Bridging the gap between protein microfluidic assay and precision oncology: developing sample preparation and assay standards for robust protein measurement

Microfluidics enables high-throughput protein measurements and holds great promise in unraveling the mechanisms underlying complex biological systems, such as the estrogen receptor signaling pathways in breast cancer. However, implementation of protein cytometry for precision oncology is stymied by insufficient classification and insight of protein isoforms, high sample losses during handling, and lack of standards that evaluate device performance. In my dissertation, I address each of these challenges by applying chemistry and physics to develop and optimize single-cell electrophoretic protein assays./nLab-on-a-disc platform for sparse sample handling: To minimize cell handling losses in sparse samples from both clinical and experimental settings, we designed and developed a multi-stage assay using a lab-on-a-disc that integrates cell handling and subsequent single-cell western blotting. As the two-layer microfluidic device rotates, the induced centrifugal force directs dissociated cells to dams, which in turn localize the cells over microwells. Taking into account cell losses from loading, centrifugation, and lysis-buffer exchange, our lab-on-a-disc device handles cell samples with as few as 200 cells with 75% cell settling efficiencies. By integrating the lab-on-a-disc cell preparation and the single-cell western blot, our platform measures proteins from sparse cell samples at single-cell resolution./n\textbf{Microparticle-delivered standards for protein sizing:} To identify different protein species (i.e. isoforms) and complexes that are spatially resolved in the separation lanes of single-cell western blot, protein sizing standards are needed for thousands of separation lane to accurately measure the protein masses. Here, we developed a technique to deliver magnetic microparticles with protein sizing standards into microwells and integrate with the single-cell western blot workflow. Using nickel-histidine chemistry, we demonstrated that 80% of protein standards are released from microparticles within a time scale of in-situ cell lysis. After characterizing release and electrophoresis of protein sizing standards in hundreds of single-cell separation lanes, we successfully analyzed lane-to-lane and chip-to-chip technical variability and extracted mass sizes of cellular endogenous proteins from individual cells./nSingle-cell western blotting for refining estrogen receptor taxonomy: Estrogen receptor (ER-alpha) is a key regulator of cancer growth, and different ER-alpha isoforms have been shown to affect hormone therapy response in breast cancer (BCa). Conventional immunoassays cannot selectively measure ER-alpha isoforms, given the high amino-acid sequence overlap between all isoforms of ER-alpha. To address this challenge, we demonstrate a high-selectivity, single-cell western blot, that distinguishes full-length ER-alpha66 from truncated ER-alpha46 in single cells by prepending an electrophoretic separation of proteins to a subsequent immunoassay. Employing this assay, we measured abundance and frequency of ER-alpha isoforms in ER-alpha+/- BCa and identified a < 10% subpopulation of BCa cells with truncated ER-alpha46. Then, by treating cells with estradiol and tamoxifen, we discovered distinctive activations of ER-alpha signaling pathways from different BCa subtypes. Our single-cell quantitation of downstream ER-alpha and downstream signaling proteins have a potential to bring new therapeutic regimes for hormone-resistant cancers./nKinetic binding measurement assay: Traditional biochemical assays suffer from low reproducibility and lengthy sample handling time to prepare a large set of samples. By using a microfluidic platform, serial preparation steps are reduced by exploiting a laminar flow and channel design to create spatial gradient and uniform concentration of proteins. Using a polyacrylamide gel as an size exclusion zone, the amount of bounded molecules over a different range of initial molecule concentrations are captured. The result of bounded fraction are plotted to find a dissociation constant rate of a receptor protein and its binding molecule./nTaken together, these advancements for robust protein measurement will provide a potential to apply microfluidic protein tools for precision oncology.

Advisor: Herr, Amy E