Selection of highly specific aptamers to Vibrio parahaemolyticus using cell-SELEX powered by functionalized graphene oxide and rolling circle amplification

Abstract

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.