Key Laboratory of Embryo Development and Reproductive Regulation, School of Biological and Food Engineering, Fuyang Normal University, Fuyang, Anhui 236037, P. R. China.
Jiaxing Key Laboratory of Molecular Recognition and Sensing, College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing, Zhejiang 314001, P. R. China.
Anal Chem. 2024 Sep 3;96(35):14205-14214. doi: 10.1021/acs.analchem.4c02795. Epub 2024 Aug 22.
Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.
级联等温核酸扩增，集成了几种不同的扩增方案，以增强分析性能，广泛应用于生物传感，特别是用于检测 microRNAs（miRNAs），与肿瘤起始和进展相关的关键生物标志物。然而，在高扩增效率和设计简单性之间取得平衡仍然是一个挑战。因此，方法实现高放大效率而不显著增加复杂性是非常有利的。在这项研究中，我们提出了一种新的 miRNA 检测方法，采用交叉引发链接的分层等温扩增（CP-HIA）来逐步激活簇状规则间隔的短回文重复（CRISPR）/Cas12a 系统。CP-HIA 方法策略地结合缺口滚环扩增（n-RCA）和回文辅助圆形链置换扩增（p-CSDA）进行 miRNA 检测。值得注意的是，这种方法仅使用两个主要探针。其关键创新在于交互交叉引发策略，其中 n-RCA 的扩增产物被回收以进一步驱动 p-CSDA，反之亦然。这个交互过程建立了一个分层的扩增，显著丰富了激活探针，用于逐步 CRISPR/Cas12a 激活和随后的靶标信号扩增。因此，该方法表现出大大增强的分析性能，包括在检测低浓度 miRNA 时具有高灵敏度和特异性。因此，可以定量检测低至 1.06 fM 的 miRNA，并且 miRNA 的线性响应范围从 10 fM 到 10 nM。这些特性表明其在早期疾病诊断和监测方面的潜力。我们预计 CP-HIA 方法将成为开发用于生物医学研究的先进分子诊断工具的有前途的平台。
