Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Immunology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Talanta. 2024 Dec 1;280:126706. doi: 10.1016/j.talanta.2024.126706. Epub 2024 Aug 13.
In this study, a porous carbon derived from a metal-organic framework (PCMOF) as a target-responsive material functionalized with Nocardia particular antisense ssDNA oligonucleotide (ssDNA capture probe) was developed to construct a simple genosensor based on biogatekeeper strategy for sensitive detection of Nocardia in complex biological samples. The PCMOF with suitable pores volume was used to encapsulate electroactive dye methylene blue (MB), and the ssDNA capture probe was used as a gatekeeper to cap PCMOF. Without the presence of Nocardia target, the electrochemical signal of trapped MB was high. Upon adding the target, the hybridization of ssDNA capture probe and target led to the formation of a probe-target double-stranded (dsDNA) structure which dissociated from PCMOF and allowed MB molecules to be released. Therefore, the electrochemical signal of the genosensor decreased. The detection of Nocardia was accomplished by observing variations in the MB peak current intensity in a dose-dependent manner. For this genosensor, a linearity range from 10 to 10 M for synthetic ssDNA target and 10 to 10 copies/mL for two standard isolates, Nocardia farcinica PTCC 1309 and Nocardia brasiliensis ATCC 19296 as well as for clinical isolates (identified as Nocardia otitidiscaviarum) was observed, respectively. The detection limit (DL) values were 0.54 aM for synthetic ssDNA target and 5, 7, and 4 copies/mL for N. farcinica, N. brasiliensis, and N. otitidiscaviarum, respectively. This genosensor was also characterized by good specificity, reproducibility, and stability.
在这项研究中，开发了一种由金属有机骨架（PCMOF）衍生的多孔碳作为目标响应材料，该材料功能化有诺卡氏菌反义 ssDNA 寡核苷酸（ssDNA 捕获探针），用于构建基于生物守门员策略的简单基因传感器，用于在复杂生物样品中灵敏检测诺卡氏菌。选择具有合适孔体积的 PCMOF 来封装电化学活性染料亚甲蓝（MB），并且 ssDNA 捕获探针用作门控物以封闭 PCMOF。在不存在诺卡氏菌靶标的情况下，捕获的 MB 的电化学信号很高。添加靶标后，ssDNA 捕获探针和靶标的杂交导致形成探针-靶标双链（dsDNA）结构，该结构从 PCMOF 上解离并允许 MB 分子释放。因此，基因传感器的电化学信号降低。通过以剂量依赖的方式观察 MB 峰电流强度的变化来完成对诺卡氏菌的检测。对于该基因传感器，对于合成的 ssDNA 靶标，线性范围为 10 至 10 摩尔，对于两个标准分离株，Nocardia farcinica PTCC 1309 和 Nocardia brasiliensis ATCC 19296 以及临床分离株（鉴定为 Nocardia otitidiscaviarum），分别为 10 至 10 拷贝/毫升。对于合成的 ssDNA 靶标，检测限（DL）值为 0.54 aM，对于 N. farcinica、N. brasiliensis 和 N. otitidiscaviarum，分别为 5、7 和 4 拷贝/毫升。该基因传感器还具有良好的特异性、重现性和稳定性。
