MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases, but their detection and quantification are currently performed through complex and time consuming procedures. Herein, we demonstrate a rapid, multiplex, homogenous 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 to 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 less than 2 hours.
The RPI enabled the characterization of equilibrium and kinetics of each individual interaction involved in this multi-step process, which allowed to model and optimize the relative concentrations and the time intervals of the assay. In particular, we found that the sensitivity for a miRNA target is affected by its sequence features, like the binding affinity and the degree of self-complementarity, which indirectly regulates probe-probe interactions. The limit of detection is ultimately determined by the kinetic constant for association between the probe and the target. Therefore, the performance of the assay may be further improved by acting on the probe architecture, favouring additional stacking interactions with the miRNA.