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A Dissertation Entitled Selection and Characterization of Ssdna Aptamers for Salivary Peptide Histatin 3 and Their Application T A Dissertation entitled Selection and Characterization of ssDNA Aptamers for Salivary Peptide Histatin 3 and Their Application Towards Assay and Point-of-Care Biosensing by Yagya Raj Ojha Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Engineering ___________________________________________ Brent D. Cameron, PhD, Committee Chair ___________________________________________ David R. Giovannucci, PhD, Committee Member ___________________________________________ Dong-Shik Kim, PhD, Committee Member ___________________________________________ Scott M. Pappada, PhD, Committee Member ___________________________________________ Casey W. Pirnstill, PhD, Committee Member ___________________________________________ Amanda C. Bryant-Friedrich, PhD, Dean College of Graduate Studies The University of Toledo December 2019 Copyright 2019, Yagya Raj Ojha This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Selection and Characterization of ssDNA Aptamers for Salivary Peptide Histatin 3 and Their Application Towards Assay and Point-of-Care Biosensing by Yagya Raj Ojha Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Engineering The University of Toledo December 2019 The development of detection methods for the novel biomarkers can have a significant impact on the research and clinical applications such as drug discovery, disease diagnosis, and treatment monitoring. Histatin 3 (H3) is an antimicrobial salivary peptide that possesses the capability of being a therapeutic agent against oral candidiasis and has recently been linked to acute stress as a potential novel biomarker. Stress biomarkers reflect the physical and cognitive performance of an individual, and their monitoring in real-time is of vital importance for the high-risk jobs, including military, pilot, and surgeon, where higher vigilance is required for an extended period. The salivary levels of H3 also have been correlated with the HIV-infection and associated oral candidiasis. Therefore, monitoring H3 levels in human saliva can provide essential information about an individual’s health status, including HIV-infection, oral candidiasis, and acute stress. Additionally, H3 detection could serve as therapeutic drug monitoring if H3 can be established as an alternative therapeutic agent. The currently available detection techniques for H3 are gel chromatography, high- performance liquid chromatography (HPLC), mass spectrometry (MS), and antibody- iii based immunoassays. The Chromatographic and mass-spectroscopic methods are laborious, utilize expensive instrumentation, require trained personnel, and time- consuming. Whereas antibody-based immunoassays are not widely validated, expensive, sensitive to temperature, and have a short lifespan. This void in analytical methods is not just for H3 but also applies to several other biomarkers in saliva. Even though saliva is considered as an optimal biofluid, several limitations are impeding its use in diagnostic and research. The major hurdles include the deficient concentration of biomarkers, need of laboratory-based preprocessing to remove mucin and interfering particulate matters, and lack of standard sample collection methods. Also, due to the absence of easily accessible and cost-effective analytical methods, it is challenging to figure out the circadian variation of biomolecules and consequently establish biomolecules as biomarkers. All these necessities mandate the development of alternative techniques that offer more accessible, cost-effective, rapid, real-time, and probably the point-of-care (POC) testing of salivary biomarkers. The aptamers, synthetic oligonucleotides functionally similar to antibodies but with several advantages, have shown promising possibilities to fulfill the requirements in such applications. However, no aptamer for H3 and several other salivary biomarkers exist in literature. This dissertation presents a widely applicable aptamer selection method for the novel biomarkers and the application of the identified aptamers with various platforms for the detection application in human saliva. In order to identify H3 aptamers, a library immobilization version of an iterative in vitro process known as the systematic evolution of ligands by exponential enrichment (SELEX) was established. This method does not require target immobilization and offers the isolation of signaling aptamers for a target in iv its unmodified form. Also, the signaling, structure-switching upon target binding, is a highly desired attribute of the aptamers for the biosensing application. Within the SELEX process, counter-selection steps were instituted to stop the evolution of nonspecific sequences that can bind to the molecules closely related to the target. Through the repetitive rounds of selection and counter-selection, four unique aptamer candidates sharing a consensus sequence were identified. The obtained aptamers were analyzed via online servers to predict the stability and structural conformations. A superior candidate in terms of stability and structural complexity was picked for the visual confirmation of binding. The magnetic bead-based affinity capture combined with sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), confirmed the selective binding between the selected aptamer candidate and target H3, indicating the successful aptamer selection process. Following the preliminary studies, more detailed characterizations were performed by developing a direct format enzyme-linked aptamer sorbent assay (ELASA). The aptamers were compared for their binding affinity and specificity to the target molecule H3. Also, the effect of primer binding sites on the binding affinity was probed. Selected clones exhibited affinity and specificity to H3, and the sequence with the highest affinity exhibited enhanced binding following the removal of primer binding sites. Based on these results, the truncated aptamer sequence was chosen for further application with other assay platforms. Next, the signaling feature of the aptamer was employed to explore the prospect of gold nanoparticles (AuNPs)-based colorimetric assays. Two different formats of the colorimetric assays were developed. After optimizing various parameters, both assay v schemes were able to detect H3 within a salivary level of a healthy individual. Besides, with the assay-format-2, the overall assay time was reduced to just 12 minutes. Furthermore, surface-enhanced Raman scattering (SERS)-based magnetic aptasensor was developed for the ultrasensitive detection of H3. By optimizing various parameters, SERS-based atpasensor was established. Additionally, the lateral flow test strip to support POC measurements of H3 was demonstrated by utilizing the optical properties of the AuNPs and the specificity of the identified H3 aptamer. The captured AuNPs on the test line of the lateral flow device provided qualitative analysis within 5 minutes for visual observation. Finally, human saliva and the artificial saliva samples were tested with the developed assay formats to investigate the challenges and the complications associated with the saliva-based measurements. In conclusion, the work presented in this dissertation lays the foundation for the identification of the signaling aptamers and their subsequent implementation in several platforms for the detection of salivary biomarkers. Further improvements and optimizations of the assay methods will eventually lead to a fast, reliable, cost-effective, ultrasensitive, and POC detection methods for the salivary biomarkers. vi To my parents, Dhananjay and Indu, brother Dipak, and wife Dikshya for their persistent love and encouragement. vii Acknowledgements First and foremost, I would like to take this opportunity to express my deepest gratitude to my advisor Dr. Brent D. Cameron for being a great mentor. This work has only been possible with his continuous support, scientific insight, and supervision. I feel truly fortunate to have had him as my advisor. Also, my sincere thanks go to the committee members, Dr. David R. Giovannucci, Dr. Dong-Shikh Kim, Dr. Scott M. Pappada, and Dr. Casey W. Pirnstill, for their invaluable words of advice and thoughtful comments. It is my great pleasure to express my appreciation to Tamara Phares for her unconditional support throughout all these years. Further, I am thankful to the faculty, staff, and my friends, Yongsoon Hwang, Niraj K. Gupta, Mahmoud Eladawi, Ujjwal Shrestha, and Nav Raj Paneru, for providing friendly advice and useful comments. I’m deeply indebted to my father; with his support and inspiration, I have been able to make it to this point. Special thanks go to my mom, brother, wife, and other family members for caring, loving, and believing in me. I am also thankful to my relatives in my home country, Nepal, for their love, support, and encouragement throughout my journey. I would also like to thank the Air Force Office of Scientific Research (AFOSR) for sponsoring the project (grant contract FA955-13-1-0187) included in this dissertation. Finally, I would like to acknowledge the Center for Materials and Sensor Characterization (CMSC) and Electron Microscope Facility (Dr. W.T. Gunning) at the University of Toledo for the instrumentation and testing support. viii Table of Contents Abstract…………………………………………………………………………………iii
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