Microfluidic tools for single-cell protein analysis

Selective measurement of proteins from single cells is an important and challenging task in the study of cell biology. Protein signaling underlies many important cellular processes such as stem cell differentiation and tumor metastasis. However, due to the diversity of proteins and the subtlety of the chemical modifications that transduce the signals, it is challenging to selectively measure a specific protein from single cells. Further, determining the localization of proteins in individual cells is challenging due to the artifacts introduced by the chemical fixation required by current tools. To address each of these analytical gaps, we introduce two new single cell protein analysis platforms and two supporting technologies./nFirst, to eliminate fixation-induced artifacts in single-cell protein localization measurements, we developed the subcellular western blotting platform. We designed a set of buffers that enabled independent separations of the cytoplasmic and nuclear compartments of the same single cell. To enable buffer exchange without disturbing the intact cellular components, we developed a hydrogel-based lid for the delivery of lysis reagents purely by diffusion. We characterized the selectivity of the fractionation by assaying a panel of proteins with known localization and demonstrated the platform with an NF-$\kappa$B translocation assay./nNext, to enhance the selectivity of single-cell protein analysis, introduced an assay for performing single-cell isoelectric focusing. Building upon the hydrogel lid based reagent delivery system, we developed a microfluidic device that integrates sample preparation, analyte separation, and detection. We characterized the theoretical performance of the device using a fluorescent ladder and purified proteins. Further, we used the single-cell isoelectric focusing assay to separate isoforms and TurboGFP and $\beta$-tubulin from single cells. Additionally, we extended the platform by designing a lid system that generates a rectangular array of pH gradients, increasing the potential throughput of the single-cell isoelectric focusing assay./nTo enable the rapid analysis of arrayed microscale separation data, we developed a suite of image processing scripts. We wrote the scripts in a modular manner both reducing complexity and enabling facile development of new functionality. We user-tested the scripts with life science researchers with backgrounds ranging from statistics to biology. From the user feedback, we identified the quality control step to be the most susceptible to user-to-user variance. Thus, on-going work aims to develop algorithms to automate the quality control process./nFinally, we report a method to prototype microscale separation devices. Polydimethylsiloxane is a common material used for rapid prototyping of microfludic devices. However, because it is oxygen permeable, it is not suitable for polyacrylamide gel electrophoresis since oxygen inhibits free radical polymerization. Thus, we developed a method to prevent oxygen inhibition and thus enable polymerization of polyacrylamide in PDMS microchannels. We characterized the separation performance of the resulting polyacrylamide hydrogels and demonstrated separation in reversibly sealed devices./nIn summary, in this dissertation, we used our understanding of mass transport and microfabrication to design new platforms for selective single-cell protein analysis measurement. Future innovation may extend the sensitivity and multiplexing of these tools to enable highly specific measurement of all the key proteins in entire cellular signaling pathways.

Advisor: Herr, Amy E