NASA Technical Reports Server (NTRS) 20120012441: Method of fabricating lipid bilayer membranes on solid supports

US00821 1712B2

(i2) United States Patent

Cho et al.

(io) Patent No.: US 8,211,712 B2

(45) Date of Patent: Jul. 3, 2012

(54) METHOD OF FABRICATING LIPID BILAYER
MEMBRANES ON SOLID SUPPORTS

(75) Inventors: Nam-Joon Cho, Stanford, CA (US);

Curtis W. Frank, Cupertino, CA (US);
Jeffrey S. Glenn, Palo Alto, CA (US);
Kwang Ho Cheong, Giheung-Gu (KR)

2006/0068503 Al * 3/2006 Cuppoletti 436/518

2006/0068504 Al * 3/2006 Kogi 436/518

2007/0224637 Al * 9/2007 McAuliffe et al 435/7.1

2007/0224639 Al * 9/2007 Matsushita et al 435/7.1

2008/0033190 Al * 2/2008 Leeetal 554/124

2008/0125367 Al 5/2008 Glenn et al.

2011/0091864 Al* 4/2011 Karlsson et al 435/4

OTHER PUBLICATIONS

(73) Assignee: The Board of Trustees of the Leland
Stanford Junior University, Palo Alto,
CA (US)

( * ) Notice: Subject to any disclaimer, the term of this

patent is extended or adjusted under 35
U.S.C. 154(b) by 711 days.

(21) Appl.No.: 11/887,669

(22) PCT Filed: Mar. 29, 2006

(86) PCT No.: PCT/US2006/012085

§371 (c)(1),

(2), (4) Date: Apr. 23, 2009

(87) PCT Pub. No.: W02006/110350

“Intact Vesicle Adsorption and Supported Biomembrane Formation
from Vesicles in Solution: Influence of Surface Chemistry, Vesicle
Size, Temperature, and Osmotic Pressure” Reimhult et al., Langmuir,
2003, 19 (5), pp. 1681-1691 *

Reimhult et al., “Intact Vesicle Adsorption and Supported
Biomembrane Formulation from Vesicles in Solution: Influence of
Surface Chemistry, Vesicle Size, Temperature, and Osmotic Pres-
sure”, Langmuir , 2003, vol. 19, No. 5: 1681-1691.

Manoil et al., “Membrane Protein Assembly: Genetic, Evolutionary
and Medical Perspectives”, Annual Review of Genetics , 1995, vol.
29:131-150.

Srinivas, et al., “Membrane Interactions of Synthetic Peptides Cor-
responding to Amphipathic Helical Segments of the Human
Immunodeficiency Vims Type-1 Envelope Gycoprotein”, Journal of
Biological Chemistry , 1992, vol. 267:7121-7127.

Elazar et al., “Amphipathic Helix-Dependent Localization of NS5A
Mediates Hepatitis C Vims RNA Replication”, Journal of Virology,
2003, vol. 77:6055-6061.

PCT Pub. Date: Oct. 19, 2006

* cited by examiner

(65) Prior Publication Data

US 2009/0263670 Al Oct. 22, 2009

Related U.S. Application Data

(60) Provisional application No. 60/666,647, filed on Mar.
29, 2005.

(51) Int.Cl.

G01N33/543 (2006.01)

(52) U.S. Cl 436/518

(58) Field of Classification Search None

See application file for complete search history.
(56) References Cited

U.S. PATENT DOCUMENTS

5,364,851 A 11/1994 Joran

5,502,022 A 3/1996 Schwarz et al.

5,521,702 A * 5/1996 Salamonetal 356/244

5,846,814 A * 12/1998 Gallaetal 435/287.2

6,306,598 B1 10/2001 Charychetal.

6,306,958 B1 10/2001 Dirschletal.

6,344,436 B1 * 2/2002 Smith etal 514/7.4

6,756,078 B2 * 6/2004 Bookbinder et al 427/407.2

2005/0201973 Al * 9/2005 Virtanenetal 424/78.27

2005/0250158 Al* 11/2005 Parikhetal 435/7.1

Primary Examiner — .Ann Lam

(74) Attorney, Agent, or Firm — Paula A. Borden;
Bozicevic, Field & Francis LLP.

(57) ABSTRACT

The present invention provides a method of producing a pla-
nar lipid bilayer on a solid support. With this method, a
solution of lipid vesicles is first deposited on the solid sup-
port. Next, the lipid vesicles are destabilized by adding an
amphipathic peptide solution to the lipid vesicle solution.
This destabilization leads to production of a planar lipid
bilayer on the solid support. The present invention also pro-
vides a supported planar lipid bilayer, where the planar lipid
bilayer is made of naturally occurring lipids and the solid
support is made of unmodified gold or titanium oxide. Pref-
erably, the supported planar lipid bilayer is continuous. The
planar lipid bilayer may be made of any naturally occurring
lipid or mixture of lipids, including, but not limited to phos-
phatidylcholine, phosphatidylethanolamine, phosphati-
dylserine, phosphatidylinsitol, cardiolipin, cholesterol, and
sphingomyelin.

4 Claims, 4 Drawing Sheets

U.S. Patent

Jul. 3, 2012

Sheet 1 of 4

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Sheet 2 of 4

US 8,211,712 B2

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U.S. Patent

Jul. 3, 2012

Sheet 3 of 4

US 8,211,712 B2

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A. Bare f sO. surface B tuiact Vesicles C Cueuplete Bllayet

U.S. Patent

Jul.3,2012 Sheet 4 of 4

US 8,211,712 B2

US 8,21 1,712 B2

1

METHOD OF FABRICATING LIPID BILAYER
MEMBRANES ON SOLID SUPPORTS

CROSS REFERENCE TO RELATED
APPLICATIONS

This application is a national stage filing under 35 U.S.C.
§371 of International Patent Application Ser. No. PCT/
US2006/0 12085, which was filed on Mar. 29, 2006 and which
was published in English under PCT Article 21(2) as WO
2006/110350 on Oct. 19, 2006, which International Patent
Application claims benefit of priority of U.S. Provisional
Patent Application Ser. No. 60/666,647, filed Mar. 29, 2005,
which applications are incorporated herein by reference in
their entirety.

FEDERALLY-SPONSORED RESEARCH OR
DEVELOPMENT

This invention was made with Government support under
contracts NAG-8-1843 awarded by the NASA Marshall
Space Flight Center and 0213618 awarded by the National
Science Foundation. The Government has certain rights in
this invention.

FIELD OF THE INVENTION

The present invention relates generally to lipid mem-
branes. More particularly, the present invention relates to
methods of fabricating lipid bilayer membranes on solid sup-
ports.

BACKGROUND

Supported lipid bilayers formed by the fusion of small
unilamellar vesicles onto silicon oxide or organic film-modi-
fied surfaces enable the biofunctionalization of inorganic sol-
ids, such as semiconductors, gold-covered surfaces, and opto-
electronic and lab-on-a-chip devices. They have proven
valuable in the study of the characteristics and behavior of
membrane-bound proteins, membrane-mediated cellular
processes, protein-lipid interactions, and biological signal
transduction. Because of the complexity of biomembranes,
there is a clear need to develop model membrane systems,
where one or a few membrane components can be isolated
and studied. In addition, a wide range of available surface-
sensitive techniques can be used to study natural biological
systems effectively by supporting model membranes on a
solid surface. Applications of supported membranes on solid
surfaces potentially include biosensors, programmed drug
delivery, the acceleration and improvement of medical
implant acceptance, and the production of catalytic inter-
faces.

In order to mimic natural biological systems, researchers
have employed vesicle fusion methods to form supported
bilayers on substrates such as glass, mica, self-assembled
monolayers, and quartz. However, it has proven problematic
to create planar lipid bilayers on preferred solid substrates,
such as gold and Ti0 2 . For example, scientists have attempted
to modify gold surfaces using self-assembled monolayers
(SAMs), which may require special synthesis, but the struc-
ture of the SAMs that are formed may not be well-defined.
Accordingly, there is a need in the art to develop new methods
of forming supported bilayers on preferred substrates.

SUMMARY OF THE INVENTION

The present invention provides a method of producing a
planar lipid bilayer on a solid support. With this method, a

2

solution of lipid vesicles is first deposited on the solid sup-
port. Next, the lipid vesicles are destabilized by adding an
amphipathic peptide solution to the lipid vesicle solution.
This destabilization leads to production of a planar lipid
5 bilayer on the solid support. Preferably, the amphipathic pep-
tide is an alpha-helical peptide. More preferably, the alpha-
helical peptide is a polypeptide having the entirety or a por-
tion of the sequence SEQ ID NO: 1 .

The present invention also provides a supported planar
10 lipid bilayer, where the planar lipid bilayer is made of natu-
rally occurring lipids and the solid support is made of
unmodified gold or titanium oxide. Preferably, the supported
planar lipid bilayer is continuous. The planar lipid bilayer
may be made of any naturally occurring lipid or mixture of
15 lipids, including but not limited to phosphatidylcholine,
phosphatidylethanolamine, phosphatidyl serine, phosphati-
dylinositol, cardiolipin, cholesterol, and sphingomyelin.

BRIEF DESCRIPTION OF THE FIGURES

20

The present invention together with its objectives and
advantages will be understood by reading the following
description in conjunction with the drawings, in which:

FIG. 1 shows a method of producing a planar lipid bilayer
25 according to the present invention.

FIG. 2 shows quartz crystal microbalance-dissipation
(QCM-D) analysis of planar lipid bilayer formation on a gold
substrate according to the present invention.

FIG. 3 shows QCM-D analysis of planar lipid bilayer for-
30 mation on a Ti0 2 substrate according to the present invention.

FIG. 4 shows atomic force microscopy (AFM) analysis of
planar lipid bilayer formation on a Ti0 2 substrate according
to the present invention.

35 DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates a schematic of a method according to the
present invention. FIG. 1A shows lipid vesicles 110 that have
been deposited onto solid substrate 120. The vesicles have
40 adsorbed to solid substrate 120. A large amount of water,
indicated by circles 130, is trapped within the intact vesicles
as well as between vesicles adsorbed on the surface of solid
substrate 120. After addition of an amphipathic peptide (AP)
solution (FIG. IB), the vesicles are destabilized and ruptured,
45 allowing the ruptured vesicles to fuse and form planar bilayer
112. When planar bilayer 112 forms, water 130 trapped
within vesicles 112 is dispersed to form water layer 132.

The amphipathic peptide is preferably an alpha-helical
peptide. More preferably, the amphipathic peptide is the AH
50 peptide of the HCV nonstructural protein NS 5 A. This peptide
is conserved across HCV isolates and has the sequence SEQ
ID NO: 1. Either the entire peptide, amino acids 1-16 of the
peptide (AH_S1), or amino acids 17-31 of the peptide
(AH_S2) may be used to destabilize the lipid vesicles. Alter-
55 natively, the peptide may not have the exact sequence of SEQ
ID NO: 1, as long as its amphipathic alpha-helical nature is
preserved. For example, as shown by circular dichroism, the
peptide may have a sequence that is at least about 80% iden-
tical to SEQ ID NO: 1, while still maintaining alpha -helicity.
60 Preferably, the concentration of amphipathic peptide in the
peptide solution is between about 0.05 jag/ml to 0.5 jug/ml.
The amphipathic peptide may be contained in a variety of
solvents, including biological buffers (such as Tris buffer,
PBS buffer, and HEPES Buffer) and dimethyl sulfoxide
65 (DMSO).

Lipid vesicles suitable for the present invention are prefer-
ably between about 25 nm and about 80 nm in diameter. The

US 8,21 1,712 B2

3

vesicles may be prepared using any method known in the art,
including but not limited to extrusion methods. The vesicles
are preferably at a concentration of about 0.05 mg/ml to about
5 mg/ml in a biological buffer, such as Tris, PBS, and HEPES
buffer, with NaCl concentration of about 1 00 mM to about 5
250 mM. Any lipid or mixture of lipids may be used to form
the lipid vesicles, including but not limited to phospholipids.
Preferred lipids are phosphatidylcholine, phosphatidyletha-
nolamine, phosphatidylserine, phosphatidylinositol, cardio-
lipin, cholesterol, and sphingomyelin.

Any solid support may be used according to the present
invention. Example materials include, but are not limited to
silicon-containing materials, gold, platinum, and titanium
oxide. 15

The present invention also provides supported planar lipid
bilayers produced using the method of the present invention.
Preferably, the lipid bilayer is composed of naturally occur-
ring lipids and the solid support is made of unmodified gold or
titanium oxide. Any naturally occurring lipid may be used for 20
the bilayer, such as phospholipids. Preferred lipids are phos-
phatidylcholine, phosphatidylethanolamine, phosphati-
dylserine, phosphatidylinositol, cardiolipin, cholesterol, and
sphingomyelin. Preferably, planar lipid bilayers according to
the present invention are continuous, i.e. there are no gaps in 2 5
the layer.

EXAMPLES

Formation of a Planar Lipid Bilayer on a Sold Substrate 3Q
Bilayer formation from intact vesicles was characterized
using a quartz crystal microbalance-dissipation (QCM-D)
instrument. To interpret the QCM-D results, a linear relation-
ship between Af and adsorbed mass (Am) derived from the
classical Sauerbrey equation was employed: „

C Equation (1)

Am = A /

where C is the mass -sensitivity constant with value 17.7
ngcm _2 Hz _1 for the QCM-D crystal at 5 MHz, and n is the
overtone number (n=l for the fundamental and 3,5,7 for the
overtones). The QCM-D has been used in numerous studies
of the vesicle fusion process, where the dissipation is used to 45
distinguish between rigid lipid bilayers and monolayers and
soft deformable vesicles (see, e.g., Keller and Kasemo, “Sur-
face specific kinetics of lipid vesicle adsorption measured
with a quartz crystal microbalance”, Biophys J. 1998 Sep;
75(3): 1397-1402). 50

In order to investigate the ability of AH peptides to rupture
vesicles, we tested unilamellar vesicles of l-palmitoyl-2-ole-
oyl-sn-glycero-3-phosphocholine (POPC) extruded through
30 nm polycarbonate etch-tracked (PEC) membranes on a
gold surface in the absence of the AH peptide, then applied the 55
peptide to form a bilayer. When vesicles adsorb, a large
amount of trapped water exists within the intact vesicles as
well as between vesicles adsorbed on the surface. This
trapped water is able to dissipate a large amount of energy,
unlike the water that rests on top of a bilayer. This change in 60
energy dissipation can in turn be used to track the transition
between an intact vesicle and a bilayer.

In FIG. 2, Af(t) (triangles) and AD(t) (circles) show change
in frequency and change in dissipation. FIG. 2A shows
vesicle adsorption on an oxidized gold surface. After 10 min 65
(arrow 1) of stabilizing the frequency signal, the POPC
vesicle solution (0.1 mg/ml, 0 3Onm PE c = 59 nm ±0.2 nm) was

4

injected into a liquid cell. After 50 and 55 min (arrows 2 and

3) , the same buffer that was used to dilute the vesicles (10 mM
Tris (pH 7.5), 150 mM NaCl solution with 1 mM EDTA in
1 8.2 MQ-cm MilliQ water (MilliPore, Oreg., USA) was used
to wash the substrate twice and the stability of the intact
vesicles on the gold surface was observed. As shown in FIG.
2B, at 60 min (arrow 4), an amphipathic_-helix peptide (AH
peptide) solution was added (0.05 jag/ml ) to the intact vesicles
(030 nm pec = $9 nm ±0.2 nm) on the gold surface. The peptide
destabilized and ruptured the vesicles, making a complete
bilayer. This is reflected in a decrease of frequency of 25 .5 Hz
±0.5, with a maximum decrease of dissipation of as much as
0.08x1 0 -6 observed. After 120 and 140 min (arrows 5 and 6),
the vesicle buffer was used to wash the sub strate twice and the
stability of the bilayers on the gold surface was observed.
According to the Sauerbrey equation, from which the bilayer
thickness can be calculated, these QCM-D parameters indi-
cate the transition of the vesicles to a thin and rigid bilayer
film.

In FIG. 2C, the effect of a non-amphipathic non-helical
peptide (NH peptide) was examined. Unlike the AH peptide,
the NH peptide has three charged amino acids spaced at
intervals along the predicted N-terminal helix such that no
sustained hydrophobic patch remains. The NH peptide has an
Asp rather than a Val at residue 8, a Glu instead of an He at
residue 12, and an Asp instead of a Phe at residue 19 of SEQ
ID NO: 1 . In FIG. 2C, after 1 0 nm (arrow 1) of stabilizing the
frequency signal, the POPC vesicle solution (0.1 mg/ml,
0 3O nm pec = $9 mn ±0.2 nm) was applied to the liquid cell.
After 60 and 70 min (arrows 2 and 3), the vesicle buffer was
used to wash the substrate twice and the stability of the intact
vesicles on the gold surface was observed. At 85 min (arrow

4) , the NH peptide solution was added (0.05 |ig/ml) to the
intact vesicles on the gold surface. The NH peptide does not
show any evidence of having destabilized and ruptured the
vesicles. After 1 60 min (arrow 5), the vesicle buffer was used
to wash the substrate twice and the stability of the intact
vesicles on the gold surface was observed.

Formation of a Planar Lipid Bilayer on a Ti0 2 Substrate

In order to investigate the ability of AH peptides to rupture
vesicles on a Ti0 2 surface, we tested unilamellar vesicles of
POPC extruded through 30 nm PEC membranes on a Ti0 2
surface in the absence of the AH peptide, then applied the
peptide to form a bilayer (FIG. 3). In FIG. 3, Af(t) (triangles)
and AD(t) (circles) show change in frequency and change in
dissipation. FIG. 3A shows vesicle adsorption on a Ti0 2
surface. After 10 min (arrow 1) of stabilizing the frequency
signal, the POPC vesicle solution (0. 1 mg/ml, 0 3Qmin pec 59
nm ±0.2 nm) was injected into the liquid cell. After 60 and 65
min (arrows 2 and 3), the same buffer that was used to dilute
the vesicles (10 mM Tris (pH 7.5); 150 mM NaCl solution
with 1 mM EDTA in 18.2 MQ-cm MilliQ water (MilliPore,
Oreg., USA) was used to wash the substrate twice and the
stability of the intact vesicles on the Ti0 2 surface was
observed. As shown in FIG. 3B, at 70 min (arrow 4), the AH
peptide solution was added (0.05 jag/ml) to the intact vesicles
(030 nm pec = $9 nm ±0.2 nm) on the Ti0 2 surface (FIG. 2B).
The peptide destabilized and ruptured the vesicles, making a
complete bilayer. After 270 min (arrow 5), the vesicle buffer
was used to wash the substrate twice and the stability of the
bilayers on the Ti0 2 surface was observed.

In FIG. 3C, the effect of the NH peptide was examined. In
FIG. 3C, after 10 min (arrow 1) of stabilizing the frequency
signal, the POPC vesicle solution (0.1 mg/ml, 0 3Onm pec 59
nm ±0.2 nm) was applied to the liquid cell. After 40 and 50
min (arrows 2 and 3), the vesicle buffer was used to wash the
substrate twice and the stability of the intact vesicles on the

US 8,21 1,712 B2

5

Ti0 2 surface was observed. At 60 min (arrow 4), the NH
peptide solution was added (0.05 jag/ml) to the intact vesicles
on the Ti0 2 surface. The NH peptide does not show any
evidence of having destabilized and ruptured the vesicles.
After 270 min (arrow 5), the vesicle buffer was used to wash 5
the substrate twice and the stability of the intact vesicles on
the Ti0 2 surface was observed.

6

the AH peptides ruptured vesicles to form bilayers (P
^iO.OOl). These results correlate with QCM-D kinetic data.

As one of ordinary skill in the art will appreciate, various
changes, substitutions, and alterations could be made or oth-
erwise implemented without departing from the principles of
the present invention. Accordingly, the scope of the invention
should be determined by the following claims and their legal
equivalents.

SEQUENCE LISTING

<16 0> NUMBER OF SEQ ID NOS: 1

<210> SEQ ID NO 1
<2 1 1 > LENGTH: 31
<2 12 > TYPE: PRT

<213> ORGANISM: Hepatitis C virus
<4 0 0 > SEQUENCE: 1

Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp lie Cys Thr Val Leu
15 10 15

Thr Asp Phe Lys Thr Trp Leu Gin Ser Lys Leu Asp Tyr Lys Asp
20 25 30

AFM Analysis of Lipid Bilayer Formation According to
the Present Invention

AFM was utilized in order to confirm and directly display
rupture of vesicles and bilayer formation by the destabilizing
agent the AH peptide. FIG. 4 shows the results of this analy-
sis. For each column, the top image shows a top view in 2-D,
the middle image shows a top view in 3-D, and the graph
shows measurements taken along the black line shown in the
top image. The images are presented in Height mode. Scans
were taken in the direction indicated by the white arrow on
FIG. 4A, top image.

In FIG. 4A, AFM was conducted on a bare Ti0 2 surface in
Tris buffer (150 mM NaCl, 10 mM Tris [pH 7.5], 1 mM
EDTA) as a control. The bare Ti0 2 surface showed an average
root mean square roughness (Rq) of 1.63 ±0.12 nm (±S.E.
n=15). 59 nm ±0.2 nm diameter POPC vesicles (0.1 mg/ml)
were carefully added through the injection system, incubated
for 30 minutes, and thoroughly rinsed three times with Tris
buffer. Intact vesicles, such as vesicle 410, were clearly iden-
tified by AFM and the average Rq increased to 2.70 ±0.15 nm
(±S.E. n=15), as shown in FIG. 4B. A grain analysis was
applied in order to identify and count the vesicles. In order to
minimize the effects of particles other than vesicles, only
diameters between 50 to 100 nm hyperbolar- shaped objects
were counted. The AFM images, as shown in FIG. 4B, and
grain analysis were used to identify the sizes of thirteen
vesicles. The vesicles had an average diameter of 74.57 ±4.07
nm (±S.E. n=13) and average volume of 3.32x10-5 pm 3
±6.16x10-6 pm 3 (±S.E. n=13). Cross-sectional analysis dis-
plays the height of the vesicles to be approximately 1 5 nm.

The AFM images in FIG. 4C show the effect of the AH
peptide on the vesicles as a destabilizing agent, which was
examined by injecting the peptide (0.05 pg/ml) and incubat-
ing the solution for 2 hours prior to scanning the images.
These images clearly confirm the QCM-D data, indicating
that vesicles were ruptured as a result of the treatment by AH
peptide at 0.05 pg/ml concentration after 120 minutes. The
average Rq of 1.67 ±0.12 nm (±S.E. n=15) indicated the
roughness became similar to the bare Ti0 2 surface. Grain
analysis identified no vesicle-like structures, indicating that

What is claimed is:

1. A supported planar lipid bilayer comprising:

a) a planar lipid bilayer comprising naturally occurring

30 lipids; and

b) a solid support comprising unmodified gold or titanium
oxide;

wherein said lipid bilayer is supported by said solid sup-
port.

35 2. The supported planar lipid bilayer as set forth in claim 1,

wherein said lipids are phospholipids.

3 . The supported planar lipid bilayer as set forth in claim 1 ,
wherein said lipids are selected from the group consisting of
phosphatidylcholine, phosphatidylethanolamine, phosphati-

40 dylserine, phosphatidylinositol, cardiolipin, cholesterol, and
sphingomyelin.

4. The supported planar lipid bilayer as set forth in claim 1,
wherein said planar lipid bilayer is continuous.

4 < * * * * *