U.S. patent number RE42,315 [Application Number 11/825,298] was granted by the patent office on 2011-05-03 for nanostructured separation and analysis devices for biological membranes.
This patent grant is currently assigned to STC.UNM. Invention is credited to Steven R. J. Brueck, Linnea K. Ista, Gabriel P. Lopez.
United States Patent |
RE42,315 |
Lopez , et al. |
May 3, 2011 |
Nanostructured separation and analysis devices for biological
membranes
Abstract
The present invention provides a nanostructured device
comprising a substrate including nanotroughs therein; and a lipid
bilayer suspended on or supported in the substrate. A separation
method is also provided comprising the steps of supporting or
suspending a lipid bilayer on a substrate; wherein the substrate
comprises nanostructures and wherein the lipid bilayer comprises at
least one membrane associated biomolecule; and applying a driving
force to the lipid bilayer to separate the membrane associated
biomolecule from the lipid bilayer and to drive the membrane
associated biomolecule into the nanostructures.
Inventors: |
Lopez; Gabriel P. (Albuquerque,
NM), Brueck; Steven R. J. (Albuquerque, NM), Ista; Linnea
K. (Albuquerque, NM) |
Assignee: |
STC.UNM (Albuquerque,
NM)
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Family
ID: |
27372379 |
Appl.
No.: |
11/825,298 |
Filed: |
July 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10073935 |
Feb 3, 2004 |
6685841 |
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60347002 |
Jan 11, 2002 |
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60268365 |
Feb 14, 2001 |
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Reissue of: |
10338654 |
Jan 9, 2003 |
06913697 |
Jul 5, 2005 |
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Current U.S.
Class: |
210/644;
435/287.1; 210/650; 210/638; 422/527; 210/651; 210/511; 977/714;
977/704; 977/702; 210/649; 436/178; 210/645; 210/500.21; 530/414;
530/412; 977/920; 977/705; 435/287.3; 435/287.2; 977/924; 210/656;
210/198.2; 204/450; 435/7.1; 422/502; 436/177; 977/715; 977/713;
435/6.1; 422/503 |
Current CPC
Class: |
G01N
27/44773 (20130101); B82Y 30/00 (20130101); Y10T
436/25375 (20150115); G01N 30/88 (20130101); Y10S
977/717 (20130101); G01N 30/00 (20130101); G01N
2030/8813 (20130101); Y10S 977/845 (20130101); Y10T
436/255 (20150115) |
Current International
Class: |
B01D
15/34 (20060101) |
Field of
Search: |
;210/500.21,263,321.84,511,634,638,644,649-651,198.2,656,660
;422/502,503,527,534,70,100,101 ;436/161,177,178
;977/702-705,713-717,914,920,924,DIG.1 ;530/412-414
;435/4,6,7.1,7.2,287.1,287.2,287.3 ;204/450,451 |
References Cited
[Referenced By]
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WO 2006/078470 |
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|
Primary Examiner: Drodge; Joseph W
Attorney, Agent or Firm: Sudol; R. Neil Coleman; Henry D.
Sapone; William J.
Government Interests
GOVERNMENT INTEREST STATEMENT
This invention is made with government support under grant number
DAAD19-99-1-0196 awarded by the United States Army Research Office.
The government has certain rights in this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is an application for reissue of U.S. Pat. No. 6,913,697. More
than one reissue application has been filed for the reissue of U.S.
Pat. No. 6,913,697. The reissue applications include the present
application and co-pending U.S. patent application Ser. Nos.
12/215,893, 12/217,113, and 12/217,114 all of which are Divisional
Reissue Applications, filed as divisionals of the present
application, filed as a Reissue of U.S. Pat. No. 6,913,697 issued
on Jul. 5, 2005, which was filed as application Ser. No. 10/338,654
entitled "Nanostructured Separation and Analysis Devices for
Biological Membranes," on Jan. 9, 2003, as a Continuation-in-Part
of U.S. patent application Ser. No. 10/073,935, entitled
"Nanostructured Devices for Separation and Analysis," filed on Feb.
14, 2002, now U.S. Pat. No. 6,685,841 issued on Feb. 3, 2004, which
claims priority to U.S. Provisional patent application Ser. No.
60/268,365, entitled "Nanostructured Devices for Separation and
Analysis," filed on Feb. 14, 2001. This application also claims
priority to U.S. Provisional patent application Ser. No.
60/347,002, entitled "Nanostructured Devices," filed on Jan. 11,
2002. The entire contents and disclosures of the above applications
are hereby incorporated by reference.
Claims
What is claimed is:
1. A nanostructured device comprising: a substrate including an
upper face or surface and at least one elongate nanotrough therein
extending downwardly into said substrate from said upper face or
surface, said at least one elongate nanotrough having continuous or
uninterrupted opposing walls along at least a substantial length
thereof; and a separation and analysis platform in the form of a
singular continuous lipid bilayer in part disposed on said upper
face or surface and in part suspended over or supported in said at
least one elongate nanotrough, said lipid bilayer having sufficient
fluidity to allow for mobility of biomolecules embedded within said
lipid bilayer.
2. The nanostructured device of claim 1, wherein said lipid bilayer
comprises a simple bilayer.
3. The nanostructured device of claim 1, wherein said lipid bilayer
comprises a hybrid bilayer.
4. The nanostructured device of claim 3, wherein said hybrid
bilayer comprises a self-assembled monolayer hybrid bilayer.
5. The nanostructured device of claim 1, wherein said
nanostructured device further comprises an array of nanostructures
arranged so that said arrayat least one nanotrough is filled with
at least one fluid.
6. The nanostructured device of claim 1, wherein said
nanostructured device further comprises an array of nanostructures
arranged so that said arrayat least one elongate nanotrough has a
gradient property.
7. The nanostructured device of claim 1, wherein said
nanostructured device further comprises at least one nanostructured
channel.
8. The nanostructured device of claim 1, wherein said substrate
comprises Si.
9. The nanostructured device of claim 1, wherein said substrate
comprises a semiconductor chip.
10. The nanostructured device of claim 1, wherein said
nanostructured device comprises a biochip.
11. A nanostructured device comprising: a substrate including a
nanostructured matrix with an upper face or surface and at least
one elongate nanotrough thereinextending downwardly into said
substrate from said upper face or surface; and a separation and
analysis platform in the form of at least one singular continuous
lipid bilayer in part disposed on said upper face or surface and in
part supported in or suspended over at least one ofsaid at least
one elongate nanotroughs so as to allow biomolecules to pass from
said at least one lipid bilayer into said at least one respective
nanotrough, said lipid bilayer having sufficient fluidity to allow
for mobility of biomolecules embedded within said device so that
said biomolecules are moveable to effectuate a separation of said
biomolecules within said lipid bilayer.
12. The nanostructured device of claim 11, wherein said lipid
bilayer comprises a simple bilayer.
13. The nanostructured device of claim 11, wherein said lipid
bilayer comprises a hybrid bilayer.
14. The nanostructured device of claim 13, wherein said hybrid
bilayer comprises a self-assembled monolayer hybrid bilayer.
15. The nanostructured device of claim 11, wherein said
nanostructured device further comprises an array of nanostructures
arranged so that said arraymatrix has a gradient property.
16. The nanostructured device of claim 11, wherein said
nanostructured device further comprises at least one nanostructured
channel.
17. The nanostructured device of claim 11, wherein said substrate
comprises Si.
18. The nanostructured device of claim 11, wherein said substrate
comprises a semiconductor chip.
19. The nanostructured device of claim 11, wherein said
nanostructured device comprises a biochip.
20. A separation method comprising the steps of: (a) supporting or
suspending a separation and analysis platform in the form of a
lipid bilayer on a substrate; wherein said substrate comprises at
least one nanostructureelongate nanotrough, said nanotrough having
continuous or uninterrupted opposing walls along at least a
substantial length thereof, and wherein said lipid bilayer
comprises at least onehas sufficient fluidity to allow for mobility
of membrane associated biomolecules embedded within said separation
and analysis platform; and (b) applying a driving force to said
lipid bilayer to separate said at least onemembrane associated
biomolecules from said lipid bilayereach other within said
separation and analysis platform and to drive said at least
onemembrane associated biomolecules within and parallel to or in a
plane of said lipid bilayer intoseparation and analysis platform by
virtue of the fluidity of said lipid bilayer, and guided by said at
least one nanostructurenanotrough.
.[.21. The method of claim 20, wherein said at least one
nanostructure comprises at least one nanotrough..].
22. The method of claim 20, wherein said at least one nanotrough is
filled with at least one fluid.
23. The method of claim 20, wherein said at least one
nanostructurenanotrough comprises at least one channel.
24. The method of claim 20, wherein said at least one
nanostructurenanotrough further comprises at least onetwo elongate
protrusions.
25. The method of claim 20, wherein said substrate comprises
Si.
26. The method of claim 20, wherein said lipid bilayer comprises a
simple bilayer.
27. The method of claim 20, wherein said lipid bilayer comprises a
hybrid bilayer.
28. The method of claim 27, wherein said hybrid bilayer 25
comprises a self-assembled monolayer hybrid bilayer.
29. The method of claim 20, wherein said at least
onenanostructurenanotrough comprises an array of nanostructures
arranged so that said arrayhas a gradient property.
30. The method of claim 20, wherein at least one of said at least
onemembrane associated biomolecules comprises a transmembrane
protein.
31. The method of claim 20, wherein said at least one membrane
associated biomoleculesaid lipid bilayer principally comprises a
one or more membrane phospholipids.
32. The method of claim 20, wherein at least one of said at least
onemembrane associated biomolecules comprises a lipophilic
biomolecule.
33. The method of claim 20, wherein said driving force comprises an
electrophoresis.
34. The method of claim 20, wherein said driving force comprises an
externally applied pressure.
35. The method of claim 20, wherein said driving force comprises
capillarity.
36. The method of claim 20, wherein said driving force comprises
diffusion.
37. The method of claim 20, wherein said driving force comprises
osmosis.
38. A separation method comprising: providing a nanostructured
device comprising (i) a substrate including a nanostructured matrix
with at least one elongate nanotrough therein, (ii) a separation
and analysis platform in the form of a lipid bilayer suspended over
or supported in said nanostructured matrix and said at least one
elongate nanotrough, said lipid bilayer being derived from cell
organelles, and (iii) a plurality of membrane-associated
biomolecules occurring within said device and extending in said at
least one elongate nanotrough, said lipid bilayer having sufficient
fluidity to allow for mobility of biomolecules embedded within said
device; and applying a driving force to said lipid bilayer to drive
said biomolecules within said device, by virtue of the fluidity of
said lipid bilayer, and in said at least one elongate nanotrough,
to separate said biomolecules from each other in said device under
a screening action of said nanostructured matrix.
39. The method of claim 38, wherein said lipid bilayer comprises a
simple bilayer.
40. The method of claim 38, wherein said lipid bilayer comprises a
hybrid bilayer.
41. The method of claim 40, wherein said hybrid bilayer comprises a
self-assembled monolayer hybrid bilayer.
42. The method of claim 38, wherein said at least one elongate
nanotrough is filled with at least one fluid.
43. The method of claim 38, wherein said at least one elongate
nantrough has a gradient property.
44. The method of claim 38, wherein said nanostructured device
further comprises at least one nanostructured channel.
45. The method of claim 38, wherein said substrate comprises
Si.
46. The method of claim 38, wherein said substrate comprises a
semiconductor chip.
47. The method of claim 38, wherein said nanostructured device
comprises a biochip.
48. The nanostructured device of claim 6, wherein said at least one
elongate nanotrough has a varying width.
49. The method of claim 48 wherein said at least one elongate
nanotrough has a continuously decreasing width.
50. The nanostructured device of claim 15, wherein said at least
oen elongate nanotrough has a varying width.
51. The nanostructured device of claim 50, wherein said at least
one elongate nanotrough has a continuously decreasing width.
52. The method of claim 29, wherein said at leat one elongate
nanotrough has a varying width.
53. The method of claim 52, wherein said at least one elongate
nanotrough has a continuously decreasing width.
54. The method of claim 43, wherein said at least one elongate
nanotrough has a varying width.
55. The method of claim 54, wherein said at least one elongate
nanotrough has a continuously decreasing width.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the fabrication of nanostructured
matrices for use in supporting lipid bilayers for the separation
and analysis of membrane-associated molecules.
2. Description of the Prior Art
The demand for precise separation of molecules using small sample
volumes is increasing. Currently, polyacrylamide gel
electrophoresis (PAGE) remains the standard for protein separation
and identification in biotechnology. However, the set of separation
strategies that rely on this technique are hampered by: (1)
inconvenience of preparation of the variety of gels needed for the
separations, (2) inherent inconsistencies in production conditions;
and therefore, irreproducibility between different batches of gels,
(3) susceptibility of the polymer to degradation under high
electric fields, (4) lack of reusability, (5) difficulty in
incorporation of these techniques into strategies for development
of multi-dimensional (multi-technique) integrated separation
systems, and (6) limited resolution and dynamic range of
biomolecular separations.
Gradient PAGE techniques utilize one-dimensional filtration by
manipulating pore-size though control of crosslinker, monomer, and
solvent concentrations. Such separation matrices are recognized as
having the potential to maintain excellent resolution and dynamic
range. However, their utility is greatly hampered by the need for
cumbersome gel preparation protocols and lack of
reproducibility.
In general, the separation of molecules across matrices or
membranes has been known in the art. Such separations are typically
achieved by employing barriers that allow cut-offs at a precise
molecular weight or by size-exclusion. The art describes structures
where molecular transport and filtration take place perpendicular
to the surface of the separating material. These currently
available systems, however, suffer from a number of drawbacks: (1)
the matrices formed are generally composed of non-uniform
structures, (2) even where a gradation in size of structures is
required, they may be random or at best have to be serially and
sequentially arrayed through a cumbersome process of lithography,
(3) fabrications of separation devices pose problems in terms of
batch-to-batch variations; and consequently, poor reproducibility
of results therefrom, (4) lack of efficiency of separation, (5)
loss of sample volume, and (6) biomolecules may not be amenable to
separation by many of the available systems.
Thus far, the most relevant work has been the development of
"Brownian ratchets" in which assymetric diffusion leads to
separation of molecules based on their size (van Oudenaarden et
al., Science, 285: 1046-1052, 1999, the entire contents and
disclosure of which is hereby incorporated by reference).
Subsequently, Chou et al. (see, Chou et al., Proc. Natl. Acad.
Sci., 96, 13762-13765, 1999, the entire contents and disclosure of
which is hereby incorporated by reference) attempted separation of
DNA molecules using Microsystems formed by conventional
photolithography. Although such prior work demonstrated that
relatively simple 3-dimensional architectures could lead to
effective separation, the developments have not gained ground with
the biotechnological community. The primary reasons for this lack
of acceptance being the difficulty of preparation of the
nanofluidic systems and the associated high-cost of
fabrication.
Similarly, "artificial gels" incorporating regular arrays of
nanoscale pillars created through electron beam and/or imprint
lithography have been described, for instance, in U.S. Pat. No.
6,110,339 to Yager, et al. and by Turner, et al. (J. Vac. Sci.
Technol. B., 16 3835-3840, 1998, the entire contents and disclosure
of which is hereby incorporated by reference). Such
nanolithographically-defined structures utilize regular arrays of
uniform-sized nanostructures throughout the separation matrix.
Although these nanolithographic structures are useful in
separation, the systems suffer from drawbacks: (1) resolution
limitations, (2) flexibility limitations, and (3) difficulty in
integrating the system with other, more complex, separation
devices. Thus, the need for an efficient, highly-resolving,
flexible, cost-efficient, and reproducible molecular-separation
matrix, is largely unmet. The analysis and characterization of
biomolecules is further limited by the difficulty in separating
membrane-associated molecules. Typically, detergents are used to
remove transmembrane molecules, but even mild detergents may
denature such molecules, rendering them inactive and/or disrupting
necessary functional interactions with other membrane components
including other proteins or lipid components. Additionally, the
study of biomolecules is limited by the difficulty in fabricating a
cellular environment that allows for the interaction of molecules.
Such interactions may be useful in studying molecular transport and
communication across cell membranes.
Thus far, the most relevant work in this area is the use of
synthetic lipid bilayer membranes as separation platforms for
biomolecules. Because of their planar structure, such membranes are
more amenable to laboratory use. The separation technology is
achieved by integrating planar lipid bilayers with varied surfaces
to allow for separation of molecules. For instance, synthetic
membranes supported on a glass or silica surface allow for the
electrophoretic separation of labeled phospholipids and membrane
proteins. See, Groves, J. T. and Boxer, S. G.,
Electric-field-induced concentration gradients in planar supported
bilayers, Biophysical Journal, 69: 1972-1975 (1995), and Groves, J.
T., Wulfing, C., and Boxer, S. G., Electrical manipulation of
glycan phosphatidyl inositol tethered proteins in planar supported
bilayers, Biophysical Journal, 71: 2716-2723 (1996), the entire
contents and disclosures of which are hereby incorporated by
reference. Additionally, lipid bilayer membranes have been
incorporated into microstructured devices by
lithographically-derived features to partition the supported
membrane into separate regions to pattern the distribution of the
lipid bilayer over the surface or as a coating for microchannels.
See, Cremer, P. S., and Yang, T., Creating spatially addressed
arrays of planar supported fluid phospholipid membranes,
Proceedings of the National Academy of Sciences, U.S.A., 121:
8130-8131; Nissen, J., Jacobs, K., and Radler, J. O., Interface
dynamics of lipid membrane spreading on solid surfaces, Physical
Review Letters, 86: 1904-1907 (2001); and Yang, T. L., Jung, S. Y.,
Mao, H. B., and Cremer, P. S., Fabrication of phospholipid
bilayer-coated microchannels for on-chip immunoassays, Analytical
Chemistry, 73: 165-169 (2001), the entire contents and disclosures
of which are hereby incorporated by reference. Furthermore, lipid
bilayers have been supported on nanostructured arrays to produce
Brownian ratchets utilized in the electrophoresis of fluorescent
phospholipids. See, van Oudenaarden, A., and Boxer, S. G., Brownian
ratchets: Molecular separations in lipid bilayers supported on
patterned arrays, Science, 285: 1046-1048 (1999), the entire
contents and disclosures of which are hereby incorporated by
reference. Finally, hybrid lipid bilayers, in which one leaflet
(define leaflet) of the supported membrane is formed by an
alkane-thiol monolayer on gold, have shown promise for use in
bioseparations. See, Plant, A., Supported hybrid bilayer membranes
as rugged cell membrane mimics, Langmuir, 15: 5128-5135 (1999), and
Hui, et al., U.S. Pat. No. 5,919,576, the entire contents and
disclosures of which are hereby incorporated by reference. However,
in these techniques, the close proximity or constraint of the lower
leaflet to the supporting surface reduces their usefulness in
analyzing transmembrane proteins or interactions between
cytoplasmic and extracellular components of the membrane.
Also relevant to the technology of the present invention are
previous methods for creating suspended lipid bilayers in which
regions of the lipid bilayers are freely suspended between two
aqueous reservoirs. Such hybrid bilayers are formed so one leaflet
of the suspended region of the bilayer is replaced with a methyl
terminated self-assembled monolayer, allowing for suspension of
free bilayers over gaps as large as 100 .mu.m. See, Ogier, S. D.,
Bushby, R. J., Cheng, Y., Evans, S. D., Evand, S. W., Jenkins, T.
A., Knowles, P. F., and Miles, R. E., Langmuir, 16: 5696-5701
(2000), the entire contents and disclosures of which are hereby
incorporated by reference. Although these types of suspended
bilayers have been used for studying membrane permeability and
transmembrane protein function, the use of such suspended lipid
bilayers in the separation of transmembrane proteins has not been
examined. Thus, the need for technology that utilizes supported and
suspended lipid bilayer membranes that allow for (1) separation of
membrane-spanning complexes, and (2) cellular interaction is
largely unmet.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide an
efficient nanostructured matrix for separation and analysis of
molecules.
It is a further object of the present invention to provide a matrix
that enables gradient or non-uniform transport of molecules across
a plane parallel to the surface of the matrix.
A further object of the present invention is to enable integration
of multi-dimensional multi-technique molecular separation systems
into a single platform.
Yet another object of the present invention is to provide for
customized fabrication of a nanostructured separation matrix
including an array having a gradient property.
It is yet another object of the present invention is to provide a
nanostructured matrix that may cater to different ranges of
molecular separations, in terms of resolution and dynamics.
Another object of the present invention is to enable consistency in
the composition of the nanostructures forming the separation
matrix.
Yet another object of the present invention is to enable separation
and/or identification of a molecular species.
A further object of the present invention is to enable
calibration-free use of the separation/analysis process.
Yet another object of the present invention is to enable multiple
use of a single separation matrix.
A further object of the present invention is to enable parallel
production of separation matrices at relatively low cost.
In all of the above embodiments, it is an object to provide
enhanced reproducibility and resolution in the separation of
molecules.
According to a first broad aspect of the present invention, there
is provided a nanostructured device comprising a substrate
including at least one nanotrough therein; and a lipid bilayer
suspended on the substrate.
According to second broad aspect of the invention, there is
provided a nanostructured device comprising a substrate including
at least one nanotrough therein; and at least one lipid bilayer
supported in at least one of the at least one nanotroughs.
According to a third broad aspect of the invention, there is
provided a separation method comprising the steps of supporting or
suspending a lipid bilayer on a substrate; wherein the substrate
comprises at least one nanostructure and wherein the lipid bilayer
comprises at least one membrane associated biomolecule; and
applying a driving force to the lipid bilayer to separate the at
least one membrane associated biomolecule from the lipid bilayer
and to drive the at least one membrane associated biomolecule into
the at least one nanostructure.
Other objects and features of the present invention will be
apparent from the following detailed description of the preferred
embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described in conjunction with the
accompanying drawings, in which:
FIG. 1 is a micrograph showing a 150-nm period photoresist grating
written with 213 nm light;
FIG. 2 is a micrograph showing 30-nm photoresist lines;
FIG. 3 is a micrograph showing a 108-nm pitch photoresist grating,
written using 213 nm light, and immersion in DI water.
FIG. 4 is a micrograph showing a photoresist line interpolated
between two lines etched 360 nm apart into a nitride film
demonstrating spatial period division to exent the spatial
frequency coverage of optical lithography;
FIGS. 5A and 5B are micrographs showing transfer of interferometric
lithography patterns into deep structures in Si using KOH
anisotropic etching, with FIG. 5A showing the original period of
360 m with about 1 micrometer deep etched grooves and FIG. 5B
showing the 180 nm period, frequency-doubled structure
corresponding to the lithographic result of FIG. 4;
FIG. 6 illustrates in schematic form a nanostructured gradient
(chirped) separation matrix;
FIGS. 7A and 7B show perspective and top schematic views,
respectively, of a nanostructured matrix according to the present
invention;
FIGS. 8A, 8B and 8C show high aspect ratio nanostructures
fabricated by interferometric lithography and pattern transfer with
FIG. 8A showing dense 150 nm photoresist lines, FIG. 8B showing an
isolated 50 nm photoresist line, and FIG. 8C showing 50 nm wide
walls etched in Si;
FIG. 9 shows a lipid bilayer suspended on a nanostructure according
to an embodiment of the present invention;
FIG. 10 shows a suspended bilayer on a nanostructure according to
an embodiment of the present invention;
FIG. 11 shows a suspended lipid/self-assembled monolayer hybrid
bilayer on a nanostructure according to an embodiment of the
present invention; and
FIG. 12 shows a bilayer supported in nanotroughs of a nanostructure
according to an embodiment of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
It is advantageous to define several terms before describing the
invention. It should be appreciated that the following definitions
are used throughout this application.
Definitions
Where the definition of terms departs from the commonly used
meaning of the term, applicant intends to utilize the definitions
provided below, unless specifically indicated.
For the purposes of the present invention, the term "nanostructure"
refers to a protrusion or void having a diameter in at least one
direction of 1 to 500 nm.
For the purposes of the present invention, the term "diameter"
refers to the distance across a nanostructure through the middle
and perpendicular to the axis of the nanostructure, parallel to the
plane of the substrate (upon which the nanostructure is
located).
For the purposes of the present invention, the tern "axis" refers
to a line running along the middle of a nanostructure in the
direction the nanostructure's longest dimension parallel to the
surface of the substrate on which the nanostructure is located.
For the purposes of the present invention, the term "protrusion"
refers to a structure that protrudes from the surface of a
substrate or that protrudes from a portion of a substrate that has
been etched. The protrusions of the present invention may be any
convenient size or shape. The cross-section of a protrusion may be
circular, square, rectangular, oval, elliptical, etc.
For the purposes of the present invention, the term "channel"
refers to a gap between any two protrusions. The channels of the
present invention may be any convenient size or shape.
For the purposes of the present invention, the term "array" refers
to an arrangement of nanostructures.
For the purposes of the present invention, the term "gradient"
refers to an array where channels, protrusions or other features at
one end of the array are larger than those at an opposite end of
the array.
For the purposes of the present invention, the term "continuous
gradient" refers to a gradient where successive rows of channels,
protrusions or other features decrease in size substantially
continuously from one end of the gradient to the other end of the
gradient.
For the purposes of the present invention, the term "noncontinuous
gradient" refers to a gradient that includes regions of the
gradient having successive rows of channels, protrusions or other
features that are substantially the same size.
For the purposes of the present invention, the term "matrix" refers
to a substrate having an array of nanostructures present on or in
at least a portion of the substrate. A matrix of the present
invention preferably has at least one gradient on or in the
substrate formed by the nanostructures. Examples of a matrix of the
present invention include one or more arrays located on a chip,
such as a semiconductor chip, biochip, etc. Methods for making
biochips which may be readily adapted for use in making biochips of
the present invention are described in U.S. Pat. No. 6,174,683, the
entire contents and disclosure of which is hereby incorporated by
reference.
For the purposes of the present invention, the term
"interferometric lithography" (IL) refers to a process of
lithography that involves interference patterns of two (or more)
mutually coherent light waves. The angles between the light
propagation vectors of the waves are sufficiently large to produce
an interference pattern that has a high spatial frequency. The
resulting interference pattern may have nanoscale dimensions.
Examples of interferometric lithography techniques that may be used
in the present invention are described in Chen X L, Brueck S R J,
"Imaging interferometric lithography: approaching the limits of
optics" in Optics Letters, 24, pp. 124-126 (1999), in "Imaging
interferometric lithography: A wavelength division multiplex
approach to extending optical lithography, Chen X L, Brueck S R
J,Journal of Vacuum Science and Technology B, vol. 16, pp.
3392-3397 (1998), in U.S. Pat. No. 5,759,744 to Brueck et al., in
U.S. Pat. No. 6,233,044 to Brueck et al., and U.S. Pat. No.
6,042,998 to Brueck et al., the entire contents and disclosures of
which are hereby incorporated by reference.
For the purposes of the present invention, the term "biomolecules"
refers to biologically derived molecules such as peptides, small
polypeptides, long polypeptides, proteins, antigens, antibodies,
tagged proteins, oligonucleotides, nucleotides, polynucleotides,
aptamers, DNA, RNA, carbohydrates, etc., and complexes thereof.
For the purposes of the present invention, the term "size exclusion
separation process" refers to separating particles, such as
biomolecules, by size based on the ability of smaller particles to
pass through smaller openings or channels than larger
particles.
For the purposes of the present invention, the term "gel
electrophoretic mobility separation process" refers to any
conventional electrophoresis separation technique such as
two-dimensional polyacrylamide gel electrophoresis. Poly-acrylamide
gel electrophoresis (PAGE) is used to separate biomolecules,
usually proteins or DNA fragments, by the ratio of each
biomolecule's mass to charge. Proteins may be separated in either
their native state, or denatured by the addition of a detergent
such as SDS (Sodium Dodecyl Sulfate). Further resolution may be
obtained in some cases by making a gel with a gradient either in
the concentration of the acrylamide or in the degree of
crosslinking within the gel matrix. An array of the present
invention may be used in performing equivalent molecular weight
separations, with either electrical currents or flow as the driving
force.
For the purposes of the present invention, the term "isoelectric
focusing separation process" refers to the separation of charged
biomolecules, such as proteins and peptides, by each biomolecule's
isoelectric point. A pH gradient is generally generated using a
mixture of ampholytes within the separation matrix, usually
polycrylamide. The biomolecules in the mixture then migrate to the
region where the pH is equal to a particular biomolecule's
isoelectric point, at which time the charged biomolecule becomes
electrically neutral. This technique, combined with subsequent
separation by SDS-PAGE, is used in traditional two-dimensional gel
electrophoresis. Similar pH gradients may be generated using an
array of the present invention including a two-dimensional
gradient, using traditional isolectric focusing with soluble
ampholytes or by using chemical patterning techniques, or
immobilization of ampholytes after electrical focusing. Examples of
capillary-based isoelectric focusing separation processes suitable
for use with the present invention are described in Thorman, Tsai,
Michaud, Mosher and Bier, Capillary Isoelectric-Focusing: Effects
of Capillary, Geometry, Voltage Gradient and Addition of Linear
Polymer, J. Chromatography, 398:75-86 (1987), the entire contents
and disclosure of which are hereby incorporated by reference.
For the purposes of the present invention, the term "asymmetric
diffusion separation process" refers to a separation process in
which steric constraints drive diffusion preferentially in one
direction. Examples of asymmetric diffusion separation processes
suitable for use with the present invention are described in Van
Oudenaarden et al., Science, 285: 1046-1052 (1999), the entire
contents and disclosure of which are hereby incorporated by
reference.
For the purposes of the present invention, the term "entropic
trapping separation process" refers to separations using
nanostructured devices of alternating thin and thick regions, with
the thin regions being smaller than the radius of gyration of the
biomolecule being separated. Under an electrical field, the
molecules repeatedly change conformation, costing entropic free
energy, thus limiting mobility. An example of an entropic trapping
separation process suitable for use with the present invention is
described in Han J, Craighead H D, Separation of long DNA molecules
in a microfabricated entropic trap array, Science, 288:1026-1029
(2000), the entire contents and disclosure of which is hereby
incorporated by reference.
For the purposes of the present invention, the term "hydrophobic
interaction chromatography separation process" refers to a
technique whereby molecules are partitioned between a hydrophobic
matrix and a hydrophilic solvent. The degree of hydrophobicity of
the target molecule determines the target molecule's retention
time. The array of the present invention may be modified to
incorporate a gradient of hydrophobicities or to create a milieu in
which the hydrophobicity may be rapidly and reversibly changed,
thus providing a driving force for molecular movement.
For the purposes of the present invention, the term "affinity
chromatography separation process" refers to a chromatography
process that takes advantage of specific chemical interactions
between a target molecule and a chromatographic matrix. One of the
most widely used forms of affinity chromatography employs
immunoaffinity in which an antibody or series of antibodies are
immobilized on a support. Other affinity agents include enzymes
that interact with specific targets or receptors. Another example
of affinity chromatography is a molecular recognition separation
process such as the separation of long DNA molecules in a
microfabricated entropic trap array. An array of the present
invention may be used for both the generation of affinity matrices
and for the subsequent use of affinity matrices.
For the purposes of the present invention, the term "enantiomeric
resolution separation process" refers to a process to separate
organic particles, such as biomolecules by chirality. Enantiomeric
resolution is especially important in carbohydrate separations
where differences between different glycosides are exclusively
enantiomeric. Indeed, common chiral selectors are cyclodextrins
used in capillary electrophoresis. Macrocyclic antibiotics and
crown ethers are commonly used selectors. Selectors may be used
either globally or in zones of an array of the present invention to
confer yet another means of separation.
For the purposes of the present invention, the term "capillary
electrophoresis separation process" refers to a separation process
in which separation takes place in a liquid rather than in a gel
matrix. Capillary electrophoresis allows for separations to be done
on smaller quantities of material and with improved resolution in
comparison to conventional gel electrophoresis processes. The
channels in an array of the present invention may be arranged to
generate a capillary type arrangement in a second direction
following separations based on chemical properties (e.g., IEF,
affinity, hydrophobic interaction chromatography or enantiomeric
separation) or capillaries may be applied as a third dimension.
For the purposes of the present invention, the phrase "comprises
Si" refers to silicon and any silicon complex, compound, etc. that
includes silicon, such as SiO.sub.2, glass, etc.
For the purposes of the present invention, the term "lipid" refers
to conventional lipids, phospholipids, etc.
For the purposes of the present invention, the term "lipid bilayer"
refers to any double layer of oriented amphipathic lipid molecules
in which the hydrocarbon tails face inward to form a continous
nonpolar phase.
For the purposes of the present invention, the term "simple
bilayer" refers to a conventional lipid bilayer in which the
bilayer is formed from micelles of phospholipids with or without
membrane proteins.
For the purposes of the present invention, the term "hybrid
bilayer" refers to a bilayer that is derived from more than one
source, either through mixing of micelles before formation, or post
bilayer fusion. These also refer to bilayers in which one component
is synthetically derived, or in which one leaflet is supported on
the nanotextured surface prior to bilayer formation.
For the purposes of the present invention, the term "self-assembled
monolayer hybrid bilayer" refers to a hybrid bilayer in which the
synthetic portion is composed of a self-assembled monolayer of
silanes or .omega.-substituted alkanethilates on gold.
For the purposes of the present invention, the term "suspended"
refers to bilayers present on a nanostructure and located above
nanotroughs in a nanostructure. An example of a suspended bilayer
is shown in FIGS. 9, 10 and 11.
For the purposes of the present invention, the term "supported"
refers to bilayers located in nanotroughs of a nanostructure. An
example of a supported bilayer is shown in FIG. 12.
For the purposes of the present invention, the term "nanotrough"
refers to a trough with a void dimension of 1-500 nm, whether
uniform or not.
For the purposes of the present invention, the term "leaflet"
refers to one half of a fluid bilayer membrane composed of a single
layer of phospholipids and any included proteins.
For the purposes of the present invention, the term "filled with at
least one fluid" refers to a nanostructure, preferably a nanotrough
or channel, containing a fluid that is at least partially contained
within said nanostructure. The nanostructure does not need to be
completely filled with a fluid according to this definition.
For the purposes of the present invention, the term "membrane
associated biomolecule" refers to any membrane associated
biomolecule, such as transmembrane proteins, membrane
phospholipids, lipophilic biomolecules, complexes thereof, etc.
Description
The present invention provides, in part, for robust, inexpensive
and reproducible methods for forming separation matrices for
gradient separations based on, for example, electrophoresis and
size exclusion that includes all the positive traits of gradient
PAGE. These matrices may be adapted for a host of variant
separation strategies, including electrophoresis, detergent
solubilization, native electrophoresis, isoelectric focusing,
2D-electrophoresis, hydrophobic interaction, and affinity
chromatography. More specifically, the present invention provides
for the use of such separation matrices as support for lipid
bilayers that serve as separation platforms for membrane-associated
biomolecules. The methods of fabrication discussed herein may also
be adapted to existing microfabrication and integration
facilities.
The present invention provides for separation of molecular species
across a nanostructured matrix, a method of fabricating
nanostructures comprising the matrix and the use of such a matrix
for separation and/or analysis of molecules by defining the
physical size and/or chemical features of the nanostructures as a
means of screening. The nanostructured matrix may be used to
separate biological materials, such as proteins, carbohydrates, and
nucleic acids as well as nonbiological materials, such as synthetic
polymers. These nanostructures may be made out of a variety of
materials, including silicon, thus providing systems that may be
easily chemically modified for additional flexibility. The use of
lithography to generate nanostructured separation matrices has
advantages over other techniques (such as traditional acrylamide
gel polymerization) since it (1) creates highly ordered structures,
(2) gives the possibility of creating macroscopic arrays of
continually varying size or chemistry across one dimension, (3) is
highly reproducible, and (4) may be easily implemented in the
creation of complex, integrated separation systems that are
disposable or reusable. Furthermore, the use of lithographically
defined separation matrices lends itself to the facile
implementation of these matrices into multi-level, 3-dimensional
separation devices in which different screening mechanisms allow
enhanced separations. Particularly, the lithographic nanostructured
surfaces may be used to support lipid bilayers or hybrid lipid
bilayers for separating membrane-associated molecules and studying
cellular interactions. The present invention aims to (1) eliminate
some of the current limitations by the fabrication of highly
uniform and reproducible nanostructured separation systems prepared
by nano-and microlithography, and (2) eliminate some of the current
limitations by utilizing the lithographic nanostructured surfaces
in conjunction with lipid bilayers to produce separation platforms
for membrane-associated molecules. Nanolithographically-Defined
Gradients:
Using an advanced lithographic technique such as interferometric
lithography (IL) capable of producing nanostructures, patterns of
nanostructures may be rapidly created over wide, macroscopic areas
at low cost (compared to other techniques such as electron beam
lithography). In addition, it may be used to easily generate arrays
of nanostructures (protrusions or channels) whose dimensions vary
semi-continuously in the plane of surface of the material being
patterned. IL has advantages over other methods that might be used
to construct nanopatterned fluidic structures (e.g., electron beam
lithography, X-ray lithography, or local probe lithography) due to
the low cost of implementation and the parallel nature of the
lithographic technique. Combining IL with conventional lithography
allows for the formation of device structures in individual areas
and the addition of aperiodic features such as electronic and
fluidic connections. Imaging interferometric lithography extends
optics to fundamental, deep-subwavelength scales.
It is worthwhile at this point to consider the fundamental limits
of optical lithography. For the interference of two plane waves in
air, the period is given by .lamda./(2 sin .theta.) where .lamda.
is the optical wavelength and .theta. is the angle of incidence.
For a 213-nm laser source (fifth harmonic of YAG) this gives a
period of .about.150 nm (for .theta.=80.degree.). FIG. 1 shows an
example of a large-area, 150 nm period, photoresist grating. It is
important to realize that this limit is on the period, not on the
feature dimensions. Nonlinearities in the exposure/develop
processes and in subsequent processing may reduce the feature to
dimensions well below .lamda./4. An example in FIG. 2 shows 30-nm
developed resist lines on a 360-nm pitch written at a wavelength of
364 nm. The ultimate limit in linewidth is set by material
properties and by uniformity of the processing; linewidths as small
as 10 nm are routinely achieved. The use of immersion techniques
may further reduce the period by a factor of the refractive index,
approximately a factor of 1.5, to a period of .about.75 nm. Initial
results reproduced the 150 nm pitch of FIG. 1 at a lower angle of
incidence.
Water and higher-index liquids, including liquid Ar (n.about.1.6),
may be used to further extend these results into the sub-100-nm
period regime that will be important for biological separations.
FIG. 3 shows an initial example of immersion interferometric
lithography where the grating period has been reduced to 108 nm
with exposure by 213 nm light using immersion in deionized
water.
Nonlinear processes may be used to further reduce the period. FIG.
4 shows an example of a photoresist line interpolated between two
parallel lines that have already been transferred into a nitride
layer. FIG. 5B shows the result of transferring both of these
patterns into Si using a KOH etch process. The final period is
.about.half of the initial IL period. Extending the calculation
above with this spatial period division gives a period of .about.37
nm and a dense linewidth of .about.17 nm (.lamda./12).
Importantly, all of these results are macroscopic in scale, e.g.,
covering areas of .about.1 cm.sup.2 or larger. A strength of optics
is the parallel nature of the exposure, which may be cm's or larger
in extent. For a square lattice with a 100-nm pitch and a 1 cm
field, there are 10.sup.10 features, well beyond the realistic
capabilities of serial techniques such as e-beam and scanning
probes. In particular embodiments of the present invention, IL may
be extended deep into the nanometer regime (either to feature sizes
of .about.10 nm or nearest-neighbor distances (aperture sizes) of
<10 nm, but not both simultaneously).
A continuously varying channel spacing between nanostructures is
desired for many of the bio-separation applications such as various
nanofluidic configurations discussed herein.
One approach to a graded structure is to macroscopically vary the
intensity across the plane of exposure while keeping the other
interference conditions, such as the angles between the light
propagation vectors and the polarization, unchanged. One such
variation of intensity would be a smooth gradient in intensity of
one of the two interfering light waves. This results in
interference fringes with uniform spacing but different
intensities. The difference in intensity of the fringes leads to
differences in exposure of the photoresist used. Because the fringe
spacing is not changed, the pitch is uniform. The interference
pattern would have even better contrast if both light waves had the
same gradient in intensities.
When a positive photoresist is used, the areas corresponding to
fringes with stronger intensities leave wider cavities in the
photoresist after exposure and developing. The areas corresponding
to fringes with weaker intensities leave narrower cavities in the
photoresist. When the substrate is etched, these differing widths
translate into features in the substrate that have differing
widths. The features have the same pitch, however, because the
fringe spacing is not altered. This leads to a constant pitch, but
a varying line:space ratio. This procedure provides a continuously
decreasing channel width that may be accurately controlled over
very long distances. Such gradient separation matrices exhibit the
favorable traits of gradient gels (high resolution in separation),
without the difficulty and irreproducibility associated with their
preparation.
Similarly, this technique, when used with a negative photoresist,
leaves wider features in the areas corresponding to fringes with
weaker intensity and narrower features in the areas corresponding
to fringes with stronger intensity.
An alternative approach may produce features with a gradient in
width and pitch. This may be easily achieved with IL by using a
cylindrical lens in one of the beams, while keeping the other beam
as a plane wave. In this case the plane of exposure becomes a chord
for a number of circular wavefronts. Because the wavefronts have
different radii of curvature (spacing of an optical wavelength),
the spacing between the interference fringes, as well as the width
of the interference fringes, vary along the length of the plane
containing the interference fringes on the surface of the
photoresist coating the substrate. Similarly, curved surfaces
(sections of Newton's rings) may be formed by interfering a plane
wave and a spherical wave or two spherical waves of differing radii
of curvature.
Other types of separation systems may involve discontinuous
gradients. One such system may have differing aperture sizes that
may be produced by separate exposures with different intensities,
at different pitches through shadow masks, or by using multiple
exposure techniques to eliminate rows and/or columns of pillars in
certain areas of a previously exposed uniform nano-structured
surface.
Variations in size may also be produced chemically. For example,
increasing the oxidation of silicon in certain areas of a chip may
result in a swelling of the features, reducing the width of some
channels while conserving the pitch of the features. Similarly,
macroscopic areas may be selectively functionalized with
monolayers, reducing the width of channels in that area.
One may also electrochemically produce silicon carbide on a silicon
substrate. Silicon carbide is suitable for sublimation growth,
allowing one to control the width of the modified channels in a
certain area. Of course, silicon carbide is only one example of
surface modifications that may be performed.
One may also selectively heat a substrate, bringing it close to its
annealing temperature. At this time the substrate may be placed
under a highly controlled stress. The subsequent strain alters the
size of channels. A gradient in temperature across the substrate
results in a gradient of strain, and therefore a gradient in
channel widths. This technique would only be suitable for
substrates without a crystalline structure (such as glass or
amorphous silicon, for example).
The very high aspect ratios of FIGS. 5A and 5B were achieved using
highly anisotropic wet chemical etching of crystalline Si in KOH,
which exhibits a >400:1 etch-rate selectivity for etching the
<100> plane relative to the <111> plane of Si. Thus,
the vertical sidewalls are nearly perfect <111> Si facets.
These structures may be further modified by oxidation. This
provides insulation between the Si and the surrounding material
(allowing electrophoretic fluidic manipulation) and varies the
surface interactions between the nanostructure and the surrounding
materials for fluidic applications. Very high aspect ratio,
crystal-structure-independent etching processes have been developed
to address the need for 3D structures in MEMs technology. These
involve pulsed gas processes in which an isotropic etch process may
be alternated with a surface passivation step to reduce the
sidewall etch rate and only etch feature bottoms exposed by ion
bombardment. To date, these processes have largely been
investigated on micrometer scales. As part of the present
invention, these processes are extended to the nanostructured
regime. This greatly broadens the available classes of materials
for which deep, high aspect ratio structures suitable for
nanofluidic applications may be fabricated.
Nanostructures that exhibit a gradient in their capacity to
transport biomolecular species (through size exclusion or
otherwise) may be created by the IL processes discussed herein.
Such gradients make separation matrices feasible for highly
efficient separation of molecular species. Molecular species may be
driven in the direction of the gradient, and thus separated based
on their tendency to traverse the gradient, by a variety of driving
forces, including, but not limited to, electrophoresis,
externally-applied pressure, capillarity, diffusion, and
osmosis.
IL represents a convenient method for generating nanostructured
separation matrices that contain physical gradients that allow
selective transport of chemical species and, thus, may be used to
achieve a separation of different chemicals. When compared to other
nanolithographic methods of pattern generation (e.g., electron beam
lithography, scanning probe lithography), IL is more convenient,
efficient and inexpensive because it may be used to generate the
entire pattern in one, parallel step and is not a serial "writing"
technique. Other parallel techniques (e.g., imprint lithography)
rely on a primary patterning technique to generate a master that
may then be used to produce replicas of nanostructured features in
a parallel fashion. While IL is a preferred method to generate
nanostructured gradients for molecular separation, a variety of
methods could be employed to generate the nanostructured matrix
gradient "artificial gels" of the present invention. Gradients in
the chemistry of the separation matrix may be prepared by a variety
of methods as well, including those based on IL.
The use of IL allows such nanostructured separation matrices to be
produced easily and very inexpensively. Nanostructures in which
channels are on the order of the excluded size of dissolved
biomolecules allow an enhanced flexibility in separation. Higher
resolution may be obtained in combination with any of the following
mechanisms namely, size exclusion, electrophoretic mobility,
isoelectric point, asymmetric diffusion, entropic trapping,
hydrophobic interaction and affinity interaction (molecular
recognition), as well as others. The gradient matrices produced
allow efficient separation and identification of biomolecules such
as native proteins and protein complexes in addition to denatured
proteins and nucleic acids.
Nanolithography-generated systems have advantages over conventional
systems in terms of (1) the virtually perfect uniformity of pore
size and pore size distribution from device to device, and (2) the
flexibility to precisely define the required distribution
(gradient) of pore sizes and pore chemistries. This high degree of
reproducibility and versatility in nanofabrication will result in
the ability to construct separation devices that exhibit
unprecedented degrees of flexibility (resolution, dynamic range)
and reproducibility in their separation characteristics.
The separation gradient may be formed by a variety of means
including, for example, nanolithography (e.g., IL, electron beam,
local probe, nanoimprint) and pattern transfer (etching,
deposition, lift-off) means.
FIG. 6 shows a schematic of a nanostructured gradient (chirped)
separation matrix. The separation gradient may be formed by a
variety of means including nanolithography (e.g., IL, electron
beam, local probe, nanoimprint) and pattern transfer (etching,
deposition, lift-oft) means. FIG. 6 illustrates a graded array of
nanostructures. The aperture size between the nanostructures
approaches molecular dimensions. The arrows signify the direction
of movement of molecular species comprising the mixture to be
separated and the direction of separation. The height of the
nanostructures is preferably sufficiently larger (e.g., 100nm-1
.mu.m) than the diameter to allow for higher throughput of the
separated species.
Multiple-exposure IL moire patterns provide for cyclic gradients
that may be used for simultaneous manufacture of multiple
structures. Gradients may also be fabricated across uniform
patterns by non-uniform deposition or etching using properly
designed deposition and/or etching tools and techniques such as
oblique incidence of etch/deposition atomic/molecular species
(shadowing). Analogous techniques may be used in generation of
gradients in surface modification chemistry incorporated into the
array.
FIGS. 7A and 7B show a perspective view and a top view,
respectively, of a nanostructured matrix according to the present
invention. Matrix 700 has a plurality of protrusions 702. A sample
containing some concentration of molecules moves in the direction
of arrow 704. The diameter of channel 705 between protrusion 706
and protrusion 708 is larger than the diameter of channel 709
between protrusions 710 and 712. This change provides a gradient
such that larger molecules are inhibited from moving the entire
length of matrix 700 once the molecules encounter channels between
two protrusions that are smaller than the diameter of the molecule.
FIGS. 7A and 7B may be extended to formation of channels to
delineate the pathway for molecule movement.
As an example of channel formation according to the present
invention, IL and anisotropic wet etching of Si allow the creation
of open, parallel nanostructured channels (e.g., uncapped in the
direction perpendicular to the surface) with lateral features on
the order of biomolecular length scales (.about.1-10 nm) but with
overall dimensions reaching the microscopic (.about.100 .mu.m) or
even macroscopic (.about.1 cm or greater) scales. Depending upon
the dimensions, molecular transport mechanisms may include
diffusion, electrophoresis or bulk-flow. The relatively large
vertical scale is sufficient to allow high throughput of molecules
and external pumping using either electrokinetic or electro-osmotic
forces. Examples of high aspect ratio IL nanostructured samples are
shown in FIGS. 8A, 8B and 8C. Such architectures are applicable to
channel and post arrays that are of interest for the separation of
proteins and large DNA molecules.
Arrays of nanostructures (either of uniform size or with a gradient
of sizes) may be surface-modified with chemical species that
enhance the separation characteristics of the matrix. These
chemical species may be distributed uniformly over the
nanostructured separation matrix or may be distributed in a
gradient (continuous or discrete) in the direction of separation
over the matrix. These chemical species may include small organic
molecules, polymers, receptors or other biomolecules.
IL may be used to expose patterns on photoresist on silicon or
other materials (which may later be etched). Silicon and some other
materials may have an oxide surface that is easily modified with
silanization reagents. Synthetic strategies for modification are
also available for other materials (besides oxides), including
native silicon and noble metals (e.g., gold). Monomolecular layers
may be created from a wide range of commercially-or
synthetically-available chemical species that may enhance
separation characteristics based on the type and degree of
interaction of chemical species being separated with the walls of
the surface-modified nanostructured separation matrix. Examples of
types of surface modifications (either as gradients or uniform)
include the use of hydrophilic oligomeric and polymeric species
(e.g., poly-ethylene glycol (PEG)) to minimize interactions of
chemical species, especially proteins, with nanostructured
surfaces; use of hydrophobic molecular or oligomeric species to
elicit hydrophobic interaction of chemical species (especially
proteins) with nanostructured surfaces; use of mixtures of
hydrophobic and hydrophilic species (polar, apolar, H-bonding,
ionic) to tune interaction of different chemical species with
surfaces; use of ionic molecular species and mixtures of ionic
species to tune interaction of different chemical species with
surfaces; use of biomolecular or organic receptors to elicit
molecular recognition of small molecules, polymers, proteins, DNA,
RNA, or oligonucleotides with the surface; use of oligonucleotide
probes to tune interactions of DNA, RNA or nucleic-acid binding
proteins with the surface; use of cyclodextrins, macrocyclic
antibiotics, crown ethers and other chiral selectors to tune
enantiomeric interactions of chemical species with the surface; and
use of stimuli-responsive (smart) molecules or polymers to allow
external control of interaction of chemical species with the
nanostructured surface.
Other types of separation systems of the present invention may be
thought of as having discontinuous gradients. These separation
systems contain areas with different aperture sizes, and may be
made either by separate exposures at different intensity, at
different pitches through shadow masks, or by using multiple
exposure techniques to eliminate rows and/or columns of pillars.
Such systems are especially useful in that they will allow recovery
of separated compounds (purification).
Microfabricated Integrated Multi-Dimensional, Multi-Technique
Separation Systems
The present invention allows a variety of different separation
strategies (electrophoresis, iso-electric focusing, affinity
chromatography, hydrophobic interaction chromatography,
enantiomeric resolution) to be used on a single monolithic device,
thus allowing for ease of use and compactness of
instrumentation.
The closest existing commonly used multi-technique separation is
two-dimensional gel electrophoresis (2DGE). In traditional 2DGE,
proteins are first separated according to isoelectric point,
followed by resolution by mass-to-charge ratio using standard
polyacrylamide electrophoresis. This process requires that two
separate electrophoretic procedures be performed, each requiring
manipulation of the sample. A nanostructured matrix of the present
invention allows for sequential analysis on a single chip, thus
reducing sample loss and diffusion. The wide range of chemical
modifications and array architecture allowed by IL devices will
also permit separation of proteins by means in addition to size and
isoelectric point, either by appropriate chemical patterning and
valving of the device, or by addition of a third separation and/or
dilution dimension.
Fabrication of separation matrix systems from materials (e.g., Si
and quartz) commonly used in the fabrication of integrated circuits
is advantageous. They have unique etching and surface modification
characteristics that are well established, and may be easily
implemented in existing microfabrication facilities for the
development of complex separation and detection systems. Other
materials with advantageous characteristics may also be used.
The nanostructured matrix of the present invention may be used for
two-dimensional gel electrophoresis, and a number of other
separation techniques may be combined with size exclusion and/or
isoelectric focusing. In addition, the matrix has the capability of
expansion beyond two dimensions.
Combining two or more standard types of analysis on a single
platform may enhance the analytical potential of a nanostructured
matrix of the present invention. Among the possible combinations of
separation technologies applicable to this platform are those
analogous to PAGE, isoelectric focusing, hydrophobic interaction
chromatography, affinity chromatography, enantiomeric resolution
and capillary electrophoresis. The matrix lends itself well to
carrying out equivalent molecular weight separations, with either
electrical currents or flow as the driving force.
The present invention may be useful in proteomics by enabling
combinations of different types of analysis, e.g., size exclusion
in one dimension with chemical differentiation in the second. A
third dimension, oriented perpendicular to the two dimensional
array, may then be used for further separation, or for recovery and
further characterization of isolated spots.
The present invention may also find use in protein separations for
forensic and medical diagnostic tools and in the separation of
bioengineered proteins. Forensic analysis and diagnostics, for
example, depend heavily upon differentiation between carbohydrate
moieties on blood proteins and bacterial cells. Discovery of
clinically useful drugs often depends on identifying interactions
with specific cellular receptors, which are usually glycoproteins.
Capillary electrophoresis has been extremely useful in separations
of acid carbohydrates, with derivatization of the column. The
present invention allows for the separation of two properties, for
example glycoprotein size and carbohydrate content on a single
platform, thus eliminating the need for cumbersome recovery between
steps and increasing the yield of useful analyte.
Recently, techniques utilizing antibody-based affinity separations
have transitioned from clinical laboratories to those for
environmental monitoring. The present invention allows sequential
analysis of at least two different properties, thus increasing
sensitivity of analysis, with particular interest for environmental
monitoring.
The present invention allows for separation of a variety of sizes
of nucleic acid species, and thus, may be used for separations that
are currently done by standard and pulsed field gel
electrophoresis, as well as nucleic acid sequencing. In addition,
modification of the device by nucleic acid binding molecules (e.g.,
proteins, drugs) allows for isolation of relevant target sequences
from previously uncharacterized genomes, or for isolation of a
biocomplex formed with a nucleic acid. Because separation may be
multidimensional, these devices may be attached in series with a
reaction chamber (for example, a PCR thermocycler) and the
resultant product directly fed into the separation platform for
purification and analysis in a single device.
IL may be used to create nanostructures on a variety of substrates.
IL, in combination with other standard lithographic and
microfabrication methodologies, may be used to create a variety of
nanostructures that may be modified in many ways to produce tools
for separation of relevant biomolecules. These have advantages over
contemporary molecular separation systems because they exhibit
superior performance (resolution, sensitivity, dynamic range,
applicability, reproducibility), may be parallel-produced at
relatively low cost, and are extremely flexible in terms of
chemical modifications. They have defined features that may be
reproducibly made, enable flexible and complex separation, and may
be used with existing bioseparation and detection strategies.
Supported and Suspended Lipid Bilayers on Nanotextured Surfaces
An additional aspect of the present invention is the use of defined
lithographic nanostructures to suspend or support lipid bilayers
and hybrid lipid bilayers as a separation platform for
membrane-associated biomolecules. This invention expands upon
previous methods for (1) incorporating lipid bilayers and
nanostructured surfaces for separation techniques, and (2) creating
lipid bilayers in which regions of the lipid bilayers are freely
suspended or supported between two aqueous reservoirs.
Specifically, the present invention utilizes suspended or supported
lipid bilayer architecture in the separation of transmembrane
molecules.
FIG. 9 shows lipid bilayer 902 suspended on nanostructure 904. The
dimensions of nanotroughs 906 are such that lipid bilayer 902 may
be suspended on nanostructure 904 over the nanotrough, allowing for
domains of biomolecules 908 exterior to the membrane to segregate
into these troughs for separation. These structures are made by
spontaneous assembly of lipid bilayers from lipid micelles on a
hydrophilic surface. Transmembrane proteins and other biomolecules
may be incorporated either during the formation of the micelles or
formation of the membrane through fusion of micelles incorporating
them or membrane ghosts. Certain membrane proteins are also capable
of self-directed insertion into the membrane and these may be
incorporated by direct insertion into the membrane.
FIGS. 10, 11 and 12 show various bilayers that may be associated
with a particular nanostructure according to embodiments of the
present invention. FIG. 10 shows a generic bilayer 1002 suspended
on nanostructure 1004 over nanotroughs 1006. FIG. 11 shows a
suspended lipid/self-assembled monolayer hybrid bilayer 1102 on
nanostructure 1104 having nanotroughs 1106. This bilayer type of
structure forms spontaneously when a monolayer containing
hydrocarbon-like chains are present on the nanostructures. In this
configuration, fluidity of the lower leaflets in the supported
region is lost, as that leaflet is fixed. Transmembrane proteins
and other membrane biomolecules are expected to move preferentially
in those areas with greatest total membrane fluidity, i.e. in the
troughs. It is also anticipated by the present invention that given
formation conditions, e.g. size and curvature of the forming
micelles in relation to the nanoarchitecture, that one may also
achieve coverage of the bilayers over the entire surface, or over
selected surfaces of the nanosupport. An example is represented in
FIG. 12, which shows bilayer 1202 supported in nanotroughs 1206 in
nanostructure 1204. This sort of structure may be achieved through
selective modification of the tops of the nanostructures such that
they would not support bilayer formation (e.g., hydrophobic
modification) and through use of micelles that are smaller than the
diameter of the nanotrough.
Of particular interest are nanofabricated arrays of structures that
exhibit a gradient in their capacity to transport molecules. The
reason being that such gradients allow for separation matrices that
eliminate the requirement for detergent solubilization, and thus
denaturation, of transmembrane biomolecules prior to separation,
which is the current state of the art. Such gradient structures
allow molecular species to be driven in the direction of the
gradient, thereby separating the molecules based on their tendency
to traverse the gradient. Molecular species may be driven across
the gradient by forces such as electrophoresis, externally-applied
pressure, capillarity, diffusion, and osmosis. Depending on the
desired means of separation, several modifications of the
nanostructured support that will enhance separation within the
supported or suspended bilayer are envisioned. These include, but
are not limited to chemical modifications, such as changes in
hydrophobicity, charge, or dipole moment which will allow
interactions with protein domains exterior to the bilayer,
modification with ligands or other biomolecules that have the
potential for interacting with a target class of membrane proteins,
and other modifications that end users will deem necessary to base
separations on membrane protein function.
Two relevant methods for fabricating suspended lipid bilayers have
been reported: (1) suspending small unilamellar vesicles that are
made and applied directly to an unmodified Si surface, and (2)
generating large unilamellar vesicles with direct pipetting of
these structures onto a surface. See, Groves, J. T., Wulfing, c.,
and Boxer, S. G., Electrical manipulation of glycan phosphatidyl
inositol tethered proteins in planar supported bilayers,
Biophysical Journal, 71: 2716-2723 (1996), and Menger, F. M., and
Angelova, M. I., Accounts of Chemical Research, 31: 789-797 (1999),
the entire contents and disclosures of which are hereby
incorporated by reference. Preliminary studies included forming
suspended lipid bilayers to examine their applicability in the
present invention.
Since the electrophoretic mobility of transmembrane molecules
across suspended lipid bilayers depends on (1) the molecule's mass
to charge ratio, and (2) the size of the extramembrane domains
relative to the corrugated apertures in the nanostructured support,
it is necessary to fabricate a device that allows for preferential
movement of molecules. An embodiment of the present invention
suspends lipid bilayer membranes over a series of small gaps,
approximately 100 nm in size, and utilizes the entire supported
membrane as a separation and analysis platform. The small sizes of
the gaps between features allows the lipid bilayer membrane to be
suspended over the gaps, which allows for preferential movement of
membrane phospholipids, transmembrane proteins, and other
lipophilic biomolecules, and their complexes. More specifically,
the relative fluidity of the lower leaflet of the lipid bilayer in
the suspended regions, and resultant lack of steric hindrance of
extramembrane protein domains, results in greater mobility of
transmembrane molecules. Furthermore, by making the aperture size
on the order of the molecular size of the transmembrane molecules,
separations may be based on molecular filtering mechanisms in
addition to electrophoretic mobility. Because the areas scanning
the gaps may be supported on the underside by aqueous media, more
room may also be available for intercellular domains. In addition,
biophysical studies both of interactions between extra and
intercellular domains of a single protein, and/or of interactions
between intercellular domains of proteins within the same membrane
are provided by the present invention. Thus, suspended lipid
bilayer membranes offer several advantages over the current state
of the art, particularly in regard to the separation and
concentration of transmembrane proteins.
In a modification of the suspended lipid bilayer model,
alkane-chain terminated self-assembled monolayers may be formed on
the top surfaces of the nanostructured surfaces, either by silane
modification of Si substrates or .omega.-substitued alkanethiols
on, for example, gold. It is anticipated that these structures may
provide even greater mobility of lipophilic biomolecules in
supported and non-supported regions of the lipid bilayer membrane
due to the immobility of the chemically fixed lower leaflet in the
hybrid region.
Several nanotextured surfaces have also been explored. IL may
produce a variety of features, including posts and grooves, in
nearly infinite combinations of types and arrangements. Such
features may be arranged in a regular array, thus mimicking
standard gels, with the features either shaped or arranged in an
asymmetric manner, or as semidiscontinuous, or chirped, arrays that
vary in their size and/or separation distance along the direction
of separation. Furthermore, a combination of grooves and/or posts
may be arranged to achieve configurations that allow for two
dimensional separations. The present invention may be used for
separation of membranes from osmotically disrupted cells (cell
ghosts). This is particularly significant because no previous
isolation of membrane-associated biomolecules is necessary, thus
preserving the biomolecules native conformations and complexes.
Although the present invention is primarily concerned with the
structures described above, the nanostructured surfaces and lipid
bilayer membranes may be combined in such a way to modify only the
tops of the features, the lower surface of the nanostructured
surface, the sides of the features, or any combination thereof. In
addition, lipid bilayer membranes containing different molecules,
or derived from different organelles within a cell, may be
patterned on the nanostructured surface, thereby conferring a
certain level of selectivity within the membrane itself, either due
to innate properties of the molecules or the presence of
interactive biomolecules within a region of the pattern. Thus, the
present invention may be utilized in several manners to facilitate
the study of biomolecules.
For instance, the nanostructured surfaces supporting lipid bilayer
membranes may be utilized in biophysics to study membrane
components. Because, within the suspended regions, neither leaflet
of the membrane may be immobilized on the surface, total membrane
fluidity may be increased, thus allowing for greater mobility of
embedded biomolecules and creating an experimental milieu more
closely replicating that found in the cell. Furthermore,
interactions between cytoplasmic and extracellular domains may be
more easily studied.
In addition, the present device shows promise as a biosensor
device. Because the structure allows for proper orientation of the
intercellular domains of transmembrane proteins, natural or
engineered receptor proteins may take advantage of naturally
occurring transduction mechanisms to facilitate signal
transduction.
The present invention may further be useful in purification. The
nanostructured lipid bilayer device may provide a unique platform
on which to purify lipophilic membrane biomolecules. Because the
components may be applied from membrane cell ghosts or in lipid
micelles, the need for harsh and possibly denaturing detergent
extraction may be unnecessary. In addition, complexes of associated
proteins may be purified intact, thereby improving the study of
pharmaceutical agents.
The present device may also be useful in the crystallization of
membrane proteins to provide more pertinent information as to the
structure and function of the proteins. The nanostructured lipid
bilayer device may be manufactured to produce a gradient of
features so that the protein in question aggregates at a single
band in the device, thereby accumulating at the critical
concentration.
Finally, the present invention may allow for greater flexibility in
screening potential pharmaceutical agents. The nanostructured lipid
bilayer device may facilitate observation of interactions between
target transmembrane molecules and potential therapeutic agents
within a defined membrane milieu, as well as allow for in vitro
study of the resultant interactions between the drug-bound receptor
and other components within the target complexes.
The present invention allows for unprecedented advances in the
study of biomembranes and their associated molecules. The fact that
membrane associated biomolecules may be applied to the present
device, either via cell ghosts or vesicles, without first isolating
them in aqueous media using detergent solubilization means that
native configurations, associations and, thus, functionality may be
preserved.
EXAMPLES
Example 1
Design and construction of microscale electrophoresis cells
incorporated much of the characteristics of the present invention
into a compact system. The cell preferably has the following
characteristics: (1) electrochemical current and fluid flow is
restricted to occur only through the separation matrix; (2) loading
and stacking functions are included; (3) monitoring of mobility and
biomolecular detection is possible (e.g., through fluorescence
imaging); and (4) for certain applications, separated compounds are
recoverable. Simple methods have been used for incorporating
nanostructured silicon/silica chips into electrophoresis cells that
satisfy criteria (1-3) above. For example, simple methods of rapid
prototyping of elastomeric gasket materials have been used.
Example 2
Supported phospholipid bilayers (SPBs) of egg phosphatidyl choline
(Egg PC) were formed by vesicle fusion on nanostructured silicon
wafers containing troughs .about.180 nm in width on a 360 nm pitch.
An intercalating dye was introduced, and the membranes were imaged
by scanning laser microscopy. The resultant fluorescence
micrographs indicated that the SPBs formed uniformly over the
surface and simple FRAP measurements indicated that the bilayers
were fluid and that recovery of fluorescence was preferentially in
the direction parallel to the nanotroughs.
Example 3
Transmembrane or membrane associated proteins may be incorporated
into an SPB, from incorporation in the vesicle stage, insertion on
the membrane or through incorporation of cell ghosts (i.e., intact
membranes isolated from cells or organelles). The architecture
and/or chemistry of the underlying nanotextured support would be
then used to guide the movement of membrane proteins through the
supported or suspended bilayer, either by size exclusion of the
trough over which the membrane is supported, or by chemical
interactions with modifications on the nanostructured support.
Although the present invention has been fully described in
conjunction with the preferred embodiment thereof with reference to
the accompanying drawings, it is to be understood that various
changes and modifications may be apparent to those skilled in the
art. Such changes and modifications are to be understood as
included within the scope of the present invention as defined by
the appended claims, unless they depart therefrom.
* * * * *
References