Pei Jingwen, Li Lan, Li Chang, Li Zongying, Wu Yu, Kuang Haiyang, Ma Pan, Huang Li, Liu Jinbo, Tian Gang
Department of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University, Sichuan Province Engineering Technology Research Center of Molecular Diagnosis of Clinical Diseases, Molecular Diagnosis of Clinical Diseases Key Laboratory of Luzhou, Sichuan, 646000, China.
Department of Laboratory Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China.
Biosens Bioelectron. 2025 Apr 1;273:117190. doi: 10.1016/j.bios.2025.117190. Epub 2025 Jan 20.
Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26 fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.
结直肠癌（CRC）是全球癌症相关死亡的主要原因之一，因此需要开发灵敏且微创的诊断方法。在本研究中，我们提出了一种新型诊断策略，即将哑铃型探针介导的CRISPR/Cas13a与切口诱导的DNA级联反应（DP桥连Cas13a/NDCR）相结合，用于高灵敏检测微小RNA（miRNA）。靶标miRNA触发Cas13a介导的哑铃型探针切割，释放出一条中间链，该中间链与电极表面的亚甲基蓝标记的发夹探针杂交。切口酶切割形成的双链DNA，触发级联反应，放大电化学信号。在优化条件下，该方法对miRNA-21的检测限为8.26 fM，具有可靠的特异性和长期稳定性。此外，与使用多个miRNA标志物的机器学习模型相结合提高了诊断准确性，在临床验证队列中能够以100%的准确率区分结直肠癌与结直肠息肉及健康对照。本研究突出了DP桥连Cas13a/NDCR作为一种灵敏且准确的结直肠癌诊断工具的潜力。
