WO1999036172A1

WO1999036172A1 - Activated charcoal and open cell foam filtration material, and apparatus and methods of using the same

Info

Publication number: WO1999036172A1
Application number: PCT/US1999/000999
Authority: WO
Inventors: Richard G. Allen
Original assignee: Naiad Technologies, Inc.
Priority date: 1998-01-20
Filing date: 1999-01-19
Publication date: 1999-07-22
Also published as: AU2323899A

Abstract

The present invention provides filtration material (4, 5, 6) comprising activated charcoal and open-celled foam components, apparatus (1) using the filtration material (4, 5, 6), and methods for efficiently extracting target molecules from solution and substantially permanently binding those molecules to the filtration material (4, 5, 6). The binding is substantially permanent so that subsequent passage of additional solution will not unbind the target molecules. The filtration material (4, 5, 6) also provides an enhanced flow rate of solution therethrough.

Description

ACΗVATED CHARCOAL AND OPEN CELL FOAM FILTRAΗON MATERIAL, AND APPARATUS AND METHODS OF USING THE SAME

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to USSN 60/071,814, filed January 20, 1998, herein incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable.

FIELD OF THE INVENTION The invention provides filtration materials, apparatuses, and methods of filtering target molecules.

BACKGROUND OF THE INVENTION

There have been a number of conventional solutions to dealing with removal of unwanted chemicals from solution. However, none has proven adequate for dealing with removal and ultimate disposal of undesired molecules such as dyes and chemically labeled molecules regularly used in connection with basic and applied biological and medical research. Those molecules will be referred to in the present application as target molecules, and a partial, exemplary list of them is includes the following molecules: peptides; polypeptides; proteins; enzymes; RNA; DNA; nucleic acids; biopolymers; amino acids; nucleotides; dyes; ethidium bromide; and animal tissue, e.g., particulates such as connective tissue and fat.

The essential problem for laboratories performing research and diagnostic tests is that present environmental regulations no longer allow for simply pouring used solutions down the drain or putting them in the trash after, e.g. , they are chemically labeled, formalin fixed, or radioactively labeled according to known procedures. Examples of such labeling involve using radioactivity or fluorescence to label a desired molecule. Furthermore, laboratory solutions may contain toxic dyes such as ethidium bromide, and animal, particularly human, tissues, that are potentially hazardous. Thus, a need exists to provide filtration materials and methods of efficiently extracting target molecules from solution and substantially permanently binding to those molecules. Accordingly, it is a principal object of the present invention to provide such a material that overcomes the drawbacks of prior art materials. Another object is to provide such a material that provides for such removal and binding to a relatively wide variety of molecules such as the target molecules listed above. Yet another object is to provide such a material that has a relatively long working life. Another important object of the invention is to provide such a material that can be readily and easily disposed of after its working life is over. It is also an object of the invention to provide such a material that can be easily and cost-effectively manufactured. It is another object to provide a material with a large binding capacity and suitable flow rate.

SUMMARY OF THE INVENTION The present invention provides filtration material, apparatuses, and methods for extracting target molecules from solutions and substantially, permanently binding to those molecules. The filtration material has a surprising ability to bind a large capacity of molecules and not release these molecules despite washing at pH extremes, with detergent and organic solutions, and at high salt concentrations. Furthermore, the foam component of the filtration material surprisingly provides a high flow rate of solution through the material, as compared to filtration material lacking the foam component, providing an efficient means of filtering solutions and binding target molecules.

In one aspect, the invention provides a filtration material for extracting target molecules from solution, the material comprising: (i) a slurry component comprising an activated charcoal; and (ii) a foam component comprising an open cell foam; wherein the filtration material has at least about a 25 % increased flow rate of solution through the filtration material as compared to a filtration material that lacks the foam component. In one embodiment, the slurry component further comprises an ion exchange resin. In another embodiment, the ion exchange resin comprises an anion exchange resin and a cation exchange resin.

In another embodiment, the foam component comprises a polyether-based flexible polyurethane foam. In anther embodiment, the foam component comprises a yellow isomeric sponge.

In another embodiment, the filtration material comprises: (i) a slurry component comprising about 100% activated charcoal by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

In another embodiment, the filtration material comprises: (i) a slurry component comprising about 50-70% activated charcoal, about 15-25% anion exchange resin, about 15-25% cation exchange resin and about 0-20% neutral flow rate enhancers by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

In another embodiment, the filtration material comprises: (i) a slurry component comprising about 70-85% activated charcoal and about 15-30% anion exchange resin by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge. In another embodiment, the filtration material comprises: (i) a slurry component comprising about 70-85% activated charcoal and about 15-30% cation exchange resin by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

In another aspect, the invention provides an apparatus for extracting target molecules from solution, the apparatus comprising: (i) a filtration material as described above; and (ii) a container comprising an inlet, the container holding the filtration material; wherein the filter material has at least about a 25 % increased flow rate of solution through the filter material as compared to a filter material that lacks the foam component.

In one embodiment, the container further comprises an outlet allowing solution to pass out of the container. In another embodiment, the filtration material comprises the foam component distributed substantially among the slurry component. In another embodiment, the filtration material comprises the foam component and the slurry component distributed in alternating layers. In another embodiment, the apparatus further comprises a second foam component, wherein the solution first contacts the second foam component before contacting the filtration material.

In another aspect, the invention provides a method for extracting target molecules from solution, the method comprising the steps of: (i) contacting a filtration material as described above with the solution; and (ii) passing the solution through the filtration material; wherein the filtration material has at least about a 25% increased flow rate of solution through the filtration material as compared to filtration material that lacks the foam component.

In one embodiment, the method further comprises the step of placing the filtration material into a container comprising an inlet and an outlet. In another embodiment, the method further comprises repeating the passing step with additional solutions. In another embodiment, the method further comprises the step of first contacting a second foam component with the solution before the step of contacting the filtration material with the solution.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view of a filtration apparatus showing the filtration material as alternating layers of the slurry and foam components in a conventional water filtration apparatus.

Figure 2 is a side-sectional schematic view of a "large volume system" (LVS) cartridge showing the filtration material as alternating layers of the slurry component and the foam component.

Figure 3 is a schematic view of a filtration apparatus showing the filtration material as the foam component substantially distributed in the slurry component, with a layer of the foam component on top of the filtration material in a conventional water filtration apparatus.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention achieves the above described objects by providing a filtration material for extracting from solution more than 95% of target molecules, typically more than 98%, and substantially, permanently binding to those molecules. The binding is substantially permanent when multiple solutions are passed through the filtration material. Furthermore, the binding is substantially permanent and greater than about 95% across a pH range of about pH 3 to about pH 9. Thus, the filtration material provides a means of binding substantially all target molecules in a solution over a broad pH range. A surprising feature of the filtration material is the enhanced flow rate provided by the foam component, as compared to filtration material that lacks the foam component. The presence of the foam component increases flow rate of solution through the filtration material by about at least 25%, and more typically 300% or more. The filtration material overcomes the drawbacks of prior art materials, and provides for extraction of a relatively wide variety of molecules such as the target molecules listed above. The filtration material has a relatively long working life, and when its working life is over the material can be disposed of readily and easily. The material can also be easily and cost-effectively manufactured. The filtration material can be used to substantially bind target molecules from solutions used in a variety of technologies and settings, e.g., applied and basic medical and biological research, chemistry research, industrial applications and the like. More particularly, the filtration material has the advantage of improved efficiency of extraction over its individual components, reaching up to about 99% binding. In addition, and in contrast to the individual components, the retention of target molecules by the filtration material after subsequent washing of the material with a wide range of pH, salts, detergents, and organic materials, is 95% or more over a several month period. II. Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

"Filtration material" refers to a composition comprising a slurry component and a foam component, through which solutions are passed. In the filtration material, the foam component to slurry component is typically 5:4 by volume, although the ratio is not critical. The slurry and the foam component are present as alternating layers, or alternatively as the foam component substantially distributed in the slurry component. The filtration material removes target molecules from the solution and substantially, permanently binds to the target molecules. The filtration material also provides an enhanced flow rate of solution through the material, by about at least 25%, typicall up to 300% or more. The enhanced flow rate can be tested by comparing filtration material with and without the foam component. Filtration material with the open cell foam component provides at least a 25 % increase in flow rate of solution through the material as compared to the control filtration material lacking the open cell foam component.

"Target molecules" are those molecules and materials that are to be removed from a solution via passage of the solution through the filtration material, such molecules may or may not be labeled with a detectable moiety. Target molecules include, e.g., naturally occurring and synthetic forms of: amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, nucleic acid, DNA, RNA, enzymes, antibodies, small organic molecules, aromatic molecules, fluorescent dyes and ethidium bromide. Target molecules also refers to particulates of animal tissue, particularly human tissue, such as fat and connective tissue, which can be trapped by the filtration material and by a second foam component that acts as a mechanical filter in the filtration apparatus.

"Slurry component" refers to one of the components of the filtration material, where the slurry component comprises activated charcoal, and optionally includes ion exchange resins (either anion or cation exchange resins alone, or mixed), and further optionally includes neutral flow rate enhancers, and the like. The slurry component substantially, permanently binds to the target molecules passed through the filtration material. Typically the activated charcoal comprises 5-100% by weight of the slurry. "Ion exchange resin, " "anion exchange resin, " and "cation exchange resin" refer to a support matrix that carries a charge. In the case of a cation exchange resin, the charge is negative, and in the case of an anion exchange resin the charge is positive. In the case of ion exchange resins, the charge is either all positive or all negative, or mixture of support matrices that have either negative or positive charges. The charge carriers are commercially available resins bearing either positive or negatively charged groups. The support matrix of the resins is not critical. The particular charged groups are not critical and include phosphate, carboxyl sulfos, aminos, guanido, amino-benyzyl and the like. "Activated charcoal" is charcoal that is characterized by a large internal surface area and pore volume, which are responsible for its adsorptive properties. Activated charcoal is typically prepared via thermal activation processes, or chemical activation processes. The chemical activation processes typically involve the use of an acid such as phosphoric acid (see, e.g. , Kirk-Othmer, Encyclopedia of Chemical Technology, volume 4 (4th ed. 1994)). Activated charcoal can have a variety of particle sizes, and typically it is in a granular or powered form.

"Foam component" refers to a cellular plastic or rubber, which is a polymer that has an apparent density that is substantially decreased by the presence of numerous gas-filled cells distributed throughout the plastic or rubber (see, e.g. , Kirk- Othmer, Encyclopedia of Chemical Technology, volume 11 (4th ed. 1994)). The foam component therefore is a two-phase gas-solid system, with the gas present in the cells of the plastic. Foam and sponge are used interchangeably herein.

An "open celled" foam has communication of the gas phase between some or all of cells due to fracturing of the cell walls or membranes. An open celled structure of a foam can vary from partially open celled to 100% open celled. For the purposes of the present invention, the percent open cell is preferably 50-90%, more preferably 60- 70%. In comparison, "closed cell" refers to refers to a characteristic of the cells in a foam, where the cells are discrete and the cell membranes or walls are intact so that the gas phase of each cell is independent from all other cells (see, e.g. , Kirk-Othmer, Encyclopedia of Chemical Technology, volume 11 (4th ed. 1994)).

"Reticulated" refers to a foam that has been treated so that the cell walls or membranes have been removed, leaving a skeletal frame and joints between the cells. Reticulation can be partial or full, with up to 100% of cells that are reticulated. For the purposes of the present invention, a 100% reticulated foam is not suitable for the filtration material of the invention. An example of a 100% reticulated sponge is the grey reticulated sponge available from Crest/ Quality Foam Corporations.

"Yellow isomeric sponge" refers to a flexible polyester-based, polyurethane foam that is commercially available from the General Foam Corporation, as types A and B. The yellow isomeric sponge typically has a pore size of 10-45 pores per inch (ppi) and is approximately 60% open celled. The yellow isomeric sponge is also a double cell, partially reticulated sponge.

"Neutral flow rate enhancers" refers to non-foam filler material that is part of the slurry component. The neutral flow rate enhancers do not bind target molecules but increase flow rate, e.g., course sand, plastic beads or glass beads.

III. Filtration materials

The filtration material of the present invention comprises a foam component, which substantially enhances the flow rate of solutions through the material, and a slurry component to substantially, permanently bind target molecules from solution over a pH range of 3-9, typically binding greater than 95% of the target molecules in solution. Optionally, a second foam component is provided, which acts as a mechanical filter for paniculate matter. The foam component surprisingly substantially enhances the flow rate of solution through the filtration material. In addition, it is degradation resistant to the types of chemicals and solutions typically used in laboratories. The foam component has gas-containing cells distributed in a plastic or rubber solid. The cells provide an essential distinguishing characteristic of foams. If the cells are connected so that gas can pass from one cell to another, the foam is termed an "open celled" foam. As noted, foams can be partially or fully open celled. If the cells are discrete so that the gas phase of each cell is independent from all other cells, the foam is a "closed cell" foam (see, e.g. , Kirk-Othmer, Encyclopedia of Chemical Technology, volume 11 (4th ed. 1994)). In the present invention, the foam component is an open cell foam, preferably with SO- 90% open cells, more preferably with 60-70% open cells. Closed celled foams do not confer enhanced flow rate upon filtration materials.

A preferred embodiment of the foam component is a flexible polyurethane open cell foam. Polyurethane foams can be made by a number of processes and are, e.g., polyether- or polyester-based (see, e.g. , Kirk-Othmer, volume 11, supra). Particularly preferred embodiments of the foam component are the polyester-based polyurethane, yellow isomeric A sponge and the yellow isomeric B sponge, available from the General Foam Corporation. The yellow isomeric B sponge typically has a density of 1.0 lb/cubic feet, an ultimate elongation of 100%, a tensile strength of 10 psi, a tear resistance of 15 pounds per square inch, and an elongated cell type (as tested in accordance with ANSI/ATSM-D-3574-91). Yellow isomeric A and B sponges are generally resistant to chemical degradation.

The slurry component typically contains activated charcoal, and optionally contains other materials, e.g. , ion exchange resins such as anion and cation exchange resins. The proportion of activated charcoal to optional components such as ion exchange resins is not critical and can be readily determined by one of skill in the art. Activated charcoal can comprise from approximately 40% to 100% of the slurry component. Ion exchange resin can comprise anion exchange resin, cation exchange resin, or a mixture of both anion and cation exchange resin in equal or unequal proportions. Ion exchange resins can comprise from approximately 0% to 60% of the slurry component. Other materials such as high molecular weight polymers and neutral flow rate enhancers can also be optionally added to the slurry component. The proportion of such optional components is not critical and can be readily determined by one of skill in the art depending on the type of filtration container used. The proportion of foam to slurry is not critical and can be readily determined by one of skill in the art depending upon the type of filtration container used. A preferred ratio is 5:4 foam to slurry by volume.

Activated charcoal is available from a variety of vendors (e.g., Sigma Laboratories, St. Louis, Missouri), It is used in a powder form having the size particles 100-400 mesh, and also in a granular form. The proportion of granular to powdered activated charcoal is not critical and can be readily determined by one of skill in the art on the basis of the filtration container used. Examples of activated charcoal are marketed under the trade names NORIT^® and DARCO^®. Anion exchange resins include, e.g., Whatman DE52 pre-swollen microgranular anion exchanger (diethylaminoethyl cellulose, catalog number 4057 050) and Dowex I (a strongly basic anion exchanger, 8% crosslinked 200-400 mesh, from Sigma Labs, St. Louis, Missouri). Cation exchange resins include, e.g., Rolm & Haas Co. Amberlite CG-50 (a weakly acidic cation exchanger, carboxylic type, hydrogen form, wet mesh 100-200). Optional, additional components of the slurry include high molecular weight polymers such as dextran, methyl cellulose starch, agar, agarose, cellulose, polyglucose, methyl dextran, ethyl dextran, hydroxy propyl dextran, methyl cellulose, and ethyl cellulose. Suitable quantities of neutral filler material, such as course sand, plastic beads or glass beads may be added to increase flow rate, either as a layer or substantially distributed in the slurry component. All slurry components can be layered and are preferably substantially distributed among the activated charcoal.

Ion exchange resins are broadly classified as weak, intermediate or strong acid and bases. If one plans to use the filtration material for subtantially binding target molecules in solutions that contain, e.g., only nucleic acids at physiological pH, the overall negative charge of the nucleic acid phosphate backbone would render the cation exchange resin unnecessary in the slurry. The combination of positive and negative charged groups is preferred for general laboratory use since such solution typically contain both cations and anions. Thus, one of skill can envision altering the ratio and strengths of the ion exchange resins to optimize the slurry according to the type of solutions and target molecules for which it is used.

The filtration material works effectively over a range of pH from about 3.0 to about 9.0 and in a range of salt concentrations from 0 M to about 0.75 M while maintaining a general binding efficiency of at least about 95-97 % . The filtration material also has a relatively long working life, i.e., an amount of material with a wet volume of 100 ml continues to exhibit a binding efficiency of 98-99% after weekly use in a biological laboratory for approximately six months.

Example of preferred embodiments of the slurry component are as follows. For removal from solution (typically aqueous solution) of compounds such as nucleic acids, nucleotides, polypeptides, and amino acids, which may or may not be labeled, e.g., with radioactivity, the slurry component typically contains about 50-70% activated charcoal, 15-25% anion exchange resin, 15-25% cation exchange resin, and 0-20% flow rate enhancers. For solutions, e.g., aqueous solutions or organic solvents used in life sciences labs for tissue, cellular, and molecular staining and immunochemistry, the slurry used to remove dyes and other aromatic compounds (and compounds such as nucleic acids, nucleotides, and some polypeptides and amino acids) typically contains 70-100% activated charcoal, and optionally 15-30% of either an anion or cation exchange resin.

The slurry is typically formed by soaking activated charcoal in water for a preselected time period and then optionally adding to the charcoal an ion-exchange resin with a preselected charge-sensitivity, with mixing and hydrating. Optional components such as high molecular weight polymers and neutral flow rate enhancers are added to the slurry while mixing.

The foam component and the slurry component are then combined, typically in a filtration container of choice. As discussed below, the choice of the filtration container is made by one of skill in the art and is not critical. In one embodiment, the foam component is combined with the slurry component in alternating layers to form the filtration material. The thickness and number of the alternating layers is not critical and can be readily determined by one of skill in the art. Preferably, the foam component and slurry component layers are approximately equal in thickness, and are present in a ratio of approximately 5:4 foam to slurry by volume. In another embodiment, the foam component is combined with the slurry component by substantially distributing the foam component among the slurry component to form the filtration material. In this embodiment, the foam component is formed into pieces (e.g., by tearing or cutting) approximately the size of a pencil eraser. The size of the foam pieces in this embodiment is not critical and can be readily determined by one of skill in the art.

The filtration material is typically maintained in a wet condition throughout its working life, although if the material were to dry out, a simple rehydration step could be performed as described. The slurry component is typically used as a sediment where the mixture has been allowed to settle overnight and the fines decanted. The working slurry has approximately 1 g in 1 ml of water.

When the filtration material has exceeded its working life, it is dried, typically by aspiration, removed from filtration apparatus, and disposed of as solid waste. Any effluent from drying the filtration material may be poured down a drain if sufficiently low radioactivity or toxicity is detected. It should also be understood that if a solution typically contains, e.g., only nucleic acids, but not, e.g., proteins, the filtration material can be tailored to remove substantially and permanently the nucleic acids from solution with a slurry component containing activated charcoal and an anion exchange resin but not a cation exchange resin. Alternatively, for solutions containing only proteins, the filtration material can be tailored to contain activated charcoal and a cation exchange resin. The tailoring of the slurry simply allows for cost efficiency and conservation of materials while retaining the characteristic of removing substantially all the target molecules from solution. In one embodiment, the slurry component is assembled by placing approximately 200 g of activated charcoal in 600 ml double distilled water and stirring until homogeneous. The slurry is typically stirred at least 20 minutes and as much as overnight (if overnight, the solution is covered with foil). 400 ml of water is added to the slurry and stirred for at least 5 minutes. 100 g of anion exchange resin #1 is slowly added while continuing to stir for approximately 5 minutes in order to avoid formation of any lumps. The slurry is them stirred for another 5 minutes or more, and water is added up to 1450 ml volume. 100 g of cation exchange resin is slowly added while continuing to stir for approximately 5 minutes to avoid formation of lumps. Water is added up to 1600 ml volume, stir 5 minutes, slowly add 10 g anion exchange resin #2. The slurry is covered with aluminum foil and stirred overnight or longer.

Prior to use of the slurry, if it has settled for more than 2 days, the slurry is stirred for at least 10 minutes, settled for at least 5 hours and preferably overnight, then decanted to dispose of primarily liquid phase (approximately 400-600 ml). The desired amount of slurry is poured into a container and settled overnight before using. If liquid has already been decanted from the slurry and the slurry is dry enough that it forms chunks if disturbed with a spatula, the approximate volume desired is scraped into a new beaker and water is added to rehydrate, with manual stirring. The slurry is ready to pour into a container as soon as it is in a liquid form, and to add the foam component to form the filtration material. If liquid has already been decanted from the slurry but the slurry is still in a liquid form, the desired amount of slurry is measured and poured into a container in alternating layers with the foam component, or with the foam component substantially distributed among the slurry component. Coarse sand can be placed over a bottom filter before adding the slurry to prevent the charcoal from fouling the filter. The sand also improves the flow rate.

Suitable apparatuses are described below and the form or size of the apparatus is not critical to the invention. The filtration apparatus may be, e.g., a centrifuge bottle, a column, a cartridge, a container with a nylon or glass mesh, or any suitable container for holding the filtration material and passing solution through the filtration material. The apparatus may have one or two openings.

Examples of detectable moieties or labels may be attached to target molecules or may themselves be target molecules to be removed from solutions by filtration include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Typical detectable moieties include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent and visible dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, ethidium bromide, eosin Y, methylene blue, diamino benzidine (DAB), SYBR 1, and the like), radiolabels (e.g. , ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g. , horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

The solutions that are passed through the filtration materials vary widely and include solutions used in protein and nucleic acid purification, nucleic acid hybridization experiments, protein binding experiments, fluorescent cell sorting, western blots, in situ staining of tissue, immunohistochemistry, drug synthesis, histology and pathology experiments, and diagnostic tests. Such solutions include water based

(aqueous) solutions, formaldehyde (formalin), organic solutions such as formamide, and alcohols such as methanol and ethanol, dye solutions such as ethidium bromide solutions, and the like. The filtration materials are further characterized by their ability to retain target molecules after passage of solutions containing a detergent, organic compounds, or high salt, including, e.g., solutions of up to about 0.5% sodium dodecyl sulfate, solutions containing up to about 50% formamide, and solutions containing up to about lOx SSC or lOx TBE (for typical solutions used in molecular biology research, see, e.g. , Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed. 1989)); Methods in Enzymology, Guide to Molecular Cloning Techniques (Berger & Kimmel eds., 1987); and Current Protocols in Molecular Biology (Ausubel et al. eds. , 1987)). IV. Apparatuses

The invention also provides for a filter apparatus for removing target molecules contained in a solution. Referring to Figure 1 , the apparatus generally comprises a container 1 such as a water filtration container having an inlet 2 into which fluids having target molecules may be entered. The inlet 2 opens into a reservoir 3, which leads to a second foam component 4, which is optionally present in the filtration apparatus. The filtration material is composed of alternating layers of a slurry component 5 and a layer of a foam component 6. Attached to the container 1 is also an outlet 7 in a position allowing the fluid to pass through filtration material. The apparatus may further comprise a layer of a neutral porous medium such as nylon mesh 8 positioned between the filtration material and the outlet. The neutral porous material can be, e.g., a glass or nylon mesh or layer of plastic particles, glass particles, or sand particles. The solution is allowed to pass through the apparatus, e.g., by gravity, aspiration, centrifugal force, or by using a vacuum pump or a peristaltic pump. The apparatus typically includes a receptacle (not shown) for receiving effluent from outlet 7. The receptacle is formed with a suitable port (not shown) , which may be an aspiration port, for dispensing effluent from the receptacle. The port may be fitted with the usual control valve (not shown). It should also be understood that a suitable support screen (not shown) may be provided within container 1 to hold the filtration material in place so that the solution must pass through the filtration material after being poured through inlet 2.

The apparatus is not limited to a particular volume or size and is provided, e.g., as a small cartridge with approximately 50-200 ml of slurry component or as a preferred "large volume cartridge system" (LVS) that allows for sequential passages of large volumes of solution through the filtration material. The LVS typically contains approximately 500-1000 ml of slurry component. Larger or smaller units can be assembled according to the need of the user.

Referring to Figure 2, which shows an LVS system, an inlet 2, where the fluid enters the apparatus, is attached to the container, which is cartridge 9. The apparatus contains a second foam component 4 and then alternating layers of the slurry component 5 and the foam component 6. The filtered fluid then exits the apparatus through a nylon mesh 8 and an outlet 7. Referring to Figure 3, the slurry and foam components are in a alternative embodiment, with the foam component 10 substantially distributed through the slurry component 5.

As described above, in one embodiment, the filtration apparatus also includes a second foam component that is positioned as a layer on top of the slurry component so that the solution to be filtered first passes through a foam layer. This foam layer acts as a mechanical filter, removing particulates, e.g., fat, connective tissue, and other components of animal tissue, e.g., human tissue, from the solution. Preferably, the second foam component is used to remove such substances from samples used in pathology and histology experiments and diagnostic tests.

V. Methods

This invention further provides methods for extracting desired target molecules from solutions and substantially, permanently binding them to a filtration material. The amount of material is typically sufficient to bind greater than 95% of the target molecules. The method can also include the step of repeating the passing step with additional aqueous solution and maintaining the binding of the target molecules to the filtration material accross a pH range of about 3-9. The method can be performed using apparatuses and materials such as those described above. The method can be additionally be performed, e.g. , with centrifuge bottles, by mixing the solution and filtration material, agitating it or allowing it to settle, and then extracting the solution by centrifuging the mixture.

The filtration material, prepared as described above, is typically placed a container or water-filtration apparatus with an input and an output, and solutions containing the target molecules are passed through the filtration apparatus to capture substantially the target molecules in the filter material.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

Unless otherwise stated the following abbreviations are applicable: lx SSC is 150 mM NaCl, 15 mM sodium citrate at pH 7.0. lx TBE is 89 mM Tris base, 89 mM boric acid at pH 8.6.

Example I: Binding of radioactivity in the presence of increasing salt concentrations

In this experiment, γ-³²P-ATP in 2.5x SSC was passed over a single large volume system (LVS) cartridge containing alternating layers of slurry component (750 ml) and yellow isomeric sponge. The slurry component contained 50-70% activated charcoal, 15-25% anion exchange resin, 15-25% cation exchange resin, and 0-20% neutral flow rate enhancers. The results are shown in Table 1 below. With increasing salt concentrations passed over the column, the binding was greater than 98 % and the flow rate averaged 200 ml/minute with a peristaltic pump.

TABLE 1

2.5X SSC + gamma ATP Flow Rate Application Input counts Output counts Background % Bound CUM % Bound ml/min ι l I Rehydration 1 liter 303

2| I Rehydration 1 liter 241

3 81 ,615 58 29| 99.93% 99.93% 226

4 81.615 436 291 99.47% 99.70% | 212

5 81 ,615 930 291 98.86% 99.42% 189

6 81.615 1.242 29| 98.48% 99.18% | 186

7 96.153 1.210 291 98.74% 99.10% | 230

8 | 96, 1531 1726 291 98.20% 98.95% | 21 1

9| 96.153 1 ,875 291 98.05% 98.82% | 186

10 96.1531 2.072 291 97.85% 98.70% | 208

1 1 105.6481 2.121 291 97.99% 98.62% | 220

12 105.6481 2.398 291 97.73% 98.53% 211

13 105.6481 2.548 291 97.59% 98.44% 190

14 105.6481 2.634 291 97.51 % 98.37% 178

15| 219.9701 3.051 291 98.61 % 98.39% | 206-

16 | 219.9701 3.801 291 98.27% 98.38% 185

17| 219,9701 4,104 29| 98.13% 98.36% 169

18| 219.9701 4,332| 291 98.03% 98.34% 157

Row Rate

Application Input counts Output counts Background % Off CUM %Off ml/miπ

Wash Cycles 4 liters of R/O H20 I I

19| 37| 2J30I | 0.14% 0.14% 246

201 371 2691 I 0.01 % 0.07% 197

21 | 37| 111 I 0.00% 0.07% 170

221 37| 71 0.00% 0.01 % 164

Wash Cycles |4 liters of 2.5X SSC

23 25 4.71 1 0.24% 0.08% 167

24 25 4.620 0.23% 0.06% 168

25 25 3.056 0.15% 0.08% 168

26 25 2.308 0.12% 0.09% 160

Wash Cycles 4 liters of 5X SSC I

27 14| 2.574 0.13% 0.1 1 % 220

28 14| 3.567| 0.18% 0.12% 196

29 14 2.691 0.14% 0.09% 196

30 14 2.291 I 0.12% 0.09% 186

1 liter R/O H20 with gamma ATP Fiow Rate

Application Iπout counts Output counts Background % Bound CUM % Bound ml/miπ.

41 19.056 73 99.62% | 98.42% | 180

Average | 200

Totals Input Out

Hot + Wasπes | 2,033,281.000 79,277.000 | 36.10%! 141 liters

Hot Counts | 2,032,600.000 34,611.000 98.30 %j (16 liters

Washes | 702.000 44,666.0001 -1.57V.I 122 liters

43,964,000

Example II: Binding capacity for ethidium bromide

In this experiment, ethidium bromide solution at a concentration of 0.1 g/gallon (lOx working solution) was passed over a single LVS cartridge containing alternating layers of slurry component (1000 ml) and yellow isomeric sponge. The slurry component contained 70-100% activated charcoal and 0-30% of either an anion or cation exchange resin. The results are shown in Table 2 below. Almost 100% of the ethidium bromide was bound when at least up to 15 gallons (60 liters) of ethidium bromide was passed over the cartridge, with an average flow rate of 706 ml/minute (with a peristaltic pump).

TABLE 2

Claims

WHAT IS CLAIMED IS:

1. A filtration material for extracting target molecules from solution, the material comprising: (i) a slurry component comprising an activated charcoal; and (ii) a foam component comprising an open cell foam; wherein the filtration material has at least about a 25 % increased flow rate of solution through the filtration material as compared to a filtration material that lacks the foam component.

2. The filtration material of claim 1 , wherein the slurry component further comprises an ion exchange resin.

3. The filtration material of claim 2, wherein the ion exchange resin comprises an anion exchange resin and a cation exchange resin.

4. The filtration material of claim 1, wherein the foam component comprises a poly ether-based polyurethane foam.

5. The filtration material of claim 4, wherein the foam component comprises a yellow isomeric sponge.

6. A filtration material for extracting target molecules from solution, the material comprising: (i) a slurry component comprising about 100% activated charcoal by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

7. A filtration material for extracting target molecules from solution, the material comprising: (i) a slurry component comprising about 50-70% activated charcoal, about 15-25% anion exchange resin, about 15-25% cation exchange resin, and about 0-20% neutral flow rate enhancers by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

8. A filtration material for extracting target molecules from solution, the material comprising: (i) a slurry component comprising about 70-85 % activated charcoal and about 15-30% anion exchange resin by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

9. A filtration material for extracting target molecules from solution, the material comprising: (i) a slurry component comprising about 70-85% activated charcoal and about 15-30% cation exchange resin by weight; and (ii) a foam component comprising a yellow isomeric sponge; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

10. An apparatus for extracting target molecules from solution, the apparatus comprising: (i) a filtration material comprising: (a) a slurry component comprising an activated charcoal; and (b) a foam component comprising an open cell foam; and (ii) a container comprising an inlet, the container holding the filtration material; wherein the filter material has at least about a 25 % increased flow rate of solution through the filter material as compared to a filter material that lacks the foam component.

11. The apparatus of claim 10, wherein the container further comprises an outlet allowing solution to pass out of the container.

12. The apparatus of claim 10, wherein the filtration material comprises the foam component distributed substantially among the slurry component.

13. The apparatus of claim 10, wherein the filtration material comprises the foam component and the slurry component distributed in alternating layers.

14. The apparatus of claim 10, further comprising a second foam component, wherein the solution first contacts the second foam component before contacting the filtration material.

15. The apparatus of claim 10, wherein the slurry component further comprises an ion exchange resin.

16. The apparatus of claim 15, wherein the ion exchange resin comprises an anion exchange resin and a cation exchange resin.

17. The apparatus of claim 10, wherein the foam component comprises a poly ether-based polyurethane foam.

18. The apparatus of claim 17, wherein the foam component comprises a yellow isomeric sponge.

19. An apparatus for extracting target molecules from solution, the apparatus comprising: (i) a filtration material comprising: (a) a slurry component comprising about 100% activated charcoal by weight; and (b) a foam component comprising a yellow isomeric sponge; wherein the slurry component and the foam component are distributed in alternating layers; and (ii) a container comprising an inlet and an outlet, the container holding the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to filtration material that lacks the yellow isomeric sponge.

20. An apparatus for extracting target molecules from solution, the apparatus comprising: (i) a filtration material comprising: (a) a slurry component comprising about 50-70% activated charcoal, about 15-25% anion exchange resin, about 15-25% cation exchange resin, and about 0-20% neutral flow rate enhancers by weight; and (b) a foam component comprising a yellow isomeric sponge; wherein the slurry component and the foam component are distributed in alternating layers; and (ii) a container comprising an inlet and an outlet, the container holding the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to filtration material that lacks the yellow isomeric sponge.

21. An apparatus for extracting target molecules from solution, the apparatus comprising: (i) a filtration material comprising: (a) a slurry component comprising about 70-85 % activated charcoal and about 15-30% anion exchange resin by weight; and (b) a foam component comprising a yellow isomeric sponge; wherein the slurry component and the foam component are distributed in alternating layers; and (ii) a container comprising an inlet and an outlet, the container holding the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to filtration material that lacks the yellow isomeric sponge.

22. An apparatus for extracting target molecules from solution, the apparatus comprising: (i) a filtration material comprising: (a) a slurry component comprising about 70-85 % activated charcoal and about 15-30% cation exchange resin by weight; and (b) a foam component comprising a yellow isomeric sponge; wherein the slurry component and the foam component are distributed in alternating layers; and (ii) a container comprising an inlet and an outlet, the container holding the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to filtration material that lacks the yellow isomeric sponge.

23. A method for extracting target molecules from solution, the method comprising the steps of: (i) contacting a filtration material with the solution, the filtration material comprising: (a) a slurry component comprising an activated charcoal; and (b) a foam component comprising an open cell foam; and (ii) passing the solution through the filtration material; wherein the filtration material has at least about a 25 % increased flow rate of solution through the filtration material as compared to filtration material that lacks the foam component.

24. The method of claim 23, further comprising the step of placing the filtration material into a container comprising an inlet and an outlet.

25. The method of claim 23, further comprising repeating the passing step with additional solutions.

26. The method of claim 23, further comprising the step of first contacting a second foam component with the solution before the step of contacting the filtration material with the solution.

27. The method of claim 23, wherein the slurry component further comprises an ion exchange resin.

28. The method of claim 27, wherein the ion exchange resin comprises an anion exchange resin and a cation exchange resin.

29. The method of claim 23, wherein the foam component comprises a poly ether-based polyurethane foam.

30. The method of claim 29, wherein the foam component comprises a yellow isomeric sponge.

31. A method for extracting target molecules from solution, the method comprising the steps of: (i) placing in a container having an inlet and an outlet a filtration material comprising: (a) a slurry component comprising about 100% activated charcoal by weight; and (b) a foam component comprising a yellow isomeric sponge; (ii) contacting the filtration material with the solution; and (iii) passing the solution through the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

32. A method for extracting target molecules from solution, the method comprising the steps of: (i) placing in a container having an inlet and an outlet a filtration material comprising: (a) a slurry component comprising about 50-70 % activated charcoal, about 15-25% anion exchange resin, about 15-25% cation exchange resin, and about 0-20% neutral flow rate enhancers by weight; and (b) a foam component comprising a yellow isomeric sponge; (ii) contacting the filtration material with the solution; and (iii) passing the solution through the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

33. A method for extracting target molecules from solution, the method comprising the steps of: (i) placing in a container having an inlet and an outlet a filtration material comprising: (a) a slurry component comprising about 70-85 % activated charcoal and about 15-30% anion exchange resin by weight; and (b) a foam component comprising a yellow isomeric sponge; (ii) contacting the filtration material with the solution; and (iii) passing the solution through the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.

34. A method for extracting target molecules from solution, the method comprising the steps of: (i) placing in a container having an inlet and an outlet a filtration material comprising: (a) a slurry component comprising about 70-85 % activated charcoal and about 15-30% cation exchange resin by weight; and (b) a foam component comprising a yellow isomeric sponge; (ii) contacting the filtration material with the solution; and (iii) passing the solution through the filtration material; wherein the filtration material has at least about a 300% increased flow rate of solution through the filtration material as compared to a filtration material that lacks the yellow isomeric sponge.