Hydroxyl-terminated Self-assembled Monolayers Research Articles

Molecular Understanding of the Distinction between Adhesive Failure and Cohesive Failure in Adhesive Bonds with Epoxy Resin Adhesives.

In the development of adhesives, an understanding of the fracture behavior of the bonded joints is inevitable. Two typical failure modes are known: adhesive failure and cohesive failure. However, a molecular understanding of the cohesive failure process is not as advanced as that of the adhesive failure process. In this study, research was developed to establish a molecular understanding of cohesive failure using the example of a system in which epoxy resin is bonded to a hydroxyl-terminated self-assembled monolayer (SAM) surface. Adhesive failure was modeled as a process in which an epoxy molecule is pulled away from the SAM surface. Cohesive failure, on the other hand, was modeled as the process of an epoxy molecule separating from another epoxy molecule on the SAM surface or breaking of a covalent bond within the epoxy resin. The results of the simulations based on the models described above showed that the results of the calculations using the model of cohesive failure based on the breakdown of intermolecular interactions agreed well with the experimental results in the literature. Therefore, it was suggested that the cohesive failure of epoxy resin adhesives is most likely due to the breakdown of intermolecular interactions between adhesive molecules. We further analyzed the interactions at the adhesive failure and cohesive failure interfaces and found that the interactions at the cohesive failure interface are mainly accounted for by dispersion forces, whereas the interactions at the adhesive failure interface involve not only dispersion forces but also various chemical interactions, including hydrogen bonds. The selectivity between adhesive failure and cohesive failure was explained by the fact that varying the functional group density affected the chemical interactions but not the dispersion forces.

Controllable functionalization of hydroxyl-terminated self-assembled monolayers via catalytic oxa-Michael reaction.

Traditional strategies for the functionalization of materials displaying hydroxyl groups either require active esterification reagents or involve the nucleophilic attack of the hydroxyl group toward electrophilic groups. The former tends to hydrolyze in aqueous solutions while the latter occurs under harsh conditions. Herein, the authors reported a new method for the functionalization of hydroxyl groups on the surface via catalytic oxa-Michael addition with vinyl sulfones. Using hydroxyl group terminated self-assembled monolayers as a model surface, a series of organocatalysts were screened and triphenylphosphine stood out for the best catalytic activity. The catalytic reaction on the surface was characterized by x-ray photoelectron spectroscopy. The information of reaction kinetics was obtained using static water contact angle measurements. Once conjugated with ligands onto the functionalized surfaces, the multivalence binding of proteins was investigated by quartz crystal microbalance experiments. By varying the reaction conditions, e.g., catalyst types and reaction times, ligands can be anchored with a controllable density, which would be helpful to establish the relationships between ligand density and bioactivity.

Computer simulations of underwater oil adhesion of self-assembled monolayers on Au (111)

ABSTRACTMolecular dynamics and steered molecular dynamics (SMD) simulations are used to investigate the underwater adhesive forces of 1, 2-dichloroethane (DCE) on ionic and non-ionic self-assembled monolayers (SAMs) grafted on Au (111). The non-ionic SAMs containing hydroxyl group include ethanolamine-terminated SAM (ETA-SAM), dopamine-terminated SAM (DOPA-SAM), hydroxyl-terminated SAM (OH-SAM) and oligo(ethylene glycol)-terminated SAM (OEG-SAM); the ionic SAMs contain mixed-charged zwitterionic SAM and zwitterionic phosphorylcholine-terminated SAM . Simulation results show that the order of underwater oleophobicity of SAMs is OEG-SAM < ETA-SAM < OH-SAM < DOPA-SAM < < , while the order of underwater DCE oil droplet adhesion forces on different SAMs is < < ETA-SAM < DOPA-SAM < OH-SAM < OEG-SAM. The ionic SAMs show stronger underwater oleophobicity and lower oil adhesion than non-ionic SAMs. Generally, a more underwater oleophobic surface is less adhesive. The underwater oil droplet desorption behaviours on ionic and non-ionic SAMs are also different. With the oil droplet being pulled away from ionic SAMs, the oil contact lines gradually shortened and disappeared finally; whereas the oil contact lines on non-ionic SAMs almost kept an initial state even the main part of oil droplet separated from the SAMs. This work systematically proved that a zwitterionic surface was less underwater oil adhesive than a non-ionic surface and provided a new insight for their oil desorption behaviours. The above results will be helpful for the design of underwater oleophobic surfaces with tuneable adhesion.

Copper oxide surfaces modified by alkylphosphonic acids with terminal pyridyl-based ligands as a platform for supported catalysis

Self-assembled monolayer (SAM) films of phosphonates have been successfully formed via reaction of 11-hydroxyundecylphosphonic acid or 4,4′-di(methylenephosphonic acid)-2,2′-bipyridine with the oxide layer of copper via the Tethering by Aggregation and Growth (TBAG) deposition method. The hydroxyl-terminated SAM was further modified with isonicotinic acid or 4,4′-dicarboxy-2,2′-bipyridine through a Steglich esterification reaction. These three surfaces derivatized with pyridyl-based ligands are potential platforms for supported catalysis. As a proof of concept, [Ru(CO)3Cl2]2 was bound to the surfaces through the pyridyl-based ligands to yield tethered analogs of the known carbon dioxide reduction catalyst, [Ru(bpy)(CO)2Cl2]. Surface modification reactions were confirmed through specular reflectance infrared (IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Characteristic core binding energies were observed in the XPS analyses for phosphorus (P 2p), nitrogen (N 1s), and ruthenium (Ru 3p and Ru 3d), verifying the presence of the various surface functionalizations. IR and XPS data indicate that the phosphonate binding to the copper surface is tridentate in nature.

Probing redox reaction of azurin protein immobilized on hydroxyl-terminated self-assembled monolayers with different lengths

Immobilization of proteins with controlled electron transfer properties is an important task for future biomedical applications. In this work, immobilization of azurin on gold electrodes modified with 6-mercaptohexanol (MCH), and 11-mercaptoundecanol (MUD) self-assembled monolayers (SAMs) was studied by electrochemical techniques. The immobilized azurin showed a quasi-reversible redox response on MCH/Au and MUD/Au electrodes, with E1/2=0.11V vs. Ag/AgCl and ∆Ep=10mV and 40mV, respectively. Cyclic voltammograms (CVs) of azurin at different pHs revealed that proton-coupled redox reaction of azurin is a one-electron, three-proton process. The tunneling electron transfer rate constant (k0) of immobilized azurin on MCH/Au electrode was estimated 1.35 (±0.05)×103s−1 using a fast scan CV method, and that on MUD/Au electrode was measured as 120±10s−1 by scanning electrochemical microscopy (SECM). In SECM studies, the approach curves were recorded at different substrate overpotentials with different surface coverages of azurin. For extraction of kinetic parameters, azurin was reduced on MUD/Au electrode via tunneling electron transfer across the SAM, and oxidized back via solution-phase tip-generated ferrocyanide. The bimolecular electron transfer rate constant (kBI) between immobilized azurin and [Fe(CN)6]4− mediator was estimated as 3×108mol−1cm3s−1. Based on distribution of hydrophilic and hydrophobic amino acids on azurin structure, and the distinct voltammetric behavior of azurin on MCH and MUD SAMs as hydroxyl-terminated SAMs vs. methyl- and thiol-terminated SAMs, possible orientations of azurin on different SAMs were also assessed.

Structure of aqueous water films on textured -OH-terminated self-assembled monolayers.

We report the thickness and interfacial molecular structure of thin (1-3 nm) aqueous films supported on hydroxyl-terminated self-assembled monolayers over a silver substrate. The water film structure is studied as a function of varying the monolayer's methylene chain lengths. Analysis techniques include ellipsometry, contact angle, and polarization modulation reflection adsorption infrared spectroscopy. The aqueous film thicknesses follow 4-mercaptobutanol (4-MBU) > 11-mercaptoundecanol (11-MUD) > 6-mercaptohexanol (6-MHE) > 9-mercaptononanol (9-MNO). Water contact angle measurements across the same surfaces are very similar; however, vibrational spectroscopic analysis of the films shows that intermolecular bonding patterns of D2O are significantly different from those of bulk D2O. This evokes unique interfacial molecular architectures for each of these films. The structural differences depend on the nature of the SAM structure and resulting water-SAM interactions, which are evident from PM-IRRAS data. Spectroscopic peak intensity ratios of ν(O-D) modes suggest more asymmetric hydrogen-bonded D2O character near 9-MNO surfaces, whereas 4-MDU, 6-MHE, and 11-MUD surfaces exhibit increasingly symmetric hydrogen-bonded D2O character. From this, we propose a model for film structure.

Modulation of cyanobacterial photosystem I deposition properties on alkanethiolate Au substrate by various experimental conditions

We present results from atomic force microscopy (AFM) images indicating various experimental conditions, which alter the morphological characteristics of self-assembled cyanobacterial PS I on hydroxyl-terminated self-assembled alkanethiolate monolayers (SAM/Au) substrates. AFM topographical images of SAM/Au substrates incubated in solutions containing different PS I concentrations solubilized with Triton X-100 as the detergent reveal large columnar aggregates (∼100 nm and hence, much taller than a single PS I trimer) at high PS I concentrations. Depositions from dilute PS I suspensions reveal fewer aggregates and relatively uniform surface topography (∼10 nm). Confocal fluorescence microscopy analysis of fluorescently tagged PS I deposited on to SAM/Au substrates using electric field and gravity driven techniques reveal preliminary indications of directionally aligned PS I attachments, besides corroborating a uniform monolayer formation, for the former deposition method. The complex attachment dynamics of PS I onto SAM substrates are further investigated from the AFM images of PS I/SAM/Au substrates prepared under different experimental conditions using: 1) PS I isolated as monomers and trimers 2) adsorption at elevated temperatures, and 3) different detergents with varying pH values. In each of the cases, the surface topology indicated distinct yet complex morphological and phase characteristics. These observations provide useful insight into the use of experimental parameters to alter the morphological assembly of PS I on to SAM substrates en route to successful fabrication of PS I based biohybrid photoelectrochemical devices.

Hyperthermal Collision-induced Dissociation of Bromotoluene Radical Cations at Self-Assembled Monolayer Surfaces

Hyperthermal ion/surface collisions of bromotoluene radical cations were studied using perfluorinated (F-SAM) and hydroxyl-terminated (OH-SAM) self-assembled monolayer surfaces in a tandem mass spectrometer with BEEQ geometry. The isomers were differentiated by ion abundance ratios taken from surface-induced dissociation (SID). The dissociation rate followed the order of ortho > meta > para isomers. The peak abundance ratio of m/z 51 to m/z 65 showed the best result to discern the isomers. A dissociation channel leading to tolylium ion was suggested to be responsible for the pronounced isomeric differences. The capability of SID to provide high-energy activation with narrow internal energy distribution may have channeled the reaction into the specific dissociation pathway, also facilitating small differences in reaction rates to be effective in the spectral time window of this experiment. All of the molecular ions experiencing reactive collisions with the F-SAM surface undergo transhalogenation, in which a fluorine atom on the surface replaces the bromine in the incoming ions. This reactive collision was dependent on the laboratory collision energy occurring in ca. 40.75 eV range.

Soft-Landing Deposition of Al17− on a Hydroxyl-Terminated Self-Assembled Monolayer

Small aluminum clusters (<30 atoms) have been the subject of extensive study, demonstrating markedly different properties as even a single atom is added or removed. Successfully depositing and characterizing mass-selected clusters are the next steps in producing precise surfaces or cluster-assembled materials that demonstrate properties that differ from those of bulk materials. This proves to be difficult due to the reactivity of these all-metal clusters and their susceptibility to agglomerate in bulk form. In the present study, we have successfully deposited Al17− onto self-assembled monolayers using reactivity previously characterized in the gas phase.

Wetting of hydrophobic substrates by nanodroplets of aqueous trisiloxane and alkyl polyethoxylate surfactant solutions

Abstract Trisiloxane surfactants at low concentrations promote the complete and rapid wetting of aqueous droplets on very hydrophobic (hydrocarbon) substrates. This behavior has not been demonstrated by any other surfactant which explains why the trisiloxanes are referred to as superspreaders. Despite many experimental and theoretical investigations the mechanism of superspreading is not fully understood. Molecular dynamics simulations using all-atom force fields have been conducted to attempt to elucidate the mechanism of superspreading. Spherical nanodroplets containing approximately 10,000 water molecules in the bulk and 475 surfactant molecules at the liquid–vapor interface were placed in the vicinity of a graphite substrate and allowed to spread freely at room temperature. In the trisiloxane case the droplet was found to spread very little, although randomly removing 175 surfactant molecules lowered the final contact angle from 110 ∘ to 80 ∘ . In contrast, an alkyl polyethoxylate surfactant-laden droplet was found to spread significantly further, with the equilibrium contact angle reaching 55 ∘ . Similar results for the two surfactant systems were found for cylindrical nanodroplets spreading on a self-assembled monolayer (SAM). The reasons for the lack of spreading in the trisiloxane case and the simulation challenges associated with these systems are discussed. In support of our arguments we demonstrate that the surfactant molecules of an initially uniform aqueous trisiloxane solution self-assemble into a bilayer in tens of nanoseconds on a graphite substrate. Lastly, in a final set of simulations, neat trisiloxane droplets at 450 K are found to arrange into a layered structure on a methyl-terminated SAM and to form a sand pile-shape on a hydroxyl-terminated SAM.

Sulfonation of Surface-Initiated Polynorbornene Films

We report the sulfonation of surface-initiated polynorbornene with acetyl sulfate to produce ultrathin ionomer films. The complete process consists of exposure of a hydroxyl-terminated self-assembled monolayer (SAM) on gold to a norbornenyl diacid chloride, attachment of Grubbs first generation catalyst, ring-opening metathesis polymerization (ROMP), and sulfonation. Structural and chemical changes in the film upon sulfonation are confirmed by RAIRS, contact angle goniometry, ellipsometry, optical microscopy, and electrochemical impedance spectroscopy. Sulfonation of surface-initiated polynorbornene results in a highly nonuniform surface morphology which can be relaxed to a more uniform film through exposure to dimethyl sulfoxide at room temperature. The sulfonated polynorbornene films have an intermediate surface energy (θA(H2O) ≈ 75°) and a low resistance against proton transport (Rf ≈ 1.6 Ω·cm2), which is 6 orders of magnitude lower than that of the original polynorbornene film. The sulfonated films are far more stable than the original polynorbornene films because of a ∼95% diminution of olefin content within the film. Sulfonated poly(butylnorbornene) films were prepared analogously to demonstrate the versatility of this approach toward ionomer films.

Drug delivery from gold and titanium surfaces using self-assembled monolayers

Currently available drug-eluting stents (DES) use polymers for coating and releasing drugs. Increasing evidence suggests that inflammatory and hypersensitive reactions are caused by such polymer coatings. This study focused on developing new techniques for delivering drugs directly from metal implant surfaces. Hydroxyl-terminated self-assembled monolayers (SAMs) were coated on Au and Ti surfaces. Therapeutic self-assembled monolayers (TSAMs) were prepared by chemically attaching the model drug, flufenamic acid, to SAM coated metal surfaces. Three different methods of esterification (acid chloride esterification, dry heat esterification, and direct esterification) were explored to attach flufenamic acid to SAMs. TSAMs were characterized using X-ray photoelectron spectroscopy, fluorescence microscopy, atomic force microscopy, and contact angle goniometry. These techniques collectively confirmed the attachment of drug onto SAM coated metal surfaces. In vitro drug release was investigated by immersing TSAM coated metal specimens in tris-buffered saline (TBS) at 37 °C for 28 days. TBS was analyzed at 1, 3, 7, 14, 21, and 28 days for the amount of drug eluted using high performance liquid chromatography. Large data scatter was observed for the release profiles of TSAMs prepared by acid chloride esterification. TSAMs prepared by dry heat and direct esterification methods showed an initial burst release of the drug followed by a sustained slow release for up to 2 weeks. Thus, this study suggests the potential for using self-assembled monolayers as an alternate system for delivering drugs from coronary stents and other metal implants.

Pure and Molecularly Mixed Methyl- and Hydroxyl-Terminated Self-Assembled Dialkylammonium Monolayers on Mica: Wettability and Conformational Order

Muscovite mica surfaces were coated with methyl- and hydroxyl-terminated dialkyldimethylammonium monolayers via ion exchange by immersion of the substrates into solutions of the corresponding salts. The samples were examined by water contact-angle measurements and near edge X-Ray absorption fine structure spectroscopy (NEXAFS). The purely methyl-terminated species was found to form self-assembled monolayers (SAMs) of comparable quality to those of a similar thiol/gold system as has been reported earlier. The hydroxyl-terminated surfactant established films with a lower degree of orientation under the same preparation conditions and showed a high water contact angle value for a hydrophilic surface (47°). The molecularly mixed film where one alkyl chain of the surfactant is methyl terminated and one hydroxyl terminated had a significantly lower wettability but established a similar degree of orientation as the film prepared from the purely OH-terminated species.

Influence of self-assembled monolayer surface chemistry on Candida antarctica lipase B adsorption and specific activity

Immobilization of Candida antarctica B lipase was examined on gold surfaces modified with either methyl- or hydroxyl-terminated self-assembled alkylthiol monolayers (SAMs), representing hydrophobic and hydrophilic surfaces, respectively. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance. Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy. The extent of lipase adsorption was nearly identical on either surface (approximately 240 ng cm −2), but its specific activity was sixfold higher on the methyl-terminated SAM, showing no activity loss upon immobilization. A uniform, 5.5 nm high, highly packed monolayer of CALB formed on the methyl-terminated SAM, while the adsorbed protein was disordered on the hydroxyl-terminated SAM. Hydrophobic surfaces thus may specifically orient the lipase in a highly active state.

Time Lapse Confocal Microscopy Studies of Bacterial Adhesion to Self-Assembled Monolayers and Confirmation of a Novel Approach to the Thermodynamic Model

In this study, we use thermodynamic theory to develop a novel model that allows for the quantitative determination of the Gibbs free energy of adhesion for the initial bacterial attachment process. This model eliminates the need to calculate interfacial free energies and instead relies on easily measurable contact angles to determine DeltaG(adh). We experimentally verify our model using real-time observation of the initial attachment of Pseudomonas putida to methyl- and hydroxyl-terminated self-assembled monolayers. We also test the applicability of our model to a variety of experimental conditions using data available in the literature. We show that the initial attachment process is governed by dispersion forces and is accurately predicted by our model. Also, we find that our model is simple to apply and accurate for a variety of experimental conditions.

Mechanical Stabilization Effect of Water on a Membrane-like System

The penetration resistance of a prototypical model-membrane system (HS-(CH2)11-OH self-assembled monolayer (SAM) on Au(111)) to the tip of an atomic force microscope (AFM) is investigated in the presence of different solvents. The compressibility (i.e., height vs tip load) of the HS-(CH2)11-OH SAM is studied differentially, with respect to a reference structure. The reference consists of hydrophobic alkylthiol molecules (HS-(CH2)17-CH3) embedded as nanosized patches into the hydrophilic SAM by nanografting, an AFM-assisted nanolithography technique. We find that the penetration resistance of the hydrophilic SAM depends on the nature of the solvent and is much higher in the presence of water than in 2-butanol. In contrast, no solvent-dependent effect is observed in the case of hydrophobic SAMs. We argue that the mechanical resistance of the hydroxyl-terminated SAM is a consequence of the structural order of the solvent-SAM interface, as suggested by our molecular dynamics simulations. The simulations show that in the presence of 2-butanol the polar head groups of the HS-(CH2)11-OH SAM, which bind only weakly to the solvent molecules, try to bind to each other, disrupting the local order at the interface. On the contrary, in the presence of water the polar head groups bind preferentially to the solvent that, in turn, mediates the release of the surface strain, leading to a more ordered interface. We suggest that the mechanical stabilization effect induced by water may be responsible for the stability of even more complex, real membrane systems.

Lateral Nanomechanics of Cartilage Aggrecan Macromolecules

To explore the role of the brush-like proteoglycan, aggrecan, in the shear behavior of cartilage tissue, we measured the lateral resistance to deformation of a monolayer of chemically end-attached cartilage aggrecan on a microcontact printed surface in aqueous NaCl solutions via lateral force microscopy. The effects of bath ionic strength (IS, 0.001–1.0 M) and lateral displacement rate (∼1–100 μm/s) were studied using probe tips functionalized with neutral hydroxyl-terminated self-assembled alkanethiol monolayers. Probe tips having two different end-radii ( R ∼50 nm and 2.5 μm) enabled access to different length-scales of interactions (nano and micro). The measured lateral force was observed to depend linearly on the applied normal force, and the lateral force to normal force proportionality constant, μ, was calculated. The value μ increased (from 0.03 ± 0.01 to 0.11 ± 0.01) with increasing bath IS (0.001–1.0 M) for experiments using the microsized tip due to the larger compressive strain of aggrecan that resulted from increased IS at constant compressive force. With the nanosized tip, μ also increased with IS but by a smaller amount due to the fewer number of aggrecan involved in shear deformation. The variations in lateral force as a function of applied compressive strain ɛ n and changes in bath IS suggested that both electrostatic and nonelectrostatic interactions contributed significantly to the shear deformational behavior of the aggrecan layers. While lateral force did not vary with lateral displacement rate at low IS, where elastic-like electrostatic interactions between aggrecan dominated, lateral force increased significantly with displacement rate at physiological and higher IS, suggestive of additional viscoelastic and/or poroelastic interactions within the aggrecan layer. These data provide insights into molecular-level deformation of aggrecan macromolecules that are important to the understanding of cartilage behavior.

Density functional theory studies of reaction mechanisms for titanium alkylamide incorporation onto functionalized aromatic self-assembled monolayers

We have studied the reaction kinetics of the initial layer deposition of titanium-based alkylamide organometallic precursors on aromatic self-assembled monolayers (SAMs) possessing different terminal groups. Using density functional theory and Moller–Plesset (MP2) perturbation theory, we found the initial reaction rate constant for deposition to decrease in the order: OH > SH > NH2, similar to results we found previously for alkyl SAMs. Aromatic amine- and hydroxyl-terminated SAMs were kinetically and thermodynamically slightly more favorable than alkyl counterparts, with a very small (2 kcal mol−1) reduction in activation barrier. In contrast, the reactivity of thiol-terminated aromatic ligands was similar to corresponding alkyl SAMs due to the thiol group's small electronegativity. Branching on the surface functional group reduced the kinetic favorability of both aromatic and alkyl amine-terminated SAMs. Fluorine substitution in the aromatic backbone of amine-terminated SAMs is kinetically and thermodynamically much more favorable than corresponding unsubstituted aromatic SAMs and fluoryl-substituted alkyl SAMs. Investigation of the kinetics of several competing side unimolecular decomposition reactions led to the discovery of new kinetically favorable pathways that are competitive with transamination at temperatures as low as 250 K and which lead to a Ti : N ratio of 1 : 3 in agreement with experiments by Dube et al. (A. Dube, A. R. Chadeayne, M. Sharma, P. T. Wolczanski and J. R. Engstrom, J. Am. Chem. Soc., 2005, 127, 14299–14309). Significant enhancement in kinetic favorability was shown to result from the steric hindrance of large groups.

Electrochemistry and photoelectrochemistry of photosystem I adsorbed on hydroxyl-terminated monolayers

Direct electrochemistry studies on Photosystem I (PSI) were performed using cyclic voltammetry and square wave voltammetry. PSI centers stabilized in aqueous solution by Triton X-100 surfactant were adsorbed on hydroxyl-terminated hexanethiol modified gold electrodes. We have identified the electron donor, P700, and the electron acceptor sites, F A / F B , based on the previously reported preferred orientation for P700 on hydroxyl-terminated self-assembled monolayers. The reported potential values ( E P700 = +0.51 V vs. NHE; E FA/FB = −0.36 V vs. NHE) correlate very well with the established literature for P700, while the weaker signal for F A / F B lies within previous literature values. We were able to identify both redox centers on the same voltammogram. The P700 center clearly shows reversible electrochemical behavior. The expected F A / F B reduction is small in comparison, reflecting the dominant orientation of PSI with the F A / F B centers farther away and the P700 center nearer the electrode surface. As a molecular diode, PSI does not permit reverse direction conductivity to the F B so the small F A / F B peaks reflect other orientations besides the predominant one. PSI adsorbed on a hydroxyl-terminated hexanethiol modified gold substrate displays a photoenhanced reduction current for the P700 + center in the presence of light and an electron acceptor, methyl viologen. This photoelectrochemical response demonstrates protein functionality after adsorption.

Kinetic, Thermodynamic, and Mechanistic Patterns for Free (Unbound) Cytochrome c at Au/SAM Junctions: Impact of Electronic Coupling, Hydrostatic Pressure, and Stabilizing/Denaturing Additives

Combined kinetic (electrochemical) and thermodynamic (calorimetric) investigations were performed for an unbound (intact native-like) cytochrome c (CytC) freely diffusing to and from gold electrodes modified by hydroxyl-terminated self-assembled monolayer films (SAMs), under a unique broad range of experimental conditions. Our approach included: 1) fine-tuning of the charge-transfer (CT) distance by using the extended set of Au-deposited hydroxyl-terminated alkanethiol SAMs [-S-(CH(2))(n)-OH] of variable thickness (n=2, 3, 4, 6, 11); 2) application of a high-pressure (up to 150 MPa) kinetic strategy toward the representative Au/SAM/CytC assemblies (n=3, 4, 6); 3) complementary electrochemical and microcalorimetric studies on the impact of some stabilizing and denaturing additives. We report for the first time a mechanistic changeover detected for "free" CytC by three independent kinetic methods, manifested through 1) the abrupt change in the dependence of the shape of the electron exchange standard rate constant (k(o)) versus the SAM thickness (resulting in a variation of estimated actual CT range within ca. 15 to 25 A including ca. 11 A of an "effective" heme-to-omega-hydroxyl distance). The corresponding values of the electronic coupling matrix element vary within the range from ca. 3 to 0.02 cm(-1); 2) the change in activation volume from +6.7 (n=3), to approximately 0 (n=4), and -5.5 (n=6) cm(3) mol(-1) (disclosing at n=3 a direct pressure effect on the protein's internal viscosity); 3) a "full" Kramers-type viscosity dependence for k(o) at n=2 and 3 (demonstrating control of an intraglobular friction through the external dynamic properties), and its gradual transformation to the viscosity independent (nonadiabatic) regime at n=6 and 11. Multilateral cross-testing of "free" CytC in a native-like, glucose-stabilized and urea-destabilized (molten-globule-like) states revealed novel intrinsic links between local/global structural and functional characteristics. Importantly, our results on the high-pressure and solution-viscosity effects, together with matching literature data, strongly support the concept of "dynamic slaving", which implies that fluctuations involving "small" solution components control the proteins' intrinsic dynamics and function in a highly cooperative manner as far as CT processes under adiabatic conditions are concerned.