U.S. patent application number 10/155883 was filed with the patent office on 2003-08-07 for molecular recognition of materials.
Invention is credited to Belcher, Angela M..
Application Number | 20030148380 10/155883 |
Document ID | / |
Family ID | 27668129 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030148380 |
Kind Code |
A1 |
Belcher, Angela M. |
August 7, 2003 |
Molecular recognition of materials
Abstract
The present invention includes methods for selective binding of
inorganic materials and the compositions that made up of the
selecting agent and the target materials. One form of the present
invention is a method for selecting crystal-binding peptides with
binding specificity including the steps of contacting one or more
amino acid oligomers with one or more single-crystals of a
semiconductor material so that the oligomers may bind to the
crystal and eluting the bound amino acid oligomers from the
single-crystals.
Inventors: |
Belcher, Angela M.;
(Lexington, MA) |
Correspondence
Address: |
Edwin S. Flores
Gardere Wynne Sewell LLP
3000 Thanksgiving Tower
1601 Elm Street, Suite 3000
Dallas
TX
75201-4767
US
|
Family ID: |
27668129 |
Appl. No.: |
10/155883 |
Filed: |
May 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60296013 |
Jun 5, 2001 |
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Current U.S.
Class: |
435/7.1 ;
435/287.2; 436/518 |
Current CPC
Class: |
G01N 33/6803 20130101;
G01N 33/543 20130101; G01N 33/68 20130101 |
Class at
Publication: |
435/7.1 ;
435/287.2; 436/518 |
International
Class: |
G01N 033/53; C12M
001/34; G01N 033/543 |
Claims
What is claimed is:
1. A method for selecting crystal-binding peptides with binding
specificity comprising the steps of: contacting one or more amino
acid oligomers with one or more single-crystals of a semiconductor
material so that the oligomers may bind to the crystal; and eluting
the bound amino acid oligomers from the single-crystals.
2. The method recited in claim 1, further comprising the step of
contacting the eluted amino acid oligomers with one or more
semiconductor crystals, and repeating the eluting step.
3. The method recited in claim 1, wherein the nucleic acid sequence
underlying eluted oligomers is amplified.
4. The method recited in claim 3, wherein the amplification is
accomplished using a polymerase chain reaction.
5. The method recited in claim 1, further comprising the step of
determining the sequence of the eluted amino acid oligomers.
6. The method recited in claim 1, wherein the semiconductor
material is chosen from the group consisting of gallium arsenide,
indium phosphide, gallium nitride, zinc sulfide, cadmium sulfide,
aluminum arsenide, gallium stibinide, aluminum gallium arsenide,
aluminum stibinide, cadmium selenide, zinc selenide, cadmium
telluride, zinc selenide, indium arsenide, aluminum arsenide and
silicon.
7. The method recited in claim 1, wherein the semiconductor
material is chosen from the group consisting of a Group II-Group V
semiconductor and a Group III-Group VI semiconductor.
8. The method recited in claim 1, wherein the one or more amino
acid oligomers that are contacted with the single-crystal further
comprise a phage-display library.
9. The method recited in claim 1, wherein the one or more amino
acid oligomers that are contacted with the single-crystal further
comprise an antibody expression library.
10. The method recited in claim 1, wherein the one or more amino
acid oligomers that are contacted with the single-crystal further
comprise a bacterial expression library.
11. The method recited in claim 1, where the amino acid oligomers
are about 12 amino acids in length.
12. The method recited in claim 1, wherein the one or more amino
acid oligomers that are contacted with the single crystal further
comprise a library of random amino acid sequences.
13. The method recited in claim 12, where the amino acid oligomers
are about 12 amino acids in length.
14. The method recited in claim 12, wherein the amino acid
oligomers are about 7 amino acids in length.
15. The method recited in claim 12, wherein the amino acid
oligomers are between about 7 to 15 amino acids in length, and
disulfide constrained.
16. The method recited in claim 1, wherein the eluting is done at
high stringency.
17. The method recited in claim 1, wherein the eluting is done at
moderate stringency.
18. The method recited in claim 1, wherein the eluting is done at
low stringency.
19. An amino acid sequence for the binding GaAs (100) chosen from
the group consisting of Seq. ID Nos. 1 through 11, inclusive.
20. A method for selecting amino acid sequences with binding
specificity comprising the steps of: contacting one or more amino
acid oligomers with one or more single-crystals of a magnetic,
mineral or optical material so that the oligomers may bind to the
crystal; and eluting the bound amino acid oligomers from the
single-crystals.
21. The method recited in claim 20, further comprising the step of
contacting the eluted amino acid oligomers with one or more
single-crystals of a magnetic material, and repeating the eluting
step.
22. The method recited in claim 20, wherein nucleic acid sequence
underlying the eluted oligomers is amplified.
23. The method recited in claim 22, wherein the amplification is
accomplished using a polymerase chain reaction.
24. The method recited in claim 20, further comprising the step of
determining the sequence of the eluted amino acid oligomers.
25. The method recited in claim 20, wherein the magnetic, mineral
or optical material is chosen from the group consisting of FePd,
cobalt, manganese, lithium niobate, iron oxides and calcium
carbonate.
26. The method recited in claim 20, wherein the one or more amino
acid oligomers that are contacted with the single-crystal further
comprise a phage-display library.
27. The method recited in claim 26, where the amino acid oligomers
are about 12 amino acids in length.
28. The method recited in claim 20, wherein the one or more amino
acid oligomers that are contacted with the single crystal further
comprise a library of random amino acid sequences.
29. The method recited in claim 28, where the amino acid oligomers
are about 12 amino acids in length.
30. The method recited in claim 28, wherein the amino acid
oligomers are between about 7 and 15 amino acids in length.
31. The method recited in claim 28, wherein the amino acid
oligomers are 7 amino acids in length, and disulfide
constrained.
32. The method recited in claim 20, wherein the eluting is done at
moderate stringency.
33. The method recited in claim 20, wherein the eluting is done at
low stringency.
34. A method for selecting crystal-bonding amino acids comprising
the steps of: contacting one or more amino acid oligomers with one
or more crystals of a target material so that the oligomers may
bind to the crystal; and eluting the bound amino acid oligomers
from the crystals.
35. The method recited in claim 34, further comprising the step of
contacting the eluted amino acid oligomers with one or more
semiconductor crystals, and repeating the eluting step.
36. The method recited in claim 34, wherein nucleic acid sequence
underlying the eluted oligomers is amplified.
37. The method recited in claim 36, wherein the amplification is
accomplished using a polymerase chain reaction.
38. The method recited in claim 34, further comprising the step of
determining the sequence of the eluted amino acid oligomers.
39. The method recited in claim 34, wherein the semiconductor
material is chosen from the group consisting of gallium arsenide,
indium phosphide, gallium nitride, zinc sulfide, cadmium sulfide,
aluminum arsenide, gallium stibinide, aluminum gallium arsenide,
aluminum stibinide, aluminum arsenide, cadmium selenide, zinc
selenide, cadmium telluride, zinc selenide, indium arsenide and
silicon.
40. The method recited in claim 34, wherein the semiconductor
material is chosen from the group consisting of a Group III-Group V
semiconductor and a Group II-Group VI semiconductor.
41. The method recited in claim 34, wherein the one or more amino
acid oligomers that are contacted with the single-crystal further
comprise a phage-display library.
42. The method recited in claim 41, where the amino acid oligomers
are about 12 amino acids in length.
43. The method recited in claim 20, wherein the one or more amino
acid oligomers that are contacted with the single crystal further
comprise a library of random amino acid sequences.
44. The method recited in claim 43, where the amino acid oligomers
are about 12 amino acids in length.
45. The method recited in claim 43, wherein the amino acid
oligomers are about 7 to 15 amino acids in length.
46. The method recited in claim 43, wherein the amino acid
oligomers are 7 amino acids in length, and disulfide
constrained.
47. An amino acid sequence for the binding GaAs (100) chosen from
the group consisting of Seq. ID Nos. 1 to 11.
48. A specificity structure comprising: one or more single crystals
of gallium arsenide; and a selective binding amino acid
sequence.
49. The specificity structure recited in claim 48, wherein the
selective binding amino acid sequence is chosen from the group
consisting of Seq. ID Nos. 1,2,3,4,5,6,7,8,9,10, 11 and 117.
50. A specificity structure comprising: one or more single crystals
of cadmium sulfide; and a selective binding amino acid
sequence.
51. The specificity structure recited in claim 50, wherein the
selective binding amino acid sequence is chosen from the group
consisting of Seq. ID Nos. 12 through 82.
52. A specificity structure comprising: one or more single crystals
of zinc sulfide; and a selective binding amino acid sequence.
53. The specificity structure recited in claim 52, wherein the
selective binding amino acid sequence is chosen from the group
consisting of Seq. ID Nos. 83 through 116.
54. A specificity structure comprising: one or more single crystals
of lead sulfide; and a selective binding amino acid sequence.
55. The specificity structure recited in claim 54, wherein the
selective binding amino acid sequence is chosen from the group
consisting of Seq. ID Nos. 118 through 158.
56. A crystal binding amino acid oligomers comprising the sequence
motif
(ser/tyr/thr)-(arg/asp/ser)-Xaa-(ser/asn/glu/arg/thr)-Xaa-Xaa-(ser/thr/gl-
u/asp)-(ser/thr/tyr).
57. A crystal binding amino acid oligomers comprising the sequence
motif Xaa-Xaa-(ser/tyr/thr)-(arg/asp/ser)-Xaa
-(ser/asn/glu/arg/thr)-Xaa-Xaa-(s- er/thr/glu/asp)-(ser/thr/tyr)
-(ser/thr/his)-Xaa-Xaa.
58. A method of determining a binding motif comprising the steps:
contacting a binding library with one or more crystals of a target
material to allow components of the library to bind via a binding
region to the crystals; eluting off bound components; and
sequencing the eluted components.
59. The method recited in claim 58, wherein the library is
comprised of peptides.
60. The method recited in claim 58, wherein the library is a phage
display library.
61. The method recited in claim 58, wherein the library is an
antibody display library.
62. The method recited in claim 58, wherein the library is
comprised of chimeric proteins with protein cleavage sites adjacent
to the binding region.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to the selective
recognition of inorganic materials in general and specifically
toward surface recognition of single crystals of semiconductor and
magnetic materials using small organic molecules.
BACKGROUND OF THE INVENTION
[0002] 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 crystallites and other nanoscale building blocks into
complex structures required for biological function.
[0003] 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.
[0004] 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
[0005] The ability to direct the assembly of nanoscale components
into controlled and sophisticated structures has motivated intense
efforts to develop assembly methods that mimic or exploit the
recognition capabilities and interactions found in biological
systems. Of particular value would be methods that could be applied
to materials with interesting electronic or optical properties, but
natural evolution has not selected for interactions between
biomolecules and such materials.
[0006] 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.
[0007] The present invention includes methods for selective binding
of inorganic materials and the compositions that are made up of the
selecting agent and the target materials. One form of the present
invention is a method for selecting crystal-binding peptides with
binding specificity and includes the steps of contacting one or
more amino acid oligomers with one or more single-crystals of a
semiconductor material so that the oligomers may bind to the
crystal and eluting the bound amino acid oligomers from the
single-crystals.
[0008] Another form of the present invention is a method for
selecting crystal-binding peptides with binding specificity and
includes the steps of contacting one or more amino acid oligomers
with one or more crystals of a semiconductor, such as a Group III-V
or II-VI material; or a magnetic material, such an iron oxide, so
that the oligomers may bind to the crystal and eluting the bound
amino acid oligomers from the single-crystals.
[0009] Another form of the present invention is a peptide sequence
for the binding GaAs (100) chosen from the group consisting of Seq.
ID Nos. 1 through 11.
[0010] Still another form of the present invention is a method for
selecting polymeric organic molecules, lipids or nucleic acids with
binding specificity. A method of the present invention begins by
contacting one or more oligomers with one or more single-crystals
of a magnetic material so that the oligomers may bind to the
crystal and eluting the bound peptide oligomers from the
single-crystals. The sequence of the organic polymer is then
determined by direct or indirect sequencing.
[0011] Another form of the present invention is a method for
selecting crystal-bonding amino acids including the steps of
contacting one or more amino acid oligomers with one or more
crystals of a target material so that the oligomers may bind to the
crystal and eluting the bound amino acid oligomers from the
crystals.
[0012] Another form of the present invention is a specificity
structure made up of one or more single crystals of gallium
arsenide, indium phosphide, mercury cadmium telluride, zinc
sulfide, cadmium sulfide, aluminum-gallium-arsenide, zinc selenide,
cadmium selenide, cadmium telluride, zinc telluride, aluminum
arsenide, indium arsenide and the like and a selective binding
amino acid sequence.
[0013] Another form of the present invention is a crystal binding
amino acid oligomer made up of the sequence motif
(ser/tyr/thr)-(arg/asp/ser)-X-
aa-(ser/asn/glu/arg/thr)-Xaa-Xaa-ser/thr/glu/asp)-(ser/thr/tyr)
(SEQ. ID NO. 159) or
Xaa-Xaa-(ser/tyr/thr)-(arg/asp/ser)-Xaa-(ser/asn/glu/arg/thr)-
-Xaa-Xaa-(ser/thr/glu/asp)-(ser/thr/tyr)-(ser/thr/his)-Xaa-Xaa
(SEQ. ID NO 160).
[0014] The motifs and other polymers referred to in the
descriptions of various embodiments of the present invention may be
free molecules, e.g. amino acid oligomers, or they may be part of a
chimera, such as a phage display.BRIEF
DESCRIPTION OF THE DRAWINGS
[0015] 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:
[0016] FIG. 1 depicts selected random amino acid sequences in
accordance with the present invention;
[0017] FIG. 2 depicts XPS spectra of structures in accordance with
the present invention;
[0018] FIG. 3 depicts phage recognition of heterostructures in
accordance with the present invention; and
[0019] FIGS. 4-8 depict specific amino acid sequences in accordance
with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0020] 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.
[0021] 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
natural evolution has not selected for interactions between
biomolecules and such materials.
[0022] 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.
[0023] 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 coliphage, provided 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.
[0024] 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).
[0025] 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 FIG. 1, revealing similar amino-acid sequences among peptides
exposed to GaAs. 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.
[0026] 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 FIG. 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.
[0027] Phage, tagged with streptavidin-labelled 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 (FIG. 2a-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. 2a). In complementary fashion the S1 clone, screened
against the (100) Si surface, showed poor binding to the (100) GaAs
surface.
[0028] 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.
[0029] The preferential 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. 2b, 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.
[0030] The intensity of Ga 2 p electrons against the binding energy
from substrates that were exposed to the G1-3 phage clone is
plotted in 2c. As expected from the results in FIG. 2b, the Ga 2 p
intensities observed on the GaAs (100), (111)A and (111)B surfaces
are inversely proportional to the gold concentrations. The decrease
in Ga 2 p intensity on surfaces with higher gold-strptavidin
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.
[0031] 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 there are many factors involved in substrate
recognition, including atom size, charge, polarity and crystal
structure.
[0032] 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. 4e).
[0033] 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
spacings in between each line (FIGS. 3a, 3b). 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 red lines and the center dot, in FIG. 3b, 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. 3a). The same result
was obtained using non-phage bound G12-3 peptide.
[0034] The GaAs clone G12-3 was observed to be substrate-specific
for GaAs over AlGaAs (FIG. 3c). 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.
[0035] 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. 3c). 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. 3d, a model for the discrimination of phage
for semiconductor heterostructures, as seen in the fluorescence and
SEM images (FIGS. 3a-c).
[0036] The present invention demonstrates the power 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. Bivalent synthetic peptides with
two-component recognition (FIG. 4e) 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.
EXAMPLES
[0037] 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 h 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 titred and binding efficiency was
compared.
[0038] The phage eluted after third-round substrate exposure were
mixed with their Escherichia coli ER2537 host and plated on LB
XGal/IPTG plates. Since the library phage were derived from the
vector M13mp19, which carries the laczA gene, phage plaques were
blue in color when plated on media containing Xgal
(5-bromo-4-chloro-3-indoyl-.beta.-D-galac- toside) 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.
[0039] 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.
[0040] Multilayer substrates of GaAs and of Al.sub.0.98Ga.sub.0.02
As were grown by molecular beam epitaxy onto (100) GaAs. The
epitaxially grown layers were Si-doped (n-type) at a level of
5.times.10.sup.-7 cm.sup.-3.
[0041] Antibody and Gold Labeling. For the XPS, SEM and AFM
examples, substrates were exposed to phage for 1 h 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 minute 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.
[0042] 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 (100) GaAs surface was exposed to (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.
[0043] 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.
[0044] 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 -1
driven near their resonant frequency of 200.+-.400 kHz. Scan rates
were of the order of 1.+-.5 mms -1. Images were leveled using a
first-order plane to remove sample tilt.
[0045] 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 minute,
centrifuged to separate particles from unbound phage, rinsed with
TBS, and resuspended in TBS. Samples were stained with 2% uranyl
acetate.
[0046] Scanning Electron Microscopy (SEM). The G12-3 phage (diluted
1:100 in TBS) were incubated with a freshly cleaved
hetero-structure surface for 30 minute 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.
[0047] Although this invention has been described in reference to
illustrative embodiments, this description is not intended to be
construed in a limiting sense. Various modifications and
combinations of the illustrative embodiments, as well as other
embodiments of the invention, will be apparent to persons skilled
in the art upon reference to the description. It is therefore
intended that the appended claims encompass any such modifications
or embodiments.
Sequence CWU 1
1
95 1 12 PRT artificial sequence peptide binding sequence retrieved
from phag biopanning 1 Ala Met Ala Gly Thr Thr Ser Asp Pro Ser Thr
Val 1 5 10 2 12 PRT artificial sequence peptide binding sequence
retrieved from phage biopanning 2 Ala Ala Ser Pro Thr Gln Ser Met
Ser Gln Ala Pro 1 5 10 3 12 PRT artificial sequence peptide binding
sequence retrieved from phage biopanning 3 His Thr His Thr Asn Asn
Asp Ser Pro Asn Gln Ala 1 5 10 4 12 PRT artificial sequence peptide
binding sequence retrieved from phage biopanning 4 Asp Thr Gln Gly
Phe His Ser Arg Ser Ser Ser Ala 1 5 10 5 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 5 Thr Ser
Ser Ser Ala Leu Gln Pro Ala His Ala Trp 1 5 10 6 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning 6
Ser Glu Ser Ser Pro Ile Ser Leu Asp Tyr Arg Ala 1 5 10 7 12 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 7 Ser Thr His Asn Tyr Gln Ile Pro Arg Pro Pro Thr 1 5 10
8 12 PRT artificial sequence peptide binding sequence retrieved
from phage biopanning 8 His Pro Phe Ser Asn Glu Pro Leu Gln Leu Ser
Ser 1 5 10 9 12 PRT artificial sequence peptide binding sequence
retrieved from phage biopanning 9 Gly Thr Leu Ala Asn Gln Gln Ile
Phe Leu Ser Ser 1 5 10 10 12 PRT artificial sequence peptide
binding sequence retrieved from phage biopanning 10 His Gly Asn Pro
Leu Pro Met Thr Pro Phe Pro Gly 1 5 10 11 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
11 Arg Leu Glu Leu Ala Ile Pro Leu Gln Gly Ser Gly 1 5 10 12 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 12 Cys His Ala Ser Asn Arg Leu Ser Cys 1 5 13 12 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 13 Ser Met Asp Arg Ser Asp Met Thr Met Arg Leu Pro 1 5
10 14 12 PRT artificial sequence peptide binding sequence retrieved
from phage biopanning 14 Gly Thr Phe Thr Pro Arg Pro Thr Pro Ile
Tyr Pro 1 5 10 15 12 PRT artificial sequence peptide binding
sequence retrieved from phage biopanning 15 Gln Met Ser Glu Asn Leu
Thr Ser Gln Ile Glu Ser 1 5 10 16 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 16 Asp Met
Leu Ala Arg Leu Arg Ala Thr Ala Gly Pro 1 5 10 17 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
17 Ser Gln Thr Trp Leu Leu Met Ser Pro Val Ala Thr 1 5 10 18 12 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 18 Ala Ser Pro Asp Gln Gln Val Gly Pro Leu Tyr Val 1 5
10 19 12 PRT artificial sequence peptide binding sequence retrieved
from phage biopanning 19 Leu Thr Trp Ser Pro Leu Gln Thr Val Ala
Arg Phe 1 5 10 20 12 PRT artificial sequence peptide binding
sequence retrieved from phage biopanning 20 Gln Ile Ser Ala His Gln
Met Pro Ser Arg Pro Ile 1 5 10 21 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 21 Ser Met
Lys Tyr Asn Leu Ile Val Asp Ser Pro Tyr 1 5 10 22 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
22 Gln Met Pro Ile Arg Asn Gln Leu Ala Trp Pro Met 1 5 10 23 12 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 23 Thr Gln Asn Leu Glu Ile Arg Glu Pro Leu Thr Pro 1 5
10 24 12 PRT artificial sequence peptide binding sequence retrieved
from phage biopanning 24 Tyr Pro Met Ser Pro Ser Pro Tyr Pro Tyr
Gln Leu 1 5 10 25 12 PRT artificial sequence peptide binding
sequence retrieved from phage biopanning 25 Ser Phe Met Ile Gln Pro
Thr Pro Leu Pro Pro Ser 1 5 10 26 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 26 Gly Leu
Ala Pro His Ile His Ser Leu Asn Glu Ala 1 5 10 27 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
27 Met Gln Phe Pro Val Thr Pro Tyr Leu Asn Ala Ser 1 5 10 28 12 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 28 Ser Pro Gly Asp Ser Leu Lys Lys Leu Ala Ala Ser 1 5
10 29 12 PRT artificial sequence peptide binding sequence retrieved
from phage biopanning 29 Gly Tyr His Met Gln Thr Leu Pro Gly Pro
Val Ala 1 5 10 30 12 PRT artificial sequence peptide binding
sequence retrieved from phage biopanning 30 Ser Leu Thr Pro Leu Thr
Thr Ser His Leu Arg Ser 1 5 10 31 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 31 Thr Leu
Thr Asn Gly Pro Leu Arg Pro Phe Thr Gly 1 5 10 32 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
32 Leu Asn Thr Pro Lys Pro Phe Thr Leu Gly Gln Asn 1 5 10 33 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 33 Cys Asp Leu Gln Asn Tyr Lys Ala Cys 1 5 34 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 34 Cys Arg His Pro His Thr Arg Leu Cys 1 5 35 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 35 Cys Ala Asn Leu Lys Pro Lys Ala Cys 1 5 36 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 36 Cys Tyr Ile Asn Pro Pro Lys Val Cys 1 5 37 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 37 Cys Asn Asn Lys Val Pro Val Leu Cys 1 5 38 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 38 Cys His Ala Ser Lys Thr Pro Leu Cys 1 5 39 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 39 Cys Ala Ser Gln Leu Tyr Pro Ala Cys 1 5 40 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 40 Cys Asn Met Thr Gln Tyr Pro Ala Cys 1 5 41 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 41 Cys Phe Ala Pro Ser Gly Pro Ala Cys 1 5 42 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 42 Cys Pro Val Trp Ile Gln Ala Pro Cys 1 5 43 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 43 Cys Gln Val Ala Val Asn Pro Leu Cys 1 5 44 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 44 Cys Gln Pro Glu Ala Met Pro Ala Cys 1 5 45 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 45 Cys His Pro Thr Met Pro Leu Ala Cys 1 5 46 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 46 Cys Pro Pro Phe Ala Ala Pro Ile Cys 1 5 47 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 47 Cys Asn Lys His Gln Pro Met His Cys 1 5 48 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 48 Cys Phe Pro Met Arg Ser Asn Gln Cys 1 5 49 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 49 Cys Gln Ser Met Pro His Asn Arg Cys 1 5 50 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 50 Cys Asn Asn Pro Met His Gln Asn Cys 1 5 51 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 51 Cys His Met Ala Pro Arg Trp Gln Cys 1 5 52 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 52 His Val His Ile His Ser Arg Pro Met 1 5 53 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 53 Leu Pro Asn Met His Pro Leu Pro Leu 1 5 54 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 54 Leu Pro Leu Arg Leu Pro Pro Met Pro 1 5 55 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 55 His Ser Met Ile Gly Thr Pro Thr Thr 1 5 56 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 56 Ser Val Ser Val Gly Met Lys Pro Ser 1 5 57 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 57 Leu Asp Ala Ser Phe Met Gln Asp Trp 1 5 58 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 58 Thr Pro Pro Ser Tyr Gln Met Ala Met 1 5 59 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 59 Tyr Pro Gln Leu Val Ser Met Ser Thr 1 5 60 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 60 Gly Tyr Ser Thr Ile Asn Met Tyr Ser 1 5 61 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 61 Asp Arg Met Leu Leu Pro Phe Asn Leu 1 5 62 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 62 Ile Pro Met Thr Pro Ser Tyr Asp Ser 1 5 63 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 63 Met Tyr Ser Pro Arg Pro Pro Ala Leu 1 5 64 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 64 Gln Pro Thr Thr Asp Leu Met Ala His 1 5 65 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 65 Ala Thr His Val Gln Met Ala Trp Ala 1 5 66 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 66 Ser Met His Ala Thr Leu Thr Pro Met 1 5 67 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 67 Ser Gly Pro Ala His Gly Met Phe Ala 1 5 68 9 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 68 Ile Ala Asn Arg Pro Tyr Ser Ala Gln 1 5 69 7 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 69 Val Met Thr Gln Pro Thr Arg 1 5 70 7 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
70 His Met Arg Pro Leu Ser Ile 1 5 71 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 71 Leu Thr
Arg Ser Pro Leu His Val Asp Gln Arg Arg 1 5 10 72 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
72 Val Ile Ser Asn His Ala Glu Ser Ser Arg Arg Leu 1 5 10 73 7 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 73 His Thr His Ile Pro Asn Gln 1 5 74 7 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
74 Leu Ala Pro Val Ser Pro Pro 1 5 75 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 75 Cys Met
Thr Ala Gly Lys Asn Thr Cys 1 5 76 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 76 Cys Gln
Thr Leu Trp Arg Asn Ser Cys 1 5 77 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 77 Cys Thr
Ser Val His Thr Asn Thr Cys 1 5 78 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 78 Cys Pro
Ser Leu Ala Met Asn Ser Cys 1 5 79 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 79 Cys Ser
Asn Asn Thr Val His Ala Cys 1 5 80 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 80 Cys Leu
Pro Ala Gln Gly His Val Cys 1 5 81 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 81 Cys Leu
Pro Ala Gln Val His Val Cys 1 5 82 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 82 Cys Pro
Pro Lys Asn Val Arg Leu Cys 1 5 83 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 83 Cys Pro
His Ile Asn Ala His Ala Cys 1 5 84 9 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 84 Cys Ile
Val Asn Leu Ala Arg Ala Cys 1 5 85 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 85 Thr Met
Gly Phe Thr Ala Pro Arg Phe Pro His Tyr 1 5 10 86 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
86 Ala Thr Gln Ser Tyr Val Arg His Pro Ser Leu Gly 1 5 10 87 12 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 87 Thr Ser Thr Thr Gln Gly Ala Leu Ala Tyr Leu Phe 1 5
10 88 12 PRT artificial sequence peptide binding sequence retrieved
from phage biopanning 88 Asp Pro Pro Trp Ser Ala Ile Val Arg His
Arg Asp 1 5 10 89 12 PRT artificial sequence peptide binding
sequence retrieved from phage biopanning 89 Phe Asp Asn Lys Pro Phe
Leu Arg Val Ala Ser Glu 1 5 10 90 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 90 His Gln
Ser His Thr Gln Gln Asn Lys Arg His Leu 1 5 10 91 12 PRT artificial
sequence peptide binding sequence retrieved from phage biopanning
91 Thr Ser Thr Thr Gln Gly Ala Leu Ala Tyr Leu Phe 1 5 10 92 12 PRT
artificial sequence peptide binding sequence retrieved from phage
biopanning 92 Lys Thr Pro Ile His Thr Ser Ala Trp Glu Phe Gln 1 5
10 93 12 PRT artificial sequence peptide binding sequence retrieved
from phage biopanning 93 Asp Leu Phe His Leu Lys Pro Val Ser Asn
Glu Lys 1 5 10 94 12 PRT artificial sequence peptide binding
sequence retrieved from phage biopanning 94 Lys Pro Phe Trp Thr Ser
Ser Pro Asp Val Met Thr 1 5 10 95 12 PRT artificial sequence
peptide binding sequence retrieved from phage biopanning 95 Pro Trp
Ala Ala Thr Ser Lys Pro Pro Tyr Ser Ser 1 5 10
* * * * *