PiP-plex: A Particle-in-Particle System for Multiplexed Quantification of Secreted Proteins 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 a particles-in-particle (PiPs) concept comprising fluorescence intensity barcoded microparticles (BMPs) co-entrapped with a single cell inside an alginate hydrogel particle for multiplex protein secretion analysis (PiP-plex) and imaged by confocal microscopy. We developed a seven-plex fluorescent barcoding and concomitant sandwich immunoassay in PiPs with limits of detection ranging from 0.8 pg/mL to 2 ng/mL depending on the protein. PiP-plex assays were benchmarked with bulk immunoassays and found to rival or outperform them. We applied PiP-plex to analyze protein secreted by THP-1 cells upon exposure to lipopolysaccharide and detected varying cell responses, with a significant increase in MIP-1, TNF-{gamma}, and IL-17A. Multivariate analysis revealed that the majority of stimulated cells secreted either MIP-1 or IL-17A, while other cytokines were typically co-secreted. PiP-plex enables the analysis of ~1000 cell/h, while retaining >90 % cellular viability, showcasing its potential for characterizing cells and cell-based therapeutics for cancer immunotherapies.