Method of electrowetting droplet operations for protein crystallization

• US 7,763,471 B2
• Filed: 08/16/2007
• Issued: 07/27/2010
• Est. Priority Date: 04/18/2006
• Status: Expired due to Fees

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Srinivasan, Vijay, Pollack, Michael G.
• Primary Examiner(s): Gakh; Yelena G
• Assistant Examiner(s): ADAMS, MICHELLE
• Application Number:
  US11/839,762
• Publication Number:
  US 20080050834A1
• Time in Patent Office: 1,076 Days
• Field of Search: 436/86
• US Class Current: 436/86
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/06 Crystallising dishes
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: G01N 23/20 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

Methods of using protein crystallization droplet actuators are provided. Protein sample droplets and reagent droplets are dispensed, transported, and merged to yield an array of crystallization conditions by electrowetting droplet operations in a gap comprising oil filler fluid. The oil filler fluid is doped with a surfactant that enhances droplet operations using the protein sample.

Claims

1. A method of providing an array of crystallization conditions, the method comprising:

(a) providing a droplet actuator comprising substrates arranged to form a gap, wherein;

(i) the gap comprises;

(1) sample comprising a protein analyte in a concentration suitable for preparing a protein crystallization array;

(2) reagents comprising crystallization reagents; and

(3) an oil filler fluid comprising a surfactant in an amount selected to enhance droplet operations using the sample; and

(ii) one or more of the substrates comprise electrodes arranged for conducting droplet operations in the gap by electrowetting using;

(1) sample droplets comprising aliquots of the sample; and

(2) reagent droplets comprising aliquots of the reagents; and

(b) providing a processor;

(c) using the processor to control the electrodes to conduct the droplet operations comprising;

(i) dispensing sample droplets from the sample and reagent droplets from the reagents, wherein the dispensing comprises aliquoting a volume of;

(1) sample into smaller sample droplets; and

(2) reagents into smaller reagent droplets;

(ii) transporting the sample droplets and reagent droplets; and

(iii) merging sample droplets with reagent droplets;

to yield an array of crystallization conditions.

2. The method of claim 1 further comprising:

(a) permitting the array of crystallization conditions to remain intact for a predetermined period of time; and

(b) identifying droplets in the array in which crystallization results.

3. The method of claim 1 wherein the sample comprises a protein concentration which exceeds about 10 mg/mL.

4. The method of claim 1 wherein the sample comprises a protein concentration which exceeds about 50 mg/mL.

5. The method of claim 1 wherein the sample comprises a protein concentration which exceeds about 100 mg/mL.

6. The method of claim 1 wherein the crystallization reagents comprise coarse grid reagents.

7. The method of claim 1 wherein the crystallization reagents comprise fine grid reagents.

8. The method of claim 1 wherein the crystallization reagents comprise one or more reagents selected from the group consisting of salts, pH buffers, precipitants, and water.

9. The method of claim 1 wherein the oil filler fluid comprises the surfactant in an amount which ranges from about 0.001 to about 10% w/w.

10. The method of claim 1 wherein the oil filler fluid comprises the surfactant in an amount which ranges from about 0.001 to about 1% w/w.

11. The method of claim 1 wherein the oil filler fluid comprises the surfactant in an amount which ranges from about 0.001 to about 0.1% w/w.

12. The method of claim 1 wherein the sample droplets have a volume which does not exceed about 100 nL.

13. The method of claim 1 wherein the sample droplets have a volume which does not exceed about 50 nL.

14. The method of claim 1 wherein the sample droplets have a volume which does not exceed about 20 nL.

15. The method of claim 1 wherein the sample droplets have a volume which does not exceed about 10 nL.

16. The method of claim 1 wherein the enhancement in droplet operations comprises a reduction in protein contamination of droplet microactuator surfaces.

17. The method of claim 1 wherein the surfactant enables the dispensing of the sample droplets.

18. The method of claim 1 wherein the surfactant enables the splitting of the sample droplets.

19. The method of claim 1 wherein the dispensing of the sample droplets is effected from on-actuator sample reservoirs.

20. The method of claim 1 wherein the dispensing of the sample droplets is effected using voltages less than 50 V.

21. The method of claim 1 wherein the oil filler fluid comprising a surfactant increases compatible protein concentration by more than 3 orders of magnitude relative to oil filler fluid in the absence of the surfactant.

22. The method of claim 1 wherein the surfactant increases the concentration of protein that can be reliably dispensed on the droplet microactuator.