Characterization of Fiber-Forming Peptides and Proteins by Means of Atomic Force Microscopy

The atomic force microscope (AFM) is widely used in biological sciences due to its ability to perform imaging experiments at high resolution in a physiological environment, without special sample preparation such as fixation or staining. AFM is unique, in that it allows single molecule information of mechanical properties and molecular recognition to be gathered. This review sets out to identify methodological applications of AFM for characterization of fiber-forming proteins and peptides. The basics of AFM operation are detailed, with in-depth information for any life scientist to get a grasp on AFM capabilities. It also briefly describes antibody recognition imaging and mapping of nanomechanical properties on biological samples. Subsequently, examples of AFM application to fiber-forming natural proteins, and fiberforming synthetic peptides are given. Here, AFM is used primarily for structural characterization of fibers in combination with other techniques, such as circular dichroism and fluorescence spectroscopy. More recent developments in antibody recognition imaging to identify constituents of protein fibers formed in human disease are explored. This review, as a whole, seeks to encourage the life scientists dealing with protein aggregation phenomena to consider AFM as a part of their research toolkit, by highlighting the manifold capabilities of this technique

A Regenerable Biosensing Platform for Bacterial Toxins

Waterborne diarrheal diseases such as travelers’ diarrhea and cholera remain a threat to public health in many countries. Rapid diagnosis of an infectious disease is critical in preventing the escalation of a disease outbreak into an epidemic. Many of the diagnostic tools for infectious diseases employed today are time-consuming and require specialized laboratory settings and trained personnel. There is hence a pressing need for fit-for-purpose point-of-care diagnostic tools with emphasis in sensitivity, specificity, portability, and low cost. We report work toward thermally reversible biosensors for detection of the carbohydrate-binding domain of the Escherichia coli heat-labile enterotoxin (LTB), a toxin produced by enterotoxigenic E. coli strains, which causes travelers’ diarrhea. The biosensing platform is a hybrid of two materials, combining the optical properties of porous silicon (pSi) interferometric transducers and a thermoresponsive multivalent glycopolymer, to enable recognition of LTB. Analytical performance of our biosensors allows us to detect, using a label-free format, sub-micromolar concentrations of LTB in solution as low as 0.135 μM. Furthermore, our platform shows a temperature-mediated “catch-and-release” behavior, an exciting feature with potential for selective protein capture, multiple readouts, and regeneration of the sensor over consecutive cycles of use

Fabrication of silicon nanowire arrays by near-field laser ablation and metal-assisted chemical etching

We present an elegant route for the fabrication of ordered arrays of vertically-aligned silicon nanowires with tunable geometry at controlled locations on a silicon wafer. A monolayer of transparent microspheres convectively assembled onto a gold-coated silicon wafer acts as a microlens array. Irradiation with a single nanosecond laser pulse removes the gold beneath each focusing microsphere, leaving behind a hexagonal pattern of holes in the gold layer. Owing to the near-field effects, the diameter of the holes can be at least five times smaller than the laser wavelength. The patterned gold layer is used as catalyst in a metal-assisted chemical etching to produce an array of vertically-aligned silicon nanowires. This approach combines the advantages of direct laser writing with the benefits of parallel laser processing, yielding nanowire arrays with controlled geometry at predefined locations on the silicon surface. The fabricated VA-SiNW arrays can effectively transfect human cells with a plasmid encoding for green fluorescent protein

Nanoporous Anodic Alumina Photonic Crystals for Optical Chemo- and Biosensing: Fundamentals, Advances, and Perspectives

Optical sensors are a class of devices that enable the identification and/or quantification of analyte molecules across multiple fields and disciplines such as environmental protection, medical diagnosis, security, food technology, biotechnology, and animal welfare. Nanoporous photonic crystal (PC) structures provide excellent platforms to develop such systems for a plethora of applications since these engineered materials enable precise and versatile control of light–matter interactions at the nanoscale. Nanoporous PCs provide both high sensitivity to monitor in real-time molecular binding events and a nanoporous matrix for selective immobilization of molecules of interest over increased surface areas. Nanoporous anodic alumina (NAA), a nanomaterial long envisaged as a PC, is an outstanding platform material to develop optical sensing systems in combination with multiple photonic technologies. Nanoporous anodic alumina photonic crystals (NAA-PCs) provide a versatile nanoporous structure that can be engineered in a multidimensional fashion to create unique PC sensing platforms such as Fabry–Pérot interferometers, distributed Bragg reflectors, gradient-index filters, optical microcavities, and others. The effective medium of NAA-PCs undergoes changes upon interactions with analyte molecules. These changes modify the NAA-PCs’ spectral fingerprints, which can be readily quantified to develop different sensing systems. This review introduces the fundamental development of NAA-PCs, compiling the most significant advances in the use of these optical materials for chemo- and biosensing applications, with a final prospective outlook about this exciting and dynamic field

Nanotopography mediated osteogenic differentiation of human dental pulp derived stem cells

Advanced medical devices, treatments and therapies demand an understanding of the role of interfacial properties on the cellular response. This is particularly important in the emerging fields of cell therapies and tissue regeneration. In this study, we evaluate the role of surface nanotopography on the fate of human dental pulp derived stem cells (hDPSC). These stem cells have attracted interest because of their capacity to differentiate to a range of useful lineages but are relatively easy to isolate. We generated and utilized density gradients of gold nanoparticles which allowed us to examine, on a single substrate, the influence of nanofeature density and size on stem cell behavior. We found that hDPSC adhered in greater numbers and proliferated faster on the sections of the gradients with higher density of nanotopography features. Furthermore, greater surface nanotopography density directed the differentiation of hDPSC to osteogenic lineages. This study demonstrates that carefully tuned surface nanotopography can be used to manipulate and guide the proliferation and differentiation of these cells. The outcomes of this study can be important in the rational design of culture substrates and vehicles for cell therapies, tissue engineering constructs and the next generation of biomedical devices where control over the growth of different tissues is required

Effectiveness of porous silicon nanoparticle treatment at inhibiting the migration of a heterogeneous glioma cell population

BACKGROUND: Approximately 80% of brain tumours are gliomas. Despite treatment, patient mortality remains high due to local metastasis and relapse. It has been shown that transferrin-functionalised porous silicon nanoparticles (Tf@pSiNPs) can inhibit the migration of U87 glioma cells. However, the underlying mechanisms and the effect of glioma cell heterogeneity, which is a hallmark of the disease, on the efficacy of Tf@pSiNPs remains to be addressed. RESULTS: Here, we observed that Tf@pSiNPs inhibited heterogeneous patient-derived glioma cells’ (WK1) migration across small perforations (3 μm) by approximately 30%. A phenotypical characterisation of the migrated subpopulations revealed that the majority of them were nestin and fibroblast growth factor receptor 1 positive, an indication of their cancer stem cell origin. The treatment did not inhibit cell migration across large perforations (8 μm), nor cytoskeleton formation. This is in agreement with our previous observations that cellular-volume regulation is a mediator of Tf@pSiNPs’ cell migration inhibition. Since aquaporin 9 (AQP9) is closely linked to cellular-volume regulation, and is highly expressed in glioma, the effect of AQP9 expression on WK1 migration was investigated. We showed that WK1 migration is correlated to the differential expression patterns of AQP9. However, AQP9-silencing did not affect WK1 cell migration across perforations, nor the efficacy of cell migration inhibition mediated by Tf@pSiNPs, suggesting that AQP9 is not a mediator of the inhibition. CONCLUSION: This in vitro investigation highlights the unique therapeutic potentials of Tf@pSiNPs against glioma cell migration and indicates further optimisations that are required to maximise its therapeutic efficacies

Transferrin-targeted porous silicon nanoparticles reduce glioblastoma cell migration across tight extracellular space

Mortality of glioblastoma multiforme (GBM) has not improved over the last two decades despite medical breakthroughs in the treatment of other types of cancers. Nanoparticles hold tremendous promise to overcome the pharmacokinetic challenges and off-target adverse effects. However, an inhibitory effect of nanoparticles by themselves on metastasis has not been explored. In this study, we developed transferrin-conjugated porous silicon nanoparticles (Tf@pSiNP) and studied their effect on inhibiting GBM migration by means of a microfluidic-based migration chip. This platform, designed to mimic the tight extracellular migration tracts in brain parenchyma, allowed high-content time-resolved imaging of cell migration. Tf@pSiNP were colloidally stable, biocompatible, and their uptake into GBM cells was enhanced by receptor-mediated internalisation. The migration of Tf@pSiNP-exposed cells across the confined microchannels was suppressed, but unconfined migration was unaffected. The pSiNP-induced destabilisation of focal adhesions at the leading front may partially explain the migration inhibition. More corroborating evidence suggests that pSiNP uptake reduced the plasticity of GBM cells in reducing cell volume, an effect that proved crucial in facilitating migration across the tight confined tracts. We believe that the inhibitory effect of Tf@pSiNP on cell migration, together with the drug-delivery capability of pSiNP, could potentially offer a disruptive strategy to treat GBM

Effectiveness of porous silicon nanoparticle treatment at inhibiting the migration of a heterogeneous glioma cell population

Background: Approximately 80% of brain tumours are gliomas. Despite treatment, patient mortality remains high due to local metastasis and relapse. It has been shown that transferrin-functionalised porous silicon nanoparticles (Tf@pSiNPs) can inhibit the migration of U87 glioma cells. However, the underlying mechanisms and the effect of glioma cell heterogeneity, which is a hallmark of the disease, on the efficacy ofTf@pSiNPs remains to be addressed.Results: Here, we observed that Tf@pSiNPs inhibited heterogeneous patient-derived glioma cells' (WK1) migration across small perforations (3 mu m) by approximately 30%. A phenotypical characterisation of the migrated subpopulations revealed that the majority of them were nestin and fibroblast growth factor receptor 1 positive, an indication of their cancer stem cell origin. The treatment did not inhibit cell migration across large perforations (8 mu m), nor cytoskeleton formation. This is in agreement with our previous observations that cellular-volume regulation is a mediator of Tf@pSiNPs'cell migration inhibition. Since aquaporin 9 (AQP9) is closely linked to cellular-volume regulation, and is highly expressed in glioma, the effect of AQP9 expression on WK1 migration was investigated. We showed that WK1 migration is correlated to the differential expression patterns of AQP9. However, AQP9-silencing did not affect WK1 cell migration across perforations, nor the efficacy of cell migration inhibition mediated by Tf@pSiNPs, suggesting that AQP9 is not a mediator of the inhibition.Conclusion: This in vitro investigation highlights the unique therapeutic potentials ofTf@pSiNPs against glioma cell migration and indicates further optimisations that are required to maximise its therapeutic efficacies.</div