Mechanisms for covalent immobilization of horseradish peroxi-dase on ion beam treated polyethylene

The mechanism that provides the observed strong binding of biomolecules to polymer sur-faces modified by ion beams is investigated. The surface of polyethylene (PE) was modified by plasma immersion ion implantation with nitrogen ions. Structure changes including car-bonization and oxidation were observed in the modified surface layer of PE by Raman spec-troscopy, FTIR ATR spectroscopy, atomic force microscopy, surface energy measurement and XPS spectroscopy. An observed high surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with stor-age time after PIII treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish per-oxidase was covalently attached onto the modified PE surface. The enzymatic activity of co-valently attached protein remained high. A mechanism based on the covalent attachment by the reaction of protein with free radicals in the modified surface is proposed. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The long-term activity of the modified layer to attach protein (at least 2 years) is explained by stabilisa-tion of unpaired electrons in sp2 carbon structures. The native conformation of attached pro-tein is retained due to hydrophilic interactions in the interface region. A high concentration of free radicals on the surface can give multiple covalent bonds to the protein molecule and de-stroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers

Tomographic interferometry of a filtered high-current vacuum arc plasma

Tomography of a plasma enables the distribution of electron density to be visualized. We report on the design of two tomographic interferometer systems used to measureplasma electron density distributions in a high-current pulsed cathodic vacuum arc. The method is shown to be capable of microsecond time resolution. The spatial resolution of the quasioptical interferometer operating at 2 mm wavelength is 20 mm and the spatial resolution of the waveguide-based interferometer operating at 8 mm wavelength is 50 mm. In both cases the resolution achieved depends on the launching and receiving geometries. We developed criteria for assessing the tomogram for artifacts arising from limited sampling. First results of the spatial and temporal history of plasma in a high-current vacuum arc guided by a curved magnetic filter are presented and indicate poloidal field fluctuations reminiscent of magnetohydrodynamic instabilities in pinches. The applicability of the tomographic interferometry method to optimize plasma transport through the filter is also demonstrated.This work was in part supported by the Australian Research Council

Plasma Activation of Microplates Optimized for One-Step Reagent-Free Immobilization of DNA and Protein

Activated microplates are widely used in biological assays and cell culture to immobilize biomolecules, either through passive physical adsorption or covalent cross-linking. Covalent attachment gives greater stability in complex biological mixtures. However, current multistep chemical activation methods add complexity and cost, require specific functional groups, and can introduce cytotoxic chemicals that affect downstream cellular applications. Here, we show a method for one-step linker-free activation of microplates by energetic ions from plasma for covalent immobilization of DNA and protein. Two types of energetic ion plasma treatment were shown to be effective: plasma immersion ion implantation (PIII) and plasma-activated coating (PAC). This is the first time that PIII and PAC have been reported in microwell plates with nonflat geometry. We confirm that the plasma treatment generates radical-activated surfaces at the bottom of wells despite potential shadowing from the walls. Comprehensive surface characterization studies were used to compare the PIII and PAC microplate surface composition, wettability, radical density, optical properties, stability, and biomolecule immobilization density. PAC plates were found to have more nitrogen and lower radical density and were more hydrophobic and more stable over 3 months than PIII plates. Optimal conditions were obtained for high-density DNA (PAC, 0 or 21% nitrogen, pH 3–4) and streptavidin (PAC, 21% nitrogen, pH 5–7) binding while retaining optical properties required for typical high-throughput biochemical microplate assays, such as low autofluorescence and high transparency. DNA hybridization and protein activity of immobilized molecules were confirmed. We show that PAC activation allows for high-density covalent immobilization of functional DNA and protein in a single step on both 96- and 384-well plates without specific linker chemistry. These microplates could be used in the future to bind other user-selected ligands in a wide range of applications, for example, for solid phase polymerase chain reaction and stem cell culture and differentiation

Substrate-Regulated Growth of Plasma-Polymerized Films on Carbide-Forming Metals

Although plasma polymerization is traditionally considered as a substrate-independent process, we present evidence that the propensity of a substrate to form carbide bonds regulates the growth mechanisms of plasma polymer (PP) films. The manner by which the first layers of PP films grow determines the adhesion and robustness of the film. Zirconium, titanium, and silicon substrates were used to study the early stages of PP film formation from a mixture of acetylene, nitrogen, and argon precursor gases. The correlation of initial growth mechanisms with the robustness of the films was evaluated through incubation of coated substrates in simulated body fluid (SBF) at 37° for 2 months. It was demonstrated that the excellent zirconium/titanium-PP film adhesion is linked to the formation of metallic carbide and oxycarbide bonds during the initial stages of film formation, where a 2D-like, layer-by-layer (Frank–van der Merwe) manner of growth was observed. On the contrary, the lower propensity of the silicon surface to form carbides leads to a 3D, island-like (Volmer–Weber) growth mode that creates a sponge-like interphase near the substrate, resulting in inferior adhesion and poor film stability in SBF. Our findings shed light on the growth mechanisms of the first layers of PP films and challenge the property of substrate independence typically attributed to plasma polymerized coatings

Plasma ion implantation enabled bio-functionalization of PEEK improves osteoblastic activity

Slow appositional growth of bone in vivo is a major problem associated with polyether ether ketone (PEEK) based orthopaedic implants. Early stage promotion of osteoblast activity, particularly bone nodule formation, would help to improve contact between PEEK implantable materials and the surrounding bone tissue. To improve interactions with bone cells, we explored here the use of plasma immersion ion implantation (PIII) treatment of PEEK to covalently immobilize biomolecules to the surface. In this study, a single step process was used to covalently immobilize tropoelastin on the surface of PIII modified PEEK through reactions with radicals generated by the treatment. Improved bioactivity was observed using the human osteoblast-like cell line, SAOS-2. Cells on surfaces that were PIII-treated or tropoelastin-coated exhibited improved attachment, spreading, proliferation, and bone nodule formation compared to cells on untreated samples. Surfaces that were both PIII-treated and tropoelastin-coated triggered the most favorable osteoblast-like responses. Surface treatment or tropoelastin coating did not alter alkaline phosphatase gene expression and activity of bound cells but did influence the expression of other bone markers including osteocalcin, osteonectin, and collagen I. We conclude that the surface modification of PEEK improves osteoblast interactions, particularly with respect to bone apposition, and enhances the orthopedic utility of PEEK

Plasma‐Activated Coated Glass: A Novel Platform for Optimal Optical Performance and Cell Culture Substrate Customization

Borosilicate glass surpasses polystyrene in optical quality; however, it is less frequently used for cell culture due to poor protein and cell adhesion. To overcome this impasse, the surface of glass coverslips requires functionalization to enable facile covalent attachment of proteins to promote cell attachment and differentiation. Herein, a novel approach is presented to covalently attach proteins to glass by depositing a thin layer of radical‐rich carbon film using a plasma polymerization process. The surface chemistry of these plasma‐activated coatings can be controlled by varying the gas composition used during the deposition. Mass spectrometry reveals different protein profiles attached to functionalized glass coverslips when they are exposed to cell culture media. Mouse embryonic stem cell adhesion and subsequent differentiation into neural lineage on plasma‐treated coverslips are significantly enhanced compared to bare coverslips. Importantly, the coatings are in the nanometer range, preserve the optical properties of the glass coverslips for imaging, and remain stable for at least 4 weeks in simulated body fluid. These results demonstrate the utility of covalently attaching proteins to glass for enhanced cell attachment and stem cell differentiation and provide a promising technique to achieve better outcomes in cell culture in a range of biomedical applications

Nanosecond Responses of Proteins to Ultra-High Temperature Pulses

Observations of fast unfolding events in proteins are typically restricted to <100°C. We use a novel apparatus to heat and cool enzymes within tens of nanoseconds to temperatures well in excess of the boiling point. The nanosecond temperature spikes are too fast to allow water to boil but can affect protein function. Spikes of 174°C for catalase and ∼290°C for horseradish peroxidase are required to produce irreversible loss of enzyme activity. Similar temperature spikes have no effect when restricted to 100°C or below. These results indicate that the “speed limit” for the thermal unfolding of large proteins is shorter than 10(−8) s. The unfolding rate at high temperature is consistent with extrapolation of low temperature rates over 12 orders of magnitude using the Arrhenius relation

Temperature Activated Diffusion of Radicals through Ion Implanted Polymers

Plasma immersion ion implantation (PIII) is a promising technique for immobilizing biomolecules on the surface of polymers. Radicals generated in a subsurface layer by PIII treatment diffuse throughout the substrate, forming covalent bonds to molecules when they reach the surface. Understanding and controlling the diffusion of radicals through this layer will enable efficient optimization of this technique. We develop a model based on site to site diffusion according to Fick’s second law with temperature activation according to the Arrhenius relation. Using our model, the Arrhenius exponential prefactor (for barrierless diffusion), <i>D</i>₀, and activation energy, <i>E</i>_A, for a radical to diffuse from one position to another are found to be 3.11 × 10^–17 m² s^–1 and 0.31 eV, respectively. The model fits experimental data with a high degree of accuracy and allows for accurate prediction of radical diffusion to the surface. The model makes useful predictions for the lifetime over which the surface is sufficiently active to covalently immobilize biomolecules and it can be used to determine radical fluence during biomolecule incubation for a range of storage and incubation temperatures so facilitating selection of the most appropriate parameters

Correlation between film structures and potential limits for hydrogen and oxygen evolutions at a-C:N film electrochemical electrodes

A correlation between film structures and the width of the potential windows defined by the dihydrogen and dioxygen evolutions in aqueous media at nitrogen-doped amorphous carbon thin film electrodes prepared using a filtered cathodic vacuum arc system is reported. A range of film structures were obtained by changing the nitrogen mass flow rate during deposition, and the film structures were determined using electron energy-loss spectroscopy. For the film electrodes, the width of the potential windows in 0.1 M NaOH and in 0.1 M H₂SO₄ at a current density of 100 μA/cm² exceed those for glassy carbon electrodes, and increase with an increase in the sp³C fraction in the a-C:N materials. A film electrode with a rich sp²C content, has a lower electrical resistance, but still possesses a relatively large potential window. These features combined allow the materials to be tailored for particular electroanalysis