xMAP array microspheres based stem-loop structured probes as conformational switches for multiplexing detection of miRNAs.

We have designed and evaluated novel stem-loop-structured probes for fluorescence detection of multiple microRNA (miRNA) targets. In the initial stage, the probes are in a closed stem conformation, shielding sterically a biotin label from being accessible to a fluorescence reporter. After hybridizing with target miRNAs, the probes undergo a conformational switch, restoring accessibility of the biotin to streptavidin-phycoerythin (SA-PE) for signal readout. Apparently, the bulky nature of the reporter SA-PE facilitates shielding of the biotin label in the absence of the target, thereby the stem-loop-structured probes allow sensitive detection of unlabeled miRNA targets, and xMAP array microspheres further realize simultaneous detection of multiple analytes using one fluorescence dye, SA-PE, for final readout. Here we demonstrated a successful multiplex assay for quantitative measurement of miRNA21, miRNA222, miRNA20a, and miRNA223, which are associated with nonsmall cell lung cancer. The approach can be extended to detecting an increasing number of targets for various indications. We believe such advancements represent a significant improvement for early disease diagnosis and prognosis.