Magnetic particles for use in separations

Claim 2

A magnetically-responsive particle comprising a magnetic metal oxide core generally surrounded by a silane coat to which molecules can be covalently coupled, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (b) a ninetyfive-percent-turbidity-decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

Claim 3

The magnetically-responsive particle of claim 1 wherein the metal oxide core includes a group of superparamagnetic crystals.

Claim 4

The magnetically-responsive particle of claim 2 wherein the superparamagnetic crystals are comprised of iron oxide including divalent and trivalent iron cations.

Claim 5

The magnetically-responsive particle of claim 1 wherein the silane coat generally surrounding the core comprises a bifunctional silane polymeric material bearing a first set of functionalities capable of adsorptively or covalently binding to the metal oxide core and a second set of functionalities capable of covalently coupling to organic molecules.

Claim 6

The magnetically-responsive particle of claim 4 wherein the silane polymeric material bears organofunctionalities selected from the group consisting of aminophenyl, amino, carboxylic acid, hydroxyl, sulfhydryl, phenolic, aliphatic, hydrophobic and amphipathic moieties.

Claim 7

The magnetically-responsive particle of claim 4 wherein the silane polymeric material is formed from silane monomers selected from the group consisting of p- aminophenyltrimethoxysilane, 3- aminopropyltrimethoxysilane, N- 2- aminoethyl- 3- aminopropyltrimetboxysilane, n- dodecyltriethoxysilane and n- hexyltrimethoxysilane.

Claim 8

The magnetic particle of claim 4 wherein the mean diameter thereof as measured by light scattering is between about 0.1 µ and about 1.5 u.

Claim 9

The magnetic particle of claim 7 wherein the surface area thereof as measured by nitrogen gas adsorption is at least about 100 m 2 /gm.

Claim 10

A magnetically-responsive comprising a superparamagnetic iron oxide core generally surrounded by a coat of polymeric silane to which molecules can be covalently coupled, the iron oxide core including group of crystals of iron oxide, the particle having a mean diameter as measured by light scattering between about 0.1 µ and about 1.5 p and a surface area as measured by nitrogen gas adsorption of at least about 100 m 2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (a) a fifty-percent- tubidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (b) a ninetyfive- percent- turbidity- decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

Claim 11

A magnetically-responsive comprising a superparamagnetic iron oxide core generally surrounded by a coat of polymeric silane to which molecules can be covalently coupled, the iron oxide core including group of crystals of iron oxide, the particle having a mean diameter as measured by light scattering between about 0.1 µ and about 1.5 p and a surface area as measured by nitrogen gas adsorption of at least about 100 m 2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (a) a fifty-percent- tubidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (b) a ninetyfive- percent- turbidity- decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

Claim 12

A magnetically-responsive particle comprising a ferromagnetic metal oxide core generally surrounded by a coat of polymeric silane to which molecules can be covalently coupled, the metal oxide core including a group of crystals of metal oxide the particle having a mean diameter as measured by light scattering between about 0.1 p and about 1.5 p and a surface area as measured by nitrogen gas adsorption of at least about 100 m 2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (b) a ninetyfive-percent-turbidity- decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

Claim 13

A magnetically-responsive particle comprising a ferromagnetic metal oxide core generally surrounded by a coat of polymeric silane to which molecules can be covalently coupled, the metal oxide core including a group of crystals of metal oxide the particle having a mean diameter as measured by light scattering between about 0.1 p and about 1.5 p and a surface area as measured by nitrogen gas adsorption of at least about 100 m 2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (b) a ninetyfive-percent-turbidity- decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

Claim 14

ll. The magnetically-responsive particle of claim 1, 9 or 10 wherein the silane coat is covalently coupled to at least one type of bioaffinity adsorbent.

Claim 15

The magnetically-responsive particle of claim 11 wherein the bioaffinity adsorbent is selected from the group consisting of antibodies, antigens, haptens, enzymes, apoenzymes, enzymatic substrates, enzymatic inhibitors, cofactors, binding proteins, carrier proteins, compounds bound by binding proteins, compounds bound by carrier proteins, lectins, monosaccharides, polysaccharides, hormones, receptors, repressors and inducers.

Claim 16

The magnetically- responsive particle of claim 9 or 10 wherein the coat is covalently coupled to antibodies selected from the group consisting of anti-thyroxine, anti-triiodothyronine, anti-thyroid stimulating hormone, anti-thyroid binding globulin, anti-thyroglobulin, anti- digoxin, anti-cortisol, anti-insulin, anti-theophylline, anti- vitamin B 12 , anti-folate, anti-ferritin, anti-human chroinic gonadotropin, anti-follicle stimulating hormone, anti-leutenizing hormone, anti-progesterone, anti- testosterone, anti-estriol, anti-estradiol, anti-prolactin, anti-human placental lactogen, anti- gastrin and anti- human growth hormone antibodies.

Claim 17

The magnetically-responsive particle of claim 9 wherein the silane coat is a p- aminophenyltrimethoxysilane polymer, which polymer is covalently coupled by diazotization to anti-thyroxine antibodies.

Claim 18

The magnetically-responsive particle of claim 9 wherein the coat is a p- aminophenyltrimethoxysilane polymer, which polymer is covalently coupled by diazotization to anti-theophylline antibodies.

Claim 19

The magnetically-responsive particle of claim 9 wherein the coat is a carboxylic-acid terminated glutaric- anhydride treated 3- aminopropyltrimethoxysilane polymer, which polymer is covalently coupled by carbodiimide coupling to vitamin B 12 binding protein.

Claim 20

The magnetically-responsive particle of claim 9 wherein the coat is a N-2-aminoethyl-2-aminopropy]-silane polymer, which polymer is coupled by glutaraldehyde coupling to anti-triiodothyronine antibodies.

Claim 21

The magnetically-responsive particle of claim 9 wherein the coat is a N- 2- amfnoethyl- 3- aminopropylsilane polymer, which polymer is coupled by glutaraldehyde coupling to anti-thyroid stimulating hormone antibodies.

Claim 22

The magnetically-responsive particle of claim 9 wherein the coat is a N- 2- aminoethyl-3- aminopropylsilane polymer, which polymer is coupled by glutaraldehyde coupling to alkaline phosphatase or ß-galactosidase.

Claim 23

A process for preparing magnetically-responsive particles with mean diameters between about 0.1 µ and about 1.5 µ as measured by light scattering, which process comprises: a) precipitating divalent and trivalent transition metal salts in base; b) washing the precipitate to approximate neutrality; c) washing the precipitate in an electrolyte; d) resuspending the precipitate in a solution of silane monomer capable of forming a polymeric coat adsorptively or covalently bound to the precipitate to which molecules can be covalently coupled; and e) causing the silane polymeric coat to become adsorptively or covalently bound to the precipitate.

Claim 24

A process for preparing magnetically-responsive particles with mean diameters between about 0.1 µ and about 1.5 µ as measured by light scattering, which process comprises: a) precipitating divalent and trivalent transition metal salts in base; b) washing the precipitate to approximate neutrality; c) washing the precipitate in an electrolyte; d) resuspending the precipitate in a solution of silane monomer capable of forming a polymeric coat adsorptively or covalently bound to the precipitate to which molecules can be covalently coupled; and e) causing the silane polymeric coat to become adsorptively or covalently bound to the precipitate.

Claim 25

A process for preparing superparamagnetic particles with mean diameters between about 0.1 p and about 1.5 µ as measured by light scattering, which process comprises: a) precipitating divalent and trivalent iron cations of divalent and trivalent iron salts in base; b) washing the precipitate in water to approximate neutrality; c) washing the precipitate in an electroyte; and d) coating the washed precipitate with an adsorptively or covalently bound silane polymer.

Claim 26

A process for preparing superparamagnetic particles with mean diameters between about 0.1 p and about 1.5 µ as measured by light scattering, which process comprises: a) precipitating divalent and trivalent iron cations of divalent and trivalent iron salts in base; b) washing the precipitate in water to approximate neutrality; c) washing the precipitate in an electroyte; and d) coating the washed precipitate with an adsorptively or covalently bound silane polymer.

Claim 27

The process of claim 21 wherein the divalent and trivalent iron salts are FeCl 2 and FeCl 3 .

Claim 28

The process of claim 21 wherein the divalent and trivalent iron cations are used in an Fe 2+ /Fe 3+ ratio of about 4/1 to about 1/2.

Claim 29

The process of claim 21 wherein the washings are performed by redispersing the precipitate in water and electrolyte and magnetically collecting the precipitate between washings.

Claim 30

The process of claim 21 wherein the precipitate is coated with the silane polymer by deposition of said silane polymer from acidic aqueous solution.

Claim 31

The process of claim 21 wherein the precipitate is coated with the silane polymer by deposition of said silane polymer from acidic organic solution.

Claim 32

The process of claim 26 wherein the deposition of silane polymer from acidic organic solution comprises: a) suspending the washed precipitate in an organic solvent containing about 1% (V/V) water; b) adding an acidic solution of a silane monomer c) homogenizing the precipitate at high speed; d) mixing the precipitate with a wetting agent, which agent is miscible in the organic solvent and water; e) heating to a temperature sufficient to evaporate water and organic solvent; and f) washing the wetting agent from the precipitate.

Claim 33

The process of claim 27 wherein the organic solvent is methanol and the wetting agent is glycerol.

Claim 34

The process of claim 27 wherein the solution of silane monomer is made acidic with orthophosphorous acid or glacial acetic acid.

Claim 35

The process of claim 21 wherein said silane polymer is formed from a silane monomer selected from the group cosisting of p-aminophenyltrimethoxysilane, 3-aminopropyltrimethoxysilane, N- 2- aminoethyl- 3-aminopropyltrimethoxysilane, n-dodecyltriethoxysilane, and n-hexyltrimethoxysilane.

Claim 36

A process for preparing magnetically-responsive particles comprising superparamagnetic iron oxide cores generally surrounded by a silane polymeric coat to which molecules can be covalently coupled, which particles have a mean diameters between about 0.1 u and about 1.5 p as measured by light scattering, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (b) a ninetyfive-percent-turbidity- decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel, which process comprises: a) precipitating FeCl 2 and FeCl 3 in an Fe 2+ /Fe 3+ ratio of about 2/1 with sodium hydroxide; b) washing the precipitate in water to approximate neutrality by redispersing and magnetically separating the precipitate; c) washing the precipitate in a sodium chloride solution by redispersing and magnetically separating the precipitate; d) suspending the washed precipitate in methanol containing about 1% (V/V) water; e) adding an acidic solution of a silane monomer to the suspension of precipitate; f) homogenizing the precipitate at high speed; g) mixing the precipitate with glycerol; h) heating the precipitate and glycerol to a temperature in the range from about 160° to about 170°C; and i) washing glycerol from the precipitate.

Claim 37

A process for preparing magnetically-responsive particles comprising superparamagnetic iron oxide cores generally surrounded by a silane polymeric coat to which molecules can be covalently coupled, which particles have a mean diameters between about 0.1 u and about 1.5 p as measured by light scattering, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (b) a ninetyfive-percent-turbidity- decrease separation time of less than about 10 minutes in the presence of a magnetic field, the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel, which process comprises: a) precipitating FeCl 2 and FeCl 3 in an Fe 2+ /Fe 3+ ratio of about 2/1 with sodium hydroxide; b) washing the precipitate in water to approximate neutrality by redispersing and magnetically separating the precipitate; c) washing the precipitate in a sodium chloride solution by redispersing and magnetically separating the precipitate; d) suspending the washed precipitate in methanol containing about 1% (V/V) water; e) adding an acidic solution of a silane monomer to the suspension of precipitate; f) homogenizing the precipitate at high speed; g) mixing the precipitate with glycerol; h) heating the precipitate and glycerol to a temperature in the range from about 160° to about 170°C; and i) washing glycerol from the precipitate.

Claim 38

The process of claim 31 wherein the silane monomer is selected from the group consisting of p- aminophenyltrimethoxysilane, aminiopropyltrimethoxy- silane, N- 2- aminoethyl- 3- aminopropyltrimethoxysilane, n- dodecyltriethoxysilane, and n- hexyltrimethoxysilane.,

Claim 39

A method for determining the concentration of a ligate in a solution which comprises: a) reacting the solution, a known amount of labeled ligate, and magnetically- responsive particles of claim 9 or 10 to which a ligand specific for the ligate in solution is covalently coupled, to form ligand/ligate complexes; b) magnetically separating the magnetically- responsive particles from the reaction solution; c) measuring the amount of label associated with the magnetically-responsive particles or remaining free in solution; d) relating the amount of label measured in step (c) to a standard curve to determine ligate concentration.

Claim 40

The method of claim 33 wherein the ligand is an antibody.

Claim 41

The method of claim 34 wherein the antibody is selected from the group consisting of anti-thyroxine, anti-triiodothyronine, anti- thyroid stimulating hormone, anti- thyroid binding globulin, anti-thyroglobulin, anti-digoxin, anti- cortisol, anti-insulin, anti- theophylline, anti-vitamin B 12 , anti-folate, anti-ferritin, anti-human chorionic gonadotropin, anti-follicle stimulating hormone, anti-leutenizing hormone, anti-progesterone, anti- testosterone, anti-estriol, anti-estradiol, anti-prolactin, anti-human placental lactogen, anti-gastrin and anti-human growth hormone antibodies.

Claim 42

A method for carrying out an enzymatic reaction which comprises: a) contacting an enzyme covalently coupled to the magnetically-responsive particles of claim 9 with a reaction medium to form a reaction mixture in a reaction vessel to effect an enzymatic reaction; b) allowing the enzymatic reaction to occur; and c) removing the enzyme from the reaction mixture by applying a magnetic field.

Claim 43

The method of claim 36 wherein the enzyme is alkaline phophatase or B-galactosidase.

Claim 44

The method of claim 36 which further comprises recycling the enzyne by redispersing the enzyme- coupled magnetically- responsive particle in a fresh reaction medium.

Claim 45

An affinity chromatography method for isolating ligates from solution which comprises: a) contacting a ligand covalently coupled to the magnetically- responsive particles of claim 9 with a solution or suspension containing a ligate and other substances in a vessel; b) allowing the ligand and ligate to bind or interact; c) magnetically separating the magnetically- responsive particles with ligate bound thereto from the solution or suspension; and d) recovering the ligate from the ligand by desorbing it from the magnetically- responsive particles.