UC Berkeley

Proteins drive nearly all cellular processes, and direct quantitation of protein abundance from single-cells is essential to understanding heterogeneous cell states.1,2 Immunoassays are widely accepted tools for performing single-cell protein detection,3,4 but protein detection by immunoaffinity alone is insufficient for precision protein characterization, as proteins with similar bindingkineticscanhavedifferentbiologicalimpacts,includingindisease.5,6,7 Toprovideacross- validation tool for protein characterization, electrophoretic cytometry immunoassays have been developed to characterize proteins by both immunoaffinity and molecular-mass through electrophoresis.8,9 Central to these assays performance is a multifunctional gel matrix that acts as a protein sieving matrix during electrophoresis and a protein scaffolding matrix during in-gel immunoblotting. In-gel immunoblotting of target proteins is widely accomplished by diffusively-driven immunoprobing, yet, this detection strategy suffers from reduced probe access to in-gel immobilized proteins via size-exclusion partitioning. Specifically, reduced probe delivery to the gel matrix in which target proteins are immobilized both (i) adversely impacts equilibrium immunocomplex formation and thus protein detection sensitivity and (ii) extends overall assay run time.In this dissertation, to improve the analytical detection capabilities and improve assay throughput in electrophoretic cytometry assays, we present methods to enhance immunoprobe delivery to hydrogel matrices, we introduce an assay design to improve throughput in single-cell immunoblotting, and we investigate reengineered sample handling designs for reduced protein losses before immobilization. Overall, we apply fundamentals in materials science, transport and reaction phenomena, and engineering design principles for the advancement of targeted protein detection assays. We see these advancements as contributing to the broader goal of improving our understanding of cell- state in healthy and disease conditions.