Characterising a Custom-Built Radio Frequency PECVD Reactor to Vary the Mechanical Properties of TMDSO Films.

Plasma-polymerised tetramethyldisiloxane (TMDSO) films are frequently applied as coatings for their abrasion resistance and barrier properties. By manipulating the deposition parameters, the chemical structure and thus mechanical properties of the films can also be controlled. These mechanical properties make them attractive as energy adsorbing layers for a range of applications, including carbon fibre composites. In this study, a new radio frequency (RF) plasma-enhanced chemical vapour deposition (PECVD) plasma reactor was designed with the capability to coat fibres with an energy adsorbing film. A key characterisation step for the system was establishing how the properties of the TMDSO films could be modified and compared with those deposited using a well-characterized microwave (MW) PECVD reactor. Film thickness and chemistry were determined with ellipsometry and X-ray photoelectron spectroscopy, respectively. The mechanical properties were investigated by nanoindentation and atomic force microscopy with peak-force quantitative nanomechanical mapping. The RF PECVD films had a greater range of Young's modulus and hardness values than the MW PECVD films, with values as high as 56.4 GPa and 7.5 GPa, respectively. These results demonstrated the varied properties of TMDSO films that could in turn be deposited onto carbon fibres using a custom-built RF PECVD reactor.

Three-Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy.

Three-dimensional (3D) cell culture has developed rapidly over the past 5-10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.

Controlled release from PCL-alginate microspheres via secondary encapsulation using GelMA/HAMA hydrogel scaffolds.

Controlling the release of bioactive agents has important potential applications in tissue engineering. While microspheres have been investigated to manipulate release rates, the majority of these investigations have been based on delivery into aqueous media, whereas the cellular environment in tissue engineering is more typically a hydrogel scaffold. If drug-loaded microspheres are introduced within scaffolds to deliver biologically active substances in situ, it is crucial to understand how the release rate is influenced by interactions between the microspheres and the scaffold. Here, we report the fabrication and characterization of a biodegradable scaffold that contains composite microspheres and is suitable for biological applications. Our approach evaluates the influence on the release profile of a model drug (FITC-dextran sulfate) from alginate and PCL-alginate microspheres within a hydrogel construct forming a secondary encapsulation. Increasing the degree of crosslinking in the secondary encapsulation matrix led to a slower cumulative release from 36% to 15%, from the alginate microspheres, whereas a decrease from 26% to 6% was observed for the PCL-alginate microspheres. These results suggest that the release of bioactive molecules can be fine tuned by independently engineering the properties of the scaffold and microspheres.

Versatile SERS sensing based on black silicon.

Black Si (b-Si) with gold or silver metal coating has been shown to be an extremely effective substrate for surface-enhanced Raman scattering (SERS). Here, we demonstrate that it is also a highly versatile SERS platform, as it supports a wide range of surface functionalizations. In particular, we report the use of a molecularly imprinted polymer (MIP) coating and a hydrophobic coating on b-Si to establish two different sensing modalities. First, using a MIP layer on Au-coated b-Si, we show selective sensing of two closely related varieties of tetracycline. Second, a hydrophobic coating was used to concentrate the analyte adsorbed on gold colloidal nanoparticles, thus increasing the sensitivity of the measurement by an order of magnitude. In this experiment, Au nanoparticles and analyte were mixed just before SERS measurements and were concentrated by drop-drying on the super-hydrophobic b-Si. These approaches are promising for SERS measurements that are sensitive to the aging of bare plasmonic metal-coated substrates.

Laser exposure of gold nanorods can increase neuronal cell outgrowth.

The usage of gold nanoparticles (Au NPs) in biological applications has risen significantly over the last 10 years. With the wide variety of chemical and biological functionalization available and their distinctive optical properties, Au NPs are currently used in a range of biological applications including sensing, labeling, drug delivery, and imaging applications. Among the available particles, gold nanorods (Au NRs) are particularly useful because their optical absorption can be tuned across the visible to near infrared region. Here, we present a novel application of Au NRs associated with low power laser exposure of NG108-15 neuronal cells. When cells were irradiated with a 780 nm laser, the average number of neurons with neurites increased. A similar stimulatory effect was observed for cells that were cultured with poly-(4-styrenesulfonic acid)-coated and silica-coated Au NRs. Furthermore, when the NG108-15 cells were cultured with both bare and coated Au NRs and then irradiated with 1.2-7.5 W/cm(2) at 780 nm, they showed a neurite length increase of up to 25 µm versus control. To the best of our knowledge, this effect has never been reported before. While the pathways of the stimulation is not yet clear, the data presented here demonstrates that it is linked to the absorption of light by the Au NRs. These initial results open up new opportunities for peripheral nerve regeneration treatments and for novel approaches to addressing central nervous system axons following spinal cord injury.

Arginine functionalization of hydrogels for heparin binding--a supramolecular approach to developing a pro-angiogenic biomaterial.

Our aim was to synthesize a biomaterial that stimulates angiogenesis for tissue engineering applications by exploiting the ability of heparin to bind and release vascular endothelial growth factor (VEGF). The approach adopted involved modification of a hydrogel with positively charged peptides (oligolysine or oligoarginine) to achieve heparin binding. Precursor hydrogels were produced from copolymerization of N-vinyl pyrolidone, diethylene glycol bis allyl carbonate and acrylic acid (PNDA) and functionalized after activation of the carboxylic acid groups with trilysine or triarginine peptides (PNDKKK and PNDRRR). Both hydrogels were shown to bind and release bioactive VEGF165 with arginine-modified hydrogel outperforming the lysine-modified hydrogel. Cytocompatibility of the hydrogels was confirmed in vitro with primary human dermal fibroblasts and human dermal microvascular endothelial cells (HUDMECs). Proliferation of HUDMECs was stimulated by triarginine-functionalized hydrogels, and to a lesser extent by lysine functionalized hydrogels once loaded with heparin and VEGF. The data suggests that heparin-binding hydrogels provide a promising approach to a pro-angiogenic biomaterial.

Imaging approaches for monitoring three-dimensional cell and tissue culture systems.

The past decade has seen an increasing demand for more complex, reproducible and physiologically relevant tissue cultures that can mimic the structural and biological features of living tissues. Monitoring the viability, development and responses of such tissues in real-time are challenging due to the complexities of cell culture physical characteristics and the environments in which these cultures need to be maintained in. Significant developments in optics, such as optical manipulation, improved detection and data analysis, have made optical imaging a preferred choice for many three-dimensional (3D) cell culture monitoring applications. The aim of this review is to discuss the challenges associated with imaging and monitoring 3D tissues and cell culture, and highlight topical label-free imaging tools that enable bioengineers and biophysicists to non-invasively characterise engineered living tissues.

Extending In-Plane Impedance Measurements from 2D to 3D Cultures: Design Considerations.

Three-dimensional (3D) cell cultures have recently emerged as tools for biologically modelling the human body. As 3D models make their way into laboratories there is a need to develop characterisation techniques that are sensitive enough to monitor the cells in real time and without the need for chemical labels. Impedance spectroscopy has been shown to address both of these challenges, but there has been little research into the full impedance spectrum and how the different components of the system affect the impedance signal. Here we investigate the impedance of human fibroblast cells in 2D and 3D collagen gel cultures across a broad range of frequencies (10 Hz to 5 MHz) using a commercial well with in-plane electrodes. At low frequencies in both 2D and 3D models it was observed that protein adsorption influences the magnitude of the impedance for the cell-free samples. This effect was eliminated once cells were introduced to the systems. Cell proliferation could be monitored in 2D at intermediate frequencies (30 kHz). However, the in-plane electrodes were unable to detect any changes in the impedance at any frequency when the cells were cultured in the 3D collagen gel. The results suggest that in designing impedance measurement devices, both the nature and distribution of the cells within the 3D culture as well as the architecture of the electrodes are key variables.

Polymersome production on a microfluidic platform using pH sensitive block copolymers.

Development of pH sensitive biocompatible block copolymer polymersomes, which are stable in physiological conditions, is enabling the intracellular delivery of water soluble drugs and proteins. As a result, it is becoming increasingly important to develop robust production methods to enhance the polymersome encapsulation efficiency. One way that this could be achieved is through production in microfluidic devices that potentially offer more favourable conditions for encapsulation. Here a flow focussing microfluidic device is used to induce self-assembly of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacrylate) (PMPC-b-PDPA) block copolymer by changing the pH of the flows within the microchannels. The laminar flow conditions within the device result in a pH gradient at either interface of the central flow, where diffusion of hydrogen ions enables the deprotonation of the PDPA block copolymer and results in self-assembly of polymersomes. Dynamic light scattering reveals hydrodynamic diameters in the range of 75-275 nm and double membrane structures visualized using transmission electron microscopy indicate that polymersome nanostructures are being produced. The encapsulation efficiency for Bovine Serum Albumin (BSA) was calculated by measuring the spectroscopic absorbance at 279 nm and indicates that the encapsulation efficiency produced in the microfluidic device is equivalent to the standard in solution production method. Critically, the microfluidic system eliminates the use of organic solvents, which limit biological applications, through the pH induced self-assembly process and offers a continuous production method for intracellular delivery polymersomes.

Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics.

Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4-20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of α-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (µIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This µIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.

Plasma polymerization of maleic anhydride: just what are the right deposition conditions?

Maleic anhydride plasma polymers enable amine-containing biomolecules and polymers to be covalently coupled to a surface from an aqueous solution without any intermediate chemistry. The challenge in developing these functionally active plasma polymers lies in determining the optimal deposition conditions for producing a stable, highly active film. Unlike many previous studies that explore highly varied pulsed and continuous wave (CW) deposition conditions, this paper focuses on the comparison of films deposited under the same low nominal power conditions (1 W) and compares a range of CW, millisecond, and microsecond pulsing parameters that can be used to produce this power condition. The use of attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) has enabled the quantitative examination of the effects of processing parameters on the chemical functionality of the films. For the first time, the molecular specificity, surface sensitivity, and high mass resolution of time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) has been exploited to compare these films and multivariate analysis techniques used to explore the relationships between plasma processing parameters and surface chemistry. The results of the studies clearly demonstrate that a range of conditions can produce maleic anhydride films, with optimal functionality seen under microsecond pulsing regimes. Critically, the study demonstrates that the tight control and monitoring of the deposition parameters is critical if these films are to be manufactured with optimal functionality, stability, and minimum processing time.

Physical vs photolithographic patterning of plasma polymers: an investigation by ToF-SSIMS and multivariate analysis.

Physical and photolithographic techniques are commonly used to create chemical patterns for a range of technologies including cell culture studies, bioarrays and other biomedical applications. In this paper, we describe the fabrication of chemical micropatterns from commonly used plasma polymers. Atomic force microscopy (AFM) imaging, time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) imaging, and multivariate analysis have been employed to visualize the chemical boundaries created by these patterning techniques and assess the spatial and chemical resolution of the patterns. ToF-SSIMS analysis demonstrated that well-defined chemical and spatial boundaries were obtained from photolithographic patterning, while the resolution of physical patterning via a transmission electron microscopy (TEM) grid varied depending on the properties of the plasma system including the substrate material. In general, physical masking allowed diffusion of the plasma species below the mask and bleeding of the surface chemistries. Multivariate analysis techniques including principal component analysis (PCA) and region of interest (ROI) assessment were used to investigate the ToF-SSIMS images of a range of different plasma polymer patterns. In the most challenging case, where two strongly reacting polymers, allylamine and acrylic acid were deposited, PCA confirmed the fabrication of micropatterns with defined spatial resolution. ROI analysis allowed for the identification of an interface between the two plasma polymers for patterns fabricated using the photolithographic technique which has been previously overlooked. This study clearly demonstrated the versatility of photolithographic patterning for the production of multichemistry plasma polymer arrays and highlighted the need for complementary characterization and analytical techniques during the fabrication plasma polymer micropatterns.

Studies of electroosmotic flow and the effects of protein adsorption in plasma-polymerized microchannel surfaces.

This paper presents a study of EOF properties of plasma-polymerized microchannel surfaces and the effects of protein (fibrinogen and lysozyme) adsorption on the EOF behavior of the surface-modified microchannels. Three plasma polymer surfaces, i.e. tetraglyme, acrylic acid and allylamine, are tested. Results indicate EOF suppression in all plasma-coated channels compared with the uncoated glass microchannel surfaces. The EOF behaviors of the modified microchannels after exposure to protein solutions are also investigated and show that even low levels of protein adsorption can significantly influence EOF behavior, and in some cases, result in the reversal of flow. The results also highlight that EOF measurement can be used as a method for detecting the presence of proteins within microchannels at low surface coverage (<1 ng/cm(2) on glass). Critically, the results illustrate that the non-fouling tetraglyme plasma polymer is able to sustain EOF. Comparison of the plasma-polymerized surfaces with conventionally grafted polyelectrolyte surfaces demonstrates the stabilities of the plasma polymer films, enabling multiple EOF runs over 3 days without deterioration in performance. The results of this study clearly demonstrate that plasma polymers enable the surface chemistry of microfluidic devices to be tailored for specific applications. Critically, the deposition of the non-fouling tetraglyme coating enables stable EOF to be induced in the presence of protein.

Fabrication of a Biocompatible Liquid Crystal Graphene Oxide-Gold Nanorods Electro- and Photoactive Interface for Cell Stimulation.

For decades, electrode-tissue interfaces are pursued to establish electrical stimulation as a reliable means to control neuronal cells behavior. However, spreading of electrical currents in tissues limits its spatial precision. Thus, optical cues, such as near-infrared (NIR) light, are explored as alternatives. Presently, NIR stimulation requires higher energy input than electrical methods despite introduction of light absorbers, e.g., gold nanoparticles. As potential solution, NIR and electrical costimulation are proposed but with limited interfaces capable of sustaining this stimulation technique. Here, a novel electroactive nanocomposite with photoactive properties in the NIR range is constructed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysulfosuccinimide sodium (EDC)/NHS conjugation of liquid crystal graphene oxide (LCGO) to protein-coated gold nanorods (AuNR). The liquid crystal graphene oxide-gold nanorod nanocomposite (LCGO-AuNR) is fabricated into a hydrophilic electrode-coating via drop-casting, making it appropriate for versatile electrode-tissue interface fabrication. UV-vis spectrophotometry results demonstrate that LCGO-AuNR presents an absorbance peak at 798 nm (NIR range). Cyclic voltammetry measurements further confirm its electroactive capacitive properties. Furthermore, LCGO-AuNR coating supports cell adhesion, proliferation, and differentiation of NG108-15 neuronal cells. This biocompatible interface is anticipated, with ideal electrical and optical properties for NIR and electrical costimulation, to enable further development of the technique for energy-efficient and precise neuronal cell modulation.

Multitechnique investigation into the aqueous behavior of plasma polymers.

Plasma polymers are often used in applications requiring aqueous immersion; therefore, it is important to understand how this exposure affects the physical and chemical properties of the films. Three different plasma polymer films were deposited at different distances from the electrode, and the film properties were characterized using contact angle, ellipsometry, and x-ray photoelectron spectroscopy. The film behaviors in aqueous solutions were studied via quartz crystal microbalance with dissipation (QCM-D). Exposure to buffer solutions produced significant swelling of the plasma polymerized acrylic acid films, with swelling increasing with distance from the powered electrode, results that could be correlated with changes in film chemistry. Plasma polymerized octadiene and allylamine exhibited little swelling. These films exhibited changes in thickness and contact angle with respect to distance from the electrode, but this had little influence on their behavior in aqueous solution. By combining QCM-D with the more traditional surface chemical analysis techniques, the authors have been able to explore both swelling behavior and the effect that sample position and thus deposition parameters have on film properties and aqueous behavior. This approach gives the authors the basis to define deposition parameters to assist the engineering of thin films for applications such as biosensing and tissue engineering applications where specific chemistries and film behaviors are desired.

Toward Immunocompetent 3D Skin Models.

3D human skin models provide a platform for toxicity testing, biomaterials evaluation, and investigation of fundamental biological processes. However, the majority of current in vitro models lack an inflammatory system, vasculature, and other characteristics of native skin, indicating scope for more physiologically complex models. Looking at the immune system, there are a variety of cells that could be integrated to create novel skin models, but to do this effectively it is also necessary to understand the interface between skin biology and tissue engineering as well as the different roles the immune system plays in specific health and disease states. Here, a progress report on skin immunity and current immunocompetent skin models with a focus on construction methods is presented; scaffold and cell choice as well as the requirements of physiologically relevant models are elaborated. The wide range of technological and fundamental challenges that need to be addressed to successfully generate immunocompetent skin models and the steps currently being made globally by researchers as they develop new models are explored. Induced pluripotent stem cells, microfluidic platforms to control the model environment, and new real-time monitoring techniques capable of probing biochemical processes within the models are discussed.

Characterization of fibrinogen adsorption onto glass microcapillary surfaces by ELISA.

Adsorption of biomolecules onto microchannel surfaces remains a critical issue in microfluidic devices. This paper investigates the adsorption of fibrinogen on glass microcapillaries using an immunoassay method (ELISA) and X-ray photoelectron spectroscopy (XPS). Various adsorption conditions such as protein concentrations and incubation times, buffer pH, buffer ionic strengths and effects of flow are presented. ELISA is successfully demonstrated as a facile and robust technique to examine these phenomena. The highest adsorption level occurs near the isoelectric point of fibrinogen (pH 5.0) and low buffer ionic strengths (0-8 mM). Microchannel surface saturation was achieved at a fibrinogen solution concentration of approximately 50 microg ml(-1). Fibrinogen adsorption under flow was always higher than that seen in static systems. The importance of diffusion phenomena in microchannels on protein adsorption was demonstrated. ELISA experiments using fused silica and PEEK have also confirmed significant adsorption on these mass spectrometer transfer line materials.

Non-fouling microfluidic chip produced by radio frequency tetraglyme plasma deposition.

This Technical Note presents the direct surface modification of a glass/PTFE hybrid microfluidic chip, via radio frequency glow discharge plasma polymerisation of tetraethlylene glycol dimethylether (tetraglyme), to produce hydrophilic, non-fouling, PEO-like surfaces. We use several techniques including X-ray photoelectron spectroscopy (XPS), direct enzyme-linked immunosorbent assays (ELISA) and immunofluorescent imaging to investigate the channel coatings. Our results indicate the successful deposition of a PEO-like coating onto microchannel surfaces that has both solution and shelf stability (>3 months) and is capable of preventing fibrinogen adsorption to the microchannel surfaces.

Synthesis and characterization of immobilized PAMAM dendrons.

This communication describes the synthesis and characterization of immobilized PAMAM dendrons onto a surface modified silicon wafer substrate (functionalized using plasma polymerized PAA) using a "growing from" strategy.

pH-dependent lipid vesicle interactions with plasma polymerized thin films.

Model lipid vesicle and supported lipid bilayer (SLB) systems are used in a variety of applications including biosensing, cell membrane mimics, and drug delivery. Exposure of a surface to a vesicle solution provides a straightforward method for creating such systems via vesicle adsorption and collapse. However, this process is complex and the relationship between the surface physicochemical properties and vesicle collapse is poorly understood. Plasma polymers are thin conformal films that can be applied to a variety of materials to modify surface properties. This paper uses quartz crystal microbalance with dissipation and fluorescence recovery after photobleaching (FRAP) to explore lipid vesicle interactions with plasma polymerized acrylic acid (ppAAc), allylamine (ppAAm), and ppAAc/ppAAm micropatterns. Vesicle interactions were dependent on plasma polymer chemistry and pH of the buffer solution. Vesicles readily and stably adsorbed to ppAAm over a wide pH range. ppAAc demonstrated limited interactions at pH 7 and vesicle adsorption at pH 4. Vesicle collapse and SLB formation could be induced using a pH change. FRAP was used to explore the fluidity of the lipid structures on both the patterned and unpatterned plasma polymer films. On ppAAm/ppAAc micropatterns, pH transitions combined with the presence of chemically distinct regions on the same substrate enabled immobile lipid islands on ppAAc to be surrounded by fluid lipid regions on ppAAm. This work demonstrates that plasma polymer films could enable spatially controlled vesicle adsorption and SLB formation on a wide variety of different substrates.