Microparticle Delivery of Protein Markers for Single‐Cell Western Blotting from Microwells

Abstract Immunoblotting confers protein identification specificity beyond that of immunoassays by prepending protein electrophoresis (sizing) to immunoprobing. To accurately size protein targets, sample analysis includes concurrent analysis of protein markers with known molecular masses. To incorporate protein markers in single‐cell western blotting, microwells are used to isolate individual cells and protein marker‐coated microparticles. A magnetic field directs protein‐coated microparticles to >75% of microwells, so as to 1) deliver a quantum of protein marker to each cell‐laden microwell and 2) synchronize protein marker solubilization with cell lysis. Nickel‐coated microparticles are designed, fabricated, and characterized, each conjugated with a mixture of histidine‐tagged proteins (42.3–100 kDa). Imidazole in the cell lysis buffer solubilizes protein markers during a 30 s cell lysis step, with an observed protein marker release half‐life of 4.46 s. Across hundreds of individual microwells and different microdevices, robust log‐linear regression fits (R² > 0.97) of protein molecular mass and electrophoretic mobility are observed. The protein marker and microparticle system is applied to determine the molecular masses of five endogenous proteins in breast cancer cells (GAPDH, β‐TUB, CK8, STAT3, ER‐α), with <20% mass error. Microparticle‐delivered protein standards underpin robust, reproducible electrophoretic cytometry that complements single‐cell genomics and transcriptomics.