Optimizing the functionalization of super-paramagnetic nanobeads for droplet-based protein secretion profiling of immune cells

The extremely dynamic functions of an active immune response are mainly determined by the presence of various soluble factors. The secreted factors organize the individual immune cells into functional tissues regulating the immune response. Dynamical single-cell studies are therefore fundamental to investigate the heterogeneity of immune cells’ behaviors over time. The available tools lack either of single cell resolution or time resolution; new tools are required to access both single cell and time resolution in order to better understand these dynamical single cell mechanisms. The powerful resolution and sensitivity improvement provided by droplet-based microfluidics deciphering the dynamic processes at the single-cell level could help to describe the fundamental mechanisms underlying immunity, develop new strategies for vaccination and cancer immunotherapy, or diagnose inflammatory diseases. The use of functionalized paramagnetic nanoparticles is a key feature of our droplet-based immunoassay. This work describes the optimization and comparison of various functionalization methods for the integration of ready-to-use super-paramagnetic nanobeads into our droplet-based assay. Among the three functionalization tested methods, the most promising one is based on boronic acid chemistry, in terms of both compatibility with the droplet microfluidic system and detection sensitivity. It was successfully applied to the characterization of TNFα secretion from human single monocytes.