Encoded hydrogel microparticles mounting DNA probes are powerful tools for high-performance microRNA (miRNA) detection in terms of sensitivity, specificity, and multiplex detection capability. However, several particle rinsing steps in the assay procedure present challenges for rapid and efficient detection. To overcome this limitation, we encapsulated dense magnetic nanoparticles to reduce the rinsing steps and duration via magnetic separation. A large number of magnetic nanoparticles were encapsulated into hydrogel microparticles based on a discontinuous dewetting technique combined with degassed micromolding lithography. In addition, we attached DNA probes targeting three types of miRNAs related to preeclampsia to magnetically encoded hydrogel microparticles by post-synthesis conjugation and achieved sensitivity comparable to that of conventional nonmagnetic encoded hydrogel microparticles. To demonstrate the multiplex capability of magnetically encoded hydrogel microparticles while maintaining the advantages of the simplified rinsing process when addressing multiple samples, we conducted a triplex detection of preeclampsia-related miRNAs. In conclusion, the introduction of magnetically encoded hydrogel microparticles not only allowed efficient miRNA detection but also provided comparable sensitivity and multiplexed detectability to conventional nonmagnetic encoded hydrogel microparticles.

Multiplex detection of protein biomarkers in biological fluids facilitates high-throughput detection using small-volume samples, thereby enhancing efficacy of diagnostic assays and proteomic studies. Graphically encoded hydrogel microparticles conjugated with capture antibodies have shown great potential in multiplex immunoassays by providing superior sensitivity and specificity, a broad dynamic range, and large encoding capacity. Recently, the process of post-synthesis conjugation of reduced capture antibodies to unreacted acrylate moieties in hydrogel particles has been developed to efficiently prevent the aggregation of capture antibodies inside particles, which occurs when using conventional conjugation methods. This direct conjugation process yielded robust assay performance through homogeneous conjugation of the capture antibodies, and avoided the use of hydrolytically unstable linker additives. However, no research has been conducted to optimize the process of conjugating capture antibodies to the particles. We here present a strategy to optimize capture antibody conjugation based on the finding that excessive addition of capture antibodies during incubation can rather lower the amount of capture antibodies conjugated to the particles for some types of capture antibodies. Based on our optimized capture antibody conjugation process, a singleplex immunoassay for a selected target was conducted. Enhanced sensitivity compared with previous studies was confirmed. We also validated the increased specificity of multiplex detection through our optimization process. We believe that the optimization process presented herein for capture antibody conjugation will advance the field of encoded hydrogel microparticle-based immunoassays.