Salt-Induced Swelling and Electrochemical Property Change of

The salt-induced swelling of {HA/Mb}n films facilitated the transportation of counterions, and ... For a more comprehensive list of citations to this ...
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1984

J. Phys. Chem. B 2007, 111, 1984-1993

Salt-Induced Swelling and Electrochemical Property Change of Hyaluronic Acid/Myoglobin Multilayer Films Haiyun Lu and Naifei Hu* Department of Chemistry, Beijing Normal UniVersity, Beijing 100875, People’s Republic of China ReceiVed: August 27, 2006; In Final Form: December 2, 2006

The ionic strength in supporting electrolyte solution had a significant influence on the electrochemical and electrocatalytic behaviors of myoglobin (Mb) in {HA/Mb}n films, which were assembled layer-by-layer on pyrolytic graphite (PG) electrodes with oppositely charged hyaluronic acid (HA) and Mb. The results of cyclic voltammetry (CV), quartz crystal microbalance (QCM), scanning electron microscopy (SEM), rotating disk voltammetry (RDV), and electrochemical impedance spectroscopy (EIS) showed that after incubation with testing solution at high concentration of salt (CKCl), the {HA/Mb}n films swelled and the film permeability was enhanced, suggesting that the external salt ions and accompanied water molecules in the exposure solution are incorporated into the films. Systematic investigation of the type and size effect of counterions in supporting electrolyte solution on the electrochemical responses for the {HA/Mb}n films and the positive shift of the formal potential (E°′) with CKCl suggest that it is cationic rather than anionic counterions that control the electrode process of {HA/Mb}n films at PG electrodes with electron hopping mechanism. The salt-induced swelling of {HA/Mb}n films facilitated the transportation of counterions, and then accelerated the electron transfer of Mb in the films with the underlying electrodes, making the film electrodes show better CV responses. The comparative study showed that only Mb layer-by-layer films assembled with “soft” and flexible polyions could demonstrate the salt-induced effect and that the {HA/Mb}n films showed better swelling capability than {PSS/Mb}n films (PSS ) poly(styrenesulfonate)) due to the unique character of HA.

Introduction Since the 1990s, a novel technique for fabricating ultrathin films, named as layer-by-layer assembly, has been developed and has aroused increasing interests among researchers.1,2 The method is mainly based on alternate adsorption of oppositely charged species in solution onto solid surfaces and shows the advantages over other film-forming methods in the precise control of thickness at molecular or nanometer level according to a predesigned architecture, as well as its simplicity and versatility.2 The layer-by-layer assembly was first used to fabricate polyelectrolyte multilayer films, and since then, the properties of polyelectrolyte multilayer films, the assembly conditions controlling the film growth, and the factors influencing the film properties have been extensively studied. Among the various properties of polyelectrolyte multilayer films, swelling is one of the most important ones. When a polyelectrolyte layer-by-layer film is brought into contact with an electrolyte solution, the solute and water may diffuse into the film matrix and make the film swell.3-6 The swelling property of different polyelectrolyte multilayer films and capsules has been studied and found to be influenced by various factors, such as pH and ionic strength of assembly solutions,7-11 pH and salt concentration of the electrolyte solution for film incubation,9,12-19 and solvents.20,21 For example, Burke and Barrett investigated the swelling of {HA/PAH}n layer-by-layer films, where HA represents polyanionic hyaluronic acid and PAH stands for positively charged poly(allylamine hydrochloride), and found that the swelling extent was considerably dependent on the assembly pH and swelling medium.9b * Corresponding author. Tel.: +86 10 5880 5498. Fax: +86 10 5880 2075. E-mail: [email protected].

The swelling of polyelectrolyte layer-by-layer films is not only affected significantly by external conditions, but also highly related to the structure and property of the film constituents.22 Films prepared from different polyelectrolytes can exhibit different swelling behaviors due to different charge density of the constituents. Generally, higher charge density on the polyelectrolyte chains in the films may result in more ionic cross-linking, less swelling, and lower permeability.23-25 Particularly, the salt-induced swelling of polyelectrolyte multilayers has also been observed and investigated. When the layer-by-layer films assembled mainly by electrostatic interaction are incubated in salt solution, the counterions and accompanied water molecules in solution are usually incorporated into the films, making the films swell, and higher concentration of salt in exposure solution often causes the swelling to a greater extent.9,16-19 The swelling often leads to the change in film morphology26-30 and enhancement of the film permeability.17,18a,22,31,32 For example, Schlenoff and co-workers18a investigated the transport of electroactive probe ions through the multilayer films, showing that the salt addition in solution significantly accelerates the mass transport of the probe due to the enhancement of the film permeability by salt-induced swelling. The mechanism of salt-induced swelling of polyelectrolyte multilayer films has also been studied by Schlenoff and coworkers.18,28,33 Before swelling, the internal charges of layerby-layer films are balanced by matched numbers of positive and negative polymer repeat units, which is termed as “intrinsic” compensation.18,28,33,34 After incubation with the external salt solution, the counterions in solution would be “doped” or

10.1021/jp065551b CCC: $37.00 © 2007 American Chemical Society Published on Web 02/03/2007

Hyaluronic Acid/Myoglobin Multilayer Films incorporated into the films, forcing some “extrinsic” compensation to participate in charge neutralization. The process can be represented by the following equilibrium:18

Pol+Pol-(film) + yNa+(s) + yCl-(s) S (1 - y)Pol+Pol-(film) + yPol+Cl-(film) + yPol-Na+(film) (1) +

where Pol and Pol- are positive and negative polyelectrolyte repeat units, respectively, y is the fraction of the multilayer in the extrinsic form, and the subscripts “film” and “s” stand for the film and solution phases, respectively. The higher salt concentration in incubation solution would lead to the higher doping level (y) and result in the greater extent of film swelling.33a The incorporation of salt ions and accompanied water molecules into the films could weaken the ionic crosslinking that holds the layer together and make the films become thicker.18b However, the studies of salt-induced swelling up to now were all focused on polyelectrolyte multilayer films. To the best of our knowledge, the swelling of protein layer-by-layer films induced by external ionic strength has not been reported until now. In recent years, the layer-by-layer assembly has been extended to constructing protein films with polyions or nanoparticles.35 The direct electrochemistry of some redox enzymes or proteins in these multilayer films on electrodes has also been realized.36,37 The study of direct electron transfer of proteins with underlying electrodes can provide a model for the mechanistic study of electron transfer between enzymes in real biological systems and establish a foundation for fabricating the new type of biosensors, bioreactors and biomedical devices without using mediators.38,39 In the present work, the electroactive myoglobin (Mb) was selected as the model protein and redox probe, and assembled layer-by-layer with HA, forming {HA/Mb}n films. The assembly of the {Mb/HA}n layer-by-layer films was studied by our group recently.40 The stable {Mb/HA}n films provided a favorable microenvironment for Mb to transfer electron directly with electrodes. HA is a linear polyion consisting of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine, demonstrating the relatively low charge density even when its all carboxylic acid groups are ionized.41 HA has good biocompatibility and can absorb a large amount of water and form hydrogels with a distinct swelling capability.41,42 Thus, the {HA/Mb}n films were chosen in this work to investigate the salt-induced swelling behavior of the films. We expected that the swelling of {HA/Mb}n films caused by increasing the salt concentration in supporting electrolyte solution would lead to the better responses in electrochemistry and electrocatalysis. Different electrochemical approaches, such as cyclic voltammetry (CV), amperometry, rotating disk voltammetry (RDV), and electrochemical impedance spectroscopy (EIS), and other techniques including quartz crystal microbalance (QCM), UV-vis spectroscopy, and scanning electron microscopy (SEM), were used in the investigation. Rusling et al. investigated the effect of ionic strength of external solution on the direct electrochemistry of Mb in cast didodecyldimethylammonium bromide (DDAB) films.43 Calvo and co-workers studied the influence of external salt on the electrochemistry of glucose oxidase mediated by ferrocene in poly(allylamine) hydrogel films.44 However, the swelling of protein layer-bylayer films caused by salt in electrolyte solution and the influence of electrolyte concentration on the direct electrochemistry and electrocatalysis of the protein in the films were reported for the first time in the present work. The mechanism of salt-

J. Phys. Chem. B, Vol. 111, No. 8, 2007 1985 induced swelling of the {HA/Mb}n films was discussed in detail. The understanding of the relationship between the film swelling and the protein electrochemistry in the films may guide us to control the electroactivity of the redox protein in the films by adjusting the salt concentration in incubation solution and using a suitable polyelectrolyte in the assembly. Experimental Section 1. Chemicals. Horse heart or equine skeletal muscle myoglobin (Mb, MW 17 800) was from Sigma. Titanium n-butoxide (Ti(OBu)4, 97%), poly(diallyldimethylammonium) (PDDA, 20%), poly(styrenesulfonate) (PSS, MW ∼ 70 000), and 3-mercapto-1-propanesulfonate (MPS) were from Aldrich. Hyaluronic acid (HA, MW ∼ 400 000) was from Fluka. Tetraethylammonium bromide (TEABr), tetra-n-propylammonium bromide (TPABr), tetra-n-butylammonium bromide (TBABr), sodium tetrafluoroborate (NaBF4), and sodium p-toluenesulfonate (NaTS) were from Alfa Aesar. TiO 2 nanoparticles (NP-TiO2, rutile, average diameter 20 nm) were from Zhoushan Nanoparticle Technology. K3Fe(CN)6 and K4Fe(CN)6 were from Beijing Chemical Plant. All other chemicals were reagent-grade. All solutions were prepared with twice-distilled water. 2. Film Assembly. For electrochemical studies, clean and rough basal plane pyrolytic graphite disk (PG, Advanced Ceramics, geometric area 0.16 cm2) electrodes were first immersed in 3 mg mL-1 PDDA solution for 20 min to adsorb a PDDA precursor layer, and make the surface become positively charged. For assembly of {HA/Mb}n layer-by-layer films, the PG/PDDA electrodes were alternately immersed in HA and Mb solutions (all at 1 mg mL-1 and pH 5.0 containing 0.1 M NaCl) for 20 min with intermediate water washing and nitrogen stream drying until the desired number of bilayers (n) was reached. {NP-TiO2/Mb}n and {PSS/Mb}n films were assembled with the similar procedure but using 3 mg mL-1 TiO2 nanoparticle dispersions and 3 mg mL-1 PSS solutions, respectively. For assembly of {SG-TiO2/Mb}n films with vapor-surface sol-gel deposition of TiO2 (SG-TiO2), the process was described in detail previously.45 In brief, 5 µL water was deposited onto the PG/PDDA surface, and the electrode was then suspended upside down above liquid Ti(OBu)4 in a sealed flask at ambient temperature for ∼3.5 h, resulting in the slow formation of a titania sol-gel on PG/PDDA surface. The electrode was then immersed in the Mb solution at pH 5.0 for 20 min to adsorb an Mb layer. This procedure was repeated for the desired number of cycles (n), forming {SG-TiO2/Mb}n films. The TiO2 sol-gel and TiO2 nanoparticles are designated as SG-TiO2 and NP-TiO2 in this work, respectively, to distinguish them in expression. For QCM studies, gold QCM electrodes were first pretreated with piranha solution (Caution: the piranha solution should be handled with extreme care, and only small volumes should be prepared at any time). After being washed with water, the gold QCM electrodes were immersed in 4 mM MPS solution for 24 h to chemisorb an MPS monolayer and make the surface become negatively charged. The PDDA precursor layer and subsequent {HA/Mb}n films were then assembled on the Au/MPS surface with the same procedure as that used on the PG electrodes. After each deposition step, the QCM electrodes were washed thoroughly with water and dried under a nitrogen stream, and the frequency was measured by QCM in air. QCM was also used to investigate the swelling property of the {HA/Mb}n films. After incubation of the films in KCl solutions at different concentration for a certain period of time, the QCM resonators with the films were rinsed with water for 20-30 s, dried under

1986 J. Phys. Chem. B, Vol. 111, No. 8, 2007 a nitrogen stream, and the frequency was then measured by QCM in air. 3. Apparatus. A CHI 660A electrochemical workstation (CH Instruments) was used for all electrochemical measurements at ambient temperature (22 ( 3 °C). A saturated calomel electrode (SCE) was used as the reference, a platinum wire as the counter electrode, and the PG disk electrode with films was employed as the working electrode. In CV studies, the supporting electrolyte solutions were purged with high-purity nitrogen for at least 15 min prior to measurements, and a nitrogen environment was maintained during the CV scans. For RDV experiments, a rotator and speed controller (Pine Instruments) were utilized. EIS was performed in the presence of 5 mM K4Fe(CN)6/K3Fe(CN)6 (1:1, containing KCl at different concentration). A sinusoidal potential modulation with amplitude of (5 mV was superimposed on the formal potential of the redox couple of Fe(CN)64-/3- (0.17 V vs SCE), and the applied frequency was from 105 to 0.1 Hz. QCM was performed with a CHI 420 electrochemical analyzer. On the basis of the Sauerbrey equation,46 ∆F = -2F02A-1(µF0)-1/2∆m, where F0 is resonant frequency of the fundamental mode of the quartz crystal (8 MHz), µ is the shear modulus of quartz (2.947 × 1011 g cm-1 s-2), F0 is the density of the crystal (2.648 g cm-3), and A is the geometric area of the QCM electrode (0.196 cm2), the frequency shift, ∆F (Hz), would be proportional to the adsorbed mass, ∆m (g), by taking into account the properties of quartz resonator used in this work. Thus, a 1 Hz of frequency decrease corresponds to 1.35 ng of mass increase. The surface concentration of Mb in each bilayer (Γ, mol cm-2) can then be estimated based on ∆m. The nominal thickness, d (cm), can be expressed by d ) (-3.4 × 10-9) ∆F/ F, where F is the density of the adsorbed layer material (g cm-3). For the polymer HA, the density was around 1.2 ( 0.1 g cm-3,47 while for the protein, the density was about 1.3 ( 0.1 g cm-3.48 A Cintra 10e UV-visible spectrophotometer (GBC) was used for UV-vis spectroscopy. SEM was performed with a HITACHI S-4800 field emission scanning electron microscope with an acceleration voltage of 5 kV. Results 1. Influence of CKCl in Supporting Electrolyte Solution on Electrochemical Responses of {HA/Mb}n Films. Mb has isoelectric point at 6.8,49 while HA, as a weak polyacid, has pKa at 2.9.41 Thus, at pH 5.0, negatively charged HA and positively charged Mb can be alternatively adsorbed on PG/ PDDA surface via electrostatic interaction between them, forming {HA/Mb}n layer-by-layer films. The assembly of {HA/ Mb}n films and the direct electrochemistry of Mb in the films have been realized and confirmed by our previous work.40 In the present work, the effect of ionic strength or the concentration of KCl (CKCl) in supporting electrolyte solution on the electrochemical behaviors of {HA/Mb}n films was examined. Figure 1 shows the CV responses of {HA/Mb}7 films in KCl solutions at different CKCl. A well-defined, quasi-reversible reductionoxidation peak pair was observed at about -0.3 V versus SCE, characteristic of Mb heme FeIII/FeII redox couple.40,50 With increasing CKCl, both reduction and oxidation peak potentials shifted positively, and the peak currents increased significantly, showing a sequence of 0.01 < 0.1 < 0.5 < 1 M. For a given {HA/Mb}n film under the fixed CKCl, its CV showed nearly symmetric peak shapes and roughly equal heights of reduction and oxidation peaks, and the peak currents increased linearly with scan rates from 0.05 to 2.0 V s-1, indicating the diffusionless and surface-confined voltammetric behavior.51 In this case, integration of CV reduction peak would give the

Lu and Hu

Figure 1. CVs of {HA/Mb}7 film electrodes at 0.2 V s-1 in KCl solutions with CKCl at (a) 0.01, (b) 0.1, (c) 0.5, and (d) 1 M.

charge (Q) for full reduction of electroactive Mb in the films, and the Q value could be further converted to the surface concentration of electroactive Mb (Γ*, mol cm-2) in the films. At a certain CKCl, the Γ* value of {HA/Mb}n films initially increased with the number of bilayers (n), and then reached the steady state (Figure 2A), suggesting that the Mb can only demonstrate its electroactivity in a few layers closest to electrode surface. If Γ*max is defined as the maximum surface concentration of electroactive Mb when the Γ* reached the steady state and nmax is defined as the number of bilayers when Γ* began to reach the steady state, both Γ*max and nmax values increased with CKCl in supporting electrolyte solution up to CKCl ) 1 M, also showing the sequence of 0.01 < 0.1 < 0.5 < 1 M. At KCl concentration higher than 1 M, Γ*max began to decrease (Figure 2A). The total surface concentration or the total adsorption amount of Mb in each bilayer (Γ, mol cm-2) for {HA/Mb}n films was estimated by QCM. For the first bilayer, Γ was a little bit smaller than the Γ* value estimated by CV, which was also observed in our previous work,40 and the difference between Γ and Γ* was attributed to the different roughness of PG and gold QCM electrodes. These results suggest that the Mb molecules in the first bilayer of {HA/Mb}n films are nearly 100% electroactive. When n g 2, Γ* was always smaller than Γ, indicating that only a fraction of Mb molecules in the following bilayers is electrochemically accessible. The fraction of electroactive Mb in the films (Γ*/Γ) decreased exponentially with n (Figure 2B). For {HA/Mb}n films with a fixed n, the ratio of Γ*/Γ increased with the ionic strength of the supporting electrolyte solution, also showing the sequence of 0.01 < 0.1 < 0.5 < 1 M. The electrochemical parameters for {HA/Mb}n films at different CKCl are listed in Table 1 for comparison. The formal potential (E°′) of the films for Mb FeIII/FeII redox couple, estimated as the midpoint of CV reduction and oxidation peak potentials, showed a positive shift as the KCl concentration was increased from 0.01 to 1.5 M. Similar results were also observed for other electroactive films.44,52-57 The reason for the shift will be discussed later. In addition, the peak-to-peak separation (∆Ep) showed a general decrease trend as the ionic strength increased, suggesting that the higher ionic strength of the testing solution may improve the reversibility of electron transfer for Mb in the films. The Mb in {HA/Mb}n films also showed good electrocatalytic properties toward reduction of H2O2. When H2O2 was added in KCl solution, the great increase of RDV reduction peak current was observed at {HA/Mb}7 film electrodes, accompanied by the decrease or disappearance of the oxidation peak (Figure 3A).

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J. Phys. Chem. B, Vol. 111, No. 8, 2007 1987

Figure 2. Influence of the number of bilayers (n) on (A) the surface concentration of electroactive Mb (Γ*) and (B) the fraction of electroactive Mb (Γ*/Γ) for {HA/Mb}n films. Γ* was measured by CV at 0.2 V s-1 in KCl solutions with CKCl at (a) 0.01, (b) 0.1 (c) 0.5, (d) 1, and (e) 1.5 M. Γ was estimated by QCM.

Figure 3. (A) RDVs at 0.2 V s-1 and 2000 rpm of {HA/Mb}7 films in 0.1 M KCl solutions (a) without H2O2, and (b) with 20 µM H2O2. (B) Amperometric responses of {HA/Mb}7 films assembled on RDEs at -0.1 V and 2000 rpm with injecting 10 µM H2O2 every 40 s in KCl solutions with CKCl at (a) 0.01, (b) 0.1, (c) 0.5, and (d) 1 M.

TABLE 1: Electrochemical Parameters for {HA/Mb}n Films with n ) nmax Estimated by CV at 0.2 V s-1 in KCl Solutions of Different Concentrations CKCl/M nmax 0.01 0.1 0.5 1 1.5

7 9 11 13 13

Epca/V

Epaa/V

∆Ep/mV

E°/V

Γ*max/(mol cm-2)

-0.390 -0.375 -0.310 -0.297 -0.284

-0.292 -0.282 -0.210 -0.208 -0.197

98 93 100 89 87

-0.341 -0.328 -0.260 -0.252 -0.240

1.03 × 10-10 1.56 × 10-10 1.92 × 10-10 2.73 × 10-10 2.56 × 10-10

a E and E stand for cathodic and anodic peak potential, respecpc pa tively.

To further investigate the influence of ionic strength in supporting electrolyte solution, the electrocatalytic behavior of {HA/Mb}7 films toward H2O2 reduction was studied by amperometry at rotating disk electrodes (RDE) with the constant potential at -0.1 V versus SCE (Figure 3B). At a certain concentration of KCl in testing solution, the injection of H2O2 caused a stepped increase in reduction current for the {HA/ Mb}7 films, confirming the electrocatalytic activity of Mb in the films toward H2O2 reduction. The increase of CKCl in substrate solution led to an obvious improvement in testing sensitivity with the same sequence of 0.01 < 0.1 < 0.5 < 1 M. 2. Effect of CKCl in Supporting Electrolyte Solution on Swelling of {HA/Mb}n Films. The salt-induced swelling of polyelectrolyte layer-by-layer films can be considered as a transfer process from an “intrinsically” charge-compensated polyelectrolyte complex, where internal charge is balanced by polymer segments only, to an “extrinsically” charge-compensated form, where salt counterions from external electrolyte solution participate in charge neutralization.18,28 During the swelling process, salt ions and accompanied water molecules would enter the films and make the film mass increase. In this work, the investigation of salt-induced swelling was extended to protein/polyelectrolyte layer-by-layer films, and QCM was

Figure 4. QCM frequency shift (∆F) with immersing time (t) of {HA/ Mb}10 films in KCl solutions with CKCl at (a) 0.1, (b) 0.5, (c) 1, and (d) 1.5 M.

used to measure the swelling of {HA/Mb}n films and study the influence of CKCl in supporting electrolyte solution on the film swelling. The frequency decrease (-∆F) or the corresponding mass increase (∆m) with immersing time (t) in KCl solution was observed for all studied systems at different CKCl (Figure 4). Since the QCM measurement in this work was performed in air for dry films, the influence of incorporated water would be very limited, and the mass increase observed for {HA/Mb}n films after being soaked in KCl solution would most probably be ascribed to the incorporation of salt ions. Moreover, while the water rinsing after the incubation in KCl solution may lead to the desorption of some incorporated salt ions from the films, the desorption amount of salt would be very limited since the rinsing time in water was only 20-30 s, much less than the incubation time in KCl solution (at least 30 min). At the same immersing time (t) in KCl solution, the frequency decrease (-∆F) of the protein films increased with the ionic strength, demonstrating the sequence of 0.1 < 0.5 < 1 < 1.5 M. This implies that the higher concentration of KCl solution would facilitate the swelling process and make more counterions enter into the {HA/Mb}n films. The values of QCM frequency shift

1988 J. Phys. Chem. B, Vol. 111, No. 8, 2007

Figure 5. SEM top views of (A) {HA/Mb}10 films assembled on Au/ MPS/PDDA substrates, (B) {HA/Mb}10 films after being exposed in 0.5 M KCl solution for 10 h, and (C) {HA/Mb}10 films after being exposed in 1.5 M KCl solution for 10 h.

could be converted to the mass increase per unit area (∆m/A) according to the Sauerbrey equation,46 and the maximum values of ∆m/A for different KCl concentration were in the range of 1.4-7.4 µg cm-2, in the same order of magnitude as those in the literature.54d,58 The effect of salt incorporation and film swelling on surface morphology of the {HA/Mb}n films was examined by SEM (Figure 5). While the morphology of dry films observed by SEM would be different from that of wet films in KCl solution, some structure information could still be obtained. After exposure in KCl solution for 10 h, the rough and particle-aggregated morphology of {HA/Mb}10 films assembled on Au/MPS/PDDA substrates became smoother. Compared with the films exposed in 0.5 M KCl, the films exposed in 1.5 M KCl for the same 10 h demonstrated much smoother surface with almost complete disappearance of particle aggregation. All these suggest that saltinduced swelling of {HA/Mb}n films would greatly influence the surface morphology even for their dry form. Similar results have been observed in polyelectrolyte layer-by-layer films by Schlenoff et al.18b and Goh et al.,27 and the smoothing of the films after incubation in salt solution was attributed to the swelling of the films caused by the penetration of salt ions into the films. The incorporation of external counterions in the films

Lu and Hu may free some of the polyelectrolyte segments in their intrinsic complexes, allow the polyelectrolytes to inter-diffuse or intermix, and lead to the smoothing of the surface. 3. Permeability of {HA/Mb}n Films Studied by RDV and EIS. The permeability of {HA/Mb}n films incubated in KCl solution at different CKCl was first investigated by RDV using Fe(CN)63- as the electroactive probe (Figure 6A). At all CKCl in supporting electrolyte solution studied in this work, the RDV reduction limiting current of Fe(CN)63- (Ilim) decreased with increasing the number of bilayers (n) at {HA/Mb}n film electrodes, indicating that thicker films would demonstrate greater hindering effect on the transportation of Fe(CN)63through the films and then limit the reduction of Fe(CN)63- on electrode surface. However, the decrease rate or tendency of Ilim with n for the films at different CKCl was quite different, demonstrating the sequence of 0.01 > 0.1 > 0.5 > 1 M. With the same n, the increase of KCl concentration usually resulted in the increase of Ilim, indicating that the salt-induced swelling of the {HA/Mb}n films leads to the loosening of film structure and enhancement of their permeability toward the Fe(CN)63probe. In this study, the rotating disk electrode was used instead of its stationary counterpart because the RDE could greatly increase the transportation of the probe in solution phase and then amplify the permeation difference of the films at different CKCl. EIS was also performed to study the salt effect on the permeability of {HA/Mb}n films with Fe(CN)63-/4- redox couple as the electroactive probe at its formal potential (0.17 V vs SCE). Figure 6B shows the EIS spectra in the form of Nyquist diagrams for Fe(CN)63-/4- in KCl solutions at different concentration at {HA/Mb}5 film electrodes. At lower ionic strength, a semicircle was obviously observed in the high-frequency domain. The diameter of the semicircle decreased with increasing CKCl in supporting electrolyte solution. At CKCl ) 1 M, the EIS response showed a Warburg line in a very wide frequency range. The diameter of the EIS semicircle usually equals the electron-transfer resistance (Rct) and reflects the electron-transfer rate of the redox probe at the electrode interface.59 In this particular case, Rct mainly reflects the restricted diffusion of the probe through the films, and relates directly to the accessibility of the underlying electrode or the film permeability.60 The Rct values for the systems were estimated according to the Randles equivalent circuit61 and showed an order of 0.01 M (6.1 × 103 Ω) > 0.1 M (2.6 × 103 Ω) > 0.5 M (1.3 × 103 Ω), qualitatively in agreement with the results of RDV. This indicates again that the permeability of the {HA/Mb}n films would be greatly enhanced in KCl solution at higher concentration. 4. Size Effect of Counterions in Supporting Electrolyte Solution on Electrochemical Responses of {HA/Mb}n Films. To maintain the electroneutralization of {HA/Mb}n films, counterions in supporting electrolyte solution have to diffuse into the films and/or the counterions already existing in the films need to diffuse out of the films when electron transfer occurs. For example, during the reduction of Mb heme FeIII in the films, the produced extra negative charges in the {HA/Mb}n films have to be balanced by either uptake of cations from external solution into the films or expulsion of anions from the films into the solution. To investigate which type of counterions, cations or anions, controls the electron-transfer process during the reduction/oxidation of {HA/Mb}n films, a series of supporting electrolytes with the same anionic Br- but differently sized cations, or with the same cationic Na+ but differently sized anions, was selected and CVs were performed. For the study

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J. Phys. Chem. B, Vol. 111, No. 8, 2007 1989

Figure 6. (A) Influence of the number of bilayers (n) on RDV reduction limiting current (Ilim) of 1 mM K3Fe(CN)6 in KCl solutions with CKCl at (a) 0.01, (b) 0.1, (c) 0.5, and (d) 1 M for {HA/Mb}n films on PG/PDDA electrodes at 0.2 V s-1 and 4000 rpm. (B) Electrochemical impedance spectra of 5 mM Fe(CN)64-/3- at 0.17 V in KCl solutions with CKCl at (a) 1, (b) 0.5, (c) 0.1, and (d) 0.01 M for {HA/Mb}5 films at PG/PDDA electrodes.

Figure 7. CVs of {HA/Mb}7 film electrodes at 0.2 V s-1 in 0.1 M solutions of (A) (a) TBABr, (b) TPABr, (c) TEABr, and (d) KBr, and (B) (a) NaCl, (b) NaBF4, and (c) NaTS.

Figure 8. Amperometric responses of {HA/Mb}7 films assembled on RDEs at 2000 rpm with injecting 10 µM H2O2 every 40 s in 0.1 M solutions of (A) (a) TBABr, (b) TPABr, (c) TEABr, (d) and KBr at -0.1 V, and (B) (a) NaCl at -0.1 V, (b) NaBF4 at 0 V, and (c) NaTS at -0.2 V.

of cationic counterions, CV reduction peak currents of {HA/ Mb}7 films showed a sequence of TBA+ < TPA+ < TEA+ < K+, well consistent with the reverse sequence of their sizes (TBA+ > TPA+ > TEA+ > K+) (Figure 7A). This is understandable since the larger-sized cations would be more difficult to diffuse into/out of the films than the smaller ones if the cationic counterions control the kinetics of electron transfer. On the contrary, for anionic counterions, there was no correlation between their size and the CV peak currents of {HA/Mb}7 films. The size of anions is in the order of Cl- < BF4- < TS-, while CV peak currents showed the sequence of Cl- ≈ TS- < BF4(Figure 7B). In addition, the CVs of {HA/Mb}7 films in 0.1 M solutions of KCl, NaCl, and KBr were performed and compared, showing very similar peak heights. Since the size difference between K+ and Na+ is relatively small, its influence on CV peak currents may not be detectable. On the other hand, the similar CV peak heights of the Mb films for Cl- and Brsystems are qualitatively consistent with the results of Figure 7B.

To further explore the influence of the type and size of supporting electrolytes on the electron transfer of {HA/Mb}n films, the electrocatalytic reduction of H2O2 by amperometry at {HA/Mb}7 film modified RDEs was investigated. Compared with the stationary counterpart, the rotating electrodes would greatly accelerate the mass transportation of the counterions and the substrates in solution phase and amplify the effect of differently sized cationic or anionic counterions. Figure 8 shows the amperometric responses of {HA/Mb}7 films at RDEs with injecting the same amount of H2O2 each time in supporting electrolyte solutions with different cations or anions at the same concentration. The catalytic sensitivity demonstrated the sequence of TBA+ < TPA+ < TEA+ < K+ for cations and Cl≈ TS- < BF4- for anions, respectively, in good agreement with CV results (Figure 7). The influence of counterions on electrochemistry of Mb in the films would be complicated, and the exact mechanism is not very clear yet. Nevertheless, the above results support the speculation that it is the cationic instead of anionic counterions in supporting electrolyte solution that

1990 J. Phys. Chem. B, Vol. 111, No. 8, 2007

Lu and Hu

Figure 10. Effect of CKCl in electrolyte solution on surface concentration of electroactive Mb (Γ*) for (a) {SG-TiO2/Mb}7, (b) {HA/Mb}7, (c) {PSS/Mb}7, and (d) {NP-TiO2/Mb}7 films. Γ* was measured by CV at 0.2 V s-1.

Figure 9. UV-vis spectra on quartz slides for (a) dry hemin films, (b) dry Mb films, and {HA/Mb}10 films in KCl solutions with CKCl at (c) 0.1, (d) 0.5, (e) 1, (f) 1.5 M, and (g) dry {HA/Mb}10 films.

diffuse into/out of the {HA/Mb}n films in the Mb reduction/ oxidation process at electrodes. 5. Conformational Study for Swollen {HA/Mb}n Films. The Soret absorption band of heme proteins may provide information on conformational integrity of the proteins, especially in their heme region.62 UV-vis spectroscopy was used in this work to investigate the possible denaturation of Mb in {HA/Mb}n films under different conditions. Both dry Mb and {HA/Mb}10 films on quartz slides showed the Soret band at 410 nm (Figure 9, curves b and g), suggesting that Mb in dry {HA/Mb}10 films retains its near-native structure. In contrast, the spectrum of dry hemin films displayed the Soret band at the wavelength less than 400 nm with a much broader shape (Figure 9, curve a). To investigate the influence of ionic strength of exposure solution on Mb conformation, the UV-vis spectra of {HA/Mb}10 films immersed in KCl solutions at different concentration were measured. The {HA/Mb}10 films in KCl solutions at CKCl e 1 M showed the Soret band at 410 nm (Figure 9, curves c-e), the same as that of pure Mb films, indicating that Mb in {HA/Mb}10 films essentially retains its native conformation even exposed in the electrolyte solutions at high concentration. 6. Comparison Study. To further understand the essence of salt-induced swelling of {HA/Mb}n films, other kinds of Mb layer-by-layer films, including {PSS/Mb}n, {NP-TiO2/Mb}n, and {SG-TiO2/Mb}n films, were assembled, and the effect of CKCl in supporting electrolyte solution on CV responses of these Mb films was compared with that of {HA/Mb}n films. All these Mb films were reported previously to show good and quasireversible CV responses for Mb heme FeIII/FeII redox couple.37a,45,63 In the present work, the influence of ionic strength in supporting electrolyte solution on the surface concentration of electroactive Mb (Γ*) measured by CV for the four kinds of Mb layer-bylayer films was compared (Figure 10). CKCl had no obvious influence on Γ* for {SG-TiO2/Mb}7 and {NP-TiO2/Mb}7 films. This is understandable since both TiO2 sol-gel and nanoparticles have a rigid structure with negligible swelling capability. However, {PSS/Mb}7 films seemed sensitive to salt concentration in supporting electrolyte solution. Γ* increased with CKCl up to 1 M, and then showed a decrease trend at higher CKCl (1.5 M). Qualitatively, the profile of Γ* vs CKCl for {PSS/ Mb}7 films was similar to that of {HA/Mb}7 films, indicating that the effect of ionic strength on the swelling of Mb layer-

by-layer films assembled with “soft” and flexible polyelectrolytes would be similar. However, the influence of CKCl on Γ* for {HA/Mb}7 films seemed more pronounced than that for {PSS/Mb}7 films. The {HA/Mb}7 films appear to have a higher swelling capacity than the {PSS/Mb}7 films. Discussion The salt concentration (CKCl) in supporting electrolyte solution has a significant influence on the electrochemical and electrocatalytic behaviors of {HA/Mb}n films (Figures 1-3, Table 1). Higher ionic strength not only increases the maximum surface concentration of electroactive Mb (Γ*max) and the corresponding number of electroactive protein bilayers (nmax) for {HA/Mb}n films, but also enhances the sensitivity of detecting H2O2 in electrocatalysis, all showing the same sequence of 0.01 < 0.1 < 0.5 < 1 M for CKCl. Moreover, the increase of CKCl decreases the peak-to-peak separation (∆Ep) and improves the reversibility of electron transfer between Mb in the films and underlying electrodes. The reason for the influence is explored in detail in the present work. The higher concentration of KCl in supporting electrolyte solution makes the {HA/Mb}n films swell to a greater extent (Figure 4). The incorporation of more amounts of counterions and accompanied water from solution into the films would weaken the ionic bonds between oppositely charged HA and Mb that hold the layers together, make the HA chains take a more coiling conformation, and result in a looser structure of the films. In the study of polyelectrolyte layer-by-layer films by Schlenoff and co-workers,18,28,33 similar salt-induced swelling was observed and was attributed to the transition from the totally intrinsic to the partially extrinsic charge compensation as the ionic strength increased. The swollen {HA/Mb}n films demonstrate better permeability toward the electroactive probe ions (Figure 6), suggesting that higher CKCl in testing solution would be beneficial to the ion transportation through the films. The SEM top views of {HA/Mb}10 films demonstrate that after incubation in KCl solution at high concentration, the surface of dry films becomes smoother (Figure 5). These results are in agreement with those in the literature18b,27 and should not be contradictory to the better permeability of the films with salt incubation. While one may expect that the ions would diffuse more easily within rougher films, the surface roughness and morphology of the “wet” and swollen films may not be maintained after the films become “dry” and contracted. Particularly, for the {HA/Mb}n films assembled with the “soft” and flexible polyelectrolytes rather than the rigid inorganic nanoparticles or sol-gel, the change of surface roughness after vacuum-drying is more probable.

Hyaluronic Acid/Myoglobin Multilayer Films For the {HA/Mb}n layer-by-layer films, the electroactive Mb extends to 7-13 bilayers, depending on CKCl in supporting electrolyte solution (Table 1), which approximately corresponds to the dry thickness of 90-220 nm according to the QCM results. Considering that the “wet” films after swelling in solution should be thicker than the dry ones and the interlayer mixing or penetrating would happen in the assembly, which would decrease the estimated thickness, the above estimation is very rough. Nevertheless, the real thickness of electroactive layers must be at least in the order of a few tens of nanometers. It is almost impossible for the macromolecular Mb to physically diffuse through such a long distance in the film phase, reach the electrode surface, and then exchange electron with underlying electrodes in the time scale of CV scans. It is thus most probable that Mb in the films takes the electron-hopping or selfexchange mechanism to transfer electron with electrodes.64 That is, the sequential and directional electron exchange happens between neighboring Mb molecules immobilized in the films, extending from electrode surface to the interior of the films. Electron hopping between immobilized redox sites has to be accompanied by the transportation of electrochemically inert counterions so as to compensate for the charge alteration caused by electron transfer and maintain the electroneutralization of the whole films.64,65 Thus, when the films become thick with a large n value, the electron transfer of Mb in {HA/Mb}n films would be controlled by the transport of counterions into and/or out of the protein films. The higher supporting electrolyte concentration would induce the swelling of {HA/Mb}n films to a greater extent and enhance the permeability of the films, thus facilitating the transportation of counterions through the films. It is therefore understandable that higher CKCl would be favorable to the electron transfer of Mb in {HA/Mb}n films with underlying electrodes. The influence of supporting electrolytes on electron exchange has also been reported in other types of electroactive films.43,44,52-57 In addition, when the ionic strength of supporting electrolyte solution increases, the corresponding higher permeability of the films may allow H2O2 substrate to move into the films and react with Mb in the films more easily, also beneficial to the catalytic performance of the {HA/Mb}n films toward H2O2 reduction. For the {HA/Mb}n films with a given n value, the total amount of Mb in the films (Γ) is constant or fixed. On the contrary, the amount of electroactive Mb (Γ*) is smaller than Γ when n g 2 (Figure 2B). In addition, while the film growth continues with n according to QCM,40 the electroactivity of Mb can only extend to a very limited number of bilayers (Figure 2A), indicating that only those Mb molecules located in the inner bilayers closest to the electrode surface can demonstrate their electroactivity. Similar results have been observed for other protein layer-by-layer films,37,45 and are ascribed to the transport limitation of counterions through the films when n becomes large. Since the permeability of {HA/Mb}n films becomes poorer when the number of bilayers (n) or the film thickness increases (Figure 6A), the counterion transport becomes more difficult in the thicker films, therefore limiting the extension of electroactive bilayers. Thus, the favorable influence of higher CKCl in supporting electrolyte solution on electrochemistry of {HA/Mb}n films can be specifically attributed to the increase of the fraction of electroactive Mb in the films and the number of electroactive bilayers by enhancing the mobility of counterions within the swollen films. The size of cationic counterions with the same Br- anion in supporting electrolyte solution systematically influences the CV peak currents of {HA/Mb}n films and their electrocatalytic

J. Phys. Chem. B, Vol. 111, No. 8, 2007 1991

Figure 11. Dependence of formal potential (E°′) on the logarithm of KCl concentration in supporting electrolyte solutions for {HA/Mb}n films. Data are from Table 1.

behaviors toward H2O2 reduction (Figures 7A and 8A). At the same concentration, larger-sized cations always demonstrate lower CV responses and catalytic sensitivity compared with the smaller-sized ones. In contrast, the size of anionic counterions does not show any pronounced effect on the electrochemistry and electrocatalysis of the Mb films (Figures 7B and 8B). These observations imply that the electron transfer of Mb at the {HA/ Mb}n film electrodes is most probably accompanied by the incorporation/release of cationic counterions into/out of the films, which can be expressed as

MbFeIII(film) + e- + M+(s) S MbFeII M+(film)

(2)

where M+ represents the cationic counterion and the subscripts “film” and “s” stand for the film and solution phases, respectively. This speculation is further supported by the positive shift of the formal potential (E°′) of the Mb films with the ionic strength in supporting electrolyte solutions (Table 1). According to the Nernst equation for a reversible electron transfer in film phase,52,55,66 if the uptake/release of cationic counterions into/ out of the films controls the electrode process (eq 2), the linear positive shift of E°′ with log CM would be observed with a slope of 58 mV/log CM at 20 °C, where CM is the concentration of M+ cation. On the other hand, if the loading/unloading of anionic counterions into/out of the films becomes the limiting factor, the negative shift of E°′ with log CL would be observed, where CL is the concentration of L- anion.44,52,54,67,68 For {HA/ Mb}n films, the E°′- log CKCl plot demonstrates a roughly straight line with a positive slope of 50 mV/log CM (Figure 11), reasonably close to the theoretical value of 58 mV/log CM at 20 °C.52 This indicates that the uptake/release of cationic rather than anionic counterions controls the electron transfer of Mb in {HA/Mb}n films. Similar phenomenon has also been observed for other electroactive films,55-57 such as C60/Pd films,55 in which the electrochemical reaction was governed by doping/undoping of cationic supporting electrolyte, while was not affected by the anions. However, further comparative experiments demonstrate that not all Mb layer-by-layer films are sensitive to the change of supporting electrolyte concentrations in their electrochemical responses. Those Mb multilayer films assembled with TiO2 solgel and nanoparticles are difficult to swell in salt solution and show the CV responses nearly independent of salt concentration (Figure 10). This is mainly attributed to the “hard” and rigid structure of inorganic titanium oxide that seldom swells in electrolyte solution. Only those Mb films assembled with “soft” and flexible polyelectrolytes, such as HA or PSS, can demonstrate the salt-induced swelling and corresponding increase of Γ* with higher CKCl (Figure 10).

1992 J. Phys. Chem. B, Vol. 111, No. 8, 2007 While Γ* values for both {HA/Mb}n and {PSS/Mb}n films increase with CKCl in the range of 0.01-1 M, the {HA/Mb}n films seem more sensitive to CKCl than {PSS/Mb}n films (Figure 10). This indicates that at the same CKCl, {HA/Mb}n films may swell to a more extent than {PSS/Mb}n films. As a weak polyelectrolyte, HA has rather low charge density since only one residue from the two monomer units has a carboxylic acid group that can dissociate at pH 5.0, while PSS is a strong polyelectrolyte with one negative sulfonate group for each monomer unit. Thus, compared with {PSS/Mb}n films, the {HA/ Mb}n films with weaker ionic cross-linking between HA and Mb may incorporate counterions more easily when incubated in salt solution. Bruening and co-workers have reported the correlation between the swelling capability of polyelectrolyte multilayer films and the charge density of the component polyelectrolytes,22,23 showing that lower charge density of polyions leads to fewer ionic cross-links and a higher swelling extent in their layer-by-layer films. Salt-induced swelling of {HA/Mb}n films may become an effective way to enhance the electroactivity of immobilized protein. However, the concentration of salt in supporting electrolyte solution should not be too high, since extremely high ionic strength may lead to the deconstruction of the whole films.18b,c For the {HA/Mb}n films, when CKCl ) 1.5 M, while the swelling can still be observed (Figure 4), the Γ* value of Mb are smaller than that at CKCl ) 1.0 M (Table 1, Figures 2A and 10b). This may be due to partial removal or dissociation of Mb from the multilayer films. In addition, the protein may denature in salt solution at the higher concentration, which should also be taken into account. Conclusion The increase of salt concentration in supporting electrolyte solution significantly enhances the amount of electroactive Mb in {HA/Mb}n layer-by-layer films on PG electrodes. This interesting result is explained to be related to the swelling of the films induced by salt in the testing solution. The higher concentration of salt in exposure solution may weaken the electrostatic interactions between oppositely charged Mb and HA in the films by “extrinsic” compensation from external ions, making the films swell to a greater extent by incorporating more amounts of counterions and water. The salt-induced swelling of {HA/Mb}n films leads to better film permeability and easier transportation of counterions within the films. For electron hopping mechanism, in order to maintain the electroneutralization of the films during electron exchange, the diffusion of counterions into and/or out of the protein films becomes the key factor that controls the electron transfer between the protein in films and the underlying electrodes. In this particular case, cationic instead of anionic counterions are incorporated/released into/out of the {HA/Mb}n films in the electrode process. The better swelling capability of {HA/Mb}n films than {PSS/Mb}n films may be related to the lower charge density and unique structure of HA, which makes the {HA/Mb}n films demonstrate higher electrochemical responses than {PSS/Mb}n films. This study combines the salt-induced swelling of protein layer-bylayer films and the direct electrochemistry of redox protein, and may provide a general approach to control the electrochemical and electrocatalytic activity of the protein in the films by tailoring the concentration of salt in supporting electrolyte solution and the type of polyions for assembly. Acknowledgment. The financial support from the National Natural Science Foundation of China (NSFC 20475008 and 20275006) is acknowledged.

Lu and Hu References and Notes (1) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831-835. (2) Decher, G. Science 1997, 277, 1232-1237. (3) Tanchak, O. M.; Barrett, C. J. Chem. Mater. 2004, 16, 2734-2739. (4) Losche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893-8906. (5) Kugler, R.; Schmitt, J.; Knoll, W. Macromol. Chem. Phys. 2002, 203, 413-419. (6) Wong, J. E.; Rehfeldt, F.; Haenni, P.; Tanaka, M.; Klitzing, R. V. Macromolecules 2004, 37, 7285-7289. (7) Carriere, D.; Krastev, R.; Schonhoff, M. Langmuir 2004, 20, 11465-11472. (8) Dubas, S. T.; Schlenoff, J. B. Macromolecules 1999, 32, 81538160. (9) (a) Burke, S. E.; Barrett, C. J. Biomacromolecules 2003, 4, 17731783. (b) Burke, S. E.; Barrett, C. J. Biomacromolecules 2005, 6, 14191428. (10) Richert, L.; Arntz, Y.; Schaaf, P.; Voegel, J.-C.; Picart, C. Surf. Sci. 2004, 570, 13-29. (11) Blomberg, E.; Poptoshev, E.; Caruso, F. Langmuir 2006, 22, 41534157. (12) Burke, S. E.; Barrett, C. J. Macromolecules 2004, 37, 5375-5384. (13) Kim, B.-S.; Vinogradova, O. I. J. Phys. Chem. B 2004, 108, 81618165. (14) (a) Mendelsohn, J. D.; Barrett, C. J.; Chan, V. V.; Pal, A. J.; Mayes, A. M.; Rubner, M. F. Langmuir 2000, 16, 5017-5023. (b) Hiller, J.; Rubner, M. F. Macromolecules 2003, 36, 4078-4083. (c) Itano, K.; Choi, J.; Rubner, M. F. Macromolecules 2005, 38, 3450-3460. (d) Lee, D.; Nolte, A. J.; Kunz, A. L.; Rubner, M. F.; Cohen, R. E. J. Am. Chem. Soc. 2006, 128, 8521-8529. (15) (a) Mauser, T.; Dejugnat, C.; Sukhorukov, G. B. Macromol. Rapid Commun. 2004, 25, 1781-1785. (b) Dejugnat, C.; Sukhorukov, G. B. Langmuir 2004, 20, 7265-7269. (16) Sukhorukov, G. B.; Schmitt, J.; Decher, G. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 948-953. (17) Harris, J. J.; Bruening, M. L. Langmuir 2000, 16, 2006-2013. (18) (a) Farhat, T. R.; Schlenoff, J. B. Langmuir 2001, 17, 1184-1192. (b) Dubas, S. T.; Schlenoff, J. B. Langmuir 2001, 17, 7725-7727. (c) Dubas, S. T.; Schlenoff, J. B. Macromolecules 2001, 34, 3736-3740. (19) Gao, C.; Leporatti, S.; Moya, S.; Donath, E.; Mohwald, H. Chem. Eur. J. 2003, 9, 915-920. (20) Wang, L.; Schonhoff, M.; Mohwald, H. J. Phys. Chem. B 2004, 108, 4767-4774. (21) Kim, B.-S.; Lebedeva, O. V.; Koynov, K.; Gong, H.; Glasser, G.; Lieberwith, I.; Vinogradova, O. I. Macromolecules 2005, 38, 5214-5222. (22) Miller, M. D.; Bruening, M. L. Chem. Mater. 2005, 17, 53755381. (23) (a) Stanton, B. W.; Harris, J. J.; Miller, M. D.; Bruening, M. L. Langmuir 2003, 19, 7038-7042. (b) Miller, M. D.; Bruening, M. L. Langmuir 2004, 20, 11545-11551. (24) Krasemann, L.; Tieke, B. Mater. Sci. Eng., C 1999, C8-C9., 513518. (25) Sun, Q.; Tong, Z.; Wang, C.; Ren, B.; Liu, X.; Zeng, F. Polymer 2005, 46, 4958-4966. (26) Fery, A.; Scholer, B.; Cassagneau, T.; Caruso, F. Langmuir 2001, 17, 3779-3783. (27) McAloney, R. A.; Dudnik, V.; Goh, M. C. Langmuir 2003, 19, 3947-3952. (28) Jomaa, H. W.; Schlenoff, J. B. Macromolecules 2005, 38, 84738480. (29) Lulevich, V. V.; Vinogradova, O. I. Langmuir 2004, 20, 28742878. (30) Lebedeva, O. V.; Kim, B. S.; Vasilev, K.; Vinogradova, O. I. J. Colloid Interface Sci. 2005, 284, 455-462. (31) Ibarz, G.; Dahne, L.; Donath, E.; Mohwald, H. AdV. Mater. 2001, 13, 1324-1327. (32) Antipov, A. A.; Sukhorukov, G. B.; Mohwald, H. Langmuir 2003, 19, 2444-2448. (33) (a) Farhat, T. R.; Schlenoff, J. B. J. Am. Chem. Soc. 2003, 125, 4627-4636. (b) Jaber, J. A.; Schlenoff, J. B. J. Am. Chem. Soc. 2006, 128, 2940-2947. (34) (a) Schlenoff, J. B.; Ly, H.; Li, M. J. Am. Chem. Soc. 1998, 120, 7626-7634. (b) Schlenoff, J. B.; Dubas, S. T. Macromolecules 2001, 34, 592-598. (35) (a) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwald, H., Eds.; Marcel Dekker: New York, 2000; pp 125-167. (b) Lvov, Y. In Nanostructured Materials, Micelles and Colloids; Nalwa, R. W., Ed.; Handbook of Surfaces and Interfaces of Materials 3; Academic Press: San Diego, 2001; pp 170-189.

Hyaluronic Acid/Myoglobin Multilayer Films (36) (a) Lvov, Y.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (b) Lvov, Y.; Munge, B.; Giraldo, O.; Ichinose, I.; Suib, S.; Rusling, J. F. Langmuir 2000, 16, 8850-8857. (37) (a) Ma, H.; Hu, N.; Rusling, J. F. Langmuir 2000, 16, 4969-4975. (b) He, P.; Hu, N.; Zhou, G. Biomacromolecules 2002, 3, 139-146. (c) Zhou, Y.; Li, Z.; Hu, N.; Zeng, Y.; Rusling, J. F. Langmuir 2002, 18, 85738579. (d) He, P.; Hu, N. J. Phys. Chem. B 2004, 108, 13144-13152. (38) Chapin, M. F.; Bucke, C. Enzyme Technology; Cambridge University Press: Cambridge, U.K., 1990. (39) Armstrong, F. A.; Wilson, G. S. Electrochim. Acta 2000, 45, 26232645. (40) Liu, H.; Hu, N. J. Phys. Chem. B 2006, 110, 11494-11502. (41) Lapcik, L. Jr.; Lapcik, L.; De Smedit, S.; Demeester, J.; Chabrecek, P. Chem. ReV. 1998, 98, 2663-2684. (42) Mathe, G.; Albersdorfer, A.; Neumaier, K. R.; Sackmann, E. Langmuir 1999, 15, 8726-8735. (43) Nassar, A.-E. F.; Rusling, J. F.; Kumosinski, T. F. Biophys. Chem. 1997, 67, 107-116. (44) Battaglini, F.; Calvo, E. J.; Danilowicz, C.; Wolosiuk, A. Anal. Chem. 1999, 71, 1062-1067. (45) Lu, H.; Yang, J.; Rusling, J. F.; Hu, N. Electroanalysis 2006, 18, 379-390. (46) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (47) Brandrup, J.; Immergut, E. Polymer Handbook; Wiley: New York, 1975; p 5. (48) Creighton, T. E. Protein Structure, A Practical Approach; IRL Press: New York, 1990; p 43. (49) Bellelli, A.; Antonini, G.; Brunori, M.; Springer, B. A.; Sligar, S. J. J. Biol. Chem. 1990, 265, 18898-18901. (50) (a) Rusling, J. F.; Zhang, Z. In Biomolecules, Biointerfaces, and Applications; Nalwa, R. W., Ed.; Handbook of Surfaces and Interfaces of Materials 5; Academic Press: San Diego, 2001; pp 33-71. (b) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369. (51) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 191-368. (52) Levi, M. D.; Lopez, C.; Vieil, E.; Vorotyntse, M. A. Electrochim. Acta 1997, 42, 757-769.

J. Phys. Chem. B, Vol. 111, No. 8, 2007 1993 (53) Calvo, E. J.; Wolosiuk, A. J. Am. Chem. Soc. 2002, 124, 84908497. (54) (a) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708-2716. (b) Calvo, E. J.; Etchenique, R. J. Phys. Chem. B 1999, 103, 8944-8950. (c) Calvo, E. J.; Forzani, E.; Otero, M. J. Electroanal. Chem. 2002, 538-539, 231-241. (d) Forzani, E. S.; Perez, M. A.; Teijelo, M .L.; Calvo, E. J. Langmuir 2002, 18, 9867-9873. (55) Winkler, K.; de Bettencourt-Dias, A.; Balch, A. L. Chem. Mater. 1999, 11, 2265-2273. (56) Wang, X.; Wang, L.; Wang, J.; Chen, T. J. Phys. Chem. B 2004, 108, 5627-5633. (57) Chasse, T. L.; Smith, J. C.; Carroll, R. L.; Gorman, C. B. Langmuir 2004, 20, 3501-3503. (58) Barbero, C.; Calvo, E. J.; Etchenique, R.; Morales, G. M.; Otero, M. Electrochim. Acta 2000, 45, 3895-3906. (59) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913-947. (60) (a) Sabatani, E.; Rubinstein, I. J. Phys. Chem. 1987, 91, 66636669. (b) Sabatani, E.; Cohen-Boulakia, J.; Bruening, M.; Rubinstein, I. Langmuir 1993, 9, 2974-2981. (61) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; Wiley: New York, 2001. (62) (a) Theorell, H.; Ehrenberg, A. Acta Chem. Scand. 1951, 5, 823848. (b) George, P.; Hanania, G. I. H. Biochem. J. 1953, 55, 236-243. (63) Lu, H.; Hu, N. Electroanalysis 2006, 18, 1511-1522. (64) Majda, M. In Molecular Design of Electrode Surfaces; Murray, R. W., Ed.; Wiley: New York, 1992; pp 159-206. (65) (a) Saveant, J. M. J. Phys. Chem. 1988, 92, 1011-1013. (b) Saveant, J. M. J. Phys. Chem. 1988, 92, 4526-4532. (66) Naegeli, R.; Redepenning, J.; Anson, F. C. J. Phys. Chem. 1986, 90, 6227-6232. (67) Doblhofer, K.; Vorotynstev, M. In ElectroactiVe Polymer Electrochemistry. Part 1. Fundamentals; Lyons, M. E. G., Ed.; Plenum Press: New York, 1994; Chapter 3. (68) Forster, R. J.; Vos, J. G. Langmuir 1994, 10, 4330-4338.