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

Microfluidic Dialysis Protein Crystallization Jiang Huang GN Biosystems, Inc. March 26, 2009. Dialysis Protein Crystallization Method. Pro: scans a wide concentration range, the reagent composition can be easily altered during the course of the experiment.

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

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  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

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