JP2005533646A - Size exclusion ion exchange particles - Google Patents

Abstract

The particles comprise an ion exchange core microencapsulated by a shell, the shell providing size-excluded ion exchange particles containing a polymerization product of reactive monomers, and an apparatus using the particles. The shell may be at least partially a crosslinked polymer. The shell can exclude, for example, molecules larger than 10 ntss DNA molecules, or molecules larger than 100 ntss DNA molecules, and the core is capable of ion exchange. A method for producing size-excluded ion exchange particles is provided along with a method for purification using the particles.

Description

(Citation of related application)
This application includes US Provisional Patent Application No. 60 / 398,852, filed July 26, 2002, and US Patent Application Nos. 10 / 414,179, 10 / 413,797, and No. 10 / 413,935, all of which are filed on Apr. 14, 2003, all of which are incorporated herein by reference in their entirety. is there.

(Field)
The present invention relates to an apparatus and method for filtering and / or purifying samples by using ion exchange techniques.

(background)
For example, purification of reaction products obtained from polymerase chain reaction (PCR) or sequencing reactions presents several challenges in subsequent downstream processing. Impurities can cause artifacts in subsequent processing steps. Many purification steps to remove artifacts can be cumbersome and inefficient. In addition, purification by size exclusion chromatography or ion exchange chromatography, as well as accurate sample introduction techniques, requires an appropriate resin bed without cracks, bubbles, or channels, respectively. The resin bed used in purification, for example, purification by size exclusion, needs to be 10 times the size of the sample volume, requires more space and increases purification costs. Also, ion exchange resins often have undesirable interactions with target analytes, as opposed to the interaction of the resin with the ions to be removed. Furthermore, purification using ion exchange materials requires the use of high salt eluents to elute the analyte and may be undesirable for subsequent analysis and / or reactions. There is a need for purification methods that address these and other problems associated with the prior art of purification.

(Summary)
In various embodiments, size-exclusion ion-exchange (SEIE) particles are provided that can include a core and a shell, the core comprising an ion exchange material, and the shell comprising a size exclusion material. .

  In various embodiments, a method for forming size exclusion ion exchange particles is provided. The method can include providing an ion exchange core and microencapsulating the ion exchange core having a size exclusion material. In various embodiments, the core can be placed in a specific location in an emulsion comprising a polymerizable monomer, and the core can be microencapsulated by polymerizing the monomer to a predetermined size. Shells with pores that can exclude large materials can be formed.

  In various embodiments, a method for purifying a sample is provided. The method includes providing such a sample, contacting a sample with one or more size exclusion ion exchange particles to form a purified sample, and removing / purifying the purified sample from the size exclusion ion exchange particles. Separating.

  Additional features and advantages of the various embodiments will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of the various embodiments. The objectives and other advantages of various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description herein and appended claims.

  It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to describe various embodiments of the present invention. Is.

(Description of various embodiments)
In this specification and the appended claims, unless otherwise indicated, all amounts representing component amounts, ratios or ratios of substances, reaction conditions, and other numerical values used in the specification and claims are used. The numbers should be understood as being modified by the term “about” in all cases. For this reason, unless indicated to the contrary, the numerical parameters shown in the following specification and the appended claims are approximate values that vary depending on the desired characteristics obtained by the present invention. Represents that. At least, it is not intended to limit the application of the doctrine of equivalents to the claims of this application, and at least each numeric parameter takes into account the number of reported significant digits and by applying ordinary rounding techniques. Should be interpreted.

  Although numerical ranges and numerical parameters representing the broad scope of the present invention are approximate, this numerical value representing a particular embodiment is reported as accurately 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 include any sub-ranges subsumed herein. For example, the range “1-10” includes any subrange between the minimum value 1 and the maximum value 10 (including the minimum and maximum values), ie, all the subranges are the minimum The value is 1 or more and the maximum value is 10 or less, for example, 5.5 to 10.

  As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” have the plural meanings, except where expressly limited to one reference. Includes instructions. Thus, for example, “a monomer” indicates that two or more monomers are included.

  In various embodiments, size exclusion ion exchange (SEIE) particles provide an ion exchange core microencapsulated by a shell having size exclusion capability. “Microencapsulation”, “microencapsulated”, or similar terms means an encapsulation process at the individual particle level. In one embodiment, the liquid, solid, and / or gas core is microencapsulated by a shell for controlling access to the core. In various other embodiments, microencapsulation can cover the entire outer surface (and optionally the inner surface) of the core, or a portion of the outer surface (and optionally the inner surface) of the core. In various other embodiments, core microencapsulation can provide a permanent irreversible coating of the core, or the core can be released following dissolution of the reversible coating. In various embodiments, microencapsulation can include encapsulation of core material aggregates within a shell. This agglomerate can be melted with the core material, sintered, pressed, compressed, or otherwise formed. In various embodiments, the core material can be a single particle rather than an aggregate. As used herein, the term “core” or “core material” refers to a single particle or an aggregate of particles. The term “shell” refers to a coating on any portion of the outer and / or inner surface of the core. The dimensions and formation of the shell are described below. The term “substance” refers to any substance at the molecular level or in bulk. As used below, the material can be liquid and / or solid, eg, an emulsion or resin.

  “Mixture” as used herein refers to more than one SEIE particle used together, for example, in packed columns, mixed beds, homogeneous beds, fluidized beds, static columns with continuous flow, or batch mixtures. . In this mixture, size exclusion cation exchange particles and size exclusion anion exchange particles, size exclusion cation exchange particles and anion exchange particles, size exclusion cation exchange particles and cation exchange particles, SEIE particles and inert components, or Combinations of these can be included. The mixture can include any physical structure known in the art for separation, and any chemical mixture known in the art for ion exchange.

  Small molecules such as inorganic ions and nucleotides can penetrate or permeate the size exclusion shell and can be retained or ion exchanged by the ion exchange core. This shell can prevent larger ions, such as DNA fragments, from penetrating or permeating through the shell and reacting with the ion exchange core.

  In various embodiments, SEIE particles can be used in a variety of ways, such as in biomolecule purification. Applications include, for example, purification of polymerase chain reaction (PCR) products, purification of DNA sequencing reactions, and purification of RNA. SEIE particles can be used for purification and / or separation of, for example, oligonucleotides, ligase chain reaction products, proteins, antibody binding reaction products, oligonucleotide ligation analysis products, hybridization products, and antibodies. SEIE particles can also be used for desalting biologicals or reaction mixtures.

  SEIE particles according to various embodiments combine the advantages of ion exchange chromatography (IEC) and size exclusion chromatography (SEC). Both SEC and IEC can be used for biomolecule purification. In various embodiments, SEC can separate molecules based on hydrodynamic volume and requires less eluent for larger molecules, for example less elution than smaller molecules. In SEC, larger molecules can be eluted first. SEC can be used in a “spin column” format for biomolecule purification, which does not need to be eluted continuously, but can be eluted at a constant volume, eg, in a sample. is there. In the spin column format SEC, larger molecules can be eluted from the column while smaller molecules remain on the column and can be discarded later. SEC can be used for group separation. For example, in purification of DNA sequencing reactions or purification of PCR products, desired products such as sequencing ladders or PCR while unwanted products such as undesired primers and ions are captured on the column The product can be passed through the column. SEC can also be used for desalting applications where salt is retained on the column.

  For example, IEC may differ from SEC in the elution order of chemical species from the column. In various embodiments, the selectivity of the IEC can be based, for example, on the charge of the analyte. Larger molecules have a higher charge and can therefore have a higher affinity for IEC resins than smaller molecules. In biopolymers such as DNA, the charge of this species increases in proportion to the size of the molecule so that larger molecules can have a higher charge than small molecules, so it has a high affinity for IEC resins. It has sex. Smaller, less charged species such as salts and nucleotides can be quickly eluted from the IEC column, while larger species such as, for example, PCR products or DNA sequencing ladders, Binds tightly and elutes late or not at all. Thus, SEC may elute larger molecules first, IEC elutes small molecules first, and IEC and SEC may exhibit different elution orders.

  In various embodiments, the combined use of the SEC and IEC effects enables high-quality separation of biomolecules. In SEIE particles, a size exclusion shell can be used to limit the ability of large molecules to interact with the ion exchange core. SEIE particles can combine the advantages of SEC size exclusion with the high selectivity and binding capacity of IEC resins. A small molecule that can penetrate the size exclusion shell of the SEIE particle can interact with the ion exchange core and allow it to be retained on the core. For larger, highly charged species, the particle size exclusion shell can limit the interaction with the ion exchange core. Such large, highly charged species can remain in solution without binding to the ion exchange core. Larger species remaining in solution can be eluted. Since the bound material is retained on the ion exchange core of this SELE particle, the bound material may not elute due to the additional volume in the SEIE column or bed, so the SELE eluting particles may differ from the SEC column elution. And can react with the eluent and be prevented from being removed from the column or bed.

  An example of a SEIE particle interaction is shown in FIGS. 1 and 2 are not scaled and displayed, the relationship between objects such as the relationship between the core pore diameter and the shell pore diameter in the diagram is not scaled, and the core pore diameter is larger than the shell pore diameter. In fact, it may be reversed. As can be seen in FIG. 1, large molecules such as long single stranded DNA (ssDNA) fragments and double stranded DNA (dsDNA) pass through the pores 25 of the size exclusion shell 20 in the size exclusion anion exchange particles 30. Is too big. Instead, it can slip through or bounce off the outer surface 21 of the shell 20 and remain in the solution without being ion exchanged with the anion exchange particles 30. Small molecules such as deoxynucleotide triphosphates (dNTPs), dye-labeled deoxynucleotide triphosphates, dideoxynucleotide triphosphates (ddNTPs), dye-labeled dideoxynucleotide triphosphate triphosphates, and small ions such as chloride ions are It can pass through the pores 25 of the size exclusion shell 20 and is ion-exchanged by the anion exchange resin 13 at or near the interface 23 of the shell and the ion-exchange core 11 or in the pores of the ion-exchange core 11. Can be made. FIG. 1 shows a partial skin cutting portion 33 representing the surface of the ion exchange core 11 covered with the anion exchange resin 13. Anion exchange resin 13, for example, a macroporous copolymer of cross-linked methyl methacrylate and 2-hydroxy-3-methacryloxypropyltrimethylammonium chloride must be present on all inner and outer surfaces of solid core material 12. Can do. The anion exchange resin and the solid core material or support 12 together form the anion exchange core 11. The counter anion liberated from the anion exchange core 11, such as a hydroxide, reacts with a counter cation of the cation exchanger 35 that can be provided in a mixture comprising the SEIE particles 30, such as hydronium ions. Can produce neutral molecules such as water. A cut away view of the shell and core structure is shown in FIG.

  The SEIE particles can be used in a mixture of particles, a mixed bed, or a homogeneous bed. There, a homogeneous bed of anionic or cationic SEIE particles is used, and the counter ions are released directly into the sample solution following ion exchange. In some cases, the presence of counter ions in the sample solution does not affect further processing or reaction of the sample.

  In various embodiments, the selectivity of the SEIE particles can be determined by the properties of the size exclusion shell, the charge of the ion exchange core, and the properties of the counter ion. The properties of the size exclusion shell can be altered, for example, by selecting appropriate synthesis conditions that can affect the pore size of the resulting shell. By controlling the effective pore size of the size exclusion shell, it is possible to optimize the SEIE particles for other size exclusion applications.

  In various embodiments, the size exclusion shell may be a cross-linked or polymerized monomer such as a hydrogel. As used herein, “polymerization”, “polymerization”, “polymerize”, “cross-linked product”, “cross-linking”, “cross-linking”, and other similar terms are: Unless otherwise specified, it is meant to include both polymerization products and methods and cross-linked products and methods, for example, as opposed to linear polymers, the resulting products have a three-dimensional structure. Have. The term “polymer” refers to oligomers, homopolymers, and copolymers. The degree of cross-linking of the shell can be changed to change the pore size of the shell. For example, the pore size of the shell can be large enough to allow relatively small ions, such as chloride ions, nucleotides, or other small molecules, to pass through the shell. The pore size of the shell can be made sufficiently small to prevent any relatively large molecule such as DNA from passing through the shell. In various embodiments, the shell can be made hydrophilic, for example, to reduce passive adsorption or absorption of biomolecules such as ssDNA fragments.

  In various embodiments, the shell may be a cross-linked product of two or more reactive monomer units. The monomer unit may be a water-soluble monomer unit. As used herein, the term “water-soluble” includes materials that have any water solubility from slightly water soluble to highly water soluble and materials that swell in water. The monomer unit may contain nitrogen, oxygen, or both. The shell may be a homopolymer, copolymer, ternary copolymer, or other polymer. The shell may be a reaction product of acrylamide such as polyacrylamide. The shell may be the reaction product of acrylamide and N, N'-methylenebisacrylamide or acrylamide and acetic acid 2,2-bis (acrylamide). The shell may be a reaction product of poly (ethylene glycol) (meth) acrylate and poly (ethylene glycol) diacrylate. In various embodiments, representative water-soluble polymers are poly (meth) (acrylamide); poly (N-methyl (methyl) acrylamide); poly (N, N-dimethyl (methyl) acrylamide); poly (N- Poly (Nn-propyl (meth) acrylamide); Poly (N-iso-propyl (meth) acrylamide); Poly (N-ethyl-N-methyl (meth) acrylamide); Poly ( N, N-diethyl (meth) acrylamide); poly (N-vinylformamide); poly (N-vinylacetamide); poly (N-methyl-N-vinylacetamide); poly (vinyl alcohol); poly (2-hydroxy Ethyl (meth) acrylate); poly (3-hydroxypropyl (meth) acrylate); poly (vinyl) Poly (ethylene oxide); poly (vinyl methyl ether); poly (N- (meth) acrylylcinnamide); poly (vinyl oxazolidone); poly (vinyl methyl oxazolidone); poly (2-methyl-2-oxazoline) Poly (2-ethyl-2-oxazoline); water-soluble polysaccharides such as hydroxymethyl cellulose and hydroxyethyl cellulose; polymers of poly (ethylene glycol) diacrylate; polymers of poly (ethylene glycol) methacrylate; Other suitable polymers capable of binding; or combinations thereof, but are not limited to these. The shell may be neutral, anionic or cationic. In various embodiments, the shell may be hydrophilic. The shell may be a cross-linked polymer network of polymers that can swell in water, such as a hydrogel. Exemplary hydrogels are described, for example, in US Pat. No. 6,380,456 B1, which is incorporated herein by reference in its entirety. Once formed, the shell can become water insoluble. In various embodiments, the shell can prevent adsorption of ssDNA fragments and / or double-stranded DNA (dsDNA) fragments. In various embodiments, polymerization can be initiated thermally, photochemically, by ions, or by any means known to those skilled in the art of polymer chemistry. In various embodiments, the polymerization can be condensation (or process) polymerization, radical polymerization, atom transfer free radical polymerization, living free radical polymerization or ring opening polymerization.

  In various embodiments, the shell can be formed with pores of a predetermined size. The various pores of a single SEIE particle can be the same size or different sizes. The pores can function as a size exclusion opening to prevent molecules larger than a certain size from passing through the shell and reaching the ion exchange core microencapsulated by the shell. In various embodiments, the shell can be formed by cross-linking of one or more reactive monomers by the addition of a cross-linking agent such as N, N-methylenebisacrylamide or 2,2-bis (acrylamide) acetate. Based on the number of moles of reactive monomer units that can be cross-linked, an amount of crosslinker or initiator from 0.1 mol% to 95 mol% can be added. In various embodiments, 0.1 mol% to 70 mol%, 2.0 mol% to 50 mol%, 5 mol% to 30 mol%, or 10 mol based on the number of moles of reactive monomer units that can be cross-linked. Crosslinkers can be added from% to 20 mol%. The amount of crosslinker used to form the shell determines the pore size of the shell and can be a factor in at least one of the shell's size exclusion capabilities. In various embodiments, the cross-linking amount of the shell can be controlled by selecting a cross-linking agent and / or selecting reaction conditions. For example, a variety of multifunctional crosslinkers having various amounts of functionality can be used. The appropriate amount of crosslinker used to form the desired shell pore size can be determined by one skilled in the art based on the functionality of the selected crosslinker, the reaction conditions, and other factors known to those skilled in the art. . The shell pore diameter is preferably 10 ssDNA or less. The shell pore diameter is preferably 100 ssDNA or less.

  In various embodiments, the shell can be formed by any two of the following reactions that provide a cross-linked reaction product; N, N′-methylenebisacrylamide, 2,2-bis (acrylamide) acetate ), Poly (ethylene glycol) (meth) acrylate, poly (ethylene glycol) diacrylate, N, N′-di (meth) acryloylpiperazine, tri (meth) acryloyl perhydro-s-triazine.

  According to various embodiments, wherein 50% or more of the pores of the size exclusion shell have a pore size that can exclude molecules larger than 10 nt ssDNA, the shell is a nucleotide, an oligonucleotide primer of less than 100 nt in size And buffer salts can be passed through the ion exchange core but are deflected by the shell for sizes greater than 100 nt. Here, 50% or more of the pores of the size exclusion shell have a pore size that can exclude molecules having a size of 100 nt ssDNA or more, and use SEIE particles to purify biological samples, for example, PCR products, Larger DNA, such as dsDNA, is separated from ssDNA, free nucleotides, salts and ions. When the shell pore size is larger than 100 ssDNA, for example, 300 nt, 800 nt, or 1500 nt, the shell can pass nucleotides, oligonucleotides, salts and primers of less than 300 nt, 800 nt, or 1500 nt, respectively. Become.

  According to various embodiments, in which more than 50% of the pores of the size exclusion shell have a pore size that can exclude molecules larger than 100 nt ssDNA, in this shell, salts and smaller nucleotides are contained in the size exclusion shell. Can react with the ion exchange core. In embodiments where more than 50% of the pores of the size exclusion shell have pore sizes that can exclude molecules larger than 10 nt ssDNA, SEIE can be used to purify biological samples, eg, samples obtained from sequencing reactions. Particles can be used. For purification of sequencing reaction samples, the labeled dideoxynucleotide and salt components can pass through this shell and react with the ion exchange core, and the amount of ssDNA relative to the pre-filtered amount, ie 70% or more, 80% or more, By leaving a purified sample containing 90% or more, or 95% or more, such components can be removed from the sequencing reaction sample. If the shell pore size is greater than 10 nt ssDNA, for example 30 nt, the shell can pass less than 30 nt nucleotides, oligonucleotides, salts and primers. The shell pore size can be less than 10 nt as long as the pore size allows passage of primers with chain terminators, dyes, or other deposits that need to be separated from the sample. For example, the pore size can be 5 nt ssDNA or more.

  It can be seen that whether the particles pass or is deflected does not necessarily have to be exactly the predetermined “pore diameter”. Passing through or excluding this pore is based on the actual pore size (shell pores can have different sizes), steric hindrance factors, ionic attraction, polarization, and similar factors. Based on many factors. Thus, a measure of the “pore diameter” average, which provides guidance that particles larger than the pore diameter have a higher chance of not passing through the shell, while smaller particles have a higher chance of passing through the shell.

  In various embodiments, the shell can be formed simultaneously with the microencapsulation of the ion exchange core. For example, the shell can be formed by inverse emulsion polymerization of a water soluble reactive monomer, such as acrylamide, to form a hydrogel. As shown in FIG. 3, the ion exchange core 10 can be placed in an aqueous monomer solution in a container 40. The ion exchange core 10 may be a surface activated (interface activated) ion exchange core. The aqueous monomer solution can be, for example, one or more monomers such as acrylamide or N-vinyl pyrrolidone, a crosslinking agent such as 2,2-bis (acrylamide) acetate, and / or a free radical initiator such as sodium persulfate, potassium persulfate. Or an azo compound. The solution can further include one or more catalysts, terminators, chain terminators, chain transfer agents, promoters, buffers, and promoters.

  The oil phase 42 can then be added to the container 40. The oil phase 42 can include oil and a surfactant, or an oil that includes a surfactant. A typical oil phase 42 is the petroleum special Petroleum Special (Fluka, Buch SG, Switzerland), a surfactant having a low hydrophilic lyophilic balance (HLB) value, such as sorbitan monooleate (HLB = 4). .3). The size exclusion ion exchange particles 30 having the ion exchange core 10 microencapsulated by a size exclusion shell 20 that can be subjected to inverse emulsification and polymerization and partially crosslinked in an aqueous solution 46 of the container 40. Is formed. The SEIE particles produced by inverse emulsion polymerization can be spherical or nearly spherical, or other shapes depending on the shape of the ion exchange core.

  In various embodiments, the shell is formed by passing a heated ion exchange core impregnated with initiator, catalyst, or both through a fluidized bed of one or more reactive monomers suitable to form the shell. be able to. The fluidized bed can include, for example, acrylamide and N, N'-methylenebisacrylamide. The fluid bed monomer and / or polymer may be in powder form and can flow and / or melt in and on the ion exchange core. The SEIE particles produced by the fluidized bed reaction can be irregularly shaped.

  Other methods of forming a shell surrounding the ion exchange core can be used. The size exclusion characteristics of the shell can be adjusted, modified, changed, or otherwise changed by changing the reaction parameters of the process used to form the shell.

  In various embodiments, the shell is ion exchanged such that the shell has a thickness of 2% to 300% of the diameter of the ion exchange core, or a thickness of 25% to 200% of the diameter of the ion exchange core. It can be formed on the core. The thickness of the shell may be different on the surface of the ion exchange core, or the thickness of the shell may be uniform over the entire surface of the ion exchange core.

  The shell can at least partially microencapsulate the ion exchange core. The shell material can at least partially embed one or more pores or surface properties of the ion exchange core, such as pores, cracks, recesses, wall holes, channels, holes, depressions, or grooves. For example, all the inner and outer surfaces of the ion exchange core can be covered with a monomer suitable for forming a shell. This monomer reacts with one or more second monomers, catalysts, or initiators to form a size exclusion polymer that is cross-linked to all surfaces of the ion exchange core and reacts with the outermost surface of the size exclusion polymer; The outermost surface of the size exclusion shell can be formed. Due to the presence of a polymer that forms a size exclusion shell on all surfaces of the ion exchange core, large molecules cannot contact the ion exchange core.

  In various embodiments, the ion exchange core may be an anionic or cationic material. The ion exchange core may be a polymer, a crosslinked polymer, or an inorganic material such as silica. The ion exchange core may be a solid core material capable of ion exchange or a solid core material treated with an ion exchange resin. The ion exchange core can be surface activated. The ion exchange core may be non-magnetic, paramagnetic, or magnetic. Exemplary anionic ion exchange core materials include those listed below. Other ion exchange materials may also be used including cationic ion exchange materials and materials that can constitute ion exchange functions known to those skilled in the art of ion exchange.

  Table 1 shows examples of ion exchange materials that can be purchased from various manufacturers. Table 1 lists the composition of the core material, the particle size in microns, the anion exchange volume in microequivalent units per ml, the protein binding capacity in milligrams per milliliter of BSA, and the ionic form of the material. It will be apparent to those skilled in the art that this is the only subset of ion exchange materials that can be purchased or manufactured for this application.

  In various embodiments where the ion exchange core comprises a solid core material capable of ion exchange, the solid core material comprises macroporous silica, controlled pore glass (CPG), macroporous polymer microspheres with internal pores, ion exchange. Other porous materials known to those skilled in the art of separation, or combinations thereof, are possible. The solid core material may have various surface features such as, for example, pores, cracks, recesses, wall holes, channels, holes, indentations, or grooves. The solid core material can include sodium oxide, silicon dioxide, sodium borate, or combinations thereof. The solid core material can be modified to allow ion exchange, for example, cation exchange or anion exchange. The modification of the solid core material can include treatment of the solid core material to form cationic or anionic substituents on the surface of the solid core material. As used herein, the term “surface” includes an outer surface and / or an inner surface. The inner surface may be, for example, the surface of voids or pores in the solid core material. The solid core material may include one or more quaternary functional groups, at least one carboxylic acid group, at least one sulfonic acid group, other cationic or anionic functional groups known to those skilled in the art for ion exchange separation, or these The combination can be modified on the surface of the solid core material.

  In various embodiments, the solid core material may be microporous, mesoporous, and / or macroporous. The solid core material may have an average pore size of 1000 angstroms or less, such as 100 angstroms to 1000 angstroms, or 100 angstroms or less. The average diameter of the solid core material may be, for example, from 0.1 μm to 500 μm, from 1 μm to 250 μm, from 1 μm to 150 μm, from 1 μm to 100 μm, or from 2 μm to 20 μm. The average diameter of the solid core material may be 250 μm or less, 150 μm or less, 100 μm or less, 50 μm or less, or 20 μm or less.

  In various embodiments, the solid core material can adsorb an ion exchange resin to the outer surface, the inner surface, or both the outer and inner surfaces of the solid core material to form an ion exchange core. As used herein, the term “resin” includes a resin or gel. The ion exchange resin may be a cation exchange resin or an anion exchange resin. The ion exchange resin includes one or more quaternized functional groups, one or more non-quaternized amine groups, at least one carboxylic acid group, at least one sulfonic acid group, or combinations thereof. In view of this disclosure, one skilled in the art of ion exchange will know preferred anion exchange resins and cation exchange resins.

  In various embodiments, the ion exchange resin can be confined within the pores of the solid core material, such as macroporous silica particles. Compared to conventional ion exchange resins, the ions of the SEIE particles are filled by filling at least some of the pores of the solid core material and / or coating the outer surface of the solid core material with the ion exchange resin. The exchange volume can be increased. The ion exchange volume of the SEIE particles can be improved by increasing the amount of ion exchange resin, such as a quaternary ammonium resin, on the outer and / or inner surface of the pores of the ion exchange core. By appropriate selection of cationic or anionic functional groups on the outer surface, the inner surface, or both the outer and inner surfaces of the solid core material, the ion exchange volume of the SEIE particles can be improved.

  In various embodiments, the ion exchange resin can be formed in situ on the solid core material. For example, as shown in FIG. 4, the first monomer 16, prepolymer or polymer can be impregnated into the solid core material 12 so as to contact the surface of the pores 32. The impregnated first monomer 16, prepolymer or polymer is added to the ion-exchange resin 14 cross-linked in situ within and on the solid core material 12 including the inner surface of the pores 32. Monomers, compounds, or prepolymers, and optionally an initiator. An ion exchange core 10 is produced. In various embodiments, the ion exchange resin can be the product of one or more monomers, one or more prepolymers and / or polymers, or combinations thereof. For example, the formed ion exchange core 10 can be microencapsulated by the shell 20 having the pores 25 by inverse emulsification and polymerization, and the size exclusion ion exchange particles 30 are formed.

In various embodiments, the anion exchange core is formed by a shell and is microencapsulated. To form the anion exchange core, polyethyleneimine can be impregnated on the surface of the solid core material, including on the inner surface of the pores of the solid core material. For example, a SiO ₂ solid core material having an average pore size of 1000 angstroms, a void volume of 0.95 cc / g, and a diameter of 5 μm is added to the polyethyleneimine methanol solution, and within this core and substantially all of the inner surface of the solid core material. And can be incubated for a time sufficient to impregnate the outer surface with polyethyleneimine. In various embodiments, polyethyleneimine can be adsorbed on the solid core material by hydrogen bonding with silanol groups in the solid core material. Subsequently, the adsorbed polyethyleneimine is reacted with a second compound, such as 1,3-dibromopropane, in a solvent, for example dioxane, to form a crosslinked network that functions as an anion exchanger. be able to. When water is added, this cross-linked network forms a swollen gel. In various embodiments, the anion exchanger is quaternized by reaction of a cross-linked network with an alkyl halide, resulting in a strong anion exchanger. The resulting anion exchange core can be microencapsulated with a shell, such as polyacrylamide, to form size-excluded anion exchange particles. Impregnating other anion exchange resins or cation exchange resins on at least the inner surface portion, at least the outer surface portion, or at least all the surface portions of the solid core material of the ion exchange core by methods known to those skilled in the art of ion exchange separation Can be held.

  The ion exchange core may be a thin film ion exchange core prepared by surface aggregation. For example, large sulfonated cation exchange beads can be covered with, for example, a 0.1 μm layer of high volume anion exchange particles, forming a thin shell of high volume anion exchange material on the surface of the cation exchange beads. Can be made. A method for forming a thin film ion exchange core is described in, for example, J. Org. Weiss, Handbook of Ion Chromatography, 1986, and C.I. Horvath, Pellular Ion Exchangers Bonded Stationary Phases in Chromatography, Ann Arbor Science, 1974, both of which are hereby incorporated by reference in their entirety.

  In various embodiments, the ion exchange core can be surface activated to enhance or assist shell formation around the ion exchange core. Surface activation of the ion exchange core includes, for example, derivatization of ion exchange core functional groups with monomers, absorption of polyanions on the ion exchange core by ion interaction with ion exchange resin of the ion exchange core, neutrality and water solubility Alternatively, passive adsorption of at least partially water soluble oligomers, polymers, or copolymers on the ion exchange core, charged initiators by ionic interactions or combinations thereof, and the like are included.

In various embodiments, the ion exchange core can be surface activated by derivatization of the ion exchange core functionality with acrylic acid or acryloyl chloride. For example, as shown in FIG. 5, the ion exchange core of Macro-Prep ^™ anion exchange resin from BioRad can be derivatized. This anion exchange resin 100 is formed of an anion exchange quaternary ammonium group bonded to the main chain through an epoxy comonomer bond. The hydroxyl group remains on the anion exchange resin 100 after quaternization, and the hydroxyl group is further modified. Reaction of the anion exchange resin 100 with acryloyl chloride 105 provides a surface activated anion exchange resin 110 having acryloyl groups useful for graft polymerization of a shell polymer onto the anion exchange core. As shown in FIG. 5, anion exchange is performed by adsorbing negatively charged acrylic acid 115 or its anion version by ionic interaction on the surface of anion exchange resin 100 having a positive charge. The resin 100 is also surface activated. This surface activation provides a surface activated anion exchange resin 120 that can be co-polymerized with the prepolymer or monomer and forms a shell around the anion exchange core. Other derivatizations known to those skilled in the art of polymer chemistry can also be used to surface activate the cationic or anionic ion exchange core.

  In various embodiments, the ion exchange core can be surface activated by absorption of a charged or neutral polymer onto the surface of the ion exchange resin that forms at least a portion of the ion exchange core. For example, anion exchange resins can be surface activated by adsorption of negatively charged polymers such as polyanions. Examples of the polyanion include poly (meth) acrylic acid, (meth) acrylic acid copolymer having one or more (meth) acrylamides, N-methyl (meth) acrylamide, N-ethyl (meth) acrylamide, N-iso · Propyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N, N′-dimethyl (meth) acrylamide, N-ethyl-N-methyl (meth) acrylamide, N, N-diethyl (meth) acrylamide, N -Vinylpyrrolidone, N-vinylacetamide, N-vinylformamide, N-methyl N-vinylacetamide, 2-hydroxyethyl (meth) acrylate, 3-hydroxypropyl (methyl) acrylate, poly (ethylene glycol) acrylate, poly (ethylene glycol) ) Methacryl Salts, vinyl methyl ether, vinyl alcohol precursors, vinyl oxazolidone, vinyl methyl oxazolidone, N- (meth) acrylic yl Basswood bromide; and the like or combinations thereof. The acrylic component of the polyanion can fix the polyanion on the surface of the anion exchange resin by ionic interaction. Although not theoretically known, in various embodiments where the polyanion is a (meth) acrylamide component or an N, N′-dimethyl (meth) acrylamide component, these components are monomers, comonomers, And their polymerization products, such as oligomers, are believed to be possible. These distributed monomers, comonomers, and their polymerization products are an integral part of the shell in the microencapsulation process.

As shown in FIG. 6, for example, the reactive (polymerizable) polyanion 150 can be formed by copolymerization of acrylamide 130, acrylic acid 135, and NHS ester of acrylic acid 140. As shown in FIG. 7, CO ₂ negatively charged ^- reactive polyanion 150 obtained having a group (denoted as delta), can be fixed on the surface of the anion exchange resin of the anion-exchange core 12, This resin has ⁺ NX ₃ and / or ⁺ NHY ₂ plus charge groups (indicated by *) in the pores of the surface and / or core 12, where X and Y are independent of C _n H _{2n + 1} It is selected, n represents 1 or more integers, and this chloride resin negative charge in the pores of the core ion Cl ^- having a group (shown by white squares). By reverse emulsification, a water shell or water jacket 200 is formed around the anion exchange core 12 and the reactive polyanion 150. The water shell 200 has a reactive monomer, a comonomer, a crosslinker, and an initiator dissolved therein. By the polymerization of the water shell 200, the shell 20 is formed around the ion exchange core 12, and the SEIE particles 30 are formed. The reactive allyl or acrylamide group of the polyanion 150 is provided for graft polymerization of the shell on the anion exchange resin of the ion exchange core 12.

  In various embodiments, the ion exchange core can be surface activated by passive adsorption of neutral, water soluble, or organic soluble polymers on the ion exchange core. Neutral, water-soluble, or organic soluble polymers include, for example, poly (N-vinylpyrrolidone) or copolymers thereof; poly (vinyl acetate-co-vinyl alcohol); polyacrylamide or copolymers thereof; poly (N, N′— Dimethylacrylamide) or copolymers thereof; poly (N-vinylamide) or copolymers thereof; poly (ethylene oxide-co-propylene oxide); amphiphilic diblocks and amphiphilic block copolymers such as poly (styrene-b-2-hydroxy) Ethyl (meth) acrylate), poly (styrene-b-acrylamide (methyl)), poly (styrene-bN, N-dimethyl (meth) acrylamide), poly (styrene-b-ethylene oxide), poly (dimethylsiloxane) b-2-hydroxyethyl ( Acrylate), poly (methyl (meth) acrylate-b-acrylamide (meth)), poly (methyl (meth) acrylate-bN (N-dimethyl (meth) acrylamide)), poly (methyl (meth) acrylate -B-ethylene oxide), and poly (methyl (meth) acrylate-b-2-hydroxyethyl acrylate); or combinations thereof, and the like.

In various embodiments, the ion exchange core can be surface activated by ionic interaction with a charge initiator, eg, a photoinitiator that includes one or more negatively charged functional groups, or a thermal initiator. For example, as shown in FIG. 8, the initiator 155 can be provided in the form of 4,4′-azobis (4-cyanopentanoic acid) containing two carboxylic acid groups per molecule. Initiator 155 with ionic group CO ₂ ⁻ (indicated by Δ) is the surface of anion exchange core 12 having ionic groups of ⁺ NME ₃ (indicated by *) and Cl ⁻ (indicated by white squares). The surface activated ion exchange core 160 can be formed. In order to form a water shell or water jacket 200 around the surface activated ion exchange core 160, the surface activated ion exchange core 160 may be inversely emulsified. The water shell 200 has a reactive monomer, a comonomer, and a crosslinker dissolved therein. The water shell 200 and the core 160 form a structure 170 that is thermally polymerized to form SEIE particles 190 having the shell 20. As shown in FIG. 8, according to various embodiments, the initiator 155 on the surface of the ion exchange core 160 may be an aqueous or non-aqueous containing monomer such as N, N-dimethylacrylamide, a crosslinker such as 2,2-bisacrylamide acetic acid. A structure 180 having surface-bound free radicals (indicated by white circles) that undergoes thermal decomposition in an aqueous monomer solution to initiate free radical polymerization to form SEIE particles 190 having shells 20 is formed.

  Purification of the sample in the ion exchange core is by ion exchange. Each charge equivalent that can be absorbed on the ion exchange core can release an equivalent charge into the appropriate solution. In various embodiments, displacement of the counter ion from the ion exchange core liberates a large amount of counter ion into the sample solution. For example, in the case of a chloride ion type ion exchange core, the sample solution may increase the chloride ion concentration. For ions to be removed from the sample solution, the selectivity of the ion exchange core can be greater than the counterion of the ion exchange core. For example, if the ion exchange core is an anion exchange core in the form of a chloride ion, the counter ion is a chloride ion to displace the chloride ion in the resin and release the chloride ion to the sample solution. The ions to be removed from the sample solution have a higher affinity for the anion exchange core than the chloride counterion. When the counter ion is a chloride ion, some ions, such as hydroxide or phosphate, can be anionized in smaller amounts because these anions have a lower affinity for the anion exchange core than chloride ions. It can be held in an ion exchange core.

  In various embodiments, the anion exchange core has a hydroxide counter ion such that hydroxide is liberated when ions having an affinity for the anion exchange core are retained in the anion exchange core. May be. When hydroxide counter ions are used, chloride ions adhere weakly to the anion exchange core because chloride and hydroxide have a similar affinity for charge. Protonated forms of cation exchangers or size-exclusion cation-exchange particles are used with size-exclusion anion-exchange particles with hydroxide counterions to promote the positive reaction of the sample solution, such as chloride ions The retention of low affinity ions can be improved.

  In various embodiments, the ion exchange core can include a lower mobility counter ion, such as octane sulfonate. The ion exchange core can include a volatile counter ion, for example, acetate for anion exchange material, or ammonium for cation exchange material. Volatile counterions such as acetate or ammonium can later be removed from the sample solution. The counter ion for the anion exchange core may be, for example, a halide or hydroxide. The counter ion for the cation exchange core may be, for example, hydrogen.

  In various embodiments, a mixed bed comprising SEIE particles and an ion exchange resin is preferred to facilitate removal of ions such as salt ions and counter ions released from the SEIE particles from the sample solution. For example, the counterion liberated from the SEAE particles reacts with the counterion liberated from the cation exchange resin to form a neutral compound such as water or volatile material that can be transferred from the mixed bed during the reaction. In addition, size exclusion anion exchange (SEAE) particles can be combined with a bare cation exchange resin. Generation of such inert components or separable products, whether neutral or volatile, advances the retention of ions by the SEAE particles from the sample liquid. A mixed bed of size exclusion cation exchange (SECE) particles and bare anion exchange resin can be used. SEAE particles and SECE particles can form a mixed bed.

  In various embodiments, the mixed bed ion exchanger can include SEAE particles having an anionic ion exchange core in hydroxide form. The mixed bed can be used for bulk phase desalting of sample liquids. To form a mixed bed, SEAE particles can be prepared in hydroxide form and mixed with SECE particles or cation exchange resin prepared in hydrogen form. The anion of the sample solution can displace hydroxide ions from the SEAE particles and binds to the ion exchange core. There is a possibility that the sample solution becomes basic due to hydroxide ions. Hydroxide ions and cation exchange resins or SECE particles released from SEAE particles so that the mixed bed continuously removes hydroxide and hydronium ions from the sample solution and maintains the low ionic strength of the sample solution Hydronium ions liberated from irreversibly react to form water. The mixed bed can facilitate the removal of lower affinity anions, such as chloride, from the solution. The mixed bed can maintain a stable pH value of the sample liquid. The mixed bed can facilitate the adsorption of anions and cations from the sample liquid onto the respective SEAE and SECE particles. The mixed bed can desalinate the sample solution. In various embodiments, SEAE particles with hydroxide form ion exchange cores can be mixed with hydrogen form stoichiometric equivalents of cation exchange resin or SECE particles.

  In various embodiments, a mixture of SEAE particles, for example, in the hydroxide form, is increased by a conventional cation exchange resin or protonated SECE particles that are protonated in an anion exchange resin, such as the hydroxide form. A volume of desalting / cleaning agent can be formed. The volume of the desalting / cleaning agent is a function of the ionization capacity of the resin and is less than the volume of the sample solution, affecting the desalting / washing of the sample solution.

  In various embodiments, the mixed bed can be, for example, a stoichiometric equivalent amount of SEAE particles and SECE particles, such as a stoichiometric equivalent amount of SEAE particles and a cation exchange resin, or such as a stoichiometric amount. Equivalent amounts of SECE particles and anion exchange resin. In a mixed bed, the amount of anion-exchangeable material, such as SEAE particles or anion exchange resin, is stoichiometrically different from the amount of cation-exchangeable material, such as SECE particles or cation exchange resin. Also good.

  In various embodiments, the mixed bed comprises, for example, stoichiometrically non-equivalent amounts of SEAE particles and SECE particles, such as stoichiometrically non-equivalent amounts of SEAE particles and cation exchange resin, or such as stoichiometric amounts. A theoretically non-equivalent amount of SECE particles and anion exchange resin can be included. Stoichiometrically non-equivalent amounts of mixed ion exchangers provide excess capacity for either anion exchange or cation exchange.

  A typical PCR product solution can include amplification target sequences (amplicons), buffer salts, metal ions, enzymes (eg, polymerization enzymes), nucleotides, oligonucleotide primers, and other components. In various embodiments, the PCR product can be analyzed, or the PCR product can be used in subsequent enzymatic reactions that are sensitive to at least some artifacts found in a sample solution containing the PCR product. it can. For example, free nucleotide and oligonucleotide primers can interfere with downstream enzymatic reactions. The size exclusion limit of SEIE particles can be 100 ntss DNA, for example. SEIE particles can, for example, capture and remove nucleotides, oligonucleotide primers, and buffer salts that are less than 100 nt or equivalent size. The resulting solution contains the purified PCR product in a desalted environment and can be used for downstream reactions and analyses. PCR purification separates larger double-stranded DNA (dsDNA) from smaller ssDNA and dsDNA (eg, primer-dimers, unwanted side reaction products that are dsDNA), free nucleotides, and salts. Can be aimed at. PCR product purification using SEIE particles can, for example, isolate 250-600 bp amplicons and remove 44 nt primers, nucleotides, or both.

  The DNA sequencing reaction solution can include, for example, buffer salts, metal ions, polymerizing enzymes, nucleotides, oligonucleotide primers, and other components. However, the SEIE particles for purification of the sequencing reaction solution may be different from those used in the purification of the PCR reaction solution. For example, in various embodiments, the sequencing reaction product can be removed from the remaining unincorporated dye-labeled dideoxynucleotide (terminator) that can be removed prior to electrophoresis and DNA sequencing or basecalling. Can be included. Failure to remove such compounds creates “stains” that can cause errors in DNA sequencing or basecalling. In the capillary base sequence analyzer, electrokinetic injection can be used as a means for introducing a DNA base sequencing reaction sample. The presence of salt in the sample can affect the introduction of the sample into the capillary, and the salt concentration needs to be reduced for introduction into the capillary. The DNA sequencing reaction sample can be desalted by purification with SEIE particles. The sample solution purified with the SEIE particles may have a salt concentration of 100 μM or less. The sample solution purified with SEIE particles may have a salt concentration of 50 μM or less. A sample solution purified by SEIE particles is preferred for electrokinetic capillary injection. For this and other purposes, the SEIE particles have a size exclusion limit of, for example, less than 10 ntssDNA and allow small ions such as salt from the sample solution and dye-labeled nucleotides to remain while free ssDNA remains in solution. Can be removed. Sequencing reaction purification using SEIE particles can be used to separate ssDNA having a size of, for example, 10 nt to 1500 nt, or larger, from small components such as dye-labeled nucleotides and salts.

  In various embodiments, DNA purification can be performed in bulk mode using SEIE particles. A suitable chromatographic bed of SEIE particles is not always necessary.

  In various embodiments, an apparatus comprising SEIE particles as described herein is provided. The device is a microfluidic device having one or more pathways, wherein at least a portion of at least one pathway comprises SEIE particles. The device has an inlet and an outlet in fluid communication with the SEIE particles. This particle is present, for example, in a column. As used herein, a column is oriented horizontally and vertically, or at any position between the horizontal and vertical directions. The column may include cavities, chambers, storage sites, reaction zones, beds, indentations, or other containers suitable for containing or holding SEIE particles and sample solutions. The column may contain one or more SEIE particles. The outlet of the device may be provided in a fluid communication comprising a container such as a purified sample well, a tube, a glass plate, or another means for collecting the purified sample.

  In various embodiments, a method of using such an apparatus is provided for sample solution purification. The apparatus includes size exclusion ion exchange particles, such as size exclusion anion exchange particles, size exclusion cation exchange particles, or combinations thereof. This method includes the process of adding particles to the column of the apparatus. Naked cation exchange resin, anion exchange resin, or both can optionally be added to the column of the apparatus. A sample, such as a PCR purified product solution or sequencing reaction product solution, can be placed or introduced into the inlet of the device. The sample can travel from the inlet to a column containing SEIE particles and an optional additional ion exchange resin. The sample is subjected to a combination of size exclusion separation and ion exchange, and the sample is filtered and / or purified. The filtered and / or purified solution can be eluted or removed from the column to the outlet and directed to a vessel for analysis and / or subsequent processing. The sample can move through the column by centripetal force.

  In various embodiments, purification of a sample using SEIE particles is sufficient to provide an adequate ion exchange volume for ion exchange of at least 80%, at least 90%, or at least 95% of the sample. Can be achieved using. Purification can be performed in 10 minutes or less, 5 minutes or less, or 2 minutes or less. Purification includes the process of contacting the sample with the SEIE particle for a time sufficient for the SEIE particle to ion exchange with the sample and removing the purified sample from the SEIE particle.

  In various embodiments, the process of separating the purified sample from the particles includes removing the purified sample from the particles, removing the particles from the purified sample, and / or extracting the purified sample from a mixture of particles and purified sample. . An example of a process for extracting a purified sample from a mixture of particles and purified sample includes a process of analyzing the product in a test tube by immersing the capillary directly in the test tube and injecting it into a sequencing device.

  Exemplary embodiments of various aspects of the invention are described herein. The foregoing description and the following examples are representative only, and various modifications and substitutions described herein with respect to methods and materials will be apparent to those skilled in the art and are included in the description herein. And

(Formation of SEIE particles and mixed bed :)
The solution was 2.4957 g (35.11 mmol) of acrylamide (99 +% purity, Aldrich Chemical Co., St. Louis, Mo.) in 11.9391 g of water (Milli Q Water System, Millipore Billerica, Mass.), And N, N′-methylenebis. Prepared from 0.7021 g (4.55 mmol) of acrylamide (purity 99+, Aldrich Chemical). While stirring, a suspension of 0.7158 g of MACRO-PREP HQ macroporous anion exchange core (BioRad, Hercules, Calif.) In 2.0075 g of water (MilliQ Water System, Millipore) was added to this solution. A suspension of MACRO-PREP HQ was prepared with occasional stirring 30 minutes before use. A mixture of monomer solution and MACRO-PREP suspension containing 1.0072 g of SPAN-80 (sorbitan monooleate, Flukabuuf, Switzerland) in 20.10 g of PETROLEUM SPECIAL (bp 180-220 ° C., Fluka) The mixture was poured into 3600 rpm and stirred. Emulsification was carried out at room temperature for 2.5 minutes. The emulsion was added to a 24/40 three-necked round bottom flask equipped with a water-cooled condenser, purge breathing tube, and mechanical stirrer equipped with a 1 ″ stainless steel three-blade propeller. At a stirring speed of 100 rpm, The emulsion was purged with ultra high purity helium for 30 minutes at a flow rate of 100 mL / min 30. At the start of the purge, ammonium sulfate (purity 99.99 +%, (Aldrich Chemical) 0.5 mL (0.044 mmol) of a solution containing 0.4507 g was added.At the end of purging, cooling in an oil bath at 45 ± 1 ° C. and N, N, N′N′-tetramethyleneethylenediamine 50 μL (0.33 mmol) was added to the reaction flask and polymerization was carried out at 45 ± 1 ° C. for 5.5 hours. The reaction mixture was transferred to a container containing 150 mL of acetone and stirred for 5 minutes The reaction mixture was filtered with suction through a 5 micron filter paper, washed with excess PETROLEUM SPECIAL and acetone, dried with air suction, and powdered SEAE. 3.92 g (quantitative yield) of particles were obtained, and the dried SEAE particles were mixed with 50 mM phosphate buffer (0.5% by weight polyvinyl alcohol (80% hydrolysis, molecular weight 9-10 Kda, Aldrich Chemical) ( (pH = 7, JT Baker Philipsburg, NJ) Dispersed in 25 mL The phosphate buffer was filtered through a 0.2 micron filter membrane prior to use.

SEAE particles were converted to the hydroxide form by repeated washing with 1M NH ₄ OH (ultra high purity grade, Gibbstown, NJ). The volume of acid or base required to convert a given amount of resin from one form to another can be calculated by knowing the acid or base concentration, and the ion volume of the resin, Converted in a 10-fold excess of the acid or base required. According to the method of Example, 100 μL of particles were mixed with 800 μL of 1M NH ₄ OH, centrifuged at 1000 × g with a microcentrifuge, and the supernatant was removed with a pipette. This process was repeated 10 times. Although this step is optional, the particles were transferred to a spin column and washed three times with 100 μL aliquots of 1M NH ₄ OH. The resin bed was washed repeatedly with deionized (DI) water until the pH of the eluate was neutral (washed approximately 10 times with 500 μL aliquots of DI water).

SECE particles for the mixed bed were prepared by a similar process. A protonated form of a commercially available macroporous cation exchange core, AG5OW-X8, 400 mesh (BioRad, Hercules, Calif.) Was obtained. Note that although the resin is provided in protonated form, better results are obtained when the resin is converted again. Use the same process described to convert SEAE particles to the hydroxide form except that they are washed with 1M HCl (reagent grade, JT Baker) instead of NH ₄ OH. Converted to hydrogen ion form. Following the HCl wash, water wash was performed until the pH of the eluate was neutral. SEAE particles and SECE particles were semi-dried by spinning at 1000 × g for 1 minute. SEAE particles and SECE particles were each slurried with 500 μL of DI water. The particles were mixed together at a theoretical mixing ratio based on the ion volume of the anion and cation exchange beads, respectively, and vortexed to form a mixed bed slurry. To minimize the adsorption of DNA into the bead mixture, the mixed bed was washed twice with a sheared salmon sperm DNA solution (Eppendorf AG, Hamburg, Germany) prepared at a concentration of 1 mg / mL in DI water. The volume of sheared salmon sperm DNA was approximately the same as the volume of the resin bed for each wash. The bead bed was washed 3 times with 1000 μL aliquots of DI water. The slurry was semi-dried by spinning at 1000 × g for 1 minute and stored at 4 ° C. Note that the mixed bed is washed with salmon sperm DNA prior to use, as described herein. However, washing the constituent beads before mixing is also effective. If SEAE and SECE resins are used alone (ie not in a mixed bed), it is equally useful to wash with salmon sperm DNA.

(PCR purification :)
For PCR purification, it contains 102 μL of PCR master mixture, 4 μL of FAM-labeled forward primer (20 pmol / μL), 4 μL of reverse primer (20 pmol / μL), 20 μL of hgDNACEPH1347-02 (50 μg / μL), and 70 μL of deionized water (DI). Samples were prepared. A certain amount of 20 μL / well volume of this master solution was added to the wells in the thermal cycler plate. The mixture was heated at 95 ° C. for 5 minutes, followed by 40 cycles of heating, each cycle being heated at 95 ° C. for 30 seconds followed by 60 ° C. for 120 seconds.

To carry out the purification, 24 mg of mixed bed SEIE particles were formed as described in the above example (semi-dry, as described above) and weighed in a microcentrifuge tube. 30 μL of the original PCR product was added and the mixture was shaken for 5 minutes. The microcentrifuge tube was spun at 5000 × g and 5 μL of supernatant was removed and pipetted into a 96-well plate for analysis by the ABI 3100 sequencing apparatus. Samples were analyzed by ABI 3100 using a 50 cm array with POP6 polymer and GeneScan ^™ analysis software, both from Applied Biosystems, Foster City, California.

(DNA base sequencing reaction purification :)
For DNA sequencing reaction purification, BIGDYE TERMINATOR READY REACTION MIX (Applied Biosystems, Foster City, Calif.) (V.3) 400 μL, M13 universal reverse primer (3.2 pmol / μL) 50 μL, template amplicon ( About 100 μg / μL) A sample containing 25 μL and DI water 525 μL was prepared. A certain amount of 20 μL / well volume of this master solution was added to the wells in the thermal cycler plate. The mixture is heated for 25 cycles, each cycle comprising 95 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 120 seconds.

  To carry out the purification, 24 mg of mixed bed SEIE particles were formed as described in the above example (semi-dry, as described above) and weighed in a microcentrifuge tube. 30 μL of the original PCR product was added and the mixture was shaken for 5 minutes. The microcentrifuge tube was spun at 5000 × g and 5 μL of supernatant was removed and pipetted into a 96-well plate for analysis by the ABI 3100 sequencing apparatus. Samples were analyzed by ABI 3100 using a 50 cm array, Applied Biosystems POP6, and a standard sequencing module.

  The base sequencing reaction can also be purified on a small scale. For purification of DNA sequencing reaction, dRhodamine Ready Reaction Mix (Applied Biosystems, Foster City, Calif.) 8 μL, M13 universal reverse primer (3.2 pmol / μL) 1.0 μL, template amplicon (about 100 μg / μL) ) A sample containing 0.5 μL and 10.5 μL of DI water was prepared. This solution was placed in the GeneAmpPCR system 9700 (Applied Biosystems). This mixture is heated for 25 cycles, each cycle comprising 95 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 120 seconds. A SEAE / cation exchange mixed bed was prepared as previously described. This mixture was prepared as a deposition bed by depositing beads followed by removal of all water on the floor. The deposited bed was vortexed and a 1 μL aliquot was transferred to a 50 μL MicroAmp tube (Applied Biosystems). 1.0 μL of the already prepared base sequencing reaction was added to the beads and pipetted for 15 seconds. The tube was transferred to a vortexer and vortexed for 1 minute. To this tube, 6 μL of DI water was added, mixed by pipette, and centrifuged at 500 × g for 30 seconds with a bench centrifuge. The supernatant is removed with a pipette and the ABI 3100 DNA sequencing apparatus is operated. Because the sample is very highly desalted, the injection potential and time on the sequencing apparatus was reduced by 4 binding factors of 4 so that the data did not scale out.

(Experimental result:)
The PCR purification results were analyzed using GeneScan software and are shown in FIG. This experiment was designed such that removal of the majority of the dye-labeled 38 nt primer from the 560 bp PCR product was observed while retaining the double stranded DNA amplicon. As the data show, this primer was initially present in a high concentration in the sample (see graph (a) in FIG. 9), but the concentration decreased upon contact with the SEIE particles. As seen in graphs (a)-(d) of FIG. 9, the peak from 5000-7000 scans labeled 50 is derived from the FAM-labeled 38 nt primer, and the peak of 23,000 scans labeled 60 is FAM. Labeled PCR product. Contact with the SEIE particles for 2 minutes (FIG. 9 graph (b)) removed most of the primers without affecting the PCR product. Contact with SEIE particles for 5 minutes (FIG. 9 graph (c)) or several minutes (FIG. 9 graph (d)) reduced the amount of primer below the detection limit. Comparison of ROX-labeled 7 nt single-stranded DNA internal standards spiked into each sample showed that the degree of desalting was the same between the samples, and the amount injected into each capillary was almost equal. These results show that a brief contact with the mixed bed SEIE resin demineralizes the solution containing the PCR product to remove the primer and has minimal effect on the target amplicon concentration.

  DNA sequencing of PCR products purified using SEIE particles is shown in FIGS. 10a and 10b. PCR products that were purified and spiked with excess primers and nucleotides in the unpurified were sequenced. In both cases, the sequencing reaction was purified using a CentriSep spin column (Princeton Separation, Princeton, NJ, USA). FIG. 10a illustrates the sequence detected in the crude PCR product. This graph contains a lot of noise and a large number of arrays. Some miscalls and ambiguities are observed and the overall quality is low. FIG. 10b shows a graph of sequences detected with the same PCR product but with the product purified by contacting the PCR product with the SEIE resin for 10 minutes prior to sequence analysis. The arrangement of FIG. 10b is clearly readable, unlike the arrangement of FIG. 10a.

  The data in FIGS. 11a-d shows the application of SEIE to DNA sequencing reaction product purification. The graph shows that the intact DNA sequencing reaction product sequence (FIG. 11a) has large artifacts (“stains”) that obscure the sequence data. FIG. 11b shows the processing result of the DNA sequencing reaction using a standard CentriSep column. Obviously, the data of FIG. 11b is clearer than FIG. 11a. FIGS. 11c and 11d show the results of treating DNA sequencing reaction products with mixed bed SEIE particles 3 minutes prior to sequence analysis. As can be seen in FIGS. 11c and 11d, the SEIE purification eliminates most of the “stains” present in FIG. 11a, and better peak separation is observed when compared to the graph shown in FIG. 11b. It is done. Note that the 295 nt spike in FIG. 11c was caused by bubbles in the capillary. As shown in FIG. 11c, the results show that SEIE purification filters out small reaction product fragments, as reflected at the beginning of the graph. FIG. 11d shows the results relating to the purification of the d-rhodamine base sequence reaction described above. This purification was performed on different batches of SEIE resin and was performed on a smaller scale (1 μL reaction with 1 μL resin). This result shows that excellent purification can be achieved even when the ratio of sample to resin is less than 1.

  12 and 13 show the increase in size between the ion exchange core and the size exclusion ion exchange particles. FIG. 12 shows the size difference between MACRO-PREPHQ anion exchange resin (radius 25 μm) and SEIE particles made with this resin. FIG. 13 shows the size difference between MACRO-PREP25Q anion exchange resin (average radius 12.5 μm) and SEIE particles made with this resin according to an embodiment (average radius 19 μm). 12 and 13, it can be seen that the SEIE particles have a larger size with respect to the ion exchange core.

  It will be apparent to those skilled in the art that various modifications and variations can be made to the various embodiments described herein without departing from the spirit and scope of the present teachings. Accordingly, the various embodiments described herein are intended to cover other modifications and variations within the scope of the appended claims and their equivalents.

  The following figure should be construed as not representing the actual size. The relationship between objects does not represent the actual size, and the size in the figure may actually be reversed. This figure is for the purpose of understanding and clarifying the structure of each displayed object, and therefore some characteristics may be exaggerated to illustrate specific structural characteristics.

FIG. 1 shows a schematic diagram of the interaction of anionic species with size-excluded ion exchange particles in various embodiments. FIG. 2 is a schematic diagram of a core-shell structure of a size exclusion ion exchange core, which has a cutaway view to show the surface of the core and the inside of the core. FIG. 3 is a schematic diagram illustrating the formation of a shell on an ion exchange core by inverse emulsification and polymerization in various embodiments. FIG. 4 is a diagram illustrating the formation of size exclusion ion exchange particles in various embodiments. FIG. 5 is a diagram showing derivatization of an ion exchange resin. FIG. 6 shows the formation of reactive polyanions. FIG. 7 is a schematic diagram illustrating the formation of size-excluded ion exchange particles in various embodiments, where one step is surface activation of the ion exchange core. FIG. 8 is a schematic diagram illustrating two pathways for the formation of size-excluded ion exchange particles in various embodiments, with one common step being surface activation of the ion exchange core. FIG. 9 shows (a) data obtained from a raw sample comprising a mixture of dye-labeled amplicon and dye-labeled primer, and in various embodiments, size-excluded ion exchange particles to remove the dye-labeled primer. Is a series of graphs for data obtained after incubating a sample with (b) for 2 minutes, (c) for 5 minutes, and (d) for 10 minutes. Figures 10a and 10b show the base sequence analysis results of the PCR product purified using size-exclusion ion exchange particles, Figure 10a is the base sequence analysis data of the raw PCR product, and Figure 10b shows the various implementations. It is the base sequence analysis data of the PCR product refine | purified by the contact with the size exclusion ion exchange particle according to a form. FIGS. 11a-d show purification of the sequencing reaction by contact with size-excluded ion exchange particles compared to standard purification techniques, FIG. 11a is raw data of an unpurified sample, and FIG. 11b is purified on a CentriStep column. 11c and 11d are data from samples purified by contact with size-excluded ion exchange particles according to various embodiments. FIG. 12 is a graph showing the size difference in particle count between an anion exchange core according to various embodiments and the resulting SEIE particles comprising an anion exchange core, FIG. 13 is a graph showing the size difference in particle count between an anion exchange core according to various embodiments and the resulting SEIE particles comprising an anion exchange core.

Claims (60)

  A size exclusion ion exchange particle comprising a core and a shell, wherein the core comprises an ion exchange material and the shell further comprises a size exclusion material.   The size exclusion ion exchange particle of claim 1, wherein the shell comprises a polymer that is at least partially cross-linked.   The size exclusion ion exchange particle of claim 2, wherein the polymer is at least partially covalently crosslinked.   The particle of claim 1, wherein the ion exchange core comprises a solid core material.   The particle of claim 4, wherein the solid core material comprises at least one of macroporous silica, controlled pore glass, microporous polymer microspheres, mesoporous polymer microspheres, and macroporous polymer microspheres.   The particle of claim 4, wherein the solid core material is ion exchangeable.   The particle of claim 6, wherein the solid core material comprises at least one of a tertiary ammonium group, a quaternary ammonium group, a carboxylic acid group, and a sulfonic acid group.   The particle of claim 4, wherein the solid core material is porous.   The particle of claim 4, wherein the solid core material has an average diameter of 500 μm or less.   The particle of claim 4, wherein the solid core material is coated with an ion exchanger.   The particle of claim 1, wherein the core comprises a neutral, water soluble polymer, or organic soluble polymer.   The core comprises a poly (N-vinylpyrrolidone) polymer material, a poly (vinyl acetate-co-vinyl alcohol) material, a polyacrylamide material, a poly (N, N-dimethylacrylamide) material, a poly (N-vinylamide) material, a poly 12. The particle of claim 11, comprising one or more of (ethylene oxide-co-propylene oxide) material, amphiphilic diblock copolymer, and amphiphilic block copolymer.   The particle of claim 1, wherein the ion exchange core is surface activated.   The particle of claim 1, wherein the ion exchange core comprises agglomeration.   The particle of claim 1, wherein the shell comprises a cross-linked polymerization product of a water soluble monomer.   The size-exclusion ion-exchange particle according to claim 1, wherein the shell is a poly ((meth) acrylamide) material, a poly (N-methyl (meth) acrylamide) material, or a poly (N, N-dimethylacrylamide) material. Poly (N-ethyl (meth) acrylamide) material, poly (Nn-propyl (meth) acrylamide) material, poly (N-iso-propyl (meth) acrylamide) material, poly (N-ethyl-N-methyl) (Meth) acrylamide) material, poly (N, N-diethyl (meth) acrylamide) material, poly (N-vinylformamide) material, poly (N-vinylacetamide) material, poly (N-methyl-N-vinylacetamide) Substance, poly (vinyl alcohol) substance, poly (2-hydroxyethyl (meth) acrylate) substance, poly (3- Droxypropyl (meth) acrylate) material, poly (vinyl pyrrolidone) material, poly (ethylene oxide) material, poly (vinyl methyl ether) material, poly (N- (meth) acrylylcinnamide) material, poly (vinyl oxazolidone) Substances, poly (vinylmethyloxazolidone) substances, poly (2-methyl-2-oxazoline) substances, poly (2-ethyl-2-oxazoline) substances, water-soluble polysaccharides, poly (ethylene glycol) acrylate polymers, poly ( Size exclusion ion exchange particles comprising at least one of a polymer of (ethylene glycol) methacrylate, a water soluble polysaccharide, and combinations thereof.   The particle of claim 1, wherein the shell comprises a cross-linked hydrogel.   The particle of claim 1, wherein the shell comprises a reaction product of acrylamide and N, N′-methylenebisacrylamide.   The size exclusion ion exchange particle of claim 1, wherein the shell comprises a reaction product of acrylamide and 2,2-bis (acrylamide) acetic acid.   The size exclusion ion exchange particle of claim 1, wherein the shell comprises a reaction product of poly (ethylene glycol) (meth) acrylate and poly (ethylene glycol) diacrylate.   The particle of claim 1, wherein the shell comprises a plurality of pores, and at least 50% of the pores can exclude molecules having a size of 10 ntss DNA or more.   A mixture comprising an ion exchange material comprising size-excluded ion exchange particles according to claim 1, wherein the mixture comprises a cationic ion exchange material and an anion ion exchange material.   23. The mixture of claim 22, wherein the size exclusion ion exchange particles are cationic and the mixture comprises anionic size exclusion ion exchange particles.   23. A mixture according to claim 22, wherein the mixture is in the form of a mixed bed.   23. The mixture of claim 22, wherein the cationic ion exchange material and the anionic ion exchange material are present in stoichiometric equivalent amounts.   A purification apparatus comprising a container and the size-excluded ion exchange particles of claim 1 disposed in the container.   23. A purification device comprising a container and the mixture of claim 22 disposed in the container.   A microfluidic device comprising one or more columns, wherein the particles according to claim 1 are arranged in at least one of the one or more columns.   30. The apparatus of claim 28, wherein each of the one or more columns comprises an inlet and an outlet.   23. A microfluidic device comprising one or more columns, wherein the mixture of claim 22 is disposed in at least one of the one or more columns. A method of forming size-excluded ion exchange particles comprising:
Providing an ion exchange core; and microencapsulating the ion exchange core with a size exclusion material;
A method of forming size-excluded ion exchange particles. 32. The method of claim 31, comprising:
The step of microencapsulating the ion exchange core comprises:
Forming an aqueous water jacket comprising at least one monomer, prepolymer, or copolymer around the ion exchange core and by reverse emulsification of the at least one monomer, prepolymer, or copolymer in the aqueous water jacket; Forming a shell around the ion exchange core;
Including the method. 32. The method of claim 31, comprising:
Providing the core comprises:
Providing a solid core material with an outer surface;
Attaching a first monomer to the outer surface; and contacting the bound first monomer to a second monomer to form an ion exchange material on at least a portion of the outer surface. 34. The method of claim 33, wherein:
The solid core further has an inner surface, and the bonding step further includes the step of bonding the first monomer on the inner surface;
Contacting the bound first monomer with a second monomer further comprises forming an ion exchange material on at least a portion of the inner surface;
Method. 32. The method of claim 31, comprising:
Providing the core comprises:
Providing a solid core material with an outer surface; and
Coating the outer surface with an ion exchange material;
Method. 36. The method of claim 35, wherein:
The solid core further comprises an inner surface;
The method of coating includes the step of coating at least a portion of the inner surface with an ion exchange material. Providing the core includes surface activating the core;
32. The method of claim 31. 38. The method of claim 37, comprising:
The step of surface activating the core includes a step of adsorbing a neutral water-soluble polymer or an organic soluble polymer on the surface of the core.
Method. 40. The method of claim 38, wherein the surface activated core is
Poly (N-vinylpyrrolidone) polymer material, poly (vinyl acetate-co-vinyl alcohol) material, polyacrylamide material, poly (N, N-dimethylacrylamide) material, poly (N-vinylamide) material, poly (ethylene oxide-co-) A method comprising one or more of a propylene oxide) material, an amphiphilic diblock copolymer, and an amphiphilic block copolymer. Providing the core includes aggregating the core material.
32. The method of claim 31. 32. The method of claim 31, comprising:
The step of microencapsulating the ion exchange core includes:
Contacting the core with a polymerizable monomer; and reacting the monomer to form the size exclusion material.
Method. 42. The method of claim 41, comprising:
Contacting the core with a polymerizable monomer comprises:
Heating the core;
Contacting the core with at least one of an initiator and a catalyst; and contacting the core with at least one of acrylamide and N, N-methylenebisacrylamide.
Method. 32. The method of claim 31, comprising:
The step of microencapsulating the ion exchange core includes:
Placing the core in an emulsion comprising a polymerizable monomer;
Polymerizing the monomer and microencapsulating the core; and
Method. 44. The method of claim 43, comprising:
The polymerization step comprises
A method comprising the step of inverse emulsion polymerization.   44. The method of claim 43, wherein the polymerizable monomer is water soluble. The step of polymerizing includes the step of contacting a crosslinking agent with the monomer,
Wherein the crosslinker is present in an amount of 1 to 95 weight percent based on the weight of the monomer,
44. The method of claim 43. The polymerizing step includes covalently cross-linking the monomer.
44. The method of claim 43. A method of forming size-excluded ion exchange particles comprising:
Providing an ion exchange core;
Contacting the core with an emulsion comprising a polymerizable monomer;
Polymerizing the monomer to microencapsulate the core, wherein the polymerization forms a shell with a plurality of pores, wherein the plurality of pores is larger than a predetermined size Including the step of eliminating.   49. The method of claim 48, wherein the shell is at least partially covalently crosslinked.   49. The method of claim 48, wherein the predetermined size is 10 nt ssDNA or more. A method for purifying a sample comprising:
Providing a plurality of size exclusion ion exchange particles, each of the particles comprising a core for ion exchange and a shell for size exclusion;
Contacting the sample with the particles to form a purified sample; and separating the purified sample from the particles.
Method. 52. The method of claim 51, wherein the purified sample comprises a substance having a molecular size of 10 nt ssDNA or greater.   52. The method of claim 51, wherein the purified sample comprises a substance having a molecular size of 100 nt ssDNA or greater. The contacting comprises passing the sample through the plurality of particles using centripetal force;
52. The method of claim 51. 52. The method of claim 51, comprising:
The plurality of size exclusion ion exchange particles have a first volume;
The biological sample has a second volume;
The first volume is less than or equal to the second volume;
Method.   52. The method of claim 51, wherein the purified sample has a salt concentration of 50 [mu] M or less. 52. The method of claim 51, comprising:
The plurality of size exclusion ion exchange particles are
Comprising a stoichiometric equivalent amount of size-excluded anion-exchange particles and size-excluded cation-exchange particles,
Method. The size-excluded ion exchange particle according to claim 1,
The shell includes a reaction product of acrylamide and N, N′-di (meth) acryloylpiperazine.
Size exclusion ion exchange particles. The shell includes a reaction product of acrylamide and tri (meth) acryloyl perhydro-s-triazine.
The size exclusion ion exchange particle according to claim 1. The shell is composed of acrylamide, N, N′-methylenebisacrylamide, acetic acid 2,2-bis (acrylamide), poly (ethylene glycol) (meth) acrylate, poly (ethylene glycol) diacrylate, N, N′-di ( The size exclusion ion exchange particle of claim 1, comprising at least two reaction products of (meth) acryloylpiperazine, tri (meth) acryloylperhydro-s-triazine.