Development and Characterisation of High Surface Energy Microstructured Sol-gel Coatings for Sensing Applications

This study investigates the development of high surface energy photoreactive organic inorganic hybrid sol-gel coatings for the microstructuration of high-resolution microfluidic platforms and optofluidic biosensor platforms by standard photolithography processes. To achieve this, the first step of our work consisted of identifying the fundamental physico chemical processes governing the structuration and surface properties of hybrid organic inorganic sol-gel coatings. For this purpose, a reference material based on the combination of an organosilane (3-Methacryloxypropytrimethoxysilane, MAPTMS) and a transition metal (zirconium propoxide, ZPO), was firstly developed and characterised. It was highlighted that chemical, physical and combined physical and chemical processes can be performed to impact the structure, morphology and surface properties of hybrid sol-gel coatings. Therefore, our work progressed towards the investigations of chemical strategies that may impact the general properties of hybrid coatings, with a specific objective on the alteration of their surface properties. For this purpose, 3 strategies have identified including (1) to alter the content of transition metal, (2) to vary the hydrolysis degree and (3) to form core-shell nanoparticle by the surface functionalisation of the reference material during its preparation along with the curing process of the coatings. The materials were characterised employing a set of structural, thermal and surface characterisations techniques namely Contact Angle measurements (CA), DLS, DSC, FTIR, 29Si-NMR. Fundamentally, a triangular relationship between the wettability, the condensation and curing process of the coatings was taking place. More specifically, the wettability was governed by the occurrence of parallel and competitive hydroxylation and condensation processes of the coatings. Having performed the identified chemical strategies, our work has progressed towards the investigations of physical and physico-chemical treatments of the final coatings. Here, the effects of air-plasma, nitrogen-plasma and plasma treatments combined with post-silane ii surface functionalisation were performed and the durability of the treatments investigated. Although hydrophobic recovery was observed for all materials, it was found that air-plasma enabled to achieve the most stable surface properties due to the formation of hydrophilic hydroxyl groups at the surface of the coatings. The next step of the work focussed on the microstructuration fabrication of a microfluidic platform. The photolithography fabrication conditions were established to enable the successful preparation of well-defined microchannels with resolutions ranging from 50 to 500 microns. Having developed our microfluidic platform, our work concentrated on developing strategies to integrate an optical transducer onto the platform to enable the fabrication of an optofluidic device that may be applied as biosensor, thus demonstrating the potential of our technology for biosensing applications. The biosensor design we proposed consisted of integrated optical waveguides onto microfluidics that would also be fabricated employing a photolithography process. The fabrications conditions of the optofluidic platform were established by considering the required optical conditions that enable efficient light propagation in the waveguides, which can be used as an optical excitation to fluorophores located within sensor spots in the microchannels. The successful demonstration of concept of the optofluidic-based biosensor concept was successfully performed by recording optical emissions of biomolecules fluorophores under optical excitations with the optical waveguides integrated on the microfluidic platform. The work reported in this thesis has been multidisciplinary requiring chemistry, physics, biotechnology and engineering competencies which have been synergised for the development of the first “whole hybrid sol-gel optofluidic biosensor platform”. It is also showing the potential of the proposed technology for applications where functional microstructured coatings are required

The effect of curing and zirconium content on the wettability and structure of a silicate hybrid sol-gel material

Functional hybrid sol-gel coatings have been developed for numerous applications with a wide range of wettabilities. This study proposes to investigate the relationship between the structure and the wetting properties of a zirconium modified silicate hybrid sol-gel coating. The structures of the coatings were altered by varying the content of zirconium, and the curing process, while keeping the sol-gel preparation conditions identical. The structure of the materials was characterized by FTIR, 29Si NMR and SEM. The thermal properties and the wettability are identified by DSC and contact angle measurements, respectively. By corroborating the structural and wettability analyses, it is shown that the transition metal minimizes the thermally-assisted condensation of the silicate network and enables to stabilize the wetting properties at higher hydrophilic values. This article highlights that the wettability of hybrid sol-gel coatings can be controlled by both the curing process and transition metal content

Selection Signatures in Worldwide Sheep Populations

The diversity of populations in domestic species offers great opportunities to study genome response to selection. The recently published Sheep HapMap dataset is a great example of characterization of the world wide genetic diversity in sheep. In this study, we re-analyzed the Sheep HapMap dataset to identify selection signatures in worldwide sheep populations. Compared to previous analyses, we made use of statistical methods that (i) take account of the hierarchical structure of sheep populations, (ii) make use of linkage disequilibrium information and (iii) focus specifically on either recent or older selection signatures. We show that this allows pinpointing several new selection signatures in the sheep genome and distinguishing those related to modern breeding objectives and to earlier post-domestication constraints. The newly identified regions, together with the ones previously identified, reveal the extensive genome response to selection on morphology, color and adaptation to new environments

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep

Stefan Hiendleder is a member of the International Sheep Genomics ConsortiumIn sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal’s health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization–time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.Michael P. Heaton, Theodore S. Kalbfleisch, Dustin T. Petrik, Barry Simpson, James W. Kijas, Michael L. Clawson, Carol G. Chitko-McKown, Gregory P. Harhay, Kreg A. Leymaster, the International Sheep Genomics Consortiu

A Complete Mitochondrial Genome Sequence from a Mesolithic Wild Aurochs (Bos primigenius)

Background The derivation of domestic cattle from the extinct wild aurochs (Bos primigenius) has been well-documented by archaeological and genetic studies. Genetic studies point towards the Neolithic Near East as the centre of origin for Bos taurus, with some lines of evidence suggesting possible, albeit rare, genetic contributions from locally domesticated wild aurochsen across Eurasia. Inferences from these investigations have been based largely on the analysis of partial mitochondrial DNA sequences generated from modern animals, with limited sequence data from ancient aurochsen samples. Recent developments in DNA sequencing technologies, however, are affording new opportunities for the examination of genetic material retrieved from extinct species, providing new insight into their evolutionary history. Here we present DNA sequence analysis of the first complete mitochondrial genome (16,338 base pairs) from an archaeologically-verified and exceptionally-well preserved aurochs bone sample. Methodology DNA extracts were generated from an aurochs humerus bone sample recovered from a cave site located in Derbyshire, England and radiocarbon-dated to 6,738±68 calibrated years before present. These extracts were prepared for both Sanger and next generation DNA sequencing technologies (Illumina Genome Analyzer). In total, 289.9 megabases (22.48%) of the post-filtered DNA sequences generated using the Illumina Genome Analyzer from this sample mapped with confidence to the bovine genome. A consensus B. primigenius mitochondrial genome sequence was constructed and was analysed alongside all available complete bovine mitochondrial genome sequences. Conclusions For all nucleotide positions where both Sanger and Illumina Genome Analyzer sequencing methods gave high-confidence calls, no discrepancies were observed. Sequence analysis reveals evidence of heteroplasmy in this sample and places this mitochondrial genome sequence securely within a previously identified aurochsen haplogroup (haplogroup P), thus providing novel insights into pre-domestic patterns of variation. The high proportion of authentic, endogenous aurochs DNA preserved in this sample bodes well for future efforts to determine the complete genome sequence of a wild ancestor of domestic cattle