Microfluidic processes for protein separations

MICROFLUIDIC PROCESSES FOR PROTEIN SEPARATIONS LEE SU HUI, SOPHIA (B. Eng. (Hons), NUS) (M. Sc. (SMA-MEBCS), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMICAL AND PHARMACEUTICAL ENGINEERING (CPE) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2012 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _______________ Lee Su Hui, Sophia 30 June 2012 Acknowledgements First and foremost, I would like to express my sincere gratitude to my thesis advisors, Dr Saif A. Khan and Prof. T. Alan Hatton for their guidance and support. I would also like to thank my thesis committee members, Prof Raj Rajagopalan, Prof Patrick S. Doyle and Associate Prof Yang Kun Lin for their time and suggestions. I am also thankful for a great lab with positive and supportive members (Pravien, Suhanya, Swee Kun, Carl, Zahra, AJ, Dr Rahman, Reno, Arpi, and Prasanna) who are there to help me and cheer me up whenever I have difficulties or feel discouraged. I would also like to take this opportunity to thank my FYPs (Swee Kun, Carl, Loren, Taurus, Irma, and Ray) for their help and the fun times I had with them. In addition, I need to thank our collaborators for the HSSP project (Khalid and Prof Choi) for their generous help. Lastly, I must thank my wonderful family members. I am most thankful for my husband, Akasta, who has been a constant source of support and help for me throughout the course of my Ph.D. Thank you for going through all my manuscripts, and for giving me great suggestions and ideas. To my grandma, grandpa, mum and dad, thank you all for your love and for the sacrifices you have made throughout my life to give me the best. Finally, I thank National University of Singapore and Singapore-MIT Alliance for their financial support in my research. i Table of Contents Acknowledgements Summary i viii List of Tables x List of Figures xi Introduction 1.1 Protein Separation 1.2 Magnetic Separation 1.3 Liquid-Liquid Extraction 1.4 Motivation: Microfluidics for Separation 1.5 Thesis Objectives and Layout Literature Review 2.1 Protein Separation 2.1.1 Liquid-Liquid Extraction 2.1.2 Chromatography 2.2 Magnetic Particles 2.2.1 Chemical Synthesis of Magnetic Nanoclusters 10 2.2.2 Applications of Magnetic Particles 12 2.3 Aqueous Two-Phase Systems 13 2.3.1 Applications of ATPS 15 2.3.2 Mixing of Immiscible Liquids 18 2.4 Microfluidic Continuous Separations 23 2.4.1 Pinched Flow Fractionation 24 ii 2.4.2 Hydrodynamic Filtration 25 2.4.3 Continuous Flow Filtration 27 2.4.4 Deterministic Lateral Displacement 28 2.4.5 Inertial Lift and Dean Flow 29 2.4.6 Split Flow Thin (SPLITT) Fractionation 30 2.4.7 Liquid-Liquid Extraction 31 2.4.8 Free-flow Focussing Electrophoresis and Free-flow 32 2.4.9 Separation by Sound Pressure 34 2.4.10 Separation by Optical Forces 35 2.4.11 Separation by Gravity 36 2.4.12 Separation by Magnetic Fields 37 2.5 Droplet Microfluidics Isoelectric 39 2.5.1 Device Considerations 40 2.5.2 Droplet Formation 41 2.5.3 Droplet Fusion 44 2.5.4 Mixing in Droplets 46 2.5.5 Applications of Droplets Microfluidics 49 Microfluidic Continuous Magnetophoretic Protein Separation 52 3.1 Introduction 52 3.2 Focus of the Chapter 55 3.3 Concept 56 3.4 Materials and Methods 58 3.4.1 Synthesis of Magnetic Nanoclusters (MNCs) 58 iii 3.4.2 Silica Coating of MNCs 58 3.4.3 Particle Characterisation 59 3.4.4 Microfluidic Devices 59 3.4.5 Microfluidic Protein Separation 59 3.4.6 Quantification of Collected Proteins 61 3.5 Theory and Calculations 3.5.1 Calculation of Particle Trajectories 62 62 3.5.2 Finite Element Modeling (FEM) of Transverse Migration of BSA using COMSOL Multiphysics 3.3a 65 3.6 Results and Discussion 66 3.6.1 Superparamagnetic Nanoparticles 66 3.6.2 Experimental Results for Microfluidic Protein Separation 69 3.6.3 Formation of SMNC-Hb Aggregates 73 3.6.4 Calculation of Hb Recovery 75 3.6.5 Calculation of BSA Recovery 78 3.7 Enhancements in Separation Performance 79 3.8 Summary 81 Aqueous Two-Phase Microdroplets with Tunable Spatial Heterogeneity in Structure and Composition 83 4.1 Introduction 83 4.2 Focus of the Chapter 84 4.3 Concept 85 4.4 Materials and Methods 86 4.4.1 Materials 86 4.4.2 Microfluidic Devices 86 iv 4.4.3 Microfluidic Device Setup and Operation 87 4.4.4 Viscosity Measurements 88 4.5 Theory and Calculations 88 4.5.1 Calculation of Critical Thread Diameter and Comparison with Established Theory: 88 4.5.2 Calculation of Interface Thickness at Equilibrium: 4.6 Results and Discussion 4.6.1 Morphologies of ATPS Microdroplets 91 96 96 4.6.2 Construction of Dynamic Morphology Diagram 101 4.6.3 Fluid Filaments in Reticulate Microdroplets 105 4.6.4 Equilibrium Interfacial Thickness 108 4.7 Summary 109 Aqueous Two-Phase Microdroplets for Protein Partitioning 110 5.1 Introduction 110 5.2 Focus of the Chapter 111 5.3 Concept 111 5.4 Materials and Methods 112 5.4.1 Materials 112 5.4.2 Batch Partitioning 113 5.4.3 Image Intensity Calibration 115 5.4.4 Microfluidic Device Setup and Operation 117 5.4.5 Intensity Measurements within Droplets 118 5.5 Theory and Calculations 119 5.5.1 Calculation of Protein Partitioning using Two-resistance Theory224 119 v 5.6 Results and Discussion 122 5.6.1 Protein Partitioning in Batch System 122 5.6.2 Protein Partitioning in ATPS Microdroplets 124 5.6.3 Calculation of Protein Partitioning (CC) 126 5.7 Summary 128 Hierarchical Materials Synthesis at Soft All-Aqueous Interfaces 129 6.1 Introduction 129 6.2 Focus of the Chapter 130 6.3 Concept 131 6.4 Materials and Methods 134 6.4.1 Synthesis of HSSP on Hybrid Hydrophilic-superhydrophobic Nanostructured Silicon Surfaces 134 6.4.2 Characterization of HSSP 135 6.4.3 Viscosity Measurements 135 6.4.4 Microfluidic Devices 135 6.4.5 Microfluidic Device Setup and Operation 136 6.5 Results and Discussion 137 6.5.1 Hierarchically Structured Superparamagnetic Iron Oxide Particles 137 6.5.2 PAA-Fe Complex Formation: Role of PAA 139 6.5.3 Microreactor Synthesis of HSSP 144 6.6 Summary 147 Summary and Outlook 148 7.1 Thesis Contributions 148 7.2 Research Opportunities 149 vi 7.2.1 Continuous Magnetophoretic Separation Process 149 7.2.2 Microfluidic Aqueous Two-Phase System 150 7.2.3 Hierarchical Materials Synthesis at Soft All-Aqueous Interfaces 151 References 153 Publications 169 vii Summary This thesis demonstrates microfluidic-based approaches for chemical/biomolecular separation. Traditional separation methods are usually processes such as chromatography, electrophoresis, ultrafiltration or precipitation. Microfluidic continuous-flow separation techniques offer attractive alternatives to more conventional batch-based methods, and several such methods based on a variety of separation principles have been developed in recent years. Advantages of microfluidic continuous separation include continuous sample injection, continuous results readout, and integration with upstream and downstream process units. In this thesis, microfluidic continuous magnetophoretic protein separation using nanoparticle aggregates and aqueous two-phase microdroplets for protein partitioning are explored. 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Khan. “The Dynamics of High Throughput Protein Partitioning within Nanoliter Biphasic Droplets in Microfluidics” Manuscript in preparation Conferences 1. Su Hui Sophia Lee, T. Alan Hatton, and Saif A. Khan. “Functional Colloids for Continuous Separation Processes” SMA Symposium – 2009. 2. Su Hui Sophia Lee, T. Alan Hatton, and Saif A. Khan. “Microfluidic Protein Chromatography with Magnetic Nanoclusters” SMA Symposium – 2010. 3. S. H. S. Lee, and S. A. Khan. “Microfluidic moving-bed protein chromatography with silica-coated magnetic nanoclusters” 11th International Conference on Microreaction Technology, Japan – 2010. 4. Su Hui Sophia Lee, T. Alan Hatton, and Saif A. Khan. “Microfluidic Protein Chromatography with Silica-Coated Magnetic Nanoclusters” The 5th SBE International Conference on Bioengineering and Nanotechnology, Singapore – 2010. 169 5. Saif A. Khan, Su Hui Sophia Lee, Pengzhi Wang, and Swee Kun Yap. “Stirring a Cahn-Hilliard fluid in moving microdroplets” American Physical Society, 63rd Annual Meeting of the APS Division of Fluid Dynamics, California – 2010. 6. Su Hui Sophia Lee, T. Alan Hatton, and Saif A. Khan. “Microfluidic Protein Separation with Silica-coated Magnetic Nanoclusters” SMA Symposium – 2011 7. Su Hui Sophia Lee, T. Alan Hatton, and Saif A. Khan. “Dynamic field responsive nanoparticle aggregates for continuous microfluidic protein separations” International Conference on Materials for Advanced Technologies, Singapore – 2011. 8. Su Hui Sophia Lee, Pengzhi Wang, Swee Kun Yap, and Saif A. Khan. “Stirring immiscible liquids in nanoliter cavities” The 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Seattle Washington - 2011 170 [...]... often applied for separation of materials.2 The advantages of this method include high capacity, ease of scale-up and continuous operation Aqueous polymeric solutions or reversed micelle systems are commonly applied for protein separation, and separation is based on the relative partitioning of the protein and impurities in these aqueous phases 2 1.4 Motivation: Microfluidics for Separation Microfluidic. .. conditions, making them ideal for a broad range of biological applications such as extractive bioconversions28 and separation of biomolecules.29 In chapter 2, literature review on protein separation, magnetic particles, aqueous two–phase systems, microfluidic continuous separations, and droplet microfluidics, will be presented A microfluidic continuous magnetophoretic protein separation process will... interface.29, 32 This method can be applied for separating proteins from nucleic acids, polysaccharides, or other proteins, or for extracting proteins from cell debris.2 The purification procedure often involves partitioning of protein and impurities between the polymeric phases, followed by phase separation of the polymers, and polymer recovery Subsequent recovery of protein from the polymer is often carried... be applied for protein separation.33 Typically, aryl- or alkyl- ligands are conjugated to the adsorbents in HIC for protein capture, and separation is based on van der Waals interaction The density of the ligand is in the range of 5-50 mol/ml gel, and is considered low when compared to RPC This is beneficial for preserving the structure of the protein In RPC, the hydrophobic parts of the protein bind... The application of ATPS for protein refolding has been demonstrated by several groups.55-60 ATPS is attractive for protein refolding as the immiscible phases also enable separation of the refolded protein from the denatured and aggregated forms.60 Another promising application of much recent interest involves encapsulating ATPS in lipid vesicles to create experimental cell models for emulating complex... 146 xvii 1 Introduction 1.1 Protein Separation Separation and purification of proteins, peptides and other biomolecules is of major importance to the biosciences and biotechnology industries Traditional separation methods are usually processes such as chromatography, electrophoresis, ultrafiltration or precipitation.1 Macroscale continuous processes for purification of proteins include adsorptive and... of droplet flows for separation.23-25 Mary et al have demonstrated that extraction/purification process in microfluidic systems were orders of magnitude faster than conventional methods.23 1.5 Thesis Objectives and Layout The goal of this thesis is to explore the application of microfluidic systems for separation of proteins Specifically, the two systems studied in this thesis include microfluidic- magnetophoretic... isolation and concentration of proteins from materials such as salts and organic molecules, and the common techniques used are precipitation, extraction and chromatography Lastly, the fine purification of proteins is conducted by chromatography to resolve different proteins Ion-exchange, hydrophobic interaction/reverse phase and affinity chromatography are often used for separating proteins.2 In the following... include microfluidic- magnetophoretic and aqueous two-phase microdroplets for protein separation Microfluidic magnetophoresis is usually carried out under relatively mild conditions, which preserve the natural state of biological entities.8 3 Hence, it has been widely applied for cell separation, immunoassays, blood cleansing, protein extraction, and purification of carbon nanotubes.7-22 In aqueous... Chromatography Ion exchange chromatography is commonly applied for protein separation and separation is based on Coulombic interaction.33 The charged amino acid 6 residues on different protein surfaces allow their adsorptive properties to be easily manipulated by pH The adsorbed protein can be eluted by increasing the ionic strength of the buffer As most protein surface contain some hydrophobic patches, hydrophobic . MICROFLUIDIC PROCESSES FOR PROTEIN SEPARATIONS LEE SU HUI, SOPHIA (B. Eng. (Hons), NUS) (M. Sc. (SMA-MEBCS), NUS) A THESIS SUBMITTED FOR THE DEGREE OF. magnetophoretic protein separation using nanoparticle aggregates and aqueous two-phase microdroplets for protein partitioning are explored. In microfluidic continuous magnetophoretic protein separation,. The particle and protein concentrations used for this visualization experiment are nearly 10 times those used in the experimental runs for protein separation. (c) Separation performance of Hb,