Development of polymerization-based signal amplification for detection of biomolecular recognition.

摘要

The characterization of molecular biomarkers associated with the onset of disease combined with the development of molecular diagnostic biosensing technologies holds significant potential to improve disease detection and treatment. A primary challenge in molecular diagnostics involves identifying minute quantities of biological targets, often from complex mixtures, thus requiring detection methodologies that are highly sensitive and specific. Routine implementation at point-of-care settings also requires rapid, inexpensive detection assays producing reliable responses that are readily detected using minimal instrumentation.;The focus of this thesis is to develop the use of photopolymerizations towards detection of biological targets. By coupling the formation of high molecular weight polymer growth to biomolecular recognition events, a method of signal amplification is achieved. The rapid and highly amplified nature inherent in free radical polymerizations combined with its inexpensive and robust characteristics offers a unique and potentially advantageous approach to detection.;Central to this method is the use of Type I and Type II photoinitiators capable of binding to complementary biomolecules. The synthesis and characterization of these molecules is performed, followed by an investigation of their use in surface-initiated polymerization reactions to signal binding. To identify the assay requirements, sensitivities, and potentially novel aspects of this method, the amplification process is investigated on high-throughput, array-based biochips directly fabricated with the targets of interest. The potential for quantitative measurements of DNA targets is next investigated. Amounts of polymer generated from varied surface concentrations of DNA targets are measured to identify regions of dynamic signal amplification. Methods of coupling fluorescent or colorimetric signals with dynamic polymer growth are developed in effort to reduce the cost and complexity of instrumentation required for quantitative measurement. Finally, steps are taken towards implementing polymerization-based amplification in clinically relevant systems. Detection capabilities are developed towards multiplexed and base-specific detection of nucleic acid targets, as well as detection from complex mixtures. From the developments highlighted in this research, polymerization-based amplification holds unique potential for attaining signal amplification for the purpose of biodetection.