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

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 350ml/rxn), not HT compatible.

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

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

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

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

7. Microfluidic Dialysis Plate – Sample Loading

8. mFD 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. mFD 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. mFD 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 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 Manufacture Model# Ultimate Vacuum BOC/Edwards RV3 1×10-6mmHg BOC/Edwards EVA480-16-941 8×10-3mmHg Welch 1400B-01 1×10-4mmHg Welch 1399B-01 0.02mmHg Brinkmann V-500 0.01mmHg Thermo-Electron 3178712 3.8×10-3mmHg Thermo-Electron 3178707 1×10-4mmHg

12. mFD 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. mFD 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. Translation and Scale-up screening optimization growth dia.=240mm dia.=500mm dia.=1.6mm depth=400mm depth=400mm depth=400mm 50mg/ml Lysozyme vs. HCS1 #10 via diameter: 1.2mm (570nl volume) via diameter: 0.3mm (20nl volume) crystal size: up to 500mm long crystal size: up to 50mm long

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

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