WO1991012904A1 - Method and device for moving molecules by the application of a plurality of electrical fields - Google Patents
Method and device for moving molecules by the application of a plurality of electrical fields Download PDFInfo
- Publication number
- WO1991012904A1 WO1991012904A1 PCT/US1991/001307 US9101307W WO9112904A1 WO 1991012904 A1 WO1991012904 A1 WO 1991012904A1 US 9101307 W US9101307 W US 9101307W WO 9112904 A1 WO9112904 A1 WO 9112904A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- medium
- movement area
- particles
- electrical fields
- moving
- Prior art date
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
- B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F33/00—Other mixers; Mixing plants; Combinations of mixers
- B01F33/30—Micromixers
- B01F33/3031—Micromixers using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/24—Extraction; Separation; Purification by electrochemical means
- C07K1/26—Electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
- G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
- G03F7/0002—Lithographic processes using patterning methods other than those involving the exposure to radiation, e.g. by stamping
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F33/00—Other mixers; Mixing plants; Combinations of mixers
- B01F33/30—Micromixers
Definitions
- This invention relates generally to the fields of electrophoresis and photolithography which is applied in a manner so as to integrate technological innovations in the fields of biochemistry, polymer science, molecular genetics and electronics. More specifically, the invention relates to a method of moving charged molecules or particles in a medium by the simultaneous or sequential application of a plurality of electrical fields and devices for carrying out that method.
- Electrophoresis is an analytical technique to separate and identify charged particles, ions, or molecules. It involves the imposition of an electric field to move charged species in a liquid medium. The most often studied species are bio-macromolecules, such as proteins and DNA fragments, which are usually polyelectrolytes. However, electrophoresis can be used to separate any charged materials including various cells, bacteria and viral materials. At a fixed pH and ionic strength, a given polyelectrolyte acquires a certain number of net charges. Such particles are surrounded by counter-ions and have various charges, sizes (volume and shape) which effect movement. Molecules are separated by their different mobilities under an applied electric field. The mobility variation derives from the different charge and frictional resistance characteristics of the molecules.
- the present invention relates to moving charged particles such as charged molecules within a medium in response to a plurality of electrical fields which are continuously applied simultaneously and/or sequentially along the medium containing the charged molecules in order to move the charged molecules in a precise and controlled fashion.
- the movement of the electrical fields can be accurately controlled both spatially and temporally.
- Charged particles in the medium can be moved so as to separate particular types of charged particles away from one another and thus provide a highly defined analytical technique. Further, specific charged molecules can be moved towards each other into precisely defined regions in order to react particular types of molecules together in a synthesis or sequencing protocol.
- a charged particle moving device such as an electrophoresis device produced by any of a variety of procedures such as photolithography silk-screening, LASER technologies, or vapor deposition which results in a patterning of electrical circuitry.
- a "movement area" which includes a medium in which the charged particles such as charged molecules are to be moved.
- the movement area is positioned so that it can be continuously subjected to a plurality of electrical fields in a simultaneous or sequential manner.
- the electrical fields effecting the movement area are activated so as to move charged molecules in a controlled manner through the medium in the movement area. Accordingly, mixtures of different types of charged molecules can be separated away from each other in order to provide an analytical technique.
- the different fields connected to the movement area can be applied so as to move specific types of charged molecules into contact with other types of charged molecules in order to react the molecules and carry out any number of different reaction protocols.
- the electrical connections contacting the movement area are preferably in the form of intelligent integrated circuitry which is interactive with a computer system capable of activating the fields in any given manner so as to create precise types of separation of molecules for analysis or combinations of molecules for reaction.
- a primary object of the present invention is to provide a device which is capable of moving charged particles through a medium in a precise controlled fashion in response to a plurality of different electrical fields, which fields are preferably generating forces which vary in time and space simultaneously.
- Another object of the present invention is to provide a device which separates mixtures of charged particles such as charged molecules within a medium by the application of a plurality of electrical fields to the medium in a simultaneous and/or sequential fashion.
- a feature of the present invention is that a plurality (preferably large numbers) of different electrical fields are applied to a medium in order to move molecules within the medium in a precise manner.
- An advantage of the present invention is that molecules can be moved within a given medium so as to provide finer separations ' of molecules than is possible with conventional separation techniques.
- Yet another advantage of the present invention is that devices of the invention can be efficiently and economically produced.
- Yet another advantage of the present invention is the minimization or elimination of electroendosmosis by the utilization of polymeric substrates, such as polymethylmethacrylate.
- Another feature of the devices of the present invention is the use of movement areas which have a cross-sectional shape which includes flattened or slab-like regions which regions allow for the efficient accurate use of spectrometer devices which can be used in connection with the invention.
- Yet another feature of the invention is the inclusion of branched movement areas in which it is possible to move together and separate from each other charged particles in order to carry out complex reaction and/or separate schemes.
- Yet another advantage of the present invention is the use of inert polymeric substrate materials on components which might contact charged particles to be separated or combined which materials minimize protein absorption and loss of sample materials being separated and/or combined.
- Still another advantage of the present invention is that it makes possible the use of substantially smaller voltages due to the small spacing of the electrodes thus providing for a safer device for laboratory use as well as conserving power.
- Figure 1 is a plain front schematic view of a particular embodiment of the invention.
- Figure 2 is a perspective schematic view of a second embodiment of the invention.
- the device is on a Card 1 which may be comprised of a number of different types of materials such as various polymeric materials generally referred to as plastics.
- the Card 1 may be in a variety of different sizes.
- the card could be produced in the size of a conventional credit card.
- the Card 1 includes a hollowed-out area or Trench 2 to which, again, may be of any size but for convenience might preferably be produced on the credit card size Card 1 so that the Trench 2 is about 1-10 centimeters in length and has a depth of about 5-25 microns.
- the cross-sectional shape (not shown) of the Trench 2 may vary and be rectangular, oval, circular or otherwise. It is preferably a flattened oval with the flat surface providing desired optical properties.
- the Trench 2 is filled with a medium 3 which may be a buffer solution, polymeric solution, surfactant micellular dispersion or gel of the type generally used in connection with analytical separation techniques.
- a medium 3 which may be a buffer solution, polymeric solution, surfactant micellular dispersion or gel of the type generally used in connection with analytical separation techniques.
- polyacrylamide gel used in PAGE analytical procedures is extremely useful in connection with the present invention.
- a variety of material may be used alone or in combination with other materials which materials should provide frictional resistance to the charged particles and not substantially interfere with the electrical fields.
- the Card 1 has plated thereon a plurality of electroplated finger-like electrodes 4-10. Only 7 electrodes are shown on the Card 1 for purposes of simplicity. However, photoelectroplating technology could be utilized to provide hundreds of different electrodes along the length of even a relatively small (1-10 cm) Trench 2.
- the electrodes can be spaced apart from each other at any given interval. In connection with this embodiment of the invention, there are preferably 400 to 800 electrodes and they are preferably placed at regular intervals approximately 1-100 microns apart. Some preferred embodiments of the device include 5-25 electrical fields, 50-100 electrical fields, and 500 to over 1000 electrical fields. The electrodes creating these fields may be placed apart from one another at a distance 0.01 to 10 centimeters, but are more preferably placed at a range of about 1-100 microns apart from each other.
- the electrodes 4-10 are either simultaneously biased by the application of different voltages to each of the electrodes 4-10 or sequentially biased by the application of different voltages which are biased in a programmed manner. Since the spacing of the electrodes 4-10 is small, it is possible to attain relatively high field strength between the electrodes even while applying relatively low voltages. This is a substantial advantage of the present invention over prior art methods which utilize only one electrical field over the entire medium (having a large dimension) and thus require the application of substantially large voltages.
- the electrodes 4-10 are biased or fired simultaneously or sequentially and the magnitude of the field applied across any given electrode or all of the electrodes is adjustable over any given range at any given instant in time.
- the Card 1 as well as the Trench 2 and electrode connections 4-10 can be readily and economically produced by standard microelectronic fabrication techniques. Accordingly, multiple copies of nearly identical cards can be readily reproduced with a high degree of accuracy. The fidelity and economy of production are important features of the invention. Since the substrate of the card is preferably a rigid polymeric material such as polymethylmethacrylate, polycarbonate, polyethylene terepthalate, polystyrene or styrene copolymers, the card itself does not have a surface charge.
- the problem of electroend- osmosis which is a substantial problem in connection with high performance capillary electrophoresis techniques which utilize glass capillaries which generally must be coated with a polymer in order to suppress the electroend-osmosis.
- the polymer material can be made substantially non-porous. Accordingly, the charged particles such as proteins are not absorbed and loss of sample during separation is minimal. It is important to note that the gel-filled channel 2 on the Card 1 does not have to contain cross- linked gels tethered to the wall. This greatly relieves the stress formed during polymerization and cross-linking.
- the gels need not be tethered to the wall because only a small fraction of the gel 3 in the Trench 2 is under applied electrical field at any given time. Since only a small portion of the gel 3 in the Trench 2 is subjected to the field at a given moment the field does not extrude the gel 3 out of the Trench 2 in any fashion.
- a substrate support such as a polymethylmethacrylate card approximately the size of a convention credit card is provided.
- the surface of the card itself is not electrically conducting nor is the card.
- a thin layer of an electrically conducting material On the card is first deposited a thin layer of an electrically conducting material.
- the coating may be applied by a variety of different techniques known to those skilled in the art and may be comprised of a variety of different types of materials provided they are capable of conducting electricity.
- the layer is preferably extremely thin on the order of 100 angstroms to a few microns of thickness.
- the electrically conducting layer is then coated with a layer of material which is both light-sensitive and non-conducting.
- a mask is applied to the surface of the light-sensitive, non ⁇ conducting layer. After the mask covers the layer, it is exposed to light after which the mask is removed from the light-sensitive, non- conducting layer. Since the light-sensitive layer has been exposed to light at certain portions not covered by the mask, these exposed portions can be removed by conventional technology which renders these portions easily removable. When the exposed areas have been removed, the underlying electrically conductive layer is exposed. These exposed portions will, of course, provide the plurality of electrode connections to the Trench 2.
- the mask utilized in the above production procedure can be produced so as to provide hundreds of different electrode connections to the Trench 2.
- other techniques such as employing various types of laser technologies and/or other technologies such as silkscreening and vapor deposition which make it possible to provide extremely small (in size) and large numbers of electrodes to the Trench 2.
- the greater the number of electrodes the less voltage which needs to be supplied to each electrode and the more accurately it is possible to control the motion of the charged particles within the trench.
- Trench 2 must be filled with a medium 3 which is preferably in the form of a polyacrylamide gel material or a buffered solution with or without a synthetic polymer; alone or in combination with a surfactant.
- a sample of material is then placed at one end of the medium and time-dependent and/or variable position-dependent voltages are applied to the electrodes.
- all of the electrodes positioned along the Trench 2 may be biased simultaneously but have different voltages depending on the electrode spacing and position of any given electrode.
- the voltages supplied to any given electrode may also be changed continuously over time so as to create different wave-like force affects on the charged particles within the medium and move the particles through the medium at different rates based on factors such as the size, shape and charge of the particles being moved through the medium.
- the embodiment described above can be modified in a variety of different fashions. For example, it is possible for the electrodes to have opposing ends on either side of the Trench 2. If the device is constructed in this fashion, charged particles will be moved through the medium 3 in a zig-zag fashion as the different electrodes are activated.
- two cards can be produced wherein one card is substantially the mirror image of the other.
- the two cards are placed in facing abutment to each other so that the Trench 2 forms an enclosed column.
- the electrode lines do not end at the edge of the Trench 2, but rather continued across the trench on both the top and the bottom.
- electrical potential will permeate around the column formed at a plurality of different spaced intervals along the column.
- the electrodes on the device may be fired simultaneously in accordance with a predetermined scheme which will create a complex voltage profile across the entire length of the column.
- the voltage profile will create forces on the charged particles within the column and can be changed over time in order to obtain precise resolution of different species or groups of charged particles within a sample being resolved.
- the electrodes it is preferable for the electrodes to be connected to an electronic computer which computer has programmed software dedicated to providing the moving waves or voltage profile along the Trench 2.
- Various different types of software can be provided so as to obtain the best possible resolution with respect to separating various types of charged particles from one another.
- the computer software which is connected to the electrodes can be made interactive with an optical detection device such as an ultra violet or fluorescence spectrometer.
- the spectrometer can be focused singly or at various points along the medium 3 in the Trench 2.
- the ultra violet spectrometer reads different types of particles being moved to different portions of the medium 3, the information can be sent to the computer which can adjust the speed of the waves or voltage distribution profiles being generated in order to more precisely fine-tune the resolution of the charged particles being moved through the medium 3.
- the Trench 2 can be in any shape. More specifically, the Trench 2 can be fashioned so that it has a plurality of branches thereon. Each of the branches of the Trench 2, along with the trench itself can be filled with a buffer solution. Thereafter, the base of each of the branches can be supplied with a particular charged reactant material. The charged reactant materials can then be moved into contact with one another by utilizing the moving electrical wave generated by the computer. Accordingly, sophisticated computer programs can be set up in order to provide for synthesis or sequencing protocols of a variety of different types of molecules. For example, different nucleotides can be reacted to form DNA and different amino acids can be reacted to form proteins. These reactions can be carried out at greatly increased speeds as compared with conventional mechanical technologies. In addition to increased speeds, the yield is vastly improved due to the precision with which the reactants can be moved.
- a mixture of components can be separated into a variety of pure groups and moved along parallel tracks.
- the desired components can be guided by the electrical wave fields in lateral directions at a given precise moment in time and caused to react with a given neighboring reactant.
- selected components may be guided to trenches filled with antigen-antibodies reactive with given charged particles being moved in the medium or moved into contact with complimentary components, dyes, fluorescent tags, radio tags, enzyme-specific tags or other types of chemicals for any number of purposes such as various transformations which are either physical or chemical in nature.
- bacterial or mammalian cells, or viruses may be sorted by complicated trench networks which networks are in connection with a plurality of electrodes capable of generating fields in a variety of different ways in order to move the cells or viruses through the fields based on the size, charge or shape of the particular material being moved. Separated cells or viruses may be analyzed or modified subsequently.
- the embodiment shown within figure 2 is generally utilized for the purification of large quantities of bio-macromolecules including proteins and DNA as well as charged molecules, polyelectrolytes, bacteria and viruses.
- the separation results obtained utilizing this embodiment are based on the different mobilities of charged particles in a given medium when the particles in the medium are subjected to an applied electrical field.
- the invention may be carried out utilizing a single pair of electrodes by creating relative motion between the medium holding the sample and the electrodes.
- the cylindrical rod 11 passes through a pair of electrodes 12 and 12' which are connected to a power source 13 for supplying the necessary voltage.
- the electrodes 12 and 12 are in the form of parallel plates with holes centrally located therein which enables the imposition of an electrical field which creates a force on the charged molecules within a medium 14 inside the cylinder 11.
- the cylinder 11 and electrodes 12 and 12' are moved relative to each other in a controlled manner. Only the portion of the rod 11 between electrodes 12 and 12' is under the influence of any forces from the electrical field. Accordingly, only charged particles within the medium 14 residing in the particular segment under the influence of the field will be caused to move within the cylinder 11. Since any given sample of materials injected into the medium 14 will exhibit different mobilities within the medium 14 the charged particles in the medium can be separated away.
- the voltage 13 supplied to the electrodes 12 and 12' can, of course, be turned on and off as desired.
- cylinder 11 can be moved in the direction of the arrow through electrodes 12 and 12, until the end of the cylinder 11 is reached.
- the power can then be turned off and the rod returned to its original position and the process repeated as many times as necessary in order to obtain the desired separation.
- the medium such as the medium of 14 present in the cylinder 11 can be in any shape.
- the medium may be in the form of a rectangular slab of gel and one or more pairs of electrodes creating one or more different kinds of electric fields can be moved relative to the slab in order to create moving electrical fields which will move particles through the gel to obtain precise separation of different types of charged particles in a sample.
- the medium 14 can be a variety of different types of materials of the type described above in connection with the embodiment shown in Figure 1 which includes the medium material 3.
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/487,021 US5126022A (en) | 1990-02-28 | 1990-02-28 | Method and device for moving molecules by the application of a plurality of electrical fields |
US487,021 | 1990-02-28 |
Publications (1)
Publication Number | Publication Date |
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WO1991012904A1 true WO1991012904A1 (en) | 1991-09-05 |
Family
ID=23934086
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1991/001307 WO1991012904A1 (en) | 1990-02-28 | 1991-02-28 | Method and device for moving molecules by the application of a plurality of electrical fields |
Country Status (6)
Country | Link |
---|---|
US (1) | US5126022A (en) |
EP (1) | EP0521911A1 (en) |
JP (2) | JP2601595B2 (en) |
AU (1) | AU637895B2 (en) |
CA (1) | CA2075969A1 (en) |
WO (1) | WO1991012904A1 (en) |
Cited By (9)
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WO1993025899A1 (en) * | 1992-06-17 | 1993-12-23 | Beckman Instruments, Inc. | Capillary electrophoresis using time-varying field strength |
EP0629853A2 (en) * | 1993-06-15 | 1994-12-21 | Hewlett-Packard Company | Capillary electrophoresis with tracking separation field |
EP0644420A2 (en) * | 1993-09-09 | 1995-03-22 | The University Of North Carolina At Chapel Hill | Method and apparatus for gel electrophoresis |
WO1996042013A1 (en) * | 1995-06-08 | 1996-12-27 | Visible Genetics Inc. | Microelectrophoresis chip for moving and separating nucleic acids and other charged molecules |
US6110339A (en) * | 1995-06-08 | 2000-08-29 | Visible Genetics Inc. | Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same |
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US7105354B1 (en) | 1998-06-12 | 2006-09-12 | Asahi Kasei Kabushiki Kaisha | Analyzer |
US8702950B2 (en) | 2007-05-31 | 2014-04-22 | Sharp Kabushiki Kaisha | Device for electrophoresis, device for transfer, device for electrophoresis and transfer, chip for electrophoresis and transfer, and method for electrophoresis, method for transfer, and method for electrophoresis and transfer |
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EP0356187A2 (en) * | 1988-08-23 | 1990-02-28 | The Board Of Trustees Of The Leland Stanford Junior University | Electrophoresis using contour-clamped electric fields |
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Also Published As
Publication number | Publication date |
---|---|
JPH08327597A (en) | 1996-12-13 |
JP3103031B2 (en) | 2000-10-23 |
US5126022A (en) | 1992-06-30 |
AU637895B2 (en) | 1993-06-10 |
AU7467591A (en) | 1991-09-18 |
JP2601595B2 (en) | 1997-04-16 |
CA2075969A1 (en) | 1991-08-29 |
EP0521911A1 (en) | 1993-01-13 |
JPH05504628A (en) | 1993-07-15 |
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