Device And Method For Separating, Mixing And Concentrating Magnetic Particles With A Fluid And Use Thereof In Purification Methods

• US 20070215554A1
• Filed: 07/22/2005
• Published: 09/20/2007
• Est. Priority Date: 07/26/2004
• Status: Active Grant

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Beerling, Bernardus Jozef, Kreuwel, Hermannus, Spee, Franciscus, Verwimp, Emiel Gerebern
• Granted Patent: US 7,972,516 B2
• Time in Patent Office: Days
• US Class Current: 210/695
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0858 Side walls
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/0332 using permanent magnets
• 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 or biological applications
• CPC Class Codes: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• 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

Abstract

The present invention is related to a process for manipulating magnetic particles that are suspended in a fluid, possibly containing a biological entity of interest, the magnetic particles being able to bind the entity of interest, the fluid being contained in a reaction vessel constituted by a large upper compartment with a funnel shape, an elongate lower compartment with a substantially constant cross-section and a closed base. The process consists of: a) subjecting the magnetic particles to two magnetic fields applied simultaneously to separate all said magnetic particles present in at least the upper compartment of the vessel from the fluids b) transferring the separated magnetic particles from the upper compartment to the elongated lower compartment, c) removing the fluid from the vessel, d) adding a washing liquid to the lower compartment), e) subjecting the rest of the fluid to at least two magnetic fields applied successively with different and changing directions to wash all the magnetic particles present in the lower compartment, and concentrating said magnetic particles in said lower compartment The according to the present invention are especially useful in methods for the extraction of nucleic acid to enable them for further processing.

Claims

1. A process for manipulating magnetic particles that are suspended in a fluid, the magnetic particles being able to bind an entity of interest, the fluid being contained in a reaction vessel constituted by a large upper compartment with a funnel shape, an elongate lower compartment with a substantially constant cross-section and a closed base, the process comprising:

a) subjecting the magnetic particles to two magnetic fields applied simultaneously to separate all said magnetic particles present in at least the upper compartment of the vessel from the fluid,

b) transferring the separated magnetic particles from the upper compartment to the elongated lower compartment,

c) removing the fluid from the vessel,

d) adding a washing liquid to the lower compartment,

e) subjecting the rest of the fluid to at least two magnetic fields applied successively with different and changing directions to wash all the magnetic particles present in the lower compartment, and

f) concentrating said magnetic particles in said lower compartment.

2. A process according to claim 1, wherein the two magnetic fields applied simultaneously are generated by at least two magnets, the pole axis of the magnets forming together an angle different from 180°

.

3. A process according to any of claim 1, wherein the at least two magnetic fields applied successively are generated by at least two magnets, the pole axis of the magnets being parallel one to the other.

4. A process according to claim 3, wherein the successive magnetic fields with different and changing directions are applied to the vessel by moving the magnets with respect to position of the vessel and/or by moving the vessel with respect to the position of the magnets.

5. A process according to claim 1, wherein the subjecting the magnetic particle to two magnetic fields is carried out using at least two magnets, with at least one magnet positioned on an opposing side of the vessel from at least one other magnet.

6. A process according to claim 5, wherein the magnets are interdependent with one support.

7. A process according to claim 1, wherein the subjecting the magnetic particle to two magnetic fields is carried out using at least two magnets, and wherein the magnets intended to the separation are also used to mix the fluid in the vessel.

8. A process according to claim 7, wherein the magnets are interdependent with one support, and wherein the support can be moved in rotation around, to pass from separation to mixing configurations, and longitudinally along, to realize the mixing step, an axle passing through each magnet, the axle being parallel to moving of the vessel with respect to the magnets and/or the magnets with respect to the vessel, and perpendicularly to magnet'"'"'s pole axis.

9. A process according to claim 1, wherein the subjecting the magnetic particle to two magnetic fields is carried out using at least two magnets, and wherein magnets configured to separate the magnetic particles are different from magnets configured to mix the fluid in the vessel.

10. A process according to claim 1, wherein the lower compartment is constituted by:

one medium compartment to which the magnetic fields are applied successively, and

one bottom compartment in which the magnetic particles are concentrated.

11. A process according to claim 10, wherein between step e) and step f) of claim 1 the following intermediate steps are realized, and wherein the washing in step e) comprises mixing the magnetic particles in the fluid:

e1) transferring the separated and mixed magnetic particles from said medium compartment to said bottom compartment,

e2) removing the washing liquid from the vessel, and

e3) adding an elution buffer to the bottom compartment.

12. A process according to claim 11, wherein the following further steps are realized after step f) of claim 1:

g) transferring the magnetic particles from said bottom compartment to the medium compartment or to the upper compartment,

h) removing the elution buffer present in the bottom compartment and containing the entity of interest for further processing.

13. A process according to claim 10, wherein the volume of the medium compartment is smaller compared to the upper compartment and bigger compared to the bottom compartment.

14. A process according to claim 1, wherein the transfer of magnetic particles is sustained by the magnet.

15. A process according to claim 1, wherein the entity of interest comprises molecules constituted by nucleic acids (RNA and/or DNA).

16. A process according to claim 15, wherein the binding of the nucleic acids is non-specific and realized directly onto the surface of the magnetic particles.

17. A process according to claim 15, wherein the binding of the nucleic acids is specific and realized directly onto capture probes carried by the surface of the magnetic particles.

18. Device for extracting possible molecules of interest from a fluid to which is added a suspension of magnetic or (super) paramagnetic particles contained in at least one reaction vessel and capable to bind the molecules of interest comprising:

at least one separating station to capture the magnetic particles present in a large upper compartment of each reaction vessel,

at least one washing station to mix said magnetic particles present in a medium compartment of each reaction vessel,

at least one concentrating station to mix said magnetic particles present in a bottom compartment of each reaction vessel, and

at least one pipetting device to dispense and/or to remove part or all of the fluid, the washing liquid and output buffer used in an extraction process.

19. Device according to claim 18, characterized in that the at least one separating station comprises at least two magnets, a pole axis of the magnets forming together an angle different from 180°

.

20. Device according to claim 18, characterized in that the at least one washing station comprises at last two magnets, a pole axis of the magnets being parallel one to the other.

21. Reaction vessel adapted for use in the device of claim 18, comprising:

top aperture,

one upper compartment with a funnel shape,

one medium compartment with a substantially constant cross-section,

one bottom compartment with a substantially constant cross-section,

a closed base, and

a longitudinal axis defined by the lower compartment.

22. Reaction vessel according to claim 21, wherein the at least one separating station and the at least one washing station cooperate with at least two magnets, each having a pole axis, and wherein each of the opposite walls of the upper compartment, constituting the funnel shape, is perpendicular to the pole axis of the magnets that is present with respect to this wall.

23. Reaction vessel according to claim 22, characterized in that the opposite walls of the upper compartment form together an angle different from 180°

.

24. Reaction vessel according to claim 21, wherein the volume of the medium compartment is smaller compared to the upper compartment and bigger compared to the bottom compartment.

25. Reaction vessel according to claim 21, wherein each compartment has an associated volume and wherein a ratio between the volumes of the medium compartment and the upper compartment or between the volumes of the bottom compartment and the medium compartment is between 1:

2 to 1;

100.

31. Reaction vessel according to claim 23, wherein the angle is between about 60 and 150°

.

32. Reaction vessel according to claim 23, wherein the angle is between about 80 and 120°

.

33. Reaction vessel according to claim 25, wherein the ratio is between about 1:

5 to 1;

20.

34. Reaction vessel according to claim 25, wherein the ratio is about 1:

10.

26. Set of reaction vessels constituted by at least two vessels, said vessels being arranged symmetrically along one line.

27. Set of reaction vessels according to claim 26, wherein the set of vessels cooperates with at least two tips, said tips constituting:

a first pipetting device to dispense and/or to remove part or all of the fluid,

a second pipetting device to dispense and/or to remove part or all of the washing liquid, and

a third pipetting device to dispense and/or to remove part or all of output buffer.

28. Set of reaction vessels according to claim 27, wherein the tips have free ends, and wherein the free ends of the tips constituting the first, the second or the third pipetting device being arranged symmetrically along one line or two lines, the two lines of each arrangement being parallel one to the other.

29. Set of reaction vessels according to claim 28, wherein the free ends of:

the tips constituting the first pipetting device being arranged symmetrically along one line,

the tips constituting the second pipetting device being arranged symmetrically along one or two lines, and

the tips constituting the third pipetting device being arranged symmetrically along two lines.

30. Set of reaction vessels according to claim 28, wherein the first pipetting device, the second pipetting device and the third pipetting device constitute at least one different pipetting device.