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Development of New Biological Nanopores and Their Application for Biosensing and Disease Detection

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Development of New Biological Nanopores and Their Application for Biosensing and Disease Detection University of Kentucky UKnowledge Theses and Dissertations--Pharmacy College of Pharmacy 2016 Development of New Biological Nanopores and Their Application for Biosensing and Disease Detection Shaoying Wang University of Kentukcy, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2016.428 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Wang, Shaoying, "Development of New Biological Nanopores and Their Application for Biosensing and Disease Detection" (2016). Theses and Dissertations--Pharmacy. 64. https://uknowledge.uky.edu/pharmacy_etds/64 This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Shaoying Wang, Student Dr. Peixuan Guo, Major Professor Dr. David Feola, Director of Graduate Studies DEVELOPMENT OF NEW BIOLOGICAL NANOPORES AND THEIR APPLICATION FOR BIOSENSING AND DISEASE DETECTION ____________________________ DISSERTATION ____________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky By Shaoying Wang Lexington, Kentucky Co-Directors: Dr. Robert Lodder, Professor of Pharmaceutical Sciences, Lexington, Kentucky and Dr. Peixuan Guo, Professor of Pharmaceutics & Pharmaceutical Chemistry, Columbus, Ohio 2016 Copyright © Shaoying Wang 2016 ABSTRACT OF DISSERTATION DEVELOPMENT OF NEW BIOLOGICAL NANOPORES AND THEIR APPLICATION FOR BIOSENSING AND DISEASE DETECTION Nanopore technology has recently emerged as a new real-time single molecule sensing method. The current dominant technologies, such as mass spectrometry and immunoassay, for protein analysis is still slow and complex, which can’t meet the urgent need and fields of use. Development of a highly simple, portable and sensitive detection system for pathogen detection, disease diagnosis, and environmental monitoring is in great need. Membrane embedded Phi29 connector nanopore, the first protein nanopore coming from bacteriophage, was mainly focusing on DNA and RNA translocation in previous studies. Here, Phi29 connector nanopore was first time established for antibody detection by engineering Epithelial Cell Adhesion Molecule peptide as a probe. The results demonstrate that the specific antibody can be detected in presence of diluted serum or non-specific antibody. To enable detecting more different types of analytes with high sensitivity, developing new nanopore with various properties, such as size, charge, hydrophilic/hydrophobic and physical dimension, is needed. In this work, besides Phi29 nanopore, several new protein nanopores that derived from T3, T4, and SPP1 bacteriophages were developed. A shared property of three step conformational change among these portal channel has been discovered. Elucidating the sequence and oligomeric states of proteins and peptides is critical for understanding their biological functions. Here, SPP1 nanopore was used to characterize the translocation of TAT peptide with dimer and monomer forms. Translocation of the peptide was confirmed by optical single molecule imaging for the first time, and analyzed quantitatively. The dynamics of peptide oligomeric states were clearly differentiated based on their characteristic electronic signatures. Main challenge for probing protein structure, folding, detection and sequencing using nanopore is the ultra-fast translocation speed which normally beyond electronic detection limit. In this work, the peptides translocation was slowed in SPP1 nanopore by changing the charge shielding of the channel. A 500-fold reduction was observed for TAT peptide translocation. By using this method, arginine chain peptide as short as two arginine can be detected first time. Further improving the bandwidth may lead to single amino acid detection and has the potential for protein sequencing. Compared with protein nanopore, de novo designed nanopore can provide numerous advantages, such as tunable size and functionality, ease of construction, scale up and modification. In the final study, an RNA-based biomimetic nanopore was first time constructed. The insertion of RNA nanopore into lipid bilayer and cell membrane were characterized and translocation of short amino acids through RNA nanopore was detected. This new artificial nanopore has the potential to be used for sensing, disease diagnosis, and even protein sequencing. KEYWORDS: Biological nanopore, peptide translocation, bacteriophage, RNA Nanotechnology, protein sequencing Shaoying Wang Student’s Signature October 4th, 2016 Date DEVELOPMENT OF NEW BIOLOGICAL NANOPORES AND THEIR APPLICATION FOR BIOSENSING AND DISEASE DETECTION By Shaoying Wang Dr. Peixuan Guo Co-Director of Dissertation Dr. Robert Lodder Co-Director of Dissertation Dr. David.Feola Director of Graduate Studies October 4th, 2016 To my wife, parents and family for their continued support ACKNOWLEDGMENTS Firstly, I would like to express my deepest gratitude and best regards to my thesis advisor, Dr. Peixuan Guo, for his continual guidance and support during my PhD studies. I sincerely appreciate Dr. Guo’s invaluable advice, experience, and guidance. Also, I would greatly appreciate my committee members, Dr. Robert Lodder, Dr. David Rodgers, Dr. Paul Bummer, Dr.Chang-Guo Zhan, and Dr. Jianhang Jia. Their instruction through my graduate studies proved indispensable in all aspects of my graduate work. Their advices and instructions through my research, qualifying exam, and dissertation defense are indispensable to my study. I would like to give special thanks to Dr.Dan Shu, Dr. Farzin Haque, Dr.Hui Zhang, Dr. Jia Geng for their training and valuable insight in my experiments and data throughout my research. In addition, I would like to specially thanks to Hui Li and Fengmei Pi for their encouragement, and support through my PhD studies. I would have not made it through this endeavor without their assistance and kindness. I also would like to thank all past and present members of the Guo laboratory; Dr.Yi Shu, Dr.Zhi Zhou, Dr. Mario Vieweger, Dr. Ashwani Sharma, Dr. Taek Lee, Dr. Zhanxi Hao, Dr. Chad Schwartz, Dr. Huaming Fang, Fengmei Pi, Hui Li, Zhengyi Zhao, Danny Jasinski, Erfu Yan, Yanxi Xie, Zhouxiang Ji, Concong Xu, Hongran Yin, Sijin Guo, Zhefeng Li, Le Zhang, Hongzhi Wang, and Megan Heitkemper. Without their support I truly believe my success would not have happened. I would like to acknowledge all other faculty and staff members from College of Pharmacy at University of Kentucky throughout my graduate career, especially graduate coordinator Catina Rossol and current director of graduate studies Dr. David Feola and previous director of graduate studies Dr. Jim Pauly. iii Finally, I would like to show my appreciation to my wife Dr.Rongrong Zhang, my parents for their continued encouragement, love and support through my everyday struggles. The work in the thesis dissertation was supported by the National Institute of Health under grants R01 EB012135 to Peixuan Guo. iv TABLE OF CONTENTS Acknowledgments .............................................................................................................. iii Table of Contents .................................................................................................................v List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii List of Abbreviations ......................................................................................................... ix Chapter 1: Introduction and Literature Review ...................................................................1
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