适体
滚动圆复制
化学
副溶血性弧菌
组合化学
指数富集配体系统进化
环介导等温扩增
纳米技术
石墨烯
DNA
细菌
分子生物学
生物化学
材料科学
生物
基因
聚合酶
核糖核酸
遗传学
作者
Shixi Song,Xingyu Wang,Ke Xu,Qiao Li,Lufang Ning,Xingbin Yang
标识
DOI:10.1016/j.aca.2018.11.047
摘要
Cell-SELEX is a powerful tool to screen aptamers binding to living cellular organisms such as bacteria, fungi and even oncocytes. Here, we developed an advanced cell-SELEX strategy featuring functionalized graphene oxide (GO) and isothermal rolling circle amplification (RCA) to select aptamers against a prevailing foodborne pathogen, Vibrio parahaemolyticus. Polyethyleneglycol (PEG) and chitosan (CTS) were grafted onto the sheet-like GO molecules to synthesize a PC-GO material. TEM and FT-IR characterization demonstrated that the PC-GO composites were near-nanometric scale and tethered with PEG and CTS moieties, a property that significantly improved its solubility in biological buffer solutions used in cell-SELEX process. PC-GO could bind with ssDNAs with lower affinities to target cells, therefore the selection efficiency is greatly enhanced. The cell-binding aptamer candidates (CACs) were amplified by 107 fold using complementary ring mediated (CRM-RCA), a created amplification method that generate single-stranded products, which could be directly used in the next round selection. As fueled by PC-GO and CRM-RCA, four highly specific aptamers with lowest Kd value of 10.3 ± 2.5 nM were obtained. Flow cytometry analysis showed that all the four aptamers exhibited more than 75% binding affinity to V. parahaemolyticus than to other foodborne bacteria (less than 18%). Simple procedure, high efficiency, and free from expensive thermal cycler (required by PCR amplification) will enable the established strategy to find its applications in aptamer selecting against fungi, stem and cancerous cells as well.
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