Open centrifugal microfluidic chip with selective surface modification for droplet-based single-cell granzyme B activity profiling

This is the authors' abstract. We don't add key points for this paper.

Monitoring immune function at the single-cell level is essential for understanding immune heterogeneity and evaluating responses to immunotherapies. Several microfluidic platforms capable of single-cell assays require external equipment and specially trained operators, limiting accessibility and routine laboratory use. This work introduces a simple, pump-free microfluidic platform designed for high-throughput single-cell trapping and functional enzymatic monitoring within isolated microenvironments. We present an open centrifugal microfluidic chip featuring an array of hexagonal microchambers with selectively oxygen-plasma-treated surfaces that enable droplet-based compartmentalization of single cells. Centrifugal actuation drives cell loading, trapping, and droplet formation, thus simplifying the operation. By optimizing loading conditions, the device achieved efficient single-cell occupancy and supported short-term live-cell analysis. Fluorescence calibration using 5-FAM established a detection limit of 3.15 pmol, demonstrating the system’s suitability for single-cell enzymatic analysis. Using PBMCs from healthy donors, the device revealed heterogeneous GzmB activity at the single-cell level and detected increased enzymatic activity following IL-2 stimulation. Immunofluorescence staining enabled the simultaneous identification of immune cell subsets, showing a corresponding increase in the proportion of CD8⁺ T cells. Moreover, its open configuration enables direct retrieval of selected cells for downstream analyses. Our open centrifugal microfluidic chip combines ease of operation, analytical sensitivity, and single-cell resolution to enable comprehensive immune profiling. By revealing functional heterogeneity within immune populations and facilitating downstream cell analysis, it holds significant potential for immune monitoring, disease prognosis, and the development of next-generation precision immunotherapies.