The optical properties of amyloid fibers are often distinct from those of the source protein in its non-fibrillar form.These differences can be utilized for label-free imaging or characterization of such structures, which is particularly important for understanding amyloid fiber related diseases such as Alzheimer's and Parkinson's disease.We demonstrate that two amyloid forming proteins, insulin and β-lactoglobulin (β-LG), show intrinsic fluorescence with emission spectra that are dependent on the excitation wavelength.Additionally, a new fluorescence peak at about 430 nm emerges for β-LG in its amyloid state.The shift in emission wavelength is related to the red edge excitation shift (REES), whereas the additional fluorescence peak is likely associated with charge delocalization along the fiber backbone.Furthermore, the spherulitic amyloid plaque-like superstructures formed from the respective proteins were imaged label-free with confocal fluorescence, multiphoton excitation fluorescence (MPEF), and second-harmonic generation (SHG) microscopy.The latter two techniques in particular yield images with a high contrast between the amyloid fiber regions and the core of amorphously structured protein.Strong multiphoton absorption (MPA) for the amyloid fibers is a likely contributor to the observed contrast in the MPEF images.The crystalline fibrillar region provides even higher contrast in the SHG images, due to the inherently ordered non-centrosymmetric structure of the fibers together with their non-isotropic arrangement.Finally, we show that MPEF from the insulin spherulites exhibits a spectral dependence on the excitation wavelength.This behavior is consistent with the REES phenomenon, which we hypothesize is the origin of this observation.The presented results suggest that amyloid deposits can be identified and structurally characterized based on their intrinsic optical properties, which is important for probe-less and label-free identification and characterization of amyloid fibers in vitro and in complex biological samples.

Analyzing molecules at aqueous interfaces in situ, in vitro, or even in vivo without the need for labels and/or disruptive sample preparation is crucial for the understanding and optimization of material’s interactions with its surrounding. In this context, a central theme is the ability to differentiate between molecules in the respective bulk phases and those that are located at the interface. Here we introduce vibrational sum-frequency generation (SFG) spectroscopy, a nonlinear optical technique that is capable to selectively probe molecules at interfaces. SFG spectroscopy can be applied under ex vacuo conditions and allows to record vibrational spectra from molecules at interfaces. Though this technique holds great potential in research themes involving aqueous interfaces, the data analysis of SFG spectra can get quite complex and often requires a comprehensive understanding of the underlying nonlinear optical processes. This chapter introduces experimental and theoretical aspects of SFG spectroscopy with a strong focus on data analysis. It is meant for scientists new to the field of SFG spectroscopy who like to explore its applicability and theoretical background or are starting to apply SFG spectroscopy in their own research.

Conspectus Stimuli-responsive surfaces have sparked considerable interest in recent years, especially in view of their biomimetic nature and widespread biomedical applications. Significant efforts are continuously being directed at developing functional surfaces exhibiting specific property changes triggered by variations in electrical potential, temperature, pH and concentration, irradiation with light, or exposure to a magnetic field. In this respect, electrical stimulus offers several attractive features, including a high level of spatial and temporal controllability, rapid and reverse inducement, and noninvasiveness. In this Account, we discuss how surfaces can be designed and methodologies developed to produce electrically switchable systems, based on research by our groups. We aim to provide fundamental mechanistic and structural features of these dynamic systems, while highlighting their capabilities and potential applications. We begin by briefly describing the current state-of-the-art in integrating electroactive species on surfaces to control the immobilization of diverse biological entities. This premise leads us to portray our electrically switchable surfaces, capable of controlling nonspecific and specific biological interactions by exploiting molecular motions of surface-bound electroswitchable molecules. We demonstrate that our self-assembled monolayer-based electrically switchable surfaces can modulate the interactions of surfaces with proteins, mammalian and bacterial cells. We emphasize how these systems are ubiquitous in both switching biomolecular interactions in highly complex biological conditions while still offering antifouling properties. We also introduce how novel characterization techniques, such as surface sensitive vibrational sum-frequency generation (SFG) spectroscopy, can be used for probing the electrically switchable molecular surfaces in situ. SFG spectroscopy is a technique that not only allowed determining the structural orientation of the surface-tethered molecules under electroinduced switching, but also provided an in-depth characterization of the system reversibility. Furthermore, the unique support from molecular dynamics (MD) simulations is highlighted. MD simulations with polarizable force fields (FFs), which could give proper description of the charge polarization caused by electrical stimulus, have helped not only back many of the experimental observations, but also to rationalize the mechanism of switching behavior. More importantly, this polarizable FF-based approach can efficiently be extended to light or pH stimulated surfaces when integrated with reactive FF methods. The interplay between experimental and theoretical studies has led to a higher level of understanding of the switchable surfaces, and to a more precise interpretation and rationalization of the observed data. The perspectives on the challenges and opportunities for future progress on stimuli-responsive surfaces are also presented.

Vibrational sum-frequency generation (SFG) spectroscopy has become an established technique for in situ surface analysis. While spectral recording procedures and hardware have been optimized, unique data analysis routines have yet to be established. The SFG intensity is related to probing geometries and properties of the system under investigation such as the absolute square of the second-order susceptibility χ((2)) (2). A conventional SFG intensity measurement does not grant access to the complex parts of χ((2)) unless further assumptions have been made. It is therefore difficult, sometimes impossible, to establish a unique fitting solution for SFG intensity spectra. Recently, interferometric phase-sensitive SFG or heterodyne detection methods have been introduced to measure real and imaginary parts of χ((2)) experimentally. Here, we demonstrate that iterative phase-matching between complex spectra retrieved from maximum entropy method analysis and fitting of intensity SFG spectra (iMEMfit) leads to a unique solution for the complex parts of χ((2)) and enables quantitative analysis of SFG intensity spectra. A comparison between complex parts retrieved by iMEMfit applied to intensity spectra and phase sensitive experimental data shows excellent agreement between the two methods.

A 24 factorial design was used to optimize the activators regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP) grafting of sodium styrene sulfonate (NaSS) films from trichlorosilane/10-undecen-1-yl 2-bromo-2-methylpropionate (ester ClSi) functionalized titanium substrates. The process variables explored were: (1) ATRP initiator surface functionalization reaction time; (2) grafting reaction time; (3) CuBr2 concentration; and (4) reducing agent (vitamin C) concentration. All samples were characterized using x-ray photoelectron spectroscopy (XPS). Two statistical methods were used to analyze the results: (1) analysis of variance with [Formula: see text], using average [Formula: see text] XPS atomic percent as the response; and (2) principal component analysis using a peak list compiled from all the XPS composition results. Through this analysis combined with follow-up studies, the following conclusions are reached: (1) ATRP-initiator surface functionalization reaction times have no discernable effect on NaSS film quality; (2) minimum (≤24 h for this system) grafting reaction times should be used on titanium substrates since NaSS film quality decreased and variability increased with increasing reaction times; (3) minimum (≤0.5 mg cm-2 for this system) CuBr2 concentrations should be used to graft thicker NaSS films; and (4) no deleterious effects were detected with increasing vitamin C concentration.

Protein fibers play a crucial role in many disease related phenomena and biological systems. A structural analysis of fibrous proteins often requires labeling approaches or disruptive sample preparation while it lacks chemical specificity. Here we demonstrate that the technique of vibrational sum-frequency scattering (SFS) provides a label-free pathway for the chemical and structural analysis of protein fibers in solution. By examining collagen, the most abundant protein in mammals, we demonstrate that the SFS signal of fibers can be detected in the NH, CH stretching and bending, and amide I regions. SFS spectra were found to depend on the scattering angle, which implies the possibility to selectively probe various features of the fibers. The fitting of the data and maximum entropy method analysis revealed a different phase for side-chains and carbonyl contributions, which helps to identify these otherwise overlapping spectral peaks and provides the possibility to perform orientational analysis. Our findings suggest that SFS allows for the greater understanding of protein fibers in solution, which is important when, for example, designing scaffolds in tissue engineering or developing cures for diseases associated with protein fibers.

High-dielectric constant side-chain polymers that show reduced non-geminate recombination in heterojunction solar cells are reported. Active layer polymers with high dielectric side-chains show increased performance over alkyl side-chain polymers. The doubling in efficiency is attributed to suppressed recombination. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In situ sum-frequency-generation spectroscopy is used for the first time to study changes in molecular orientations in charged biofunctionalized self-assembled monolayers, in response to an applied electrical potential. The findings presented here unravel the mechanism by which charged biomolecules control biomolecular interactions, for example, protein binding affinities, and lay the foundation for future studies aiming to explore molecular conformational changes in response to electrical stimuli. Control and reversibility of biomolecular interactions at engineered interfaces presents opportunities to develop highly efficient substrates and devices for a wide range of biomedical applications.1-4 A major challenge nowadays in the field of stimuli-responsive interfaces is to acquire a molecular understanding of the changes occurring at the biointerface upon external stimulation. Herein, we used in situ Sum-Frequency-Generation (SFG) spectroscopy to study changes in molecular orientations in electrically switchable biofunctionalized self-assembled monolayers (SAMs). The bioactivity of a mixed SAM on gold consisting of a biotin-terminated positively charged oligopeptide (biotin-KKKKC) and a tri(ethylene glycol)-terminated thiol is shown to be related to a switch between upward exposure and random orientation of the biotin group in response to positive and negative applied potentials, respectively. The findings reported here support the mechanism by which charged biomolecules control biomolecular interactions, for example, protein binding affinities, and lay the foundation for future studies aiming to explore molecular conformational changes in response to electrical stimuli. Dynamic surfaces are particularly attractive for biomedical applications and are playing an increasingly important part in the development of highly sensitive biosensors,5-7 novel drug delivery systems,8 and tissue engineering scaffolds.9 Stimuli-responsive SAMs have garnered much interest since they can provide a high level of molecular organization and control over the surface properties.10 To date, stimuli-responsive SAMs have been able to selectively respond to external inputs such as electrical,11-13 temperature,14, 15 pH,16 and light.17, 18 Switchable SAMs used to control biomolecular interactions via an electrical stimulus are particularly appealing because of their fast response times, ease of creating multiple individually addressable switchable regions on the same surface, as well as low-driven voltage and electric fields that are compatible with biological systems.19 Electrically switchable SAMs have been demonstrated to modulate the interactions of surfaces with proteins,11-13 DNA,20, 21 and mammalian19 and bacterial22 cells. For instance, an electrically switchable mixed SAM on gold that comprised a positively charged oligopeptide (biotin-KKKKC) and a shorter tri(ethylene glycol)-terminated thiol (TEGT) (Figure 1) was previously13 demonstrated by us to be able to control the bioactivity of biotin on the surface and its binding to a specific protein, Neutravidin. High protein binding was observed for an applied positive potential (+0.3 V, bio-active state), while minimal binding was detected for an applied negative potential (-0.4 V, bio-inactive state). While the charged molecular backbone or the end group on the structure of the reported electrically switchable SAMs are the hypothesized basis for controlling such biomolecular interactions,23 the mechanistic principles underpinning these electrically-driven systems are not fully experimentally proven. Without such proven mechanistic detail, designing novel biologically relevant surfaces, and understanding the potential and limitations of present ones is haphazard at best. Herein, we address this challenge by studying the biotin-KKKKC:TEGT mixed SAM as a model system and report the first observation of biotin orientations in the charged mixed SAM in response to an applied potential, using in situ SFG spectroscopy. This technique takes advantage of even-order non-linear optical selection rules that precludes signals from isotropic environments or molecular arrangements that possess inversion symmetry.24 At interfaces, the symmetry is necessarily broken making even-order processes, such as second-order SFG, intrinsically surface specific.25 In a typical SFG experiment, IR and visible laser pulses are overlapped in time and space at an interface to eventually generate a sum-frequency signal. Spectra are recorded as a function of IR frequency; SFG signals are resonantly enhanced when the IR light is exciting vibrational states that are both IR and Raman active. In the realm of SAMs used in electrochemical settings, SFG spectroscopy has been applied to study chemical, orientational, and conformational changes within SAMs at electrified interfaces.26-35 While the majority of these studies focus on the use of aliphatic and aromatic compounds, herein we use for the first time in situ SFG spectroscopy to investigate changes in molecular orientations in charged biofunctionalized SAMs in response to an applied electrical potential. This is particularly challenging due to the comparably lower density of charged biomolecules on the surface and a resulting greater degree of conformational freedom. Both effects decrease SFG signals, which are depending on density and order. The SFG characterization was performed with an easy to assemble and purpose built electrochemical cell that allowed recording of SFG spectra while applying an electrical potential. Furthermore, the conformational changes of the biotin-KKKKC:TEGT SAMs observed by SFG at negative and positive potential are compared with densely packed biotin-KKKKC SAMs to understand the importance of the presence of TEGT as a spacer group. SPR data are also discussed to further strengthen our findings. Finally, the reversibility of the conformational changes is verified. The switching properties of the mixed SAMs were investigated by applying an electrical potential as an external stimulus. SAMs of biotin-KKKKC:TEGT were formed directly onto a 15 nm gold coated side of an equilateral CaF2 prism (size 25 mm). The prism was then placed onto a purpose built Teflon electrochemical cell (Figure 2) containing PBS buffer (pH = 7.4). Applied potentials were measured versus a Ag/AgCl reference electrode (FLEXREF, WPI, USA). Reference and counter electrode (Pt wire) were placed about 3–5 mm below the gold coated prism, which was used as the working electrode. Details on the SAM preparation and picosecond SFG setup (EKSPLA, Lithuania) can be found in the Supporting Information. SFG measurements were performed at static potentials. First, a potential of +0.3 V was applied to the biotin-KKKKC:TEGT SAM and the corresponding SFG spectra was recorded. Subsequently, a potential of –0.4 V was applied to the same substrate while the SFG setup was left unchanged. The corresponding spectra (normalized to the IR and visible intensities of the incoming beams) are shown in Figure 3 for the region between 3150 and 3350 cm−1. While changing the potential from positive to negative values, SFG signals nearly overlap, except for spectral contributions around 3245 cm−1. While NH vibrations of the peptide are occurring at frequencies centered above 3280 cm−136 and CH vibrations are significantly lower (below 3000 cm−1), the peak around 3245 cm−1 can be attributed to molecular vibrations within the heterocyclic imidazole moiety of the biotin group.37 Besides the narrow band visible in Figure 3, a rather steep incline in the overall spectral shape is observable that is associated to non-resonant signals from electronic transitions within the Au substrate, Fresnel coefficients that change with frequency, and typically broad OH contributions from water.38 SFG control studies performed with the single component biotin-KKKKC SAM show that no switching occurs, presumably due to the high level of packing of the oligolysine chains that are constrained in one conformation (Supporting Information). Further evidence of this non-switching behaviour is provided by electrochemical SPR (Supporting information). An analysis of the relative phase in between the resonant and non-resonant signals can retain information on the orientation of this particular group. A dip in the spectrum is related to a mean orientation of the corresponding transition dipole moment (TDM) away from the substrate (destructive interference), while a peak is indicating an orientation towards the substrate (constructive interference). Applied to the spectra in this present research, the biotin moiety contributing in this spectral region has its mean TDM orientation pointing away from the substrate at positive potential resulting in a dip in the spectrum. In this scenario, the positively charged peptide chain is prone to adopt a conformation that will extend itself away from the substrate due to electrostatic repulsion resulting in an anisotropic upright orientation of the biotin group. At negative potential, the peptide chains are likely to adopt a collapsed folded conformation due to electrostatic attraction between the negative potential of the surface and the positive charges on the peptide backbone, which appears to have resulted in a disordered biotin group since the SFG signal is no longer visible (isotropic molecular ordering cannot generate SFG signals), Figure 3. Oftentimes in situ SFG spectra of biomolecules at surfaces are rather complex and relatively weak due to the isotropic nature coming with a less tightly packed arrangement of molecules. The mixed biotin-KKKKC:TEGT SAM used in our study has been previously13 characterised by X-ray photoelectron spectroscopy (XPS) and an average ratio on the surface of 1:16 ± 4 was observed. It is remarkable that at such a small surface coverage, SFG signals of biomolecules in solution still deliver a significant contribution above the noise ratio. So, our experiments are highlighting the importance of following changes in SFG spectra while changing external parameters such as surface potentials. In this respect, the combination of electrochemistry and SFG, as applied in this study, provides a powerful platform when it comes to in situ spectral analysis utilizing SFG in the context of biointerfaces. Furthermore, the applied potential can be switched back and forth to reproducibly cycle between the two spectral states. Moreover, the observed spectral features might be only slightly above noise level, but the reproducibility strengthens evidence and provides statistical means to an otherwise only singular event. Cycling the external parameter also allows investigating the reversibility of molecular conformations as discussed in the following paragraphs. Figure 4a shows baseline corrected normalized SFG spectra that have been recorded at +0.3 V, −0.4 V, and back to +0.3 V applied potential. The ability to turn on and off the upwards orientation with the applied potential allows us to monitor the molecular reorientation of the biotin group. The reappearance of biotin peaks at positive potential shows that the biotin group can be reversibly switched from being isotropically oriented at negative potential towards an anisotropic orientation at positive potential.39 The corresponding fitted intensities for the spectra shown in Figure 4a can be found in Figure 4b. Positive and negative potentials are clearly separated demonstrating that the identification of 2 states (upright and random orientation) is above noise level. Additionally, mean values at repeating positive potential are within the error of the spectral fit quantifying the reversible nature of the switching process. The conformational change of the biotin-KKKKC peptide is further illustrated by SPR measurements of the binding events between the biotin end-group of the biotin-KKKKC:TEGT SAM and the neutrally charged protein Neutravidin at different applied potential (Figure 4c). For biotin-KKKKC:TEGT SAM, the binding process is favoured at +0.3 V when the oligolysine backbones are in an extended conformation and the biotin end-groups are exposed. In contrast, at –0.4 V the interaction between the biotin and the Neutravidin is prevented due to the folded conformation of the backbones which makes the biotin unavailable. On the other hand, when the same experiment is performed on a pure biotin-KKKKC SAM no difference in binding events is observed (see Supporting Information), indicating that despite the different potentials applied no molecular conformational changes are occurring. Although it is important to study the switching behaviour of SAMs with different experimental detection techniques, no information at the molecular level can be gained by SPR. Furthermore, the reversibility of the switching monitored by SPR implies the use of specific analytes which could lead to several issues such as the occurrence of non-specific binding and the irreversible chemical bonds between the analyte and the ligand. Both these circumstances have an impact on the reversibility performance and therefore on its analysis. However, these difficulties are overcome by using SFG spectroscopy where no extra binding processes are needed (Figures 4a and b). We have demonstrated that SFG spectroscopy is a highly sensitive tool able to provide an in depth characterisation of the reversibility of electrically switchable biotin-KKKKC:TEGT SAMs. By studying the orientation of the SFG peak's characteristics of the biotin end-group, the determination of the structural orientation under electro-induced switching was ascertained. The switchable process and its reversibility were assessed by repeatedly switching between positive and negative surface potentials. Monolayers of single component biotin-KKKKC SAMs exhibited no conformational change upon the application of an electrical potential, indicating that due to the high level of packing the oligolysine chains are constrained to one extended conformation (anisotropic molecular ordering). On the contrary, when a negative potential of –0.4 V is applied to the mixed biotin-KKKKC:TEGT SAM, a change in conformation occurs owing to the presence of the spacer, TEGT, presumably allowing the folding of the oligolysine backbones (isotropic molecular ordering). Furthermore, the reverse phase of the SFG signal in the region between 3200–3300 cm−1 at –0.4 V suggest that the biotin end-group is facing in the opposite direction compared to the initial measurement (+0.3 V). Such information has the potential to positively impact the field of biosensors as well as surface engineering where the direct knowledge of the structure and the geometry of the molecules at interfaces are vitally important. The work was funded by the European Community Framework Programme 7, SEACOAT (No 237997), HYSENS (No 263091), Leverhulme Trust grant number F/00 094/BD and Wellcome Trust grant number WT091285MA. This research was also supported through Birmingham Science City: Innovative Uses for Advanced Materials in the Modern World (West Midlands Centre for Advanced Materials Project 2), supported by Advantage West Midlands (AWM) and part funded by the European Regional Development Fund (ERDF). P.K. acknowledges support from NIH grant EB-002027 to the National ESCA and Surface Analysis Center for Biomedical Problems. We are grateful to Sanghun Song for his assistance with the SFG experiments. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Bacterial adhesion can be controlled by applying electrical potentials to surfaces incorporating well-spaced negatively charged 11-mercaptoundecanoic acids. When combined with electrochemical surface plasmon resonance, these dynamic surfaces become powerful for monitoring and analysing the passage between reversible and non-reversible cell adhesion, opening new opportunities to advance our understanding of cell adhesion processes.

The adsorption and structure of sodium dodecyl sulfate (SDS) layers onto positively charged films have been monitored in situ with vibrational sum-frequency-generation (SFG) spectroscopy and surface plasmon resonance (SPR) sensing. Substrates with different charge densities and polarities used in these studies include CaF2 at different pH values as well as allylamine and heptylamine films deposited onto CaF2 and Au substrates by radio frequency glow discharge deposition. The SDS films were adsorbed from aqueous solutions ranging in concentration from 0.067 to 20 mM. In general the SFG spectra exhibited well resolved CH and OH peaks. However, at SDS concentrations between 1 and 8 mM the SFG CH and OH intensities decreased close to background levels. Combined data sets from molecular conformation, orientation, and order sensitive SFG with mass sensitive SPR suggest that the observed changes in SFG intensities above 0.2 mM are related to structural arrangements in the SDS layer. A model is proposed where the SFG intensity minimum between 1 and 8 mM is associated with a monolayer containing two headgroup orientations, one pointing toward the substrate and one pointing toward the solution phase. The SFG peaks observed at concentrations below 0.2 mM are dominated by the presence of adsorbed contaminants such as fatty alcohols (e.g., dodecanol), which are more surface active than SDS. As SDS solution concentration is increased above 1 mM SDS molecules are incorporated in the surface layer, with dodecanol continuing to be present in the surface layer for solution concentrations up to at least the critical micelle concentration.

Vibrational sum-frequency-generation (SFG) spectroscopy experiments at electrified interfaces involve incident laser radiation at frequencies in the IR and near-IR/visible regions as well as a static electric field on the surface. Here we show that mixing the three fields present on the surface can result in third-order effects in resonant SFG signals. This was achieved for closed packed self-assembled monolayers (SAMs) with molecular groups of high optical nonlinearity and surface potentials similar to those typically applied in cyclic voltammograms. Broadband SFG spectroscopy was applied to study a hydrophobic well-ordered araliphatic SAM on a Au(111) surface using a thin-layer analysis cell for spectro-electrochemical investigations in a 100 mM NaOH electrolyte solution. Resonant contributions were experimentally separated from non-resonant contributions of the Au substrate and theoretically analyzed using a fitting function including third-order terms. The resulting ratio of third-order to second-order susceptibilities was estimated to be [Formula: see text](10-10) m/V.

Electroreductive desorption of a highly ordered self-assembled monolayer (SAM) formed by the araliphatic thiol (4-(4-(4-pyridyl)phenyl)phenyl)methanethiol leads to a concurrent rapid hydrogen evolution reaction (HER). The desorption process and resulting interfacial structure were investigated by voltammetric techniques, in situ spectroscopic ellipsometry, and in situ vibrational sum-frequency-generation (SFG) spectroscopy. Voltammetric experiments on SAM-modified electrodes exhibit extraordinarily high peak currents, which differ between Au(111) and polycrystalline Au substrates. Association of reductive desorption with HER is shown to be the origin of the observed excess cathodic charges. The studied SAM preserves its two-dimensional order near Au surface throughout a fast voltammetric scan even when the vertex potential is set several hundred millivolt beyond the desorption potential. A model is developed for the explanation of the observed rapid HER involving ordering and pre-orientation of water present in the nanometer-sized reaction volume between desorbed SAM and the Au electrode, by the structurally extremely stable monolayer, leading to the observed catalysis of the HER.

Nonfouling surfaces capable of reducing protein adsorption are highly desirable in a wide range of applications. Coating of surfaces with poly(ethylene oxide) (PEO), a water-soluble, nontoxic, and nonimmunogenic polymer, is most frequently used to reduce nonspecific protein adsorption. Here we show how to prepare dense PEO brushes on virtually any substrate by tethering PEO to polydopamine (PDA)-modified surfaces. The chain lengths of hetero-bifunctional PEOs were varied in the range of 45-500 oxyethylene units (M-n = 2000-20 000). End-tethering of PEO chains was performed through amine and thiol headgroups from reactive polymer melts to minimize excluded volume effects. Surface plasmon resonance (SPR) was applied to investigate the adsorption of model protein solutions and complex biologic medium (human blood plasma) to the densely packed PEO brushes. The level of protein adsorption of human serum albumin and fibrinogen solutions was below the detection limit of the SPR measurements for all PEO chains end-tethered to PDA, thus exceeding the protein resistance of PEO layers tethered directly on gold. It was found that the surface resistance to adsorption of lysozyme and human blood plasma increased with increasing length and brush character of the PEO chains end-tethered to PDA with a similar or better resistance in comparison to PEO layers on gold. Furthermore, the chain density, thickness, swelling, and conformation of PEO layers were determined using spectroscopic ellipsometry (SE), dynamic water contact angle (DCA) measurements, infrared reflection-absorption spectroscopy (IRRAS), and vibrational sum-frequency-generation (VSFG) spectroscopy, the latter in air and water.

N-Acyl-L-homoserine lactones (AHLs) are small cell-to-cell signaling molecules involved in the regulation of population density and local gene expression in microbial communities. Recent evidence shows that contact of this signaling system, usually referred to as quorum sensing, to living eukaryotes results in interactions of AHL with host cells in a process termed "interkingdom signaling". So far details of this process and the binding site of the AHLs remain unknown; both an intracellular and a membrane-bound receptor seem possible, the first of which requires passage through the cell membrane. Here, we used sum-frequency-generation (SFG) spectroscopy to investigate the integration, conformation, orientation, and translocation of deuterated N-acyl-L-homoserine lactones (AHL-d(n)) with varying chain length (8, 12, and 14 C atoms) in lipid bilayers consisting of a 1:1 mixture of POPC:POPG supported on SiO(2) substrates (prepared by vesicle fusion). We found that all AHL-d(n) derivatives are well-ordered within the supported lipid bilayer (SLB) in a preferentially all-trans conformation of the deuterated alkyl chain and integrated into the upper leaflet of the SLB with the methyl terminal groups pointing downward. For the bilayer system described above, no flip-flop of AHL-d(n) from the upper leaflet to the lower one could be observed. Spectral assignments and interpretations were further supported by Fourier transform infrared and Raman spectroscopy.

N-Acyl-l-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-l-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum’s acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein.

The effects of target sequence mismatches and target length mismatches on the orientation and ordering of surface-bound, poly(dT).poly(dA) hybrids on gold were investigated using a combination of high-resolution X-ray photoelectron spectroscopy and near-edge X-ray absorption fine structure spectroscopy. It was found that the overall orientation of the probe and target strands remained upright and relatively unchanged despite the presence of up to four sequence mismatches in the target strands, although the total number of hybrids decreased with higher numbers of mismatches. Upright probes were also observed in hybrids formed with targets of shorter length, with a greater number of hybrids present in these films as compared to those formed with longer targets. These results suggest that in DNA films of relatively high densities, such as those tested here, the presence of sequence or length mismatches in target strands does not result in a large disruption of orientation and ordering in surface-bound hybrids. This finding may be useful for those seeking to maintain consistent DNA strand orientation for precise nanotechnological applications or those using DNA microarrays for biosensing purposes.

A combination of X-ray photoelectron spectroscopy (XPS), high-resolution XPS, near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and sum-frequency-generation (SFG) spectroscopy was used to monitor two types of ssDNA films on Au(111) before and after hybridization. As probe systems, films of thiolated and block-oligonucleotides were used, taking thiolated thymine d(T) homo-oligonucleotides and thymine-adenine d(A-T) diblock-oligonucleotides as representative examples. In accordance with previous work, hybridization of the shorter and more densely packed thiolated ssDNA films produced fewer (if any) hybrids, whereas the longer and less densely packed layers exhibited a larger hybridization yield. The above effects were less pronounced in the case of the d(A-T) films where the hybridization yield of the less densely packed monolayers was significantly lower. This was presumably due to the formation of internal dimeric hybrids in the immobilization step of the probe molecules, resulting in the generation of fewer probe-target hybrids upon exposure to the target molecules. In all ssDNA films displaying a reasonable number of hybrids present, significant orientational changes were observed and could be monitored in detail. These results suggest that the given combination of spectroscopic techniques can be a valuable tool to gain molecular-level information about hybrids at interfaces.