Microfabricated two-pin system for biomolecule crystallization

• US 20030170146A1
• Filed: 12/23/2002
• Published: 09/11/2003
• Est. Priority Date: 12/21/2001
• Status: Active Grant

• Current Assignee: Cytonome/St, Llc (Cytonome Inc.)
• Original Assignee: Teragenics, Inc.
• Inventors: Gilbert, John, Trikha, Jaishree
• Granted Patent: US 7,153,699 B2
• Time in Patent Office: Days
• US Class Current: 422/100
• CPC Class Codes: B01L 2200/142 Preventing evaporation
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2400/0439 ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 3/0244 using pins
• CPC Class Codes: B01L 3/0248 Prongs, quill pen type dispenser
• CPC Class Codes: B01L 3/0255 characterized by the form or material of the pin tip
• CPC Class Codes: B01L 3/06 Crystallising dishes
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: G01N 2035/1037 Using surface tension, e.g. pins or wires
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

A two-pin liquid handling system for crystallizing biomolecules comprises a pair of interacting pins for holding a droplet of liquid therebetween. Each pin includes a tip spaced predetermined distance from the other pin to define a sample acquisition region. The two-pin liquid handling system includes a temperature and humidity control element for controlling the temperature of the sample acquisition region to cause transpiration of water to and from a droplet of biomolecule solution to promote crystallization of the biomolecule.

Claims

1. A system for crystallizing biomolecules comprising:

a holder;

a first pin coupled to the holder and having a first tip;

a second pin coupled to the holder and having a second tip spaced a predetermined separation distance from the first tip; and

a temperature control element for controlling the temperature of said first and second pin to promote crystallization of a biomolecule held between the first and second pin.

2. The system of claim 1, wherein the second tip is spaced from the first tip to form a cavity for receiving the biomolecule to be crystallized.

3. The system of claim 2, wherein, the cavity has a cylindrical shape.

4. The system of claim 2, wherein the cavity has a conical shape.

5. The system of claim 2, wherein the cavity has a spherical shape.

6. The system of claim 2, wherein the cavity has a volume of between about 0.1 nanoliters and about 50 nanoliters.

7. A system for crystallizing biomolecules comprising:

a holder;

a first pin coupled to the holder and having a first tip; and

a second pin coupled to the holder and having a second tip spaced a predetermined separation distance from the first tip, wherein the first tip and the second tip are shaped to promote biomolecule crystallization.

8. The system of claim 7, wherein the first tip and second tip form an open cavity for holding a sample.

9. The system of claim 8, wherein the open cavity has a cylindrical shape.

10. The system of claim 8, wherein the open cavity has a conical shape.

11. The system of claim 8, wherein the open cavity has a spherical shape.

12. The system of claim 8, wherein the open cavity has a volume of between about 0.1 nanoliters and about 50 nanoliters.

13. A liquid handling system, comprising:

a pin-type liquid handling assembly comprising a plurality of components; and

a temperature control element for controlling the temperature one of said components of the pin-type liquid dispensing assembly, whereby a temperature of a liquid to be handled by the pin-type liquid dispensing assembly is controlled so as to control evaporation.

14. The liquid dispensing system of claim 13, wherein the cooling element is a Peltier element.

15. The liquid dispensing system of claim 13, wherein the pin-type liquid dispensing assembly comprises a base, a first pin coupled to the base having a first tip and a second pin coupled to the base having a second tip.

16. The liquid dispensing system of claim 15, wherein the second tip is spaced from the first tip to form a cavity for receiving a liquid volume.

17. The liquid dispensing system of claim 16, wherein the cavity retains the liquid volume by capillary forces.

18. The liquid sample dispensing system of claim 16, wherein the cooling element reduces evaporation of the liquid volume held in the cavity.

19. The liquid dispensing system of claim 13, wherein the pin-type liquid dispensing assembly is manufactured by microfabrication technology.

20. The liquid dispensing system of claim 13, wherein the cooling element is attached to one of said components.

21. The liquid dispensing system of claim 13, wherein the pin-type liquid dispensing assembly includes a holder and the cooling element cools the holder.

22. The liquid dispensing system of claim 13, further comprising a temperature control system for controlling the temperature of the liquid dispensing system.

23. The sample dispensing system of claim 22, wherein the temperature control system comprises:

a temperature sensor for measuring an actual temperature of the system; and

a controller for comparing the actual temperature to a reference temperature and triggering the cooling element to adjust the actual temperature to equal the reference temperature.

24. The liquid dispensing system of claim 23, wherein the temperature sensor comprises a thin-film resistor.

25. A method for crystallizing biomolecules, comprising the steps of:

providing a liquid handling assembly comprising at least one pin;

acquiring a droplet of a, biomolecule solution on the liquid carrying element;

transpiring water between the biomolecule solution and the environment to concentrate the biomolecule, thereby causing crystallization of the biomolecule to occur.

26. The method of claim 25, wherein the pin-type liquid handling assembly comprises a first pin having a first tip and a second pin having a second tip and said droplet is held between the first tip and the second tip.

27. The method of claim 25, wherein the liquid carrying element forms an open cavity for holding the droplet of biomolecule solution.

28. The method of claim 27, wherein the cavity has a capacity of between about 0.1 nanoliters and about 50 nanometers.

29. The method of claim 25, wherein the liquid carrying element comprises a capillary uptake pin having a channel for holding a droplet of liquid by capillary force.

30. The method of claim 25, further comprising the step of:

observing formation of a crystal.

31. The method of claim 30, wherein the step of observing comprises detecting biomolecule aggregation by laser scattering

32. The method of claim 30, wherein the step of observing comprises using a an optical device selected from the group consisting of a polarization microscope, a CCD camera, and a video camera.

33. The method of claim 25, further comprising the step of freezing the crystal on the pin.

34. The method of claim 25, wherein the step of acquiring a droplet of biomolecule solution comprises dipping the pin in a well containing a supply of the biomolecule solution and a crystallization reagent.

35. The method of claim 25, wherein the step of transpiring comprises controlling the temperature of the liquid carrying element.

36. A method of crystallizing biomolecules, comprising:

providing a crystallizing structure for growing a crystal from a biomolecule solution;

forming a biomolecule crystal on the structure; and

freezing the crystal on the structure.

37. The method of claim 36, further comprising the step of:

observing the crystal on the structure.

38. The method of claim 36, wherein the crystallizing structure comprises a first pin having a first tip and a second pin having a second tip spaced a predetermined separation distance from the first tip for holding the biomolecule solution therebetween.

39. The method of claim 36, wherein the crystallizing structure includes a temperature control element for controlling the temperature of the biomolecule solution to promote formation of the biomolecule crystal.

40. A method of crystallizing a biomolecule comprising

providing a crystallizing structure for growing a crystal from a biomolecule solution;

forming a biomolecule crystal on the structure under a set of crystallization conditions; and

observing the crystal on the structure.

41. The method of claim 40, further comprising the step of altering the crystallization conditions in response to the step of observing.

42. The method of claim 41, wherein the step of altering comprising varying the temperature of the crystallizing structure.

43. A system for crystallizing biomolecules, comprising:

an array of wells containing a supply of biomolecule solution;

an array of biomolecule crystallization devices , wherein each biomolecule crystallization devices comprises a first pin having a first tip and a second pin having a second tip spaced from the first tip to define a sample acquisition region.

44. The system of claim 43, further comprising an effector setting system for varying effectors in the array of wells to set crystallization conditions.

45. The system of claim 44, wherein the effector setting system comprises a membrane for dialyzing effectors in the array of wells.

46. The system of claim 45, wherein the effectors comprise ions, ligands that are exchanged through the membrane.

47. A system for crystallizing biomolecules, comprising:

an array of wells containing a supply of biomolecule solution; and

an array of biomolecule crystallization cavity devices, wherein each biomolecule crystallization cavity device includes a cavity that can pick up between about 0.1 and about 50η

l of biomolecule solution for crystallization of the biomolecule solution in the cavity.

48. The system of claim 47, wherein the at least two biomolecule crystallization cavity devices in the array are substantially identical.

49. The system of claim 47, wherein two or more of the biomolecule crystallization cavity devices are different.

50. The system of claim 47, wherein each biomolecule crystallization cavity device comprises a first pin having a first tip and a second pin spaced from the second tip to form said cavity.

51. The system of claim 47, wherein the concentration of the protein solution in the wells is modified by modifying effectors in each well.

52. The system of claim 51, wherein the effectors are modified by adding concentrated effector solutions to the on modifying effectors in wells.

53. The system of claim 51, wherein each well includes a biomolecule impermeable membrane, wherein the protein impermeable membrane is used to dialyze effectors into the protein solution.

54. A method of determining optimal conditions for promoting crystallization of a biomolecule, comprising:

providing an array of devices for crystallizing biomolecules, wherein each device comprises two interacting pins for holding a sample therebetween;

forming an array of crystals in the devices by controlling crystallization conditions;

recording the crystallization conditions; and

observing the array of crystals formed in the devices.

55. A system for crystallizing a biomolecule, comprising:

a pin assembly for handling the biomolecules, and

a temperature control element coupled to the pin assembly for controlling the temperature of said pin assembly to promote crystallization of a biomolecule.

56. The system of claim 55, wherein said pin assembly comprises a first pin having a tip, and a second pin having a tip, said tips of the first and second pins forming a cavity sized and configured for receiving the biomolecule to be crystallized.

57. The system of claim 56, wherein said pin assembly further comprises a holder coupled to said first and second pins.

58. The system of claim 56, wherein said pin assembly comprises a relaxation region coupled to one of said pins.

59. The system of claim 56, wherein said pin assembly further comprises an actuator for actuating at least one of said pins.

60. The system of claim 59, wherein said pin assembly further comprises a sensor for sensing movement of one of said pins.

61. The system of claim 55, further comprising a temperature sensor for sensing the temperature of the assembly, and a controller in circuit with the temperature control element and the temperature sensor for controlling the temperature of the pin assembly.

62. The system of claim 56, wherein said first pin is stationary and said second pin is movable, said pin assembly further comprising a fulcrum region coupled to said movable pin.

63. The system of claim 62, further comprising a driver coupled to said fulcrum region for moving said second pin.

64. The system of claim 56, wherein the cavity has a volume of between about 0.1 nanoliters and about 50 nanoliters.

65. The system of claim 56, wherein the temperature control element reduces evaporation of a fluid volume held in the cavity.

66. A method for crystallizing a biomolecule, comprising:

providing a pin assembly for handling the biomolecules, and

controlling the temperature of said pin assembly to promote crystallization of a biomolecule.

67. The method of claim 66, further comprising the step of evaporating a fluid containing the biomolecule to crystallize the biomolecule.

68. The method of claim 66, further comprising the step of transpiring a fluid contianing the biomolecule to concentrate the biomolecule, thereby promoting crystallization of the biomolecule.

69. The method of claim 66, further comprising the step of observing the biomolecule during crystallization.