Recordings of transmembrane ion currents of a single channel embedded in artificial lipid bilayer membranes (BLMs), i.e., single-channel recordings, are useful for electrophysiological and kinetic studies of biological channels, and also for the development of channel-based sensors that afford amplified detection of analytes.
1,2The majority of single-channel recordings has been performed using planar BLMs with two separated aqueous phases, which were formed by a variety of techniques, such as painting, 3 monolayer folding 4,5 and tip-dip 6 methods. Planar BLMs have been utilized for the design of various kinds of electrochemical sensors.
1On the other hand, various supported bilayer membranes have been reported, including those formed on metals, 7 tethered ones formed on gold 8,9 and a peptide, 10 those formed on porous supports 11 and those on hydrogels, such as agarose 12-14 and agar. [15][16][17] These BLMs have attracted increasing interest due to their mechanical stability, which affords fabricating robust chemical sensors and molecular electronic devices.7 However, the metal-supported BLMs lack an aqueous phase on the metal support side, and hence ion translocation across the BLMs is not feasible, and the microscopically rough metal surface causes a local disturbance of the membrane structure, and consequently leaks. 17 The recordings of ion translocation so far reported as changes in the transmembrane ion currents [12][13][14][16][17][18] and the membrane potential, 15 have utilized mostly hydrogel-supported BLMs in which bioelements were embedded. However, few studies 13,14 have reported single-channel recordings, where a high electric resistance is essential for discriminating a singlechannel current at the level of a few pA from the background one. Ide et al. succeeded in making simultaneous optical and electric recording of single channels, such as a BK-channel 14 and alametithin, 13 using painted BLMs formed on agarosecoated glasses. For the design of channel-based sensing devices, excellent ion-blocking properties and a long lifetime along with an easy procedure for BLM formation become essential.In the present paper, we describe agarose-supported BLMs formed by the tip-dip and painting methods, which enable single-channel recordings of gramicidin. Gramicidin channels have been utilized for designing different types of channelbased BLM sensors. 8,9,19,20 The recording of a single-channel current is essential for the design of an ion-channel sensor.
19,21The properties of the supported BLMs are compared with those of unsupported ones in terms of a lifetime of the membranes, probability of single-channel recordings and open-close kinetics of gramicidin channels.
Experimental
MaterialsPhosphatidylcholine (PC, purity >99%, chloroform solution), phosphatidylethanolamine (PE, chloroform solution), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(2,4-dinitrophenyl) (DNP-PE) and 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPOPC, >99%, chloroform solution) were purchased from Avanti Polar Lipids, Inc...
