Enzyme-free and copper-free strategy based on cyclic click chemical-triggered hairpin stacking circuit for accurate detection of circulating microRNAs.
Enzyme-free and copper-free strategy based on cyclic click chemical-triggered hairpin stacking circuit for accurate detection of circulating microRNAs.
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DOI：
10.1016/j.aca.2021.339282
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发表时间：
2021-11
影响因子：
6.2
通讯作者：
Shuang Zhao;Sha Yang;Han Xu;Xiaoqi Tang;Hongwei Wang-;Lianyu Yu;Xiaopei Qiu;Yunxia Wang;Mingxuan Gao;Kai Chang;Ming Chen
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文献类型：
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作者：
Shuang Zhao;Sha Yang;Han Xu;Xiaoqi Tang;Hongwei Wang-;Lianyu Yu;Xiaopei Qiu;Yunxia Wang;Mingxuan Gao;Kai Chang;Ming Chen
循环microRNA（miRNAs）的准确检测在各种疾病的诊断中起着至关重要的作用。然而，无酶扩增检测仍然具有挑战性。在这里，我们报告了一种无酶的荧光共振能量转移测定法，称为“3C-ASK”（循环点击化学触发发夹堆叠试剂盒），用于检测循环miRNA。在这种策略中，miRNA可以启动无铜点击化学连接反应，然后连接产物触发另一个发夹堆积回路。第一次信号放大是通过点击化学连接回路中靶miRNA的再循环实现的，第二次信号放大是通过热力学驱动的发夹堆叠回路中连接的探针的再循环实现的。通过荧光信号值对miRNAs触发的两步链式反应事件进行定量，从而实现对靶miRNAs的准确检测。3C-task很容易控制，因为在整个过程中没有酶参与。虽然简单，但该策略显示出8.63 pM的检测限的灵敏度和区分具有单碱基变异的miRNA序列的特异性。此外，通过检测稀释血浆样本中的靶miRNA，验证了该方法在复杂生物样本中的适用性。该方法实现了对miRNA的灵敏、特异检测，为无酶化学反应和DNA电路在生物传感中的广泛应用提供了新的视角。
Accurate detection of circulating microRNAs (miRNAs) plays a vital role in the diagnosis of various diseases. However, enzyme-free amplification detection remains challenging. Here, we report an enzyme-free fluorescence resonance energy transfer assay termed “3C-TASK” (cyclic click chemical-triggered hairpin stacking kit) for the detection of circulating miRNA. In this strategy, the miRNA could initiate copper-free click chemical ligation reactions and the ligated products then trigger another hairpin stacking circuit. The first signal amplification was achieved through the recycling of the target miRNA in the click chemical ligation circuit, and the second signal amplification was realized through the recycling of ligated probes in a hairpin stacking circuit driven by thermodynamics. The two-step chain reaction event triggered by miRNAs was quantified by the fluorescence signal value so that accurate detection of target miRNA could be achieved. The 3C-TASK was easily controlled because no enzyme was involved in the entire procedure. Although simple, this strategy showed sensitivity with a detection limit of 8.63 pM and specificity for distinguishing miRNA sequences with single-base variations. In addition, the applicability of this method in complex biological samples was verified by detecting target miRNA in diluted plasma samples. Hence, our method achieved sensitive and specific detection of miRNA and may offer a new perspective for the broader application of enzyme-free chemical reaction and DNA circuits in biosensing.