U.S. patent application number 10/665721 was filed with the patent office on 2005-03-24 for peptide mediated synthesis of metallic and magnetic materials.
This patent application is currently assigned to Semzyme. Invention is credited to Belcher, Angela M., Mao, Chuanbin, Reiss, Brian D., Solis, Daniel J..
Application Number | 20050064508 10/665721 |
Document ID | / |
Family ID | 34312933 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050064508 |
Kind Code |
A1 |
Belcher, Angela M. ; et
al. |
March 24, 2005 |
Peptide mediated synthesis of metallic and magnetic materials
Abstract
The present invention includes methods for producing magnetic
nanocrystals by using a biological molecule that has been modified
to possess an amino acid oligomer that is capable of specific
binding to a magnetic material.
Inventors: |
Belcher, Angela M.;
(Lexington, MA) ; Reiss, Brian D.; (Boston,
MA) ; Mao, Chuanbin; (Austin, TX) ; Solis,
Daniel J.; (Boston, MA) |
Correspondence
Address: |
STEPHEN B MAEBIUS
FOLEY AND LARDNER
3000 K STREET N W SUITE 500
WASHINGTON
DC
20007-5109
US
|
Assignee: |
Semzyme
|
Family ID: |
34312933 |
Appl. No.: |
10/665721 |
Filed: |
September 22, 2003 |
Current U.S.
Class: |
435/7.1 ; 257/40;
436/526; 506/14; 506/22; 977/902 |
Current CPC
Class: |
G01N 33/54326 20130101;
C07K 7/06 20130101; C07K 7/08 20130101; G01N 33/531 20130101; B82Y
5/00 20130101; C07K 7/00 20130101; C12N 15/1037 20130101; C40B
30/04 20130101; B82Y 30/00 20130101 |
Class at
Publication: |
435/007.1 ;
436/526 |
International
Class: |
G01N 033/53; G01N
033/553 |
Goverment Interests
[0002] The research carried out in the subject application was
supported in part by grants from the Army Research Office, Grant
No. DADD19-99-0155, the government may own certain rights.
[0003] In addition, a nucleotide and/or amino acid sequence listing
is incorporated by reference of the material on computer readable
form.
Claims
What is claimed is:
1. A method of making a magnetic material comprising the steps of:
providing a molecule comprising a portion that binds specifically
to the surface of a magnetic material; and contacting one or more
magnetic material precursors with the molecule under conditions
that permit formation of the magnetic material.
2. The method according to claim 1, wherein the magnetic material
is a particle.
3. The method according to claim 1, wherein the magnetic material
is a nanoparticle.
4. The method according to claim 1, wherein the magnetic material
is a ferromagnetic material.
5. The method according to claim 1, wherein the magnetic material
is a ferromagnetic nanoparticle.
6. The method recited in claim 1, wherein the molecule comprises an
amino acid oligomer as the portion that binds specifically to the
surface of the magnetic material.
7. The method recited in claim 6, wherein the oligomer is between
about 7 and about 100 amino acids long.
8. The method recited in claim 6, wherein the oligomer is between
about 7 and about 20 amino acids long.
9. The method recited in claim 1, wherein the molecule is selected
from a combinatorial library screen.
10. The method recited in claim 7, wherein the magnetic material
comprises Co, SmCo5, CoPt or FePt.
11. The method recited in claim 1, further comprising the steps of
isolating the magnetic material.
12. The method recited in claim 11, wherein the magnetic material
is attached to a substrate.
13. The method recited in claim 1, wherein the molecule is defined
further as peptide that comprises a portion of a self-assembling
molecule.
14. The method recited in claim 13, wherein the self-assembling
molecule is a phage.
15. The method recited in claim 13, wherein the self-assembling
molecule is grown in a bacterium.
16. A method of making a magnetic material comprising the step of:
contacting a molecule that initiates magnetic material formation
with magnetic material precursor and a reducing agent.
17. The method recited in claim 16, wherein contacting is carried
out at about room temperature.
18. The method recited in claim 16, wherein contacting is carried
out at about 350.degree. C. or less.
19. The method recited in claim 16, wherein the molecule is an
amino acid oligomer.
20. The method recited in claim 16, wherein the molecule is an
amino acid oligomer comprising between about 7 and about 100 amino
acids.
21. The method recited in claim 16, wherein the molecule is an
amino acid oligomer comprising between about 7 and about 20 amino
acids.
22. The method recited in claim 16, wherein the molecule further
comprises a self-assembling viral particle.
23. The method recited in claim 16, wherein the magnetic material
comprises Co, CoPt, SmCo5, or FePt magnetic material.
24. The method recited in claim 16, wherein the magnetic material
is a magnetic quantum dot.
25. The method recited in claim 22, wherein the self-assembling
viral particle is used to produce a casting film.
26. The method according to claim 16, wherein the magnetic material
is a particle.
27. The method according to claim 16, wherein the magnetic material
is a nanoparticle.
28. The method according to claim 16, wherein the magnetic material
is a ferromagnetic material.
29. The method according to claim 16, wherein the magnetic material
is a ferromagnetic nanoparticle.
30. A magnetic material made by the method of claim 1.
31. A magnetic material made by the method of claim 16.
32. A method of making a magnetic material comprising the steps of:
linking a magnetic material binding molecule to a substrate;
contacting one or more magnetic material precursors with the
magnetic material binding molecule under conditions that form the
magnetic material; and forming the magnetic material.
33. The method according to claim 32, wherein the magnetic material
is a ferromagnetic material.
34. The method according to claim 32, wherein the magnetic material
is a particle.
35. The method according to claim 32, wherein the magnetic material
is a nanoparticle.
36. The method according to claim 32, wherein the magnetic material
is a ferromagnetic nanoparticle.
37. The method of claim 32, wherein the magnetic material binding
molecule comprises a chimeric protein that exposes one or more
magnetic material binding amino acid oligomers on its surface.
38. The method of claim 32, wherein the magnetic material binding
molecule is an amino acid oligomer.
39. The method of claim 32, wherein the magnetic material binding
molecule comprises between about 7 and about 100 amino acid
oligomers.
40. The method of claim 32, wherein the magnetic material binding
molecule comprises between about 7 and about 20 amino acids.
41. The method of claim 32, wherein the magnetic material binding
molecule is linked chemically to the substrate.
42. The method of claim 32, wherein the magnetic material binding
molecule comprises a chimeric protein with a self-assembling viral
particle.
43. The method of claim 32, wherein the magnetic material comprises
Co, CoPt, SmCo5 or FePt.
44. The method of claim 32, wherein the method is used to produce a
film.
45. The method of claim 32, wherein substrate comprises a patterned
surface, and the magnetic material binding molecule is fixed only
on the patterns of the patterned surface.
46. A magnetic material made by the method of claim 32.
47. A magnetic material formed using a binding molecule and one or
more magnetic material precursors.
48. The magnetic material according to claim 47, wherein the
binding molecule comprises a binding amino acid oligomer
portion.
49. The magnetic material according to claim 48, wherein the
oligomer comprises between about 7 and about 100 amino acids.
50. The magnetic material according to claim 48, wherein the
binding oligomer portion comprises between about 7 and about 20
amino acids.
51. The magnetic material according to claim 47, wherein the
magnetic material is a ferromagnetic material.
52. The magnetic material according to claim 47, wherein the
magnetic material comprises particles.
53. The magnetic material according to claim 47, wherein the
magnetic material comprises nanoparticles.
54. The magnetic material according to claim 47, wherein the
magnetic material comprises ferromagnetic nanoparticles.
55. The magnetic material of claim 47, wherein the magnetic
material is formed at a temperature of less than 350 degrees
centigrade.
56. The magnetic material of claim 47, wherein the magnetic
material is selected from the group consisting of Co, CoPt, SmCo5,
and FePt.
57. A magnetic material formed using a magnetic material specific
binding molecule in the presence of a metal salt and a reducing
agent.
58. The magnetic material according to claim 57, wherein the
material is a ferromagnetic material.
59. The magnetic material according to claim 58, wherein the
material is a ferromagnetic nanoparticle material.
60. The magnetic material according to claim 59, wherein the
binding molecule comprises a binding amino acid oligomer
portion.
61. A composition comprising peptide that binds specifically to
.epsilon.-Co.
62. A composition according to claim 61, wherein the peptide binds
specifically to a crystalline surface of .epsilon.-Co.
63. A composition according to claim 61, wherein the peptide binds
comprises the sequence of ALSPHSAPLTLY (SEQ ID NO.:15).
64. A composition comprising peptide that binds specifically to
CoPt.
65. A composition according to claim 64, wherein the peptide binds
specifically to a crystalline surface of CoPt.
66. A composition according to claim 64, wherein the peptide
comprises the sequence of NAGDHAN (SEQ ID NO.:12).
67. A composition according to claim 64, wherein the peptide
comprises the sequence of SVSVGMKPSPRP(SEQ ID NO.:16).
68. A composition comprising peptide that binds specifically to
FePt.
69. A composition according to claim 68, wherein the peptide binds
specifically to a crystalline surface of FePt.
70. A composition according to claim 68, wherein the peptide
comprises the sequence of SKNSNIL (SEQ ID NO.:13).
71. A composition according to claim 68, wherein the peptide
comprises the sequence of HNKHLPSTQPLA (SEQ ID NO.:17).
72. A composition comprising peptide that binds specifically to
SmCo5.
73. A composition according to claim 72, wherein the peptide binds
specifically to a crystalline surface of SmCo5.
74. A composition according to claim 72, wherein the peptide that
binds to SmCo5 comprises the sequence of TKPSVVQ (SEQ ID
NO.:14).
75. A composition according to claim 72, wherein the peptide that
binds to SmCo5 comprises the sequence of WDPYSHLLQHPQ (SEQ ID
NO.:18).
76. A composition comprising peptide that binds specifically to a
ferromagnetic surface.
77. A composition according to claim 76, wherein the peptide binds
specifically to a crystalline surface of a ferromagnetic
surface.
78. A method of isolating a molecule that binds specifically to a
magnetic material comprising the steps of: contacting a library of
molecules with a magnetic material; removing non-binding molecules
from the library; and eluting the bound molecules from the magnetic
material.
79. The method of claim 78, further comprising the step of
determining the molecular structure of the molecules that bind the
magnetic material.
80. The method of claim 78, wherein the molecular library is
further defined as comprising a phage library.
81. The method of claim 78, wherein the molecular library is
further defined as comprising a phage display library.
82. The method of claim 78, wherein the molecular library is
further defined as comprising a combinatorial chemistry
library.
83. The method of claim 78, wherein the molecular library is
further defined as comprising a peptide library.
84. The method according to claim 78, wherein the magnetic material
is ferromagnetic.
85. The method according to claim 78, wherein the molecules
comprise amino acid oligomers which specifically bind the magnetic
material.
86. A method of preparing a particle film comprising the steps of:
adding a solution of particles to a surface; wherein the particles
are synthesized with use of binding molecules; evaporating the
solution of nanoparticles on the surface; and annealing the
particles to the surface to create a film of particles.
87. The method according to claim 86, wherein the particles are
magnetic.
88. The method according to claim 86, wherein the particles are
ferromagnetic.
89. The method according to claim 86, wherein the particles are
nanoparticles.
90. The method according to claim 86, wherein the particles are
ferromagnetic nanoparticles.
91. The method of claim 86, wherein the solution of nanoparticles
are magnetic nanoparticles in a solvent and are selected from the
group consisting of .epsilon.-Co, Co, SmCo5, CoPt, FePt, and
combinations thereof.
92. The method of claim 86, wherein the solvent is evaporated.
93. The method of claim 86, wherein the surface is a
microfabricated solid surface to which molecules may attach through
either covalent or non-covalent bonds and selected from the group
consisting of Langmuir-Bodgett films, glass, functionalized glass,
germanium, silicon, PTFE, polystyrene, gallium arsenide, gold,
silver, or any materials comprising amino, carboxyl, thiol or
hydroxyl functional groups incorporated onto a surface.
94. The method of claim 86, wherein the annealing is at a
temperature of least about 700 degrees Centigrade for at least
about 30 minutes under an inert gas.
95. A particle film prepared by the method of claim 86.
96. A method of making a metal material comprising the steps of:
providing a molecule comprising a portion that binds specifically
to a metal surface; and contacting one or more metal material
precursors with the molecule under conditions that permit formation
of the metal material.
97. The method of claim 96, wherein the formation of the metal
material occurs in the presence of a reducing agent.
98. The method according to claim 96, wherein the molecule
comprises an amino acid oligomer portion which binds specifically
to the metal surface.
99. The method recited in claim 96, wherein the oligomer portion
which binds specifically is between about 7 and about 100 amino
acids long.
100. The method recited in claim 96, wherein the oligomer portion
which binds specifically is between about 7 and about 20 amino
acids long.
101. The method of claim 96, wherein the metal material is
magnetic.
102. The method of claim 96, wherein the metal material is
ferromagnetic.
103. The method of claim 96, wherein the metal material is a
particle.
104. The method of claim 96, wherein the metal material is a
nanoparticle.
105. The method of claim 96, wherein the metal material is a
ferromagnetic nanoparticle and the molecule comprises an amino acid
oligomer.
106. A method of making a magnetic material comprising the step of
contacting a molecule which binds specifically to the magnetic
material with a magnetic material precursor at a temperature of
300.degree. C. or below to form the magnetic material.
107. The method according to claim 106, wherein the temperature is
200.degree. C. or below.
108. The method according to claim 106, wherein the temperature is
100.degree. C. or below.
109. The method according to claim 106, wherein the molecule
comprises a peptide which binds specifically to the magnetic
material.
110. The method according to claim 106, wherein the molecule
comprises an oliogopeptide which binds specifically to the magnetic
material.
111. The method according to claim 106, wherein the magnetic
material is a crystalline material.
112. The method according to claim 106, wherein the magnetic
material is a ferromagnetic material.
113. The method according to claim 106, wherein the magnetic
material comprises particles.
114. The method according to claim 106, wherein the magnetic
material comprises nanoparticles.
115. The method according to claim 106, wherein the contacting and
formation are carried out in solution and the magnetic material
does not precipitate out of solution.
116. A method of making a metallic material comprising the step of
contacting a molecule which binds specifically to the metallic
material with a metallic material precursor at a temperature of
300.degree. C. or below to form the metallic material.
117. The method according to claim 116, wherein the temperature is
200.degree. C. or below.
118. The method according to claim 116, wherein the temperature is
100.degree. C. or below.
119. The method according to claim 116, wherein the molecule
comprises a peptide.
120. The method according to claim 116, wherein the molecule
comprises an oliogopeptide.
121. The method according to claim 116, wherein the metallic
material is a crystalline material.
122. The method according to claim 116, wherein the metallic
material is a ferromagnetic material.
123. The method according to claim 116, wherein the metallic
material comprises particles.
124. The method according to claim 116, wherein the metallic
material comprises nanoparticles.
125. The method according to claim 116, wherein the contacting and
formation are carried out in solution and the metallic material
does not precipitate out of solution.
126. A composition comprising an oligopeptide and a magnetic
material, wherein the oligopeptide is specifically bound to the
magnetic material.
127. The composition according to claim 126, wherein the magnetic
material is a ferromagnetic material.
128. The composition according to claim 126, wherein the magnetic
material comprises magnetic alloy.
129. The composition according to claim 126, wherein the magnetic
material comprises particles.
130. The method according to claim 126, wherein the magnetic
material comprises nanoparticles.
131. A composition comprising SmCo.sub.5 nanoparticles.
132. The composition according to claim 131, wherein the SmCo.sub.5
nanoparticles are HCP nanoparticles.
133. A metal material comprising binding molecule-synthesized metal
particles.
134. A metal material of claim 133, wherein the particles are
magnetic.
135. A metal material of claim 133, wherein the particles are
ferromagnetic.
136. A metal material of claim 133, wherein the particles are
nanoparticles.
137. A metal material of claim 133, wherein the binding molecule
synthesized particles are free from biological molecules by removal
by heating.
138. A metal material of claim 133, wherein the metal particles
have an aspect ratio greater than 50.
139. A metal material of claim 133, wherein the metal particles are
elongated and comprise functionalized regions at either long
end.
140. A metal material of claim 133, wherein the metal particles are
elongated and have regions at either long end to facilitate
binding.
141. A metal material of claim 133, wherein the material is
localized prior to heating.
142. A metal material comprised of a collection of binding molecule
synthesized metal nanoparticles that are annealed from
polycrystalline to single crystalline materials.
143. A metal material comprising of a collection of binding
molecule synthesized metal nanoparticles that are synthesized
independently from viruses.
Description
RELATED APPLICATIONS
[0001] This application claims benefit of provisional patent
application Ser. No. 60/411,804 filed Sep. 18, 2002 to Belcher et
al., which is hereby incorporated by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0004] The present invention is directed to organic materials
capable of binding to inorganic materials, and specifically, toward
specific peptide sequences that tightly and directly bind to metal
materials including magnetic materials.
BACKGROUND OF THE INVENTION
[0005] In biological systems, organic molecules exert a remarkable
level of control over the nucleation and mineral phase of inorganic
materials such as calcium carbonate and silica, and over the
assembly of building blocks into complex structures required for
biological function.
[0006] Materials produced by biological processes are typically
soft, and consist of a surprisingly simple collection of molecular
building blocks (i.e., lipids, peptides, and nucleic acids)
arranged in astoundingly complex architectures. Unlike the
semiconductor industry, which relies on a serial lithographic
processing approach for constructing the smallest features on an
integrated circuit, living organisms execute their architectural
"blueprints" using mostly non-covalent forces acting simultaneously
upon many molecular components. Furthermore, these structures can
often elegantly rearrange between two or more usable forms without
changing any of the molecular constituents.
[0007] The use of "biological" materials to process the next
generation of microelectronic devices provides a possible solution
to resolving the limitations of traditional processing methods. The
critical factors in this approach are identifying the appropriate
compatibilities and combinations of biological-inorganic materials,
and the synthesis of the appropriate building blocks.
SUMMARY OF THE INVENTION
[0008] The present inventors have designed constructs and produced
biological materials that direct and control the assembly of
inorganic materials, including metallic and magnetic materials,
into controlled and sophisticated structures. Of particular
interest are ferromagnetic materials, and particulate materials
including nanoparticulate materials. The use of biological
materials to create and design materials that have interesting
electrical, magnetic or optical properties may be used to decrease
the size of features and improve the control of, e.g., the
opto-electical properties of the material, as well as control of
material fabrication. For example, room temperature methods have
been developed in the present invention for preparing materials
which formerly involved high temperature preparation methods.
[0009] A combinatorial peptide phage display library expressing a
large collection of bacterial phage that expresses millions of
different peptide sequences on their surfaces was combined with
biopanning techniques to select specific peptide sequences that
tightly and directly bind to metal materials including magnetic
materials (e.g., Co, CoPt SmCo5, or FePt). The present inventors
have found that these metal and magnetic material binding
molecules, including peptides, can be used to control the
nucleation of inorganic materials, as has been demonstrated in
nature and with II-VI semiconductors. If proteins can be used to
control the nucleation of metal, including magnetic, materials,
then magnetic nanoparticles and their applications could be
prepared much cheaper and easier than using traditional methods.
The nanomolecular metals, including magnets and magnetic material,
may be used, e.g., for micro or nanomachines, dynamos, generators,
magnetic storage or any other applications for materials that are
magnetic or may be magnetized. Another use for these materials is
to modify the surface of metal, including magnetic, materials. The
peptides can act as linkers for attaching over materials to the
surface of the magnetic material, allowing the self-assembly of
complex nanostructures, which could form the basis of novel
electronic devices.
[0010] The present inventors have recognized that this approach of
selecting binding peptides (using combinatorial peptide libraries
and panning techniques) may also be used to form and control the
nucleation of metal materials, including magnetic materials. Other
techniques being researched to synthesize metal particles,
including magnetic nanoparticles, are based on a high temperature
synthesis that must be performed in an inert atmosphere using
expensive reagents and often require further processing and
purification after synthesis to fabricate particles, including
nanoparticles, with the desired shape and crystallinity. The result
is that preparing magnetic nanoparticles in the traditional fashion
is expensive and not conducive to large scale and/or volume
production. The approach presented herein is generally performed at
room temperatures using inexpensive reagents yielding nanoparticles
with controlled crystallinity, reducing the cost for the synthesis
of metal particles, including magnetic nanoparticles, with
controlled crystal structure and orientation.
[0011] Peptide-mediated synthesis of metal materials, including
magnetic materials, provides a much cheaper and environmentally
friendly approach to the synthesis of metal materials, including
magnetic nanoparticles. Current protocols for preparing metal
nanoparticles, including magnetic nanoparticles, are time
consuming, expensive and yield nanoparticles coated with organic
surfactants. These surfactants are not amicable to further
modification of the nanoparticles. Advances in the field of
molecular biology enable the functionalization of peptides,
therefore, particles and nanoparticles grown from peptides will
also be easily functionalized. Peptide functionalization
facilitates their incorporation into electronic devices and
integration into magnetic memory devices.
[0012] One form of the present invention is a method for using
self-assembling biological molecules, e.g., bacteriophage, that are
genetically engineered to bind to metals, nanoparticles-, and
magnetic or other materials and to organize well-ordered
structures. These structures may be, e.g., nanoscale arrays of
particles and nanoparticles. Using bacteriophage as an example,
self-assembling biological materials can be selected for specific
binding properties to particular surfaces (e.g., semiconductor),
and thus, the modified bacteriophage and the methods taught herein
may be used to create well-ordered structures of the materials
selected.
[0013] More particularly, the present invention includes
compositions and methods for creating metal materials, including
magnetic materials, particles, and nanoparticles. One embodiment is
a method of making a metal particle, including magnetic particle,
including the steps of; providing a molecule comprising a portion
that binds specifically to a metal surface, including a magnetic
surface, and contacting one or more metal material precurosrs,
including magnetic material precursors, with the molecule under
conditions that permit formation of the metal material, including
the magnetic particle. The molecule may be, e.g., a biological
molecule such as an amino acid oligomer or peptide. The oligomer
may be, for example, between about 7 and about 100 amino acids
long, and more particularly, between about 7 and about 30 amino
acids long, and more particularly about 7 and about 20 amino acids
long, and may form part of a combinatorial library and/or include a
chimeric molecule.
[0014] The types of metal materials, including magnetic particles,
that are disclosed herein may be formed from, e.g., Co, CoPt,
SmCo5, and/or FePt. Another method of the present invention
includes a method for identifying molecules that bind through
non-magnetic interactions with a magnetic material including the
steps of contacting an amino acid oligomer library with a magnetic
material to select oligomers that bind specifically to the magnetic
material and eluting those oligomers that bind specifically to the
magnetic material. The oligomer library may be a library of
self-assembling molecules, e.g., a phage library such as an M13
phage library. The library may even be contained in a bacterium and
may be assembled externally.
[0015] A method of making a magnetic particle may also include the
step of contacting a molecule that initiates magnetic molecule
formation with magnetic material precursors and a reducing agent.
The molecule that initiates magnetic molecule formation with
magnetic material precursors may be contacted at, e.g., room
temperature or below a temperature of, e.g., 100, 200 or even 300
degrees centigrade. The molecule may be an amino acid oligomer of,
e.g., between about 7 and 20 amino acids long. The magnetic
particle may be a Co, CoPt, SmCo5, or FePt magnetic particle in the
form of a magnetic quantum dot or even a film. The skilled artisan
will recognize that combinations or one or more of the magnetic
particles disclosed herein may be positioned in a wide assortment
of one-, two- and three-dimensional locations, shapes, and the like
for particular uses.
[0016] The present invention also includes magnetic particles,
e.g., nanoparticles made by the methods disclosed herein. These
magnetic particles may form a portion of an integrated circuit made
by fixing a magnetic material binding peptide to a substrate;
contacting one or more magnetic material precursors with the
magnetic material binding peptide under conditions that form a
magnetic particle; and forming a magnetic crystal on the substrate.
The magnetic material binding peptide may be linked chemically to a
substrate, e.g., silicon or other semiconductor substrate. The
magnetic particles of the present invention may be used to make
memory, short- or long-term storage, identification systems or any
use that the skilled artisan will recognize may be made of these
particles. Examples of other used for the magnetic micro-, nano-
and femto-particles of the present invention include, micro or
nano-motors, dynamos and the like.
[0017] Another form of the present invention is a method of
creating nanoparticles that have specific alignment properties.
This is accomplished by creating, e.g., an M13 bacteriophage that
has specific binding properties, amplifying the bacteriophage to
high concentrations (e.g., incubation of phage library with
bacterial host culture to allow infection, replication, and
subsequent purification of virus), and resuspending the phage.
[0018] This same method may be used to create bacteriophage that
have three liquid crystalline phases, a directional order in the
nemetic phase, a twisted nemetic structure in the cholesteric
phase, and both directional and positional order in smectic phase.
In one aspect the present invention is a method of making a
polymer, e.g., a film, comprising the steps of, amplifying a
self-assembling biological molecule comprising a portion that binds
a specific semiconductor surfaces to high concentrations and
contacting one or more semiconductor material precursors with the
self-assembling biological molecule to form or direct the formation
of a crystal.
[0019] Another form of the present invention is method for creating
nanoparticles that have differing cholesteric pitches by using,
e.g., an M13 bacteriophage that has been selected to bind to
semiconductor surfaces and resuspending the phage to various
concentrations. Another form of the present invention is a method
of preparing a casting film with aligned nanoparticles by using,
e.g., genetically engineered M13 bacteriophage and re suspending
the bacteriophage.
[0020] Still another form of the present invention is a method of
preparing a nanoparticle film comprising the steps of adding a
solution of nanoparticles to a surface, evaporating the solution of
nanoparticles on the surface, and annealing the nanoparticles to
the surface, where the nanoparticles are magnetic molecules. The
surface may include any microfabricated solid surface to which
molecules may attach through either covalent or non-covalent bonds,
such Langmuir-Bodgett films, glass, functionalized glass,
germanium, silicon, PTFE, polystyrene, gallium arsenide, gold,
silver, or any materials comprising amino, carboxyl, thiol or
hydroxyl functional groups incorporated onto a surface. Annealing
generally occurs by high temperatures under an inert gas (e.g.,
nitrogen). Another form of the present invention is a nanoparticle
film prepared by the method just described.
BRIEF DESCRIPTION OF THE FIGURES
[0021] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
FIGURES in which corresponding numerals in the different FIGURES
refer to corresponding parts and in which:
[0022] FIG. 1 are X-ray photoelectron spectroscopy (XPS) elemental
composition determination of phage-substrate interactions through
the intensity of a gold 4f-electron signal (A-C), model of phage
discrimination for semiconductor heterostructures (D), and examples
of bivalent synthetic peptides with two-component recognition
attachments (E-F);
[0023] FIG. 2 depicts schematic diagrams of the smectic alignment
of M13 phages in accordance with the present invention;
[0024] FIG. 3 include images of the A7-ZnS suspensions using (A-B)
POM, (C) AFM, (D) SEM, (E) TEM, and (F) TEM image with electron
diffraction insert;
[0025] FIG. 4 include images of the M13 bacteriophage nanoparticle
as (A) photograph of the film, (B) schematic diagram of the film
structure, (C) AFM image, (D) SEM image, (E-F) TEM images along the
x-z and z-y planes;
[0026] FIG. 5 is (A) TEM image of annealed SmCo5 nanoparticles, (B)
TEM image with the selected area electron diffraction pattern and
(C) STEM image of annealed SmCo5 nanoparticles;
[0027] FIG. 6 are examples of binding assays illustrating (A) the
specificity of the Co-specific phage for Co and (B) an isotherm of
the Co-specific phage on Co in accordance with the present
invention;
[0028] FIG. 7 includes a series of high resolution TEM images of
CoPt nanoparticles prepared using (A) phage that express the
7-constrained-peptide that selectively binds to CoPt, (B) phage
that express a random peptide, and (C) wild-type phage;
[0029] FIG. 8 is (A) high resolution TEM image of Co nanoparticles
that have been grown using a 12mer peptide that selectively bind to
Co and (B) the corresponding electron diffraction pattern;
[0030] FIG. 9 are (A) high resolution TEM image of FePt
nanoparticles that have been grown using phage that express a 12
mer peptide and are selective for FePt, wherein (B) shows the
electron diffraction pattern both of which are compared to (C) FePt
nanoparticles grown using wild-type phage;
[0031] FIG. 10 is (A) high resolution TEM image of SmCo5
nanoparticles grown using a 12mer that selectively binds SmCo5 as a
template, (B) an electron diffraction pattern of a selected area of
(A) and (C) SmCo5 nanoparticles grown using wild-type phage as a
control;
[0032] FIG. 11 is (A) an AFM image of Co-specific phage with Co
nanoparticles bound to its P3 protein and (B) the corresponding MFM
image;
[0033] FIG. 12 is (A) a hysteresis loop of biologically prepared
FePt nanoparticles and (B) a higher resolution scan of the central
portion of the loop to clarify the coercivity;
[0034] FIG. 13 is (A) a hysteresis loop of biologically prepared
SmCo5 nanoparticles and (B) the central portion of the loop plotted
on a smaller axis to clarify the coercivity; and
[0035] FIG. 14 include (A) TEM of CoPt nanoparticles grown using a
phage that has been genetically engineered to express a CoPt
specific 12mer sequence on their P8 proteins, (B) higher resolution
TEM image of the same CoPt nanoparticles, (C) the corresponding
electron diffraction pattern, (D) STEM image of similarly prepared
particles, (E) STEM mapping for Pt, and (F) STEM mapping for Co in
accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] This application claims benefit of provisional patent
application Ser. No. 60/411,804 filed Sep. 18, 2002 to Belcher et
al., which is hereby incorporated by reference in its entirety
including the figures, summary, detailed description, working
examples, claims, and sequence listing.
[0037] Although making and using various embodiments of the present
invention are discussed in detail below, it should be appreciated
that the present invention provides many applicable inventive
concepts that can be embodied in a wide variety of specific
contexts. The specific embodiments discussed herein are merely
illustrative of specific ways to make and use the invention, and do
not delimit the scope of the invention.
[0038] To facilitate the understanding of this invention, a number
of terms are described further below. As used herein, "metal
material" can be, for example, a substance that encompasses, but is
not limited to, metal alloys, metal oxides, and pure metals, that
may or may not have the magnetic and/or ferromagnetic properties,
may be crystalline, polycrystalline or amorphous. Metal materials
may also exist in several spatial forms, including particles,
patterned surfaces or layered films. The term "particle" can refer
to the size and shape of said materials, and includes but is not
limited to micron-scaled particles, nano-scaled particles (called
nanoparticles), single molecule of metal materials and other sizes
and shapes here unsaid but controlled by the described biological
methods.
[0039] The term binding molecule is hereby defined as a molecule
that binds, recognizes or directs the growth of a metal material.
Examples of binding molecules includes but are not limited to
peptides, amino acid oligomers, and nucleic acid oligomers. These
binding molecules may be selected from combinatorial library
screening, or synthesized, conjugated or formulated independently
from such libraries. These binding molecules may be coupled to a
substrate, i.e. conjugated to a surface or to scaffolds, such as
M13 viruses where the binding molecules are displayed on viral
coats or various binding molecule-conjugated structures.
[0040] The inventors have previously shown that peptides can bind
to semiconductor materials. In the present invention, the inventors
demonstrate that binding molecules, including peptides, can
specifically bind to metal materials, including magnetic materials.
These peptides have been further developed into a way of nucleating
nanoparticles and directing their self-assembly. The main features
of the peptides are their ability to recognize and bind
technologically important materials with face specificity, to
nucleate size-constrained crystalline semiconductor materials, and
to control the crystallographic phase of nucleated nanoparticles.
The peptides can also control the aspect ratio of the nanoparticles
and therefore, the optical properties.
[0041] Briefly, the facility with which biological systems assemble
immensely complicated structure on an exceedingly minute scale has
motivated a great deal of interest in the desire to identify
non-biological systems that can behave in a similar fashion. Of
particular value would be methods that could be applied to
materials with interesting electronic or optical properties, but of
which natural evolution has not selected for interactions between
biomolecules and such materials.
[0042] The present invention is based on recognition that
biological systems efficiently and accurately assemble nanoscale
building blocks into complex and functionally sophisticated
structures with high perfection, controlled size and compositional
uniformity.
[0043] Peptide Sequence Selection
[0044] One method of providing a random organic polymer pool is
using a Phage-display library, based on a combinatorial library of
random peptides containing between 7 and 12 amino acids fused to
the pIII coat protein of M13 bacteriophage, providing different
peptides that were reacted with crystalline semiconductor
structures. Five copies of the pIII coat protein are located on one
end of the phage particle, accounting for 10-16 nm of the particle.
The phage-display approach provided a physical linkage between the
peptide substrate interaction and the DNA that encodes that
interaction. The examples described here used as examples, five
different single-crystal semiconductors: GaAs (100), GaAs (111)A,
GaAs(111)B, InP(100) and Si(100). These substrates allowed for
systematic evaluation of the peptide substrate interactions and
confirmation of the general utility of the methodology of the
present invention for different crystalline structures.
[0045] Protein sequences that successfully bound to the specific
crystal were eluted from the surface, amplified by, e.g., a
million-fold, and reacted against the substrate under more
stringent conditions. This procedure was repeated five times to
select the phage in the library with the most specific binding.
After, e.g., the third, fourth and fifth rounds of phage selection,
crystal-specific phage were isolated and their DNA sequenced.
Peptide binding has been identified that is selective for the
crystal composition (for example, binding to GaAs but not to Si)
and crystalline face (for example, binding to (100) GaAs, but not
to (111)B GaAs).
[0046] Twenty clones selected from GaAs(100) were analyzed to
determine epitope binding domains to the GaAs surface. The partial
peptide sequences of the modified pIII or pVIII protein are shown
in TABLE 1, revealing similar amino-acid sequences among peptides
exposed to GaAs.
[0047] With increasing number of exposures to a GaAs surface, the
number of uncharged polar and Lewis-base functional groups
increased. Phage clones from third, fourth and fifth round
sequencing contained on average 30%, 40% and 44% polar functional
groups, respectively, while the fraction of Lewis-base functional
groups increased at the same time from 41% to 48% to 55%. The
observed increase in Lewis bases, which should constitute only 34%
of the functional groups in random 12-mer peptides from our
library, suggests that interactions between Lewis bases on the
peptides and Lewis-acid sites on the GaAs surface may mediate the
selective binding exhibited by these clones.
[0048] The expected structure of the modified 12-mers selected from
the library may be an extended conformation, which seems likely for
small peptides, making the peptide much longer than the unit cell
(5.65 A.degree.) of GaAs. Therefore, only small binding domains
would be necessary for the peptide to recognize a GaAs crystal.
These short peptide domains, highlighted in TABLE 1, contain
serine- and threonine-rich regions in addition to the presence of
amine Lewis bases, such as asparagine and glutamine. To determine
the exact binding sequence, the surfaces have been screened with
shorter libraries, including 7-mer and disulphide constrained 7-mer
libraries. Using these shorter libraries that reduce the size and
flexibility of the binding domain, fewer peptide-surface
interactions are allowed, yielding the expected increase in the
strength of interactions between generations of selection.
[0049] Phage, tagged with streptavidin-labeled 20-nm colloidal gold
particles bound to the phage through a biotinylated antibody to the
M13 coat protein, were used for quantitative assessment of specific
binding. X-ray photoelectron spectroscopy (XPS) elemental
composition determination was performed, monitoring the phage
substrate interaction through the intensity of the gold 4f-electron
signal (FIGS. 1A-C). Without the presence of the G1-3 phage, the
antibody and the gold streptavidin did not bind to the GaAs(100)
substrate. The gold-streptavidin binding was, therefore, specific
to the phage and an indicator of the phage binding to the
substrate. Using XPS it was also found that the G1-3 clone isolated
from GaAs(100) bound specifically to GaAs(100) but not to
Si(100)(see FIG. 1A). In complementary fashion the S1 clone,
screened against the (100) Si surface, showed poor binding to the
(100) GaAs surface.
[0050] Some GaAs clones also bound the surface of InP (100),
another zinc-blende structure. The basis of the selective binding,
whether it is chemical, structural or electronic, is still under
investigation. In addition, the presence of native oxide on the
substrate surface may alter the selectivity of peptide binding.
[0051] The preferential specific binding of the G1-3 clone to
GaAs(100), over the (111)A (gallium terminated) or (111)B (arsenic
terminated) face of GaAs was demonstrated (FIG. 1B, C). The G1-3
clone surface concentration was greater on the (100) surface, which
was used for its selection, than on the gallium-rich (111)A or
arsenic-rich (111)B surfaces. These different surfaces are known to
exhibit different chemical reactivities, and it is not surprising
that there is selectivity demonstrated in the phage binding to the
various crystal faces. Although the bulk termination of both 111
surfaces give the same geometric structure, the differences between
having Ga or As atoms outermost in the surface bilayer become more
apparent when comparing surface reconstructions. The composition of
the oxides of the various GaAs surfaces is also expected to be
different, and this in turn may affect the nature of the peptide
binding.
[0052] The intensity of Ga 2p electrons against the binding energy
from substrates that were exposed to the G1-3 phage clone is
plotted in FIG. 1C. As expected from the results in FIG. 1B, the Ga
2p intensities observed on the GaAs (100), (111)A and (111)B
surfaces are inversely proportional to the gold concentrations. The
decrease in Ga 2p intensity on surfaces with higher
gold-streptavidin concentrations was due to the increase in surface
coverage by the phage. XPS is a surface technique with a sampling
depth of approximately 30 angstroms; therefore, as the thickness of
the organic layer increases, the signal from the inorganic
substrate decreases. This observation was used to confirm that the
intensity of gold-streptavidin was indeed due to the presence of
phage containing a crystal specific bonding sequence on the surface
of GaAs. Binding studies were performed that correlate with the XPS
data, where equal numbers of specific phage clones were exposed to
various semiconductor substrates with equal surface areas.
Wild-type clones (no random peptide insert) did not bind to GaAs
(no plaques were detected). For the G1-3 clone, the eluted phage
population was 12 times greater from GaAs(100) than from the
GaAs(111)A surface.
[0053] The G1-3, G12-3 and G7-4 clones bound to GaAs(100) and
InP(100) were imaged using atomic force microscopy (AFM). The InP
crystal has a zinc-blende structure, isostructural with GaAs,
although the In--P bond has greater ionic character than the GaAs
bond. The 10-nm width and 900-nm length of the observed phage in
AFM matches the dimensions of the M13 phage observed by
transmission electron microscopy (TEM), and the gold spheres bound
to M13 antibodies were observed bound to the phage (data not
shown). The InP surface has a high concentration of phage. These
data suggest that many factors are involved in substrate
recognition, including atom size, charge, polarity and crystal
structure.
[0054] The G1-3 clone (negatively stained) is seen bound to a GaAs
crystalline wafer in the TEM image (not shown). The data confirms
that binding was directed by the modified pIII protein of G1-3, not
through non-specific interactions with the major coat protein.
Therefore, peptides of the present invention may be used to direct
specific peptide-semiconductor interactions in assembling
nanostructures and heterostructures (FIG. 1E).
[0055] X-ray fluorescence microscopy was used to demonstrate the
preferential attachment of phage to a zinc-blende surface in close
proximity to a surface of differing chemical and structural
composition. A nested square pattern was etched into a GaAs wafer;
this pattern contained 1-.mu.m lines of GaAs, and 4-.mu.m SiO.sub.2
spacing in between each line (FIGS. 1A-1B). The G12-3 clones were
interacted with the GaAs/SiO2 patterned substrate, washed to reduce
non-specific binding, and tagged with an immuno-fluorescent probe,
tetramethyl rhodamine (TMR). The tagged phage were found as the
three lighter lines (red, if in color) and the center dot, in FIG.
1B, corresponding to G12-3 binding only to GaAs. The SiO.sub.2
regions of the pattern remain unbound by phage and are dark in
color. This result was not observed on a control that was not
exposed to phage, but was exposed to the primary antibody and TMR
(FIG. 1A). The same result was obtained using non-phage bound G12-3
peptide.
[0056] The GaAs clone G12-3 was observed to be substrate-specific
for GaAs over AlGaAs (FIG. 1C). AlAs and GaAs have essentially
identical lattice constraints at room temperature, 5.66 A.degree.
and 5.65 A.degree., respectively, and thus ternary alloys of
AlxGa1-xAs can be epitaxially grown on GaAs substrates. GaAs and
AlGaAs have zinc-blende crystal structures, but the G12-3 clone
exhibited selectivity in binding only to GaAs. A multilayer
substrate was used, consisting of alternating layers of GaAs and of
Al.sub.0.98Ga.sub.0.02As. The substrate material was cleaved and
subsequently reacted with the G12-3 clone.
[0057] The G12-3 clones were labeled with 20-nm gold-streptavidin
nanoparticles. Examination by scanning electron microscopy (SEM)
shows the alternating layers of GaAs and Al.sub.0.98Ga.sub.0.02As
within the heterostructure (FIG. 1C). X-ray elemental analysis of
gallium and aluminum was used to map the gold-streptavidin
particles exclusively to the GaAs layers of the heterostructure,
demonstrating the high degree of binding specificity for chemical
composition. In FIG. 1D, a model is depicted for the discrimination
of phage for semiconductor heterostructures, as seen in the
fluorescence and SEM images (FIGS. 1A-C).
[0058] The present invention demonstrates the powerful use of
phage-display libraries to identify, develop and amplify binding
between organic peptide sequences and inorganic semiconductor
substrates. This peptide recognition and specificity of inorganic
crystals has been extended to other substrates, including GaN, ZnS,
CdS, Fe.sub.3O.sub.4, Fe.sub.2O.sub.3, CdSe, ZnSe and CaCO.sub.3
using peptide libraries.
[0059] Bivalent synthetic peptides with two-component recognition
(FIGS. 1E-F) are currently being designed; such peptides have the
potential to direct nanoparticles to specific locations on a
semiconductor structure. These organic and inorganic pairs should
provide powerful building blocks for the fabrication of a new
generation of complex, sophisticated electronic structures.
[0060] Metallic and Magnetic Materials
[0061] In the present invention, specific binding and recognition
of binding molecules is extended in unexpected ways to metal
materials including but not limited to magnetic and ferromagnetic
materials, including particles and nanoparticles. A combinatorial
peptide phage display library expressing a large collection of
bacteriophage that expresses millions of different peptide
sequences on their surfaces was combined with biopanning techniques
to select specific peptide sequences that tightly and directly bind
to and recognize metal materials, including magnetic materials,
(e.g., Co, SmCo5, CoPt and FePt). The present inventors have found
that these magnetic material binding peptides can be used to
control the nucleation of inorganic materials, as has been
demonstrated in nature and in the III-V and II-VI semiconductors.
If proteins can be used to control the nucleation of magnetic
materials, then magnetic nanoparticles could be prepared much
cheaper and easier than using traditional methods. The
nanomolecular magnets and magnetic material may be used, e.g., for
micro or nanomachines, dynamos, generators, magnetic storage or any
other applications for material that are magnetic or may be
magnetized. Another use for these materials is to modify the
surface of magnetic materials. The peptides can act as linkers for
attaching other materials to the surface of the magnetic material,
allowing the self-assembly of complex nanostructures, which could
form the basis of novel electronic devices.
[0062] The present inventors have recognized that this approach of
selecting binding peptides (using combinatorial peptide libraries
and panning techniques) has not been used with magnetic materials,
and peptides have never been used to control the nucleation of
magnetic materials. There are currently many other techniques being
researched to synthesize magnetic nanoparticles. All of these
efforts are based on a high temperature synthesis that must be
performed in an inert atmosphere using expensive reagents and often
require further processing and purification after synthesis to
fabricate nanoparticles with the desired shape and crystallinity.
The result is that preparing magnetic nanoparticles in the
traditional fashion is very expensive and not conducive to scale
up. The approach presented herein can be performed at room
temperatures using inexpensive reagents yielding nanoparticles with
controlled crystallinity, making it a much cheaper approach to the
synthesis of magnetic nanoparticles. This approach may also be used
to control crystal structure and crystal orientation.
[0063] Peptide-mediated synthesis of magnetic materials provides a
much cheaper and environmentally friendly approach to the synthesis
of magnetic nanoparticles. The current protocol for preparing
magnetic nanoparticles is both time-consuming and expensive. In
addition, the current protocol yields nanoparticles that are coated
with organic surfactants. These surfactants are not amicable to
further modification of the nanoparticle. Advances in the field of
molecular biology have enabled the functionalization of peptides,
suggesting that nanoparticles grown from peptides will also be
easily functionalized, which facilitates their incorporation into
electronic devices and integration into magnetic memory
devices.
[0064] Current techniques for preparing magnetic nanoparticles are
expensive and time consuming requiring high temperatures, inert
atmospheres, expensive reagents, cumbersome purifications, and post
synthetic modifications. This new technique for preparing magnetic
nanoparticles using peptides to mediate particle formation
alleviates all of these concerns allowing much more rapid and
inexpensive particle synthesis. In addition, better control of
crystal structure and orientation is achievable.
[0065] Known techniques may be used to produce enough peptide to
prepare large quantities of nanoparticles. Genetically designed
organisms may be used to produce the peptide or peptides of
interest. The peptide(s) may be manufactured in one of the coat
proteins of, e.g., M13 bacteriophage. The bacteriophage may be
further designed or engineered to express the protein in additional
coat proteins. Furthermore, bacteria, such as E. coli, may be
engineered to express the peptides of interest in one or more
designs or at locations of interest. One distinct advantage of
using peptides for localizing or positioning the magnetic materials
made herein is that they do not have the limitations inherent in
semiconductor processing, which is generally limited to two
dimensions, e.g., using photolithography. The peptide(s) of the
present invention may be used in or about a matrix that permits the
three-dimensional positioning or synthesis of the peptides. These
peptides may then be formed as a film, in lines or striations,
layers, dots, in grooves, on the surface, sides or bottom of an
opening and the like.
[0066] Magnetic nanostructures have a variety of applications,
including memory devices, sensors, ferrofluids, etc. The materials,
particles, and nanoparticles described herein are applicable to all
of these fields.
[0067] Still further, the metallic and magnetic materials of the
invention can be used in methods of use in applications which
include the following. Additional applications include
therapeutics, diagnostics, engineering, chemical engineering
processing of reactions, cellular, and environmental applications.
For example, magnetic separations can be carried out (including
bulk separations in large scale processing of reaction processes).
Other applications include purifications, therapeutics,
biocompatibility, drug delivery, imaging contrast agents,
localization (in vivo) of magnetics which are externally
addressable. Drugs delivery can include the coupling of particles
to drugs or chemotherapeutics followed by localization in the body
by magnetic fields. Proper particle design can yield cellular
penetration. Another application is blood-urine detection. In
engineering applications, display devices can be made with
controlled aspect ratio magnetic particles coupled to optoactive
materials including fluorescent and birefringent materials. Sensor
devises can be made wherein binding events change the moment of
inertia for magnetic particles coupled to binding elements. The
moment of inertia change can be detected through polarization
decay, including use of a coupled optically active agent. Another
application is in storage. For example, memory can be made wherein
the readout involves response to time varying magnetic field. The
writing step may involve binding of a specific moiety to a specific
address. Cellular applications include cell modifications and cell
triggering. In cellular modification, the size of the magnetic
particle can be adjusted to allow penetration into the cell,
wherein the particle is coupled with a reagent. Magnetic fields can
be used as a motive force for penetration. This can be useful for
transfection procedures. In cellular triggering, the reagent
coupled with the magnetic particle can enter the cell and then time
varying magnetic fields can be used to trigger a reponse in the
cell.
[0068] Examples of magnetic separation include classical affinity
based separations in-vitro and localization of reagents in-vivo. In
affinity based separation, the magnetic nanoparticles can have an
advantage because of the smaller size and large aspect ratio, and
good control over size and shape distribution. Another advantage is
if the particles have high magnetic permitivity. The particle can
be long and can rotate in the magnetic field, thus generating
additional forces from the shape effect. More powerful separation
forces can be achieved per mg of reagent. In localization of
reagents in vivo, magnetic particles can be injected or ingested
coupled with reagents. External, spatially varying field can be
applied to a subject causing particles to collect in the region of
highest gradient B. Small size of particle plus reagent can allow
for reagent to access tissues or even penetrate cells.
[0069] More particularly, the present inventors have used
combinatorial peptide phage display libraries (i.e., large
collections of bacterial phage that express millions of different
peptide sequences on their surfaces) and biopanning techniques to
select specific peptide sequences that tightly bind directly to
magnetic materials (.epsilon.-Co, CoPt, FePt). By selecting and
identifying specific peptide sequences that interact with high
affinity to magnetic materials, one can quickly and easily identify
peptides that can potentially be used to control the nucleation of
magnetic nanostructures. Using peptides to control the nucleation
of magnetic nanoparticles enables the synthesis of magnetic
nanostructures under ambient conditions. The traditional protocols
for preparing magnetic nanoparticles often require elaborate
synthetic schemes and extensive purification, implying that
peptide-mediated nucleation would provide a much cheaper
alternative to nanoparticle synthesis.
[0070] One of the special advantages of the present invention is
that the peptides selected by this approach permit peptides to be
selected to bind specifically and directly to magnetic materials.
These peptides have demonstrated an ability to nucleate selectively
magnetic nanostructures with controlled crystallinity. To date, Co
nanoparticles have been prepared of hexagonally close packed Co,
and CoPt and FePt nanoparticles have been prepared with the layered
crystallinity traditionally associated with the Invar alloys. These
crystal structures exhibit the largest magnetic susceptibility of
their respective materials, and that these materials retain their
desirable magnetic properties at the nanometer length scale. These
properties make these materials excellent candidates for the
fabrication of next generation magnetic memory devices. Currently
memory devices are prepared using a CoCr alloy with a density of
16.3 Gb/in2. The smaller size of these nanoparticles conceivably
allows the construction of memory devices with a density in the
terabit/in2 range. With the present invention, SmCo5 nanoparticles
are prepared that possess HCP P6/mm crystallinity.
[0071] Using peptides to control the nucleation of the
nanoparticles also facilitates further functionalization of the
nanoparticles. Nanoparticles prepared in the traditional fashion
are often coated with hydrophobic surfactants making further
functionalization (activity or active group attachments) a
laborious process. Nanoparticles prepared as disclosed herein may
be coated with peptides, which are relatively easy to functionalize
using a variety of chemical and biological techniques, as known to
those of skill in the art. Further functionalization of these
nanoparticles allows their self-assembly into complex architectures
and memory devices.
[0072] The particles and nanoparticles prepared using peptides to
control their crystallinity possess the ability to revolutionize
the magnetic recording industry due to their small size, high
magnetic susceptibility and ease of preparation.
Example I
Peptide Preparation, Isolation, Selection and Characterization
[0073] Peptide selection. The phage display or peptide library was
contacted with the semiconductor, or other, crystals in
Tris-buffered saline (TBS) containing 0.1% TWEEN-20, to reduce
phage-phage interactions on the surface. After rocking for 1 hour
at room temperature, the surfaces were washed with 10 exposures to
Tris-buffered saline, pH 7.5, and increasing TWEEN-20
concentrations from 0.1% to 0.5%(v/v). The phage were eluted from
the surface by the addition of glycine-HCl (pH 2.2) 10 minute,
transferred to a fresh tube and then neutralized with Tris-HCl (pH
9.1). The eluted phage were titered and binding efficiency was
compared.
[0074] The phage eluted after third-round substrate exposure were
mixed with their Escherichia coli (E. coli) ER2537 host and plated
on LB XGal/IPTG plates. Since the library phage were derived from
the vector M13mp19, which carries the lacZ.alpha. gene, phage
plaques were blue in color when plated on media containing Xgal
(5-bromo-4-chloro-3-indoyl-.be- ta.-D-galactoside) and IPTG
(isopropyl-.beta.-D-thiogalactoside). Blue/white screening was used
to select phage plaques with the random peptide insert. Plaques
were picked and DNA sequenced from these plates.
[0075] Substrate preparation. Substrate orientations were confirmed
by X-ray diffraction, and native oxides were removed by appropriate
chemical specific etching. The following etches were tested on GaAs
and InP surfaces: NH.sub.4OH:H.sub.2O (1:10), HCl:H.sub.2O (1:10),
H.sub.3PO.sub.4:H.sub.2O.sub.2:H.sub.2O (3:1:50) at 1 minute and 10
minute etch times. The best element ratio and least oxide formation
(using XPS) for GaAs and InP etched surfaces was achieved using
HCl: H.sub.2O for 1 minute followed by a deionized water rinse for
1 minute. However, since an ammonium hydroxide etch was used for
GaAs in the initial screening of the library, this etch was used
for all other GaAs substrate examples. Si(100) wafers were etched
in a solution of HF:H.sub.2O 1:40 for one minute, followed by a
deionized water rinse. All surfaces were taken directly from the
rinse solution and immediately introduced to the phage library.
Surfaces of control substrates, not exposed to phage, were
characterized and mapped for effectiveness of the etching process
and morphology of surfaces by AFM and XPS.
[0076] Multilayer substrates of GaAs and of Al.sub.0.98Ga.sub.0.02
As were grown by molecular beam epitaxy onto GaAs(100). The
epitaxially grown layers were Si-doped (n-type) at a level of
5.times.10.sup.17 cm.sup.-3.
[0077] Antibody and Gold Labeling. For the XPS, SEM and AFM
examples, substrates were exposed to phage for 1 hour in
Tris-buffered saline then introduced to an anti-fd
bacteriophage-biotin conjugate, an antibody to the pIII protein of
fd phage, (1:500 in phosphate buffer, Sigma) for 30 minutes and
then rinsed in phosphate buffer. A streptavidin-20-nm colloidal
gold label (1:200) in phosphate-buffered saline (PBS, Sigma) was
attached to the biotin-conjugated phage through a
biotin-streptavidin interaction; the surfaces were exposed to the
label for 30 minutes and then rinsed several times with PBS.
[0078] X-ray Photoelectron Spectroscopy (XPS). The following
controls were done for the XPS examples to ensure that the gold
signal seen in XPS was from gold bound to the phage and not
non-specific antibody interaction with the GaAs surface. The
prepared GaAs(100) surface was exposed to three conditions: (1)
antibody and the streptavidin-gold label, but without phage; (2)
G1-3 phage and streptavidin-gold label, but without the antibody;
and (3) streptavidin-gold label, without either G1-3 phage or
antibody.
[0079] The XPS instrument used was a Physical Electronics Phi ESCA
5700 with an aluminum anode producing monochromatic 1,487-eV
X-rays. All samples were introduced to the chamber immediately
after gold-tagging the phage (as described above) to limit
oxidation of the GaAs surfaces, and then pumped overnight at high
vacuum to reduce sample outgassing in the XPS chamber.
[0080] Atomic Force Microscopy (AFM). The AFM used was a Digital
Instruments Bioscope mounted on a Zeiss Axiovert 100s-2tv,
operating in tip scanning mode with a G scanner. The images were
taken in air using tapping mode. The AFM probes were etched silicon
with 125-mm cantilevers and spring constants of 20.+-.100 Nm.sup.-1
driven near their resonant frequency of 200.+-.400 kHz. Scan rates
were of the order of 1.+-.5 mms.sup.-1. Images were leveled using a
first-order plane to remove sample tilt.
[0081] Transmission Electron Microscopy (TEM). TEM images were
taken using a Philips EM208 at 60 kV. The G1-3 phage (diluted 1:100
in TBS) were incubated with GaAs pieces (500 mm) for 30 minutes,
centrifuged to separate particles from unbound phage, rinsed with
TBS, and resuspended in TBS. Samples were stained with 2% uranyl
acetate.
[0082] Scanning Electron Microscopy (SEM). The G12-3 phage (diluted
1:100 in TBS) were incubated with a freshly cleaved
hetero-structure surface for 30 minutes and rinsed with TBS. The
G12-3 phage were tagged with 20 nm colloidal gold. SEM and
elemental mapping images were collected using the Norian detection
system mounted on a Hitachi 4700 field emission scanning electron
microscope at 5 kV.
EXAMPLE II
Biofilms
[0083] The present inventors have recognized that organic-inorganic
hybrid materials offer new routes for novel materials and devices.
Size controlled nanostructures give optically and electrically
tunable properties of semiconductor materials and organic additives
modify the inorganic morphology, phase, and nucleation direction.
The monodispersed nature of biological materials makes the system
compatible for highly ordered smectic-ordering structure. Using the
methods of the present invention, highly ordered nanometer scale as
well as multi-length scale alignment of II-VI semiconductor
material using genetically engineered, self-assembling, biological
molecules, e.g., M13 bacteriophage that have a recognition moiety
of specific semiconductor surfaces were created.
[0084] Using the compositions and methods of the present invention
nano- and multi-length scale alignment of semiconductor materials
was achieved using the recognition and self-ordering system
described herein. The recognition and self-ordering of
semiconductors may be used to enhance micro fabrication of
electronic devices that surpass current photolithographic
capabilities. Application of these materials include:
optoelectronic devices such as light emitting displays, optical
detectors and lasers; fast interconnects; and nano-meter scale
computer components and biological sensors. Other uses of the
biofilms created using the present invention include well-ordered
liquid crystal displays and organic-inorganic display
technology.
[0085] The films, fibers and other structures may even include
high-density sensors for detection of small molecules including
biological toxins. Other uses include optical coatings and optical
switches. Optionally, scaffoldings for medical implants or even
bone implants; may be constructed using one or more of the
materials disclosed herein, in single or multiple layers or even in
striations or combinations of any of these, as will be apparent to
those of skill in the art.
[0086] Other uses for the present invention include electrical and
magnetic interfaces, or even the organization of 3D electronic
nanostructures for high-density storage, e.g., for use in quantum
computing. Alternatively, high density and stable storage of
viruses for medical application that can be reconstituted, e.g.,
biologically compatible vaccines, adjuvants and vaccine containers
may be created with the films and or matrices created with the
present invention. Information storage based on quantum dot
patterns for identification, e.g., department of defense friend or
foe identification in fabric of armor or coding. The present
nanofibers may even be used to code and identify money.
[0087] Building well-ordered, well-controlled, two and three
dimensional structure at the nanolength scale is the major goal of
building next generation optical, electronic and magnetic materials
and devices. Current methods of making specific nanoparticles are
limited in terms of both length scale and the types of materials.
The present invention exploits the properties of self-assembling
organic or biological molecules or particles, e.g., M13
bacteriophage to expand the alignment, size, and scale of the
nanoparticles as well as the range of semiconductor materials that
can be used.
[0088] The present inventors have recognized that monodisperse
biomaterials having anisotropic shapes are an alternative way to
build well-ordered structures. Nano- and multi-length scale
alignment of II-VI semiconductor material were accomplished using
genetically engineered M13 bacteriophage that possess a recognition
moiety (a peptide or amino acid oligomer) for specific
semiconductor surfaces.
[0089] Seth and coworkers have characterized Fd virus smectic
ordering structures that have both a positional and directional
order. The smectic structure of Fd virus has potential application
in both multi-scale and nanoscale ordering of structures to build
2-dimensional and 3-dimensional alignment of nanoparticles.
Bacteriophage M13 was used because it can be genetically modified,
has been successfully selected to have a shape identical to the Fd
virus, and has specific binding affinities for II-VI semiconductor
surfaces. Therefore, M13 is an ideal source for smectic structure
that can serve in multi-scale and nanoscale ordering of
nanoparticles.
[0090] The present inventors have used combinatorial screening
methods to find M13 bacteriophage containing peptide inserts that
are capable of binding to semiconductor surfaces. These
semiconductor surfaces included materials such as zinc sulfide,
cadmium sulfide and iron sulfide. Using the techniques of molecular
biology, bacteriophage combinatorial library clones that bind
specific semi-conductor materials and material surfaces were cloned
and amplified up to concentrations high enough for liquid crystal
formation.
[0091] The filamentous bacteriophage, Fd, has a long rod shape
(length: 880 nm; diameter: 6.6 nm) and monodisperse molecular
weight (molecular weight: 1.64.times.10.sup.7). These properties
result in the bacteriophage's lyotropic liquid crystalline behavior
in highly concentrated solutions. The anisotrophic shape of
bacteriophage was exploited as a method to build well-ordered
nanoparticle layers by use of biological selectivity and
self-assembly. Monodisperse bacteriophage were prepared through
standard amplification methods. In the present invention, M13, a
similar filamentous bacteriophage, was genetically modified to bind
nanoparticles such as zinc sulfide, cadmium sulfide and iron
sulfide.
[0092] Mesoscale ordering of bacteriophage has been demonstrated to
form nanoscale arrays of nanoparticles. These nanoparticles are
further organized into micron domains and into centimeter length
scales. The semiconductor nanoparticles show quantum confinement
effects, and can be synthesized and ordered within the liquid
crystal.
[0093] Bacteriophage M13 suspension containing specific peptide
inserts were made and characterized using AFM, TEM, and SEM.
Uniform 2D and 3D ordering of nanoparticles was observed throughout
the samples.
[0094] AFM. Includes Digital Instruments Bioscope mounted on a
Zeiss Axiovert 100s-2tv, operating in tip scanning mode with a G
scanner. The images were taken in air using tapping mode. The AFM
probes were etched silicon with 125 mm cantilevers and spring
constants of 20.+-.100 Nm.sup.-1 driven near their resonant
frequency of 200.+-.400 kHz. Scan rates were of the order of 1.+-.5
mms.sup.-1. Images were leveled using a first-order plane to remove
sample tilt. FIGS. 2A and 2B are schematic diagrams of the smectic
alignment of M13 phages observed using AFM.
[0095] TEM. TEM images were taken using a Philips EM208 at 60 kV.
The G1-3 phage (diluted 1:100 in TBS) were incubated with
semiconductor material for 30 minutes, centrifuged to separate
particles from unbound phage, rinsed with TBS, and resuspended in
TBS. Samples were stained with 2% uranyl acetate.
[0096] SEM. The phage (diluted 1:100 in TBS) were incubated with a
freshly cleaved hetero-structure surface for 30 minutes and rinsed
with TBS. The G12-3 phage were tagged with 20 nm colloidal gold.
SEM and elemental mapping images were collected using the Norian
detection system mounted on a Hitachi 4700 field emission scanning
electron microscope at 5 kV.
[0097] Genetically engineered M13 bacteriophage that had specific
binding properties to semiconductor surfaces was amplified and
purified using standard molecular biological techniques. 3.2 mL of
bacteriophage suspension (concentration: .about.10.sup.7
phages/.mu.L) and 4 mL of overnight culture were added to 400 mL LB
medium for mass amplification. After amplification, .about.30 mg of
pellet was precipitated. The suspensions were prepared by adding
Na.sub.2S solutions to ZnCl.sub.2 doped A7 phage suspensions at
room temperature. The highest concentration of A7-phage suspension
was prepared by adding 20 .mu.L of 1 mM ZnCl.sub.2 and Na.sub.2S
solutions, respectively into the -30 mg of phage pellet. The
concentration was measured using extinction coefficient of 3.84
mg/mL at 269 nm.
[0098] As the concentration of the isotropic suspension is
increased, nemetic phase that has directional order, cholesteric
phase that has twisted nemetic structure, and smectic phase that
has directional and positional orders as well, are observed. These
phases had been observed in Fd viruses that did not have
nanoparticles.
[0099] Polarized optical microscopy (POM). M13 phage suspensions
were characterized by polarized optical microscope. Each suspension
was filled to glass capillary tube of 0.7 mm diameter. The highly
concentrated suspension (127 mg/mL) exhibited iridescent color [5]
under the paralleled polarized light and showed smectic texture
under the cross-polarized light (FIG. 3A). The cholesteric pitches
in FIG. 3B can be controlled by varying the concentration of
suspension as shown in TABLE 2. The pitch length was measured and
the micrographs were taken after 24 hours later from the
preparation of samples.
1TABLE 2 Cholesteric pitch and concentration relationship.
Concentration Pitch length (mg/ml) (um) 76.30 31.9 71.22 51.6 56.38
84.8 50.52 101.9 43.16 163.7 37.04 176.1 27.54 259.7
[0100] AFM. For AFM observation, 5 .mu.L of M13 suspension
(concentration: 30 mg/mL) of M13 bacteriophage suspension was dried
for 24 hours on the 8 mm.times.8 mm mica substrate that was silated
by 3-amino propyl triethyl silane for 4 hours in the dessicator.
Images were taken in air using tapping mode. Self-assembled
ordering structures were observed due to the anisotropic shape of
M13 bacteriophage, 880 nm in length and 6.6 nm in width. In FIG.
3C, M13 phage lie in the plane of the photo and form smectic
alignment.
[0101] SEM. For SEM observation, the critical point drying samples
of bacteriophage and ZnS nanoparticles smectic suspension
(concentration of bacteriophage suspension 127 mg/mL) were
prepared. In FIG. 3D, nanoparticles rich areas and bacteriophage
rich areas were observed. The length of the separation between
nanoparticles and bacteriophage correspond to the length of
bacteriophage. The ZnS wurzite crystal structure was confirmed by
electron diffraction pattern using dilution sample of the smectic
suspension with TEM (FIGS. 3E and 3F).
[0102] Preparation of the biofilm. Bacteriophage pellets were
suspended with 400 .mu.L of Tris-buffered saline (TBS, pH 7.5) and
200 .mu.L of 1 mM ZnCl.sub.2 to which 1 mM Na.sub.2S was added.
After rocking for 24 hours at room temperature, the suspension
(contained in a 1 mL eppindorff tube) was slowly dried in a
dessicator for one week. A semi-transparent film .about.15 .mu.m
thick was formed on the inside of the tube. This film, shown in
FIG. 4A, was carefully taken using a tweezers. A schematic diagram
of the biofilm is shown in FIG. 4B.
[0103] SEM observation of biofilm. Nanoscale bacteriophage
alignment of the A7-ZnS film were observed using SEM. In order to
carry out SEM analysis the film was cut then coated via vacuum
deposition with 2 nm of chromium in an argon atmosphere. Highly
close-packed structures, FIG. 4D were observed throughout the
sample. The average length of individual phage, 895 nm is
reasonable analogous to that of phage, 880 nm. The film showed the
smectic like A- or C-like lamellar morphologies that exhibited
periodicity between the nanoparticle and bacteriophage layers. The
length of periodicity corresponded to that of the bacteriophage.
The average size of nanoparticle is .about.20 nm analogous to the
TEM observation of individual particles.
[0104] TEM observation of biofilm. ZnS nanoparticle alignment was
investigated by embedding the film in epoxy resin (LR white) for
one day and polymerized by adding 10 .mu.l of accelerator. After
curing, the resin was thin sectioned using a Leica Ultramicrotome.
These .about.50 nm sections were floated on distilled water, and
picked up on blank gold grids. Parallel-aligned nanoparticles in a
low, which corresponded to x-z plane in the schematic diagram, were
observed, FIG. 4E-F. Since each bacteriophage had 5 copies of the
A7 moieties, each A7 recognize one nanoparticle (2.about.3 nm size)
and aligned approximately 20 nm in a width and extended to more
than two micrometers in length. The two micrometers by 20 nm bands
formed in parallel each band separated by .about.700 nm. This
discrepancy may come from the tilted smectic alignment of the phage
layers with respect to observation in the TEM, which is reported by
Marvin group. A y-z axis like nanoparticle layer plane was also
observed similar to that shown in FIG. 1F. The SAED patterns of the
aligned particles showed that the ZnS particles have the wurzite
hexagonal structure.
[0105] AFM observation of biofilm: The surface orientation of the
viral film was investigated using AFM. In FIG. 4C, phage were shown
to have formed an parallel aligned herringbone pattern that have
almost right angle between the adjacent director normal
(bacteriophage axis) on most of surface that is named as smectic O.
The film showed long range ordering of normal director that is
persistent to the tens of micrometers. In some of areas where two
domain layers meet each other, two or three multi-length scale of
bacteriophage aligned paralleled and persistent to the smectic C
ordering structure.
[0106] Nano and multi-length scale alignment of semiconductor
materials using the recognition and as well as self-ordering system
enhances the future microfabrication of electronic devices. These
devices have the potential to surpass current photolithographic
capabilities. Other potential applications of these materials
include optoelectronic devices such as light-emitting displays,
optical detectors, and lasers, fast interconnects, nano-meter scale
computer component and biological sensors.
EXAMPLE III
Formation of Metallic and Magnetic Materials
[0107] A phage display technique was used to discover novel
peptides that bind selectively to magnetic materials. In these
particular studies, films of the magnetic materials were prepared
by first synthesizing colloidal dispersions of the magnetic
materials. These colloidal solutions were then drop coated onto Si
wafers and annealed under N.sub.2 to generate the desired crystal
structure. Phage display was then performed on these films
(.epsilon.-Co, CoPt, and FePt), and peptides were discovered that
bind selectively to each substrate. These peptides were then used
to nucleate unique nanoparticles by mixing the phage expressing the
peptide of interest, the metal salt, and a reducing agent.
[0108] The synthesis of nanoparticles with controlled size and
composition is of fundamental and technological interest. In the
last few years there has been a flurry of papers describing the
synthesis of nanoparticles composed of metals and semiconductors
with remarkable control over the size and shape of the resulting
nanoparticles. Recently it has been shown that peptides identified
via phage display can bind selectively to inorganic surfaces and
can be used to control the nucleation of semiconducting
nanoparticles. In this case, the peptides can control the size,
shape, composition, and even the crystallinity of the resulting
nanoparticles. Due to the success of peptides in controlling the
synthesis of semiconducting nanoparticles, there is a great deal of
interest in applying the technology to other materials of
interest.
[0109] One particularly interesting and commercially useful class
of materials is ferromagnets, including particles and
nanoparticles. Ferromagnetic materials are the cornerstone of the
billion dollar per year magnetic recording industry. Current
devices use a CoCr alloy for data storage because of the high
magnetic susceptibility and ease of preparation. Other materials
are currently in development. One such material is metallic Co,
which has a magnetic anisotropy in the range of 10.sup.7
ergs/cm.sup.3. This high magnetic anisotropy suggests that
particles as small as 10 nm in diameter, can act as single domains
and function as memory elements. Current technology uses memory
elements with a domain size that is in the range of hundreds of
nanometers, so generating Co nanoparticles in the 10 nm size range
would be a dramatic improvement that would lead to much denser
memory devices. More interesting ferromagnetic materials are the
magnetic alloys of Pt, specifically FePt and CoPt. These materials
have very large magnetic anisotropies (10.sup.8 ergs/cm.sup.3), due
to the Invar effect, in which perturbations in the lattice constant
caused by the layering of Fe and Pt atoms causes the Pt to develop
a magnetic state. The large anisotropy possessed by these systems
suggests that nanoparticles as small as 2 nm can act as
ferromagnets at room temperature, implying that they can be used in
the development of very high-density memory devices.
[0110] Due to the large magnetic anisotropies of these systems, a
great deal of effort has been invested in the synthesis of
particles and nanoparticles composed of these materials. Several
different synthetic protocols have been developed for .epsilon.-Co,
FePt and CoPt and they all possess the same fundamental weaknesses.
All of these synthetic strategies rely on the restricted
precipitation of nanoparticles in the presence of surfactants at
elevated temperatures. All of these nanoparticle preparations must
be performed in an inert atmosphere with expensive reagents, making
them very expensive and not amicable to scale up. Furthermore,
these preparations often require further modifications of the
particles, including high temperature annealing to attain the
desired crystallinity, and size selective precipitation to acquire
monodisperse populations of particles. These extra synthetic steps
increase the cost of these synthetic strategies.
[0111] Since these materials are commercially important, a novel
synthetic strategy was desired. Applying the principle of
peptide-mediated synthesis to magnetic materials provides such an
alternative. In these studies phage display selection was performed
on the magnetic materials of interest (Co, CoPt, SmCo5, and FePt)
to identify peptides that specifically bind to the magnetic
materials with high affinity. After characterization, these
peptides were then used to control the nucleation of magnetic
nanoparticles. In these studies, phage expressing the peptides of
interest were mixed with the metallic salts of the metals of
interest. A reducing agent (NaBH4) was then added to generate the
nanoparticles. The nanoparticles were formed and characterized
using TEM. The synthesis of the present invention was performed
under ambient conditions to provide a much cheaper alternative to
existing synthetic strategies for generating magnetic
nanoparticles.
[0112] X-Ray Diffraction Analysis of Magnetic Nanoparticles
[0113] Magnetic surfaces had to be generated to use as substrates
in the phage display. To accomplish this, magnetic nanoparticles
were prepared in the traditional fashion, and drop coated onto Si
wafers. Before the phage display studies were begun, the surfaces
were characterized with x-ray diffraction (XRD) to ensure the
material possessed the appropriate crystallinity.
[0114] The XRD pattern obtained for .epsilon.-Co correlated well
with patterns obtained from the literature, displaying a triplet of
peaks between 45 degrees and 50 degrees that are particularly
distinctive because they correspond to the (221), (310), and (311)
crystal planes of .epsilon.-Co. The FePt and CoPt patterns also
agreed with the literature spectra for FePt11 with peaks
corresponding to the (001), (110), (111), (200), (002), (210),
(112), and (202) planes of FePt and CoPt. The XRD on SmCo5 agreed
with literature values for HCP SmCo5 with peaks representing the
(101), (110), and (111) facets. This is the first reported
synthesis of HCP SmCo5 nanoparticles. FIG. 5A is a high resolution
TEM image of a SmCo5 nanoparticle and FIG. 5B is a selected area of
the TEM image showing the electron diffraction pattern. Several
spots in the diffraction pattern correlate well with the known
facets of HCP SmCo5 (FIG. 51B). FIG. 5C is a STEM image of the
annealed SmCo5 nanoparticles and illustrates their size, shape, and
overall morphology.
[0115] Sequence Analysis and Binding Assays of Binding Phage
[0116] TABLE 3 lists all of the peptides that were selected using
phage display for their ability to bind to the magnetic materials
of interest.
2TABLE 3 Selected clones with magnetic binding properties.
7-Constrained Material Sequence 12mer Sequence .epsilon.-Co *
ALSPHSAPLTLY (SEQ ID NO. :15) CoPt NAGDHAN SVSVGMKPSPRP (SEQ ID NO.
:12) (SEQ ID NO. :16) FePt SKNSNIL HNKHLPSTQPLA (SEQ ID NO. :13)
(SEQ ID NO. :17) SmCo5 TKPSVVQ WDPYSHLLQHPQ (SEQ ID NO. :14) (SEQ
ID NO. :18) *No consensus sequence was obtained for the
7-constrained library on .epsilon.-Co.
[0117] All of the selected sequences appear to be valid sequences
that should possess high affinity for the metallic surfaces.
Histidine residues appear in several of the sequences. Due to its
imidazole side group, histidine is an excellent ligand for metals,
so its presence in these sequences is expected. With the exception
of the 7-constrained sequence on CoPt, all of the sequences
isolated for the Pt alloys contain a lysine residue. Lysine-Pt
interactions are believed to be important in the function of
cisplatin, an important anticancer drug. The Lysine-Pt interaction
suggests that these sequences bind selectively to these materials,
however, the present invention is not limited to any mechanism of
interaction, known or unknown.
[0118] Specific Binding Assays. To determine the affinity of the
isolated phage for the magnetic substrate, two studies were
performed. In the first study several different peptide-containing
phage were exposed to a Co surface including our Co specific phage,
a random phage, and wild type phage.
[0119] Additionally, the Co-specific phage was exposed to several
different material surfaces. The results are depicted in FIG. 6.
The Co-specific phage possessed a relative higher affinity for Co
than either the wild-type phage or a random phage library sequence
(FIG. 6A). Additionally, the Co-specific phage displayed a greater
affinity for Co than for Si, suggesting they bound preferentially
to the Co surface.
[0120] In the second study a Co surface was immersed into a
solution of the Co-specific phage. This study was repeated at
several different concentrations of phage. Plotting the amount of
adsorbed phage vs. the concentration of phage (FIG. 6B) indicated
that the adsorption of phage onto the Co surface followed the
Langmuir model for adsorption of analytes on a surface. Since the
adsorption is Langmuirian, generating a reciprocal plot revealed a
linear correlation between the adsorbed phage and the concentration
(not shown). The slope of this line is equal to the binding
constant, and in the case of Co, the phage possessed a k.sub.ads of
2.times.10.sup.-12 M. This is the first measurement of the
thermodynamic properties associated with the binding between a
phage and an inorganic surface, making it difficult to interpret,
but the magnitude of this binding constant is comparable to several
other biological interactions. This approach may be used for the
CoPt and FePt systems.
[0121] Both studies showed that the peptides selected using phage
display screening possessed specific binding towards Co and not
towards other materials. It is this specificity that can be used to
direct metal materials formation, including magnetic materials.
[0122] TEM Analysis of Nanoparticles Prepared Through
Peptide-Mediated Nucleation
[0123] In one embodiment of the present invention, nanoparticles
were prepared using peptides to modify and/or control
crystallinity. High resolution TEM images of CoPt nanoparticles
grown using the 7-constrained sequence are shown in TABLE 3 were
also taken (not shown). These nanoparticles had lattice spacings of
0.19 and 0.22 nm, which correlates with the lattice spacing of L10
CoPt.
[0124] High resolution TEM images of nanoparticles grown using wild
type phage were also taken as were images of CoPt nanoparticle
grown using phage with a random peptide insert (not depicted). In
both control studies, nanoparticles still form, but they lacked the
crystallinity that the particles grown with the CoPt selective
peptide possess. Nanoparticles grown in the absence of phage
aggregate and precipitate out of solution, making TEM imaging
nearly impossible.
[0125] High-resolution TEM images were also taken of FePt
nanoparticles grown using the phage that expresses the 12mer
peptide, which is selective for FePt (not depicted). These
nanoparticles exhibited similar lattice spacing to the CoPt
nanoparticles suggesting they are composed of L10 FePt. Electron
diffraction patterns were taken of these same particles, e.g., FePt
nanoparticles grown in the presence of wild type phage (not
depicted). Again, these nanoparticles lack the crystallinity of the
nanoparticles grown with the FePt selective phage. Also,
nanoparticles grown in the absence of phage aggregate and
precipitate out of solution before they could be imaged.
[0126] High resolution TEM images of CoPt nanoparticles grown using
the 7-constrained sequence from Table 1 are shown in FIG. 7. The
lattice spacing in these nanoparticles is at or about 0.22 nm and
correlating well with literature values for HCP Co of approximately
0.19 nm (FIG. 7A) and with the lattice spacing of L1.sub.0 CoPt. A
selected area was also used to observe the electron diffraction
pattern of the nanoparticles (not shown). Several bands were
present in the diffraction pattern that correlate with the facets
of HCP Co and indicate that the nanoparticles were, in fact,
composed of HCP Co. In control experiments with either wild-type
phage (FIG. 7C), nonspecific phage (FIG. 7B), nanoparticles still
form, but lack the crystallinity that the particles grown with the
CoPt selective peptide possess. Nanoparticles grown in the absence
of phage aggregate and precipitate out of solution, making TEM
imaging nearly impossible.
[0127] FIG. 8 shows high resolution TEM images of Co nanoparticles
grown using the phage that expressed the 12 mer peptide that binds
specifically to Co (FIG. 8A). The lattice spacing in these
particles is 0.2 nm, which correlates well with the literature
values for HCP Co (0.19 nm). A selected area is chosen for electron
diffraction pattern for these nanoparticles (FIG. 8B). Several
bands are present in the diffraction pattern that correlate with
the facets of HCP Co, indicating that the nanoparticles are
composed of HCP Co. In control experiments involving either
wild-type phage, nonspecific phage, or no phage, Co particles
aggregate and sediment out of solution (not shown).
[0128] FIG. 9A shows a high resolution TEM image of FePt
nanoparticles grown using phage that expressed a 12mer peptide
selective for FePt. These nanoparticles exhibit similar lattice
spacing to the CoPt nanoparticles and were likely composed of
L1.sub.0 FePt. FIG. 9B is the corresponding electron diffraction
pattern, and FIG. 9C an image of FePt nanoparticles grown in the
presence of wild type phage. In the absence of wild-type phage,
nanoparticles lacked the crystallinity of the nanopaticles grown
with the FePt-selective phage. In addition, nanoparticles grown in
the absence of phage aggregated and precipitated out of solution
before they can be imaged.
[0129] High resolution TEM images were also taken of SmCo5
nanoparticles grown using phage that expresses the 12 mer peptide
that is specific to SmCo5 (FIG. 10A). A selected area was used to
observe the electron diffraction pattern (FIG. 10B). Again, the
diffraction pattern showed several bands that correlated with the
facets of HCP SmCo5. Control experiments performed with the SmCo5
system yielded results similar to that observed for the Co system,
such that nanoparticles aggregated and/or precipitated out of
solution when nonspecific phage were used. TEM images of such
particles showed some crystalline domains, but the majority of the
material was amorphous.
[0130] MFM Characterization of Nanoparticles
[0131] Magnetic Force Microscopy (MFM) was used to characterize the
magnetic properties of the nanoparticles. Atomic force images of
phage that were used to nucleate Co nanoparticles were first taken
(FIG. 11A). A large aggregate of nanoparticles was evident at the
end of the phage, indicating that the P3 proteins were controlling
the nucleation of the nanoparticles as expected. Corresponding MFM
image was taken to confirm these results (FIG. 11B)). Here, the
phage could not be seen because they were non-magnetic, but the
aggregate of nanoparticles was still clearly visible, indicating
the nanoparticles possess a high degree of magnetic anisotropy.
[0132] SQUID. In one embodiment of the present invention, the
magnetic properties of the nanoparticles may be quantified using a
Superconducting Quantum Interference Device (SQUID) magnetometer.
SQUID magnetrometry was used to further characterize the particles.
With SQUID, a room temperature hysteresis loop for FePt
nanoparticles grown using the 12mer peptide expressed on phage was
taken (FIG. 12A). A high-resolution hysteresis loop of the central
portion of the scan was also taken to clarify the presence of the
coercivity (FIG. 12B). These samples possessed relatively low
coercivity (approximately 50 Oe). The data represents the first
example of ferromagnetic nanoparticles grown under ambient
conditions. Hysteresis loops were also measured on biologically
prepared SmCo.sub.5 nanoparticles (FIG. 13). The hystersis was much
larger for these nanoparticles (400 Oe). This result was expected
since macroscopic samples of SmCo.sub.5 typically display higher
coercivity values than FePt.
[0133] Magnetic-Specific Peptides on P8 Coat Proteins
[0134] In one embodiment of the present invention, nanoparticles
with magnetic behaviors are prepared using the material-specific
phage that were expressed on the p3 protein of M13 bacteriophage.
The p3 protein is only present on one end of the rod-shaped phage
and is present in limited numbers (3-5 copies per phage).
Alternatively, the p8 coat protein is expressed along the length of
the phage, and there are hundreds of copies per phage. For this
reason, the p8 protein was engineered to express a CoPt-specific
peptides, and CoPt nanoparticles were nucleated along the length of
the phage. One example of the material preparation is presented
below. Other methodologies apparent to those of ordinary skill in
the art of material and biologic sciences may be used without undue
experimentation.
[0135] Upon nucleation of magnetic materials, including magnetic
particles and nanoparticles, the peptides, with or without phage,
can be heated to sufficiently high temperatures to burn off and
eliminate the binding molecules associated with the scaffold in a
high temperature annealing process. For example, heating to
500.degree. C. or 1,000.degree. C. can be carried out for times
which provide optimum burn off and elimination. The temperatures
can be also in the range for metal annealing, whereby
polycrystalline domains can fuse into single crystalline
domains.
[0136] Methodology.
[0137] Materials. Samarium (III) Chloride, Platinum (II)
Acetylacetonate (Pt(Acac).sub.2, Dihydrogen Hexachloroplatinate
(H.sub.2PtCl.sub.6), and Cobalt Octacarbonyl (CO.sub.2(CO).sub.8)
were purchased from Alfa Aesar. Iron Pentacarbonyl (Fe(CO).sub.5),
Cobalt (II) Chloride (COCl.sub.2), Iron (II) Chloride (FeCl.sub.2),
Trioctylphosphine oxide (TOPO), Sodium Borohydride (NaBH.sub.4),
oleyl amine, and oleic acid were purchased from Aldrich.
[0138] Nanoparticle Synthesis of .epsilon.-Co. Co nanoparticles
were prepared by first dissolving 0.6 g of CO.sub.2(CO).sub.8 in 5
mL of o-dichlorobenzene. This mixture was stirred for one hour to
dissolve the Co and 20 mL of o-dichlorobenzene, 0.416 g of TOPO,
and 0.2 mL of oleic acid were mixed in a 500 mL three-necked
reaction vessel under Ar. This mixture was then heated to 100
degrees Centigrade. The mixture was then exposed to vacuum for 5
minutes to remove any dissolved O.sub.2 and H.sub.2O. The mixture
was then heated to boiling (180 degrees Centigrade), and Co
solution was added. The mixture turned black and generated a cloud
of CO gas. After 20 minutes of refluxing, the reaction was cooled
to room temperature. To purify the particles, 3 mL of Co
nanoparticle solution was mixed with 3 mL of ethanol. After 1 hour,
the mixture was centrifuged at 10,000 rpm for 5 minutes. The
precipitant was resuspended in 3 mL of CH.sub.2Cl.sub.2 followed by
3 ml of ethanol and the centrifugation step was repeated. The
precipitant was then resuspended in 3 mL of CH.sub.2Cl.sub.2.
[0139] Nanoparticle Synthesis of FePt. 20 mL of phenyl ether, 0.205
g of Pt(Acac).sub.2, and 0.358 g of 1,2-tetradecanediol were mixed
and heated to 100 degrees Centigrade under Ar after which 0.16 mL
of oleic acid, 0.17 mL of oleyl amine, and 0.13 mL of Fe(CO).sub.5
were added. The mixture was heated to 300 degrees Centigrade and
refluxed for 30 minutes and allowed to cool to room temperature.
FePt nanoparticles were purified in a similar fashion to the Co
nanoparticles
[0140] Nanoparticle Synthesis of CoPt. Preparation was identical to
FePt, except 0.16 g of CO.sub.2(CO).sub.8 was substituted for 0.13
mL of Fe(CO).sub.5.
[0141] Nanoparticle Synthesis of SmCo5. An arrested precipitation
approach was taken to prepare nanoparticles of SmCo5. This
technique was adapted from previous efforts at preparing
nanoparticles. 38.75 mg of CoCl.sub.2 was mixed with 16.0 mg of
SmCl.sub.3 and dissolved in 20 mL of phenyl ether. 0.357 mL of
oleic acid was then added to the mixture, which was then heated to
100 degrees Centigrade under Ar. 1.35 mL of trioctylphosphine was
then added. The mixture was then exposed to vacuum for ten minutes
to remove any remaining dissolved O.sub.2 or H.sub.2O from
solution. After purging the solution with vacuum, it was heated to
a 290 degrees Centigrade to boil the phenyl ether. 1 mL of
superhydride solution was then added. The solution turns from blue
to black immediately. The black mixture was then refluxed for 20
minutes and allowed to cool to room temperature
[0142] Film Formation. To prepare films for phage display
selection, a colloidal solution of nanoparticles was drop coated
onto a Si slide. The solvent was allowed to evaporate. In the case
of FePt and CoPt, the slides were then annealed at 700 degrees
Centigrade for 30 minutes under N.sub.2 to form the L10 phase. XRD
analysis was performed on all of these slides to ensure they were
the proper material.
[0143] Peptide Selection. The use of a phage display library
technique was used to find peptides that bind exclusively to
.epsilon.-Co, and the L10-phase of CoPt and FePt. Specifically, the
Ph.D.-12(tm) and Ph.D.-7 CTM Phage Display Peptide Library Kits
were used beginning with 1 .mu.L (or an initial amount) of phage
display library to initiate selection against the magnetic
substrates (in 1 mL of TBS). For .epsilon.-Co, selections were
performed in a 10 mM solution of NaBH.sub.4 in TBST. After five
rounds of panning, peptides and DNA of the peptides were isolated
and sequences were obtained from the University of Texas DNA Core
Facility. These sequences, which correspond to the peptides
displayed on the bacteriophage, underwent analysis to determine
consensus sequences. Analysis of the DNA sequences consisted of
percent abundance of amino acid per position. Because of the
possibility of non-specific binding in the first two rounds,
analysis was only performed on the last three rounds of
panning.
[0144] Binding Affinity. To determine that the peptides bind
specifically to .epsilon.-Co, CoPt, and FePt, binding affinity was
determined. Titer counts were obtained from consensus peptide
panning studies and compared to titer counts of WT and random
peptides not raised to .epsilon.-Co, CoPt, and FePt. Panning
studies were then performed using varying concentrations of phage
to determine the binding constant of the phage to the metallic
surface of interest.
[0145] Peptide-Mediated Nucleation of Co. Approximately 880 ul of
H.sub.2O were mixed with 100 .mu.L of 1 mM CoCl.sub.2 and 20 .mu.L
of phage solution (pfu=1011). The mixture was gently agitated for
30 minutes, and then 100 .mu.L of 100 mM NaBH.sub.4 was added. The
solution was vortexed, and allowed to incubate for another 5
minutes. 100 mL of a solution of TOPO and oleic acid dissolved in
CH.sub.2Cl.sub.2 was then added. The mixture was vortexed and
gently agitated for 1 hour. Over this time period the
CH.sub.2Cl.sub.2 layer changed to dark grey. This was repeated with
several different phage, including Co-1, Co-2, wild type phage, and
a TBS solution containing no phage.
[0146] Peptide-Mediated Nucleation of CoPt. For nucleation, 50
.mu.L of 1 mM CoCl.sub.2 solution was mixed with 50 .mu.L of 1 mM
H.sub.2PtCl.sub.6 solution. 10 ml of phage solution was then added
(pfu=1011). The mixture was agitated gently for 30 min, and 20
.mu.L of 100 mM NaBH.sub.4 was then added. The solution was
immediately vortexed and placed on a tumbler for 30 min. The final
solution was yellow in color.
[0147] Peptide-Mediated Nucleation of FePt. FePt was prepared in a
similar fashion to CoPt, except a FeCl.sub.2 solution was used in
place of COCl.sub.2.
[0148] Peptide-Mediated Nucleation of SmCo5. Identical to Co
synthesis except 100 .mu.L of 1 mM CoCl.sub.2 was replaced with
16.7 .mu.L of 1 mM SmCl.sub.3 and 83 .mu.l of 1 mM CoCl.sub.2.
[0149] P8 Expression of Peptides. Genetically modified E. coli were
amplified overnight in 20 mL LB media, diluted 1:100 and then grown
to O.D.=0.6. Tetracycline-HCl (1000.times.) and 100 mM IPTG was
added to a final concentration of 1 mM. The IPTG triggers the
production of the modified p8 protein within the cell for their
incorporation into the viral coat during assembly. The mixture is
allowed to rest for 1 hour without shaking. Infection by the helper
phage after 1 hour is then followed by shaking overnight at 39
degrees Centigrade. Phage are then separated and purified by
centrifugation and PEG precipitation. The amplified phage pellet is
resuspended into 10 mL of TBS (pH 7.5) and dialyzed in 18 MW water.
0.5 mL of both 5 mM CoCl.sub.2 and 5 mM H.sub.2PtCl.sub.6 is added
to 1 mL of amplified phage stock which has been spun down and the
supernatant removed. This is allowed to shake for 60 minutes, after
which 0.5 mL of 100 mM NaBH4 is added as a reducing agent.
[0150] TEM images of the nanoparticles were taken along with the
selected area electron diffraction pattern that showed many bands
corresponding to the expected values for the CoPt facets. An STEM
image of one of these phage with CoPt nanoparticle grown along its
P8 proteins was also taken. The length of this structure correlates
to the length of a phage (800 nm). FIG. 14A depicts the TEM image
of the nanoparticles, and FIG. 14B the resolution image with the
selected area electron diffraction pattern (FIG. 14C) showing many
bands corresponding to the expected values for the CoPt facets. The
STEM image of one of these phage with CoPt nanoparticles grown
along its P8 proteins is shown in FIG. 14D. The EDS mapping for Pt
(FIG. 14E) and Co (FIG. 14F) indicate that Co and Pt are both found
along the length of the structure in equal concentrations.
[0151] The present invention illustrates phage display may be used
to identify peptides that bind to magnetic materials. The
identification is rapid and cost-effective and requires few
additional materials. These peptides may then be used to control
the nucleation of magnetic nanoparticles, granting the user control
over the size, composition, and crystallinity of the resulting
nanoparticles. These peptides allow the synthesis of nanoparticles
under ambient conditions, making them a desirable alternative to
current synthetic strategies.
[0152] Phage display libraries and experimental methods for using
them in biopanning are further described, for example, in the
following U.S. patent publications to Belcher et al.: (1)
"Biological Control of Nanoparticle Nucleation, Shape, and Crystal
Phase"; 2003/0068900 published Apr. 10, 2003; (2) "Nanoscale
Ordering of Hybrid Materials Using Genetically Engineered Mesoscale
Virus"; 2003/0073104 published Apr. 17, 2003; (3) "Biological
Control of Nanoparticles"; 2003/0113714 published Jun. 19, 2003;
and (4) "Molecular Recognition of Materials"; 2003/0148380
published Aug. 7, 2003.
[0153] Applications of the present invention, including methods of
use, are described in the following references. Use of
superparamagnetic materials in magnetic resonance imaging is
described in, for example, U.S. Pat. No. 5,262,176 to Palmacci et
al. (Nov. 16, 1993), including use of colloids and
superparamagnetic metal oxide covered with a polymer, which is
hereby incorporated by reference in its entirety. Superparamagnetic
materials are also described in, for example, Lee Josephson et al.,
Bioconjugate Chem., 1999, 10, 186-191, including biocompatible
dextran coated superparamagnetic iron oxide particles derivatized
with a peptide sequence, and is hereby incorporated by reference in
its entirety. Applications include magnetic resonance imaging and
magnetic separations. J. Manuel Perez et al., J. Am. Chem. Soc.,
2003, 125, 10192-10193, describes viral-induced self-assembly of
magnetic nanoparticles for use in magnetic nanosensors, including
MRI, capable of detecting a variety of targets including nucleic
acids and proteins. This reference is incorporated by reference in
its entirety.
[0154] Finally, surfaces can be patterned by a variety of methods
known in the art including microlithography and nanolithography and
use of resists and self-assembled monolayers, including
functionalized self-assembled monolayers.
[0155] Although making and using various embodiments of the present
invention are discussed in detail below, it will be appreciated
that the present invention provides many applicable inventive
concepts that can be embodied in a wide variety of specific
contexts. The specific embodiments discussed herein are merely
illustrative of specific ways to make and use the invention, and do
not delimit the scope of the invention.
Sequence CWU 1
1
18 1 12 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 1 Ala Met Ala Gly Thr Thr Ser Asp Pro Ser Thr Val
1 5 10 2 12 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 2 Pro Ala Gln Ser Met Ser Gln Thr Pro
Ser Ala Ala 1 5 10 3 12 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 3 His Thr His Thr Asn Asn Asp
Ser Pro Asn Gln Ala 1 5 10 4 12 PRT Artificial Sequence Description
of Artificial Sequence Synthetic peptide 4 Asp Thr Gln Gly Phe His
Ser Arg Ser Ser Ser Ala 1 5 10 5 12 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 5 Thr Ser Ser
Ser Ala Leu Gln Pro Ala His Ala Trp 1 5 10 6 12 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 6 Ser
Glu Ser Ser Pro Ile Ser Leu Asp Tyr Arg Ala 1 5 10 7 12 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 7 Ser Thr His Asn Tyr Gln Ile Pro Arg Pro Pro Thr 1 5 10 8
12 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 8 His Pro Phe Ser Asn Glu Pro Leu Gln Leu Ser Ser
1 5 10 9 12 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 9 Ser Ser Leu Phe Ile Gln Gln Asn Ala
Leu Thr Gly 1 5 10 10 12 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 10 Gly Pro Phe Pro Thr Met
Pro Leu Pro Asn Gly His 1 5 10 11 12 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 11 Gly Ser Gly
Gln Leu Pro Ile Ala Leu Glu Leu Arg 1 5 10 12 7 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 12
Asn Ala Gly Asp His Ala Asn 1 5 13 7 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 13 Ser Lys Asn
Ser Asn Ile Leu 1 5 14 7 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 14 Thr Lys Pro Ser Val Val
Gln 1 5 15 12 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 15 Ala Leu Ser Pro His Ser Ala Pro Leu
Thr Leu Tyr 1 5 10 16 12 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 16 Ser Val Ser Val Gly Met
Lys Pro Ser Pro Arg Pro 1 5 10 17 12 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 17 His Asn Lys
His Leu Pro Ser Thr Gln Pro Leu Ala 1 5 10 18 12 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 18
Trp Asp Pro Tyr Ser His Leu Leu Gln His Pro Gln 1 5 10
* * * * *