High throughput functional genomics

Abstract

This invention focuses on the marriage of solid-state electronics and neuronal function to create a new high-throughput electrophysiological assay to determine a compound's acute and chronic effect on cellular function. Electronics, surface chemistry, biotechnology, and fundamental neuroscience are integrated to provide an assay where the reporter element is an array of electrically active cells. This innovative technology can be applied to neurotoxicity, and to screening compounds from combinatorial chemistry, gene function analysis, and basic neuroscience applications. The system of the invention analyzes how the action potential is interrupted by drugs or toxins. Differences in the action potentials are due to individual toxins acting on different biochemical pathways, which in turn affects different ion channels, thereby changing the peak shape of the action potential differently for each toxin. Algorithms to analyze the action potential peak shape differences are used to indicate the pathway(s) affected by the presence of a new drug or compound; from that, aspects of its function in that cell are deduced. This observation can be exploited to determine the functional category of biochemical action of an unknown compound. An important aspect of the invention is surface chemistry that permits establishment of a high impedance seal between cell and a metal microelectrode. This seal recreates the interface that enables functional patch-clamp electrophysiology with glass micropipettes, and allows extracellular electrophysiology on a microelectrode array. Thus, the invention teaches the feasibility of using living cells as diagnostics for high throughput real-time assays of cell function.

What is claimed is:  
     
         1 . A system capable of identifying one or more ion channels of a cell, which channels are affected by a test substance, comprising a device and accompanying software, 
 in which said device comprises: 
 (a) a solid state microelectrode;  
 (b) a cell culture comprising one or more electrically active cells having a cell membrane including one or more ion channels, which one or more cells are capable of providing a measurable action potential that exhibits one or more perceptible characteristics; and  
 (c) an intervening layer which (i) comprises a surface modifying agent, and (ii) is positioned between said microelectrode and the one or more cells of said cell culture, such that a high impedance seal is provided at least in the vicinity of said one or more cells of said cell culture;  
   and in which said accompanying software comprises instructions that can be implemented by a computer and which are capable of relating changes in the one or more characteristics exhibited by said action potential to one or more ion channels of said one or more cells upon exposure of said one or more cells to a test substance.    
     
     
         2 . The system of  claim 1  in which the one or more characteristics exhibited by said action potential is manifested in its waveform or a derivative thereof.  
     
     
         3 . The system of  claim 2  in which the one or more characteristics include at least one of after potential, time to cessation of activity, frequency, amplitude, shape, spike rate, or time constant.  
     
     
         4 . The system of  claim 1  in which the data processing instructions are further capable of receiving input data comprising data on ion flux through ion channels selected from the group consisting of sodium channels, potassium channels, calcium channels, or combinations thereof.  
     
     
         5 . The system of  claim 1  in which the microelectrode is planar or flexible.  
     
     
         6 . The system of  claim 1  in which the microelectrode is a field effect transducer.  
     
     
         7 . The system of  claim 1  which further comprises an insulator selected from the group consisting of silicon, modified silicon dioxide, silicon nitride, silicon carbide, germanium, silica, gallium, arsenide, epoxy resin, polystyrene, polysulfone, alumina, silicone, fluoropolymer, polyester, acrylic copolymers, polylactate, or combinations thereof.  
     
     
         8 . The system of  claim 1  in which said electrically active cell comprises a neuronal cell or a cardiac cell.  
     
     
         9 . The system of  claim 8  in which the neuronal cell is a hippocampal cell.  
     
     
         10 . The system of  claim 1  in which the cell culture comprises a stem cell, a transformed stem cell, their respective progeny, or combinations thereof.  
     
     
         11 . The system of  claim 10  in which the stem cell is exposed to a differentiating factor.  
     
     
         12 . The system of  claim 1  in which the surface modifying agent comprises a self-assembling monolayer or monolayers.  
     
     
         13 . The system of  claim 12  in which the self-assembling monolayer comprises a silane, a thiol, isocyanide, polyelectrolyte or similar molecules or combinations thereof.  
     
     
         14 . The system of  claim 1  in which the data processing instructions is further capable of receiving input data comprising data on the changes in the temporal description of the action potential.  
     
     
         15 . The system of  claim 1  in which the intervening layer further comprises cell anchorage molecules.  
     
     
         16 . The system of  claim 15  in which the cell anchorage molecules comprise antibodies, antigens, receptor ligands, receptors, lectins, carbohydrates, enzymes, enzyme inhibitors, biotin, avidin, streptavidin, cadherins, RGD-type peptides, integrins, cadherins, modified lipids, or combinations thereof.  
     
     
         17 . The system of  claim 1  in which the intervening layer comprises a high viscosity mixture comprising alcohols, ethers, esters, ketones, amides, glycols, amino acids, saccharides, carboxymethylsaccharides, carboxyethylsaccharides, aminosaccharides, acetylaminosaccharides, polymers thereof, or combinations thereof.  
     
     
         18 . The system of  claim 1  in which the one or more cells of the cell culture comprise one or more cells transfected with endogenous or exogenous nucleic acid.  
     
     
         19 . The system of  claim 18  in which the nucleic acid comprises a nucleotide sequence associated with known or unknown function.  
     
     
         20 . The system of  claim 1  in which the cell culture is coated with a polymer.  
     
     
         21 . The system of  claim 20  in which the polymer comprises cellulose, methylcellulose, dextran and the like.  
     
     
         22 . The system of  claim 1  in which the intervening layer can be characterized as either an attractive layer or a repulsive layer.  
     
     
         23 . The system of  claim 1  which further comprises a detector circuit.  
     
     
         24 . A method of determining one or more ion channels that are affected by a test substance comprising: 
 (a) contacting a substance to be tested with a device comprising a solid state microelectrode; a cell culture including one ore more cells having a cell membrane including one or more ion channels, which one or more cells are capable of providing a measurable action potential that exhibits one or more perceptible characteristics; and an intervening layer that is acting as a high impedance seal and which is positioned between said microelectrode and said cell culture;    (b) collecting data on the action potential, the one or more characteristics thereof, or one or more changes therein; and    (c) determining from said data the one or more ion channels that are affected by the test substance.    
     
     
         25 . The method of  claim 24  which the test substance comprises a toxin, a drug, a pathogen, a neurotransmitter, a nerve agent, or mixtures thereof.  
     
     
         26 . The method of  claim 24  in which the determining step includes deconvoluting the action potential or its derivative, the one or more characteristics thereof, or the one or more changes therein.  
     
     
         27 . The method of  claim 24  in which the deconvolution leads to information on pathways or functional categories affected in the cell.  
     
     
         28 . A system having a high throughput capacity to determine potential physiological effects of a test substance comprising a device and accompanying software, 
 in which said device comprises a solid state microelectrode and a cell culture comprising one ore more cells, which are capable of providing a measurable action potential that exhibits one or more perceptible characteristics,    and in which said accompanying software comprises data processing instructions capable of relating changes in the one or more characteristics exhibited by said action potential to one or more potential physiological effects exerted by a test substance upon exposure of the test substance to the one or more cells of said cell culture.    
     
     
         29 . The system of  claim 28  in which said device further comprises an intervening layer that is acting as a high impedance seal and which is positioned between said microelectrode and the one or more cells of said cell culture.  
     
     
         30 . The system of  claim 28  in which said accompanying software comprises instructions for manipulating one or more system parameters to alter one or more conditions of a given experiment, for interpreting the outcome of such manipulations, or for both.  
     
     
         31 . The system of  claim 30  in which such manipulations include the addition of a compound of interest to said cell culture or the removal thereof from said cell culture.  
     
     
         32 . The system of  claim 31  in which the compound of interest is a nutritive material or a cell modulator.  
     
     
         33 . The system of  claim 28  in which said data processing instructions includes a temporal analysis of the action potential or the changes observed therein.  
     
     
         34 . The system of  claim 28  in which said data processing instructions is capable of providing an output suggestive of the involvement of one or more cellular pathways or receptors of interest.  
     
     
         35 . The system of  claim 34  which is capable of determining a mode of action of a test substance based on the one or more cellular pathways or receptors of interest involved.  
     
     
         36 . The system of  claim 28  in which said accompanying software includes instructions for a feedback loop.  
     
     
         37 . The system of  claim 28  in which the test substance comprises a toxin, a drug candidate, a pathogenic agent, a neurotransmitter, a nerve agent, a gene, a gene product, or mixtures thereof.  
     
     
         38 . A system for determining one or more potential functions of an isolated nucleic acid, its expression product, or one or more active fragments of said nucleic acid or expression product, comprising a device and accompanying software, 
 in which said device comprises a solid state microelectrode and a cell culture comprising one ore more cells that are capable of providing a measurable action potential that exhibits one or more perceptible characteristics and which cells have been either transfected with an isolated nucleic acid or exposed to its expression product,    and in which said accompanying software comprises data processing instructions capable of relating changes in the one or more characteristics exhibited by said action potential to one or more potential functions of said isolated nucleic acid, its expression product, or one or more active fragments of said nucleic acid or expression product.    
     
     
         39 . The system of  claim 38  in which said device further comprises an intervening layer that is acting as a high impedance seal and which is positioned between said microelectrode and the one or more cells of said cell culture.  
     
     
         40 . The system of  claim 38  in which said accompanying software comprises instructions for manipulating one or more system parameters to alter one or more conditions of a given experiment, for interpreting the outcome of such manipulations, or for both.  
     
     
         41 . A method of determining one or more potential functions of an isolated nucleic acid, its expression product, or one or more active fragments of said nucleic acid or expression product comprising 
 (a) providing a device comprising a solid state microelectrode; a cell culture comprising one ore more cells that are capable of providing a measurable action potential that exhibits one or more perceptible characteristics and which cells have been either transfected with an isolated nucleic acid or exposed to its expression product,    (b) collecting data on the action potential, the one or more characteristics thereof, or one or more changes therein; and    (c) determining from said data the one or more potential functions of said isolated nucleic acid, its expression product, or one or more active fragments of said nucleic acid or expression product.    
     
     
         42 . The method of  claim 41  in which the determining step includes deconvoluting the action potential, the one or more characteristics thereof, or the one or more changes therein.  
     
     
         43 . The method of  claim 41  which further comprises manipulating one or more parameters to alter one or more conditions of a given experiment.  
     
     
         44 . The method of  claim 43  which further comprises interpreting the outcome of such manipulations.  
     
     
         45 . The method of  claim 41  in which said device further comprises an intervening layer serving as a high impedance seal, which is positioned between said microelectrode and the one or more cells of said cell culture.  
     
     
         46 . A computer readable medium encoding a program that includes instructions for execution by a computer, which instructions comprise data processing steps that relate changes in one or more characteristics exhibited by an observed action potential to one or more ion channels of one or more cells of a cell culture upon exposure of the one or more cells to a test substance.  
     
     
         47 . The computer readable medium of  claim 46  in which said data processing steps comprise a deconvolution step by which the changes in the one or more characteristics exhibited by the observed action potential are compared with stored information from past observations allowing the computer to attribute the changes to the one or more ion channels of said one ore more cells.  
     
     
         48 . The computer readable medium of  claim 46  in which said data processing steps do not include a spectral analysis.  
     
     
         49 . The computer readable medium of  claim 48  in which said spectral analysis makes use of a Fourier transform.