Aqueous Two-Phase Systems for Next-Generation Biotechnological Assays.

Abstract

Next-generation biotechnological assays, described here as those that increase throughput, improve upon physiological relevance, accomplish previously impossible biological and/or engineering tasks, or some combination thereof, will play increasingly crucial roles in the healthcare, biotechnology, and pharmaceutical industries in the next several decades. Unfortunately, although many of the recent technological innovations that have been achieved accomplish these goals, they are also commonly burdensome, technologically challenging, and perform highly niche tasks, thereby making them difficult and sometimes impossible to adopt into the healthcare, biotechnology, and pharmaceutical industries that would benefit most from them. This dissertation has four chapters, each of which describes the application of an aqueous two-phase system (ATPS) for next-generation biotechnological assays. The importance and relevance of these assays is discussed in the context of drug development in the pharmaceutical industry, where there has been decreasing return-on-investment despite the influx of billions of dollars in research and development. First, ATPS-immunocytochemistry was used to stain for multiple biomarkers from a single cell monolayer and do so more rapidly and with higher signal intensity than traditional cell staining. An ATPS of 5% polyethylene glycol (PEG) and 12.8% dextran (DEX) was then determined to be the optimal system with which to perform cell monoculture patterning for high-throughput screening analysis of cell migration and to perform co-culture patterning to achieve more physiologically relevant cell behavior that can be used as a toxicological and/or functional screening assay. ATPS was further used to create an assay that localizes trypsin to achieve reproducible and high-throughput in vitro wounding on transwell inserts. Finally, an ATPS-enzyme-linked immunosorbent assay (ELISA) was developed to pattern detection antibodies and quantify 4 biomarkers of graft-versus-host-disease without antibody cross-reactivity and with greater sensitivity compared to traditional ELISA. Such next-generation technologies will provide a launching point for the development of user-friendly, easily adoptable, and scalable assays that can be utilized by both basic science researchers and for-profit biotechnology industries to better characterize diseases and develop therapeutics.