WO2004100887A2 - Surfaces d'extraction en phase solide tridimensionnelles - Google Patents

Surfaces d'extraction en phase solide tridimensionnelles Download PDF

Info

Publication number
WO2004100887A2
WO2004100887A2 PCT/US2004/014321 US2004014321W WO2004100887A2 WO 2004100887 A2 WO2004100887 A2 WO 2004100887A2 US 2004014321 W US2004014321 W US 2004014321W WO 2004100887 A2 WO2004100887 A2 WO 2004100887A2
Authority
WO
WIPO (PCT)
Prior art keywords
extraction
capillary
polymer
capillary channel
analyte
Prior art date
Application number
PCT/US2004/014321
Other languages
English (en)
Other versions
WO2004100887A3 (fr
Inventor
Douglas T. Gjerde
Christopher P. Hanna
Liem Nguyen
Leon Yengoyan
Original Assignee
Phynexus, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/434,713 external-priority patent/US20040126890A1/en
Priority claimed from US10/733,534 external-priority patent/US7879621B2/en
Priority claimed from US10/754,775 external-priority patent/US20040224329A1/en
Application filed by Phynexus, Inc. filed Critical Phynexus, Inc.
Publication of WO2004100887A2 publication Critical patent/WO2004100887A2/fr
Publication of WO2004100887A3 publication Critical patent/WO2004100887A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption

Definitions

  • This invention relates generally to capillary channels coated with three- dimensional solid phase extraction matrices, and the use of such capillary channels for the extraction of analytes from solution.
  • Analytes of particular interest include biomolecules such as polypeptides and polynucleotides.
  • Solid phase extraction is one of the primary tools for preparing protein samples prior to this sort of analysis.
  • a particularly powerful form of this technology described in U.S. Patent Application No. 10/434,713 (filed May 8, 2003), employs solid-phase extraction capillaries to purify and enrich samples of proteins and other analytes.
  • the subject invention provides solid-phase extraction capillaries having three-dimensional solid phases extraction matrices. These extraction capillaries find utility in the methods described in the 10/434,713 application, as well as in other application described herein. It has been found that these three-dimensional matrices provide powerful advantages relative to a corresponding two-dimensional extraction surface.
  • the subject invention provides an extraction capillary channel, wherein a substantial portion of the channel is coated with a 3 -dimensional solid phase extraction surface that binds an analyte.
  • the analyte binding capacity of the 3 -dimensional solid phase extraction surface is greater than could be achieved by a corresponding 2- dimensional solid phase extraction surface.
  • the solid-phase extraction surface comprises a polymer, which can be attached to the surface of the capillary channel by one or more covalent bonds , one or more non-covalent interaction, or a combination of covalent and non-covalent interactions.
  • An example of non-covalent interaction is an electrostatic interaction.
  • the polymer can be attached to the capillary channel by electrostatic interaction to a second polymer, wherein the second polymer is attached to the capillary channel.
  • Polymers of the invention can be cross-linked or non-cross- linked, can be in the form of a bead, e.g., a latex bead.
  • examples of polymers include polysaccharides, such as dextran.
  • the 3-D extraction surface is accessible to penetration by relatively large biomolecules, e.g., biomolecules of a mass of about 2000 Da.
  • an extraction agent is attached to the solid-phase extraction surface.
  • extraction agent include an immobilized metal, a protein, or an antibody, e.g., Ni-NTA, Protein A or Protein G.
  • the extraction agent can be covalently attached to the polymer.
  • the analyte is a biomolecule, such as a protein.
  • the capillary channel is fused silica capillary tubing.
  • the invention further provides a method for preparing an extraction capillary channel having a 3 -dimensional extraction surface, comprising the steps of: providing a capillary channel bearing a first attachment group; and attaching an extraction polymer to said capillary channel by an interaction between said first attachment group and a second attachment group on said extraction polymer, wherein said extraction polymer bears an affinity group having an affinity for an analyte.
  • said extraction polymer is attached to said capillary channel by formation of a covalent bond between said first and second attachment groups, e.g., by formation of an amide bond, an isourea bond or thioether bond.
  • said extraction polymer is attached to said capillary channel by an electrostatic interaction between said first and second attachment groups.
  • the invention further provides a method for molecular open tubular solid phase extraction, the method comprising the steps of: adsorbing analyte molecules in a sample solution to the extraction surface of a fused silica extraction capillary tubing of claim 1, the capillary tubing having a total capillary volume; and desorbing a substantial portion of the analyte molecules from the extraction surface with a desorbent liquid passed through the capillary channel.
  • the analyte molecules is desorbed with a Tube Enrichment Factor of at least 1.
  • the direction of passage of the desorption solution through the column reversed during the desorption step.
  • a wash solution is passed through the capillary channel between steps (a) and (b).
  • the wash solution is any liquid present in the capillary channel is substantially displaced from the capillary channel by a gas before step (b).
  • the direction of passage of the gas through the column is reversed during displacement of the liquid.
  • the extraction surface has an affinity binding agent bound thereto, and the affinity binding agents is: a chelated metal having a binding affinity for a biomolecule analyte; a protein having a binding affinity for a protein analyte; an organic molecule or group having a binding affinity for a protein analyte; a sugar having a binding affinity for a protein analyte; a nucleic acid having a binding affinity for a protein analyte; a nucleic acid or a sequence of nucleic acids having a binding affinity for a nucleic acid analyte; or a small molecule binding agent having a binding affinity for a small molecule analyte.
  • the analyte concentration is increased at least 100 times.
  • the analyte molecules are desorbed with a Tube Enrichment Factor from within a range from 1 to 400.
  • the subject invention provides, inter alia, capillary channels coated with three-dimensional solid phase extraction matrices, and methods and reagents for using such capillary channels for the extraction of analytes from solution.
  • Analytes of particular interest include biomolecules, such as polypeptides and polynucleotides.
  • the subject invention provides capillary channel devices useful for the purification and enrichment of analytes, particularly biomolecules, as well as methods and reagents for their use and production, and kits that include the same.
  • capillary channel devices useful for the purification and enrichment of analytes, particularly biomolecules, as well as methods and reagents for their use and production, and kits that include the same.
  • the subject devices and reagents are described first in greater detail, followed by a review of representative methods in which they find use and a review of the kits that include the subject compounds.
  • the capillary channel can be single or bundled tubing, or it can be one or more channels in a block or chip.
  • the channels can be straight. They can be non-linear shapes in the form of coils or other curved shapes which will promote agitated flow through the channels.
  • the channels can be straight wall, undulating, knitted, circular, knotted, coiled, a combination of coiling and reverse coiling or filled with large bead to promote transport to the tube surface, or a monolithic structure. Coiled tubes can be cut to length for a specific application single sample use, eliminating cross- contamination.
  • the capillary channel may be composed of a number of different materials. These include fused silica, polypropylene, polymethylmethacrylate, polystyrene, nylon, (nickel) metal capillary tubing, and carbon nanotubes. Polymeric tubes are available as straight tubing or multihole tubing (Paradigm Optics, Inc., Pullman, WA). Nickel tubing is available from Valco Instrument, Inc., Houston, TX. Formation of carbon nanotubes has been described in a number of publications including Kenichiro Koga, et al., Nature, 412:802 (2001).
  • the capillary channel is an element of a chip or disk, for example of the type commercially available from vendors such as Tecan Systems, Inc. (San Jose, CA) and Gyros, Inc. (Monmouth Junction, NJ). Extraction via a disk- based preconcentrator is described, for example, by Tomlinson et al., J. Chromatography A, 1996, 744:3-15.
  • the capillary channel is a capillary tubing.
  • Fused silica capillary tubing i.e., fused silica capillaries
  • capillaries comprising synthetic fused silica
  • the fused silica provides numerous silanol groups which serve as useful attachment points for the extraction surface chemistries described herein.
  • Fused silica capillaries that are suitable for the purposes of this invention include those produced by Polymicro Technologies, LLC of Phoenix, AZ and SGE Inc. of Ringwood, Australia.
  • coiling of capillary tubing is not necessary, it can be advantageous in certain embodiments of the invention. Coiling results in a more tortuous flow path, which can improve the efficiency of the extraction process. Another benefit of coiling is that it allows for the production of a relatively compact extraction device that would not otherwise be feasible, due to the length of the extraction capillary tubing.
  • capillary tubing having a total outside diameter in the range of 90 to 3500, 90 to 1500, 90 to 850, 150 to 850 or 238 to 435 microns.
  • Capillary channel internal diameters are typically in the range of about 2 to 3000 microns, about 2 to 1000 microns, about 10 to 700 microns, about 25 to 400 microns, or about 100 to 200 microns.
  • the outer surface of the capillary tubing is coated with a flexible coating material, typically a polymer or resin.
  • Preferred coating materials include polyimide, silicone, polyacrylate, aluminum or fluoropolymer, especially semiconductor grade polyimide.
  • all diameter refers to the total diameter of a capillary, including coating if any.
  • outer diameter refers to outer diameter of capillary minus any coating, e.g., the outer diameter of a fused silica capillary tubing.
  • the length of the capillary channel can vary greatly depending upon the desired capacity, contemplated sample size and desired enrichment. Because fused silica tubing can be coiled, relatively compact extraction devices can be constructed that include 1 meter or more of capillary tubing. Alternatively, in some embodiments very short lengths of capillary can be employed, down to 1 mm in length or even shorter.
  • fused silica capillary tubing particularly where the tubing is coiled, it is important to exert all possible care to coil avoid the introduction of nicks or other breakage that can lead to breakdown of extraction function. For instance, it is usually better not to introduce any twisting of the tubing during the coiling process, as this twisting will itself introduce stress into the tubing beyond that introduced by the coiling.
  • Other precautions that will reduce breakage include minimizing nicks on inner and outer surface of capillary, the use of a thicker coating, preferably a polyimide coating, and minimizing exposure of the capillary surface to base.
  • the capillary channel can be a single tube or be formed as a block of multiple tubes or a multichannel block (multicapillary format).
  • the subject invention provides extraction capillaries having channel surfaces coated with a three-dimensional solid phase extraction surface that binds an analyte of interest.
  • the surface bind tightly and specifically to a biomolecule (or class of biomolecules) of interest, especially relatively large biological macromolecules (e.g., polynucleotides, polypeptides and polysaccharides having a MW of greater than about 1000 Da, including, for example, in the range of 1000 to 10,000,000 Da or more, or more typically in the range of 5000 to 500,000 Da).
  • relatively large biological macromolecules e.g., polynucleotides, polypeptides and polysaccharides having a MW of greater than about 1000 Da, including, for example, in the range of 1000 to 10,000,000 Da or more, or more typically in the range of 5000 to 500,000 Da.
  • the three-dimensional solid phase extraction surface forms a biocompatible porous surface.
  • the porosity of the surface allows for the penetration of biomolecules such as proteins into the surface, and interaction of the biomolecules with affinity groups present in the surface.
  • the extraction surface is based upon a fluidic, hydrogel-type environment. Such an environment is particularly suited for the extraction and purification of proteins, since it mimics the properties of bulk solution and can help stabilize the protein in its active form, i.e, the conditions are non-denaturing.
  • suitable surface materials for providing the 3- D structure include porous gold, sol gel materials, polymer brushes and dextran surfaces.
  • the three-dimensional surface layer typically has a thickness of from a few angstroms to thousands of angstroms. In some embodiments the surface is between 5 to 10,000 angstroms thick, e.g., 5 to 1000 angstroms. The thickness of the surface can be adjusted as desired based on factors including the dimensions of the capillary channel, the nature of the analyte or analytes of interest, the nature of an affinity group or extraction reagent present in the surface, the desired binding capacity, etc.
  • the 3-D solid phase extraction surface is a hydrogel formed from a polymer, e.g., a polysaccharide or a swellable organic polymer.
  • the polymer should be compatible with the analyte of interest and with a minimal tendency towards nonspecific interactions.
  • suitable polysaccharides include agarose, sepharose, dextran, carrageenan, alginic acid, starch, cellulose, or derivatives of these such as, e.g., carboxymethyl derivatives.
  • polysaccharides of the dextran type which are non-crystalline in character, in contrast to e.g., cellulose, are very suitable for use in the subject invention.
  • water-swellable organic polymer examples include polyvinyl alcohol, polyacrylic acid, acrylate, polyacrylamide, polyethylene glycol, functionalized styrenes, such as amino styrene, and polyamino acids.
  • exemplary polyamino acids include both poly-D-amino acids and poly-L-amino acids, such as polylysine, polyglutamic acid, polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid, co-polymers of lysine with alanine, tyrosine, phenylalanine, serine, tryptophan, and/or proline.
  • Desirable functional attributes of the 3-D surface would include that it should have minimal tendency to interact non-specifically with biomolecules, it should be chemically resistant to the media employed, it should be compatible with proteins and other biomolecules and should not interact with any molecules other than those desired. Furthermore, it should be capable of providing for covalent binding of such a large number of affinity groups as is required for a general applicability of this technique to a variety of analytical problems .
  • dextran, dextran-derivatives and dextran-like materials are particularly suited for use as the backbone molecules in the subject 3-D extraction surfaces.
  • the resulting hydrogel layer is highly flexible, largely non-cross linked and typically extends 100-200 nm from coupling surface under physiological buffer conditions.
  • Dextran can be derivatized, e.g., via carboxymethylation or vinylsulfonation, to incorporate additional reactive handles for activation and covalent attachment of affinity groups.
  • Non-limiting examples of coupling chemistries that can be used with these and related backbone molecules include thiol, amine, aldehyde and streptavidin. See, e.g., F.
  • the polymer used to form the extraction surface can be cross-linked, e.g., cross-linked dextran.
  • the degree of cross-linking can be varied to adjust the porosity and hence accessibility of the extraction surface, particularly to larger molecules such as biological macromolecules.
  • extractions surface backbones that have no or low degree of cross-linking can be used, resulting in greater accessibility of the extraction surface to analyte, particularly high MW biomolecules.
  • extraction surfaces comprising a polymer backbone that is, for example, less than 0.1% crosslinked, about 0.1 to 0.5% crosslinked, about 0.5 to 1% crosslinked, about 1 to 2% crosslinked, about 2 to 3 % crosslinked, about 3 -5% crosslinked, about 5-7% crosslinked, about 7-10% crosslinked, or even greater than 10% crosslinked can be used.
  • the acceptable degree of crosslinking varies depending upon the nature of the polymer backbone (e.g., swellability of the polymer) and the nature of the analyte (e.g., size and structure of a biomolecule, the molecules hydration volume). Because crosslinking is not required, a variety of backbone chemistries may be employed that would not be appropriate for use in a conventional chromatography bead.
  • the interior of the 3-D extraction surface is accessible to analyte, such that analyte molecules are able to penetrate and adsorb to the surface in 3-dimensions.
  • some embodiments are accessible to relatively large biological macromolecules, e.g., polynucleotides, polypeptides and polysaccharides having a MW of greater than about 1000 Da, including, for example, in the range of 1000 to 10,000,000 Da or more, or more typically in the range of 5000 to 500,000 Da (e.g., biomolecules of 1000 Da, 2000 Da, 5000 Da, 10,000 Da, 50,0000 Da, 100,000 Da, 500,000 Da, 1,000,000 Da, etc.).
  • biomolecule complexes e.g., complexes comprising two or more proteins bound to one another by covalent or non- covalent interactions, a protein bound to a polynucleotide, etc. It is known that many clinically relevant biomolecules function as part of such complexes, which can in some cases be quite large. Thus, one advantage of the subject invention is that it facilitates the study of such complexes.
  • the invention provides methods for purifying and characterizing such complexes.
  • a complex of interest can be adsorbed to the extraction surface, and then components of the complex selectively desorbed and collected, and optionally subjected to further characterization, e.g, by MS, NMR or SPR.
  • further characterization e.g, by MS, NMR or SPR.
  • Properties of a 3-D extraction surface of the invention can be modified by varying the MW (or MW range) of the polymer backbone.
  • Polymers in the MW range from about 500 to several million can be used, preferably at least 1000, for example in the range of 10,000 to 500,000. In some cases an increase in MW can result in improved performance, e.g., higher capacity.
  • dextran is available in a variety of MW ranges, allowing for modification of physical characteristics of the resulting hydrogel.
  • Properties of the hydrogel can also be modified by variation of functional groups, extent and nature of cross-linking, etc.
  • the 3-D surface In order to function as a solid phase extraction medium, the 3-D surface should have an affinity for an analyte of interest.
  • the affinity is strong and selective, resulting in a substantially single equilibrium absorption of the analyte under extraction conditions.
  • the affinity can be inherent to the surface itself, or more typically the result of attachment of an affinity group to the surface backbone.
  • affinity group refers to a chemical entity having an affinity for an analyte of interest, e.g spiral a biomolecule.
  • Types of affinity groups include ion exchangers, which can be strong or weak (e.g., acids, bases, quaternary amines).
  • Other types of affinity groups include polar or non-polar groups, e.g., hydrophobic or reverse phase groups.
  • the affinity group is often an extraction agent able to bind specifically to a biomolecule analyte of interest, e.g., a specific polypeptide or class of polypeptides, a specific polynucleotide or class of polynucleotides, a polysaccharide, a lipid, a metabolite or other small molecule.
  • a biomolecule analyte of interest e.g., a specific polypeptide or class of polypeptides, a specific polynucleotide or class of polynucleotides, a polysaccharide, a lipid, a metabolite or other small molecule.
  • an analyte and an affinity group can be specific or relatively non-specific, e.g., attraction based on electrostatic or hydrophobic interaction
  • an extraction agent is characterized by more specific interaction.
  • Extraction agents include various ligands such as metal chelators (and the corresponding immobilized metal ions), antibodies, proteins, polynucleotides, etc.
  • metal chelators and the corresponding immobilized metal ions
  • affinity groups can be used in a like manner with the 3-D extraction matrices of the subject invention.
  • Non-limiting examples of some particularly useful extraction agents include metal chelators used in immobilized metal affinity chromatography (e.g, metal-IDA, metal-NTA, metal-CMA), Protein A, Protein G, avidin or streptavidin (monomeric or multimeric), calmodulin, glutathione, maltose and antibodies having an affinity for an epitope tag.
  • the extraction surface contains a functional group or groups suitable for use in the attachment of an affinity group, e.g., an extraction agent. Representative examples of such groups include hydroxyl, carboxyl, amino, aldehyde, ketone, carbonyl, activated ester, epoxy and vinyl groups.
  • These functional groups are also useful for attachment of the extraction surface to the surface of the capillary channel, or can be used for cross-linking the matrix itself.
  • two or more different functional groups are used, e.g., one for attachment of extraction agent or extraction agents, another for attachment of matrix to channel surface.
  • the same functional group can be used for attachment of the matrix to extraction agents and channel surface. If a desired functional group is not inherently present in the 3 -dimensional matrix backbone it can be introduced synthetically, e.g., the carboxymethylation of dextran to introduce carboxyl groups, as described in the appended examples.
  • an extraction matrix including a functional group in an activated form e.g., an activated carboxyl.
  • This activation facilitates the coupling of an extraction agent of interest to the matrix, e.g,. via formation of an amide bond.
  • an activated carboxyl group can take any of a number of forms, including but not limited to activated reactive esters, hydrazides, thiols or reactive disulfide-containing derivatives.
  • a reactive ester can be prepared in any of a number of ways known to one of skill in the art, including by reaction with a carbodiimide.
  • the activated functional group is a 2-aminoethanethiol derivative.
  • the activated functional group is a vinyl sulfone.
  • a hydrazide function can be created in dextran matrix for binding ligands containing aldehyde groups, for example antibodies in which the carbohydrate chain has been oxidized so that it then contains an aldehyde function.
  • the dextran matrix is initially modified with, e.g., carboxymethyl groups, which are subsequently reacted to form hydrazide groups.
  • carboxyl groups in carboxymethyl- modified dextran are modified so as to give reactive ester functions, e.g., by treatment with an aqueous solution of N-hydroxysuccinimide and N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide hydrochloride.
  • Ligands containing amine groups such as, for example, proteins and peptides may then be coupled to the dextran matrix by covalent bonds.
  • the aforesaid reactive ester is utilized for reaction with a disulfide-containing compound such as for instance 2-(2- pyridinyldithio) ethanamine; in this manner a matrix is obtained which contains disulfide groups, and these can be employed for coupling thiol-containing ligands such as for example reduced F(ab) fragments of immunoglobulins.
  • a disulfide-containing compound such as for instance 2-(2- pyridinyldithio) ethanamine
  • the thiol modified surface formed can be used for coupling of a disulfide-containing ligand such as, for instance, N-succinimidyl 3-(2-pyridinyldithio) propionate (SPDP) modified proteins.
  • a disulfide-containing ligand such as, for instance, N-succinimidyl 3-(2-pyridinyldithio) propionate (SPDP) modified proteins.
  • the invention provides methods for preparing extraction capillary channels having 3 -dimensional extraction surfaces.
  • the extraction surface is prepared by attaching an extraction polymer (e.g., a polymer bearing an affinity group as described herein) to a capillary channel.
  • the attachment is accomplished by means of an interaction between complementary attachment groups on the polymer and channel.
  • complementary refers to the ability of the attachment groups to interact with one another in such a way as to result in attachment of the polymer to the channel. Examples of such interactions include electrostatic attraction (e.g., where the attachment groups are oppositely charged ions) and hydrophobic interactions (e.g., where the attachment groups are non-polar groups that are attracted to one another in a polar environment.
  • the interaction can be one that results in the formation of a covalent bond
  • the complementary attachment groups are functional groups capable of forming covalent bonds, e.g. , a carboxyl group and an amide group are complementary functional groups capable of reacting to form an amide bond, vinyl and thiol are complementary functional groups capable of reacting to form a thioether bond.
  • Other examples of complementary groups are cyanogen bromide and the amine group, which can react to form an isourea bond (Porath et al. (1973) J. Chromatograph. 86:53; and Kohn and Wilchek (1984) Appl Biochem. Biotechnol.
  • maleimide and thiol which can react to form a thioether bond
  • the maleimide reaction is particularly useful in certain embodiments of the invention for attaching a group to a polydextran matrix with minimal crosslinking of the matrix.
  • the maleimide group is relatively specific for the thiol group, and not prone to unintended reaction with the dextran matrix.
  • maleimide group as a linker is exemplified further in the examples, where preparation of a polymaleimide dextran is described.
  • This polymaleimide dextran can be a particularly low-crosslinked matrix, which can be more easily penetrated by some larger molecules, as described elsewhere herein.
  • the attachment of an extraction polymer to a capillary channel can be direct, but more typically is accomplished by one or more linker molecule that serves as intermediaries bridging the polymer and the surface of the extraction channel.
  • Attachments between polymer and linker, linker and channel surface, and/or linker to linker can be covalent or non-covalent.
  • the linker molecule can itself be a polymer, or not.
  • the linker molecule can be a polymer that interacts with the capillary channel and with the extraction polymer, bridging the two.
  • the capillary channel is silica, for example, surface of the channel is normally covered with silanol groups, resulting in a net negative charge to the surface.
  • a bridge molecule having a positive charge e.g., a polymer, such as a strong base anion exchanger
  • An extraction polymer having a negative charge can then be attached to the surface through the bridging molecule, in this case by electrostatic attraction to the positively charged bridging polymer.
  • this embodiment involves the successive stacking of layers of polymer having opposite charge on the capillary surface.
  • the number of layers can be one, two or more.
  • successive layers of oppositely charged polymers can be coated on the surface of the capillary channel, with the last applied (or top) layer constituting the extraction surface.
  • the extraction polymer and/or bridge polymers are beads. These beads can be held together by cross-linking (or not). Latex beads are used for this purpose in some of the Examples.
  • silica capillary When employing a silica capillary, it is often convenient to covalently couple the matrix to the capillary through free silanol groups on the channel surface. This is typically accomplished through a linking molecule bridging the silanol group and matrix backbone, e.g., polymer.
  • a linking molecule bridging the silanol group and matrix backbone e.g., polymer.
  • reactive thiol or amino groups can be attached via reaction with a thiosilane or aminosilane, respectively.
  • a carboxyl group can be introduced on the capillary surface by reaction of amino-functionalized capillary with an anhydride, e.g., succinic anhydride.
  • a three-dimensional matrix can be attached to a capillary surface through a self-assembled monolayer.
  • the capillary is metal, e.g., gold.
  • the attachment of a matrix to a metal surface through a self-assembled monolayer has been described elsewhere, see, for example U.S. Patent Nos. 5,242,828; 6,472,148; 6,197,515 and 5,620,850.
  • a 3-D polymer matrix can be attached through the SMIL (successive multiple ionic-polymer) approach as described by Katayama et al. (1998) Analytical Sciences 14:407-409.
  • An advantage of the 3-D extraction surfaces of the subject invention is their high surface area relative to a corresponding 2-D extraction surface (i.e., monolayer), which allows for improved analyte binding capacity. That is, the 3-D matrix allows for denser placement of affinity groups (e.g., extraction agents) per surface area of the capillary channel (or length of capillary channel), and/or for denser binding of analyte.
  • affinity groups e.g., extraction agents
  • the support coated capillaries prepared by Cai et al. using a colloidal silica solution do not exhibit the increased capacity of the preferred 3-D extraction matrices of the subject invention, since the silica coating is not swellable (i.e., does not take up water or solvent like polysaccharide polymer such as dextran) and cannot be substantially penetrated by high MW biological macromolecules.
  • the concept of a 2-D monolayer does not necessarily imply a flat surface, since a monolayer surface can be rough or have contours that in some cases can provide some increase in capacity.
  • a 3-D matrix is penetrable.
  • the capacity of a 2-D binding surface will depend on the diameter of the analyte molecule and the ability of the molecules to "close pack” together.
  • “Close pack” refers to the situation where sides of the analyte molecules are touching or nearly touching each other on a 2-D surface.
  • One way of considering the subject invention is that a 3-D binding phase allows for packing of analyte molecules on a 3 rd dimension. This packing can be a close pack or approach a close pack in three dimensions.
  • the magnitude of the increased capacity compared to a monolayer follows from the ability of the binding phase to capture analyte molecules in the third dimension.
  • the three-dimensional nature of the matrix is particularly advantageous in that it allows for much higher binding capacity of large biomolecules such as proteins.
  • a globular protein analyte to a 2-dimensional, monolayer extraction surface.
  • the binding of the globular protein creates a "footprint" on the surface where no other protein is able to bind.
  • the protein can bind in the matrix at varying distances from the channel surface, allowing for a staggering of the proteins and the capacity to bind many more proteins than would be possible on a 2-D surface in a capillary channel of comparable dimensions.
  • Representative data demonstrating the substantial improvement in protein binding capacity of a 3-D extraction matrix relative to a corresponding 2-D extraction matrix is provided in the Examples.
  • the term "corresponding" refers to matrices sharing the same affinity group (e.g., extraction agent), the difference between the corresponding matrices being that one is 2-D while the other is 3-D.
  • 3-D extraction surface can provide a more gentle and hospitable environment for delicate biomolecules (e.g., large proteins and protein complexes) compared to a 2-D surface.
  • the 3-D matrix allows for the creation of an environment that more closely mimic the properties of bulk solution. This biomolecule-friendly environment can promote protein stability and the retention of native biological activity.
  • the extraction at a temperature and under conditions that stabilize the functional protein, e.g., non-denaturing conditions.
  • most proteins are more stable at moderate to low temperatures, e.g., at a temperature of less than 60° C, preferably in a range of around 0 to 40° C, 0 to 25° C, 0 to 10° C, 0 to 4° C, 2 to 40° C, 2 to 25° C, 2 to 10° C, 2 to 4° C, 4 to 20° C, or 4 to 10° C.
  • Functional proteins can also be stabilized by control of pH using a buffer adjusted to a pH range suitable for the analyte of interest (if known). In many cases a neutral pH (pH 7) or pH around neutral (pH 4 to pH 10) will be best, but this can vary depending upon the nature of the analyte, e.g, the pi of a protein.
  • the extraction capillary is a component of one of the extraction devices described in U.S. Patent Application No. 10/434,713.
  • the extraction capillary can be used as an open tubular chromatography column by adapting conventional chromatographic methodologies to the capillary.
  • An advantage of performing extractions in a capillary channel as opposed to a conventional packed column is that solvent can flow through the column at a much higher linear velocity. For example, in a typical Protein A affinity packed column of dimensions 0.7 x 2.5 cm (1 mL) about half the column volume is taken up by resin. Therefore for a (typical) flow rate of 1.0 mL/min the linear velocity of the fluid flow is 5 cm/min. For a 200 ⁇ m i.d.
  • the column volume is 33 ⁇ L.
  • the flow 0.1 mL/min, corresponding to a linear velocity of 300 cm/min.
  • the effective capacity of a column will decrease as the flow rate is increased. See, for example, Samuelson, O., "Ion Exchange Separations in Analytical Chemistry” (John Wiley and Sons, 1963) page 97 et seq.; and Kunin, R., "Ion Exchange Resins, 2 nd Ed.” (John Wiley and Sons, 1958) page 339 et seq. This is especially true for gel resins.
  • the capillaries can also be used in multiplexed or parallel operations, especially when used in conjunction with automated and/or computer-controlled apparatuses, e.g., robotic instruments.
  • the extraction capillary, or a device comprising same is used in a separation method or procedure as described in U.S. Patent Application No. 10/434,713.
  • TEF tube enrichment factor
  • solid phase extraction tube enrichment factor or "TEF” is defined as the ratio of the volume of a channel, to the volume of the liquid segment containing the desorbed analyte.
  • liquid segment is defined herein as a block of liquid in a channel, bounded at each end by a block of liquid or gas.
  • the subject extraction capillaries can be adapted for use as open tubular chromatography column for use in conventional chromatographic applications.
  • TEFs of one are higher can be achieved using the methods of the invention, for example TEFs in the range of 1 to 10, 1 to 100, 1 to 400, 1 to 1000 or even higher in some cases.
  • the term "solid phase extraction enrichment factor" is defined as the ratio of the volume of a sample to the volume of liquid segment containing the desorbed analyte.
  • the enrichment factor takes advantage not only of the TEF, but also the ability to run a large volume of sample through the capillary during the loading step, and optionally to run the sample back and forth through the capillary multiple times during the sample adsorption step. This is particularly advantageous where the analyte of interest is present at a low level in the sample solution.
  • the extraction capillaries and devices of the inventions can be used in a variety of methods for extraction, typically resulting in purification and/or concentration of the analyte. These methods can be performed by loading the sample into the capillary channel from either end, washing the capillary channel from either end, and desorbing with a segment of solvent from either end, where the segment containing desorbed protein(s) or biomolecules(s) is directed to or deposited on a target.
  • the target can be a spot on a protein chip device.
  • the method used involves attaching one end of an extraction capillary to a pump capable of pumping liquid and/or gas, and introducing sample solution containing an analyte of interest into the second end of the capillary by contacting the second end with a sample solution and activating the pump.
  • the volume of sample solution can be much larger than that of the capillary, or in some cases smaller.
  • the ability to pass a larger volume of sample solution through the capillary can be useful in the case where the analyte is present at low concentration.
  • the sample solution may be desirable to pass back and forth through the capillary, allowing for increased exposure to extraction surface and potentially greater extent of binding.
  • the flow rate can also be reduced to allow more time for interaction between the analyte and matrix.
  • An advantage of the invention is that generally higher flow rates can be used than with a corresponding conventional packed bed extraction matrix.
  • Displacement is typically achieved by introducing a gas into the capillary, e.g., a pump can be used to blow or suck air through the capillary, or centrifugation or a vacuum pump could be used to achieve a like result.
  • the sample can be displaced by a wash solution.
  • the wash solution is useful for removing unwanted contaminants prior to the desorption step.
  • Gas can be run through the column prior to and/or subsequent to the wash step, to remove any residual liquid from the capillary.
  • the passage of gas through the column allows for improved purification and concentration of the sample.
  • Gas can be run through the capillary in one direction, or the direction of gas flow can be reversed one or more times during the process.
  • the gas is nitrogen gas, run through the capillary at a sufficient pressure and for as sufficient time to substantially remove any liquid from the capillary, e.g., at 50-60 psi for 30-60 seconds.
  • any liquid solution passed through the capillary can run through the capillary in either or both directions, and flow can be reversed one or more times.
  • sample, wash and/or desorption solutions enter and exit the channel through the same opening, as opposed to flowing in one end of the capillary and out the other as in other forms of chromatography.
  • the analyte is desorbed by introduction of desorption solution into the capillary, preferably flowing the desorption solution through the capillary one or more times throughout the entire length of the capillary to which analyte is adsorbed.
  • a small plug of desorption solution having a volume equal to or less than that of the capillary can be used to achieve a Tube Enrichment Factor of one or greater.
  • the desorption solution can enter and exit the capillary through same opening. In some cases analyte recovery can be improved by running the plug of desorption solution back and forth through the capillary one or more times.
  • a pump that is capable of precisely aspirating a small slug of desorption solution (of desired quantity) and accurately manipulate the slug in the capillary so as to achieve maximal elution of analyte in a minimal volume.
  • high recovery of concentrated sample is particularly desirable when the analyte is to be subjected to further analysis, e.g., by MS, X-ray crystallography, NMR, SPR, etc..
  • the invention also provides a device comprising an extraction capillary channel having a first end and a second end, the first end being connected to a pump for pumping liquid and gas.
  • the pump can be, e.g., a syringe, pressurized container, centrifugal pump, electrokinetic pump, or an induction based fluidics pump.
  • the second end can be connected to an interface for a protein chip sample applicator or a mass spectrometer.
  • the subject invention also includes kits including one or more of the subject extraction capillaries, and optionally including ancillary reagents and devices for use in conjunction with said capillaries, such as wash, loading and/or elution solutions, pumps, etc.
  • Fused silica capillaries (204 ⁇ m ID, 362 ⁇ m OD; 50 meters x 2; obtained from Polymicro Inc. (Phoenix, AZ, lot #PBW04A) were etched by flowing 100 mM NaOH through the capillary at a flow rate of 0.05 mL/minfor 50 minutes. The capillaries were then washed with water (6.0 mL), 0.1N HC1 (2 mL), water (10 mL) and acetonitrile (6 mL), after which they were dried with nitrogen gas.
  • a four meter length of the amino-functionalized capillary described in Example 2 was filled with a solution containing succinic anhydride (125 mg; 1.25 mmol), DMAP (20 mg), pyridine (25 ⁇ L) in DMF (400 ⁇ L) and acetonitrile (900 ⁇ L).
  • the capillary was placed in a 65°C oven and the reaction continued for 15 hrs with the flow of the succinic anhydride solution adjusted to 0. 6 ⁇ L/min. The capillary was then washed with acetonitrile (2000 ⁇ L).
  • NTA Nitrilotriacetic Acid
  • N,N-Bis-(carboxymethyl) lysine (commonly referred to as "Nitrilotriacetic acid,” or “NTA”) was synthesized as follows based the procedure reported by Hochuli et al. (Journal of Chromatography, 411:177-184 (1987)).
  • the solid product (Z-protected NTA) was filtered off and recrystalized by re-dissolving the solid in 1M NaOH, then neutralized with the same amount of 1 M HC1.
  • the Z-protected NTA was collected by filtration and dried.
  • a four meter length of the carboxyl-functionalized capillary described in Example 3 was activated by filling the capillary with a solution of N- hydroxysuccinimide (115 mg, 1.0 mmol), and EDAC (191.7 mg, 1.0 mmol) in acetonitrile (1500 ⁇ L). The reaction continued for 3 hrs at RT with the flow of the above solution through the capillary adjusted to 5 ⁇ L/min. (The reaction can also be carried out for about 14 hrs with the flow of the reagents solution adjusted to 0. 6 ⁇ L/min.)
  • the activated capillary was washed with acetonitrile (1000 ⁇ L), then treated with a solution of NTA (described in Example 4) in water (200 mM, pH ⁇ 8, 1.0 mL). The reaction continued for 14 hrs at RT with the flow rate adjusted to about 1 ⁇ L/min. The capillary was further reacted with 0.5% ethanolamine in water for 2 min before it was washed with water (4 mL).
  • An extraction capillary coated with NTA monolayer as described in Example 5 was washed by flowing 500 ⁇ L of 100 mM NaHC0 3 through the capillary at a fast flow rate.
  • the washed capillary was then charged with 10 mM NiS0 4 for 20 min (flow rate ⁇ 20 ⁇ L/min).
  • the charged capillary was then washed with water (1 mL at a fast flow rate), followed by 10 mM NaCl (500 ⁇ L; 50 ⁇ L/min), and then a final water wash (6 mL; 100 ⁇ L/min).
  • Toward the end of the final water wash the effluent was spot checked with PAR reagent (pyridine azoresorcinol) for the presence of any Ni + (see Example 18).
  • the capillary was then cut into 1 meter lengths each for use in extraction procedures. Capillaries that have been used in extractions can be recharged using the same procedure. Prior to recharging a capillary it should be washed with 50 mM Na 2 EDTA (500 ⁇ L; fast with about 1 min of incubation).
  • Dextran (ICN Cat# 101507; MW. 15000-20000; 3 g) was dissolved in 60 mL of water (with the help of a heat gun) and bromoacetic acid (9.3g) was added followed by Ag 2 0 (8.6g). The reaction was allowed to continue at RT for 24 hrs. The Ag 2 0 was not completely dissolved, so the reaction looked like it contained charcoal. This charcoal color eventually turned to milky-brown. The reaction stopped and solid material was filtered over celite. The filtrate was dialyzed then lyophilized to dried powder.
  • the dextran treated capillary was washed with water (0.5mL; flow rate 100 ⁇ L/min) before a solution of NTA in water (200 mM; pH ⁇ 8.0; 0.5 mL, as described in Example 4) was pumped through the capillaries. The reaction continued for 4 hrs at RT with the flow rate adjusted to 0.20 mL/h. The capillaries were washed with water (2 mL) before one meter of capillary was removed and charged with Ni2+ as described in Example 6 (single activation).
  • the capillary was quickly washed with slightly acidic water before being treated with a solution of N-hydroxysuccinimide (170 mg; 1.5 mmol) and EDAC (290 mg; 1.5 mmol) in water (1.5 mL) for 6 hrs with a flow rate of 0.15 mL/h.
  • the capillary was washed with water (0.5 mL; flow rate 0.10 mL/min), then a solution of NTA in water (200 mM; pH ⁇ 8.0; 0.5 mL) was introduced into the capillary.
  • the reaction continued for 14h at RT with the flow rate adjusted 1 ⁇ L/min.
  • the capillary was then washed with water (4 mL).
  • the washed capillary was charged with 10 mM NiS0 4 for 20 min as described in Example 6 (double activation).
  • the combined colorless product solution was added to a solution of NTA (see Example 4; 175 mM; pH ⁇ 8.2; 30 mL; 5.25 mmol; this solution was purged with nitrogen for about 10 min prior to the reaction) and the pH of the reaction mixture adjusted to 8.65 with IN NaOH. The reaction continued for 3 hrs at RT under nitrogen. The pH of the reaction mixture was readjusted to 2.5 with 6M HC1 before filtering. The total volume is 50 mL and assuming 100% yield, the concentration of this solution is 100 mM.
  • Etched capillaries were prepared as described in Example 1 and were filled with a solution of (MeO) 3 Si(CH 2 ) 3 SH (20% in toluene) before being placed in an oven at about 125°C. The reaction continued for 16 hrs with the flow of the silanization solution through the capillary adjusted to 0.15 mL/h. The capillaries were washed with toluene (3000 ⁇ L), acetonitrile (2000 ⁇ L), water (4 mL), acetonitrile (3000 ⁇ L), and dried with nitrogen.
  • Dextran (Fluka, St. Louis, MO. #31387; MW. 15000-20000; 2g) was dissolved in water (60 mL) and phosphate buffer (pH 11.5, 400 mM Na 3 P0 4 , 20 mL) was added to NaBH 4 (40 mg), followed by divinylsulfone (5.5 mL; 74 mmol; added all at once). The reaction continued at RT for 27 minutes, then quenched by adjusting the pH to 6 with 6M HC1. The light yellow reaction mixture was dialyzed and lyophilized.
  • Thiol functionalized capillaries (Example 10; ⁇ 50meters x2) were filled with the above solution using 450 psi (it took ⁇ 25min) and the reaction was allowed to continue for 1 hr at a flow rate through the capillary of 0.5 mL/h.
  • the dextran treated capillaries were washed with water (2.5 mL each) before reacting with a solution of HSCH 2 CO-NTA (Example 9; 100 mM; readjusted to pH 8.5; 3.0 mL per capillary). The reaction continued for 1 hr at RT at a flow rate of 0.4 mL/h. The capillaries were then washed with water (2.5mL each) and charged with 25 mM NiS0 4 for 20 minutes followed by a solution of 5 mM NiS0 4 in 10% MeOH- H 2 0 which was used to displace the 25 mM NiS0 4 solution (Example 6). The capillaries are stored at 4°C filled with 5 mMNiS0 4 in 10% MeOH-H 2 0 solution.
  • a Ni 2+ rNTA capillary of interest is dried with N 2 , then loaded with a 20 ⁇ L sample plug of a 2500 ⁇ g/mL stock solution of His-GST protein (described in U.S. Patent Application No. 10/434,713).
  • the sample plug is moved through the capillary two complete cycles with about 2-5 mins of incubation before being expelled from the capillary.
  • the capillary is then washed with water (500 ⁇ L; fast flow rate), followed by PBS (20mM phosphate pH7 + 140mM NaCl, 500 ⁇ L with about lmin of incubation) and water (500 ⁇ L; fast flow rate).
  • the capillary is then dried with nitrogen for about 1-2 mins.
  • the protein is eluted off the capillary with 200 mM imidazole (15 ⁇ L).
  • the imidazole plug is moved through the capillary with two complete cycles with about 2-5 mins of incubation before being expelled from the capillary and collected. 15 ⁇ L of water is then added to the collected sample.
  • the amount of protein in the sample is determined by running sample on an HP1050 HPLC system using a non-porous C-18 column, a gradient of 25% B to 75% B in 5 mins. (solvent A: 0.1%TFA in water and solvent B: 0.1%TFA in acetonitrile) with the detection wavelength of 214 nm, and integrating the protein absorbance peak.
  • a calibration standard is used, which is made by adding 15 ⁇ L of a 125 ⁇ g/mL protein solution with 15 ⁇ L of 200 mM imidazole.
  • Example 5 The capacity of a monolayer extraction capillary as described in Example 5 was determined using the method of Example 13. A one meter long section of the capillary was found to bind 1.4 ⁇ g of His-GST.
  • Example 5 A number of 3-D extraction capillaries as described in Example 5 (of the same length) were tested in the same manner, and were found to typically bind about 10-15 ⁇ g of protein. Thus, the 3-D extraction surface results in a substantial improvement in protein binding capacity.
  • Vinylsulfone Dextran Assay The purpose of this assay is to determine the amount of vinylsulfone groups in vinylsulfone dextran that are available for further reaction with any nucleophilic thiol group.
  • This assay is based on the reaction between excess sodium thiosulphate and the available vinyl groups of vinylsulfone dextran. This reaction produces hydroxide ions which can be titrated with hydrochloric acid to determine the level of vinylsulfone substitution for a given amount of vinylsulfone dextran (Journal of Chromatography (1975) 103:49-62).
  • Example 11 A number of different samples of vinylsulfone dextran were prepared using the method described in Example 11 and assayed using the procedure described in Example 15.
  • the vinylsulfone dextran samples were also used to synthesize 3-D extraction capillaries as described in Example 12 and assayed for His-GST binding capacity using the method of Example 13.
  • the following table provides the mass yield for the vinylsulfonation reactions, the results of vinylsulfone dextran assay for each sample, and the GST capacity for the capillaries corresponding to each sample.
  • reaction variables include: the integrity of the GST protein as it was shown to degrade over time, the integrity of the thiol-NTA reagent, the amount of available thiol groups on the capillaries, and the experimental variables such as MW of the starting dextran and reaction time.
  • Ni 2+ ions bound to capillary surface by chelation to the NTA moieties.
  • the assay is performed on an extraction capillary that has been loaded with
  • N :i2+ as described above.
  • a 20 ⁇ l slug of 0.01 M HCl is passed through the capillary four times, dissolving the Ni-NTA complex.
  • the sample is analyzed at 495 nm on a FIA flow injection system. Quantification is done via a "one-point" calibration, using l.OxlO ⁇ M NiS0 in 0.25M HCl as the standard solution.
  • Ni 2+ capacity and protein capacity was determined for several different capillaries (see Table), using the procedures of Examples 13 and 18.
  • Capillary 042203Ni is a Ni-NTA monolayer capillary that was prepared as described in Examples 5 and 6.
  • Capillaries D042303 and D042403Ni were prepared using the double activation method of Example 8.
  • Capillary D041003Ni was made by the same procedure as D042303Ni, but the carboxymethyl dextran was used before dialysis and lyophilization.
  • Capillary D042503Ni was produced by the same procedure as D042303Ni, with the exception that the solvent in the reactivation reaction of the attached carboxymethyl dextran was done in acetonitrile instead of water.
  • a 100 ⁇ m ID 50 cm etched fused silica capillary (Polymicro, Inc.) is attached to a syringe pump containing an aqueous 0.1% (v/v) suspension of Biocryl BPA 1000 strong anion exchanger latex (Rohm and Haas, Inc.) and latex is pumped through the capillary at the rate of 100 ⁇ L/min for 10 minutes. Then the capillary is flushed with deionized water for 10 minutes, removing the residual anion exchanger.
  • a 0.1% (v/v) aqueous suspension of strong acid cation exchanger, SPR-H (Sarasep, Inc.) is pumped through the capillary at the rate of 100 ⁇ L/min for 10 minutes. The capillary is flushed with deionized water for 10 minutes and then put into a refrigerator for storage.
  • Biocryl 1050 Rohm and Haas, Inc. is used in place of Biocryl BPA 1000.
  • Biocryl 1050 latex contains both strong base and weak base anion exchanger sites.
  • Example 20 The process as described in Example 20 is repeated except Polybrene ® (1,5- dimethyl-l,5-diazaundecamethylene polymethobromide, hexadimethrine bromide) Part Number 10.768-9 /Sigma Aldrich, Inc. is used in place of Biocryl BPA 1000 Polybrene ® is a linear strong base anion exchanger polymer.
  • HSCH 2 CO-NTA (100 mM; 5 mL, see Example 9) is added to the vinylsulfone dextran solution.
  • the pH of the resulting solution is adjusted to about 8.5 with 1M NaOH.
  • the reaction continues for 1 hr at RT before the pH readjusted to about 6 with 1M HCl and the whole reaction mixture is dialyzed and freeze dried.
  • Example 26 Preparation of a NTA chelator
  • Example 23 The processes as described in Examples 23 are repeated except the polymer suspension prepared according to Example 24 or 25 is used in place of SPR-H.
  • a 1% (w/v) aqueous suspension of the polymer is pumped through the coated capillary at a rate of 100 ⁇ L/min for 10 mins and then washed with DI water for 10 mins.
  • the capillary is charged with 10 mM NiS0 for 10 mins and then washed with DI water for 10 mins.
  • N-(N'-tert-Butyloxycarbonyl)ethylenemaleimide [M. A. Walker (Tett. Lett., 1994, 35, 665] is treated with trifluoroacetic acid to remove the BOC protecting group. The resulting aminoethylenenialeimide is then acylated with bromoacetylchloride to form N-(N'-bromoacetyl)ethylenemaleimide.
  • Poly-maleimide dextran is synthesized using an analogous synthetic scheme as was used to synthesize polycarboxymethyl dextran in Example 7, with N-(N'- bromoacetyl)ethylenemaleimide used in place of bromoacetic acid.
  • the poly-maleimide dextran is then reacted with a protein containing a reactive cysteine group, forming a covalent attachment of the protein to the 3- dimensional dextran matrix (Wang et al. (2003) Bioorganic and Medicinal Chemistry 11 :159-6; Toyokuni et al. (2003) Bioconjugate Chem. 14:1253-59; Frisch et al. (1996) Bioconjugate Chem. 7:180-86).

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

La présente invention se rapporte à des capillaires d'extraction, dans lesquels une partie importante du canal est revêtue d'une surface d'extraction en phase solide tridimensionnelle, qui se lie à une substance à analyser. Dans certains modes de réalisation, la matrice d'extraction contient un squelette polymère auquel est lié un agent d'extraction. Parmi les substances à analyser d'intérêt particulier, on compte des biomolécules, telles que des protéines, des polynucléotides, des lipides et des polysaccharides. L'invention a également trait à des dispositifs comprenant les capillaires d'extraction, à des réactifs destinés à être utilisés conjointement avec les capillaires et les dispositifs, et à des procédés de production et d'utilisation des capillaires et des dispositifs.
PCT/US2004/014321 2003-05-08 2004-05-06 Surfaces d'extraction en phase solide tridimensionnelles WO2004100887A2 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US10/434,713 2003-05-08
US10/434,713 US20040126890A1 (en) 2002-06-10 2003-05-08 Biomolecule open channel solid phase extraction systems and methods
US52351803P 2003-11-18 2003-11-18
US60/523,518 2003-11-18
US10/733,534 US7879621B2 (en) 2003-05-08 2003-12-10 Open channel solid phase extraction systems and methods
US10/733,534 2003-12-10
US10/754,775 2004-01-08
US10/754,775 US20040224329A1 (en) 2003-05-08 2004-01-08 Three-dimensional solid phase extraction surfaces

Publications (2)

Publication Number Publication Date
WO2004100887A2 true WO2004100887A2 (fr) 2004-11-25
WO2004100887A3 WO2004100887A3 (fr) 2005-05-19

Family

ID=33459124

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/014321 WO2004100887A2 (fr) 2003-05-08 2004-05-06 Surfaces d'extraction en phase solide tridimensionnelles

Country Status (1)

Country Link
WO (1) WO2004100887A2 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2780705A4 (fr) * 2011-11-16 2015-09-02 Becton Dickinson Co Procédés et systèmes de détection d'un analyte dans un échantillon
US9649061B2 (en) 2015-03-10 2017-05-16 Becton, Dickinson And Company Biological fluid micro-sample management device
US9678065B2 (en) 2013-01-11 2017-06-13 Becton, Dickinson And Company Low-cost point-of-care assay device
US9693723B2 (en) 2014-10-14 2017-07-04 Becton, Dickinson And Company Blood sample management using open cell foam
US9797899B2 (en) 2013-11-06 2017-10-24 Becton, Dickinson And Company Microfluidic devices, and methods of making and using the same
US10018640B2 (en) 2013-11-13 2018-07-10 Becton, Dickinson And Company Optical imaging system and methods for using the same
CN109564147A (zh) * 2016-06-03 2019-04-02 普度研究基金会 用于分析使用吸附材料从样品中提取的分析物的系统和方法
US10578606B2 (en) 2015-09-01 2020-03-03 Becton, Dickinson And Company Depth filtration device for separating specimen phases
US11298061B2 (en) 2014-10-14 2022-04-12 Becton, Dickinson And Company Blood sample management using open cell foam

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5453163A (en) * 1993-10-29 1995-09-26 Yan; Chao Electrokinetic packing of capillary columns
US20020062309A1 (en) * 2000-09-08 2002-05-23 Jin-Soo Lee Multimedia searching method using histogram

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5453163A (en) * 1993-10-29 1995-09-26 Yan; Chao Electrokinetic packing of capillary columns
US20020062309A1 (en) * 2000-09-08 2002-05-23 Jin-Soo Lee Multimedia searching method using histogram

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9523682B2 (en) 2011-11-16 2016-12-20 Becton, Dickinson And Company Methods and systems for detecting an analyte in a sample
EP2780705A4 (fr) * 2011-11-16 2015-09-02 Becton Dickinson Co Procédés et systèmes de détection d'un analyte dans un échantillon
EP3441142A1 (fr) * 2011-11-16 2019-02-13 Becton, Dickinson and Company Procédés et systèmes de détection d'un analyte dans un échantillon
US9678065B2 (en) 2013-01-11 2017-06-13 Becton, Dickinson And Company Low-cost point-of-care assay device
US10073093B2 (en) 2013-11-06 2018-09-11 Becton, Dickinson And Company Microfluidic devices, and methods of making and using the same
US9797899B2 (en) 2013-11-06 2017-10-24 Becton, Dickinson And Company Microfluidic devices, and methods of making and using the same
US10663476B2 (en) 2013-11-13 2020-05-26 Becton, Dickinson And Company Optical imaging system and methods for using the same
US10018640B2 (en) 2013-11-13 2018-07-10 Becton, Dickinson And Company Optical imaging system and methods for using the same
US10219731B2 (en) 2014-10-14 2019-03-05 Becton, Dickinson And Company Blood sample management using open cell foam
US9693723B2 (en) 2014-10-14 2017-07-04 Becton, Dickinson And Company Blood sample management using open cell foam
US10595762B2 (en) 2014-10-14 2020-03-24 Becton, Dickinson And Company Blood sample management using open cell foam
US10888261B2 (en) 2014-10-14 2021-01-12 Becton, Dickinson And Company Blood sample management using open cell foam
US11134875B2 (en) 2014-10-14 2021-10-05 Becton, Dickinson And Company Blood sample management using open cell foam
US11298061B2 (en) 2014-10-14 2022-04-12 Becton, Dickinson And Company Blood sample management using open cell foam
US9873117B2 (en) 2015-03-10 2018-01-23 Becton, Dickinson And Company Biological fluid micro-sample management device
US9649061B2 (en) 2015-03-10 2017-05-16 Becton, Dickinson And Company Biological fluid micro-sample management device
US10578606B2 (en) 2015-09-01 2020-03-03 Becton, Dickinson And Company Depth filtration device for separating specimen phases
US11366095B2 (en) 2015-09-01 2022-06-21 Becton, Dickinson And Company Depth filtration device for separating specimen phases
US11808757B2 (en) 2015-09-01 2023-11-07 Becton, Dickinson And Company Depth filtration device for separating specimen phases
CN109564147A (zh) * 2016-06-03 2019-04-02 普度研究基金会 用于分析使用吸附材料从样品中提取的分析物的系统和方法
CN109564147B (zh) * 2016-06-03 2022-02-18 普度研究基金会 用于分析使用吸附材料从样品中提取的分析物的系统和方法

Also Published As

Publication number Publication date
WO2004100887A3 (fr) 2005-05-19

Similar Documents

Publication Publication Date Title
Mallik et al. Affinity monolith chromatography
US8399055B2 (en) Open channel solid phase extraction systems and methods
US7122640B2 (en) Open channel solid phase extraction systems and methods
Moser et al. Immunoaffinity chromatography: an introduction to applications and recent developments
US7151167B2 (en) Open channel solid phase extraction systems and methods
US7667010B2 (en) Open channel solid phase extraction systems and methods
US20070196833A1 (en) Open channel solid phase extraction systems and methods
US20040126890A1 (en) Biomolecule open channel solid phase extraction systems and methods
US8362219B2 (en) Open channel solid phase extraction systems and methods
US20040224425A1 (en) Biomolecule open channel solid phase extraction systems and methods
EP1715948B1 (fr) Produit chromatographique pour l'absorption de proteines a une force ionique physiologique
US20110118452A1 (en) Open Channel Solid Phase Extraction Systems and Methods
Gunasena et al. Organic monoliths for hydrophilic interaction electrochromatography/chromatography and immunoaffinity chromatography
Platonova et al. Quantitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography
US20040224329A1 (en) Three-dimensional solid phase extraction surfaces
US20040241721A1 (en) Open channel solid phase extraction systems and methods
US20050029196A1 (en) Packing materials for separation of biomolecules
Jadaun et al. HPLC for Peptides and Proteins: Principles, Methods and Applications.
US5043062A (en) High performance affinity chromatography column comprising non-porous, nondisperse polymeric packing material
WO2004100887A2 (fr) Surfaces d'extraction en phase solide tridimensionnelles
Pichon Aptamer-based and immunosorbents
Hedhammar et al. Chromatographic methods for protein purification
CA2025487A1 (fr) Resine d'avidine monomesique d'affinite pour la clhp
Acikara et al. Affinity chromatography and importance in drug discovery
Jackson et al. Principles and applications of immunoaffinity chromatography

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase