Coreactant-free polyfluorene-based electrochemiluminescence (ECL) systems have attracted much attention and shown a good application prospect in various analyses. However, the signal regulators currently used are generally limited to hydrogen peroxide (HO). In order to expand the application of coreactant-free polyfluorene-based ECL systems, it is necessary to develop more signal regulators that are stable, cheap, efficient and easy to be immobilized. This work exploited copper ions (Cu) as highly effective ECL quenchers for coreactant-free poly[(9,9-dioctylfluorenyl-2,7-diyl)--(1,4-benzo-{2,1’,3}-thiadazole)] nanoparticles (PFBT NPs). Cu-quenched PFBT NPs coupled with tetra-way strand displacement amplification (TW-SDA)-activated CRISPR-Cas12a system for detecting microRNA-132 (miRNA-132). In the absence of miRNA-132, bidirectional SDA was triggered, producing only one functional strand S5. In this case, the trans-cleavage activity of CRISPR-Cas12a system could not be activated to cut S5. As a consequence, the hybrid chain reaction (HCR) on the electrode was triggered by S5 and Cu was enriched on the electrode surface via A-Cu-T bond, thus obtaining “signal off” state. When miRNA-132 was present, TW-SDA was triggered to produce a single-strand S5 and target strand (TS). TS activated trans-cleavage activity of CRISPR-Cas12a system for indiscriminately cutting S5. As a result, HCR on the electrode cannot be triggered and the quencher Cu cannot be brought to the electrode surface via A-Cu-T bond, thus recovering the ECL for miRNA-132 detection. The integration of Cu-quenched PFBT NPs and TW-SDA strategy endowed the ECL biosensor wide linear range (1.0 fM∼1.0 nM) and low limit of detection (147 aM). Cu-quenched PFBT NPs created a very promising ECL platform for bioassay.

中文翻译：

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Cu2+淬灭的聚芴纳米粒子耦合四路链置换扩增激活的 CRISPR-Cas12a 系统，用于无共反应物电化学发光分析

无共反应剂的聚芴基电化学发光（ECL）系统引起了广泛关注，并在各种分析中显示出良好的应用前景。然而，目前使用的信号调节器通常仅限于过氧化氢（H2O）。为了扩大无共反应剂的聚芴基ECL系统的应用，有必要开发更多稳定、廉价、高效且易于固定的信号调节剂。这项工作利用铜离子 (Cu) 作为高效 ECL 猝灭剂，用于无共反应物的聚[(9,9-二辛基芴基-2,7-二基)-(1,4-苯并-{2,1',3}-噻达唑）]纳米颗粒（PFBT NP）。 Cu 淬灭的 PFBT NP 与四路链置换扩增 (TW-SDA) 激活的 CRISPR-Cas12a 系统相结合，用于检测 microRNA-132 (miRNA-132)。在没有 miRNA-132 的情况下，双向 SDA 被触发，仅产生一条功能链 S5。在这种情况下，无法激活CRISPR-Cas12a系统的反式切割活性来切割S5。结果，S5触发了电极上的混合链式反应（HCR），Cu通过A-Cu-T键富集在电极表面，从而获得“信号关闭”状态。当 miRNA-132 存在时，TW-SDA 被触发产生单链 S5 和目标链 (TS)。 TS 激活 CRISPR-Cas12a 系统的反式切割活性，用于无差别切割 S5。结果，电极上的HCR无法被触发，猝灭剂Cu无法通过A-Cu-T键带到电极表面，从而恢复用于miRNA-132检测的ECL。 Cu淬灭的PFBT NPs和TW-SDA策略的集成赋予ECL生物传感器较宽的线性范围(1.0 fM∼1.0 nM)和低检测限(147 aM)。 Cu 淬火 PFBT NP 创建了一个非常有前途的 ECL 生物测定平台。