Microfluidic Dialysis Protein Crystallization

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
plate18nl 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 110-6mmHg BOC/Edwards EV
A480-16-941 810-3mmHg Welch 1400B-01 110-4mmHg
Welch 1399B-01 0.02mmHg Brinkmann V-500 0.01mmHg
Thermo-Electron 3178712 3.810-3mmHg Thermo-Elec
tron 3178707 110-4mmHg
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 depth400mm depth400mm dep
th400mm
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