Schematic of a complex microfluidic purification system by combining two different types of the separation process (two-phase extraction process and three-flow desalting system). (a) Two-phase extraction process (ATPS and ILTPS) was performed to purify BR from the cell lysate sample. PEG-rich (or ionic liquid phase) and water-rich phases were injected through both sides inlet ports by pressure-driven flow. Subsequently, sample solution was introduced into the middle inlet channel. Under the formation of the laminar-flow extraction system, impurities diffuse away from the cell lysate stream depending on the difference in hydrophobicity and biomolecular interactions. (b) Three-flow desalting dialysis was applied for the removal of contaminated proteins and excessive sucrose after fractionation of the sample stream from the laminar-flow extraction process.
Photograph of microfluidic device and the formation of the stable three-phase stream in the microchannel. (a) Center region near the inlets of the microchannel, (b) middle area of the microchannel, and (c) center region near the outlets of the microchannel. The flow rates of the PEG-rich and the salt-rich phases were 0.2 and 3.0 ml/h, respectively, and that of the sample phase kept at 0.7 ml/h.
Effect of pH on (a) the recovery rate and (b) the purity of BR using two kinds of purification techniques (ATPS and ILTPS) in the microfluidic device.
Effect of flow rate of both buffer phases on the recovery rate and purity of BR in the microdialysis system.
Effect of pH on the recovery rate of BR and the removal efficiency of sucrose using the three-flow dialysis system in the microfluidic device.
Characterization of purified BR proteins by (a) SDS-PAGE. SM, molecular size marker; lane 1, BR; lane 2, BR; lane 3, BR; lane 4, BR; black arrow, target BR. (b) MALDI-TOF analysis recorded with the DHB matrix from the BR sample purified through the microfluidic device. The purified target BR was analyzed as a protein sample having a molecular weight of 27 088.9 Da.
Recovery and purity of BR, and the removal of sucrose with different pHs in the microdialysis system.
Purification profile related to each step.
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