Design and Optimization of a Rapid, Multiplex miRNA Assay without Washing Steps

MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases, but the procedures for their detection and quantification are currently complex and time consuming. We demonstrate a rapid, multiplex, one-pot detection method based on two-step amplification of the signal measured by a recent label-free optical biosensor, Reflective Phantom Interface (RPI). The specific capture with surface DNA probes is combined with mass amplification by an antibody targeting DNA–RNA hybrids and polyclonal secondary antibody, all performed without washing steps. Through this method, we achieved linear, sub-pM quantification of different miRNAs in 1.5 h. The RPI enabled the characterization of equilibrium and kinetics of each individual interaction involved in this multi-step process, which allowed us to model and optimize the relative concentrations and the time intervals of the assay.