ELECTROSONIC CELL MANIPULATION DEVICE AND METHOD OF USE THEREOF

• US 20060223185A1
• Filed: 03/28/2006
• Published: 10/05/2006
• Est. Priority Date: 03/30/2005
• Status: Active Grant

• Current Assignee: Georgia Tech Research Corporation (University System of Georgia)
• Original Assignee: Georgia Tech Research Corporation (University System of Georgia)
• Inventors: Fedorov, Andrei G, Degertekin, Fahrettin L.
• Granted Patent: US 7,704,743 B2
• Time in Patent Office: Days
• US Class Current: 435/461
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Abstract

In method of injecting a substance into a living cell having a cell membrane, the substance, the cell and a liquid are placed into a tapering passage. Energy is applied to the cell, thereby inducing poration. To sort cells, a cellular suspension is placed in a tapering passage, including a narrow end that defines an opening that has a dimension corresponding to a cell size. An acoustic wave is applied, thereby forcing cells having a cell size smaller than the selected cell size through the opening, with a portion of the cells having a cell size not smaller than the selected cell size not forced through the opening. To extract material from a cell, an electric field and an acoustic wave are applied, thereby causing the cell membrane to allow the material to pass out of the cell.

Claims

1. A method of injecting a substance into a living cell having a cell membrane, comprising the steps of:

a. placing the substance, the cell and a liquid into a tapering passage; and

b. applying an energy to the cell sufficient to induce poration of the cell.

2. The method of claim 1, wherein the step of applying an energy to the cell, comprises applying an electric field to the cell, thereby inducing electroporation in the cell.

3. The method of claim 2, wherein the step of applying an electric field to the cells comprises applying an oscillating electric field.

4. The method of claim 1, wherein the step of applying an energy to the cell, comprises applying an acoustic pressure wave to the liquid, thereby inducing sonoporation in the cell.

5. The method of claim 4, wherein the step of applying an acoustic pressure wave further comprises the step of forming a standing wave within the tapering passage.

6. The method of claim 4, wherein the acoustic wave comprises an ultrasonic wave.

7. The method of claim 4, wherein the tapering passage includes a narrow end that defines an opening passing therethrough, wherein the method further comprises the step of forcing the cell through the opening so that as the cell passes through the opening the cell membrane allows the substance to pass therethrough.

8. The method of claim 1, wherein the step of applying an energy to the cell comprises the step of focusing the energy within the tapering passage, thereby applying a highly localized energy gradient to the cell.

9. The method of claim 1, wherein the substance comprises genetic material.

10. The method of claim 1, wherein the substance comprises a predetermined chemical composition.

11. A method of sorting cells, comprising the steps of:

a. suspending the cells in a liquid, thereby creating a cellular suspension;

b. placing the cellular suspension in a tapering passage, the tapering passage including a wide end and an oppositely-disposed narrow end that defines an opening, the opening having a dimension corresponding to a selected cell size; and

c. applying an acoustic wave to the cells, thereby forcing cells having a cell size smaller than the selected cell size through the opening and so that at least a portion of the cells having a cell size not smaller than the selected cell size are not forced through the opening.

12. The method of claim 11, further comprising the steps of:

a. placing the cellular suspension into a passage prior to the step of applying a standing acoustic wave; and

b. focusing the acoustic wave with the passage so that the acoustic wave has a predetermined compression geometry relative to the passage.

13. A method of extracting material from a cell, having a cell membrane, comprising the steps of:

a. suspending the cell in a liquid, thereby creating a cellular suspension;

b. applying a predetermined electric field to the cell; and

c. applying a acoustic wave to the cell,

wherein the electric field and the acoustic wave cause the cell membrane to allow the material to pass out of the cell.

14. The method of claim 13, wherein the material comprises DNA.

15. The method of claim 13, wherein the step of applying an acoustic wave includes applying a standing acoustic wave, the method further comprising the step of focusing the standing acoustic wave by placing the cellular suspension in a tapered passage.

16. The method of claim 13, further comprising the steps of:

a. placing the cellular suspension into a passage prior to the step of applying a predetermined electric field to the cell; and

b. focusing the standing acoustic wave with the passage so that the standing acoustic wave has a predetermined compression geometry relative to the passage.

17. An apparatus for manipulating cells, comprising:

a. a substrate, having a first side and an opposite second side, the substrate defining at least one tapering passage passing therethrough, the tapering passage opening to the first side with a wide end and also opening to the second side with a narrow end, the narrow end having a size corresponding to a predetermined characteristic of a selected cell;

b. a first poration electrode and a spaced-apart second poration electrode disposed so as to impart a predetermined electrical field on the passage when an electrical potential difference is applied between the first poration electrode and the second poration electrode;

c. a fluid driving structure that drives fluid through the opening;

d. an oscillating circuit that applies an oscillating electric potential to the ultrasonic transducer, thereby causing the ultrasonic transducer to generate a standing wave in the tapering passage,

wherein the standing wave and the electrical field impart energy on at least a portion of the cells so as to cause a predetermined action on the cells.

18. The apparatus of claim 17, wherein the fluid driving structure comprises an ultrasonic transducer, spaced apart from the wide end.

19. The apparatus of claim 17, wherein the fluid driving structure comprises an ultrasonic transducer that is disposed adjacent to the substrate.

20. The apparatus of claim 17, wherein the fluid driving structure comprises a fluid pump.

21. The apparatus of claim 17, wherein the substrate comprises a silicon wafer.

22. The apparatus of claim 17, wherein the tapering passage comprises a pyramidal passage.

23. The apparatus of claim 17, wherein the tapering passage comprises a frusto-conical passage.

24. The apparatus of claim 17, wherein the first poration electrode and the second poration electrode each comprise a conductive substance disposed on an inside surface of the tapering passage.

25. The apparatus of claim 17, wherein the second poration electrode is disposed adjacent to the second side of the substrate.

26. The apparatus of claim 25, further comprising an insulator layer disposed adjacent to the second poration electrode and wherein the first poration electrode is disposed adjacent to the insulator layer.

27. The apparatus of claim 17, wherein the substrate includes a dopant so that the substrate comprises the second poration electrode.

28. The apparatus of claim 17, wherein the ultrasonic transducer comprises a capacitive transducer.

29. The apparatus of claim 28, wherein the capacitive transducer comprises a first transducer electrode and spaced-apart second transducer electrode so that when a potential is applied between the first transducer electrode and the second transducer electrode, the second transducer electrode moves relative to the first transducer electrode.

30. The apparatus of claim 17, wherein the ultrasonic transducer comprises a piezoelectric transducer.

31. The apparatus of claim 30, wherein the piezoelectric transducer comprises a layer of a piezoelectric material disposed between a first transducer electrode and an opposite second transducer electrode and oriented so that when an electric field is applied between the first transducer electrode and the second transducer electrode, a dimensional characteristic of the layer of piezoelectric material changes.

32. The apparatus of claim 17, wherein the substrate defines an array of tapering passages passing therethrough.

33. The apparatus of claim 32, wherein the array of tapering passages includes a first plurality of tapering passages and a spaced-apart second plurality of tapering passages, the apparatus further comprising a wall extending from the substrate so as to separate the first plurality of tapering passages from the second plurality of tapering passages.

34. The apparatus of claim 32, wherein the second poration electrode comprises a plurality of sub-electrodes, wherein each sub-electrode is disposed adjacent to a different set of the tapering passages and wherein each sub-electrode may be driven by a different electrical potential.