Finding new drugs which have specific modulatory effects on ion channels requires methods for measuring and manipulating the membrane potential of cells with the ion channels present in the membrane. A number of methods exist today that can be used to measure cell transmembrane potentials and to measure the activities of specific ion channels. Probably the best known approach is the patch clamp, originally developed by Neher, Sakmann, and Steinback. (The Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranes”, Pfluegers Arch. 375; 219-278, 1978; Hamill et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391(2); 85-100, (1981)). Other methods include optical recording of voltage-sensitive dyes or proteins (Cohen et al., Annual Reviews of Neuroscience 1:171-82, 1978), extracellular recording of fast events using metal (Thomas et al., Exp. Cell Res. 74:61-66, 1972) or field effect transistors (FET) (Fromherz et al., Science 252:1290-1293, 1991) electrodes, or by modulating the transmembrane potential with applied electric fields and the measurement of this change using fluorescent dyes (U.S. Pat. No. 6,686,193). Still other methods include using extracellular electrodes (Thomas et al., Exp. Cell Res. 74:61-66, 1972), radioactive flux assays, the expression of endogenous proteins or the use of reporter genes or molecules.
The patch clamp technique is significantly limited by its low throughput. Further, it is not easily amenable to automation. Similarly, optical detection systems are limiting in that they require the use of one or more dyes and do not provide the ability to regulate, or clamp, the transmembrane potential of a cell during the measurement.
Dielectric spectroscopy (DS) can be used to study the electrical properties of living cell suspensions. There has been significant interest over the time in measuring the complex dielectric function of cells in suspensions. There are several dielectric spectroscopy techniques that have been applied directly to biological systems (See G. R. Facer, et al., Appl. Phys. Lett. 78 (2001), no. 7, 996-998; H. E. Ayliffe, et al., J. Microelectromechanical Systems 8 (1999), no. 1, 50-56; G. De Gasperis, et al, Measurement Science and Technology 9 (1998), no. 3, 518-529; G. Smith, et al., J. Pharmaceutical Sciences 84 (1995), no. 9, 1029-1044; E. Gheorghiu, Bioelectrochemistry and Bioenergetics 40 (1996), no. 2, 133-139; and E. Gheorghiu and K. Asami, Bioelectrochemistry and Bioenergetics 45 (1998), no. 2, 139-143). However, most either become non-reliable in the low frequency range or involve extremely large electric fields.
The frequency dependent permittivity and/or conductivity of a material or a living organism has been measured using linear dielectric spectroscopy. (See S. Gawad, K. Cheung, U. Seger et al., Dielectric spectroscopy in a micromachined flow cytometer: theoretical and practical considerations, Lab on a Chip 4 (2004), 241-251; and E. Gheorghiu, Measuring living cells using dielectric spectroscopy, Bioelectrochemistry 40 (1996), 133-139). However, dielectric spectroscopy has not been shown to be useful for measuring membrane potential of mammalian cells, particularly in a screening or high throughput assay.
For the low frequency range the alpha dispersions are known to provide information on cell behavior by observing the evolution of electrical and morphological parameters during cell cycle progressions. This data has theoretically known to provide information on the transmembrane potential of the cell (Gheorghiu (1996). Characterizing cellular systems by means of dielectric spectroscopy. Bioelectromagnetics 17:475-482). (C. Prodan and E. Prodan (1999), The Dielectric Behavior of Living Cell Suspensions, J. Phys. D: Appl. Phys. 32 335-343). However, the theory has not been sufficient to accurately and quickly calculate the membrane potential until now.
USPTO Backgrounds
USPTO Backgrounds