1. Microfluidic Dialysis Protein Crystallization Jiang Huang GN Biosystems, Inc. March 26, 2009

2. Dialysis Protein Crystallization Method Pro: scans a wide concentration range, the reagent composition can be easily altered during the course of the experiment. Con: difficult to setup, protein consumption too high (5 to 350l/rxn), not HT compatible.

3. 7 10 6 MICROBATCH HANGING DROP FD Venn Diagrams 3 1 Method Hits VDX 20 MB 8 FD 23 00 3 0 1 3 MICROBATCH HANGING DROP 1 8 4 Method Hits VDX 9 MB 15 FD 7 FD 0 3 0 2 MICROBATCH HANGING DROP 1 7 0 Method Hits VDX 1 MB 10 FD 5 FD Lysozyme Glucose isomerase Catalase

4. Microfluidic Dialysis Plate - Design open bottom microtiter plate protein inlet film dialysis membrane discs microfluidic plate adhesive sealing tape

5. protein inlet vacuum port dialysis membrane reagent well Microfluidic Dialysis Plate - Design adhesive film

6. Microfluidic Dialysis Plate Design Top View Dialysis chambers dimensions: Screening plate:18nl per chamber (240m dia., 400m deep). Optimization plate: 80nl per chamber (500m dia., 400m deep). Growth plate: 1l per chamber (1.6mm dia., 400m deep) Bottom View Vacuum port Dialysis chamber Microfluidic channel Protein port

7. Microfluidic Dialysis Plate – Sample Loading

8. FD Method Highlights Protein consumption as low as 15nl per dialysis chamber Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers Low capital equipment costs to begin running experiments High-throughput compatibility with standard dispensing robotics Easy translation and scale-up designs Chemically compatible with commercial reagent kits The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation

9. FD Method Highlights Protein consumption as low as 15nl per dialysis chamber Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers Low capital equipment costs to begin running experiments High-throughput compatibility with standard dispensing robotics Easy translation and scale-up designs Chemically compatible with commercial reagent kits The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation

10. FD Method Highlights Protein consumption as low as 15nl per dialysis chamber Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers Low capital equipment costs to begin running experiments High-throughput compatibility with standard dispensing robotics Easy translation and scale-up designs Chemically compatible with commercial reagent kits The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation

11. Equipment and Accessories Needed 1.Vacuum pump or house vacuum with an ultimate vacuum ≤ 0.1mmHg *Air bubbles in every dialysis chamber will result due to insufficient vacuum A List of Qualified Vacuum Pumps at under $2,000 ManufactureModel#Ultimate Vacuum BOC/EdwardsRV31×10 -6 mmHg BOC/EdwardsEVA480-16-9418×10 -3 mmHg Welch1400B-011×10 -4 mmHg Welch1399B-010.02mmHg BrinkmannV-5000.01mmHg Thermo-Electron31787123.8×10 -3 mmHg Thermo-Electron31787071×10 -4 mmHg

12. FD Method Highlights Protein consumption as low as 15nl per dialysis chamber Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers Low capital equipment costs to begin running experiments High-throughput compatibility with standard dispensing robotics Easy translation and scale-up designs Chemically compatible with commercial reagent kits The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation

13. FD Method Highlights Protein consumption as low as 15nl per dialysis chamber Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers Low capital equipment costs to begin running experiments High-throughput compatibility with standard dispensing robotics Easy translation and scale-up designs Chemically compatible with commercial reagent kits The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation

14. via diameter: 1.2mm (570nl volume)via diameter: 0.3mm (20nl volume) crystal size: up to 500m longcrystal size: up to 50m long 50mg/ml Lysozyme vs. HCS1 #10 Translation and Scale-up screeningoptimizationgrowth dia.=240mdia.=500m dia.=1.6mm depth=400mdepth=400mdepth=400m

15. #2 #6 #7 #9 #13 #14 15  l dialysis bottom Optimization Plate Screening Plate Hampton Crystal Screen I 90x 20x Translation and Scale-up

16. FD Method Highlights Protein consumption as low as 15nl per dialysis chamber Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers Low capital equipment costs to begin running experiments High-throughput compatibility with standard dispensing robotics Easy translation and scale-up designs Chemically compatible with commercial reagent kits The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation

17. FD Method Highlights Chemically compatible with commercial reagent kits (materials used: PMMA, epoxy, dialysis membrane) M. W. Toepke, D. J. Beebe, PDMS absorption of small molecules and consequences in microfluidic applications, Lab Chip, 2006, 6: 1484-1486

18. FD Method Highlights Protein consumption as low as 15nl per dialysis chamber Fast set-up in as little as 4 minutes for 96 or 384 dialysis chambers Low capital equipment costs to begin running experiments High-throughput compatibility with standard dispensing robotics Easy translation and scale-up designs Chemically compatible with commercial reagent kits The dialysis membrane can be conveniently to allow easy loop access for crystal manipulation

19. protein inlet vacuum port dialysis membrane reagent well Microfluidic Dialysis Plate - Design adhesive film