Rapid Single‐Cell Proteomics Using Nanoconfined Enzyme Reactors on a Microscale Digital Microfluidics Platform

Abstract Multicellular organisms exhibit cellular heterogeneity, crucial for understanding physiological and pathological processes. Single‐cell proteomics (SCP) enables exploration of this diversity but faces challenges such as sample loss due to nonspecific adsorption and relies on free protease solutions for enzymatic digestion. Here, a microfluidic platform is reported that enhances proteomic analysis of single cells by integrating nanoconfined enzyme reactors with digital microfluidics (DMF). Trypsin immobilized on NHS‐activated magnetic beads via click chemistry (Try@Fe 3 O 4 ) shows improved stability and enzyme loading, reducing autolysis risks. Using DMF‐Try@Fe 3 O 4 , it achieves over twice the sequence coverage and four times the peptide matches for standard proteins in 10 min compared to conventional 10‐h methods. The densely packed enzymes in the nanoscale microenvironment enhance reaction rates. This system identifies 3,916 and 1,849 protein groups from 50 HeLa cells and single cells, respectively, showing 27% and 201% increases over tube digestion. The platform also classifies leukocyte subtypes (HL‐60, Jurkat, and Raji, with N = 20 for each) with SCP and identifies key upregulated proteins. Proteomic analysis of gemcitabine‐treated PANC‐1 cells reveal alterations consistent with known drug mechanisms. This approach enhances protein digestion efficiency and identification rates, offering a rapid, automated SCP solution for high‐throughput applications and broader biological investigations.