U.S. patent application number 12/850771 was filed with the patent office on 2010-11-25 for method of patterning molecules on a substrate using a micro-contact printing process.
This patent application is currently assigned to Sony Deutschland GmbH. Invention is credited to Gregor Kron, Dirk Mayer, Andreas Offenhaeusser, Daniel Schwaab, Jurina WESSELS, Akio Yasuda.
Application Number | 20100298169 12/850771 |
Document ID | / |
Family ID | 36096436 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100298169 |
Kind Code |
A1 |
WESSELS; Jurina ; et
al. |
November 25, 2010 |
METHOD OF PATTERNING MOLECULES ON A SUBSTRATE USING A MICRO-CONTACT
PRINTING PROCESS
Abstract
The present invention relates to a method of patterning
molecules on a substrate using a micro-contact printing process, to
a substrate produced by said method and to uses of said substrate.
It also relates to a device for performing the method according to
the present invention.
Inventors: |
WESSELS; Jurina; (Stuttgart,
DE) ; Kron; Gregor; (Stuttgart, DE) ; Yasuda;
Akio; (Esslingen, DE) ; Schwaab; Daniel;
(Wesseling, DE) ; Mayer; Dirk; (Frechen, DE)
; Offenhaeusser; Andreas; (Eynatten, BE) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Sony Deutschland GmbH
Koeln
DE
|
Family ID: |
36096436 |
Appl. No.: |
12/850771 |
Filed: |
August 5, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11477655 |
Jun 30, 2006 |
7802517 |
|
|
12850771 |
|
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Current U.S.
Class: |
506/18 ;
428/195.1; 428/209; 428/210; 435/395; 436/501; 436/518 |
Current CPC
Class: |
Y10T 428/24926 20150115;
B01J 2219/00605 20130101; B01L 3/0258 20130101; B05C 1/027
20130101; Y10S 977/793 20130101; Y10S 977/887 20130101; B01J
2219/0063 20130101; B01J 2219/0061 20130101; B01J 2219/00659
20130101; B01J 2219/00612 20130101; B01J 2219/00626 20130101; B41M
3/006 20130101; B01J 2219/00637 20130101; Y10S 977/789 20130101;
B01J 2219/00725 20130101; B01J 2219/00527 20130101; B82Y 10/00
20130101; B82Y 40/00 20130101; B01J 2219/00382 20130101; B81C
1/00206 20130101; Y10T 428/24802 20150115; Y10T 428/24917 20150115;
G03F 7/0002 20130101 |
Class at
Publication: |
506/18 ;
428/195.1; 428/209; 428/210; 436/518; 436/501; 435/395 |
International
Class: |
C40B 40/10 20060101
C40B040/10; B32B 3/10 20060101 B32B003/10; G01N 33/543 20060101
G01N033/543; G01N 33/53 20060101 G01N033/53; C12N 5/00 20060101
C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 2, 2005 |
EP |
05 023 880.7 |
Claims
1-30. (canceled)
31. A substrate comprising a pattern of molecules thereon, which
molecules have retained their function and/or activity and/or
native conformation, the substrate produced by a method of
patterning the molecules on the substrate using a micro-contact
printing process, whereby the molecules to be patterned are kept in
solvent or are covered by solvent during the entire micro-contact
printing process, said method comprising: providing molecules to be
patterned in a solvent and providing a patterned surface, said
molecules being first immobilized on an ink-pad within said
solvent; transferring said molecules to be patterned onto said
patterned surface and immobilizing them thereon while keeping said
molecules in said solvent or covered by said solvent, said ink-pad
having said molecules immobilized thereon is brought into conformal
contact with said patterned surface in a first solvent environment
containing said molecules to be patterned and said solvent, thereby
transferring said molecules onto said patterned surface and
immobilizing them thereon; and providing said substrate in a second
solvent environment, transferring said patterned surface having
said molecules immobilized thereon to said second solvent
environment, and bringing said patterned surface having said
molecules immobilized thereon into conformal contact with said
substrate, thereby creating a pattern of said molecules on said
substrate, while keeping said molecules in said solvent or covered
by said solvent, wherein said molecules to be patterned are protein
molecules, wherein said micro-contact printing process occurs in
the absence of a drying step.
32. The substrate according to claim 31, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 180
min after immobilizing said molecules to be patterned on said
patterned surface.
33. The substrate according to claim 31, wherein, after the
providing a substrate, said patterned surface is lifted from said
substrate, thereby leaving behind a substrate having a pattern of
said molecules thereon.
34. The substrate according to claim 33, wherein said substrate
having a pattern of molecules thereon is kept in or covered by
solvent containing a buffer.
35. The substrate according to claim 31, wherein said molecules to
be patterned retain their function and/or activity and/or native
conformation throughout the entire process, due to their being kept
in solvent or covered by solvent during the entire micro-contact
printing process.
36. The substrate according to claim 31, further comprising a
spacer layer and/or a binding layer which facilitates binding of
said substrate to said molecules to be patterned through covalent
binding, electrostatic forces, van der Waals forces, H-bonding,
London forces or any combination of the foregoing.
37. The substrate according to claim 31, selected from the group
consisting of: metals and semi metals, single or polycrystalline
materials; single or polycrystalline metals and semi metals, gold,
platinum, or silicon, or; composite materials of single or
polycrystalline composites, siliconoxide, or GaAs, or amorphous
composite materials or glass; plastics, or elastomers or
polydimethylsiloxane, or plastomers, or polyolefines, or ionomers,
or resist materials, or UV-NIL resists.
38. The substrate according to claim 31, wherein said patterned
surface is made from a material selected from the group consisting
of: single-crystalline materials and polycrystalline materials,
silicon, silicon oxide, layered composite systems, silicon oxide on
silicon, metal layers on silicon or metal layers on silicon oxide;
amorphous materials, glass; plastics, elastomers,
polydimethylsiloxane, plastomers, polyolefines (POP, polyolefinic
plastomers), ionomers, resist materials, or UV-NIL-resists.
39. The substrate according to claim 31, wherein at least a surface
of said ink-pad is made from a material selected from the group
consisting of: single-crystalline materials and polycrystalline
materials, silicon, silicon oxide, layered composite systems,
silicon oxide on silicon, metal layers on silicon/siliconoxide;
amorphous materials, glass; plastics, elastomers,
polydimethylsiloxane, plastomers, polyolefines (POP, polyolefinic
plastomers), ionomers, resist materials, or UV-NIL-resists.
40. The substrate according to claim 31, wherein said molecules to
be patterned are selected from the group consisting of: redox
proteins, nucleic-acid binding proteins, metallo-proteins,
cytochrome c, azurin, cytoskeleton-proteins.
41. The substrate according to claim 31, wherein said molecules to
be patterned have one or plural lysine residues, and wherein said
substrate is Au, or Au having a spacer layer on its surface so as
to avoid denaturation of said protein, said spacer layer having a
thickness in the range of from 0.5 nm to 200 nm.
42. The substrate according to claim 31, wherein the pattern
comprises features having a length in the range of from
approximately 10 nm to 500 .mu.m.
43. The substrate according to claim 31, wherein said providing a
patterned surface includes providing a patterned surface in the
form of a stamp.
44. The substrate according to claim 31, wherein said providing
molecules to be patterned in a solvent includes providing molecules
first immobilized on an ink-pad in the form of a non-patterned
surface within said solvent.
45. The substrate according to claim 31, wherein said first solvent
environment also includes a buffer.
46. The substrate according to claim 31, wherein said second
solvent environment also includes a buffer.
47. The substrate according to claim 31, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 120
min after immobilizing said molecules to be patterned on said
patterned surface.
48. The substrate according to claim 31, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 10 min
after immobilizing said molecules to be patterned on said patterned
surface.
49. The substrate according to claim 31, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 1 min
after immobilizing said molecules to be patterned on said patterned
surface.
50. The substrate according to claim 31, wherein the pattern
comprises features having a length in the range of from
approximately 10 nm to .ltoreq.200 nm.
51. The substrate according to claim 31, wherein the pattern
comprises features having a length in the range of from
approximately 10 nm to .ltoreq.150 nm.
52. The substrate according to claim 31, wherein the substrate is
modified with a molecular layer.
53. The substrate of claim 52, wherein the molecular layer is a
self assembling monolayer (SAM).
54. The substrate of claim 53, wherein the molecules of the SAM
have two termini, a first terminus for binding to the substrate,
and a second terminus for binding the protein molecules to be
patterned.
55. The substrate of claim 54, wherein the substrate comprises gold
and the first terminus comprises a thiol for binding to the
gold.
56. The substrate of claim 54, wherein the substrate comprises
silicon oxide and the first terminus comprises a silane for binding
to the silicon oxide.
57. The substrate according to claim 54, wherein the SAM is
selected from the group consisting of: a SAM with mercapto- or
amino-groups for binding metals, a SAM with carboxy-groups for
electrostatic binding, a SAM with mercapto-groups for binding
metals, with plain alkyl chains having methylene groups for van der
Waals interaction, with --COOH, --OH or vinyl-groups for covalent
coupling, or SAMs with antibodies for binding corresponding
antigens, or SAMs with antigens for binding corresponding
antibodies, or SAMs with receptors for specific binding of
molecules.
58. The substrate according to claim 52, wherein the molecular
layer includes antibodies for binding antigens or includes antigens
for binding corresponding antibodies.
59. The substrate according to claim 52, wherein the molecular
layer includes receptors for specific binding of molecules.
60. The substrate according to claim 31, wherein the substrate is
gold modified with a mercapto undecanoic acid layer (MUA).
Description
[0001] The present invention relates to a method of patterning
molecules on a substrate using a micro-contact printing process, to
a substrate produced by said method and to uses of said substrate.
It also relates to a device for performing the method according to
the present invention.
[0002] During the past decade, soft lithography has developed to a
versatile technique for fabricating chemically micro- and
nanostructured surfaces [1,2]. Among several techniques known
collectively as soft lithography, micro-contact printing (.mu.CP)
has become the most commonly used method [1]. A patterned polymer
stamp is covered with an ink of molecules using either contact
inking or wet inking. In contact inking the solvent is reduced to
the dry state while the molecules self assemble on an inkpad. The
molecules are transferred onto the stamp under ambient conditions
by bringing the stamp and the inkpad into conformal contact. In the
wet inking process, the ink is poured over the stamp and then
reduced under a stream of nitrogen to a dry state. In both cases
the molecules are on the stamp prior to the transfer onto a
substrate. For the transfer of the ink onto the substrate, stamp
and substrate are brought into conformal contact with a substrate
for the transfer of the molecules from the stamp to the substrate
[3,4].
[0003] Recently, also proteins have been transferred to a variety
of substrates [5-7]. The advantage of .mu.CP thereby is the direct,
fast and gentle transfer of proteins, however, all
.mu.CP-techniques reported so far ultimately lead to a denaturation
of the printed proteins. Native proteins immobilized onto modified
surfaces are of major interest for sensor technology, cell
culturing and micro-biology. One application is e.g., the
patterning of growth factor proteins on silicon oxide for guiding
cell growth [8].
[0004] A critical issue for the immobilization of biomolecules,
e.g. proteins, nucleic acids etc. on surfaces is their denaturation
and hence the loss of the functionality after their immobilization.
The functionality, as e.g. in the case of cytochrome c (cyt c), may
depend on the orientation and conformation of the protein on the
surface. So far, the immobilization and redox activity of cyt c has
been investigated on chemically modified Au surfaces [9-11] and on
ITO [12]. Runge et al. reported a process for the transfer for cyt
C molecules onto ITO surfaces, in which the proteins are dried on
the stamp [12]. For ITO-surfaces it could be demonstrated that the
reactivity of the proteins depend on the surface modification of
the stamps used for the process [11].
[0005] In addition to transferring proteins, a method for
transfer-printing of highly aligned DNA nanowires has been
described by Nakao et al. [13] using PDMS stamps. In this method
hydrodynamic forces are used to align DNA on PDMS. After the
alignment step the PDMS stamp is brought into conformal contact
with a mica sheet for the transfer of DNA onto mica. AFM images
showed that the apparent height of the as transferred DNA is
between 0.27 and 0.35 nm, indicating that the DNA molecules are
probably elongated and possibly sheared as a result of the
hydrodynamic forces.
[0006] However, all the above described inking methods used in the
prior art cause denaturation of the protein(s) and loss of their
activity.
[0007] Accordingly, it was an object of the present invention to
provide for a method allowing the immobilization and patterning of
molecules on a substrate, whereby the molecules to be patterned and
immobilized retain their function and/or native conformation and/or
activity. Furthermore, it was an object of the present invention to
provide for a method of patterning molecules on a substrate that is
easy to perform even with biological macromolecules whilst
maintaining their functionality. Furthermore, it was an object of
the present invention to provide for a method of patterning
molecules on a substrate whereby pattern features .ltoreq.200 nm
can be achieved.
[0008] All these objects are solved by a method of patterning
molecules on a substrate using a micro-contact printing process,
whereby the molecules to be patterned are kept in solvent or are
covered by solvent during the entire micro-contact printing
process.
[0009] Such a micro-contact printing process in which the molecules
to be patterned are kept in solvent or are covered by solvent
during the entire process is herein also sometimes referred to as
"in-situ printing process" or "in-situ stamping process". The term
"in-situ printing process" or "in-situ stamping process" as used
herein, is meant to denote a micro-contact printing process whereby
the molecules to be patterned retain their functionality and/or
conformation as a result of being kept in solvent or being covered
by solvent during the entire printing process.
[0010] In a preferred embodiment such "in-situ printing process is
meant to denote a micro-contact printing process in the entire
course of which the molecules to be patterned are kept in their
respective physiological conditions that allow them to retain their
native functionality and/or conformation. It should be emphasized
that the term "physiological conditions" will depend on the type of
molecules to be patterned. For example, if the molecules to be
patterned are molecules of an oxygen transporting protein, the
"physiological conditions" for such a molecule will preferably
include a pH-value in the range of from 7.0 to 7.8, preferably
around pH 7.4. If, on the other hand, the molecules to be patterned
are molecules of a gastric enzyme, the "physiological conditions"
for such a molecule will include a pH-value in the range of from
1.8 to 4. Hence, overall the term "physiological conditions" will
include pH-values that may range from 1 to 10.
[0011] In one embodiment said micro-contact printing process occurs
in the absence of a drying step.
[0012] In one embodiment, the method according to the present
invention comprises the following steps:
[0013] a) providing molecules to be patterned in a solvent and
providing a patterned surface, preferably in the form of a
stamp,
[0014] b) transferring said molecules to be patterned onto said
patterned surface and immobilizing them thereon whilst keeping said
molecules in said solvent or covered by said solvent,
[0015] c) providing a substrate and bringing said patterned surface
having said molecules immobilized thereon into conformal contact
with said substrate, thereby creating a pattern of said molecules
on said substrate, whilst keeping said molecules in said solvent or
covered by said solvent.
[0016] The term "to bring into conformal contact with" is meant to
denote a contact between two entities, e.g. surfaces, allowing the
transfer of molecules that were on one entity before the contact,
to the other entity. In some embodiments, exertion of pressure is
needed for such transfer to occur, and in these instances, the term
"to bring into conformal contact with" is to be equated with "to
press on(to)".
[0017] The term "to immobilize a molecule on a surface", as used
herein, is meant to denote an activity by which a molecule becomes
attached to a surface, It does not mean that a molecule thus
immobilized will be unable to move completely. For example, parts
of a molecule thus immobilized may still rotate about certain
chemical bonds and/or may "swing" within the solvent covering the
surface. "Immobilization", as used herein, merely implies some kind
of attachment of a molecule to a surface which attachment prevents
the molecule to diffuse freely from said surface. In its simplest
form, such immobilization of molecules on a surface may occur by
exposing said surface to said molecules.
[0018] In one embodiment in step a), said molecules are provided in
said solvent and are first immobilized on an ink-pad, preferably in
the form of a non-patterned surface, within said solvent, wherein,
preferably, in step b), said ink-pad having said molecules
immobilized thereon is brought into conformal contact with said
patterned surface in a first solvent environment containing said
molecules to be patterned, said solvent and, optionally, a buffer,
thereby transferring said molecules onto said patterned surface and
immobilizing them thereon, and wherein, more preferably, in step
c), said substrate is provided in a second solvent environment
containing said solvent and, optionally a buffer, and wherein said
patterned surface having said molecules immobilized thereon, after
step b), is transferred to said second solvent environment and is
brought into conformal contact with said substrate. The first and
second and subsequent solvent environments contain a solvent and
may, in addition thereto, also contain a solute, such as a salt,
preferably a buffer, more preferably a buffer by the presence of
which physiological conditions are established or conditions are
established which mimic a physiological state. The second and
subsequent solvent environments initially contain no molecules to
be patterned or only a very small amount thereof As soon as the
patterned surface or the ink-pad has been transferred to said
second, third, fourth etc. solvent environment, however, there will
be some molecules to be patterned present in said solvent
environment.
[0019] The term "ink-pad", as used herein, is meant to signify any
surface that is capable of acting as a transfer-facilitating
surface for molecules to be patterned. In its simplest form it may
simply be a non-patterned surface. Under certain conditions,
however, it may also be some sort of surface that has a pattern on
it, and/or that has the capacity of absorbing molecules to be
patterned and the capacity of releasing some of these molecules
upon bringing a stamp into conformal contact with said ink-pad.
[0020] In one embodiment in step b), said ink-pad having said
molecules immobilized thereon is brought into conformal contact
with said patterned surface in a second solvent environment
containing said solvent and, optionally, a buffer, after transfer
of said ink-pad having said molecules immobilized thereon to said
second solvent environment, thereby transferring said molecules
onto said patterned surface and immobilizing them thereon, wherein,
preferably, in step c) said substrate is provided in a third
solvent environment containing said solvent and, optionally, a
buffer, and wherein said patterned surface having said molecules
immobilized thereon, after step b), is transferred to said third
solvent environment and is brought into conformal contact with said
substrate.
[0021] In one embodiment in step a), said molecules are provided in
said solvent and, in step b), said molecules are immobilized on
said patterned surface within said solvent, wherein, preferably,
step b) occurs by immersing said patterned surface in said solvent,
and wherein, more preferably, in step c), said substrate is
provided in a fourth solvent environment containing said solvent
and, optionally, a buffer and wherein said patterned surface having
said molecules immobilized thereon, after step b), is transferred
to said fourth solvent environment and is brought into conformal
contact with said substrate.
[0022] In one embodiment in step c), said substrate is provided
without a solvent environment, and wherein said patterned surface
having said molecules immobilized thereon is brought into conformal
contact with said substrate whilst keeping said molecules covered
by said solvent, and wherein said patterned surface is transferred
to a fifth solvent environment containing said solvent and,
optionally a buffer, whilst being in contact with said substrate,
said transfer of said patterned surface and said substrate
occurring immediately after said patterned surface is brought into
conformal contact with said substrate, so as to avoid a drying of
said patterned surface on said substrate.
[0023] In one embodiment, said step b) is performed over a period
in the range of from 1 s to 60 min. Step b) may be considered an
"inking step".
[0024] Preferably, the bringing into conformal contact of said
patterned surface with said substrate of step c) occurs in a period
not longer than 180 min after immobilizing said molecules to be
patterned on said patterned surface, preferably not longer than 120
min, more preferably not longer than 10 min, most preferably not
longer than 1 min after immobilizing said molecules to be patterned
on said patterned surface.
[0025] As used in this context, the term "occurs in a period not
longer than . . . after immobilizing . . . " is meant to denote
that said bringing into conformal contact must take place within a
period of 180 min at a maximum, said period commencing from the
time that said molecules to be patterned are immobilized on said
patterned surface.
[0026] In one embodiment after step c), said patterned surface is
lifted from said substrate, thereby leaving behind a substrate
having a pattern of said molecules thereon, wherein, preferably,
said substrate having a pattern of molecules thereon is kept in or
covered by solvent optionally containing a buffer.
[0027] In one embodiment said molecules to be patterned are
selected from the group comprising proteins, nucleic acids,
preferably DNA or RNA, lipids and combinations of any of the
foregoing, wherein, preferably, said molecules to be patterned are
protein molecules.
[0028] Preferably, said molecules to be patterned retain their
function and/or activity and/or native conformation throughout the
entire process, due to their being kept in solvent or covered by
solvent during the entire micro-contact printing process, wherein,
more preferably, said molecules to be patterned are kept under
physiological conditions, as measured by, for example, pH and
salinity, throughout the entire micro-contact printing process.
[0029] The term "solutes" as used in this context, does not exclude
the presence of solutes within the solvent. In fact, these may be
preferred in order to establish the desired physiological
conditions. Such solutes, without being limited thereto, include
salts and their ion-components, buffers, proteins, nucleic acids
and lipids.
[0030] In one embodiment said substrate has a hydrophilic surface
if said molecules to be patterned are hydrophilic, and wherein said
substrate has a hydrophobic surface if said molecules to be
patterned are hydrophobic.
[0031] Preferably, said substrate comprises a spacer layer and/or a
binding layer which facilitates binding of said substrate to said
molecules to be patterned through covalent binding, electrostatic
forces, van der Waals' forces, H-bonding, London forces or any
combination of the foregoing.
[0032] In one embodiment said substrate is selected from the group
comprising metals and semi metals, single or polycrystalline
materials; preferably single or polycrystalline metals and semi
metals (most preferably gold, platinum, silicon) or; composite
materials preferably single or polycrystalline composites (most
preferably (siliconoxide, GaAs) or amorphous composite materials
(most preferably glass); plastics, preferably elastomers (most
preferably polydimethylsiloxane), preferably plastomers (most
preferably polyolefines), preferably ionomers, preferably resist
materials (most preferably UV-NIL resists); any of the afore
mentioned materials modified with molecular layers, preferably SAMs
(self assembling monolayers), for direct binding or indirect
binding, SAMs for indirect binding will be with one or multiple
chemicals or treatments to achieve the desired binding site;
[0033] most preferably SAMs with two termini: one for attaching the
molecule to the substrate such as a thiol-headgroup for binding on
gold; most preferably SAMs with a silane-headgroup terminus for
binding on siliconoxide; the second terminus for coupling the ink,
such as SAMs with mercapto- or amino-groups for binding metals,
SAMs with carboxy-groups for electrostatic binding, most preferably
SAMs with mercapto-groups for binding metals, with plain
alkylchains having methylene groups for van der Waals interaction,
with --COOH, --OH or vinyl-groups for covalent coupling; or SAMs
with antibodies for binding corresponding antigens, or SAMs with
antigens for binding corresponding antibodies, or SAMs with
receptors for specific binding of molecules; or any of the
aforementioned materials modified with molecular layers, with
antibodies for binding corresponding antigens, or modified with
molecular layers with antigens for binding corresponding
antibodies, or modified with molecular layers with receptors for
specific binding of molecules; most preferably gold modified with a
mercapto undecanoic acid layer (MUA).
[0034] Preferably, said patterned surface is made from a material
selected from the group comprising single-crystalline materials and
polycrystalline materials, such as silicon, silicon oxide, layered
composite systems, such as silicon oxide on silicon, metal layers
on silicon/silicon oxide; amorphous materials, such as glass;
plastics, such as elastomers, preferably polydimethylsiloxane,
plastomers, preferably polyolefines (POP, polyolefinic plastomers),
ionomers, resist materials, such as UV-NIL-resists.
[0035] In one embodiment said ink-pad surface is made from a
material selected from the group comprising single-crystalline
materials and polycrystalline materials, such as silicon, silicon
oxide, layered composite systems, such as silicon oxide on silicon,
metal layers on silicon/silicon oxide; amorphous materials, such as
glass; plastics, such as elastomers, preferably
polydimethylsiloxane, plastomers, preferably polyolefines (POP,
polyolefinic plastomers), ionomers, resist materials, such as
UV-NIL-resists.
[0036] Preferably, said molecules to be patterned are selected from
the group comprising protein molecules, such as redox proteins,
nucleic-acid binding proteins, enzymes, metallo-proteins, such as
cytochrome c, azurin, cytoskeleton-proteins, antibodies, nucleic
acids, such as DNA, RNA, PNA, lipids, such as phospholipids and
sphingolipids.
[0037] In one embodiment said molecules to be patterned are protein
molecules having one or several lysine residues, and wherein said
substrate is Au, preferably having a spacer layer on its surface so
as to avoid denaturation of said protein, said spacer layer
preferably having a thickness in the range of from 0.5 nm to 200
nm.
[0038] In one embodiment the pattern comprises features having a
length in the range of from approximately 10 nm to 500 .mu.m,
preferably approximately 10 nm to .ltoreq.200 nm, more preferably
approximately 10 nm to .ltoreq.150 nm. It is clear that the size of
the actual features printed by the method according to the present
invention depends on the intended application of the pattern thus
printed. For example, if the intended application lies in the field
of nucleic acid chips or sensor applications, the average size of
the printed features is likely to be in the range of from 1 .mu.m
to 500 .mu.m. If the intended application lies in the field of
molecular electronics, the average size of the printed features is
likely to be in the range of from approximately 10 nm to
.ltoreq.200 nm, preferably approximately 10 nm to .ltoreq.150
nm.
[0039] The objects of the present invention are also solved by a
substrate produced by the method according to the present invention
and comprising a pattern of molecules thereon which molecules
retain their function and/or activity and/or native
conformation.
[0040] The objects of the present invention are also solved by use
of a substrate according to the present invention in a sensor, a
bioreactor or for guiding cell growth.
[0041] The objects of the present invention are also solved by a
device for performing the method according to the present
invention, comprising [0042] a first means holding a solution of
molecules to be patterned, [0043] a patterned surface, preferably
in the form of a stamp, [0044] a substrate, kept in a solvent or
covered by a solvent, [0045] a second means to transfer said
molecules to be patterned
[0046] from said first means to said patterned surface, [0047] a
third means to transfer said molecules to be patterned from said
patterned surface to said substrate, [0048] a fourth means to
ensure that said molecules to be patterned are kept in a solvent or
are covered by a solvent during transfer from said first means to
said patterned surface to said substrate.
[0049] In a preferred embodiment of the device according to the
present invention, the second means is an ink-pad, preferably a
non-patterned surface.
[0050] The inventors have surprisingly found that it is possible to
perform a micro-contact printing process using molecules,
preferably biological macromolecules, such as proteins, nucleic
acids and/or lipids, and keeping these biological macromolecules in
solution or under solvent, preferably aqueous solvent all the time.
The method according to the present invention can be performed
using various schemes outlined further below.
[0051] As used herein, the term "molecules" is meant to denote any
molecule which may have a biological relevance. It includes nucleic
acids, including oligonucleotides, proteins, including peptides,
and lipids. The molecules may be of synthetic or natural origin. In
the case of proteins or nucleic acids, they may have sequences
occurring in nature or they may have artificial sequences.
[0052] Hence the present inventors describe an in-situ stamping
process that prevents drying or denaturation of the molecules, e.g.
proteins on the stamp after the inking process. In this .mu.CP
process, the stamp, the inkpad (if present) and the substrate are
kept during all process steps in a solvent environment, e.g. a
buffer solution, or at least covered by a buffer solution. Thus all
steps can be performed under in-situ physiological conditions.
[0053] In one aspect, the inventive method, can be described by
various processes which are explained further as follows:
[0054] Process 1 (See Scheme 1 of FIG. 4):
[0055] An ink-pad is immersed in the solution of the desired
molecules. After several hours a stamp is immediately brought into
contact with the ink-pad for a few minutes in the same container.
The stamp is then rapidly transferred into a container with pure
buffer solution, so that the stamp's surface does not dry. This
buffer solution contains the substrate, on which the molecules
should be transferred. The stamp is brought into contact with the
substrate for a few minutes. Finally the modified substrate is
inserted into a buffer solution, that is free of molecules to be
printed/stamped, for storage.
[0056] Process 2 (See Scheme 2 of FIG. 4):
[0057] An ink-pad is immersed in the solution of the desired
molecules. After several hours the ink-pad is rapidly transferred
into a container with pure buffer solution, so that the ink-pad
surface does not dry. A stamp is immediately brought into contact
with the ink-pad for a few minutes. The stamp is than rapidly
transferred into another container with pure buffer solution, which
contains the target substrate. The stamp is pressed onto the
substrate for a few minutes. Finally the modified substrate is
inserted into a buffer solution for storage, that is free of
molecules to be printed/stamped.
[0058] Process 3 (See Scheme 3):
[0059] A stamp is immersed in the solution of the desired
molecules. The molecules adsorb to the stamp surface. After several
hours the stamp is rapidly transferred into a container with pure
buffer solution, so that the stamp's surface does not dry. This
buffer solution contains the substrate, on which molecules should
be transferred. The stamp is brought into contact with the
substrate for a few minutes. Finally the modified substrate is
inserted into a protein free buffer solution for storage, that is
free of molecules to be printed/stamped.
[0060] Process 4 (See Scheme 4):
[0061] The stamp of schemes 1, 2 or 3, onto which molecules to be
stamped have been immobilized is brought into contact with a
substrate under ambient conditions directly after removing it from
the ink-solution. This has to be done, as long the stamp is wet.
The stamp with the attached substrate is put immediately into a
container with pure buffer solution. After a few minutes the
transfer is finished. Finally the modified substrate is inserted
into a buffer solution for storage, that is free of molecules to be
printed/stamped.
[0062] In the following reference is made to the figures wherein
the figures show the following:
[0063] FIG. 1: SEM image of cyt c on (MUA)/gold. The lines are 1
.mu.m to 150 nm with equal gaps in between. The dark lines are cyt
c molecules.
[0064] FIG. 2: Cyclic Voltammograms (scan rate: 50 mV/s; reference
electrode: SCE) of cyt c on MUA/gold. Reference substrate without
cyt c (-), cyt c absorbed from solution (.tangle-solidup.), cyt c
after in-situ stamping or printing (.box-solid.), and cyt c after
ambient stamping or printing (.cndot.).
[0065] FIG. 3: Cyclic Voltammograms (scan rate: 50 mV/s; reference
electrode: SCE) of cyt c on MUA/gold. Comparison of different times
for which the stamp remains in a buffer reservoir before contacting
the substrate, namely 5 s (.box-solid.), 10 min (.tangle-solidup.)
and 2 h (-).
[0066] FIG. 4: shows a schematic representation of the various
schemes 1-4 that are specific embodiments of the present
invention.
[0067] More specifically, FIG. 4 and the schemes shown therein can
be summarized as follows:
[0068] Scheme 1:
[0069] Schematic presentation of the in-situ micro-contact printing
process. An inkpad is put into a buffered solution of Cytochrome c
for 2 h. The stamp is placed on the inkpad for 2 min. The stamp is
removed and rapidly brought into a buffer solution without drying.
Immersed into the buffer solution is a gold substrate covered with
a mercaptoundecanoic acid SAM. The stamp is brought into conformal
contact with the substrate for 2 min and is released.
[0070] Scheme 2:
[0071] Schematic presentation of the in-situ micro-contact printing
process. An inkpad is immersed into the solution containing the
desired molecules. After several hours the ink-pad is rapidly
transferred into a container with buffer solution. A stamp is
immediately brought into contact with the ink-pad for a few
minutes. Subsequently the stamp is transferred into a container
with buffer solution, which contains also the target substrate. The
stamp is pressed onto the substrate for a few minutes and is
subsequently released.
[0072] Scheme 3:
[0073] Schematic presentation of the in-situ micro-contact printing
process. A stamp is immersed in the solution of the desired
molecules. The molecules adsorb to the stamp surface. After several
hours the stamp is rapidly transferred into a container with pure
buffer solution containing the target substrate. The stamp is
brought into conformal contact with the substrate for a few minutes
and is subsequently released.
[0074] Scheme 4:
[0075] The wet stamp prepared according to Scheme 1, 2 or 3 is
brought into contact with a substrate under ambient conditions
directly after removing it from the ink-solution. The stamp with
the attached substrate is immediately immersed into a container
with pure buffer solution. After a few minutes the stamp is
released from the substrate.
[0076] Furthermore, reference is made to the following examples
which are given to illustrate, not to limit the invention.
EXAMPLE
[0077] A) Stamps and Functional/Structural Investigations
[0078] It is clear to someone skilled in the art that the choice of
substrate and stamp depends on the molecules to be patterned. For
example it is clear to someone skilled in the art that for printing
nucleic acids, hydrophilic and hydrophobic substrates are suitable.
Depending on the substrate hydrophilicity the DNA may be
immobilized in from of bundles (hydrophobic surface) or as
individual strands (hydrophilic substrate). It is also clear to
someone skilled in the art that for the transfer of proteins
containing cystein groups onto gold a spacerlayer (e.g
Mercapto-SAM) has to be used to cover the bare gold surface in
order to prevent the binding of cystein to gold which may denature
the protein. On the other hand it is clear to someone skilled in
the art that a hydrophilic polypeptide (with undefined tertiary and
quaternary structure) like polylysin can be printed to a
hydrophilic siliconoxide surface. It is also clear to someone
skilled in the art that the choice of the stamp material depends on
the pattern size. The minimum pattern size is strongly dependent on
the tensile modulus of the materials, e.g PDMS with a tensile
modulus of 1 MPa is good for printing patterns down to 300 nm,
while for patterns below 300 nm Polyolefine with a tensile modulus
of 1 GPa may be suitable. It is also clear to someone skilled in
the art that for printing patterns on large areas a flexible stamp
made out of a flexible plastic material is more preferable than
made of any other material, because the flexible stamp is able to
make a conformal contact on the whole area. It is also clear to
someone skilled in the art that the hydrophilicity of the stamp and
the substrate and the solubility of the biomolecules to be
transferred determine the interaction of the biomolecules with the
inkpad, the stamp and the substrate.
[0079] The stamps for the process can generally be made from
elastomers, plastomers, ionomers, resist materials, and also from
hard materials such as crystalline and polycrystalline materials.
It is also possible to use a combination of these materials for the
preparation of composite stamps (soft-soft, soft-hard, and
hard-hard). The stamps are prepared either by drop casting and
thermal- or photo-induced curing or by hot embossing techniques
from masters that are, if required, passivated with a release
layer, e.g. a monolayer of
(1,1,2,2,-tridecafluoro-octyl)-trichlorosilane, or sodium dodecyl
sulfate (SDS). The surface of the stamp surface can be chemically
modified by exposing the stamp surface to e.g. an oxygen plasma or
by chemically reacting it.
[0080] For the specifically disclosed embodiments further below,
POP (polyolefine plastomer) (Affinity VP 8770G) from Dow Chemicals
was used. POP was heated up to 85.degree. C. and pressed with 90
kPa into a silicon oxide (Sioxide) master, which is passivated with
monolayer of (1,1,2,2,-tridecafluoro-octyl)-trichlorosilane (Sigma
Aldrich).
[0081] All specific embodiments were performed using horse heart
cytochrome c as a model system.
[0082] The redox activity of the proteins was investigated using a
PAR Model 283 potentiostat controlled by a PC running version 2.4
of CorrWare software. The working electrode was an Au(111) single
crystal cylinder with a diameter of 3.5 mm. For the measurement the
hanging meniscus method was applied. In this method the particular
metal plane of the single crystal is brought into contact with the
electrolyte by forming a meniscus. The Au crystal was cleaned in
sulfuric acid. After flame annealing the Au(111) crystals was
placed for 10 min into mercaptoundecanoic acid (MUA)
(Sigma-Aldrich) and subsequently rinsed with ethanol and MilliQ
water (18.2 M.OMEGA., total amount of carbon 3-4 ppm). A standard
calomel electrode (SCE) was used as reference electrode; the
counter electrode is a platinum coil. The setup was placed in a
Faraday cage to reduce electronic noise.
[0083] For SEM imaging, a 5 nm chromium and 50 nm gold layer was
evaporated onto a piece of silicon oxide wafer. The chip was also
cleaned with sulfuric acid, flame annealed and placed into a 10 mM
ethanolic solution of MUA (Sigma-Aldrich) for 10 min.
[0084] B) Preparation and Printing
[0085] A sodium phosphate solution
(Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4) (Merck) at pH 7 with a
concentration of 3.26 mM was used as a buffer to prepare a 12.6
.mu.M hourse heart cyt c (Sigma-Aldrich) solution. A
Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) inkpad was
immersed into the cyt c solution. After 2 h, the stamp was
introduced into the solution and gently pressed onto the inkpad for
2 min. Immediately after the separation of the inkpad and the
stamp, the stamp was introduced into the buffer solution containing
the substrate without drying, in order to cover the proteins with a
thin wet film maintaining the desired physiological conditions
during the transfer of the stamp from one solution to the other.
The transfer time was less than 5 s. After transferring the stamp
into the solution containing the substrate, the stamp was brought
into conformal contact with the substrate for 2 min by applying
gentle finger pressure, for the transfer of the proteins from the
stamp onto the MUA modified Au substrate. The immobilization of cyt
c onto a MUA SAM is based on electrostatic interaction.
[0086] The acid group of MUA is deprotonated and thus negatively
charged, while cyt c has a positive net charge. Since the positive
charge of the lysine groups are located on one side of the cyt c,
the orientation of cyt c on the surface is always the same. FIG. 1
shows a SEM image of the transferred pattern. The four 150 nm broad
lines are dearly separated by a 150 nm wide gap (right side). The
interspacing between transferred lines is free of molecules. No
common drawbacks or shortcoming of .mu.CP like sagging or diffusion
of the ink molecules can be seen.
[0087] C) Cyclic Voltammetry Measurements
[0088] The redox activity of proteins stamped with an unstructured
stamp onto a Au/Cr coated Si/Sioxide wafer was measured with cyclic
voltammetry. The proteins were stamped in the same way as described
under B). After transferring the proteins onto the substrate, the
substrate was directly transferred into the measurement cell. FIG.
2 shows cyclic voltammograms of cyt c after the in-situ .mu.CP
process ("in-situ printing") according to the present invention in
comparison with cyt c stamped under ambient conditions, i.e. where
cyt c was dried on the stamp ("printing under ambient conditions)
and a voltammogramm of cyt c adsorbed from solution ("cyt c in
solution"). In all cases a distinct and reversible redox peak
occurred at a redox potential of E.sup.0=-60 mV. The symmetry of
the peak indicates that cyt c is adsorbed onto a surface. The
current at the redox potential is comparable for the samples
prepared by in-situ printing and by adsorption from solution, while
the current observed for the proteins transferred under ambient
conditions is 70% smaller. This could be either due to a lower
protein density or result from a partial loss in functionality due
to the drying process.
[0089] One, important aspect for the process appears to be the time
duration the stamp is exposed to the buffer solution without being
in conformal contact with the substrate. FIG. 3 shows redox
activity of transferred proteins after the stamp was exposed to
buffer solution for 5 s to 120 min (5s, 10 min and 2 h) prior to
the transfer process. The longer the stamp was exposed to the
buffer solution, the lower was the current at the redox potential.
The reduction in the current is due to a decrease in the surface
coverage on the stamp. Since the proteins are only weakly adsorbed
by London forces to the stamp surface, the concentration gradient
drives desorption of the proteins into the buffer solution.
[0090] The process according to the present invention allows to
pattern biomolecules to dimensions down to 150 nm, while preserving
their structural integrity and functionality by using an in-situ
process.
[0091] The features of the present invention disclosed in the
specification, the claims and/or in the accompanying drawings, may,
both separately, and in any combination thereof, be material for
realizing the invention in various forms thereof.
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