Acoustofluidic Innovations for Cellular Processing On-Chip

Abstract

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.