Liu Shiwu, Chen Shanquan, Tian Longzhi, He Qizhi, Wang Xiaoqi, Lu Fangguo, Ning Yi
Department of Microbiology, The Medicine School of Hunan University of Chinese Medicine, Changsha, Hunan, 410208, People's Republic of China.
Department of General Education, The School of Humanities and Social Science of the Chinese University of Hong Kong (Shenzhen Campus), Shenzhen, Guangdong, 518172, People's Republic of China.
Talanta. 2023 Jul 1;259:124549. doi: 10.1016/j.talanta.2023.124549. Epub 2023 Apr 11.
We describe a graphene oxide (GO)-based bioassay for the fluorometric determination of norA gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This approach is based on Nb.BbvCI-assisted target recycling (NATR) and T7 exonuclease (T7 Exo)-triggered cascade dual-recycling signal amplification (TTCDRSA). The system included GO, a capture probe (CP), an assistant probe (AP), two carboxyfluorescein (FAM)-labeled hairpins (HP1 and HP2), endonuclease Nb.BbvcI, and exonuclease T7. In the presence of a target, AP, together with the target RNA, can hybridise with CP via partial complementarity to one another and open its hairpin structure to form a triple complex that is recognised by Nb.BbvCI. Once the CP is cleaved, the released AP and target RNA can walk on the carboxylated graphene oxide (CGO) surface to bind with another CP which induces the next round of cleavage, accumulating many trigger probes (TPs). The TPs then activate TTCDRSA with the assistance of T7 Exo, HP1, and HP2 to produce large amounts of free FAMs. These free FAMs are repelled by GO and exhibit enhanced fluorescence signals at excitation/emission wavelengths of 480/514 nm. The limit of detection (LOD) of the bioassay was calculated to be 0.37 fM, and the linear range of the method ranged from 1 fM to 1 nM. More importantly, the bioassay also exhibited high sensitivity and selectivity for target RNA detection in real samples, which may open a new promising avenue for monitoring drug efflux and studying the mechanisms of drug actions.
我们描述了一种基于氧化石墨烯（GO）的生物测定法，用于荧光测定耐甲氧西林金黄色葡萄球菌（MRSA）中的norA基因转录（mRNA）。该方法基于Nb.BbvCI辅助的靶标循环（NATR）和T7核酸外切酶（T7 Exo）触发的级联双循环信号放大（TTCDRSA）。该系统包括GO、捕获探针（CP）、辅助探针（AP）、两个羧基荧光素（FAM）标记的发夹（HP1和HP2）、核酸内切酶Nb.BbvcI和核酸外切酶T7。在存在靶标的情况下，AP与靶标RNA一起可以通过彼此部分互补与CP杂交，并打开其发夹结构形成三元复合物，该复合物被Nb.BbvCI识别。一旦CP被切割，释放的AP和靶标RNA可以在羧基化氧化石墨烯（CGO）表面上移动，与另一个CP结合，从而诱导下一轮切割，积累许多触发探针（TP）。然后，TP在T7 Exo、HP1和HP2的辅助下激活TTCDRSA，产生大量游离FAM。这些游离FAM被GO排斥，并在480/514 nm的激发/发射波长处表现出增强的荧光信号。该生物测定法的检测限（LOD）计算为0.37 fM，该方法的线性范围为1 fM至1 nM。更重要的是，该生物测定法对实际样品中的靶标RNA检测也表现出高灵敏度和选择性，这可能为监测药物外排和研究药物作用机制开辟一条新的有前景的途径。
