Temperature-Responsive Agarose-Based Digital Microfluidics: An In-Chip Sample Preparation To Mass Spectrometry Analysis for Trace Cells and Single-Cell Proteomics

Mass spectrometry (MS)-based single- and trace-cell proteomics provides critical insights into cellular phenotypes, but widespread use is limited by the cost and complexity of advanced MS systems. We present a cost-effective, accessible workflow compatible with standard MS platforms and scalable for multiomics. The temperature-responsive agarose-based digital microfluidics (TRA-DMF) platform enables one-step sample processing, including lysis, reduction/alkylation, and digestion, in a parallel four-channel format. Unlike conventional droplet-based microfluidics or fluorescence-activated cell sorting (FACS) approaches, our DMF system ensures real-time visualization and confirmation of single-cell capture and 98.3% sample recovery, minimizing losses through nonpipetting transfer. The TRA-DMF system also overcomes the MS-incompatibility of oil-phase microfluidics, allowing high-efficiency droplet transfer to MS vials. The entire TRA-DMF for single cell proteomics (TRA-DMF-SCP) workflow is completed in ∼3 h (including single-cell capture), with seamless sample introduction into standard LC-MS/MS systems. Using a regular benchtop MS instrument Orbitrap Fusion, we identified over 4000 protein groups (PGs) from 50 293T cells with excellent reproducibility and robustness. This system offers a practical and scalable solution for trace- and single-cell proteomics and holds strong potential for integration into routine multiomics workflows.