“One-to-many” signal-output strategy-based CRISPR/Cas12a system for sensitive label-free fluorescence detection of HBV-DNA

清脆的 DNA 滚动圆复制 荧光染料 生物物理学 化学 生物 实时聚合酶链反应 DNA聚合酶 生物化学 基因
作者
Bingxin Liu,Yanli Li,Lei Du,Fengqi Zhang,Yeling Liu,Jiuming Sun,Qi Zhang,Chenzhong Li,Xia Li,Qingwang Xue
出处
期刊:Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy [Elsevier BV]
卷期号:304: 123338-123338 被引量:3
标识
DOI:10.1016/j.saa.2023.123338
摘要

Although CRISPR/Cas12a systems significantly enhance the analytical accuracy and flexibility of fluorescent biosensors, their sensitivity is limited by traditional "one-to-one" mediation types and ineffective signal-output turnover routes. Herein, we demonstrate a "one-to-many" signal-output strategy-based CRISPR/Cas12a systems resembling a "seaweed" to enhance the sensitivity. Based on dendrimer DNA from high-dimensional hybridization chain (HCR) of three hairpin-free DNA building blocks, the 3D magnetic DNA machine was created. The HBV-DNA initiates the rolling circle amplification (RCA) reaction and produces DNA nanowires to activate the CRISPR/Cas12a system. The trans-cleavage of the "seaweed root" by CRISPR/Cas12a system left dendrimer DNA in solution, thus, adding SYBR Green I (SG I) to the high-density DNA duplexes, achieving multiple-turnover label-free fluorescence signal output demonstrated and a low LOD (1.502 pM). However, in the absence of target, the blocked RCA failed to activate the CRISPR/Cas12a system, resulting in complete separation from substrate and negligible fluorescence signals. Moreover, the mandatory RCA-based pre-amplification of the DNA activator could efficiently trigger the multiple-turnover trans-cleavage activity of Cas12a. it can cleave one single-stranded linker of "seaweed-like" DNA machine, thereby releasing massive DNA duplex-enriched dendrimer DNA with a "one-to-many" signal-output turnover. By coupling the periodically extended Cas12a activator generated by RCA with hyperbranched DNA duplex by high-dimensional HCR, compact 3D extension structures were formed, achieving high-density fluorescence distribution in focal volume, avoiding signal dilution and ensuring high enhancement. Additionally, spiked recoveries in physiological media exceeded 95%, demonstrating the potential application of such platforms in clinical diagnosis.
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