Transduction of antigens into amplifiable DNA signals using structure switching aptamers

Abstract

Detection of specific antigens has one vital step in common: detection of biomarkers. Diagnostic testing that is rapid and reliable is unavailable in limited resource and rural settings. The solution to this need must be simple, inexpensive, robust, rapid and not require highly trained personnel to operate. Aptamers are capable of delivering those needs when matched with a novel high gain amplification method. This thesis focuses on important aspects of a novel protein detection assay that uses aptamers. Aspects that play an important role on the assay's success were investigated; aptamer selection and design of structure switching aptamers, designing DNA templates that will transduce the signal created by the aptamers, solid phase selection, aptamer immobilization on the solid phase, protein capture, and amplification of the signal. The first step was to find aptamers that were proven to specifically target clinically relevant targets and modify them to suit the needs of the assay. It is important to validate the aptamers' performance. The second important step was finding a solid phase that is compatible with the novel nucleotide amplification reaction that will be used to amplify the signal produced by the aptamers. Paramagnetic microbeads, membranes and polyacrylamide hydrogels were potential candidates for solid phases. Non-specific interaction of the target protein with the solid phase surface will not have negative effects while running the assay due to the structure switching of the aptamers however, it prevented the accurate quantification of the protein capture by aptamers. There is a need for the development of a blocking buffer that is specific to the solid phase. Washing of the excess DNA templates that are not bound to target-bound aptamers plays an important role in the assay's accuracy. The results presented here show the preliminary work that has been done for the novel protein detection assay that uses structure switching aptamers. This assay has the potential to detect diseases at point-of-care in low resource settings.