Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-powered biosensor with a G-quadruplex (G4) reporter offer the benefits of simplicity and sensitivity, making them extensively utilized in detection applications. However, these biosensors used for monitoring pollutants in environmental water samples may face the problem of high background signal and easy interference due to the “signal-off” output. It is obvious that a biosensor based on the CRISPR/Cas12a system and G4 with a “signal on” output mode needs to be designed for detecting environmental pollutants. By using phosphorothioate-modified G4 as a reporter and catalytic hairpin assembly (CHA) integrated with Cas12a as an amplification strategy, a “signal-on” colorimetric/photothermal biosensor (psG4-CHA/Cas) for portable detection of environmental pollutants was developed. With the help of functional nucleotides, the target pollutant (kanamycin or Pb) triggers a CHA reaction to produce numerous double-strand DNA, which can activate Cas12a′s -cleavage activity. The active Cas12a cleaves locked DNA to release caged psG-rich sequences. Upon binding hemin, the psG-rich sequence forms a psG4/hemin complex, facilitating the oxidation of the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into the blue photothermal agent (oxTMB). The smartphone was employed for portable colorimetric detection of kanamycin and Pb. The detection limits were found to be 100 pM for kanamycin and 50 pM for Pb. Detection of kanamycin and Pb was also carried out using a portable thermometer with a detection limit of 10 pM for kanamycin and 8 pM for Pb. Sensitive, selective, simple and robust detection of kanamycin and Pb in environmental water samples is achieved with the psG4-CHA/Cas system. This system not only provides a new perspective on the development of efficient CRISPR/Cas12a-based “signal-on” designs, but also has a promising application for safeguarding human health and environmental monitoring.

中文翻译：

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硫代磷酸酯修饰的 G-四链体作为信号双模式报告基因，用于基于 CRISPR/Cas12a 的便携式环境污染物检测


具有 G 四链体 (G4) 报告基因的簇状规则间隔短回文重复序列 (CRISPR)/Cas12a 驱动的生物传感器具有简单性和灵敏度的优点，使其广泛用于检测应用。然而，这些用于监测环境水样中污染物的生物传感器由于“信号关闭”输出，可能面临背景信号高、易干扰的问题。显然，需要设计一种基于CRISPR/Cas12a系统和具有“信号开启”输出模式的G4的生物传感器来检测环境污染物。通过使用硫代磷酸酯修饰的 G4 作为报告基因，并使用与 Cas12a 集成的催化发夹组装（CHA）作为放大策略，开发了一种用于便携式环境污染物检测的“信号开启”比色/光热生物传感器（psG4-CHA/Cas）。在功能核苷酸的帮助下，目标污染物（卡那霉素或Pb）触发CHA反应，产生大量的双链DNA，从而激活Cas12a的β-裂解活性。活性 Cas12a 裂解锁定的 DNA，释放出富含 psG 的笼状序列。结合氯化血红素后，富含 psG 的序列形成 psG4/氯化血红素复合物，促进无色 3,3',5,5'-四甲基联苯胺 (TMB) 氧化成蓝色光热剂 (oxTMB)。该智能手机用于卡那霉素和铅的便携式比色检测。卡那霉素的检测限为 100 pM，Pb 的检测限为 50 pM。还使用便携式温度计检测卡那霉素和 Pb，卡那霉素的检测限为 10 pM，Pb 的检测限为 8 pM。 psG4-CHA/Cas 系统可对环境水样中的卡那霉素和 Pb 进行灵敏、选择性、简单且稳定的检测。 该系统不仅为开发基于CRISPR/Cas12a的高效“信号开启”设计提供了新的视角，而且在保障人类健康和环境监测方面也具有广阔的应用前景。