Method of mixing magnetic particles

• US 20060201887A1
• Filed: 05/15/2006
• Published: 09/14/2006
• Est. Priority Date: 02/21/1995
• Status: Active Grant

• Current Assignee: Sigris Research Incorporated
• Original Assignee: Iqbal Waheed Siddiqi
• Inventors: Siddiqi, Iqbal Waheed
• Granted Patent: US 7,517,457 B2
• Time in Patent Office: Days
• US Class Current: 210/695
• CPC Class Codes: B01F 33/451 wherein the mixture is directly exposed to an electromagnetic field without use of a stirrer, e.g. for material comprising ferromagnetic particles or for molten metal
• CPC Class Codes: B01F 33/81 Combinations of similar mixers, e.g. with rotary stirring devices in two or more receptacles
• CPC Class Codes: B01F 33/813 mixing simultaneously in two or more mixing receptacles
• CPC Class Codes: B01L 9/523 for multisample carriers, e.g. used for microtitration plates
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/12 with magnets moving during operation; with movable pole pieces
• CPC Class Codes: B03C 1/14 with non-movable magnets
• CPC Class Codes: B03C 1/24 with material carried by travelling fields
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

An apparatus and method for carrying out the affinity separation of a target substance from a liquid test medium by mixing magnetic particles having surface immobilized ligand or receptor within the test medium to promote an affinity binding reaction between the ligand and the target substance. The test medium with the magnetic particles in a suitable container is removably mounted in an apparatus that creates a magnetic field gradient in the test medium. This magnetic gradient is used to induce the magnetic particles to move, thereby effecting mixing. The mixing is achieved either by movement of a magnet relative to a stationary container or movement of the container relative to a stationary magnet. In either case, the magnetic particles experience a continuous angular position change with the magnet. Concurrently with the relative angular movement between the magnet and the magnetic particles, the magnet is also moved along the length of the container causing the magnetic field gradient to sweep the entire length of the container. After the desired time, sufficient for the affinity reaction to occur, movement of the magnetic gradient is ended, whereby the magnetic particles are immobilized on the inside wall of the container nearest to the magnetic source. The remaining test medium is removed while the magnetic particles are retained on the wall of the container. The test medium or the particles may then be subjected to further processing.

Claims

1. A method of mixing and separating non-colloidal paramagnetic or super paramagnetic particles for purifying molecular or cellular entities by affinity binding on said particles from a liquid comprising:

a. providing one or more container means having a liquid and said particles therein;

b. providing magnet means disposed outside of said container means to generate a magnetic field gradient within said container means to aggregate said particles on the inner wall of said container means adjacent to magnet means;

c. providing moving means for continuously changing the relative angular position between said magnet means and said aggregated particles to direct the magnetic flux from a different and changing direction to break up and disperse said aggregated particles within said liquid while the said liquid in said container means remains relatively motionless; and

d. providing means for forming a deposit of said magnetic particles adhering in the agglomerated state to internal face of the side wall of said container means by immobilizing said magnetic means outside said container means wherein said magnetic particles separate on the side wall from the said liquid;

2. The method of claim 1 wherein the said continuous change in the relative angular position between said magnets and said aggregated particles is by means of a rotary motion in a circular path around a substantially horizontal or vertical axis.

3. The method of claim 2 wherein said rotary motion is stepwise angular displacement of the magnetic flux in opposite directions of about 180 degrees at an acceleration of about 21 rads/s² or higher with a delay time of about 0.25 s to about 120 s or over 120 seconds between successive angular changes.

4. The method of claim 1 wherein the said change in the relative angular position between said magnets and said aggregated particles is by means of a linear motion in substantially horizontal or vertical planes.

5. The method of claim 4 wherein said linear motion is stepwise and oscillating to direct magnetic flux from opposite directions of about 180 degrees wherein stepwise motion is at an acceleration of about 0.2 m/s² to about 1 m/s² with a delay time of about 0.25 s to about 120 s or over 120 seconds between successive angular changes.

6. The method of claim 1 wherein said moving means comprises at least one electric motor operated and controlled by electrical and electronic means.

7. The method of claim 1 further comprising repeatedly moving said magnetic means along the vertical axis of said container means to a predetermined stroke length and simultaneously dispersing the said aggregated particles by repeatedly changing the direction of magnetic flux to opposite sides of said containers.

8. The method of claim 1 further comprising moving said magnet means along the vertical axis of said container means to transport said separated particles to a desired position on the inside wall of said container means.

9. The method of claim 1 further comprising displacing the said magnet means to a position where its influence on said magnetic particles is substantially reduced or becomes negligible.

10. The method of claim 1 wherein at least one container is in a fixed position and providing means for rotating at least two magnet mounted in orthogonal positions on two separate supports fixed on a common hub to direct an oscillating magnetic flux acting on said particles from opposite sides of said container at about 180 degrees to break up and disperse said aggregated particles in said liquid while said liquid in said containers remains relatively motionless.

11. The method of claim 10 wherein the rotation of said magnet support is stepwise at an angular acceleration of about 21 rads/m/s² or higher with a delay time of about 0.25 s to about 120 s or over 120 seconds between successive angular changes.

12. A method of purifying a desired molecular or cellular species from a sample in a liquid medium by affinity binding on non-colloidal paramagnetic or super paramagnetic particles comprising:

a. providing means for holding a plurality of said containers in at least one circular array, with a liquid medium and said particles therein;

b. providing means for holding a plurality of magnet in at least two circular arrays sufficiently spaced apart to position said container array there between whereby each magnet means is alternately disposed on the opposite side of said containers and said circular arrays of containers and magnets are concentric;

c. providing means for transferring said particles and said sample in liquid medium to said containers whereby said particles become aggregated on internal face of the side wall of said containers adjacent to said magnets;

d. providing rotating means in a circular path around a horizontal axis for repetitively changing the relative angular position between said magnets and said aggregated particles to alternately subject said particles to magnetic flux from an opposite directions of about 180 degrees to break up and disperse said aggregated particles within said liquid for sufficient time to promote affinity binding on said particles while said liquid in said containers remains relatively motionless;

e. providing means to immobilize magnets of said magnet arrays outside containers in said container array to deposit of said particles adhering in an agglomerated state on internal face of the side walls of containers in said container array thereby separating them from the said liquid;

f. providing means to remove most of sample and liquid mixture from containers in said container array while said particles remain immobilized on inner wall of containers in said container array;

g. providing means for adding washing liquid to containers in said container array and repeating steps “

d”

mixing and “

e”

separation as many times as desired; and

h. providing means to dissociate and elute the affinity bound cellular or molecular species from said magnetic particles in a liquid medium.

13. The method of claim 12 wherein said rotational change in angular position between said magnets and said aggregated particles is stepwise at an acceleration of about 21 rads/m/s² or higher with a delay time of about 0.25 s to about 120 s or over 120 seconds between successive angular changes.

14. The method of claim 12 wherein at least one electric motor operated and controlled by electrical and electronic means is used for said rotational means.

15. The method of claim 12 further comprising means of repeatedly moving magnet arrays aligned along the vertical axis of containers in said container array in predetermined stroke lengths while simultaneously dispersing the said aggregated particles by repeatedly changing the direction of magnetic flux to opposite sides of said containers.

16. The method of claim 12 further comprising means of moving said magnet arrays aligned along the vertical axis of containers in said container array to transport said separated particles to a desired position on the inside walls of said containers.

17. The method of claim 12 further comprising displacing magnets in said magnet arrays to a position where the influence of magnetic flux on said magnetic particles is substantially reduced or becomes negligible.

18. A method of mixing and separating non-colloidal paramagnetic or super paramagnetic particles for purifying molecular or cellular entities by affinity binding on said particles from a liquid comprising:

a. providing one at least one 96-well microplate held on a support means;

b. providing at least two linear arrays of magnet mounted on independently moveable support means of 6×

4 and 7×

5 arrays of evenly spaced magnets aligned and disposed outside 96 wells of said microplate to generate a magnetic field gradient within said wells to aggregate said particles on the inner wall of said wells adjacent to magnets;

c. providing means for successive linear movements along a vertical axis of said support means with mounted magnet array to alternately generate a magnetic field gradient on the opposite sides of each well in said microplate wherein reciprocating movement of said movable supports repeatedly changes the relative angular position between said magnets and said aggregated particles thereby alternately subjecting said particles to magnetic flux from an opposite directions of about 180 degrees to break up and disperse said aggregated particles in said liquid inside each well while the liquid in said wells remains relatively motionless;

d. providing means for forming a deposit of said magnetic particles adhering in the an agglomerated state to internal face of the side wall of said well by immobilizing said magnets on the outside wall of said wells wherein said magnetic particles separate on the inside wall of said wells from said liquid; and

e. providing means to move said magnet supports to a distance away from said 96-well microplate where the influence of said magnet flux acting on said particles is substantially reduced or becomes negligible.

19. The method of claim 18 wherein said reciprocating movement of movable supports to repeatedly displace said magnetic flux at opposite orientation of said wells of about 180 degrees is carried out at an acceleration of about 0.2 m/s² to about 1 m/s² with a delay time of about 0.25 s to about 120 s or over 120 seconds between successive movements of said movable supports.

20. The method of claim 18 wherein said linear movement of said supports along a vertical axis is carried out by at least one electric motor operated and controlled by electrical and electronic means.