Electrokinetic Detection of Sepsis Biomarkers in Dehydrated/Rehydrated Hydrogel

Abstract

According to the third international consensus definition (sepsis-3), sepsis is characterized as life-threatening organ dysfunction resulting from an uncontrolled host response to infection. Sepsis stands as a prominent contributor to worldwide mortality. A study revealed approximately 50 million reported cases of sepsis and 11 million associated deaths worldwide, constituting nearly 20% of all global fatalities. Various biomarkers have been investigated for sepsis prognosis including Procalcitonin (PCT), C-reactive protein (CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and protein C. In addition to proteomic markers genomic biomarkers have also been investigated for sepsis. For instance, research indicates a substantial rise in plasma cell-free DNA (cfDNA) and total circulating histones levels during sepsis, correlating with its severity and mortality. The complexity arises in creating a measurement tool for sepsis, given the diverse nature of these biomarkers, each requiring distinct detection methods. The objective of this doctoral thesis is to develop a low-cost fully integrated microfluidic device for detecting a genomic biomarker (cfDNA) and a proteomic biomarker (total circulating histones) using a new method for integration of hydrogels inside microfluidic devices during the fabrication process. This method involves using porous and fibrous membranes as scaffolds to support gels. The scaffold facilitates the drying and reconstitution of these gels without any loss of shape or leakage, making it advantageous in various applications, especially in point-of-care (POC) devices where long-term storage of gels inside the device is required. This hydrogel integration method was applied to demonstrate gel electrophoretic concentration and isoelectric trapping of cfDNA and histones respectively in rehydrated agarose gates with proper pH embedded in a porous membrane in a microfluidic device. Then, these two detections were performed in a single fully integrated microfluidic device. Additionally, nonspecific fluorescent dyes were incorporated within the device, eliminating the necessity for off-chip sample preparation. This enables direct testing of plasma samples without the need to label DNA and histones with fluorescent dyes beforehand. In all the fabrication steps of the microfluidic device, xurography, a cost-effective and rapid fabrication method, was utilized. This device demonstrated the effective separation of cfDNA and histones in the agarose gates in a total time of 20 minutes, employing 10 and 30 Volts for cfDNA and histone accumulation, respectively. This device could be further developed to create a POC device for the quantification of cfDNA and histones simultaneously in severe sepsis patients plasma sample.