Ka Lok Hong

Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Letha J Sooter

Committee Co-Chair

Erik A Bey

Committee Member

Yon Rojanasakul

Committee Member

Grazyna D Szklarz

Committee Member

Linda Vona-Davis

Abstract

Molecular recognition elements (MREs) are biomolecules such as single-stranded DNA (ssDNA), RNA, small peptides and antibody fragments that can bind to user defined targets with high affinities and specificities. This binding property allows MREs to have a wide range of applications, including therapeutic, diagnostic, and biosensor applications. The identification of MREs can be achieved by using the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This process begins with a large library of 109 to 1015 different random molecules, molecules that bind to the user defined target or positive target are enriched in the process. Subsequently, this process can be modified and tailored to direct the enriched library away from binding to related targets or negative targets, and thus increasing the specificity. Single-stranded DNA (ssDNA) MREs are particularly favorable for biosening applications due to their relative stability, reusability and low cost in production. This work investigated the identification and application of ssDNA MREs to detect different bacterial toxins and pesticide.;In Chapter 1, it begins by reviewing recent discovery and advancement in the SELEX technique for the identification and biosensing application of ssDNA MREs specific for bacteria, viruses, their related biomolecules, and selected environmental toxins. It is then followed by a brief discussion on major biosensing principles based upon ssDNA MREs. In Chapter 2, the pilot project of this work, ssDNA MRE specific for Pseudomonas aeruginosa exotoxin A was identified. In this chapter, a novel variation of SELEX called Decoy-SELEX, previously developed by our laboratory is described in greater detail. Additionally, the development of a ssDNA MRE modified enzyme-linked immunosorbent assay (ELISA) for the exotoxin A detection is also discussed. In Chapter 3, similar methodology was applied to identify a ssDNA MRE specific for the second target, Clostridium difficile toxin B. Subsequently, similar ssDNA MRE modified ELISA was developed for target detection in clinically relevant samples. In Chapter 4, ssDNA MRE specific for alpha toxin of Staphylococcus aureus was identified, and it was applied for sensitive detection of the target in clinically relevant samples. In Chapter 5, the overall conclusion and potential future studies as a result from this work is discussed. Lastly, in Appendix, the project of identifying and potential future application of ssDNA MREs specific for a pesticide, Fipronil is described.;Overall, this work has shown the proof-of-principle of using ssDNA MREs in biosensing application for target detections in clinically relevant samples. The work will be useful in the development of potential point-of-care diagnostic tools for rapid diagnosis of bacterial infections.

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