Biomacromolecules 2000, 1, 202-207
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Calcification-Resistant Nafion/Fe3+ Assemblies for Implantable Biosensors Izabela Galeska,† Debjit Chattopadhyay,† Francis Moussy,‡ and Fotios Papadimitrakopoulos*,† Department of Chemistry, Polymer Science Program, Nanomaterials Optoelectronic Laboratory, Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269; and Center for Biomaterials & Surgical Research Center, University of Connecticut Health Center, Farmington, Connecticut 06030-1615 Received January 3, 2000
An electrostatic layer-by-layer deposition technique was employed for the formation of thin films consisting of alternating layers of perfluorinated ionomer (Nafion) and ferric ions. UV-vis spectroscopic and ellipsometric data indicate a stepwise growth that in certain cases is as high as 47 nm per dip cycle. The growth characteristics of these assemblies can be correlated with Nafion’s hydrodynamic radius, iron content, as well as the ionic strength and pH of Nafion and the wash solution. When these assemblies were compared to cast Nafion films, they exhibit the following advantages: (i) increased hydrolytic stability, attained without thermal treatment required for pristine Nafion films, and (ii) resistance to calcification, by more than an order of magnitude. These results, along with the ability to control glucose permeability by varying the number of Nafion/Fe3+ layers, could prove vital in prolonging the lifetime of implantable biosensors. Introduction Nafion is a perfluorinated polyelectrolyte that has found various applications in areas such as fuel cells,1,2 polymeric catalysts,3 and ion exchange membranes intended for various separation and purification systems.4 Recently, Nafion has attracted considerable attention as semipermeable membranes for sensor applications.5-7,8 The good mechanical properties and thermal stability (up to 200 °C)9 and its chemical and biological inertness exalts Nafion to one of the premier systems capable of operating in harsh biological environments.10,11 Moreover, the above features have established Nafion as a leading material for semipermeable membranes in a number of electrochemical sensors.12-18 Biocompatibility studies on presterilized solution-cast and commercially available Nafion membranes have shown no significant acute or chronic foreign body response, for duration of up to 3 months.19 The biological response of Nafion was comparable to that of silicone rubber (Silastic). On the other hand, the strong ion-exchange properties of Nafion result in significant calcification, in both in vitro and in vivo environments.20 The sulfonate (R-SO3H) groups in the hydrophilic domains of the Nafion membrane act as nucleating sites for calcium phosphate crystals. Apart from inhibiting glucose transport, these crystals also tend to embrittle the Nafion membranes, causing them to crack and thus contribute to sensor failure. Spin-coated Nafion membranes preincubated in ferric chloride have shown a significant decrease in calcareous * To whom correspondence should be addressed. E-mail: papadim@ mail.ims.uconn.edu. † Institute of Materials Science, University of Connecticut. ‡ Center for Biomaterials & Surgical Research Center, University of Connecticut Health Center.
deposits.21 However, glucose oxidase based sensors suffer from enzyme denaturation upon prolonged exposure to the low pH of FeCl3 solution (ca. 2), necessary for iron infiltration.21 In addition, infiltrated ferric ions and its hydroxides impart local stresses that could eventually lead to membrane rapture. To address the aforementioned issues, the electrostatic self-assembly of Nafion and Fe3+ from dilute solutions is presently investigated. This procedure should reduce stress levels within these films and saturate the sulfonate groups in Nafion with Fe3+, thereby preventing calcification. Since Fe3+ complexes with sulfonate groups are more stable when compared to Ca2+,22 the driving force for calcification is expected to decrease significantly, if not to be eliminated completely. Alternating electrostatic self-assembly, based on the attraction of oppositely charged species is a powerful tool for building a variety of layered and multilayered structures. The pioneering work on sequential deposition of nanoparticle colloids by Iler23 was successfully extended by Decher and co-workers to polyanions and polycations.24-26 Since then this technique has found applications in a number of charged systems such as proteins and biopolymers,27-29 nanoparticles30-32 and multicomponent films.33,34 The layer thickness and other microstructural attributes of these assemblies were found to be sensitive to the type of charged species, their concentration, molecular weight, ionic strength and pH. Although the ease for automation exists, electrostatic selfassemblies have been viewed unfavorably for the construction of thick films. This paper describes an approach to construct thick Fe3+/Nafion films intended for use in biomedical applications, based on their controlled glucose permeability and enhanced resistance to calcification.
10.1021/bm0002813 CCC: $19.00 © 2000 American Chemical Society Published on Web 04/15/2000
Calcification-Resistant Nafion/Fe3+ Assemblies
Experimental Section Reagents and Materials. Nafion was obtained from Aldrich as a 5% w/v mixture in water and lower aliphatic alcohols (1100 equivalent weight; i.e., 1100 g of polymer per mole of -SO3H groups). Iron(III) chloride hexahydrate (FeCl3‚H2O) was also acquired from Aldrich. A.C.S. certified KCl was purchased from Fischer and used without further purification. 28-30 wt % aqueous solution of NH4OH (Acros) and 35-38% hydrochloric acid (J. T. Baker) were used as a 1% dilution to adjust pH. Millipore quality deionized water (resistivity >18 MΩ) was utilized in all experiments. Silicon wafers with native oxide and microscope glass slides (Fisher) were used as substrates for self-assembly. These were cleaned in pirahana solution (H2SO4/H2O2 (7:3)), rinsed with deionized water and methanol, kept in deionized water overnight, and used for the self-assembly growth without further surface modification. A 1 mg/mL (0.9 mM, based on the repeat unit molecular weight) Nafion solution was prepared by diluting the as received solution in a (9:1) methanol/water mixture and used for all experiments. The pH of these solutions was adjusted with aqueous NH4OH solution. The ionic strength of Nafion solutions was modified with KCl. Then 0.5 g of FeCl3‚6H2O was solubilized in 100 mL of water to produce 5 mg/mL (18.5 mM) solution. The pH of this solution was in the range of 1.9-2.0. No further attempts were made to vary the pH of the FeCl3 solution. Experimental Techniques. An HMS Series programmable slide stainer from Carl Zeiss, Inc., was used for the layer-by-layer assembly of Nafion with Fe3+. The sample holder in the HMS Series slide stainer was covered to reduce solvent evaporation, thereby improving film quality. Each dip cycle consisted of 8 steps. First the substrates were immersed in Nafion solution for 15 min followed by three consecutive washing steps, of 1 min each in Millipore deionized water. Subsequently, the substrates were dipped into ferric chloride solution for 15 min followed by three washes as before. Twelve subsequent dip cycles were usually employed in this study. The substrates were constantly agitated throughout the dip cycle to improve film quality. After completion of the desired number of dip cycles, the substrates were removed, rinsed with Millipore water and methanol, and air-dried. Characterization Techniques. Ultraviolet-visible (UV/vis) spectroscopy was performed on the Perkin-Elmer Lambda 6 spectrophotometer. Ellipsometric data was acquired by a variable angle WVASE32 spectroscopic ellipsometer; J. A. Woolman Co., Inc. Films were scanned in the range 250-1000 nm at incident angles of 65, 70, and 75°. Data were collected every 10 nm at 10 revolutions per measurement. A standard Cauchy model was used to fit for thickness and optical constants in the entire optical range. Dynamic light scattering experiments were performed to determine the hydrodynamic radius of Nafion in solution. Four different Nafion solutions, pH 3 and 5.5, with and without salt were used in the DLS experiments. The INNOVA “multiple-angle” dynamic light scattering apparatus was used. All the samples were filtered through 0.45 µm low-protein-binding Durapore membranes prior to the scan. A dialysis chamber was used to determine permeability of 1 µm glass fiber membranes (Gelman Sciences) coated with selfassembled Nafion/Fe3+ layers. The interior of the dialysis chamber was filled with 1 mL of 1 M glucose in 0.9% NaCl solution. Subsequently, the concentration of glucose that diffused out from the dialysis chamber to the dialysis buffer (50 mL of 0.9% NaCl solution, no glucose) was determined by a Beckman glucose analyzer. The experimental procedure has been described elsewhere.21 The apparent diffusion coefficients of glucose through the
Biomacromolecules, Vol. 1, No. 2, 2000 203 Scheme 1. Schematic Representation of the Growth of Alternating Nafion/Fe3+ Multilayer Assembliesa
a
This simplified illustration is further elaborated on in the text.
self-assembled membranes were derived based on the following assumptions: (i) the glass membrane can be considered extremely porous and does not contribute to the resistance, and hence, the flux can be assumed to be through the self-assembled layer; (ii) the flow rate did not effect the flux of glucose through the membrane; (iii) rapid diffusion was assumed through the boundary layers. The equations used for the apparent diffusion coefficient calculation (DApp) were as follows:35-37
dC ) NA A dt
NA )
-V
(1)
DApp [[CG]i - [CG]f] L
(2)
where NA represents the flux, L and A represent the thickness and the area of the membrane with the self-assembled film, and V is the volume of the dialysis chamber (1 cm3). At time t, the change of glucose concentration (dC/dt) in the dialysis chamber is related to the difference between glucose concentration in the dialysis chamber [CG]i and dialysis buffer [CG]f. A Kevex quantum energy dispersive X-ray analyzer (EDX), operating with an AMRAY 1000A scanning electron microscope (SEM) was utilized to determine the degree of mineralization via elemental analysis.
Results and Discussion Solubility studies in a series of solvents have shown that, depending on the dielectric constant of the solvent or solvent mixture, Nafion can form homogeneous mixtures, colloids or precipitates.38 On the basis of the 9/1 methanol/water solvent ratio used in this study ( ∼ 38), Nafion is expected to attain a micellar conformation with the polar sulfonate groups located on the surface and the hydrophobic fluorocarbon backbone buried inside.38 The self-assembly process is schematically depicted in Scheme 1. Upon immersion in the acidic Nafion solution (pH ∼ 3), the substrate silanol groups (Si-OH) are partially protonated,23,32 providing an electrostatic force to attract the negatively charged Nafion micelles. The degree of micellar spreading during assembly will be addressed in the following section. After being rinsed in water to remove loosely bound
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Figure 1. Ellipsometrically determined thickness vs dip cycle for alternating Nafion/Fe3+ assemblies as a function of pH of Nafion solution. (A) pH ) 3, (B) pH ) 4.5, and (C) pH ) 5.5. The pH values of FeCl3 and wash solutions were kept constant at 2 and 7, respectively. Table 1. Dynamic Light Scattering (DLS) Deduced Hydrodynamic Radius RH and Diffusion Coefficient DH of Nafion Solutions as a Function of pH and Ionic Strength Nafion w/o KCl
Nafion 0.01 M KCl
pH
RH (nm)
D (cm2 s-1)
RH (nm)
D (cm2 s-1)
3.0 5.5
113.5 115.9
3.83 × 10-8 3.75 × 10-8
45.8 51.0
9.5 × 10-8 8.5 × 10-8
species, the substrate is dipped into ferric chloride solution. Ferric ions are attracted by the sulfonate groups, facilitating surface charge reversal and thereby restoring the original surface charge. The entire process is repeated until the desired thickness is achieved. A number of deposition parameters influence the microstructure and growth rate of such assemblies. Figure 1 illustrates the ellipsometrically determined film thickness as a function of dip cycles. Maintaining the pH of FeCl3 and wash solutions constant, the pH of the Nafion solution was found to have a profound influence on film growth. A relatively fast growth was observed at pH 3, corresponding to ca. 40 nm per dip cycle. At higher pH a significantly lower deposition is observed (i.e., 6.7 and 6.3 nm/dip cycle for pH values of 4.5 and 5.5 respectively). Table 1 illustrates the hydrodynamic radius RH and diffusion coefficient DH of Nafion solutions as determined by dynamic light scattering (DLS). The influence of pH on the hydrodynamic radius of Nafion appears to be negligible for pH 3 and 5.5. This concurs with the strong acidic character of sulfonate groups. Nafion’s acidity (-Ho ∼ 12 on Hammett’s scale) is comparable with that of 100% sulfuric acid,39 implying a nearly complete degree of ionization for both pH 3 and 5.5. On the other hand, the formation of insoluble hydroxides occurs around pH g 4.3 based on the solubility product of Fe(OH)3 (Ksp ∼ 6 × 10-39).40 This transformation of adsorbed Fe3+ to Fe(RSO3)x(OH)3-x (x ) 1, 2, 3; RSO3 stands for Nafion) results in increasing the basicity of the substrate. On the basis of these observations, the transition to significantly lower growth rate of the Nafion/ Fe3+ assemblies above pH 4.5 could be associated with surface spreading of Nafion induced by acid-base neutralization. The thickness of the Nafion/Fe3+ assemblies, as a function
Figure 2. Ellipsometrically determined thickness vs dip cycle for alternating Nafion/Fe3+ assemblies as a function of pH and ionic strength of Nafion solution: (A) pH ) 3, 0.01 M KCl; (B) pH ) 3, no salt; (C) pH ) 4.5, 0.01 M KCl; (D) pH ) 4.5, no salt. The pH values of FeCl3 and wash solutions were kept constant at 2 and 7, respectively.
of the pH and ionic strength of Nafion solution is depicted in Figure 2. The addition of 0.01 M KCl was found to have a profound effect on film growth. The influence of salt concentration on the thickness of the deposited films was also investigated, with the above value determined as optimum ionic strength based on film quality. Using 0.1 M KCl, no film deposition was observed and salt was preferentially precipitating on the surface. A similar effect of screening-reduced adsorption or dehydration of the surface at high salt content was observed by Hammond et al.41,42 The well documented charge screening effect in polyelectrolytes,43-48 results in diminishing repulsive interactions between the negatively charged sulfonate groups upon addition of positively charged ions (i.e., K+) and allows Nafion to attain a more compact conformation. This results in a nearly 60% reduction in the hydrodynamic volume compared to salt-free Nafion solutions (see Table 1). The corresponding increase in diffusion coefficient (D) of Nafion micelles implies greater diffusion rate to the assembly surface. Surprisingly enough, the average growth of 47 nm/dip cycle (Figure 2A: pH 3, 0.01 M KCl) roughly corresponds to the hydrodynamic radius in solution (Table 1). This indicates that surface adsorption is accompanied by minimum Nafion surface spreading, relative to the salt-free case, where a nearly 65% spreading results in growth of 40 nm/dip cycle. Introduction of salt results in higher growth for different Nafion pH. This is further influenced by the relative strength of the surface-induced interactions that tend to flatten the micelles and the charge-screening forces, which attempt to keep the conformation intact. Nevertheless, it appears that at pH 4.5 the basicity of the Fe(RSO3)x(OH)3-x surface overpowers the charge-screening forces and causes Nafion spreading. The pronounced optical absorbance of ferric ions (bright yellow films) provides additional insight into the film growth and degree of iron incorporation. Figure 3 shows the gradual increase in the intensity of UV/vis spectra of a Nafion/Fe3+ assembly (pH ) 4.5 and 0.01 M KCl) as a function of dip cycles. The profound shoulder at 350 nm is associated with iron(III) absorption and increases linearly with the number of layers. The absorbance values at 350 nm, when plotted
Calcification-Resistant Nafion/Fe3+ Assemblies
Figure 3. UV/vis spectra of Nafion/Fe3+ assemblies for pH ) 4.5 and 0.01 M KCl for increasing number of deposited layers. Insert shows the optical absorbance at 350 nm vs number of dip cycles for films deposited at: (A) pH ) 3, 0.01 M KCl; (B) pH ) 3, no salt; (C) pH ) 4.5, 0.01 M KCl; (D) pH ) 4.5, no salt. The pH values of FeCl3 and wash solutions were kept constant at 2 and 7, respectively.
Figure 4. Ellipsometrically determined thickness vs dip cycle for alternating Nafion/Fe3+ assemblies deposited at pH ) 3, 0.01 M KCl and with (A) neutral (pH ) 7) and (B) acidic (pH ) 3) wash. The pH of the FeCl3 solution was kept constant at 2.
against the number of dip cycles, follow linear growth pattern (see insert in Figure 3) similar to that of the ellipsometrically determined thickness shown in Figure 2. The sharp transition of adsorbed Fe3+ to Fe(RSO3)x(OH)3-x above pH of 4.3 prompted us to investigate the effect of pH of the wash cycle on film growth characteristics. Figure 4 illustrates the ellipsometrically determined Nafion/Fe3+ multilayer thickness vs dip cycle for an acidic (pH ) 3) and neutral (pH ) 7) wash. In general, washing at neutral pH results in thicker films, regardless of the pH and ionic strength for the Nafion solution. This is attributed to iron precipitation in neutral wash, which tends to incorporate larger amounts of iron in these assemblies, thus explaining the faster growth. Surprisingly, assemblies obtained using a neutral wash appear to be smoother and more uniform than those obtained by an acidic wash, when investigated by optical (differential interference contrast) and scanning electron microscopy. Evidently, the pH ) 3 wash stimulates not only excess Fe3+ removal but also surface migration that leads to the formation of large domains (on the order of 500 nm) that contribute to light scattering. On the other hand, the assemblies obtained by the neutral wash tend to immobilize the Fe(III) ions in the form of hydroxides
Biomacromolecules, Vol. 1, No. 2, 2000 205
Figure 5. Relative iron concentration vs layer number for Nafion/ Fe3+ assembled with FeCl3 and Nafion at pH 2 and 3, respectively: (A) 0.01 M KCl; water wash (B) no salt; water wash (C) 0.01 M KCl; acidic wash (D) no salt; acidic wash (see text for details).
(Fe(RSO3)x(OH)3-x), which yield highly transparent and scattering-free assemblies. A measure of iron incorporation can be derived from the correlation of the optical absorbance at 350 nm (arising from Fe(III)) and film thickness. Figure 5 illustrates the dip cycle dependence of the relative iron concentration, as derived by dividing the absorbance at 350 nm by the ellipsometrically determined film thickness. The initial iron concentration appears to start from a low value and rapidly increases within six bilayers to a fairly constant level. The early stage deviation from equilibrium could be attributed to various surface effects and film inhomogeneities. For example, imperfect surface coverage by Nafion in the early stages significantly contributes to error associated with the ellipsometrically derived thickness. The lower refractive indices for the initial bilayers (ranging from 1.22 to 1.28) relative to thicker films (1.35-1.41) adds certain validity to this argument. The iron concentration plateau value appeared to be dependent only on the wash pH and was independent of the pH and ionic strength of Nafion solutions. This reinforces the argument that Fe(III) precipitates in neutral wash, resulting in higher iron incorporation in these assemblies as compared to the acidic wash. The hydrolytic stability of these Nafion/Fe3+ assemblies is of great importance for long-term implantable devices. Figure 6 illustrates the UV/vis spectra of a 12 layer Nafion/ Fe3+ assembly (Nafion pH ) 3, 0.01 M KCl) immersed in water for 6 weeks. Although this experiment reflects only the Fe(III) content, Fourier Transform infrared (FTIR) spectroscopy suggests that the Nafion to Fe(III) ratio remains unchanged throughout the length of this experiment. During the course of 1 week, only a slight decrease of iron (and subsequently Nafion) was observed, reaching a constant value thereafter for the duration of the experiment (6 weeks). The initial iron loss (ca. 3.5% based on integrated intensity) is attributed to losely attached Nafion moieties, presumably the low molecular weight tail of this polymer. In contrast, cast Nafion films require annealing above Tg (109 °C for protonated Nafion) to ensure insolubility.49 The control of small molecule diffusion through a membrane is one of the most important characteristics for biosensor development.20,50 Figure 7 illustrates glucose
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Figure 6. UV/vis spectra of 12 layers Nafion/Fe3+ film (pH ) 3, 0.01 M KCl) immersed in water for one to six weeks. Inset shows the absorbance at 310 nm as a function of immersion time.
Figure 7. Effect of annealing on glucose permeability through selfassembled Nafion/Fe3+ films on 1 µm glass fiber membrane: (A) empty membrane; (B) one layer, annealed; (C) one layer, not annealed; (D) two layers, annealed; (E) two layers, not annealed; (F) three layers, annealed; (G) four layers, annealed.
permeability studies on Nafion/Fe3+ assemblies supported on 1 µm glass fiber membranes as a function of deposited bilayers and annealing treatment at 120 °C (for 30 min). The permeability of glucose through the Nafion/Fe3+ assembly on the membrane decreases constantly with increasing number of bilayers. Annealing results in higher glucose permeability, which can be attributed to film shrinkage and increased porosity. The abrupt transition observed from the second to the third bilayer is associated with closing of the micron size pores of the substrate membrane. Since alternative electrostatic assembly takes place on the entire surface area of the membrane, which includes the inner pores, insufficient removal of excess reagents via the wash could explain the accelerated clogging of the composite structure. For the first and second layer assemblies, the large permeability might be attributed to the percolated pores of the supporting membrane. These pores progressively decrease in size and by the third and fourth layer they are finally filled. Thus, we have chosen the third and fourth layer to calculate the apparent diffusion coefficient (DApp). On the basis of the model discussed in the Experimental Section, the apparent diffusion coefficients were determined to be 1.37 × 10-10 and 1.34 × 10-10 cm2 s-1, assuming steady state conditions and film thickness corresponding to three and four dip cycles
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Figure 8. Extent of calcification studied by EDX. Comparison of calcium line intensity for self-assembled Nafion/Fe3+ and spin-coated and annealed (120 °C) Nafion films deposited on polycarbonate membrane and treated in DMEM nutrient mixture for a period of 4 weeks.
respectively (i.e. 120 and 160 nm). Since these films were self-assembled on a glass fiber membrane, their thickness and microstructure greatly influence the diffusion coefficient. In certain regions the film thickness could be smaller than this obtained from one dip cycle (i.e. 40 nm) resulting in glucose diffusion coefficients lesser then 10-11 cm2 s-1. The above values for the self-assembled Nafion/Fe3+ films show a marked reduction in the diffusion coefficient when compared to the diffusion coefficient for unannealed Nafion film (3.4 × 10-8 cm2 s-1).51 For polyelectrolyte selfassembled films (with thickness of 8-40 Å per dip cycle) the diffusion coefficient of Rhodamine have been reported to be on the order of 10-15 cm2 s-1.52,53 Klitzing et al. estimated that for smaller molecules such as TEMPOL (structurally similar to glucose) the diffusion coefficient should be greater than 10-13 cm2 s-1.52,53 On the basis of the growth rate of these assemblies, the resulting film structure is expected to be more compact, thus less penetrable to small molecules such as TEMPOL as compared to self-assembled Nafion/Fe3+ films. Moreover, the increased diffusion in the self-assembled Nafion/Fe3+ films could be attributed to annealing, which modifies the microstructure by inducing hydrophilic channels in the Nafion assembly thereby facilitating transport of glucose through the self-assembled films.54-56 Kevex quantum energy dispersive X-ray was utilized to investigate the degree of calcification in these assemblies. Self-assembled Nafion/Fe3+ (10 layers, 400 nm; pH ) 3, water wash) and spin-coated Nafion films (ca. 400 nm)20,21 deposited on 10 µm pore size polycarbonate membrane were immersed in Dulbecco’s modified Eagle’s medium (DMEM) for 4 weeks. This cell culture medium was previously shown to be a good in vitro model solution to study Nafion calcification.21 Subsequently, EDX elemental analysis indicated a more than 20-fold decrease in intensity of adsorbed calcium as compared to spin-coated and annealed (120 °C) Nafion films (see Figure 8). These results suggest that the Nafion/Fe3+assemblies may be a viable alternative in reducing calcification in implantable devices. Further in Vitro and in ViVo studies are presently underway in order to prove the superiority of these structures in prolonging sensor’s life span in terms of calcification.
Calcification-Resistant Nafion/Fe3+ Assemblies
Conclusions We have described a simple and effective method for fabrication of calcification-resistant semipermeable Nafionbased membranes intended for implantable sensors and devices. This technique is also promising for application in different systems of ionic character as a means of preventing mineralization. These Nafion/Fe3+ assemblies feature controlled film thickness and ability to grow relatively thick films (i.e., 47 nm/dip cycle). pH and ionic content affect the growth of Nafion/Fe(III) bilayer assemblies, resulting in different thickness and varying degree of Fe3+/Fe(OH)1-3 incorporation. The self-assembled films impart hydrolytic stability making this method superior to traditional dip- or spin-coated Nafion films, which require thermal treatment to ensure stability and insolubility. The diffusion of glucose through the resulting assemblies appears to be at least 2 orders of magnitude smaller than dip-coated Nafion films51 and can be further varied by the number of deposited layers. Acknowledgment. This work was supported by a grant from the National Institute of Health (#R01RR14171). The authors wish to thank our collaborators: Drs. D. J. Burgess, S. Huang, J. T. Koberstein, D. Kreutzer, and T. K. Kim for stimulating discussions and Drs. T. A. P. Seery and A. Segal for assistance with the dynamic light scattering experiments. The authors would also like to thank T. Valdes for her help in the calcification studies. References and Notes (1) Lee, S. J.; Mukerjee, S.; McBreen, J.; Rho, Y. W.; Kho, Y. T.; Lee, T. H. Electrochim. Acta 1998, 3693-3701. (2) Wilson, M. S.; Gottesfeld, S. J. Appl. Electrochem. 1992, 22, 1-7. (3) Harmer, M. A.; Farneth, W. E.; Sun, Q. J. Am. Chem. Soc. 1996, 118, 7708-7715. (4) Nafion Perfluorinated Membranes; Product Bulletin, Du Pont Company: Wilmington, DE, 1983; pp 1-4. (5) Ishiji, T.; Kudo, K.; Kaneko, M. Sens. Actuators B 1994, 22, 205210. (6) Huang, Q.; Lu, Z.; Rusling, J. F. Langmuir 1996, 12, 5472-5480. (7) Chan, C. M.; Fung, C. S.; Wong, K. Y.; Lo, W. Analyst 1998, 123, 1843-1847. (8) Barton, S. A. C.; Murach, B. L.; Fuller, T. F.; West, A. C. J. Electrochem. Soc. 1998, 145, 3783-3788. (9) Nafion Resins; Aldrich Technical Bulletin; Aldrich: Milwaukee, WI, 1988; Vol. 163, pp 1-5. (10) Wilkins, E.; Atanasov, P.; Muggenburg, B. A. Biosens. Bioelect. 1995, 10, 485-494. (11) Madaras, M. B.; Buck, R. P. Anal. Chem. 1996, 68, 3832-3839. (12) Hodgson, A. W. E.; Jacquinot, P.; Hauser, P. C. Anal. Chem. 1999, 71, 2831-2837. (13) Jacquinot, P.; Hodgson, A. W. E.; Hauser, P. C.; Muller, B.; Wehrli, B. Analyst 1999, 124, 871-876. (14) Sotiropoulos, S.; Wallgren, K. Anal. Chim. Acta 1999, 388, 51-62. (15) Sun, L. X.; Xu, F.; Okada, T. Chem. Sens. 1998, 14, 149-152. (16) Villeneuve, N.; Bedioui, F.; Voituriez, K.; Avaro, S.; Vilaine, J. P. J. Pharmacol. Toxicol. Methods 1998, 40, 95-100.
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