High-Sensitivity Amperometric Biosensors Based on Ferrocene

Apr 21, 2009 - Amperometric biosensors for glucose and hydrogen peroxide have been built by immobilizing glucose oxidase (GOX) and horseradish peroxid...
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High-Sensitivity Amperometric Biosensors Based on Ferrocene-Modified Linear Poly(ethylenimine) Stephen A. Merchant,† Tu O. Tran,† Matthew T. Meredith,‡ Tracy C. Cline,† Daniel T. Glatzhofer,‡ and David W. Schmidtke*,†,§ †

School of Chemical, Biological, and Materials Engineering and ‡Department of Chemistry and Biochemistry and §University of Oklahoma Bioengineering Center, University of Oklahoma, Norman, Oklahoma 73019 Received February 9, 2009. Revised Manuscript Received March 24, 2009

Amperometric biosensors for glucose and hydrogen peroxide have been built by immobilizing glucose oxidase (GOX) and horseradish peroxidase (HRP) in cross-linked films of ferrocene-modified linear poly(ethylenimine). At pH 7, the glucose sensors generated limiting catalytic current densities of 1.2 mA/cm2. These current densities are approximately 4 times higher than those with other ferrocene-based redox polymers and are comparable to the highest reported values for osmium-based redox polymers with GOX. Because of the high sensitivity of these films (73 nA/cm2 3 μM), glucose concentrations in the micromolar range could be detected. Similarly, sensors were constructed with HRP-generated current densities of 0.9 mA/cm2 under saturation conditions and sensitivities of 500 nA/cm2 3 μM. The results show that the ability of Fc-LPEI to effectively communicate with a variety of enzymes has potential applications in measuring low substrate concentrations in implantable biosensors and producing high current outputs in enzymatic biofuel cells.

Introduction The bioelectrocatalysis of redox enzymes by redox polymers has attracted significant attention in a number of fields. In the area of health care, redox-polymer-based biosensors are being designed for the in vivo monitoring of glucose in diabetes,1-3 glutamate and ascorbate in brain tissue,4,5 and peroxides in neurodegenerative disorders.6 Similar approaches to redox polymer bioelectrocatalysis are also being utilized in the detection of hybridization reactions in RNA and DNA assays7,8 and antigenantibody binding in immunoassays.9-12 In the food industry, redox polymer-enzyme films are being utilized for detecting fish freshness13 and ethanol content in wine fermentation.14,15 Finally, redox polymer bioelectrocatalysis is emerging as a promising *Corresponding author. Tel: (405) 325-7944. Fax: (405) 325-5813. E-mail: [email protected]. (1) Csoregi, E.; Schmidtke, D. W.; Heller, A. Anal. Chem. 1995, 67, 1240–1244. (2) Ishikawa, M.; Schmidtke, D. W.; Raskin, P.; Quinn, C. A. J. Diabetes Complicat. 1998, 12, 295–301. (3) Feldman, B.; Brazg, R.; Schwartz, S.; Weinstein, R. Diabetes Technol. Ther. 2003, 5, 769–779. (4) Kulagina, N. V.; Shankar, L.; Michael, A. C. Anal. Chem. 1999, 71, 5093– 5100. (5) Oldenziel, W. H.; Dijkstra, G.; Cremers, T. I.; Westerink, B. H. Anal. Chem. 2006, 78, 3366–3378. (6) Kulagina, N. V.; Michael, A. C. Anal. Chem. 2003, 75, 4875–4881. (7) Campbell, C. N.; Gal, D.; Cristler, N.; Banditrat, C.; Heller, A. Anal. Chem. 2002, 74, 158–162. (8) Kavanagh, P.; Leech, D. Anal. Chem. 2006, 78, 2710–2716. (9) Vreeke, M.; Rocca, P.; Heller, A. Anal. Chem. 1995, 67, 303–306. (10) Lu, B.; Iwuoha, E. I.; Smyth, M. R.; OKennedy, R. Anal. Chim. Acta 1997, 345, 59–66. (11) Lopez, M. A.; Ortega, F.; Dominguez, E.; Katakis, I. J. Mol. Recognit. 1998, 11, 178–181. (12) Calvo, E. J.; Danilowicz, C.; Lagier, C. M.; Manrique, J.; Otero, M. Biosens. Bioelectron. 2004, 19, 1219–1228. (13) Niculescu, M.; Nistor, C.; Frebort, I. I.; Pec, P.; Mattiasson, B.; Csoregi, E. Anal. Chem. 2000, 72, 1591–1597. (14) Niculescu, M.; Mieliauskiene, R.; Laurinavicius, V.; Csoregi, E. Food Chem. 2003, 82, 481–489. (15) Niculescu, M.; Erichsen, T.; Sukharev, V.; Kerenyi, Z.; Csoregi, E.; Schuhmann, W. Anal. Chim. Acta 2002, 463, 39–51.

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technology for developing miniaturized biofuel cells16-19 that may be implanted in biological systems. For the biosensors described above to be useful in real world situations, they must be portable, simple to operate, and able to make measurements in a natural environment. To make these systems portable, it is often necessary to miniaturize these systems, which necessitates that the sensor produces a signal that is measurable with low-cost, portable electronics. Furthermore, in a number of sensing situations it is necessary to detect small quantities of target analyte in small volumes. Therefore, there is a need to develop sensors that exhibit high sensitivity and low detection limits. Similarly, in the field of biofuel cells there is a need for high power output. For an enzymatic biofuel cell to generate the maximum amount of power, it must produce both high current and high potential. High current in the case of enzymatic reactions necessitates the efficient transfer of electrons between the enzyme’s redox center and the electrode surface. In the case of redox polymer bioelectrocatalysis, the sensor sensitivity or the biofuel cell power output is dependent upon the flow of electrons between the analyte/metabolite and the electrode surface (Figure 1). Depending upon the system design, any of these transduction steps may limit the overall reaction rate and consequently the signal output. Previous studies20-22 have suggested that normally either step 3 or 4 is rate-determining. The rate at which electrons are transferred between the enzyme and the mediator is dependent on the distance between them and (16) Chen, T.; Barton, S. C.; Binyamin, G.; Gao, Z. Q.; Zhang, Y. C.; Kim, H. H.; Heller, A. J. Am. Chem. Soc. 2001, 123, 8630–8631. (17) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2003, 125, 6588–6594. (18) Tamaki, T.; Ito, T.; Yamaguchi, T. J. Phys. Chem. B 2007, 111, 10312– 10319. (19) Barriere, F.; Kavanagh, P.; Leech, D. Electrochim. Acta 2006, 51, 5187– 5192. (20) Aoki, A.; Heller, A. J. Phys. Chem. 1993, 97, 11014–11019. (21) Aoki, A.; Rajagopalan, R.; Heller, A. J. Phys. Chem. 1995, 99, 5102–5110. (22) Rajagopalan, R.; Aoki, A.; Heller, A. J. Phys. Chem. 1996, 100, 3719–3727.

Published on Web 04/21/2009

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Figure 1. Schematic of the transduction steps involved in the bioelectrocatalytic oxidation of the substrate in a cross-linked redox polymer-enzyme-coated electrode.

whether an electrostatic complex forms between the redox polymer and enzyme.22,23 The rate at which electrons are transported through the film is dependent on charge propagation along the polymer’s backbone (through σ and other chemical bonds), electron self-exchange between identical redox centers, collisions between polymer segments, and counterion movement.24 Increasing the charge density on the polymer backbone results in (a) making the polymer films more hydrophilic and allowing for enhanced permeation of substrates and products, (b) increases in the strength of the electrostatic complex formed with the enzyme, and (c) increases in the rate of electron transport through the films, all of which result in increased current density.20,22 In addition, the rate of electron transfer between the enzyme and the mediator is significantly enhanced when (a) the polymer backbone is flexible, (b) the spacer arm coupling the redox center to the polymer backbone is flexible,25 and (c) the length of the spacer arm is increased.25,26 Recently, we reported the synthesis and characterization of a novel redox polymer based on attaching ferrocene (Fc) redox centers to a linear poly(ethylenimine) (LPEI) backbone.27 Electrochemical impedance spectroscopy (EIS) measurements of the product cD1/2 e (where De is the electron diffusion coefficient and c is the concentration of redox centers in the film) showed that the rate of electron transport through cross-linked films of Fc-LPEI without enzyme ranged from 10-8 to 10-9 mol/cm2 3 s1/2 and was dependent on the electrolyte. This initial study suggested that LPEI-Fc redox polymers could be used as molecular wires to connect the redox centers of an enzyme (GOX) to an electrode surface. The current densities at saturating glucose concentrations (∼480 μA/cm2) were significantly higher than those reported (13.5-275 μA/cm2) for other ferrocene-based redox polymers28-30 and compared favorably with osmium-based redox polymers.31-33 We initially attributed these high current densities to the high rate of electron transfer in these films. However, we did not measure electron diffusion coefficients for cross-linked redox polymer films that contained enzyme. In this article, the relationship between electron diffusion through cross-linked LPEI-Fc films containing enzyme and the (23) Katakis, I.; Ye, L.; Heller, A. J. Am. Chem. Soc. 1994, 116, 3617–3618. (24) Rusling, J. F.; Forster, R. J. J. Colloid Interface Sci. 2003, 262, 1–15. (25) Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2003, 125, 4951–4957. (26) Schuhmann, W.; Ohara, T. J.; Schmidt, H. L.; Heller, A. J. Am. Chem. Soc. 1991, 113, 1394–1397. (27) Merchant, S. A.; Glatzhofer, D. T.; Schmidtke, D. W. Langmuir 2007, 23, 11295–11302. (28) Calvo, E. J.; Etchenique, R.; Danilowicz, C.; Diaz, L. Anal. Chem. 1996, 68, 4186–4193. (29) Bu, H. Z.; Mikkelsen, S. R.; English, A. M. Anal. Chem. 1995, 67, 4071– 4076. (30) Hale, P. D.; Boguslavsky, L. I.; Inagaki, T.; Karan, H. I.; Lee, H. S.; Skotheim, T. A.; Okamoto, Y. Anal. Chem. 1991, 63, 677–682. (31) Gregg, B. A.; Heller, A. Anal. Chem. 1990, 62, 258–263. (32) de Lumley-Woodyear, T.; Rocca, P.; Lindsay, J.; Dror, Y.; Freeman, A.; Heller, A. Anal. Chem. 1995, 67, 1332–1338. (33) Kenausis, G.; Taylor, C.; Katakis, I.; Heller, A. J. Chem. Soc., Faraday Trans. 1996, 92, 4131–4136.

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sensor response was studied. We also investigated the effects of pH, enzyme fraction, and film thickness on the sensor response to glucose. We report that a slight modification of our synthesis procedure of LPEI-Fc results in a dramatic increase in the current density for glucose oxidase-based sensors (imax = 1.2 mA/cm2). The high sensitivity of these films (73 nA/μM 3 cm2) allowed for the detection of glucose in the micromolar range. We also demonstrate the versatility of Fc-LPEI as a “wire” for other redox enzymes by cross-linking the polymer with horseradish peroxidase to produce highly sensitive (imax = 0.94 mA/cm2) hydrogen peroxide sensors.

Experimental Section Chemicals and Solutions. Glucose oxidase (GOX) from Aspergillus niger (EC 1.1.3.4, type X-S, 246 units/mg of solid, 75% protein), horseradish peroxidase (HRP; EC 1.11.1.7, type VI, 250-300 untis/mg), ferrocenecarboxaldehyde, poly(2-ethyl-2oxazoline), and all salts and acids were purchased from SigmaAldrich. Ethylene glycol diglycidyl ether (EGDGE) was purchased from Polysciences Inc., Warrington, PA. Stock solutions of 2 M glucose were allowed to mutarotate for 24 h before use and were subsequently kept refrigerated at 4 °C whereas solutions of hydrogen peroxide were prepared by diluting a 50% solution. All other chemicals and solvents were reagent grade and used as received. Redox Polymer Synthesis. The redox polymer designated as Fc-LPEI was synthesized by coupling ferrocenecarboxaldehyde to linear poly(ethylenimine) (LPEI) by using a modified version of our previously published protocol.27 Linear poly(ethylenimine) (LPEI) (avg MW ca. 86 000) was obtained by acidic hydrolysis of poly(2-ethyl-2-oxazoline) (avg MW 200 000), followed by neutralization with sodium hydroxide.34 In a round-bottomed flask, 0.252 g of LPEI was dissolved in 10 mL of methanol, and a solution of 0.187 g (0.87mmol) of ferrocenecarboxaldehyde dissolved in 3 mL of methanol was added to it dropwise under constant agitation. The resulting dark-red solution was stirred for 2 h and cooled in an ice bath. Sodium borohydride (0.033 g, 0.87mmol) was added, upon which the solution lightened in color. After 1 h, the methanol was removed under vacuum, and the residue was extracted overnight with diethyl ether to remove any nonreacted aldehyde and ferrocenylmethanol. The ether was decanted, and the residue was washed with diethyl ether before being dried under vacuum. At this point, the procedure was changed from the previous protocol.27 Instead of extracting the residue with benzene, the polymer was redissolved in absolute methanol and filtered to remove any residue, and the methanol was removed under reduced pressure. This dissolution/filtration/drying sequence was repeated two more times and resulted in a cleaner polymer, the structure of which was verified by 1H NMR spectroscopy.27 Approximately 20% of the nitrogen was substituted with ferrocene. Enzyme Sensor Construction. Prior to use, all electrodes were polished successively on three grades of alumina (5, 1, and (34) York; Frech, S.; Snow, R.; Glatzhofer, A. D. Electrochim. Acta 2001, 46, 1533–1537.

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0.3 μm) and washed thoroughly with Nanopure water after each polishing step. The Fc-LPEI polymer was dissolved in water by the addition of a 0.1 M HCl solution until the final concentration of the polymer solution was 10 mg/mL and the pH was 5. Unless otherwise noted, glucose sensors were prepared by cross-linking glucose oxidase to Fc-LPEI to form enzymatic redox hydrogels: 14 μL of polymer solution (10 mg/mL), 6 μL of glucose oxidase solution (10 mg/mL), and 0.75 μL of EGDGE solution (10% v/v) were mixed; 3 μL aliquots were placed onto the glassy carbon electrode surface; and the mixture was allowed to cure for 1824 h. Hydrogen peroxide sensors were prepared by combining 14 μL of polymer solution (10 mg/mL), 6 μL of horseradish peroxidase solution (10 mg/mL), and 0.75 μL of EGDGE solution (10% v/v). Aliquots of this mixture (3 μL) were then placed on the glassy carbon electrode surface and allowed to cure. Electrochemical Measurements. Cyclic voltammetry and constant potential experiments were performed with a bipotentiostat (model 832, CH Instruments, Austin, TX), and electrochemical impedance measurements were performed with a Solartron SI 1260 impedance/gain-phase analyzer in conjunction with a SI 1287 potentiostat. Rotating disk electrode experiments were performed with a Pine Instruments AFMSRX rotater. Unless otherwise noted, experiments were conducted in a three-electrode cell configuration with a saturated calomel reference electrode (SCE) and a platinum wire counter electrode with either 0.1 M NaH2PO4 or a mixture of 0.05 M NaH2PO4 + 0.05 M NaCl as the background electrolyte. Constant temperature (25 ( 1 °C) was maintained during the experiments by using a water-jacketed electrochemical cell connected to a circulating water bath. Calculations and Statistics. Values are presented as mean ( standard error of the mean (SEM) unless otherwise specified.

Results and Discussion Electrochemistry of Cross-Linked Films of Fc-LPEI and GOX. Figure 2 shows the cyclic voltammograms for 3 mm glassy carbon electrodes coated with cross-linked films of Fc-LPEI and GOX. In a 0.1 M phosphate solution at pH 5 with no glucose, the film exhibited a stable and reversible oxidation peak at ∼355 mV (Figure 2A). However, at pH 7 the films exhibited multiple oxidation waves at ∼340 and ∼550 mV (Figure 2B). When 20 mM glucose was added to each solution, there was an increase in the oxidation peak and a diminished reduction peak (Figure 2A,B). This behavior is indicative of redox polymer mediation of the GOX-catalyzed oxidation of glucose: GOXðFADÞ þ glucosefGOXðFADH2 Þ þ gluconolactone ðrxn1Þ GOXðFADH2 Þ þ 2LPEIFc þ fGOXðFADÞ þ 2LPEIFc þ 2H þ ðrxn2Þ 2LPEI  Fcf2LPEIFc þ þ 2e -

ðrxn3Þ

Previously, we attributed this multiwave behavior to the morphological confinement of ferrocene centers due to deswelling or collapse of the polymer gels.27,35 At pH g7, monobasic -2 phosphate (H2PO4 ) begins to form dibasic phosphate (HPO4 ), 36,37 and cause the and phosphate has been shown to bind to PEI (35) Okazaki, Y.; Ishizuki, K.; Kawauchi, S.; Satoh, M.; Komiyama, J. Macromolecules 1996, 29, 8391–8397. (36) Birnbaum, E. R.; Rau, K. C.; Sauer, N. N. Sep. Sci. Technol. 2003, 38, 389– 404. (37) Glatzhofer, D. T.; Erickson, M. J.; Frech, R.; Yepez, F.; Furneaux, J. E. Solid State Ionics 2005, 176, 2861–2865.

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Figure 2. Effect of pH on the electrochemical response. Cyclic voltammograms of Fc-LPEI/glucose oxidase enzymatic redox hydrogels with no glucose and with 20 mM glucose added to 0.1 M phosphate at (A) pH 5 and (B) pH 7. Scan rate = 5 mV/s and T = 25 °C.

deswelling or collapse of polymer gels.27,35 This would produce two apparent populations of ferrocenes and hence two anodic peaks because the more confined ferrocene molecules would require more energy to oxidize. It is worth noting that for the pH 7 case, when glucose is present and the ferrocinium is quickly reduced by the enzyme, the second redox wave is reduced or eliminated. This might suggest alternatively that the second redox peak is associated with the formation of a second type of ferricinium ion that potentially interacts with the phosphate dianion. Interactions between monobasic38,39 and dibasic40 phosphate and ferrocinium have been reported in the literature. Further studies on determining the cause of this multiwave behavior are in progress. Effects of pH on Glucose Response and Electron Transport. In addition to pH affecting the electrochemical response of the redox polymer, the solution pH can also potentially influence both the activity of the enzyme and the electrical communication between the redox polymer and the enzyme’s FAD redox center. The effect of pH on the steady-state enzymatic response of the sensors to glucose was investigated by holding the electrodes at 0.4 V versus SCE and measuring the output current as aliquots of 2 M glucose were added to a well-stirred solution of 0.1 M phosphate. Figure 3A shows the calibration curves for the glucose response between 0 and 100 mM as a function of pH. At low glucose (38) Alonso, E.; Labande, A.; Raehm, L.; Kern, J. M.; Astruc, D. C. R. Acad. Sci., Ser. II 1999, 2, 209–213. (39) Valerio, C.; Fillaut, J. L.; Ruiz, J.; Guittard, J.; Blais, J. C.; Astruc, D. J. Am. Chem. Soc. 1997, 119, 2588–2589. (40) Ikeda, S.; Oyama, N. Anal. Chem. 1993, 65, 1910–1915.

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electrochemical impedance spectroscopy (EIS) at a dc potential of E = 0.35 V and an ac perturbation of 10 mV as previously described.27 In the low-frequency range, the impedance response was analyzed using the Randles circuit by plotting the imaginary impedance, Im(Z), versus the inverse square root of frequency, w-1/2, with a slope equal to the Warburg coefficient, sw: ImðZÞ ¼ sw w -1=2

ð4Þ

The value of cD1/2 e was determined directly from sw: sw ¼ RT=n2 F 2 cDe 1=2

Figure 3. Effect of pH on the enzymatic response to glucose and electron transport. (A) Glucose calibration curves in buffered solutions of different pH values. (B) Plot of the limiting glucose electrooxidation current density and electron transport as a function of pH.

concentrations (1 mA/cm2) to glucose. The fact that the FcLPEIGOX hydrogels had glucose sensitivities as high as the osmium redox polymers but an order of magnitude lower electron transport through the films suggests that the enhanced sensitivity of the FcLPEI films was due to something other then electron transport. On the basis of these results, we hypothesize that the high current densities that were observed in response to glucose with the Fc-LPEI/GOX films are due to enhanced electrical communication between the enzyme and the redox polymer. The high density of amines on the PEI backbone should lead to better complexation with the anionic glucose oxidase, and a shorter electron-transfer distance between the FAD site of GOX and the ferrocene redox centers. This reduced electron-transfer distance would result in a higher efficiency and rate of electron transfer and hence increased current densities. In support of this idea are the (41) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5970–5975. (42) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1993, 65, 3512–3517. (43) Katakis, I.; Heller, A. Anal. Chem. 1992, 64, 1008–1013. (44) Voet, J. G.; Coe, J.; Epstein, J.; Matossian, V.; Shipley, T. Biochemistry 1981, 20, 7182–7185. (45) Nowak, R. J.; Schultz, F. A.; Umana, M.; Lam, R.; Murray, R. W. Anal. Chem. 1980, 52, 315–321. (46) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Chem. Soc., Faraday Trans. 1993, 89, 377–384.

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facts that (a) GOX has an isoelectric point of 4.2 and the charge (Z) on GOX increases from Z ≈ -20 at pH 5 to Z ≈ -80 at pH 7.744 and (b) the pKa of LPEI has been reported to be between 7.947 and 8.7448 and consequently a significant portion of the amino groups will be protonated at physiological pH. Thus, as the pH is increased the electrostatic binding should be enhanced. In addition, PEI appears to enhance several enzyme properties. PEI by itself has been shown to enhance the long-term storage stability of enzymes in solution and during freeze drying49 as well as increasing the sensitivity and stability of enzymes in a number of biosensor designs.50-54 Effects of Enzyme Weight Percent and Film Thickness on the Glucose Response. Because the response of “wired” enzyme systems is dependent upon the polymer/enzyme mass ratio,22,23,33 we prepared a set of sensors in which the amount of GOX in the cross-linked films was varied from 10 to 50 wt %. Individual responses for the different enzyme loadings over the entire glucose concentration range are presented in the Supporting Information (Figure S-1). At enzyme concentrations of less than 30 wt %, the sensor response was limited by the enzyme activity of the film (Figure 4A), which was insufficient to supply enough electrons to reduce the ferrocene redox sites of the film. However, when the amount of enzyme was excessive (>30 wt %), collisions between oxidized and reduced ferrocene redox centers were reduced, and the rate of electron transfer through the matrix was decreased. To the best of our knowledge, the saturating current density observed here (1180 μA/cm2 at pH 7) is among the highest current densities achieved for wired glucose oxidase-based sensors on 3-mmdiameter electrodes. Current densities of the same order of magnitude have been obtained using either pyrroloquinoline quinone (PQQ)-modified glucose dehydrogenase,55 ultramicro electrodes where radial diffusion at the electrode surface is possible,25,56,57 or the incorporation of graphite particles58 or single-walled carbon nanotubes into the hydrogel matrix.59 In all of the studies mentioned above, osmium-modified redox polymers were used to wire the enzyme. Typical results in the literature for glucose sensors based on ferrocene-modified polymers including polyallylamine,28 polyacrylamide,29 polysiloxane,30 and branched poly(ethylenimine)60 range from ∼10 to ∼300 μA/cm2. The effect of film thickness on the glucose response was studied by fixing the film composition and varying the amount of material (14-684 μg/cm2) that was applied to the electrode surface. Figure 4B is a plot of the limiting glucose current measured in the presence of 100 mM glucose versus film loading. Individual responses for the different film loadings over the entire glucose concentration range are presented in the Supporting Information, (47) Brissault, B.; Kichler, A.; Guis, C.; Leborgne, C.; Danos, O.; Cheradame, H. Bioconjugate Chem. 2003, 14, 581–587. (48) Kobayashi, S.; Hiroishi, K.; Tokunoh, M.; Saegusa, T. Macromolecules 1987, 20, 1496–1500. (49) Andersson, M. A.; Hatti-Kaul, R. J. Biotechnol. 1999, 72, 21–31. (50) Mcmahon, C. P.; Rocchitta, G.; Serra, P. A.; Kirwan, S. M.; Lowry, J. P.; O’Neill, R. D. Analyst 2006, 131, 68–72. (51) Mcmahon, C. P.; Rocchitta, G.; Kirwan, S. M.; Killoran, S. J.; Serra, P. A.; Lowry, J. P.; O’Neill, R. D. Biosens. Bioelectron. 2007, 22, 1466–1473. (52) Gorton, L.; Jonssonpettersson, G.; Csoregi, E.; Johansson, K.; Dominguez, E.; Markovarga, G. Analyst 1992, 117, 1235–1241. (53) Jezkova, J.; Iwuoha, E. I.; Smyth, M. R.; Vytras, K. Electroanalysis 1997, 9, 978–984. (54) Wang, J.; Liu, J.; Chen, L.; Lu, F. Anal. Chem. 1994, 66, 3600–3603. (55) Ye, L.; Hammerle, M.; Olsthoorn, A. J. J.; Schuhmann, W.; Schmidt, H. L.; Duine, J. A.; Heller, A. Anal. Chem. 1993, 65, 238–241. (56) Pishko, M. V.; Michael, A. C.; Heller, A. Anal. Chem. 1991, 63, 2268–2272. (57) Mano, N.; Mao, F.; Heller, A. Chem. Commun. 2004, 2116–2117. (58) Binyamin, G.; Cole, J.; Heller, A. J. Electrochem. Soc. 2000, 147, 2780– 2783. (59) Joshi, P. P.; Merchant, S. A.; Wang, Y.; Schmidtke, D. W. Anal. Chem. 2005, 77, 3183–3188. (60) Chuang, C. L.; Wang, Y. J.; Lan, H. L. Anal. Chim. Acta 1997, 353, 37–44.

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Figure 4. Effect of GOX weight fraction and film loading on the enzymatic response to glucose. (A) Dependence of the limiting glucose electrooxidation current density on the GOX weight fraction for sensors with a film loading of 410 μg/cm2. (B) Dependence of the limiting glucose electrooxidation current density on the total amount of material deposited for sensors containing 30 wt % GOX. Electrodes positioned at 0.4 V vs SCE in a 0.1 M phosphate buffer, T = 25 °C, 100 mM glucose.

Figure S-2. Assuming that the thickness of the films is proportional to the amount of material deposited, the current density initially increased with film thickness and then plateaued at 1200 μA/cm2 for thicknesses of 205 μg/cm2 and above. This plateau in the response suggests that the reaction layer of the film does not extend through the entire film for very thick films. It was also observed that as the amount of material deposited on the electrode increased there was generally an increase in the background current (Supporting Information Figure S-3). We believe that this was caused by the increased thickness of the films, which limited the diffusion of electrons from ferrocene centers at the outer film surface. When an oxidizing potential is applied to a redox-polymer-coated sensor, there is a transient spike in the current that decays with time. This current spike is made up of both faradaic and nonfaradaic currents. The nonfaradaic (or charging) current decays exponentially with time. The faradaic current, which is due to the conversion of ferrocene to ferricinium in the film, also decays with time but at a slower rate. When the films were thin (1 mA/cm2. These current densities are among the highest ever reported for redox polymers with GOX. Electrochemical impedance spectroscopy (EIS) measurements showed that the incorporation of enzyme into the cross-linked polymer films did not significantly change the rate of electron transport through the films as compared to films without enzyme. A comparison of electron-transport rates with the enzymatic response suggested that the glucose electrooxidation current densities do not correlate with electron transport alone at low pH but that other factors are involved. We hypothesize that the high density of protonated amines on the LPEI backbone promotes electrostatic complexation between the Fc-LPEI and GOX, which leads to enhanced electrical communication with the FAD redox center. We demonstrate that the high sensitivity of these films (73 nA/μM 3 cm2) (66) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708–2716. (67) Wang, Y.; Joshi, P. P.; Hobbs, K. L.; Johnson, M. B.; Schmidtke, D. W. Langmuir 2006, 22, 9776–9783. (68) Flexer, V.; Forzani, E. S.; Calvo, E. J.; Luduena, S. J.; Pietrasanta, L. I. Anal. Chem. 2006, 78, 399–407. (69) Sirkar, K.; Revzin, A.; Pishko, M. V. Anal. Chem. 2000, 72, 2930–2936. (70) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117–6123. (71) Rodriguez, M. C.; Rivas, G. A. Electroanalysis 2004, 16, 1717–1722.

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allow us to measure glucose concentrations in the micromolar range. Finally, we demonstrate the versatility of Fc-LPEI as a wire for enzyme electrodes by cross-linking the polymer with horseradish peroxidase. The hydrogen peroxide sensors also exhibited current densities of around 0.9 mA/cm2. The exceptional ability of Fc-LPEI to communicate with a variety of enzymes has potential applications in measuring low analyte concentrations in implantable biosensors and producing significant amounts of power for portable biofuel cells. In addition, the high density of positive charges on the Fc-LPEI backbone makes it an excellent candidate for layer-by-layer sensor construction where the sensor components are electrostatically complexed66-69 and unmodified PEI is routinely used.70,71 Acknowledgment. This work was supported in part by an NSF Career Award to D.W.S (BES-0547619) Supporting Information Available: Dependence of the glucose electrooxidation current density on the weight fraction of glucose oxidase for sensors with a film loading of 410 μg/cm2. Dependence of the glucose electrooxidation current density and background current density on the amount of material deposited for sensors containing 30 wt % GOX. This material is available free of charge via the Internet at http://pubs.acs.org.

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