Apparatus and method for rapid ultrasonic disruption of cells or viruses

• US 6,431,476 B1
• Filed: 12/21/1999
• Issued: 08/13/2002
• Est. Priority Date: 12/21/1999
• Status: Expired due to Term

• Current Assignee: Cepheid (Danaher Corporation), Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Cepheid (Danaher Corporation)
• Inventors: Belgrader, Phillip, Chang, Ronald, McMillan, William A., Pourahmadi, Farzad, Dority, Douglas B., Taylor, Michael T.
• Primary Examiner(s): Rosenbaum, Mark
• Application Number:
  US09/469,724
• Time in Patent Office: 966 Days
• Field of Search: 241/1, 241/2, 241/301, 241/172, 435/306.1, 435/259
• US Class Current: 241/1
• CPC Class Codes: B01L 2300/042 Caps; Plugs
• CPC Class Codes: B01L 2300/046 Function or devices integrated in the closure
• CPC Class Codes: B01L 2300/14 Means for pressure control
• CPC Class Codes: B01L 2400/0439 ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0644 rotary valves
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/50825 Closing or opening means, corks, bungs closures for containers B65D; means for removing stoppers B67B7/02
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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

Abstract

The present invention provides an apparatus and method for disrupting cells or viruses to release the nucleic acid therefrom. The apparatus includes a container having a chamber for holding the cells or viruses. The apparatus also includes an ultrasonic transducer for contacting a wall of the chamber and for transmitting ultrasonic energy into the chamber through the wall. A support structure holds the container and the transducer against each other such that the transducer contacts the wall of the chamber. The support structure includes an elastic body, such as a spring, for applying to the container or to the transducer a substantially constant force to press together the transducer and the wall. The chamber also preferably contains beads for enhancing the disruption of the cells or viruses. The apparatus performs rapid and consistent lysis of cells or viruses, often in as little time as 5 to 10 seconds.

Claims

1. An apparatus for disrupting cells or viruses, the apparatus comprising:

a) a container having a chamber for holding the cells or viruses, wherein the container includes at least one wall defining the chamber;

b) an ultrasonic transducer for contacting an external surface of the wall and for transmitting ultrasonic energy into the chamber through the wall; and

c) a support structure for holding the container and the transducer against each other such that the transducer contacts the external surface of the wall and for applying to the container or to the transducer a substantially constant force to press together the transducer and the wall.

2. The apparatus of claim 1, wherein the support structure comprises:

a) a base;

b) a first holder attached to the base for holding the transducer;

c) a second holder slidably mounted to the base for holding the container and for positioning the container against the transducer such that the external surface of the wall contacts the transducer; and

d) at least one elastic body for applying to the second holder the substantially constant force to press the wall against the transducer.

3. The apparatus of claim 2, wherein the elastic body comprises a spring, and wherein the apparatus further comprises means for adjusting the preload on the spring.

4. The apparatus of claim 1, wherein the chamber is defined by:

a) a first wall for contacting the transducer, wherein the first wall is sufficiently flexible to conform to a surface of the transducer;

b) a second wall spaced apart from the first wall, wherein the second wall is sufficiently flexible to move inwardly or outwardly as ultrasonic energy is received in the chamber; and

c) a rigid frame defining the side walls of the chamber, wherein the side walls connect the flexible walls to each other;

d) and wherein the support structure includes a holder for holding the frame such that the flexible walls are substantially unrestrained by the holder.

5. The apparatus of claim 4, wherein the holder comprises a body having a recess for receiving the frame and having an opening adjacent the recess, the opening being positioned to permit the outward expansion of the second wall when the container is positioned in the recess.

6. The apparatus of claim 4, wherein each of the flexible walls comprises a film of polymeric material, and wherein each of the flexible walls has a thickness in the range 0.025 to 0.1 mm.

7. The apparatus of claim 4, wherein the spacing between the first and second flexible walls is less than or equal to 5 mm.

8. The apparatus of claim 1, wherein the transducer comprises an ultrasonic horn having a tip for contacting the wall, and wherein the thickness of the chamber is less than or equal to half of the diameter of the tip.

9. The apparatus of claim 1, wherein the transducer comprises an ultrasonic horn having a tip for contacting the wall, and wherein the wall is sufficiently flexible to conform to a surface of the tip.

10. The apparatus of claim 9, wherein the container includes a port and a channel connecting the port to the chamber, and wherein the apparatus further comprises a plunger that is inserted into the channel to increase pressure in the chamber.

11. The apparatus of claim 10, wherein the plunger has a pressure stroke sufficient to increase the pressure in the chamber to at least 2 psi above the ambient pressure external to the container.

12. The apparatus of claim 10, wherein the plunger has a pressure stroke sufficient to increase the pressure in the chamber to at least 8 psi above the ambient pressure external to the container.

13. The apparatus of claim 1, wherein the wall comprises a film of polymeric material, and wherein the wall has a thickness in the range 0.025 to 0.1 mm.

14. The apparatus of claim 1, wherein the force is in the range of 0.25 to 4 lbs.

15. The apparatus of claim 1, wherein the force is at least 1 lb.

16. The apparatus of claim 1, further comprising beads in the chamber.

17. The apparatus of claim 1, wherein the ratio of the width of the chamber to the thickness of the chamber is at least 2:

1.

18. The apparatus of claim 17, wherein the ratio of the width of the chamber to the thickness of the chamber is at least 4:

1.

19. The apparatus of claim 17, wherein the thickness of the chamber is less than 5 mm.

20. The apparatus of claim 1, further comprising means for increasing the pressure in the chamber to at least 2 psi above the ambient pressure external to the container.

21. The apparatus of claim 1, further comprising means for increasing the pressure in the chamber to at least 8 psi above the ambient pressure external to the container.

22. The apparatus of claim 1, wherein the support structure includes a spring for applying the force, and wherein the force is in the range of 0.25 to 4 lbs.

23. The apparatus of claim 1, wherein the support structure includes a spring for applying the force, and wherein the force is at least 1 lb.

24. The apparatus of claim 1, wherein the support structure includes a spring for applying the force, the spring having a spring rate less than 4 lbs/inch.

25. The apparatus of claim 1, wherein the support structure includes at least one elastic body for applying the force, the at least one elastic body being selected from the group consisting of a coil spring, wave spring, torsion spring, spiral spring, leaf spring, elliptic spring, and half-elliptic spring.

26. The apparatus of claim 1, wherein the transducer comprises an ultrasonic horn having a tip for contacting the wall.

27. The apparatus of claim 1, wherein the transducer comprises piezoelectric material.

28. An apparatus for holding a container for disrupting cells or viruses that are placed in a chamber of the container, the apparatus comprising:

a) an ultrasonic transducer for transmitting ultrasonic energy to the chamber through a wall of the container; and

b) a support structure for coupling the transducer to an external surface of the wall and for applying to the container or the transducer a substantially constant force to press together the transducer and the wall.

29. The apparatus of claim 28, further comprising beads in the chamber.

30. The apparatus of claim 28, wherein the support structure comprises:

a) a base;

b) a first holder attached to the base for holding the transducer;

c) a second holder slidably mounted to the base for holding the container and for positioning the container against the transducer such that the wall contacts the transducer; and

d) at least one elastic body for applying to the second holder the substantially constant force to press the wall against the transducer.

31. The apparatus of claim 28, wherein the transducer comprises an ultrasonic horn having a tip for contacting the wall.

32. The apparatus of claim 28, wherein the force is in the range of 0.25 to 4 lbs.

33. The apparatus of claim 28, wherein the force is at least 1 lb.

34. The apparatus of claim 28, wherein the support structure includes a spring for applying the force, and wherein the apparatus further comprises means for adjusting the preload on the spring.

35. The apparatus of claim 28, further comprising means for increasing the pressure in the chamber to at least 2 psi above the ambient pressure external to the container.

36. The apparatus of claim 28, further comprising means for increasing the pressure in the chamber to at least 8 psi above the ambient pressure external to the container.

37. The apparatus of claim 28, wherein the support structure includes a spring for applying the force, and wherein the force is in the range of 0.25 to 4 lbs.

38. The apparatus of claim 28, wherein the support structure includes a spring for applying the force, and wherein the force is at least 1 lb.

39. The apparatus of claim 28, wherein the support structure includes a spring for applying the force, the spring having a spring rate less than 4 lbs/inch.

40. The apparatus of claim 28, wherein the support structure includes at least one elastic body for applying the force, the at least one elastic body being selected from the group consisting of a coil spring, wave spring, torsion spring, spiral spring, leaf spring, elliptic spring, and half-elliptic spring.

41. The apparatus of claim 28, wherein the transducer comprises piezoelectric material.

42. A method for disrupting cells or viruses, the method comprising the steps of:

a) holding a liquid or gel containing the cells or viruses in the chamber of a container;

b) holding an ultrasonic transducer against a wall of the chamber;

c) applying to the container or to the transducer a substantially constant force to press together the transducer and the wall; and

d) transmitting ultrasonic energy from the transducer into the chamber.

43. The method of claim 42, further comprising the step of placing beads in the chamber prior to transmitting ultrasonic energy into the chamber.

44. The method of claim 42, wherein the ultrasonic energy is transmitted to the chamber for a duration of time less than 30 seconds.

45. The method of claim 42, wherein the transducer comprises an ultrasonic horn having a tip for contacting the wall of the chamber, and wherein the wall is sufficiently flexible to conform to the surface of the tip.

46. The method of claim 42, wherein the transducer comprises an ultrasonic horn having a tip for contacting the wall of the chamber, and wherein the thickness of the chamber is less than or equal to half of the diameter of the tip.

47. The method of claim 42, further comprising the step of pressurizing the chamber prior to transmitting ultrasonic energy into the chamber.

48. The method of claim 47, wherein the chamber is pressurized to at least 2 psi above the ambient pressure external to the container.

49. The method of claim 47, wherein the chamber is pressurized to at least 8 psi above the ambient pressure external to the container.

50. The method of claim 42, wherein the force is in the range of 0.25 to 4 lbs.

51. The method of claim 42, wherein the force is at least 1 lb.

52. The method of claim 42, wherein the force is applied by a spring having a spring rate less than 4 lbs/inch.

53. The method of claim 42, wherein the force is applied by at least one elastic body selected from the group consisting of a coil spring, wave spring, torsion spring, spiral spring, leaf spring, elliptic spring, and half-elliptic spring.

54. The method of claim 42, wherein the transducer comprises an ultrasonic horn having a tip that is held against the wall.

55. The method of claim 42, wherein the transducer comprises piezoelectric material.

56. A method for disrupting cells or viruses, the method comprising the steps of:

a) providing a container having a chamber defined by;

i) a first wall having an external surface for contacting an ultrasonic transducer, wherein the first wall is sufficiently flexible to conform to a surface of the transducer;

ii) a second flexible wall spaced apart from the first wall; and

iii) side walls connecting the flexible walls to each other, wherein the ratio of the width of the chamber to the thickness of the chamber is at least 2;

1;

b) holding in the chamber;

i) a liquid or gel containing the cells or viruses; and

ii) beads;

c) placing the ultrasonic transducer in contact with the external surface of the first wall of the chamber; and

d) transmitting ultrasonic energy from the transducer into the chamber.

57. The method of claim 56, further comprising the step of applying to the container or to the transducer a substantially constant force to press together the transducer and the first wall of the chamber.

58. The method of claim 57, wherein the force is applied by a spring having a spring rate less than 4 lbs/inch.

59. The method of claim 57, wherein the force is applied by at least one elastic body selected from the group consisting of a coil spring, wave spring, torsion spring, spiral spring, leaf spring, elliptic spring, and half-elliptic spring.

60. The method of claim 57, wherein the force is in the range of 0.25 to 4 lbs.

61. The method of claim 57, wherein the force is at least 1 lb.

62. The method of claim 56, wherein the transducer comprises an ultrasonic horn having a tip for contacting the first wall of the chamber, and wherein the thickness of the chamber is less than or equal to half of the diameter of the tip.

63. The method of claim 56, further comprising the step of pressurizing the chamber prior to transmitting ultrasonic energy into the chamber.

64. The method of claim 63, wherein the chamber is pressurized to at least 2 psi above the ambient pressure external to the container.

65. The method of claim 63, wherein the chamber is pressurized to at least 8 psi above the ambient pressure external to the container.

66. The method of claim 56, wherein the transducer comprises an ultrasonic horn having a tip that is placed in contact with the first wall.

67. The method of claim 56, wherein the transducer comprises piezoelectric material.