CRISPR/Cas12a-Responsive Smart DNA Hydrogel for Sensitive Electrochemiluminescence Detection of the Huanglongbing Outer Membrane Protein Gene

IF 6.7 1区化学 Q1 CHEMISTRY, ANALYTICAL

Analytical Chemistry Pub Date : 2024-06-29 DOI:10.1021/acs.analchem.4c02489

Yutian Zhang, Can Hu, Yashi Yin, Kejing Ren, Yingsi He, Yanru Gao, Heyou Han*, Chengzhou Zhu* and Wenjing Wang*,

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Abstract

Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the CLas outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the trans-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (I₀ – I) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.

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用于灵敏电化学发光检测黄龙兵外膜蛋白基因的 CRISPR/Cas12a 响应型智能 DNA 水凝胶

柑橘黄龙病（HLB）被称为 "柑橘癌症"，其中最常见的致病菌是亚洲自由杆菌（CLas）。在这项研究中，我们报告了一种新型电化学发光（ECL）生物传感器，通过将滚动圈扩增（RCA）与 CRISPR/Cas12a 响应型智能 DNA 水凝胶相结合，实现了对 CLas 外膜蛋白（Omp）基因的高灵敏度检测。在目标存在的情况下，RCA 会产生大量扩增子。扩增子通过与 crRNA 杂交激活 CRISPR/Cas12a 的反式裂解活性，触发智能 DNA 水凝胶释放电极上封装的 AuAg 纳米团簇（AuAg NCs），从而导致 ECL 信号降低。在 50 fM 至 5 nM 范围内，ECL 强度变化（I0 - I）与目标物的浓度呈正相关，检测极限为 40 fM。我们还用 10 份柑橘活叶样本（5 份 HLB 阴性样本和 5 份 HLB 阳性样本）对传感器的性能进行了评估，结果与黄金标准 qPCR 结果非常吻合。该传感策略扩展了 ECL 的多功能性，可以高灵敏度地检测植物中不同水平的 dsDNA 或 ssDNA。

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来源期刊

Analytical Chemistry 化学-分析化学

CiteScore

12.10

自引率

12.20%

发文量

1949

审稿时长

1.4 months

期刊介绍： Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.