Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2002-04-29
2004-07-06
Bell, Mark L. (Department: 1755)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C435S006120, C436S526000, C204S465000, C204S600000, C204S554000, C204S571000
Reexamination Certificate
active
06758953
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of combining free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, proteins, and solutes in useful quantities at high concentrations.
DESCRIPTION OF THE PRIOR ART
Conventional particle separation techniques typically include centrifugation, which is limited due to its specificity, capacity, speed, energy consumption, biological impact, and microgravity environment disturbances.
Electrophoresis is a leading method for resolving mixtures of cells or charged macromolecules (proteins and nucleic acids). The electrophoretic separation of proteins without gels has been a long-standing goal of separations research. The process of electrophoresis has so far been unable to “graduate” from an analytical tool to a viable unit operation. This is primarily because of various problems such as thermal convection, electro osmosis, particle sedimentation, droplet sedimentation, particle aggregation, and electro hydrodynamic zone distortion have been found to be major obstacles to scale-up. The traditional approach has been to devise density gradients or elaborate flowing devices to counteract these problems; however incorporation of such methods has been unable to address these problems effectively. Also, their addition has caused the process to become cumbersome, thus further reducing the appeal of electrophoresis.
Without the need to prepare density gradients and/or use elaborate flowing systems, free electrophoresis can enjoy much more widespread use because it is a high-resolution method that does not require adsorption to solid media and the subsequent solids handling. It can separate both particles (cells) and solutes (macromolecules) with equal ease. Some specific applications for electrophoresis include the separation of different cells of peripheral blood and bone marrow in hematological and immunological research. Other potential applications include clinical therapeutics and the separation of proteins from body fluids, tissue extracts and fermentation broths.
A mixing problem encountered during free electrophoresis is the mixing caused by the release of gases at the electrodes. However, the use of either non-gassing electrodes such as described in (Agarwala 1994) and incorporated herein or membrane-separated electrodes such as described in (Cole et. al. 1995) and incorporated herein will effectively solve this problem. Experiments performed using palladium electrodes have demonstrated our ability to solve this problem.
Free electrophoresis is a process in which a sample is introduced into a liquid buffer, static or flowing, in a zone and subjected to an electric field in which separands migrate according to their surface charge properties. Fluid instabilities result because longer migration paths are exposed for longer times to developing instabilities.
Timmerman A. Tulp and M. G. Barnhoorn in
Preparative Density Gradient electrophoresis of Cells and Cell Organelles A New Separation Chamber, Electroporesis
(1982), teaches that a short electrophoretic migration path in a non-moving buffer avoids exposure of migrating separands to unstable buffer flows. Tulp designed a reorienting, free electrophoresis device consisting of a flat disk-shaped container with thin sample bands and a short vertical migration distance. The bottom and the top electrode fluids served as the coolant, the total height of the separation column was 1-2 cm, and its diameter was greater than 15 cm. The distance between the unrelated separands was 1-2 mm, and this distance was increased during fractionation after electrophoresis by re-orienting the disk so that it became a narrow vertical column.
In a different field of separations, Albertsson et al. teaches that multistage extraction processes can proceed in a multistage separator consisting of two sets of cavities facing each other around the periphery of a pair of plates. Further, conventional electrophoresis devices rely on the use of gels, paper or flowing channels to stabilize the electrophoresis buffer in which separands migrate. The devices are limited in capacity and, in the case of flowing channels, difficult for the user to operate and maintain. Obviously, prior art is not meeting the needs of separations by free electrophoresis. Further, applications of free electrophoresis in low gravity require a gravity independent means of collecting electrophoretically separated fractions of the sample.
The present invention fulfills this requirement and is hence ideal for applications in space-flight electrophoresis experiments and applications. For instance, the overall efficacy of electrophoresis as a unit operation can be greatly improved if the migration distance is greatly reduced and the process is multistaged.
SUMMARY OF THE INVENTION
A thin-layer countercurrent distribution apparatus is designed and constructed so that up to 20 fractions can be collected on the basis of electrophoretic mobility by application of an electric field. The multistage electrophoretic separation and purification of cells, particles, proteins, and solutes utilize an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating and/or purifying living cells, particles and proteins in useful quantities and at high concentrations. The mixture to be separated starts in a bottom cavity, and successive top cavities, collect fractions as separand particles or molecules are electrophoresis upward out of the bottom cavity. Mathematical models of this process have been developed, and experiments performed to verify the predictions of the models by collecting and counting particles in each cavity after fractionation. The process depends on the electrophoretic mobility of separands, and is gravitationally stabilized so that it functions in laboratories on earth and in space.
Moreover, an electrophoresis device is disclosed which separates cells, particles, proteins and other separands by collecting samples of decreasing electrophoretic mobility in a train of inverted cavities while an electric field is applied between said inverted cavities and one or more sample cuvettes containing a mixture of cells, particles, proteins or other separands. One circular plate is provided for the one or more sample cuvettes, and one circular plate is provided for the multiple collection cavities. The invention utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, and proteins in useful quantities at high concentrations. The purification method includes a method for dealing with electrolysis products, a technique for controlling the electrical energy input, and an approach for keeping the process isothermal. The invention solves many separation applications problems on earth and also in reduced gravity in space flight.
The multistage electrophoretic purification of cells, particles, and proteins, utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles and proteins in useful quantities and at high concentrations. The isothermal process depends on the electrophoretic mobility of separands, and is gravitationally stabilized so that it functions in laboratories on earth and in space. The purification method includes a method for dealing with electrolysis, a technique for transporting and varying electrical energy, and an approach for keeping the process isothermal. The electrophoretic technology resolves many unique separation applications on earth as well as in reduced gravity environments in space flight.
The instant invention is extremely well suited to immunological research, pharmaceutical delivery, biomedical applications, cell biology, and cell separation problems associated with clinical, animal, and plant research. The separation process is well suited to space flight, specifically for on-orbit cell s
Sengupta Shramik
Thomas Nathan A.
Todd Paul W.
Vellinger John C.
Bell Mark L.
Brown Jennine
Carrithers David W.
Carrithers Law Office
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