Zhou Xudong, Zhang Rui, Cai Luguang, Fu Hang, Wang Ding, Yuan Ruo, Yuan Yali
Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P.R. China.
School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China.
Anal Chem. 2025 Aug 5;97(30):16355-16363. doi: 10.1021/acs.analchem.5c02014. Epub 2025 Jul 28.
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.
传统CRISPR/Cas12a切割系统的信号放大效率有限，主要是由于crRNA的结构限制和单触发激活。在此，开发了一种高效的靶标诱导熵驱动交叉双环链置换反应（SDR）级联不对称CRISPR/Cas12a切割，以构建一种超灵敏且可靠的信号关闭型电化学生物传感器。理想的熵驱动调节能够自发地进行具有自我加速能力的交叉双环反应，有效提高链置换速率，避免使用额外的燃料链，并产生两种丰富且不同的DNA输出，显著提高靶标转换效率。更重要的是，所有靶标和两种不同的DNA输出都可以在不对称CRISPR/Cas12a系统中同时充当激活剂，它们协同结合到切割型和全长crRNA上，从而实现电极上二茂铁标记的单链DNA（Fc报告分子）的高效释放，因此与传统传感器相比，显著提高了检测灵敏度和可靠性。实验结果表明，所提出的生物传感器具有1 fM至1 nM的宽线性范围，检测限低至0.23 fM。通过将熵驱动放大与CRISPR增强的信号转导相结合，这项工作建立了一种用于早期癌症诊断和精确生物分子检测的通用且强大的分析工具。
