Absolute quantification of microRNAs (miRNAs) or other nucleic acid biomarkers is an important requirement for molecular and clinical biosensing. Emerging technologies with beneficial features concerning simplicity and multiplexing present an attractive route for advancing diagnostic tools toward rapid and low-cost bioanalysis. However, the actual translation into the clinic by miRNA quantification in human samples is often missing. Here, we show that implementing time-gated Förster resonance energy transfer (TG-FRET) into a catalytic hairpin assembly (CHA) can be used for the simultaneous quantification of two miRNAs with a single measurement from total RNA extracts of human tissues. A single terbium–dye FRET pair was conjugated at two specific distances within target-specific CHA hairpin probes, such that each miRNA resulted in distinct amplified photoluminescence (PL) decays that could be distinguished and quantified by TG PL intensity detection. Enzyme-free amplification in a separation-free assay format and the absence of autofluorescence background allowed for simple, specific, and sensitive detection of miR-21 and miR-20a with limits of detection down to 1.8 pM (250 amol). Reliable duplexed quantification of both miRNAs at low picomolar concentrations was confirmed by analyzing total RNA extracts from different colon and rectum tissues with single- and dual-target CHA-TG-FRET and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for comparison. These simple and multiplexed nucleic acid biomarker assays present a capable method for clinical diagnostics and biomolecular research.

中文翻译：

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使用催化发夹组装和Förster共振能量转移对人组织中的MicroRNA进行单次测量的定量分析。

microRNA（miRNA）或其他核酸生物标记物的绝对定量是分子和临床生物传感的重要要求。具有简单性和多路复用性的有益功能的新兴技术为将诊断工具推向快速且低成本的生物分析提供了一条诱人的途径。但是，通常缺少通过人类样品中的miRNA定量将其实际翻译到临床中的方法。在这里，我们显示了实现时间门控的Förster共振能量转移（TG-FRET）到催化发夹组件（CHA）中可用于同时定量两个miRNA，而只需一次测量即可从人体组织的总RNA提取物中进行。在靶标特异性CHA发夹探针中，在两个特定距离处结合了一个ter染料FRET对，这样，每个miRNA都会导致明显的扩增光致发光（PL）衰变，可以通过TG PL强度检测来区分和定量。无分离测定形式的无酶扩增和无自体荧光背景允许对miR-21和miR-20a进行简单，特异性和灵敏的检测，检测限低至1.8 pM（250 amol）。通过用单靶和双靶CHA-TG-FRET和逆转录定量聚合酶链反应（RT-qPCR）分析来自不同结肠和直肠组织的总RNA提取物，确认了低皮摩尔浓度下两种miRNA的可靠双链定量。 。这些简单且多重的核酸生物标志物测定方法为临床诊断和生物分子研究提供了一种有效的方法。