Electrokinetic manipulation of microparticles and fluids for single-cell assays in a microchamber array
Date
2023-05
Authors
Pagariya, Darshna Lalit
Major Professor
Advisor
Anand, Robbyn K
Anderson, Jared L
Nilsen-Hamilton, Marit
Reuel, Nigel F
Gundlach-Graham, Alexander
Committee Member
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Abstract
Single-cell studies have the potential to greatly improve cancer treatment by providing a more comprehensive understanding of the genetic and cellular processes that contribute to cancer growth and progression and by enabling the development of more targeted and effective therapies. For example, single-cell studies have been used to identify subpopulations of cancer cells with unique genetic profiles resistant to chemotherapy. By targeting these specific subpopulations with alternative treatments, such as immunotherapy or targeted therapies, researchers may be able to improve the effectiveness of cancer treatment. Furthermore, single-cell studies can identify new drug targets and biomarkers for cancer treatment. By analyzing the gene expression profiles of individual cancer cells, researchers can identify genes and pathways that are specifically upregulated in cancer cells compared to healthy cells. These genes and pathways can then be targeted with new therapies. Clinically tested single-cell analysis platforms such as 10X genomics, illumina are limited to nonselective cell isolation and require different instruments to perform distinct functions, making integration with resource-limited settings difficult.
In this dissertation, we address the challenges of single-cell studies by utilizing microfluidics technology with cell confinement. Specifically, we employ a chamber-based microfluidic approach that integrates wireless bipolar electrodes (BPEs). These BPEs act identical if they are identical in shape and size, enabling us to create large-scale chamber-based devices with access to BPEs for each chamber. By applying an electric current to the BPEs through the solution in which they are suspended, we can create local electric field maxima and minima in the device. At high frequencies, the BPEs act as a capacitor holding the charges; this creates a non-uniformity at the tip of each BPE that allows for the implementation of dielectrophoresis (DEP). When a dielectric material is suspended in a dielectric solution under a non-uniform electric field, the material gains a net DEP force either negatively, i.e., away from the high electric point, or positively, i.e., towards the high electric point. As the DEP response is frequency dependent, we can polarize the cell of interest and move it to a specific location based on the device geometry. This method enables us to capture single cells in a label-free, chamber-based platform.
The first section of this dissertation addresses the ongoing challenge of cancer diagnosis caused by the loss of less abundant cells. The lack of understanding of mutation detection at the single-cell level creates poor drug therapy, leading to cancer relapse. Therefore, recent trends in treatment focus on single-cell studies to increase the chances of detecting low-abundance mutations in tumors. To detect the genomic content of a single K562 cell, we use a single-cell nucleic acid amplification method, reverse transcription loop-mediated isothermal amplification (RT-LAMP). We optimized the assay mixture to account for the nonspecific adsorption of polymerase on a microfluidic device with a high surface area. Next, we tested chamber stability at elevated temperatures, which was achieved by modifying the surface and creating better sealing protocols. Finally, we tested the assay efficiency with K562 and MDA-MB-231 cells, showing a ~7-fold higher signal increase for the device with K562 than that with MDA-MB-231 cells. The results of this section will aid in better drug therapy in resource-limited settings where access to sequencing is limited and urgent results are needed to timely monitor tumor evolution.
The second section of this dissertation aims to broaden the device's scope by enabling the capture of nonpolarizable particles, specifically streptavidin-coated polystyrene beads (SPS). SPS beads are highly sought after due to their exceptional ligand affinity, surface area, and versatility. Our approach combines electroosmotic flow and electrophoretic force to capture nonpolarizable beads into chamber platforms, a unique aspect of this study. We experimented with various buffer compositions, concentrations, and bead populations to optimize the capture and transfer rate of beads in our device. Additionally, we demonstrated the co-encapsulation efficiency of cells and silver-coated glass beads using DEP. We then utilized our EP+EO method in conjunction with DEP to capture SPS and cells into the chamber, respectively. This study expands the device's versatility to manipulate conductive and non-conductive particles, providing numerous opportunities to test tunable cell-bead pairs for multiplex secretion profiling at the single-cell level.
The final chapter of this dissertation focuses on developing a versatile microfluidic chip capable of meeting the fluidic requirements of various bioassays without requiring a complete device redesign. We demonstrate the ability to achieve a range of fluid movements using electrokinetics and compare the rates to ubiquitous pressure-driven flow (PDF). We also show the rapid injection and ejection of tracers into selective chambers. We tested the electrokinetic solution exchange method in the presence of cells and beads and measured the viability and retention rates. These solution exchanges are particularly useful for bioassays such as immunoassays or nucleic acid amplification assays where sequential loading steps and delay in uniform loading can result in a variable signal at the end of the assay. Additionally, we created on-chip gradients by changing the time of DC application and flow rate, which are great for monitoring a drug's lethal dose 50 at single-cell resolution. These techniques allow researchers to test nascent ideas without designing a new microfluidic chip for each application.
Finally, our preliminary work on a single-cell immunobead-based assay is presented, using MDA-MB-231 cells incubated with anti-interleukin-8 (IL-8) coated beads. This study aims to enhance our understanding of protein secretion at the single-cell level, in line with emerging trends toward discovering new immunotherapy drugs for better cancer treatment. Our results demonstrate the potential for future research in multiplex secretory protein detection. The bead capture method can be combined with lysed single cells to capture mRNA on the bead surface, which can be further used for single-cell sequencing. Overall, the work presented in this dissertation represents promising advances in single-cell studies, with broad applications in drug discovery, signaling pathway monitoring, and the ability to perform a range of bioassays using a tunable selection of microparticles, all by using the same microfluidic platform.
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