基于AND逻辑门的交替PER-Cas12a信号放大系统，用于sev的超灵敏检测。

IF 6.1 1区化学 Q1 CHEMISTRY, ANALYTICAL

Talanta Pub Date : 2025-12-01 Epub Date: 2025-05-27 DOI:10.1016/j.talanta.2025.128411

Man Wang, XuZhen Zhang, Jiayi Fan, Cuiling Zhang, Yuezhong Xian

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引用次数: 0

摘要

乳腺癌来源的小细胞外囊泡（BC-sEVs）的蛋白质生物标志物在液体活检中具有很大的前景。然而，由于其固有的异质性和低丰度，仍然具有挑战性。在此，我们开发了一种基于AND逻辑门的DNA级联信号扩增策略，称为交替引物交换反应激活Cas12a (Alt-PER-Cas12a)，用于临床样品中bc - sev的超灵敏检测。该双蛋白识别系统采用EpCAM/ muc1特异性捕获探针释放两个DNA发夹（Hep和Hmu）作为Alt-PER的and门输入。相应的Hep和Hmu发夹可以用大量引物启动Alt-PER，生成重复单位交替的长单链DNA产物。每个重复单元都充当CRISPR激活剂，诱导Cas12a的反式切割活性，并使级联信号放大。构建的策略具有良好的灵敏度，LOD为2.6 × 103个粒子/mL。在临床验证中，该方法已成功用于区分乳腺癌患者和健康供体（AUC = 0.992），并在液体活检中显示出巨大的潜力。

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AND logic gate-based alternating PER-Cas12a signal amplification system for ultrasensitive detection of sEVs.

Protein biomarkers on breast cancer-derived small extracellular vesicles (BC-sEVs) hold great promise in liquid biopsy. However, it remains challenging due to their inherent heterogeneity and low abundance. Herein, we developed an AND logic gate-based DNA cascade signal amplification strategy, termed Alternating Primer Exchange Reaction-activated Cas12a (Alt-PER-Cas12a), for the ultrasensitive detection of BC-sEVs in clinic samples. This dual-protein recognition system employs EpCAM/MUC1-specific capture probes to release two DNA hairpins (Hep and Hmu) as AND gate inputs in Alt-PER. The corresponding Hep and Hmu hairpins can initiate the Alt-PER with a large amount of primers to generate long single-stranded DNA products with alternating repeat units. Each repeating unit serves as a CRISPR activator, inducing the trans-cleavage activity of Cas12a and enabling cascade signal amplification. The as-constructed strategy exhibits excellent sensitivity with LOD of 2.6 × 10³ particles/mL. It has been successfully used to discriminate breast cancer patients from healthy donors (AUC = 0.992) in clinical validation, and shows great potential for liquid biopsy.

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来源期刊

Talanta 化学-分析化学

CiteScore

12.30

自引率

4.90%

发文量

861

审稿时长

29 days

期刊介绍： Talanta provides a forum for the publication of original research papers, short communications, and critical reviews in all branches of pure and applied analytical chemistry. Papers are evaluated based on established guidelines, including the fundamental nature of the study, scientific novelty, substantial improvement or advantage over existing technology or methods, and demonstrated analytical applicability. Original research papers on fundamental studies, and on novel sensor and instrumentation developments, are encouraged. Novel or improved applications in areas such as clinical and biological chemistry, environmental analysis, geochemistry, materials science and engineering, and analytical platforms for omics development are welcome. Analytical performance of methods should be determined, including interference and matrix effects, and methods should be validated by comparison with a standard method, or analysis of a certified reference material. Simple spiking recoveries may not be sufficient. The developed method should especially comprise information on selectivity, sensitivity, detection limits, accuracy, and reliability. However, applying official validation or robustness studies to a routine method or technique does not necessarily constitute novelty. Proper statistical treatment of the data should be provided. Relevant literature should be cited, including related publications by the authors, and authors should discuss how their proposed methodology compares with previously reported methods.