Efficient Entropy-Driven Cross-Double-Loop Reaction Cascade Asymmetric CRISPR/Cas12a Cleavage for Ultrasensitive Electrochemical Biosensing

化学 级联 清脆的 生物传感器 劈理(地质) 组合化学 纳米技术 电化学 色谱法 电极 生物化学 物理化学 材料科学 岩土工程 断裂(地质) 工程类 基因
作者
Xudong Zhou,Rui Zhang,Liang Cai,Haibing Fu,Ding Wang,Ruo Yuan,Yali Yuan
出处
期刊:Analytical Chemistry [American Chemical Society]
标识
DOI:10.1021/acs.analchem.5c02014
摘要

The limited signal amplification efficiency of the conventional CRISPR/Cas12a-cleavage system was primarily due to the structural constraints of crRNA and single-trigger activation. Herein, an efficient target-induced entropy-driven cross-double-loop strand displacement reaction (SDR) cascade asymmetric CRISPR/Cas12a cleavage was developed to construct an ultrasensitive and reliable signal-off electrochemical biosensor. The desirable entropy-driven modulation could spontaneously undergo a cross-double-loop reaction that possessed self-accelerating ability, effectively improving the rate of chain replacement and avoiding the usage of extra fuel chains with generation of two abundant distinct DNA outputs, significantly improving target conversion efficiency. More importantly, all the targets and two distinct DNA outputs could simultaneously act as activators in the asymmetric CRISPR/Cas12a system, which cooperatively bound to both split and full-sized crRNAs to accomplish the highly efficient discharge of ferrocene-labeled single-stranded DNA (Fc-reporter) on the electrode, thereby markedly improving the detection sensitivity and reliably compared to that of traditional ones. The experimental results suggested that the proposed biosensor had a wide linear range spanning from 1 fM to1 nM with a detection limit as low as 0.23 fM. By integrating entropy-driven amplification with CRISPR-enhanced signal transduction, this work established a versatile and robust analytical tool for early cancer diagnosis and precision biomolecular detection.
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