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

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@Fe₃O₄) shows improved stability and enzyme loading, reducing autolysis risks. Using DMF-Try@Fe₃O₄, 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.