Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms By
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Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms by C. Wyatt Shields IV Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ W. Monty Reichert, Chair ___________________________ Gabriel P. López, Supervisor ___________________________ Benjamin B. Yellen ___________________________ Stefan Zauscher ___________________________ Andrew J. Armstrong Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2016 ABSTRACT Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms by C. Wyatt Shields IV Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ W. Monty Reichert, Chair ___________________________ Gabriel P. López, Supervisor ___________________________ Benjamin B. Yellen ___________________________ Stefan Zauscher ___________________________ Andrew J. Armstrong An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2016 Copyright by C. Wyatt Shields IV 2016 Abstract Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management. The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs iv via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis. The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments. v Dedication To my family, and especially my mother, for teaching me the most important things in life. vi Contents Abstract ......................................................................................................................................... iv List of Tables .............................................................................................................................. xiii List of Figures .............................................................................................................................xiv Acknowledgements ............................................................................................................... xxvii Specific Aims ............................................................................................................................. xxx 1. Introduction to Microfluidic Cell Sorting .............................................................................. 1 1.1 Background and significance of microfluidic cell sorting .......................................... 1 1.2 Existing approaches for microfluidic cell sorting ........................................................ 4 1.2.1 Fluorescent label-based cell sorting .......................................................................... 4 1.2.1.1 Electrokinetics ....................................................................................................... 5 1.2.1.2 Acoustophoresis ................................................................................................. 10 1.2.1.3 Optical systems .................................................................................................. 14 1.2.1.4 Mechanical systems ........................................................................................... 16 1.2.2 Bead-based sorting .................................................................................................... 18 1.2.2.1 Magnetophoresis ................................................................................................ 19 1.2.2.2 Acoustophoresis ................................................................................................. 22 1.2.2.3 Electrokinetics ..................................................................................................... 24 1.2.3 Label-free cell sorting ................................................................................................ 26 1.2.3.1 Acoustophoresis ................................................................................................. 27 1.2.3.2 Electrokinetics ..................................................................................................... 29 vii 1.2.3.3 Magnetophoresis ................................................................................................ 31 1.2.3.4 Optical systems .................................................................................................. 33 1.2.3.5 Passive cell sorting ............................................................................................. 34 1.3 Commercialization of existing microfluidic cell sorters ........................................... 52 1.4 Barriers to commercialization and clinical translation .............................................. 56 1.4.1 Device-related barriers ............................................................................................. 56 1.4.2 Commercialization-related barriers ........................................................................ 57 1.5 Overcoming the barriers to commercialization .......................................................... 60 1.5.1 Device-related solutions ........................................................................................... 60 1.5.2 Commercialization-related solutions ..................................................................... 62 1.6 Conclusions ..................................................................................................................... 64 1.7 Acknowledgements ........................................................................................................ 64 2. Elastomeric Particles for Use in Acoustofluidic Devices ................................................... 66 2.1 Nucleation and growth synthesis of elastomeric particles ....................................... 66 2.2 Methods for elastomeric particle preparation ............................................................ 69 2.3 Means of controlling particle size ................................................................................ 72 2.4 Means of controlling particle composition ................................................................. 76 2.5 Means of controlling particle surface properties and biofunctionality .................. 78 2.6 Means of measuring and controlling particle acoustic contrast factor ................... 82 2.7 Qualitative verification of particle acoustic contrast factor