; Schlenoff, J. B. Langmuir 1997, 13, 1552. (c) Chuang, C. L.; Wang, Y. J.; Lan, H. L. Anal. Chim. Acta 1997, 353, 37. (d) Sun, C.; Li, W.; Sun, Y.; Zhang, X.; Shen, J. Electrochim. Acta 1999, 44, 3401. (e) Li, W.; Wang, Z.; Sun, C.; Xian, M.; Zhao, M. Anal. Chim. Acta 2000, 418, 225. (6) (a) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo ¨hwald, H., Eds.; Marcel Dekker: New York, 1999; p 125. (b) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (c) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163. (d) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073. (e) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427. (f) Caruso, F.; Mo ¨hwald, H. J. Am. Chem. Soc. 1999, 121, 6039. (g) Brynda, E.; Houska, M.; Skvor, J.; Ramsden, J. J. Biosens. Bioelectron. 1998, 13, 165. (h) Anzai, J.; Nishimura, M. J. Chem. Soc., Perkin Trans. 2 1997, 1887. (i) Anzai, J.; Kobayashi, Y.; Nakamura, N.; Nishimura, M.; Hoshi, T. Langmuir 1999, 15, 221. (j) Anzai, J.; Nakamura, N. J. Chem. Soc., Perkin Trans. 2 1999, 2413. (k) Anzai, J.; Hoshi, T.; Nakamura, N. Langmuir 2000, 16, 6306. (7) (a) Lvov, Y.; Decher, G.; Sukhorukov, G. B. Macromolecules 1993, 26, 5396. (b) Decher, G.; Lehr, B.; Lowack, K.; Lvo, Y, Schmitt, J. Biosens. Bioelectron. 1994, 9, 677. (c) Sukhorukov, G. B.; Montrel, M. M.; Petrov, A. I.; Shabarchina, L. I.; Sukhorukov, B. I. Biosens. Bioelectron. 1996, 11, 913. 10.1021/ac010605t CCC: $20.00

© 2001 American Chemical Society Published on Web 09/28/2001

solution, depending on the type of polymeric materials and the number of layers.8 Bruening and co-workers studied the permeability of Fe(CN)63-/4- and Ru(NH3)62+/3+ ions through PEM films using PEM film-coated electrodes and found that the permeability depends on the solution pH, the number of layers in the film, and the type of polymeric materials used.8a They also reported the possibility of controlling the permeability of PEM films through chemical derivatizations.8b Willner and co-workers reported electron transfer on the PEM film-coated electrodes.8c They demonstrated that PEM films generate a swollen, electrically neutral, porous structure and that the permeation of Fe(CN)63-/4anion and protonated N,N-dimethylaminomethylferrocene cations depends decisively on the polarity of electric charge on the outermost surface of the PEM film. These findings strongly suggest that the permeability of PEM films can be tuned by designing the layered structure of the film satisfactorily using suitable materials. In this context, we reported a preliminary result that a PEM film-coated platinum electrode can be used successfully for detecting hydrogen peroxide in the presence of possible interferences such as ascorbic acid, uric acid, and acetaminophen.9 It is known that ascorbic acid, uric acid, and acetaminophen are oxidized electrochemically on the surface of a Pt electrode at 0.6 V versus Ag/AgCl, causing an interference in the amperometric measurements of enzyme biosensors.10 On the contrary, the response of the Pt electrode coated with a PEM film composed of a few layers of poly(allylamine) and poly(vinyl sulfate) to the interfering compounds was suppressed almost completely, while the modified electrode still exhibited an appropriate response to H2O2.9 Thus, the H2O2 selectivity of the electrode was substantially improved by coating the PEM film on the surface. The high selectivity of the electrode was rationalized based on the selective permeation of H2O2 over the interferences due to a size exclusion mechanism. In the present paper, the amperometric responses of the PEM film-coated electrodes to H2O2 and the interferences are described in detail in relation to the type of polymeric materials used and the number of layers in the film. On the basis of the selective permeation of H2O2 through the PEM films, a possible use of the modified electrode for constructing enzyme biosensors is proposed. EXPERIMENTAL SECTION Materials. An aqueous solution (20%) of poly(allylamine) hydrochloride [PAA; average molecular weight (MW), ∼10 000], a 30% aqueous solution of poly(ethyleneimine) (PEI; MW, 60 00080 000), and a 20% aqueous solution of poly(diallyldimethylammonium chloride) (PDDA; MW, 100 000-200 000) were purchased from Nittobo Co. (Tokyo, Japan), Nakalai Tesque Co. (Kyoto, Japan), and Aldrich Chemical Co. (Milwaukee, WI), (8) (a) Harris, J. J.; Bruening, M. L. Langmuir 2000, 16, 2006. (b) Dai, J.; Jensen, A. W.; Mohanty, D. K.; Erndt, J.; Bruening, M. L. Langmuir 2001, 17, 931. (c) Pardo-Yissar, V.; Katz, E.; Lioubashevski, O.; Willner, I. Langmuir 2001, 17, 1110. (d) Farhat, T. R.; Schlenoff, J. B. Langmuir 2001, 17, 1184. (9) Hoshi, T.; Saiki, H.; Kuwazawa, S.; Kobayashi, Y.; Anzai, J. Anal. Sci. 2000, 16, 1009. (10) (a) Maidan, R.; Heller, A. Anal. Chem. 1992, 64, 2889, (b) Harrison, D. J.; Moussy, F.; Jakeway, S.; Fan, Z.; Rajotte, R. V. In Interfacial Design and Chemical Sensing; Mallouk, T. E., Harrison, D. J., Eds.; ACS Symposium Series 561: American Chemical Society: Washigton, DC, 1994; p 255. (c) Anzai, J.; Takeshita, H.; Kobayashi, Y.; Osa, T.; Hoshi, T. Anal. Chem. 1998, 70, 811.

Figure 1. Chemical structures of polyelectrolytes used.

respectively. Poly(potassium vinyl sulfate) (PVS; MW, 242 000) and poly(sodium 4-styrenesulfonate) (PSS; MW, 35 000) were obtained from Nakalai Tesque Co. and Pressure Chemical Co. (Pittsburgh, PA). The chemical structures of the polymeric materials are shown in Figure 1. Concanavalin A (Con A) was obtained from Seikagaku Kogyo (Tokyo, Japan). Glucose oxidase (GOx; EC 1.1.3.4, from Aspergillus niger) was purchased from Sigma Co. (St. Louis, MO). GOx was purified by gel filtration before use. All other reagents were of the highest grade available and were used without further purification. Preparation of PEM Film-Coated Electrodes. The PEM films were prepared on the surface of a platinum disk electrode (3-mm diameter) according to the reported procedure.1g The electrode was polished thoroughly using alumina powder and rinsed with distilled water before use. The bathing solutions from which the polyelectrolytes were deposited were prepared using Dulbecco’s phosphate-buffered saline unless otherwise noted. Dulbecco’s phosphate buffer used contains 8 g L-1 NaCl, 0.2 g L-1 KCl, 0.2 g L-1 KH2PO4, and 1.15 g L-1 Na2HPO4. The concentrations of the polyelectrolyte solutions were 2 mg mL-1 for PAA, PDDA, PVS, and PSS and 1.5 mg mL-1 for PEI. The Pt electrode was immersed in the polycation solution for 20 min and rinsed in the working buffer for 5 min. The polycation-coated electrode was then immersed in the polyanion solution for 20 min to deposit the polycation/polyanion bilayer film. To deposit further layers, the above procedure was repeated. The preparation of PEM films was carried out without drying between the depositions. Preparation of GOx Biosensors. The GOx biosensors were prepared using PEM film-modified and unmodified Pt electrodes according to the reported procedure.11 The electrodes were immersed in a Con A solution (0.1 mg mL-1 in Dulbecco’s phosphate-buffered saline, pH 7.4) for 30 min to deposit the first layer of Con A on the electrode surface. After being rinsed with PBS to remove any weakly adsorbed Con A, the electrode was immersed in a GOx solution for 30 min to immobilize the enzyme on the Con A-modified surface, through biological affinity between Con A and sugar residues intrinsically located on the surface of GOx molecule.11 The deposition was repeated to prepare Con A/GOx multilayers. Electrochemical Measurements. The electrochemical response of PEM film-modified electrode was measured with a (11) (a) Anzai, J.; Kobayashi, Y.; Hoshi, T.; Saiki, H. Chem. Lett. 1999, 365. (b) Anzai, J.; Kobayashi, Y. Langmuir 2000, 16, 2851.

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conventional three-electrode system using a Ag/AgCl as a reference electrode. A Pt wire (1-mm diameter) was used as an auxiliary electrode. An amperometric measurement of the electrode was carried out at 0.6 V versus Ag/AgCl with stirring the solution gently. Cyclic voltammetry (CV) was carried out under a nitrogen atmosphere. A 0.1 M phosphate buffer solution (pH 6.8) was used for the electrochemical measurements. All measurements were carried out at room temperature (∼20 °C). Gravimetric Measurements. A quartz crystal microbalance (QCM) was employed to determine the loading of polymers in the PEM films. A 9-MHz AT-cut quartz resonator coated with a thin platinum layer was used as a probe. The probe was immersed in the polymer solutions (0.1 mg mL-1 in Dulbecco’s phosphatebuffered saline) for 20 min to deposit the polymer layer and then rinsed in water. The probe was dried in air until the frequency showed a steady-state value. RESULTS AND DISCUSSION The oxidizable compounds such as ascorbic acid, uric acid, and acetaminophen often interfere with an amperometric measurement of enzyme biosensors.10 For example, glucose biosensors, which are constructed using GOx, suffer from interference caused by the oxidizable compounds dissolved in biological fluids because GOx-based biosensors detect oxidation current of enzymatically generated H2O2 at ∼0.6 V versus Ag/AgCl (eqs 1 and 2), where the oxidizable compounds are also oxidized electroGOx

glucose + O2 98 gluconolactone + H2O2 -2e-

H2O2 98 O2 + 2H+

(1) (2)

chemically to induce bias current. To eliminate the interference, polymeric materials including cellulose,12 Nafion,13 and electrochemically synthesized polymers14 have been extensively used as coatings on the electrode surface so that the interferences could not reach the electrode surface. Response of PEM Film-Coated Electrodes to H2O2, Ascorbic Acid, Uric Acid, and Acetaminophen. We use here PEM films as the coating of the electrode to eliminate the amperometric interference induced by the oxidizable compounds. Prior to constructing GOx biosensors, the amperometric response of a PEM film-coated Pt electrode to H2O2 was studied. Figure 2 shows the response current of the modified and unmodified electrodes to H2O2 as a function of concentration. The PEM filmcoated electrode is sensitive to H2O2 although the response of the electrode is slightly lower than those of the unmodified electrode. These results imply that the PEM film-coated electrode can be used for constructing amperometric biosensors. The response of the PEM film-coated electrode to ascorbic acid, uric acid, and H2O2 was studied by cyclic voltammetry. Figure (12) (a) Wang, J.; Hutchins, L. D. Anal. Chem. 1985, 57, 1536. (b) Kuhn, L. S.; Weber, S. G.; Ismail, K. Z. Anal. Chem. 1989, 61, 303. (13) (a) Ji, H.; Wang, J. J. Chromatogr. 1987, 410, 111. (b) Pan, S.; Arnold, M. A. Talanta 1996, 43, 1157. (14) (a) Sasso, S. V.; Pierce, R. J.; Walla, R.; Yacynych, A. M. Anal. Chem. 1990, 62, 1111. (b) Palmisano, F.; Guerrieri, A.; Quinto, M.; Zambonin, P. G. Anal. Chem. 1995, 67, 1005. (c) Manowitz, P.; Stoecker, P. W.; Yacynych, A. M. Biosens. Bioelectron. 1995, 10, 359. (d) Yang, Q.; Atanasov, P.; Wilkins, E. Sens. Actuators, B 1998, 46, 249. (e) Garjonyte, R.; Malinauskas, A. Sens. Actuators, B 1999, 56, 85.

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Figure 2. Amperometric response of unmodified (b) and (PAA/ PVS)2PAA film-modified Pt electrodes (O) to H2O2. Electrode potential: +0.6 V vs Ag/AgCl.

3 shows the CVs of ascorbic acid and uric acid recorded on the PEM film-coated and bare Pt electrodes. The bare electrode afforded well-defined CVs which show irreversible oxidation reaction of the compounds (Figure 3A). On the contrary, the anodic peaks were suppressed almost completely after the electrode was coated with a (PAA/PVS)2PAA film (Figure 3B), suggesting that the permeation of the compounds was suppressed probably due to a diffusional barrier of the PEM film. Thus, the (PAA/PVS)2PAA film-coated electrode may be used for determining H2O2 in the presence of the interferences. On the other hand, the (PEI/PVS)2PEI film did not block the permeation of the compounds (Figure 3C). Both the PEM film-coated and the uncoated Pt electrodes showed similar CVs to H2O2 (Figure 3D), confirming that H2O2 is permeable through the PEM films. The amperometric response of the modified electrodes was studied systematically as a function of the type of polyelectrolytes used and the number of layers in the films. All measurements were carried out at 0.6 V versus Ag/AgCl because amperometric enzyme sensors are usually operated at this potential to detect enzymatically generated H2O2. Figure 4 illustrates typical response of the bare and (PAA/PVS)2 film-coated electrodes to H2O2, ascorbic acid, uric acid, and acetaminophen. The bare Pt electrode exhibited oxidation current for all substrates; the response currents to ascorbic acid, uric acid, and acetaminophen were ∼40-65% of the response to the same concentration of H2O2 (Figure 4A). It is clear that bare Pt electrode cannot be used for amperometric determination of H2O2 in the presence of the oxidizable compounds. On the other hand, after the electrode was coated with the (PAA-PVS)2 film, responses of the electrode to ascorbic acid, uric acid, and acetaminophen were eliminated almost completely, while the response to H2O2 still remained (Figure 4B). The response current for the oxidizable compounds is less than 1% of the response to H2O2, suggesting that the (PAAPVS)2 film excludes ascorbic acid, uric acid, and acetaminophen and passes H2O2. These results are consistent with the CV data in Figure 3. The response of the (PAA/PVS)2 film-coated electrode to H2O2 was satisfactorily fast and the PEM film gave virtually no effect on the response time, probably due to a thin nature of the PEM film. Effects of the Number of Layers in the PEM Film on the Amperometric Response. Table 1 summarizes the effects of the number of layers in PAA/PVS films on the amperometric response of the electrode, where the PEM films were deposited from polymer solutions prepared using Dulbecco’s phosphate buffer or pure water. The response of the PAA/PVS film-coated electrode depended significantly on the number of layers in the PEM film.

Figure 4. Amperometric responses of a bare Pt (A) and (PAA/PVS)2 film-coated Pt electrodes (B) to H2O2 (a), ascorbic acid (b), uric acid (c), and acetaminophen (d). Concentration of the substrates, 0.1 mM; electrode potential, +0.6 V vs Ag/AgCl. Table 1. Amperometric Response of PAA/PVS Film-Coated Electrodes as a Function of the Number of Layers in the Film response to 0.1 mM substrates/µAa

Figure 3. Cyclic voltammograms of ascorbic acid, uric acid, and H2O2 recorded on PEM film-coated and uncoated Pt electrodes. (AC) CVs of ascorbic acid (s) and uric acid (- - -) recorded on uncoated Pt (A), (PAA/PVS)2PAA film-coated Pt (B), and (PEI/PVS)2PEI filmcoated Pt electrode (C). (D) CVs of H2O2 recorded on uncoated Pt (a), (PAA/PVS)2PAA film-coated Pt (b), and (PEI/PVS)2PEI filmcoated Pt electrode (c). Sample solutions: 5 mM ascorbic acid, 5 mM uric acid or 1 mM H2O2 in 10 mM phosphate buffer (pH 6.8) containing 100 mM KCl. Scan rate, 50 mV s-1.

The response current to ascorbic acid, uric acid, and acetaminophen was decreased remarkably after the electrode was coated with a PAA/PVS bilayer film, (PAA/PVS), whereas the coating had a lesser effect to the response to H2O2. An additional coating of the third layer of PAA induced a further decrease in the response to the oxidizable compounds. After the electrode was coated with two-bilayer film, (PAA/PVS)2, the electrode exhibited a highly selective response to H2O2 over the oxidizable compounds; the interfering current was suppressed to ∼1% or less of the response to H2O2. We checked the effects of ionic strength of the PAA and PVS solutions from which the films were deposited. When the films were deposited from the buffer solution, the electrode showed a lower current than the electrodes modified in water solutions, suggesting the former films blocked the permeation of the compounds more effectively than the latter films

electrodeb

H2O2

ascorbic acid

uric acid

acetaminophen

bare Pt Pt/(PAA/PVS)c Pt/(PAA/PVS)PAAc Pt/(PAA/PVS)2c Pt/(PAA/PVS)2PAAc

4.9 4.0 4.0 3.7 3.7

2.5 0.2 0.1 0.02 0.08

3.2 0.9 0.08 0.02 0.02

2.1 0.4 0.02 0.03 0.01

Pt/(PAA/PVS)d Pt/(PAA/PVS)PAAd Pt/(PAA/PVS)2d Pt/(PAA/PVS)2PAAd

4.5 4.5 4.2 4.0

1.8 1.0 0.2 0.1

1.7 0.7 0.2 0.05

0.9 0.4 0.2 0.03

a The current was measured at 0.6 V vs Ag/AgCl. b The average values of three preparations are listed. These values contain ∼(10% error. c The films were deposited from Dulbecco’s phosphate buffer solutions. d The films were deposited from water solutions.

did. This is reasonable because thicker films tend to form when the polyelectrolytes are deposited from high ionic strength solutions.1a,e,g,2b It should be noted here that, even if the film was deposited from water solutions, H2O2-selective response was eventually attained by coating the electrode surface with the (PAA/PVS)2PAA film. A size exclusion effect may be responsible for the selective permeation of H2O2 through the PAA/PVS films, in view of the fact that the molecular size of H2O2 is much smaller (MW, 34) than those of ascorbic acid, uric acid, and acetaminophen (MW, 176, 168, and 151, respectively). This view is further supported Analytical Chemistry, Vol. 73, No. 21, November 1, 2001

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Table 2. Amperometric Response of PAA/PSS, PEI/PVS, and PDDA/PVS Film-Coated Electrodes as a Function of the Number of Layers in the Film response to 0.1 mM substrates/µAa H2O2

ascorbic acid

uric acid

acetaminophen

Pt/(PAA/PSS) Pt/(PAA/PSS)PAA Pt/(PAA/PSS)2 Pt/(PAA/PSS)2PAA

4.5 4.2 4.6 4.1

1.6 0.5 0.3 0.2

1.6 0.2 0.2 0.03

1.0 0.2 0.2 0.1

Pt/(PEI/PVS) Pt/(PEI/PVS)PEI Pt/(PEI/PVS)2 Pt/(PEI/PVS)2PEI Pt/(PEI/PVS)4PEI

4.7 4.2 2.6 2.6 2.1

2.9 3.3 2.6 1.8 0.3

2.9 2.7 3.0 1.8 0.1

1.7 1.4 1.3 0.7 0.2

Pt/(PDDA/PVS) Pt/(PDDA/PVS)PDDA Pt/(PDDA/PVS)2 Pt/(PDDA/PVS)2PDDA

4.5 5.2 5.2 5.3

2.4 2.4 2.5 2.3

3.3 3.2 3.4 3.1

1.7 1.3 1.8 2.1

electrodeb

a The current was measured at 0.6 V vs Ag/AgCl. b The average values of three preparations are listed. These values contain ∼(10% error. The films were deposited from Dulbecco’s phosphate buffer solutions.

by the fact that the amperometric response of the electrode decreased linearly with the increasing number of layers and did not depend on the polarity of electric charge on the outermost surface of the PEM film. For example, the (PAA/PVS) film suppressed the permeation of ascorbic acid and uric acid, which are charged negatively under the experimental conditions, less effectively than the (PAA/PVS)PAA film did, in spite the fact that the outermost surface of the (PAA/PVS) film contains negative charges originating from SO3- groups in PVS. In other words, an electrostatic exclusion may not be a main factor contributing to the suppressed permeation of the compounds. Recently, Mizutani and co-workers reported selective permeation of H2O2 through polyelectrolyte complex (PEC) films, which were prepared by coprecipitating aqueous polyelectrolytes as polyion complexes.15 They demonstrated a size exclusion as an origin of the permselectivity. The PEM and PEC films are suggested to have a structure closely related to each other.16 For both films, the permselectivity presumably originates from a compact structure of the films arising from the densely packed polyelectrolyte chains through ion paring in the films. It should be noted that PEM films enable us to optimize the permeability at the molecular level by systematically regulating the number of layers and changing the polymeric materials used. Another merit of PEM films lies in the thinness of each layer (thickness of a single layer is typically a few nanometers, depending on the conditions of the bathing solution).1d,1g, 1h Effects of the Type of Polyelectrolyte in the PEM Film on the Amperometric Response. Table 2 lists the amperometric response of the electrodes coated with PAA/PSS, PEI/PVS, and PDDA/PVS films as a function of the number of polymer layers. (15) (a) Mizutani, F.; Yabuki, S.; Hirata, Y. Denki Kagaku 1995, 63, 100. (b) Mizutani, F.; Yabuki, S.; Hirata, Y. Anal. Chim. Acta 1995, 314, 233. (c) Mizutani, F.; Sato, Y.; Hirata, Y.; Sawaguchi, T.; Yabuki, S. Anal. Chim. Acta 1998, 363, 173. (d) Mizutani, F.; Sato, Y.; Sawaguchi, T.; Yabuki, S.; Iijima, S. Sens. Actuators, B 1998, 52, 23. (16) Farhat, T.; Yassin, G.; Dubas, S. T.; Schlenoff, J. B. Langmuir 1999, 15, 6621.

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The PAA/PSS film-coated electrodes exhibited a behavior similar to those of PAA/PVS film-coated electrodes, although blocking by the PSS-based films was slightly less effective than that by the PVS-based films. This may originate from different molecular packing of the polyelectrolytes in the PAA/PVS and PAA/PSS films. The bulky aromatic rings in PSS presumably induce somewhat loose packing of the polymer chains in the film. The PAA/PSS film-coated electrodes nevertheless may be useful for the selective determination of H2O2. On the other hand, the effects of the type of polycation are significant. Table 2 contains the data for PEI/PVS and PDDA/ PVS film-coated electrodes. The response of the PEI/PVS filmcoated electrodes decreased moderately with increasing number of polymer layers. However, the (PEI/PVS)2PEI film-coated electrode exhibited appreciable responses to all substances tested. This is in contrast to the H2O2-selective response of the (PAA/ PVS)2PAA film-coated electrode. Even if the electrode was coated with a thicker (PEI/PVS)4PEI film, the amperometric responses to ascorbic acid, uric acid, and acetaminophen were still not negligible. For the PDDA/PVS film-coated electrodes, the film coating did not induce any suppression in the response current. In Table 2, one can find a few exceptional results in which the response current for the PEM film-coated electrodes is slightly higher than those for uncoated Pt electrode. In other words, some of the PEI/PVS and PDDA/PVS film-coated electrodes showed slightly higher response to ascorbic acid and uric acid, respectively, than the bare Pt electrode did. Conceivably, in these cases, the substrates may be concentrated to some extent in the PEM films. In any case, these observations suggest that both H2O2 and the oxidizable compounds penetrate the PEI/PVS and PDDA/ PVS films. Thus, the PEI- and PDDA-based PEM films cannot be used as an electrode coating for the selective determination of H2O2. The different permeabilities among the PEM films may be ascribed to the different molecular geometry and flexibility of PAA, PEI, and PDDA, which in turn result in the different film structures. The structural features of PAA and PEI are as follows. PEI has a random branched structure (the ratio of primary, secondary, and tertiary amino groups is nominally ∼1:2:1) whereas PAA consists of a linear chain without branched structure (Figure 1). Amino groups in PAA may form ion pairs complementarily with sulfate anions on the linear chain of PVS, resulting in a compact film in which the polymer chains are densely packed. In contrast, PEI and PVS may form a loosely packed film due to a geometrical mismatching of the branched and linear chains. PAA and PEI have been used frequently as the cationic counterpart of PEM films, and some reports also suggested a different structure between PAA and PEI films.6b,h,i A feature of PDDA is the ring structure of the diallyldimethylammonium residue (Figure 1), which would result in the loss of flexibility of the polymer chain in the film. In other words, PDDA chains may be stretched in the film. It is known that PEM films containing stretched polyelectrolytes are thinner than those prepared with the same polyelectrolytes in a coiled conformation under higher ionic strength.1a,e,g,2b To check the surface density of polyelectrolyte in the films, gravimetric measurements of the films were carried out using QCM. It was found that the loading of polyelectrolytes in the PAA/

Figure 5. Typical calibration graphs of glucose sensors parepared using unmodified Pt (A) and (PAA/PVS)2PAA film-modified Pt electrodes (B). The sample solution contains glucose only (b), glucose + 0.1 mM ascorbic acid (2), or glucose + 0.5 mM uric acid (9).

PVS film is much higher than those in the PEI/PVS and PDDA/ PVS films; the mass loadings being ∼3.6 µg cm-2 for the (PAA/ PVS)2PAA film, ∼2.5 µg cm-2 for the (PEI/PVS)2PEI film, and ∼1.1 µg cm-2 for the (PDDA/PVS)2PDDA film. Assuming the density of the films to be ∼1.2 g cm-3, the thickness of the films is estimated to be ∼30, ∼21, and ∼9 nm for the (PAA/PVS)2PAA, (PEI/PVS)2PEI, and (PDDA-/PVS)2PDDA films, respectively.1g These results are qualitatively consistent with the different permeability among the PEM films, although the permeability of the films may depend not only on the thickness but also on other factors such as molecular packing of the polymer chains in the film. Glucose Biosensors Based on (PAA/PVS)2PAA FilmCoated Electrodes. Two kinds of glucose biosensors were fabricated using a bare Pt electrode and a (PAA/PVS)2PAA filmcoated electrode. Both electrodes were coated with a (Con A/GOx)10 film, which is composed of 10 Con A/GOx bilayers prepared by a layer-by-layer deposition of Con A and GOx according to the reported procedure.11 Figure 5 illustrates typical calibration graphs of the sensors to glucose over the concentration range of 1 × 10-4-1 × 10-1 M in the presence and absence of a physiological level of ascorbic acid (0.1 mM) and uric acid (0.5 mM). In the absence of ascorbic acid and uric acid, both sensors showed useful calibration response to glucose in this concentration range, which covers both normal and diabetic blood levels of glucose (5-20 mM). Both sensors responded rapidly to glucose and reached a steady-state current in ∼10 s. In the presence of

ascorbic acid and uric acid, the calibration graph of the (Con A/GOx)10 film-coated sensor without a PEM film deviated significantly from that obtained in the absence of the interferences (Figure 5A). On the contrary, for the glucose sensor based on the PEM film-coated electrode, the calibration graphs in the presence and absence of the interferences are practically identical to each other (or deviation is 5% or less) in the glucose concentration range of >1 mM (Figure 5B). The improved response of the glucose sensor originates from the selective permeation of enzymatically generated H2O2 through the (PAA/ PVS)2PAA film. Thus, the PEM film-based glucose sensor can be used for the determination of glucose in the samples containing a physiological level of ascorbic acid and uric acid. The stability of the PEM film-based glucose sensor was checked by measuring glucose, ascorbic acid, and uric acid every 3 days and keeping the sensor in buffer at 4 °C when not in use. The response of the sensor to ascorbic acid and uric acid was still negligible after 2 months, whereas the response to glucose was decreased to 4050% of the original activity. CONCLUSIONS PEM films prepared on the surface of a Pt electrode by a layerby-layer deposition of polyelectrolyte showed a permselectivity to H2O2 over ascorbic acid, uric acid, and acetaminophen. The permeability of the film depended significantly on the type of polyelectrolyte and the number of layers in the film. The PAA/ PVS and PAA/PSS films showed higher selectivity than PEI- and PDDA-based films. The permeability of the PAA-based films to ascorbic acid, uric acid, and acetaminophen decreased with the increasing number of layers, and the permeation was suppressed almost completely by (PAA/PVS)2PAA and (PAA/PSS)2PAA films. A size exclusion was suggested as an origin of the selective permeation of the films. The (PAA/PVS)2PAA film-coated electrode is useful for constructing a glucose biosensor, in which interference originating from the oxidizable compounds can be eliminated. The PAA-based PEM films would be applicable to the fabrication of other amperometric biosensors. ACKNOWLEDGMENT This work was supported in part by Grant-in-Aid (11480252) from the Ministry of Education, Sciences, Sports and Culture of Japan. The Casio Sciecnce Promotion Foundation is also acknowledged for financial support. Received for review May 30, 2001. Accepted August 14, 2001. AC010605T

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