ENGINEERING THE BIO-ELECTRODE INTERFACE FOR ELECTROCHEMICAL BIOSENSORS WITH SENSITIVITY, ACCURACY AND SIMPLICITY By YIFAN DAI Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemical and Biomolecular Engineering CASE WESTERN RESERVE UNIVERSITY May, 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Yifan Dai candidate for the degree of Doctor of Philosophy. Committee Chair Chung Chiun Liu Committee Member Robert F. Savinell Committee Member Harihara Baskaran Committee Member Blanton S. Tolbert Date of Defense 03/2020 *We also certify that written approval has been obtained for any proprietary material contained therein. 1 Great thanks to my families and friends and all the inspiring mentors! I want to thank my advisor, Prof. Chung Chiun Liu. I started doing research with Prof. Liu since I was an undergraduate. I have been deeply appreciated for the trust and opportunities he gave me. He guided me through all my major decisions in my early career with great patience. Prof. Liu shows me more than how to be a delicate scientist. Most importantly, he shows me a great demonstration on how to be a nice individual with patience and love. It has been a great pleasure and honor for me to work with him in the past years. I also want to thank all my collaborators and professors who inspire me a lot. I had no background about biology or biochemistry before I got into the biosensing field. All the knowledge was learnt from my collaborators and many great papers published by the experts in the field. Those works make learning so enjoyable and have taught me so much throughout my career. Lastly, I want to thank my great friends. Yu Fei and his families have made my time in Cleveland so joyful. I am very lucky to have you guys. Special thanks to my best friend of life, Xintong Cao. You are always such an inspiring person for me with great courage and kindness. Thanks for always being there for me. Our time together has always been enjoyable. Finally, I want to thank my parents for giving me such a happy life. I love you all. Yifan 2 Table of Contents Abstract .............................................................................................................................. 5 Chapter 1 Introduction ................................................................................................... 6 Chapter 2 CRISPR based Biosensing Platforms ........................................................ 8 2.1 Development of CRISPR based Universal Biosensing System ......................... 8 2.1.1 Introduction ....................................................................................................... 8 2.1.2 Verification of E-CRISPR on Nucleic Acid Detection ............................... 11 2.1.3 Evaluation of the Optimized Condition for On-chip Trans-Cleavage Activity ...................................................................................................................... 12 2.1.4 E-CRISPR for Nucleic Acid Detection ......................................................... 19 2.1.5 Aptamer based E-CRISPR Cascade for Protein Detection........................ 21 2.1.6 Conclusions ..................................................................................................... 25 2.1.7 Experimental Procedures.............................................................................. 26 2.2 CRISPR Mediated E-DNA Sensor ....................................................................... 29 2.2.1 Introduction ..................................................................................................... 29 2.2.2 Optimization of the E-DNA Sensor ............................................................. 34 2.2.3 Evaluation of CRISPR Cas9 mediated E-DNA Sensor .............................. 36 2.2.4 Comparison of the Cas9 with Cas12a mediated E-DNA Sensor ............. 38 2.2.5 Probing mismatches with CRISPR mediated DNA Electrochemistry .... 42 2.2.6 Conclusions ..................................................................................................... 47 2.2.7 Experimental Procedures.............................................................................. 47 Chapter 3 Bio-electrode Interface for Protein Detection ....................................... 50 3.1 Enhancing the Simplicity for the Construction of Bioelectrode Interface .... 50 3.1.1 Evaluation of the Effectiveness of the Bioconjugation Method ............... 52 3.1.2 Surface Analysis of Au-S Formation ............................................................ 55 3.1.3 Electrochemical Detection of TDP-43 .......................................................... 56 3.1.4 Experimental Procedures............................................................................... 62 3 3.2 Enhancing the Sensitivity through Electrostatic Interaction .......................... 66 3.2.1 NCI Construction for Analyte with Acidic pI ............................................ 70 3.2.2 NCI Construction for Analyte with Basic pI .............................................. 72 3.2.3 NCI Development for HE4 Detection .......................................................... 73 3.2.4 NCI Development for Tau Detection ........................................................... 79 3.2.5 Conclusions ...................................................................................................... 83 3.2.6 Experimental Procedures............................................................................... 84 Chapter 4 Conclusion .................................................................................................... 87 References ....................................................................................................................... 89 4 Engineering The Bio-Electrode Interface For Electrochemical Biosensors With Sensitivity, Accuracy And Simplicity Abstract By YIFAN DAI The rapid development of personalized medicine highlights the need for a robust point- of-care diagnostic system. To address this need, in my doctoral research, I focus on engineering the bio-electrode interface for the development of sensitive, accurate and simple electrochemical biosensing systems. The developed biosensing strategies have been applied for the detection of nucleic acids and proteins. These advancements can be of especially high-utility for the biosensing community, achieving the ultimate goal of point-of-care diagnosis. 5 Chapter 1 Introduction The advancement in understanding of molecular pathways on the regulation of diseases promotes the development of point-of-care diagnosis.1-12 The information from broad assessments of biomolecular signatures from nucleic acid, protein and cell throughout the disease progression is utilized as a general standard to quantitatively evaluate the severity of the disease.13-17 The existence of specific biomolecules in human fluids is recognized as the confirmation of a corresponding disease, such as cancers,13, 18, 19 neurodegenerative disorders.20, 21 Therefore, techniques on early detection of selected biomolecules are extremely critical for medical industry, especially meaningful for disease diagnosis and drug treatment.22-29 Current techniques for biomolecular quantification involve the use of mass spectrometry,30 ELISA,31-34 Western blot,35 and Northern blot.36 However, these techniques are expensive, complex and time-consuming, therefore not suitable for the contemporary needs of point-of-care disease evaluation. With the unresolved medical demands on an accurate, simple, cost-effective, time-efficient and sensitive biomarker detection system, recent scientific endeavors lead to many novel biosensing systems, such as field-effect transistor,37, 38 surface plasmon resonance sensor,39 and electrochemical biosensor.7, 9, 10, 40- 45 Among these emerging diagnostic platforms, electrochemical biosensor stands out for wide developments toward point-of-care diagnostics owing to the advantages discussed below.9, 46-50 Every sensing system consists of three essential elements: a sensing element, 6 a bio-recognition element, and a transduction element. For an electrochemical biosensing system, it possesses a simple and cost-effective sensing element (cost less than $1 for each sensor), such as a single-use sensor,51 or a sensor array.52 The processing method of bio- recognition element in an electrochemical biosensing system is relatively simple and efficient, delivering an effective bio-interface without complex operations. Furthermore, the cost-effectiveness of the electric (signal) transduction system, such as linear sweep voltammetry and differential pulse voltammetry, making the whole sensing system applicable in undeveloped area. Thus, the development of electrochemical biosensing system has created a positive and robust impact on global health. For the development of ideal integrated point-of-care system, many technical challenges remain lingering, limiting the utilizations of electrochemical biosensing system. First, for the detection of early-stage disease, the abundance of biomarkers in human fluid is relatively low comparing with that of irrelevant biomolecules, hence, requiring high-sensitivity and high-differentiation ability for accurate analysis. Second, the matrix effect caused by the biomolecules (other than the target) from human fluid interferes the target recognition process (during mass transportation), delivering a high- possibility of false positive