The CRISPR/Cas12a system has emerged as a powerful signal switch that converts target molecules into activators for Cas12a activation, enabling the transformation of target information into detectable signals. Herein, we engineered a tweezer-shaped DNA generator for auto-cycling proximity recording (APR) amplification via DNA tetrahedron scaffold to immobilize double-stranded DNA (dsDNA) templates for the production of abundant activators, which could efficiently convert targets into activated Cas12a for signal transduction. Compared to the random hairpin generator, the tetrahedron scaffold enhanced local concentration and precise orientation of dsDNA templates due to its programmability and rigid structure, improving APR amplification efficiency. Furthermore, we prepared the electrochemiluminescence (ECL) emitters of dual-ligand metal-organic framework loaded with Pd nanoparticles (d-MOF@Pd NPs) to construct highly sensitive ECL biosensors for apurinic/apyrimidinic endonuclease 1 (APE1) detection. Initially, the dsDNA templates with locked sticky ends were anchored on the vertices of the DNA tetrahedron with apurinic/apyrimidinic (AP) sites. The target APE1 could effectively recognize AP sites to expose sticky ends, which then triggered the APR to produce numerous dsDNA activators for Cas12a activation. In parallel, dopamine-modified quenching probes (QPs) were immobilized on the functional electrode (d-MOF@Pd NPs/GCE) to achieve a low initial signal. Subsequently, the activated Cas12a triggered nonspecific cleavage of the QPs, effectively restoring the ECL signal. The APE1 biosensor exhibited a linear detection ranging from 1 × 10^-10 to 1 × 10^-4 U/μL with a detection limit of 2.06 × 10^-11 U/μL. This work provides a valuable reference for effectively transforming target information into signal output in molecular diagnostics.

中文翻译：

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一种工程化的镊子形 DNA 生成器，通过自循环邻位记录扩增激活 Cas12a，用于 APE1 活性测定


CRISPR/Cas12a 系统已成为一种强大的信号开关，可将靶分子转化为 Cas12a 激活的激活剂，从而能够将靶标信息转化为可检测的信号。在此，我们设计了一种镊子形 DNA 发生器，用于通过 DNA 四面体支架进行自循环接近记录 （APR） 扩增，以固定双链 DNA （dsDNA） 模板以产生丰富的激活剂，从而有效地将靶标转化为激活的 Cas12a 进行信号转导。与随机发夹发生器相比，四面体支架由于其可编程性和刚性结构增强了 dsDNA 模板的局部浓度和精确定向，从而提高了 APR 扩增效率。此外，我们制备了载有 Pd 纳米颗粒 （d-MOF@Pd NPs） 的双配体金属有机框架的电化学发光 （ECL） 发射器，以构建用于脱嘌呤/脱嘧啶核酸内切酶 1 （APE1） 检测的高灵敏度 ECL 生物传感器。最初，具有锁定粘性末端的 dsDNA 模板锚定在具有嘌呤/无嘧啶 （AP） 位点的 DNA 四面体的顶点上。靶标 APE1 可以有效地识别 AP 位点以暴露粘性末端，然后触发 APR 产生大量 dsDNA 激活剂以激活 Cas12a。同时，将多巴胺修饰的淬灭探针 （QP） 固定在功能电极 （d-MOF@Pd NPs/GCE） 上，以获得低初始信号。随后，激活的 Cas12a 触发了 QP 的非特异性切割，有效地恢复了 ECL 信号。APE1 生物传感器表现出 1 × ^10-10 到 1 × ^10-4 U/μL 的线性检测范围，检测限为 2.06 × ^10-11 U/μL。 这项工作为分子诊断中将靶点信息有效转化为信号输出提供了有价值的参考。