pH-Induced changes in adsorbed cytochrome c. voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.     

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.     

Effect of mono-CDNP substitution of lysine residues on the redox reaction of cytochrome c electrostatically adsorbed on a mercaptoheptanoic acid modified Au(111) surface

The effect of charge-inverting modification of single surface lysine residue on the electron transfer (ET) reaction of horse heart cytochrome c (cyt c) is examined for 12 different types of mono-4-chloro-2,5-dinitrobenzoic acid substituted cyt c (mCDNPc) adsorbed on a Au(111) electrode modified with a self-assembled monolayer (SAM) of 7-mercapto-heptanoic acid (MHA). A negative shift in the redox potential by 10-35 mV as compared to that of native cyt c and a monolayer coverage in the range of 13-17 pmol cm(-2) are observed for electroactive mCDNPc's. The magnitude of the decrease in the ET rate constant (k(et)) of mCDNPc's compared with that of native cyt c depends on the position of the CDNP substitution. For mCDNPc's in which the modified lysine residue is outside of the interaction domain of cyt c with the SAM, the ratio of the k(et) of mCDNPc to that of native cyt c is correlated to the change in the dipole moment vector of cyt c due to the CDNP modification. This correlation suggests that the dipole moment of cyt c determines its orientation of adsorption on the SAM of MHA and significantly affects the rate of the ET. The CDNP modification of lysine residues at the interaction domain significantly decreases the rate, demonstrating the importance of the local charge environment in determining the rate of ET.     

Impact of surface immobilization and solution ionic strength on the formal potential of immobilized cytochrome C

Four different self-assembled monolayer (SAM) electrode systems were examined electrochemically in order to better understand surface charge effects on the redox thermodynamics of immobilized horse heart cytochrome c (cyt c). Neutralization of protein surface charge upon adsorption on anionic COOH-terminated SAMs was found to cause substantial changes in the formal potential, as determined by cyclic voltammetry. For cyt c immobilized on negatively charged surfaces, the formal potential shifted to more negative values as the ionic strength was decreased, which is opposite to the trend displayed by solution cyt c. In contrast, immobilization to uncharged interfaces resulted in an ionic strength dependence for cyt c that is similar to its solution behavior. The results provide insight into the importance of surface charge on the formal potential of cyt c.     

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.     

SERRS study of [Ru(CN)<Subscript>5</Subscript>(pyS)]<Superscript>4−</Superscript> SAM and cytochrome c: A suggestion toward the heterogeneous molecular recognition

Surface-Enhanced Raman Scattering (SERS) spectra of [Ru(CN)₅(pyS)]⁴⁻ (RupyS) complex self-assembled monolayer (SAM) were obtained on gold and silver surfaces at 632.8 and 413.1 nm excitation radiations, respectively. The bands assigned to the heme iron of the cytochrome c (cyt c) metalloprotein group were observed by using the RupyS SAM on silver at 413.1 nm. The Surface-Enhanced Resonance Raman Scattering (SERRS) spectra of the RupyS SAM on silver in the cyt c solution obtained at −0.2 and +0.2 V present bands at 1,365 and 1,374 cm⁻¹ characteristic of the heme group, indicating the reduced and oxidized states of this protein, respectively. The bands observed at 1,464, 1,504, and 1,638 cm⁻¹ are used to confirm the redox state of cyt c. The presence of the oxidized and reduced bands in function of different applied potential is an evidence that the protein is interacting with the modifier. This paper is dedicated to Prof. Francisco Nart, in memoriam.     

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.     

银电极表面异亮氨酸单分子膜结构和表面性质的表面增强拉曼光谱研究

利用表面增强拉曼光谱(SERS)技术研究了在粗糙化银电极表面吸附的异亮氨酸自组装单层膜结构及其表面性质随溶液酸碱性和电极电位改变的特征.研究结果表明溶液pH值的变化并没有显著改变异亮氨酸分子在银电极表面以去质子化羧基吸附为主的特征.借助于高氯酸根离子这一SERS光谱探针,对异亮氨酸单分子膜的表面酸碱性质进行了表征和分析.而就电位改变对该单分子膜结构的影响而言,在所研究的电位范围内,单分子膜中的异亮氨酸分子是通过去质子化羧基与氨基两个位点而吸附的,且吸附作用随电位负移而呈现有规律的变化.     

Voltammetric Study of Methylene Blue at Thiol SAMs‐Modified Gold Electrodes

The voltammetric behavior of methylene blue (MB) at thiol self‐assembled monolayers modified gold electrodes (SAMs/Au) has been investigated. MB exhibited a redox peak at about ?0.35 V (vs.SCE) in alkaline solution at bare gold electrodes. When the gold electrodes were modified with thiol SAMs, the peak grew due to the accumulation of MB at SAMs. With the solution pH rising, more MB was accumulated, hence the peak height increased, which differed from that at bare gold electrodes. The electrode process at SAMs/Au featured the characteristics of adsorption and/or electrode reaction controlled. The enhancing action of glutathione monolayer (GSH SAM), 3‐mercaptopropionic acid monolayer (3MPA SAM) and other thiol SAMs was compared. Among these, GSH SAM made the MB peak increase more. At GSH SAM/Au, the peak height varied linearly with MB concentration over the range of 2 μM to 400 μM. So this can be developed for the determination of MB and studies concerned. The accumulation behavior caused by GSH SAM and native fish sperm dsDNA was compared. The interaction between DNA and MB was also discussed under this condition.     

Electrochemical study of self-assembled monolayer adsorption

The electrochemical behaviour of self-assembled monolayer (SAM) of aliphatic hexadecanethiol was studied by cyclic voltammetry (CV), elimination voltammetry with linear scan (EVLS) and crystal quartz microbalance (QCM). SAMs were electrochemically created on gold-coated QCM crystal through the sulphur in 1-hexadecanethiol molecule head group. The effect of thiol concentration and potential scan rate on the SAM formation was studied. Formation of SAM was confirmed by CV and QCM. EVLS results revealed the kinetically controlled process followed with electrode reaction in adsorbed state characteristic for SAM formation at lower concentration. The electrode reaction of a totally adsorbed electroactive species was indicated by means of a peak-counter peak signal at higher thiol concentration.     

Orientation of 6-mercaptopurine SAMs at the silver electrode as studied by Raman mapping and in situ SERS

Self-assembled monolayers (SAMs) of 6-mercaptopurine (6MP) on a silver electrode in acid and alkaline media were investigated by a combination protocol of the SERS technique with Raman mapping, and it was found that the adsorption mode of 6MP SAMs changed with the pH value of the environment. Quantum calculations for the vibrational mode were performed by the BLYP/6-31G method. 6MP was adsorbed on the silver electrode with a tilted orientation via S, N1, and N7 atoms in acid medium, while the SAMs adopted head-on adsorption modes with the S atom and the N1 atom anchoring the silver surface in alkaline medium. However, 6MP SAMs turned to the same upright orientation on the electrode through the S and N7 atoms when either acid or basic solution was removed. Stability of 6MP SAMs was observed by in situ SERS spectroelectrochemical measurements. The results reveal that the desorption potentials of 6MP SAMs formed under acid and alkaline conditions from the Ag electrode were at ca. -1.3 V and -1.6 V vs SCE, respectively.     

DNA-modified electrodes; part 4: optimization of covalent immobilization of DNA on self-assembled monolayers

The covalent immobilization of DNA onto self-assembled monolayer (SAM) modified gold electrodes (SAM/Au) was studied by X-ray photoelectron spectrometry and electrochemical method so as to optimize its covalent immobilization on SAMs. Three types of SAMs with hydroxyl, amino, and carboxyl terminal groups, respectively, were examined. Results obtained by both X-ray photoelectron spectrometry and cyclic voltammetry show that the largest covalent immobilization amount of dsDNA could be gained on hydroxyl-terminated SAM/Au. The ratio of amount of dsDNA immobilized on hydroxyl-terminated SAMs to that on carboxyl-terminated SAMs and to that on amino-terminated SAMs is (3-3.5): (1-1.5): 1. The dsDNA immobilized covalently on hydroxyl-terminated SAMs accounts for 82.8-87.6% of its total surface amount (including small amount of dsDNA adsorbed). So the hydroxyl-terminated SAM is a good substrate for the covalent immobilization of dsDNA on gold surfaces.     

Voltammetry of immobilized cytochrome c on novel binary self-assembled monolayers of thioctic acid and thioctic amide modified gold electrodes

Horse heart cytochrome c (cyt c) was adsorbed on the binary self-assembled monolayers (SAMs) composed of thioctic acid (T-COOH) and thioctic amide (T-NH₂) at gold electrodes via electrostatic interaction. The cyt c adsorbed on the modified gold electrode exhibited well-defined reversible electrochemical behavior in 10 mM phosphate buffer solution (PBS, pH 7.0). The surface concentration (Γ) of electroactive species, cyt c, on the binary SAMs was higher than that in single-component SAMs of T-COOH, and reached a maximum value of 9.2 × 10⁻¹² mol cm⁻² when the ratio of T-COOH to T-NH₂ in adsorption solution was of 3:2, and the formal potential (E^0′=(E_pa+E_pc)/2) of cyt c was −0.032 V (vs. Ag|AgCl (3 M NaCl)) in a 10 mM PBS. The interaction between cyt c and the binary SAMs made the E^0′ shift negatively when compared with that of cyt c in solution (+0.258 V vs. NHE, i.e., +0.058 V vs. Ag|AgCl (3 M NaCl)). The fractional coverage of bound cyt c was a 0.64 theoretical monolayer. The standard electron transfer rate constant of cyt c immobilized on the binary SAMs was also higher than that on single-component SAMs of T-COOH, and the maximum value of 15.8 ± 0.6 s⁻¹ was obtained when the ratio of T-COOH to T-NH₂ in adsorption solution was at 3:2. The results suggest that the electrode modified with the binary SAMs functions better than the electrode modified with single-component SAMs of T-COOH.     

The [Ru(CN)5(pyS)](4-) complex, an efficient self-assembled monolayer for the cytochrome c heterogeneous electron transfer studies

The organothiol 4-mercaptopyridine (pyS) has been used extensively as facilitator for the assessment of heterogeneous electron transfer reaction of cytochrome c (cyt c). Its efficiency, however, is strongly affected by the instability of the adlayer due to the C-S bond cleavage. The K(4)[Ru(CN)(5)(pyS)].3H(2)O complex was synthesized and characterized aiming its utilization as an inorganic self-assembled monolayer (SAM) that would enhance the gold adlayer stability. The SAM formed by this complex onto gold (RupySAu) was characterized by spectroscopic (FTIRRAS and SERS) and electrochemical (LSV) techniques. The ex situ vibrational SERS and FTIRRAS spectra data of this SAM formed onto gold suggest a sigma interaction between the gold and sulfur atoms of the complex, inducing a perpendicular arrangement in relation to the surface normal. Additionally, SERS and FTIRRAS spectra performed for freshly prepared RupySAu adlayer and for large immersion times in the precursor solution have not shown any significant change that would reflect the degradation of the adlayer. The LSV desorption curves of this SAM indicate an enhancement in the C-S bond strength of the pyS ligand when coordinated to the [Ru(CN)(5)](3-) moiety. Comparatively to the data obtained for the desorption process of the pyS monolayer, the reductive desorption potential, E(rd), of the RupySAu presents a shift of -17 mV. This bond strength intensification leads to an increase in the stability of the monolayer. The voltammetric curves of cyt c carried out with the RupySAu electrode showed electrochemical parameters consistent with those reported for the native protein, as well as the maintenance of the electrochemical kinetic data after repetitive cycles. The results all together suggest that the pi back-bonding effect from the [Ru(CN)(5)](3-) metal center plays an important role in the stability of the RupySAu adlayer, improving the assessment of the cyt c heterogeneous electron transfer reaction.     

The Electrochemistry of Cytochrome c at a Viologen-thiol Self-Assembled Monolayer

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Factors that determine the protein resistance of oligoether self-assembled monolayers --internal hydrophilicity,terminal hydrophilicity,and lateral packing density

Protein resistance of oligoether self-assembled monolayers (SAMs) on gold and silver surfaces has been investigated systematically to elucidate structural factors that determine whether a SAM will be able to resist protein adsorption. Oligo(ethylene glycol) (OEG)-, oligo(propylene glycol)-, and oligo(trimethylene glycol)-terminated alkanethiols with different chain lengths and alkyl termination were synthesized as monolayer constituents. The packing density and chemical composition of the SAMs were examined by XPS spectroscopy; the terminal hydrophilicity was characterized by contact angle measurements. IRRAS spectroscopy gave information about the chain conformation of specific monolayers; the amount of adsorbed protein as compared to alkanethiol monolayers was determined by ellipsometry. We found several factors that in combination or by themselves suppress the protein resistance of oligoether monolayers. Monolayers with a hydrophobic interior, such as those containing oligo(propylene glycol), show no protein resistance. The lateral compression of oligo(ethylene glycol) monolayers on silver generates more highly ordered monolayers and may cause decreased protein resistance, but does not necessarily lead to an all-trans chain conformation of the OEG moieties. Water contact angles higher than 70 degrees on gold or 65 degrees on silver reduce full protein resistance. We conclude that both internal and terminal hydrophilicity favor the protein resistance of an oligoether monolayer. It is suggested that the penetration of water molecules in the interior of the SAM is a necessary prerequisite for protein resistance. We discuss and summarize the various factors which are critical for the functionality of "inert" organic films.     

Electron transfer and electrocatalytic properties of the immobilized methionine80alanine cytochrome c variant

The M80A variant of yeast iso-1-cytochrome c (cytc), which features a noncoordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM) and investigated by cyclic voltammetry at varying conditions of temperature, pH, and O2 concentration. The E degrees ' value (standard reduction potential for the heme Fe(III)/Fe(II) couple) of M80A cytc on both SAMs is of approximately -200 mV (vs the standard hydrogen electrode, SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous electron transfer (ET) are dominated by the presence of a hydroxide ion as the sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is mainly responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range of 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.     

Formation of a protein monomolecular layer by a combined technique of LB and SA methods

This paper describes a technique developed for the formation of the self-assembled protein monomolecular layer. The main idea is a direct transfer of protein molecules consisting of a Langmuir-Blodgett (LB) film onto the surface of another chemically modified metal substrate and induction of the spontaneous formation of a self-assembled monolayer (SAM) by chemisorption without protein aggregates. In the present experiments, a cytochrome c (cyt c) SAM on a gold substrate was prepared by incubating a EDC/MUA-modified gold substrate overlaid with a cyt c LB film in a phosphate buffer solution. Scanning tunneling microscopy (STM) image of a cyt c SAM shows that the size of cyt c clusters in the most part of the substrate is approximately 5 nm, indicating the cyt c monomolecular layer. The rectifying property of a cyt c monolayer was confirmed to remain by an asymmetric I-V curve in the applied bias from -1 V to 1 V.     

Modulation of direct electron transfer of cytochrome c by use of a molecularly imprinted thin film

We describe the preparation of a molecularly imprinted polymer film (MIP) on top of a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold, where the template cytochrome c (cyt c) participates in direct electron transfer (DET) with the underlying electrode. To enable DET, a non-conductive polymer film is electrodeposited from an aqueous solution of scopoletin and cyt c on to the surface of a gold electrode previously modified with MUA. The electroactive surface concentration of cyt c was 0.5 pmol cm^?2. In the absence of the MUA layer, no cyt c DET was observed and the pseudo-peroxidatic activity of the scopoletin-entrapped protein, assessed via oxidation of Ampliflu red in the presence of hydrogen peroxide, was only 30 % of that for the MIP on MUA. This result indicates that electrostatic adsorption of cyt c by the MUA–SAM substantially increases the surface concentration of cyt c during the electrodeposition step, and is a prerequisite for the productive orientation required for DET. After template removal by treatment with sulfuric acid, rebinding of cyt c to the MUA–MIP-modified electrode occurred with an affinity constant of 100,000 mol^?1 L, a value three times higher than that determined by use of fluorescence titration for the interaction between scopoletin and cyt c in solution. The DET of cyt c in the presence of myoglobin, lysozyme, and bovine serum albumin (BSA) reveals that the MIP layer suppresses the effect of competing proteins.     

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.