Hierarchical Self-Assembly of Water-Soluble Fullerene Derivatives into Supramolecular Hydrogels.

Controlling the self-assembly of nanoparticle building blocks into macroscale soft matter structures is an open question and of fundamental importance to fields as diverse as nanomedicine and next-generation energy storage. Within the vast library of nanoparticles, the fullerenes-a family of quasi-spherical carbon allotropes-are not explored beyond the most common, C60. Herein, a facile one-pot method is demonstrated for functionalizing fullerenes of different sizes (C60, C70, C84, and C90-92), yielding derivatives that self-assemble in aqueous solution into supramolecular hydrogels with distinct hierarchical structures. It is shown that the mechanical properties of these resultant structures vary drastically depending on the starting material. This work opens new avenues in the search for control of macroscale soft matter structures through tuning of nanoscale building blocks.

Enhancing nanoscale viscoelasticity characterization in bimodal atomic force microscopy.

Polymeric, soft, and biological materials exhibit viscoelasticity, which is a time dependent mechanical response to deformation. Material viscoelasticity emerges from the movement of a material's constituent molecules at the nano- and microscale in response to applied deformation. Therefore, viscoelastic properties depend on the speed at which a material is deformed. Recent technological advances, especially in atomic force microscopy (AFM), have provided tools to measure and map material viscoelasticity with nanoscale resolution. However, to obtain additional information about the viscoelastic behavior of a material from such measurements, theoretical grounding during data analysis is required. For example, commercially available bimodal AFM imaging maps two different viscoelastic properties of a sample, the storage modulus, E', and loss tangent, tan δ, with each property being measured by a different resonance frequency of the AFM cantilever. While such techniques provide high resolution maps of E' and tan δ, the different measurement frequencies make it difficult to calculate key viscoelastic properties of the sample such as: the model of viscoelasticity that describes the sample, the loss modulus, E'', at either frequency, elasticity E, viscosity η, and characteristic response times τ. To overcome this difficulty, we present a new data analysis procedure derived from linear viscoelasticity theory. This procedure is applied and validated by performing amplitude modulation-frequency modulation (AM-FM) AFM, a commercially available bimodal imaging technique, on a styrene-butadiene rubber (SBR) with known mechanical behavior. The new analysis procedure correctly identified the type of viscoelasticity exhibited by the SBR and accurately calculated SBR E, η, and τ, providing a useful means of enhancing the amount of information gained about a sample's nanoscale viscoelastic properties from bimodal AFM measurements. Additionally, being derived from fundamental models of linear viscoelasticity, the procedure can be employed for any technique where different viscoelastic properties are measured at different and discrete frequencies with applied deformations in the linear viscoelastic regime of a sample.

Cell wall fucosylation in Arabidopsis influences control of leaf water loss and alters stomatal development and mechanical properties.

The Arabidopsis sensitive-to-freezing8 (sfr8) mutant exhibits reduced cell wall (CW) fucose levels and compromised freezing tolerance. To examine whether CW fucosylation also affects the response to desiccation, we tested the effect of leaf excision in sfr8 and the allelic mutant mur1-1. Leaf water loss was strikingly higher than in the wild type in these, but not other, fucosylation mutants. We hypothesized that reduced fucosylation in guard cell (GC) walls might limit stomatal closure through altering mechanical properties. Multifrequency atomic force microscopy (AFM) measurements revealed a reduced elastic modulus (E'), representing reduced stiffness, in sfr8 GC walls. Interestingly, however, we discovered a compensatory mechanism whereby a concomitant reduction in the storage modulus (E'') maintained a wild-type viscoelastic time response (tau) in sfr8. Stomata in intact leaf discs of sfr8 responded normally to a closure stimulus, abscisic acid, suggesting that the time response may relate more to closure properties than stiffness does. sfr8 stomatal pore complexes were larger than those of the wild type, and GCs lacked a fully developed cuticular ledge, both potential contributors to the greater leaf water loss in sfr8. We present data that indicate that fucosylation-dependent dimerization of the CW pectic domain rhamnogalacturonan-II may be essential for normal cuticular ledge development and leaf water retention.

Multifrequency AFM reveals lipid membrane mechanical properties and the effect of cholesterol in modulating viscoelasticity.

The physical properties of lipid bilayers comprising the cell membrane occupy the current spotlight of membrane biology. Their traditional representation as a passive 2D fluid has gradually been abandoned in favor of a more complex picture: an anisotropic time-dependent viscoelastic biphasic material, capable of transmitting or attenuating mechanical forces that regulate biological processes. In establishing new models, quantitative experiments are necessary when attempting to develop suitable techniques for dynamic measurements. Here, we map both the elastic and viscous properties of the model system 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers using multifrequency atomic force microscopy (AFM), namely amplitude modulation-frequency modulation (AM-FM) AFM imaging in an aqueous environment. Furthermore, we investigate the effect of cholesterol (Chol) on the DPPC bilayer in concentrations from 0 to 60%. The AM-AFM quantitative maps demonstrate that at low Chol concentrations, the lipid bilayer displays a distinct phase separation and is elastic, whereas at higher Chol concentration, the bilayer appears homogenous and exhibits both elastic and viscous properties. At low-Chol contents, the Estorage modulus (elastic) dominates. As the Chol insertions increases, higher energy is dissipated; and although the bilayer stiffens (increase in Estorage), the viscous component dominates (Eloss). Our results provide evidence that the lipid bilayer exhibits both elastic and viscous properties that are modulated by the presence of Chol, which may affect the propagation (elastic) or attenuation (viscous) of mechanical signals across the cell membrane.

Biophysical characterisation of DNA origami nanostructures reveals inaccessibility to intercalation binding sites.

Intercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecule fluorescence microscopy, atomic force microscopy, gel electrophoresis and controllable damage using low temperature plasma on synthetic DNA origami samples. Our results indicate that not all potential DNA binding sites are accessible for dye intercalation, which has implications for future DNA nanostructures designed for targeted drug delivery.

Reconfigurable T-junction DNA Origami.

DNA self-assembly allows the construction of nanometre-scale structures and devices. Structures with thousands of unique components are routinely assembled in good yield. Experimental progress has been rapid, based largely on empirical design rules. Herein, we demonstrate a DNA origami technique designed as a model system with which to explore the mechanism of assembly. The origami fold is controlled through single-stranded loops embedded in a double-stranded DNA template and is programmed by a set of double-stranded linkers that specify pairwise interactions between loop sequences. Assembly is via T-junctions formed by hybridization of single-stranded overhangs on the linkers with the loops. The sequence of loops on the template and the set of interaction rules embodied in the linkers can be reconfigured with ease. We show that a set of just two interaction rules can be used to assemble simple T-junction origami motifs and that assembly can be performed at room temperature.

Temporal evolution of mechanical properties in PDMS: A comparative study of elastic modulus and relaxation time for storage in air and aqueous environment.

Polydimethylsiloxane (PDMS) is a soft, biocompatible polymer extensively employed in biomedical research, notable for its tunable mechanical properties achieved through cross-linking. While many studies have assessed the mechanical properties of PDMS utilizing macroscopic and microscopic methods, these analyses are often limited to freshly prepared samples. However, the mechanical properties of PDMS can be expected to change during prolonged exposure to water or air, such as interface polymer chain loosening or surface hardening, which are critical considerations in applications like cell culture platforms or microfluidic devices. This paper presents a comprehensive 10-day investigation of the evolution of PDMS surface mechanical properties through AFM-based nano-indentation. We focused on the most commonly utilized crosslinker-to-base ratios of PDMS, 1:10 (r10) and 1:20 (r20), under conditions of air and deionized water storage. For r10 samples, a hardening process was detected, peaking at 2.12 ± 0.35 MPa within five days for those stored in air and 1.71 ± 0.16 MPa by the third day for those immersed in water. During indentation, the samples displayed multiple contact points, suggesting the formation of distinct regions with varying mechanical properties. In contrast, r20 samples exhibited better stability, with an observed elastic modulus averaging 0.62 ± 0.06 MPa for air-stored and 0.74 ± 0.06 MPa for water-stored samples. Relaxation experiments, interpreted via the General Maxwell Model featuring two distinct component responses, a relatively consistent fast response τ1 (on the order of 10-1 s), and a more variable, slower response τ2 (on the order of 10 s), throughout the study period. The identification of two distinct relaxation times suggests the involvement of two disparate material property regimes in the relaxation process, implying changes in the surface material composition at the interface with air/water. These variations in mechanical properties could significantly influence the long-term functionality of PDMS in various biomedical applications.

Double Electrode Experiments Reveal the Processes Occurring at PEDOT-Coated Neural Electrode Arrays.

Neural electrodes have recently been developed with surface modifications of conductive polymers, in particular poly(3,4-ethylenedioxythiophene) (PEDOT), and extensively studied for their roles in recording and stimulation, aiming to improve their biocompatibility. In this work, the implications for the design of practical neural sensors are clarified, and systematic procedures for their preparation are reported. In particular, this study introduces the use of in vitro double electrode experiments to mimic the responses of neural electrodes with a focus on signal-recording electrodes modified with PEDOT. Specifically, potential steps on one unmodified electrode in an array are used to identify the responses for PEDOT doped with different anions and compared with that of a bare platinum (Pt) electrode. The response is shown to be related to the rearrangement of ions in solution near the detector electrode resulting from the potential step, with a current transient seen at the detector electrode. A rapid response for PEDOT doped with chloride (ca. 0.04 s) ions was observed and attributed to the fast movement of chloride ions in and out of the polymer film. In contrast, PEDOT doped with poly(styrenesulfonate) (PSS) responds much slower (ca. 2.2 s), and the essential immobility of polyanion constrains the direction of current flow.

Nanoscale Rheology: Dynamic Mechanical Analysis over a Broad and Continuous Frequency Range Using Photothermal Actuation Atomic Force Microscopy.

Polymeric materials are widely used in industries ranging from automotive to biomedical. Their mechanical properties play a crucial role in their application and function and arise from the nanoscale structures and interactions of their constitutive polymer molecules. Polymeric materials behave viscoelastically, i.e., their mechanical responses depend on the time scale of the measurements; quantifying these time-dependent rheological properties at the nanoscale is relevant to develop, for example, accurate models and simulations of those materials, which are needed for advanced industrial applications. In this paper, an atomic force microscopy (AFM) method based on the photothermal actuation of an AFM cantilever is developed to quantify the nanoscale loss tangent, storage modulus, and loss modulus of polymeric materials. The method is then validated on styrene-butadiene rubber (SBR), demonstrating the method's ability to quantify nanoscale viscoelasticity over a continuous frequency range up to 5 orders of magnitude (0.2-20,200 Hz). Furthermore, this method is combined with AFM viscoelastic mapping obtained with amplitude modulation-frequency modulation (AM-FM) AFM, enabling the extension of viscoelastic quantification over an even broader frequency range and demonstrating that the novel technique synergizes with preexisting AFM techniques for quantitative measurement of viscoelastic properties. The method presented here introduces a way to characterize the viscoelasticity of polymeric materials and soft and biological matter in general at the nanoscale for any application.

Transcending Markov: non-Markovian rate processes of thermosensitive TRP ion channels.

The Markov state model (MSM) is a popular theoretical tool for describing the hierarchy of time scales involved in the function of many proteins especially ion channel gating. An MSM is a particular case of the general non-Markovian model, where the rate of transition from one state to another does not depend on the history of state occupancy within the system, i.e. it only includes reversible, non-dissipative processes. However, an MSM requires knowledge of the precise conformational state of the protein and is not predictive when those details are not known. In the case of ion channels, this simple description fails in real (non-equilibrium) situations, for example when local temperature changes, or when energy losses occur during channel gating. Here, we show it is possible to use non-Markovian equations (i.e. offer a general description that includes the MSM as a particular case) to develop a relatively simple analytical model that describes the non-equilibrium behaviour of the temperature-sensitive transient receptor potential (TRP) ion channels, TRPV1 and TRPM8. This model accurately predicts asymmetrical opening and closing rates, infinite processes and the creation of new states, as well as the effect of temperature changes throughout the process. This approach therefore overcomes the limitations of the MSM and allows us to go beyond a mere phenomenological description of the dynamics of ion channel gating towards a better understanding of the physics underlying these processes.

Bilayer-mediated clustering and functional interaction of MscL channels.

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

Temperature-dependent phase transitions in zeptoliter volumes of a complex biological membrane.

Phase transitions in purple membrane have been a topic of debate for the past two decades. In this work we present studies of a reversible transition of purple membrane in the 50-60 °C range in zeptoliter volumes under different heating regimes (global heating and local heating). The temperature of the reversible phase transition is 52 ± 5 °C for both local and global heating, supporting the hypothesis that this transition is mainly due to a structural rearrangement of bR molecules and trimers. To achieve high resolution measurements of temperature-dependent phase transitions, a new scanning probe microscopy-based method was developed. We believe that our new technique can be extended to other biological systems and can contribute to the understanding of inhomogeneous phase transitions in complex systems.

Communication is central to the mission of science.

The future of our species and planet hinges on our scientific creativity to tackle future challenges. However, the trust of the public in scientific processes needs to be earned and kept, which will require inclusive, self-reflecting, honest and inspiring science communication.

Mapping cellular nanoscale viscoelasticity and relaxation times relevant to growth of living Arabidopsis thaliana plants using multifrequency AFM.

The shapes of living organisms are formed and maintained by precise control in time and space of growth, which is achieved by dynamically fine-tuning the mechanical (viscous and elastic) properties of their hierarchically built structures from the nanometer up. Most organisms on Earth including plants grow by yield (under pressure) of cell walls (bio-polymeric matrices equivalent to extracellular matrix in animal tissues) whose underlying nanoscale viscoelastic properties remain unknown. Multifrequency atomic force microscopy (AFM) techniques exist that are able to map properties to a small subgroup of linear viscoelastic materials (those obeying the Kelvin-Voigt model), but are not applicable to growing materials, and hence are of limited interest to most biological situations. Here, we extend existing dynamic AFM methods to image linear viscoelastic behaviour in general, and relaxation times of cells of multicellular organisms in vivo with nanoscale resolution (~80 nm pixel size in this study), featuring a simple method to test the validity of the mechanical model used to interpret the data. We use this technique to image cells at the surface of living Arabidopsis thaliana hypocotyls to obtain topographical maps of storage E' = 120-200 MPa and loss E″ = 46-111 MPa moduli as well as relaxation times τ = 2.2-2.7 µs of their cell walls. Our results demonstrate that (taken together with previous studies) cell walls, despite their complex molecular composition, display a striking continuity of simple, linear, viscoelastic behaviour across scales-following almost perfectly the standard linear solid model-with characteristic nanometer scale patterns of relaxation times, elasticity and viscosity, whose values correlate linearly with the speed of macroscopic growth. We show that the time-scales probed by dynamic AFM experiments (microseconds) are key to understand macroscopic scale dynamics (e.g. growth) as predicted by physics of polymer dynamics.

Biotechnology, nanotechnology and medicine.

The 1980s mark the starting point of nanotechnology: the capacity to synthesise, manipulate and visualise matter at the nanometre scale. New powers to reach the nanoscale brought us the unprecedented possibility to directly target at the scale of biomolecular interactions, and the motivation to create smart nanostructures that could circumvent the hurdles hindering the success of traditional pharmacological approaches. Forty years on, the progressive integration of bio- and nanotechnologies is starting to produce a transformation of the way we detect, treat and monitor diseases and unresolved medical problems [ 1]. While much of the work remains in research laboratories, the first nano-based treatments, vaccines, drugs, and diagnostic devices, are now receiving approval for commercialisation and clinical use. In this special issue we review recent advances of nanomedical approaches to combat antibiotic resistance, treatment and detection of cancers, targeting neurodegerative diseases, and applications as diverse as dentistry and the treatment of tuberculosis. We also examine the use of advanced smart nanostructured materials in areas such as regenerative medicine, and the controlled release of drugs and treatments. The latter is currently poised to bring ground-breaking changes in immunotherapy: the advent of 'vaccine implants' that continuously control and improve immune responses over time. With the increasingly likely prospect of ending the COVID 19 pandemic with the aid of a nanomedicine-based vaccine (both Moderna and BioNTech/Pfizer vaccines are based on lipid nanoparticle formulations), we are witnessing the coming of age of nanomedicine. This makes it more important than ever to concentrate on safety: in parallel to pursuing the benefits of nanomedine, we must strengthen the continuous focus on nanotoxicology and safety regulation of nanomedicines that can deliver the medical revolution that is within our grasp.

Bioelectrical understanding and engineering of cell biology.

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo, the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.

Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy.

We have used high-speed atomic force microscopy to study the dynamics of bacteriorhodopsin (bR) molecules at the free interface of the crystalline phase that occurs naturally in purple membrane. Our results reveal temporal fluctuations at the crystal edges arising from the association and dissociation of bR molecules, most predominantly pre-formed trimers. Analysis of the dissociation kinetics yields an estimate of the inter-trimer single-bond energy of -0.9kcal/mol. Rotational motion of individual bound trimers indicates that the inter-trimer bond involves W10-W12 tryptophan residues.

AFM nanoindentation reveals decrease of elastic modulus of lipid bilayers near freezing point of water.

Cell lipid membranes are the primary site of irreversible injury during freezing/thawing and cryopreservation of cells, but the underlying causes remain unknown. Here, we probe the effect of cooling from 20 °C to 0 °C on the structure and mechanical properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using atomic force microscopy (AFM) imaging and AFM-based nanoindentation in a liquid environment. The Young's modulus of elasticity (E) at each temperature for DPPC was obtained at different ionic strengths. Both at 20 mM and 150 mM NaCl, E of DPPC bilayers increases exponentially -as expected-as the temperature is lowered between 20 °C and 5 °C, but at 0 °C E drops from the values measured at 5 °C. Our results support the hypothesis that mechanical weakening of the bilayer at 0 °C  is produced by  structural changes at the lipid-fluid interface.

Atomic force microscopy-indentation demonstrates that alginate beads are mechanically stable under cell culture conditions.

Alginate microbeads are extensively used in tissue engineering as microcarriers and cell encapsulation vessels. In this study, we used atomic force microscopy (AFM) based indentation using 20 µm colloidal probes to assess the local reduced elastic modulus (E * ) using a novel method to detect the contact point based on the principle of virtual work, to measure microbead mechanical stability under cell culture conditions for 2 weeks. The bead diameter and swelling were assessed in parallel. Alginate beads swelled up to 150% of their original diameter following addition of cell culture media. The diameter eventually stabilized from day 2 onwards. This behaviour was mirrored in E * where a significant decrease was observed at the start of the culture period before stabilization was observed at ~ 2.1 kPa. Furthermore, the mechanical properties of freeze dried alginate beads after re-swelling them in culture media were measured. These beads displayed vastly different structural and mechanical properties compared those that did not go through the freeze drying process, with around 125% swelling and a significantly higher E * at values over 3 kPa.

A simple mathematical model of allometric exponential growth describes the early three-dimensional growth dynamics of secondary xylem in Arabidopsis roots.

Unravelling the specific growth dynamics of key tissues and organs is fundamental to understand how multicellular organisms orchestrate their different growth programmes. In plants, the secondary growth (thickening) of stems and roots provides the mechanical support that plants need to achieve their developmental potential. We used conventional anatomical and microscopy techniques, image-processing software, and quantitative analysis to understand and mathematically describe the growth dynamics of the early developmental stages of secondary xylem (the main tissue developed during secondary growth). Results show that such early developmental stages are characterized by exponential expansion of secondary xylem in three dimensions in the form of an inverted cone, with a power law that describes the relationship between the area of the base and the longitudinal progression (height) of the growing secondary xylem cone over time with a scaling exponent of 2/5: the signature of allometric growth. Our work constitutes a starting point for future modelling of secondary xylem in particular and secondary growth in general.