Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-02-17
2001-07-17
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S450000, C204S451000, C204S469000, C204S600000, C204S601000, C204S605000
Reexamination Certificate
active
06261430
ABSTRACT:
BACKGROUND TO THE INVENTION
The instant invention relates to methods and apparatus for electrophoretic movement and separation of samples of charged molecular species in a microelectrophoresis chip.
The principle of separating a mixed pool of charged molecules into separate species by electrophoresis through which a separation matrix is well known in the art. Commercial apparatus are available for separation of proteins, nucleic acids and other biomolecules. In the most general format, a sample of biomolecules is loaded into a separation matrix (e.g., agarose gels and polyacrylamide gels, either with or without denaturing buffer solutions) at a loading site. Electrodes, e.g., solution electrodes, are placed at either end of the separation matrix. An electric field is the applied across the separation matrix, which causes electrically charged molecules in the sample migrate away from the loading site. The direction and extent of sample migration is a function of charge, size and shape of the molecule. (Current Protocols in Molecular Biology (ed. Janssen, K) John Wiley & Sons, Inc., 1987-94).
Electrophoresis call also be performed in tubes as described in International Patent Application No. WO91/12904 and European Patent Application No. 0 653 631 A2. Materials can be moved through these tubes in what amounts to a one-dimensional separation using variable electric fields generated by a plurality of electrodes disposed along the length of the tube.
Molecular species separated by electrophoresis may be simply observed and discarded (recorded patterns of separation provide important information such as in DNA sequencing: see Sanger et al. Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) or, in some applications, species are recovered from the separation matrix for further investigation. Recovery can be achieved in several well known ways. One method requires slicing the separation matrix and physically removing matrix fragments containing the subject species. Such matrix fragments are then processed on glass or Sephacryl S-300 columns, or electroeluted using buffer-filled dialysis bags, followed by concentration and purification. Alternatively, molecular species may be electrophoresed directly onto NA-45 paper, or another adsorption membrane, which may then be removed from the gel for further investigation of sample. (Current Protocols, supra)
Though electrophoresis is a well studied phenomena, researchers have not developed methods or apparatus for using electrophoresis in conjunction with recently developed miniature components for analyzing biomolecules. Small sized electrophoresis apparatus such as the 43×50×0.45 mm gels of the PhastSystem of Pharmacia Biotech (Uppsala, Sweden) do not offer enough sensitivity for the micromanipulation of tiny samples of charged molecules. Biosensors such as those disclosed in U.S. Pat. No. 5,063,081, U.S. Pat. No. 5,200,051, U.S. Pat. No. 5,212,050 and U.S. Pat. No. 5,112,455 granted to Cozzette et al., which are incorporated herein by reference, operate on the scale of a few square millimeters. Other researchers have developed tiny silicon wafers, related to computer chips in their appearance, that can be used for chemical and enzymatic manipulation of biomolecules. Miniature components that can be used for detection, measurement or modification of biomolecules are also found in the following patents and patent applications which are incorporated herein by reference: WO 93/22680; WO 93/09668; WO 92/10587; WO 92/10092; U.S. Pat. No. 5,384,261; U.S. Pat. No. 5,320,725; U.S. Pat. No. 5,286,364; U.S. Pat. No. 5,264,105; U.S. Pat. No. 5,264,104; U.S. Pat. No. 5,262,305; and U.S. Pat. No. 4,975,175.
An electrophoresis system which is capable of very small scale movement and separation of nucleic acids and other charged molecules could be extremely useful in conjunction with miniature components to accomplish rapid and cost effective diagnoses of medical conditions, to conduct complex experiments using tiny quantities of reagents, and to generally take advantage of the development of miniature bio-analytical and bio-manipulative components, such as those listed above.
It is an object of the instant invention to provide methods and apparatus for manipulation of a sample of biomolecules such as nucleic acids and other electrically charged molecules.
It is a further object of the instant invention to provide methods and apparatus for the movement and separation of samples of charged molecular species in a miniature electrophoresis apparatus.
It is a further object of the instant invention to provide a process for the fabrication of a miniature electrophoresis apparatus.
These and other objects of the invention are obtained according to the following specification
SUMMARY OF THE INVENTION
In accordance with the disclosure, the instant invention relates to a method and apparatus for moving and separating charged molecules such as nucleic acids, and to a separation matrix useful in use a method and apparatus. The instant disclosure also relates to processes for the fabrication of such a separation matrix, in the form of a small scale chip.
The separation matrix of the invention comprises a substrate in which there are formed one or more channels, one channel for each sample to be evaluated. The channels extend for the length of the chip, a distance of generally around 1 cm, and are about 1 to 10 &mgr;m wide and 1 to 10 &mgr;m in depth. The channels are filled with a homogeneous separation matrix which acts as an obstacle to the electrophoretic migration of the charged molecules. Microelectrodes disposed in the channels are used to induce an electric filed within the homogeneous separation medium. When a voltage is applied across two or more of the microelectrodes, the charged molecules are induced to move and separate according to the electric field density, the type of solvent film, and the charge, shape and size of the charged molecule.
The separation matrix may further comprise detectors, such as light polarization detectors, fluorescence emission detectors, biosensors, electrochemical sensors or other microcomponents which may include sites for enzymatic or chemical manipulation of the moved or separated charged molecules.
When the apparatus is used in conjunction with a second substrate, substantially parallel to the first substrate, electrodes may be located on either or both substrates. The gap between the substrates is filled with the solvent film. Ultra-thin films on the order of 0.5-100 microns thickness, preferably 2-10 microns thickness are effective for rapid, energy efficient separations of nucleic acids and other charged molecules. Such arrangements may include sample loading and sample collection sites, as well as sites for enzymatic or chemical manipulation of the moved or separated charged molecules.
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Izmailov Alexandre M.
Larson Marina T.
Maruzzo Bruno C.
Stevens John K.
Waterhouse Paul
Oppedahl & Larson LLP
Starsiak Jr. John S.
Visible Genetics Inc.
Wallenhorst Maureen M.
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