Wang Xiaoqi, Liu Shiwu, Xiao Rong, Hu Jue, Li Ling, Ning Yi, Lu Fangguo
Department of Microbiology, The Medicine School of Hunan University of Chinese Medicine, Changsha, Hunan, 410208, People's Republic of China.
Experimental Center of Molecular Biology, The Medicine School of Hunan University of Chinese Medicine, Changsha, Hunan, 410208, People's Republic of China.
Talanta. 2022 Dec 1;250:123714. doi: 10.1016/j.talanta.2022.123714. Epub 2022 Jun 25.
Herein, we report the development of a graphene-oxide-based (GO-based) fluorescent bioassay for determining agrC gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). The design is based on nicking-enzyme-assisted (Nb.BbvcI-assisted) target recycling amplification (NATR) and a hybridization chain reaction (HCR). The system consists of a helper probe (HP), a molecular beacon (MB) probe, four hairpins, and endonuclease Nb.BbvcI, which plays a role in target recycling and signal amplification. In the absence of the target, all of the carboxyfluorescein-labeled (FAM-labeled) hairpins are adsorbed through π-stacking interactions onto the surface of GO, resulting in FAM signal quenching. When the target is added, three nucleic acid chains hybridize together to form a triple complex that is recognized by Nb.BbvCI. The MB probe is then cleaved by Nb.BbvCI to generate an HP/target complex and two new DNA fragments; the former is hybridized to another MB probe and enters the next round of reaction. The two newly reproduced DNA fragments induce a HCR with the assistance of hairpins 1-4 to create double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and generate strong fluorescence at excitation/emission wavelengths of 480/514 nm. Importantly, synergy between FAM and the dsDNA-SYBR Green I duplex structure led to significantly amplified fluorescence and enhanced sensitivity. The bioassay showed a detection limit of 7.5 fM toward the target and a good linearity in the 10 fM to 100 pM range. The developed method was applied to monitor biofilm formation and study the mechanism of drug action, with satisfactory results obtained.
在此，我们报道了一种基于氧化石墨烯（GO）的荧光生物测定法的开发，用于测定耐甲氧西林金黄色葡萄球菌（MRSA）中的agrC基因转录（mRNA）。该设计基于切口酶辅助（Nb.BbvcI辅助）的靶标循环扩增（NATR）和杂交链式反应（HCR）。该系统由一个辅助探针（HP）、一个分子信标（MB）探针、四个发夹结构和内切核酸酶Nb.BbvcI组成，后者在靶标循环和信号放大中发挥作用。在没有靶标的情况下，所有羧基荧光素标记（FAM标记）的发夹结构通过π-堆积相互作用吸附在GO表面，导致FAM信号淬灭。当加入靶标时，三条核酸链杂交形成一个三元复合物，该复合物被Nb.BbvCI识别。然后，MB探针被Nb.BbvCI切割，产生一个HP/靶标复合物和两个新的DNA片段；前者与另一个MB探针杂交并进入下一轮反应。这两个新产生的DNA片段在发夹1-4的辅助下引发HCR，产生双链DNA（dsDNA）产物。这些dsDNA产物被GO排斥，并在激发/发射波长为480/514 nm时产生强烈荧光。重要的是，FAM与dsDNA-SYBR Green I双链体结构之间的协同作用导致荧光显著放大并提高了灵敏度。该生物测定法对靶标的检测限为7.5 fM，在10 fM至100 pM范围内具有良好的线性。所开发的方法被应用于监测生物膜形成和研究药物作用机制，获得了满意的结果。
