Cholate Adsorption Behavior at Carbon Electrode Interface and Its Promotional Effect in Laccase Direct Bioelectrocatalysis

Fast electron transfer between laccase (Lac) and single-walled carbon nanotubes (SWCNTs) can be achieved at a cholate-modified SWCNT interface. Furthermore, the catalytic reduction of O2 starts at a high potential, close to the equilibrium redox potential of the O2/H2O couple. A sodium cholate (SC)-modified electrode interface provides suitable conditions for Lac direct bioelectrocatalysis. In the present study, the SC promotional effect in Lac direct bioelectrocatalysis was investigated using various types of electrode materials. The fully hydrophilic surface of indium tin oxide and an Au electrode surface did not show a SC promotional effect, because SC did not bind to these surfaces. A carbon surface with a large number of defects was unsuitable for SC binding because of hydrophilic functional groups at the defect sites. Carbon surfaces with few defects, for example, basal-plane highly oriented pyrolytic graphite (HOPG), gave a SC promotional effect.


Introduction
Steroid biosurfactants such as cholate, deoxycholate, taurocholate, and deoxycholate are large, rigid, planar, hydrophobic moieties that have a steroid nucleus with two or three hydroxyl groups.These biosurfactants have been used as solubilizers for nucleotide-and membrane-binding proteins [1].Furthermore, these biosurfactants, especially sodium cholate (SC, Figure 1), are outstanding dispersing agents for nanocarbon, especially single-walled carbon nanotubes (SWCNTs) [2][3][4].Direct electron transfer reactions between proteins (enzymes) and electrodes have been extensively studied from the viewpoints of both, understanding the fundamental features and for applications as biosensors and biofuel cell [5][6][7][8][9][10][11].Typically, enzymatic biofuel cell has been focused because of their possibility to harvest energy out of non-toxic and non-combustible compounds like sugar opened the way for glucose biofuel cells [5][6][7][8][9][10][11][12][13][14][15][16].However, the transfer of electrons involved in the redox reactions to an external circuit is a challenging theme.This difficulty is due to the redox Direct electron transfer reactions between proteins (enzymes) and electrodes have been extensively studied from the viewpoints of both, understanding the fundamental features and for applications as biosensors and biofuel cell [5][6][7][8][9][10][11].Typically, enzymatic biofuel cell has been focused because of their possibility to harvest energy out of non-toxic and non-combustible compounds like sugar opened the way for glucose biofuel cells [5][6][7][8][9][10][11][12][13][14][15][16].However, the transfer of electrons involved in the redox reactions to an external circuit is a challenging theme.This difficulty is due to the redox center of the enzyme
Plastic formed carbon (PFC) plate (electrode area: 0.28 cm 2 , Mitsubishi Pencil Co., Ltd., Tokyo, Japan) was obtained from the Tsukuba Materials Information Laboratory, Ltd. (Tsukuba, Japan).Prior to use, the surface was polished with 0.05 mm alumina slurry, followed by sonication in Milli-Q water for 10 min.HOPG plate (electrode area: 0.28 cm 2 ) was obtained from Veeco Instruments, New York, NY, USA.Prior to use, the surface of the basal plane was peeled off with adhesive tape to expose a fresh basal plane.A tin-doped indium oxide (ITO) electrode (electrode area: 0.25 cm 2 , tin-doped indium oxide thickness: ca.30 nm), which was used as a metallic oxide, was obtained from the Kinoene Optics Co., Tokyo, Japan.The ITO electrode surface was cleaned with a UV-ozone system (OC-2503 model, Eye Graphics Co., Ltd., Tokyo, Japan) for 3 min to remove organic contaminants [30].An Au disk electrode (electrode area: 0.20 cm 2 ) was obtained from the BAS Co., Ltd.(Tokyo, Japan).Prior to use, the disk surface was polished with 0.05 µm alumina slurry, followed by sonication in water.
The electrode with a fresh surface was immersed in 0.2% (w/v) SC aqueous solution for 30 min.For Lac modification, the electrode was immersed in 0.1 mol•dm −3 acetate buffer solution (pH 5) containing 5 µmol•dm −3 Lac at 8 • C for 30 min.Finally, each electrode surface was gently rinsed with 0.1 mol•dm −3 acetate buffer solution.
Voltammetric measurements were performed with an electrochemical analyzer (ALS/CHI model 600A) with a conventional three-electrode cell.The reference electrode was Ag|AgCl|saturated KCl (+199 mV vs. NHE) and a Pt plate was used as the counter electrode.All potentials are reported with respect to Ag|AgCl|saturated KCl at 25 • C. For the Lac bioelectrocatalysis experiments, 0.1 mol dm −3 acetate buffer solution (pH 5) was used as the electrolyte; it was purged with high-purity argon before measurements were performed.A steady-state sigmoidal shape (with a plateau current) in the voltammograms of Lac adsorbed on the electrodes was obtained by stirring the buffer solution with a magnetic stirrer.The cell temperature was controlled at 25 • C by a thermostated incubator.The redox waves of K 3 [Fe(CN) 6 ] and [Ru(NH 3 ) 6 ]Cl 3 were examined in 0.1 mol dm −3 acetate buffer solution.
Raman spectroscopy was performed with a HORIBA LabRAM HR-800 instrument (HORIBA Jobin Yvon, Les Ulis, France) with 514 nm (2.41 eV) laser excitation.All images were captured with a digital charge-coupled device camera.Wavenumbers were calibrated against the 520 cm −1 emission of the silica slides used for analysis.A 50× long lens was used to focus the laser at 2 µm, with a laser power of 0.2 mW.

Dependence of SC Effect on Electrode Type
To investigate the SC promotion effect against the different type of planar electrode, we used two types of carbon electrodes of HOPG and PFC, ITO, and Au electrodes.Figure 2 shows cyclic voltammograms for HOPG, PFC, ITO, and Au electrodes after immersion in SC solution and then Lac solution.A catalytic O 2 reduction current was observed at the SC-treated HOPG electrode from +0.65 V; this arises from direct electron transfer between immobilized Lac and the HOPG electrode.However, the O 2 reduction potential from +0.65 was lower potential than the previously obtained result at SC-modified SWCNT interface.This direct bioelectrocatalytic current was not observed at the bare HOPG electrode (i.e., HOPG without SC treatment).This clearly indicates that SC plays an important role in promoting direct electron transfer from Lac at the HOPG interface.In contrast, direct bioelectrocatalytic currents were not observed at PFC, ITO, and Au electrodes, although these electrode surfaces were immersed in SC solution.It should be noted that the PFC did not give the SC promotional effect although it is a carbon material.Possible reasons for these results are as follows.The SC molecules were not adsorbed on the PFC, ITO, and Au electrode interfaces.Alternatively, the SC molecules were adsorbed on the electrode surfaces, but the direct electron transfer reaction with Lac did not occur.

SC Adsorption Behavior
The dependence of the SC promotional effect on the electrode type was clarified by investigating SC adsorption phenomena, with redox complexes [Fe(CN) 6 ] 3− and [Ru(NH 3 ) 6 ] 3+ as probes.The redox reactions of [Fe(CN) 6 ] 3− and [Ru(NH 3 ) 6 ] 3+ are strongly affected by electrode interface conditions [32].It was expected that the redox behavior would change when SC was adsorbed on the interface.Figure 3 shows the changes in the redox behaviors of [Fe(CN) 6 ] 3− and [Ru(NH 3 ) 6 ] 3+ at the HOPG electrode before and after surface modification with SC.As described above, the HOPG electrode gave a SC promotional effect in direct bioelectrocatalysis.This clearly indicates that SC was adsorbed on its surface.Figure 3a shows that the redox reaction of [Fe(CN) 6 ] 3− was inhibited at the SC-modified HOPG interface, but that of [Ru(NH 3 ) 6 ] 3+ was not affected.The results obtained with [Fe(CN) 6 ] 3− and [Ru(NH 3 ) 6 ] 3+ differed because the SC molecule has a negatively charged carboxylic acid group bound to the steroid framework; therefore, electrostatic repulsion should occur between [Fe(CN) 6 ] 3− and the SC-modified HOPG interface, but not at the interface with [Ru(NH 3 ) 6 ] 3+ .These results show that [Fe(CN) 6 ] 3− is a suitable indicator for monitoring SC adsorption.The redox reactions of [Fe(CN) 6 ] 3− at the PFC, ITO, and Au electrodes were also investigated.No change in the redox behavior was observed at these electrodes, which indicates that SC molecules did not adsorb on the PFC, ITO, and Au interfaces.This suggests that the fully hydrophilic surfaces of the ITO and Au interfaces did not interact with the hydrophobic steroid framework.Moreover, electrostatic interactions between the negatively charged carboxylic acid bound to the steroid framework and the electrode interface is not expected because the ITO and Au interfaces are slightly negatively charged.
To investigate the SC promotion effect against the different type of planar electrode, we used two types of carbon electrodes of HOPG and PFC, ITO, and Au electrodes.Figure 2 shows cyclic voltammograms for HOPG, PFC, ITO, and Au electrodes after immersion in SC solution and then Lac solution.A catalytic O2 reduction current was observed at the SC-treated HOPG electrode from +0.65 V; this arises from direct electron transfer between immobilized Lac and the HOPG electrode.However, the O2 reduction potential from +0.65 was lower potential than the previously obtained result at SC-modified SWCNT interface.This direct bioelectrocatalytic current was not observed at the bare HOPG electrode (i.e., HOPG without SC treatment).This clearly indicates that SC plays an important role in promoting direct electron transfer from Lac at the HOPG interface.In contrast, direct bioelectrocatalytic currents were not observed at PFC, ITO, and Au electrodes, although these electrode surfaces were immersed in SC solution.It should be noted that the PFC did not give the SC promotional effect although it is a carbon material.Possible reasons for these results are as follows.The SC molecules were not adsorbed on the PFC, ITO, and Au electrode interfaces.Alternatively, the SC molecules were adsorbed on the electrode surfaces, but the direct electron transfer reaction with Lac did not occur.

SC Adsorption Behavior
The dependence of the SC promotional effect on the electrode type was clarified by investigating SC adsorption phenomena, with redox complexes [Fe(CN)6] 3− and [Ru(NH3)6] 3+ as probes.The redox reactions of [Fe(CN)6] 3− and [Ru(NH3)6] 3+ are strongly affected by electrode interface conditions [32].It was expected that the redox behavior would change when SC was adsorbed on the interface.Figure 3 shows the changes in the redox behaviors of [Fe(CN)6] 3− and [Ru(NH3)6] 3+ at the HOPG electrode before and after surface modification with SC.As described above, the HOPG electrode gave a SC promotional effect in direct bioelectrocatalysis.This clearly indicates that SC was adsorbed on its surface.Figure 3a shows that the redox reaction of [Fe(CN)6] 3− was inhibited at the SC-modified HOPG interface, but that of [Ru(NH3)6] 3+ was not affected.The results obtained with [Fe(CN)6] 3− and [Ru(NH3)6] 3+ differed because the SC molecule has a negatively charged carboxylic acid group bound to the steroid framework; therefore, electrostatic repulsion should occur between [Fe(CN)6] 3− and the SC-modified HOPG interface, but not at the interface with [Ru(NH3)6] 3+ .These results show that [Fe(CN)6] 3− is a suitable indicator for monitoring SC adsorption.The redox reactions of [Fe(CN)6] 3− at the PFC, ITO, and Au electrodes were also investigated.No change in the redox behavior was observed at these electrodes, which indicates that SC molecules did not adsorb on the PFC, ITO, and Au interfaces.This suggests that the fully hydrophilic surfaces of the ITO and Au interfaces did not interact with the hydrophobic steroid framework.Moreover, electrostatic interactions between the negatively charged carboxylic acid bound to the steroid framework and the electrode interface is not expected because the ITO and Au interfaces are slightly negatively charged.The PFC electrode material is carbon, which is the same materials as HOPG.Carbon defects are important to decide its surface characteristics.Figure 4 shows Raman spectra of HOPG and PFC.Raman spectroscopy can be used to investigate sp 2 -hybridized structures in carbon materials, and yields information on defects and the crystalline structure [33,34].The prominent features in the Raman spectra of HOPG and PFC are the G-band at ca. 1585 cm −1 and the D-band at ca. 1350 cm −1  [33,34].The G-band is a doubly-degenerate phonon Raman active mode in sp 2 -structured carbon networks, whereas the D-band is localized where the lattice structure is not perfect, mostly at the edges, and at defects in the sp 2 -hybridized carbon structure.A D-band is not observed for HOPG.This indicates that the sp 2 -hybridized carbon structure HOPG is highly crystalline, with almost no defects at the detection level of this method.In contrast, the G/D intensity ratio for PFC was calculated to be ca.0.65.The intensity of the D-band was higher than that of the G-band.Usually, defects in carbon are caused by cleavage of carbon sp 2 -bonds and oxidation by O2 at cleavage sites.Hydrophilic functional groups such as -C=O, -COOH, -C-OH, and -C-O-C-are present at defect sites [35][36][37].The Raman results indicate that the PFC surface is more hydrophilic than HOPG.In The PFC electrode material is carbon, which is the same materials as HOPG.Carbon defects are important to decide its surface characteristics.Figure 4 shows Raman spectra of HOPG and PFC.Raman spectroscopy can be used to investigate sp 2 -hybridized structures in carbon materials, and yields information on defects and the crystalline structure [33,34].The prominent features in the Raman spectra of HOPG and PFC are the G-band at ca. 1585 cm −1 and the D-band at ca. 1350 cm −1 [33,34].The G-band is a doubly-degenerate phonon Raman active mode in sp 2 -structured carbon networks, whereas the D-band is localized where the lattice structure is not perfect, mostly at the edges, and at defects in the sp 2 -hybridized carbon structure.A D-band is not observed for HOPG.This indicates that the sp 2 -hybridized carbon structure HOPG is highly crystalline, with almost no defects at the detection level of this method.In contrast, the G/D intensity ratio for PFC was calculated to be ca.0.65.
The intensity of the D-band was higher than that of the G-band.Usually, defects in carbon are caused by cleavage of carbon sp 2 -bonds and oxidation by O 2 at cleavage sites.Hydrophilic functional groups such as -C=O, -COOH, -C-OH, and -C-O-C-are present at defect sites [35][36][37].The Raman results indicate that the PFC surface is more hydrophilic than HOPG.In fact, the contact angles of HOPG and PFC were obtained to be 71 • (±2 • ) and 58 • (±2 • ), which clearly indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does not adsorb on the PFC interface.
Colloids Interfaces 2018, 2, x FOR PEER REVIEW 5 of 10 fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.Figure 5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1 obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
In Equation ( 1), B is the adsorption coefficient, CA is the SC concentration, a is an interaction coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentration.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN) 6 ] 3− as a probe.Figure 5a shows the redox reaction of [Fe(CN) 6 ] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k Colloids Interfaces 2018, 2, x FOR PEER REVIEW 5 of 10 fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.Figure 5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1 obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
In Equation ( 1), B is the adsorption coefficient, CA is the SC concentration, a is an interaction coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentration.fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), w indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be w not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotio the electrode type.The SC promotional effect was observed only at the HOPG interfa investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− Figure 5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol• buffer solution in the presence of SC.The redox reaction was rapidly inhibited, eve concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') o under these conditions was investigated by cyclic voltammogram digital simulation.Th k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Ad nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value cm•s −1 obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited adsorbed easily on the HOPG interface.The degree of inhibition became almost co concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•d concluded that binding of SC molecules to the HOPG interface reached a maxim concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was use the SC adsorption behavior [38].
In Equation ( 1), B is the adsorption coefficient, CA is the SC concentration, a is a coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' v concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC co ' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3  SC caused the k W 5 of 10 d PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly PFC was higher than that of HOPG.This could be why SC does .The adsorption behavior of SC was analyzed by plotting centration (Figure 6a) [38].The k˚' value decreased quickly with ca. 10 nmol dm −3 , and then decreased slowly until ca.40 st constant or decreased slightly at 40-4000 nmol•dm −3 .We thus lecules to the HOPG interface reached a maximum at a SC rumkin adsorption isotherm (Equation ( 1)) was used to analyze ption coefficient, CA is the SC concentration, a is an interaction erage of the HOPG interface by SC.If we assume that electron coverage ratio of SC at the HOPG interface, θ can be described tial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC k˚' is the obtained value at a given value of the SC concentration.
' of [Fe(CN) 6 ] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1  obtained with no SC (Figure 5b).The reaction of [Fe(CN) 6 ] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k Colloids Interfaces 2018, 2, x FOR PEER REVIEW fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which cl indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effe the electrode type.The SC promotional effect was observed only at the HOPG interface.We investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a p Figure 5a  obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited becaus adsorbed easily on the HOPG interface.The degree of inhibition became almost constant a concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plo the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until c nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We concluded that binding of SC molecules to the HOPG interface reached a maximum at concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to an the SC adsorption behavior [38].
In Equation (1), B is the adsorption coefficient, CA is the SC concentration, a is an intera coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that ele transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be desc by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentra fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.Figure 5a  obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
In Equation (1), B is the adsorption coefficient, CA is the SC concentration, a is an interaction coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentration.
' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k was inhibited because SC degree of inhibition became almost constant at SC adsorption behavior of SC was analyzed by plotting (Figure 6a) [38].The k˚' value decreased quickly with mol dm −3 , and then decreased slowly until ca.40 t or decreased slightly at 40-4000 nmol•dm −3 .We thus the HOPG interface reached a maximum at a SC sorption isotherm (Equation ( 1)) was used to analyze icient, CA is the SC concentration, a is an interaction e HOPG interface by SC.If we assume that electron ratio of SC at the HOPG interface, θ can be described ined in the absence of SC, kf˚' is the k˚' value at a SC tained value at a given value of the SC concentration.
' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
In Equation (1), B is the adsorption coefficient, C A is the SC concentration, a is an interaction coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described by Equation (2), where k i EER REVIEW 5 of 10 OPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly ilicity of PFC was higher than that of HOPG.This could be why SC does rface. .The adsorption behavior of SC was analyzed by plotting of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with on up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 ere almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus f SC molecules to the HOPG interface reached a maximum at a SC m −3 . The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze r [38].
' is the initial k ces 2018, 2, x FOR PEER REVIEW 5 of 10 ntact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly hat the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does on the PFC interface.was inhibited because SC asily on the HOPG interface.The degree of inhibition became almost constant at SC ons higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting es as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus that binding of SC molecules to the HOPG interface reached a maximum at a SC on of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze rption behavior [38].
' obtained in the absence of SC, k f Colloids Interfaces 2018, 2, x FOR PEER REVIEW 5 of 10 fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.Figure 5a  obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
' is the k Colloids Interfaces 2018, 2, x FOR PEER REVIEW 5 of 10 fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.Figure 5a  obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
' value at a SC concentration of 40 nmol•dm −3 , and k 018, 2, x FOR PEER REVIEW 5 of 10 ct angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does the PFC interface.was inhibited because SC ly on the HOPG interface.The degree of inhibition became almost constant at SC higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting s a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 e k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus t binding of SC molecules to the HOPG interface reached a maximum at a SC of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze tion behavior [38].Figure 6b shows the obtained θ values as a function of BCA, and the simulated curve when various a values were used.The figure clearly shows that the obtained results fitted the simulated curve well according to Equation 1, when a was 0.5 and B was 1.4 (±0.4) × 10 8 .Usually, in the Frumkin adsorption isotherm, the a value indicates the following: a > 0, an attractive force between molecules; a = 0, no interactions between molecules; and a < 0, a repulsive force between molecules.For SC, a (=0.5) > 0, which indicates slight attraction between SC molecules and the HOPG interface.Direct bioelectrocatalysis of Lac at the HOPG interface was also observed when SC and Lac were both present (Figure 7b), but the catalytic current was much smaller than that obtained for only HOPG surface modification with SC (Figure 7a).This could be because of the slow electron transfer rate of Lac.The electron transfer rate is strongly affected by the electron tunneling distance.The electron transfer rate decreases in proportion to exp (−βR), where R is the edge-to-edge electron tunneling distance and β is proportional to the square root of the barrier height [39,40].SC molecules act as an insulating barrier and inhibit fast electron transfer when the SC molecules form a multilayer on the HOPG interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.Figure 5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1 obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
In Equation ( 1), B is the adsorption coefficient, CA is the SC concentration, a is an interaction coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentration.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe. .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
In Equation ( 1), B is the adsorption coefficient, CA is the SC concentration, a is an interaction coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentration.Figure 6b shows the obtained θ values as a function of BCA, and the simulated curve when various a values were used.The figure clearly shows that the obtained results fitted the simulated curve well according to Equation 1, when a was 0.5 and B was 1.4 (±0.4) × 10 8 .Usually, in the Frumkin adsorption isotherm, the a value indicates the following: a > 0, an attractive force between molecules; a = 0, no interactions between molecules; and a < 0, a repulsive force between molecules.For SC, a (=0.5) > 0, which indicates slight attraction between SC molecules and the HOPG interface.Direct bioelectrocatalysis of Lac at the HOPG interface was also observed when SC and Lac were both present (Figure 7b), but the catalytic current was much smaller than that obtained for only HOPG surface modification with SC (Figure 7a).This could be because of the slow electron transfer rate of Lac.The electron transfer rate is strongly affected by the electron tunneling distance.The electron transfer rate decreases in proportion to exp (−βR), where R is the edge-to-edge electron tunneling distance and β is proportional to the square root of the barrier height [39,40].SC molecules act as an insulating barrier and inhibit fast electron transfer when the SC molecules form a multilayer on the HOPG interface.fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clearly indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC does not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on the electrode type.The SC promotional effect was observed only at the HOPG interface.We then investigated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe. .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze the SC adsorption behavior [38].
In Equation ( 1), B is the adsorption coefficient, CA is the SC concentration, a is an interaction coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentration.fact, the contact angles of HOPG and PFC were obtained to be 71° (±2°) and 58° (±2°), which clear indicated that the hydrophilicity of PFC was higher than that of HOPG.This could be why SC do not adsorb on the PFC interface.

Analysis of SC Adsorption Phenomena at HOPG Interface
In the Section 3. .The Frumkin adsorption isotherm (Equation ( 1)) was used to analy the SC adsorption behavior [38].
In Equation (1), B is the adsorption coefficient, CA is the SC concentration, a is an interactio coefficient, and θ is the surface coverage of the HOPG interface by SC.If we assume that electro transfer rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be describe by Equation (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a S concentration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentratio Figure 6b shows the obtained θ values as a function of BC A , and the simulated curve when various a values were used.The figure clearly shows that the obtained results fitted the simulated curve well according to Equation 1, when a was 0.5 and B was 1.4 (±0.4) × 10 8 .Usually, in the Frumkin adsorption isotherm, the a value indicates the following: a > 0, an attractive force between molecules; a = 0, no interactions between molecules; and a < 0, a repulsive force between molecules.For SC, a (=0.5) > 0, which indicates slight attraction between SC molecules and the HOPG interface.Direct bioelectrocatalysis of Lac at the HOPG interface was also observed when SC and Lac were both present (Figure 7b), but the catalytic current was much smaller than that obtained for only HOPG surface modification with SC (Figure 7a).This could be because of the slow electron transfer rate of Lac.The electron transfer rate is strongly affected by the electron tunneling distance.The electron transfer rate decreases in proportion to exp (−βR), where R is the edge-to-edge electron tunneling distance and β is proportional to the square root of the barrier height [39,40].SC molecules act as an insulating barrier and inhibit fast electron transfer when the SC molecules form a multilayer on the HOPG interface.The electron transfer rate constant (k˚') for Lac at the SC-modified HOPG was evaluated by previously reported methods [25][26][27].Figure 7a shows the catalytic current steady-state voltammograms and their fittings with simulated plots (Supplementary Material).The obtained onset potential (+0.65 V) for O2 reduction was similar to the previously reported value, suggesting that the T1 Cu site is the primary electron acceptor from the HOPG electrode, and electrons are shuttled to the T2/T3 Cu site, at which O2 is fully reduced to water (Figure 7c,d) [7].The simulated curve fitted the experimental bioelectrocatalytic current curves when the individual values of the determined parameters were used as shown in Table 1.The residual slope obtained potential of ca.0.3-0 V in Figure 7a could be a result of random orientation effect of Lac.The determined k˚' value was 30-115 s −1 , which is much smaller than the value obtained at the SC-modified SWCNT electrode.The reason for the difference is as yet unclear, but it may come from the differences between the morphologies of the atomically flat HOPG surface and the atomically one-dimensional SWCNTs.SC molecule binding would be suited to a slightly curved surface because SC has a slightly curved steroid framework.Г: total surface concentration of the electrically active laccase (Lac); k˚: the heterogeneous electron transfer rate constant (s −1 ) at the E˚′ between the adsorbed Lac and the electrode intreface; E˚′ is the formal redox potential for the T1 Cu site of Lac; α: transfer coefficient.SC-sodium cholate; HOPG-highly oriented pyrolytic graphite.
Roughly, without accounting endergonic tunneling compensation, we used Equation (3) to evaluate the electron tunneling distance between the T1 Cu site of Lac and the modified electrode surface from the obtained k˚' values [41].where ket is the electron tunneling rate; ρ is the packing density of protein atoms in the volume between the redox centers, to account for the β variations in exergonic electron tunneling rates; R is the edge-to-edge distance; λ is the energy required to reorganize the nuclear coordinates on electron   , which is similar to the previously reported value [32].Addition of 40 −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 btained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC d easily on the HOPG interface.The degree of inhibition became almost constant at SC rations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting alues as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with ng SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus ed that binding of SC molecules to the HOPG interface reached a maximum at a SC ration of 40 nmol•dm −3 .The Frumkin adsorption isotherm (Equation ( 1)) was used to analyze dsorption behavior [38].
Equation ( 1), B is the adsorption coefficient, CA is the SC concentration, a is an interaction nt, and θ is the surface coverage of the HOPG interface by SC.If we assume that electron rate is linearly related to the coverage ratio of SC at the HOPG interface, θ can be described tion (2), where ki˚' is the initial k˚' obtained in the absence of SC, kf˚' is the k˚' value at a SC ration of 40 nmol•dm −3 , and k˚' is the obtained value at a given value of the SC concentration.

'
) of [Fe(CN) 6 ] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k Colloids Interfaces 2018, 2, x FOR PEER REVIEW

3 ,
PG and (b) PFC surfaces.Excitation laser wavelength: 514.5 nm ena at HOPG Interface the reasons for the dependence of the SC promotional effect on onal effect was observed only at the HOPG interface.We then ena at the HOPG interface by using [Fe(CN)6] 3− as a probe. of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate SC.The redox reaction was rapidly inhibited, even when the −as shown by the increasing potential difference between the ls.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− ated by cyclic voltammogram digital simulation.The calculated imilar to the previously reported value [32].Addition of 40 CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 re 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC rface.The degree of inhibition became almost constant at SC l•dm −3
shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 ac buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference betwee positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN under these conditions was investigated by cyclic voltammogram digital simulation.The calcu k˚' was 6.3 × 10 cm•s −1, which is similar to the previously reported value[32].Addition nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 cm•s −1
shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1

5 of 10 e
obtained to be 71° (±2°) and 58° (±2°), which clearly igher than that of HOPG.This could be why SC does PFC surfaces.Excitation laser wavelength: 514.5 nm G Interface s for the dependence of the SC promotional effect on was observed only at the HOPG interface.We then e HOPG interface by using [Fe(CN)6] 3− as a probe.)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate dox reaction was rapidly inhibited, even when the n by the increasing potential difference between the terogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− clic voltammogram digital simulation.The calculated the previously reported value [32].Addition of 40 ecrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 e reaction of [Fe(CN)6] 3− a of (a) HOPG and (b) PFC surfaces.Excitation laser wavelength: 514.5 nm on Phenomena at HOPG Interface clarified the reasons for the dependence of the SC promotional effect on promotional effect was observed only at the HOPG interface.We then n phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.x reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate sence of SC.The redox reaction was rapidly inhibited, even when the 0 nmol•dm −3 , as shown by the increasing potential difference between the k potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− s investigated by cyclic voltammogram digital simulation.The calculated hich is similar to the previously reported value [32].Addition of 40 k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC OPG interface.The degree of inhibition became almost constant at SC 400 nmol•dm −3

4 .
Raman spectra of (a) HOPG and (b) PFC surfaces.Excitation laser wavelength: 514.5 nm ).s of SC Adsorption Phenomena at HOPG Interface Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on de type.The SC promotional effect was observed only at the HOPG interface.We then d SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate tion in the presence of SC.The redox reaction was rapidly inhibited, even when the on of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the d negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− e conditions was investigated by cyclic voltammogram digital simulation.The calculated × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 ined with no SC (Figure 5b).The reaction of [Fe(CN)6] 3−
shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1
shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1 aman spectra of (a) HOPG and (b) PFC surfaces.Excitation laser wavelength: 514.5 nm SC Adsorption Phenomena at HOPG Interface tion 3.2, we clarified the reasons for the dependence of the SC promotional effect on ype.The SC promotional effect was observed only at the HOPG interface.We then C adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.ws the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate in the presence of SC.The redox reaction was rapidly inhibited, even when the of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the egative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− nditions was investigated by cyclic voltammogram digital simulation.The calculated 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 d with no SC (Figure 5b).The reaction of [Fe(CN)6] 3−

Figure 6 .
Figure 6.(a) Heterogeneous electron transfer rate (k˚') as function of SC concentration.The k˚' value was estimated from the simulated analysis of the cyclic voltammograms of [Fe(CN)6] 3− at HOPG in 0.1 mol•dm −3 acetate buffer solution in the presence of various concentrations of SC at potential sweep rate of 20 mV•s −1 .(b) SC coverage (θ) as a function of BCA (B: adsorption coefficient, CA: SC concentration) and curves (broken line) simulated by Frumkin adsorption isotherm at various values of the interaction coefficient (a).The θ value was estimated from Equation (2).

Figure 5 .
Figure 5. (a) Cyclic voltammograms of [Fe(CN) 6 ] 3− at HOPG electrode in 0.1 mol•dm −3 acetate buffer solution in the absence (broken line) and presence of 40 (solid black line) and 400 (solid gray line) nmol•dm −3 SC at potential sweep rate of 20 mV•s −1 .(b) Simulated cyclic voltammograms (open circle) fitted to experimental voltammograms at HOPG electrode in the absence (broken line) and presence (solid line) of 40 nmol•dm −3 SC.The electrochemical parameters for the simulated voltammograms were as follows: electrode area 0.28 cm 2 ; heterogeneous electron transfer rate constants (k

Figure 3 .
Figure 5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1 obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3.We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3

Figure 6 .
Figure 6.(a) Heterogeneous electron transfer rate (k˚') as function of SC concentration.The k˚' value was estimated from the simulated analysis of the cyclic voltammograms of [Fe(CN)6] 3− at HOPG in 0.1 mol•dm −3 acetate buffer solution in the presence of various concentrations of SC at potential sweep rate of 20 mV•s −1 .(b) SC coverage (θ) as a function of BCA (B: adsorption coefficient, CA: SC concentration) and curves (broken line) simulated by Frumkin adsorption isotherm at various values of the interaction coefficient (a).The θ value was estimated from Equation (2).

Figure 3 .
Figure 5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 acetate buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when the concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between the positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− under these conditions was investigated by cyclic voltammogram digital simulation.The calculated k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 40 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 10 cm•s −1 obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because SC adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at SC concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plotting the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly with increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca.40 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thus concluded that binding of SC molecules to the HOPG interface reached a maximum at a SC concentration of 40 nmol•dm −3

'
) as function of SC concentration.The k Colloids Interfaces 2018, 2, x FOR PEER REVIEW 5 of

3 , 1 , 3 .
Figure 5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm −3 aceta buffer solution in the presence of SC.The redox reaction was rapidly inhibited, even when th concentration of SC was 40 nmol•dm −3 , as shown by the increasing potential difference between th positive and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6 under these conditions was investigated by cyclic voltammogram digital simulation.The calculate k˚' was 6.3 × 10 cm•s −1 , which is similar to the previously reported value [32].Addition of 4 nmol•dm −3 SC caused the k˚' of [Fe(CN)6] 3− to decrease to 6.3 × 10 cm•s −1 from the value of 11.5 × 1 cm•s −1 obtained with no SC (Figure 5b).The reaction of [Fe(CN)6] 3− was inhibited because S adsorbed easily on the HOPG interface.The degree of inhibition became almost constant at S concentrations higher than 400 nmol•dm −3 .The adsorption behavior of SC was analyzed by plottin the k˚' values as a function of SC concentration (Figure 6a) [38].The k˚' value decreased quickly wi increasing SC concentration up to ca. 10 nmol dm −3 , and then decreased slowly until ca. 4 nmol•dm −3 .The k˚' values were almost constant or decreased slightly at 40-4000 nmol•dm −3 .We thu concluded that binding of SC molecules to the HOPG interface reached a maximum at a S concentration of 40 nmol•dm −3 ' value was estimated from the simulated analysis of the cyclic voltammograms of [Fe(CN) 6 ] 3− at HOPG in 0.1 mol•dm −3 acetate buffer solution in the presence of various concentrations of SC at potential sweep rate of 20 mV•s −1 .(b) SC coverage (θ) as a function of BC A (B: adsorption coefficient, C A : SC concentration) and curves (broken line) simulated by Frumkin adsorption isotherm at various values of the interaction coefficient (a).The θ value was estimated from Equation (2).
lysis of SC Adsorption Phenomena at HOPG Interface the Section 3.2, we clarified the reasons for the dependence of the SC promotional effect on trode type.The SC promotional effect was observed only at the HOPG interface.We then ated SC adsorption phenomena at the HOPG interface by using [Fe(CN)6] 3− as a probe.5a shows the redox reaction of [Fe(CN)6] 3− at the HOPG interface in 0.1 mol•dm−3 acetate olution in the presence of SC.The redox reaction was rapidly inhibited, even when the ration of SC was 40 nmol•dm−3 , as shown by the increasing potential difference between the and negative peak potentials.The heterogeneous electron transfer rate (k˚') of [Fe(CN)6] 3− hese conditions was investigated by cyclic voltammogram digital simulation.The calculated 6.3 × 10 cm•s−1

Table 1 .
Estimated parameters from experiments and simulation.