Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China.
Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China.
Sci Total Environ. 2024 Feb 10;911:168708. doi: 10.1016/j.scitotenv.2023.168708. Epub 2023 Nov 20.
Freshwater lead pollution has posed severe threat to the environment and human health, underscoring the urgent necessity for accurate and user-friendly detection methods. Herein, we introduce a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) sensor for highly sensitive Pb detection. To accomplish this, we designed a dual-functional deoxyribozyme (df-DNAzyme) probe that functions as an activator for the CRISPR-Cas12a system while also recognizing Pb. The df-DNAzyme probe was subsequently combined with gold nanoparticles (AuNPs) to fabricate a DNAzyme/AuNP nanoprobe, facilitating the activation of CRISPR-Cas12a in a one-to-multiple manner. Upon exposure to Pb, the df-DNAzyme is cleaved, causing disintegration of the DNAzyme/AuNP nanoprobe from magnetic beads. The degraded DNAzyme/AuNP containing multiple double-stranded DNA activators efficiently triggers CRISPR-Cas12a activity, initiating cleavage of fluorescence-quenched reporter DNA and generating amplified signals accordingly. The amplified fluorescence signal is accurately quantified using a quantitative polymerase chain reaction (qPCR) instrument capable of measuring 96 or 384 samples simultaneously at the microliter scale. This technique demonstrates ultra-sensitive detection capability for Pb at concentrations as low as 1 pg/L within a range from 1 pg/L to 10 μg/L, surpassing limits set by World Health Organization (WHO) and United States Environmental Protection Agency (US EPA) guidelines. This study offers an ultrasensitive and high-throughput method for the detection of Pb in freshwater, thereby advancing a novel approach towards the development of precise and convenient techniques for detecting harmful contaminants.
淡水铅污染对环境和人类健康构成了严重威胁，因此迫切需要准确且易于使用的检测方法。在此，我们介绍了一种用于高灵敏度 Pb 检测的新型 Clustered Regularly Interspaced Short Palindromic Repeats（CRISPR-Cas）传感器。为此，我们设计了一种双功能脱氧核酶（df-DNAzyme）探针，该探针可用作 CRISPR-Cas12a 系统的激活剂，同时也能识别 Pb。随后，将 df-DNAzyme 探针与金纳米粒子（AuNPs）结合，制备 DNAzyme/AuNP 纳米探针，以多对一的方式激活 CRISPR-Cas12a。暴露于 Pb 后，df-DNAzyme 被切割，导致 DNAzyme/AuNP 纳米探针从磁珠上解体。包含多个双链 DNA 激活剂的降解 DNAzyme/AuNP 有效地触发了 CRISPR-Cas12a 的活性，引发荧光猝灭报告 DNA 的切割，并相应地产生放大信号。使用能够同时在微升规模上测量 96 或 384 个样品的定量聚合酶链反应（qPCR）仪器准确地定量放大的荧光信号。该技术在 1pg/L 至 10μg/L 的范围内，对浓度低至 1pg/L 的 Pb 具有超灵敏的检测能力，超过了世界卫生组织（WHO）和美国环境保护署（US EPA）指南规定的限值。本研究提供了一种用于淡水 Pb 检测的超灵敏高通量方法，为开发精确便捷的有害污染物检测技术提供了新方法。
