Protein Crystallization Screening and Optimization Droplet Actuators, Systems and Methods

• US 20080044914A1
• Filed: 08/16/2007
• Published: 02/21/2008
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee, Srinivasan, Vijay, Pollack, Michael
• Granted Patent: US 8,007,739 B2
• Time in Patent Office: Days
• US Class Current: 436/86
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/00587 High throughput processes
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C07K 1/1136 by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• 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: Y10T 436/2575 : Volumetric liquid transfer

Abstract

Protein crystallization screening and optimization droplet actuators, systems and methods are provided. According to one embodiment, a screening droplet actuator is provided and includes: (a) a port for introduction of one or more crystallization reagents and/or one or more protein solutions; and (b) a substrate including: (i) an array of two or more mixing wells; and (ii) electric field mediated microfluidics for moving droplets comprising the crystallization reagents and protein solutions into the mixing wells. Optimization droplet actuators, systems including screening droplet actuators, methods of screening protein crystallization conditions, and methods of testing conditions for growing a crystal are also provided.

Claims

1. A screening droplet actuator comprising:

(a) a port for introduction of one or more crystallization reagents and/or one or more protein solutions; and

(b) a substrate comprising;

(i) an array of two or more mixing wells; and

(ii) electric field mediated microfluidics for moving droplets comprising the crystallization reagents and protein solutions into the mixing wells.

2. The screening droplet actuator of claim 1 wherein two or more of the mixing wells are pre-loaded with crystallization reagents for creating differing crystallization conditions in the two or more mixing wells.

3. A system comprising the screening droplet actuator of claim 1 wherein:

(a) the droplet actuator is loaded with;

(i) one or more dispensing wells comprising protein solution loaded therein; and

(ii) two or more mixing wells comprising crystallization droplets loaded therein; and

(b) the system is programmed to execute steps comprising;

(i) dispensing one or more sample droplets from the protein solution;

(ii) transporting one or more of such sample droplets to a mixing well;

(iii) dispensing one or more reagent droplets from the crystallization reagents;

(iv) transporting one or more of such reagent droplets to at least a portion of the mixing wells to yield an array of wells comprising crystallization droplets, said array representing multiple crystallization conditions; and

(v) combining a sample droplet with each crystallization reagent to potentially yield one or more crystallization droplets comprising one or more crystals.

4. The system of claim 3 wherein the protein solution has a concentration which exceeds about 1 mg/mL.

5. The system of claim 3 wherein the protein solution has a concentration which exceeds about 10 mg/mL.

6. The system of claim 3 wherein the protein solution has a concentration which exceeds about 50 mg/mL.

7. The system of claim 3 wherein the protein solution has a concentration which exceeds about 100 mg/mL.

8. The system of claim 3 wherein the volume of protein solution transported to each mixing well in the array of mixing wells comprising crystallization droplets does not exceed about 100 nL.

9. The system of claim 3 wherein the volume of protein solution transported to each mixing well in the array of mixing wells comprising crystallization droplets does not exceed about 50 nL.

10. The system of claim 3 wherein the volume of protein solution transported to each mixing well in the array of mixing wells comprising crystallization droplets does not exceed about 25 nL.

11. The system of claim 3 wherein the volume of protein solution transported to each mixing well in the array of mixing wells comprising crystallization droplets does not exceed about 10 nL.

12. A system comprising the screening droplet actuator of claim 1 wherein:

(a) the droplet actuator further comprises;

(i) one or more dispensing wells comprising protein sample loaded therein; and

(ii) one or more dispensing wells comprising crystallization reagents loaded therein; and

(b) the system is programmed to execute steps comprising conducting droplet operations to;

(i) produce sample droplets and crystallization reagent droplets; and

(ii) combine sample droplets and crystallization reagent droplets into two or more of the mixing wells to produce a unique crystallization condition in each of the two or more mixing wells.

13. A system comprising the screening droplet actuator of claim 1 wherein:

(a) the droplet actuator comprises;

(i) one or more dispensing wells comprising protein solution loaded therein; and

(ii) one or more mixing wells comprising crystallization droplets loaded therein, said crystallization droplets representing multiple crystallization conditions; and

(b) the system is programmed to execute steps comprising;

(i) dispensing one or more sample droplets from the protein solution; and

(ii) transporting at least a portion of the dispensed sample droplets to the mixing wells, wherein at least a portion of the mixing wells each receives one or more of the sample droplets, thereby yielding an array of crystallization reagents comprising protein sample and having the capacity to one or more protein crystals.

14. The system of claim 13 wherein the protein solution has a concentration which exceeds about 1 mg/mL.

15. The system of claim 13 wherein the protein solution has a concentration which exceeds about 10 mg/mL.

16. The system of claim 13 wherein the protein solution has a concentration which exceeds about 50 mg/mL.

17. The system of claim 13 wherein the protein solution has a concentration which exceeds about 100 mg/mL.

18. The system of claim 13 wherein the volume of protein solution transported to each mixing well to provide the array of crystallization reagents comprising protein sample does not exceed about 100 nL.

19. The system of claim 13 wherein the volume of protein solution transported to each mixing well to provide the array of crystallization reagents comprising protein sample does not exceed about 50 nL.

20. The system of claim 13 wherein the volume of protein solution transported to each mixing well to provide the array of crystallization reagents comprising protein sample does not exceed about 25 nL.

21. The system of claim 13 wherein the volume of protein solution transported to each mixing well to provide the array of crystallization reagents comprising protein sample does not exceed about 10 nL.

22. A system comprising the screening droplet actuator of claim 1 wherein the droplet actuator is programmed to separate a protein solution sample into multiple droplets and move, using droplet operations, each such droplet to a mixing well where it is combined with a pre-loaded crystallization reagent to yield a sample potentially comprising one or more crystals.

23. The screening droplet actuator of claim 22 wherein the sample droplets are surrounded by a filler fluid comprising silicone oil.

24. The screening droplet actuator of claim 23 wherein silicone oil is doped with a surfactant.

25. The screening droplet actuator of claim 23 wherein silicone oil is doped with a lipophilic surfactant.

26. The screening droplet actuator of claim 23 wherein silicone oil is doped with Triton X-15.

27. A device comprising the screening droplet actuator of claim 22 and a means for automatically analyzing each sample for the presence and/or type of crystal.

28. A device comprising the screening droplet actuator of claim 22 and a means for manually analyzing each sample for the presence and/or type of crystal.

29. A device comprising the screening droplet actuator of claim 22 and a means for crystal harvesting.

30. The device of claim 29 wherein the means for crystal harvesting comprises microfluidics for transport of crystal-laden droplets to bring said droplets into contact with one or more capillaries.

31. The device of claim 30 wherein the droplets enter the one or more capillaries by capillary action.

32. The device of claim 30 wherein the droplets enter the one or more capillaries by active vertical axis electrostatic actuation.

33. The screening droplet actuator of claim 1 wherein the droplet actuator comprises a grid of wells which is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

34. The screening droplet actuator of claim 1 wherein the droplet actuator comprises a grid of wells which includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990 or 2000 reagent conditions.

35. The screening droplet actuator of claim 1 wherein the droplet actuator comprises a grid consisting of 96, 384 or 1586 wells.

49. A method of testing conditions for growing a crystal comprising:

(a) providing the screening droplet actuator of claim 22;

(b) providing droplets in the crystal comprising a variety of potential conditions and the target molecule; and

(c) observing the droplets to identify crystal growth.

50. The method of claim 49 further comprising:

(a) observing the droplets to identify nucleation;

(b) performing on-droplet actuator dilutions after nucleation is observed to favor growth; and

(c) stopping further nucleation.

36. A method of screening protein crystallization conditions, the method comprising:

(a) providing a screening droplet actuator comprising;

(i) one or more ports for introduction of one or more crystallization reagents and one or more protein solutions; and

(ii) a substrate comprising;

(1) an array of two or more mixing wells; and

(2) electric field mediated microfluidics for moving droplets comprising the crystallization reagents and protein solutions into the mixing wells;

(b) loading the screening droplet actuator with reagents comprising;

(i) sample comprising protein for crystallization; and

(ii) crystallization reagents;

(c) conducting droplet operations to produce and combine sample droplets and crystallization reagent droplets into mixing wells in varying ratios to create a variety of crystallization conditions in the mixing wells; and

(d) identifying mixing wells in which crystals are formed and identifying the associated crystallization conditions.

37. An optimization droplet actuator comprising:

(a) a port for introduction of one or more crystallization reagents and/or one or more protein solutions; and

(b) a substrate comprising;

(i) one or more reagent wells in fluid connection with a port;

(ii) one or more sample wells in fluid connection with a port;

(iii) two or more mixing wells; and

(iv) microfluidics for moving;

(1) droplets comprising the crystallization reagents from the reagent wells into the mixing wells; and

(2) droplets comprising the protein solutions into the mixing wells.

38. The optimization droplet actuator of claim 37 wherein the one or more reagent wells are pre-loaded with crystallization reagents.

39. The optimization droplet actuator of claim 37 wherein the one or more reagent wells are pre-loaded with 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different crystallization reagents.

40. The optimization droplet actuator of claim 39 wherein the crystallization reagents comprise reagents selected from salt solutions, pH buffer solutions, organic acids, and polyethylene glycol.

41. The optimization droplet actuator of claim 39 wherein the droplet actuator is programmed to create systematically varied crystallization conditions by performing steps comprising (in any order):

(a) separate a protein solution sample from a sample well into multiple sample droplets and move each such sample droplet to a mixing well;

(b) separate one or more reagents from a reagent well into multiple reagent droplets and move each such reagent droplet into a mixing well; and

(c) thereby combining one or more reagent droplets with one or more sample droplets to yield a crystallization sample potentially forming one or more crystals.

42. A device comprising the optimization droplet actuator of claim 41 and a means for automatically analyzing each sample for the presence and/or type of crystal.

43. A device comprising the optimization droplet actuator of claim 41 and a means for manually analyzing each sample for the presence and/or type of crystal.

44. A device comprising the optimization droplet actuator of claim 41 wherein the device is programmed to permit a user to control mixing of reagents and protein sample so as to systematically vary conditions in each well according to parameters set by the user.

45. A device comprising the optimization droplet actuator of claim 41 wherein the device is programmed to automatically select parameter variables for use in the optimization droplet actuator based on results from a screening droplet actuator.

46. The optimization droplet actuator of claim 37 wherein the droplet actuator comprises a grid of wells which is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

47. The optimization droplet actuator of claim 37 wherein the droplet actuator comprises a grid of wells which includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990 or 2000 reagent conditions.

48. The optimization droplet actuator of claim 37 wherein the droplet actuator comprises a grid 96, 384 or 1586 wells.