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. 2007 Mar 15;79(6):2207-13.
doi: 10.1021/ac0614285. Epub 2007 Feb 9.

Stochastic sensing on a modular chip containing a single-ion channel

Ji Wook Shim  1 , Li Qun Gu
Affiliations

Stochastic sensing on a modular chip containing a single-ion channel

Ji Wook Shim et al. Anal Chem. .

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 microfluidic system, and be made into a microarray for both biomedical detection and membrane protein research.

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Figures

Figure 1
Figure 1
Composition, fabrication, usage and prototype of the modular ion channel chip. (A) The chip is composed of two flat compartments that are separated by a partition, with each compartment enclosing a thin layer of ultra-low gelling temperature agarose which has been premixed with salt at the desired concentration. The two agarose layers on both sides of the partition sandwich a lipid bilayer membrane over a 100 μm-aperture in the center of partition, where single protein pores are embedded as sensor elements; (B) Usage of the modular ion channel chip as an independent device. The analyte can be added from the sample cell on the back of either compartment and delivered to the sensor element through agarose between the sample cell and the membrane; (C) Usage of the chip as a pluggable device; (D) Pictures of prototype chips made from Teflon (left) and silicone elastomer polydimethylsiloxane (PDMS, middle). The partition is a 25-μm Teflon film. The quarter (right) is to compare the chip dimension.
Figure 2
Figure 2
(A) A 65-hour continuous current recording of long-lived single Kcv channels in the chip. The trace is screen-captured. Agarose layers contain 1 M KCl, 10 mM Tris, and 1.5% ultra-low gelling temperature agarose (pH7.2). Detailed current recordings at the 3rd and 55th hour in (A) are expanded and screen-captured in (B) and (C) respectively.
Figure 3
Figure 3
Pictures captured from a movie showing the portability of the chip (Movie S1 in Supplementary Materials). (A) Visualizing a single potassium channel Kcv in the chip. The gating profile is marked with a yellow circle; (B) Disconnecting the chip from the electrical recording devices and moving the chip out of the testing box; (C) Handling the chip and transporting it around the building; (D) Re-connecting the chip to the electrical system; (E) Recovery of single channel current from the chip.
Figure 4
Figure 4
Stochastic sensing on the modular ion channel chip. (A) Time-dependent transportation of analyte from the sample cell to the sensor element in the chip. Single pores of wild-type αHL were incorporated into the sandwiched bilayer as the sensor element. 40 μM βCD was added to the chip from the sample cell (Fig.1B). Agarose layers contained 1 M KCl, 10 mM Tris, and 1.5% ultra-low gelling temperature agarose (pH7.2). Left panel shows current traces recorded after 5, 30 and 40 minutes after addition of βCD. Right panel is the time-dependent block occurrence with βCD; (B) Detection of second messenger IP3 on chips. Mutant αHL, M113R/T145R, was used as the single pore sensor element. Agarose layers contained the same components as in A, with the addition of 0.5 mM MgCl2. All traces were recorded at +30 mV. Trace (I), control test without analyte; Trace (II), 500 nM IP3 was loaded into the sample cell of the same chip; Trace (III) 0.3 mM ATP was added in a separate control test; and Trace (IV), mixture of 500 nM IP3 and 0.3 mM ATP was loaded in the chip. All traces were captured after the block occurrence reached the maximum.
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