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abstract: Demand for biosensor research applications is growing steadily. According to a new report by Frost & Sullivan, the biosensor market is expected to reach $14.42 billion by 2016. Clinical diagnostic applications continue to be the largest market for biosensors, and this demand is likely to continue through 2016 and beyond. Biosensor technology for use in clinical diagnostics, however, requires translational research that moves bench science and theoretical knowledge toward marketable products. Despite the high volume of academic research to date, only a handful of biomedical devices have become viable commercial applications. Academic research must increase its focus on practical uses for biosensors. This dissertation is an example of this increased focus, and discusses work to advance microfluidic-based protein biosensor technologies for practical use in clinical diagnostics. Four areas of work are discussed: The first involved work to develop reusable/reconfigurable biosensors that are useful in applications like biochemical science and analytical chemistry that require detailed sensor calibration. This work resulted in a prototype sensor and an in-situ electrochemical surface regeneration technique that can be used to produce microfluidic-based reusable biosensors. The second area of work looked at non-specific adsorption (NSA) of biomolecules, which is a persistent challenge in conventional microfluidic biosensors. The results of this work produced design methods that reduce the NSA. The third area of work involved a novel microfluidic sensing platform that was designed to detect target biomarkers using competitive protein adsorption. This technique uses physical adsorption of proteins to a surface rather than complex and time-consuming immobilization procedures. This method enabled us to selectively detect a thyroid cancer biomarker, thyroglobulin, in a controlled-proteins cocktail and a cardiovascular biomarker, fibrinogen, in undiluted human serum. The fourth area of work involved expanding the technique to produce a unique protein identification method; Pattern-recognition. A sample mixture of proteins generates a distinctive composite pattern upon interaction with a sensing platform consisting of multiple surfaces whereby each surface consists of a distinct type of protein pre-adsorbed on the surface. The utility of the "pattern-recognition" sensing mechanism was then verified via recognition of a particular biomarker, C-reactive protein, in the cocktail sample mixture. / Dissertation/Thesis / Ph.D. Electrical Engineering 2011

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This dissertation describes the elucidation and implementation of derivatisation in the quantification of biologically active vitamin D metabolites in limited volume serum and dry blood spot samples (DBS) using the liquid chromatography tandem-mass spectrometry (LC-MS/MS) analytical technique. This manuscript describes in detail the development and validation of an analytical methodology, highlighting the role derivatisation and mass spectrometry plays in the structural characterisation and quantification of vitamin D metabolites. The first chapter reviews comprehensively, the history of vitamin D biosynthesis discovery as an anti-rickets agent, the biochemistry of vitamin D, its metabolic pathway, functions in the different biological systems and the consequences of its deficiency in the body. The second chapter reviews the current methods and techniques utilised for the detection and characterization of vitamin D metabolites, with specific emphasis based on the contribution made by derivatisation and mass spectrometry. A brief introduction to derivatisation is provided, with specific focus on PTAD and Amplifex diene reagents (Cooksontype reagents) used in this study. The importance of sensitivity and selectivity of targeted analytes is described first in detail for underivatised analytes, followed by PTAD and Amplifex derivatised samples. Chapter 2 also describes the importance of vitamin D quantification using liquid chromatography, the strengths and limitations of LC-MS/MS when used in isolation and after derivatisation. Also discussed, is how combining these techniques can overcome inherent limitations in LCMS/MS and enhance analytical performance. In Chapter 3 the materials and methods used and the study design is laid out, describing a brief introduction of the routinely used clinical diagnostics assay enzyme-linked immunosorbent assay (ELISA) as a reference method and is compared to an LC-MS/MS assay, to ascertain discrepancies and agreement between both methodologies from the same volunteer samples. Chapters 3 and 4 describes the comprehensive development, optimisation and validation of the highly sensitive PTAD derivatives LC-MS/MS assay for the quantification of active vitamin D metabolites, as well as the development of method using Amplifex diene derivatisation. Also discussed, is sample preparation optimisation of DBS and Mitra micro-samples. A holistic approach was taken to the development of the methodologies to provide data from which the required analytical information can be obtained for method evaluation and statistical analysis. The validated PTAD derivatives method is applied to the quantification of vitamin D metabolites in limited volume (100 μL) clinical human serum samples from 30 volunteers compared to results obtained using the clinical diagnostics ELISA technique. In Chapter 4 data analysis is described and the results are further discussed and a conclusion made based on the findings from the study. This study envisaged that combination of limited sample volume and DBS, derivatisation and LCMS/ MS is a powerful tool in vitamin D metabolite analysis and provided evidence of a positive increase in sensitivity and selectivity between derivatised compared to underivatised samples. A 10-fold increase in signal-to-noise-ratio (S/N) was observed when comparing PTAD derivatised, and Amplifex diene derivatised versus underivatised samples. Chapter 5 presents suggested future directions and considerations in the areas of vitamin D metabolite derivatisation and DBS sampling technique analysis using LC-MS/MS research based on the results presented in this dissertation. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates. Furthermore, the utility of MDA products in comparative genomic hybridisation (CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities.

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Title: Obesity monitoring in middle aged men in Hradec Kralove Targets: The targets of thesis is to determine prevalence of the obesity in men at age 30 - 50 years in Hradec Kralove. The necessary data for this empirical research will be obtained on the basis of the questionnaires distributed among 30 active athletes and 30 pacients of prof. Martinik's diabetology office, who suffer from obesity and undergo treatment in his office. Subsequently, I will perform a deep analysis of all the data obtained from the questionnaries, in order to identify hazard factors for obesity, stress management, physical aktivity, fixed daily routine and life management or the prevalence of the genetic load in the group of surveyed athletes and surveyed obese patients of prof. Martiník's diabetology office. These data will be then compared in order to identifily the main differences between active athletes and obese patients. Methods: The empirical research was conducted at 30 randomly selected active athletes (at age 30 - 50 years), who live in Hradec Kralove and at 30 random patients (also at age 30 - 50 years) of prof. Martiník, who also live in Hradec Kralove and undergo medical treatment on the basis of the questionnaire, which I created myself and filled personally with the patients and athletes in order to...