Sink-dFCM: Microparticle Floating-to-Sinking-Enabled Digital Flow Cytometric Platform for Multiplexed microRNA Sensing

Abstract Digital bioassays, which quantify targets by binarily counting individual microreactors, render the biomarker detection at the single-molecule level, holding great promise in biomedical applications. However, the existing digital bioassays are predominantly achieved with high reliance on complex signal amplification procedures, proprietary experiment consumables, and specialized instruments for signal readout. Here, we present a new microparticle floating-to-sinking-enabled digital flow cytometric platform, termed Sink-dFCM, for the sensitive and multiplexed microRNA (miRNA) analysis with simple operation. Specifically, the polymer microparticles (MPs), acting as the single-molecule reaction carriers, float on the surface of the liquid that is at a critical density just a little bit higher than that of the MPs. While even a single miRNA-mediated one-step click chemical ligation of magnetic nanoparticles can provide enough downward force to drag the floating MPs to sink, enabling the “one miRNA-one sunken ligation complex” signal transduction. Accordingly, by immobilizing the fluorescently encoded MPs with miRNA-specific click chemical probes, different miRNA targets induce different colors of sunken events. Therefore, the multiplexed miRNA analysis can be achieved via profiling the fluorescence and number information of colored sunken events with the versatile flow cytometer. By eliminating the need for multistep signal amplification procedures, proprietary experiment necessities and dedicated instruments, Sink-dFCM not only presents a unique concept toward rapid digital multiplexed biosensing, but also extends the diagnostic toolbox with outstanding clinical translational potential.