Protein crystallization screening and optimization droplet actuators, systems and methods

• US 8,007,739 B2
• Filed: 08/16/2007
• Issued: 08/30/2011
• 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: Eckhardt, Allen E., Pamula, Vamsee K., Srinivasan, Vijay, Pollack, Michael G.
• Primary Examiner(s): Gordon; Brian R
• Application Number:
  US11/839,744
• Publication Number:
  US 20080044914A1
• Time in Patent Office: 1,475 Days
• Field of Search: 422/99, 422/100, 422/63, 422 81- 82, 422/500, 422502-504, 422/509, 436/180
• US Class Current: 422/509
• 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 droplet actuator system comprising:

a computer processor electronically coupled to a screening droplet actuator;

one or more input devices electronically coupled to the computer processor;

one or more output devices electronically coupled to the computer processor; and

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 in fluid communication with the port, the 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;

wherein the droplet actuator comprises;

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

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

further wherein the system is programmed to execute steps mediated by electrowetting comprising;

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

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

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

(d) 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

(e) combining a sample droplet from the protein solution with each crystallization reagent in the mixing wells to potentially yield one or more crystallization droplets comprising one or more crystals.

2. The system 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. The system of claim 1 wherein the protein solution has a concentration of protein which exceeds about 50 mg/mL.

4. The system of claim 1 wherein the volume of the one or more sample droplets from the protein solution transported to each mixing well in the array of mixing wells does not exceed about 100 nL.

5. The system of claim 1 wherein the droplet actuator comprises a grid of at least 4 wells.

6. The system of claim 1 wherein the droplet actuator comprises a grid of wells which includes at least 2 reagent conditions.

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

8. A droplet actuator system comprising:

a computer processor electronically coupled to a screening droplet actuator;

one or more input devices electronically coupled to the computer processor;

one or more output devices electronically coupled to the computer processor; and

a screening droplet actuator comprising;

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

(b) a substrate in fluid communication with the port, the substrate comprising;

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

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

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

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

wherein the system is programmed to execute steps mediated by the electric field microfluidics comprising conducting droplet operations on the substrate to;

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

(b) 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.

9. A droplet actuator system comprising:

a computer processor electronically coupled to a screening droplet actuator;

one or more input devices electronically coupled to the computer processor;

one or more output devices electronically coupled to the computer processor; and

a screening droplet actuator comprising;

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

(b) a substrate in fluid communication with the port, the substrate comprising;

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

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

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

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

wherein the system is programmed to execute steps by electrowetting on the substrate, the steps comprising;

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

(b) 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 droplets comprising protein sample and having the capacity to yield one or more protein crystals.

10. The system of claim 9 wherein the protein solution has a concentration of protein which exceeds about 50 mg/mL.

11. The system of claim 9 wherein the volume of the one or more sample droplets from the protein solution transported to each mixing well to provide the array of crystallization reagents comprising protein sample does not exceed about 100 nL.

12. A droplet actuator system comprising:

a computer processor electronically coupled to a screening droplet actuator;

one or more input devices electronically coupled to the computer processor;

one or more output devices electronically coupled to the computer processor; and

a screening droplet actuator comprising;

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

(b) a substrate in fluid communication with the port, the 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;

wherein the system is programmed to separate by electrowetting on the substrate a protein solution sample into multiple protein solution sample droplets and move, using droplet operations, each of the protein solution sample droplets to a mixing well where it is combined with a pre-loaded crystallization reagent to yield a sample potentially comprising one or more crystals.

13. The system of claim 12 wherein the droplet actuator further comprises a filler fluid comprising silicone oil, wherein the sample droplets are surrounded by the filler fluid.

14. The system of claim 13 wherein the silicone oil is doped with a surfactant.

15. The system of claim 13 wherein the silicone oil is doped with a lipophilic surfactant.

16. The system of claim 12 further comprising a means for automatically analyzing each sample for the presence and/or type of crystal.

17. The system of claim 12 further comprising a means for manually analyzing each sample for the presence and/or type of crystal.