WO2010002462A1 - Porous asymmetric membranes - Google Patents

Abstract

The invention relates to asymmetric porous exiting membranes having a plurality of unbound size-exclusion particles retained within the membrane pores, for the separation and retention of analytes of interest, contained in a liquid sample, by size-exclusion, as the sample flows through the membrane. The size-exclusion properties of the porous membrane can be tuned or adjusted by controlling the shape and/or size of the unbound size-exclusion particles retained within the membrane pores. Devices, kits and methods of using and making porous asymmetrical membranes having a plurality of unbound size-exclusion particles within the membrane pores are also provided.

Description

POROUS ASYMMETRIC MEMBRANES

CROSS-REFERENCED TO RELATED APPLICATIONS [001] This application claims the benefit of U.S. Provisional Patent

Application No.: 61/133,885, filed on July 3, 2008, the entire contents of which are incorporated by reference herein.

Field of the Invention

[002] The present invention relates generally to porous membranes for separating and retaining analytes of interest present in liquid samples. In certain specific embodiments, the invention provides porous asymmetrical membranes having a plurality of unbound particles retained within the membrane pores. Devices, kits and methods relating to detecting, filtering, preparing, identifying, separating, purifying, characterizing, and/or enriching one or more analytes of interest from liquid samples are also provided, as are methods of making the same.

Background of the Invention

[003] A number of methods for separating, purifying or preparing biological and/or chemical liquid samples currently exist. Fluidized bed chromatography, and separation medium filled chromatography columns have all been employed with varying success to separate and/or purify biological and/or chemical substances of interest from liquid samples with respect to yield, time consumption, purity and cost.

[004] Fluidized bed chromatography is a means of separating substances from liquid mixtures, commonly utilized in the chemical industries. The advantages of a fluidized bed are higher flow rates at lower pressures as compared to fixed bed chromatography. Although the higher flow rates offer certain advantages to the chromatographic separation, the method has several shortcomings, such as less surface binding capacity. To minimize the loss of surface area and decrease density, the fluidized bed resins are highly porous structures. The most significant problem of the fluidized bed is mixing, wherein the high flow rate and limited mixing inhibit the uniform phase change required during elution of the product from the resin. [005] Purification of reaction products obtained from, for example, a polymerase chain reaction (PCR) or a sequencing reaction, such as by size- exclusion chromatography or ion-exchange chromatography, requires a well- formed resin bed, without cracks, bubbles, or channels, as well as correct sample-loading techniques. Resin beds used for purification by size-exclusion can be ten times or more the volume of the sample in size, requiring much space and increasing the cost of purification. In addition, ion-exchange resins often interact undesirably with target analytes as opposed to interacting with the ions the resins are supposed to remove. Further, purification using ion- exchange materials can require elution of the analyte using a high salt eluant, which can be undesirable for subsequent analysis and/or reactions. See for example, U.S. Patent Application Publication No. 2006/0051583 to Lau which teaches a chromatographic mixed bed ion exchanger incorporating size- exclusion ion-exchange particles.

[006] Biopharmaceutical companies and biopharmaceutical manufacturers typically use resin- or gel- packed stainless steel columns for high-volume capture steps. Column chromatography, however, requires cleaning and cleaning validation, is limited by slow diffusion factors, non- uniformity or irregular flow during processing, fluid channeling, compression of the packed resin (particularly in the case of softer gels such as agarose), inefficient capacity for handling large bio-molecules and the like, and can be costly for scale-up operations. A particular problem in this respect is the cleaning of fixed bed chromatography columns, in which irregular flow channels tend to form through the chromatography resin. These irregular flow channels present a particular problem in the purification of biological substances, since a failure to completely clean the column can result in the contamination of subsequent batches. These frequently observed "rat tunnels" which can present problems for validation of the cleaning process also negate a significant portion of the capacity and resolution capability of chromatography columns. Tunneling and compression prevent uniform distribution of the elution liquid, resulting in imprecise separation of the target substance from contaminants which have similar elution profiles to the target product as well as to randomly eluted contaminants entrapped in the compressed media. As a result, the purity of the target substance is reduced because high purity requires uniform elution of the target substance. [007] Many different types of separation media are used in separation columns including, but not limited to chromatography materials such as gel- filtration, affinity, ion-exchange, reverse-phase, and silica or modified-silica materials. In separation columns the liquid sample flows by diffusion flow, which can slow the rate at which a sample passes through the column. [008] An alternative to conventional separation columns comprises a sample preparation device comprising one or more pipette tips having particles of a separation medium directly embedded and directly bound to the inner walls of the pipette tip. The pipette tips are free of any filters or solid matrices that can potentially slow the rate of sample preparation and result in sample loss. See for example U.S. Patent No. 6,416,716 to Shukla et al. However because the separation particles are actually bound to the inner wall of the pipette tip, the device has limited flexibility because of the inability to "tune" or adjust the device by simply adding additional particles of different sizes and/or made from different materials, hi this way, the end user is not able to "tune" the device pursuant to a substance of interest contained in a liquid sample.

[009] As an alternative to column chromatography, membrane chromatography overcomes the challenges inherent in column chromatography by eliminating slow diffusion times, increasing throughput, handling high flow rates and high loading concentrations for proteins, DNA, plasmids, viruses and the like thereby speeding up purification processes, and lowering overall labor and manufacturing costs. Single-use membrane chromatography also eliminates the need for cleaning and cleaning validation, further reducing manufacturing costs as well as risks of cross-contamination. [010] Membranes offer dynamic binding capacities equivalent to column chromatography, but they are not affected by changes in volumetric flow rates, therefore allowing much higher processing throughputs and predictable scale-up. Membrane chromatography supports the use of exceptionally high flow rates because molecules access all binding sites by direct fluid convection, rather than fluid diffusion, as is the case with column assemblies or an array of columns. Since binding is not diffusion limited as it is in column assemblies or even arrays of columns, high dynamic binding and sharp breakthrough curves are achieved over a wide range of flow rates and molecular sizes. This offers significant advantages over resin- or gel- packed columns, especially when capturing or removing large molecules, or when high flow rates must be achieved. For separation and purification of large particles and bio-molecules, membrane systems are also sized smaller than columns, making them easier to handle than large column assemblies. [011] Porous membranes have a first porous surface, a second porous surface, and a continuous porous structure that extends throughout the membrane from the first to the second surface. Porous membranes can be classified as "microporous" membranes or "ultrafiltration" membranes based on the size of the pores of the membrane. Generally, the range of pore sizes for microporous membranes is considered to be from approximately 0.05 micron to approximately 10.0 microns, whereas the range of pore sizes for ultrafiltration membranes is considered to be from approximately 0.002 micron to about 0.05 micron. These pore sizes refer to pore diameter for circular or approximately circular pores, or to a characteristic dimension for non-circular pores.

[012] The pore size of a membrane can be denominated by the size of the smallest species (particle or molecule) that cannot pass through the membrane above a specified fractional passage. A common rating is below 10% passage, which corresponds to a 90% cutoff or retention. Other methods are known to those skilled in the art, including image analysis of scanning electron microscopy to obtain pore size distribution characteristics.

[013] Asymmetric membranes are characterized by having the pore size of the membrane vary as a function of location within the thickness of the membrane. The most common asymmetric exiting membrane has a gradient structure, in which pore size gradually and continually increases from one surface (often referred to as the "tight" side) to the other (often referred to as the "open" side). Asymmetric membranes have a higher flux than comparable symmetric membranes. When used in the configuration with their larger pore side upstream, these membranes typically have greater throughput as compared to symmetric membranes. See, for example, U.S. Pat. No. 4,261,834 to de Winter. Asymmetrical membranes are used in a variety of applications such as food and beverage filtration, pharmaceutical and biopharmaceutical manufacture, laboratory filtration, water filtration and the like.

[014] While most asymmetric membranes work satisfactorily on water or aqueous based solutions, they tend to prematurely clog and have poor throughput capacity when used with viscous or heavily loaded streams or the like, such as blood or plasma sample fluids, even when used in the "open side" upstream configuration.

[015] Obtaining high quality intact nucleic acids suitable for analysis is thus often a useful and desirable starting point for subsequent analyses. While a variety of techniques for isolating nucleic acids from a sample have been described (see e.g., U.S. Patent Nos. 6,274,308; 6,958,392; 6,953,686; and 6,992,182; and U.S. Patent Application Publication No. 2006/0024701), it would be, nonetheless, beneficial to provide a method of isolating a nucleic acid from a sample that was fast, economical, and produced high yields. It would also be useful if the method minimized the required manipulation of the sample, permitted the use of primarily liquid handling steps and could be performed using a single porous membrane device.

[016] Accordingly, it would be desirable to have a porous asymmetric membrane, devices, kits and methods for using the same, for the rapid, inexpensive, and efficient isolation of one or more analytes of interest from a fluid sample.

[017] Additionally, the porous asymmetric membrane would also be readily scalable, adaptable to process volumes of sample fluids ranging from milliliters to the thousands of liters, and capable of use with liquids containing analytes of interest, including contaminants and/or unwanted byproducts, having a wide array of properties, including viscous complex solutions, and is "tunable" or adjustable such that properties like pore size exclusion of the membrane can be controlled based upon the intended end use of the porous membrane. The invention is directed to these, as well as other objectives.

SUMMARY OF THE INVENTION

[018] A new asymmetric porous exiting membrane having a plurality of unbound particles retained within the membrane pores is provided herein in response to the above needs and problems associated with reduced throughput capacity and size-exclusion permeability when separating analytes contained in liquid samples having a wide range of physical, chemical and biological properties using known column, bed, bead, and monolithic chromatography devices. Thus, in accordance with the present invention, a new asymmetric porous exiting membrane, that enables the control of the size-exclusion properties of the membrane, is provided having improved convective flow characteristics and throughput capacity permeability when separating analytes contained within liquid samples having a wide range of physical, chemical and/or biological properties.

[019] In accordance with the present invention, the pores on the surface of the asymmetric exiting membrane on the inlet side of the membrane ("open" side) have a larger pore size than the pores located on the opposing surface of the outlet side of the membrane ("tight" side), and a continuous porous structure of membrane pore channels extends throughout the membrane from the open side to the tight side. The fluid sample containing the analyte(s) of interest enters the membrane through the pores on the inlet side surface of the membrane, wherein the continuous porous structure which extends throughout the membrane separates and retains the analytes of interest contained in the liquid sample based on size exclusion properties, as the liquid sample flows through and exits the porous membrane via the membrane outlet surface, and the analyte(s) of interest remains trapped within the membrane by the unbound particles blocking the membrane pores to the outlet surface based on the size-exclusion properties of the particles retained therein.

[020] In certain embodiments, the present invention provides a porous asymmetric exiting membrane having plurality of unbound particles located within the continuous porous structure of membrane pore channels which extends throughout the membrane and constitutes the membrane pores, and associated novel devices, methods, and kits that find particular utility for detecting, filtering, preparing, identifying, separating, purifying, characterizing, and/or enriching one or more analytes of interest present in a liquid sample.

[021] In certain embodiments, the present invention provides a porous asymmetric exiting membrane having a plurality of unbound particles retained within the continuous porous structure of membrane pore channels which extends throughout the membrane and constitutes the membrane pores,, such that size exclusion properties of membrane filtration can be "tuned" or adjusted to allow a liquid sample to pass through the membrane, while preventing an analyte(s) of interest contained in the sample from exiting the membrane. The ability to adjust and control the size selectivity properties of the membranes can be achieved by varying the size, shape and/or other physical and chemical properties of the unbound particles located within the continuous porous structure of membrane pore channels which extends throughout the membrane and constitutes the membrane pores.

[022] In still other embodiments, the invention provides devices and methods for using these devices, comprising a porous asymmetric exiting membrane having a plurality of unbound particles located within the membrane pore channels in the continuous porous structure which extends throughout the membrane, contacting a liquid sample containing, or suspected of containing, one or more analytes of interest with the porous asymmetric membrane; separating the liquid sample and the analyte(s) of interest by size- exclusion such that the liquid sample flows through and exits the porous membrane via the membrane outlet surface; and isolating the analyte(s) of interest by trapping the analyte(s) within the membrane by the unbound size- exclusion particles selectively blocking the membrane pores to the outlet surface. The analyte(s) of interest can undergo additional processing steps while either retained in the membrane pores and/or after removal from the membrane by elution and the like, including but not limited to, hybridization, amplification, lysis, denaturing, and any combinations thereof.

[023] In another embodiment, the invention provides methods, procedures and techniques for using a separation device comprising: a) contacting a liquid sample containing a wide variety of analytes with the inlet surface of a separation device comprising a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles retained within the membrane pores for determining, purifying, concentrating, enriching, separating and/or detecting the analytes contained within the liquid sample; b) separating the liquid sample and the analyte(s) of interest by size- exclusion as the liquid sample flows through and exits the porous membrane via the membrane outlet surface, while the analyte(s) of interest remains trapped within the membrane by the unbound size-exclusion particles selectively blocking the membrane pores to the outlet surface. The analytes of interest include, but are not limited to, organisms, cells, proteins, nucleic acid, RNA, DNA, carbohydrates, viruses and virus particles, bacteria, fungi, parasites prions, chemicals, biochemicals, and environmental and food contaminants.

[024] In still other embodiments, the invention provides a separation device and a method of isolating a nucleic acid from a liquid sample comprising: a) contacting a liquid sample containing a nucleic acid with the inlet surface of a separation device comprising a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles retained within the membrane pores; b) lysing the liquid sample and nucleic acid; c) separating the liquid sample and nucleic acid by passing the liquid through the porous membrane, whereby the liquid exits through the outlet surface of the membrane, and isolating the nucleic acid by trapping it within the membrane by the unbound size-exclusion particles blocking the membrane pores to the outlet surface. The liquid sample may be a prokaryotic and/or eukaryotic cellular sample, and/or a viral sample, wherein one or more these samples may be lysed. Optionally, the isolated nucleic acid trapped within the membrane pores may be eluted from the porous membrane.

[025] In another embodiment, the invention provides a separation device and a method of isolating a nucleic acid from a protein sample comprising: a) contacting a protein liquid sample containing nucleic acids with the inlet surface of a separation device comprising a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles retained within the membrane pores; b) lysing the protein sample containing the nucleic acids; c) separating the protein sample and nucleic acids by passing the protein liquid sample through the porous membrane and exiting through the membrane outlet surface, and isolating the nucleic acid by selectively trapping the nucleic acid within the membrane by the unbound size-exclusion particles blocking the membrane pores to the outlet surface. Optionally, the isolated nucleic acid trapped within the membrane pores may be eluted from the porous membrane. [026] In another embodiment, the invention provides a separation device and a method of isolating DNA from a liquid sample comprising: a) contacting a liquid sample containing DNA with the inlet surface of a separation device comprising a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles retained within the membrane pores; b) lysing the liquid sample and DNA; c) separating the liquid sample and DNA by passing the liquid through the porous membrane and exiting through the outlet surface of the membrane, and isolating the DNA by selectively trapping it within the membrane by the unbound size-exclusion particles blocking the membrane pores to the outlet surface. The liquid sample may be a prokaryotic and/or eukaryotic cellular sample, and/or a viral sample, wherein one or more these samples may be lysed. Optionally, the isolated DNA trapped within the membrane pores may be eluted from the porous membrane.

[027] In another embodiment, the invention provides a separation device and a method of isolating RNA from a liquid sample using the device comprising: a) contacting a liquid sample containing RNA with the inlet surface of a separation device comprising a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles retained within the membrane pores; b) lysing the liquid sample and RNA; c) separating the liquid sample and RNA by size-exclusion as the liquid sample flows through and exits the porous membrane via the membrane outlet surface, and isolating the RNA by selectively trapping it within the membrane by the unbound size-exclusion particles blocking the membrane pores to the outlet surface. The liquid sample may be a prokaryotic and/or eukaryotic cellular sample, and/or a viral sample, wherein one or more these samples may be lysed. Optionally, the isolated RNA selectively trapped within the membrane pores may be eluted from the porous membrane.

[028] In another embodiment, the invention provides methods for using a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles located within the membrane pores for chemically and/or physically changing the liquid sample, and/or analyte of interest present in the liquid sample. An example is the desalting of a sample, where some or substantially all of the salt (or other constituent) in a sample is removed or replaced by a different salt (or non-salt constituent). The removal of potentially interfering salt from a sample prior to analysis is important in a number of analytical techniques, e.g., mass spectroscopy. Another example would include heating the membrane, wherein the unbound size-exclusion particles are metallic, and the membrane would heat up much more quickly at the location of the beads.

[029] In another embodiment, the invention provides separation devices and methods for identifying analytes of interest present in a liquid sample using the devices, wherein the device includes a porous asymmetric exiting membrane having plurality of unbound size-exclusion indicator particles retained within the membrane. The unbound size-exclusion indicator particles can be any known in the art, such as dyes and the like. The size- exclusion indicator particles can be porous or non-porous. The size-exclusion indicator particles can be coated with a chemical dye or can be porous indicator particles that contain a dye. The dye can be a solid or a liquid. The size-exclusion indicator particles can be of any size or shape as described herein. In this embodiment, the inlet surface of the separation device is contacted with the liquid sample containing the analyte of interest for the separation, retention and identification of the analyte by size-exclusion, as the liquid sample flows through and exits the membrane via the membrane outlet surface. Contact between the analyte and the size-exclusion indicator particles will activate an indicator dye to produce a color change on and/ or in the indicator particles. This color change will indicate the presence of an analyte of interest. If there is no color change, the analyte of interest was not present in the sample.

[030] In certain embodiments, the invention provides separation devices and methods of separating one or more cellular or viral analytes from a liquid sample comprising: a) contacting a liquid sample having one or more cellular and/or viral analytes with the inlet surface of a separation device comprising a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles retained within the membrane; b) separating the liquid sample from the one or more cellular or viral analytes by size- exclusion as the liquid sample flows through and exits the membrane via the membrane outlet surface, isolating the cellular and/or viral analytes by selectively trapping them within the membrane by the unbound size-exclusion particles blocking the membrane pores to the outlet surface. Optionally, the isolated one or more cellular or viral analytes may undergo lysing, eluting, identifying, further processing, and any combination thereof.

[031] In another embodiment the invention provides a separation device and a method of isolating RNA from a liquid sample comprising: a) contacting a liquid sample containing RNA with the inlet surface of a separation device comprising a porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles retained within the membrane pores; b) lysing the liquid sample and RNA; c) separating the lysed liquid sample and RNA by size-exclusion as the lysed liquid sample flows through and exits the porous membrane via the membrane outlet surface, while the RNA remains selectively trapped within the membrane by the unbound size- exclusion particles blocking the membrane pores to the outlet surface. The liquid sample may be a prokaryotic and/or eukaryotic cellular sample, and/or a viral sample, wherein one or more these samples may be lysed. Optionally, the isolated RNA selectively trapped within the membrane pores may be eluted from the porous membrane.

[032] In other embodiments, the invention provides a method of isolating DNA and/or RNA from a cellular sample comprising: a) lysing the cellular sample to obtain a cellular lysate; b) optionally clarifying the cellular lysate (e.g., by centrifugation); c) contacting the liquid lysate sample with the inlet surface of a separation device comprising a porous membrane having a plurality of unbound size-exclusion porous particles having a first pore size, and a plurality of unbound size-exclusion porous particles having a second pore size that are different from the first pore size, and retained within the membrane pores, wherein the RNA binds to the size-exclusion particles having a first pore size, and the DNA binds to the size-exclusion particles having a second pore size; d) separating the liquid lysate sample from the bound DNA and RNA, wherein the liquid lysate passes through the porous membrane and exits through the membrane outlet surface, isolating the particle bound DNA and RNA by selectively trapping them within the membrane by the size-exclusion particles blocking the membrane pores to the outlet surface; e) washing the first and/or second size-exclusion particles bound to their respective RNA and DNA with one or more suitable buffers; f) eluting the RNA and/or DNA from their respective size-exclusion particles they are bound to with a suitable elution buffer.

[033] In certain embodiments, the invention provides a device and a method of isolating a first and second species of nucleic acid from a biological liquid sample comprising: a) contacting a biological liquid sample containing a first and second species of nucleic acid with the inlet surface of a separation device comprising a porous membrane having a plurality of unbound porous size-exclusion particles having a first pore size, and a plurality of unbound size-exclusion porous particles having a second pore size that are different from the first pore size, retained within the membrane pores; b) separating the first and second species of nucleic acid from the biological liquid sample, and from each other, by size-exclusion, as the biological liquid sample flows through and exits the porous membrane via the membrane outlet surface, while the first species of nucleic acid binds to the size-exclusion particles having the first pore size, and the second species of nucleic acid binds to the size-exclusion particles having the second pore size, such that each of nucleic acid bound to a size-exclusion particle remains selectively trapped within the membrane by the size-exclusion particles blocking the membrane pores to the outlet surface; c) washing the first and/or second size-exclusion particles bound to their respective nucleic acid species with one or more suitable buffers, d) eluting the first and/or second nucleic acid species from their respective size-exclusion particles they are bound to with a suitable elution buffer.

[034] In another embodiment, the invention provides methods for detecting and/or correlating analyte(s) retained within the pores of an asymmetric membrane having a plurality of unbound size-exclusion particles retained within the membrane pores, in order to determine the concentration of the analyte(s), (i.e., a heavy metal), in a liquid sample, (i.e., water), having a known volume.

[035] In yet other embodiments, the invention provides a kit for isolating nucleic acid from a liquid sample comprising: a) an asymmetric exiting membrane having plurality of unbound size-exclusion particles located within the membrane pores; b) one or more buffers, reagents, and eluents; c) at least one container. [036] In still other embodiments, the invention provides a kit for isolating RNA, DNA, proteins, prokaryotic cells, eukaryotic cells, viruses and any combination thereof from a liquid sample comprising: a) an asymmetric exiting membrane having plurality of unbound size-exclusion particles located within the membrane pores; b) one or more buffers, reagents, and eluents; and c) one or more containers.

[037] In another embodiment, the invention provides for kits including filtration and separation devices comprising porous asymmetric membranes having a plurality of unbound size-exclusion particles retained within the membrane, and used as a tool for reducing the cost of conducting reactions or reducing the time required to conduct reactions of one or more analytes of interest contained in a liquid sample.

[038] Additional features and advantages of the invention will be set forth in the detailed description and claims, which follows. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. It is to be understood that the foregoing general description and the following detailed description, the claims, as well as the appended drawings are exemplary and explanatory only, and are intended to provide an explanation of various embodiments of the present teachings. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[001] Fig. 1 is a line graph of the measured particle size distributions.

[002] Fig. 2 is a line graph of the measured filtered volume over time during filtration by each membrane loaded with particles for determining flow rate and flux loss.

[003] Fig. 3 is a line graph of the measured membrane pressure over time during filtration for each membrane loaded with particles.

[004] Fig. 4 is a photograph of a scanning electron micrograph showing a porous membrane with latex beads retained with the membrane pores.

[005] Fig. 5 shows a closed version of a filter device of the invention: vented.

[006] Fig. 6 is a line graph of the measured signal emergence and detection above noise values after the transcription mediated amplification of Pseudomonas aeruginosa nucleic acids from membranes loaded with beads and membranes without beads.

[007] Figs. 7 and 8 are bar graphs depicting and comparing data results between filter devices of the invention against comparative filter devices.

DESCRIPTION OF THE EMBODIMENTS

[008] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about".

[009] Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[010] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass all subranges subsumed therein. For example, a range of "1 to 10" includes any and all subranges between (and including) the minimum value of 1 and the maximum value of 10, that is, any and all subranges having a minimum value of equal to or greater than 1 and a maximum value of equal to or less than 10, e.g., 5.5 to 10. [Oi l] Before describing the present invention in further detail, a number of terms will be defined. Use of these terms does not limit the scope of the invention but only serve to facilitate the description of the invention.

[012] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[013] As used herein the phrase "analyte(s) of interest" includes, but is not limited to, molecules or substances to be detected in a liquid sample when present in the sample. Analytes of interest include, but are not limited to, cellular sources including eukaryotic cells, and prokaryotic cells. Examples of eukaryotic cells may include cells derived from any mammal, e.g., humans, non-human primates, horses, goats, sheep, rats, rabbits, mice, guinea pigs. Examples of prokaryotic cells include gram negative bacteria and gram positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcal aureus. Exemplary analytes of interest include, but are not limited to biomolecules, DNA, RNA, nucleic acids, nucleotides, polynucleotides, oligonucleotides, aptamers; allergens of all types; products or components of normal or malignant cells; carbohydrates, proteins, peptides, parasites, fungi, prions, effector molecules, a receptor, a signal-generating molecule, a structural molecule, amino acids, carbohydrates, polymers, ligands, enzymes, antibodies, dyes, bacteria, cells, prokaryote cells, eukaryote cells, tissues, cyclodextrins, lectins, metal ions, antigens, small organic molecules such as medicinal pharmaceuticals, macromolecules, and other chemical compounds, chemical moieties, or biologies, petroleum-based chemicals and synthetic petro-chemical additives, toxins, pesticides, herbicides, fungicides; environmental and food contaminants, and biowarfare agents. The analyte of interest preferably originates from a liquid sample. The analyte of interest can, for example, be dissolved and/or suspended in a liquid sample.

[014] As used herein the term "antibody" includes, but is not limited to an immunoglobulin that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art, or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies. Antibodies may include a complete immunoglobulin or fragment thereof, and human or humanized versions of immunoglobulin molecules or fragments thereof. The immunoglobulins include the various classes and isotopes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab').sub.2, Fab¹, and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular molecule is maintained.

[015] As used herein the term "antigen" includes, but is not limited to, substances that upon administration are capable of eliciting an immune response, thereby stimulating the production and release of antibodies that bind specifically to the antigen. Antigens include molecules and/or moieties that are bound specifically by an antibody to form an antigen/antibody complex. Examples of antigens include, but are not limited to, peptides, polypeptides, proteins, nucleic acids, DNA, RNA, and combinations thereof, fractions thereof, or mimetics thereof.

[016] The term "asymmetric membrane" means a membrane in which the average pore size is not constant across the membrane cross-section. For example, in asymmetric membranes pore sizes can vary smoothly or discontinuously as a function of location through the membrane cross-section. As will be appreciated, included within the definition of "asymmetric membranes" are membranes that have a ratio of pore sizes on one external surface to those on the opposite external surface that are substantially greater than one.

[017] As used herein the term size-exclusion "particle" includes, but is not limited to, spherically shaped particles, cube shaped particles, other three dimensional polygonal shapes, as well as cylinders, ovals and irregular shaped particles. The particles can be magnetic, can have a magnetic core, and/or can be magnetizable. Such an embodiment requires the use of a magnet or magnetic field outside the membrane.

[018] As used herein the phrase "effector molecules," also referred to as "effector species," "effectors," and "molecular effectors" include, but are not limited to molecules capable of transforming energy into work, work into energy, work or energy, or information into work or energy and include, but are not limited to, signal-generating species, stimulus-response molecules, response-generating molecules, enzymes, synthetic enzymes, drugs, catalytic antibodies, catalysts, transport proteins, regulatory proteins, cytochromes, electroactive compounds, photoactive compounds, and shape-memory structures, or any derivative or variant thereof.

[019] As used herein the term "fluid" includes, but is not limited to gases or liquid, such as a suspension or a solution, e.g. aqueous, or organic solutions, blood, any body fluid or biofluid for life science applications such as fluids in biological, diagnostic or biotechnological applications e.g. buffer solutions, infusion fluids, or dialysis fluids, fluids for nutrition and fluids for industrial use.

[020] As used herein, the term "fluid sample" includes, but is not limited to both gases and liquids, preferably the latter. The terms "fluid sample" or "liquid sample" refer to a liquid containing, or suspected of containing, one or more analytes of interest. The liquid sample may be an aqueous solution containing particles, cells, microorganisms, ions, or small and large molecules, such as proteins and nucleic acids, etc., or combinations thereof. A fluid sample includes, but are not limited to, biological samples, such as samples from taken from animals (e.g., saliva, whole blood, serum, and plasma, urine, tears and the like), tissue or cell cultures, plant tissue or cell cultures, preparations containing bacteria, viruses, fungi, spores, and lysed ingredients thereof, and any combinations thereof. A fluid sample also includes environmental (e.g., water), industrial, chemical processing streams, food, waste water, natural waters, soil extracts, radioactive samples, and any combinations thereof. It is understood that a solid sample containing an analyte can be homogenized or otherwise put into solution to facilitate the analysis of the sample. Additionally, samples may require diluting, filtering, centrifuging or stabilizing prior to use with the invention. For the purposes herein, "sample" refers to the either the raw sample or a sample that has been prepared or otherwise pretreated.

[021] As used herein the term "functional group" includes, but is not limited to mean a specific atom, or group of atoms, that gives a molecule a positive or a negative charge, hydrophobicity or hydrophilicity, and/or any other physio-chemical force, e.g. van der Waals forces and .pi.-.pi.- interactions among aromatic groups or a capacity to form further bonds, such as hydrogen bonds or covalent bonds.

[022] As used herein the term "ion" includes, but is not limited to alkali metal ions, alkali earth metal ions, transition metal ions, or lanthanide metal ions.

[023] As used herein a "ligand" may be a full length protein or a functional variant of a full length protein. The ligand may be a monomer, dimer or multimer of a full length protein or functional variant. Examples of ligands include, but are not limited to, receptor agonists, partial agonists, mixed agonists, antagonists, drugs, hormones, regulatory factors, antigens, haptens, structural molecules, effector molecules,and analogs, competitors, derivatives of these molecules, and any combination thereof. Nonlimiting examples of protein ligands include Protein A, Protein G, the Fc receptor of an antibody, and a receptor for a hormone or growth factor. See, e.g., U.S. Patent Nos. 5,084,559; 5,260,373; PCT Publication No. WO 95/19374. In other embodiments the protein ligand may be an immunoglobulin, e.g. IgG, IgM, IgA, IgD, IgE or a functional variant thereof. The ligand may be a naturally occurring molecule or an engineered molecule. In a specific embodiment the ligand is Protein A, or a functional variant thereof. Protein A or a functional variant thereof may be recombinantly produced, e.g., in a prokaryotic cell such as E. coli. In another embodiment the ligand may be Protein A, obtained from Staphylococcus aureus. In some embodiments, the ligand may comprise one or more domains, e.g., A, B, C, D, or E domains of a native Protein A, or a functional variant thereof. In other embodiments the ligand may be Protein Z, an altered B domain of Protein A. See for example, U.S. Patent Publication No. 2005/0143566. Modifications which improve the performance capability of the protein A ligand are also contemplated. The ligand may be a monomer of Protein A, or a functional variant thereof or a multimer of Protein A, or a functional variant thereof.

[024] As used herein, the term "nucleic acid" includes, but is not limited to any synthetic or naturally occurring nucleic acid, such as DNA or RNA, in any possible configuration, i.e., in the form of double-stranded nucleic acid, single-stranded nucleic acid, or any combination thereof. A "nucleic acid" includes, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA). The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Where the target is DNA, the DNA may be, plasmid DNA, vector DNA, genomic DNA, including mitochodrial DNA, or a fragment of any of the preceding. Where the target is comprised of RNA the RNA may be mRNA, tRNA or rRNA e.g. 16s RNA, 23 s RNA or any combination thereof. The nucleic acid may be comprised of nucleotide analogs, e.g., chain terminators. The size of the target nucleic acid may range from 2-30 nucleotides or may be in the kilobase or larger range.

[025] As used herein the term "receptor" includes, but is not limited to a molecule capable of specifically binding to a ligand. Receptors include, but are not limited to, biological, synthetic or engineered membrane receptors, hormone receptors, drug receptors, transmitter receptors, stimulus-response coupling or receptive molecules, antibodies, antibody fragments, antibody mimics or mimetics, molecular mimics, adhesion molecules, and congeners, analogs, competitors, derivatives of these molecules, and combinations thereof.

[026] As used herein the phrase "signal-generating molecules" and "signal-generating species" include, but are not limited to molecules capable of generating a detectable signal or enhancing or modulating the detectability of a substance or transducing an energy, activity, output or signal of a substance into a qualitatively, quantitatively or detectably different energy, activity, output, signal, state or form. Alternatively, signal-generating molecules can interact with an analyte of interest to produce an analyte capable of localization. Signal-generating molecules include, but are not limited to, molecules, conjugates and complexes comprising detectable (and optionally dyed, modified, conjugated, labeled or derivatized) tags, tracers, radioisotopes, labels, photosynthetic molecules, signal transduction pathways, dyes, and other photon-absorbing, photon-emitting and photosensitive molecules, including molecules or groups that enhance, attenuate, modulate or quench the photon-absorbing or photon-emitting properties of another molecule or group, energy transfer donors and acceptors, enzymes, coenzymes, cofactors, catalysts, molecular mimics and mimetics, luminescent, electroluminescent, chemiluminescent and bioluminescent molecules, electron transfer donors and acceptors, oxidizing and reducing compounds.

[027] As used herein the term "structural molecule" includes, but is not limited to, elements, atoms, molecules, ions, and compounds comprising surfaces, inorganic and organic materials, inorganic crystals, selected solvents, selected solutes, fibers, filaments, nanotubes , fullerenes, buckyballs, semiconductors, insulators, metals, plastics, polymers, detergents, lubricants, waxes, oils, fillers, excipients, dendrimers, electrolytes, hydrocarbons, ceramics, fatty acids, surfactants, amino acids, peptides, proteins, sugars, starches, cellulose, and conjugates thereof.

[028] It is to be understood that the size-exclusion particles admitted to or deflected from the porous membrane are not necessarily exactly the "pore size" given. That is, admittance to or exclusion from the membrane pores is based on many factors, including actual pore size, steric hindrance factors, ionic attractions, and the like. As used herein a "pore size" is a mean measurement, providing a guideline that particles larger than the pore size have a higher chance of not passing into the pores of the membrane, while smaller particles have a larger chance of passing into the membrane pores.

[029] Described herein are methods for separating, concentrating, and filtering one or more analytes of interest that may be present, or absent, in a fluid sample. Typically the fluid sample will have a known volume, which facilitates certain types of analysis of the analyte of interest. Separating an analyte of interest from a liquid sample means removing or separating the analyte from the liquid sample. The amount and precision of analyte separation from the liquid sample can vary. In one aspect, qualitative determination for the presence or absence of the analyte may be performed on less precise and less efficient analyte separation from the liquid sample. In another aspect, the analyte of interest is separated with increased precision and efficiency. In this aspect, the separated analyte can be subjected to further manipulation, such as quantification determination.

Particles

[030] The size-exclusion particles used in the invention can be porous or non-porous. The size-exclusion particles can be made of one or more inert materials. Exemplary inert materials include, but are not limited to, polymers, organic materials, inorganic materials, metals, metal oxides, ceramics, siliceous material, and the like. Exemplary polymers that can be used to make the particles include, but are not limited to, cellulose, cross-linked dextrans, agarose, thermoset plastics such as epoxy, and thermoplastic resins such as polytetrafluoroethylenes (e.g., TEFLON.RTM. from DuPont), polysulfones, polyethersulfones, acetates, polystyrenes, polypropylene, polyvinylchlorides, polycarbonates, polystyrene/acrylonitrile copolymers, polyvinylidenefluorides, or mixtures of two or more thereof. Exemplary inorganic materials that can be used to make the particles include, but are not limited to, controlled siliceous material, chromia, tin oxide, steel, gold, silver, aluminum and copper. The siliceous material includes, but is not limited to particulate glass, controlled porous glass, colloidal silica, wollastonite, silica gel and bentonite. The particles can be cation-exchange particles, anion-exchange particles, SEIE particles or any combinations thereof. The particles can be magnetic, magnetizable and/or have a magnetic core.

[031] The size-exclusion particles can be of substantially any shape and/or size can be used in the present invention so long as the size-exclusion particles can enter the porous asymmetric membrane through the inlet pores on the "open" surface of the membrane, and are not able to exit the membrane through the outlet pores on the "tight" surface of the membrane. The size- exclusion particles typically are from about 0.01 microns to less than about 10 microns in average diameter By way of nonlimiting examples only, the particles shape can be regular bead shape, spherical, cubical, cylindrical, oval, irregular, and any combinations thereof. Particles, in particular beads, having a generally spherical geometry are particularly well suited for use herein. Such beads are available from a variety of sources, including Thermo Fisher Scientific, formerly Duke Scientific, Fremont, CA.

[032] Additionally, the size-exclusion particles can carry any desired reagent. As used herein, the term "reagent" can refer to a single substance, or a grouping of substances. Porous asymmetric membranes

[033] By membrane what is meant is a structure having lateral dimensions much greater than its longitudinal dimensions, through which mass transfer may occur under a variety of driving forces.

[034] The pores on the surface of the asymmetric membrane on the inlet side of the membrane ("open" side) have a larger size than the pores located on the opposing surface of the outlet side of the membrane ("tight" side). When a liquid sample containing an analyte of interest enters the membrane through the pores on the inlet side, the liquid sample flows through the membrane such that only the liquid sample exits the membrane through the pores on the outlet side surface of the membrane, while the analyte of interest is trapped within the pores, due to the plurality of unbound particles blocking the path of the analyte from exiting the outlet side of the membrane.

[035] The porous membranes can be microporous or ultraporous Microporous pore sizes preferably are from about 0.05 microns to about 10 microns. Ultraporous pore sizes are typically below 0.05 microns. The selection of the suitable pore size depends upon the application for which the product is used. Ultraporous membranes are preferred for diafiltration, nanofiltration, dialysis and reverse osmosis while microporous membranes are typically used for clarification, purification and size separation processes.

[036] The asymmetrical membranes may have a pore size gradient of from about 2:1 to about 1000:1, preferably from about 2:1 to about 100:1. This asymmetry is measured by comparing the average pore size on one major surface of the membrane with the average pore size of the other major surface of that membrane. In accord with the invention, one can create two or more asymmetrical layers, each having a different or similar asymmetry.

[037] The size-exclusion particles are loaded or placed into the membrane pores through the inlet surface of the membrane, where upon reaching a point where the diameter of the pore channels prevents the particles from traveling any further into the membrane; the particles become trapped and retained within the pores. The size-exclusion particles are considered to be unbound to any surface or pore of the membrane, as well as to each other, because the size-exclusion particles are not placed into the membrane by any chemical or physical coating, bonding, adhering, embedding, or fastening processes, including, but not limited to, applying or using a resin, binding matrix, affinity layer, adhesive, epoxy, and the like to attach the particles the membrane. The size-exclusion particles are retained within the pores because the diameter and/or shape of the size-exclusion particles prevent them from exiting the outlet side of the membrane surface. The membrane can be "tuned" or adjusted so as to allow a liquid sample to pass through the membrane while preventing the analyte(s) of interest contained in the sample from exiting the membrane. The ability to adjust and control the size-selective permeability of the porous membranes can be achieved by varying the size and/or shape and/or other physical and chemical properties of the unbound size-exclusion particles located within the membrane pore channels.

[038] The porous asymmetric exiting membrane may be made from polymeric materials including, but not limited to, PVDF (polyvinylidene fluoride), polytetrafluoroethylene (e.g., TEFLON.RTM. by DuPont), polyethylene, polypropylene, nylons, polyamides, polyimides, polyethersulfones, polysulfones, polyarylsulfones, PVC, polycarbonates, polystyrene/acrylonitrile copolymer, cellulose, regenerated cellulose, cellulose esters, cellulose acetate, polystyrenes, polyetherimides, acrylic polymers, methacrylic polymers, copolymers of acrylic or methacrylic polymers, as well as glass, metal, silica, paper, cardboard, and any combinations thereof

[039] Additionally, one can vary the thickness of the membrane and, if two or more membranes are used, the thickness of each membrane may vary within a wide range and still obtain a self-supporting, integral multi-layered structure. The membranes of the invention may have thickness ranging from 0.1 mm to 10 mm. Thickness refers to the distance from one outer surface to another outer surface and corresponds to the distance a sample will travel as it traverses the membrane when a force, e.g. gravity, a vacuum, is applied to the membrane. Chromatography systems

[040] In some embodiments the invention provides a system for isolating an analyte of interest from a liquid sample mixture. The system includes a device comprising porous asymmetric exiting membrane having a plurality of unbound size-exclusion particles located within the membrane pores. The system may also include a housing suitable for containing the membrane. The system may include one or more pumps to facilitate flow of the mixture to the membranes. Suitable pumps include peristaltic pumps, pulsed pumps and/or positive displacement pumps. The system may take the form of a high pressure liquid chromatography system (HPLC), medium pressure liquid chromatography system (MPLC) or a low pressure liquid chromatography system (LPLC). The system may include one or more means to detect the contents of an eluant from the membrane. The detector may be a light based detector which relies on multi-wavelength detection or single wavelength detection. Suitable detectors include a spectrophotometer capable of detecting visible wavelengths of light, a UV absorption detector, a fluorescence detector. The detector may be a light scattering detector which relies on a laser source or an electrochemical detector which responds to substances that are either oxidizable or reducible and the electrical output is an electron flow generated by a reaction that takes place at the surface of the electrodes. The system may also include one or more printers for providing chromatograms of the eluted material from the chromatography media. The system may also include one or more personal computers. The personal computer may be suitable for recording data, such as the absorbance or fluorescence of an elution fraction. Additionally the computer may be equipped with suitable software to calculate the concentration of a target molecule in an elution fraction. The computer may also be used to automate the process of performing affinity chromatography such that liquid samples that may contain an analyte of interest are applied to the asymmetric porous membrane, the membrane is washed with one or more suitable buffers and the target molecule retained by the size-exclusion particles is optionally eluted off of the membrane with a suitable elution buffer.

Method of use

[041] The invention may be used for filtering, separating, preparing, identifying, enriching, detecting, and/or purifying analytes of interest from liquid samples using a porous asymmetric exiting membrane having a plurality of unbound particles located within the membrane pores.

[042] The liquid sample containing analytes of interest may be introduced into the membrane of the invention by a variety of means and techniques, manual or automated, well known in the art. Examples would include use of a pump (e.g., a syringe, pressurized container, centrifugal pump, electrokinetic pump, peristaltic pump, or an induction based fluidics pump), gravity, centrifugal force, capillary action, or gas pressure to move fluid through the capillary. [043] The analyte of interest retained within the membrane pores can be collected and analyzed by a number of techniques well known in the art. Examples of ways to collect the analyte include, but are not limited to, eluting the analyte from the particles by altering one or more conditions, e.g. by applying an elution buffer, one or more wash steps. The use of eluants is well known to one of ordinary skill in the art and vary depending upon the type or types of particles used, and the analyte to be captured and recovered. Typical eluants include, but are not limited to, water, alcohols, acids, bases, organic, detergents, salt solutions and blends thereof.

[044] Examples of subsequent ways of analyzing the analyte, include but is not limited to, mass spectrometry, electrospray mass spectrometry, thin layer chromatography, electrophoresis, infrared spectroscopy, fluorescent spectroscopy, gas chromatography, atomic absorption, amino acid sequence analysis, nucleic sequence analysis, matrix assisted laser desorption/ionization (MALDI), surface enhanced laser desorption ionization (SELDI) or high performance liquid chromatography (HPLC).

[045] The size-exclusion of the porous asymmetric membrane can be adjusted or "tuned" by controlling the shape and/or size of the particles retained in the membrane pores. Controlling the effective pore size of the membrane pores can be achieved by varying the size and/or shape of the unbound size-exclusion particles located within the membrane pore channels, thereby optimizing the porous membrane for different size-exclusion applications. The size-exclusion characteristics of the membrane can be tailored, modified, changed, or otherwise varied by changing the desired properties of the size-exclusion particles retained within the membrane pores. For example, a liquid sample that may contain an analyte(s) of interest can be filtered, separated and/or purified by adding the liquid sample and analyte(s) to the "open" surface (i.e., inlet side) of the asymmetric membrane, such that the liquid sample and analyte enters the membrane through the pores on the inlet side, passing through the membrane such that only the liquid sample exits the membrane through the pores on the outlet side of the membrane, while the analyte(s) of interest are retained within the pores due to the plurality of size- exclusion particles blocking the path of the analyte from exiting the outlet side of the membrane.

[046] Solvents (e.g., weak eluting solvents) may then be added to the membrane to remove the impurities from the analyte. After the impurities have been removed, the purified analyte of interest may be eluted from the asymmetric porous membrane with an appropriate solvent or buffer (e.g., relatively stronger eluting solvent or buffer).

[047] The asymmetric porous membrane of the invention may be use in any repetitive chemical process requiring synthesis or degradation. For example, the asymmetric porous membrane may be used in the synthesis of a variety of oligomers, such as polypeptides, polysaccharides, and oligonucleotides. The asymmetric porous membrane of the invention may also be used for preparing biomolecules (e.g., oligonucleotides, peptides, DNA, RNA, proteins). For example, oligonucleotides may be prepared using the asymmetric porous membrane of the invention. Alternatively, the initial protected nucleoside may be added to the asymmetric porous membrane which has been made to contain appropriate unbound plurality of particles that will retain the nucleoside within the pores of the membrane.

[048] In another embodiment, reagents and solvents may be added to the asymmetric porous membrane of the present invention to consecutively remove and add sugar protecting groups to generate specific chemical moieties to provide a stepwise addition to the growing oligonucleotide chain. The steps for preparing oligonucleotides, e.g., deblocking, activating/coupling, oxidating, capping, are known in the art and may be followed to produce oligonucleotides in the asymmetric porous membrane of the invention. Once the oligonucleotides are formed, they may be removed from the asymmetric porous membrane using known reagents.

[049] In another embodiment, cell lines (including hybridomas) can be cultured in the asymmetric porous membrane of the invention, including, for example, cell lines available from the ATCC and the ECACC. The cell cultures can be grown from normal, embryonic and malignant tissues. For adherent cells, the plurality of unbound particles in the asymmetric porous membrane may have a suitable surface on which the cells may adhere. For growing adherent cells, the asymmetric porous membrane and the plurality of unbound particles may preferably comprise polystyrenes, polypropylene, polytetrafluoroethylenes, polyvinylchlorides, polycarbonates, and/or titanium. [050] In another embodiment the asymmetric porous membrane, having a plurality of unbound particles located within the membrane pores of the invention may be used for running assays. Assays known in the art involve complementary binding pairs including, for example, enzyme-linked immunosorbent assays (ELISA), sandwich assays, competitive assays, latex agglutination assays, radioimmunoassays (RIA), fluorescent immunoassays (FIA), and the like. Quantitative and/or qualitative assays may then be performed to further study the eluted analytes. By choosing the appropriate particles, the invention may also be used to study DNA-protein interactions, protein-protein interactions, and many other interactions between biomolecules and other molecules.

[051] The present invention can be used with any sample preparation methods including, but not limited to, chromatography; high pressure liquid chromatography (HPLC); electrophoresis; gel filtration; sample centrifugation; on-line sample preparation; diagnostic kits testing; diagnostic testing; transport of chemicals; transport of biomolecules; high throughput screening; affinity binding assays; purification of a liquid sample; size-based separation of the components of the fluid sample; physical properties based separation of the components of the fluid sample; chemical properties based separation of the components of the fluid sample; biological properties based separation of the components of the fluid sample; electrostatic properties based separation of the components of the fluid sample; and, combinations thereof. Also, the porous membrane of the present invention can be part of a larger device. Kits

[052] The invention also provides kits which may be used to isolate analytes of interest from a liquid sample. The kit may comprise, for example, one or more filtration devices according to the instant invention and one or more containers. The kit may contain one or more controls or sample analytes of interest and may optionally include various buffers useful in the methods of the invention. As an example the kit may include a lysis buffer suitable for lysising viral particles or cells. Wash buffers for eliminating reagents or non- specifically retained or bound material may optionally be included in the kit. Other optional kit reagents include an elution buffer for eluting a bound target nucleic acid from a membrane. Each of the buffers may be provided in a separate container as a solution. Alternatively the buffers may be provided in dry form or as a powder and may be made up as a solution according to the users desired application. In this case the buffers may be provided in packets. The kit may provide a power source in instances where the device is automated as well as a means of providing an external force such as a vacuum pump. The kit may also include instructions for using the device and/or loading the asymmetric membrane with the unbound particles and/or for making up reagents suitable for use with the device and methods according to the instant invention. Optional software for recording and analyzing data obtained while practicing the methods of the invention or while using the device of the invention may also be included. [053] The term "kit" includes, for example, each of the components combined in a single package, the components individually packaged and sold together, or the components presented together in a catalog (e.g., on the same page or double-page spread in the catalog).

[054] The invention will be further clarified by the following examples which are intended to be exemplary of the invention.

Examples

Example 1 :

Isolation and purification of rRNA from Pseudomonas aeruginosa

[055] The following solutions were formulated:

[056] Solution 1 (8 L of 0.22 μm filtered MiIIiQ water; 800 μL of 0.2 μm gold nanospheres, 7.OxIO⁸ particles/mL, Corpuscular, lot # 5597; 335 μL of 0.25 μm silica nanospheres, 5% solids, Corpuscular, lot # MO 19; 575 μL of 0.3 μm titania nanospheres, 25 mg/mL, Corpuscular (no lot number); 128 μL of 0.5 μm silica nanospheres, 5% solids, Corpuscular, lot # M02).

[057] Solution 2 (8 L of 0.22 μm filtered MiIIiQ water; 480 μL of 0.25 μm silica nanospheres, 5% solids, Corpuscular, lot # M019;240 μL of 0.5 μm silica nanospheres, 5% solids, Corpuscular, lot # M02).

[058] As depicted in Fig. 1, particle size distributions were measured using a Liqualiz S02 from Particle Measuring Systems

[059] Four arrangements of two 47 mm membrane disk holders for holding membranes were connected in series with a pressure transducer in between the two were setup. Two arrangements, designated Sartopore 2, had Sartopore 0.45 (Sartorius, lot# 311053) feeding into Sartopore 0.2 (Sartorius, lot# 311053). The other two arrangements, designated SHC, had HEPP (0.5 micron, Millipore Corporation, Billerica, MA, lot# KT03075TE) feeding into GEPP 0.2 micron, Millipore Corporation, Billerica, MA, lot# 100703TE134). Each of the solutions described above were fed into 1 Sartopore 0.45 over Sartopore 0.2 (Sartopore 2) arrangement or 1 HEPP over GEPP arrangement.

[060] The solution were pressurized to 10 psi. The volume filtered was measured by collecting filtrates in buckets on load cells. Intermediate pressures were recorded to determine which membrane layer in each train maintained highest flow while becoming retained with particles.

[061] As depicted in Fig. 2, both mixtures (Millipore Corporation, Billerica, MA) SHC filtered more volume than Sartorius Sartopore 2. With Solution 1, SHC had 76% flux loss, and Sartopore 2 had 80% flux loss. With Solution 2, SHC had 61% flux loss, and Sartopore 2 had 64% flux loss. Therefore, all the membranes trapped some particles, resulting in the flux decline, but all had capacity for more particles, especially those challenged with the second (more dilute) particle mixture. Particle concentrations could easily be adjusted or "tuned" to provide higher or lower degrees of particle loading within the membrane pores.

[062] Intermediate pressures gave an indication of which of the two layers in each train became more heavily loaded by particles. As depicted in Fig. 3, for Millipore Corporation, Billerica, MA, SHC membranes, the pressure was steady or rising over of the course of the filtration, indicating that the majority of the pressure drop was over the downstream layer and that GEPP was becoming more particle loaded than HEPP. On the other hand, pressures between the Sartorius Sartopore 2 layers decreased during the run, indicating that most of the pressure drop is over the upstream layer and that Sartopore 0.45 became particle loaded more readily than Sartopore 0.2.

[063] As depicted in Fig. 4, scanning electron micrograph for the (Millipore Corporation, Billerica, MA) HEPP membrane loaded with 0.25 micron fluorescent latex beads in a similar fashion as described above indicated a defined and discrete layer of unbound beads had been formed within the membrane.

[064] Pseudomonas aeruginosa, ATCC 9027, cells were grown in Trypticase Soy Broth at 35⁰C overnight. The overnight culture of Pseudomonas aeruginosa (~lX10⁸cells/ml) cells were serially diluted (1:10) in 0.1% peptone to an estimated 1000 colony forming units per one milliliter (cfu/ml). To determine actual cfu/ml, one hundred microliters of the estimated dilution of lOOOcfu/ml were plated onto Trypticase Soy Agar (TSA) plates at 35⁰C overnight. After approximately 18 hrs, bacterial colonies were counted to obtain confirmed cfu/ml values. For each of the comparative experiments described here, a total of 5,700 cfus of Pseudomonase aeruginosa were used per experiment.

[065] Silica (0.5 micron) beads were loaded into asymmetric exiting membranes, HEPP (0.45 micron) and GEPP (0.2 micron) Sartopore 0.45 (0.45 micron) and Sartopore 0.2 (0.2 micron), which were then inserted into a 25mm closed sealed vented Millipore device, (Millipore Corporation, Billerica, MA), as depicted in Fig. 5.

[066] With a 10ml syringe attached to the 25mm device, 5mls of 0.1% peptone solution (Millipore Corporation, Billerica, MA) was filtered through membrane in the device to prewet the membrane.

[067] Next, one milliliter of diluted Pseudomonas aeruginosa cell suspension was filtered through membrane in the device. Bacterial cells were captured on the membrane. Air was pushed air through membranes to remove remaining liquid in the device. One ml of lysis solution (chaotropic salts; RLT buffer from Qiagen RNeasy kit, Qiagen, Valencia, CA) was slowly filtered through the membrane in the device to lyse cells and release nucleic acid from cell. One ml of wash solution (RWl buffer from Qiagen RNeasy kit, Qiagen, Valencia CA) was filtered through the device and membrane to wash away other cellular components. One ml of another wash solution (RPE buffer from Qiagne RNeasy kit, Qiagen Valencai, CA) was filtered through the device and membrane to wash away salts. Air was pushed air through the device several times to remove remaining liquid. Five hundred microliters of water was used to elute purified nucleic acid from the beads retained in the membrane. The eluted 500 microliters containing the nucleic acid was concentrated by drying to a pellet overnight in a speed- vac (ThermoFisher DNA 120 SpeedVac Concentrator). The dried pellet was resuspended in 50 microliters of water. [068] Two controls were also used for the present evaluation. The first control comprised a closed sealed doomed Millipore 25mm device with two membranes: the Pseudomonas aeruginosa retentive membrane 0.5 micron HEPP (Millipore Corporation, Billerica, MA) stacked over a fiber glass filter (APFF, 0.7micron, Millipore Corporation, Billerica, MA) that serves as a nucleic acid affinity membrane. This device was treated in the same manner as the bead loaded membrane devices described above. The second control consisted of ImI of diluted Pseudomonas aeruginosa cell suspension (5,700 cfu) processed for nucleic acid sample prep as described in the Qiagen RNeasy kit, (Qiagen, Valencia, CA). For this second control the elution volume was 50 microliters.

[069] To 50 microliters of purified Pseudomonas aeruginosa nucleic acid derived from the various sample preps was added ImI of GenProbe PAE Lysis Solution and transcription mediated amplification was performed according to MILLIPROBE System for Pseudomonas aeruginosa (Millipore Corporation, Billerica, MA).

[070] As depicted in Fig. 6, the data demonstrates that the signal emergence and detection above noise values obtained from the bead loaded membrane capture and nucleic acid purification come up earlier, indicating greater amount of nucleic acid than the Qiagen devices and also Millipore devices with retentive HEPP membrane stacked over nucleic acid affinity fiberglass APFF filter. The use of silica beads loaded into asymmetric Millipore or Sartorius membranes offers an improvement over the stacked membrane HEPP over APFF configurations or the Qiagen RNeasy kit (Qiagen, Vaslencia, CA) RNA purification approach for the nucleic acid based detection of contaminant bacteria in a sample.

[071] Example 2 : Isolation and purification of rRNA from Pseudomonas aeruginosa

[072] The following solution was formulated : Solution 1 (2 L of 0.22 μm filtered MiIIiQ water; 1.2mL of 1 μm silica nanospheres, 5% solution by weight, Sigma # 56798

[073] A 192 mm membrane disk holder for holding membranes was connected in series with a pressure transducer. A 192mm HEPP (0.5 micron, Millipore Corporation, Billerica, MA) was placed in the filter holder. The solutions described above was fed into filter holder with the filter. The solution were pressurized to 10 psi. The 2L volume filtered and beads were embedded into the HEPP membrane.

[074] Silica (0.5 micron) beads were loaded into asymmetric exiting membranes, HEPP (0.45 micron) were then inserted into a 25mm closed sealed vented Millipore device, (Millipore Corporation, Billerica, MA), as depicted in Fig. 5.

[075] Pseudomonas aeruginosa, ATCC 9027, cells were grown in Trypticase Soy Broth at 35⁰C overnight. The overnight culture of Pseudomonas aeruginosa (3.4X10 cells/ml) cells were serially diluted (1:10) in 0.1% peptone to an estimated 1000 colony forming units per one milliliter (CFU/ml). To determine actual CFU/ml, one hundred microliters of the estimated dilution of 100 and lOOOCFU/ml were plated onto Trypticase Soy Agar (TSA) plates at 35⁰C overnight. After approximately 18 hrs, bacterial colonies were counted to obtain confirmed CFU/ml values. For each of the comparative experiments described here, a total of 340 CFUs of Pseudomonas aeruginosa were used per experiment.

[076] With a 10ml syringe attached to the 25mm device, lOmls of 0.1% peptone solution (Millipore Corporation, Billerica, MA) was filtered through membrane in the device to prewet the membrane.

[077] Next, 10ml of diluted Pseudomonas aeruginosa cell suspension was filtered through membrane in the device. Bacterial cells were captured on the membrane. Air was pushed air through membranes to remove remaining liquid in the device. One ml of lysis solution (chaotropic salts; a 1 : 1 mixture of 100% Ethanol and RLT buffer from Qiagen RNeasy kit, Qiagen, Valencia, CA) was slowly filtered through the membrane in the device to lyse cells and release nucleic acid from cell. One ml of wash solution (RWl buffer from Qiagen RNeasy kit, Qiagen, Valencia CA) was filtered through the device and membrane to wash away other cellular components. One ml of another wash solution (RPE buffer from Qiagne RNeasy kit, Qiagen Valencia, CA) was filtered through the device and membrane to wash away salts. Air was pushed air through the device several times to remove remaining liquid. One milliliter of nuclease-free water was used to elute purified nucleic acid from the beads retained in the membrane. The eluted ImI containing the nucleic acid was concentrated by drying to a pellet overnight in a speed-vac (ThermoFisher DNA 120 SpeedVac Concentrator). The dried pellet was resuspended in 50 microliters of water.

[078] Two controls were also used for the present evaluation. The first control comprised a closed sealed doomed Millipore 25mm device with two membranes (stacked): the Pseudomonas aeruginosa retentive membrane 0.5 micron HEPP (Millipore Corporation, Billerica, MA) stacked over a Mixed esters of cellulose(MCE, 1.25micron, Millipore Corporation, Billerica, MA) that serves as a nucleic acid affinity membrane. This device was treated in the same manner as the bead loaded membrane devices described above. The second control consisted of ImI of diluted Pseudomonas aeruginosa cell suspension (340 CFU) processed for nucleic acid sample prep as described in the Qiagen RNeasy kit, (Qiagen, Valencia, CA). For this second control the elution volume was 50 microliters.

[079] The following 23 S Pseudomonas aeruginosa specific primers were purchased for Quantitative Reverse Transcriptase Polymerase Chain Reaction(qRT-PCR) assay:

PAF 1-23 S, forward primer, AAT AAA TCA TAA GCA GGC CTA ACA CAT GCA AGT( Applied Biosystems, Inc., Foster City, CA) PARl -23 S, reverse primer, AAT AAA TCA TAA TCC GCC GCT GAA TCC A (Applied Biosystems, Inc., Foster City, CA)

PAP1-23S, probe 6FAM - ATG AAG GGA GCT TGC T MGBNFQ (Applied Biosystems, Inc., Foster City, CA). A one-step RT-PCR master mix kit (ABI) was used. To a final volume of 25 microliters, forward primer (30OnM) was mixed with reverse primer (30OnM), probe (25OnM), 2X RT-PCR mix, reverse transcriptase, and 5 microliters of eluant RNA from samples. In an ABI 7000 cycler (Applied Biosystems, Inc., Foster City, CA), the following cycling program was executed: 1. 50°C, 30:00 minutes

2. 95°C, 10:00 minutes

3. 95°C, 0: 15 minutes, 40 cycles

4. 60°C, 1 :00 minutes

[080] As depicted in Fig. 7, the data demonstrates that the signal emergence and detection above noise values obtained from the bead loaded membrane capture and nucleic acid purification come up earlier than the controls with purification in a tube by Qiagen methodology and the device with 2 layers of membranes without beads(stacked), indicating greater amount of nucleic acid than the Qiagen devices and also Millipore devices with retentive HEPP membrane stacked over nucleic acid affinity MCE filter. The use of silica beads loaded into asymmetric Millipore membrane offers an improvement over the stacked membrane HEPP over MCE configurations or the Qiagen RNeasy kit (Qiagen, Valencia, CA) RNA purification approach for the nucleic acid based detection of contaminant bacteria in a sample. Example 3 : Isolation and purification of rRNA from Pseudomonas aeruginosa in samples containing Chinese Hamster Ovary (CHO) cells. [081] The bead-embedded membrane and device were made the same way as described in example 2 above.

[082] Pseudomonas aeruginosa, ATCC 9027, cells were grown in Trypticase Soy Broth at 35⁰C overnight. The overnight culture of Pseudomonas aeruginosa (7.7X10⁸cells/ml) cells were serially diluted (1:10) in 0.1% peptone to an estimated 1000 colony forming units per one milliliter (CFU/ml). To determine actual CFU/ml, one hundred microliters of the estimated dilution of 100 and lOOOCFU/ml were plated onto Trypticase Soy Agar (TSA) plates at 35⁰C overnight. After approximately 18 hrs, bacterial colonies were counted to obtain confirmed CFU/ml values. For each of the comparative experiments described here, a total of 770 CFUs of Pseudomonas aeruginosa were used per experiment.

[083] With a 10ml syringe attached to the 25mm device, lOmls of 0.1% peptone solution (Millipore Corporation, Billerica, MA) was filtered through membrane in the device to prewet the membrane.

[084] Next, 10ml of diluted Pseudomonas aeruginosa (770 CFU) in a CHO cell suspension containing 10⁵, 10⁶ or 10⁷ cells was filtered through membrane in the device. Bacterial and CHO cells were captured on the membrane. Ten milliliters of CHO cell suspension containing 10⁷ cells was passed through a device as a control. Another control was passing 10ml of only Pseudomonas aeruginosa suspension (770 CFU) through the device. Air was pushed air through membranes to remove remaining liquid in the device. One ml of lysis solution (chaotropic salts; a 1:1 mixture of 100% Ethanol and RLT buffer from Qiagen RNeasy kit, Qiagen, Valencia, CA) was slowly filtered through the membrane in the device to lyse cells and release nucleic acid from cell. One ml of wash solution (RWl buffer from Qiagen RNeasy kit, Qiagen, Valencia CA) was filtered through the device and membrane to wash away other cellular components. One ml of another wash solution (RPE buffer from Qiagen RNeasy kit, Qiagen Valencia. CA) was filtered through the device and membrane to wash away salts. Air was pushed air through the device several times to remove remaining liquid. One milliliter of nuclease- free water was used to elute purified nucleic acid from the beads retained in the membrane. The eluted 1ml containing the nucleic acid was concentrated by drying to a pellet overnight in a speed-vac (ThermoFisher DNA 120 SpeedVac Concentrator). The dried pellet was resuspended in 50 microliters of water.

[085] Tube controls were also used for the present evaluation. The first type of control was 10ml of CHO and Pseudomonas cell suspension control (10⁵ or 10⁶or 10⁷ CHO cells with 770 CFU Pseudomonas). The other type consisted of 10ml of diluted Pseudomonas aeruginosa cell suspension (770 CFU) and the third type was 10⁷ CHO cells. These samples were processed by centrifuging in a table top centrifuge at 4000rpm for 10 min. The supernatant was discarded and samples were processed for nucleic acid sample prep as described in the Qiagen RNeasy kit, (Qiagen, Valencia, CA). These control samples were eluted in 50 microliters of nuclease-free water. [086] The following 23 S Pseudomonas aeruginosa specific primers were purchased for Quantitative Reverse Transcriptase Polymerase Chain Reaction(qRT-PCR) assay:

PAF1-23S, forward primer, AAT AAA TCA TAA GCA GGC CTA ACA CAT GCA AGT(Applied Biosystems, Inc., Foster City, CA) PARl -23S, reverse primer, AAT AAA TCA TAA TCC GCC GCT GAA TCC A (Applied Biosystems, Inc., Foster City, CA)

PAP 1-23 S, probe 6FAM - ATG AAG GGA GCT TGC T MGBNFQ (Applied Biosystems, Inc., Foster City, CA).

A one-step RT-PCR master mix kit (ABI) was used. To a final volume of 25 microliters, forward primer (30OnM) was mixed with reverse primer (30OnM), probe (25OnM), 2X RT-PCR mix, reverse transcriptase, and 5 microliters of eluant RNA from samples. In an ABI 7000 cycler (Applied Biosystems, Inc., Foster City, CA), the following cycling program was executed: 1. 5O⁰C, 30:00 minutes

2. 95°C, 10:00 minutes

3. 95°C, 0:15 minutes, 40 cycles

4. 60°C, 1 :00 minutes

[087] As depicted in Fig. 8, the data demonstrates that the signal emergence and detection above noise values obtained from the bead loaded membrane capture and nucleic acid purification come up earlier than the controls with purification in a tube by Qiagen methodology. This indicates greater recovery of nucleic acid. The performance of the bead-embedded membrane is better than tube controls even in the presence of up to 10⁷ CHO cells. Increasing the concentration of CHO cells in the samples from 10⁵ to 10⁷ does not effect the emergence time indicating that the binding capacity of the bead-embedded membranes has not exceeded.

[088] The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in applications claiming priority from this or a related application. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.

Claims

WHAT IS CLAIMED IS:

1. A porous asymmetric membrane for isolating at least one substance of interest from a fluid sample comprising: a porous asymmetric membrane having a first surface and a second surface, each surface having a plurality of pores, wherein the pores on the first surface have a larger nominal pore size than the pores on the second surface, and a plurality of size-exclusion particles retained within the membrane, wherein the plurality of particles are sized to enter the membrane through the larger pores on the first surface, but not exit the membrane through the smaller pores on the second surface, wherein the plurality of size-exclusion particles are not bonded to the membrane or to each other.

2. The membrane of claim 1 , wherein the membrane is composed of a material selected from the group consisting of a hydrophilic material, hydrophobic material, oleophobic material, oleophilic material, and combinations thereof.

3. The membrane of claim 1, wherein the plurality of size-exclusion particles are composed of a material selected from the group consisting of a hydrophilic material, hydrophobic material, porous material, oleophobic material, oleophilic material, and combinations thereof.

4. The membrane of claim 1 , wherein the plurality of size-exclusion particles are composed of a material selected from the group consisting of agarose, silica, gold, titinia, latex, glass, controlled porous glass, metal, metal oxide, and combinations thereof.

5. The membrane of claim 1, wherein the size-exclusion particle is a spherical bead.

6. The membrane of claim 1, wherein the size-exclusion particle is a porous bead.

7. The membrane of claim 1, wherein the membrane is composed of a material selected from the group consisting of polypropylene, polysaccharide, polyethersulfone nitrocellulose, polyethylene, polypropylene, polytetrafluoroethylene, polysulfone, polyetherstlfone, cellulose acetate, polystyrene, polystyrene/acrylonitrile copolymer, PVDF, glass, metal, silica, and combinations thereof.

8. A filter device suitable for isolating nucleic acids from a liquid sample comprising: a porous asymmetric membrane having a first surface and a second surface, each surface having a plurality of pores, wherein the pores on the first surface have a larger nominal pore size than the pores on the second surface, and a plurality of size-exclusion particles retained within the membrane, wherein the plurality of size-exclusion particles are sized to enter the membrane through the larger pores on the first surface, but not exit the membrane through the smaller pores on the second surface, wherein the plurality of size-exclusion particles are not bonded to the membrane or to each other.

9. The device of claim 8, wherein the plurality of size-exclusion particles are composed of a material selected from the group consisting of agarose, silica, gold, titinia, latex, glass, controlled porous glass, metal, metal oxide, and combinations thereof.

10. The device of claim 8, wherein the size-exclusion particle is a spherical bead.

11. The device of claim 8, wherein the size-exclusion particle is a porous bead.

12. The device of claim 8, wherein the membrane is composed of a material selected from the group consisting of polypropylene, polysaccharide, polyethersulfone nitrocellulose, polyethylene, polypropylene, polytetrafluoroethylene, polysulfone, polyetherstlfone, cellulose acetate, polystyrene, polystyrene/acrylonitrile copolymer, PVDF, glass, metal, silica, and combinations thereof.

13. A method of isolating at least one substance of interest from a liquid sample comprising: a) a porous asymmetric membrane having a first surface and a second surface, each surface having a plurality of pores, wherein the pores on the first surface have a larger nominal pore size than the pores on the second surface, and a plurality of particles retained within the membrane, wherein the plurality of size-exclusion particles are sized to enter the membrane through the larger pores on the first surface, but not exit the membrane through the smaller pores on the second surface, and the plurality of size-exclusion particles are not bonded to the membrane or to each other; and b) contacting the liquid sample having at least one substance of interest with the porous membrane such that the liquid sample and at least one substance of interest contact the size-exclusion particles retained within the membrane pores, and the size-exclusion particles prevent the substance of interest from exiting the membrane, but permit the liquid sample to exit the membrane.

14. The method of claim 13, further comprising contacting the membrane with one or more buffers or wash reagents.

15. The method of claim 13, further comprising contacting the membrane with an elution buffer.

16. The method of claim 13, wherein the membrane is composed of a material selected from the group consisting of a hydrophilic material, hydrophobic material, oleophobic material, oleophilic material, and combinations thereof.

17. The method of claim 13, wherein the plurality of size-exclusion particles are composed of a material selected from the group consisting of a hydrophilic material, hydrophobic material, porous material, oleophobic material, oleophilic material, and combinations thereof.

18. The method of claim 13 , wherein the plurality of size-exclusion particles are composed of a material selected from the group consisting of agarose, silica, gold, titinia, latex, glass, metal, metal oxide, and combinations thereof.

19. The method of claim 13, wherein the size-exclusion particle is a bead.

20. The method of claim 19, wherein the size-exclusion particle is a porous bead.

21. The method of claim 13, wherein the size-exclusion particles have a shape selected from the group consisting of spherical, cubical, cylindrical, oval, irregular, and combinations thereof.

22. The method of claim 13, wherein the substance of interest is a nucleic acid.

23. The method of claim 22, wherein the nucleic acid is DNA or RNA.

24. The method of claim 13, wherein the porous membrane is composed of a material selected from the group consisting of polypropylene, polysaccharide, polyethersulfone nitrocellulose, polyethylene, polypropylene, polytetrafluoroethylene, polysulfone, polyetherstlfone, cellulose acetate, polystyrene, polystyrene/acrylonitrile copolymer, PVDF, glass, metal, silica, and combinations thereof.

25. The method of claim 13, wherein the liquid sample comprises at least two substances of interest.

26. The method of claim 25, wherein each substance of interest is different from the other substance of interest.

27. The method of claim 13, wherein the liquid sample is selected from the group consisting of a biofluid, biological sample, chemical sample, cellular sample, bacteria culture, viral culture, fungal culture, food sample, and combinations thereof.

28. The method of claim 13, wherein the at least one substance of interest is selected from the group consisting of a biomolecule, a nucleic acid, a protein, an organic small-molecule compound, a polymer, an antibody, a ligand, a receptor, a signal-generating molecule, a structural molecule, an ion, an antigen, a heavy metal, a bacteria, a virus, a fungus, a parasite, and combinations thereof.

29. The method of claim 13, further comprising contacting the liquid sample with an additional porous exiting membrane.