The measurement of intracellular reactive oxygen species (ROS) levels provides critical insights into cell health and pathophysiology. Here, we present a semi-quantitative, plate reader-based protocol using CellROX Green that can be used for the real-time measurement of general ROS levels of viable suspension mammalian cells. With menadione being the oxidative stress inducer, a cell concentration of 100,000 cells per well, a CellROX Green concentration of 10 µM, and a CellROX Green incubation time of 1 h was shown to provide the most accurate measurement of intracellular ROS levels in viable human monocyte leukemia (THP-1) cells. Unlike plate reader protocols using other general ROS-sensitive fluorogenic probes, this plate reader protocol eliminates the washing step of the unbound probe prior to measuring fluorescence, making it more suitable for suspension cells. Using the presented protocol, the fluorescence of up to 96 samples can be measured in under 1 min, making it ideal for rapid screening applications where moderate to large differences in oxidative stress levels are to be measured.

HYPOTHESIS:Liquid metals (LMs) and alloys such as GaCu, GaZn, and GaBi may possess previously unresolved nanoscale structures that govern their physicochemical behaviour. We hypothesise that such structuring is alloy-dependent and thermally sensitive, arising from differences in material properties, growth kinetics, and thermodynamic behaviours, and that the presence of nanoscale structures can be detected and resolved using in-situ neutron scattering, supported by atomistic simulations. EXPERIMENTS:In-situ Small Angle Neutron Scattering (SANS) was performed across a q-range of 0.004-0.5 Å-1 at 30, 60, and 90 °C on GaCu, GaZn, and GaBi alloys at 1, 2, and 5 wt%. Complementary Molecular Dynamics (MD) simulations were utilised to interpret structural evolution at the atomic scale. FINDINGS:The investigation demonstrates the presence of nanostructures in 1, 2 and 5 wt% GaCu alloys in the measured SANS range at 30 °C. Interestingly, these structures do not persist at elevated temperatures of 60 and 90 °C, indicating thermally driven homogenisation. In contrast, GaZn and GaBi alloys at similar concentrations do not exhibit any nanostructures, irrespective of the measured temperatures, highlighting alloy-specific structuring behaviour. These results provide new insight into nanoscale organisation within LMs and inform rational design for catalysis, electronics, and additive manufacturing applications.

Bacterial infections continue to impact billions of lives worldwide. Current detection methods are struggling to provide both rapid and accurate bacterial detection. Therefore, innovative approaches to rapid disease detection are urgently required. Recently, there has been much interest in the space of 2D material-based sensing. In particular, transition metal dichalcogenides (TMDs) have emerged as suitable biosensor candidates, due to their unique properties. These materials are relatively easy to make, non-toxic and have distinct charge characteristics. This study explores the integration of Molybdenum disulfide (MoS2) into a monoclonal antibody (mAb) functionalized biosensor which successfully detects methicillin-resistant Staphylococcus aureus (MRSA) using both Raman and photoluminescence (PL) spectroscopy. 2D nanoflakes of MoS2 were deposited onto a Silicon oxide (SiO2) substrate via chemical vapour deposition (CVD). The resulting MoS2 chips were functionalized with F598, a mAb that binds to the polysaccharide poly-N-acetyl-glucosamine (PNAG) found in various microbes, including many diverse species of bacteria. MRSA was incubated onto the MoS2 chips for 30 min, before rinsing with phosphate buffered saline (PBS), then PL and Raman spectroscopy were performed on the samples. Our results show accurate and rapid detection of MRSA using both PL and Raman spectroscopy, based on spectral changes that occur because of bacterial adhesion onto the chip surfaces. Machine learning was then utilized in a convolutional neural network (CNN) to distinguish between the different classes of spectra (controls and MRSA). Using a CNN an accuracy of 86% and 96% was found for the Raman and PL data, respectively. Thus, with a platform of MoS2 nanoflakes, generalized detection of PNAG-expressing bacteria (in this case MRSA) using spectroscopic techniques is successful. As this platform's detection is based on the antibody used, the platform can be modified to detect various species of bacteria by using different monoclonal antibodies.

Early disease diagnosis has significant benefits in saving human lives by detecting various biomarkers existing in various organs, such as the liver, heart, and kidney. Therefore, point-of-care diagnostic devices are the need of the hour. Acute myocardial infarction (AMI) is a condition in which reduced or obstructed coronary blood flow leads to oxygen deprivation, ultimately causing irreversible damage to cardiac tissue. Out of the major cardiac biomarkers, myoglobin (Mb) has higher concentrations in blood; therefore, it is important to detect Mb for cardiac conditions. There exist various approaches for detecting myoglobin, yet direct resistive approach is yet to be explored. Hence, this present work reports a novel way of detecting cardiac biomarker myoglobin, by developing a flexible and cost-effective graphene-based resistor named here as Graphene BioResistor (GBR). The GBR masquerades antibodies of specific biomarkers on its surface to grasp only the antigen of its own kind providing a highly selective way of detection of cardiac biomarkers. The device analyzes the behavior of the concentration of analytes to the resistance of the sensor. The device is cost-effective, flexible, and user-friendly because of its ease of fabrication and customizable surface properties. The multilayer porous 3-D graphene surface provides the platform for the bioanalyte to settle on the pores with impressive stability. The signal to noise ratio of the GBR is found to be 8.48. The limit of detection and limit of quantification of the device are 27.96 and 53.79 ng/ml, respectively, which are well within the ranges for AMI detection. The reproducibility of GBR at a certain concentration is found to be 99.19% and repeatability is at 80.1%. This fabrication process of GBR can be utilized to detect several other biomarkers present in human body at a very minimal cost and ease of fabrication.

Gallium-based liquid metal (LM) nanoparticles hold an exceptional promise for catalysis, energy storage, and printed electronics due to their high conductivity, fluidity, and dynamic catalytic surfaces. However, maintaining their mechanical and chemical stability remains a major challenge, as LM nanoparticles tend to agglomerate due to their high surface tension and are susceptible to chemical degradation, such as dissolution or leaching in reactive environments. Surface modification and encapsulation techniques are employed to enhance the mechanical and functional stability of these particles. Previously, methane pyrolysis has been considered as a route to produce high-purity hydrogen and carbon. In this work, we employ methane pyrolysis as a controllable route to synthesise carbon-encapsulated Ga-based alloy nanoparticles (NPs), where catalytic activity serves as the driving mechanism for shell formation rather than the ultimate function of the material. During pyrolysis, trimetallic Cu-Pt-Ga NPs act as transient catalytic sites that initiate carbon growth, while the resulting graphitic shell provides mechanical confinement, prevents agglomeration, and enhances resistance to leaching. By tuning alloy composition, the rate and morphology of carbon formation can be modulated, enabling precise control over the resulting core-shell architecture. Overall, the primary contribution of this work is the demonstration of a robust and general method for producing carbon-coated liquid-metal nanomaterials with tailored structural and functional properties for applications beyond catalysis.

Antimicrobial resistance (AMR) is a global health challenge responsible for millions of deaths annually. Hence, there is an urgent need for improved strategies to combat AMR. Nanoparticle (NP)-based drug delivery has shown promise for enhancing the efficacy of conventional antibiotic treatments. Moreover, lipid functionalization of NP surfaces can enhance drug loading, colloidal stability, and specificity. Cell membrane vesicles as the outer shell coating for NPs provokes a unique interaction between fabricated NPs and their respective parent bacteria. Despite numerous studies having investigated the use of bacterial extracellular vesicle coatings for drug delivery, to the best of our knowledge, the potential of isolated bacterial membrane lipids has not yet been explored. This study investigates how particle-cell adhesion changes when gold NPs (AuNPs) coated with bacterial membrane lipids are re-introduced to their original parent cells. Hence, bacterial lipid coated AuNPs (BLC-AuNPs) were constructed using AuNPs in conjunction with membrane lipids harvested from Escherichia coli. Compared with bare AuNPs, BLC-AuNPs showed significant increase in particle-cell adhesion upon re-exposure confirmed by confocal and electron microscopy. Lipid coating also improved particle distribution and surface coverage on bacterial cells. These findings suggest that bacterial membrane lipid coating provides an effective biomimetic strategy, enhancing drug deliveryto drug resistant pathogens.

Liquid metal systems are increasingly important due to their versatility in applications, including catalysis, flexible electronics, and synthesis processes. The ultra-thin native oxide skin that forms at the liquid metal-air interface can influence these applications positively or negatively, depending on whether a dynamic or static interface is required. We hypothesise that a deeper understanding of the oxide layer's behaviour and interaction forces could enable advancements in liquid metal applications. We developed a method to create liquid metal micro-islands-hemi-spherical regions of liquid metal adhered to solid substrates with a fluidic nature and an outer native oxide layer. Using atomic force microscopy (AFM), including surface-normal and lateral force spectroscopy, we investigated the forces between the oxide layer and the underlying liquid metal. Our AFM measurements reveal that the scalar force needed to delaminate the oxide layer from the liquid metal interior is similar to 1 mu N, with a force per unit area of similar to 69 nN/nm(2). Moreover, this work suggests that the developed protocol for creating liquid metal micro-islands could serve as a platform technology for in-situ analysis of liquid metal systems, facilitating further research in this area.

In this study, the novel use of high-resolution force-curve imaging is demonstrated using Atomic Force Microscopy (AFM) to continuously map topographical and biomechanical changes in individual bone marrow-derived hMSCs simultaneously with chemical stimulation. The cell's effective stiffness, quantified as Young's modulus, is largely determined by the cytoskeletal structure within the individual cell. Chemical treatments that modulate cytoskeletal dynamics induced measurable changes in the effective stiffness, reflecting changes in cytoskeletal components such as the actin and microtubule networks. Cytochalasin D, blebbistatin, and nocodazole all reduced the effective stiffness of the cells, while colchicine increased the effective stiffness, and this is quantified across whole cells and individual actin fibers over a 200 min period. Performing these measurements continually enabled a temporal analysis of distinct locations and features across the cell with a high degree of spatial confidence.

The presence of solvated metallic elements with high mobility enables many applications of liquid metals (LMs) such as in catalysis, phase change materials, welding, soldering, and additive manufacturing. While the processes of crystallisation and dissolution in LM resemble those of traditional solvents, they have not been studied in great detail. Furthermore, crucial observations of solute mass transport speeds (flux) in LM have not been evaluated yet, despite being discovered within catalytic environments. Thereby for processes like phase change materials and additive manufacturing, the fundamental chemistry is underexplored. We hypothesise that a deeper understanding of these fundamental properties is obtained by studying the phase-change behaviour of solid metal in liquid metal colloidal systems. We have employed transmission electron microscopy (TEM) to examine the dissolution and crystallisation processes of a Ga-Cu colloidal system. This approach involves in-situ heating and cooling TEM analysis with a specialised TEM holder. TEM analysis shows that upon heating, the Ga2Cu crystal begins to dissolve from its outermost layers, progressing until complete dissolution occurs. Recrystallisation then occurs in a supercooled environment, where the solute rapidly crystallises. This crystallisation process involves substantial mass transport within the LM, allowing for the estimation of the directional atom flux of Cu (valued -0.02 mol/m2s at 178 °C).

Hypothesis: The small size of the nanoparticles used to obtain high surface area photocatalysts makes their removal from solution difficult. Producing photocatalysts on substrates would alleviate this limitation. Adding heterojunctions to photocatalysts, for example, TiO2/Ag, could improve photocatalytic performance due to Schottky junction formation and introduce antibacterial properties.Experiments: TiO2 nanorod arrays were synthesised on a substrate via a hydrothermal approach, on which Ag nanoparticles were deposited using an electroless plating technique with varied deposition times and metal precursor concentrations. Photocatalytic performance was evaluated by monitoring Rhodamine B (RhB) degradation under ultraviolet light and antibacterial properties of the films tested using Methicillin-resistant Staphylococcus aureus.Findings: The Ag nanoparticle content was controlled by the Ag deposition process. The TiO2/Ag nanorod array containing 6.6 atomic% Ag as nanoparticles of ∼ 25 nm in diameter degraded 88 % of the RhB in 6 h compared to 54 % degradation for bare TiO2 nanorods under the same reaction conditions. Decreased photoluminescence with heterojunction formation would indicate electron transfer from the TiO2 into the Ag nanoparticles, thereby reducing charge carrier recombination. The antibacterial test conducted in the dark revealed enhanced performance for the TiO2/Ag sample compared to TiO2 nanorods against Methicillin-resistant Staphylococcus aureus after 16 h exposure with a death rate of 84 %.

Developing long-term corrosion protection through inhibitor molecules requires in-depth understanding of dynamic film formation processes under corrosive media. To this end, this study explores time-dependent film growth of inhibitor 2-mercaptobenzimidazole (2-MBI) upon Al alloy 6061 (AA6061) in saline solution through both in-situ and ex-situ surface analytical approaches. Electrochemical study reveals that the inhibition efficiency increases to the 94 % after treatment by 2-MBI for 90 min, associating with the growth of porosity-free elastic film formed over AA6061 as evidenced by high-resolution AFM imaging. The self-assembly film grew from 30 min with a Young's modulus value of approx. 8000 MPa which has increased to nearly 30000 MPa after 90 min, indicating pronounced surface film cohesion. A subsequent film softening with extended treatment was observed in which nano-/micro-sized pores emerged significantly in the film structure from 12 to 24 h, resulting in reduced inhibition efficiency to 71 %. XPS results suggest that the presence of S and N heteroatoms promote active chemical adsorption. Further inhibitor exposure gives rise to physisorption process thickening the inhibitor layer with increased porosity. However, the defective nature of subsequent layer formation casts detrimental effects by channeling electrolyte and metal substrate for local film breakdown, leading to degraded inhibition performance.

Nanomaterials are revolutionizing the development of novel therapies, with applications ranging from drug delivery and diagnostics to controlling specific biological processes. However, the specific interactions that govern nanomaterial behavior in biological systems remain difficult to elucidate due to the complex dynamic nature of the lipid bilayer environment. Here, a combination of atomic force microscopy and molecular dynamics simulations is used to discover the precise mechanisms by which various ligand‐capped 5 nm gold nanoparticles (AuNPs) interact with supported lipid bilayers of pure fluid phospholipids (1,2‐di(9Z‐octadecenoyl)‐sn‐glycero‐3‐phosphocholine (DOPC)). When the ligand capping agent is altered, differences in adsorption and bilayer disruption as a function of capping agent size and charge are observed. Weakly physiosorbed ligands enable the absorption of the AuNP into the bilayer's hydrophobic core, whereas more strongly adsorbed ligands inhibit the complete insertion of the AuNP. However, ligand‐dependent headgroup interactions can lead to interfacial adhesion or inhibition of adsorption. These results reveal that the interaction of AuNPs with biological membranes varies depending on the specific capping agent. Notably, the mechanisms may involve cooperative (or synergistic) effects with membrane components, highlighting the importance of understanding these interactions at molecular resolution.

Innovations in nanostructured surfaces have found a practical place in the medical area with use in implant materials for post-operative infection prevention. These textured surfaces should be dual purpose: (1) bactericidal on contact and (2) resistant to biofilm formation over prolonged periods. Here, hydrothermally etched titanium surfaces were tested against two highly antimicrobial resistant microbial species, methicillin-resistant Staphylococcus aureus and Candida albicans. Two surface types - unmodified titanium and nanostructured titanium - were incubated in a suspension of each microbial strain for 1 day and 7 days. Surface topography and cross-sectional information of the microbial cells adhered to the surfaces, along with biomass volume and live/dead rate, showed that while nanostructured titanium was able to kill microbes after 1 day of exposure, after 7 days, the rate of death becomes negligible when compared to the unmodified titanium. This suggests that as biofilms mature on a nanostructured surface, the cells that have lysed conceal the nanostructures and prime the surface for planktonic cells to adhere, decreasing the possibility of structure-induced lysis. Synchrotron macro-attenuated total reflection Fourier transform infrared (macro ATR-FTIR) micro-spectroscopy was used to elucidate the biochemical changes occurring following exposure to differing surface texture and incubation duration, providing further understanding into the effects of surface morphology on the biochemical molecules (lipids, proteins and polysaccharides) in an evolving and growing microbial colony.

Additively manufactured titanium implant materials are rapidly advancing prosthetics and orthopaedic devices by making them more cost-effective and customisable. However, the surface finish of materials printed via Selective Laser Melting (SLM) currently limits their integration into the medical device field. Printing parameters, such as build angle inclination, can cause variations in the surface roughness of a part, often exceeding what is suitable for implant materials. Excessive roughness can promote microbial attachment and proliferation, potentially leading to implant rejection. Nanostructuring titanium has previously demonstrated success in mitigating bacteria and fungi via a mechanomicrobiocial mechanism on traditionally flat titanium and complex SLM-made parts but its effectiveness on the inherent roughness of three-dimensional (3D) printed titanium remains unexplored. This study examines the surface roughness of 3D-Ti at three build angles (0, 40 and 90 degrees), before and after nanostructuring. Surfaces were assessed against methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans, representative antimicrobial resistant pathogens. Results showed the nanostructures were more effective against MRSA, but microbial attachment increase with steeper angles, regardless of the presence of nanostructures. This study investigates how surface roughness of 3D printed titanium substrates impacts bacterial and fungal adhesion and the resulting nanomorphology of the surface post-hydrothermal modification.

Advanced tissue engineering (TE) strategies are vital to address challenging musculoskeletal conditions, such as volumetric muscle loss. These disorders impose a considerable economic burden and affect individuals' quality of life, highlighting the need for innovative treatments, such as TE, to address these challenges. Here, we examine how scaffold fibre orientation influences mechanical properties and cellular behaviour by utilising melt electrowriting (MEW) as a high-resolution 3D printing technique that combines aspects of electrospinning and melt based polymer deposition. In this work, we investigated the effects of fibre orientation in MEW scaffolds, and its effect on the scaffold mechanical properties as well as cell orientation and alignment. MEW scaffolds were mechanically characterised through uniaxial strain testing to determine critical parameters, including strain at failure, ultimate tensile strength, Young's modulus (E), fatigue rate, recovery time, and yield strain. These mechanical properties were analysed to define an optimal strain regime for transitioning from static to dynamic culture conditions under muscle-like cyclic loading, relevant to muscle's viscoelastic behaviour. In parallel, static cultures of primary human skeletal muscle myoblasts and normal human dermal fibroblasts (NHDFs) were grown on MEW scaffolds, with varying architectures, to study the effects of fibre aspect ratio on cell alignment. Cell alignment was visualised using DAPI/phalloidin staining and quantified with the ImageJ directionality plugin, enabling a systematic comparison of scaffold designs. This approach evaluates the potential of supportive scaffold architectures to promote aligned cell growth, offering insights into designing effective scaffolds for tissue regeneration.

Interactions between nanomaterials and biointerfaces are of great interest across many research fields, including chemistry, physics, engineering, and biology, with applications from diagnostics to therapeutics, and in drug delivery systems. By tailoring nanomaterial properties via functionalisation, their efficacy can be enhanced for a variety of biomedical scenarios. Nanomaterial-based therapeutics, diagnostics, and theranostics display a common need to interact with biological tissue, i.e., they must make contact with, and often transverse, the external membrane of a cell or organism to elicit the desired response. As such, understanding nanoparticle (NP)-bio-membrane interactions is paramount to designing and optimising nanoparticle-based therapies and technologies for biological applications. Historically, in vivo investigations have proven to be challenging due to the complexity and intricate interactions involved with complete cell membranes. This has led researchers to employ model bio-membranes, which can be investigated using a range of in vitro experimental and theoretical techniques, to isolate the underpinning interaction mechanisms of NPs at the bio-interface. This review aims to unify the current literature regarding the biophysical interactions between inorganic and organic NPs and bio-membrane interfaces, including living and synthetic systems. The article will explore the role of membrane composition, NP morphology and chemistry, and the forces involved in interactions between the two entities. Identifying the fundamental influences of NP-membrane interactions, primarily synthetic NPs as these are more clinically applied, will allow for the more effective design of novel biomedical agents for future therapies.

Stem cell mechanosensitivity to their external environment is a key process in regulating and governing lineage commitment. The dynamic changes stem cells undergo due to mechanotransduction can reveal the processes triggering signaling pathways, however it is a highly complex methodology to capture these transient changes in living stem cells. Here, the use of new biophysical techniques to capture these changes in situ is discussed, namely emerging atomic force microscopy techniques, which are capable of high resolution and gentle biomechanical analysis.

Black phosphorus (BP) nanoflakes have attracted interest as an antimicrobial material for wound healing and implant-associated infections due to their bactericidal activity without the use of antibiotics. Hydrogels are frequently used as a delivery system; however, most research uses photonic activation in the form of near-infrared (NIR) laser stimulation to cause rapid BP degradation, reactive oxygen species (ROS) generation, and a localized photothermal effect. For implant-coating applications, using NIR laser stimulation could be challenging in practice, especially for porous orthopedic implants. This article investigates whether BP nanoflakes, suspended in Pluronic F127 (F127) hydrogels, remain effective against Staphylococcus aureus without photonic activation. The experimental results showed 89.4 ± 7.6% bacterial inhibition from BP nanoflakes at a 5120 µg/mL concentration via passive diffusion in F127; however, it could not kill all the bacteria present. It is hypothesized that the F127 gel interface could create a barrier between the bacteria, which continue to multiply in media, and the antimicrobial black phosphorus compound, which degrades in the F127.

Liquid metal (LM) alloys have attracted significant interest as exceptional functional materials due to their wide range of applications. Although significant theoretical advancements have been made, the experimental investigation of surface oxides in complex metal alloys remains largely unexplored. This study investigates the formation of surface oxide in eutectic zinc (Zn)-tin (Sn) alloy to increase the understanding of composite metal oxides and enable new technological applications. The study reveals the formation of zinc tin composite metal oxide (ZTCMO) with ≈82.7 at% ZnO and ≈17.3 at% SnO2 by utilizing the liquid metal-based van der Waals (vdW) printing approach. Structural characterizations confirm the formation of highly crystalline ZTCMO nanosheets with a wide bandgap of 3.3 eV. The ultrathin nanosheets demonstrate their practicality as an ultraviolet (UV) optical sensor with maximum responsivities of 3.84 A W-1 at 285 nm and 1.31 A W-1 at 365 nm, achieved at a low power density of 0.1 mW cm-2. Additionally, ZTCMO nanosheets exhibit excellent room temperature ammonia (NH3) gas sensing with high sensitivity and selectivity, detecting concentrations as low as 50 parts per million under UV light illumination. These findings highlight the potential of composite metal oxide-based devices with the capability of multifunctional sensing.