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Interfacing to Biological Systems Using Microfluidics University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2018 Interfacing to Biological Systems Using Microfluidics Peter Golden Shankles University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Shankles, Peter Golden, "Interfacing to Biological Systems Using Microfluidics. " PhD diss., University of Tennessee, 2018. https://trace.tennessee.edu/utk_graddiss/5315 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Peter Golden Shankles entitled "Interfacing to Biological Systems Using Microfluidics." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Energy Science and Engineering. Scott T. Retterer, Major Professor We have read this dissertation and recommend its acceptance: Steven M. Abel, Mitchel J. Doctycz, Jennifer L. Morrell-Falvey Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Interfacing to Biological Systems Using Microfluidics A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Peter Golden Shankles December 2018 Copyright © 2018 by Peter Golden Shankles All rights reserved. ii DEDICATION I dedicate this work to David Shankles. I hope to live my life as intensely and as fully as he lived the 19 years he was given on this Earth. iii ACKNOWLEDGEMENTS This dissertation would not be possible without all the people that helped me along the way. I would like to thank my family for supporting me through the years with steady encouragement, a positive disposition, and reminders that success is multifaceted. I would also like to thank my girlfriend, Jenna, who has been my biggest supporter through the ups and downs of grad school. And to my lab mates and classmates, community and sharing ideas have added depth to the papers that makeup this dissertation. Finally, thank you to my advisor who taught me to ask important questions and build knowledge to share with others. iv ABSTRACT Biological systems operate on scales ranging from nanoscale chemical reactions to the global flow of nutrients and energy. Building knowledge of each level requires techniques and technologies that can address the biological system at the chosen level of interest. On the cellular and community levels, microfluidics are able to replicate the spatial scales of the natural system from the cellular, to community through the local microenvironment while providing engineering solutions to control flow through the system and interfaces with the system through microscopy and chemical sampling. Herein, biological interfaces were created using microfluidics to control cellular interactions and chemical reactions. At the subcellular scale, molecular exchange bioreactors enhanced the protein production of a cell-free protein synthesis system by using a microscale serpentine channel to reduce lateral diffusion distances. Size dependent transport of reactants into, and byproducts out of, the reaction channel through the nanoporous barrier extended the reaction time and enhanced protein yield. Nanoporous membranes were also developed for studying cellular interactions. Membranes confined cells within culture chambers while allowing transport of nutrients and signal molecules between the chambers and support channels. Quorum sensing within the microfluidic chambers was modeled using a quasi- steady-state PDE based approach to estimate relative concentrations. The platform facilitated the use of brightfield imaging and analysis to characterize morphological changes of a growing biofilm as the oral microbe Streptococcus gordonii formed aggregates only when co-cultured adjacent to Fusobacterium nucleatum. The investment of capital and time to start incorporating microfluidic into research can be prohibitive. To combat this, tools were created to provide researchers the ability to create microfluidics using 3D printing to simplify the process and remove the need for cumbersome and expensive cleanroom facilities. The technique was used in two common microfluidic applications of v chemical gradient and droplet formation in addition to building 3D fluidics that cannot be replicated directly with microfabrication techniques. These microfluidics controlled the spatiotemporal environment on the scales of biological systems to enhance the effectiveness of protein synthesis, give insight to morphological effects of cell signaling, and introduced technology to enable others to do the same. vi TABLE OF CONTENTS CHAPTER 1 INTRODUCTION ................................................................... 1 Microfluidics ............................................................................................ 1 Cell-Free Protein Synthesis Systems ..................................................... 3 3D Printed Microfluidics .......................................................................... 4 Microfluidic Cell Culture .......................................................................... 6 Cell Signaling .......................................................................................... 8 Biofilms ................................................................................................. 10 Oral Microbiome ................................................................................... 11 Modeling Bacterial Growth and Signaling ............................................. 13 Research Aims ..................................................................................... 14 CHAPTER 2 FABRICATION OF NANOPOROUS MEMBRANES FOR TUNING MICROBIAL INTERACTIONS AND BIOCHEMICAL REACTIONS ...... 17 Abstract ................................................................................................ 20 Introduction ........................................................................................... 21 Experimental ......................................................................................... 24 Nanoporous Exchange Device .......................................................... 24 Electron Beam Lithography ........................................................... 26 Microchannels ............................................................................... 26 Reactive Ion Etching of Fluidic Network and Membrane ............... 27 Silicon Dioxide Coating of Nanopores ........................................... 27 Device Testing ............................................................................... 28 Cell Culture Device ........................................................................... 29 Photolithography ........................................................................... 29 PDMS Casting and Device Bonding .............................................. 31 Device Testing and Cell Culture .................................................... 32 Results and Discussion ........................................................................ 33 Nanoporous Exchange Device .......................................................... 33 vii Cell Culture Device ........................................................................... 37 Summary and Conclusions ................................................................... 40 Acknowledgements .............................................................................. 41 CHAPTER 3 A NANOSTRUCTURED CO-CULTURE ENVIRONMENT ENABLED STUDY OF CHEMICAL SIGNALING BETWEEN BACTERIA ........... 42 Abstract ................................................................................................ 43 Introduction ........................................................................................... 44 Materials and Methods ......................................................................... 48 Cell Culture ....................................................................................... 48 Microfluidic Device Fabrication ......................................................... 49 Well-Plate Microfluidics Fabrication .................................................. 50 Well-Plate Preparation ................................................................... 51 APTES Bonding Procedure ........................................................... 52 Assembly ....................................................................................... 52 Uncured PDMS Procedure ............................................................ 53 Imaging ............................................................................................. 56 Modeling ........................................................................................... 56 Experimental Procedure .................................................................... 60 Characterization of Chemical Gradients ........................................ 60 Microbial Signaling Experiments ................................................... 60 Results and Conclusions .....................................................................
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