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A Microfluidic Investigation of Calcium Oxalate

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A Microfluidic Investigation of Calcium Oxalate A MICROFLUIDIC INVESTIGATION OF CALCIUM OXALATE CRYSTALLIZATION By JINNIE ANSTICE A thesis submitted to the Graduate School-Camden Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Master of Science Graduate Program in Chemistry Written under the direction of Dr. George Kumi And approved by ______________________________ Dr. George Kumi ______________________________ Dr. Alex J. Roche ______________________________ Dr. Hao Zhu Camden, New Jersey May 2018 THESIS ABSTRACT A microfluidic investigation of calcium oxalate crystallization By JINNIE ANSTICE Thesis Director: Dr. George Kumi Calcium oxalate crystallization is widely studied due to the prevalence of this substance in various biomineralization processes, especially the formation of kidney stones. Bulk crystallization studies are a common and popular method for investigating calcium oxalate in particular. Crystallization studies using microfluidic platforms are becoming more popular because of the simplicity of use, cost effectiveness and enhanced control of system variables that these systems offer. Microfluidic systems using a two-input, one- output design results in crystallization at the entrance of the microchannel. This could lead to clogging of the device, and clogging makes it difficult to study crystallization over long lengths of time using these devices. In this study a three-input, three-output microfluidic channel system was designed and a protocol was established that minimized bubble occurrence and synthesized calcium oxalate crystals within the device; crystals were analyzed ex-situ. Equimolar input salt concentrations (CaCl2, K2C2O4) of 20, 40 and 60 mM were used in these experiments. Evidence of crystallization within the device was a line forming in the center channel that grew (i.e., darkened) over time. As the concentration of input salt solution increased, the time that it took for the line to be visible decreased but the length and darkness of the line increased. In order to re-use devices hydrochloric acid was tested as a cleaning solvent, and it proved to be an ii appropriate solvent that did not alter the crystallization process. Experiments in which sodium polyacrylate (a common additive in calcium oxalate crystallization experiments) was added to the system showed that the additive inhibited the growth of crystals formed within the microfluidic device. Analysis of collected crystals was done by optical and scanning electron microscopy. iii Dedication I would like to dedicate this thesis to my amazing husband Shane. Without his support, encouragement and sacrifice I could not have reached this goal. For ten years he has been picking me up when I fall down, wiping my tears, brushing me off and pushing me to get back on track. Thank you for all that you do. I also dedicate this work to my children. They have watched me spend many hours in front of the computer, reading a textbook or practicing flashcards. They have tolerated me working at their extracurricular activities and have understood the commitment necessary to achieve this goal. I wish them all the best and don’t ever give up on your dreams. Finally, I dedicate this thesis to my family and friends especially my brother, Kevin. Thank you for not letting me fall. To my firehouse family, my campground family and all of my wonderful friends thank you for your understanding and patience throughout this process. iv Acknowledgements Rutgers-Camden, specifically the science department, has been a second home to me for the last ten years. I came to Rutgers as a non-traditional student with high hopes of achieving my goals. Rutgers- Camden was a welcoming and supportive environment especially for students in complicated situations or with family obligations. It was wonderful to have access to professors, tutors, and multiple programs that assist students, especially non-traditional students. First, I would like to thank Dr. Alex J. Roche, who tolerated my endless office visits while trying to wrap my head around organic chemistry. I am also thankful he accepted the task of being on my thesis committee. Lastly, I want to thank Dr. Roche for recommending me as a work study student for Dr. Arbuckle-Keil which opened up numerous opportunities for me. Dr. Arbuckle-Keil and I had an instant connection almost a decade ago. I wanted to learn, and she wanted to teach. Together we have tackled many things over the years. Thank you for believing in me and pushing me to be the best that I can be not only as a student and colleague but as a person. It is always a joy to see her in the lab, especially when she is determined to fix an instrument. Dr. Arbuckle-Keil is a role model and strong advocate for women in science. Thank you for providing me with the many opportunities that I had at Rutgers. I look forward to our talks after this chapter of my life has closed. Ms. Mary Craig is another woman that I have had the pleasure of working with throughout my time at Rutgers-Camden. Ms. Craig has been an integral part of this process and my success as a student, colleague, educator, parent, and spouse. She always v knows what to say no matter what the situation. I would not have made it without relying upon her simple but meaningful words of wisdom. I would also like to thank Dr. Zhu for being on my committee. Thank you for making me see bigger connections. Thank you to Sarah Belh and Todd Grover for the endless hours of entertainment in the lab. Thank you to Peter Fazzino and Janet Caruso for their help in the office (and Janet for sharing my birthday). My deepest thanks go to Dr. Kumi for taking me into his group and committing to be my advisor. It is always such a pleasure to see Dr. Kumi interact with his students, both undergraduate and graduate. He has a genuine interest in seeing his students succeed. I appreciate your talent to explain things and value your wisdom and expertise and an advisor and an educator. Thank you for your patience and understanding throughout this long process. vi Table of Contents Title .................................................................................................................................i Abstract ......................................................................................................................... ii Dedication ...................................................................................................................... iv Acknowledgements ......................................................................................................... v Table of Contents ..........................................................................................................vii List of Figures ................................................................................................................ x List of Tables ................................................................................................................ xv Chapter 1: Introduction ................................................................................................... 1 1.A. Urolithiasis ................................................................................................... 1 1.B. Calcium Oxalate Contribution ...................................................................... 2 1.C. Microfluidics ................................................................................................ 5 1.D. Research Objective ..................................................................................... 10 Chapter 2: Experimental Method and Materials ............................................................ 12 2.A. Microfluidic Device Fabrication ................................................................. 12 2.A.1. Glass Substrate Preparation .......................................................... 12 2.A.2. Master Structure Fabrication ........................................................ 13 2.A.3. Replica Molding .......................................................................... 17 2.B. Device Operation ........................................................................................ 18 2.C. Establishing the Crystallization Protocol ..................................................... 19 vii 2.C.1.Bubble Reduction .......................................................................... 19 2.C.2. Device Flow Characterization ...................................................... 21 2.C.3. CaOx Crystallization .................................................................... 22 2.C.4. Crystal Collection and Analysis.................................................... 23 2.C.5. Establishing re-usability of device ................................................ 24 2.C.6. Additives (addition of NaPA) ....................................................... 24 Chapter 3: Results and Discussion ................................................................................. 26 3.A. CaOx crystallization and analysis ............................................................... 26 3.A.1. Establishing reproducibility using the same master structure. ....... 28 3.A.2. Establishing reproducibility using different master structures. ...... 31 3.A.2.a. 60 mM concentration inputs ........................................... 32 3.A.2.b. 40 mM concentration inputs .......................................... 34 3.A.2.c. 20 mM concentration inputs ........................................... 36 3.A.3. Summary of results .....................................................................
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