METHOD FOR ISOLATING WEAKLY INTERACTING MOLECULES FROM A FLUIDIC SAMPLE

• US 20120178096A1
• Filed: 01/10/2011
• Published: 07/12/2012
• Est. Priority Date: 01/10/2011
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Beebe, David, Burgess, Richard, Strotman, Lindsay, Berry, Scott
• Granted Patent: US 8,993,243 B2
• Time in Patent Office: Days
• US Class Current: 435/6.19
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54326 Magnetic particles

Abstract

Methods of isolating weakly interacting molecules in a fluidic sample using an immiscible phase filtration technique are disclosed. A complex is formed between a solid phase substrate, a molecule immobilized on the solid phase substrate, and at least one target molecule present in the fluidic sample. The complex is transferred into an immiscible phase by applying an external force to the solid phase substrate. The methods eliminate the need for complex and time consuming washing steps.

Claims

1. A method for isolating weakly interacting molecules from a fluidic sample, the method comprising:

a) forming a mixture comprising the fluidic sample and a solid phase substrate, wherein the solid phase substrate comprises at least one immobilized molecule thereon;

b) incubating the mixture under conditions sufficient for the immobilized molecule to interact with a target molecule in the fluidic sample to form a solid phase substrate-immobilized molecule-target molecule complex, wherein the immobilized molecule and the target molecule have an interaction half-life of about 10 minutes or less; and

c) transferring the solid phase substrate-immobilized molecule-target molecule complex into an immiscible phase in about 1 second or less.

2. The method of claim 1, wherein the solid phase substrate-immobilized molecule-target molecule complex is transferred into the immiscible phase in about 10 milliseconds or less.

3. The method of claim 1, wherein the immobilized molecule and the target molecule have an interaction half-life of about 2 seconds or less.

4. The method of claim 1, further comprising transferring the solid phase substrate-immobilized molecule-target molecule complex through the immiscible phase and into a solution.

5. The method of claim 4, wherein the solid phase substrate-immobilized molecule-target molecule complex is transferred from the mixture, through the immiscible phase, and into the solution in about 10 seconds or less.

6. The method of claim 4, wherein the solution is selected from the group consisting of an elution buffer, a downstream process reagent, a detection reagent, and combinations thereof.

7. The method of claim 4, further comprising transferring the complex from the solution through a second immiscible phase and into a second solution.

8. The method of claim 4, further comprising forming and incubating the mixture in an input zone of a device comprising:

i) the input zone;

ii) a zone comprising the immiscible phase;

iii) an output zone comprising the solution, wherein the mixture in the input zone and the solution in the output zone are separated by the zone comprising the immiscible phase; and

iv) a pathway interconnecting the input zone and the output zone, wherein a first constriction separates the input zone from the zone comprising the immiscible phase and a second constriction separates the zone comprising the immiscible phase from the output zone; and

wherein the solid phase substrate-immobilized molecule-target molecule complex is transferred from the mixture in the input zone through the pathway through the zone comprising the immiscible phase and into the solution in the output zone.

9. The method of claim 1, wherein the immobilized molecule-target molecule interaction is selected from the group consisting of a protein-protein interaction, a nucleic acid-nucleic acid interaction, a protein-protein complex interaction, a protein-small molecule interaction, a nucleic acid-small molecule interaction, a small molecule-small molecule interaction, a cell-protein interaction, a cell-nucleic acid interaction, a cell-small molecule interaction, a cell-protein complex interaction, and combinations thereof.

10. The method of claim 1, wherein the immobilized molecule is selected from the group consisting of a protein, nucleic acid, a small molecule, a cell, and combinations thereof.

11. The method of claim 1, wherein the target molecule is selected from the group consisting of a protein, a protein complexed with at least one additional molecule, a nucleic acid, a nucleic acid complexed with at least one additional molecule, a small molecule, a small molecule complexed with at least one additional molecule, a cell, a cell complexed with at least one additional molecule, and combinations thereof.

12. The method of claim 1, wherein the solid phase substrate is responsive to an external force.

13. The method of claim 12, wherein the external force is selected from the group consisting of a magnetic field, air, gravitational force, centrifugal force, inertial force, mechanical force, pumping, and combinations thereof.

14. The method of claim 1, wherein the solid phase substrate is selected from the group consisting of a non-magnetic particle, a paramagnetic particle, a superparamagnetic particle, a magnetic particle, a ferromagnetic particle, a porous particle, a non-porous particle, a membrane, a rod, a wire, and combinations thereof.

15. The method of claim 1, wherein the immiscible phase is selected from the group consisting of an organic solvent, a liquid wax, an oil, a gas, and combinations thereof.

16. A method for isolating weakly interacting molecules from a fluidic sample, the method comprising:

a) forming a mixture comprising the fluidic sample and a solid phase substrate, wherein the solid phase substrate comprises at least one immobilized molecule thereon;

b) incubating the mixture under conditions sufficient for the immobilized molecule to interact with a target molecule in the fluidic sample to form a solid phase substrate-immobilized molecule-target molecule complex, wherein the immobilized molecule and the target molecule have an interaction half-life of about 5 seconds or less; and

c) transferring the solid phase substrate-immobilized molecule-target molecule complex into an immiscible phase.

17. The method of claim 16, wherein the immobilized molecule and the target molecule have an interaction half-life of about 2 seconds or less.

18. The method of claim 16, wherein the solid phase substrate-immobilized molecule-target molecule complex is transferred into the immiscible phase in about 1 second or less.

19. The method of claim 16, further comprising transferring the solid phase substrate-immobilized molecule-target molecule complex through the immiscible phase and into a solution.

20. The method of claim 19, wherein the solid phase substrate-immobilized molecule-target molecule complex is transferred from the mixture, through the immiscible phase, and into the solution in about 10 seconds or less.

21. The method of claim 19, wherein an interfacial energy between the mixture and the immiscible phase and the immiscible phase and the solution is from about 0.2 mN/m to about 30 mN/m.

22. The method of claim 19, wherein the solution is selected from the group consisting of an elution buffer, a downstream process reagent, a detection reagent, and combinations thereof.

23. A method for isolating weakly interacting molecules from a fluidic sample, the method comprising:

a) forming a mixture comprising the fluidic sample and a solid phase substrate, wherein the solid phase substrate comprises at least one immobilized molecule thereon;

b) incubating the mixture under conditions sufficient for the immobilized molecule to interact with a target molecule in the fluidic sample to form a solid phase substrate-immobilized molecule-target molecule complex, wherein the target molecule interacts with at least one additional molecule in the fluidic sample, and wherein the target molecule and the at least one additional molecule have an interaction half-life of about 10 minutes or less; and

c) transferring the solid phase substrate-immobilized molecule-target molecule complex into an immiscible phase in about 1 second or less.

24. A method for isolating weakly interacting molecules from a fluidic sample, the method comprising:

a) forming a mixture comprising the fluidic sample and a solid phase substrate, wherein the solid phase substrate comprises at least one immobilized molecule thereon;

b) incubating the mixture under conditions sufficient for the immobilized molecule to interact with a target molecule in the fluidic sample to form a solid phase substrate-immobilized molecule-target molecule complex, wherein the target molecule interacts with at least one additional molecule in the fluidic sample, and wherein the target molecule and the at least one additional molecule have an interaction half-life of about 5 seconds or less; and

c) transferring the solid phase substrate-immobilized molecule-target molecule complex into an immiscible phase.