Method for preparation of microarrays for screening of crystal growth conditions

• US 20030232967A1
• Filed: 10/23/2002
• Published: 12/18/2003
• Est. Priority Date: 04/06/1999
• Status: Active Grant

• Current Assignee: UAB Research Foundation (University of Alabama System)
• Original Assignee: UAB Research Foundation (University of Alabama System)
• Inventors: Chait, Arnon, Belgovskiy, Alexander, DeLucas, Lawrence, Stoops, Brad
• Granted Patent: US 7,244,396 B2
• Time in Patent Office: Days
• US Class Current: 506/12
• CPC Class Codes: B01L 3/06 Crystallising dishes
• CPC Class Codes: C07K 14/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: C07K 14/43 Sweetening agents, e.g. thaumatin, monellin
• CPC Class Codes: C12N 9/0016 with NAD or NADP as acceptor (1.4.1)
• CPC Class Codes: C12N 9/2442 Chitinase (3.2.1.14)
• CPC Class Codes: C12N 9/6427 Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
• CPC Class Codes: C12Y 302/01014 Chitinase (3.2.1.14)
• CPC Class Codes: C30B 29/58 Macromolecular compounds
• CPC Class Codes: C30B 7/00 Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions from molten solvents C30B9/00; by normal or gradient freezing C30B11/00; under a protective fluid C30B27/00
• CPC Class Codes: G01N 21/01 Arrangements or apparatus for facilitating the optical investigation
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures

Abstract

A method of screening solution conditions suitable for the crystallization of macromolecules with a picogram to a microgram of protein using picoliter or nanoliter volumes is provided. A preferred method comprises preparing a plurality of recipe solutions in milliliter volumes, aspirating protein and recipe solutions, respectively, from a plurality of wells, and dispensing the protein and recipe solutions into a plurality of wells, covering the combined protein and recipe solutions with oil, and maintaining the solution wells until crystallization or precipitation occur therein.

Claims

1. ) A method for screening protein crystal growth conditions comprising the steps of:

(a) providing a microarray with a plurality of wells in the microarray;

(b) dispensing a volume of protein solution containing a protein into at least one of the wells;

(c) dispensing a recipe solution into at least one of the wells, wherein the recipe solution was prepared from bulk ingredients prior to being dispensed into the wells and was stored in a plurality of containers or in a second multiwell plate;

(d) subjecting the protein solution, or a combination solution formed by the combination of the protein solution with the recipe solution, to environmental conditions effective to form protein crystals; and

(e) observing the growth of protein crystals or precipitation of protein.

2. ) The method of claim 1, wherein a solution selected from the group consisting of the protein solution, the recipe solution and the combination solution is covered by a first oil.

3. ) The method of claim 1, wherein the recipe solutions are prepared in a volume of greater than or equal to 1 microliter.

4. ) The method of claim 1, wherein the recipe solutions are prepared in a volume of less than 5 milliliters.

5. ) The method of claim 1, wherein the recipe solutions are mixed by a method selected from the group consisting of vortexing, use of magnetic stirring elements, agitation and shaking.

6. ) The method of claim 1, wherein fractions of the recipe solutions are transferred from the containers or the second multiwell plate into further containers or further multiwell plates.

7. ) The method of claim 1, wherein the dispensing of the protein or the recipe solutions is conducted using a dispensing device comprising fast solenoid technology.

8. ) The method of claim 7, wherein the dispensing device comprises the use of adjustable constant back pressure.

9. ) The method of claim 7, wherein the dispensing device uses fast solenoid technology with syringe dispensing.

10. ) The method of claims 7, wherein the dispensing device is adapted to use a dispenser tip.

11. ) The method of claim 1, wherein the recipe solutions are prepared from the bulk ingredient using a dispensing device comprising fast solenoid technology.

12. ) The method of either claim 11, wherein the dispensing device comprises the use of adjustable constant back pressure.

13. ) The method of claim 11, wherein the dispensing device uses fast solenoid technology with syringe dispensing.

14. ) The method of claims 11, wherein the dispensing device is adapted to use a dispenser tip.

15. ) The method of claim 1, wherein the dispensing of the protein or the recipe solutions is conducted using a dispensing device comprising piezo technology.

16. ) The method of claims 15, wherein the dispensing device is adapted to use a dispenser tip.

17. ) The method of claim 1, wherein the recipe solutions are prepared from the bulk ingredient using a dispensing device comprising piezo technology.

18. ) The method of claims 17, wherein the dispensing device is adapted to use a dispenser tip.

19. ) The method of claim 1, wherein the dispensing device is adapted to use a dispenser tip and wherein the tip is calibrated for fluids which the dispensing device is used to dispense the fluids dispensed.

20. ) The method of claim 19, wherein the tip is calibrated by;

i) dispensing a designated quantity of the fluid;

ii) determining the actual delivered volume; and

iii) adjusting indicated values for designated quantity of fluid to correspond with the actual volume delivered.

21. ) The method of claim 20, wherein the volume is determined by visually determining the actual delivered volume.

22. ) The method of claim 20, wherein the volume is determined by quantifying a detectable agent contained in the designated quantity of the fluid, wherein the concentration of the detectable agent is known.

23. ) The method of claim 22, wherein the detectable agent is a dye that can be detected by quantifying its absorbance or by its fluorescence.

24. ) A method for screening protein crystal growth conditions comprising the steps of:

(a) providing an array of at least two wells;

(b) dispensing a volume of protein solution containing a protein into at least one of the wells;

(c) dispensing a recipe solution into at least one of the wells, wherein the recipe solution was prepared from bulk ingredients prior to being dispensed into the wells and was stored in at least one of a plurality of containers or in a second multiwell plate;

(d) subjecting the protein solution, or a combination solution formed by the combination of the protein solution with the recipe solution, to environmental conditions effective to form protein crystals; and

(e) observing the growth of protein crystals or precipitation of protein.

25. ) The method of claim 24, wherein a solution selected from the group consisting of the protein solution, the recipe solution and the combination solution is covered by a first oil.

26. ) The method of claim 25, wherein the first oil is water-permeable.

27. ) The method of claim 26, further comprising an additional layer of oil, wherein the additional layer of oil overlays the first oil.

28. ) The method of claim 27 wherein the additional layer of oil is added after the first oil is added.

29. ) The method of claim 28 wherein the additional layer of oil is added after the volume of the solution over which the additional layer of oil is added has been reduced by evaporation.

30. ) The method of claim 29 wherein the additional layer of oil is less permeable to water than the first oil.

31. ) The method of claim 25, wherein the first oil is water-impermeable.

32. ) The method of claim 31, further comprising an additional layer of oil, wherein the additional layer of oil overlays the first oil.

33. ) The method of claim 32, wherein the additional layer of oil is added after the first oil is added.

34. ) The method of claim 33, wherein the additional layer of oil is added after the volume of the solution over which the additional layer of oil is added has been reduced by evaporation.

35. ) The method of claim 34, wherein the additional layer of oil is less permeable to water than the first oil.

36. ) The method of claim 25, wherein step (d) comprises subjecting the solutions to a defined or selected humidity or range of humidities.

37. ) The method of claim 24, wherein a combination solution is formed in the wells of a multiwell plate.

38. ) The method of claim 37, wherein the protein solution is dispensed into the wells of the multiwell plate prior to the recipe solution.

39. ) The method of claim 24, wherein the conditions effective to form crystals must evaporate greater than 10% of the protein or the combination solution.

40. ) The method of claim 39, wherein the conditions effective to form crystals must evaporate greater than 20% of the protein or the combination solution.

41. ) The method of claim 40, wherein the conditions effective to form crystals must evaporate greater than 30% of the protein or the combination solution.

42. ) The method of claim 41, wherein the conditions effective to form crystals must evaporate greater than 40% of the protein or the combination solution.

43. ) The method of claim 42, wherein the conditions effective to form crystals must evaporate greater than 50% of the protein or the combination solution.

44. ) The method of claim 43, wherein the conditions effective to form crystals must evaporate greater than 60% of the protein or the combination solution.

45. ) The method of claim 44, wherein the conditions effective to form crystals must evaporate greater than 75% of the protein of the combination solution.

46. ) The method of claim 24, wherein at least a pair of the protein and the recipe solutions are in vapor-vapor contact with each other.

47. ) The method of claim 46, wherein the protein solution, the recipe solution or both the protein solution and the recipe solution are overlayed with an oil.

48. ) A crystallization system, comprising a first recipe solution preparation and storage sub-system used to prepare a plurality of recipe solutions for storage until said recipe solutions are required for use within the crystallization system, and a second aspiration and dispensing sub-system used to aspirate and dispense a plurality of protein solutions and of said recipe solutions, respectively, into a plurality of wells.

49. ) The recipe preparation and storage sub-system of claim 48, comprising at least one fluid dispenser tip, at least one syringe, and at least one distribution valve, wherein the total number of fluid dispenser tips provided is less than the total number of a plurality of available bulk solutions from which the recipe solutions are prepared, and wherein the total number of distribution valves used to connect different ones of said bulk solutions to the at least one fluid dispenser tip and the at least one syringe, respectively, is minimized.

50. ) The recipe preparation and storage sub-system of claim 48, further comprising a plurality of multi well plates, each said well plate having a volume ranging from about 10 microliter to about 10 milliliters.

51. ) A crystallization experiment preparation system, comprising:

a) a dispense head provided with plurality of fluid dispenser tips;

b) a video camera and a long range microscope each carried on said dispense head;

c) a stage constructed and arranged to move the dispense head, the video camera, and the long range microscope together in the X, the Y, and the Z directions, respectively;

d) a deck area positioned with respect to the stage, said deck area having at least one fixture for receiving at least one recipe plate, at least one protein plate, at least one crystallization plate, a quality control area, and a wash system, respectively, thereon.

52. ) A crystallization experiment preparation system, comprising:

a) a dispense head provided with plurality of fluid dispenser tips;

b) a video camera and a long range microscope each carried on said dispense head;

c) a deck area positioned with respect to the dispense head, said deck area having at least one fixture for receiving at least one recipe plate, at least one protein plate, at least one crystallization plate, a quality control area, and a wash system, respectively, thereon;

d) wherein the dispense head, the video camera, and the long range microscope are held together in a stationary position with respect to a deck area; and

e) wherein the deck area is constructed and arranged to be moved with respect to the dispense head, the video camera, and the long range microscope by a stage movable in the X, the Y, and the Z, directions, respectively.

53. ) A crystallization experiment preparation system, comprising:

a) a dispense head provided with plurality of fluid dispenser tips;

b) a video camera and a long range microscope each carried on said dispense head;

c) a first stage on which the dispense head is positioned, said first stage being constructed and arranged to move the dispense head in the Z direction;

d) a deck area positioned with respect to the dispense head, said deck area having at least one fixture for receiving at least one recipe plate, at least one protein plate, at least one crystallization plate, a quality control area, and a wash system, respectively, thereon; and

e) a second stage adapted to carry said deck area in the X and the Y directions, respectively.

54. ) The crystallization experiment preparation system of claim 53, said dispense head further comprising an aspiration and dispensing system.

55. ) The crystallization experiment preparation system of claim 54, said aspiration and dispensing system comprising a constant pressure fast solenoid.

56. ) The crystallization experiment preparation system of claim 54, said aspiration and dispensing system comprising a piezo device.

57. ) The crystallization experiment preparation system of claim 54, said aspiration and dispensing system comprising a syringe-driven fast solenoid.

58. ) The crystallization experiment preparation system of claim 53, further comprising a dispenser tip fluidics system, said dispenser tip fluidics system comprising a plurality of fluid dispenser tips.

59. ) The crystallization experiment preparation system of claim 58, wherein each dispenser tip of the tip fluidics system is individually calibrated.

60. ) The crystallization experiment preparation system of claim 59, wherein each said individually calibrated dispenser tips has an individual calibration curve used to dispense a measured and predetermined volume of a protein solution or a recipe solution, respectively, therethrough.

61. ) The crystallization experiment preparation system of claim 53, further comprising at least one droplet array, each said at least one droplet array comprising at least one protein solution and at least one recipe solution, respectively, prepared in blocks of protein and recipe solutions, respectively.

62. ) The crystallization experiment preparation system of claim 61, wherein the total time required to prepare each said protein solution and recipe solution block, respectively, ranges from about one to about sixty seconds.