Rapid Prototyping of Purification Platforms

Transcription

1 Rapid Prototyping of Purification Platforms Tom Huang, Nathan Mosier, Michael Ladisch Department of Agricultural and Biological Engineering Weldon School of Biomedical Engineering Laboratory of Renewable Resources Engineering Purdue University W. Lafayette, IN

2 Acknowledgments Material in this work was supported by: USDA (ARS ) Food Safety Engineering Center Agricultural Research Programs, Purdue University National Defense University (DAB J29-03-P-0022) LORRE members

3 Bioseparations Challenges 1. Obtaining biomolecules for testing 2. Selecting media and surface chemistry 3. Packing column and running 4. Scaling up a. Particle size b. Column dimensions c. Sample volumes d. Flowrates, gradients

4 Microfluidics Movement of fluids at microscopic level Micron-sized channels and features Applications Biosensors Bioassays Micro-bioseparations Pathogen detection

5 Questions Can separations media be selected or developed using nanoliter sample volumes? Can nanoliter scale separations be rapidly prototyped (in minutes to hours) if only simple channels are needed? Can microfluidic flow effects be related to macroscale dispersion behavior encountered in LC columns?

6 Rationale Diffusion is limiting for most interactions between target species and surfaces of separations media (particles, gels, pores) Diffusivity of solutes in fluid within pores or gels is less than diffusivity in an unbounded solution Solute / adsorbent interactions in boundary layers approximates local equilibrium.

7 Momentum/Mass Diffusivity Reduced velocity 2Rvε b ReSc = < 1 D m R = average radius of stationary phase v = interstitial fluid velocity ε b = void fraction between particles (extra-particulate) D m = solute diffusivity in unbounded solution (Ladisch, 2001)

8 Diffusivity (in free solution) D m = T µ R o T = temperature, K R g = radius µ o = viscosity of water at 20 C = cp = 0.01 g cm sec (based on 86 random-coil proteins, Tyn and Gusek, 1990)

9 Rapid Prototyping Enables rapid selection of purification techniques and chip configuration Uses very small sample, 50 to 200 nano-liters, to select appropriate stationary phase Configures different media chemistries without packing columns Requires labeled proteins or target species

10 The Need Simple and rapid fabrication Defined surface chemistries Method to rapidly form channels Rapid prototyping of different separations

11 Microfibers form channels in minutes Press-fit Devices PDMS sheet SiO 2, glass, or PDMS substrate Press together Glass fiber (12 µm dia) Labeled avidin (green) SiO 2 substrate (a) (b) (c) PDMS Fill by capillary action After 3 minutes SiO 2 Glass fiber Flow channel 1 nl/mm channel Channel outlet Huang et al., AICHE J.

12 Microfiber-directed boundary flow Inlet 10 mm PDMS Pipette in water; t= 0 Glass fiber (~12 µm diameter) Tubing 180 OTS PDMS To vacuum 127 mm Hg OTS Pull fluid by applying vacuum t= ~3 seconds t= ~1 minute t= ~1 minute PDMS PDMS PDMS Water Air Water Air Water Air Air PDMS Air PDMS Air PDMS Huang et al., In review

13 Corner micro-channel fabricated using glass fiber and PDMS on SiO2 substrate

14 T-junction micro-channel fabricated using glass fiber and PDMS on SiO2 substrate Elastomer : crosslinker 10 : 1

15 Crossing Fibers form Well Color image for mixing of BSA (red color) and avidin (green color)

16 Effect of Crosslinker (PDMS) Elastomer : crosslinker (10:1) Elastomer : crosslinker (20:1) Width= 91µm Width= 40 µm

17 Self assembly of microbeads by capillary force during solvent evaporation 22 micron Aminex cation exchange resin, 100 x magnification

18 Press-fit microbeads module with inlet and outlet channels

19 Press-fit beads Module with multiple inlets and outlets

20 Microfluidic, 2-D packed bed

21 Dip-coating Microfibers with Protein or Glass fiber Derivatized Particles EtOH Ultra-sonic cleaning in EtOH for 5 min Dip-coat in protein or particle slurry Glass fiber coated with particles

22 Glass Fiber Coated with Streptavidin Beads (biotin anchors beads) Streptavidin microbeads, 800 nm Biotin + Streptavidin Glass fiber pre-coated with biotin-bsa

23 Glass Fiber Coated with Dimethylamino Beads (positively charged) Dimethyl-amino microbeads, 800 nm CH 3 H N CH 3 + OH OH OH Bare glass fiber, 12 µm dia

24 Micro-scale Separation Derivatize channels with ion exchange or bioreceptor (in bead form) Assemble micro-device Carry out separation through capillary action Sample size < 1 µl

25 Separation of labeled BSA from avidin on DEAE Beads Labeled avidin (green; 10 µg/ml) and BSA (red; 10 µg/ml) liquid mixture (ph 7.4); t=0 PDMS Glass substrate Glass fiber coated with dimethylamino microbeads (a) t= ~3 minutes

26 Separation of IgG from BSA on Protein A coated fiber Labeled IgG (green; 10 µg/ml) and BSA (red; 10 µg/ml) liquid mixture; t=0 (c) Glass substrate PDMS t= ~3 minutes Glass fiber coated with Protein A microbeads

27 Techniques apply to other commercially available micro/nano particles Type Surface Chemistry Size (nm) Silica Hydroxyl -OH Polystyrene Hydroxyl -OH 700 to 790 Polystyrene Carboxyl -COOH 700 to 790 Polystyrene Sulfonate -SO to 790 Polystyrene Amino -NH to 790 Polystyrene Dimethylamino -NH(CH 3 ) to 790 Data from Spherotec, Inc. Libertyville IL. Surface charge

28 Protein Coated Micro/Nano Particles Type Surface pi Size (nm) Polystyrene Antibody (IgG) NA 700 to 7900 Polystyrene Avdin to 790 Polystyrene Streptavidin to 790 Polystyrene Biotin NA 700 to 790 Polystyrene Protein A to 790 Polystyrene Protein G to 790 Surface Affinity Binds protein A, G Binds to biotin Binds to bitoin Binds to strept/avidin Binds to IgG antibody Binds to IgG antibody Data from Spherotec, Inc. Libertyville IL.

29 Advantages of Bead-Based Surfaces Commercially available Sizes ranging from 100 nm to 8 µm Various types of chemistry Anchors onto surface through surface chemistry Easily identifiable in microfluidic chips Provides increased surface area Various shapes Controlled porosities

30 Conclusions Rapid fabrication of microchannels demonstrated Hydrophobic surfaces direct fluid into boundary layer Separations surface chemistry provided by beads Small sample volumes of labeled proteins separated Availability of micro-beads provide link between chip based prototype and column separation

31 Thank you!

32 Outline Background Rationale Forming channels using fibers (Press-fit devices) Surface chemistry Example of separation Conclusions

33 C18 Surface Coating O H O H O H O H O SiO 2 Si Si H O H O H O H O H Cl Hydrophilic microchip surface with a contact angle of ~ 2 º Si Cl Cl Octadecyltrichlorosilane (OTS) Si Si Si Si O O OO O OO O OO O O SiO 2 Si Hydrophobic microchip surface modified with C18 with a contact angle of ~ 110 º Ladisch MR. Bioseparations Engineering, 2001 p

34 Materials and Methods Glass wool fiber (diameter: ~12 µm) PDMS silicon elastomer base: curing agent ratio of 10:1 Microbead: 22 µm diameter Aminex cation exchange resin(bio-rad) Glass slide: Dow corning - 5 x 7.5 cm

35 Methods for Press Fit Devices Materials used Fiber used: glass wool fiber (outer diameter: 12 µm) PDMS: silicon elastomer : cross linker (10:1) Substrate used: C18 derivatized silicon dioxide Glass slide: Dow corning glass slide (3x2 in)

36 2.8 micron Streptavidin beads on biotinylated BSA adsorbed onto C18 surface ~14 nm Biotinylated antibody ~2 µm Streptavidin coated microbeads (2.8 µm) Biotinylated BSA ~4 nm Glass fiber surface Drawing not to scale.

37 Micro assembly of 800 nm beads Protein ~4 nm Dimethylamino or polystyrene microbeads (800 nm) 800 nm Glass fiber surface Drawing not to scale.

38 Separation of avidin from BSA on Biotinylated fiber Labeled avidin (green; 10 µg/ml) and BSA (red; 10 µg/ml) liquid mixture; t=0 PDMS Glass substrate Glass fiber coated with biotinylated BSA (b) t= 5 minutes Huang et al., AICHE J.