Separation of macromolecules using ultrathin silicon membranes

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

http://www.dow.com/liquidseps/prod/uf_index.htm

http://www.fumatech.com/EN/Membrane-technology/Membrane-processes/Ultrafiltration/

http://www3.ntu.edu.sg/home/DDSun/research.html

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

http://www.dow.com/liquidseps/prod/uf_index.htm

http://www.mymedicalsuppliers.com/dialysis-equipment-and-supplies/


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

http://www.appliedmembranes.com/about_ultrafiltration.htm

http://www.hydrotech.cn/English/mofenli.asp


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

http://www.appliedmembranes.com/about_ultrafiltration.htm


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)


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

http://www.kochmembrane.com/mww_purification.html

OUTLINE




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


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


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


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)



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



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


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.


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


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


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




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


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



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



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


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



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)



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


OUTLINE




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




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

Documents

Separation of macromolecules using ultrathin silicon membranes