Single-Receptor Interfaces for Real-Time Kinetics
Abstract
The purpose of this research is to develop a label-free, field-deployable electronic interface to membrane proteins used as biosensing elements. We developed and demonstrated a new method for single-molecule electrophysiology, measuring changes in microwave-frequency transmission through a pore in a planar lipid bilayer simultaneously with conventional single-channel DC recordings. The results indicated that an increase in capacitance of order 0.5-1 fF was observed with blocking of the pore. The system consists of two sharpened coaxial probes aligned across a glass support with a lipid bilayer and a single pore-forming protein. Transmission through the pore was measured with a microwave spectrum analyzer functioning as a tuned receiver, whose output was recorded simultaneously with that of a patch-clamp amplifier. The protein staphylococcal a-hemolysin (aHL) was used as the pore. This molecule is a heptameric pore which allows the passage of ions, small globular molecules (< 2000 D) or long polymer chains such as DNA. The aHL pore can be blocked by the molecular adapter a-cyclodextrin, which lodges in the pore's lumen. These blocking events were observed simultaneously by decreases in transmembrane current and by increases in the microwave transmission at 900 MHz, due to increases in capacitance. The superior bandwidth of the spectrum analyzer (> 1 MHz) over that of the patch clamp amplifier (approx. 10 kHz) ultimately promises higher temporal resolution, and the use of high frequencies moves the measurement away from the 1/f noise characteristics that dominate DC measurements. Finally, the use of near-field microwave transmission using low-impedance probes obviates the need for a giga-ohm seal in patch clamping.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2005
- Accession Number
- ADA435098
Entities
People
- Daniel Van Der Weide
- Robert H Blick
Organizations
- University of Wisconsin–Madison