Bilayer lipid membrane formation on surface assemblies with sparsely distributed tethers.

A combined computational and experimental study of small unilamellar vesicle (SUV) fusion on mixed self-assembled monolayers (SAMs) terminated with different deuterated tether moieties (-(CD2)7CD3 or -(CD2)15CD3) is reported. Tethered bilayer lipid membrane (tBLM) formation of synthetic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine was initially probed on SAMs with controlled tether (d-alkyl tail) surface densities and lateral molecular packing using quartz crystal microbalance with dissipation monitoring (QCM-D). Long time-scale coarse-grained molecular dynamics (MD) simulations were then employed to elucidate the mechanisms behind the interaction between the SUVs and the different phases formed by the -(CD2)7CD3 and -(CD2)15CD3 tethers. Furthermore, a series of real time kinetics was recorded under different osmotic conditions using QCM-D to determine the accumulated lipid mass and for probing the fusion process. It is shown that the key factors driving the SUV fusion and tBLM formation on this type of surfaces involve tether insertion into the SUVs along with vesicle deformation. It is also evident that surface densities of the tethers as small as a few mol% are sufficient to obtain stable tBLMs with a high reproducibility. The described "sparsely tethered" tBLM system can be advantageous in studying different biophysical phenomena, such as membrane protein insertion, effects of receptor clustering, and raft formation.

Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices.

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase (PTK2) and activated ACTC1 and TNNT2. Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds.

PURPOSE: To investigate the efficacy of recombinant human collagen type I (RHC I) and collagen-like peptide (CLP) hydrogels as alternative carrier substrates for the cultivation of limbal epithelial stem cells (LESC) under xeno-free culture conditions. METHODS: Human LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified RHC I, (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked CLP (CLP-12 EDC), (4) DMTMM- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium-) crosslinked CLP (CLP-12), (5) fibronectin-patterned CLP-12, (6) "3D limbal niche-mimicking" CLP-12, and (7) DMTMM-crosslinked CLP made from higher CLP concentration solution. Cell proliferation, cell morphology, and expression of LESC markers were analyzed. All data were compared to cultures on human amniotic membrane (HAM). RESULTS: Human LESC were successfully cultivated on six out of seven hydrogel formulations, with primary cell cultures on CLP-12 EDC being deemed unsuccessful since the area of outgrowth did not meet quality standards (i.e., inconsistence in outgrowth and confluence) after 14 days of culture. Upon confluence, primary LESC showed high expression of the stem cell marker ΔNp63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-ß4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-α1, and collagen type IV. Cells showed low expression of differentiation markers KRT3 and desmoglein 3 (DSG3). Significantly higher gene expression of KRT3 was observed for cells cultured on CLP hydrogels compared to RHC I and HAM. Surface patterning of hydrogels influenced the pattern of proliferation but had no significant effect on the phenotype or genotype of cultures. Overall, the performance of RHC I and DMTMM-crosslinked CLP hydrogels was equivalent to that of HAM. CONCLUSION: RHC I and DMTMM-crosslinked CLP hydrogels, irrespective of surface modification, support successful cultivation of primary human LESC using a xeno-free cultivation protocol. The regenerated epithelium maintained similar characteristics to HAM-based cultures.

A Tautoleptic Approach to Chiral Hydrogen-Bonded Supramolecular Tubular Polymers with Large Cavity.

A new strategy towards tubular hydrogen-bonded polymers based on the self-assembly of isocytosine tautomers in orthogonal directions is proposed and experimentally verified, including by 1 H fast magic-angle spinning (MAS) solid-state NMR. The molecular tubes obtained possess large internal diameter and tailor-made outer functionalities rendering them potential candidates for a number of applications.

Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications.

The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes for the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500µm thick RHCIII-MPC constructs comprising over 85% water are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, unlike the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30µm wide fibronectin stripes enhanced cell attachment and showed the highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas.

Nanobiochips.

The actual progress towards biological chip devices consisting of nanostructured functional entities is summarized. The practical aspects of molecular nanobiochips are discussed, including the main surface chemistry platforms, as well as conventional and unconventional fabrication tools. Several successful biological demonstrations of the first generation of nanobiochip devices (mainly, different nanoarrays) are highlighted with the aim of revealing the potential of this technology in life sciences, medicine, and related areas.

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules.

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.

Long-chain alkylthiol assemblies containing buried in-plane stabilizing architectures.

A series of alkylthiol compounds were synthesized to study the formation and structure of complex self-assembled monolayers (SAMs) consisting of interchanging structural modules stabilized by intermolecular hydrogen bonds. The chemical structure of the synthesized compounds, HS(CH(2))(15)CONH(CH(2)CH(2)O)(6)CH(2)CONH-X, where X refers to the extended chains of either -(CH(2))(n)CH(3) or -(CD(2))(n)CD(3), with n = 0, 1, 7, 8, 15, was confirmed by NMR and elemental analysis. The formation of highly ordered, methyl-terminated SAMs on gold from diluted ethanolic solutions of these compounds was revealed using contact angle goniometry, null ellipsometry, cyclic voltammetry, and infrared reflection absorption spectroscopy. The experimental work was complemented with extensive DFT modeling of infrared spectra and molecular orientation. New assignments were introduced for both nondeuterated and deuterated compounds. The latter set of compounds also served as a convenient tool to resolve the packing, conformation, and orientation of the buried and extended modules within the SAM. Thus, it was shown that the lower alkyl portion together with the hexa(ethylene glycol) portion is stabilized by the two layers of lateral hydrogen bonding networks between the amide groups, and they provide a structurally robust support for the extended alkyls. The presented system can be considered to be an extension of the well-known alkyl SAM platform, enabling precise engineering of nanoscopic architectures on the length scale from a few to approximately 60 A for applications such as cell membrane mimetics, molecular nanolithography, and so forth.

Protein-protein interactions in reversibly assembled nanopatterns.

We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures.

Self-assembled monolayers containing terminal mono-, bis-, and tris-nitrilotriacetic acid groups: characterization and application.

We have undertaken a structural and functional study of self-assembled monolayers (SAMs) formed on gold from a series of alkylthiol compounds containing terminal multivalent chelators (MCHs) composed of mono-, bis-, and tris-nitrilotriacetic acid (NTA) moieties. SAMs were formed from single-component solutions of the mono-, bis-, and tris-NTA compounds, as well as from mixtures with a tri(ethylene glycol)-terminated alkylthiol (EG(3)). Contact angle goniometry, null ellipsometry, and infrared spectroscopy were used to explore the structural characteristics of the MCH SAMs. Ellipsometric measurements show that the amount of the MCH groups on surfaces increases with increasing mol % of the MCH thiols in the loading solution up to about 80 mol %. We also conclude that mixed SAMs, prepared in the solution composition regime 0-30 mol % of the MCH thiols, consist of a densely packed alkyl layer, an amorphous ethylene glycol layer, and an outermost layer of MCH groups exposed toward the ambient. Above 30 mol %, a significant degree of disorder is observed in the SAMs. Finally, functional evaluation of the three MCH SAMs prepared at 0-30 mol% reveals a consistent increase in binding strength with increasing multivalency. The tris-NTA SAM, in particular, is enabled for stable and functional immobilization of a His6-tagged extracellular receptor subunit, even at low chelator surface concentrations, which makes it suitable for applications when a low surface density of capturing sites is desirable, e.g., in kinetic analyses.

Piezo dispensed microarray of multivalent chelating thiols for dissecting complex protein-protein interactions.

The fabrication of a novel biochip, designed for dissection of multiprotein complex formation, is reported. An array of metal chelators has been produced by piezo dispensing of a bis-nitrilotriacetic acid (bis-NTA) thiol on evaporated gold thin films, prestructured with a microcontact printed grid of eicosanethiols. The bis-NTA thiol is mixed in various proportions with an inert, tri(ethylene glycol) hexadecane thiol, and the thickness and morphological homogeneity of the dispensed layers are characterized by imaging ellipsometry before and after back-filling with the same inert thiol and subsequent rinsing. It is found that the dispensed areas display a monotonic increase in thickness with increasing molar fraction of bis-NTA in the dispensing solution, and they are consistently a few Angströms thicker than those prepared at the same molar fraction by solution self-assembly under equilibrium-like conditions. The bulkiness of the bis-NTA tail group and the short period of time available for chemisorption and in-plane organization of the dispensed thiols are most likely responsible for the observed difference in thickness. Moreover, the functional properties of this biochip are demonstrated by studying multiple protein-protein interactions using imaging surface plasmon resonance. The subunits of the type I interferon receptor are immobilized as a composition array determined by the surface concentration of bis-NTA in the array elements. Ligand dissociation kinetics depends on the receptor surface concentration, which is ascribed to the formation of a ternary complex by simultaneous interaction of the ligand with the two receptor subunits. Thus, multiplexed monitoring of binding phenomena at various compositions (receptor densities) offers a powerful tool to dissect protein-protein interactions.

Selective recruitment of membrane protein complexes onto gold substrates patterned by dip-pen nanolithography.

Dip-pen nanolithography (DPN) is employed to develop a generic array platform for the selective recruitment of membrane protein complexes. An atomic force microscope tip inked with HS(CH2)16NH2 is used to generate amino-terminated domains on gold. These domains can be arranged into microscopic and submicroscopic patterns, and the untreated gold substrate is subsequently blocked with HS(CH2)2CONH(CH2CH2O)15CH3, a compound known to resist the unspecific binding of proteins and cells. The patterned gold substrate is exposed to an enriched membrane fraction from mutant Rhodobacter sphaeroides, which contains photosynthetic core complexes consisting of the reaction center and the light-harvesting complex LH1. The selective recruitment to the patterned domains, governed primarily by electrostatic interactions, is confirmed by contact mode atomic force microscopy.

Ice nucleation and phase behavior on oligo(ethylene glycol) and hydroxyl self-assembled monolayers: simulations and experiments.

The nucleation and phase behavior of ultrathin D2O-ice overlayers have been studied on oligo(ethylene glycol) (OEG)-terminated and hydroxyl self-assembled monolayers (SAMs) at low temperatures in ultrahigh vacuum. Infrared reflection-absorption spectroscopy (IRAS) is used to characterize the ice overlayers, the SAMs, and the interactions occurring between the ice and the SAM surfaces. Spectral simulations, based on optical models in conjunction with Maxwell Garnett effective medium theory, point out the importance of including voids in the modeling of the ice structures, with void fractions reaching 60% in some overlayers. The kinetics of the phase transition from amorphous-like to crystalline-like ice upon isothermal annealing at 140 K is found to depend on the conformational state of the supporting OEG SAM surface. The rate is fast on the helical OEG SAMs and slow on the corresponding all-trans SAMs. This difference in kinetics is most likely due to a pronounced D2O interpenetration and binding to the all-trans segments of the ethylene glycol portion of the SAM. No such penetration and binding was observed on the helical OEG SAM.

Structural and kinetic properties of laterally stabilized, oligo(ethylene glycol)-containing alkylthiolates on gold: a modular approach.

The formation of highly ordered self-assembled monolayers (SAMs) on gold from an unusually long and linear compound HS(CH(2))(15)CONH(CH(2)CH(2)O)(6)CH(2)CONH(CH(2))(15)CH(3) is investigated by contact angle goniometry, ex situ null ellipsometry, cyclic voltammetry and infrared reflection-absorption spectroscopy. The molecules are found to assemble in an upright position as a complete monolayer within 60 min. The overall structure of the SAM reaches equilibrium within 24 h as evidenced by infrared spectroscopy, although a slight improvement in water contact angles is observed over a period of a few weeks. The resulting SAM is 60 A thick and it displays an advancing water contact angle of 112 degrees and excellent electrochemical blocking characteristics with typical current densities about 20 times lower as compared to those observed for HS(CH(2))(15)CH(3) SAMs. The dominating crystalline phases of the supporting HS(CH(2))(15) and terminal (CH(2))(15)CH(3) alkyl portions, as well as the sealed oligo(ethylene glycol) (OEG) "core," appear as unusually sharp features in the infrared spectra at room temperature. For example, the splitting seen for the CH(3) stretching and CH(2) scissoring peaks is normally only observed for conformationally trapped alkylthiolate SAMs at low temperatures and for highly crystalline polymethylenes. Temperature-programmed infrared spectroscopy in ultrahigh vacuum reveals a significantly improved thermal stability of the SAM under investigation, as compared to two analogous OEG derivatives without the extended alkyl chain. Our study points out the advantages of adopting a "modular approach" in designing novel SAM-forming compounds with precisely positioned in plane stabilizing groups. We demonstrate also the potential of using the above set of compounds in the fabrication of "hydrogel-like" arrays with controlled wetting properties for application in the ever-growing fields of protein and cell analysis, as well as for bioanalytical applications.

High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: a generic platform for protein chip technologies.

Protein micro-/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressable protein immobilization on solid supports. Here we present multivalent metal-chelating thiols that are suitable for stable binding of histidine-tagged proteins on biocompatible self-assembled monolayers (SAMs). The architectures and physicochemical properties of these SAMs have been probed by various surface-sensitive techniques such as contact angle goniometry, ellipsometry, and infrared reflection-absorption spectroscopy. The specific molecular organization of proteins and protein complexes was demonstrated by surface plasmon resonance, confocal laser scanning, and atomic force microscopy. In contrast to the mono-NTA/His6 tag interaction, which has major drawbacks because of its low affinity and fast dissociation, drastically improved stability of protein binding by these multivalent chelator surfaces was observed. The immobilized histidine-tagged proteins are uniformly oriented and retain their function. At the same time, proteins can be removed from the chip surface under mild conditions (switchability). This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies.

First-principle DFT and MP2 modeling of infrared reflection-absorption spectra of oriented helical ethylene glycol oligomers.

First-principle modeling is used to obtain a comprehensive understanding of infrared reflection absorption (RA) spectra of helical oligo(ethylene glycol) (OEG) containing self-assembled monolayers (SAMs). Highly ordered SAMs of methyl-terminated 1-thiaoligo(ethylene glycols) [HS(CH2CH2O)(n)CH3, n = 5, 6] on gold recently became accessible for systematic infrared analyses [Vanderah et al., Langmuir, 2003, 19, 3752]. We utilized the quoted experimental data to validate the first-principle modeling of infrared RA spectra of HS(CH2CH2O)(5,6)CH3 obtained by (i) DFT methods with gradient corrections (using different basis sets, including 6-311++G) and (ii) HF method followed by a Møller-Plesset (MP2) correlation energy correction. In focus are fundamental modes in the fingerprint and CH-stretching regions. The frequencies and relative intensities in the calculated spectra for a single molecule are unambiguously identified with the bands observed in the experimental RA spectra of the corresponding SAMs. In addition to confirming our earlier assignment of the dominating peak in the CH-stretching region to CH2 asymmetric stretching vibrations, all other spectral features observed in that region have received an interpretation consistent (but not in all cases coinciding) with previous investigations. The obtained results provide an improved understanding of the orientation and conformation of the molecular building blocks within OEG-containing assemblies, which, in our opinion, is crucial for being able to predict the folding and phase characteristics and interaction of OEG-SAMs with water and proteins.