Circular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. Herein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. This represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

中文翻译：

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四面体DNA纳米结构修饰的微流控芯片传感circRNA


环状核糖核酸 (circRNA) 是一类具有疾病相关表达的共价闭合非编码 RNA，使其成为有前景的诊断和预后生物标志物。生物样品中circRNA的准确定量是其临床应用的必要条件。迄今为止，开发的检测circRNA的方法包括RNA印迹、逆转录定量聚合酶链反应（RT-qPCR）、微阵列分析和RNA测序。这些方法普遍存在样本消耗大、流程繁琐、选择性低等缺点，导致circRNA定量不准确。人们认为上述缺点可以通过微流体传感器的构造来消除。在此，首次利用四面体DNA纳米结构（TDN）作为识别探针的骨架，以靶标引发的杂交链式反应（HCR）作为信号放大策略，构建了用于circRNA分析的微流控传感器。在存在 circRNA 的情况下，识别探针以 circRNA 特异性反向剪接点 (BSJ) 为目标。然后捕获的 circRNA 通过与末端用 6-FAM 标记的两个发夹物种反应来触发 HCR，产生带有丰富荧光标记的长 DNA 链。通过使用circ_0061276作为模型circRNA，该方法已被证明能够检测阿托摩尔浓度的circRNA。它还消除了线性 RNA 对应物的干扰，显示出对 circRNA 的高选择性。检测过程可以等温进行，不需要昂贵的复杂仪器。此外，该生物传感器在分析从癌细胞提取的总RNA中的circRNA靶标方面表现出良好的性能。 这代表了第一个用于检测 circRNA 的微流体系统。该生物传感器具有使用方便、成本低、样品消耗少、灵敏度和特异性高、在复杂生物基质中可靠性好等优点，为床旁应用场景中的circRNA分析和相关疾病诊断提供了便捷的工具。