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Separation of macromolecules using ultrathin silicon membranes

Separation of macromolecules using ultrathin silicon membranes. By Mary Coan Chemical Engineering Ph.D. Outline. Ultra-filtration (UF) Membranes Nanofabricated Membranes Ultrathin Porous Nanocrystalline Silicon ( pnc -Si) Membranes Fabrication Physical Properties Tunability

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Separation of macromolecules using ultrathin silicon membranes

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  1. Separation ofmacromolecules using ultrathin silicon membranes By Mary Coan Chemical Engineering Ph.D.

  2. Outline • Ultra-filtration (UF) Membranes • Nanofabricated Membranes • Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes • Fabrication • Physical Properties • Tunability • Molecular Separation • Proposed Future Work • Conclusion

  3. Ultra-filtration (UF) Membranes • Pressure driven membrane separation process (Image 1) • Separates particulate matter from soluble components in the carrier fluid • Water • PEG • Blood • Pore sizes typically range from 0.01 - 0.10 µm (Image 2) • High removal capability for bacteria and most viruses, and colloids • Smaller pore sizes result in higher removal capabilities http://www.dow.com/liquidseps/prod/uf_index.htm Image #1: http://www.fumatech.com/EN/Membrane-technology/Membrane-processes/Ultrafiltration/ Image #2: http://www3.ntu.edu.sg/home/DDSun/research.html Used For water Reclamation

  4. Ultra-filtration (UF) Membranes Membrane used for bacteria removal • Most materials that are used in UF are polymeric and are naturally hydrophobic • Polysulfone (PS) • Polyethersulfone (PES) • Polypropylene (PP) • Polyvinylidenefluoride (PVDF) • Materials are blended with hydrophilic agents to decrease hydrophoicity (Image 1) • Potentially reduces the membranes ability to be cleaned with high strength disinfectants • Impacts removal of bacterial growth http://www.dow.com/liquidseps/prod/uf_index.htm Image #1: http://www.mymedicalsuppliers.com/dialysis-equipment-and-supplies/

  5. Ultra-filtration (UF) Membranes • Four types of UF membrane modules • plate-and-frame (Image1), spiral-wound (Image2), tubular (Image3) and hollow fiber (Image3) configurations • Suited for one or more specific applications • Many applications can use more than one configuration • For high purity water • spiral-wound and hollow fiber configurations • For more concentrated solutions • plate-and-frame and tubular configurations  http://www.appliedmembranes.com/about_ultrafiltration.htm , Image #1-4: http://www.hydrotech.cn/English/mofenli.asp

  6. Ultra-filtration (UF) Membranes • The selection of the proper configuration depends on the type and concentration of colloidal material or emulsion • It must take into account the flow velocity, pressure drop, temperature, power consumption, membrane fouling and module cost http://www.appliedmembranes.com/about_ultrafiltration.htm Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  7. Ultra-filtration (UF) Membranes • Limitations of typical UF membranes: • broad pore size distributions • < 1,000 times thicker than the molecules they are designed to separate • Results in poor size cutoff properties, filtrate loss within the membranes, and low transport rates 1. Tong, H. D. et al. Silicon nitride nanosieve membrane. Nano Lett. 4, 283–287 (2004). 2. Kuiper, S., van Rijn, C. J. M., Nijdam, W. & Elwenspoek, M. C. Development and applications of very high flux microfiltration membranes. J. Membr. Sci. 150, 1–8 (1998)

  8. Ultra-filtration (UF) Membranes • Nanofabricated membranes offer more precise structural control, yet transport is also limited by μm-scale thicknesses • New class of ultrathin nanostructured membranes (Image1) • Membrane thickness ≈ the size of the molecules being separated (10 nm) • Membrane fragility, complex and expensive fabrication processes have prevented the use of ultrathin membranes for molecular separations in commercial use 1. Yamaguchi, A. et al. Self-assembly of a silica-surfactant nanocomposite in a porous alumina membrane. Nature Mater. 3, 337–341 (2004). 2. Lee, S. B. & Martin, C. R. Electromodulated molecular transport in goldnanotubule membranes. J. Am. Chem. Soc. 124, 11850–11851 (2002). 3. Tong, H. D. et al. Silicon nitride nanosieve membrane. Nano Lett. 4, 283–287. 4. Martin, F. et al. Tailoring width of microfabricatednanochannels to solute size can be used to control diffusion kinetics. J. Control. Release 102, 123–133 (2005).. 5. http://www.kochmembrane.com/mww_purification.html

  9. Outline • Ultra-filtration (UF) Membranes • Nanofabricated Membranes • Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes • Fabrication • Physical Properties • Tunability • Molecular Separation • Proposed Future Work • Conclusion

  10. Nanofabricated Membranes • Part of the Ultrafilitration Membranes • Fabricated using typical microelectronic techniques • Lithography • Focused Ion Beam • Reactive Ion Etching • Sputtering • Chemical Vapor Deposition http://www.homecents.com/h2o/ro/index.html

  11. Nanofabricated Membranes • Silicon Nitride Nanoseive Membrane • Nanopores, 25 nm in diameter, were directly drilled by FIB in a 10-nm SiN membrane (110 Kx, scale bar: 50 nm).

  12. Nanofabricated Membranes • Perspective SEM of a filter • Square holes in the top layer are the entrance ports • Hexagonal outline on the surface is the result of structurally reinforcing trenches defined in the first phase of fabrication • Channels revealed in the cross section are formed by the removal of silicon dioxide grown between the layers of polysilicon.

  13. Nanofabricated Membranes • Molecule-Nanofilter Interaction at the Micro(Macro)-Nano-Micro junction • Various factors are in play to affect the transport of biomolecules (with various shapes and sizes) through a nanopore or a nanofluidic filter

  14. Outline • Ultra-filtration (UF) Membranes • Nanofabricated Membranes • Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes • Fabrication • Physical Properties • Tunability • Molecular Separation • Proposed Future Work • Conclusion

  15. Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes • An UF Nanofabricated Membrane • Ultrathin: 15 nm thick • Prepared using typical silicon fabrication techniques • Lithography • Etching • Left Image: TEM image of the porous nanostructure of a 15-nm-thick membrane • Pores appear as bright spots • Nanocrystalline silicon is in grey or black contrast. Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  16. Ultrathin pnc-Si Membranes:Fabrication • Silicon fabrication techniques provide control over average pore sizes from 5nm to 25 nm, are fully understood and readily available • Uses precision silicon deposition and etching techniques to create the ultrathin membrane (next slide, animation) • Instead of directly patterning pores, voids are formed spontaneously as nanocrystals nucleate and grow in a 15-nm-thick amorphous silicon (a-Si) film during a rapid thermal annealing step Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  17. Ultrathin pnc-Si Membranes:Fabrication Process Pnc-Si Membrane Oxide 500 nm thermal oxide a-Si a-Si a-Si a-Si a-Si a-Si a-Si Oxide Oxide Oxide (100) Silicon Wafer ~ 500 μm Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532 500 nm thermal oxide Step 1: Grow 500nm thick Thermal Oxide Step 2: Pattern Backside Step 3: Remove front oxide and deposit a 3-layer oxide/a-Si/oxide film stack Step 4: Rapid Thermal Anneal Step 5: Anisptropic Etching of (100) Si Wafer using EDP Step 6: Remove Oxide Masks

  18. Ultrathin pnc-Si Membranes:Fabrication • Voids span the molecularly thin membrane to create pores • The resulting membranes cover openings several hundred μm across in a rigid crystalline silicon frame • Can be easily handled and used Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  19. Ultrathin pnc-Si Membranes:Physical Properties • Several characterization techniques were used to confirm/determine the properties of the pnc-Si membranes • Transmission Electron Microscopy (TEM) • Refractive Index • Atomic Force Microscopy (AFM) • Mechanical Stability using a customized holder and Optical Microscope • Refractive Index (Right Image) • For a 15-nm-thick silicon film after deposition (a-Si) and after crystallization (pnc-Si) Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  20. Ultrathin pnc-Si Membranes:Physical Properties 2) Shift in optical Properties • Sputtered a-Si: • High optical density, comparable to microelectronic quality a-Si deposited with chemical vapor deposition (CVD) • Exhibits a clear shift in optical properties after crystallization • Resonance peaks similar to crystalline silicon after crystallization • Results are indicative of high purity silicon films with smooth interfaces • TEM images of the as-deposited a-Si show no distinguishable voids or crystalline features Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532 3) Similar Peaks

  21. Ultrathin pnc-Si Membranes:Physical Properties • Membranes were transferred onto polished quartz • Atomic Force Microscopy (AFM) • confirm the accuracy of the Refractive Index data • Measured the step height of the membrane edge • Confirmed the 15nm thickness of a sample membrane • Showed highly smooth surface morphology Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  22. Ultrathin pnc-Si Membranes:Physical Properties • Important characteristic of pnc-Si membranes is their remarkable mechanical stability • Mechanically Stability: • Used a customized holder to apply pressure to one side of the membrane while an optical microscope was used to monitor deformation • Right Top and Bottom Images are optical micrographs of a 200 μm x 200 μm x 15nm membrane • no applied pressure (Top) • more than 1 atm of differential applied pressure across it for ~ 5 minutes (Bottom) • With no differential pressure, the membrane is extremely flat (Top), and at maximum pressure (Bottom) the membrane elastically deforms but maintains its structural integrity throughout the duration of test. Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  23. Ultrathin pnc-Si Membranes:Physical Properties • pnc-Si membranes exhibit no plastic deformation • Immediately return to their flat state when the pressure is removed • Pressurization tests were cycled three times with no observable membrane degradation • Due to their smooth surfaces and random nanocrystal orientation • inhibit the formation and propagation of cracks Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  24. Ultrathin pnc-Si Membranes:Tunability • Pore size distributions in pnc-Si membranes are controlled by the Rapid Thermal Annealing Process (RTP) • Nanocrystal nucleation and growth are Arrhenius-like processes that exhibit strong temperature dependence above a threshold crystallization temperature of approximately 700ºC in a-Si • Existing crystallization models fail to predict void formation, and must be extended to account for how volume contraction and material strain lead to pore formation in ultrathin membranes Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  25. Ultrathin pnc-Si Membranes:Tunability • Pore size tunability: • 3 wafers with 15-nm-thick pnc-Si membranes were processed identically, except for the annealing temperature • Annealed at 715ºC resulted in an average pore size of 7.3 nm • Annealed at 729ºC resulted in an average pore size of 13.9nm • Annealed at 753ºC resulted in an average pore size of 21.3 nm • Pore size and density increase monotonically with temperature Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  26. Ultrathin pnc-Si Membranes:Tunability • Another sample annealed at 700ºC exhibited no crystalline structure and resulted in no voids • strong morphological dependence on temperature near the onset of crystallization • With the ability to “tune” the average pore size pnc-Si Membranes are well suited for: • size-selective separation of large biomolecules • Examples: proteins and DNA Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  27. Ultrathin pnc-Si Membranes:molecular Separation • Two common blood proteins of different molecular weight (MW) and hydrodynamic diameter (D) were used to test the molecular capabilities of the pnc-Si Membrane • Bovine serum albumin, BSA (MW=67,000 (67K), D=6.8 nm), fluorescently labelled with Alexa 488 • Immunoglobulin-c, IgG (MW=150 K, D=14 nm), fluorescently labelled with Alexa 546 • Free Alexa 546 dye was used as an additional low molecular weight (MW=1 K, D < 1 nm) species Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  28. Ultrathin pnc-Si Membranes:molecular Separation Well thin diffusion chamber 50 mm bead spacer 15nm thick membrane Fluorescent Mixture PBS Glass Coverslip Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532 Step 1: Fill the Diffusion Chamber with 50ml Clean Buffer solution (PBS) Step 2: Fill the Well with 3 ml of a fluorescent mixture containing BSA and Free Alexa 546 dye Taking a closer look at the membrane interface as time passes one can see the Alexa 546 dye (Species 1) flows through the pnc-Si Membrane into the diffusion chamber while the larger Protein (BSA, Species 2) remains in the well

  29. Ultrathin pnc-Si Membranes:molecular Separation • Images of the membrane edge were taken every 30s • Spreading of the fluorescence signal from the membrane edge to the diffusion chamber during separation, is illustrated in the two false-color images below Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532 False Color images

  30. Ultrathin pnc-Si Membranes:molecular Separation Alexa Dye vs BSA BSA vsIgG • Results from the separation of free Alexa 546 dye and BSA using membrane A • Dye passes freely through the membrane while BSA is almost completely blocked. • Results from the separation of IgG and BSA through membrane B at 1 mM concentration • BSA diffuses through the membrane 0.4 times more rapidly than IgG Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  31. Ultrathin pnc-Si Membranes:molecular Separation • BSA diffuses through the membrane more rapidly than IgG • The diffusion coefficients for these molecules are within 25% of each other • The measured rate difference indicates that pnc-Si membranes hinder IgG diffusion relative to BSA diffusion • The increased cut-off size of membrane B allows for a increase in BSA diffusion by 15x compared to membrane A • BSA and IgG were retained behind membranes with maximal pore sizes 2x as large as their reported hydrodynamic diameters • electrostatic interactions and protein adsorption might create an effective pore size smaller than that measured by TEM Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  32. Ultrathin pnc-Si Membranes:molecular Separation • Negatively charged Alexa 488 dye in the presence and absence of high salt concentrations during separation • diffusion of the Alexa dye drops by a factor of 10 when experiments are conducted in deionized water • electrostatic repulsion between the dye and a negatively charged native oxide layer on the surface of the pnc-Si membranes • High salt concentrations increase throughput by screening surface and solute charges Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  33. Ultrathin pnc-Si Membranes:molecular Separation • Charge effects • Modified membranes to carry abundant negative and positive surface charges (Image 1) • In low ionic strength solutions • Positively charged membranes blocked only positively charged dyes • Negatively charged membranes blocked only negatively charged dyes • In high ionic strength phosphate buffered saline solutions • Stronger electrostatic interactions that reduce the effective pore size were expected • Results in pnc-Si membranes that can be functionalized to separate similarly sized molecules on the basis of their charge Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  34. Ultrathin pnc-Si Membranes:molecular Separation • Factors affecting the Effective Pore Size of the pnc-Si Membrane • Protein adsorption to the pore walls will reduce the effective pore size • BSA adsorption shrinks, but does not occlude, the largest membrane pores by as much as 7nm • Charge Effects • Uncertain relationship between a protein’s physical size and hydrodynamic dimensions may reduce effective pore size • Behavior of water (hydrogen bonding) in nanoscale pores may reduce pore size Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  35. Ultrathin pnc-Si Membranes:molecular Separation • Given the long passage-times of molecules through thick membranes, it is significant that filtrate molecules appear downstream of pnc-Si filters within minutes • Quantified the transport through pnc-Si membranes • fluorescence microscopy experiments with bench-top experiments • Easily remove and assay the Alexa 546 dye that diffused across membrane A from a 100 mM starting concentration using a similar unstirred geometry Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  36. Ultrathin pnc-Si Membranes:molecular Separation • Dye diffuses over 9x faster through pnc-Si membrane A than dialysis membranes • pnc-Si membrane A exhibits an initial transport rate of 156 nmol cm-2h-1 that slows as the 3 ml source volume depletes • Due to the lowering of the concentration gradient across the barrier • For membrane C an increase of 10% in dye transport was measured relative to membrane A, despite porosities differing by 29x (0.2% versus 5.7%) Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532 Dye diffusion through pnc-Si membranes compared to diffusion through standard regenerated cellulose dialysis membranes (Spectra/Por 7 dialysis membrane, molecularweight cut-off550K)

  37. Ultrathin pnc-Si Membranes:molecular Separation • Dye or small molecule transport is essentially unhindered by pnc-Si membranes • as porosities far lower than that of membrane A should theoretically allow greater than half-maximal diffusion through an infinitely thin porous barrier • Diffusion through the commercial membrane is the rate-limiting transport process • Due to the observed increase in the diffusion rate over conventional dialysis membranes • Diffusion through the bulk solution is rate-limiting for the pnc-Si membrane experiment • Enhancement of the transport rate is expected in systems that implement active mixing, or forced flow Christopher C. Striemer, Thomas R. Gaborski, James L. McGrath & Philippe M. Fauchet “Charge- and size-based separation of macromolecules using ultrathin silicon membranes” Nature, Vol 445| 15 February 2007| doi:10.1038/nature05532

  38. Outline • Ultra-filtration (UF) Membranes • Nanofabricated Membranes • Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes • Fabrication • Physical Properties • Tunability • Molecular Separation • Proposed Future Work • Conclusion

  39. Proposed Future Work • More robust study of separation • Not limited to only two proteins at one time • Test using proteins commonly found in blood • Determine the effects of different concentrations of proteins • Increase concentrations to those similar in Blood and beyond • Integration into microfluidicdevices • Silicon-based platform opens several avenues for future developments • surface functionalization using well-established chemistries • modify surface charge • reduce protein adsorption • protect the silicon from chemical attack in harsh environments.

  40. Proposed Future Work • Effects of large scale production on the physical properties of the device • Determine low-cost feasibility • Environmental effects • Separation properties of the membrane • Physical properties of the membrane • Determine methods to “clean” the membranes • if high-cost production Image: http://www.rikenresearch.riken.jp/eng/frontline/4950

  41. Outline • Ultra-filtration (UF) Membranes • Nanofabricated Membranes • Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes • Fabrication • Physical Properties • Tunability • Molecular Separation • Proposed Future Work • Conclusion

  42. Conclusion • First use of ultrathin nanomembranes for size-based molecular separations • Separation of BSA and IgG suggests that pnc-Si can be used for membrane-based protein fractionation • Are too close in size to be efficiently separated using conventional membrane processes • Standard membranes cause a lot of the filtrate species to be lost • Due to the high surface area and tortuous porosity • pnc-Si membranes should allow for recovery of both the retentate and filtrate fractions to enable membrane-based chromatography

  43. Conclusion • pnc-Si membranes are expected to be highly efficient for separation processes • Due to the thickness and minimal filter surface area • Diffusion transport rate of 156 nmolcm-2 h-1for Alexa 546 dye • More than 10x faster than thick nanofabricated membranes • 0.9 x faster than the authors measurements through dialysis membranes • pnc-Si membranes with fixed charges • Can be used to separate similarly sized molecules with different charges • adds another dimension of control for highly efficient molecular separations

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