Browsing by Author "Yellen, Benjamin B"

The advent of increasingly proficient cell handling tools has led to a drastic maturation of our understanding of life on the microscale. Thus far, impressive strides have been made towards creating efficient and compact systems for manipulating cells on-chip. Multiple exciting cell handling methodologies have been explored and incorporated into cell separation and analysis tools ranging from passive hydrodynamic based systems to active magnetic and optoelectronic systems. Of these tools, acoustofluidic technologies, which employ the use of sound waves to manipulate cells in microfluidic environments, show great promise. These technologies offer a myriad of benefits including labeled or unlabeled cell manipulation, gentle handling of cells, long rang manipulation of cells, and simplified fabrication compared to other active systems (e.g. magnetics, optoelectronics). Accordingly, in this dissertation I develop novel acoustofluidic tools that can combine with existing technologies and expand upon the array of systems available to scientists in biology and medicine for cell handling.

In my first experimental chapter, I characterize and develop elastomeric magnetic microparticles that can be used in future applications for multi-target cell separation from complex mixtures. These particles are comprised of a varying loading of magnetic nanoparticles evenly and stably distributed throughout a silicone matrix. Consequently, these particles uniquely exhibit a “dual contrast”, whereby they undergo positive magnetophoresis and negative acoustophoresis in water. Further, I show that these particles can be functionalized with biomolecules via chemical modification by linking a biotin group to the surface-accessible amine groups on the particles using carbodiimide chemistry. These functionalized particles can then non-specifically or non-covalently bind to streptavidin molecules. Additionally, I characterize both the magnetic and acoustic properties of these particles by quantifying the magnetic susceptibilities and extent of acoustic focusing after 3 seconds for each particle formulation (i.e. 3 wt. % magnetite, 6 wt. % magnetite, 12 wt. % magnetite, 24 wt. % magnetite, and 48 wt. % magnetite in solids), respectively. Finally, I demonstrate a simple ternary separation of my 12 wt. % formulation from unlabeled human umbilical vein cells (HUVEC, non-magnetic) and magnetic beads that exhibit a positive acoustic contrast using magnetic and acoustic-based separations to highlight the unique properties of the mNACPs.

In my second experimental chapter I characterize and develop a novel acoustofluidic chip that employs the use of a trap and transfer approach to organize a high-density array of single cells in spacious compartments. My approach is based on exploiting a combination of microfluidic weirs and acoustic streaming vortices to first trap single cells in specific locations of a microfluidic device, and then transfer the cells into adjacent low shear compartments with an acoustic switch. This highly adaptable, compact system allows for imaging with standard bright field and fluorescence microscopes, and can array more than 3,000 individual cells on a chip the size of a standard glass slide. I optimize the hydrodynamic resistance ratios through the primary trap site, the bypass channel, and the adjacent compartment region such that particles first enter the trap, subsequent particles enter the bypass, and particles enter the compartment regions of a clean acoustofluidic chip upon acoustic excitation. Further, I optimize the acoustic switching parameters (e.g. frequency and voltage), and prove that acoustic switching occurs due to the generation of steady streaming vortices using particle tracking methods. Uniquely, my system demonstrates for the first time the manipulation of single cells with an array of streaming vortices in a highly parallel format to compartmentalize cells and generate a single cell array.

Finally, for my third experimental chapter, I demonstrate the biological relevance of the acoustofluidic chip I designed in my third chapter. First, I determine the trapping and arraying efficiencies of cells in my acoustofluidic chip to be 80 and 67 % respectively. Here, the arraying efficiency represents the percentage of single cells in the compartment regions and is dependent on both the trapping efficiency and the acoustic switching efficiency (which is roughly 84 %). Additionally, I observe the adhesion, division, and escape of single PC9 cells from the compartment regions of my acoustofluidic chip at 8 hour increments over 24 hours and identify potential obstacles for quantitative analysis of cell behavior for motile populations. In these studies, I found that it is possible to incubate arrayed single cells on-chip. Finally, I demonstrate that single cells can be stained on chip in a rapid and facile manner with ~ 100 % efficiency either before or after adhesion to the surface of the microfluidic chip.

While the studies described herein address but a small fraction of the wider need for next- generation cellular manipulation and analysis tools, I present meaningful knowledge that can expand our understanding of the utility of acoustofluidic devices. Importantly, I (i) characterize for the first time a new type of particle that exhibits both negative acoustic contrast and positive magnetic contrast and (ii) develop a novel acoustofluidic chip that exploits the use of steady acoustic streaming vortices to generate a single cell array.

Pattern formation is a mysterious phenomenon occurring at all scales in nature. The beauty of the resulting structures and myriad of resulting properties occurring in naturally forming patterns have attracted great interest from scientists and engineers. One of the most convenient experimental models for studying pattern formation are colloidal particle suspensions, which can be used both to explore condensed matter phenomena and as a powerful fabrication technique for forming advanced materials. In my thesis, I have focused on the study of colloidal patterns, which can be conveniently tracked in an optical microscope yet can also be thermally equilibrated on experimentally relevant time scales, allowing for ground states and transitions between them to be studied with optical tracking algorithms.

In particular, I have focused on systems that spontaneously organize due to particle-surface and particle-particle interactions, paying close attention to systems that can be dynamically adjusted with an externally applied magnetic or acoustic field. In the early stages of my doctoral studies, I developed a magnetic field manipulation technique to quantify the adhesion force between particles and surfaces. This manipulation technique is based on the magnetic dipolar interactions between colloidal particles and their "image dipoles" that appear within planar substrate. Since the particles interact with their own images, this system enables massively parallel surface force measurements (>100 measurements) in a single experiment, and allows statistical properties of particle-surface adhesion energies to be extracted as a function of loading rate. With this approach, I was able to probe sub-picoNewton surface interactions between colloidal particles and several substrates at the lowest force loading rates ever achieved.

In the later stages of my doctoral studies, I focused on studying patterns formed from particle-particle interaction, which serve as an experimental model of phase transitions in condensed matter systems that can be tracked with single particle resolution. Compared with other research on colloidal crystal formation, my research has focused on multi-component colloidal systems of magnetic and non-magnetic colloids immersed in a ferrofluid. Initially, I studied the types of patterns that form as a function of the concentrations of the different particles and ferrofluid, and I discovered a wide variety of chains, rings and crystals forming in bi-component and tri-component systems. Based on these results, I narrowed my focus to one specific crystal structure (checkerboard lattice) as a model of phase transformations in alloy. Liquid/solid phase transitions were studied by slowly adjusting the magnetic field strength, which serves to control particle-particle interactions in a manner similar to controlling the physical temperature of the fluid. These studies were used to determine the optimal conditions for forming large single crystal structures, and paved the way for my later work on solid/solid phase transitions when the angle of the external field was shifted away from the normal direction. The magnetostriction coefficient of these crystals was measured in low tilt angle of the applied field. At high tilt angles, I observed a variety of martensitic transformations, which followed different pathways depending on the crystal direction relative to the in-plane field.

In the last part of my doctoral studies, I investigated colloidal patterns formed in a superimposed acoustic and magnetic field. In this approach, the magnetic field mimics "temperature", while the acoustic field mimics "pressure". The ability to simultaneously tune both temperature and pressure allows for more efficient exploration of phase space. With this technique I demonstrated a large class of particle structures ranging from discrete molecule-like clusters to well ordered crystal phases. Additionally, I demonstrated a crosslinking strategy based on photoacids, which stabilized the structures after the external field was removed. This approach has potential applications in the fabrication of advanced materials.

My thesis is arranged as follows. In Chapter 1, I present a brief background of general pattern formation and why I chose to investigate patterns formed in colloidal systems. I also provide a brief review of field-assisted manipulation techniques in order to motivate why I selected magnetic and acoustic field to study colloidal patterns. In chapter 2, I present the theoretical background of magnetic manipulation, which is the main technique used in my research. In this chapter, I will introduce the basic knowledge on magnetic materials and theories behind magnetic manipulation. The underlining thermodynamic mechanisms and theoretical/computational approaches in colloidal pattern formation are also briefly reviewed. In Chapter 3, I focus on using these concepts to study adhesion forces between particle and surfaces. In Chapter 4, I focus on exploring the ground states of colloidal patterns formed from the anti-ferromagnetic interactions of mixtures of particles, as a function of the particle volume fractions. In Chapter 5, I discuss my research on phase transformations of the well-ordered checkerboard phase formed from the equimolar mixture of magnetic and non-magnetic beads in ferrofluid, and I focus mainly on phase transformations in a slowly varying magnetic field. In Chapter 6, I discuss my work on the superimposed magnetic and acoustic field to study patterns formed from monocomponent colloidal suspensions under vertical confinement. Finally, I conclude my thesis in Chapter 7 and discuss future directions and open questions that can be explored in magnetic field directed self-organization in colloidal systems.

This thesis will investigate previously unexplored concepts in magnetic manipulation including controlling the assembly of magnetic and nonmagnetic particles either in bulk fluid or near a substrate. Both uniform glass interfaces and substrates with magnetic microstructures are considered. The main goal of this work is to discuss new strategies for implementing magnetic assembly systems that are capable of exquisitely controlling the positions and orientations of single-component as well as multi-component particle suspensions, including both magnetic and non-magnetic particles. This work primarily focuses on controlling spherical particles; however, there are also several demonstrations of controlling anisotropically shaped particles, such as microrods and Janus colloids.

Throughout this work, both conventional magnetophoresis and inverse magnetophoresis techniques were employed, the latter relying on ferrofluid, i.e. a suspension of magnetic nanoparticles in a nonmagnetic carrier fluid, which provides a strong magnetic permeability in the surrounding fluid in order to manipulate effectively non-magnetic materials. In each system it was found that the dimensionless ratio between magnetic energy and thermal energy could be successfully used to describe the degree of control over the positions and orientations of the particles. One general conclusion drawn from this work is that the ferrofluid can be modeled with a bulk effective permeability for length scales on the order of 100 nm. This greatly reduces modeling requirements since ferrofluid is a complex collection of discrete nanoparticles, and not a homogenous fluid. It was discovered that the effective magnetic permeability was often much larger than expected, and this effect was attributed to particle aggregation which is inherent in these systems. In nearly all cases, these interactions caused the ferrofluid to behave as though the nanoparticles were clustered with an effective diameter about twice the real diameter.

The principle purpose of this thesis is to present novel systems which offer the ability to manipulate and orient multi-component spherical or anisotropic particle suspensions near surfaces or in the bulk fluid. First, a novel chip-based technique for transport and separation of magnetic microparticles is discussed. Then, the manipulation of magnetic nanoparticles, for which Brownian diffusion is a significant factor, is explored and modeled. Parallel systems of nonmagnetic particles suspended in ferrofluid are also considered in the context of forming steady state concentration gradients. Next, systems of particles interacting with planar glass interfaces are analyzed, modeled, and a novel application is developed to study the interactions between antigen-antibody pairs by using the self-repulsion of non-magnetic beads away from a ferrofluid/glass interface. This thesis also focuses on studying the ability to manipulate particles in the bulk fluid. First, simple dipole-dipole aggregation phenomenon is studied in suspensions of both nonmagnetic polystyrene particles and endothelial cells. For the sizes of particles considered in these studies, currently accepted diffusion limited aggregation models could not explain the observed behavior, and a new theory was proposed. Next, this thesis analyzed the interactions that exist in multi-component magnetic and nonmagnetic particle suspensions, which led to a variety of novel and interesting colloidal assemblies. This thesis finally discusses the manipulation of anisotropic particles, namely, the ability to control the orientation of particles including both aligning nonmagnetic rods in ferrofluid as well as achieving near-holonomic control of Janus particles with optomagnetic traps. General conclusions of the viability of these techniques are outlined and future studies are proposed in the final chapter.

Single-cell analysis (SCA) tools have important applications in the analysis of phenotypic heterogeneity, which is difficult or impossible to analyze in bulk cell culture or patient samples. SCA tools thus have a myriad of applications ranging from better credentialing of drug therapies to the analysis of rare latent cells harboring HIV infection or in Cancer. However, existing SCA systems usually lack the required combination of programmability, flexibility, and scalability necessary to enable the study of cell behaviors and cell-cell interactions at the scales sufficient to analyze extremely rare events. To advance the field, I have developed a novel, programmable, and massively-parallel SCA tool which is based on the principles of computer circuits. By integrating these magnetic circuits with microfluidics channels, I developed a platform that can organize a large number of single particles into an array in a controlled manner.

My magnetophoretic circuits use passive elements constructed in patterned magnetic thin films to move cells along programmed tracks with an external rotating magnetic field. Cell motion along these tracks is analogous to the motion of charges in an electrical conductor, following a rule similar to Ohm’s law. I have also developed asymmetric conductors, similar to electrical diodes, and storage sites for cells that behave similarly to electrical capacitors. I have also developed magnetophoretic circuits which use an overlaid pattern of microwires to switch single cells between different tracks. This switching mechanism, analogous to the operation of electronic transistors, is achieved by establishing a semiconducting gap in the magnetic pattern which can be changed from an insulating state to a conducting state by application of electrical current to an overlaid electrode. I performed an extensive study on the operation of transistors to optimize their geometry and minimize the required gate currents.

By combining these elements into integrated circuits, I have built devices which are capable of organizing a precise number of cells into individually addressable array sites, similar to how a random access memory (RAM) stores electronic data. My programmable magnetic circuits allow for the organization of both cells and single-cell pairs into large arrays. Single cells can also potentially be retrieved for downstream high-throughput genomic analysis.

In order to enhance the efficiency of the tool and to increase the delivery speed of the particles, I have also developed microfluidics systems that are combined with the magnetophoretic circuits. This hybrid system, called magnetomicrofluidics, is capable of rapidly organizing an array of particles and cells with the high precision and control. I have also shown that cells can be grown inside these chips for multiple days, enabling the long-term phenotypic analysis of rare cellular events. These types of studies can reveal important insights about the intercellular signaling networks and answer crucial questions in biology and immunology.

Phase transitions represent a fascinating condensed matter physics phenomenon; however, the study of phase transitions at the microscopic level is challenging because of the difficulty in tracking individual atoms, which cannot be resolved in an optical microscope. To solve this problem, colloidal particles are often used to model these phase transitions because of the ease in tracking individual particles with optical microscopy and their ability to equilibriate at room temperature on experimentally accessible time scales (i.e., minutes to hours). However, most of the existing colloidal systems are not easily tunable, which makes it difficult to control and study phase transitions.

The goal of my thesis is to develop a magnetically tunable system for studying phase transitions using a monolayer of magnetic colloidal particles, which self-assemble under the influence of an external time-varying magnetic field. In this dissertation, I have both an engineering goal of developing experimental techniques that can grow sufficiently large colloidal crystals, and a scientific goal of studying the kinetics of phase transitions, paying particularly close attention to the early crystallization dynamics which my system is uniquely poised to probe. I have used this experimental apparatus to study the phase transitions in densely packed mono-component and bi-component colloidal monolayers. In both of these systems I have used magnetic fields to adjust the interactions between colloidal particles and image tracking algorithms to follow the system dynamics.

In the following chapters, I will describe the methods I have used to characterize crystal growth rates, and the mechanisms for how crystals heal, with the key points are summarized as follows. First, the ability to form large single crystals is fundamentally limited by impurities, such as the presence of random large or small particle contaminants, particle doublets, and particles that are randomly pinned to the substrate. When these impurities or defects are present even at concentrations as low as a few percent, it dramatically reduces the size of the attainable crystals. Second, I have showed that long-range magnetic interactions can produce complex phase diagrams that have both critical points and triple points, and that it is possible to move between the different phases on the fly by adjusting the strength of the magnetic field. This ability can be used to study the early dynamics of melting and solidification processes. Finally, I have used the system to find unique pathways that occur during the healing of colloidal crystal. One of these mechanisms involves both lattice slip and rotation, which does not appear to have been reported previously. This colloidal system thus has many potential applications both as a method to fabricate new materials and as a fundamental model for materials science.