A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

MicroRNAs (miRNAs) are small non-coding RNA regulators linked to various human diseases incl. heart disease, a leading cause of death in Western countries. Their alterations signify the need for early detection methods. We devised a diffusion microbead assay, combining it with antibody-based miRNA detection. Diffusion involves co-diffusion of miRNAs and antibodies in a microchamber. Randomly ordered size and dye encoded microbeads loaded with specific capture probes target heart disease-associated miRNAs. MiRNA detection is time- and dose-dependent using an anti-DNA:RNA hybrid antibody. The miRNAs are successively exposed to randomly ordered microbeads, which leads to microbeads that become saturated with the target molecules first in front rows. Unbound miRNAs diffuse further and bind to microbeads with free binding sites. Our assay provides real-time multiplex detection of multiple miRNA within 2 h in an isothermal amplification-free environment, with low detection limits (miR-21-5p: 0.21 nM; miR-30a-3p: 0.03 nM; miR-93-5p: 0.43 nM). This study presents a miRNA detection principle that differs from other microbead assays where all microbeads are simultaneously mixed with the sample solution, so that all target molecules bind to microbeads equally, ultimately resulting in an averaged signal intensity.