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Details for Patent: 4,698,302

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Details for Patent: 4,698,302

Title: Enzymatic reactions using magnetic particles
Abstract:A process is provided for the preparation of magnetic particles to which a wide variety of molecules may be coupled. The magnetic particles can be dispersed in aqueous media without rapid settling and conveniently reclaimed from media with a magnetic field. Preferred particles do not become magnetic after application of a magnetic field and can be redispersed and reused. The magnetic particles are useful in biological systems involving separations.
Inventor(s): Whitehead; Roy A. (Hingham, MA), Chagnon; Mark S. (Lowell, MA), Groman; Ernest V. (Brookline, MA), Josephson; Lee (Arlington, MA)
Assignee: Advanced Magnetics, Inc. (Cambridge, MA)
Filing Date:Jun 13, 1985
Application Number:06/744,457
Claims:1. A method for carrying out an enzymatic reaction which comprises:

(a) contacting an enzyme covalently coupled to magnetically-responsive particles 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,

wherein the magnetically-responsive particles of step (a) individually comprise a magnetic metal oxide core generally surrounded by a coat of polymeric silane, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (i) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (ii) a ninty-five-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.

2. A method for carrying out an enzymatic reaction which comprises:

(a) contacting an enzyme covalently coupled to magnetically-responsive particles 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,

wherein the magnetically-responsive particles of step (a) individually comprise a superparamagnetic iron oxide core generally surrounded by a coat of polymeric silane, the iron oxide core including a group of crystals of iron oxide, an individual particle having a mean diameter as measured by light scattering between about 0.1.mu. and about 1.5.mu. and a surface area as measured by nitrogen gas adsorption of at least about 100 m.sup.2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (i) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (ii) a ninety-five-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.

3. A method for carrying out an enzymatic reaction which comprises:

(a) contacting an enzyme covalently coupled to magnetically-responsive particles 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,

wherein the magnetically-responsive particles of step (a) individually comprise a ferromagnetic metal oxide core generally surrounded by a coat of polymeric silane, the metal oxide core including a group of crystals of metal oxide, an individual particle having a mean diameter as measured by light scattering between about 0.1.mu. and about 1.5.mu. and a surface area as measured by nitrogen gas adsorption of at least about 100 m.sup.2 /gm, a mass of the particles being dispersable in aqueous media to form an aqueous dispersion having (i) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field, and (ii) a ninety-five-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.

4. The method of claim 1, 2 or 3 wherein the enzyme is alkaline phosphatase.

5. The method of claim 2 which further comprises recycling the enzyme by redispersing the enzyme-coupled magnetically-responsive particles in a fresh reaction medium.

6. The method of claim 1, 2 or 3 wherein the the enzyme is .beta.-galactosidase.

7. The method of claim 1, 2 or 3 wherein the enzyme is amylo-glucosidase.

8. The method of claim 1, 2 or 3 wherein the enzyme is glucose oxidase.

9. The method of claim 1, 2 or 3 wherein the enzyme is glucoamylase.

10. The method of claim 1, 2 or 3 wherein the enzyme is .beta.-amylase.

11. The method of claim 1, 2 or 3 wherein the enzyme is invertase.

12. The method of claim 1, 2 or 3 wherein the enzyme is glucose isomerase.

13. The method of claim 1, 2 or 3 wherein the enzyme is lactase.

14. The method of claim 1, 2 or 3 wherein the enzyme is trypsin.

15. The method of claim 1, 2 or 3 wherein the enzyme is aminoacylase.

16. The method of claim 1, 2 or 3 wherein the enzyme is lysozyme.
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