Brain natriuretic peptide (BNP) is considered to be an important biomarker of heart failure (HF) attracting attention. However, its low concentration and short half-life in blood lead to a low-sensitivity detection of BNP, which is a challenge that has to be overcome. In this work, we propose a highly specific, highly sensitive T7 RNA polymerase-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system to detect BNP via an electrochemiluminescence (ECL) sensing platform and incorporate exonuclease III (Exo III)-hairpin and dumbbell-shaped hybridization chain reaction (HCR) technologies. In this detection scheme, the ECL sensing platform possesses low background signal and high sensitivity. Firstly, the T7 promoter-initiated T7 RNA polymerase acts as a signal amplification technique to generate large amounts of RNAs that can activate CRISPR/Cas13a activity. Secondly, CRISPR/Cas13a is able to -cleave the surrounding trigger strand to produce DNA1. Thirdly, DNA1 is involved in the co-amplification reaction of Exo III and hairpin DNA, which subsequently triggers a dumbbell-shaped HCR technology. Eventually, a large number of Ru (II) molecules are inserted into the interstitial space of the dumbbell-shaped HCR to generate a strong ECL signal. The CRISPR/Cas13a possesses outstanding specificity for a single base and increased sensitivity. The tightly conformed dumbbell-shaped HCR provides higher sensitivity than the traditional linear HCR amplification technique. Ultimately, the clever combination of several amplification reactions enables the limit of detection (LOD) as low as 3.2 fg/mL. It showed promise for clinical sample testing, with recovery rates ranging from 98.4% to 103% in 5% human serum samples. This detection method offered a valuable tool for early HF detection, emphasizing the synergy of amplification strategies and specificity conferred by CRISPR/Cas13a technology.

中文翻译：

---

探索 T7 RNA 聚合酶辅助 CRISPR/Cas13a 扩增通过电化学发光传感平台检测 BNP

脑钠肽（BNP）被认为是引起关注的心力衰竭（HF）的重要生物标志物。然而，其在血液中浓度低、半衰期短，导致BNP检测灵敏度低，这是一个必须克服的挑战。在这项工作中，我们提出了一种高度特异性、高度敏感的 T7 RNA 聚合酶辅助的成簇规则间隔短回文重复序列 (CRISPR)/Cas13a 系统，通过电化学发光 (ECL) 传感平台检测 BNP，并结合核酸外切酶 III (Exo III)-发夹和哑铃形杂交链式反应（HCR）技术。在该检测方案中，ECL传感平台具有低背景信号和高灵敏度。首先，T7启动子启动的T7 RNA聚合酶充当信号放大技术，产生大量可以激活CRISPR/Cas13a活性的RNA。其次，CRISPR/Cas13a 能够切割周围的触发链以产生 DNA1。第三，DNA1参与Exo III和发夹DNA的共扩增反应，随后触发哑铃形HCR技术。最终，大量Ru(II)分子被插入到哑铃形HCR的间隙中，产生强烈的ECL信号。 CRISPR/Cas13a 对单碱基具有出色的特异性和更高的灵敏度。紧密贴合的哑铃形 HCR 比传统的线性 HCR 扩增技术具有更高的灵敏度。最终，多种扩增反应的巧妙组合使检测限 (LOD) 低至 3.2 fg/mL。它在临床样品测试中显示出良好的前景，5% 人血清样品的回收率范围为 98.4% 至 103%。该检测方法为早期 HF 检测提供了一种有价值的工具，强调了扩增策略和 CRISPR/Cas13a 技术赋予的特异性的协同作用。