ATPS as an Efficient Method for Separation of Bionanoparticles: Investigation and Optimization of Partition Behavior of pDNA

In this paper, the efficiency of an aqueous two-phase system (ATPS) for purification of nanometer-sized bioparticles, plasmid DNA (pDNA), was studied. Polymer-salt ATPS consisting of polyethylenglycol (PEG)-K2HPO4 was used for the purification of 7 kb and 14 kb plasmid vectors. PEG-300 and PEG-1450 were applied to investigate the influence of different molecular mass of PEG on partitioning behavior of pDNA. The Taguchi design of experiments has been applied in order to optimize the significant system characteristics including PEG/salt ratio, temperature, lysate mass fraction and size of plasmid for pDNA separation by using ATPS. The results indicated that PEG/salt ratio has a considerable contribution on pDNA recovery both in the presence of PEG-300 and PEG-1450. It is also obtained that the size of pDNA in the range of 7 kb to 14 kb is not a significant factor on its partitioning. Furthermore, pDNA is easily partitioned to polymer-rich top phase in PEG300/salt system; and in salt-rich bottom phase in PEG1450/salt system. Under optimum conditions, pDNA was extracted in top phase of PEG-300/K2HPO4 with mass percent of 26 : 17 at 25 °C with a recovery percent of 85.