抄録
Introduction
Recently, we found that, in addition to the conventional transmembrane voltage, the voltage applied laterally to the membrane can reactivate once inactivated human voltage-gated Na ion channel membrane proteins using an artificial cell membrane system [1]. This finding suggests that lateral voltage may be an important novel parameter for promoting the activity of ion channels prone to inactivation, which are difficult to measure by conventional methods. However, the short electrode lifetime and low yield of lateral voltage-applying devices are major bottlenecks in the establishment of a new lateral voltage-based ion channel function analysis system. The usable lifetime of the lateral voltage application devices is as short as 20 minutes due to oxidation of the Ti electrodes, and the fabrication yield is less than 10% due to cracks in the SiO2 layer used as the insulating layer. In this study, to address these issues, we aimed to develop a highly efficient fabrication process for more stable and longer-lived devices by using Au, a non-oxidizing metal, as the electrodes and CYTOP, a flexible insulating coating, as the insulating layer.
Method
A micropore was formed in a Teflon film by an electric spark. The Teflon film was covered with a Ni mask with an electrode pattern, and a Ti/Au thin film used for electrodes was formed on the film by sputtering. To form an insulating layer on the electrodes, the Teflon film was then dipped into CYTOP (CTL-109AE) except for the electrode contact region, pulled up by a dip coater at a speed of 1 mm/s, and baked. We evaluated the electrical properties of the fabricated devices by measuring the resistance between the electrodes and the contact resistance of the electrode region that was connected to the lateral voltage source. In addition, the effect of lateral voltage on ion channels was evaluated using the fabricated devices. Artificial cell membranes were formed in the micropores of the devices by the monolayer-folding method, and human Na-ion channels were embedded in the membranes via proteoliposome fusion. Ion channel currents were measured and compared with and without the application of the lateral voltage.
Results and discussion
To investigate the electrode lifetime of the fabricated devices, the contact resistance was measured after that lateral voltage was applied to the devices for a given time. The result showed that the contact resistance of the Au-based electrodes remained low (< 10 Ω) even after the lateral voltage was applied for more than 2 hours, which was sufficient for the functional analysis of ion channels. We next investigated the insulation property of the CYTOP layer through the measurement of resistance between the fabricated electrodes in buffer solutions. When the devices were coated with a single CYTOP layer, we observed current leakage through the buffer solution. When the devices were coated twice with the CYTOP layers, the resistance between the electrodes increased to more than 250 GΩ even in the buffer. Thus, it was found that the double CYTOP coating was suitable for obtaining a high resistance of the insulating layer. The concentration of the CYTOP was also important to improve the fabrication yield. By adjusting these conditions, we were able to improve the device fabrication yield to 60%. Using the fabricated devices based on the Au electrodes and CYTOP layers, we applied lateral voltage to inactivated Na channels and observed that the channel functions were re-activated by the lateral voltage. It was demonstrated that the devices fabricated by the new process could be useful as a novel tool for the analysis of inactivated ion channels.
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