Improved Multiprotein Microcontact Printing on Plasma Immersion Ion Implanted Polystyrene.

Multiprotein micropatterning allows the creation of complex, controlled microenvironments for single cells that can be used for the study of the localized effects of various proteins and signals on cell survival, development, and functions. To enable analysis of cell interactions with microprinted proteins, the multiprotein micropattern must have low cross-contamination and high long-term stability in a cell culture medium. To achieve this, we employed an optimized plasma ion immersion implantation (PIII) treatment to provide polystyrene (PS) with the ability to covalently immobilize proteins on contact while retaining sufficient transparency and suitable surface properties for contact printing and retention of protein activity. The quality and long-term stability of the micropatterns on untreated and PIII treated PS were compared with those on glass using confocal microscopy. The protein micropattern on the PIII treated PS was more uniform and had a significantly higher contrast that was not affected by long-term incubation in cell culture media because the proteins were covalently bonded to PIII treated PS. The immunostaining of mouse pancreatic ß cells interacting with E-cadherin and fibronectin striped surfaces showed phosphorylated paxillin concentrated on cell edges over the fibronectin stripes. This indicates that multiprotein micropatterns printed on PIII treated PS can be used for high-resolution studies of local influence on cell morphology and protein production.

Hybrid Carbon-Based Nanostructured Platforms for the Advanced Bioreactors.

Mankind faces several global challenges such as chronic and acute hunger, global poverty, energy deficiency and environment conservation. Common biotechnologies based on batch, fluidbed and other similar processes are now extensively used for the production of a wide range of products such as antibiotics, biofuels, cultured and fermented food products. Unfortunately, these processes suffer from low efficiency, high energy demand, low controllability and rapid biocatalyst degradation by microbiological attack, and thus still are not capable of seriously addressing the global hunger and energy deficiency challenges. Moreover, sustainable future technologies require minimizing the environmental impact of toxic by-products by implementing the "life produces organic matter, organic matter sustains life" principle. Nanostructure-based biotechnology is one of the most promising approaches that can help to solve these challenges. In this work we briefly review the unique features of the carbon-based nanostructured platforms, with some attention paid to other nanomaterials. We discuss the main building blocks and processes to design and fabricate novel platforms, with a focus on dense arrays of the vertically-aligned nanostructures, mainly carbon nanotubes and graphene. Advantages and disadvantages of these systems are considered.

Orientation and conformation of anti-CD34 antibody immobilised on untreated and plasma treated polycarbonate.

The conformation and orientation of proteins immobilised on synthetic materials determine their ability to bind their antigens and thereby the sensitivity of the microarrays and biosensors employing them. Plasma immersion ion implantation (PIII) of polymers significantly increases both their wettability and protein binding capacity. This paper addresses the hypothesis that a PIII treated polymer surface modifies the native protein conformation less significantly than a more hydrophobic untreated surface and that the differences in surface properties also affect the protein orientation. To prove this, the orientation and conformation of rat anti-mouse CD34 antibody immobilized on untreated and PIII treated polycarbonate (PC) were investigated using ToF-SIMS and FTIR-ATR spectroscopy. Analysis of the primary structure of anti-CD34 antibody and principal component analysis of ToF-SIMS data were applied to detect the difference in the orientation of the antibody attached to untreated and PIII treated PC. The difference in the antibody conformation was analysed using deconvolution of the Amide I peak (in FTIR-ATR spectra) and curve-fitting. It was found that compared to the PIII treated sample, the antibody immobilized on the untreated PC sample has a secondary structure with a lower fraction of ß-sheets and a higher fraction of α-helices and disordered fragments. Also, it was found that anti-CD34 antibody has a higher tendency to occur in the inactive 'tail-up' orientation when immobilized on an untreated PC surface than on a PIII treated surface. These findings confirm the above hypothesis.

Effective AC needleless and collectorless electrospinning for yarn production.

Nanofibrous materials are essential components for a wide range of applications, particularly in the fields of medicine and material engineering. These include protective materials, sensors, cosmetics, hygiene, filtration and energy storage. The most widely used and researched technology in these fields is electrospinning. This method for producing fibers yields highly promising results thanks to its versatility and simplicity. Electrospinning is employed in multiple forms, among which needle and needleless direct current (DC) variants are the most distinctive. The former is based on the generation of just one single jet from a nozzle; hence this fabrication process is not very productive. The latter uses the destabilization of free liquid surfaces by means of an electric field, which enhances the throughput since it produces numerous jets, emitted from the surfaces of rollers, spheres, strings and spirals. However, although some progress in total producibility has been achieved, the efficiency of the DC method still remains relatively low. A further drawback of DC electrospinning is that both variants need a collector, which makes it difficult to combine DC electrospinning easily with other technologies due to the presence of the high field strength within the entire spinning zone. This paper describes our experiments with AC electrospinning. We show that alternating current (AC) electrospinning based on a needleless spinning-electrode provides a highly productive smoke-like aerogel composed of nanofibers. This aerogel rises rapidly from the electrode like a thin plume of smoke, without any need for a collector. Our work shows that AC needleless electrospinning gains its efficiency and collector-less feature thanks to the creation of a perpetually charge-changing virtual counter-electrode composed of the nanofibers emitted. High-speed camera recordings demonstrate the formation mechanism of the nanofibrous plume, which is wafted by an electric wind. This wind's velocity field is experimentally investigated. One potential use of AC needleless electrospinning is demonstrated here by spinning it into a yarn.

Cluster of differentiation antibody microarrays on plasma immersion ion implanted polycarbonate.

Plasma immersion ion implantation (PIII) modifies the surface properties of polymers, enabling them to covalently immobilize proteins without using linker chemistry. We describe the use of PIII treated polycarbonate (PC) slides as a novel platform for producing microarrays of cluster of differentiation (CD) antibodies. We compare their performance to identical antibody microarrays printed on nitrocellulose-coated glass slides that are currently the industry standard. Populations of leukocytes are applied to the CD microarrays and unbound cells are removed revealing patterns of differentially immobilized cells that are detected in a simple label-free approach by scanning the slides with visible light. Intra-slide and inter-slide reproducibility, densities of bound cells, and limits of detection were determined. Compared to the nitrocellulose-coated glass slides, PIII treated PC slides have a lower background noise, better sensitivity, and comparable or better reproducibility. They require three-fold lower antibody concentrations to yield equivalent signal strength, resulting in significant reductions in production cost. The improved transparency of PIII treated PC in the near-UV and visible wavelengths combined with superior immobilization of biomolecules makes them an attractive platform for a wide range of microarray applications.

Biointerface: protein enhanced stem cells binding to implant surface.

The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment.

Comment on 'Shear stiffness in nanolaminar Ti3SiC2 challenges ab initio calculations'.

In a recent publication by Kisi et al (2010 J. Phys.: Condens. Matter 22 162202) the authors present experimentally measured elastic constants for the M(n + 1)AX(n) (MAX) phase, Ti(3)SiC(2), that differ from density functional theory calculations. They then conclude that 'prediction [by ab initio calculation] of the full elasticity tensor for Ti(3)SiC(2) has not been successful'. However the authors do not compare with previous experimental work in which Finkel et al measure the elastic moduli (Finkel et al 2000 J. Appl. Phys. 87 1701). The predictions of ab initio calculations (Yu et al 2005 J. Mater. Res. 20 1180) agree with the measurements of Finkel et al as well as with most other experimentally measured elastic moduli for MAX phases (Cover et al 2008 10 935; Cover et al 2009 J. Phys.: Condens. Matter 21 305403). The unrealistically high value of the C(44) constant obtained by Kisi et al, which would mean that Ti(3)SiC(2) is almost as resistant to shear as diamond, undermines confidence in their results.

Fizeau interferometer system for fast high resolution studies of spectral line shapes.

A monochromator∕Fizeau interferometer∕intensified CCD camera system is described that was developed for the measurement of the shape of spectral lines that are rapidly time varying. The most important operating parameter that determines the performance of the instrument is the size of the entrance aperture as this determines both the light throughput and the effective interferometer wavelength resolution. This paper discusses, both theoretically and experimentally, the effect of the finite source area on the instrumental resolution to assist in optimizing the choice of this parameter. A second effect that often produces a practical limit to the quality of the spectra is drift of the interferometer plates. Measurements of the shapes of spectral lines of ions and atoms ejected from the cathode spot of continuous and pulsed cathodic arcs are presented to demonstrate the utility of this instrument.

A comparison of covalent immobilization and physical adsorption of a cellulase enzyme mixture.

This paper reports the first use of a linker-free covalent approach for immobilizing an enzyme mixture. Adsorption from a mixture is difficult to control due to varying kinetics of adsorption, variations in the degree of unfolding and competitive binding effects. We show that surface activation by plasma immersion ion implantation (PIII) produces a mildly hydrophilic surface that covalently couples to protein molecules and avoids these issues, allowing the attachment of a uniform monolayer from a cellulase enzyme mixture. Atomic force microscopy (AFM) showed that the surface layer of the physically adsorbed cellulase layer on the mildly hydrophobic surface (without PIII) consisted of aggregated enzymes that changed conformation with incubation time. The evolution observed is consistent with the existence of transient complexes previously postulated to explain the long time constants for competitive displacement effects in adsorption from enzyme mixtures. AFM indicated that the covalently coupled bound layer to the PIII-treated surface consisted of a stable monolayer without enzyme aggregates, and became a double layer at longer incubation times. Light scattering analysis showed no indication of aggregates in the solution at room temperature, which indicates that the surface without PIII-treatment induced enzyme aggregation. A model for the attachment process of a protein mixture that includes the adsorption kinetics for both surfaces is presented.

van der Pauw method for measuring resistivity of a plane sample with distant boundaries.

In the paper, we derive an algorithm which follows from the original van der Pauw's technique for measuring resistivity with the added advantage of allowing contacts to be positioned a distance away from the boundary. For a large sample area, we show that the resistivity calculated by our algorithm is equivalent to the resistivity calculated by the original van der Pauw's method. In practice, this configuration is easier to achieve and can eliminate errors associated with contacts that are not placed exactly at the edge.

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy.

Alterations in dihydropyrimidine dehydrogenase gene (DPYD) coding for the key enzyme (DPD) of fluoropyrimidines (FPs) catabolism contribute to the development of serious FPs-related toxicity. We performed mutation analysis of DPYD based on cDNA sequencing in 76 predominantly colorectal cancer patients treated by FPs with early development of high (grade 3-4) hematological and/or gastrointestinal toxicity. Six previously described [85T>C (C29R), 496A>G (M166V), 775A>G (K259E), 1601G>A (S534N), 1627A>G (I543V), IVS14+1G>A, 2194G>A (V732I)] and two novel [187A>G (K63E) and 1050 G>A (R357H)] non-synonymous DPYD variants were found in 56/76 (73.7%) high-toxicity patients. Subsequently, these alterations were analyzed in 48 patients with excellent long-term tolerance of FPs and in 243 controls and were detected in 37/48 (77.1%) and 166/243 (68.3%) cases, respectively. Analysis of these alterations as risk factors for development of toxicity in pooled FPs-treated population demonstrated that C29R negatively correlated with overall gastrointestinal toxicity (OR = 0.48; 95%CI 0.23-1.0) and M166V in women protected against overall hematological toxicity and neutropenia (both OR = 0.26; 95%CI 0.07-0.89), whereas IVS14+1G>A (found in five high-toxicity patients only) increased risk of mucositis in overall population (OR = 7.0; 95%CI 1.1-44.53), and thrombocytopenia in women (OR = 10.8; 95%CI 1.24-93.98). Moreover, we identified a strong association of V732I with leucopenia (OR = 8.17; 95%CI 2.44 - 27.31) and neutropenia (OR=2.78; 95% CI 1.03-7.51). Our data enabled characterization of "high risk" haplotypes (carriers of IVS14+1G>A or V732 lacking M166V) representing small (22% female and 11% male patients), population in high risk of serious hematological toxicity development, and in patients with "lower risk" that unlikely develop serious hematological toxicity [carriers of M166V without IVS14+1G>A and V732I in females (32% women), and non-carriers of C29R, M166V, IVS14+1G>A, and V732I in males (46% men)]. Our results indicate that genotyping of several DPYD variants may lead to stratification of patients with respect to the risk of serious hematological toxicity development during FPs treatment.

Enhancing the hardness of Al/W nanostructured coatings.

Two-component multilayer thin films frequently show hardness enhancements at specific repeat periods above that of the constituent layers. This study of hardness enhancements in W/Al nanostructured coatings provides strong new evidence that hardness enhancements in this system arise not only from the presence of a layered structure, but also from the presence of defects introduced by changing the deposition conditions. Samples with well defined layers of W and Al were produced by sputtering to cover a wide range of periods from 10 to 200 nm. No evidence of enhanced hardness in these films was found by nanoindentation. On the other hand, samples deposited from cathodic arc sources showed strong hardness enhancement above that of pure W. However, the samples of highest hardness did not contain Al layers for much of their thickness. The hardening mechanism therefore could not be attributed to the presence of a multilayer structure. Examination of the microstructure showed that the interruptions to the W deposition caused by operation of the Al source introduced defects which acted as pinning sites for dislocations. The nanoindentation hardness data were well described using a modified Hall-Petch relation.

A comprehensive survey of M(2)AX phase elastic properties.

M(2)AX phases are a family of nanolaminate, ternary alloys that are composed of slabs of transition metal carbide or nitride (M(2)X) separated by single atomic layers of a main group element. In this combination, they manifest many of the beneficial properties of both ceramic and metallic compounds, making them attractive for many technological applications. We report here the results of a large scale computational survey of the elastic properties of all 240 elemental combinations using first-principles density functional theory calculations. We found correlations revealing the governing role of the A element and its interaction with the M element on the c axis compressibility and shearability of the material. The role of the X element is relatively minor, with the strongest effect seen in the in-plane constants C(11) and C(12). We identify several elemental compositions with extremal properties such as W(2)SnC, which has by far the lowest value of C(44), suggesting potential applications as a high-temperature dry lubricant.

Dielectric functions of a growing silver film determined using dynamic in situ spectroscopic ellipsometry.

The dielectric functions of plasma deposited silver on SiO2 through all stages of Volmer-Weber growth at room temperature and 150 degrees C were determined unambiguously by applying a model-independent inversion method to dynamic in situ spectroscopic ellipsometric data. The results show large differences in the localized plasmon resonance and the percolation threshold at the two temperatures. Using these model-independent dielectric functions we assess the effectiveness of modelling the plasmon resonance by fitting a Lorentz oscillator. The methods show agreement for the position of the plasmon resonance below the percolation threshold and for the effective film thickness up to 5.6 nm at room temperature and 11.5 nm at 150 degrees C, however the line shape of the resonance is described by the Lorentzian only in the early stages of film growth.

Plasma immersion ion implantation treatment of polyethylene for enhanced binding of active horseradish peroxidase.

Robust attachment of active proteins to synthetic surfaces underpins the development of biosensors and protein arrays. This paper presents the results of experiments in which energetic ions, extracted from an inductively coupled nitrogen plasma, are used to modify the surface of ultrahigh molecular weight polyethylene (UHMWPE). The ability of the surface to bind active horseradish peroxidase (HRP) is significantly enhanced by the plasma treatment. The amide signal in infrared spectroscopy indicates an increased quantity of surface-attached protein on the modified surface. The activity of the bound HRP remains high compared with that of protein attached to the untreated surface, after repeated washing in buffer solution. Although Tween 20 was an effective blocking agent for the unmodified polyethylene surface, binding of HRP to the modified surface is not inhibited by its presence. We propose that the treatment produces new binding sites on the surface and that the function of the HRP is retained because the treated surface is substantially more hydrophilic.

Amorphous and crystalline phases in thermal quench simulations of alumina.

The authors report molecular dynamics simulations of alumina (Al2O3) during crystallization from the melt. Using liquid quench methods, they investigate the effect of cooling rate on the structural evolution of the alpha, kappa, and the bixbyite phases. A critical temperature window is identified, where the time spent in this window is crucial in determining the extent to which the systems approach crystallinity. A strong dependence is observed between the final structure and the quench rate, which is most pronounced for the alpha phase and to lesser extent for the other phases. The results show that the different phases have different tendencies to crystallize that are determined by energetics, complexity of crystal structure, and the number of metastable states.

Comparison of protein surface attachment on untreated and plasma immersion ion implantation treated polystyrene: protein islands and carpet.

Surface attachment of the enzyme horseradish peroxidase (HRP) was studied on untreated and ion beam implanted polystyrene (PS) films. The PS films of 100 nm thickness on a silicon wafer were treated using the plasma immersion ion implantation (PIII) technique, with argon ions of energy 20 keV and fluences of up to 2 x 10(16) ions/cm2. Differential transmittance Fourier transform infrared (FTIR) spectra confirmed the presence of proteins on the PS surfaces by detection of the amide A, I, and II protein vibrations. Spectroscopic ellipsometry over the UV-vis spectral region provided the optical constants and thickness of the protein layer, while tapping mode atomic force microscopy (AFM) was used to image the protein distribution on the surface. The combination of AFM, ellipsometry, and FTIR analysis showed that, on the untreated PS surface, HRP formed islands 8 nm in height and 30 nm in lateral size, covering approximately 27% of the PS surface. After PIII modification of the PS surface, the protein covered 100% of the surface area.

Dynamic spectroscopic ellipsometry determination of nanostructural changes in plasmonic silver films.

Dynamic in situ spectroscopic ellipsometry is used to probe post-deposition nano-structural changes in silver films at room temperature in the pre- and post-coalescence stages of Volmer-Weber growth. In the island growth phase the Maxwell-Garnett theory is used to determine structural changes in the island film. Changes in the plasmon resonance frequency indicate an increased distance between islands which explain pre-coalescence resistivity changes. Post-coalescence changes in the resistivity are determined to be due to grain growth. A reduction in film thickness of 0.2 - 0.3 nm is also observed. The results are used to evaluate recent competing theories based on in situ stress measurements.

Real-time verification of HDR brachytherapy source location: implementation of detector redundancy.

Independent treatment verification for high dose rate (HDR) brachytherapy is needed to ensure that the treatment proceeds as prescribed. In this paper, we investigate the feasibility of a proposed real-time source position verification process. This process provides immediate confirmation of the source position during the treatment, so that the treatment can be aborted and modified if necessary. We show that an array of dosimeters placed on the patient's skin can independently verify the position in three dimensions. This verification was demonstrated by using a diamond detector placed in several locations on the surface of an anthropomorphic phantom. A mathematical algorithm was constructed to estimate the location of the source given a measured data set in the presence of tissue heterogeneity. The accuracy of the source localization was found to increase with the number of detectors used to compute the estimation of the source position. The resolution to which the 12 detectors can identify the location of the source was within 3 mm.