Estimation of Bound Carbonyls in PMMA-r-PS Copolymers Adsorbed on Silica

The adsorption of random copolymers on solid surfaces is essential for developing good properties in composite materials. A good control and understanding of interfaces allows tuning of the properties of the individual components. Interfacial polymers are important in many industrial applications such as photoresists, protective coatings, metal insulator and semi-conductor insulator junctions, metal-filled polymer composites and polymer-lined metal containers for protective food packaging. The effect of the drying process,1 configuration and tacticity,2 molecular mass, density of surface hydroxyls,3 and adsorbed amounts4 have already been studied for interfacial polymers. The thermal characterization for polystyrene,5 PMMA11 and PMMA-r- PS6 copolymers adsorbed on silica have been studied recently by our research group. One of the important considerations for adsorbed copolymer composites was the composition of the copolymer and the individual surface affinities of monomer units on the substrate. The dependence of chain length, blockiness and self-association of the chain was also a consideration for these composites.7 Bound carbonyls are the ones which hydrogen bond directly with surface hydroxyls that, in our case, are the hydroxyls on silica. Transmission FTIR is one of the simplest techniques used to estimate the number of bound carbonyls. It was shown that the bound fractions decreased with increased adsorbed amounts while molecular weights8 indicated a flattened configuration for lower adsorbed amounts and shorter chains. However, the dependence of bound fractions on molecular mass9 was dramatically less

Enhancing the resolution of micro free flow electrophoresis through spatially controlled sample injection

Free flow electrophoresis is a versatile technique for the continuous separation of mixtures with both preparative and analytical applications. Microscale versions of free flow electrophoresis are particularly attractive strategies because of their fast separation times, ability to work with small sample volumes and large surface area to volume ratios facilitating rapid heat transfer, thus minimising the detrimental effects of Joule heating even at high voltages. The resolution of microscale free flow electrophoresis, however, is limited by the broadening of the analyte beam in the microfluidic channel - an effect that becomes especially pronounced when the analyte is deflected significantly away from its original position. Here we describe and demonstrate how by spatially restricting the sample injection and collection to the regions where the gradients in the velocity distribution of the carrier medium are the smallest, this broadening effect can be substantially suppressed and hence the resolution of microscale free flow electrophoresis devices increased. To demonstrate this concept we fabricated microfluidic free flow electrophoresis devices with spatially restricted injection nozzles implemented via the use of multilayer soft-photolithography and further integrated quartz based observation areas for fluorescent detection and imaging. With these devices we demonstrated a five fold reduction in the beam broadening extent compared to conventional free flow electrophoresis approaches with non-restricted sample introduction. The manifold enhancement in the achievable resolution of microscale free flow electrophoresis devices opens up the possibility of rapid separation and analysis of more complex mixtures

Scalable integration of nano-, and microfluidics with hybrid two-photon lithography

Abstract: Nanofluidic devices have great potential for applications in areas ranging from renewable energy to human health. A crucial requirement for the successful operation of nanofluidic devices is the ability to interface them in a scalable manner with the outside world. Here, we demonstrate a hybrid two photon nanolithography approach interfaced with conventional mask whole-wafer UV-photolithography to generate master wafers for the fabrication of integrated micro and nanofluidic devices. Using this approach we demonstrate the fabrication of molds from SU-8 photoresist with nanofluidic features down to 230 nm lateral width and channel heights from micron to sub-100 nm. Scanning electron microscopy and atomic force microscopy were used to characterize the printing capabilities of the system and show the integration of nanofluidic channels into an existing microfluidic chip design. The functionality of the devices was demonstrated through super-resolution microscopy, allowing the observation of features below the diffraction limit of light produced using our approach. Single molecule localization of diffusing dye molecules verified the successful imprint of nanochannels and the spatial confinement of molecules to 200 nm across the nanochannel molded from the master wafer. This approach integrates readily with current microfluidic fabrication methods and allows the combination of microfluidic devices with locally two-photon-written nano-sized functionalities, enabling rapid nanofluidic device fabrication and enhancement of existing microfluidic device architectures with nanofluidic features

Rapid two-dimensional characterisation of proteins in solution

Abstract: Microfluidic platforms provide an excellent basis for working with heterogeneous samples and separating biomolecular components at high throughput, with high recovery rates and by using only very small sample volumes. To date, several micron scale platforms with preparative capabilities have been demonstrated. Here we describe and demonstrate a microfluidic device that brings preparative and analytical operations together onto a single chip and thereby allows the acquisition of multidimensional information. We achieve this objective by using a free-flow electrophoretic separation approach that directs fractions of sample into an on-chip analysis unit, where the fractions are characterised through a microfluidic diffusional sizing process. This combined approach therefore allows simultaneously quantifying the sizes and the charges of components in heterogenous mixtures. We illustrate the power of the platform by describing the size distribution of a mixture comprising components which are close in size and cannot be identified as individual components using state-of-the-art solution sizing techniques on their own. Furthermore, we show that the platform can be used for two-dimensional fingerprinting of heterogeneous protein mixtures within tens of seconds, opening up a possibility to obtain multiparameter data on biomolecular systems on a minute timescale

Accelerating Reaction Rates of Biomolecules by Using Shear Stress in Artificial Capillary Systems.

Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Biotechnology and Biological Sciences Research CouncilFunder: Centre for Misfolding Diseases, University of CambridgeFunder: Wellcome TrustBiomimetics is a design principle within chemistry, biology, and engineering, but chemistry biomimetic approaches have been generally limited to emulating nature's chemical toolkit while emulation of nature's physical toolkit has remained largely unexplored. To begin to explore this, we designed biophysically mimetic microfluidic reactors with characteristic length scales and shear stresses observed within capillaries. We modeled the effect of shear with molecular dynamics studies and showed that this induces specific normally buried residues to become solvent accessible. We then showed using kinetics experiments that rates of reaction of these specific residues in fact increase in a shear-dependent fashion. We applied our results in the creation of a new microfluidic approach for the multidimensional study of cysteine biomarkers. Finally, we used our approach to establish dissociation of the therapeutic antibody trastuzumab in a reducing environment. Our results have implications for the efficacy of existing therapeutic antibodies in blood plasma as well as suggesting in general that biophysically mimetic chemistry is exploited in biology and should be explored as a research area

Resolving protein mixtures using microfluidic diffusional sizing combined with synchrotron radiation circular dichroism

Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer’s and Parkinson’s diseases

Massively parallel C. elegans tracking provides multi-dimensional fingerprints for phenotypic discovery.

BACKGROUND: The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan. Manual assays, however, are time consuming and limited in their scope providing a strong motivation for automation. NEW METHOD: We describe the development and application of an advanced machine vision system for characterising the behaviour of C. elegans, the Wide Field-of-View Nematode Tracking Platform (WF-NTP), which enables massively parallel data acquisition and automated multi-parameter behavioural profiling of thousands of worms simultaneously. RESULTS: We screened more than a million worms from several established models of neurodegenerative disorders and characterised the effects of potential therapeutic molecules for Alzheimer's and Parkinson's diseases. By using very large numbers of animals we show that the sensitivity and reproducibility of behavioural assays is very greatly increased. The results reveal the ability of this platform to detect even subtle phenotypes. COMPARISON WITH EXISTING METHODS: The WF-NTP method has substantially greater capacity compared to current automated platforms that typically either focus on characterising single worms at high resolution or tracking the properties of populations of less than 50 animals. CONCLUSIONS: The WF-NTP extends significantly the power of existing automated platforms by combining enhanced optical imaging techniques with an advanced software platform. We anticipate that this approach will further extend the scope and utility of C. elegans as a model organism

Multistep Inhibition of α-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine.

The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders