Li Shuofeng, Wang Fangfang, Hao Lin, Zhang Pengbo, Song Guangyi, Zhang Yawen, Wang Chun, Wang Zhi, Wu Qiuhua
College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China; College of Science, Hebei Agricultural University, Baoding 071001, China.
Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes, College of Life Sciences, Hebei Agricultural University, Baoding 071001, China.
Int J Biol Macromol. 2024 Dec;283(Pt 1):137594. doi: 10.1016/j.ijbiomac.2024.137594. Epub 2024 Nov 12.
CRISPR/Cas13a with precise and controllable programming of endonuclease activity has been served as powerful tool for RNA sensing. Although with high sensitivity, existing CRISPR/Cas13a-based biosensors need complex amplification procedure or special equipment that limited quantification capability. Here, Mn-doped NiCoO (Mn/NiCoO) nanozyme with enhanced peroxidase activity was synthesized and combined with CRISPR/Cas13a-based reaction to develop a simple, sensitive and universal biosensor for RNA detection, which is achieved through target recognition that activates Cas enzymes to cleave RNA reporter for inhibiting Mn/NiCoO nanozyme to assemble on microplate. The Mn/NiCoO nanozyme assembled on microplate can be monitored through colorimetric and fluorometric approaches. On one hand, Mn/NiCoO nanozyme offers ideal peroxidase activity to catalyze colorimetric reaction, and as low as dozens of amol level of RNA target can be sensitively detected by naked eyes without any amplification procedures. On the other hand, Mn/NiCoO can be also served as a signal amplifier to produce large amount of Co, Mnand Ni to quench the fluorescence of calcein. The fluorescent approach can achieve higher sensitivity (about 40-fold) than colorimetric method. More importantly, the proposed biosensor can work well for multiple RNA detection in real biological samples, showing a great potential for monitoring non-coding RNA-related diseases.
具有精确且可控的内切核酸酶活性编程的CRISPR/Cas13a已成为RNA传感的强大工具。尽管现有基于CRISPR/Cas13a的生物传感器具有高灵敏度，但需要复杂的扩增程序或特殊设备，这限制了其定量能力。在此，合成了具有增强过氧化物酶活性的锰掺杂镍钴氧化物（Mn/NiCoO）纳米酶，并将其与基于CRISPR/Cas13a的反应相结合，开发了一种用于RNA检测的简单、灵敏且通用的生物传感器，该传感器通过靶标识别激活Cas酶切割RNA报告基因来抑制Mn/NiCoO纳米酶组装在微孔板上实现。组装在微孔板上的Mn/NiCoO纳米酶可通过比色法和荧光法进行监测。一方面，Mn/NiCoO纳米酶具有理想的过氧化物酶活性以催化比色反应，无需任何扩增程序，肉眼即可灵敏地检测低至数十个阿摩尔水平的RNA靶标。另一方面，Mn/NiCoO还可作为信号放大器产生大量的钴、锰和镍以淬灭钙黄绿素的荧光。荧光法比色法可实现更高的灵敏度（约40倍）。更重要的是，所提出的生物传感器可很好地用于实际生物样品中的多种RNA检测，显示出监测非编码RNA相关疾病的巨大潜力。
