Multiplexed protein analysis using encoded antibody-conjugated microbeads

We describe a method for multiplexed analysis of proteins using fluorescently encoded microbeads. The sensitivity of our method is comparable to the sensitivity obtained by enzyme-linked immunosorbent assay while only 5 µl sample volumes are needed. Streptavidin-coated, 1 µm beads are encoded with a combination of fluorophores at different intensity levels. As a proof of concept, we demonstrate that 27 microbead populations can be readily encoded by affinity conjugation using three intensity levels for each of three different biotinylated fluorescent dyes. Four populations of encoded microbeads are further conjugated with biotinylated capture antibodies and then combined and immobilized in a microfluidic flow cell for multiplexed protein analysis. Using four uniquely encoded microbead populations, we show that a cancer biomarker and three cytokine proteins can be analysed quantitatively in the picogram per millilitre range by fluorescence microscopy in a single assay. Our method will allow for the fabrication of high density, bead-based antibody arrays for multiplexed protein analysis using integrated microfluidic devices and automated sample processing.