3.
A Method to Create Tethered Membrane Structures
3.1
Introduction
3.1.1
Chapter Summary
This chapter describes the work done towards the goal of creating a
tethered membrane. This structure could be made in two general ways, by
physical or chemical patterning of a surface. Both were attempted in this study,
although a successful bilayer was deposited only on the physically patterned
surface.
Chemically patterned surfaces were made by a combination of
microcontact printing and self-assembly techniques. The patterned surface was
then selectively ionized, and polyelectrolyte solutions were used to sequentially
deposit anionic and cationic polyelectrolyte layers on one region of the pattern,
creating “posts” on the substrate. These posts could then be used to support the
membrane in a tent-like configuration. These polyelectrolyte-adsorbed surfaces
were characterized by AFM to determine the height of the built up structures. It
was found that the polyions absorbed well to the ionized silane, but the CH3terminated silane used for the stamping also absorbed a significant amount of
polyelectrolyte. The cleanest structures were formed after the deposition of 10
bilayers of polyelectrolyte, but increased number of layers caused build-up
everywhere and a loss of contrast in the pattern. We had hoped to create “posts”
approximately a micrometer high, which would entail depositing at least 30 – 40
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bilayers. New methods to resist the unilateral build up are currently being
studied. These include using PEG-silanes as the polyelectrolyte-resistant region
on the surface, or completely changing to the thiol-gold system currently used by
other groups.
The physically patterned surface were made by etching “holes” into the
substrate, and then depositing a bilayer to span over the open region. The holes
in the substrate were made in two ways. The first method was using traditional
microlithography to pattern the surface, and then wet etching holes into the
substrate. The second method was by a combination of fission fragment
bombardment and wet etching of mica to create nanometer to micrometer size
holes. Bilayers were deposited by a combination of the RLS and LS techniques.
The first layer was deposited by the reverse Langmuir- Schaefer technique, then
the second layer was added by deposition using the traditional Langmuir-Schaefer
technique. These membranes have been characterized by fluorescence optical
microscopy, and have been found to span the open holes in the substrate.
3.1.2
Supported Membranes
Supported membranes on solid surfaces are an important tool for many
reasons. First, they enable biofunctionalization of inorganic solids (i.e.,
semiconductors, metal-covered surfaces) and polymeric materials. Secondly,
they allow the immobilization of proteins under more natural conditions and in
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well-defined orientations. One can also prepare ultra-thin, high-electrical
resistance layers for the incorporation of receptors for use as biosensors. And
finally, supported lipid-protein bilayers separated from the solid surface by a thin
layer of material (such as a polymer cushion) can maintain the thermodynamic
and structural properties of a free bilayer (E. Sackmann, 1996). It is this last
application that forms the basis of this project.
To attach a membrane to a solid surface, it is necessary to provide a
secure anchor that strongly binds to the surface and provides a compatible link to
the membrane. Both the substrate and the anchoring group must be smooth at the
nanometer scale to provide defect-free coverage (D.K. Schwartz et. al., 1992c; E.
Sackmann, 1996) and to allow for accurate measurements with surface-sensitive
techniques, such as the SFA (J. Israelachvili et. al., 1994; T.L. Kuhl et. al., 1994;
J.N. Israelachvili, 1994; S. Chiruvolu et. al., 1995) and the AFM (J.A.
Zasadzinski et. al., 1994a; S.W. Hui et. al., 1995). While self-assembly (SA) and
LB techniques seem to work well for supported membranes, for specialized
systems such as membranes containing proteins or for highly fluid or multicomponent bilayers, LB and SA are not as successful. This is mainly because
most proteins denature at the air-water interface, or interact unfavorably with
solid substrates. Also, when membrane fluctuations or in-plane diffusion of
membrane components is important, a more realistic cell membrane model is
needed (Y.L. Chen and J.N. Israelachvili, 1992), since the proximity of the
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substrate can eliminate many dynamic features of the membrane. To address
these concerns, new methods for discretely anchoring membranes to a patterned
substrate are proposed that will allow for partial attachment to the surface, but
also allow for a significant fraction of the bilayer to be free from the substrate.
Supported membranes have been used for many years to study membrane
properties, and to look at membrane-protein interactions (E. Sackmann, 1996). In
recent years, some interesting applications of supported membranes have been
introduced. These include the study of lipid diffusion by electrophoresis (M.
Stelzle et. al., 1992; J.T. Groves and S.G. Boxer, 1995), the use of membranes to
adsorb proteins for study with the AFM (A.L. Weisenhorn et. al., 1992; J. Mou
et. al., 1995a; J. Mou et. al., 1995b), and electrical characterization of hybrid
membranes (M. Stelzle et. al., 1993; A. Plant, 1993; C. Steinem et. al., 1996).
However, the use of supported membranes have drawbacks for systems
containing membrane-spanning proteins. The diffusion of the proteins in the
membrane can be hindered as compared with vesicle systems, due to the presence
of the surface (A.A. Brian and H.M. McConnell, 1984; J. Salafsky et. al., 1996).
The proteins may also have groups extending out of the membrane, which may
interact unfavorably with the solid substrate. Still, for surface-sensitive
techniques like the SFA and the AFM, a solid surface is necessary to support the
membrane under investigation. This led us to consider an intermediate structure,
a semi-supported or tethered membrane, which would include the benefits of both
195
the supported and free membrane, and allow these systems to be studied with the
techniques commonly used in our laboratory.
3.1.3
Microcontact Printing
From previous work in our laboratory, it has been shown that silanes, such
as OTS, can form robust monolayers on mica (D.K. Schwartz et. al., 1992b) and
silicon by self-assembly. Since we were interested in patterning the substrate
surface, a technique that combined self-assembly and patterning was needed.
A new technique, called microcontact printing, has been used to pattern thiols on
gold (A. Kumar and G.M. Whitesides, 1993), silver (Y. Xia et. al., 1996a),
copper (Y. Xia et. al., 1996b), and also silanes on oxides (Y. Xia et. al., 1995;
N.L. Jeon et. al., 1995a; N.L. Jeon et. al., 1995b). The microcontact printing
process begins with a poly(dimethylsiloxane) (PDMS) rubber stamp made from a
master pattern, which can be a TEM grid, photoresist pattern on a Si wafer, or an
etched surface. The stamp made of PDMS is then inked with the desired solution
(thiol or silane) and brought into contact with the surface (metal or oxide). Just
like with a regular ink stamp, only the portions that are "raised" actually touch the
surface and create the design. This process is shown schematically in Figure 3.1.
We can now produce a surface that has bare regions, and regions that have been
derivatized by our ink solution. Baking (curing) allows for further surface bond
formation and crosslinking, thus producing a robust, patterned surface with
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PDMS
PDMS
~ 5 mm
PDMS
peeled away
from master
Si
“ink”
“ink”stamp
stampand
and
press
presstotosurface
surface
~ 3 nm
Si / Glass
remove
PDMS
stamp
Si / Glass
Figure 3.1. Microcontact Printing Technique. The PDMS elastomeric mixture is
poured over the master, which could be a TEM grid, photoresist pattern on a Si
wafer, or an etched surface. The PDMS is then cured and pulled away from the
master. The “ink” solution is applied onto the patterned side of the stamp with a
swab, and this is pressed against the desired surface. The stamp is removed from
the surface, and the pattern in the “ink” is left behind.
which we can eventually use to attach our membrane. The bare regions can also
be derivatized by the self-assembly of other molecules by immersing the
patterned substrate into a different silane solution (A. Kumar et. al., 1994; R.
Singhvi et. al., 1994; S. Palacin et. al., 1996).
The thiols on gold technology has been shown to have many potential
applications (A. Kumar at. al., 1995). These include optical diffraction gratings
(A. Kumar and G.M. Whitesides, 1994), microelectrodes (N.L. Abbott et. al.,
1994), molecular wires (J.M. Tour et. al., 1995), biosensors (M. Mrksich and
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G.M. Whitesides, 1995), arrays of channel waveguides (E. Kim et. al., 1996),
circuits (C.B. Gorman et. al., 1995) and protein adsorption and cell attachment
(R. Singhvi et. al., 1994; M. Mrksich et. al., 1995). Recent studies have looked at
different metal surfaces, mainly copper and silver. Copper has similar edge
resolution to the gold surfaces (~100 nm), while the silver surfaces provide much
improved resolution (~20 nm). These surfaces have been characterized by
surface plasmon resonance (M. Mrksich et. al., 1995), X-ray photoelectron
spectroscopy (S. Tam-Chang et. al., 1995), infrared external reflection
spectroscopy (J. Tour et. al., 1995), ellipsometry (P.T. Hammond and G.M.
Whitesides, 1995), Scanning electron microscopy (H.A. Biebuyck and G.M.
Whitesides, 1994), cyclic voltametry (N.L. Abbott et. al., 1994), Auger electron
spectroscopy (N.L. Jeon et. al., 1995a), contact angle (J. Drelich et. al., 1996),
optical microscopy (Y. Xia et. al., 1995), and AFM (J.L. Wilbur et. al., 1995).
Continued work is being done to broaden the applicability and scope of this work.
When microcontact printing silanes on silicon, it was found that the edge
resolution with this system is inferior to the thiols on gold, ~200 nm compared to
~100 nm (Y. Xia et. al., 1995). This could be due to the decreased reaction speed
of the silane molecules with the surface. However, since our anchoring points
from the stamp are not smaller than about 5 m (photolithographic limitation),
the decreased edge resolution should not be important.
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The pattern height from the surface can be enhanced by the sequential
layering of polyions onto the surface pattern (P.T. Hammond and G.M.
Whitesides, 1995). By patterning with a uncharged silane, then filling the bare
spots with a charge silane (or vice versa), ionic multilayers can be built up on the
charged silane. This process is shown schematically in Figure 3.2. This results
in a patterned substrate upon which the bilayer can be deposited, utilizing the
electrostatic interaction to the ionic regions. The distance between posts can be
adjusted to provide an optimal support for a particular bilayer.
3.1.4
Physical Patterning Techniques
• Microlithography and Etching
Microlithography is the process by which patterns are transferred onto a
solid substrate. The steps involved in microlithography include spin coating the
resist onto the surface, irradiating the surface through a mask, and developing the
resist. After the surface has been patterned by the resist, etching of the surface by
chemical or plasma treatments can be done to create the actual pattern in the
surface. These steps are shown schematically in Figure 3.3. First, a thin layer of
polymeric resist material is spin coated onto the surface of interest. This film is
usually about 1 m thick after solvent evaporation and baking (S.M. Sze, 1988).
The next step involves the irradiation of the resist material through a mask of the
desired pattern. The irradiation can be done using photons (photolithography),
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a
b
c
–
+
Figure 3.2. Schematic of the Polyion Deposition Process (G. Decher, 1997).
(a) Shows that the process can take place in sequential beakers. The first and
third beaker contain the polyions, while the second and fourth are washing steps.
These steps are repeated until the desired number of layers are applied. (b) From
a charged surface, alternating layers of polyanion and polycation are deposited.
The surface charge is reversed after the deposition of each layer. (c) Chemical
structure of the two polyions used in this study (PDAC and SPS), which are
discussed in the next section.
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Si
(a) The photoresist is spin coated onto the substrate,
usually a silicon wafer.
Irradiation
Source
Si
(b) The photoresist is irradiated through a pattern mask.
The exposed parts of the photoresist are altered by the
irradiation.
Si
(c) The photoresist is developed in a solution which
selectively removes the photoresist to expose the
pattern on the surface.
Figure 3.3. Schematic of the Microlithography Process. The irradiation can be
done with a variety of sources, such as photons, electrons, x-rays, or ions. The
photoresist can be either positive or negative. For a positive photoresist, the
irradiated regions are dissolved away by the developer, as shown in the
schematic. The opposite is true for a negative resist.
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electrons (e-beam lithography), x-rays (x-ray lithography), or ions (ion
lithography). The smallest linewidths can be written by e-beam lithography,
which can be as small as 100 Å (S. Middleman and A.K. Hochberg, 1993). The
irradiation of the photoresist causes it to react chemically, and makes the exposed
areas more or less soluble to a given solvent system. During the development of
the photoresist, the exposed areas are dissolved away for a positive resist, while
the opposite it true for the negative resist. Once the development is done, areas
of the surface are now exposed, and selective etching of the surface can be done.
Etching processes are classified as wet or dry etching. Wet etching involves the
use of solutions for the isotropic etching of the surface. This process often leads
to poor control of the pattern geometry, and overetching often occurs. However,
this process is usually faster and more selective. Dry etching is also called
plasma etching, and involves the use of an ionized gas (plasma). This process is
highly anisotropic, and gives unidirectional etching of the surface. This process
is commonly used in semiconductor processing today. Once the surface is
etched, the remaining resist material can be removed using an organic solvent,
and the surface is ready for use.
• Etching Holes in Mica
This process was discovered historically from naturally occurring fission
tracks in mica, which contained inclusions rich in uranium. When the mica was
immersed in HF acid, etch pits were created (P.B. Price and R.M. Walker, 1962).
202
In the current version of this technique, spontaneous fission fragments from 252Cf
are used to create pores in mica. The mica is cleaved and placed a small distance
away from the radiation source. Fragments from the source travel through the
mica, and create damage paths perpendicular to the surface. Etching with a HF
acid solution removes the damaged material in the irradiation path, and creates
cylindrical pores in the mica. Both the irradiation and etch time can be controlled
to affect the size of the pores formed. The irradiation time controls the average
distance between the tracks, while the etching time controls the size of the pores
formed. Prolonged etching causes the pores to grow in width, although the shape
of the pores changes from circles to parallelograms (C.P. Bean et. al., 1970).
Images of mica surfaces with small and large holes are shown in Figure 3.4.
One possible use for this process is the creation of an inorganic
membrane. The pores etched in the mica could be kept as small as those inferred
to exist in biological membranes (C.P. Bean et. al., 1970). The very small pores
in the mica are not easily imaged with optical microscopy, as they are rather
shallow. AFM allows for easier imaging and the determination of the depth of
the holes.
3.1.5
Bilayer Deposition
Bilayers can be deposited onto solid substrates in a variety of ways.
Symmetric and asymmetric bilayers can be made by a single technique, or by a
203
a
50 m
b
1 m
Figure 3.4. Optical and AFM images of mica with holes made by the
californium irradiation technique. (a) Optical image of 50 m holes made by
short irradiation and long etch times. The irradiation was 1 second and the etch
time was 41 hours. (b) AFM image (5 m X 5 m) of approximately 250 nm
holes made by long irradiation and short etch times. The irradiation time was 3
hours and the etch time was 8 minutes. The shape of holes changes dramatically
as the etch time increases.
204
combination of the following techniques. Figure 3.5 schematically describes the
methods for bilayer deposition.
• Vesicle Adsorption
Bilayers can be easily made on hydrophilic surfaces by spreading vesicles.
For vesicle spreading to occur, the surface of the support must be attractive to the
molecules in the vesicles, or the vesicles must be under high tension (E.
Sackmann, 1996). Spontaneous fusion of the vesicles to the solid substrate then
occurs, with a thin water layer (~10 Å) separating the membrane from the surface
(T.M. Bayerl and M. Bloom, 1990; B.W. Koenig et. al., 1996). Because of this
water layer, the membrane is able to maintain its lateral fluidity. Using this
technique alone, symmetric bilayers can be created. By combining this technique
with a self-assembled monolayer (thiols on gold or silanes on silicon oxide),
hybrid bilayers can be created, with only the top leaflet of the bilayer coming
from the molecules in the vesicles.
• Langmuir-Blodgett Technique
The LB technique was described in detail in Chapter 1. The desired lipid
or lipid and protein molecules are spread at the air-water interface, and two layers
are deposited onto the solid substrate. Asymmetric membranes can be easily
created using this technique. It can also be combined with other techniques, such
as vesicle spreading or the Langmuir-Schaefer technique.
One problem with the LB technique is that many proteins do not transfer
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(a) Vesicle breaking onto a solid surface. The vesicles
touch the surface and break open, spreading a bilayer
across the surface.
(b) LB deposition onto a solid surface. To deposit a
bilayer, two passes are done through the interface.
(c) LS deposition onto a solid surface. An initial layer
is deposited by LB, then the surface is pushed down
through the interface to deposit the second layer.
Figure 3.5. Schematic of the methods to create bilayers on solid surfaces.
206
well to the air-water interface, where some of their domains may be exposed to
the air. A combination of LB (the first layer) and vesicle spreading has been used
to maintain partial activity of some proteins (E. Kalb and L.K. Tamm, 1992).
• Langmuir-Schaefer and Reverse Langmuir-Schaefer Techniques
While the LB technique is a vertical transfer of material from the airwater interface to a solid substrate, the Langmuir-Schaefer (LS) technique is the
corresponding horizontal transfer technique (I. Langmuir and V.J. Schaefer,
1938). A substrate is positioned horizontally above the air-water interface, and
the substrate is then pushed through the interface, depositing a monolayer of
material. We call the reverse process, where a horizontal substrate is lifted from
under the subphase up through the interface, the reverse Langmuir-Schaefer
(RLS) technique (K.Y.C. Lee et. al., 1998). The LS technique is often used in
combination with the LB technique for the deposition of the second leaflet of the
bilayer (L.K. Tamm and H.M. McConnell, 1985).
3.2
Experimental Methods
3.2.1
Microcontact Printing
The stamping procedure was derived from a similar one used by the
Whitesides' group for patterning silanes on oxide surfaces (N.L. Jeon et. al.,
1995a). First, a master with a desired pattern is chosen. Presently, we have used
TEM grids, etched silicon, and patterned photoresist as masters to make the
elastomeric stamps. The surface of the master is fluorinated so the stamp and
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master do not stick to each other (G.P. Lopez et. al., 1993). The master was put
into a desiccator with approximately 30 L of (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane, and left under vacuum for 20 minutes. The remaining
fluorosilane was then removed and the desiccator was evacuated by continuous
pumping for 1 hour. The master is now ready for use as a stamp pattern.
The PDMS elastomeric (Dow Corning) mixture is a two component
system mixed in a 10:1 weight ratio of prepolymer to binder. After the two
components are thoroughly mixed, the mixture is pumped under vacuum to
remove the air bubbles that form (otherwise your stamp surface will be bumpy).
Next, the mixture is poured over the master in a petri dish, and allowed to sit
overnight to cure. We make the stamps approximately 5 mm high. Before the
master is removed, if the PDMS feels tacky to the touch, the stamp is baked at
65 °C for 2 hours.
Initially, the stamps were cut to the size of the pattern, because we
208
thought the extra PDMS around the pattern would bleed the ink solution onto the
patterned regions. However, using this procedure meant that the stamps were
extremely small and difficult to handle, especially the TEM grid stamps. Our
current cutting method allows for extra area to be left around the stamp pattern.
The larger stamps are easier to hold and maneuver, and no bleeding into the
patterned region has been observed.
The substrates used for stamping were either silicon pieces or glass
coverslips. The substrates were cleaned in piranha solution (see Chapter 1.2.1),
then dried in a stream of nitrogen prior to use.
The concentration of the stamping solution and the printing (contact) time
are important parameters for achieving good quality patterns. We use a solution
concentration of 10 mM silane in hexane, and the printing time is 1 minute. The
stamp is inked with a moistened lint-free cotton swab, then the surface of the
stamp is blown dry with nitrogen (or allowed to air dry). The stamp is then
pressed down onto the silicon or mica substrate. At this point, the stamp should
stick to the substrate, and a wetting front should travel across the stamp-substrate
interface. The stamp should not be too wet or else the stamp will slip and slide
off the surface. After 1 minute, the stamp is removed, the substrate is washed
with hexane, then baked at 90 °C for a minimum of 30 minutes. The surfaces
were then rinsed again with hexane and blown dry. The stamps themselves are
rinsed after use first with hexane, then sonicated in soap and water.
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3.2.2
Polyion Adsorption
• Vinyl Group Oxidation
Self-assembly of a vinyl terminated silane molecule was done to the
unpatterned regions of the surface. The vinyl silane used in this study was (7octen-1-yl)trimethoxysilane, which was about half the length of the stamping
silane (OTS, C18). The self-assembled vinyl silane monolayer was prepared
following the same procedure created for mica surfaces (D.K. Schwartz et. al.,
1992b). A 15 mM solution in dry hexadecane was poured over the stamped
samples and left for at least 3 hours. The samples were then rinsed in
chloroform, dried with nitrogen, and baked at 90 °C for at least 3 hours.
The oxidation of the vinyl group to form the carboxylic acid was done
following a procedure created for self-assembled monolayers (S.R. Wasserman
et. al., 1989). Stock solutions of 5 mM potassium permanganate (KMnO4), 195
mM sodium periodate (NaIO4), and 18 mM potassium carbonate (KCO3) in ultrapure water were prepared. Immediately prior to the oxidation, 1 mL of each
solution was combined with 7 mL of ultra-pure water to form the oxidizing
solution. The samples were left in the solution for 2 hours (R.U. Lemieux and E.
von Rudloff, 1955). After removal from the oxidizing solution, the samples were
rinsed in 20 mL each of 0.3 M sodium bisulfite (NaHSO3), water, 0.1 N HCl,
water, and ethanol. The samples were then dried with nitrogen and used within a
few days.
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• Layer-by-Layer Deposition
The layering processes was done using aqueous solutions of
poly(diallydimethylammonium chloride) (PDAC) and sodium poly(styrene
sulfonate) (SPS). The PDAC is the positively charged polyelectrolyte, with a
molecular weight of ~150,000 (Mw). The SPS is the negatively charged
polyelectrolyte with a molecular weight of 100,000 (Mw). Solutions with
concentrations of 20 mM were made for both polyelectrolytes in ultra-pure water.
All polymer concentrations reported are based on the number of repeat units.
Sodium chloride was added to each solution at a concentration of 0.4 M. This
was shown to increase the selectivity of the adsorption and increase the thickness
of the deposited layers, likely by increasing the ionization of the acid surface
(S.L. Clark et. al., 1997). Both solutions were filtered through a 0.22 m MillexGS filter unit (Millipore) before use to remove particulates. The sample was first
submerged in the PDAC solution for 20 minutes, then rinsed with water. The
sample was next immersed in the SPS solution for 20 minutes, rinsed with water,
and then sonicated for 1 – 5 minutes. This is to remove loosely bound
polyelectrolyte, and also to prevent the material from building up on the CH3terminated regions. The deposition of the bilayer structure was repeated for up to
20 bilayers. After deposition of approximately 7 bilayers, the pattern was
observable by optical microscopy.
The final layered samples were imaged with the AFM in contact mode.
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The largest scanner (J) was used, since most of the stamp features were quite
large. The optical tip positioning setup (K.Y.C. Lee et. al., 1998) was used to
find the best area on the sample to image.
3.2.3
Vesicle Adsorption
Dilauroylphosphatidylcholine (DLPC) was used to make fluorescently-
labeled vesicles for adsorption onto the patterned surface. A vesicle solution
concentration of 10 mg/mL with 0.05 mole % N-(Texas Red sulfonyl)-1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt
(Texas Red-DHPE) was made by well-established procedures (S.A. Walker,
1996). The lipid/dye mixture was dissolved in chloroform, then dried to a lipid
cake in flowing nitrogen gas. The lipid cake was allowed to dry overnight in a
vacuum oven. The cake was hydrated with buffer (0.1 mM Na2HPO4, 0.1 mM
NaH2PO4, 0.2 M NaCl, 0.02 wt. % NaN3, pH = 7.2) in a water bath at 37 °C for
at least a day. The mixture was then put through 5 freeze-thaw cycles, first
freezing in liquid nitrogen, then thawing in warm water (~60 °C). The resulting
solution of large unilamellar vesicles was extruded through 200 nm then 50 nm
Nuclepore filters. This resulting solution is clear, with vesicles approximately the
size of the filter pores (S.A. Walker, 1996).
The procedure for the vesicle adsorption was modified from a procedure
for the adsorption of planar lipid bilayers (J.T. Groves and S.G. Boxer, 1995). A
212
small drop (80 L) of the vesicle solution is placed on the bottom of a
crystallizing dish, and the patterned sample is placed pattern side down over the
drop. After a few minutes, the dish was carefully filled with water, making sure
the pattern side stays facing the bottom of the dish (the sample sometimes floats
on the interface). The sample is then gently shaken to remove the excess
unbroken vesicles, and a glass coverslip of the appropriate size is used to create a
sandwich. The sample/coverslip sandwich can then be removed from the water
and imaged with optical microscopy.
3.2.4
Etched Holes in Mica
Mica samples are prepared in the same way as for use in the AFM or for
reverse LS deposition. The mica is cleaved, then placed within
1
16
inch of the
irradiation source, separated by a teflon spacer ring. The mica is irradiated
anywhere from 1 second to a few hours, and then etched with a 34 % HF acid
solution. The etching time varied from 2 minutes to 45 hours to create holes
from 200 Å to 50 microns. The irradiation time controls the average distance
between the tracks, while the etching time controls the size of the holes. A long
irradiation time and a short etch time will give many small holes close together,
while a short irradiation time and a long etch time will give a few large holes
farther apart.
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3.2.5
Membrane Deposition
Initially, deposition was done by using the LB deposition technique for
both layers, which was shown to be moderately successful for spanning large
holes in surfaces (T.D. Osborn and P. Yager, 1995). We did not have
reproducible success with this method, and proceeded to devise a better one. The
new procedure combines the RLS technique with the regular LS technique. The
monolayer used for the initial experiments was the Tanaka mixture
(DPPC/POPG/PA, described in Chapter 2.2.1, with no added protein). This was
used because the mixture makes distinctive domain structures that could be easily
seen and monitored via fluorescence microscopy. The monolayer was deposited
and compressed to a pressure of 20 mN/m before deposition. At this pressure the
flower shaped solid phase domains are quite large and close together. The first
layer was deposited by the RLS technique. Once the monolayer was deposited,
the sample was inverted and pushed down through another monolayer with the
same composition. The waiting time between the first and second layer
deposition is important, and should be kept as short as possible to minimize
leakage of the entrapped water. These samples were imaged with fluorescence
optical microscopy.
Samples for the bilayer deposition were either etched mica or etched
silicon. The mica samples were described previously. The silicon samples were
made by Phil Infante at the Cornell Nanofabrication Facility. A single square
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hole was etched into the center of the substrate using a KOH etch. The hole
extended completely through the sample, and was smaller on one side. In general
the holes were between 10 – 30 m on one side of the Si, and 200 – 300 m on
the other side of the Si. These samples were also cleaned using the piranha
solution, then dried with nitrogen. The samples were then epoxied to a glass
coverslip or the AFM steel discs (these were also cleaned). Care was taken to
keep the epoxy away from the hole in the center of the sample. This sample
could now be used with the RLS apparatus.
3.3 Microcontact Printing with Silanes
The stamping technique was chosen as a way to pattern surfaces for
bilayer deposition. The silane chemistry was chosen as the molecule for
stamping because initially these samples were to be used in the AFM and
possibly the surface forces apparatus (SFA). Our group also had some past
experience with self-assembly of silanes (D.K. Schwartz et. al., 1992b), so this
seemed like a good system to work with initially. From our early work on the
stamping with silanes, we found that longer chained molecules work best to
transfer the pattern onto the surface. Shorter chained molecules tended to
assemble all over the surface. From imaging with the AFM, the edge resolution
of our patterns was not very good, and material tended to be deposited in the
unpatterned regions. However, we were able to transfer the patterns onto the
surface and image the surfaces with AFM.
215
• Different Chain Length Silanes
We tried stamping with five different silanes, octadecyltrichlorosilane
(CH3(CH2)17SiCl3, OTS), dodecyltrichlorosilane (CH3(CH2)11SiCl3, DTS), (3Glycidyloxypropyl)-trimethoxysilane ((CH2CHO)CH2O(CH2) 3Si(OCH3) 3, GTS),
(3-Thiocyanatopropyl)-triethoxysilane ((CH3CH2O)3SiCH2CH2SCN, TTS), and
(7-octen-1-yl)trimethoxysilane (H2C=(CH2)7Si(OCH3)3, vinyl silane). When
doing the stamping with these different chain lengths, we found that only the
longest chain silane, OTS, gave distinct features. The others formed aggregates
of material on the surface without pattern transfer. Recently, stamping with a
PEG-silane (350 and 550 molecular weight PEG) was attempted. This was also
found to form aggregates of material on the surface, and the stamp pattern was
not seen.
• AFM Imaging of the Stamped Surfaces
The stamps we have were made from patterned photoresist on silicon,
done by Mike McGehee in the Physics Department, or from 400 and 200 mesh
TEM grids. Figure 3.6 shows optical images of a couple of the PDMS stamps
we used.
We tried stamping these patterns on silicon, glass, and mica surfaces. We
got better (cleaner) patterns on silicon and glass, but we did see patterns on mica.
For AFM imaging purposes, we were able to get the best patterns from the stamp
with the lines. The TEM grid spacing is quite large, and realistically only the 400
216
mesh grid with the smaller holes was easily imaged. Figure 3.7 shows some
representative pictures of what we were able to see with the stamping technique.
Sometimes the features transferred to the surface got distorted from the original
stamp. This is due to the amount of pressure exerted on the stamp during the
process. The measured step height of the OTS pattern on the surface was
approximately 3 nm, which is the value measured previously for the OTS
monolayer (D.K. Schwartz et. al., 1992b).
217
a
50m
b
50m
Figure 3.6. Optical images of the PDMS stamps. (a) Stamp made from a
pattern photoresist deposited on a silicon wafer. The lines are approximately
10 m wide and 8 m apart. (b) Stamp made from a 400 mesh TEM grid. The
squares are 46 m on a side and 16 m apart.
218
a
20 m
b
20 m
Figure 3.7. AFM images (100 m X 100 m) of the stamped surfaces.
(a) Glass surface, using the stamp in Figure 3.6a. (b) Silicon surface, using the
stamp in Figure 3.6b. The pattern is often covered by aggregated OTS
molecules, which show up as white spots on the AFM images. This is due to the
fast reaction of the OTS with water in the solution and the air, which forms
polymerized aggregates.
219
20 m
3.4
Lipid Adsorption to Stamped Surfaces
After the stamping procedure and imaging was accomplished, deposition
of bilayers onto the stamped surfaces was desired. Small fluorescently-labeled
vesicles can be used to easily create bilayers on solid surfaces, which can be
partitioned using lithographically patterned substrates (J.T. Groves et. al., 1997).
We were interested as to whether we could use the fluorescent vesicles to
selectively pattern our stamped surfaces. We found that the lipid bilayers formed
preferentially on the hydrophilic (unstamped) regions of the surface. However, a
small amount, most likely a monolayer, is formed over the stamped pattern. The
deposition dynamics were further investigated using the reverse LS technique,
whereby we were able to watch the deposition process in situ.
3.4.1
Vesicle Adsorption
The fluorescently-labeled vesicles were spread onto the patterned surfaces
and imaged with FM. The adsorption was done on both Si and glass surfaces,
and the images are shown in Figure 3.8. The image of the patterned glass surface
shows the expected behavior. The fluorescence is brighter in the unstamped
regions, which correspond to the hydrophilic part of the surface. This was a
much faster and easier way for us to image our surfaces and also to tell if the
220
stamp application was done properly. The vesicles adsorbed to the patterned
silicon surface did not show the behavior we expected. The fluorescence looked
221
a
100m
b
100m
Figure 3.8. FM images of vesicle adsorption onto the patterned surface.
(a) Adsorption onto a glass coverslip patterned with a 200 mesh TEM stamp.
The vesicles spread onto the unpatterned, or hydrophilic regions of the surface.
(b) Adsorption onto a Si wafer patterned with a 400 mesh TEM stamp. The
vesicles still spread onto the unpatterned regions, but the thin native oxide layer
quenches the fluorescence, and looks dark. A monolayer adsorbs onto the
patterned OTS region, and this looks brighter.
222
brighter in the stamped, or hydrophobic part of the surface. In general, the
surface chemistry of the two samples is the same, so we expected similar
behavior. Upon further analysis, we found that the thin native oxide layer was
quenching the fluorescence near the silicon surface (A. Lambacher and P.
Fromherz, 1996). Since the stamped regions were slightly further away from the
silicon surface, the fluorescence of the molecules deposited on the OTS appeared
brighter. Quenching is a common problem for bilayers deposited on silicon
wafers, and a thick oxide layer (~800 nm) is usually thermally grown to prevent
this (L.K. Tamm and H.M. McConnell, 1985).
3.4.2
Reverse Langmuir-Schaefer Deposition
Using the RLS deposition technique, we were able to watch the
deposition of a monolayer of fluorescently-labeled lipids onto our stamped
surface. When the floating monolayer came into contact with the stamped
surface, water was trapped between the surface and the monolayer. Because of
this, we could watch the water front dry, and leave lipid molecules behind in
specific regions on the stamped surface. This is shown in Figure 3.9. On the
glass surface, the molecules were captured in the unstamped region, and were
seen to avoid the OTS regions. However, some fluorescent material was seen
trapped on the OTS regions due to the movement of the water front. When
completely dry, the surface looks just like the surface with the adsorbed vesicles.
223
a
100m
b
100m
Figure 3.9. FM images of the RLS deposition of a monolayer onto a patterned
surface. (a) Monolayer deposited onto a glass surface patterned with a 400 mesh
TEM stamp. The water is draining beneath the monolayer, leaving the lipid
molecules behind. (b) After the water has completely dried, the fluorescence is
bright in the unstamped, or hydrophilic regions.
224
These experiments, along with the AFM done in the previous section,
helped us to determine that the OTS stamped surfaces were not far enough away
from the surface to be a bilayer tether. The 3 nm separation distance would most
likely result in the monolayer conforming to the surface instead of being
supported. Also, the bilayers were being built up in the wrong areas. The
bilayers were supposed to interact with the tether regions, which is the opposite
of the current system. Thus the next section discusses a possible method for
building up the pattern to keep the bilayer a sufficient distance away from the
surface, and to provide a charged surface which would allow for electrostatic
interactions with the bilayer.
3.5
Adsorption of Polyions to Patterned Surfaces
In the previous sections, patterning of silanes onto oxide surfaces was
performed, and the resulting samples were characterized by both the AFM and
FM. The problem with the technique so far is the height of the resulting pattern.
The OTS molecules used for the patterning are only 3 nm high, and this short
distance would lead to the bilayer conforming to instead of suspending above the
surface. A recent technique used to build up polyelectrolyte multilayers onto
gold-thiol patterned surfaces has been modified for our oxide-silane surfaces.
The procedure includes the initial patterning of the surface by microcontact
printing with OTS, then the self-assembly of a vinyl-terminated silane to fill in
the remaining unpatterned regions. The vinyl silane was then oxidized to the
225
carboxylic acid, and the polyion deposition proceeded on this charged endgroup.
We encountered a problem with the selectivity of the adsorption process. We
found that the OTS surface could not totally prevent polyion deposition;
significant amounts of the material was depositing onto the area.
• Optical and AFM Imaging of the Layered Surfaces
The applied patterns after the stamping and oxidation steps are not visible
with the optical microscope, but become visible after deposition of approximately
7 bilayers. As additional bilayers are adsorbed to the surface, the pattern
becomes more distinct, and can be seen with the naked eye.
In order to determine if the structures are building up to a sufficient
226
height, AFM was done of the surface to measure the height difference between
the patterned and unpatterned regions. We found that the quality of the original
stamping was a critical factor in determining how well the layering resulted. The
best sample we have made had 10 bilayers and a height of 300 – 400 nm. The
AFM image and a trace of the height profile are shown in Figure 3.10. While
this was encouraging, a subsequent 10 bilayer sample only had a 30 – 60 nm
height difference, with significantly more material deposited in the patterned
region (Figure 3.11). In some situations, the entire sample had the same height
except for a small edge around the boundary for the patterned/unpatterned region
(Figure 3.12).
A comparison of the AFM imaged samples under the optical microscope
gave us an idea of what a sample with better height contrast would look like,
without having to actually do the AFM. This would give us the opportunity to
discard bad samples without having to do the AFM imaging. Two comparison
images of the samples are shown in Figure 3.13. The contrast for the good
image (large height difference) is much better than for the bad image (no height
difference). This now allowed us a simple way to evaluate samples before
proceeded too far in the process.
In order to eliminate possible problems from the deposition and oxidation
steps, we varied the experimental parameters to determine their effect on the
resulting patterned surface. The length of sonication, oxidation time, drying, and
227
30 m
Figure 3.10. AFM image (130 m X 130 m) and height trace of a 10 bilayer
PDAC/SPS adsorbed sample. The stamped pattern was the 200 mesh TEM grid.
The black line on the image shows the region of the line trace. The arrows on the
height trace show the approximate height difference, which is 300 nm.
228
20 m
Figure 3.11. AFM image (120 m X 120 m) and height trace of a 10 bilayer
PDAC/SPS adsorbed sample. The stamped pattern was the 400 mesh TEM grid.
The black line on the image shows the region of the line trace. The arrows on the
height trace show the approximate height difference, which is 60 nm.
229
30 m
Figure 3.12. AFM image (150 m X 150 m) and height trace of an 8 bilayer
PDAC/SPS adsorbed sample. The stamped pattern was the 400 mesh TEM grid.
The black line on the image shows the region of the line trace. The arrows on the
height trace show the approximate height difference, which is negligible.
230
a
100 m
b
100 m
Figure 3.13. Optical microscope images of a good and bad sample, determined
after imaging with AFM. The stamped pattern was the 400 mesh TEM grid.
(a) This image shows good contrast between the squares and the lines, which
corresponded to a significant height difference using AFM. The additional lines
in the image are tweezer scratch marks. (b) This image does not show much
contrast between the squares and the lines, just the edge of the line. This image
had almost no height difference from the AFM image.
231
the surface charge were changed, but none were found to change the resulting
height difference. The only parameter that had a significant effect on the polyion
deposition was the quality of the original stamping. This step is difficult to
quantify, and also difficult to know until at least 7 bilayers have been deposited
and the sample can be imaged under the optical microscope. AFM imaging of
each sample is also not desirable because the stamped regions are very hard to
find, making tip positioning difficult and time consuming. This makes fixing the
problem very difficult.
A parameter we have tried to change is the adsorption to the stamped
(OTS terminated) regions. The similar work done with thiols on gold use a long
chained carboxylic acid terminated thiol to stamp, and an ethylene glycolterminated thiol to fill in the spaces (S.L. Clark et. al., 1997). We have not found
an equivalent carboxylic acid-terminated silane, or a long chained vinyl silane
molecule. We have tried stamping with a PEG-silane molecule (H(OCH2CH2) n –
Si – (OCH2CH3) 3), where n is approximately 8 – 12. This was also unsuccessful,
most likely due to the slow reaction of the ethoxy silane group to the surface.
3.5
Adsorption of Polyions to Patterned Surfaces
In the previous sections, patterning of silanes onto oxide surfaces was
performed, and the resulting samples were characterized by both the AFM and
FM. The problem with the technique so far is the height of the resulting pattern.
The OTS molecules used for the patterning are only 3 nm high, and this short
232
distance would lead to the bilayer conforming to instead of suspending above the
surface. A recent technique used to build up polyelectrolyte multilayers onto
gold-thiol patterned surfaces has been modified for our oxide-silane surfaces.
The procedure includes the initial patterning of the surface by microcontact
printing with OTS, then the self-assembly of a vinyl-terminated silane to fill in
the remaining unpatterned regions. The vinyl silane was then oxidized to the
carboxylic acid, and the polyion deposition proceeded on this charged endgroup.
We encountered a problem with the selectivity of the adsorption process. We
found that the OTS surface could not totally prevent polyion deposition;
significant amounts of the material was depositing onto the area.
• Optical and AFM Imaging of the Layered Surfaces
The applied patterns after the stamping and oxidation steps are not visible
with the optical microscope, but become visible after deposition of approximately
7 bilayers. As additional bilayers are adsorbed to the surface, the pattern
becomes more distinct, and can be seen with the naked eye.
In order to determine if the structures are building up to a sufficient
233
height, AFM was done of the surface to measure the height difference between
the patterned and unpatterned regions. We found that the quality of the original
stamping was a critical factor in determining how well the layering resulted. The
best sample we have made had 10 bilayers and a height of 300 – 400 nm. The
AFM image and a trace of the height profile are shown in Figure 3.10. While
this was encouraging, a subsequent 10 bilayer sample only had a 30 – 60 nm
height difference, with significantly more material deposited in the patterned
region (Figure 3.11). In some situations, the entire sample had the same height
except for a small edge around the boundary for the patterned/unpatterned region
(Figure 3.12).
A comparison of the AFM imaged samples under the optical microscope
gave us an idea of what a sample with better height contrast would look like,
without having to actually do the AFM. This would give us the opportunity to
discard bad samples without having to do the AFM imaging. Two comparison
images of the samples are shown in Figure 3.13. The contrast for the good
image (large height difference) is much better than for the bad image (no height
difference). This now allowed us a simple way to evaluate samples before
proceeded too far in the process.
In order to eliminate possible problems from the deposition and oxidation
steps, we varied the experimental parameters to determine their effect on the
resulting patterned surface. The length of sonication, oxidation time, drying, and
234
30 m
Figure 3.10. AFM image (130 m X 130 m) and height trace of a 10 bilayer
PDAC/SPS adsorbed sample. The stamped pattern was the 200 mesh TEM grid.
The black line on the image shows the region of the line trace. The arrows on the
height trace show the approximate height difference, which is 300 nm.
235
20 m
Figure 3.11. AFM image (120 m X 120 m) and height trace of a 10 bilayer
PDAC/SPS adsorbed sample. The stamped pattern was the 400 mesh TEM grid.
The black line on the image shows the region of the line trace. The arrows on the
height trace show the approximate height difference, which is 60 nm.
236
30 m
Figure 3.12. AFM image (150 m X 150 m) and height trace of an 8 bilayer
PDAC/SPS adsorbed sample. The stamped pattern was the 400 mesh TEM grid.
The black line on the image shows the region of the line trace. The arrows on the
height trace show the approximate height difference, which is negligible.
237
a
100 m
b
100 m
Figure 3.13. Optical microscope images of a good and bad sample, determined
after imaging with AFM. The stamped pattern was the 400 mesh TEM grid.
(a) This image shows good contrast between the squares and the lines, which
corresponded to a significant height difference using AFM. The additional lines
in the image are tweezer scratch marks. (b) This image does not show much
contrast between the squares and the lines, just the edge of the line. This image
had almost no height difference from the AFM image.
238
the surface charge were changed, but none were found to change the resulting
height difference. The only parameter that had a significant effect on the polyion
deposition was the quality of the original stamping. This step is difficult to
quantify, and also difficult to know until at least 7 bilayers have been deposited
and the sample can be imaged under the optical microscope. AFM imaging of
each sample is also not desirable because the stamped regions are very hard to
find, making tip positioning difficult and time consuming. This makes fixing the
problem very difficult.
A parameter we have tried to change is the adsorption to the stamped
(OTS terminated) regions. The similar work done with thiols on gold use a long
chained carboxylic acid terminated thiol to stamp, and an ethylene glycolterminated thiol to fill in the spaces (S.L. Clark et. al., 1997). We have not found
an equivalent carboxylic acid-terminated silane, or a long chained vinyl silane
molecule. We have tried stamping with a PEG-silane molecule (H(OCH2CH2) n –
Si – (OCH2CH3) 3), where n is approximately 8 – 12. This was also unsuccessful,
most likely due to the slow reaction of the ethoxy silane group to the surface.
3.7
Discussion
The new technique described for bilayer formation has some advantages
over the LB and vesicle adsorption techniques currently used. First, the
continuous monitoring of the surface morphology is useful to determine the
success of the deposition. This is difficult to do with the LB technique, and
239
samples are usually just imaged after the deposition is completed. Also, since the
sample is initially kept parallel to the surface and the monolayer is placed gently
onto the substrate, distortion of the monolayer features does not usually occur
unless the sample is not very level or if the monolayer is vibrating. The LB
technique is known to distort the features of lipid films from what was present at
the air-water interface (K. Spratte et. al., 1994). Currently, our method, black
lipid membranes, and the LB technique are the only ways to make hole spanning
bilayers. Vesicle deposition will not allow bilayer formation across a large hole,
and would instead coat the entire sample surface with a bilayer. One concern we
had with our technique was that the entrapped water in the hole would completely
drain when the sample was inverted for the LS deposition, but so far we have
seen only slight water loss due to leakage, not from the inversion of the sample.
The next step after the creation of the bilayer is the inclusion of protein
molecules into the bilayer. Techniques involving the air-water interface are not
often used for this purpose, since most proteins denature at this interface, where
some of their domains may be exposed. A more common technique for making
supported bilayers with proteins is vesicle adsorption to the surface (A.A. Brian
and H.M. McConnell, 1984; J. Salafsky et. al., 1996), although in those cases the
proteins were rendered immobile due to unfavorable interactions with the
underlying surface. In another study, a combination of LB deposition and vesicle
adsorption was able to maintain partial mobility of the protein (E. Kalb and L.K.
240
Tamm, 1992). We could also try and utilize the RLS monolayer deposition with
vesicle adsorption to create bilayers with embedded proteins, with possible full
mobility over the free membrane region. This would allow us to utilize
techniques such as FRAP (fluorescence recovery after photobleaching) and
microelectrophoresis to measure diffusivities of the lipids and proteins in the free
membrane. These techniques have already been used to measure diffusivities in
supported membranes (B.A. Smith and H.M. McConnell, 1978; M. Stelzle et. al.,
1992; J.T. Groves and S.G. Boxer, 1995).
Progress in the formation of the chemically-patterned surfaces was stalled
due to the difficulties with the selectivity of the polyion adsorption. Current
techniques using the CH3/COOH-terminated silanes so far have not consistently
built up structures of very large height differences. This is due to the large
amount of the polyion that sticks to the CH3 endgroup. A new combination of
PEG/COOH-terminated silanes may allow for more selectivity of the polyion
deposition, with the PEG moiety resisting polyion adsorption, as seen with the
thiol systems (S.L. Clark and P.T. Hammond, 1998).
Nonetheless, the larger question for this system is whether the polymer
“posts” we build will be sufficiently high enough to prevent sagging of the
membrane once it is deposited. The aspect ratio of the holes we currently make
are at best 50 m wide by 0.5 m high. This means that the posts are at least a
hundred times farther apart then they are high. An analogy would be a tent that
241
was 6 feet long supported by posts that were less than an inch off the ground.
Depending on how heavy the tent was, the center will probably just sag to the
ground. This would not be the desired structure, and higher posts (or smaller
separation distances) are needed. Smaller separation distances are also not
especially useful, since distances that are too small would make viewing under
optical microscopy difficult. This may necessitate the use of the physicallyetched samples over the chemically-patterned due to the above constraints.
3.8
Conclusions
The goal of constructing a tethered membrane was approached from two
directions. The first combined microcontact printing of a surface with polyion
layering to create patterned “posts” on the surface. The second method used
conventional photolithography techniques to create a physically-etched hole in
the surface. Currently, we have been able to deposit a membrane over the
physically-etched surface, spanning a hole approximately 300 m wide.
The chemically patterned surfaces were stamped using silane chemistry.
The surfaces were microcontact printed using OTS, then the unpatterned regions
were filled with a vinyl-terminated silane. The vinyl silane was then oxidized to
the carboxylic acid, and this charged molecule was used to initiate polyion
deposition. We were able to create a surface with posts 300 – 400 nm high
(although not consistently), but the quality of the layering depended on the quality
of the initial stamping. Also, the polyions were found to deposit onto the OTS
242
regions on the sample, and this significantly lessened the height difference
between the two regions.
The physically etched samples were made using either mica or silicon.
The holes in the silicon went completely through the substrate, while the holes in
mica were approximately 10 m deep. Monolayers were first deposited by the
RLS technique, then the second layer was deposited by the regular LS technique.
The FM images of the monolayer showed that the domain shape from the airwater interface was maintained over the free region, while the domain shape over
the free region in the bilayer became smaller and rounded. We think this may be
due to a drop in the surface pressure of the bilayer over the hole. This occurred
because of leakage of the entrapped water out from the substrate-glue interface,
during the interval between the two depositions. This caused the bilayer to sag
down into the hole, and may also be the cause of the domain shape change.
243