Cells are the smallest structural unit of biological organisms that are capable of independent functioning, consisting of a semipermeable cell membrane enclosing cytoplasm, a nucleus, and various organelles. Cell membranes are important to the functions of a cell, with variety of important biological processes occurring at or within the membrane, including the absorption of nutrients, the secretion of metabolic products, the control of cell volume, and the communication with the outside environment.
Thus, it is not surprising that the biological function of cell membranes and especially of membrane proteins has become an area of active research. Signal transduction processes in general, including nerve conduction, and neuroreceptors in particular have been shown to be influenced by pharmacologically active ingredients, making them obvious targets for drug development.i Ion channels and ion transporters also have been shown to be an important class of therapeutic targets. In fact, interactions with ion channels have become a major potential source of adverse effects when administering a therapeutic agent, leading the Food and Drug Administration (FDA) and other government regulatory agencies to require safety profiling of potential therapeutics against certain ion channels.
This understanding of the interactions between potential drugs and cell membrane components is beginning to play a crucial role in modem drug development. In view of the increasing number of known receptors and the rapidly growing libraries of potential pharmaceutical ingredients, there clearly is a need for highly sensitive screening methods that permit the analysis of a large number of different substances with high assay throughput per unit time, otherwise known as “high throughput screening” (or “HTS”). In particular, there is a need for automated and/or high throughput screening methods that are relevant to cell membrane components.
At present, relatively traditional methods are used for the screening of pharmaceutical ingredients. Such methods include ligand binding assays and receptor function tests that are performed separately.ii Although binding assays are relatively inexpensive, and amenable to high throughput, they require labeled high-affinity ligands, and generally are limited to assays for ligands that can compete effectively for labeled ligand. Fluorescent or fluorogenic reagents generally are compatible with high throughput assays, including the analysis of ion channels using fluorescent calcium indicators, and the evaluation of membrane potential effects with potential-sensitive dyes. However, such reagents typically are not sensitive enough for single cell measurements, and generally can provide only indirect measurements of the membrane component of interest.
The patch clamp was introduced by Neher and Sakmann in the early 1980s as a powerful technique for the direct study of drug effects on single receptors. In recognition of the strength of the method, Neher and Sakmann were awarded the Nobel prize in 1991. Classical patch-clamp methods often are used in conjunction with functional membrane receptor assays, including receptors coupled to G-proteins and ion channel-forming receptors.iii This method is highly specific and extremely sensitive: it can, in principle, be used to measure the channel activity of individual receptor molecules. In doing so, glass micropipettes with an opening diameter of typically 1-0.1 μm are pressed on the surface of a biological cell. The membrane surface that is covered by the micropipette is called a “patch.” If the contact between the glass electrode and the cell membrane surface is sufficiently electrically isolating, the ion flow over the membrane patch can be measured electrically with the aid of microelectrodes, one placed in the glass pipette and the other placed in the milieu opposite the membrane.iv A significant advantage of this electrophysiological method is that it makes directly accessible the function of the corresponding channel-forming proteins or receptors coupled to channel-forming proteins via the measured electrical characteristics of the channel-forming proteins.
Unfortunately, several major limitations have prevented patch-clamp technology from revolutionizing receptor science and pharmaceutical drug development. For example, to produce high quality results, the patch-clamp method requires a tremendous effort in technical installation and highly qualified operators. Moreover, in addition to being expensive, a standard patch-clamp setup may require a long set-up time and have a high failure rate.
Thus, there is a need for a system for positioning and/or analyzing cells that is rapid, facile, and suitable for multiarray analysis, such as the system provided by the invention.