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Hydrogen peroxide (H2O2), a strong oxidizer, is frequently used for chemical sterilization in aseptic food processing. Due to their inherent robustness, bacterial spores, i.e. spores of Bacillus atrophaeus, are commonly employed to evaluate the efficacy of the sterilization procedure [1]. Despite its widespread use, however, the details of the sporicidal action of H2O2 are not yet fully understood, with various mechanisms being discussed in the literature [2,3]. Here, we employ optical trapping Raman microscopy to elucidate the sporicidal mechanism of H2O2. Optical trapping Raman microscopy allows us to isolate, investigate, and manipulate individual spores from a sample (Fig. 1A). Combining optical microscopy with the real-time, label-free chemical information obtained from Raman spectroscopy directly reveals the sequence of steps that lead to oxidative spore death after H2O2 exposure (Fig. 1B). We find that spore degradation begins with the fast, cooperative release of dipicolinic acid (DPA), a key spore biomarker and a major component of spore’s core. This indicates a breach of the inner membrane surrounding the core. The onset of DPA release depends on the concentration of H2O2. DPA outflux is accompanied by the formation of oxidation products (mostly carbonyl compounds) and oxidation continues after exchange of DPA with the surrounding medium is complete. The remaining spore shell then slowly disintegrates and continues to shrink on a timescale of hundreds of seconds. Simultaneous observation of spore morphology and size by optical microscopy and particle tracking corroborates this three-step mechanism. The presented assay allows on-line monitoring of spore composition and morphology, which directly reveals the mechanism underlying the sporicidal action of H2O2. In addition, trapped spores can be exposed to different chemical or physical stimuli (temperature, light, chemical composition of the surrounding medium, etc.) to investigate a variety of industrially relevant sterilization conditions. Jildeh, Z. B., Wagner, P. H. & Schöning, M. J. Sterilization of Objects, Products, and Packaging Surfaces and Their Characterization in Different Fields of Industry: The Status in 2020. physica status solidi (a) 218, 2000732 (2021). Setlow, P. & Christie, G. What’s new and notable in bacterial spore killing. World J Microbiol Biotechnol 37, 144 (2021). Setlow, P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101, 514-525 (2006). Figure 1

Surface-enhanced Raman spectroscopy (SERS) combines the unique advantages of Raman spectroscopy – namely detection of specific molecular fingerprints without requiring prior labelling – with enhanced sensitivity. SERS takes advantage of the amplification of the incident electric field by localized surface plasmons on noble metal nanoparticle surfaces, which allows the rapid, non-destructive detection of a wide variety of analytes ranging from small molecules to entire microorganisms. The data obtained from SERS experiments, however, can be difficult to interpret. Spectra are dominated by the chemical species in closest contact with the plasmonic field, and consequently spectra recorded at different points in time from the same sample can exhibit considerable differences. The rich chemical information contained in the spectral data therefore is often not utilized to its full potential or lost altogether. Here, we combine deep convolutional neural networks (CNNs) with SERS to classify series of spectra obtained from mixtures of different sample species (1). We find that CNNs outperform standard chemometric methods, such as principal component analysis, but also support-vector machine classifiers, shallow artificial neural networks, or even trained human experts while requiring minimal data preprocessing, such as background subtraction or smoothing. We discuss optimizing the classifier with regards to network topology, learning strategy, and data augmentation. In addition, we show that we can extract useful information from trained networks. In simple cases, activation maximization spectra, which represent the spectral features that are most relevant to discriminate between different sample classes, can provide a good approximation of the spectra in the original sample. We also train generative adversarial networks (GANs) (2) to generate / extract characteristic spectra from large, more heterogeneous datasets. These spectra can provide additional insight into the composition of the sample and might thus be relevant to guide further experiments. Deep learning and SERS are two powerful techniques that complement each other. The high sensitivity of SERS quickly generates large, but often highly complex spectral datasets, which are in turn well-suited to the analysis by CNNs. Most importantly, deep learning not only excels at classification but can be used as a more general tool to facilitate the interpretation of heterogeneous, seemingly intractable datasets. Given the tremendous attention deep learning has attracted over the past decade, we anticipate that it will play an increasingly prominent role in spectral analysis as well. Yang, J. et al., 2019. Deep learning for vibrational spectral analysis: Recent progress and a practical guide. Analytica chimica acta, 1081, pp.6–17. Odena, A., Olah, C. & Shlens, J., 2016. Conditional Image Synthesis With Auxiliary Classifier GANs. arXiv:1610.09585 [stat.ML] Figure 1

In recent years, Raman spectroscopy has evolved into a broadly used analytical method in chemistry, life sciences, and medicine. Here, we combine Raman spectroscopy with optical trapping and optical microscopy to isolate and investigate individual micron-size biological particles from a sample. In this setup, Raman spectroscopy provides a label-free chemical fingerprint while light microscopy reveals the sample morphology. In addition, elastic light scattering as well as observation of diffusive movement in the optical trap’s potential offer information on particle size and refractive index. Single particles can be captured and manipulated for tens of minutes. During this time, the stably trapped particles can be exposed to a variety of chemical and physical stimuli such as heat, light of different wavelengths, or changes in the chemical composition of the particle environment through microfluidics (Fig. 1A). In this work, we employ this optical trapping Raman spectroscopy sensor / actuator / treatment system to study the effect of hydrogen peroxide (H2O2) on individual spores of Bacillus atrophaeus, an important model system to assess the efficacy of industrial sterilization processes. Interestingly, we find B. atrophaeus spores to be stable for more than 30 minutes while optically trapped even at elevated temperatures (> 70 °C). Exposure to H2O2 results in spore degradation in a concentration-dependent manner within several minutes and is accelerated by high temperatures. Raman spectroscopy indicates that spore degradation is characterized by the rupture of the spore core resulting in the fast release of dipicolinic acid, a major spore biomarker that is considered key for spore resistance to environmental stress (Fig. 1B). Oxidative degradation of the spore shell starts before core rupture and continues after dipicolinic acid release. Simultaneous observation of spore morphology corroborates this two-phase model. Illumination with shorter (< 550 nm) wavelength light exposes the spores to oxidative stress via the generation of reactive oxygen species in the spore environment by light, thereby mimicking the H2O2 sterilization process. In the presence of small amounts (0.5%) H2O2, spore lifetime is reduced to several seconds. With the presented system, we address spore activity down to the single cellular level in a multimodal manner: trapping and manipulation of single spores over several tens of minutes (actuator functionality), stimulation of single spores by temperature, illumination, and H2O2 exposure (treatment functionality) as well as on-line monitoring of spore activity (sensor functionality). Figure 1

Self-stability in light-emitting electrochemical cells (LECs) based on Ir(III) ionic transition metal complexes (Ir-iTMC) has been long overlooked. Herein, it is demonstrated that the nature of the active layer blending an archetype Ir-iTMC as emitter and ionic electrolytes—ionic liquid (IL) or ionic polyelectrolyte (IP)—is paramount for the storage and mechanical stability of rigid/flexible LECs. Strikingly, devices with ionic polyelectrolytes (IPs) stand out compared to those with traditional configurations with or without ILs. They exhibit i) superior brightness and efficiencies in rigid/flexible devices due to the higher photoluminescence quantum yield, ii) the best performance at pulsed current driving mode under inert/ambient operation conditions due to a slower growth of the doped regions, iii) enhanced device stabilities upon ambient/inert storage, resulting in

Electrodeposition process has been widely applied to various areas to form functional thin films and nanostructures. In order to understand the deposition process from molecular level to achieve further precise control, we employ theoretical calculation and surface enhanced Raman scattering (SERS). For the theoretical calculation to study the deposition reaction mechanism, molecular orbital and density functional theory calculations were applied to investigate the behaver of the species at the electrode surface, and various factors such as solvation effect and catalytic activity of the surface were also analysed. In addition, SERS equipped with confocal microscopy was used for in situ analysis of the reaction processes at the electrode surface. For this we applied plasmon sensors [1] to achieve very high sensitivity analysis. Furthermore multi-confocal type SERS microscopy was employed for mapping and imaging analysis. By using these techniques, various systems have been analysed and modelling of their reaction processes has been carried out. This work was financially supported in part by “Development of Systems and Technology for Advanced Measurement and Analysis,” Japan Science and Technology Agency, and Grant-in-Aid for Scientific Research, MEXT, Japan. [1] M. Yanagisawa, M. Saito, M. Kunimoto, T. Homma, Appl. Phys. Express, 9 , 122002 (2016).

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric alpha-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the alpha-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the alpha-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an "all-or-none" mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the alpha-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the alpha-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.

Electrochemical deposition processes have been widely applied to form functional thin films and micro/nano structures featuring their controllability and capability to fabricate uniform deposits to wide, non-flat surfaces. For achieving further precise control, we have attempted molecular-level analysis of the deposition processes and reaction mechanisms through theoretical and experimental approaches. In this paper, we will introduce these approaches with some of our resent results. For the theoretical analysis to quantitatively understand the reaction mechanisms, we have applied density functional theory (DFT) calculations focusing on the surface species such as additives, reaction intermediates, reducing agents (for electroless deposition), and so on[1]. Effects of solvation and catalytic activity of the surfaces, etc., were also investigated and behaviours of these species, which are unique to the deposition at solid/liquid interfaces, were elucidated. While these theoretical analysis could predict rigorous mechanisms of the deposition reaction processes, acquiring such molecular-level data through experimental approach is quite challenging since the amount of these surface species are trace and they are “buried” under the electrolyte. For this, we focused on surface enhanced Raman scattering (SERS) equipped with confocal microscopy and developed plasmonic sensors with patterned nanostructures to achieve very high enhancement effect, and we applied them to analyze the electrochemical deposition processes such as the behaviour of additives. For the plasmonic sensors, we have developed “reflection” type and “transmission” type; the “reflection” type sensors consist of metals active for plasmonic enhancement such as Ag, Au, and Cu with centrifuge-grooved nanostructures or nanodot arrays for maximizing the enhancement, which can be directly used as the substrates for the deposition[2]. The “transmission” type sensors, which are single or array of the microlens coated with discrete nanoparticles of the plasmon-active metals, can be applied to various processes regardless of the substrate materials[3]. By using these sensors, we analyzed behaviours of the additives and reductants at the deposition sites. In addition, we have also developed a system for in situ analysis of the processes inside the microscopic pores such as through silicon via (TSV) [4]. A model-pore was fabricated using the sensor (to be a part of “side-wall” of the pore) with the metal nanodot array covered with the structure made of poly(dimethylsiloxane) (PDMS). It was confirmed that Raman signal of the additives could be detected in situ in high sensitivity without interference of the signals originated from PDMS, and their behaviour was investigated in detail. Furthermore, attempts on measuring local temperature as well as pH in the vicinity of the electrode were made by using the SERS measurements, which will be able to provide new insights for precise analysis of the behaviours of surface species during the deposition processes. This work was financially supported in part by “Development of Systems and Technology for Advanced Measurement and Analysis,” Japan Science and Technology Agency, and Grant-in-Aid for Scientific Research, MEXT, Japan. [1] For example, T. Homma, Electrochemistry, 83, 680 (2015). [2] B. Jiang, M. Kunimoto, M. Yanagisawa, T. Homma, J. Electrochem. Soc., 160, D366 (2013). [3] M. Yanagisawa, M. Saito, M. Kunimoto, T. Homma, Appl. Phys. Express, 9 , 122002 (2016). [4] T. Homma, A. Kato, M. Kunimoto, M. Yanagisawa, Electrochem. Commn., submitted.

Mechanical stability of bonds and protein interactions has recently become accessible through single molecule mechanical experiments. So far, mechanical information about molecular bond mechanics has been largely limited to a single direction of force application. However, mechanical force acts as a vector in space and hence mechanical stability should depend on the direction of force application. In skeletal muscle, the giant protein titin is anchored in the Z-disk by telethonin. Much of the structural integrity of the Z-disk hinges upon the titin-telethonin bond. In this paper we show that the complex between the muscle proteins titin and telethonin forms a highly directed molecular bond. It is designed to resist ultra-high forces if they are applied in the direction along which it is loaded under physiological conditions, while it breaks easily along other directions. Highly directed molecular bonds match in an ideal way the requirements of tissues subject to mechanical stress.

Single-molecule methods have given experimental access to the mechanical properties of single protein molecules. So far, access has been limited to mostly one spatial direction of force application. Here, we report single-molecule experiments that explore the mechanical properties of a folded protein structure in precisely controlled directions by applying force to selected amino acid pairs. We investigated the deformation response of GFP in five selected directions. We found fracture forces widely varying from 100 pN up to 600 pN. We show that straining the GFP structure in one of the five directions induces partial fracture of the protein into a half-folded intermediate structure. From potential widths we estimated directional spring constants of the GFP structure and found values ranging from 1 N/m up to 17 N/m. Our results show that classical continuum mechanics and simple mechanistic models fail to describe the complex mechanics of the GFP protein structure and offer insights into the mechanical design of protein materials.

The highly oriented filamentous protein network of muscle constantly experiences significant mechanical load during muscle operation. The dimeric protein myomesin has been identified as an important M-band component supporting the mechanical integrity of the entire sarcomere. Recent structural studies have revealed a long α-helical linker between the C-terminal immunoglobulin (Ig) domains My12 and My13 of myomesin. In this paper, we have used single-molecule force spectroscopy in combination with molecular dynamics simulations to characterize the mechanics of the myomesin dimer comprising immunoglobulin domains My12–My13. We find that at forces of approximately 30 pN the α-helical linker reversibly elongates allowing the molecule to extend by more than the folded extension of a full domain. High-resolution measurements directly reveal the equilibrium folding/unfolding kinetics of the individual helix. We show that α-helix unfolding mechanically protects the molecule homodimerization from dissociation at physiologically relevant forces. As fast and reversible molecular springs the myomesin α-helical linkers are an essential component for the structural integrity of the M band.

In the sarcomeric M-band, the giant ruler proteins titin and obscurin, its small homologue obscurin-like-1 (obsl1), and the myosin cross-linking protein myomesin form a ternary complex that is crucial for the function of the M-band as a mechanical link. Mutations in the last titin immunoglobulin (Ig) domain M10, which interacts with the N-terminal Ig-domains of obscurin and obsl1, lead to hereditary muscle diseases. The M10 domain is unusual not only in that it is a frequent target of disease-linked mutations, but also in that it is the only currently known muscle Ig-domain that interacts with two ligands—obscurin and obsl1—in different sarcomeric subregions. Using x-ray crystallography, we show the structural basis for titin M10 interaction with obsl1 in a novel antiparallel Ig-Ig architecture and unravel the molecular basis of titin-M10 linked myopathies. The severity of these pathologies correlates with the disruption of the titin-obsl1/obscurin complex. Conserved signature residues at the interface account for differences in affinity that direct the cellular sorting in cardiomyocytes. By engineering the interface signature residues of obsl1 to obscurin, and vice versa, their affinity for titin can be modulated similar to the native proteins. In single-molecule force-spectroscopy experiments, both complexes yield at forces of around 30 pN, much lower than those observed for the mechanically stable Z-disk complex of titin and telethonin, suggesting why even moderate weakening of the obsl1/obscurin-titin links has severe consequences for normal muscle functions.

In the past decade, single-molecule force spectroscopy has provided new insights into the key interactions stabilizing folded proteins. A few recent studies probing the effects of ligand binding on mechanical protein stability have come to quite different conclusions. While some proteins seem to be stabilized considerably by a bound ligand, others appear to be unaffected. Since force acts as a vector in space, it is conceivable that mechanical stabilization by ligand binding is dependent on the direction of force application. In this study, we vary the direction of the force to investigate the effect of ligand binding on the stability of maltose binding protein (MBP). MBP consists of two lobes connected by a hinge region that move from an open to a closed conformation when the ligand maltose binds. Previous mechanical experiments, where load was applied to the N and C termini, have demonstrated that MBP is built up of four building blocks (unfoldons) that sequentially detach from the folded structure. In this study, we design the pulling direction so that force application moves the two MBP lobes apart along the hinge axis. Mechanical unfolding in this geometry proceeds via an intermediate state whose boundaries coincide with previously reported MBP unfoldons. We find that in contrast to N-C-terminal pulling experiments, the mechanical stability of MBP is increased by ligand binding when load is applied to the two lobes and force breaks the protein-ligand interactions directly. Contour length measurements indicate that MBP is forced into an open conformation before unfolding even if ligand is bound. Using mutagenesis experiments, we demonstrate that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. This work illustrates how varying the direction of the applied force allows revealing important details about the ligand binding mechanics of a large protein.

Identifying independently folding cores or substructures is important for understanding and assaying the structure, function and assembly of large proteins. Here, we suggest mechanical stability as a criterion to identify building blocks of the 366 amino acid maltose-binding protein (MBP). We find that MBP, when pulled at its termini, unfolds via three (meta-) stable unfolding intermediates. Consequently, the MBP structure consists of four structural blocks (unfoldons) that detach sequentially from the folded structure upon force application. We used cysteine cross-link mutations to characterize the four unfoldons structurally. We showed that many MBP constructs composed of those building blocks indeed form stably folded structures in solution. Mechanical unfoldons may provide a new tool for a systematic search for stable substructures of large proteins.

Single-molecule methods such as force spectroscopy give experimental access to the mechanical properties of protein molecules. So far, owing to the limitations of recombinant construction of polyproteins, experimental access has been limited to mostly the N-to-C terminal direction of force application. This protocol gives a fast and simple alternative to current recombinant strategies for preparing polyproteins. We describe in detail the method to construct polyproteins with precisely controlled linkage topologies, based on the pairwise introduction of cysteines into protein structure and subsequent polymerization in solution. Stretching such constructed polyproteins in an atomic force microscope allows mechanical force application to a single protein structure via two precisely controlled amino acid positions in the functional three-dimensional protein structure. The capability for site-directed force application can provide valuable information about both protein structure and directional protein mechanics. This protocol should be applicable to almost any protein that can be point mutated. Given correct setup of all necessary reagents, this protocol can be accomplished in fewer than 10 d.

In recent years, single molecule force spectroscopy has opened unique possibilities to investigate the mechanical properties of single protein molecules. So far, experiments have focused on the mechanical behavior of a protein's tertiary structure. For a large number of proteins, however, multiple folded protein molecules are arranged into a multi-subunit complex. How the quaternary structure of such a complex responds to force is not clear. Here, we present a toolkit to study the mechanical properties of dimeric and trimeric protein complexes by single molecule force spectroscopy. We apply these methods to two different model systems:The dimeric α-crystallin domain is the building block of Hsp 16.5 from Methanococcus janashii. We can directly observe that this dimer dissociates at ∼ 200 pN into two metastable monomeric subunits, which subsequently unfold independently.The two most N-terminal domains of human titin are assembled into an antiparallel complex by telethonin in the Z-disk region of the sarcomere. We show that the Ig domains of titin are stabilized in the presence of telethonin, and that this stabilization is optimized to provide a high level of mechanical strength in the sarcomere. The dissociation force of the titin-telethonin complex exceeds 600 pN, making it one of the highest rupture forces known to date.Single molecule force spectroscopy allows us not only to measure directly the dissociation forces of protein complexes, but also to observe the mechanical hierarchy of the involved building blocks. Building on these results, future experiments will attempt to observe the assembly of protein complexes using single molecule force spectroscopy.