Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on cooperative binding split aptamers (CBSAs). CBSAs contain two target-binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields.

中文翻译：

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使用协同结合分裂适体和酶辅助靶标回收对小分子靶标进行灵敏检测

通过酶辅助靶标回收 (EATR) 进行信号放大为提高 DNA 检测分析的灵敏度提供了一种强大的方法，但由于适体的靶标响应不敏感，事实证明将 EATR 与基于适体的检测结合用于小分子检测具有挑战性。在这里，我们描述了一种基于协同结合分裂适体（CBSA）开发快速、灵敏的 EATR 放大小分子传感器的通用方法。CBSA 包含两个靶标结合域，与单域分割适体相比，表现出增强的靶标响应。我们在两个结合域之间引入了双链 C3 间隔区脱碱基位点，通过核酸外切酶 III 的无嘌呤核酸内切酶活性实现 EATR 信号放大。作为演示，我们设计了一种基于 CBSA 的 EATR 放大荧光测定法来检测 3-硫酸脱氢异雄酮。相对于非 EATR 检测，该检测的目标灵敏度提高了 100 倍，50% 尿液中的检测限为 1 μM。我们进一步开发了一种免仪器比色测定法，采用 EATR 介导的 CBSA 修饰金纳米粒子聚集，用于视觉检测低微摩尔浓度的可卡因。基于 CBSA 工程的普适性和 EATR 在复杂样品中的稳健性能，我们相信此类测定对于检测不同领域的小分子靶标具有价值。