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

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

Measurement and simulation of carbon nanotube’s piezoresistance property by a micro/nano combined structure

282-286In this paper, the present status of carbon nanotube’s electromechanical properties was reviewed. The relationships among the carbon nanotube’s resistance, gauge factor and the rates of change of the band gaps with strain (dEℊ/dƐ) were analyzed and simulated. Then, a micro/nano combined device and method for measuring the piezoresistance property of carbon nanotube were proposed. The device is consisted of a silicon chip and a printed circuit board which is used for loading and leading wire. The microelectrodes were fabricated on the silicon chip by FIB and a CVD-growth single-wall carbon nanotube was connected with the microelectrodes. The voltage-current characteristic of the carbon nanotube was measured using the proposed device. The relationship between the current and the voltage is basically linear, which demonstrates that the carbon nanotube is metallic. The experimental results show that the micro/nano combined device can be used for measuring the piezoresistance of carbon nanotube in our future work

The microfluidic apparatus and a sample recording of channel formation.

<p>(A) A fabricated BLM microfluidic bilayer formation “chip” with one buffer inlet, three perfusion inlets, and two Ag/AgCl electrodes. (B) Membrane conductance trace showing the formation of a ceramide channel. The BLM was formed by diffusive painting (inset) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043513#pone.0043513-Shao1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043513#pone.0043513-Hromada1" target="_blank">[19]</a>.</p

Dynamics of Ceramide Channels Detected Using a Microfluidic System

<div><p>Ceramide, a proapoptotic sphingolipid, has been shown to form channels, in mitochondrial outer membranes, large enough to translocate proteins. In phospholipid membranes, electrophysiological studies and electron microscopic visualization both report that these channels form in a range of sizes with a modal value of 10 nm in diameter. A hydrogen bonded barrel-like structure consisting of hundreds of ceramide molecules has been proposed for the structure of the channel and this is supported by electrophysiological studies and molecular dynamic simulations. To our knowledge, the mechanical strength and deformability of such a large diameter but extremely thin cylindrical structure has never been reported. Here we present evidence for a reversible mechanical distortion of the cylinder following the addition of La³⁺. A microfluidic system was used to repeatedly lower and then restore the conductance by alternatively perfusing La³⁺ and EDTA. Although aspects of the kinetics of conductance drop and recovery are consistent with a disassembly/diffusion/reassembly model, others are inconsistent with the expected time scale of lateral diffusion of disassembled channel fragments in the membrane. The presence of a residual conductance following La³⁺ treatment and the relationship between the residual conductance and the initial conductance were both indicative of a distortion/recovery process in analogy with a pressure-induced distortion of a flexible cylinder.</p> </div

Correlations between the rates of conductance increases and decreases with channel size.

<p>The initial rate of conductance decrease (nS/min) is proportional to the starting conductance (nS) (A) (r = 0.96) and the calculated initial rate of column loss (columns/min) is proportional to the starting circumference of columns (inset in (A) (r = 0.94)). The initial rate of conductance increase (nS/min) is proportional to the conductance (nS) just before EDTA perfusion (B) (r = 0.98), and the calculated initial rate of column reassembly (columns/min) is proportional to the circumference of columns before EDTA perfusion (inset in (B) (r = 0.96)).</p

There is a linear relationship between the conductance (nS) of a ceramide channel after LaCl₃ treatment and the conductance (nS) before LaCl₃ perfusion (r = 0.94).

<p>The circles are results of treatments with 50 µM LaCl₃ whereas the triangle is an experiment with 500 µM LaCl₃.</p

Lack of statistically significant correlation between the percentage of conductance recovery following EDTA treatment and the prior time of exposure to LaCl₃.

<p>Each curve is an independent experiment. The averages of the relative percentage of conductance recovery of different experiments in each group are shown in the inset. The results were grouped and normalized to the values of the “<10 min” group. No statistically significant difference was observed.</p