Abstract
Introduction Recently, we have developed a novel functional analysis system for ion channel proteins, in which we can apply a lateral voltage to the hydrophobic core of artificial cell membranes. Using this system, we reported that the activity of the human voltage-gated Na ion channels can be recovered from their non-conductive state by the application of the lateral voltages [1]. This finding suggests that lateral voltage may be a novel parameter to enhance the activity of channels that have been difficult to measure due to their susceptibility to inactivation. However, there have been two challenges with the lateral voltage devices that have prevented them from becoming established as a functional analysis system for ion channels. The short electrode lifetime (about 30 minutes) due to oxidation of the electrode material (Ti) and the low fabrication yield (less than 10%) due to cracks in an insulating SiO2 layer. In this study, we aimed to develop a highly efficient fabrication process for longer-lived and more stable devices by using Au as the electrode metal and CYTOP resin as the flexible insulating layer.
Fabrication Method
Micropores were formed in the Teflon film by electric sparking, and two Ti/Au thin films for the electrodes were sputtered around the micropores. The electrode pattern was controlled by a Ni mask. The film was then washed in chloroform using an ultrasonic cleaner. To form an insulating layer over the electrode, the Teflon film was dipped in CYTOP (CTL-109AE) except for the electrode contact area, pulled up with a dip coater at a rate of 1 mm/s, and baked. This process (dip, pull, and bake) was repeated 2-3 times. The CYTOP resin clogged in the micropores was removed by RIE (Reactive Ion Etching) using a Ni mask with a 120 µm aperture. The electric properties of the fabricated devices were evaluated by measuring the contact resistance of the electrode area connected to the lateral voltage source and the resistance between the two electrodes. In addition, the effect of lateral voltage on ion channels was evaluated. A lipid bilayer was formed in the micropore of the device by the folding method, and human Na channels were incorporated by the vesicle fusion method. Ion channel currents were measured and compared with and without the application of lateral voltage.
Results and Discussion
To investigate the lifetime of the fabricated electrode devices, the contact resistance was measured after applying a lateral voltage to the devices for a given time. The contact resistance of the Au electrode remained <10Ω even after the voltage (DC 4 V) was applied for more than 6 hours, which was found to be sufficient for the functional analysis of ion channels. Next, the resistance between the two electrodes was measured in buffer solution to examine the insulating properties of the CYTOP layer. When the device was coated twice with the CYTOP layer, the electrode-to-electrode resistance of the device exceeded 250 GΩ. After optimizing the CYTOP concentration, the device fabrication yield was improved up to 60%. Using the fabricated device, we applied the lateral voltage to lipid bilayers containing Na channels whose activities disappeared during the repetitive applications of transmembrane voltage pulses. Upon the application of the lateral voltage, the channel activities appeared again, showing frequent opening and closing events. The device fabricated by this process proved to be a useful tool for the novel analysis of inactivated ion channels. The establishment of such a system for analyzing ion channel function is expected to contribute to the discovery of new knowledge about ion channels and drug discovery.
