JPH05502944A - Organometallic coated particles for use in separations - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Magnetic separation in biological systems is used as an alternative to gravity separation or centrifugation. . B, L. Hirschbein et al., Chemtech, pp. 172-179 ( 1982): M, Pourfarzaneh, Th■@Ligand Quarterly, 5(1): 4l-47 (1982): and P. J, , and P, , Dunhi Il, Enzyme@11ii cro b, Te- chnol, 2:2-10 (1980). Enzymes, antibodies and others carriers for biological molecules, such as bioaffinity adsorbents. There are several advantages to using magnetically separable particles. For example, magnetic When air particles are used as a solid support in an immobilized enzyme system, enzymes or media (suspended solids) can be selectively recovered from the body (including the culture medium), allowing circulation in the enzyme reactor It is possible. P, J, Robinson et al., Bio-tech, Bioe ng, 15:603-606 (+973). immunoassay or When used as a solid support in other competitive binding assays, magnetic particles This allows the reaction to occur under homogeneous conditions, which promotes optimal binding kinetics and facilitates separation. It changes the analyte-adsorbent equilibrium only slightly and, especially compared to centrifugation, Facilitates separation of bound analytes from bound analytes.

Centrifugation is time-consuming, requires expensive and energy-consuming equipment, and poses radiological, biological and physical hazards. Magnetic separation is relatively quick and It is easy and requires simple equipment. For affinity chromatography system The use of non-porous adsorbents or coupled magnetic particles in chromatography system, and requires fewer samples than conventional affinity chromatography systems. resulting in contamination of materials.

The practical development of magnetic separation depends on several important properties of the magnetic particles developed to date. was hindered by. For example, large magnetic particles (e.g. 10 microns in solution) (having an average diameter larger than the average diameter of the magnetic field) responds to weak magnetic fields and magnetic field gradients. Stiff Since, they tend to settle quickly and are not suitable for reactions requiring homogeneous conditions. limits their effectiveness. Also, large particles are more restricted than small particles. surface area per weight, so less material cumulates on them It is possible. Examples of large particles are Robinson et al., Biotech, Bioe ng, 15:603-606 (1973) (these may be 0-125 microns); Mobach and And-erson, N ature, 270:259-261 (1977) (these are 60-140 microns in diameter) and Guesdon et al., J. AI Ie rgY C1in, InITlnol, 61(1):23-27(1 X78) (these are 50 to 160 microns in diameter).

Smaller magnetic particles are also disclosed. For example, Hersh and Maverbau The composite particles described in U.S. Pat. No. 3,933,997 to M. Iron (Fe+O4) carrier particles, these have a diameter of 1.5-10 microns. It was reported that it grows.

However, the reported sedimentation rate in 5 minutes and only 12 mg/g of particles Based on the protein coupling capacity of , the actual size of the particles in solution is 10 It is expected to be much larger than a micron. L, S, Hersh and S, Y averbaum, CI in Chem, Acta, 63:69-7 2 (1975).

Small magnetic particles (e.g., with an average diameter smaller than about 0.03 microns in solution) ) can be kept in solution by thermal agitation, yet have a tendency to spontaneously settle. There is no. However, the magnetic field required to remove such particles from solution is The fields and magnetic field gradients are large, and heavy and bulky magnets are required to generate these fields. They require stones, which are inconvenient for use in benchtop experiments. For example, 5oo o A magnet that can generate a magnetic field exceeding Oersted has a diameter of 0.03 microns or more. Typically needed to separate small magnetic particles.

For example, a reactive substrate may be provided with a chemical functional group that binds an enzyme or antibody. In order to achieve this, the ferromagnetic carrier particles generally need to be coated. this purpose Silane polymers are often used for this purpose. U.S. Patent No. 3.933.99 The particles described in the specification of No. 7 react with anti-digoxin antibodies and carry the antibodies. coated with a silane that can chemically couple to the ear particles. Various Silanka Pulling agents are described in U.S. Pat. No. 3,652,761 and are is incorporated herein by reference. Silanization operations known in the art are , generally the medium selected for the polymerization of the silane and its placement on the reactive surface. They differ from each other in some respects. The medium is generally an organic solvent, such as toluene (H, W., Weetall, Methods of Enzymology, Mo. 5bach (ed.), 44:134-148.140 (1976)), Meta. Nol (U.S. Pat. No. 3,933,997) or chloroform (U.S. Pat. No. 3,652,761). Also, hydroalcoholic solutions with acids and silane deposition from aqueous solutions have been used. H, W., Weetall, M. methods of Enzymology, cited above, p. 139 (1976 )checking.

Silane-coated particles have several drawbacks. For example, air drying and/or The dehydration method used to dry the coating, such as oven drying, The silanized surfaces of the silanized particles are allowed to come into contact with each other, and the bonds between the particles (silanized cross-linking between particles due to formation of particles, van der Waals interactions or physics between adjacent particles (including adhesion). This interparticle bonding is caused by the individual carrier particles Covalently or physically bonded silanized carrier particles of diameter significantly larger than Forms aggregates. Such aggregates have a low surface area per unit weight; Therefore, they have a low ability to couple with molecules such as antibodies, antigens or enzymes. Ru. Also, such aggregates have gravitational settling times that are too short for many applications. Ru.

Magnetic particles capable of binding bioaffinity reagents can be used, for example, in radioimmunoassays. It is useful for separating desired biological components from a sample. radio immunoassay A (referred to as R[A) is the concentration of the substance with the radiolabeled substance binding to the antibody. is a term used to describe a method for analyzing. radioactivity bound The amount of test substance can be altered by the presence of unlabeled test substance that can bind to the same antibody. .

The unlabeled substance, if present, has a binding site relative to the labeled substance. compete, thus reducing the amount of radioactivity bound to the antibody. The decrease in bound radioactivity is , can be correlated to the concentration of unlabeled test substance by means of a standard curve. RIA essentials The first step is the separation of bound and free label, which is necessary to quantify the bound fraction. Does it need to be done?

Various conventional separation methods (coated tube separation, particulate separation, and dual antibody separation) ) or RIAs. In U.S. Patent No. 3,646,346 Coated tubes as described can separate bound and free labels without centrifugation. There are two important issues at stake in making the separation of signs possible. Firstly, Chu The surface of the tube limits the amount of antibody that can be used in the reaction. Second, antibodies are derived from antigens. removed at a distance (as large as 0,5 cm) and slows down the reaction between antibody and antigen. Ru. G, M, Par-sons, ktechods in Enzymo Logy, J. F. Langone (ed.), 73:225 (+981) :P, m, Nayak.

The Ligand Quarterly, 4(4):34 (1981) checking ...

The antibodies were attached to a particulate system that facilitates separation. U.S. Patent No. 3.652.7 See No. 61 and No. 3.555.143. Such a system is large have a large surface area that allows almost unlimited amounts of antibodies to be used; Particulate matter often settles during the assay. Tubes are frequently agitated to ensure partial uniformity. Is it necessary to get it? P, M, Jacobs, The Ligand See Quarterly, 4(4):23-33 (1981)■B Centrifugation is additionally required to achieve complete separation of bound and free label.

Antibodies can react with labeled and unlabeled molecules, followed by a second reaction. Separation is performed using a second antibody that is increased relative to one antibody. It is called the two-antibody method. Techniques used can either obtain antibody homogeneity during reaction with a label or an incubation period for reaction, followed by a centrifugation period to pellet the antibody. Requires separation.

nortributyrin, methotrexate, digoxin, thyroxine and human fertility In an attempt to eliminate the centrifugation step in lactogen radioimmunoassays, antibody or coupled to a magnetic carrier. R,S, Kamel et al., C1in, CheIn, , 25(12): 1997-2002 (1979j: R, S, Kamel and J, Gardner, CI i n, Chem , Acta, 89:363-370 (197Bj; U.S. Patent No. 3, 933.997: C, Dawes and J, Gardener, CI in, Chem-Acts, 86:353-356 (1X78); D. S.

1thakissios et al., C1in, Chea Acta, 84 : 69-84 (1978); D, S, l thak weir@ss ios and 1.0° Kub iatowi CZ, CI in, Che+n, , 23 (11 ): 2072-2079 (+977): Nari, mye et al., CI i n, Chem.

See Acta, 69:387-396 (+976). The above teaching is included herein by reference. Such methods are suitable for large particle sizes (diameter 1 0 to 100 microns), and it is difficult to disperse and preserve the antibody during the assay. Stirring is required to ensure consistency. Substantial separation occurs by natural sedimentation in the absence of a magnetic field. therefore, these traditional methods are actually magnetically assisted gravitational separations. Only. In U.S. Pat. No. 4,177,253, Davies and Janata has a magnetic coating (2 microns to 10 microns) that covers part of the particle surface. Hollow glass or polypropylene (diameter 4-10 microns) to grow (thickness) A magnetic material was used. anti-estradiol antibodies or cambu to such particles. and demonstrated their potential efficacy in Nistranol RiA. It was. This method may have eliminated the sedimentation problem, but it still The particle size and magnetic coating limit the surface area and therefore the potential for antibody coupling. limits the availability of parts.

Magnetic separation has been applied in other biological systems besides RtA. Fluorescence immunoassay Some non-radioactive assays such as (FIA) and enzyme immunoassay (EIA) immunoassays have been developed, and these are coupled to antibodies (or antigens or molecules). (coupled) magnetic particles. The principle of competitive binding is that fluorophores and enzymes In the case of RIA and F The same applies to IA and EIA. By way of example, M. Pourfarzan Eh et al., Masaru, S., and Ron e1 et al. Cortisol and Phenyl Using Ringed Ferromagnetic Cellulose/Iron Oxide Particles We have developed a magnetizable solid-phase FIA. M., Pourfarzaneh et al. , CI in, Chem, , 26(6) Near 3O-733 (1980) :R, S, Ka high ■ attack ACl1n.

See Chem., 26(9):1281-1284 (1980).

Noncompetitive Solid Phase Sandwich Technique EIA for the Determination of IgH J. Allerg. y C1in, [mmu-nol, 61(1):23-27(+978) J. L. Guesdon. By this method, glutar Coupling to magnetic polyacrylamide agarose beads by aldehyde activation The labeled anti-1gE antibody is incubated with a test sample containing IgE to allow binding. make it possible. Bound 1gE is alkaline phosphatase or β-galactosidase quantification by adding a second labeled anti-1gE antibody. Enzyme mark The identified second antibody forms a complex with the IgE bound to the first antibody, and The particles are magnetically separated. The enzymatic activity associated with the particles (which (proportional to total IgH) is measured [gE allows quantification.

Magnetizable solid-phase non-immune radioassay for vitamin BI2, S. Itthak issios and 0. Kub iatowicz, CI i n, Chem-, 23(11): 2072-2079 (1977) ■ ■■B non-exempt The principle of competitive binding in epidemiology radioassay is the same as in RIA, and both The assay uses a radioisotope label. However, RIA contains vitamin B 12 species of biological molecules and specific or non-specific binding carriers or or based on binding or interaction with receptor proteins. On the other hand, Ith akissios and Uubiatowicz's magnetic particles are water-insoluble proteins. It contained barium ferrite particles embedded in a matrix.

In addition to their use in the solid-phase biological assays described above, magnetic particles can be used in a variety of other was used for biological purposes. For example, magnetic particles can be mixed in cell sorting systems. used to isolate and select viruses, bacteria, and other cells from collective populations. Ta. U.S. Pat. No. 3.970.518, U.S. Pat. No. 4.230.685 and U.S. Pat. See No. 267,234. These teachings are incorporated herein by reference. be caught. They are affinity chromatography systems that selectively select molecules from solution. It is particularly advantageous for purification from colloidal suspensions. be. K, Mo5bach and Shi.

Anderson, Nature 170:259-261 (1977). About Teru. This document is included herein by reference. In addition, magnetic particles are supported on a solid phase. It was used as an enzyme system immobilized in the body. coupled to magnetic particles Enzymes must be kept in contact with a substrate for a sufficient period of time to catalyze a biochemical reaction. It will be done. The enzyme can then be magnetically separated from the product, removing unreacted substrate and potentially reusing it. can. Magnetic particles can be used to bind α-chymotrypsin, β-galactosidase in an immobilized enzyme system. No. 4,152,210, which patent is incorporated herein by reference. ) and glucose isomerase (U.S. Pat. No. 4,343,901) specification, this patent is incorporated herein by reference). .

Summary of the invention The present invention provides a bioaffinity sorbent coated with an organometallic polymer capable of binding bioaffinity adsorbents. magnetically responsive particles and separation of biomolecules from surrounding media or surrounding media In the directed move+ent of these molecules in regarding their use. Organometallic coatings are adsorbed to magnetic particles or Covalently bonded to air particles. For the preparation and use of organometallic coated magnetic particles Methods and compositions are provided.

The magnetic particles are surrounded by an organometallic polymer coating that is adsorbed or covalently bonded to the particles. containing a magnetically responsive metal, metal alloy or metal oxide core. organometallic po Remer is produced from organometallic monomers, which are applied to metal particles in situ. Thermal crosslinking forms an adsorbed or covalently bonded polymer coating. organic chita preferred, but other transition metals, such as zirconium (Zr), Hafnium (Hf), vanadium (V), tantalum (Ta) and niobium (Nb) , or coordination complexes of post-transition metals such as tin (Sn) and antimony (Sb) Organometallic polymers produced from Various bioaffinity absorption Covalently bonded to the organometallic polymer coating by adhesive or selected coupling chemistries. Can be combined.

More specifically, the present invention provides a metal, metal alloy or metal oxide core and an aliphatic moiety. and organic functional groups (to which various organic and/or biological molecules can be coupled) The present invention relates to a method for preparing magnetically responsive particles comprising an organometallic coating (which can be used in the present invention).

Coupled or uncoupled particles are separated into an aqueous medium. can be dispersed to produce a colloidal dispersion, which is stable, i.e. the particles Resist force settling. Particles can be regenerated from the medium by applying a magnetic field.

Preferably, the particles are superparamagnetic, i.e. they do not form magnetic aggregates. that the particles exhibit no residual magnetization after removal of the magnetic field allowing them to be redispersed; preferable.

The organometallic-coated magnetically responsive particles of the present invention can be used with other biological components of the species. affinity for, or ability to adsorb, molecules or organic molecules; biological molecules or organic molecules by organic functional groups with the ability to interact with those molecules. Can be coupled to molecules. The thus coupled particles can be processed in a separation process or are used in various in vitro or Can be used for in vivo systems, including immunoassays, other biological assays, and biochemical reaction or enzymatic reaction, affinity chromatography purification, cell sorting and Including diagnostic and therapeutic uses.

The organometallic coated magnetic particles of the present invention have an excellent composition for use in bioseparations. , size, surface area, flexibility of coupling, sedimentation and magnetic behavior. Main departure Ming's magnetic particles can be used in many assays, enzyme immobilization, cell sorting and Suitable for affinity chromatography operations and has actually been used in the past for such operations. Solving many of the problems experienced with particle settling and reuse.

Detailed description of the invention The magnetically responsive particles of the present invention have the same size, surface area, and gravity characteristics as previously developed magnetic particles. Solve problems related to sedimentation velocity and magnetic properties. Gravity settling for more than about 24 hours This can be achieved with the magnetic particles of the present invention over time. The gravity settling time depends on the turbidity of the particle dispersion. It is defined as the time to decrease by 50% in the absence of a magnetic field gradient.

The magnetic particles of the present invention contain reactive groups or agents, e.g. chemically reactive groups, biologically reactive groups. coated with an organometallic polymer capable of binding functional groups or bioaffinity agents. a magnetically responsive metal, metal alloy or metal oxide core. organometallic poly The mer is adsorbed or covalently bonded to the magnetic particle. “Magnetically responsive particles” or “magnetic The term "air particles" refers to particles that are dispersed or suspended in an aqueous medium with little or no gravitational settling. Defined as any particle that can be separated from a suspension by application of a magnetic field. be done.

“The term magnetic core” refers to the ferrospinel structure. and trivalent and divalent atoms of the same or different transition metals or magnetic metal crystal groups. crystals or groups of crystals of transition metals, their alloys or magnetic metal oxides, including cluster). Useful as a magnetic core material in the present invention The metals, alloys and oxides in the Periodic Table include Groups 4b, 5a and 5b, Metals found in Group 7a as well as Group 7a, alloys and oxides based on these metals including. These include, for example, divalent transition metals such as iron, magnesium, manganese, Guns, cobalt, nickel, zinc and copper, alloys of these metals, e.g. Gold or oxides (e.g. iron-magnesium oxide, iron-manganese oxide, iron-cobal iron oxide, iron nickel oxide, iron zinc oxide and steel oxide), cobalt ferrite iron, samarium cobalt, barium ferrite, and aluminum-nickel - Cobalt and magnetite (Fez04), hematite (Fe203) and chromium dioxide Contains metal oxides containing aluminum (CrO□).

By way of example, the magnetic core may be a cluster of superparamagnetic crystals of iron oxide, or may contain clusters of superparamagnetic or ferromagnetic crystals of the oxide; may consist of superparamagnetic or ferromagnetic single crystals of iron oxide or metal alloys. stomach.

The particles of the present invention are about 0.003 to about 1.5 microns in diameter, and about 50 to 150 microns in diameter. It is preferred that the bioaffinity adsorbent has a surface area of m2/g. , giving a high capacity for coupling of chemically or biochemically reactive groups . Magnetic particles in this size range solve the problem of rapid settling of large particles or large magnets that generate the magnetic fields and magnetic field gradients needed to separate small particles. Eliminate the need to. For example, the magnet used to perform the magnetic particle separation of the present invention only requires generating a magnetic field of about 100 to about 1000 Oerste soto. do not have. Such a magnetic field creates a permanent magnet that is smaller than the container holding the dispersion of magnetic particles. It can be obtained in stone and is thus suitable for tabletop use.

Particles with superparamagnetic behavior are preferred. After all, superparamagnetic particles are ferromagnetic Because it does not exhibit particle-related magnetic agglomeration, yet allows for redispersion and reuse. be. The term "superparamagnetism" Iron, cobalt, nickel or other gold with a crystal size of less than about 300 defined as the magnetic behavior exhibited by alloys or metal oxides of the genus; It is characterized by responsiveness to magnetic fields without causing magnetization.

Ferromagnetic particles may be useful in certain applications of the present invention. “Ferromagnetic The term "magnetism" refers to crystals with a crystal size larger than about 500 defined as the magnetic behavior exhibited by iron, iron alloys, or iron oxides that It is characterized by its responsiveness to magnetic fields with a remanent magnetization larger than about lO after the removal of do.

The particles or crystals are then coated with organometallic monomers that can be adsorbed or co-bonded to the magnetic particles. coated with a mer material. The organometallic monomers useful in the coated particles of the present invention are selected from transition metals and/or post-transition metals (these may form stable coordination compounds) ) and organic coordination complexes of organic ligands, which are adsorbed or shared by magnetic particles. can be bonded and cross-linked in situ on the particle surface, thereby forming an organometallic polymer coating. can be formed. Organometallic monomers are used in polymers or bioaffinities formed from them. can form covalent bonds with affinity or chemical affinity adsorbents. There is a need for it to be functionalized or derivatized to make it functional. For this purpose, The machine metal polymer has an aliphatic “spacer arm” (this is a bioaffinity post-functionalization or induction with Becomes a conductor. A "spacer arm" can contain from about 2 to about 60 atoms, e.g., a carbon atom. It is an aliphatic hydrocarbon having a nitrogen atom and/or an oxygen atom. spacer The purpose of the arm is to react with chemical groups, biochemical groups or bioaffinity adsorbents. a non-reactive linker (or spacer) between the organic group and the polymer chain, and The objective is to impart a suitable hydrophilic/hydrophobic balance to the surface of the coated particles. organic The group is generally a reactive group coupled to an aliphatic or aromatic moiety, e.g. , amine group (Nl2), carboxyl group (COOH), cyanate group (CN) , phosphate group (PO3H), sulfate group (SO,H), thiol group (S H), hydroxyl group (OH), vinyl group (C=C), nitrate group (N02 ), an aldehyde group, an epoxide group, a succinamide group, or an acid anhydride group.

Particularly useful organometallic compounds include selected transition metals (e.g., Ti, Zr, Hf , ■, Zn, Cd, Mn, Te, Re, Ta, Nb) and/or Formed from post-transition metals (e.g. Sn, Sb, AI, Ga, in, Ge) It is a coordination complex. Particularly preferred are organic titanium compounds.

Organotitanium compounds that are useful include, for example, titanium-tetraisobropoquinoto, Amino-hexyl-titanium-triisopropoxide, amino-propyl-tita titanium-triisopropoxide and carboxyl-hexyl titanium-triisopropoxide Contains propoxide. In one embodiment of the invention, amino-hexyl-titanium - tri-isopropoxide is coated onto selected magnetic particles and cross-linked with heat An organic chain containing a group spacer arm (hexyl moiety) and an organic functional group (amine group) Forms a tan polymer coating.

Optionally, the coated particles can be coated with organometallic polymers or bioaffinity adsorbents or The chemical affinity allows the adsorbent to form a covalent bond with the latter. be enabled. In one aspect of martial arts, it lacks spacer arms and organic functional groups. Organotitanium polymers such as titanium-tetra-isopropoxide can be The organic titanium polymer becomes coated with air particles and adsorbed onto the particle surface. partially crosslinked at about 40°C for a period sufficient to allow .

The organic titanium-coated magnetic particles are then coated with 1-hydroxy-6-aminohex Amino-hexyl-titanium trichloride is activated by reaction with agents such as Produces isopropoxide. The coating is then crosslinked at high temperature to Organotitanium polymer coating with pacer arms and organic functional groups (i.e., amine groups) Form a covering. The functionalized particles then bind to the selected bioaffinity adsorbent. Can react or couple with adhesives.

Magnetic core particles are prepared according to the general procedure below. Precipitated in metal salts or bases This produces fine magnetic metal oxide crystals. The crystals are redispersed and then washed with water. , washed in electrolyte. If crystalline or superparamagnetic, between magnetic separation or washing Can be used to collect crystals.

In one embodiment of the invention, the superparamagnetic iron oxide particles are dissolved in a divalent iron salt in an aqueous base. (Fe2″″) and trivalent iron salts (Fe″), such as ferrous ammonium sulfate , Fe2(NO3)(SO,) and ferric sulfate, Fe2(SOJt). I can't believe it. The ratio of Fe"&Fe"" to counterions must be maintained to maintain a constant molar amount of iron. by increasing the amount of Fe”+ without causing appreciable changes in the final product. You can change it. Nitrate ion, sulfate ion, chloride ion or hydroxide ion Counterions containing ions are useful in the method.

Fe''/Fe'' ratios of about 21 to about 41 are useful in the present invention. Approximately 2:1 Fe”/ A Fe''/Fe'' ratio of 11 is particularly beneficial. /Fe” ratio, resulting in magnetic particles of slightly inferior quality to those produced by the The particle size is even larger than the magnetic particles produced from 2-1 or 4:1 Fe”/Fe”. is unevenly distributed.

In this embodiment, an aqueous solution of an iron salt or a base, e.g. ammonium hydroxide, hydroxide Crystalline superparamagnetic iron oxide mixed in sodium or potassium hydroxide This results in the formation of a precipitate. The precipitate is magnetically separated and redistributed until a neutral pH is reached. It is washed repeatedly with water by sprinkling. The precipitate is then mixed with approximately the same amount of water-miscible solvent. The garlic was dried in a molecular knob to remove all the water), e.g. , acetone, methanol or ethanol about 5 times.

The repeated use of magnetic fields to separate iron oxide from suspension during the washing process Facilitated by magnetism. Regardless of how many times the particles are exposed to the magnetic field, they are never magnetically agglomerated, so that they can be redispersed by gentle agitation. ferromagnetic Sexual particles cannot be prepared by this washing step. After all, they are exposed to magnetic fields. This is because they tend to agglomerate magnetically and cannot be uniformly redispersed.

Other divalent transition metal salts, e.g. magnesium, manganese, kohaltonisoke Magnetic gold salts, zinc and copper salts are used to replace iron salts during precipitation or milling operations. metal or metal liquefied products. For example, instead of FeCl2 in the above operation Substitution of divalent cobalt chloride (C1) CI2) yielded ferromagnetic metal oxide particles . Ferromagnetic metal oxide particles, such as those produced by CoCl2, are centrifuged between washes. or the particles are washed in the absence of a magnetic field by using conventional techniques such as filtration. It is possible to avoid magnetizing the child. Ferromagnetic metal oxide particles or water obtained as long as it is of a sufficiently small diameter to remain dispersed in the They are also used in systems requiring single magnetic separation, e.g. radioimmunoassay consisting of coated with organometallic polymers for use in bioaffinity adsorbents can be cambred to. Ferromagnetic limiting the usefulness of particles in the process.

In another embodiment of the invention, magnetic core particles are obtained by precipitating metal powders. The particle size can be reduced by milling the precipitate, for example in a ball mill. It can be made by In this method, the metal powder is, for example, Fe” salt or or Fe’“ salt from an aqueous solution with sodium borohydride. For example, , an aqueous solution of ferrous chloride (FeCI2) is converted into sodium borohydride (NaBH4) mixed with iron to form a fine iron precipitate. The properties of the resulting metal powder are selected unaffected by the valence of the counterion or iron metal salt. Complete precipitation is borohydride occurs naturally after addition of compounds. The magnetic metal powder is then recovered by filtration, Washed approximately 5 times with equal volumes of water to remove all soluble salts, then washed with equal volumes of dry water. Washed five times with dry acetone to remove any residual water. The particles are 1/4 inch Add approximately 1-25% concentration to a commercially available ball mill half-filled with a stainless steel pole. It is added as a slurry of 100 ml and pulverized for 3 to 30 days. At the end of the milling period In this case, the superparamagnetic particles described in the slurry of superparamagnetic metal or the part of the slurry produced are coated and functionalized.

In another embodiment of the invention, the magnetic core particles are metallocenes, e.g. Rocene (dicyclobentadenyliron, C3°Hl.Fe) is reacted with iron hydroxide (m). It is created by In this embodiment, aqueous ferrocene (or other An aqueous slurry of astringent iron hydroxide (11) was prepared separately. be done.

Ferrocene slurry is made by, for example, pulverizing a mixture of ferrocene and water in a ball mill. Prepared by crushing. Slurry of iron(II) hydroxide can be made of iron(II) hydroxide, for example Precipitating an aqueous slurry of monoferrous with ammonium hydroxide to produce ferrous hydroxide It can be prepared by: The two slurries are then combined and pulverized, e.g. For example, it produces fine magnetite particles. Other metallocene compounds (e.g. locene, cobaltocene) is mixed with ferrocene to form various magnetic ferrite particles. can be generated. This method was filed by M. S. Chagnon at the same time as this case. Pending U.S. Patent Application No. (Attorney Docket NαOQC9O-02) This specification is fully described in the specification, and the teachings thereof are incorporated herein by reference.

In one embodiment of the invention, the coating around the magnetic core particle is an amino-propylene It is pyru-titanium-triisopropoxide. The polymerization acetates the magnetic particles. redispersion in a solution of organic titanium, adding organic titanium monomer, and then crosslinking with heat. This is done by “Coupled Magnetically Responsive Particles” or “Force, Blind” The term “magnetic particles” refers to the use of one or more bioaffinity adsorbents or Represents any magnetic particle coupled by a covalent bond, whose covalent bond is magnetic Functions that can be used for binding both particle coatings and bioaffinity adsorbents. Depending on the functional group, amide, ester, ether sulfonamide, disulfide, azo Alternatively, it may be any other suitable organic bond.

Preferred magnetically responsive particles of the present invention maintain superparamagnetic properties and have low magnetic fields (10 A class of superparamagnetic crystals that provides effective separation of particles between 0 and 1000 oersted It has a metal oxide core containing a Particle aggregation is controlled during particle synthesis and The dispersion of particles is used for the intended biological assay or other application. sufficiency to avoid appreciable gravitational settling for a sufficient period of time to allow It produces particles that are preferably small in size. Superparamagnetic core in magnetically responsive particles The advantage of having a magnetic field is that such particles can be repeatedly exposed to magnetic fields. . Superparamagnetic particles do not exhibit remanent magnetization and have no coercive strength, so they are not magnetically It is less agglomerated and thus the particles can be redispersed and reused. Even after coating, Preferred particles of the invention having cores made of clusters of crystals have units of Significantly higher surface area per weight and correspondingly higher coupling capacity in general , which indicates that such particles have an open or porous structure.

The bioaffinity adsorbent can be used to bind the organometallic metal of the present invention by conventional coupling chemistry. can be covalently bonded to magnetic particles coated with genus. Performing several coupling reactions I can do it. for example, (a) If the ligand being coupled contains an amino group, it is activated. Can be coupled directly to organometallic polymers. If different functional groups are desired, It can be used, for example, in organometallic polymers such as titanium-tetra-impropoxy A spacer containing a functional group is created by the sequential reaction of (d) and an ω-functional high molecular weight alcohol. - Can be introduced by adding an arm. Then, the amino group of the ligand is can be coupled to a free functional group of the sirarm. or (b) the ligand or amino acid. If it contains an aldehyde group instead of a Free amino group of can (i.e. alkane spacer arm with amino functionality) can be directly coupled to

The term “bioaffinity adsorbent” refers to or any biological molecules or other organic components that may bind or interact non-specifically. child, whose binding or interaction is defined as a “ligand/ligate” binding or a complementary This can be called an interaction between antibody/antigen, antibody/habten, enzyme/substrate, and carrier. protein/substrate, lectin/carbohydrate, receptor/hormone, receptor -/effector or receptor/inducer binding or interaction. Examples include, but are not limited to.

The coupled organometallic coated magnetic particles of the present invention can be used in immunoassays. or other binding assays for the measurement of analytes in solution.

The term “immunoassay” refers to the use of polyclonal or monoclonal antibodies. determines the concentration or amount of analyte in solution based on immune binding or interaction of antigen with the body Defined as any method for measuring (a) the unbound analyte; (b) requires the separation of bound analytes and/or unbound analytes; Radioisotope drift, fluorescent labeling, and enzyme labeling as a means to measure analytes , using a chemiluminescent label or other label, and (C) a coupled measurable “Competition” occurs when the amount of labeled label is generally inversely proportional to the amount of initial analyte in solution. and the amount of measurable label bound or generally in solution for the first analysis. It may be described as "non-competitive" if it is directly proportional to the amount of interest. Label is in antigen, antibody or, in a two-antibody method, in the second antibody. Immunoah Sei is radioimmunoassay (RrA), immunoradiometric assay ( IRMA), Fluorescence Immunoassay (FIA), Enzyme Immunoassay (E RA), and sandwich immunoassays, or are limited to these. Not determined. The analyte or high affinity adsorbent is, for example, an antibody, an anti- original, habten, enzyme, apoenzyme, enzyme substrate, enzyme inhibitor, cofactor, bound by a nucleic acid, a binding protein, a carrier protein, a binding protein compounds bound by carrier proteins, lectins, monosaccharides, May include polysaccharide necks, hormones, receptors, libressors and inducers.

Such an anesthetic converts a sample containing an unknown concentration of analyte into an unlabeled analyte. and a pyoa that can bind to or interact with both the analyte and the analyte of interest. Label a known amount in the presence of magnetic particles coupled to a affinity adsorbent. The particles are mixed with the analyte, bonded or interacted with, and the particles are magnetically separated. Analyze the amount of label associated with the particles in the sample by comparing the amount of label to a standard curve. Preferably, this is done by measuring the concentration of the target.

The term “binding assay” or “non-immune assay” refers to antibody/antigen binding or or interactions between bioaffinity adsorbents and other biological molecules or Partition in solution based on specific or non-specific binding or interaction with non-organic molecules. Defined as any method for measuring the concentration or amount of an analyte; a) requires the separation of bound analytes from unbound analytes; and (b) requires the separation of bound analytes from unbound analytes; radioisotope drift as a means of measuring analytes and/or unbound analytes. label, fluorescent label, enzymatic label, chemiluminescent label or other label, and ( C) The amount of measurable label bound is generally inversely proportional to the amount of initial analyte in solution. For example, the word “competition” is used, or the amount of measurable label bound is specified. Generally, it can be described as "non-competitive" if it is in the initial solution.

The organometallic-coated magnetic particles of the present invention can be used in immobilized enzyme systems, especially for enzyme regeneration. It is available if required. The term “immobilized enzyme system” refers to any Defined as an enzyme-catalyzed biochemical transformation or synthesis or degradation, in which case the enzyme The molecule or its active site is not easily soluble or adsorbed or covalently bound to a solid support the carrier is suspended in or contacted with the surrounding medium; The dough may be regenerated or separated from the method described above. In this case, the enzymatic reaction is An enzyme is camped under conditions sufficient for a reaction between the enzyme and the substrate to occur. Magnetic particles are dispersed in a reaction mixture containing one or more substrates, and the product and unreacted groups are separated. magnetically separate the enzyme-magnetic particles from the reaction mixture containing the enzyme and, if desired, the particles. is carried out by redispersing the enzyme into a new substrate, thereby reusing the enzyme. .

Affinity stomatography separation and cell sorting can be performed using the magnetic particles of the present invention. It can be done using The term “affinity stomatography” , select molecules are attached to a solid phase support by adsorption or covalent bonding. separation from its surrounding medium based on its binding or interaction with the affinity adsorbent. defined as a method of separating, isolating, and/or purifying the carrier in the surrounding medium. suspended or in contact with a bioaffinity adsorbent or cup Solution containing molecules or cells from which ringed magnetic particles are isolated and/or purified or dispersed in suspension to bind the bioaffinity adsorbent and the desired molecules or fine particles. The particles are magnetically separated from their solution or suspension, and the isolated particles are Regeneration or separation of cells or cells from said medium by collecting them from magnetic particles It is possible.

The organometallic-coated magnetic particles of the present invention have a special Diagnostic location of cells or tissues recognized by bioaffinity adsorbents For confirmation and magnetically guided delivery of therapeutic agents coupled to particles to the pathogenic site. It is further contemplated that it may be used in an in vivo system for development.

A magnetic separation time of less than about 10 minutes or a container containing a dispersion of magnetic particles of the invention to its capacity. The magnet of the present invention is made by contacting the magnetic pole face of a magnet with a volume not larger than the volume of the container. This can be achieved with air particles. The magnetic separation time is the time for the turbidity of the dispersion to decrease by 95%. It is defined as

Additionally, as a coating surrounding the metal oxide core of the magnetic particles described herein, The use of fully functionalized organometallic polymers allows for even more limited coupling functionality. compared to other magnetic particle coatings known in the prior art having Allows coupling of a wide variety of molecules under specific coupling conditions.

The invention is further illustrated by the following examples.

Example 1 Preparation of superparamagnetic magnetic particles Ferrous chloride (■R Scientific) 200g (1.58 mol) and salt 325 g (2.0 mol) of ferric oxide was dissolved in 3 liters of water. Ammonium hydroxide (seedlings) Scientifica) 2000g of a thick liquid at 50ml/min with constant stirring The temperature of the solution was maintained at 25-40°C. ammonia hydroxide After the addition of Fe.04 was completed, the aqueous slurry of magnetic particles (Fe.04) was cooled to room temperature. Ta.

Titanium triisobroboxide (Tizol TPT DuPont, Wilmington) , Delaware) and 0.1 mole of 6-amino-1-hexanol. Add to a 0 ml beaker and stir for 1 minute at room temperature to prepare aminohexyl titanium. 0.1 mol of tri-isopropoquine was produced. The reaction mixture was heated to 70°C for 1 The inobrovir alcohol produced during the reaction was evaporated by heating for 0 minutes.

The material was cooled to room temperature and used for preparing the tetravalent titanium organometallic coating in Example 3. It was used as a monomer.

Example 3 Preparation of Magnetic Particles Coated with Amine-Functional Organotitanate According to the procedure shown, 4 mol of FeCls and 2 mol of FeClt are added to 4 liters of distilled water. and precipitated with 16 molar ammonium hydroxide. Wash the precipitate 5 times with water, and Washed three times with setone. N. N-dimethylformamide (Dlt(F)) as shown below Ratio: lOml of DlitF per Fein4Ig was added to the precipitate. the mixture The material was placed in an Eiger Mill and allowed to run for 10 minutes. It was continuously crushed. Next, transfer the mixture to a beaker and heat at 100°C for 30 minutes. The mixture was heated without stirring for a while. The amine-functional organotitanate prepared in Example 2 was prepared. Immediately after preparation, add 3 g of amine-functional organic titanium to the mixture without constant stirring. It was added at a ratio of 41 g of dry FezO.

The mixture was then heated at 65°C for 20 minutes with stirring and I went through Gar Mill twice. The resulting material was washed 5 times with water and the coated particles were coated with 2000 g. The aqueous waste solution was collected using an external magnetic field and decanted.

Example 4: Preparation of Alternating Functional Mono-Nonfunctional Organotitanate Monomers Organotitanes A comixture of amino-functional hexanol and hexanol (comi-xtur e) to produce monomers that develop reduced amine functionality. , the procedure described in Example 2 was followed. 6l molar ratio of hexanol and 6-amino -1-Hexanol was mixed in a 50 ml beaker for 1 minute. Thisol TPT and 1 mol of alcohol per 1 mol of Thizol TPT in the alcohol mixture. It was added at the ratio of The reaction mixture was stirred for 1 minute and heated to 70°C for 10 minutes. The isopropyl alcohol produced by the reaction was evaporated and cooled to room temperature. profit The compound is an organic titanate, containing alternating non-functional hexyl groups, i.e. amino groups. 6-Aminohexyrutitanium triisopropoxy with missing hexyl chain It was de. 6-amino-1-hexanol. Thisol TPT: Hexanol The weight ratio was 126.9.6.

This compound was used as a monomer for making the organotitanium coating described in Example 5. I used it.

Example 5 Preparation of amine-functional organotitanate magnetic particles Amici-functional organotitanate The procedure described in Example 3 was followed except that the sample was the material prepared in Example 4. Ta. The mixture of magnetic particles and organic titanate monomer was heated to 95°C with constant stirring. Heat for 1 hour and mill in an Eiger mill for 4 minutes.

The mixture was washed with water nine times. Adivic acid was added at 0.5 mol of adivic acid per mol of total particles. Bic acid was added at the same ratio as Bic acid. Add 1 mol of carposiimide (CDI) and mix The materials were mixed in a Pall mill for 30 minutes. 1.1 mole of 6-hexane-diamine in all particles 1.6-hexane-diamine was added at a ratio of 0.5 moles per mol of 1.6-hexane-diamine. 1 mole of C DI was added and the mixture was mixed for 30 minutes. The resulting material was washed 5 times with water and granulated. The particles were collected using an external magnetic field of 2000 Gauss and the aqueous effluent was decanted.

20% by weight of Huekousen (dicyclopentadenyl iron) in water: Strem Chemical Co. , Newbury Port, Massachusetts) was added to the Hue It was prepared by mixing locene with water. Add slurry to commercial ball mill did.

Fill the mill halfway with 1/4 inch stainless steel balls and mix the slurry for 2 hours. Finely ground over a period of time.

Make a second ferrous hydroxide (iron hydroxide (I+)) slurry by following the steps below. Ta.

An aqueous solution containing 20 g of ferrous sulfate (VWR Scientific) was mixed with hydroxide. Precipitate using 50 g of ammonium concentrate to produce gelatinous ferrous hydroxide. did. The gel was filtered and the filtrate was washed with 5-long volumes of water. Then wash The cleaned gel was made into a lO% aqueous slurry and milled for 5 hours as described above.

Mix ferrocene slurry and hydroxide slurry, and pulverize the mixture for one day. Fine FexO particles were formed. These particles are about 100 people in diameter and have a magnetic It was responsive to the situation. These particles were prepared as described in Examples 2-5 above. Can be coated.

Example 7: Preparation of subdomain nickel-ferrite particles Ferrocene slurry of Example 6 - 20% chive kerosene slurry (ncyclopentadenyl nitride) in place of 100 g Kel: Strom Chemical Co., Newbury, Massachusetts) 50 Example 6 except that a mixture of Subdomain nickel-ferrite particles were prepared according to the procedure shown. about 100 people Magnetically responsive nickel-ferrite particles with particle size are produced by this method. Built.

Example 8 Preparation of two subdomain cobalt ferrite particles Ferrocene slurry of Example 6 -20% by weight of cobalt sense slurry (dicyclobentadenyl) in place of 100g Cobalt, Strom Chemical Co., Newbury, Massachusetts) Example 6 except that a mixture of 50 g and 50 g of ferrocene slurry was used. Sub-domain cobalt ferrite particles were prepared according to the procedure described above. About 100 particles Magnetically responsive cobalt-ferrite particles having a size are produced by this method. Ta.

200g (1.58 moles) of ferrous chloride in water II! dissolved in. Dry sodium borohydride 500 g of thorium was added to the solution to form a fine iron powder precipitate. precipitation It was washed with water and collected by filtration. The filtered powder is resuspended in water and filtered again. Ta. The washing operation was repeated four more times. During the final suspension, reduce the slurry to 20% fine powder as described in Example 6 over a period of 75 days. Particles were produced that were crushed to grow an average diameter of less than 50 mm.

equivalent Those skilled in the art will be able to determine, without the use of routine experimentation, particular implementations of the invention described herein. Many equivalents of the embodiments may be recognized or ascertained. equivalent like this is intended to be encompassed by the following claims.

Summary book separation of the molecules of the species consisting of the magnetically responsive particles as well as the surrounding medium is required; or their use in desired systems is disclosed. Magnetically responsive particles are Combines organic functional groups that can be coupled with various organic molecules and/or biological molecules. a metal, metal oxide or metal alloy core coated with an organometallic polymer Consisting of These particles can be dispersed in aqueous media without rapid gravitational settling. , and can be conveniently reproduced from the medium using a magnetic field.

The magnetically responsive particles of the present invention can be with an affinity for or the ability to adsorb or interact with them. Can be coupled to biological or organic molecules. Grains coupled in this way The child involves a separation step or guided movement of the coupled molecule to a special site. various in vitro or in vivo systems (immunoassays, other biological assays, biochemical reaction or enzymatic reaction, affinity chromatography purification, cell sorting and including diagnostic and therapeutic applications).

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Claims (27)

[Claims] 1. (a) Magnetic core particles comprising a magnetically responsive metal, metal alloy or metal oxide and (b) an organometallic polymer coating covalently bonded or adsorbed to said particle. The organometallic polymer coating contains at least one bioaffinity adsorbent. Coated magnetically responsive particles characterized in that they are capable of binding. 2. Magnetic core particles include iron, magnetite, iron-magnesium oxide, iron-manganese oxide , consisting of iron cobalt oxide, iron nickel oxide, iron zinc oxide and iron copper oxide Claim 1 comprising a metal, metal alloy or metal oxide selected from the group coated magnetically responsive particles. 3. The coated magnetically responsive material according to claim 2, wherein the magnetic core particles are magnetite. Particles of response. 4. Claim 1 having a particle size of about 0.003 to about 1.5 microns in diameter. Coated magnetically responsive particles as described in Section. 5. Organometallic polymers combine organic ligands with titanium, zirconium, and hafnium. , vanadium, tantalum, niobium, tin and antimony. A coating according to claim 1 produced from a monomer that is a coordination complex with a metal. magnetically responsive particles. 6. Coated magnetically responsive particles according to claim 5, wherein the metal is titanium. . 7. Organometallic polymers include titanium-tetraisopropoxide, amino-hexyl -titanium-triisopropoxide, amino-propyl-titanium-triisopropoxide Consisting of poxide and carboxyl-hexyl-titanium-triisopropoxide The coated material according to claim 1, which is an organotitanium polymer selected from the group Magnetically responsive particles. 8. Organotitanium polymer is amino-hexyl-titanium-tri-isopropoxide Coated magnetically responsive particles according to claim 7. 9. Coated magnetically responsive particles according to claim 1, which are superparamagnetic. 10. a) magnetic core particles comprising magnetically responsive metals, metal alloys or metal oxides; b) organotitanium polymer coating covalently bonded or adsorbed to the particle (previously the organotitanium polymer has an organic functional group attached thereto); and c) Bioaffinity Covalently Coupled to Organic Functional Groups of Polymer Encapsulants adsorbent A coated magnetically responsive particle comprising: 11. Magnetic core particles contain iron, magnetite, iron-magnesium oxide, iron-manganese oxide iron cobalt oxide, iron nickel oxide, iron zinc oxide and iron copper oxide. Claim 10 is a metal, metal alloy or metal oxide selected from the group consisting of: The coated magnetically responsive particles described. 12. The coated magnetically responsive material according to claim 11, wherein the magnetic core comprises magnetite. Particles of response. 13. The organic titanium polymer deposit is amino-hexyl-titanium-tri-isopropylene. 11. The coated magnetically responsive particles of claim 10 which are poxides. 14. Bioaffinity adsorbents are used to bind antibodies, antigens, enzymes and specific binding proteins. The coated magnetically responsive material according to claim 10, which is selected from the group consisting of particles. 15. Coated magnetically responsive particles according to claim 10, which are superparamagnetic. . 16. Organic functional groups include amino groups, carboxyl groups, hydroxyl groups, and sulfate groups. A polymer selected from the group consisting of a phosphate group, a phosphate group, a cyanate group and a thiol group. The coated magnetically responsive particles according to claim 10. 17. Claim 10 having an average diameter of about 0.003 to about 1.5 microns. Subverted magnetically responsive particles as described in . 18. Organic titanium to which bioaffinity adsorbents can be covalently coupled Contains a superparamagnetic metal oxide core surrounded by a polymer; contains a group of metal oxide crystals, and the particles are about 0.003 to about 1.5 microns. Magnetically responsive particles characterized by having an average diameter. 19. a. (i) a magnetic core comprising a magnetically responsive metal, metal alloy or metal oxide; - particles; and (ii) an organometallic polymer covalently entrapped or adsorbed on said particles. Coating (the organometallic coating has a bioamount covalently coupled thereto). The bioaffinity adsorbent is used for unlabeled analysis. (capable of binding to or interacting with both the target and the labeled analyte) Label a known amount of a sample containing an unknown concentration of analyte in the presence of magnetic particles containing a step of bringing the sample into contact with the analyte; b. The mixture obtained in step (a) is heated to a sufficient level for said binding or interaction to occur. The process of maintaining under suitable conditions; c. magnetically separating the magnetic particles; and d. Measures the amount of label bound to magnetic particles and measuring the concentration of the analyte in the sample. A method for measuring an analyte in a sample, the method comprising: 20. Analyzes include antibodies, antigens, haptens, enzymes, apoenzymes, enzyme substrates, and enzyme enzymes. inhibitors, cofactors, nucleic acids, waiting proteins, carrier proteins, binding Compounds bound by a conjugate protein, bound by a carrier protein compounds, lectins, monosaccharides, polysaccharides, hormones, receptors, repressors and 20. The method of claim 19, wherein the inducer is selected from the group consisting of: 21. Magnetic core particles contain iron, magnetite, iron-magnesium oxide, iron-manganese oxide iron cobalt oxide, iron nickel oxide, iron zinc oxide and iron copper oxide. Claim 19 includes a metal, metal alloy or metal oxide selected from the group of Method described. 22. The magnetic core particles have a particle size of about 0.003 to about 1.5 microns in diameter. 22. The method according to claim 21. 23. Organometallic polymer coatings combine organic ligands with titanium, zirconium, From the group consisting of hunium, vanadium, tantalum, niobium, tin and antimony Claim 19, which is produced from monomers that are coordination complexes of selected metals. How to put it on. 24. Organometallic polymers include titanium-tetraisopropoxide, amino-hexyl Sil-titanium-triisopropoxide, amino-propyl-titanium-isopropoxide the group consisting of oxide and carboxyl-hexyl-titanium-triisopropoxide 24. The method of claim 23, wherein the organic titanium polymer is selected from: 25. 20. The method of claim 19, wherein the magnetically responsive particles are superparamagnetic. 26. Bioaffinity adsorbents can be used for antibodies, antigens, haptens, enzymes, and apoenzymes. , enzyme substrates, enzyme inhibitors, cofactors, nucleic acids, binding proteins, carriers carrier protein, compound assembled by waiting protein, carrier protein Compounds bound by quality, lectins, monosaccharides, polysaccharides, hormones, receptors , a repressor, and an inducer. The method described in. 27. Labeled analytes include radioisotopes, fluorescent compounds, enzymes, and chemiluminescence. Claim 19 marked with a label selected from the group consisting of The method described in section.