Nano-FTIR spectroscopy of surface confluent polydopamine films - What is the role of deposition time and substrate material?

Polydopamine (PDA) is a widely used anchoring layer for multiple purposes. While simple to prepare, PDA is characterized by high chemical and topological diversity, which can limit its versatility. Unraveling the formation mechanism and physicochemical properties of continuous confluent layer and adherent nanoparticles on the nanoscale is crucial to further extend the prospective applications of PDA. Utilizing nano-FTIR spectroscopy, we investigate layers of PDA on three different substrates (silicon/silicon dioxide, nitrogen-doped titanium oxide, and gold substrates) at varying times of deposition (ToD). We observed a good correlation between the nano-FTIR and macroscopic FTIR spectra that reflected the changes in the relative abundance of PDA and polymerization intermediates as ToD increased. To gain analytical power, we utilized the principal component analysis (PCA) and extracted additional information from the resulting loadings spectral curves and data distribution in the score plots. We revealed a higher variability of the spectra of ultrathin surface confluent layers compared to the adherent nanoparticles. While the spectra of nanoparticles showed no apparent dependency on either ToD or the substrate material, the spectra of layers were highly affected by the increasing ToD and exhibited a rise in the absorption of PDA. Concomitantly, the spectra of layers grouped according to the substrate material at the lowest ToD point to the fact that the substrate material affects the PDA's initial physicochemical structure. The observed separation gradually diminished with the increasing ToD as the PDA physicochemical structure became less influenced by the substrate material.

Nanoscale insights into the local structural rearrangements of amyloid-ß induced by bexarotene.

A better understanding of the abnormal protein aggregation and the effect of anti-aggregation agents on the fibrillation pathways and the secondary structure of aggregates can determine strategies for the early treatment of dementia. Herein, we present a combination of experimental and theoretical studies providing new insights into the influence of the anti-aggregation drug bexarotene on the secondary structure of individual amyloid-ß aggregates and its primary aggregation. The molecular rearrangements and the spatial distribution of ß-sheets within individual aggregates were monitored at the nanoscale with infrared nanospectroscopy. We observed that bexarotene limits the parallel ß-sheets formation, known to be highly abundant in fibrils at later phases of the amyloid-ß aggregation composed of in-register cross-ß structure. Moreover, we applied molecular dynamics to provide molecular-level insights into the investigated system. Both theoretical and experimental results revealed that bexarotene slows down the protein aggregation process via steric effects, largely prohibiting the antiparallel to parallel ß-sheet rearrangement. We also found that bexarotene interacts not only via the single hydrogen bond formation with the peptide backbone but also with the amino acid side residue via a hydrophobic effect. The studied model of the drug-amyloid-ß interaction contributes to a better understanding of the inhibition mechanism of the amyloid-ß aggregation by the small molecule drugs. However, our nanoscale findings need to meet in vivo research requiring different analytical approaches.

Elucidating the fine-scale structural morphology of nanocellulose by nano infrared spectroscopy.

Nanoscale infrared (IR) spectroscopy and microscopy, enabling the acquisition of IR spectra and images with a lateral resolution of 20 nm, is employed to chemically characterize individual cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs) to elucidate if the CNCs and CNFs consist of alternating crystalline and amorphous domains along the CNF/CNC. The high lateral resolution enables studies of the nanoscale morphology at different domains of the CNFs/CNCs: flat segments, kinks, twisted areas, and end points. The types of nanocellulose investigated are CNFs from tunicate, CNCs from cotton, and anionic and cationic wood-derived CNFs. All nano-FTIR spectra acquired from the different samples and different domains of the individual nanocellulose particles resemble a spectrum of crystalline cellulose, suggesting that the non-crystalline cellulose signal observed in macroscopic measurements of nanocellulose most likely originate from cellulose chains present at the surface of the nanocellulose particles.

Surface engineering of mixed conjugated/polyelectrolyte brushes - Tailoring interface structure and electrical properties.

HYPOTHESIS: Mixed polymer brushes (MPBs) could be synthesized by surface dilution of homopolymer brushes and subsequent grafting of other type of chains in the formed voids. Nanophase separation and dynamics of surface-grafted chains could be tailored by modification of their molecular architecture. Mixed polyelectrolyte and conjugated chains contribute synergistically to tailor properties of the coating. EXPERIMENTS: A new synthetic strategy that allowed spatially controlled grafting of poly(sodium 4-styrenesulfonate) chains (PSSNa) in close neighborhood of poly(3-methylthienyl methacrylate) (PMTM) brushes (precursors of the conjugated chains) using surface-initiated polymerizations was developed. The final mixed conjugated/polyelectrolyte brushes were prepared by template polymerization of pendant thiophene groups in PMTM chains. Surface dynamics and nanophase separation of MPBs were studied by nanoscale resolution IR imaging, SIMS profiling and AFM mapping in selective solvents. FINDINGS: Unconjugated MPBs were shown to undergo vertical, and horizontal nanophase separation, while the size and shape of the nanodomains were dependent on molar ratio of the mixed chains and their relative lengths. Generation of the conjugated chains led to diminishing of nanophase separation thanks to stronger mutual interactions of conjugated PMTM and PSSNa (macromolecular mixing). The obtained systems demonstrated tunable interfacial structure and resistance switching phenomenon desired in construction of smart surfaces or memristive devices.

Nano-Infrared Imaging of Primary Neurons.

Alzheimer's disease (AD) accounts for about 70% of neurodegenerative diseases and is a cause of cognitive decline and death for one-third of seniors. AD is currently underdiagnosed, and it cannot be effectively prevented. Aggregation of amyloid-ß (Aß) proteins has been linked to the development of AD, and it has been established that, under pathological conditions, Aß proteins undergo structural changes to form ß-sheet structures that are considered neurotoxic. Numerous intensive in vitro studies have provided detailed information about amyloid polymorphs; however, little is known on how amyloid ß-sheet-enriched aggregates can cause neurotoxicity in relevant settings. We used scattering-type scanning near-field optical microscopy (s-SNOM) to study amyloid structures at the nanoscale, in individual neurons. Specifically, we show that in well-validated systems, s-SNOM can detect amyloid ß-sheet structures with nanometer spatial resolution in individual neurons. This is a proof-of-concept study to demonstrate that s-SNOM can be used to detect Aß-sheet structures on cell surfaces at the nanoscale. Furthermore, this study is intended to raise neurobiologists' awareness of the potential of s-SNOM as a tool for analyzing amyloid ß-sheet structures at the nanoscale in neurons without the need for immunolabeling.

Nanodiamond surface chemistry controls assembly of polypyrrole and generation of photovoltage.

Nanoscale composite of detonation nanodiamond (DND) and polypyrrole (PPy) as a representative of organic light-harvesting polymers is explored for energy generation, using nanodiamond as an inorganic electron acceptor. We present a technology for the composite layer-by-layer synthesis that is suitable for solar cell fabrication. The formation, pronounced material interaction, and photovoltaic properties of DND-PPy composites are characterized down to nanoscale by atomic force microscopy, infrared spectroscopy, Kelvin probe, and electronic transport measurements. The data show that DNDs with different surface terminations (hydrogenated, oxidized, poly-functional) assemble PPy oligomers in different ways. This leads to composites with different optoelectronic properties. Tight material interaction results in significantly enhanced photovoltage and broadband (1-3.5 eV) optical absorption in DND/PPy composites compared to pristine materials. Combination of both oxygen and hydrogen functional groups on the nanodiamond surface appears to be the most favorable for the optoelectronic effects. Theoretical DFT calculations corroborate the experimental data. Test solar cells demonstrate the functionality of the concept.

Conformation in Ultrathin Polymer Brush Coatings Resolved by Infrared Nanoscopy.

Polymer brush coatings are effective in preventing blood coagulation or bacterial attachment, but their chain conformation, while vital for this effect, was never characterized in high spatial resolution. Here, we report mid-infrared spectroscopic nanoscopy studies of few-nanometer-thin poly(ethylene oxide) (PEO) films which reveal marked spectral variations along the surface at a length scale smaller than 100 nm and originating only from the physical conformation of the chains. The conformation and average orientation of the polymer chains in the layer is extracted from the spectra with the aid of theoretic modeling, confirming the spontaneous formation of a crystalline phase. This result suggests spectroscopic nanoscopy as a powerful new tool to characterize polymer brush coatings.

An Analysis of Isolated and Intact RBC Membranes-A Comparison of a Semiquantitative Approach by Means of FTIR, Nano-FTIR, and Raman Spectroscopies.

This work presents the potential of vibrational spectroscopy, Vis and NIR Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) in reflection and transmission modes, and nano-FTIR microscopy to study the biochemical alterations in membranes of isolated and intact red blood cells (RBCs). The main goal was to propose the best spectroscopic method which enabled following biochemical alterations in the RBC membranes and then to translate this spectroscopic signature of degradation to in situ analysis of RBCs. Two models corresponding to two distinct cases of RBC membrane conditions were employed, and they were derived from healthy and young mice and mature mice with advanced atherosclerosis. It was shown that each technique provided essential information about biochemical alterations of the isolated membranes as well as membranes in the intact RBCs, which can be used in the development of a rapid and in situ analytical technology. Finally, we proposed that the combination of macro- and nanoprobing implemented in IR spectroscopy provided a wide chemical characterization of the RBC membranes, including alterations in lipid and protein fractions. This study also examined the effect of the sample preparation to determine destructive factors influencing a spectroscopic analysis of isolated membranes and intact RBCs derived from healthy and disease-affected mice.

Nanoscale optical and structural characterisation of silk.

The nanoscale composition of silk defining its unique properties via a hierarchial structural anisotropy needs to be analysed at the highest spatial resolution of tens of nanometers corresponding to the size of fibrils made of ß-sheets, which are the crystalline building blocks of silk. Nanoscale optical and structural properties of silk have been measured from 100 nm thick longitudinal slices of silk fibers with ca. 10 nm resolution, the highest so far. Optical sub-wavelength resolution in hyperspectral mapping of absorbance and molecular orientation were carried out for comparison at IR wavelengths of 2-10 µm using synchrotron radiation. A reliable distinction of transmission changes by only 1-2% as the anisotropy of amide bands was obtained from nanometer-thin slices of silk.

Raman imaging highlights biochemical heterogeneity of human eosinophils versus human eosinophilic leukaemia cell line.

Eosinophils are acidophilic granulocytes that develop in the bone marrow. Although their population contributes only to approximately 1-6% of all leucocytes present in the human blood, they possess a wide range of specific functions. They play a key role in inflammation-regulating processes, when their numbers can increased to above 5 × 109 /l of peripheral blood. Their characteristic feature is the presence of granules containing eosinophil peroxidase (EPO), the release of which can trigger a cascade of events promoting oxidative stress, apoptosis or necrosis, leading finally to cell death. Raman spectroscopy is a powerful technique to detect EPO, which comprises a chromophore protoporphyrin IX. Another cell structure associated with inflammation processes are lipid bodies (lipid-rich organelles), also well recognized and imaged using high resolution confocal Raman spectroscopy. In this work, eosinophils isolated from the blood of a human donor were analysed versus their model, EoL-1 human eosinophilic leukaemia cell line, by Raman spectroscopic imaging. We showed that EPO was present only in primary cells and not found in the cell line. Eosinophils were activated using phorbol 12-myristate 13-acetate, which resulted in lipid bodies formation. An effect of cells stimulation was studied and compared for eosinophils and EoL-1.

Structure-Function Correlative Microscopy of Peritubular and Intertubular Dentine.

Peritubular dentine (PTD) and intertubular dentine (ITD) were investigated by 3D correlative Focused Ion Beam (FIB)-Scanning Electron Microscopy (SEM)-Energy Dispersive Spectroscopy (EDS) tomography, tapping mode Atomic Force Microscopy (AFM) and scattering-type Scanning Near-Field Optical Microscopy (s-SNOM) mapping. The brighter appearance of PTD in 3D SEM-Backscattered-Electron (BSE) imaging mode and the corresponding higher grey value indicate a greater mineral concentration in PTD (~160) compared to ITD (~152). However, the 3D FIB-SEM-EDS reconstruction and high resolution, quantitative 2D map of the Ca/P ratio (~1.8) fail to distinguish between PTD and ITD. This has been further confirmed using nanoscale 2D AFM map, which clearly visualised biopolymers and hydroxyapatite (HAp) crystallites with larger mean crystallite size in ITD (32 ± 8 nm) than that in PTD (22 ± 3 nm). Correlative microscopy reveals that the principal difference between PTD and ITD arises primarily from the nanoscale packing density of the crystallites bonded together by thin biopolymer, with moderate contribution from the chemical composition difference. The structural difference results in the mechanical properties variation that is described by the parabolic stiffness-volume fraction correlation function introduced here. The obtained results benefit a microstructure-based mechano-chemical model to simulate the chemical etching process that can occur in human dental caries and some of its treatments.

Label-Free Infrared Spectroscopy and Imaging of Single Phospholipid Bilayers with Nanoscale Resolution.

Mid-infrared absorption spectroscopy has been used extensively to study the molecular properties of cell membranes and model systems. Most of these studies have been carried out on macroscopic samples or on samples a few micrometers in size, due to constraints on sensitivity and spatial resolution with conventional instruments that rely on far-field optics. Properties of membranes on the scale of nanometers, such as in-plane heterogeneity, have to date eluded investigation by this technique. In the present work, we demonstrate the capability to study single bilayers of phospholipids with near-field mid-infrared spectroscopy and imaging and achieve a spatial resolution of at least 40 nm, corresponding to a sample size of the order of a thousand molecules. The quality of the data and the observed spectral features are consistent with those reported from measurements of macroscopic samples and allow detailed analysis of molecular properties, including orientation and ordering of phospholipids. The work opens the way to the nanoscale characterization of the biological membranes for which phospholipid bilayers serve as a model.

Direct Electrical Probing of Periodic Modulation of Zinc-Dopant Distributions in Planar Gallium Arsenide Nanowires.

Selective lateral epitaxial (SLE) semiconductor nanowires (NWs), with their perfect in-plane epitaxial alignment, ability to form lateral complex p-n junctions in situ, and compatibility with planar processing, are a distinctive platform for next-generation device development. However, the incorporation and distribution of impurity dopants in these planar NWs via the vapor-liquid-solid growth mechanism remain relatively unexplored. Here, we present a detailed study of SLE planar GaAs NWs containing multiple alternating axial segments doped with Si and Zn impurities by metalorganic chemical vapor deposition. The dopant profile of the lateral multi-p-n junction GaAs NWs was imaged simultaneously with nanowire topography using scanning microwave impedance microscopy and correlated with infrared scattering-type near-field optical microscopy. Our results provide unambiguous evidence that Zn dopants in the periodically twinned and topologically corrugated p-type segments are preferentially segregated at twin plane boundaries, while Si impurity atoms are uniformly distributed within the n-type segments of the NWs. These results are further supported by microwave impedance modulation microscopy. The density functional theory based modeling shows that the presence of Zn dopant atoms reduces the formation energy of these twin planes, and the effect becomes significantly stronger with a slight increase of Zn concentration. This implies that the twin formation is expected to appear when a threshold planar concentration of Zn is achieved, making the onset and twin periodicity dependent on both Zn concentration and nanowire diameter, in perfect agreement with our experimental observations.

Sub-micron phase coexistence in small-molecule organic thin films revealed by infrared nano-imaging.

Controlling the domain size and degree of crystallization in organic films is highly important for electronic applications such as organic photovoltaics, but suitable nanoscale mapping is very difficult. Here we apply infrared-spectroscopic nano-imaging to directly determine the local crystallinity of organic thin films with 20-nm resolution. We find that state-of-the-art pentacene films (grown on SiO2 at elevated temperature) are structurally not homogeneous but exhibit two interpenetrating phases at sub-micrometre scale, documented by a shifted vibrational resonance. We observe bulk-phase nucleation of distinct ellipsoidal shape within the dominant pentacene thin-film phase and also further growth during storage. A faint topographical contrast as well as X-ray analysis corroborates our interpretation. As bulk-phase nucleation obstructs carrier percolation paths within the thin-film phase, hitherto uncontrolled structural inhomogeneity might have caused conflicting reports about pentacene carrier mobility. Infrared-spectroscopic nano-imaging of nanoscale polymorphism should have many applications ranging from organic nanocomposites to geologic minerals.

New pulsed EPR methods and their application to characterize mitochondrial complex I.

Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron-sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior.

2D-REFINE spectroscopy: separation of overlapping hyperfine spectra.

We show on a mixture of three spectrally overlapping paramagnetic compounds TEMPO, BDPA and CuHis that it is possible to separate their field-swept and hyperfine spectra based on the difference in their longitudinal relaxation times T1. This was achieved in a two-dimensional experiment, where one dimension corresponds to the spectral domain and the second dimension encodes the relaxation behavior of the individual compound. Inverse Laplace Transform with respect to this domain separates the field-swept and hyperfine spectra of the individual compounds in the relaxation rate domain. This extends our formerly proposed Relaxation Filtered Hyperfine (REFINE) method to be applicable to more than two spectrally overlapping spectra by adding a further dimension to the chosen EPR experiment.