Multiplexed immunoassay using post-synthesis functionalized hydrogel microparticles

In response to a growing demand for simultaneous detection of multiple proteins in a single sample, multiplex immunoassay platforms have emerged at the forefront of proteomic analysis. In particular, detections using graphically encoded hydrogel microparticles synthesized via flow lithography have received attention for integrating a hydrogel, a substrate that can provide enhanced kinetics and high loading capacity, into the bead-based multiplex platform. Currently, the method of microparticle functionalization involves copolymerization of antibodies with the gel during particle synthesis. However, its practical operation is too precarious to be adopted because antibodies are susceptible to aggregation due to incompatibility with hydrophobic photoinitiators used in the photo-induced gel polymerization. In this work, we present a multiplex immunoassay platform that uses encoded hydrogel microparticles that are functionalized after particle synthesis by conjugating antibodies with remnant active groups readily available in the hydrogels. The method not only precludes antibody aggregation but also augments the loading density of the antibodies, which translates into enhanced detection performance. In addition to multiplexing, our platform demonstrates high sensitivity, a broad assay range, and a fast detection rate that outperform the enzyme linked immunosorbent assay (ELISA).