Quantitative Detection of Biological Nanovesicles in Drops of Saliva Using Microcantilevers.

Extracellular nanovesicles (EVs) are lipid-based vesicles secreted by cells and are present in all bodily fluids. They play a central role in communication between distant cells and have been proposed as potential indicators for the early detection of a wide range of diseases, including different types of cancer. However, reliable quantification of a specific subpopulation of EVs remains challenging. The process is typically lengthy and costly and requires purification of relatively large quantities of biopsy samples. Here, we show that microcantilevers operated with sufficiently small vibration amplitudes can successfully quantify a specific subpopulation of EVs directly from a drop (0.1 mL) of unprocessed saliva in less than 20 min. Being a complex fluid, saliva is highly non-Newtonian, normally precluding mechanical sensing. With a combination of standard rheology and microrheology, we demonstrate that the non-Newtonian properties are scale-dependent, enabling microcantilever measurements with a sensitivity identical to that in pure water when operating at the nanoscale. We also address the problem of unwanted sensor biofouling by using a zwitterionic coating, allowing efficient quantification of EVs at concentrations down to 0.1 µg/mL, based on immunorecognition of the EVs' surface proteins. We benchmark the technique on model EVs and illustrate its potential by quantifying populations of natural EVs commonly present in human saliva. The method effectively bypasses the difficulty of targeted detection in non-Newtonian fluids and could be used for various applications, from the detection of EVs and viruses in bodily fluids to the detection of molecular clusters or nanoparticles in other complex fluids.

Dual-action silver functionalized nanostructured titanium against drug resistant bacterial and fungal species.

HYPOTHESIS: Titanium and its alloys are commonly used implant materials. Once inserted into the body, the interface of the biomaterials is the most likely site for the development of implant-associated infections. Imparting the titanium substrate with high-aspect-ratio nanostructures, which can be uniformly achieved using hydrothermal etching, enables a mechanical contact-killing (mechanoresponsive) mechanism of bacterial and fungal cells. Interaction between cells and the surface shows cellular inactivation via a physical mechanism meaning that careful engineering of the interface is needed to optimse the technology. This mechanism of action is only effective towards surface adsorbed microbes, thus any cells not directly in contact with the substrate will survive and limit the antimicrobial efficacy of the titanium nanostructures. Therefore, we propose that a dual-action mechanoresponsive and chemical-surface approach must be utilised to improve antimicrobial activity. The addition of antimicrobial silver nanoparticles will provide a secondary, chemical mechanism to escalate the microbial response in tandem with the physical puncture of the cells. EXPERIMENTS: Hydrothermal etching is used as a facile method to impart variant nanostrucutres on the titanium substrate to increase the antimicrobial response. Increasing concentrations (0.25 M, 0.50 M, 1.0 M, 2.0 M) of sodium hydroxide etching solution were used to provide differing degrees of nanostructured morphology on the surface after 3 h of heating at 150 °C. This produced titanium nanospikes, nanoblades, and nanowires, respectively, as a function of etchant concentration. These substrates then provided an interface for the deposition of silver nanoparticles via a reduction pathway. Methicillin-resistant Staphylococcous aureus (MRSA) and Candida auris (C. auris) were used as model bacteria and fungi, respectively, to test the effectiveness of the nanostructured titanium with and without silver nanoparticles, and the bio-interactions at the interface. FINDINGS: The presence of nanostructure increased the bactericidal response of titanium against MRSA from âˆ¼ 10 % on commercially pure titanium to a maximum of âˆ¼ 60 % and increased the fungicidal response from âˆ¼ 10 % to âˆ¼ 70 % in C. auris. Introducing silver nanoparticles increased the microbiocidal response to âˆ¼ 99 % towards both bacteria and fungi. Importantly, this study highlights that nanostructure alone is not sufficient to develop a highly antimicrobial titanium substrate. A dual-action, physical and chemical antimicrobial approach is better suited to produce highly effective antibacterial and antifungal surface technologies.

Broad-spectrum treatment of bacterial biofilms using magneto-responsive liquid metal particles.

The formation and proliferation of bacterial biofilms on surfaces, particularly those on biomedical devices, is a significant issue that results in substantial economic losses, presenting severe health risks to patients. Furthermore, heterogeneous biofilms consisting of different bacterial species can induce the increase in pathogenicity, and the resistance to antimicrobial agents due to the synergistic interactions between the different species. Heterogeneous bacterial biofilms are notoriously difficult to treat due to the presence of extracellular polymeric substances (EPS) and, in conjunction with the rapid rise of multi-drug resistant pathogens, this means that new solutions for anti-biofilm treatment are required. In this study, we investigate the application of magneto-responsive gallium-based liquid metal (GLM-Fe) nanomaterials against a broad range of Gram-positive and Gram-negative bacterial mono-species and multi-species biofilms. The GLM-Fe particles exhibit a magneto-responsive characteristic, causing spherical particles to undergo a shape transformation to high-aspect-ratio nanoparticles with sharp asperities in the presence of a rotating magnetic field. These shape-transformed particles are capable of physically removing bacterial biofilms and rupturing individual cells. Following treatment, both mono-species and multi-species biofilms demonstrated significant reductions in their biomass and overall cell viability, demonstrating the broad-spectrum application of this antibacterial technology. Furthermore, the loss of integrity of the bacterial cell wall and membranes was visualized using a range of microscopy techniques, and the leakage of intracellular components (such as nucleic acids and protein) was observed. Insights gained from this study will impact the design of future liquid metal-based biofilm treatments, particularly those that rely on magneto-responsive properties.

Engineering the Interface: Nanodiamond Coating on 3D-Printed Titanium Promotes Mammalian Cell Growth and Inhibits Staphylococcus aureus Colonization.

Additively manufactured selective laser melted titanium (SLM-Ti) opens the possibility of tailored medical implants for patients. Despite orthopedic implant advancements, significant problems remain with regard to suboptimal osseointegration at the interface between the implant and the surrounding tissue. Here, we show that applying a nanodiamond (ND) coating onto SLM-Ti scaffolds provides an improved surface for mammalian cell growth while inhibiting colonization of Staphylococcus aureus bacteria. Owing to the simplicity of our methodology, the approach is suitable for coating SLM-Ti geometries. The ND coating achieved 32 and 29% increases in cell density of human dermal fibroblasts and osteoblasts, respectively, after 3 days of incubation compared with the uncoated SLM-Ti substratum. This increase in cell density complements an 88% reduction in S. aureus detected on the ND-coated SLM-Ti substrata. This study paves a way to create facile antifouling SLM-Ti structures for biomedical implants.

Polycrystalline Diamond Coating of Additively Manufactured Titanium for Biomedical Applications.

Additive manufacturing using selective laser melted titanium (SLM-Ti) is used to create bespoke items across many diverse fields such as medicine, defense, and aerospace. Despite great progress in orthopedic implant applications, such as for "just in time" implants, significant challenges remain with regards to material osseointegration and the susceptibility to bacterial colonization on the implant. Here, we show that polycrystalline diamond coatings on these titanium samples can enhance biological scaffold interaction improving medical implant applicability. The highly conformable coating exhibited excellent bonding to the substrate. Relative to uncoated SLM-Ti, the diamond coated samples showed enhanced mammalian cell growth, enriched apatite deposition, and reduced microbial S. aureus activity. These results open new opportunities for novel coatings on SLM-Ti devices in general and especially show promise for improved biomedical implants.

Nano-structured antimicrobial surfaces: From nature to synthetic analogues.

The scientific and industrial interest in antimicrobial surfaces has significantly increased in recent times. This interest is largely in response to the persistent microbial contamination of industrial and, importantly, medical implant surfaces. Bacterial contamination of implant surfaces often leads to infection at the implant-tissue interface, and with the prevalence of increasing levels of antimicrobial resistance, the treatment of these infections is becoming far more challenging. Recently, many naturally occurring, high-aspect-ratio surface topographies have been discovered that exhibit high levels of biocidal efficacy. These include epicuticular lipid nano-architectures that are formed on the surfaces of insect wings, such as cicadae and dragonflies. The antimicrobial activity of such surfaces has been found to be a consequence of the physical interactions between the nanoscale topography of the substrate and the attaching pathogenic cells, meaning that the activity is independent of biochemical surface functionality. Importantly, these desirable surface properties can be translated to synthetic biomimetic surfaces, which, when mimicked, lead to a substantial increase in the antimicrobial properties of such surfaces. This paper reviews the recent advances in understanding the basis of these mechanical antimicrobial mechanisms, and discusses the progress being made towards the fabrication of optimised, biocompatible, synthetic analogues.