Microfluidic Planar Phospholipids Membrane System Advancing Dynamics Studies of Ion Channels and Membrane Physics

Abstract

The interrogation of lipid membrane and biological ion channels supported within bilayer phospholipid membranes has greatly expanded our understanding of the roles membrane and ion channels play in a host of biological functions. Several key drawbacks of traditional electrophysiology systems used in these studies have long limited our effort to study the ion channels. Firstly, the large volume buffer in this system typically only allows single or multiple additions of reagents, while complete removal either is impossible or requires tedious effort to ensure the stability of membrane. Thus, it has been highly desirable to be able to rapidly and dynamically modulate the (bio)chemical conditions at the membrane site. Second, it is difficult to change temperature effectively with large thermal mass in macro device. Third, traditional PPM device host vertical membranes, therefore incompatible with confocal microscopy techniques. The miniaturization of bilayer phospholipid membrane has shown potential solution to the drawbacks stated above. A simple microfluidic design is developed to enable effective and robust dynamic perfusion of reagents directly to an on-chip planar phospholipid membrane (PPM). It allows ion channel conductance to be readily monitored under different dynamic reagent conditions, with perfusion rates up to 20 µL/min feasible without compromising the membrane integrity. It is estimated that the lower limit of time constant of kinetics that can be resolved by our system is 1 minute. Using this platform, the time-dependent responses of membrane-bound ceramide ion channels to treatments with La3+ and a Bcl-xL mutant were studied and the results were interpreted with a novel elastic biconcave distortion model. Another engineering challenge this dissertation takes on is the integration of fluorescence studies to micro-PPM system. The resulting novel microfluidic system enables high resolution, high magnification and real-time confocal microscope imaging with precise top and bottom (bio)chemical boundary conditions defined by perfusion, by integrating in situ PPM formation method, perfusion capability and microscopy compatibility. To demonstrate such electro-optical chip, lipid micro domains were imaged and quantitatively studied for their movements and responses to different physical parameters. As an extension to this platform, a double PPM system has been developed with the aim to study interactions between two membranes. Potential application in biophysics and biochemistry using those two platforms were discussed. Another important advantage of microfluidics is its lower thermal mass and compatibility with various microfabrication methods which enables potential integration of local temperature controller and sensor. A prototype thermal PPM chip is also discussed together with some preliminary results and their implication on ceramide channel assembly and disassembly mechanism.