Metal cation crosslinked, partially reduced graphene oxide membranes with enhanced stability for high salinity, produced water treatment by pervaporative separation.

Graphene oxide (GO)-based membranes hold significant promise for applications ranging from energy storage to protective coatings, to saline water and produced water treatment, owing to their chemical stability and unique barrier properties achieving a high selectivity for water permeation. However, unmodified GO membranes are not stable when submerged in liquid water, creating challenges with their commercial utilization in aqueous filtration and pervaporation applications. To mitigate this, we develop an approach to modify GO membranes through a combination of low temperature thermal reduction and metal cation crosslinking. We demonstrate that Zn2+-rGO and Fe3+-rGO membranes had the highest permeation flux of 8.3 ± 1.5 l m-2h-1and 7.0 ± 0.4 l m-2h-1, for saline water separation, respectively, when thermally reduced after metal cross-linking; These membranes maintained a high flux of 7.5 ± 0.7 l m-2h-1, and 5.5 ± 0.3 l m-2h-1for produced water separation, respectively. All the membranes had a salt rejection higher than 99%. Fe3+crosslinked membranes presented the highest organic solute rejections for produced water of 69%. Moreover, long term pervaporation testing was done for the Zn2+-rGO membrane for 12 h, and only a minor drop of 6% in permeation flux was observed, while Zn2+-GO had a drop of 24%. Both modifiers significantly enhanced the stability with Fe3+-rGO membranes displaying the highest mechanical abrasion resistance of 95% compared to non-reduced and non-crosslinked GO. Improved stability for all samples also led to higher selectivity to water over organic contaminants and only slightly reduced water flux across the membrane.

In-Vitro Study of Indium (III) Sulfate-Containing Medium on the Viability and Adhesion Behaviors of Human Dermal Fibroblast on Engineered Surfaces.

Tissues and organs consist of cells organized in specified patterns that support their function, as exemplified by tissues such as skin, muscle, and cornea. It is, therefore, important to understand how external cues, such as engineered surfaces or chemical contaminants, can influence the organization and morphology of cells. In this work, we studied the impact of indium sulfate on human dermal fibroblast (GM5565) viability, production of reactive oxygen species (ROS), morphology, and alignment behavior on tantalum/silicon oxide parallel line/trench surface structures. The viability of cells was measured using the alamarBlue™ Cell Viability Reagent probe, while the ROS levels in cells were quantified using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate. Cell morphology and orientation on the engineered surfaces were characterized using fluorescence confocal and scanning electron microscopy. When cells were cultured in media containing indium (III) sulfate, the average cell viability decreased by as much as ~32% and the concentration of cellular ROS increased. Cell geometry became more circular and compact in the presence of indium sulfate. Even though actin microfilaments continue to preferentially adhere to tantalum-coated trenches in the presence of indium sulfate, the cells are less able to orient along the line axes of the chips. Interestingly, the indium sulfate-induced changes in cell alignment behavior are pattern dependent-a larger proportion of adherent cells on structures with line/trench widths in the range of 1 µm and 10 µm lose the ability to orient themselves, compared to those grown on structures with line widths smaller than 0.5 µm. Our results show that indium sulfate impacts the response of human fibroblasts to the surface structure to which they adhere and underscores the importance of evaluating cell behaviors on textured surfaces, especially in the presence of potential chemical contaminants.

Influence of Indium (III) Chloride on Human Dermal Fibroblast Cell Adhesion on Tantalum/Silicon Oxide Nano-Composites.

Cell adhesion is an essential biological function for division, migration, signaling and tissue development. While it has been demonstrated that this cell function can be modified by using nanometer-scale surface topographic structures, it remains unknown how contaminants such as indium (III) ion might influence this specific cell behavior. Herein, the influence of indium chloride on human dermal fibroblast (GM5565) adhesion characteristics was investigated, given the frequent contact of contaminants with skin. The morphology of the adherent cells and their mitochondrial reticulum was characterized on cell culture dishes and nanopatterned surfaces by using fluorescence confocal microscopy and scanning electron microscopy. Results showed a significant proportion of cells lost their ability to align preferentially along the line axes of the nanopattern upon exposure to 3.2 mM indium chloride, with cells aligned within 10° of the pattern line axes reduced by as much as ~70%. Concurrent with the cell adhesion behaviors, the mitochondria in cells exposed to indium chloride exhibit a punctate staining that contrasts with the normal network of elongated tubular geometry seen in control cells. Our results demonstrate that exposure to indium chloride has detrimental effects on the behavior of human fibroblasts and adversely impacts their mitochondrial morphology. This shows the importance of evaluating the biological impacts of indium compounds.

Limitation in Controlling the Morphology of Mammalian Vero Cells Induced by Cell Division on Asymmetric Tungsten-Silicon Oxide Nanocomposite.

Engineered nanomaterials are often used in tissue engineering applications to influence and manipulate the behavior of cells. Recently, a number of tungsten-silicon oxide nanocomposite devices containing equal width (symmetric) tungsten and silicon oxide parallel line comb structures were developed and used by our group. The devices induced over 90% of seeded cells (Vero) to align within ±20° of the axes of 10 µm wide tungsten lines. Furthermore, a mathematical model was successfully developed to predict this alignment behavior and forecast the minimum width of isolated tungsten lines required to induce such behavior. However, the mechanism by which the widths of the symmetrical tungsten and silicon oxide lines induce the alignment behavior is still unknown. Furthermore, the model was never tested on more complex asymmetrical structures. Herewith, experiments were conducted with mammalian cells on complex asymmetrical structures with unequal tungsten and silicon oxide line widths. Results showed that the model could be extended to more complex pattern structures. In addition, cell morphology on the patterned structures reset during cell division because of mitotic rounding, which reduced the population of cells that elongated and aligned on the tungsten lines. Ultimately, we concluded that it was impossible to achieve a 100% alignment with cells having unsynchronized cell cycles because cell rounding during mitosis took precedence over cell alignment; in other words, internal chemical cues had a stronger role in cell morphology than external cues.

Nanoscale-Textured Tantalum Surfaces for Mammalian Cell Alignment.

Tantalum is one of the most important biomaterials used for surgical implant devices. However, little knowledge exists about how nanoscale-textured tantalum surfaces affect cell morphology. Mammalian (Vero) cell morphology on tantalum-coated comb structures was studied using high-resolution scanning electron microscopy and fluorescence microscopy. These structures contained parallel lines and trenches with equal widths in the range of 0.18 to 100 µm. Results showed that as much as 77% of adherent cell nuclei oriented within 10° of the line axes when deposited on comb structures with widths smaller than 10 µm. However, less than 20% of cells exhibited the same alignment performance on blanket tantalum films or structures with line widths larger than 50 µm. Two types of line-width-dependent cell morphology were observed. When line widths were smaller than 0.5 µm, nanometer-scale pseudopodia bridged across trench gaps without contacting the bottom surfaces. In contrast, pseudopodia structures covered the entire trench sidewalls and the trench bottom surfaces of comb structures with line-widths larger than 0.5 µm. Furthermore, results showed that when a single cell simultaneously adhered to multiple surface structures, the portion of the cell contacting each surface reflected the type of morphology observed for cells individually contacting the surfaces.

Pattern-Dependent Mammalian Cell (Vero) Morphology on Tantalum/Silicon Oxide 3D Nanocomposites.

The primary goal of this work was to investigate the resulting morphology of a mammalian cell deposited on three-dimensional nanocomposites constructed of tantalum and silicon oxide. Vero cells were used as a model. The nanocomposite materials contained comb structures with equal-width trenches and lines. High-resolution scanning electron microscopy and fluorescence microscopy were used to image the alignment and elongation of cells. Cells were sensitive to the trench widths, and their observed behavior could be separated into three different regimes corresponding to different spreading mechanism. Cells on fine structures (trench widths of 0.21 to 0.5 µm) formed bridges across trench openings. On larger trenches (from 1 to 10 µm), cells formed a conformal layer matching the surface topographical features. When the trenches were larger than 10 µm, the majority of cells spread like those on blanket tantalum films; however, a significant proportion adhered to the trench sidewalls or bottom corner junctions. Pseudopodia extending from the bulk of the cell were readily observed in this work and a minimum effective diameter of ~50 nm was determined for stable adhesion to a tantalum surface. This sized structure is consistent with the ability of pseudopodia to accommodate ~4⁻6 integrin molecules.

What's Happening on the Other Side? Revealing Nano-Meter Scale Features of Mammalian Cells on Engineered Textured Tantalum Surfaces.

Advanced engineered surfaces can be used to direct cell behavior. These behaviors are typically characterized using either optical, atomic force, confocal, or electron microscopy; however, most microscopic techniques are generally restricted to observing what's happening on the "top" side or even the interior of the cell. Our group has focused on engineered surfaces typically reserved for microelectronics as potential surfaces to control cell behavior. These devices allow the exploration of novel substrates including titanium, tungsten, and tantalum intermixed with silicon oxide. Furthermore, these devices allow the exploration of the intricate patterning of surface materials and surface geometries i.e., trenches. Here we present two important advancements in our research: (1) the ability to split a fixed cell through the nucleus using an inexpensive three-point bend micro-cleaving technique and image 3D nanometer scale cellular components using high-resolution scanning electron microscopy; and (2) the observation of nanometer projections from the underbelly of a cell as it sits on top of patterned trenches on our devices. This application of a 3-point cleaving technique to visualize the underbelly of the cell is allowing a new understanding of how cells descend into surface cavities and is providing a new insight on cell migration mechanisms.

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips.

Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 µm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 µm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

Bacterial Networks on Hydrophobic Micropillars.

Bacteria have evolved as intelligent microorganisms that can colonize and form highly structured and cooperative multicellular communities with sophisticated singular and collective behaviors. The initial stages of colony formation and intercellular communication are particularly important to understand and depend highly on the spatial organization of cells. Controlling the distribution and growth of bacterial cells at the nanoscale is, therefore, of great interest in understanding the mechanisms of cell-cell communication at the initial stages of colony formation. Staphyloccocus aureus, a ubiquitous human pathogen, is of specific clinical importance due to the rise of antibiotic resistant strains of this species, which can cause life-threatening infections. Although several methods have attempted to pattern bacterial cells onto solid surfaces at single cell resolution, no study has truly controlled the 3D architectures of growing colonies. Herein, we present a simple, low-cost method to pattern S. aureus bacterial colonies and control the architecture of their growth. Using the wetting properties of micropatterened poly(dimethyl siloxane) platforms, with help from the physiological activities of the S. aureus cells, we fabricated connected networks of bacterial microcolonies of various sizes. Unlike conventional heterogeneous growth of biofilms on surfaces, the patterned S. aureus microcolonies in this work grow radially from nanostrings of a few bacterial cells, to form micrometer-thick rods when provided with a nutrient rich environment. This simple, efficient, and low-cost method can be used as a platform for studies of cell-cell communication phenomena, such as quorum sensing, horizontal gene transfer, and metabolic cross-feeding especially during initial stages of colony formation.

Differential Collective- and Single-Cell Behaviors on Silicon Micropillar Arrays.

Three-dimensional vertically aligned nano- and micropillars have emerged as promising tools for a variety of biological applications. Despite their increasing usage, the interaction mechanisms of cells with these rigid structures and their effect on single- and collective-cell behaviors are not well understood for different cell types. In the present study, we examine the response of glioma cells to micropillar arrays using a new microfabricated platform consisting of rigid silicon micropillar arrays of various shapes, sizes, and configurations fabricated on a single platform. We compare collective- and single-cell behaviors at micropillar array interfaces and show that glial cells under identical chemical conditions form distinct arrangements on arrays of different shapes and sizes. Tumor-like aggregation and branching of glial cells only occur on arrays with feature diameters greater than 2 µm, and distinct transitions are observed at interfaces between various arrays on the platform. Additionally, despite the same side-to-side spacing and gaps between micropillars, single glial cells interact with the flat silicon surface in the gap between small pillars but sit on top of larger micropillars. Furthermore, micropillars induced local changes in stress fibers and actin-rich filopodia protrusions as the cells conformed to the shape of spatial cues formed by these micropillars.

Cell responses to metallic nanostructure arrays with complex geometries.

Metallic nanopillar/nanowires are emerging as promising platforms for biological applications, as they allow for the direct characterization and regulation of cell function. Herein we study the response of cells to a versatile nanopillar platform. Nanopillar arrays of various shape, size, and spacing and different nanopillar-substrate interfacial strengths were fabricated and interfaced with fibroblasts and several unique cell-nanopillar interactions were observed using high resolution scanning electron microscopy. Nanopillar penetration, engulfment, tilting, lift off and membrane thinning, were observed by manipulating nanopillar material, size, shape and spacing. These unique cell responses to various nanostructures can be employed for a wide range of applications including the design of highly sensitive nano-electrodes for single-cell probing.

Responses of Staphylococcus aureus bacterial cells to nanocrystalline nickel nanostructures.

A broad range of human diseases are associated with bacterial infections, often initiated by specific adhesion of a bacterium to the target environment. Despite the significant role of bacterial adhesion in human infectious diseases, details and mechanisms of bacterial adhesion have remained elusive. Herein, we study the physical interactions between Staphylococcus aureus, a type of micro-organism relevant to infections associated with medical implants, and nanocrystalline (nc) nickel nanostructures with various columnar features, including solid core, hollow, x-shaped and c-shaped pillars. Scanning electron microscopy results show the tendency of these bacterial cells to attach to the nickel nanostructures. Moreover, unique single bacterium attachment characteristics were observed on nickel nanostructures with dimensions comparable to the size of a single bacterium.