Voltammetric and surface-enhanced resonance Raman spectroscopic characterization of cytochrome C adsorbed on a 4-mercaptopyridine monolayer on silver electrodes

To combine voltammetric techniques with surface-enhanced resonance Raman scattering (SERRS), cytochrome c (cyt c) was immobilized on a roughened silver electrode chemically modified with a self-assembled monolayer (SAM) of 4-mercaptopyridine (PySH). All measurements were performed on the same electrode in a homemade spectroelectrochemical cell suitable for such applications. Cyt c on a PySH-SAM shows a quasi-reversible, monoelectronic, adsorption-controlled CV response with a formal reduction potential of -0.061 V (vs SCE), which is comparable to the values found for native cyt c adsorbed on different SAMs. SERRS spectra proved that cyt c adsorbed on a PySH monolayer is present in the native conformer (the B1 state). Voltammetric and SERRS experiments at high ionic strength revealed that the interaction between the SAM and the protein is electrostatic in nature. In conclusion, PySH was found to be suitable for adsorption of cyt c at SERRS-active silver surfaces. In comparison with other SAMs, PySH requires less time (10 min vs 12-18 h) to form a long-time durable and reproducible coating on the roughened electrode surface.     

Reversible Electrochemistry of Cytochrome c on Recompressed,Binderless Exfoliated Graphite Electrodes

The direct electrochemistry of cytochrome c (cyt‐c) has been investigated on exfoliated graphite (EG) electrodes. The as‐polished and roughened (using SiC emery sheet) EG surfaces are inactive for the direct electron transfer. However, when the EG electrode was sonicated before the experiment, a pair of redox waves were obtained for freely diffusing cyt‐c in the solution phase. The formal potential was found to be 0.01 V (vs. SCE) in 0.1 M phosphate buffer at a pH of 7.1. The electrochemical response for the adsorbed cyt‐c on sonicated EG electrodes, which is shown to have carbonyl functional groups on its surface, shows nearly reversible voltammograms in the same electrolyte. However, the formal potential in the adsorbed state is more negative than that observed for the solution phase cyt‐c. A structure based on an open heme conformation proposed by Hildebrandt and Stockburger is probably present on the EG surface. It is suggested that the electrochemistry at the EG electrode is essentially governed by favourable electrostatic interactions.     

Interfacial biocatalysis on charged and immobilized substrates: the roles of enzyme and substrate surface charge

An enzyme charge ladder was used to examine the role of electrostatic interactions involved in biocatalysis at the solid-liquid interface. The reactive substrate consisted of an immobilized bovine serum albumin (BSA) multilayer prepared using a layer-by-layer technique. The zeta potential of the BSA substrate and each enzyme variant was measured to determine the absolute charge in solution. Enzyme adsorption and the rate of substrate surface hydrolysis were monitored for the enzyme charge ladder series to provide information regarding the strength of the enzyme-substrate interaction and the rate of interfacial biocatalysis. First, each variant of the charge ladder was examined at pH 8 for various solution ionic strengths. We found that for positively charged variants the adsorption increased with the magnitude of the charge until the surface became saturated. For higher ionic strength solutions, a greater positive enzyme charge was required to induce adsorption. Interestingly, the maximum catalytic rate was not achieved at enzyme saturation but at an invariable intermediate level of adsorption for each ionic strength value. Furthermore, the maximum achievable reaction rate for the charge ladder was larger for higher ionic strength values. We propose that diffusion plays an important role in interfacial biocatalysis, and for strong enzyme-substrate interaction, the rate of diffusion is reduced, leading to a decrease in the overall reaction rate. We investigated the effect of substrate charge by varying the solution pH from 6.1 to 8.7 and by examining multiple ionic strength values for each pH. The same intermediate level of adsorption was found to maximize the overall reaction rate. However, the ionic strength response of the maximum achievable rate was clearly dependent on the pH of the experiment. We propose that this observation is not a direct effect of pH but is caused by the change in substrate surface charge induced by changing the pH. To prove this hypothesis, BSA substrates were chemically modified to reduce the magnitude of the negative charge at pH 8. Chemical modification was accomplished by the amidation of aspartic and glutamic acids to asparagine and glutamine. The ionic strength response of the chemically modified substrate was considerably different than that for the native BSA substrate at an identical pH, consistent with the trend based on substrate surface charge. Consequently, for substrates with a low net surface charge, the maximum achievable catalytic rate of the charge ladder was relatively independent of the solution ionic strength over the range examined; however, at high net substrate surface charge, the maximum rate showed a considerable ionic strength dependence.     

The effect of ionic strength on the electron-transfer rate of surface immobilized cytochrome C

Horse heart cytochrome c was immobilized on four different self-assembled monolayer (SAM) films. The electron tunneling kinetics were studied in the different assemblies as a function of the ionic strength of the buffer solution using cyclic voltammetry. When cytochrome c is electrostatically immobilized, the standard electron exchange rate constant k0 decreases with the increase of the solution's ionic strength. In contrast, the protein covalently attached or ligated has a rate constant independent of the ionic strength. The inhomogeneity of electrostatically immobilized cytochrome c increases with the increase of the solution's ionic strength whereas that of the covalently attached protein is independent of the ionic strength. A comparison of these different electron-transfer behaviors suggests that the thermodynamically stable geometry of cytochrome c in the electrostatic assemblies is also an electron transfer favorable one. It suggests that the surface charges of cytochrome c are capable of guiding it into geometries in which its front surface faces the electron-transfer partner. The inhomogeneity observed in this study indicates that a distribution of cytochrome c orientations and thus a distribution of electron transfer rate constants exists.     

Immobilization and electrochemical redox behavior of cytochrome c on fullerene film-modified electrodes

Two different fullerene film-modified electrodes were prepared and used for surface immobilization and electrochemical property investigation of horse heart cytochrome c (cyt c). Both a pristine fullerene film and fullerene-palladium (C(60)-Pd) polymer film-modified platinum, glassy carbon and indium-tin-oxide (ITO) electrodes were used. The immobilized cyt c was characterized by piezoelectric microgravimetry at a quartz crystal microbalance (QCM), UV-visible absorption, and X-ray photoelectron spectroscopy (XPS), as well as cyclic voltammetry (CV) techniques. The UV-visible spectral studies revealed a small blue shift of both the Soret and Q band of the heme moiety of cyt c, immobilized on the C(60)-Pd polymer film-modified ITO electrode, as compared to the bands of cyt c in solution suggesting that molecules of cyt c are densely packed onto the surface of the modified electrode. The CV studies revealed a quasi-reversible electrode behavior of the heme moiety indicating the occurrence of kinetically hindered electron transfer. A good agreement was found between the values of cyt c electrode surface coverage determined by piezoelectric microgravimetry and cyclic voltammetry. For piezoelectric microgravimetry, these values ranged from 0.5 x 10(-10) to 2.5 x 10(-10) mol cm(-2), depending upon the amount of cyt c present in solution and the time allowed for immobilization, which compared with a value of 3.6+/-0.4 x 10(-10) mol cm(-2) determined by CV. The possible mechanisms of cyt c immobilization on the C(60) film and C(60)-Pd film-modified electrodes are also discussed.     

REDOX PROTEINS AS ELECTRON ACCEPTORS FROM CHLOROPHYLL TRIPLET STATE IN LIPID BILAYER VESICLES: KINETICS OF REDUCTION OF MEMBRANE RECONSTITUTED CYTOCHROME c OXIDASE MEDIATED BY 2,5-DI-t-BUTYL BENZOQUINONE AND CYTOCHROME c

Laser flash photolysis was used to determine the kinetics of electron transfer between membrane-bound triplet chlorophyll (3C), cytochrome c (cyt c) located in the external water phase, and vesicle-reconstituted cytochrome c oxidase (CCO). 2,5-Di-t-butyl benzoquinone (2,5 TBQ) was used as an electron transfer mediator between 3C and cyt c. A light-induced cyclic electron transfer sequence between the redox components was observed (3C----2.5 TBQ----cyt c----CCO----C+.). Under optimum conditions of membrane surface charge and ionic strength, the overall efficiency of CCO reduction (based on 3C generated by the laser flash) was 14%. Under the anaerobic conditions used, CCO reoxidation (occurring via electron transfer to C+.) was quite slow (halftime approx. 1 s at 75 mM ionic strength). The multicomponent system displayed a high level of stability, as indicated by its ability to undergo many cycles of reduction and reoxidation without any apparent degradation of the components. These results demonstrate the feasibility of constructing complex electron transfer chains, including both soluble and membrane-bound redox proteins, in artificial lipid bilayers, whose properties can be readily controlled by manipulating parameters such as ionic strength and membrane composition.     

A Colloidal Au Monolayer Modulates the Conformation and Orientation of a Protein at the Electrode/Solution Interface

The orientation and conformation of adsorbed cytochrome c (cyt c) at the interface between an electrode modified with colloidal Au and a solution were studied by electrochemical, spectroscopic, and spectroelectrochemical techniques. The results indicate that the colloidal Au monolayer formed via preformation of an organic self‐assembled monolayer (SAM) can increase the electronic coupling between the SAM and cyt c in the same manner as bifunctional molecular bridges, one functional group of which is bound to the electrode surface while the other interacts with the protein surface. The approach of cyt c to the modified electrode/solution interface can be assisted by strong interactions of the intrinsic charge of colloidal particles with cyt c, while the heme pocket remains almost unchanged due to the screening effect of the negatively charged field created by the intrinsic charge. The conformational changes of cyt c induced by its adsorption at a bare glassy carbon electrode/solution interface and the effect of the electric field on the ligation state of the heme can be avoided at the colloidal‐Au‐modified electrode/solution interface. Finally, a possible model for the adsorption orientation of cyt c at the colloidal‐Au‐modified electrode/solution interface is proposed.     

Cytochrome c self-assembly on alkanethiol monolayer electrodes as characterized by AFM, IR, QCM, and direct electrochemistry

With the advantage of carbodiimide coupling chemistry, horse heart cytochrome c (cyt c) has been covalently immobilized onto self-assembled monolayers (SAMs) from 11-mercaptoundecanoic acid (MUDA) developed on single-crystal or polycrystalline gold substrate surfaces. The cyt c immobilized substrates thus prepared have been characterized by atomic force microscopy (AFM); we have succeeded in obtaining surface topographical images down to single-protein resolution. AFM imaging has also shown densely packed, uniform protein monolayer formation that is highly suggestive of self-assembly of cyt c molecules on MUDA SAMs. Covalent attachment of cyt c has been further evidenced by reflection-absorption FT-IR as well as microgravimetric analysis using a quartz crystal microbalance (QCM). In the latter, the specific MUDA and cyt c surface concentrations were determined to be 0.86 +/- 0.11 nmol cm-2 (n = 5) and 28 +/- 12 pmol cm-2 (n = 5), both of which agree fairly well with their theoretical counterparts. The obtained QCM chips having the cyt c/MUDA/Au interfacial structure were found to be capable of the direct electrochemistry of the surface-attached cyt c molecules. Cyclic voltammetric measurements on the chips gave particular redox waves showing characteristics of surface process. The electroactive protein surface concentration was determined to be 7.2 +/- 4.8 pmol cm-2 (n = 6); it was almost consistent with values found in literature, while it was limited to 26% in magnitude for the QCM data. This was deemed to have arisen from the orientation variation of the surface-confined cyt c molecules and is discussed briefly.     

The role of electrostatic interactions in protease surface diffusion and the consequence for interfacial biocatalysis

This study examines the influence of electrostatic interactions on enzyme surface diffusion and the contribution of diffusion to interfacial biocatalysis. Surface diffusion, adsorption, and reaction were investigated on an immobilized bovine serum albumin (BSA) multilayer substrate over a range of solution ionic strength values. Interfacial charge of the enzyme and substrate surface was maintained by performing the measurements at a fixed pH; therefore, electrostatic interactions were manipulated by changing the ionic strength. The interfacial processes were investigated using a combination of techniques: fluorescence recovery after photobleaching, surface plasmon resonance, and surface plasmon fluorescence spectroscopy. We used an enzyme charge ladder with a net charge ranging from -2 to +4 with respect to the parent to systematically probe the contribution of electrostatics in interfacial enzyme biocatalysis on a charged substrate. The correlation between reaction rate and adsorption was determined for each charge variant within the ladder, each of which displayed a maximum rate at an intermediate surface concentration. Both the maximum reaction rate and adsorption value at which this maximum rate occurs increased in magnitude for the more positive variants. In addition, the specific enzyme activity increased as the level of adsorption decreased, and for the lowest adsorption values, the specific enzyme activity was enhanced compared to the trend at higher surface concentrations. At a fixed level of adsorption, the specific enzyme activity increased with positive enzyme charge; however, this effect offers diminishing returns as the enzyme becomes more highly charged. We examined the effect of electrostatic interactions on surface diffusion. As the binding affinity was reduced by increasing the solution ionic strength, thus weakening electrostatic interaction, the rate of surface diffusion increased considerably. The enhancement in specific activity achieved at the lowest adsorption values is explained by the substantial rise in surface diffusion at high ionic strength due to decreased interactions with the surface. Overall, knowledge of the electrostatic interactions can be used to control surface parameters such as surface concentration and surface diffusion, which intimately correlate with surface biocatalysis. We propose that the maximum reaction rate results from a balance between adsorption and surface diffusion. The above finding suggests enzyme engineering and process design strategies for improving interfacial biocatalysis in industrial, pharmaceutical, and food applications.     

Morphology-dependent electrochemistry and electrocatalytical activity of cytochrome c

The morphology-dependent electrochemistry and electrocatalytical activity of cytochrome c (cyt. c) were investigated at pyramidal, rodlike, and spherical gold nanostructures directly electrodeposited onto sputtered gold surfaces. Direct, reversible electron transfer of cyt. c, for the first time, was realized at nanorod-like and nanopyramidal gold surfaces without any mediators or promoters, while no redox reaction was observed at the nanospherical gold electrode. The electrochemical properties of cyt. c vary with the shape of gold nanostructures with respect to the reversibility of electrode reactions, kinetic parameters, the formal potentials (E0'), and charge-transport resistance (Rct), suggesting shape-dependent mechanisms for the electrode reactions of cyt. c. The experimental results manifest that cyt. c was stably immobilized on the nanostructured gold electrodes with different conformational changes of the heme microenvironment. Consequently, not only the electroactivity, but also the inherent biological activity of the immobilized cyt. c strongly depended on the shape of the electrode surfaces. The facilitated electron transfer combined with the intrinsic catalytical activity of cyt. c substantially constructed a third-generation H2O2 biosensor with high selectivity, quick response time, large linear range, and good sensitivity. The electrocatalytical activity of the immobilized cyt. c toward H2O2 was also found to be morphology dependent, and the linear range of H2O2 detection could be tuned by means of employing the nanostructured gold surfaces with different shapes.     

Direct electrochemistry of cytochrome c on a phosphonic acid terminated self-assembled monolayers

The direct electrochemistry of cytochrome c (cyt c) on a gold electrode modified with 3-mercaptopropylphosphonic acid [HS-(CH₂)₃-PO₃H₂, MPPA] self-assembled monolayers (SAMs) was for the first time investigated. Electrochemical measurements and surface-enhanced infrared absorption spectroscopic reveal that the adsorption kinetics of cyt c on the MPPA-SAMs is very fast (saturation adsorption is completed within 5 s) and the immobilized cyt c molecules retain their native secondary protein structure. The nature of interaction between cyt c and -PO₃H₂ groups is mainly the electrostatic interaction. The direct electrochemistry of the immobilized cyt c on the -PO₃H₂ terminated SAMs with short chain is nearly reversible. Its formal potential (E⁰′ = 18 ± 3 mV vs. SCE) is very close to that of cyt c in an aqueous solution (E⁰′ = 18-22 mV vs. SCE). In addition, the electron transfer rate of cyt c immobilized on -PO₃H₂ terminated SAMs is relatively slow as compared to -SO₃H and -COOH terminated SAMs, indicating excess negative charge density on the SAMs surface will decrease the electron transfer rate of cyt c.     

Protonation of Lysozymes and Its Consequences for the Adsorption onto a Mica Surface

Several physicochemical properties of chicken egg white lysozyme (LSZ) in electrolyte solutions were determined. The hydrodynamic diameter of LSZ at an ionic strength of 0.15 M was found to be 4.0 nm. Using the determined parameters, the number of uncompensated (electrokinetic) charges, N(c), on the molecule surface was calculated from the electrophoretic mobility data. It was found that the N(c) = 2.8 at pH = 3.0 and an ionic strength of I = 0.15 M. At the lower ionic strength, I = 1 × 10(-3) M, this positive charge increased to N(c) = 5.6 at a pH = 3.0 The physicochemical characteristics were supplemented by the dynamic viscosity measurements. The intrinsic viscosity and the hydrodynamic diameter results were compared with theoretical predictions from Brenner's model. Using this approach, it was found that the effective molecule length of LSZ is equal to L(ef) = 5.6 nm. Additional information on the LSZ adsorbed films was obtained by the contact angle measurements. The notably large contact angles were measured on LSZ films formed under the conditions where both the LSZ and the mica were oppositely charged. The higher the positive zeta potential of LSZ, the greater the contact angle measured, which indicates that LSZ affinity for the adsorption on mica increases with its uncompensated charge. The adsorption dependence on the zeta potential of LSZ was explained, assuming a roughly uniform distribution of the net charge on the molecule surface. This assumption is supported by the results of depositing negatively charged, fluorescent latex particles onto the mica surface, which had been modified by LSZ adsorption. The highest latex coverage was formed on mica surfaces that had first been coated with LSZ solutions of lower pH, as a result of the increasing charge of LSZ monolayers in this condition.     

Detection of Cytochrome c with Calixarenes Incorporated into Supported Lipid Membranes Via Charge Transient Measurements

The interaction of cytochrome c (cyt c) with the calix[6]arene carboxylic acid derivative (tOct[6]CH2COOH) (CX) incorporated into supported lipid membranes (sBLM) composed of dimyristoylphosphatidylcholine (DMPC) were investigated in the metal/insulator/metal arrangement with the isothermal charge transient spectroscopy (IQTS) in local mode and with voltammetry and measurements of double layer capacitance. Cyt c was detected on hydrated DMPC layers as a distinct IQTS peak with relaxation constant below 100 ms. Incorporation of CX in DMPC increased the sensitivity to cyt c about 50%. Reliable detection limit of cyt c was 30 nM. In sBLMs the capture of cyt c increased the permittivity of both sBLMs, with and without CX, respectively. Nevertheless, cyt c was detected in voltammogram only for DMPC layer contained CX, as a wave at the potential of +0.25 V. This potential is in good agreement with the redox potential of cyt c. Hysteresis in double layer capacitance observed only for the mixed DMPC+CX layer is interpreted by reduction of the charge, which is involved in the ongoing redox reaction.     

离子强度对DNA媒介电荷转移的影响——基于Debye-Hückel 理论的实验结果分析

用电化学的方法研究了溶液离子强度对DNA媒介电荷转移的影响, 观察到[Ru(NH₃)₆]³⁺的还原峰电势随支持电解质的浓度增加向负方向移动. 分析发现微分脉冲伏安法(DPV)的峰电势与溶液离子强度间在一定范围内存在线性关系, 以式电势(E⁰')作为“桥梁”, 用Debye-Hückel理论给予了解释. 在高离子强度下, 峰电势对线性关系的偏移是由于超过了Debye-Hückel理论的适用范围, 而无强电解质存在时, DNA自身堆积的强负电荷对DNA媒介电荷转移起了推动作用.     

Direct Electrochemistry of Cytochrome c on EDTA-ZrO2 Organic-inorganic Hybrid Film Modified ElectrodesState Key Laborator3.＂ of Chemo／Biosensing and Chemometrics, Hunan Universit3; Changsha. Hunan 410082. China

A composite film of ethylenediamine tetraacetic acid (EDTA)‐ZrO₂ organic‐inorganic hybrid was prepared based on the chelation between Zr(IV) and EDTA. The direct electrochemical behavior of cytochrome c (cyt. c) at the hybrid film modified glassy carbon electrodes was investigated. The immobilized EDTA can promote the redox of heme in horse heart cyt. c which gives rise to a pair of reversible redox peaks with a formal potential of 40 mV (vs. SCE). The peak current increased linearly with the increase of cyt. c concentration in the range of 1.6 × 10^?6—8.0 × 10^?5 mol·L^?1 with the correlation coefficient of 0.996. Further investigation shows that metal ions can impede the electron transfer of cyt. c. The impediment capability of metal ions depends on their coordination capability with EDTA and their valence number.     

Direct electrochemistry of hemoglobin immobilized in the sodium alginate and SiO2 nanoparticles bionanocomposite film on a carbon ionic liquid electrode

In this paper a carbon ionic liquid electrode (CILE) was fabricated by using a room temperature ionic liquid of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) as binder. By using the CILE as basal electrode, the hemoglobin (Hb) molecule was immobilized on the surface of CILE with a sodium alginate (SA) hydrogel and SiO₂ nanoparticles organic-inorganic composite material. The direct electrochemical behaviors of Hb in the bionanocomposite film were further studied in a pH 7.0 Britton-Robinson (B-R) buffer solution. A pair of well-defined quasi-reversible cyclic voltammetric peaks of Hb was obtained on SA/nano-SiO₂/Hb/CILE with the formal potential (E^0’) at -0.355 V (vs. SCE), which was the characteristic of heme Fe(III)/Fe(II) redox couples. The formal potential of Hb Fe(III)/Fe(II) couple shifted negatively with increasing pH of solution with a slope of -45.2 mV/pH, which indicated that a one electron transfer accompanied with one proton transportation. The immobilized Hb showed good electrocatalytic manner to the reduction of trichloroacetic acid (TCA).     

Considerations for the quantitative transduction of hybridization of immobilized DNA

Immobilized single-stranded DNA (ssDNA) can be used as a selective ‘reagent’ to bind complementary DNA or RNA for applications such as the detection of pathogenic organisms, gene therapy agents and genetic mutations. The density of ssDNA on a surface will determine the charge density due to ionizable phosphate groups. Such a negatively charged interface will attract positive counter-ions from solution, which may result in a local ionic strength, pH and dielectric constant on the surface that is substantially different from that in bulk electrolyte solution. It is the local conditions which influence the thermodynamics of hybridization, and this can studied by the melt temperature (T_m) of double-stranded DNA (dsDNA). Experimental work and theoretical models have been used to examine whether hybridization reactions on a surface can cause dynamic changes in local charge density, and therefore, changes in selectivity and drift in calibration for quantitative analysis. Organosilane chemistry has been used to covalently immobilize hexaethylene glycol linkers and to control the subsequent density of dT₂₀ that was prepared by automated synthesis. Fiber-optic biosensors based on fused silica that was coated with DNA were used in a total internal reflection fluorescence instrument to determine T_m from the dissociation of duplexes of fluorescein-labeled dA₂₀ : dT₂₀. The experimental results suggest that the thermodynamic stability of duplexes that are immobilized on a surface is dependent on the density of immobilized DNA and on the extent of hybridization of DNA. The experimental results show that the thermodynamic stability of immobilized dsDNA is significantly different than that of dsDNA in bulk solution, and include observations of the variation of enthalpy at different ionic strengths, asymmetry in the melt curves, and the possibility of a reduced dielectric constant within a DNA layer relative to that in bulk solution.     

Measurements and theoretical interpretation of points of zero charge/potential of BSA protein

The points of zero charge/potential of proteins depend not only on pH but also on how they are measured. They depend also on background salt solution type and concentration. The protein isoelectric point (IEP) is determined by electrokinetical measurements, whereas the isoionic point (IIP) is determined by potentiometric titrations. Here we use potentiometric titration and zeta potential (ζ) measurements at different NaCl concentrations to study systematically the effect of ionic strength on the IEP and IIP of bovine serum albumin (BSA) aqueous solutions. It is found that high ionic strengths produce a shift of both points toward lower (IEP) and higher (IIP) pH values. This result was already reported more than 60 years ago. At that time, the only available theory was the purely electrostatic Debye-Hu?ckel theory. It was not able to predict the opposite trends of IIP and IEP with ionic strength increase. Here, we extend that theory to admit both electrostatic and nonelectrostatic (NES) dispersion interactions. The use of a modified Poisson-Boltzmann equation for a simple model system (a charge regulated spherical colloidal particle in NaCl salt solutions), that includes these ion specific interactions, allows us to explain the opposite trends observed for isoelectric point (zero zeta potential) and isoionic point (zero protein charge) of BSA. At higher concentrations, an excess of the anion (with stronger NES interactions than the cation) is adsorbed at the surface due to an attractive ionic NES potential. This makes the potential relatively more negative. Consequently, the IEP is pushed toward lower pH. But the charge regulation condition means that the surface charge becomes relatively more positive as the surface potential becomes more negative. Consequently, the IIP (measuring charge) shifts toward higher pH as concentration increases, in the opposite direction from the IEP (measuring potential).     

Effect of the electrostatic interaction on the redox reaction of positively charged cytochrome C adsorbed on the negatively charged surfaces of acid-terminated alkanethiol monolayers on a Au(111) electrode

The electrochemical properties of cytochrome c (cyt c) adsorbed on mixed self-assembled monolayers (SAMs) of 2-mercaptoethanesulfonate (MES)/2-mercaptoethanol (MEL) are compared with those on single-component SAMs of MES, MEL, and mercaptopropionic acid (MPA), using cyclic voltammetry and potential-modulated UV-vis reflectance spectroscopy. The rate constant of electron transfer (ET), k(et), of cyt c adsorbed on the SAM of MPA decreases from 1450 +/- 210 s(-1) at pH 7 to 890 +/- 100 s(-1) at pH 9. In contrast, the value of k(et) of cyt c on the SAM of MES is pH-independent at 100 +/- 15 s(-1). Those facts suggest that a large negative charge density on the SAM surface slows down the ET between cyt c and the electrode. The surface charge density of the SAM affects also the amount of electroactive cyt c, Gamma(e), which decreases from 10.0 +/- 1.0 to 5.3 +/- 1.1 pmol cm(-2) with increasing pH from 7 to 9 on the SAM of MPA. Similarly, the k(et) of cyt c adsorbed on the mixed SAMs of MES/MEL sharply decreases from 900 +/- 300 s(-1) to 110 s(-1) as the surface mole fraction of MES increases beyond 0.5, suggesting the presence of a negative surface charge threshold beyond which the rate of ET of cyt c is dramatically lowered. The decrease in the k(et) on the SAMs at high negative charge densities probably results from the confinement of adsorbed cyt c by the strong electrostatic force to an orientation that is not optimal for the ET reaction.     

The role of the activity coefficients of surface groups in the formation of surface charge of oxides. Part II: Ion exchange and ζ potentials

A new method is developed to calculate the surface charge densities and potentials of oxides in contact with electrolyte solution as functions of pH and ionic strength. For low ionic strength and not too far from p.z.c. (up to 3 pH units for 10^–3 mol dm^–3 NaCl) the previous model (Kosmulski, 1992) neglecting the ion exchange can be used but farther from p.z.c., correction for the ion exchange is necessary for some systems. This correction leads to increase of the calculated titration charge (that is not necessarily equal to the surface charge), but does not affect the diffuse charge and potential.