Microscale Tools for Targeted Protein Cytology

Direct measurement of proteins in single cells is necessary in the study of human biology and disease. Protein signaling underpins a variety of cellular processes, such as stem cell differentiation, cell migration, and cancer metastasis. Extracellular factors such as paracrine signaling, biophysical properties of the substrate, and cell-cell interactions are known to regulate a variety of signaling processes. Yet, challenges in selectively measuring protein targets exist, and measuring signaling in adherent cells remains elusive as most conventional approaches to study single cell signaling involve cell detachment. To address these measurement gaps, we introduce methods to manipulate the cell-extracellular microenvironment and design new technologies for assessing protein signaling in adherent cells./nFirst, to determine how the biophysical environment influences cell behavior, we designed hydrogels of varying stiffness and evaluated how stiffness modulates reprogramming efficiency. We fabricated tunable hydrogels with dynamic stiffness modulation using DNA crosslinkers and demonstrated how stiffness modulation can enhance reprogramming of murine fibroblasts. These findings provide new insights into biophysical regulation of cellular reprogramming./nNext, we designed and characterized a suite of novel electrophoretic cytometry tools for highly selective analysis of proteins from single cells. We first introduced a microfluidic platform to perform isoelectric focusing of single adherent cells. By spatially separating proteins by isoelectric point, we conferred higher selectivity compared to conventional immunoassays. We characterized the separation performance of the assay and demonstrated detection of protein signaling events and surface receptors that are otherwise undetectable by conventional measurement techniques. Next, we leveraged the automation and facile handling of centrifugal microfluidics to design a microscale/ndevice to capture rare cells. We demonstrated photopolymerization of polyacrylamide gels within single-cell capture arrays for enhanced cell capture and possible downstream electrophoresis. Finally, we discussed expandability of electrophoretic cytometry tools to study alternatively-spliced protein isoforms. We investigated fundamental limitations and tradeoffs in assay design parameters./nIn addition to assay design, we developed a set of image processing scripts to analyze image data from non-arrayed protein separations. Building on existing algorithms for protein separation data, we expanded the capability of the analysis scripts to first segment protein peaks and then generate defined regions of interest for peak quantitation. Quality control checks were performed, guided by user interaction./nFinally, we proposed a model for understanding physical parameters that influence analytical sensitivity of electrophoretic cytometry. Sensitivity is a key metric in single-cell assays, as high concentrations must be maintained to ensure assays have applicable lower limits of detection. We characterized fluid films formed between hydrogels and used numerical simulation to characterize protein transport within these films. These findings provide insights into engineering design for enhanced sensitivity and has broader implications in drug delivery and tissue engineering strategies./nOverall, in this dissertation, we employed fundamental principles of mass transport and micro-scale electrokinetics to design, characterize, and validate new technologies for selective cell manipulation and protein measurement. Future directions may expand on improving separation metrics, sensitivity, multiplexing, and/or interfacing with complementary analysis tools such as mass spectrometry or nucleotide analysis (e.g., RNA-Seq).

Advisor: Herr, Amy E