xMAP Array Microspheres Based Stem-Loop Structured Probes as Conformational Switches for Multiplexing Detection of miRNAs
xMAP Array Microspheres Based Stem-Loop Structured Probes as Conformational Switches for Multiplexing Detection of miRNAs
复制标题
基于 xMAP 阵列微球的干环结构探针作为构象开关，用于 miRNA 的多重检测
DOI：
10.1021/ac501989b
复制
发表时间：
2014-10-21
影响因子：
7.4
通讯作者：
Lu, Jianzhong
中科院分区：
文献类型：
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作者：
Li, Dongbei;Wang, Yinan;Lu, Jianzhong
我们设计并评估了用于荧光检测多种microRNA （miRNA）靶标的新型茎环结构探针。在初始阶段，探针呈闭茎构象，屏蔽生物素标记，使荧光报告基因无法接近。在与目标mirna杂交后，探针进行构象转换，恢复生物素对链亲和素-植青素（SA-PE）的可及性，用于信号读出。显然，报告基因SA-PE的大体积特性有助于在没有靶标的情况下屏蔽生物素标签，因此茎环结构探针可以灵敏地检测未标记的miRNA靶标，xMAP阵列微球进一步实现了使用一种荧光染料SA-PE同时检测多种分析物以获得最终读数。在这里，我们展示了一种成功的多重定量测定miRNA21、miRNA222、miRNA20a和miRNA223的方法，它们与非小细胞肺癌相关。该方法可扩展到检测各种适应症的越来越多的目标。我们相信这些进步代表了早期疾病诊断和预后的重大改善。
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.