Enhancing Micro Free Flow Electrophoresis: Detection, Application, And Fabrication

Abstract

Micro Free Flow Electrophoresis (µFFE) is a separation technique where analytes are moved through a planar separation channel via pressure driven flow. Analytes streams are deflected laterally within an electric field applied perpendicular to the flow and separated from one another based on differences in their electrophoretic mobility. Notably, µFFE is capable of continuously separating analytes making it uniquely useful for a range of applications such as multidimensional separations, microscale sample purification, and continuous on-line monitoring. This work focuses on improving the sensitivity of laser induced fluorescence (LIF) detection via modifying the laser alignment, exploring new device designs for rapid on-line buffer exchange, and developing a novel fabrication technique for hot embossing µFFE devices in cyclic olefin copolymer (COC) with the goal of expanding µFFE’s use in analytical workflows. A new laser alignment for LIF was explored, focusing the laser light through the side of the device instead of spreading it into a line and reflecting it down onto it. Improvement in the limit of detection (LOD) for fluorescein was obtained in a glass µFFE device using this alignment; however, it was found to be incompatible with 3D printed acrylonitrile butadiene styrene (ABS) due to excessive scattering of the laser light. New µFFE device designs were developed and modeled using Multiphysics modeling software to optimize a design for rapid on-line buffer exchange. This was performed with the intent of interfacing incompatible separation modes with electrospray ionization mass spectrometry (ESI-MS) to develop novel multi-attribute analysis techniques for the assessment of therapeutic monoclonal antibodies (mAbs). Lastly, a novel fabrication technique for µFFE was developed to hot emboss µFFE devices in COC utilizing a poly jet 3D printed master mold. The performance of devices produced this way were tested via a separation of three fluorescent dyes. Their LODs quantified and compared to similar ABS and glass µFFE devices.