Mechanism of Plasmon-Induced Catalysis of Thiolates and the Impact of Reaction Conditions.

The conversion of the thiols 4-aminothiophenol (ATP) and 4-nitrothiophenol (NTP) can be considered as one of the standard reactions of plasmon-induced catalysis and thus has already been the subject of numerous studies. Currently, two reaction pathways are discussed: one describes a dimerization of the starting material yielding 4,4'-dimercaptoazobenzene (DMAB), while in the second pathway, it is proposed that NTP is reduced to ATP in HCl solution. In this combined experimental and theoretical study, we disentangled the involved plasmon-mediated reaction mechanisms by carefully controlling the reaction conditions in acidic solutions and vapor. Motivated by the different surface-enhanced Raman scattering (SERS) spectra of NTP/ATP samples and band shifts in acidic solution, which are generally attributed to water, additional experiments under pure gaseous conditions were performed. Under such acidic vapor conditions, the Raman data strongly suggest the formation of a hitherto not experimentally identified stable compound. Computational modeling of the plasmonic hybrid systems, i.e., regarding the wavelength-dependent character of the involved electronic transitions of the detected key intermediates in both reaction pathways, confirmed the experimental finding of the new compound, namely, 4-nitrosothiophenol (TP*). Tracking the reaction dynamics via time-dependent SERS measurements allowed us to establish the link between the dimer- and monomer-based pathways and to suggest possible reaction routes under different environmental conditions. Thereby, insight at the molecular level was provided with respect to the thermodynamics of the underlying reaction mechanism, complementing the spectroscopic results.

Supramolecular Reorientation During Deposition Onto Metal Surfaces of Quasi-Two-Dimensional Langmuir Monolayers Composed of Bifunctional Amphiphilic, Twisted Perylenes.

Supramolecular dye structures, which are often ruled by π-π interactions between planar chromophores, crucially determine the optoelectronic properties of layers and interfaces. Here, we present the interfacial assembly of perylene monoanhydride and monoimide that do not feature a planar chromophore but contain chlorine substituents in the bay positions to yield twisted chromophores and hence modified π-stacking. The assembly of the twisted perylene monoanhydride and monoimide is driven by their amphiphilicity that ensures proper Langmuir layer formation. The shielding of the hydrophilic segment upon attaching an alkyl chain to the imide moiety yielded a more rigid Langmuir layer, even though the degrees of freedom were increased due to this modification. For the characterization of the Langmuir layer's supramolecular structure, the layers were deposited onto glass, silver, and gold substrates via Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques and were investigated with atomic force microscopy and surface-enhanced resonance Raman spectroscopy (SERRS). From the similarity between all SERR spectra of the LS and LB layers, we concluded that the perylenes have changed their orientation upon LB deposition to bind to the silver surface of the SERRS substrate via sulfur atoms. In the Langmuir layer, the perylenes, which are π-stacked with half of the twisted chromophores, must already be inclined and cannot achieve full parallel alignment because of the twisting-induced steric hindrance. However, upon rotation, the energetically most favorable antiparallel aligned structures can be formed and bind to the SERRS substrate. Thus, we present, to the best of our knowledge, the first fabrication of quasi-two-dimensional films from twisted amphiphilic perylene monoimides and their reassembly during LB deposition. The relation between the molecular structure, supramolecular interfacial assembly, and its adoption during adsorption revealed here is crucial for the fabrication of defined functionalizations of metal surfaces, which is key to the development of organic (opto)electronic devices.

Unveiling the interaction of protein fibrils with gold nanoparticles by plasmon enhanced nano-spectroscopy.

The development of various degenerative diseases is suggested to be triggered by the uncontrolled organisation and aggregation of proteins into amyloid fibrils. For this reason, there are ongoing efforts to develop novel agents and approaches, including metal nanoparticle-based colloids, that dissolve amyloid structures and prevent pathogenic protein aggregation. In this contribution, the role of gold nanoparticles (AuNPs) in degrading amyloid fibrils of the model protein lysozyme is investigated. The amino acid composition of fibril surfaces before and after the incubation with AuNPs is determined at the single fibril level by exploiting the high spatial resolution and sensitivity provided by tip-enhanced and surface-enhanced Raman spectroscopies. This combined spectroscopic approach allows to reveal the molecular mechanisms driving the interaction between fibrils and AuNPs. Our results provide an important input for the understanding of amyloid fibrils and could have a potential translational impact on the development of strategies for the prevention and treatment of amyloid-related diseases.

pH-dependent disintegration of insulin amyloid fibrils monitored with atomic force microscopy and surface-enhanced Raman spectroscopy.

Aggregation of insulin into amyloid fibrils is characterized by the conversion of the native secondary structure of the peptide into an enriched ß-sheet conformation. In vitro, the growth or disintegration of amyloid fibrils can be influenced by various external factors such as pH, temperature etc. While current studies mainly focus on the influence of environmental conditions on the growth process of insulin fibrils, the present study investigates the effect of pH changes on the morphology and secondary structure of mature fibrils. In the experiments, insulin is fibrillated at pH 2.5 and the grown mature fibrils are suspended in pH 4-7 solutions. The obtained structures are analyzed by atomic force microscopy (AFM) and surface-enhanced Raman spectroscopy (SERS). Initially grown mature fibrils from pH 2.5 solutions show a long and intertwined morphology. Increasing the solution pH initiates the gradual disintegration of the filamentous morphology into unordered aggregates. These observations are supported by SERS experiments, where the spectra of the mature fibrils show mainly a ß-pleated sheet conformation, while the amide I band region of the amorphous aggregates indicate exclusively α-helix/unordered structures. The results demonstrate that no complex reagent is required for the disintegration of insulin fibrils. Simply regulating the pH of the environment induces local changes in the protonation state within the peptide chains. This effectively disrupts the well-ordered ß-sheet structure network based on hydrogen bonds.

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes.

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

Laser spectroscopic technique for direct identification of a single virus I: FASTER CARS.

From the famous 1918 H1N1 influenza to the present COVID-19 pandemic, the need for improved viral detection techniques is all too apparent. The aim of the present paper is to show that identification of individual virus particles in clinical sample materials quickly and reliably is near at hand. First of all, our team has developed techniques for identification of virions based on a modular atomic force microscopy (AFM). Furthermore, femtosecond adaptive spectroscopic techniques with enhanced resolution via coherent anti-Stokes Raman scattering (FASTER CARS) using tip-enhanced techniques markedly improves the sensitivity [M. O. Scully, et al, Proc. Natl. Acad. Sci. U.S.A. 99, 10994-11001 (2002)].

Surface characterization of nanoscale co-crystals enabled through tip enhanced Raman spectroscopy.

Atomic Force Microscopy coupled with Tip Enhanced Raman Spectroscopy (AFM-TERS) was applied to obtain information about the structure and surface composition of single nano co-crystals. For this purpose, a co-crystalline system consisting of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazatetracyclo-[5.5.0.03,11.05,9]-dodecane (CL-20) and 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) in a molar ratio of 2 : 1 (CL-20/HMX) was chosen. CL-20/HMX nano-plates were prepared by spray flash evaporation. To ensure co-crystallinity and nanostructures, powder X-ray diffraction and AFM investigations were performed. The results demonstrate that coherence lengths and particle dimensions are on a similar level though coherence lengths appear shorter than measured particle dimensions. According to this fact, defects inside the nano co-crystals are minimized. The co-crystallinity was additionally proven by confocal Raman spectroscopy. Here, marker bands for pristine CL-20 and HMX were chosen which appear in the CL-20/HMX spectrum in an intensity ratio of ∼2.5 : 1 (CL-20 : HMX). Afterwards surface investigations of single CL-20/HMX nano-plates were performed by AFM-TERS. Due to the surface sensitivity of TERS, these experiments reveal that the ratio of the Raman intensities between CL-20 and HMX inverts at CL-20/HMX nano-plate surfaces. Therefore, it is concluded that nano co-crystal surfaces consist of molecular layers of HMX. A theoretical approximation of the normal coordinates of the investigated marker vibrations supports this conclusion since it can exclude the occurrence of the intensity ratio inversion because of the given orientation between CL-20/HMX nano-plates and the Raman scattering system. Based on this finding, an impact ignition mechanism is proposed, enabling explanation of the close impact sensitivity values of ß-HMX and CL-20/HMX.

Plasmon induced deprotonation of 2-mercaptopyridine.

Surface plasmons can provide a novel route to induce and simultaneously monitor selective bond formation and breakage. Here pH-induced protonation, followed by plasmon-induced deprotonation of 2-mercaptopyridine was investigated using surface- and tip-enhanced Raman scattering (SERS and TERS). A large difference in the deprotonation rate between SERS and TERS will be demonstrated and discussed with respect to hot-spot distribution.

Organic acids, siderophores, enzymes and mechanical pressure for black slate bioweathering with the basidiomycete Schizophyllum commune.

Although many fungi are known to be able to perform bioweathering of rocks and minerals, little information is available concerning the role of basidiomycetes in this process. The wood-rotting basidiomycete Schizophyllum commune was investigated for its ability to degrade black slate, a rock rich in organic carbon. Mechanical pressure of hyphae and extracellular polymeric substances was investigated for biophysical weathering. A mixed ß1-3/ß1-6 glucan, likely schizophyllan that is well known from S. commune, could be identified on black slate surfaces. Secretion of siderophores and organic acids as biochemical weathering agents was shown. Both may contribute to biochemical weathering in addition to enzymatic functions. Previously, the exoenzyme laccase was believed to attack organic the matter within the black slate, thereby releasing metals from the rock. Here, overexpression of laccase showed enhanced dissolution of quartz phases by etching and pitting. At the same time, the formation of a new secondary mineral phase, whewellite, could be demonstrated. Hence, a more comprehensive understanding of biophysical as well as biochemical weathering by S. commune could be reached and unexpected mechanisms like quartz dissolution linked to shale degradation.

Protein Handshake on the Nanoscale: How Albumin and Hemoglobin Self-Assemble into Nanohybrid Fibers.

Creating and establishing proof of hybrid protein nanofibers (hPNFs), i.e., PNFs that contain more than one protein, is a currently unsolved challenge in bioinspired materials science. Such hPNFs could serve as universal building blocks for the bottom-up preparation of functional materials with bespoke properties. Here, inspired by the protein assemblies occurring in nature, we introduce hPNFs created via a facile self-assembly route and composed of human serum albumin (HSA) and human hemoglobin (HGB) proteins. Our circular dichroism results shed light on the mechanism of the proteins' self-assembly into hybrid nanofibers, which is driven by electrostatic/hydrophobic interactions between similar amino acid sequences (protein handshake) exposed to ethanol-triggered protein denaturation. Based on nanoscale characterization with tip-enhanced Raman spectroscopy (TERS) and immunogold labeling, our results demonstrate the existence and heterogenic nature of the hPNFs and reveal the high HSA/HGB composition ratio, which is attributed to the fast self-assembling kinetics of HSA. The self-assembled hPNFs with a high aspect ratio of over 100 can potentially serve as biocompatible units to create larger bioactive structures, devices, and sensors.

Tip-enhanced Raman spectroscopy - from early developments to recent advances.

An analytical technique operating at the nanoscale must be flexible regarding variable experimental conditions while ideally also being highly specific, extremely sensitive, and spatially confined. In this respect, tip-enhanced Raman scattering (TERS) has been demonstrated to be ideally suited to, e.g., elucidating chemical reaction mechanisms, determining the distribution of components and identifying and localizing specific molecular structures at the nanometre scale. TERS combines the specificity of Raman spectroscopy with the high spatial resolution of scanning probe microscopies by utilizing plasmonic nanostructures to confine the incident electromagnetic field and increase it by many orders of magnitude. Consequently, molecular structure information in the optical near field that is inaccessible to other optical microscopy methods can be obtained. In this general review, the development of this still-young technique, from early experiments to recent achievements concerning inorganic, organic, and biological materials, is addressed. Accordingly, the technical developments necessary for stable and reliable AFM- and STM-based TERS experiments, together with the specific properties of the instruments under different conditions, are reviewed. The review also highlights selected experiments illustrating the capabilities of this emerging technique, the number of users of which has steadily increased since its inception in 2000. Finally, an assessment of the frontiers and new concepts of TERS, which aim towards rendering it a general and widely applicable technique that combines the highest possible lateral resolution and extreme sensitivity, is provided.

High-resolution Raman Spectroscopy for the Nanostructural Characterization of Explosive Nanodiamond Precursors.

The specific attributes of nanodiamonds have attracted increasing interest for electronics or biomedical applications. An efficient synthetic route towards nanodiamonds is via detonation of hexolite (i.e. a mixture of TNT [2,4,6-trinitrotoluene] and RDX [1,3,5-trinitro-1,3,5-triazine]). In particular, detonation of hexolite crystallized by spray flash evaporation (SFE) yields extremely small diamonds (<4 nm). To unravel the detonation mechanism, a structural characterization of the explosives is required but is challenging due to their thermal instability. We demonstrate a combination of conventional Raman spectroscopy and tip-enhanced Raman spectroscopy (TERS) for resolving morphological and structural differences of differently prepared hexolite nanocomposites. The experiments allow for the first time a structural differentiation of individual TNT and RDX crystals and 15-20 nm sized core-shell structures, consequently providing a general approach to investigate the actual composition of mixtures on the nanometer scale.

High resolution spectroscopy reveals fibrillation inhibition pathways of insulin.

Fibril formation implies the conversion of a protein's native secondary structure and is associated with several neurodegenerative diseases. A better understanding of fibrillation inhibition and fibril dissection requires nanoscale molecular characterization of amyloid structures involved. Tip-enhanced Raman scattering (TERS) has already been used to chemically analyze amyloid fibrils on a sub-protein unit basis. Here, TERS in combination with atomic force microscopy (AFM), and conventional Raman spectroscopy characterizes insulin assemblies generated during inhibition and dissection experiments in the presence of benzonitrile, dimethylsulfoxide, quercetin, and ß-carotene. The AFM topography indicates formation of filamentous or bead-like insulin self-assemblies. Information on the secondary structure of bulk samples and of single aggregates is obtained from standard Raman and TERS measurements. In particular the high spatial resolution of TERS reveals the surface conformations associated with the specific agents. The insulin aggregates formed under different inhibition and dissection conditions can show a similar morphology but differ in their ß-sheet structure content. This suggests different aggregation pathways where the prevention of the ß-sheet stacking of the peptide chains plays a major role. The presented approach is not limited to amyloid-related reasearch but can be readily applied to systems requiring extremely surface-sensitive characterization without the need of labels.

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies.

The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D'-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the ß-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.

Tip-Enhanced Raman Spectroscopy of Atmospherically Relevant Aerosol Nanoparticles.

Atmospheric aerosol nanoparticles play a major role in many atmospheric processes and in particular in the global climate system. Understanding their formation by homogeneous or heterogeneous nucleation as well as their photochemical aging and atmospheric transformation is of utmost importance to evaluate their impact on atmospheric phenomena. Single particle analysis like tip-enhanced Raman spectroscopy (TERS) opens access to a deeper understanding of these nanoparticles. Atmospherically relevant nanoparticles, formed above a simulated salt lake inside an aerosol smog-chamber, were analyzed using TERS. TERS spectra of 11 nanoparticles were studied in detail. First results of TERS on atmospherically relevant aerosol nanoparticles reveal the presence of inorganic seed particles, a chemical diversity of equally sized particles in the nucleation mode, and chemical transformation during photochemical aging. Therefore, single particle analysis by optical near-field spectroscopy such as TERS of atmospheric nanoparticles will significantly contribute to elucidate atmospheric nucleation, photochemical aging, and chemical transformation processes by uncovering single particle based properties.

Spatially resolved spectroscopic differentiation of hydrophilic and hydrophobic domains on individual insulin amyloid fibrils.

The formation of insoluble ß-sheet-rich protein structures known as amyloid fibrils is associated with numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. A detailed understanding of the molecular structure of the fibril surface is of interest as the first contact with the physiological environment in vivo and plays a decisive role in biological activity and associated toxicity. Recent studies reveal that the inherent sensitivity and specificity of tip-enhanced Raman scattering (TERS) renders this technique a compelling method for fibril surface analysis at the single-particle level. Here, the reproducibility of TERS is demonstrated, indicating its relevance for detecting molecular variations. Consequently, individual fibrils are systematically investigated at nanometer spatial resolution. Spectral parameters were obtained by band-fitting, particularly focusing on the identification of the secondary structure via the amide III band and the differentiation of hydrophobic and hydrophilic domains on the surface. In addition multivariate data analysis, specifically the N-FINDR procedure, was employed to generate structure-specific maps. The ability of TERS to localize specific structural domains on fibril surfaces shows promise to the development of new fibril dissection strategies and can be generally applied to any (bio)chemical surface when structural variations at the nanometer level are of interest.

Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism.

Amyloid fibrils are a large class of self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal ß-sheet core stabilized by hydrogen bonds, but the molecular structure of the peptide subunits exposed on the fibril surface is variable. Here we show that multimodal spectroscopy using a range of bulk- and surface-sensitive techniques provides a powerful way to dissect variations in the molecular structure of polymorphic amyloid fibrils. As a model system, we use fibrils formed by the milk protein ß-lactoglobulin, whose morphology can be tuned by varying the protein concentration during formation. We investigate the differences in the molecular structure and composition between long, straight fibrils versus short, wormlike fibrils. We show using mass spectrometry that the peptide composition of the two fibril types is similar. The overall molecular structure of the fibrils probed with various bulk-sensitive spectroscopic techniques shows a dominant contribution of the ß-sheet core but no difference in structure between straight and wormlike fibrils. However, when probing specifically the surface of the fibrils with nanometer resolution using tip-enhanced Raman spectroscopy (TERS), we find that both fibril types exhibit a heterogeneous surface structure with mainly unordered or α-helical structures and that the surface of long, straight fibrils contains markedly more ß-sheet structure than the surface of short, wormlike fibrils. This finding is consistent with previous surface-specific vibrational sum-frequency generation (VSFG) spectroscopic results ( VandenAkker et al. J. Am. Chem. Soc. , 2011 , 133 , 18030 - 18033 , DOI: 10.1021/ja206513r ). In conclusion, only advanced vibrational spectroscopic techniques sensitive to surface structure such as TERS and VSFG are able to reveal the difference in structure that underlies the distinct morphology and rigidity of different amyloid fibril polymorphs that have been observed for a large range of food and disease-related proteins.

Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies.

Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (ß-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have ß-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of ß-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface.