SEPARATION OFMACROMOLECULES USING ULTRATHIN SILICON MEMBRANES
By Mary CoanChemical Engineering Ph.D.
OUTLINE
Ultra-filtration (UF) Membranes Nanofabricated Membranes Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes
Fabrication Physical Properties Tunability Molecular Separation
Proposed Future Work Conclusion
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
ULTRA-FILTRATION (UF) MEMBRANES
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.htmImage #1: http://www.mymedicalsuppliers.com/dialysis-equipment-and-supplies/
Membrane used for bacteria removal
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
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.htmChristopher 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
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)
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 microfabricated nanochannels 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
OUTLINE
Ultra-filtration (UF) Membranes Nanofabricated Membranes Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes
Fabrication Physical Properties Tunability Molecular Separation
Proposed Future Work Conclusion
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
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).
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.
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
OUTLINE Ultra-filtration (UF) Membranes Nanofabricated Membranes Ultrathin Porous Nanocrystalline Silicon (pnc-Si)
Membranes Fabrication Physical Properties Tunability Molecular Separation
Proposed Future Work Conclusion
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
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
ULTRATHIN PNC-SI MEMBRANES:FABRICATION PROCESS
(100) Silicon Wafer
500 nm thermal oxide
500 nm thermal oxide
Step 2: Pattern Backside
Step 1: Grow 500nm thick Thermal Oxide
Step 3: Remove front oxide and deposit a 3-layer oxide/a-Si/oxide film stack
Oxide
Oxidea-Si
Step 4: Rapid Thermal AnnealStep 5: Anisptropic Etching of (100) Si Wafer using
EDP
a-Si a-Sia-Si a-Sia-Si a-Si
~ 500 μm
Step 6: Remove Oxide Masks
Oxide Oxide
Pnc-Si Membrane
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
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
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
ULTRATHIN PNC-SI MEMBRANES:PHYSICAL PROPERTIES
Sputtered a-Si:1) High optical density, comparable to
microelectronic quality a-Si deposited with chemical vapor deposition (CVD)
2) Exhibits a clear shift in optical properties after crystallization
3) 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
2) Shift in optical Properties
3) Similar Peaks
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
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
morphologyChristopher 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
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
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
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
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
a) Annealed at 715ºC resulted in an average pore size of 7.3 nm
b) Annealed at 729ºC resulted in an average pore size of 13.9nm
c) Annealed at 753ºC resulted in an average pore size of 21.3 nm
Pore size and density increase monotonically with temperatureChristopher 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
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 DNAChristopher 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
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) speciesChristopher 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
ULTRATHIN PNC-SI MEMBRANES:MOLECULAR SEPARATION
Glass Coverslip
50 mm bead spacer
thin diffusion chamberWell
Step 1: Fill the Diffusion Chamber with 50ml Clean Buffer solution (PBS)
PBS
Step 2: Fill the Well with 3 ml of a fluorescent mixture containing BSA and Free Alexa 546 dye
Fluorescent Mixture
15nm thick membrane
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 wellChristopher 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
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
False Color images
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
ULTRATHIN PNC-SI MEMBRANES:MOLECULAR SEPARATION
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
Alexa Dye vs BSA BSA vs IgG
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
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
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
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
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 sizeChristopher 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
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
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%)
Dye diffusion through pnc-Si membranes compared to diffusion through standard regenerated cellulose dialysis membranes (Spectra/Por 7 dialysis membrane, molecularweight cut-off550K)
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
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
OUTLINE
Ultra-filtration (UF) Membranes Nanofabricated Membranes Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes
Fabrication Physical Properties Tunability Molecular Separation
Proposed Future Work Conclusion
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 microfluidic devices 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.
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
OUTLINE
Ultra-filtration (UF) Membranes Nanofabricated Membranes Ultrathin Porous Nanocrystalline Silicon (pnc-Si) Membranes
Fabrication Physical Properties Tunability Molecular Separation
Proposed Future Work Conclusion
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
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 nmol cm-2 h-1 for 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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