Probing Electrocatalytic and Bioelectrocatalytic Processes by Contact

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Langmuir , 2003, 19 (13), pp 5413–5420. DOI: 10.1021/la034519v...
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Probing Electrocatalytic and Bioelectrocatalytic Processes by Contact Angle Measurements Xuemei Wang, Zoya Gershman, Andrei B. Kharitonov, Eugenii Katz, and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received March 26, 2003 Electroswitchable wetting of electrode surfaces modified with redox-active monolayers and thin films is described. Electrocatalytic and bioelectrocatalytic processes that are activated by the redox-active interfaces associated with electrodes control the hydrophobic/hydrophilic properties of the surfaces, thus allowing the probing of the chemical transformations by static contact angle measurements. A Prussian Blue film associated with an ITO electrode undergoes redox transformations between the hydrophilic reduced state, PB4-, the hydrophobic semioxidized state, PB0, and the hydrophilic fully oxidized state, PB3+. Contact angle measurements follow the reversible switching of the film between the three states. The oxidized state, PB3+, electrocatalyzes the oxidation of NADH, and thus, the ratio of PB3+/PB0 on the film interface upon the electrochemical oxidation of NADH is controlled by the cofactor concentration. This enables following the electrocatalyzed oxidation of NADH by static contact angle measurements. Similarly, the hydrophobic/hydrophilic properties of a naphthoquinone-functionalized polyethylenimine film are reversibly electroswitched by the reduction and oxidation of the film. In the reduced state of the film the naphthohydroquinone units catalyze the reduction of O2, thus leading to a hydrophobic film that originates from the high naphthoquinone/naphthohydroquinone ratio associated with the film. The hydrophobic/ hydrophilic properties of an Au electrode modified with a ferrocene monolayer are electroswitched between a hydrophilic state in the presence of the ferrocenylium (Fc+) oxidized monolayer and a hydrophobic state in the presence of the ferrocene (Fc) monolayer configuration. The ferrocenylium monolayer activates the bioelectrocatalyzed oxidation of glucose in the presence of glucose oxidase. The bioelectrocatalyzed oxidation of glucose leads to the control of the Fc+/Fc ratio associated with the monolayer by the glucose concentration in the system. This enables following the bioelectrocatalytic oxidation of glucose by static contact angle measurements.

Introduction Contact angle measurements were extensively employed to characterize the hydrophilic or hydrophobic properties of monolayer- or thin film-functionalized surfaces.1-4 The signal-controlled switching of the hydrophilic/hydrophobic properties of chemically modified surfaces attracts research efforts directed, for example, to the photochemical patterning of surfaces5 or the electroswitchable mechanical movement of solvents.6 Several studies have addressed the use of contact angle measurements for following redox-controlled7 or photochemically triggered8 hydrophobic-to-hydrophilic or hy* To whom correspondence should be addressed. Telephone: 9722-6585272. Fax: 972-2-6527715. E-mail: [email protected]. (1) (a) Chaudhury, M. K.; Whitesides, G. M. Science 1992, 256, 1539. (b) Bain, C. D.; Whitesides, G. M. Langmuir 1989, 5, 1370. (2) (a) Evans, S. D.; Sharma, R.; Ulman, A. Langmuir 1991, 7, 156. (b) Sondag-Huethorst, J. A. M.; Fokkink, L. G. J. Langmuir 1992, 8, 2560. (3) Walczak, M. M.; Chung, C. K.; Stole, S. M.; Widrig, G. A.; Porter, M. D. J. Am. Chem. Soc. 1991, 113, 2370. (4) (a) Marmur, A. J. Colloid Interface Sci. 1992, 148, 541. (b) Sharma, A. Langmuir 1993, 9, 3580. (5) (a) Welle, A.; Gottwald, E. Biomed. Microdevices 2002, 4, 33. (b) Lin, W. B.; Lin, W. P.; Wong, G. K.; Marks, T. J. J. Am. Chem. Soc. 1996, 118, 8034. (c) Stenger, D. A.; Georger, J. H.; Dulcey, C. S.; Hickman, J. J.; Rudolph, A. S.; Nielsen, T. B.; McCort, S. M.; Calvert, J. M. J. Am. Chem. Soc. 1992, 114, 8435. (6) Abbott, N. L.; Whitesides, G. M. Langmuir 1994, 10, 1493. (7) (a) Abbott, N. L.; Gorman, C. B.; Whitesides, G. M. Langmuir 1995, 11, 16. (b) Sullivan, J. T.; Harrison, K. E.; Mizzell, J. P.; Kilbey, S. M. Langmuir 2000, 16, 9797. (c) Albagli, D.; Wrighton, M. S. Langmuir 1993, 9, 1893. (8) (a) Kaczmarec, H.; Drag, R.; Swiatek, M.; Oldak, D. Surf. Sci. 2002, 507, 877. (b) Kavc, T.; Kern, W.; Ebel, M. F.; Svagera, R.; Polt, P. Chem. Mater. 2000, 12, 1053. (c) Sarkar, N.; Bhattacharjee, S.; Sivaram, S. Langmuir 1997, 13, 4142.

drophilic-to-hydrophobic transformations on surfaces.9 Here we report on the use of contact angle analyses to follow electrochemical, electrocatalytic, and bioelectrocatalytic processes on surfaces. Experimental Section Chemicals and Reagents. The enzyme glucose oxidase, GOx, (EC 1.1.3.4; type X-S from Aspergillus niger), β-D-glucose, and β-nicotinamide adenine dinucleotide, reduced form, NADH, were purchased from Sigma, cystamine dihydrochloride was from Fluka, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES), 2,3-dichloro-1,2-naphthoquinone, and polyethylenimine (PEI, MW ) ∼60 000) were purchased from Aldrich. N-2-Methylferrocenecaproic acid (1) was synthesized as described elsewhere.12 All other chemicals were obtained from Aldrich and used as supplied. Ultrapure water from Serapur PRO90CN was used throughout all the experiments. Preparation of the Prussian Blue-Modified Electrodes and Electrochemical Measurements. The indium tin oxide (ITO) electrodes were pretreated by sonication in piranha solution (50% concentrated H2O2 + 50% concentrated H2SO4), ethanol, and a 2% solution of HCl, each time for 4-5 min, and then the electrodes were sonicated three times in distilled water for 5 min each time. (Caution! The piranha solution has very strong (9) (a) William, K. W.; Murray, R. W. Anal. Chem. 1983, 55, 1139. (b) Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. Soc. 1992, 114, 1990. (10) (a) Karyakin, A. A. Electroanalysis 2002, 13, 813. (b) Chidsey, C. E.; Murray, R. W. J. Phys. Chem. 1986, 90, 1479. (c) Laviron, E. J. J. Electroanal. Chem. 1980, 112, 1. (11) (a) Komplin, G. C.; Pietro, W. J. Sens. Actuators, B 1996, 30, 173. (b) Raitman, O. A.; Katz, E.; Willner, I.; Chegel, V. I.; Popova, G. V. Angew. Chem., Int. Ed. 2001, 40, 3649. (c) Zhao, H.; Yuan, Y.; Adeloju, S.; Wallace, G. G. Anal. Chim. Acta 2002, 472, 113. (12) Willner, I.; Doron, A.; Katz, E.; Levi, S.; Frank, A. J. Langmuir 1996, 12, 946.

10.1021/la034519v CCC: $25.00 © 2003 American Chemical Society Published on Web 05/17/2003

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Wang et al. redox-active interface, respectively. For all systems, the images of the drops were recorded in the respective oxidized and reduced states of the interfaces. To extract the precise contact angle values, the drop images were fitted with a commercial software (KSV Instruments, Finland) that uses the Young-Laplace equation.14 The uncertainty in the contact angle values is estimated to be (0.5° from 10 independent measurements.

oxidizing power and is extremely dangerous to handle in the laboratory; gloves, goggles, and face shields are needed for protection.) The Prussian Blue (PB) film was deposited on the pretreated ITO electrodes according to the reported procedure.13 A mixture composed of 0.05 M HCl, 0.05 M K3[Fe(CN)6], and 0.05 M FeCl3 (1:2:2) was introduced into a conventional three-electrode cell, comprising the ITO working electrode, a glassy carbon auxiliary electrode isolated by a glass frit, and a saturated calomel reference electrode (SCE). The reference electrode was connected to the working volume via a Luggin capillary. The PB film was deposited galvanostatically by passing a current of 40 µA/cm2 through the ITO electrode for 100 s. After the film deposition, the electrode was rinsed with distilled water and kept under air. To characterize the PB-modified electrode, it was first cycled 15 times between +0.6 V and -0.2 V vs SCE in 1 M KCl (pH ) 3.2, scan rate 20 mV‚s-1) and after this, the electrode was stabilized at +0.6 V for 30 s.13 The cycling voltammogram of the resulting PB0 film was then recorded in 1 M KCl (pH ) 7.1) starting the scan at 0.6 V at a scan rate of 50 mV‚s-1. The electrode area exposed to the solution was ∼0.4 cm2. All voltammetric measurements were performed using a potentiostat (EG&G, Model 283) connected to a computer (EG&G Software PowerSuite 1.03). Modification of the Polyethylenimine-Functionalized Electrodes with 2,3-Dichloro-1,4-naphthoquinone (2). Clean glass plates coated with gold (thickness of Au layer is ∼5 nm, Analytical-µSystem, Germany) were first modified by polyethylenimine (PEI) by their immersion in an aqueous solution containing PEI (0.1 g‚mL-1) for 5 min. The PEI-modified plates were further reacted with 2,3-dichloro-1,4-naphthoquinone by immersion of the plates into a boiling saturated solution of the quinone in absolute ethanol for 3 min. The resulting electrodes were thoroughly rinsed with ethanol and distilled water and dried under a flow of nitrogen. Cyclic voltammograms of the quinone-functionalized electrode were recorded in the electrochemical cell with the electrode area exposed to the solution, ∼1 cm2. Preparation of the Ferrocene-Functionalized Electrodes. Gold-covered glass plates were pretreated by immersion in hot (60 °C) ethanol solution for 5 min followed by thorough rinsing with distilled water. The pretreated plates were reacted with a solution of cystamine dihydrochloride (0.1 M) for 2 h. The N-2-methylferrocenecaproic acid (1) (1 mM) was then coupled to the cystamine monolayer using EDC (5 mM) solution in 0.1 M HEPES buffer (pH 7.1) at room temperature for 2 h. After the modifications, the electrodes were thoroughly rinsed with distilled water and dried under a flow of nitrogen. Cyclic voltammograms of the ferrocene-functionalized electrode were recorded in the electrochemical cell with the electrode area exposed to the solution, ∼1 cm2. Contact Angle Measurements. Static contact angle in situ electrochemical measurements were performed on modified conductive surfaces using a CAM2000 optical contact angle analyzer (KSV Instruments) and an EG&G, model 283 potentiostat. A thin silver wire (φ ) 0.1 mm) and a platinum wire (φ ) 0.1 mm) were used as a quasi-reference electrode and a counter electrode, respectively. Such thin wires were chosen to prevent the distortion of the drops. The second reason for using a silver wire quasireference electrode is that chloride ions react irreversibly with ferrocene units at oxidative potentials.6 All potentials are reported here versus the silver wire quasi-reference electrode. We find that the reference potentials of an SCE and the silver wire in 0.1 M phosphate buffer, pH 7.1, have the relation VSCE ) VAg-wire + 0.07 V. For PB0-modified ITO electrodes the contact angles were first measured at 0.6 V. Then, the potential was switched to 0.3 V, and subsequently, the potential was switched to 1.2 V. In each case the potential was kept for 20 s prior to contact angle measurement to allow a drop to adjust its shape. For the ferrocene (1)-functionalized electrode, the applied potential was switched between 0.6 and 0.1 V. For the naphthoquinone-functionalized electrode, the potential was switched between the potentials -0.1 V and -0.5 V to generate the oxidized and reduced states of the

The fact that the semireduced state, PB0, is neutral while the oxidized or reduced states, PB4- or PB3+, of the film are negatively or positively charged, respectively, suggests that the system may undergo transition from the hydrophobic PB0 state to the two hydrophilic states, PB4- and PB3+. Figure 1A shows the characteristic cyclic voltammogram of the Prussian Blue film deposited on ITO electrodes. The redox wave at ∼0.2 V corresponds to the PB0/PB4- couple, whereas the redox wave at ∼0.9 V corresponds to the PB3+/PB0 redox couple. This implies that the film exists in the PB4- state at E e 0.2 V, in the PB0 form at 0.2 V < E < 0.9 V, and in the PB3+ state at E > 0.9 V. Figure 1B shows the images of a drop of phosphate buffer solution (0.1 M, pH 7.1) on the PBmodified electrode surface, upon the electrochemical switching of the film between the three redox states. The contact angle of the droplet deposited on the neutral PB0 film (image ii) corresponds to 72°. The reduction of the film to the PB4- form or the oxidation of the film to the PB3+ state results in the spreading of the droplet due to the hydrophilic properties of the interface (images i and iii, respectively). The contact angle of the droplet associated with the PB4- or PB3+ is 66° or 62°, respectively. Figure 1C shows the cyclic transformation of the droplet shapes upon the electrochemical switching of the film between its different states. Figure 2 shows the gradual changes of the contact angles of the aqueous droplet upon the stepwise changing of the potential from 0.4 to 1.2 V. For comparison, the cyclic voltammogram of the PB0 film upon its oxidation is also given. The upper horizontal axis of Figure 2 shows the calculated ratio of [PB3+]/[PB0] at the respective potentials at which the contact angles were measured. The [PB3+]/ [PB0] ratio was calculated according to the Nernst equation, assuming that the redox equilibrium is achieved at each potential applied. It can be seen that at the standard redox potential of the PB0-functionalized film, E° ) 0.87 V, although the Nernst equation states that the ratio is [PB3+]/[PB0] ) 1, the contact angle is almost unaltered as compared to that for the PB0-functionalized

(13) Carcia-Jaredo, J. J.; Benito, D.; Navarro-Laboulais, J.; Wivente, F. J. Chem. Educ. 1998, 75, 881.

(14) (a) Jennings, J. W.; Pallas, N. R. Langmuir 1988, 4, 959. (b) Van Oss, C. J.; Good, R. J.; Chaudhury, M. K. Langmuir 1988, 4, 884.

Results and Discussion Prussian Blue (PB) is an inorganic redox-active polymer that undergoes reversible redox processes between the reduced form PB4-, the semireduced neutral form PB0, and the oxidized state PB3+ (eqs 1 and 2). The electrochemical features of Prussian Blue have been studied in detail,10 and its electrocatalytic properties for the oxidation of NADH were demonstrated.11

Fe4III[FeII(CN)6]3 + 4e- + 4K+ a K4Fe4II[FeII(CN)6]3 PB0 PB4(1) Fe4III[FeII(CN)6]3 - 3e- + 3A- a PB0 Fe4III[FeIII(CN)6]3A3 (2) PB3+

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Figure 2. Potential-controlled contact angle changes of an aqueous droplet upon the oxidation of the PB0-functionalized electrode to the PB3+ state: (a) cyclic voltammogram of the PB film; (b) changes in the contact angle, ∆θ, upon stepping the potential on the PB0-functionalized electrode. The top horizontal axis shows the [PB3+]/[PB0] values at the different potentials, calculated by the Nernst equation.

Figure 1. (A) Cyclic voltammogram of the Prussian Bluemodified ITO electrode recorded in 0.1 M KCl in 0.1 M phosphate buffer solution, pH 7.1, at the scan rate 20 mV‚s-1. (B) Potentialcontrolled wetting of a drop composed of 0.1 M phosphate buffer, pH 7.1, on a Prussian Blue-modified ITO surface: (i) image of the drop while keeping the interface in the PB4- reduced state, E ) -0.3 V; (ii) Image of the drop in the PB0 neutral state of the interface, E ) 0.6 V; (iii) image of the drop in the PB3+ oxidized state of the interface, E ) 1.2 V. The volume of drops was ∼10 µL. The red curve and the blue lines show the results of the computer fitting of the droplet shape and contact angles, respectively. (C) Change in the contact angles of an aqueous drop containing 0.1 M phosphate buffer solution, pH 7.1, on a Prussian Blue-modified ITO surface upon switching the potential from 0.6 V (points i) to -0.3 V (points ii) and further to 1.2 V (points iii).

surface. Significant changes in the contact angle are observed when the ratio of [PB3+]/[PB0] is higher than 10.

The oxidized state of the PB3+ film acts as an electrocatalyst for the oxidation of NADH.14 Thus, upon the interaction of an aqueous droplet that includes NADH with the Prussian Blue-modified electrode that is subjected to the potential E ) 1.2 V, the electrocatalytic oxidation of NADH will proceed in the droplet. Provided that the electrocatalytic oxidation of NADH by PB3+ is rapid, a steady-state concentration of [PB0]/[PB3+] is generated at the film and droplet junction. The ratio of [PB0]/[PB3+] is controlled by the NADH concentration, and it is expected to be higher as the concentrations of NADH are elevated.11b Thus, at an applied potential of E ) 1.2 V, as the concentration of NADH in the droplet increases, the Prussian Blue interface will turn more hydrophobic. Figure 3A shows the cyclic voltammograms that correspond to the electrocatalyzed oxidation of NADH by the PB3+ film. As the concentration of NADH increases, the amperometric response of the electrode is higher, and it levels off to a saturation value of ∼500 µA, that corresponds to the highest electrocatalytic turnover (Figure 3A, inset). Figure 3B, curve a, shows the contact angles of the droplets containing different concentrations of NADH upon interaction with the semireduced PB0 film associated with the electrode (applied potential E ) 0.6 V). Evidently, the chemical composition of the aqueous droplet influences the contact angle, and it decreases as the concentration of NADH is elevated. Figure 3B, curve b, shows the values of the contact angles of the series of droplets that include variable concentrations of NADH upon applying a potential of E ) 1.2 V on the electrode, that results in the oxidation of the film to the oxidized PB3+ state. The contact angle of the droplet that lacks NADH decreases from 72° to 61.5°, consistent with the transformation of the film from hydrophobic PB0 to the hydrophilic PB3+ state. Assuming that the NADH concentration has a similar effect on the contact angle of the droplet in the PB0 and PB3+ forms, the calculated contact angles of the PB film

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Figure 3. (A) Cyclic voltammograms of a Prussian Blue-modified ITO electrode: (a-e) in the presence of 1, 3, 5, 10, and 20 mM of NADH, respectively. In all experiments 0.1 M phosphate buffer, pH 7.1, was used as an electrolyte solution. Scan rate 5 mV‚s-1. Inset: Electrocatalytic anodic currents observed at E ) 0.9 V in the presence of the Prussian Blue-functionalized electrode and different concentrations of NADH in the electrolyte solution. (B) Changes in the contact angles of droplets containing various concentrations of NADH in 0.1 M phosphate buffer solution, pH 7.1, on Prussian Blue-modified ITO electrodes upon the application of different potentials: (a) E ) 0.6 V, the film is in the PB0 neutral state; (b) E ) 1.2 V, the film is in the oxidized state PB3+; (c) calculated curve showing the contact angles of the droplet associated with the Prussian Blue film in the oxidized state, PB3+, and assuming that the electrocatalytic oxidation of NADH has no effect on the contact angle of the droplet and that NADH has a similar effect on the contact angle of the droplet for PB0 and PB3+ forms; (d) calculated net changes in the contact angles of the droplet as a result of the electrocatalytic oxidation of different NADH concentrations by the PB3+ film-modified electrode. The curve is calculated by the subtraction of curve c from curve b. Scheme 1 . Assembly of N-2-Methylferrocenecaprioc Acid (1) onto an Au Electrode and the Mediated Bioelectrocatalyzed Oxidation of Glucose

in its oxidized PB3+ state at variable NADH concentrations are depicted in Figure 3B, curve c. Clearly, the experimental values of the contact angles, curve b, are smaller than the calculated values. The experimental changes in the contact angles indicate that at low NADH concentrations the contact angles are very similar to the calculated values, implying that the contact angle at low NADH concentrations is mainly controlled by the composition of the droplet. At high NADH concentrations, for example, 4 × 10-2 M, the contact angle deviates substantially from the calculated value, and the contact angle is very similar to that of the PB0 film. These results are consistent with the fact that the electrocatalyzed oxidation of NADH proceeds in the droplets. As the concentration of NADH increases, the ratio of [PB0]/[PB3+] increases, and the junction between the droplet and the surface turns more hydrophobic. The more hydrophobic nature of the surface leads to a substantially higher value of the contact angle,

as expected from the mere composition of the droplet. Figure 3B, curve d, depicts the curve that corresponds to the subtraction of curve c from curve b. This curve corresponds to the net changes in the contact angles at different concentrations of NADH, that exclude the effect of chemical composition on the contact angle. It is clear that contact angle increases as the concentration of NADH increases, and at a NADH concentration of 4 × 10-2 M it reaches a value that is ∼15% of the contact angle of the PB0 film recorded at E ) 0.6 V. The fact that the contact angle of the Prussian Blue film, subjected to the oxidation potential 1.2 V in the presence of NADH, is ∼85% restored to the value of the PB0 state, and referring to Figure 2, suggests that in the presence of [NADH] ) 20 mM a steadystate ratio of [PB3+]/[PB0] ) ∼5 is generated on the electrode support. This description supports our explanation that the experimental contact angle values in the presence of NADH reflect the bioelectrocatalytic oxidation

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Figure 4. Electrochemical and wetting properties of the ferrocene (1) monolayer-modified Au electrode: (a) cyclic voltammogram of the ferrocene monolayer-functionalized electrode, recorded in 0.1 M phosphate buffer, pH 7.1, under Ar, at the scan rate 100 mV‚s-1; (b) changes in the contact angles, ∆θ, of a drop composed of 0.1 M phosphate buffer, pH 7.1, upon application of the potential steps on the monolayer-modified electrode. The top horizontal axis shows the calculated [Fc+]/ [Fc] values at the different potentials.

of NADH that proceeds in the droplet. As the concentration of NADH increases, the ratio of the [PB0]/[PB3+] states in the film increases, leading to the enhanced hydrophobicity of the interface and to higher contact angle values. The redox-controlled triggering of the hydrophobic/ hydrophilic properties of surfaces, and the analysis of bioelectrocatalytic transformations by means of contact angle measurements, was further demonstrated in the presence of a N-(2-methylferrocene)caproic acid (1) monolayer assembled on a cystamine-functionalized Au electrode (Scheme 1). The cyclic voltammogram of the resulting ferrocene monolayer reveals a quasi-reversible redox-wave at E° ) 0.38 V vs the Ag wire quasi-reference electrode (Figure 4, curve a). Coulometric assay of the redox wave indicates a surface coverage of 1.8 × 10-10 mol‚cm-2. Figure 5A shows the images of an aqueous droplet on the 1-functionalized Au electrode upon switching the monolayer between the reduced state, image i, and the oxidized ferrocenylium cation state, image ii. The contact angle changes from the value of 63° in the reduced state to 55° in the oxidized state, consistent with the redox switching of the nature of the monolayer from hydrophobic to hydrophilic. Figure 5B shows the reversible switching of the hydrophilic/hydrophobic properties of the interface upon the reversible oxidation and reduction of the monolayer-modified Au surface. Figure 4, curve b, shows the gradual changes of the contact angles of the aqueous droplet upon the stepwise changing of the potential from 0.1 to 0.6 V. The upper horizontal axis of Figure 4 shows the calculated values of the ferrocenylium (Fc+)/ferrocene (Fc) units associated with the monolayer at the respective potentials at which the contact angle measurements were performed. The values of the ratio Fc+/Fc were calculated by the Nernst equation. At the standard redox potential of the ferrocene units, E° ) 0.38 V, where [Fc+]/[Fc] ) 1,

Figure 5. (A) Potential-dependent wetting of a drop composed of 0.1 M phosphate buffer, pH 7.1, on a N-2-methylferrocenecaproic acid (1)-modified gold electrode surface: (i) image of the drop upon its interaction with the reduced state of the ferrocene monolayer, E ) 0.1 V; (ii) image of the drop in the oxidized state of the monolayer, E ) 0.6 V. The volume of drops was ∼10 µL. The red curve and the blue lines show the results of the computer fitting of the droplet shape and contact angles, respectively. (B) Change in the contact angles of an aqueous drop composed of 0.1 M phosphate buffer solution, pH 7.1, on a (1)-functionalized gold surface upon switching the potential from 0.1 V (points i) to 0.6 V (points ii).

the contact angle of the droplet is almost unaltered as compared to the case of the reduced state of the monolayer. Only at [Fc+]/[Fc] values higher than 50 are sharp changes in the contact angles observed. It should be noted that the differences in the contact angle values of the ferrocene monolayer-functionalized surface and the ferrocenyliummodified surface are substantially lower than those reported for another ferrocene-modified monolayer interface.6 Also, in contrast to the later study that reported a hysteresis effect in the contact angle values, that was attributed to the time-dependent degradation of the monolayer, we do not observe any significant hysteresis upon the application of two oxidation and reduction cycles on the surface. These apparent differences may be attributed to the differences in the structures and stabilities of the ferrocene monolayer employed in two studies: (i) In the previously reported system,6 the ferrocene units are linked to the gold surface by a C15H31 tether, thus generating a dense hydrophobic interface. In the present system the ferrocene units are linked to the electrode by a short bridging linker in a nondense configuration, where substantial domains are functionalized by the residual cystamine units. The enhanced hydrophilicity of the

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Figure 6. (A) Cyclic voltammograms of an N-2-methylferrocenecaproic acid (1)-functionalized gold electrode: (a-e) in the presence of 0, 1, 5, 15, and 30 mM glucose, respectively. The electrolyte solution was composed of glucose oxidase (1 mg‚mL-1) in 0.1 M phosphate buffer solution, pH 7.1. Data were recorded under argon, at the scan rate 2 mV‚s-1. Inset: Electrocatalytic anodic currents observed at E ) 0.6 V in the presence of various concentrations of glucose. (B) Changes in the contact angles of droplets containing glucose oxidase, 1 mg‚mL-1, and various concentrations of glucose in 0.1 M phosphate buffer solution, pH 7.1, on N-2-methylferrocenecaproic acid (1)-functionalized gold electrodes upon the application of different potentials: (a) E ) 0.1 V; (b) E ) 0.6 V; (c) calculated curve for the droplet associated with the monolayer-functionalized electrode at E ) 0.6 V, assuming that the electrocatalytic current has no effect on the contact angle of the droplet and that the changes in the glucose concentration have a similar effect on the contact angle of the droplet on the ferrocene (1)-modified surface at E ) 0.1 V and E ) 0.6 V; (d) calculated net changes in the contact angles of the droplet as a result of the electrocatalytic oxidation of different NADH concentrations by a ferrocenylium-functionalized electrode. The curve (d) is calculated by subtraction of curve c from curve b.

monolayer in the reduced ferrocene state leads to the substantially lower difference in the contact angle values of the reduced and oxidized monolayer states. (ii) We do not observe any significant degradation of the ferrocene monolayer upon the application of five oxidation/reduction cycles. This enhanced stability of our monolayer leads to the lack of any observable hysteresis. The activation of redox enzymes by electroactive relay units is well established in bioelectronics.15,16 Ferrocene derivatives act as efficient electron relays for the electrical contacting of different redox enzymes and electrodes,17 for example, glucose oxidase. Accordingly, we decided to probe the bioelectrocatalyzed oxidation of glucose by glucose oxidase, GOx, in the presence of the ferrocenemodified electrode, as schematically presented in Scheme 1. Upon the application of a potential on the electrode that oxidizes the ferrocene (Fc) monolayer to the ferrocenylium cation (Fc+) state, the mediated oxidation of the redox center of GOx occurs, and this activates the bioelectrocatalyzed oxidation of glucose. Provided that the mediated bioelectrocatalyzed oxidation of glucose is fast, a steady-state ratio of [Fc]/[Fc+] in the monolayer is generated, even though the potential of the electrode is retained at a value adequate to oxidize the ferrocene sites. The ratio [Fc]/[Fc+] and, thus, the hydrophobicity of the interface are expected to be controlled by the concentration of glucose. As the glucose concentration is elevated, the [Fc]/[Fc+] ratio should increase, and the hydrophobic (15) (a) Zakeeruddin, S. M.; Fraser, D. M.; Nazeeruddin, M.-K.; Gra¨tzel, M. J. Electroanal. Chem. 1992, 337, 253. (b) Willner, I.; Willner, B. React. Polym. 1994, 22, 267. (c) Limoges, B.; Moiroux, J.; Saveant, J. M. J. Electroanal. Chem. 2002, 521, 8. (16) (a) Willner, I. Science 2002, 298, 2407. (b) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180. (c) Willner, I.; Katz, E.; Willner, B. Electroanalysis 1997, 9, 965. (d) Willner, I.; Willner, B. Bioelectrochem. Bioenerg. 1997, 42, 43. (17) (a) Okawa, Y.; Nagano, M.; Hirota, S.; Kobayashi, H.; Ohno, T.; Watanabe, M. Biosens. Bioelectron. 1999, 14, 229. (b) Willner, I.; LionDagan, M.; Marx-Tibbon, S.; Katz, E. J. Am. Chem. Soc. 1995, 117, 6581. (c) Sakura, S.; Buck, R. P. Bioelectrochem. Bioenerg. 1992, 28, 387.

features of the interface are expected to be higher. Figure 6A shows the cyclic voltammograms of the ferrocenemodified electrode in the presence of variable concentrations of glucose and GOx, 1 mg‚mL-1. An electrocatalytic current is observed at the redox potential of the ferrocene units, consistent with the mediated bioelectrocatalyzed oxidation of glucose. The anodic current increases as the concentration of glucose increases, and it levels off to a saturation value of ∼7.2 µA at a glucose concentration of ∼3 × 10-2 M (Figure 6A, inset). Figure 6B, curve a, shows the contact angles of droplets containing GOx, 1 mg‚mL-1, in 0.1 M phosphate buffer, pH 7.1, and different concentrations of glucose upon the application of a potential on the electrode that corresponds to 0.1 V. At this potential, the monolayer exists in its reduced state, and the bioelectrocatalyzed oxidation of glucose is blocked. With no glucose in the droplet, the contact angle corresponds to 63° and as the concentration of glucose is elevated, the contact angle of the droplet decreases to 41°. Thus, the addition of glucose to the droplet affects its contact angle with the modified electrode. Figure 6B, curve b, shows the contact angle values of the droplet that includes GOx, 1 mg‚mL-1, and variable concentrations of glucose upon the application of a potential that corresponds to 0.6 V on the 1-functionalized electrode that allows the bioelectrocatalyzed oxidation of glucose. This is confirmed by parallel electrochemical experiments that monitor in situ the electrocatalytic anodic currents in the droplets. It can be seen that at the potential of 0.6 V, where the bioelectrocatalyzed oxidation of glucose proceeds, at low glucose concentrations a sharp decrease in the contact angles is observed, whereas at a high concentration of glucose, ∼4 × 10-2 M, the contact angle of the droplet is very similar to the contact angle of the same droplet on the electrode containing the reduced state of the monolayer. Figure 6B, curve c, depicts the calculated curve of the contact angle values under conditions where the electrode is biased at a potential of 0.6 V and assuming that the composition of the droplets has the sole effect on the contact angle. At

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Scheme 2 . Assembly of the Naphthoquinone/PEI-Functionalized Electrode and the Electrocatalyzed Reduction of O2 by the System

a low glucose concentration, 1 × 10-3 M, the experimental and calculated values of the contact angle are very similar, and as the concentration of glucose increases, the contact angles deviate from the calculated values that assume the pure effect of the glucose solute. These results are consistent with the fact that the bioelectrocatalyzed oxidation of glucose occurs in the droplets. At high glucose concentrations, the mediated electron transfer yields a high ratio of the [Fc]/[Fc+] monolayer composition, even though the electrode is biased at E ) 0.6 V, resulting in a more hydrophobic interface and high values of the contact angle. At low glucose concentrations, the steady-state ratio of [Fc]/[Fc+] is low, thus preserving the hydrophilic nature of the monolayer interface. Figure 6B, curve d, shows the curve obtained upon the subtraction of curve c from curve b. Thus, curve d corresponds to the net change in the contact angles of the droplets as a result of the bioelectrocatalytic processes occurring in the droplets and excluding the effect of material composition on the contact angle values. The contact angle increases as the concentration of glucose is elevated, and at a high glucose concentration, 4 × 10-2 M, the contact angle of the droplet associated with the electrode biased at 0.6 V is almost similar to the contact angle of the aqueous droplet associated with the 1-modified electrode that is biased at 0.1 V. Thus, our results indicate that the hydrophobic/ hydrophilic properties of the ferrocene-modified interface are controlled by the bioelectrocatalytic processes occurring in the droplets associated with the functionalized electrode. We see that, at a glucose concentration of 4 × 10-2 M, ∼80% of the contact value change (∆θ ) 7.8°) as a result of the oxidation of the ferrocene monolayer is depleted. Referring to Figure 4, curve b, this translates to a [Fc+]/[Fc] ratio of ∼8 that is generated on the electrode support as a result of the bioelectrocatalytic process. A further electroswitchable interface that was probed by contact angle measurements includes a naphthoquinone-functionalized polymer film associated with the electrode surface (Scheme 2). An Au electrode was modified with a polyethylenimine, PEI, film.18 The PEI is composed of primary, secondary, and tertiary amine functionalities at a 1:1:1 ratio19 (m ) n ) q in Scheme 2). Reaction of the (18) Moore, A. N. J.; Katz, E.; Willner, I. Electroanalysis 1996, 8, 1092. (19) Willner, I.; Eichen, Y.; Frank, A. J.; Fox, M. A. J. Phys. Chem. 1993, 97, 7264.

interface with 2,3-dichloro-1,4-naphthoquinone (2) yields the naphthoquinone-modified electrode with electrochemical properties similar to those described before.20 Figure 7A, curve a, shows the cyclic voltammogram of the modified electrode. Figure 7B, curve a, shows the cyclic changes of the contact angle upon the switching of the redox-active interface between the quinone and hydroquinone states, respectively. The application of a potential that corresponds to -0.1 V on the electrode preserves the interface in the quinone state, resulting in a hydrophobic surface. The contact angle of the phosphate-buffered droplet associated with the electrode corresponds to 63°. Switching the potential to -0.5 V results in the more hydrophilic interface consisting of the hydroquinone film. This results in a decrease in the contact angle by ∼6°. Further oxidation of the film to the quinone state restores the hydrophobic interface and the characteristic contact angle between the droplet and this modified surface. Figure 7A, curve b, shows the cyclic voltammogram of the naphthoquinonemodified electrode under air. An electrocatalytic cathodic current is observed at the redox potential of the quinone units, consistent with the electrocatalyzed reduction of O2 by the electrogenerated hydroquinone units. This suggests that upon the application, under oxygen, of a potential that corresponds to -0.5 V on the electrode, where the naphthoquinone interface is reduced to the naphthohydroquinone, the electrocatalytic reduction of O2 will generate the hydrophobic naphthoquinone interface. Thus, the electrocatalytic reduction of oxygen by the naphthohydroquinone is anticipated to yield the hydrophobic interface even though the negative potential, E ) -0.5 V is applied on the electrode. Figure 7B, curve b, shows that this prediction is, indeed, experimentally observed. At point ii the potential corresponding to E ) -0.5 V that reduces the naphthoquinone interface to the naphthohydroquinone is applied on the electrode under O2. Almost no changes in the contact angle are observed as compared to the oxidized state of the hydrophobic film, points i and iii. This result clearly demonstrates that the electrocatalytic reduction of O2 at the negative potential preserves the interface in the hydrophobic configuration. (20) (a) Katz, E.; Shkuropatov, A. Y.; Vagabova, O. I.; Shuvalov, V. A. J. Electroanal. Chem. 1989, 260, 53-62. (b) Katz, E.; Solov’ev, A. A. J. Electroanal. Chem. 1990, 291, 171-186.

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Figure 7. (A) Cyclic voltammograms of an Au electrode modified with polyethylenimine film and functionalized with 2,3-dichloro1,4-naphthoquinone: (a) under argon; (b) in the presence of oxygen. In all cases 0.1 M phosphate buffer (pH 7.1) was used as the electrolyte solution. Scan rate, 5 mV‚s-1. (B) Changes in the contact angles of droplets composed of 0.1 M phosphate buffer and associated with a naphthoquinone-PEI film-modified electrode at different potentials: (a) under argon, points x and z at E ) -0.1 V, point y at E ) -0.5 V; (b) under oxygen, points i and iii at E ) -0.1 V, point ii at E ) -0.5 V.

Conclusions The present study demonstrated the reversible electrochemical triggering of the hydrophobic/hydrophilic properties of surfaces that are functionalized with redoxactive films or monolayers. The hydrophobic/hydrophilic properties of the surfaces were monitored by contact angle measurements, that revealed mechanostructural changes in the droplets shape upon the reversible electrical transformation on the redox-active interfaces associated with the electrodes. An important aspect of the research was the quantitative correlation between the changes in the contact angles (or the hydrophobicity/hydrophilicity

of the interface) and the relative population of the electroactive units that contribute to the hydrophilic and hydrophobic properties of the interface. Our study has demonstrated that contact angle measurements can be applied to follow electrocatalytic and bioelectrocatalytic processes occurring at the interface between substratecontaining droplets and at electrocatalytic interfaces associated with electrodes. Acknowledgment. This research (No. 101/00) was supported by The Israel Science Foundation. LA034519V