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Microstructure apparatus and method for separating differently charged molecules using an applied electric fieldRelated Patent Categories: Chemical Apparatus And Process Disinfecting, Deodorizing, Preserving, Or Sterilizing, Analyzer, Structured Indicator, Or Manipulative Laboratory Device, Miscellaneous Laboratory Apparatus And Elements, Per Se, Including Means For Separating A Constituent; E.g., Filter, Condenser, Extractor, Etc.Microstructure apparatus and method for separating differently charged molecules using an applied electric field description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050271554, Microstructure apparatus and method for separating differently charged molecules using an applied electric field. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF INVENTION [0001] The field of the present invention relates generally to a microstructure apparatus which may be used in a high-throughput screening context to monitor the rate of reaction of an enzyme with its substrate in cases where the product of the reaction has an altered net charge. For example, the systems and methods disclosed herein may be used to detect the activity of phosphatase enzymes, proteases and kinases on charged peptide substrates. The microstructure devices of the present invention comprise a plurality of microstructures, wherein each microstructure comprises a capture matrix located between two electrodes. BACKGROUND OF THE INVENTION [0002] Protein kinases are of particular interest in drug discovery research because they have been shown to be key regulators of many cell functions, including signal transduction (Ullrich and Schlessinger, 1990), transcriptional regulation (Pawson and Bernstein, 1990), cell motility (Miglietta and Nelson, 1988) and cell division (Pines and Hunter, 1990). Protein kinases are enzymes which covalently modify proteins and peptides by the attachment of a phosphate group to one or more sites on the protein. Phosphatases perform the opposite function. Many of the known protein kinases use adenosine triphosphate (ATP) as the phosphate donor, placing the .gamma.-phosphate onto a histidine, tyrosine, serine or threonine residue in the protein. The location of the modification site and the type of residue modified by the kinase are usually specific for each particular kinase. [0003] The added phosphate alters certain structural, thermodynamic and kinetic properties of the phosphorylated protein. Generally, the phosphate adds two negative charges to the protein. This modifies the electrostatic interactions between the protein's constituent amino acids, in turn altering secondary and tertiary protein structure. The phosphate may also form up to three hydrogen bonds or salt bridges with other protein residues, or may otherwise change the conformational equilibrium between different functional states of the protein. These structural changes provide the basis, in a biological system, for altering substrate binding and catalytic activity of the phosphorylated proteins. [0004] Phosphorylation and dephosphorylation reactions, under the control of kinases and phosphatases, respectively, can occur rapidly to form stable structures. This makes the phosphorylation system ideal as a regulatory process. Phosphorylation and dephosphorylation reactions may also be part of a cascade of reactions that can amplify a signal that has an extracellular origin, such as hormones and growth factors. [0005] Methods for assaying the activity of protein kinases often utilize a synthetic peptide substrate that can be phosphorylated by the kinase protein under study. The most common mechanisms for detecting phosphorylation of the peptide substrates are 1) Incorporation of .sup.32P (or .sup.33P) phosphate from [.sup.32P].gamma.-ATP into the peptides, purification of the peptides from ATP, and scintillation or Cherenkov counting of the incorporated radionucleotide, 2) Detection of phosphoamino acids with radiolabeled specific antibodies, or 3) Purification of phosphorylated peptides from unphosphorylated peptides by chromatographic or electrophoretic methods, followed by quantification of the purified product. [0006] For example, in one widely used method, a sample containing the kinase of interest is incubated with activators and a substrate in the presence of gamma .sup.32P-ATP, with an inexpensive substrate, such as histone or casein being used. After a suitable incubation period, the reaction is stopped and an aliquot of the reaction mixture is placed directly onto a filter that binds the substrate. The filter is then washed several times to remove excess radioactivity, and the amount of radiolabelled phosphate incorporated into the substrate is measured by scintillation counting (Roskoski, 1983). [0007] The use of .sup.32P in assays, however, poses significant disadvantages. One major problem is that, for sensitive detection, relatively high quantities of .sup.32P must be used routinely and subsequently disposed. The amount of liquid generated from the washings is not small, and contains .sup.32P. Due to government restrictions, this waste cannot be disposed of easily. It must be allowed to decay, usually for at least six months, before disposal. Another disadvantage is the hazard posed to personnel working with the isotope. Shielding and special waste containers are inconvenient but necessary for safe handling of the isotope. Further, the lower detection limit of the assay is determined by the level of background phosphorylation and is therefore variable. In short, the study of protein kinases would be greatly facilitated by the development of an efficient and accurate assay that does not require the use of radioactivity. [0008] Although radioisotope methods have been applied in high throughput screening, the high cost and strict safety regulation incurred with the use of radioisotopes in high throughput screening greatly limits their use in drug discovery. For these and other reasons, it would be useful to develop alternative methods and apparatus for high throughput screening that facilitate measuring the kinase dependent phosphorylation of peptides. [0009] Recently, several analytical chemistry research groups have experimented with micro-lithographed electrophoretic separation devices. These devices typically contain four or more reservoirs connected by a cross-shaped arrangement of channels. A long, sinuous channel is usually situated at the tail of the cross, which is utilized for electrophoretic separation of charged molecules in the system. These devices rely on electro osmotic flow (EOF) forces to provide flow of the solution through the system, and thus all molecules (positive, negative, and uncharged) in the sample are transported in the same direction along the separation channel, their speed and position determined by their net charge/mass. In order to detect each molecule in the sample, a continuous detection system is used during the electrophoretic separation process. Because of their reliance on EOF forces, these devices must be manufactured to high tolerance (with small cross-section microchannels) and are designed to include a rather long separation path. SUMMARY OF THE INVENTION [0010] The devices and methods of the present invention provide a simple solution to the problem of separating differently charged molecules in a sample for the detection of a molecule of interest. These methods and devices are particularly suitable for analyzing substrate-enzyme reactions, or other simple biochemical model systems where a labeled molecule may undergo change in net charge, in a highly parallel manner. The devices of the invention utilize a capture matrix located in the electrophoretic path of the charged molecule of interest in order to capture that molecule for later analysis. Thus, continuous detection of molecules traveling along the electrophoretic path is not necessary with the devices of the present invention. Moreover, because they do not utilize migration speed in solution in order to separate the charged molecules, the devices of the invention are able to use much shorter and direct migration paths, simplifying device design and construction. [0011] Thus, in one aspect, the current invention provides novel systems for separating peptides, or other molecules in a sample having different net charges, for detection and quantification. [0012] In preferred embodiments of this aspect of the invention, the one or more samples are simultaneously assayed in the system, which contains one or more microstructures to separate the charged molecules in the sample. Each microstructure comprises a capture matrix, which, after the individual samples containing molecules of different charges are introduced into the microstructures, electrophoresed, and concentrated in the capture matrix section of the microstructure, binds or holds the molecules of interest for detection. The systems of the invention comprise: [0013] a) a microstructure plate comprising: [0014] at least one microstructure, each microstructure comprising a series of microstructure sections and channels, wherein each microstructure section is directly interconnected to at least one other microstructure section by at least one channel, the series comprising: [0015] at least one sample accepting microstructure section, wherein the sample accepting section is fluidly connected to the exterior of the microstructure plate; [0016] at least one first electrode microstructure section; [0017] at least one second electrode microstructure section; [0018] at least one capture microstructure section containing a capture matrix, wherein the capture microstructure section is between the first and second electrode microstructure sections in the series; [0019] wherein the microstructures in the microstructure plate are formed by at least two layers of material, wherein at least one layer is a sealing plate layer which seals at least one channel or microstructure section in the assembled microstructure plate; and [0020] b) an electrode assembly, the electrode assembly having at least one first and at least one second electrode, wherein each first electrode microstructure section is in electrical contact with at least one first electrode, and wherein each second electrode microstructure section is in electrical contact with at least one second electrode. [0021] Preferred embodiments of the system comprise a plurality of microstructure units, preferably arrayed in a rectangular fashion. These systems may be compatible with 96, 384, or 1536 well microtiter plate formats, and compatible with microtiter plate loaders and readers, such as absorbance or fluorescence microtiter plate readers. This allows for the simultaneous processing of a plurality of samples for high-throughput screening applications by simultaneously loading the samples into the system and simultaneously subjecting the samples to electrophoresis. Continue reading about Microstructure apparatus and method for separating differently charged molecules using an applied electric field... 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