National Key Laboratory of Agricultural Microbiology, College of Chemistry, Huazhong Agricultural University, Wuhan 430070, China.
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Anal Chem. 2024 Jul 16;96(28):11611-11618. doi: 10.1021/acs.analchem.4c02489. Epub 2024 Jun 29.
Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Liberibacter asiaticus (Las) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the Las 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 -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 ( - ) 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.
柑橘黄龙病（HLB）俗称柑橘癌症，其中亚洲韧皮杆菌（Las）是导致 HLB 的最普遍菌株。在本研究中，我们通过将滚环扩增（RCA）与 CRISPR/Cas12a 反应性智能 DNA 水凝胶偶联，报道了一种用于 Las 外膜蛋白（Omp）基因高灵敏度检测的新型电化学发光（ECL）生物传感器。在存在靶标时，通过 RCA 产生大量的扩增子。扩增子通过与 crRNA 杂交激活 CRISPR/Cas12a 的 -切割活性，触发智能 DNA 水凝胶响应以释放电极上包封的 AuAg 纳米团簇（AuAg NCs），从而导致 ECL 信号降低。ECL 强度变化（ - ）与靶标浓度在 50 fM 至 5 nM 的范围内呈正相关，检测限为 40 fM。该传感器的性能还使用 10 个活柑橘叶片样本（5 个 HLB 阴性和 5 个 HLB 阳性）进行了评估，结果与金标准 qPCR 结果非常吻合。该传感策略通过高灵敏度检测植物中不同水平的 dsDNA 或 ssDNA 扩展了 ECL 的多功能性。
