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xMAP Array Microspheres Based Stem–Loop Structured Probes as Conformational Switches for Multiplexing Detection of miRNAs
journal contribution
posted on 2014-10-21, 00:00 authored by Dongbei Li, Yinan Wang, Choiwan Lau, Jianzhong LuWe
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.