Investigating Membrane-Mediated Antimicrobial Peptide Interactions with Synchrotron Radiation Far-Infrared Spectroscopy.

Synchrotron radiation-based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio-molecular phenomena including folding-mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near-THz 400-40 cm-1 range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature-dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane-induced and folding-mediated activity of AMPs. The far-FTIR study offers a direct and information-rich probe of membrane-related antimicrobial interactions.

Compressed FTIR spectroscopy using low-rank matrix reconstruction.

Fourier transform infrared (FTIR) spectroscopy is a powerful technique in analytical chemistry. Typically, spatially distributed spectra of the substance of interest are conducted simultaneously using FTIR spectrometers equipped with array detectors. Scanning-based methods such as near-field FTIR spectroscopy, on the other hand, are a promising alternative providing higher spatial resolution. However, serial recording severely limits their application due to the long acquisition times involved and the resulting stability issues. We demonstrate that it is possible to significantly reduce the measurement time of scanning methods by applying the mathematical technique of low-rank matrix reconstruction. Data from a previous pilot study of Leishmania strains are analyzed by randomly selecting 5% of the interferometer samples. The results obtained for bioanalytical fingerprinting using the proposed approach are shown to be essentially the same as those obtained from the full set of data. This finding can significantly foster the practical applicability of high-resolution serial scanning techniques in analytical chemistry and is also expected to improve other applications of FTIR spectroscopy and spectromicroscopy.

Correction to Infrared Nanospectroscopy of Phospholipid and Surfactin Monolayer Domains.

[This corrects the article DOI: 10.1021/acsomega.7b01931.].

Melanoidin formed from fructosylalanine contains more alanine than melanoidin formed from d-glucose with L-alanine.

In this study the elemental compositions of melanoidin formed at 160 °C from d-glucose (Glc) and l-alanine (Ala) as well as from fructosylalanine - the corresponding Amadori rearrangement product - were compared. Specific chemical bonds were probed by FTIR spectroscopy. This approach tackles the different chemical pathways for melanoidin formation via the Amadori rearrangement in contrast to the reaction from Glc/Ala. Melanoidins formed from fructosylalanine contain about twice as much nitrogen and therefore amino acid as compared to melanoidin from Glc/Ala and exhibit higher absorption in the UV/Vis. Consequently, melanoidins formed from Glc/Ala contain more sugar degradation products with lower absorption due to a smaller size of the conjugated double bond network.

PCA-based identification and differentiation of FTIR data from model melanoidins with specific molecular compositions.

Melanoidins formed from different carbohydrates, such as d-glucose, d-fructose, and d-xylose, and their typical degradation products, such as hydroxymethylfurfural, furfural, glyoxal, and methylglyoxal, with l-alanine were analyzed with Fourier transform infrared spectroscopy (FTIR). Characteristic infrared bands were identified representing spectral differences between the investigated melanoidin species due to their different molecular compositions. With the help of principal components analysis (PCA) the IR data allowed for a fast discrimination between the different model melanoidins. From this study it is inferred that the intensity and relative absorption wavelength of CO single versus CO double bonds are characteristic features of the investigated melanoidins. Melanoidins formed from carbohydrates exhibit less carbonyl functions in comparison to melanoidins from the degradation products, the situation is opposite for the CO bond. The amount of CO is additionally correlated with a higher absorption at 420 nm indicating that strong colored melanoidins contain more carbonyl functions.

A methodological inter-comparison study on the detection of surface contaminant sodium dodecyl sulfate applying ambient- and vacuum-based techniques.

Biomedical devices are complex products requiring numerous assembly steps along the industrial process chain, which can carry the potential of surface contamination. Cleanliness has to be analytically assessed with respect to ensuring safety and efficacy. Although several analytical techniques are routinely employed for such evaluation, a reliable analysis chain that guarantees metrological traceability and quantification capability is desirable. This calls for analytical tools that are cascaded in a sensible way to immediately identify and localize possible contamination, both qualitatively and quantitatively. In this systematic inter-comparative approach, we produced and characterized sodium dodecyl sulfate (SDS) films mimicking contamination on inorganic and organic substrates, with potential use as reference materials for ambient techniques, i.e., ambient mass spectrometry (AMS), infrared and Raman spectroscopy, to reliably determine amounts of contamination. Non-invasive and complementary vibrational spectroscopy techniques offer a priori chemical identification with integrated chemical imaging tools to follow the contaminant distribution, even on devices with complex geometry. AMS also provides fingerprint outputs for a fast qualitative identification of surface contaminations to be used at the end of the traceability chain due to its ablative effect on the sample. To absolutely determine the mass of SDS, the vacuum-based reference-free technique X-ray fluorescence was employed for calibration. Convex hip liners were deliberately contaminated with SDS to emulate real biomedical devices with an industrially relevant substance. Implementation of the aforementioned analytical techniques is discussed with respect to combining multimodal technical setups to decrease uncertainties that may arise if a single technique approach is adopted. Graphical abstract ᅟ.

Compressed sensing FTIR nano-spectroscopy and nano-imaging.

Infrared scattering scanning near-field optical microscopy (IR s-SNOM) provides for spectroscopic imaging with nanometer spatial resolution, yet full spatio-spectral imaging is constrained by long measurement times. Here, we demonstrate the application of compressed sensing algorithms to achieve hyperspectral FTIR-based nano-imaging at an order of magnitude faster imaging speed to achieve the same spectral content compared to conventional approaches. At the example of the spectroscopy of a single vibrational resonance, we discuss the relationship of prior knowledge of sparseness of the employed Fourier base functions and sub-sampling. Compressed sensing nano-FTIR spectroscopy promises both rapid and sensitive chemical nano-imaging which is highly relevant in academic and industrial settings for fundamental and applied nano- and bio-materials research.

Infrared Nanospectroscopy of Phospholipid and Surfactin Monolayer Domains.

A main challenge in understanding the structure of a cell membrane and its interactions with drugs is the ability to chemically study the different molecular species on the nanoscale. We have achieved this for a model system consisting of mixed monolayers (MLs) of the biologically relevant phospholipid 1,2-distearoyl-sn-glycero-phosphatidylcholine and the antibiotic surfactin. By employing nano-infrared (IR) microscopy and spectroscopy in combination with atomic force microscopy imaging, it was possible to identify and chemically detect domain formation of the two constituents as well as to obtain IR spectra of these species with a spatial resolution on the nanoscale. A novel method to enhance the near-field imaging contrast of organic MLs by plasmon interferometry is proposed and demonstrated. In this technique, the organic layer is deposited on gold and ML graphene substrates, the latter of which supports propagating surface plasmons. Plasmon reflections arising from changes in the dielectric environment provided by the organic layer lead to an additional contrast mechanism. Using this approach, the interfacial region between surfactin and the phospholipid has been mapped and a transition region is identified.

A calibration procedure for a traceable contamination analysis on medical devices by combined X-ray spectrometry and ambient spectroscopic techniques.

There is a strong need in the medical device industry to decrease failure rates of biomedical devices by reducing the incidence of defect structures and contaminants during the production process. The detection and identification of defect structures and contaminants is crucial for many industrial applications. The present study exploits reference-free X-ray fluorescence (XRF) analysis as an analytical tool for the traceable characterization of surface contaminants of medical devices, in particular N,N'-ethylene-bis (stearamide), an ubiquitous compound used in many industrial applications as a release agent or friction reduction additive. Reference-free XRF analysis as primary method has been proven to be capable of underpinning all other applied methods since it yields the absolute mass deposition of the selected N,N'-ethylene-bis (stearamide) contaminant whilst X-ray absorption fine structure analysis determines the chemical species. Ambient vibrational spectroscopy and mass spectroscopy methodologies such as Fourier transform infrared, Raman, and secondary ion mass spectroscopy have been used in this systematic procedure providing an extensive range of complementary analyses. The calibration procedure described in this paper was developed using specially designed and fabricated model systems varying in thickness and substrate material. Furthermore, typical real medical devices such as both a polyethylene hip liner and a silver-coated wound dressing have been contaminated and investigated by these diverse methods, enabling testing of this developed procedure. These well-characterized samples may be used as calibration standards for bench top instrumentation from the perspective of providing traceable analysis of biomaterials and surface treatments. These findings demonstrate the potential importance and usefulness of combining complementary methods for a better understanding of the relevant organic materials.

Structural characterization of melanoidin formed from d-glucose and l-alanine at different temperatures applying FTIR, NMR, EPR, and MALDI-ToF-MS.

The aim of this study was to identify specific chemical bonds and characteristic structures in melanoidins formed from d-glucose and l-alanine between 130 and 200 °C. The results might be used to control the type and amount of melanoidin produced during food processing. For this purpose, complementary techniques, such as FTIR, NMR, EPR, and MALDI-ToF, were employed. At 160 °C color, solubility and UV/Vis absorption change characteristically and consequently, structural transformations could be observed in FTIR and NMR spectra. For example, sharp signals of N-H, C-N, and C-H oscillations in the l-alanine spectrum are prone to inhomogeneous broadening in melanoidins prepared above 150 °C. These changes are caused due to formation of heterogeneous macromolecular structures and occur during condensation reactions that lead to an increasing loss of water from the melanoidins with increasing temperatures. Additionally, MALDI-ToF-MS indicates the polymerization of glyoxal/glyoxylic acid and EPR shows the formation of radical structures.

A pilot study on fingerprinting Leishmania species from the Old World using Fourier transform infrared spectroscopy.

Leishmania species are protozoan parasites and the causative agents of leishmaniasis, a vector borne disease that imposes a large health burden on individuals living mainly in tropical and subtropical regions. Different Leishmania species are responsible for the distinct clinical patterns, such as cutaneous, mucocutaneous, and visceral leishmaniasis, with the latter being potentially fatal if left untreated. For this reason, it is important to perform correct species identification and differentiation. Fourier transform infrared spectroscopy (FTIR) is an analytical spectroscopic technique increasingly being used as a potential tool for identification of microorganisms for diagnostic purposes. By employing mid-infrared (MIR) spectral data, it is not only possible to assess the chemical structures but also to achieve differentiation supported by multivariate statistic analysis. This work comprises a pilot study on differentiation of Leishmania species of the Old World (L. major, L. tropica, L. infantum, and L. donovani) as well as hybrids of distinct species by using vibrational spectroscopic fingerprints. Films of intact Leishmania parasites and their deoxyribonucleic acid (DNA) were characterized comparatively with respect to their biochemical nature and MIR spectral patterns. The strains' hyperspectral datasets were multivariately examined by means of variance-based principal components analysis (PCA) and distance-based hierarchical cluster analysis (HCA). With the implementation of MIR spectral datasets we show that a phenotypic differentiation of Leishmania at species and intra-species level is feasible. Thus, FTIR spectroscopy can be further exploited for building up spectral databases of Leishmania parasites in view of high-throughput analysis of clinical specimens. Graphical abstract For Leishmania species discrimination, sample films of intact parasites and their extracted DNA were analyzed by FTIR micro-spectroscopy. Hyperspectral datasets that comprise mid-infrared fingerprints were submitted to multivariate analysis tools such as principal components analysis (PCA) and hierarchical cluster analysis (HCA).

Qualifying label components for effective biosensing using advanced high-throughput SEIRA methodology.

The need for technological progress in bio-diagnostic assays of high complexity requires both fundamental research and constructing efforts on nano-scaled assay recognition elements that can provide unique selectivity and design-enhanced sensitivity features. Nanoparticle induced sensitivity enhancement and its application related to multiplexed capability Surface-Enhanced InfraRed Absorption (SEIRA) assay formats are well suitable for these purposes. The potential of diverse fluorophore-antibody conjugates, being chemisorbed onto low-cost gold nanoparticulate SEIRA substrates, has been explored with respect to their spectral discriminability. These novel biolabels deliver molecular SEIRA fingerprints that have been successfully analyzed by both uni- and multivariate analyzing tools, to discriminate their multiplexing capabilities. We show that this robust spectral encoding via SEIRA fingerprints opens up new opportunities for a fast, reliable and multiplexed high-end screening in biodiagnostics.

Characterization of semiconductor materials using synchrotron radiation-based near-field infrared microscopy and nano-FTIR spectroscopy.

We describe the application of scattering-type near-field optical microscopy to characterize various semiconducting materials using the electron storage ring Metrology Light Source (MLS) as a broadband synchrotron radiation source. For verifying high-resolution imaging and nano-FTIR spectroscopy we performed scans across nanoscale Si-based surface structures. The obtained results demonstrate that a spatial resolution below 40 nm can be achieved, despite the use of a radiation source with an extremely broad emission spectrum. This approach allows not only for the collection of optical information but also enables the acquisition of near-field spectral data in the mid-infrared range. The high sensitivity for spectroscopic material discrimination using synchrotron radiation is presented by recording near-field spectra from thin films composed of different materials used in semiconductor technology, such as SiO2, SiC, SixNy, and TiO2.

Specific biomolecule corona is associated with ring-shaped organization of silver nanoparticles in cells.

We correlate the localization of silver nanoparticles inside cells with respect to the cellular architecture with the molecular information in the vicinity of the particle surface by combining nanoscale 3D cryo-soft X-ray tomography (cryo-SXT) with surface-enhanced Raman scattering (SERS). The interaction of the silver nanoparticle surface with small molecules and biopolymers was monitored by SERS in vitro over time in living cells. The spectra indicate a stable, time-independent surface composition of silver nanoparticles, despite the changing environment in the endosomal structure. Cryo-SXT reveals a characteristic ring-shaped organization of the silver nanoparticles in endosomes of different cell types. The ring-like structures inside the endosomes suggest a strong association among silver particles and with membrane structures. The comparison of the data with those obtained with gold nanoparticles suggests that the interactions between the nanoparticles and with the endosomal component are influenced by the molecular composition of the corona.

Intracellular SERS hybrid probes using BSA-reporter conjugates.

Surface-enhanced Raman scattering (SERS) hybrid probes are characterized by the typical spectrum of a reporter molecule. In addition, they deliver information from their biological environment. Here, we report SERS hybrid probes generated by conjugating different reporter molecules to bovine serum albumin (BSA) and using gold nanoparticles as plasmonic core. Advantages of the BSA-conjugate hybrid nanoprobes over other SERS nanoprobes are a high biocompatibility, stabilization of the gold nanoparticles in the biological environment, stable reporter signals, and easy preparation. The coupling efficiencies of the BSA-reporter conjugates were determined by MALDI-TOF-MS. The conjugates' characteristic SERS spectra differ from the spectra of unbound reporter molecules. This is a consequence of the covalent coupling, which leads to altered SERS enhancement and changes in the chemical structures of the reporter and of BSA. The application of the BSA-reporter conjugate hybrid probes in 3T3 cells, including duplex imaging, is demonstrated. Hierarchical cluster analysis and principal components analysis were applied for multivariate imaging using the SERS signatures of the incorporated SERS hybrid nanoprobes along with the spectral information from biomolecules in endosomal structures of cells. The results suggest more successful applications of the SERS hybrid probes in cellular imaging and other unordered high-density bioanalytical sensing.