Post-synthesis functionalized hydrogel microparticles for high performance microRNA detection

Encoded hydrogel microparticles, synthesized by Stop Flow Lithography (SFL), have shown great potential for microRNA assays for their capability to provide high multiplexing capacity and solution-like hybridization kinetics. However, due to the low conversion of copolymerization during particle synthesis, current hydrogel microparticles can only utilize ∼10% of the input probes that functionalize the particles for miRNA assay. Here, we present a novel method of functionalizing hydrogel microparticles after particle synthesis by utilizing unconverted double bonds remaining inside the hydrogel particles to maximize functional probe incorporation and increase the performance of miRNA assay. This allows covalent bonding of functional probes to the hydrogel network after particle synthesis. Because of the abundance of the unconverted double bonds and accessibility of all probes, the probe density increases about 8.2 times compared to that of particles functionalized during the synthesis. This results lead to an enhanced miRNA assay performance that improves the limit of detection from 4.9 amol to 1.5 amol. In addition, higher specificity and shorter assay time are achieved compared to the previous method. We also demonstrate a potential application of our particles by performing multiplexed miRNA detections in human plasma samples.