Single-Molecule\nDetection of Serum MicroRNAs for Medulloblastoma\nwith Biphasic Sandwich Hybridization-Assisted Plasmonic Resonant Scattering\nImaging

MicroRNA (miRNA) dysregulation is closely related to\nthe occurrence\nand progression of medulloblastoma (MB). However, the full potential\nof serum circulating miRNAs in MB diagnosis is restricted by their\nultralow abundance in peripheral blood due to blood-brain barrier.\nHere, we report the direct preamplification-free detection of aberrant\nexpression of oncogenic miRNAs in serum from MB patients by proposing\na simple yet robust single-molecule assay that combines <b>b</b>iphasic <b>s</b>andwich hybridization in nucleic acids and\nthe dark-field <b>s</b>ingle-<b>p</b>article <b>p</b>lasmonic <b>i</b>maging (B2S2PI). In this strategy, signal\nDNA was prehybridized with target miRNA in homogeneous solution to\nform sDNA-RNA complexes. Then the captured DNA strands with rationally\nadjusted surface densities could efficiently capture the sDNA-RNA\ncomplexes to generate a well-separated DNA-RNA sandwich structure.\nThe combination of homogeneous and heterogeneous reactions enabled\ninterface-mediated hybridization reactions to maintain molecular\nstability with fewer bases, making it suitable for the direct amplification-free\nassays of short miRNA targets. Labeling the DNA-RNA hybrids with plasmatic\ngold nanotags allowed nondestructive recognition and imaging of individual\nmiRNA targets under mild conditions with high signal-to-noise ratio.\nBy digitally counting and analyzing the bright plasmonic resonant\nscattering spots, B2S2PI enabled both the measurement of a low femtomolar\nconcentration of circulating miRNA-21 in 5 μL sample volume\nwithin a turnaround of 2 h and the discrimination of single base mismatches.\nMoreover, B2S2PI was universal for detecting miRNAs with different\nsequences and secondary structures. Further analysis of clinical serum\nsamples revealed that B2S2PI was capable of accurately distinguishing\nMB patients from noncancer controls with an area under the curve (AUC)\nof 0.99, which was superior to that of qRT-PCR. B2S2PI holds promise\nas a novel alternative means for single-molecule miRNA assay and sheds\nlight on the circulating nucleic acid-based liquid biopsy of intracranial\nmalignant tumors.