Computational analysis of the colonic transciptome & in vitro biomarker analysis using a novel microfluidic quantum dot linked immunoassay

Peter M. Clark

doi:10.17918/etd-3778

DNA microarray technology facilitates the high throughput analysis of transcriptional disease regulation by measuring the relative expression levels of transcripts present within a tissue. While such computational approaches have been used to study the genetic regulation of a variety of illnesses, such studies often suffer from inadequate patient sample sizes and statistical power resulting in conflicting results and lab-specific bias. In order to overcome these limitations and fully utilize the wealth of publicly available genomic data, an integrated microarray analysis method was used to analyze and interpret microarray data in the context of colonic diseases including colorectal carcinoma (CRC) and inflammatory bowel disease (IBD). The results of this work indicate widespread genetic perturbations related to IBD in which a variety of cell types are implicated including resident host enterocytes, innate and adaptive immune cells as well as native luminal microflora. Our work has identified subtle genetic differences between IBD phenotypes for the realization of disease specific therapeutic treatments as well as novel diagnostic biomarkers. Furthermore, our analysis has revealed significant overlap in the genetic regulation and predisposition to IBD, lupus, type 1 diabetes, graves disease and rheumatoid arthritis, providing the first genetic link between the enteropathic disease symptoms associated with IBD. Druggable pathways involved in these diseases as well as known therapeutic drug targets were also analyzed for the potential repositioning of existing therapeutics for the treatment of IBD. IBD patients are known to be at an elevated risk for developing colorectal carcinoma, with risk increasing with the duration of the disease. In order to better understand the phenotype shift from IBD to cancerous phenotypes, integrated microarray analysis was used to identify gene signatures, implicated pathways and novel discriminatory biomarkers for differentiating between IBD and CRC phenotypes. Our diagnostic panels were shown to accurately differentiate between phenotypes using an independent dataset for validation. In order to transition the identified biomarkers to the clinic for diagnostic use, a novel microfluidic quantum dot linked immunosorbent assay platform was developed with enhanced surface chemistry and reaction kinetics. The developed prototype has the capability of multiplexed biomarker detection within clinically relevant samples for the stratification of disease phenotypes. In order to validate our design, human samples spiked with the fecal IBD biomarker lactoferrin were analyzed. Results indicate increased sensitivity and signal to noise ratios over our predicate device, with a reduction of the limit of detection. This proof of concept device shows great promise as a portable bedside diagnostic device for multiplexed biomarker analysis within a clinical setting.

Computational analysis of the colonic transciptome & in vitro biomarker analysis using a novel microfluidic quantum dot linked immunoassay

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Computational analysis of the colonic transciptome & in vitro biomarker analysis using a novel microfluidic quantum dot linked immunoassay

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