Ultra-sensitive detection methods for capillary electrophoresis (CE) based on patch-clamp electrode technology will be developed. The project goal is to fabricate rugged biosensors which are sensitive, quantitative, and not easily desensitized.
The specific aims are 1) to analyze individual cells, with the ultimate goal of early disease diagnosis, and 2) to achieve single molecule detection. The membrane/ion-channel source may be natural, genetically engineered or synthetic, depending on what best suits the analyte and the application. A patch of cell membrane serves as the biosensor portion of the detector, in which the membrane receptors face toward the outlet end of the separation capillary. Following CE separation, an analyte molecule, such as acetylcholine (ACh), elutes upon and binds to its receptor. An ion-channel is opened, many thousands of ions are passed through the channel, and the current is recorded. Achieving single molecule detection with the patch-clamp electrode is based upon this inherent amplification, in which one or two molecules elicit a response whereby thousands of ions are measured. Its success represents an unrivaled accomplishment in single molecule detection by a non-fluorescent based method. For the analysis of individual biological cells, approaches are proposed for both indirect detection of agonist molecules (i.e., those which bind to the cell membrane receptors) and direct detection of inorganic ions (i.e., those which pass selectively through the ion-channels). The latter approach uses ACh to open the ion-channels, but the measured ions are the analytes. Although the amplification feature is lost, expected detection limits are in the 0.1 to 1 attomole range. This technique should allow for the first time the determination of intracellular divalent cations in single cells, and give detection limits for Na+ and K+ 100-1000 times better than with indirect laser-induced fluorescence.
| Kassavetis, George A; Prakash, Prachee; Shim, Eunjung (2010) The C53/C37 subcomplex of RNA polymerase III lies near the active site and participates in promoter opening. J Biol Chem 285:2695-706 |
