DEVELOPMENT OF MICROCHIP ISOTACHOPHORESIS FOR THE DETECTION OF NOVEL CARDIAC TROPONIN I BIOMARKERS
Jacroux, Tom Jacroux
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The emphasis of this dissertation consists of three major parts in relation to improving nucleic acid detection strategies and detection of novel cardiac disease protein biomarkers using microchip isotachophoresis (ITP) and immunoaffinity monoliths. The dissertation is split amongst six different chapters. The first chapter outlines the dissertation while also introducing nucleic acid detection systems that utilize molecular beacons and Enzymatic-Assisted Target Recycling (EATR) to improve current molecular beacon design. It also introduces the electrophoresis technique ITP and immunoaffinity monolith, and to preconcentrate and detect significant cardiac biomarkers such as unphosphorylated (cTnI) and phosphorylated cardiac troponin I (pcTnI). Chapter 2 looks into the development of a rapid DNA detection assay. This assay is operable under isothermal or non-isothermal conditions is wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). The second part (Chapters 3 and 4) of this dissertation looks into the development of microchip isotachophoresis (ITP), a widely used electrophoresis technique to separate, preconcentrate, and quantify protein cardiac biomarkers from abundant serum proteins and in complex mediums such as human serum. Chapter 3 illustrates how cationic ITP can be used to separate and preconcentrate cTnI from two different protein contaminants, R-Phycoerythrin (PE) and a high abundant serum protein, human serum albumin. In addition, we show how a 1D COMSOL numerical model is used to confirm our results. Chapter 4 looks into using a cascade poly (methylmethacrylate) (PMMA) microchip with reductions in cross-sectional area in conjunction with cationic ITP to preconcentrate fluorescently labeled cTnI and pcTnI in human serum samples. This technique is able to preconcencrate cTnI isoforms by almost 104. Chapter 5 looks into using a hydrophilic monolith in a simple PMMA microchannel to perform an on-chip immunoassay of both labeled and unlabeled cTnI and pcTnI. This monolith shows effective antibody attachment and also significantly reduced non-specific binding. Lastly, Chapter 6 concludes the research examined in the dissertation and comments on future work needed to improve the sensitivity of cTnI isoform diagnostic microchip assays for point-of-care diagnostics.