Development of a Microfluidic Platform for R-Phycoerythrin Purification Using an Aqueous Micellar Two-Phase System.

Temperature-dependent aqueous micellar two-phase systems (AMTPSs) have recently been gaining attention in the isolation of high-added-value biomolecules from their natural sources. Despite their sustainability, aqueous two-phase systems, and particularly AMTPSs, have not been extensively applied in the industry, which might be changed by applying process integration and continuous manufacturing. Here, we report for the first time on an integrated microfluidic platform for fast and low-material-consuming development of continuous protein purification using an AMTPS. A system comprised of a microchannel incubated at high temperature, enabling instantaneous triggering of a two-phase system formation, and a microsettler, allowing complete phase separation at the outlets, is reported here. The separation of phycobiliproteins and particularly the purification of R-phycoerythrin from the contaminant proteins present in the aqueous crude extract obtained from fresh cells of Gracilaria gracilis were thereby achieved. The results from the developed microfluidic system revealed that the fractionation performance was maintained while reducing the processing time more than 20-fold when compared with the conventional lab-scale batch process. Furthermore, the integration of a miniaturized ultrafiltration module resulted in the complete removal of the surfactant from the bottom phase containing R-phycoerythrin, as well as in nearly twofold target protein concentration. The process setup successfully exploits the benefits of process intensification along with the integration of various downstream processes. Further transfer to a meso-scale integrated system would make such a system appropriate for the separation and purification of biomolecules with high commercial interest.

DNA-Polyelectrolyte Complexation Study: The Effect of Polyion Charge Density and Chemical Nature of the Counterions.

Complexes of polycations and DNA, also known as polyplexes, have been extensively studied in the past decade, as potential gene delivery systems. Their stability depends strongly on the characteristics of the polycations, as well as the nature of the added salt. We present here a study of the DNA ionene complexation in which we used fluorescence, UV, and CD spectroscopy, combined with molecular dynamics computer simuations, to systematically examine the influence of the polycation charge density, as well as the influence of the nature of the counterion, on the stability of these systems. Ionenes as polycations, depending on their structural characteristics, have previously been found to possess low cytotoxicity, and are therefore particularly interesting as potential gene delivery agents. The results show that the DNA solutions in the presence of the polycation are more stable in the case of very large or very small ionene charge density, suggesting different mechanism of complexation. The computer simulations show that the ionenes with high charge density bind to the minor groove of the DNA molecules, while the ionenes with lower charge density bind to the major groove of the DNA. The nature of the counterions play only a minor role: precipitation of the DNA molecules occurs at slightly lower ionene concentration when fluoride counterion are present, compared to the bromide counterions.

Specific counter-ion and co-ion effects revealed in mixing of aqueous solutions of 3,3 and 6,6-ionenes with solutions of low molecular weight salts.

Enthalpies of mixing of aliphatic 3,3 and 6,6-ionene fluorides with low molecular weight salts (sodium formate, acetate, nitrate, chlorate(v), and thiocyanate), all dissolved in water, were determined. In addition, to complement our previous study (Luksicet al., Phys. Chem. Chem. Phys., 2012, 14, 2024), new measurements were performed where aqueous solutions of 3,3 and 6,6-ionene bromides were mixed with solutions of sodium fluoride, chloride, bromide, and iodide. Electrostatic theory, based on Manning's limiting law or the Poisson-Boltzmann equation, predicted the enthalpy of mixing to be endothermic in all the cases, while experiments showed that this is not always true. When an aqueous solution of 3,3-ionene fluoride was mixed with a solution of sodium fluoride (or formate and acetate) in water, the effect was indeed endothermic. For all other salts, i.e. sodium chlorate, nitrate, and thiocyanate, heat was released upon mixing. The situation was similar for 6,6-ionene fluoride solutions with an exception of mixing with sodium chlorate, where the effect was endothermic. The enthalpy of mixing was strongly correlated with the enthalpy of hydration of the counterion of the low molecular weight salt. A lyotropic series, similar to that of Hofmeister, was obtained. To examine also the effect of co-ions, ionene bromides were titrated with tetramethyl-, tetraethyl-, or tetrapropylammonium bromides. The enthalpy was exothermic for all mixtures while, somewhat unexpectedly, the co-ion specific effect was quite strong.

Influence of a hairpin loop on the thermodynamic stability of a DNA oligomer.

DSC was used to evaluate the mechanism of the thermally induced unfolding of the single-stranded hairpin HP = 5'-CGGAATTCCGTCTCCGGAATTCCG-3' and its core duplex D (5'-CGGAATTCCG-3')(2). The DSC melting experiments performed at several salt concentrations were successfully described for HP and D in terms of a three-state transition model HP↔I (intermediate state) ↔ S (unfolded single-stranded state) and two state transition model D↔2S, respectively. Comparison of the model-based thermodynamic parameters obtained for each HP and D transition shows that in unfolding of HP only the HP↔I transition is affected by the TCTC loop. This observation suggests that in the intermediate state its TCTC loop part exhibits significantly more flexible structure than in the folded state while its duplex part remains pretty much unchanged.