Multiplexed Immunoassay Using Quantum Dots to Monitor Proteins Secreted from Single Cells at Near-Single Molecule...

Single-cell secretion studies find important applications in molecular diagnostics, therapeutic target identification, and basic biology research. One increasingly important area of research is non-genetic cellular heterogeneity, a phenomenon that can be studied by assessing secretion of soluble effector proteins from single cells. This is particularly impactful for immune cells, as secreted proteins such as cytokines, chemokines, and growth factors are the gold standard for identifying phenotype. Current methods that rely upon immunofluorescence suffer from low detection sensitivity, requiring thousands of molecules to be secreted per cell. We have developed a quantum dot (QD)-based single-cell secretion analysis platform that can be used in different sandwich immunoassay formats to dramatically lower detection threshold, such that only one to a few molecules need be secreted per cell. We have also expanded this work to include multiplexing capabilities for different cytokines and employed this platform to study macrophage polarization under different stimuli at the single-cell level.