PiP‐Plex: A Particle‐in‐Particle System for Multiplexed Quantification of Proteins Secreted by Single Cells

Cell signaling is modulated by the secretion of various proteins, which can be used to infer a cell's phenotype. However, these proteins cannot be readily detected in multiplex by commonly used methods at the single‐cell level. Here we present PiP‐plex a particles‐in‐particle (PiPs) syst for multiplex protein secretion analysis by confocal microscopy. PiP‐plex‐comprises (i) fluorescence intensity barcoded microparticles (BMPs) co‐entrapped with (ii) a single cell inside an alginate hydrogel particle. We found that PiPs maintained >90% cellular viability and allowed live cells retrieval. A seven‐plex fluorescent barcoding and concomitant sandwich immunoassay in PiPs was developed with limits of detection ranging from 0.8 pg mL−1 to 2 ng mL−1 depending on the protein. PiP‐plex assays were benchmarked with bulk immunoassays and found to rival or outperform them. Proteins secreted by single THP‐1 cells upon exposure to lipopolysaccharid were measured by PiP‐plex and varying cell responses detected, including a significant increase in MIP‐1α, TNF‐α, and IL‐17A; MIP‐1α and IL‐17A were the most frequently secreted cytokines, while other cytokines were typically co‐secreted. Using PiP‐plex, we analyzed ≈750 THP‐1 cells, showcasing its potential for characterizing cells and cell‐based therapeutics for e.g. cancer immunotherapies.