MicroRNA (miRNA) dysregulation is closely related to the occurrence and progression of medulloblastoma (MB). However, the full potential of serum circulating miRNAs in MB diagnosis is restricted by their ultralow abundance in peripheral blood due to blood-brain barrier. Here, we report the direct preamplification-free detection of aberrant expression of oncogenic miRNAs in serum from MB patients by proposing a simple yet robust single-molecule assay that combines biphasic sandwich hybridization in nucleic acids and the dark-field single-particle plasmonic imaging (B2S2PI). In this strategy, signal DNA was prehybridized with target miRNA in homogeneous solution to form sDNA-RNA complexes. Then the captured DNA strands with rationally adjusted surface densities could efficiently capture the sDNA-RNA complexes to generate a well-separated DNA-RNA sandwich structure. The combination of homogeneous and heterogeneous reactions enabled interface-mediated hybridization reactions to maintain molecular stability with fewer bases, making it suitable for the direct amplification-free assays of short miRNA targets. Labeling the DNA-RNA hybrids with plasmatic gold nanotags allowed nondestructive recognition and imaging of individual miRNA targets under mild conditions with high signal-to-noise ratio. By digitally counting and analyzing the bright plasmonic resonant scattering spots, B2S2PI enabled both the measurement of a low femtomolar concentration of circulating miRNA-21 in 5 μL sample volume within a turnaround of 2 h and the discrimination of single base mismatches. Moreover, B2S2PI was universal for detecting miRNAs with different sequences and secondary structures. Further analysis of clinical serum samples revealed that B2S2PI was capable of accurately distinguishing MB patients from noncancer controls with an area under the curve (AUC) of 0.99, which was superior to that of qRT-PCR. B2S2PI holds promise as a novel alternative means for single-molecule miRNA assay and sheds light on the circulating nucleic acid-based liquid biopsy of intracranial malignant tumors.

中文翻译：

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双相夹心杂交辅助等离子体共振散射成像对髓母细胞瘤血清 MicroRNA 进行单分子检测


MicroRNA （miRNA） 失调与髓母细胞瘤 （MB） 的发生和进展密切相关。然而，由于血脑屏障，血清循环 miRNA 在 MB 诊断中的全部潜力受到其在外周血中的超低丰度的限制。在这里，我们通过提出一种简单而稳定的单分子检测方法，将核酸中的 biphasic sandwich 杂交和暗场 single-p 文章 plasmonic i 相结合，报道了 MB 患者血清中致癌 miRNAs 异常表达的无预扩增直接检测maging （B2S2PI） 的 ming 中。在该策略中，信号 DNA 与均相溶液中的靶标 miRNA 预杂交，形成 sDNA-RNA 复合物。然后，具有合理调整表面密度的捕获 DNA 链可以有效地捕获 sDNA-RNA 复合物，以产生分离良好的 DNA-RNA 夹心结构。均相和异质反应的组合使界面介导的杂交反应能够以更少的碱基保持分子稳定性，使其适用于短 miRNA 靶标的直接无扩增检测。用质体金纳米标签标记 DNA-RNA 杂交体，可以在温和的条件下以高信噪比对单个 miRNA 靶标进行无损识别和成像。通过数字计数和分析明亮的等离子体共振散射点，B2S2PI 能够在 2 小时的周转时间内测量 5 μL 样品体积中循环 miRNA-21 的低飞摩尔浓度，并区分单碱基错配。此外，B2S2PI 通常用于检测具有不同序列和二级结构的 miRNA。 对临床血清样本的进一步分析表明，B2S2PI 能够准确区分 MB 患者和非癌症对照者，曲线下面积 （AUC） 为 0.99，优于 qRT-PCR。B2S2PI 有望成为单分子 miRNA 检测的新型替代手段，并阐明了颅内恶性肿瘤的基于循环核酸的液体活检。