Stochastic Sensing on a Modular Chip Containing a Single-Ion Channel

Abstract

Engineered protein channels have many potential applications in biosensing at the single molecule level. A future generation of biosensor could be an array of target-specific ion channels, where each protein pore acts as a sensor element. An important step toward this goal is to create a portable, durable, single protein channel-integrated chip device. Here we report a versatile, modular chip that contains a single ion channel for single molecular biosensing. The core of the device is a long-lived lipid membrane that has been sandwiched between two air-insulated agarose layers which gel in situ. A single protein pore embedded in the membrane serves as the sensor element. The modular device is highly portable, allowing a single ion channel to continuously function following detachment of the chip from the instrument and independent transportation of the device. The chip also exhibits high durability, which is evidenced from long-duration continuous observation of single channel dynamics. Once engineered protein pores are installed, the chip becomes a robust stochastic sensor for real-time targeting such as detection of the second messenger IP3. This pluggable biochip could be incorporated with many applicable devices, such as a micro-fluidic system, and be made into a micro-array for both biomedical detection and membrane protein research.

Stable Lipid Bilayers Based on Micro- and Nano-Fabrication as a Platform for Recording Ion-Channel Activities

“…This lifetime was longer than that (several hours) of BLMs formed in conventional μm-scale apertures. 34 As shown in Fig. 3(d), gramicidin channel activities were clearly seen even 16 h after membrane formation, demonstrating the long-lasting functionality and stability of the present BLMs.…”

“…Thus the present BLMs could withstand a much higher voltage than the breakdown voltage (~220 -330 mV) reported for BLMs in apertures of 50 -100 μm. [32][33][34] Considering that the present BLMs have a total membrane size of ~75 μm, much higher BLM stability was achieved by supporting large membrane area with a nano-mesh structure of the porous alumina films. The membrane lifetime, defined as the duration for which BLMs retained resistance higher than 1 GΩ, was 16 -30 h (n = 2) with and without gramicidin channels.…”

“…15,38 On the other hand, Teflon and SiO2 are insulators with a similar dielectric constant to SU8, but they also have the advantage over SU8 of having superior chemical stability. In addition, both materials are well-suited for suspending BLMs, as exemplified by the folding method for Teflon 19,34,39 and the tip-dip method for SiO2 (glass). 40,41 Here, insulator layers of Teflon and SiO2 (thermal oxide) have been coated on the surface of the Si chip and background noise currents of BLMs prepared in the Si chips were examined.…”

Stable insulating tethered bilayer lipid membranes

“…Recent progress has been made by encapsulating planar bilayers in agarose gels. 35,36 It has been demonstrated that membranes with embedded proteins could be stored for weeks and regain their function after rehydration. Similarly, hydrogels have been used to protect planar bilayers.…”

Lipid Bilayers at Gel/Gel Interface for Ion Channel Recordings

“…A great deal of skill and practice is necessary in order to acquire practical data from these channel recordings meaning that although the information acquired is extremely useful, the labour is laborious and frustrating. Thus several proposals have been made for the construction of durable bilayers [1][2][3][4][5]. We have developed a practical method to produce such durable bilayers for channel measurements.…”