MAGNETIC PARTICLE BASED MICROFLUIDIC SEPARATION OF CANCER CELLS FROM WHOLE BLOOD FOR APPLICATIONS IN DIAGNOSTIC MEDICINE A Dissertation Presented by Brian Dennis Plouffe to The Department of Chemical Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Chemical Engineering Northeastern University Boston, Massachusetts April 1, 2011 ACKNOWLEDGEMENTS I wish to express sincere gratitude to my advisors, Profs. Shashi Murthy and Laura Lewis, whose enthusiasm and dedication for research drew me to the interdisciplinary field of magnet-based microfluidic cell separation. Shashi’s friendship, encouragements, and faith in me as his first graduate student greatly contributed to my research and the completion of this dissertation. I thank Laura for her patience with me as I truly learned, for the first time, the interesting field of magnetism. She definitely has challenged me and strengthened me both as a researcher and a critical thinker. Even though at first it may have seemed like we where talking two different languages, I only hope you learned as much about microfluidics as I have about magnetism and nanotechnology. I would also like to thank Dattu Nagasha along side my two advisors, as this dissertation would never have been completed without him. I owe my deepest gratitude to my committee members: Prof. Sridhar and Prof. Klapperich who agreed to serve on my dissertation committee despite their tremendously busy schedules. It is also a pleasure to thank my lab mates (past and present) for all of their support and assistance along the way. In particular I would like to thank Anil, Jimmy, and Dwayne for all the fun times. You were always there to think things out, both academically and personally. Thank to all the other who have directly or indirectly contributed to my experience here at Northeastern including graduate students: Deepa, Dayo, Adam, and Sean; high school students: Robert and Lucy; post-docs: Beili and Mariana; and collaborators: Virna, Georg, and Don ii I would like to thank my main funding source over the past several years, the NSF/NCI IGERT Nanoscience and Technology Program. The program forced me to think about every step of my project along the way and provided me with the tools (both in the classroom and in the lab) to pursue this interdisciplinary dissertation topic. My special thanks to my family, for creating an environment in which pursuing my dreams was always encouraged, as well as for their support every step of the way. Finally, I would like to thank my wife, Aimée, for her endless love and encouragement, as well as a much needed distraction on those days when I really needed one. Her continued belief in me has been a source of strength over the past 5 years. I LOVE YOU! iii ABSTRACT Metastasis, or the process in which tumor cells spread and grow from a primary tumor site to a distant secondary site, is a significant problem in cancer research today. Metastases have also been shown to causes 90% of all cancer-related deaths, i.e. half-a- million people in the US each year. Detection of circulating tumor cells (CTCs) in whole blood demonstrates that there is a connection between the primary tumor and metastases. Therefore, there is a need to create technologies to enable biological CTC studies. This could contribute to understanding of the spreading of cancer and development of various new drugs and strategies. As means to isolate these rare cells conventional magnet-activated cell separation (MACS) is carried out at the macroscale, with a large external magnet surrounding a flow channel. This technology uses labeling with antibody-coated magnetic microparticles and extraction by attractive magnetic forces in order to effectively isolate the cells of interest. In recent years, there has been tremendous interest in miniaturizing the MACS process to harness the traditional advantages of microfluidic systems, namely the ability to process microliter-size sample volumes economically and portably. However, recent device designs have typically required large permanent magnets or electromagnets. These approaches have typically followed an empirical, experimental- and device- centric approach. By contrast, this dissertation represents a “bottom-up” effort to design a microfluidic MACS system where physical force balance calculations coupled, with measurements of particle and cell parameters, lead to elements of device design. This design includes external magnet design, flow channel layout, and manipulation of multiphase flows. This approach has led directly to a prototype microfluidics MACS iv system that overcomes the current limitations on external magnetic field sources. In addition, the designed microfluidic platform achieved throughputs better than the state of the art, and efficiencies and purity comparable or better than the standards in separation today. Concurrent with the rational optimization an effort to investigate the feasibility using magnetic nanoparticle as a substitute for the microparticle and sub-micron tags currently used in MACS was conducted. Magnetite (Fe3O4) particles were synthesized using traditional thermal decomposition methods, followed by a ligand exchange using the biocompatible surfactant dopamine. Although it was ultimately determined that labeling with magnetic nanoparticles would required applied magnetic fields beyond the constraints of the mathematical optimization, an interesting increase in magnetic moment was observed following this ligand exchange. Additionally, whilst characterizing the synthesized nanoparticles’ particle diameter and distribution, a novel quantitative evaluation model of the nanoparticle ensemble was outlined solely from temperature- dependent magnetization measurements. These new insights into the characteristics of nanoparticles may allow for better understanding of the synthesized ensembles for implantation in bio-nanotechnological applications. v TABLE OF CONTENTS LIST OF FIGURES …………………………………………..………………………. ix LIST OF TABLES ………………………………………………….………………… xv 1.0 INTRODUCTION ………………………………………………………………… .1 2.0 CRITICAL LITERATURE REVIEW ………………………………….…………. 7 2.1 MAGNETIC NANOPARTICLES IN BIOMEDICINE………………………………….7 2.1.1 Impact of Ligand-Exchange……………………………………………8 2.1.2 Determination of Particle Size Attributes……………………………..10 2.2 CELL SEPARATION TECHNIQUES........................................................................ 13 2.2.1 Traditional Cell Separation Techniques……………………………..14 2.2.2 MACS Approaches in Circulating Tumor Cells...................................16 2.2.3 Microfluidic Cell Separation Methods in Circulating Tumor Cell Isolation…………………………………………………………….. 20 2.2.3.1 Size-Based Microfluidic Separation ……………………… 22 2.2.3.2 Adhesion-Based Microfluidic Separation............................ 25 2.2.3.3 Microfluidic MACS............................................................... 31 2.2.4 Model-Based Magnetic Microfluidic Design...................................... 35 2.3 SUMMARY.......................................................................................................... 36 3.0 MATERIALS AND MATERIALS…………………………………………........... 38 3.1 MAGNETIC NANOPARTICLE SYNTHESIS AND EXCHANGE ................................. 38 3.1.1 Nanoparticle Synthesis..........................................................................38 3.1.2 Seed Mediated Growth………………………………………………..40 3.1.3 Ligand Exchange.................................................................................. 42 3.1.4 Characterization................................................................................... 43 3.2 EXPERIMENTAL METHODS FOR QUANTIFICATION OF KEY DEVICE PARAMETERS.................................................................................................... 45 3.2.1 Model Cell Culture Conditions and Characteristics............................ 46 3.2.2 Magnetic Particle Diameter and Characteristics................................. 46 3.2.3 Particle-Cell Attachment Density......................................................... 47 3.2.4 Fabrication of a Validation Microfluidic Separation System.............. 48 3.2.5 Validation of Sheath Flow…………………………………………… 50 vi 3.2.6 Homogeneous Cell Suspension Validation…………………………… 51 3.3 SEPARATION OF CANCER CELLS FROM HETEROGENEOUS SUSPENSION............. 52 3.3.1 Spiked Cell Experiments…………………………………………….... 52 3.3.2 Viability of Recovered Cells…………………………………………... 53 3.4 SEPARATION ON CANCER CELLS FROM WHOLE BLOOD……………………….. 54 3.4.1 Determination of the Blood-Buffer Interface………………………… 54 3.4.2 Whole Blood Cancer Cell Isolation Experiments……………………. 55 4.0 THEORETICAL FORMULATIONS……………………………………………… 56 4.1 THERMOMAGNETIC ANALYSIS DERIVED FROM THE SIZE-DEPENDENT RESPONSE OF THE MAGNETIC NANOPARTICLES TO THERMAL FLUCTUATIONS... 59 4.2 COMPUTATIONAL DESIGN APPROXIMATION TOWARDS THE DEVELOPMENT OF A MAGNETOPHORETIC CELL ISOLATION CHAMBER……………………….... 64 4.2.1 Device Geometries……………………………………………………. 64 4.2.2 Theoretical Formulation: Forces on the Cell-particle Complex………67 4.2.2.1 Magnetic Force Determination…………………………….68 4.2.2.2 Hydrodynamic Resistance Force Determination…………. 72 4.2.2.3 Joule Heating……………………………………………… 72 4.2.3 Optimization of the Channel Length Device Designs………………….75 4.2.3.1 Generation I Microfluidic Device Design………………… 76 4.2.3.2 Generation II Microfluidic Device Design………………...78 4.3 TUNING OF THE MAGNETOPHORETIC DESIGN FOR APPLICATION WITH WHOLE BLOOD SAMPLES………………………………………………………………..80 5.0 RESULTS AND DISCUSSION……………………………………………………..83