Detection Of Sepsis Biomarkers Using Microfluidics

Abstract

Sepsis is a “life-threatening organ dysfunction caused by a dysregulated host response to infection” that has a widespread impact on human life around the world. It affects more than 1.5 million people, killing at least 250,000 each year in the US alone and affects 90,000 people annually, with estimated mortality rates of up to 30% in Canada. Our understanding of the different biochemical pathways that in the progression of sepsis has improved patient care for sepsis patients. One part of patient care is the use of biomarkers for patient prognosis that draws on the full range of relevant and available information to model the possible outcomes for an individual. Numerous biomarkers have been studied for patient prognosis that includes Procalcitonin (PCT), C-reactive protein (CRP), TNF-α, cfDNA, protein C and PAI 1. Using a panel of multiple biomarkers provided more accuracy in patient prognosis than using individual biomarkers and one such panel that was proposed used cfDNA, protein C, platelet count, creatinine, Glasgow Coma Scale [GCS] score, and lactate. Commercial, low cost POC techniques were available for the measurement of all biomarkers besides cfDNA and protein C. The objective of this doctoral thesis was chosen to develop low cost, microfluidic devices for the measurement of protein C and cfDNA using nonspecific fluorescence dyes that would enable the eventual integration of the systems and improve patient prognosis. The measurement of protein C in plasma required the separation of protein C from interfering proteins in plasma. This was done through the development of a two-stage separation process that included the development of tunable agarose isoelectric gates for separating proteins using their isoelectric point and the miniaturization of immobilized metal affinity chromatography and its extension to Barium for the selective binding of proteins using their chemical affinity. This was performed in a xurographically fabricated chip to reduce costs and enable the use of geometric focusing of the electric field to enable the operation of the device at a lower applied voltage. The challenges faced with cfDNA were different due to the different characteristics of the material and less interference from plasma. The requirement was to measure the total cfDNA content with minimal cost in comparison to currently available techniques. This was achieved through the development of thread microfluidic devices that showed the use of thread for automated aliquoting of samples by controlling length and twists of the thread. Preconcentration and use of external apparatus was avoided by showing that thread could be used to amplify fluorescence response to a range that was sufficient for the measurement of cfDNA in sepsis patients. A portable fluorescence imaging setup was developed for this purpose and was used in demonstration for the measurement of cfDNA in plasma with sufficient resolution. In conclusion, we developed technologies for rapid and low-cost measurement of protein C and cfDNA using xurographic and thread-based microfluidics that may serve as valuable in improving patient prognosis.