Functional Ion Channels in Tethered Bilayer Membranes—Implications for Biosensors

The demand for rapid in situ detection of chemical and biological analytes has increased the interest in the development of biosensors, which combine biological sensing elements with physicochemical transducers. Engineered membrane-bound ion channels are one promising class of biological receptors because they allow for highly sensitive stochastic detection of analytes, and produce a well-defined read-out that is inherently suitable for digitization. However, in order to perform stochastic sensing, it is necessary to measure the ion currents associated with single ion channel opening and closing events. Although, sensors based on supported tethered bilayers that contain various pore forming proteins have been described, there is still great limitations in creating a signal-to-noise ratio that is high enough to allow for single-channel activity detection. An alternative way to design bilayers on a chip, in which the lipid membrane covers an aperture, has been proposed. This technique has proven sensitive enough for detection of single ionchannel activity. However, this approach is fundamentally different to the tethering of bilayers onto a stable solid surface, and is likely to cause problems due to low mechanical stability. Here, we present a biosensor based on modulation of single ion-channel activity, with the ability to detect analytes in the micromolar range. The ion channels were interfaced to a gold surface, where they were reconstituted into tethered bilayer lipid membranes (tBLMs), which were in turn formed at multiple individual pixels of a microelectrode array device. The limited size of the gold sense pad surface (100;100 mm) and the electrical stability of the overlying lipid bilayer membrane made each pixel sensitive enough to measure single ion-channel currents in the picoampere range, and yet the device is convenient for monolithically integrated fabrication schemes. The biosensor is illustrated in Figure 1. Recently we were able to measure, for the first time, single ion-channel activity by using gramicidin A (gA), which was ACHTUNGTRENNUNGdirectly interfaced to a gold device surface. Even though gA can be modified to be used as a sensor there are still limitations in its use due to its relatively simple chemical struc-