Ferrocene Embedded in an Electrode-Supported Hybrid Lipid Bilayer

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Ferrocene Embedded in an Electrode-Supported Hybrid Lipid Bilayer Membrane: A Model System for Electrocatalysis in a Biomimetic Environment Ali Hosseini, James P. Collman,* Anando Devadoss, Genevieve Y. Williams, Christopher J. Barile, and Todd A. Eberspacher Department of Chemistry, Stanford University, Stanford, California 94305, United States Received July 21, 2010. Revised Manuscript Received August 25, 2010 An electrode-supported system in which ferrocene molecules are embedded in a hybrid bilayer membrane (HBM) has been prepared and characterized. The redox properties of the ferrocene molecules were studied by varying the lipid and alkanethiol building blocks of the HBM. The midpoint potential and electron transfer rate of the embedded ferrocene were found to be dependent on the hydrophobic nature of the electrolyte and the distance at which the ferrocene was positioned in the HBM relative to the electrode and the solution. Additionally, the ability of the lipid-embedded ferrocenium ions to oxidize solution phase ascorbic acid was evaluated and found to be dependent on the nature of the counterion.

Introduction Knowledge about the physical parameters that affect electron transfer to redox molecules in biomimetic environments, specifically in hydrophobic environments, is of paramount importance in understanding the functional aspects of redox proteins. While in proteins the hydrophobic pockets encompassing the redox cofactors are formed from complex amino acid chains with additional functional aspects (e.g., proton channels, hydrogen bonding, etc.), the reconstruction of such systems is extremely difficult for the purpose of systematic studies. The construction of electrode platforms in which a redox species is buried in long chain alkanes and positioned at defined distances from the electrode surface assists in studying the effects of a nonpolar environment in electron transfer reactions.1-7 Electrode-supported hybrid bilayer membranes (HBM) have been used as a platform for the immobilization of biological molecules on surfaces.8-11 Typically in a HBM system, a monolayer of lipid molecules is anchored to a self-assembled monolayer (SAM) of alkanethiols covalently attached to a gold surface, with the polar head groups orientated outward to the aqueous solution and the hydrophobic tails oriented inward to the hydrophobic *Corresponding author. E-mail: [email protected].

(1) Rowe, G. K.; Creager, S. E. J. Phys. Chem. 1994, 98, 5500–5507. (2) Rowe, G. K.; Creager, S. E. Langmuir 1994, 10, 1186–1192. (3) Creager, S. E.; Rowe, G. K. Anal. Chim. Acta 1991, 246, 233–239. (4) Rowe, G. K.; Creager, S. E. Langmuir 1991, 7, 2307–2312. (5) Creager, S. E.; Rowe, G. K. J. Electroanal. Chem. 1997, 420, 291–299. (6) Adams, D. M.; Brus, L.; Chidsey, C. E. D.; Creager, S.; Creutz, C.; Kagan, C. R.; Kamat, P. V.; Lieberman, M.; Lindsay, S.; Marcus, R. A.; Metzger, R. M.; Michel-Beyerle, M. E.; Miller, J. R.; Newton, M. D.; Rolison, D. R.; Sankey, O.; Schanze, K. S.; Yardley, J.; Zhu, X. Y. J. Phys. Chem. B 2003, 107, 6668–6697. (7) Sumner, J. J.; Creager, S. E. J. Phys. Chem. B. 2001, 105, 8739–8745. (8) Knoll, W.; Koper, I.; Naumann, R.; Sinner, E. K. Electrochim. Acta 2008, 53, 6680–6689. (9) Cremer, P. S.; Castellana, E. T. Surf. Sci. Rep. 2006, 61, 429–444. (10) Bokoch, M. P.; Devadoss, A.; Palencsar, M. S.; Burgess, J. D. Anal. Chim. Acta 2004, 519, 47–55. (11) Devadoss, A.; Burgess, J. D. J. Am. Chem. Soc. 2004, 126, 10214–10215. (12) Plant, A. L. Langmuir 1993, 9, 2764–2767. (13) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126–1133. (14) Plant, A. L.; Brighamburke, M.; Petrella, E. C.; Oshannessy, D. J. Anal. Biochem. 1995, 226, 342–348. (15) Plant, A. L. Langmuir 1999, 15, 5128–5135.

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SAM.12-15 The driving force for the formation of an HBM is the reduction in the free energy of the hydrophobic alkanethiol/water interface and is well understood.8 When a redox molecule is immobilized on the SAM, and a HBM is subsequently formed, the redox molecule is surrounded by an organic nonpolar environment. The flexibility in choosing the SAM (e.g., variations in the chain length and degree of saturation of the alkyl thiols) controls the electron transfer rate between the electrode and the redox species. There are very few examples of embedding redox-active molecules inside a HBM system.16-18 For example, Nuzzo and co-workers formed ferrocene-embedded HBM systems using ferroceneterminated alkanethiols and 1,2-dimyrisitoyl-sn-glycero-3-phosphatidylcholine (DMPC) molecules. Their analysis demonstrated that the HBM environment significantly perturbs the redox properties of ferrocene. They also used this system to study the membrane permeability of solution phase redox molecules.17 In the present study, we have formed HBM systems using ferrocene-attached SAMs and either 1,2-dilauroyl-sn-glycero-3phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the lipid layers. These reported HBM systems were used to study the effect of variations in the lipid, the thickness of the self-assembled monolayer, and the supporting electrolytes in the redox reactions of ferrocene. Additionally, the effect of the HBM systems on the electrochemical oxidation of solution phase ascorbic acid by the embedded ferrocene was also studied. Ferrocene-embedded HBM systems were constructed by a three-step process. In the first step, gold electrodes were modified with a mixed monolayer of azide-terminated thiols and alkanethiols. Next, in a postmodification step, ethynylferrocene was coupled to the azide-terminated thiols by the formation of a stable, aromatic 1,2,3-triazole linker via the Cu(I)-catalyzed “click” reaction. A lipid layer was then formed on top of the (16) Dominska, M.; Krysinski, P.; Blanchard, G. J. Langmuir 2008, 24, 8785– 8793. (17) Twardowski, M.; Nuzzo, R. G. Langmuir 2003, 19, 9781–9791. (18) Twardowski, M.; Nuzzo, R. G. Langmuir 2004, 20, 175–180.

Published on Web 10/06/2010

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Figure 1. A simple approach for embedding an electroactive species inside an electrode-supported hybrid bilayer membrane.

ferrocene-attached SAM by exposing the surface to a lipid vesicle solution. Figure 1 illustrates the steps involved in the formation of the ferrocene-embedded HBM layer. This was used as a model system to study the effect of the hydrophobic environment on the midpoint potential, the electron-transfer/ion-transfer kinetics, and electrocatalytic behavior. These studies are expected to provide a background for future analysis of redox and electrocatalytic behavior of complex molecular catalysts embedded in a biomimetic, hydrophobic environment such as HBMs and vesicle membranes.

Experimental Section Azide-terminated thiols and ethynylferrocene were prepared according to a previously reported procedure; alkanethiols were purchased from Acros Inc. and were used without further purification. Lipids (DMPC and DLPC) were purchased from Sigma-Aldrich. Ellipsometry. Ellipsometric measurements were made using a Gaertner Scientific, model L116B ellipsometer equipped with a He-Ne laser (6328 A˚) set at an incidence angle of 70°. Measurements were taken at four or five spots on the substrate surface and averaged. A two-layer transparent film model was used for the thickness calculations based on pseudosubstrate constants measured on the clean substrate. The refractive index of the organic film was fixed at 1.5. Gold Electrode. Silicon wafers (111) were cleaned in a piranha solution containing 50 mL of H2O2 and 150 mL of H2SO4 for 1 min (warning: piranha solutions are extremely corrosive and can cause explosions in the presence of organic molecules; proper precautions and safety procedures are advised), rinsed thoroughly with water, and dried in an isopropanol bath prior to metal deposition. A titanium adhesion layer (50 A˚), followed by a gold layer (500 A˚), was deposited on the cleaned silicon wafers via a home-built e-beam vacuum deposition apparatus. Electrochemical Studies. Electrochemical studies were carried out using a Pine E-chem station RDE 5 using a threeelectrode setup with platinum as the counter electrode. All the electrochemical potentials presented in this work are measured and reported using a Ag/AgCl (sat. NaCl) as the reference electrode. The midpoint potentials (Em) were calculated by using the formula (Ep,a þ Ep,c)/2, where Ep,a is the anodic peak potential and Ep,c the cathodic peak potential. Self-Assembled Monolayer. The formation of a SAM was accomplished using a method previously published by our group.19 The gold electrodes were first electrochemically cleaned in a 0.5 M HNO3 solution by cycling from 0 to 1.650 V until a stable gold (19) Collman, J. P.; Hosseini, A.; Eberspacher, T. A.; Chidsey, C. E. D. Langmuir 2009, 25, 6517–6521.

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reductive wave was observed at 0.850 V. This electrode was then immersed in the desired mixed thiol solution (0.4 mM total thiol concentration) for 1 min. The surface was then immersed in a 10 mM solution of alkanethiol for 3 h to remove any weakly adsorbed thiols. Click Reaction. Tris(benzyltriazolylmethyl)amine (TBTA) was prepared using a previously reported procedure.20-22 Click reactions were performed using a mixture of 800 μM Cu(NO3)2, 800 μM TBTA in 3:1 DMSO/water, and 1 mM ascorbic acid (to ensure that the catalyst was in the active Cu(I) oxidation state) and ethynylferrocene in the 1-20 μM range. An azide-terminated SAM was exposed to the solution for 30 min and rinsed with ethanol, dichloromethane, ethanol, and water. HBM Formation. Lipid vesicles were prepared using a previously reported procedure in the literature.23 The lipid (2 mg) of interest was dissolved in chloroform (2 mL) in a glass vial; the resulting solution was evaporated under a stream of nitrogen until a dry lipid film was formed at the base of the vial. This film was further dried in a vacuum desiccator for 30 min. The dry lipid film was subsequently dissolved in isopropyl alcohol (100 μL) and then hydrated using a pH 7 buffer (20 mL). The resulting suspension was sonicated for 30 min to obtain a clear vesicle solution that was used immediately. For electrochemical measurements during the formation of the HBM, a pH 7 buffer was used with KPF6 (100 mM) as an additional electrolyte. Cholesterol Inclusion. The following procedure was adapted from the method reported by Christian et al.24 A solution of cholesterol (2 mg) in chloroform (2 mL) was evaporated under a stream of nitrogen. To the resulting cholesterol film, a solution of cyclodextrin (60 mg) in pH 7 buffer containing 0.1 M KPF6 supporting electrolyte (20 mL) was then added. The resulting mixture was sonicated for 30 min before it was filtered through a 0.45 pm syringe filter and added to the surface immediately.

Results and Discussion HBM Formation. The formation of HBM layers were confirmed using ellipsometry. The values obtained25 are consistent (20) Collman, J. P.; Devaraj, N. K.; Chidsey, C. E. D. Langmuir 2004, 20, 1051– 1053. (21) Collman, J. P.; Devaraj, N. K.; Eberspacher, T. P. A.; Chidsey, C. E. D. Langmuir 2006, 22, 2457–2464. (22) Devaraj, N. K.; Collman, J. P. QSAR Comb. Sci. 2007, 26, 1253–1260. (23) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9–23. (24) Christian, A. E.; Haynes, M. P.; Phillips, M. C.; Rothblat, G. H. J. Lipid Res. 1997, 38, 2264–2272. (25) The thicknesses are ca. 21 A˚ for a C16SH monolayer and ca. 43 A˚ for a bilayer after immersing the SAM-modified surfaces in the DMPC solution for 18 h containing C16SH and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

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Figure 3. Schematic showing the variables affecting the midpoint potential of the ferrocene buried in the HBM. The model is based on the framework provided by White and co-workers.26

Figure 2. Shift in the ferrocene redox couple as a function of incubation time: (;) SAM, (---) after 30 min, ( 3 3 3 ) after 60 min, and (- 3 -) after 18 h exposure to vesicle solution in pH 7 buffer containing 0.1 M KPF6; scan rate 5 V s-1. All the data were obtained at the same electrode.

with the formation of a monolayer of lipid (ca. 4.3 nm) on a monolayer of alkanethiol (ca. 2.1 nm) to form a HBM. The observed change in the interfacial capacitance (8 to 6 μF/cm2) of the electrode after exposure to the lipid vesicle solution is also consistent with the formation of a HBM layer. Figure 2 illustrates the change in the voltammogram of the embedded ferrocene as a function of time, as obtained during the formation of a HBM on an azide-terminated-C16-alkanethiol/ C16-alkanethiol SAM. The midpoint potential of ferrocene immobilized on the azide-terminated SAM prior to forming a HBM was found to be 0.3 V vs Ag/AgCl. Exposure to DMPC vesicles shifted the midpoint potential to more positive values. After ∼18 h in DMPC solution, the midpoint potential of ferrocene shifted to 0.4 V vs Ag/AgCl. When the electrode surface was immersed in DMPC solution for periods longer than 18 h, no additional shift in potential was observed, suggesting the HBM formation was complete. Removing the lipid monolayer by washing the surface with ethanol, chloroform, ethanol, and then water restored the initial redox behavior of the attached ferrocene with no significant loss of ferrocene electroactivity. This demonstrates that the ferrocene embedding process is reversible and also that the Fc-SAM system is stable during the HBM formation and subsequent voltammetric studies. A ferrocene coverage of up to 15% was utilized to maintain the site-isolated nature of the redox species. The shift in the midpoint potential of ferroceneembedded HBM was found to be independent of the coverages (4-15%) of the redox species in our studies (see Supporting Information). The redox shifts observed in these studies were less pronounced than those reported for systems in which ferrocene was buried in a SAM with longer alkanethiols surrounding the immobilized ferrocene.1-5 For example, Creager and co-workers have shown that when ferrocene was embedded inside a hydrophobic layer by using a Fc-CONH-C7-SH/n-nonanethiol SAM in a solution containing KPF6 as the electrolyte, a 0.080 V shift in midpoint potential was observed.1 For the ferrocene embedded in HBM presented in this work, a shift in midpoint potential of 0.109 V was observed in a 0.1 M phosphate buffer containing 0.1 M KPF6 as an additional electrolyte. The increased shift in midpoint potential can be partially explained because ferrocene is embedded in a layer of lipids that has much longer chains than alkanethiols. 17676 DOI: 10.1021/la1029118

However, a more significant potential shift is expected (e.g., an estimate of 0.75 V for an electroactive species completely buried in an organic environment was obtained using the Born equation26) compared to the 0.109 V shift observed. This less pronounced shift in midpoint potential can be explained by a theoretical framework presented by White and co-workers.26 According to their model, the potential drop at the plane of electron transfer, ΦPET, determines the observed redox properties of the molecule buried in an organic film at the electrode. The value of ΦPET is in turn affected by various factors such as thicknesses (d1, d2) of the dielectric medium, the dielectric constants of the medium (ε1, ε2) and solvent (ε3), and the ionic strength of the solution (see Figure 3 for a schematic diagram showing all the parameters). Considering this framework, the less pronounced potential shift observed for the ferrocene embedded in a lipid layer is probably due to the higher dielectric constant of the lipid layer (ε2) compared to that of the SAM. The higher dielectric constant is likely the result of the lower packing density of the lipid molecules and their ability to allow for the migration of hydrophobic counterions such as PF6- during the redox cycling of ferrocene. This hypothesis is supported by another experiment. Exposure of excess cholesterol to the HBM completely blocked the ferrocene electron transfer (see Supporting Information). Cholesterol is a pseudo lipid that inserts into the lipid membrane forming a dense lipid layer. The role of cholesterol in the lipid membrane is to minimize the passive permeability of small molecules across the interface.27 It is logical to expect that in the current scenario cholesterol insertion decreases ε2 and halts migration of PF6- to the ferrocenium sites, thereby shifting the midpoint potential to a more positive value. Counterion Effect. Table 1 summarizes the effect of counterions in the voltammetry of the ferrocene-embedded (N3-C16SH/ C16SH/DMPC and N3-C11SH/C8SH/DMPC) HBM systems. From Table 1 it is clear that a change in the nature of the electrolyte results in a significant change in the redox behavior of the embedded ferrocene. After HBM formation, the Fc/Fcþ couple shifts by ca. þ0.08 V with N3-C16SH/C16SH when 0.1 M KPF6 was used as the electrolyte. For 0.1 M KCl, a shift in the midpoint potential of þ0.15 V was observed for the HBM system. The full width at half-maximum (fwhm) of the voltammograms increased in all cases for the ferrocene HBM systems compared to the lipid-free ferrocene SAM systems. Additionally, the increase in fwhm was greater when KCl was used as an electrolyte instead of KPF6. For instance, with ferrocene attached to N3C16SH/ C16SH in 0.1 M KPF6, the fwhms at 1 V s-1 were 0.09 V (SAM) and 0.11 V (HBM), but in 0.1 M KCl, the redox peaks broadened (26) Smith, C. P.; White, H. S. Anal. Chem. 1992, 64, 2398–2405. (27) Mouritsen, O. G.; Zuckermann, M. J. Lipids 2004, 39, 1101–1113.

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Table 1. Effects of the Electrolyte Solution on the Redox Properties of Ferrocene Clicked onto SAM Containing N3-C16SH/C16SH or N3-C11SH/ C8SH and DMPC Vesicles To Form the Lipid Monolayera azide-thiol 1 2 3 4 5 6 7 8

diluent-thiol

electrolyte

background buffer

Fc-SAM Em/V

C16SH 0.1 M KPF6 0.33 N3-C16SH C16SH 0.1 M KCl 0.37 N3-C16SH C16SH 0.1 M phosphate 0.35 N3-C16SH C16SH 0.1 M KPF6 0.1 M phosphate 0.35 N3-C16SH C8SH 0.1 M KPF6 0.33 N3-C11SH C8SH 0.1 M KCl 0.37 N3-C11SH C8SH 0.1 M phosphate 0.35 N3-C11SH C8SH 0.1 M KPF6 0.1 M phosphate 0.35 N3-C11SH a For each set of conditions, the Em values for the SAM and HBM were measured using the same surface.

Fc-HBM Em/V

ΔEm/V

0.41 0.52 0.44 0.46 0.36 0.47 0.40 0.41

0.08 0.15 0.09 0.11 0.03 0.10 0.05 0.06

Table 2. Effects of the Changes in d1 and d2 by Variations in the SAM (Diluent and Azide-Terminated Thiol) and the Lipid Used in the HBM in the Midpoint Potential of Embedded Fca azide-thiol diluent-thiol

lipid

Fc-SAM Fc-HBM Em/V Em/V ΔEm/V

1 2 3 4 5

N3-C16SH C16SH DMPC 0.35 0.46 0.11 C16SH DLPC 0.34 0.43 0.09 N3-C16SH C15SH DMPC 0.34 0.41 0.07 N3-C16SH C10SH DMPC 0.35 0.42 0.07 N3-C11SH C8SH DMPC 0.35 0.41 0.06 N3-C11SH a The voltammetry experiments were conducted in a solution containing 0.1 M KPF6 and 0.1 M phosphate electrolyte. For each set of conditions, the Em values for the SAM and HBM were measured using the same surface.

Figure 4. Shift in the ferrocene redox couple using KCl as the supporting electrolyte: (;) (SAM); (---) after overnight incubation with vesicle scan rate 5 V s-1 on the same surface.

to 0.10 V (SAM) and 0.14 V (HBM). Because the ferrocene molecules are site-isolated in this surface coverage range (1.8  1014-8.4  1013 molecules cm-2), the increase in fwhm indicates that the electron transfer rate between the electrode and embedded ferrocene is lower for Cl- compared to PF6-. The variation in the magnitude of the midpoint potential shift with a change in the anion can be explained by the hydrophobicity of the counterion. Since the chloride ion is less hydrophobic (more hydrophilic), its migration in and out of the lipid layer is more hindered compared to PF6-. While the limited migration of Clexplains the decrease in electron transfer rate between the electrode and the lipid-immersed ferrocene, it does not explain the observed shift in the midpoint potential. However, a careful consideration of the model presented by White and co-workers26 suggests that the presence of ions in the lipid layer could affect ε2, the dielectric constant in the lipid region and the potential drop between Φ PET and the solution. On the basis of this criterion, if the lipid membrane is permeated with PF6- ions, the value of ε2 would increase and the potential drop would be higher. This would lead to a lower shift in the midpoint potential compared to when the lipid membrane is permeated with fewer ions. Cyclic voltammetry analysis of ferrocene-embedded HBM revealed significantly lower redox activity (broadening of the peak and a decrease in the area of the redox peaks) in the presence of Cl- as an electrolyte, when compared to the ferroceneterminated SAM. Voltammetry of the same electrode after removal of the lipid layer from the surface revealed a decrease in the surface coverage of ferrocene by 60% with no change in the midpoint potential (Figure 4). The significant decrease in surface coverage could be due to the coordination of chloride ions with ferrocenium ions, resulting in the decomposition of ferrocene Langmuir 2010, 26(22), 17674–17678

molecules. When the voltammetry of ferrocene-attached SAM (in the absence of the lipid layer) was conducted in a solution containing Cl- ions, a decrease of less than 5% in the surface coverage of ferrocene was observed. This can be attributed to a difference in the activity of Cl- in the hydrophobic layer compared to the solution phase. Similar variations in redox behaviors were observed for PF6- and Cl- with other SAM and HBM systems. Electron Transfer Rate. Electron transfer to and from the electrode to a redox species is coupled with charge compensation.6 In this report, we present the effect of the HBM on electron transfer using Cl- and PF6- as the counterion for the ferrocenium species formed during the electrochemical oxidation of ferrocene. The rate of electron transfer is estimated using the Laviron equation.28 Previously, we used chronoamperometric techniques to measure the ket from the electrode to the Fc-terminated SAM directly. The Laviron equation estimated the ket for the Fc clicked to N3-C16SH/C16SH to be 2.1 ( 0.5 and 1.1 ( 0.4 s-1, when using 0.1 M KPF6 and 0.1 M KCl supporting electrolytes, respectively. The value obtained for 0.1 M KPF6 is comparable to the value obtained previously using chronoamperometric techniques (2.2 ( 0.2 s-1) with phosphate buffered KPF6 as the electrolyte.29 When the HBM was formed, the estimated ket (0.2 ( 0.2 s-1) in the presence of KCl decreased by an order of magnitude compared to the ferrocene-immobilized SAM. Interestingly, the ket for the HBM system in KPF6 (1.9 þ 0.2 s-1) was not significantly affected by a monolayer of DMPC. This indicates that the rate of PF6- diffusion through the lipid layer is not the rate-determining step. SAM and Lipid Effect. Reconsidering Figure 3 and the theoretical framework provided by White and co-workers, it can be conceived that varying the distance between the electrode and ferrocene (d1), or the distance between the solution and (28) Laviron, E. J. Electroanal. Chem. Interfacial Electrochem. 1979, 101, 19–28. (29) Devaraj, N. K.; Decreau, R. A.; Ebina, W.; Collman, J. P.; Chidsey, C. E. D. J. Phys. Chem. B 2006, 110, 15955–15962.

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Electrochemical Oxidation of Ascorbic Acid. Ferrocene is known to facilitate the oxidation of ascorbate30-33 when incorporated into a SAM-modified gold electrode. It is known that oxidation of ascorbic acid is a two-electron two-proton oxidation process that yields a single product.34 With ferrocene attached to N3-C11SH/C8SH, the current rises, peaks, and decreases as it is limited by the mass transport of ascorbate to the surface from the bulk solution. Figure 5 illustrates the effect of a HBM on the electrochemical conversion of 0.1 M sodium ascorbate in a solution containing 0.1 M KPF6. After HBM formation, the oxidation current remained the same in 0.1 M KCl, but decreased by 62% in 0.1 M KPF6. Since the electron transfer rate was not affected significantly by the formation of the HBM for the KPF6 case (see above discussion about electron transfer rate), it is not expected to be the rate-limiting step for oxidation of ascorbate at the surfaces. Competition between the counterion diffusion and ascorbate, which is also an anion, for neutralization of ferrocenium could account for this variation. In the case of PF6-, rapid diffusion of both the PF6- and ascorbate may be limiting the amount of ascorbate accessing the ferrocenium ions. On the contrary, in the case of Cl-, ascorbate ions are more hydrophobic and thus preferentially diffuse into the HBM and undergo oxidation. This situation can be visualized as a partitioning of hydrophobic species into the HBM systems, resulting in variations in the concentration of species available in the lipid membrane for oxidation. Similar results were obtained when the surface coverage of ferrocene was in the range of 4-15%.

Conclusion

Figure 5. Ascorbate oxidation by Fc-immobilized SAM (;) and Fc-embedded HBM (---) using (a) KCl and (b) KPF6 as the supporting electrolyte. The corresponding SAM and HBM scans were performed on the same electrode surface.

ferrocene (d2), should change the observed redox parameters of the embedded ferrocene. Table 2 summarizes the midpoint potentials observed for a range of ferrocene-embedded HBM systems. As expected, the observed midpoint potential of the ferroceneattached SAM is independent of the thiol used (i.e., in the absence of lipid layers). It is also evident from Table 2 that the variation in the lengths of the azide-terminated thiols and diluent thiols in the HBM systems changes d1 and d2, which in turn affects the observed midpoint potential of the ferrocene embedded in the HBM systems. The results illustrate that the formation of the DMPC layer on C16SH/N3-C16SH shifted the midpoint potential by 0.11 V. In comparison, when the DMPC layer was formed on the C15SH/N3-C16SH SAM, the midpoint potential shifted by 0.07 V. The same shift in midpoint potential was observed for the C10SH/N3-C11SH/DMPC HBM system. Additionally, the formation of DMPC layer on C8SH/N3-C11SH resulted in a shift in the midpoint potential of 0.06 V. In an experiment (row 2) where DLPC was used instead of the DMPC with the N3-C16SH/C16SH, a midpoint potential shift of 0.09 V was observed. This is consistent with the trend that an increase in d2 shifts the midpoint potential more positively. It is noted that d2 is affected by both the lipid layer and the choice of diluent thiol in the SAM (Figure 4). 17678 DOI: 10.1021/la1029118

In this work, we have embedded a redox species in a lipid membrane using “click” chemistry and studied the effect of the hydrophobic environment on the electrochemical properties of the redox species. The effect of variations in the diluent, azideterminated thiols, and lipids has demonstrated that the change in d1 has a more significant effect on the thermodynamic potential of the ferrocene-embedded HBM than d2. The counterion shows a significant effect on the rate of electron transfer to the ferroceneembedded HBM. The more hydrophilic anion Cl- shifts the potential more positively than the hydrophobic anion PF6-. The effects of the HBM on the electrochemical oxidation of ascorbate using ferrocene as a mediator illustrate that there is a competitive nature of the oxidation process of ascorbate with counterions. This work provides a foundation for studying electroactive species other than ferrocene inside a HBM. The application of the HBM system presented in this paper to biologically relevant electroactive molecules may have profound implications for understanding their redox properties inside a biomimetic hydrophobic environment. Acknowledgment. This material is based upon work supported by the NIH under Grant 5 R01 GM069658. We acknowledge insightful discussions with Dr Christopher E. D. Chidsey. Supporting Information Available: Experimental procedure with additional figures. This material is available free of charge via the Internet at http://pubs.acs.org. (30) Kazakeviciene, B.; Valincius, G.; Niaura, G.; Talaikyte, Z.; Kazemekaite, M.; Razumas, V.; Plausinaitis, D.; Teiserskiene, A.; Lisauskas, V. Langmuir 2007, 23, 4965–4971. (31) Zhang, S. X.; Fu, Y. Q.; Sun, C. Q. Electroanalysis 2003, 15, 739–746. (32) Degefa, T. H.; Schon, P.; Bongard, D.; Walder, L. J. Electroanal. Chem. 2004, 574, 49–62. (33) Liu, A. H.; Anzai, J. Anal. Bioanal.Chem. 2004, 380, 98–103. (34) Hu, I. F.; Kuwana, T. Anal. Chem. 1986, 58, 3235–3239.

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