Zhao Zhe, Xiong Qing, Zhu Yan, Zhang Cong, Li Zhixin, Chen Zhonghui, Zhang Ying, Deng Xiaoyu, Tao Yingzhou, Xu Shaohua
Cancer Research Center & Jiangxi Engineering Research Center for Translational Cancer Technology, Jiangxi University of Chinese Medicine & Jiangxi Province Key Laboratory for Diagnosis, Treatment and Rehabilitation of Cancer in Chinese Medicine, Nanchang, Jiangxi 330004, China.
College of Acupuncture and Tuina Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China.
Anal Chem. 2025 Jan 14;97(1):1019-1027. doi: 10.1021/acs.analchem.4c06196. Epub 2024 Dec 19.
CRISPR/Cas12a-based biosensors have garnered significant attention in the field of point-of-care testing (POCT), yet the majority of the CRISPR-based POCT methods employ fluorescent systems as report probes. Herein, we report a new CRISPR/Cas12a-enabled multicolor visual biosensing strategy for the rapid detection of disease biomarkers. The proposed assay provided vivid color responses to enhance the accuracy of visual detection. In the existence of the target, the -cleavage activity of CRISPR-Cas12a was activated. The report probe modified with magnetic beads (MBs) and horseradish peroxidase (HRP) was cleaved, and HRP was released in the supernatant. As a result, HRP mediated the etching of gold nanobipyramids (AuNBPs) under hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine and generated a vivid color response. The proposed method has been verified by the detection of the breast cancer 1 gene (BRCA1) as a proof-of-principle target. According to the different colors of AuNBPs, our experimental results have demonstrated that as low as 30 pM BRCA1 can be detected with no more than 60 min. Additionally, the proposed sensor has been successfully applied in the analysis of BRCA1 in human serum samples with satisfactory results, which indicates great potential for the sensitive determination of biomarkers and the POCT area.
基于CRISPR/Cas12a的生物传感器在即时检测（POCT）领域备受关注，然而大多数基于CRISPR的POCT方法采用荧光系统作为报告探针。在此，我们报告了一种新的基于CRISPR/Cas12a的多色视觉生物传感策略，用于快速检测疾病生物标志物。所提出的检测方法提供了生动的颜色响应，以提高视觉检测的准确性。在存在靶标的情况下，CRISPR-Cas12a的切割活性被激活。用磁珠（MBs）和辣根过氧化物酶（HRP）修饰的报告探针被切割，HRP释放到上清液中。结果，HRP在过氧化氢和3,3',5,5'-四甲基联苯胺存在下介导金纳米双锥体（AuNBPs）的蚀刻，并产生生动的颜色响应。所提出的方法已通过检测乳腺癌1基因（BRCA1）作为原理验证靶标得到验证。根据AuNBPs的不同颜色，我们的实验结果表明，在不超过60分钟的时间内可以检测到低至30 pM的BRCA1。此外，所提出的传感器已成功应用于人类血清样本中BRCA1的分析，结果令人满意，这表明在生物标志物的灵敏测定和POCT领域具有巨大潜力。
