Compartmentalized Suspension Array for the Isothermal, Digital, and Multiplex Detection of microRNAs

The sensitive and accurate detection of panels of microRNA molecules is critical to enable their use as disease biomarkers. While microarrays and next-generation sequencing allow comprehensive miRNA profiling, they generally suffer from low accuracy and sensitivity. Conversely, digital bioassays such as digital PCR provide an absolute quantification with unrivaled sensitivity, although with limited multiplexing capabilities. In this work, we describe a novel microRNA profiling procedure, termed Digiplex, that combines the multiplexing power of a DNA-grafted suspension array with the accuracy of a digital flow cytometry readout. microRNAs are first captured on fluorescently encoded DNA-grafted particle populations following a Poisson distribution. The particles are subsequently isolated in microfluidic droplets, where single captured miRNA molecules trigger an isothermal exponential amplification that ultimately activates a fluorescent probe on the particle surface. Flow cytometry analysis yields the ratio of positive to negative particles for each targeted microRNA, allowing the reconstruction of the multiplex concentration profile in one go. After optimizing the workflow on a single microRNA model, we successfully developed a 10-plex assay with femtomolar sensitivity. The Digiplex method was finally validated on total RNA samples and benchmarked against droplet digital PCR.