Ultrasensitive Raman Detection of Biomolecular Conformation at the Attomole Scale using Chiral Nanophotonics.

Understanding the function of a biomolecule hinges on its 3D conformation or secondary structure. Chirally sensitive, optically active techniques based on the differential absorption of UV-vis circularly polarized light excel at rapid characterisation of secondary structures. However, Raman spectroscopy, a powerful method for determining the structure of simple molecules, has limited capacity for structural analysis of biomolecules because of intrinsically weak optical activity, necessitating millimolar (mM) sample quantities. A breakthrough is presented for utilising Raman spectroscopy in ultrasensitive biomolecular conformation detection, surpassing conventional Raman optical activity by 15 orders of magnitude. This strategy combines chiral plasmonic metasurfaces with achiral molecular Raman reporters and enables the detection of different conformations (α-helix and random coil) of a model peptide (poly-L/D-lysine) at the ≤attomole level (monolayer). This exceptional sensitivity stems from the ability to detect local, molecular-scale changes in the electromagnetic (EM) environment of a chiral nanocavity induced by the presence of biomolecules using molecular Raman reporters. Further signal enhancement is achieved by incorporating achiral Au nanoparticles. The introduction of the nanoparticles creates highly localized regions of extreme optical chirality. This approach, which exploits Raman, a generic phenomenon, paves the way for next-generation technologies for the ultrasensitive detection of diverse biomolecular structures.

Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in monolayers interact mechanically via cell-cell and cell-substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC-CM monolayers (1) attached to glass or plastic (Young's modulus (E) >1 GPa), (2) detached (substrate-free) and (3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify the underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multiphasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate-free cultures or single cells where only simple twitch-like time-courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared with glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress-activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell-to-cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multiphasic contractions in hiPSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions. KEY POINTS: Spatiotemporal contractility analysis of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers seeded on conventional, rigid surfaces (glass or plastic) revealed the presence of multiphasic contraction patterns across the monolayer with a high variability, despite action potentials recorded in the same areas being identical. These multiphasic patterns are not present in single cells, in detached monolayers or in monolayers seeded on soft substrates such as a hydrogel, where only 'twitch'-like transients are observed. HiPSC-CM monolayers that display a high percentage of regions with multiphasic contraction have significantly increased contractile duration and a decreased lusotropic drug response. There is no indication that the multiphasic contraction patterns are associated with significant activation of the stress-activated NPPA or NPPB signalling pathways. A computational model of cell clusters supports the biological findings that the rigid surface and the differential cell-substrate adhesion underly multiphasic contractile behaviour of hiPSC-CMs.

Design and fabrication of memory devices based on nanoscale polyoxometalate clusters.

Flash memory devices--that is, non-volatile computer storage media that can be electrically erased and reprogrammed--are vital for portable electronics, but the scaling down of metal-oxide-semiconductor (MOS) flash memory to sizes of below ten nanometres per data cell presents challenges. Molecules have been proposed to replace MOS flash memory, but they suffer from low electrical conductivity, high resistance, low device yield, and finite thermal stability, limiting their integration into current MOS technologies. Although great advances have been made in the pursuit of molecule-based flash memory, there are a number of significant barriers to the realization of devices using conventional MOS technologies. Here we show that core-shell polyoxometalate (POM) molecules can act as candidate storage nodes for MOS flash memory. Realistic, industry-standard device simulations validate our approach at the nanometre scale, where the device performance is determined mainly by the number of molecules in the storage media and not by their position. To exploit the nature of the core-shell POM clusters, we show, at both the molecular and device level, that embedding [(Se(IV)O3)2](4-) as an oxidizable dopant in the cluster core allows the oxidation of the molecule to a [Se(v)2O6](2-) moiety containing a {Se(V)-Se(V)} bond (where curly brackets indicate a moiety, not a molecule) and reveals a new 5+ oxidation state for selenium. This new oxidation state can be observed at the device level, resulting in a new type of memory, which we call 'write-once-erase'. Taken together, these results show that POMs have the potential to be used as a realistic nanoscale flash memory. Also, the configuration of the doped POM core may lead to new types of electrical behaviour. This work suggests a route to the practical integration of configurable molecules in MOS technologies as the lithographic scales approach the molecular limit.

Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control.

Nanoscale organisation of receptor ligands has become an important approach to study the clustering behaviour of cell-surface receptors. Biomimetic substrates fabricated via different nanopatterning strategies have so far been applied to investigate specific integrins and cell types, but without multivalent control. Here we use DNA origami to surpass the limits of current approaches and fabricate nanoarrays to study different cell adhesion processes, with nanoscale spatial resolution and single-molecule control. Notably, DNA nanostructures enable the display of receptor ligands in a highly customisable manner, with modifiable parameters including ligand number, ligand spacing and most importantly, multivalency. To test the adaptability and robustness of the system we combined it with focused ion beam and electron-beam lithography nanopatterning to additionally control the distance between the origami structures (i.e. receptor clusters). Moreover, we demonstrate how the platform can be used to interrogate two different biological questions: (1) the cooperative effect of integrin and growth factor receptor in cancer cell spreading, and (2) the role of integrin clustering in cardiomyocyte adhesion and maturation. Thereby we find previously unknown clustering behaviour of different integrins, further outlining the importance for such customisable platforms for future investigations of specific receptor organisation at the nanoscale.

Chiral Plasmonic Fields Probe Structural Order of Biointerfaces.

The structural order of biopolymers, such as proteins, at interfaces defines the physical and chemical interactions of biological systems with their surroundings and is hence a critical parameter in a range of biological problems. Known spectroscopic methods for routine rapid monitoring of structural order in biolayers are generally only applied to model single-component systems that possess a spectral fingerprint which is highly sensitive to orientation. This spectroscopic behavior is not a generic property and may require the addition of a label. Importantly, such techniques cannot readily be applied to real multicomponent biolayers, have ill-defined or unknown compositions, and have complex spectroscopic signatures with many overlapping bands. Here, we demonstrate the sensitivity of plasmonic fields with enhanced chirality, a property referred to as superchirality, to global orientational order within both simple model and "real" complex protein layers. The sensitivity to structural order is derived from the capability of superchiral fields to detect the anisotropic nature of electric dipole-magnetic dipole response of the layer; this is validated by numerical simulations. As a model study, the evolution of orientational order with increasing surface density in layers of the antibody immunoglobulin G was monitored. As an exemplar of greater complexity, superchiral fields are demonstrated, without knowledge of exact composition, to be able to monitor how qualitative changes in composition alter the structural order of protein layers formed from blood serum, thereby establishing the efficacy of the phenomenon as a tool for studying complex biological interfaces.

Nanoimprinting of biomedical polymers reduces candidal physical adhesion.

Management of fungal biofilms represents a significant challenge to healthcare. As a preventive approach, minimizing adhesion between indwelling medical devices and microorganisms would be an important step forward. This study investigated the anti-fouling capacity of engineered nanoscale topographies to the pathogenic yeast Candida albicans. Highly ordered arrays of nano-pit topographies were shown to significantly reduce the physical adherence capacity of C. albicans. This study shows a potential of nanoscale patterns to inhibit and prevent pathogenic biofilm formation on biomedical substrates.

Integrated 3D Hydrogel Waveguide Out-Coupler by Step-and-Repeat Thermal Nanoimprint Lithography: A Promising Sensor Device for Water and pH.

Hydrogel materials offer many advantages for chemical and biological sensoring due to their response to a small change in their environment with a related change in volume. Several designs have been outlined in the literature in the specific field of hydrogel-based optical sensors, reporting a large number of steps for their fabrication. In this work we present a three-dimensional, hydrogel-based sensor the structure of which is fabricated in a single step using thermal nanoimprint lithography. The sensor is based on a waveguide with a grating readout section. A specific hydrogel formulation, based on a combination of PEGDMA (Poly(Ethylene Glycol DiMethAcrylate)), NIPAAm (N-IsoPropylAcrylAmide), and AA (Acrylic Acid), was developed. This stimulus-responsive hydrogel is sensitive to pH and to water. Moreover, the hydrogel has been modified to be suitable for fabrication by thermal nanoimprint lithography. Once stimulated, the hydrogel-based sensor changes its topography, which is characterised physically by AFM and SEM, and optically using a specific optical set-up.

Biomacromolecular Stereostructure Mediates Mode Hybridization in Chiral Plasmonic Nanostructures.

The refractive index sensitivity of plasmonic fields has been exploited for over 20 years in analytical technologies. While this sensitivity can be used to achieve attomole detection levels, they are in essence binary measurements that sense the presence/absence of a predetermined analyte. Using plasmonic fields, not to sense effective refractive indices but to provide more "granular" information about the structural characteristics of a medium, provides a more information rich output, which affords opportunities to create new powerful and flexible sensing technologies not limited by the need to synthesize chemical recognition elements. Here we report a new plasmonic phenomenon that is sensitive to the biomacromolecular structure without relying on measuring effective refractive indices. Chiral biomaterials mediate the hybridization of electric and magnetic modes of a chiral solid-inverse plasmonic structure, resulting in a measurable change in both reflectivity and chiroptical properties. The phenomenon originates from the electric-dipole-magnetic-dipole response of the biomaterial and is hence sensitive to biomacromolecular secondary structure providing unique fingerprints of α-helical, ß-sheet, and disordered motifs. The phenomenon can be observed for subchiral plasmonic fields (i.e., fields with a lower chiral asymmetry than circularly polarized light) hence lifting constraints to engineer structures that produce fields with enhanced chirality, thus providing greater flexibility in nanostructure design. To demonstrate the efficacy of the phenomenon, we have detected and characterized picogram quantities of simple model helical biopolymers and more complex real proteins.

Fluorinated ethylene-propylene: a complementary alternative to PDMS for nanoimprint stamps.

Polydimethylsiloxane (PDMS) is used by many for nanoimprint applications due to its affordability, ease of preparation, mechanical flexibility, compatibility with imprint resists and transparency to UV light. However PDMS is notoriously flexible, tacky and permeable to air. Here fluorinated ethylene-propylene (FEP) is considered as a viable and versatile alternative material for nanoimprint stamps. FEP possesses many of the desirable nanoimprint attributes associated with PDMS but crucially also features a range of complementary characteristics, including an order of magnitude more mechanical strength allowing it to handle higher loads than PDMS, an intrinsically non-stick surface and is compatible with oxygen sensitive resists. Unlike elastomeric polymers, FEP is glassy so patterning may be realised via hot embossing. Not only is this a facile and rapid means of physical structuring but it also facilitates combinatorial patterning, providing a versatility beyond that of traditional casting materials. Due to the intrinsically slow creep of FEP both micro- and nanopatterning are successfully performed sequentially. Feature sizes from 45 nm were successfully realised via the hot-embossing method. To further demonstrate the potential of the material, a modified computer numerical control machine is used. It is capable of photo-, nanoimprint- and laser lithography in conjunction with patterned FEP foils. The tool is used to perform pattern transfer into a developmental nanoimprint resist from Micro Resist Technology, mr-NIL210 XP, and Nano SU-8 3005 negative tone photo resist from MicroChem. Ultimately three-tier lithography is performed in unison and advantageous step-and-repeat performance is achieved with fabricated FEP imprint stamps as they demould more compliantly and resist pressure and contamination better than PDMS.

Characterisation of CorGlaes(®) Pure 107 fibres for biomedical applications.

A degradable ultraphosphate (55 mol % P2O5) quinternary phosphate glass composition has been characterised in terms of its chemical, mechanical and degradation properties both as a bulk material and after drawing into fibres. This glass formulation displayed a large processing window simplifying fibre drawing. The fibres displayed stiffness and strength of 65.5 ± 20.8 GPa and 426±143 MPa. While amorphous discs of the glass displayed a linear dissolution rate of 0.004 mg cm(-2) h(-1) at 37 °C, in a static solution with a reduction in media pH. Once drawn into fibres, the dissolution process dropped the pH to <2 in distilled water, phosphate buffer saline and corrected-simulated body fluid, displaying an autocatalytic effect with >90 % mass loss in 4 days, about seven times faster than anticipated for this solution rate. Only cell culture media was able to buffer the pH taking over a week for full fibre dissolution, however, still four times faster dissolution rate than as a bulk material. However, at early times the development of a HCA layer was seen indicating potential bioactivity. Thus, although initial analysis indicated potential orthopaedic implant applications, autocatalysis leads to accelerating degradation in vitro.

Mechanical compatibility of sol-gel annealing with titanium for orthopaedic prostheses.

Sol-gel processing is an attractive method for large-scale surface coating due to its facile and inexpensive preparation, even with the inclusion of precision nanotopographies. These are desirable traits for metal orthopaedic prostheses where ceramic coatings are known to be osteoinductive and the effects may be amplified through nanotexturing. However there are a few concerns associated with the application of sol-gel technology to orthopaedics. Primarily, the annealing stage required to transform the sol-gel into a ceramic may compromise the physical integrity of the underlying metal. Secondly, loose particles on medical implants can be carcinogenic and cause inflammation so the coating needs to be strongly bonded to the implant. These concerns are addressed in this paper. Titanium, the dominant material for orthopaedics at present, is examined before and after sol-gel processing for changes in hardness and flexural modulus. Wear resistance, bending and pull tests are also performed to evaluate the ceramic coating. The findings suggest that sol-gel coatings will be compatible with titanium implants for an optimum temperature of 500 °C.

"Superchiral" Spectroscopy: Detection of Protein Higher Order Hierarchical Structure with Chiral Plasmonic Nanostructures.

Optical spectroscopic methods do not routinely provide information on higher order hierarchical structure (tertiary/quaternary) of biological macromolecules and assemblies. This necessitates the use of time-consuming and material intensive techniques, such as protein crystallography, NMR, and electron microscopy. Here we demonstrate a spectroscopic phenomenon, superchiral polarimetry, which can rapidly characterize ligand-induced changes in protein higher order (tertiary/quaternary) structure at the picogram level, which is undetectable using conventional CD spectroscopy. This is achieved by utilizing the enhanced sensitivity of superchiral evanescent fields to mesoscale chiral structure.

Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate.

Stem cells respond to nanoscale surface features, with changes in cell growth and differentiation mediated by alterations in cell adhesion. The interaction of nanotopographical features with integrin receptors in the cells' focal adhesions alters how the cells adhere to materials surfaces, and defines cell fate through changes in both cell biochemistry and cell morphology. In this Review, we discuss how cell adhesions interact with nanotopography, and we provide insight as to how materials scientists can exploit these interactions to direct stem cell fate and to understand how the behaviour of stem cells in their niche can be controlled. We expect knowledge gained from the study of cell-nanotopography interactions to accelerate the development of next-generation stem cell culture materials and implant interfaces, and to fuel discovery of stem cell therapeutics to support regenerative therapies.

Nanotopographical effects on mesenchymal stem cell morphology and phenotype.

There is a rapidly growing body of literature on the effects of topography and critically, nanotopography on cell adhesion, apoptosis and differentiation. Understanding the effects of nanotopography on cell adhesion and morphology and the consequences of cell shape changes in the nucleus, and consequently, gene expression offers new approaches to the elucidation and potential control of stem cell differentiation. In the current study we have used molecular approaches in combination with immunohistology and transcript analysis to understand the role of nanotopography on mesenchymal stem cell morphology and phenotype. Results demonstrate large changes in cell adhesion, nucleus and lamin morphologies in response to the different nanotopographies. Furthermore, these changes relate to alterations in packing of chromosome territories within the interphase nucleus. This, in turn, leads to changes in transcription factor activity and functional (phenotypical) signalling including cell metabolism. Nanotopography provides a useful, non-invasive tool for studying cellular mechanotransduction, gene and protein expression patterns, through effects on cell morphology. The different nanotopographies examined, result in different morphological changes in the cyto- and nucleo-skeleton. We propose that both indirect (biochemical) and direct (mechanical) signalling are important in these early stages of regulating stem cell fate as a consequence of altered metabolic changes and altered phenotype. The current studies provide new insight on cell-surface interactions and enhance our understanding of the modulation of stem cell differentiation with significant potential application in regenerative medicine.

Development of a novel 3D culture system for screening features of a complex implantable device for CNS repair.

Tubular scaffolds which incorporate a variety of micro- and nanotopographies have a wide application potential in tissue engineering especially for the repair of spinal cord injury (SCI). We aim to produce metabolically active differentiated tissues within such tubes, as it is crucially important to evaluate the biological performance of the three-dimensional (3D) scaffold and optimize the bioprocesses for tissue culture. Because of the complex 3D configuration and the presence of various topographies, it is rarely possible to observe and analyze cells within such scaffolds in situ. Thus, we aim to develop scaled down mini-chambers as simplified in vitro simulation systems, to bridge the gap between two-dimensional (2D) cell cultures on structured substrates and three-dimensional (3D) tissue culture. The mini-chambers were manipulated to systematically simulate and evaluate the influences of gravity, topography, fluid flow, and scaffold dimension on three exemplary cell models that play a role in CNS repair (i.e., cortical astrocytes, fibroblasts, and myelinating cultures) within a tubular scaffold created by rolling up a microstructured membrane. Since we use CNS myelinating cultures, we can confirm that the scaffold does not affect neural cell differentiation. It was found that heterogeneous cell distribution within the tubular constructs was caused by a combination of gravity, fluid flow, topography, and scaffold configuration, while cell survival was influenced by scaffold length, porosity, and thickness. This research demonstrates that the mini-chambers represent a viable, novel, scale down approach for the evaluation of complex 3D scaffolds as well as providing a microbioprocessing strategy for tissue engineering and the potential repair of SCI.

Label-free segmentation of Co-cultured cells on a nanotopographical gradient.

The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents.

Graphene-Based Engineered Living Materials.

With the rise of engineered living materials (ELMs) as innovative, sustainable and smart systems for diverse engineering and biological applications, global interest in advancing ELMs is on the rise. Graphene-based nanostructures can serve as effective tools to fabricate ELMs. By using graphene-based materials as building units and microorganisms as the designers of the end materials, next-generation ELMs can be engineered with the structural properties of graphene-based materials and the inherent properties of the microorganisms. However, some challenges need to be addressed to fully take advantage of graphene-based nanostructures for the design of next-generation ELMs. This work covers the latest advances in the fabrication and application of graphene-based ELMs. Fabrication strategies of graphene-based ELMs are first categorized, followed by a systematic investigation of the advantages and disadvantages within each category. Next, the potential applications of graphene-based ELMs are covered. Moreover, the challenges associated with fabrication of next-generation graphene-based ELMs are identified and discussed. Based on a comprehensive overview of the literature, the primary challenge limiting the integration of graphene-based nanostructures in ELMs is nanotoxicity arising from synthetic and structural parameters. Finally, we present possible design principles to potentially address these challenges.

Enhancing Supercapacitor Electrochemical Performance with 3D Printed Cellular PEEK/MWCNT Electrodes Coated with PEDOT: PSS.

In this study, we examine the electrochemical performance of supercapacitor (SC) electrodes made from 3D-printed nanocomposites. These composites consist of multiwalled carbon nanotubes (MWCNTs) and polyether ether ketone (PEEK), coated with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). The electrochemical performance of a 3D-printed PEEK/MWCNT solid electrode with a surface area density of 1.2 mm-1 is compared to two distinct periodically porous PEEK/MWCNT electrodes with surface area densities of 7.3 and 7.1 mm-1. To enhance SC performance, the 3D-printed electrodes are coated with a conductive polymer, PEDOT:PSS. The architected cellular electrodes exhibit significantly improved capacitive properties, with the cellular electrode (7.1 mm-1) displaying a capacitance nearly four times greater than that of the solid 3D-printed electrode-based SCs. Moreover, the PEDOT:PSS-coated cellular electrode (7.1 mm-1) demonstrates a high specific capacitance of 12.55 mF·cm-3 at 50 mV·s-1, contrasting to SCs based on 3D-printed cellular electrodes (4.09 mF·cm-3 at 50 mV·s-1) without the coating. The conductive PEDOT:PSS coating proves effective in reducing surface resistance, resulting in a decreased voltage drop during the SCs' charging and discharging processes. Ultimately, the 3D-printed cellular nanocomposite electrode with the conductive polymer coating achieves an energy density of 1.98 µW h·cm-3 at a current of 70 µA. This study underscores how the combined effect of the surface area density of porous electrodes enabled by 3D printing, along with the conductivity imparted by the polymer coating, synergistically improves the energy storage performance.

Spatial Control of 2D Nanomaterial Electronic Properties Using Chiral Light Beams.

Single-layer two-dimensional (2D) nanomaterials exhibit physical and chemical properties which can be dynamically modulated through out-of-plane deformations. Existing methods rely on intricate micromechanical manipulations (e.g., poking, bending, rumpling), hindering their widespread technological implementation. We address this challenge by proposing an all-optical approach that decouples strain engineering from micromechanical complexities. This method leverages the forces generated by chiral light beams carrying orbital angular momentum (OAM). The inherent sense of twist of these beams enables the exertion of controlled torques on 2D monolayer materials, inducing tailored strain. This approach offers a contactless and dynamically tunable alternative to existing methods. As a proof-of-concept, we demonstrate control over the conductivity of graphene transistors using chiral light beams, showcasing the potential of this approach for manipulating properties in future electronic devices. This optical control mechanism holds promise in enabling the reconfiguration of devices through optically patterned strain. It also allows broader utilization of strain engineering in 2D nanomaterials for advanced functionalities in next-generation optoelectronic devices and sensors.

Electromagnetic Enantiomer: Chiral Nanophotonic Cavities for Inducing Chemical Asymmetry.

Chiral molecules, a cornerstone of chemical sciences with applications ranging from pharmaceuticals to molecular electronics, come in mirror-image pairs called enantiomers. However, their synthesis often requires complex control of their molecular geometry. We propose a strategy called "electromagnetic enantiomers" for inducing chirality in molecules located within engineered nanocavities using light, eliminating the need for intricate molecular design. This approach works by exploiting the strong coupling between a nonchiral molecule and a chiral mode within a nanocavity. We provide evidence for this strong coupling through angular emission patterns verified by numerical simulations and with complementary evidence provided by luminescence lifetime measurements. In simpler terms, our hypothesis suggests that chiral properties can be conveyed on to a molecule with a suitable chromophore by placing it within a specially designed chiral nanocavity that is significantly larger (hundreds of nanometers) than the molecule itself. To demonstrate this concept, we showcase an application in display technology, achieving efficient emission of circularly polarized light from a nonchiral molecule. The electromagnetic enantiomer concept offers a simpler approach to chiral control, potentially opening doors for asymmetric synthesis.