Zhang Xindan, Wu Minkang, Shi Haoran, Kim Soochan, Lu Shixiang, Wang Ping, Qin Jieling
School of Chemistry and Chemical Engineering, Beijing Institute of Technology Zhengzhou Academy of Intelligent Technology, Beijing Institute of Technology, Beijing 100081, China.
Tongji University Cancer Center, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai 200092, China.
Cyborg Bionic Syst. 2025 May 1;6:0266. doi: 10.34133/cbsystems.0266. eCollection 2025.
, a bacterium linked to colorectal cancer, possesses a specific gene called that serves as an early diagnostic biomarker. The CRISPR/Cas12a system has demonstrated marked potential for nucleic acid detection due to its satisfactory selectivity and trans-cleavage ability. However, most CRISPR/Cas-based sensors suffer from problems such as probe entanglement or local aggregation, reducing the Cas enzyme efficiency. In this study, an amplification-free biosensing platform for ultrasensitive detection of was developed by integrating the highly specific CRISPR/AsCas12a with an improved electrochemiluminescence (ECL) biosensor. Different from the conventional 1- or 2-dimensional probes, the platform was constructed by tetrahedral DNA nanostructure (TDN) probes conjugated with quenchers and coralliform gold (CFAu) functionalized with luminescent agents. The TDN serves as an exceptional scaffold to modulate the recognition unit, substantially enhancing the recognition and cleavage efficiency of AsCas12a toward the probes. Furthermore, the high surface area of CFAu provides extensive landing sites for the luminescent agents, thereby improving the detection sensitivity. The prepared ECL biosensor exhibited a wider linear range (10 fM to 100 nM) and was capable of detecting down to 1 colony-forming unit/ml. Additionally, the high mismatch sensitivity of AsCas12a to protospacer adjacent motifs and nearby areas provides a strategy for distinguishing mutant from wild-type sequences. Finally, by designing CRISPR RNA (crRNA), this diagnostic method can also be easily modified to detect other bacteria or biomarkers for the early diagnosis of various diseases.
一种与结直肠癌相关的细菌，拥有一种名为 的特定基因，可作为早期诊断生物标志物。由于其令人满意的选择性和反式切割能力，CRISPR/Cas12a系统在核酸检测方面显示出显著潜力。然而，大多数基于CRISPR/Cas的传感器存在诸如探针缠结或局部聚集等问题，降低了Cas酶的效率。在本研究中，通过将高度特异性的CRISPR/AsCas12a与改进的电化学发光（ECL）生物传感器相结合，开发了一种用于超灵敏检测 的无扩增生物传感平台。与传统的一维或二维探针不同，该平台由与淬灭剂共轭的四面体DNA纳米结构（TDN）探针和用发光剂功能化的珊瑚状金（CFAu）构建而成。TDN作为一种特殊的支架来调节识别单元，显著提高了AsCas12a对探针的识别和切割效率。此外，CFAu的高表面积为发光剂提供了广泛的着陆位点，从而提高了检测灵敏度。所制备的ECL生物传感器表现出更宽的线性范围（10 fM至100 nM），能够检测低至1个菌落形成单位/毫升的 。此外，AsCas12a对原间隔相邻基序和附近区域的高错配敏感性为区分突变体和野生型序列提供了一种策略。最后，通过设计CRISPR RNA（crRNA），这种诊断方法也可以很容易地修改以检测其他细菌或生物标志物，用于各种疾病的早期诊断。
