| Summary: | Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015. === Cataloged from PDF version of thesis. === Includes bibliographical references (pages 124-135). === Polymerization-based signal amplification is an approach to biosensing that leverages the amplification inherent to radical polymerization to enhance signal associated with molecular recognition. This versatile technique has been implemented with a number of radical polymerization chemistries, including atom-transfer radical polymerization (ATRP), photopolymerization, reversible addition-fragmentation chain transfer polymerization (RAFT), and enzyme-mediated redox polymerization. This thesis focuses on the development of photopolymerization-based signal amplification (PBA) as a platform technology for use at the point-of-care. We sought to build a mechanistic understanding of the system, specifically examining the effects of non-ideal binding reactions and oxygen. One of the greatest barriers to wider implementation of polymerization-based signal amplification is the susceptibility of radical polymerization reactions to oxygen inhibition. Oxygen reacts with initiating and propagating radicals to form peroxy radicals that are unreactive towards propagation, and thus have the effect of terminating the reaction. Chapter 2 describes the development of an air-tolerant monomer solution that allows interfacial photopolymerization reactions to proceed under ambient conditions in as little as 35 seconds where previous implementations of PBA required inert gas purging to remove oxygen and reaction times of 20 minutes or longer. We showed that the inclusion of submicromolar concentrations of eosin, the photoinitiator, in the monomer solution mitigated the effects of oxygen. The ability to perform these reactions under ambient conditions increases their clinical utility by simplifying the procedure and by eliminating purging gases that might be detrimental in some biological applications, specifically those involving cells. Intrigued by eosin's ability to overcome over 1000-fold excess of oxygen, we performed spectroscopic measurements in order to elucidate the mechanisms underlying eosin's resistance towards oxygen inhibition. A dual-monitoring system for measuring eosin consumption and monomer conversion was used to corroborate the hypothesized regeneration of eosin in the presence of oxygen (Chapter 3). This required the development of a method for analyzing absorbance data for polymerizing hydrogels. We further examined the photoactivation kinetics of the eosin/tertiary amine system and the effect of oxygen using absorbance spectroscopy and kinetic modeling (Chapter 4). The spectroscopic investigation revealed that, in addition to the previously postulated reactions in which eosin is regenerated by oxygen, additional reactions between oxygen and eosin in its triplet state and semireduced form occur and must be taken into account. The formation and consumption of the semireduced species informed the construction of a kinetic model, for which the importance of considering the reaction between triplet state eosin and the tertiary amine as two separate steps was clearly demonstrated. Transitioning away from an examination of the amplification chemistry, we next considered the molecular recognition event, exploring the concept of the amplification threshold by investigating the impact of the binding affinity of the molecular recognition event on the limit of detection (Chapter 5). We showed that improvements in binding affinity enhance detection sensitivity. A mass action kinetics based model was used to accurately predict experimental findings and identify the key parameters for predicting the performance of PBA reactions: surface capture probe density, incubation time, concentration and binding affinity of the target molecule. We evaluated the commonly proposed strategy of developing polymeric macrophotoinitiators for improving the sensitivity of photopolymerization-based signal amplification reactions (Chapter 6). Building on earlier work, in which solubility limits were encountered coupling eosin and neutravidin to a poly (acrylic acid-co-acrylamide) backbone, we synthesized a more water-soluble polymeric macrophotoinitiator based on a generation 7 poly (amidoamine) dendrimer scaffold. Although the solubility was improved, a new quenching limitation was identified, demonstrating the complexity of designing polymeric macrophotoinitiators that incorporate eosin as the photoinitiator. In lieu of viable photoinitiator alternatives to eosin, we concluded that future efforts to design polymeric macrophotoinitiators should include features that increase the distance between eosin molecules. We used photopolymerization-based signal amplification to selectively encapsulate a target population of cells in a co-culture (Chapter 7). PBA allows for the selective growth of an immunoprotective hydrogel only at the surfaces of the labeled cells, even in closely contacted cell aggregates. The hydrogel protects the cells against subsequent lysis, allowing for nucleic acid extraction from the unlabeled cells without the need for cell sorting. Finally, we consider the vast, unexplored parameter space for photopolymerization-based signal amplification, surveying alternative photoinitiation chemistries, new approaches to signal interpretation, and future applications. === by Kaja Kaastrup. === Ph. D.
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