Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
  • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
  • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
  • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
  • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
  • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
  • A61K49/1872—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid coated or functionalised with a polyamino acid, e.g. polylysine, polyglutamic acid
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

• nucleotide and/or amino acid sequence listing is incorporated by reference of the material on computer readable form.
• 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.
• 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.
• 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 astonishingly complex architectures.
• molecular building blocks i.e., lipids, peptides, and nucleic acids
• living organisms execute their architectural "blueprints" using mostly non-covalent forces acting simultaneously upon many molecular components.
• these structures can often elegantly rearrange between two or more usable forms without changing any of the molecular constituents.
• 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.
• inorganic materials including metallic and magnetic materials
• 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.
• room temperature methods have been developed in the present invention for preparing materials which formerly involved high temperature preparation methods.
• 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) .
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• the present invention includes compositions and methods for creating metal materials, including magnetic materials, particles, and nanopar icles.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 .
• 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.
• FIGURE 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) ;
• XPS X-ray photoelectron spectroscopy
• FIGURE 2 depicts schematic diagrams of the smectic alignment of M13 phages in accordance with the present invention
• FIGURE 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;
• FIGURE 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;
• FIGURE 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;
• FIGURE 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
• FIGURE 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;
• FIGURE 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;
• FIGURE 9 are (A) high resolution TEM image of FePt nanoparticles that have been grown using phage that express a 12mer 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;
• FIGURE 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;
• FIGURE 11 is (A) an AFM image of Co-specific phage with Co nanoparticles bound to its P3 protein and (B) the corresponding MFM image;
• FIGURE 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;
• FIGURE 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
• FIGURE 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.
• 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
• F STEM mapping for Co in accordance with the present invention.
• 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.
• particle can refer to the size and shape of said materials, and includes but is not limited to micron-scaled particles, nano-scaled particles
• binding molecule is hereby defined as a molecule that binds, recognizes or directs the growth of a metal material .
• 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.
• peptides can bind to semiconductor materials.
• binding molecules including peptides
• 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.
• 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.
• Phage-display library based on a combinatorial library of random peptides containing between 7 and 12 amino acids fused to the pill coat protein of M13 bacteriophage, providing different peptides that were reacted with crystalline semiconductor structures.
• Five copies of the pill 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.
• 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-speci ic 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 (lll)B GaAs).
• 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°) 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.
• 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.
• 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.
• XPS X-ray photoelectron spectroscopy
• elemental composition determination was performed, monitoring the phage substrate interaction through the intensity of the gold 4f-electron signal (FIGURES 1A-C) .
• FIGURES 1A-C intensity of the gold 4f-electron signal
• 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.
• the presence of native oxide on the substrate surface may alter the selectivity of peptide binding.
• the intensity of Ga 2p electrons against the binding energy from substrates that were exposed to the Gl-3 phage clone is plotted in FIGURE IC.
• the Ga 2p intensities observed on the GaAs (100), (lll)A and (lll)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.
• 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.
• the Gl-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 pill protein of Gl-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 (FIGURE IE) .
• 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- ⁇ m lines of GaAs, and 4- ⁇ m Si0 2 spacing in between each line (FIGURES 1A-1B) .
• the G12-3 clones were interacted with the GaAs/Si02 patterned substrate, washed to reduce non-specific binding, and tagged with an immuno- fluorescent probe, tetramethyl rhodamine (TMR) .
• TMR tetramethyl rhodamine
• the tagged phage were found as the three lighter lines (red, if in color) and the center dot, in FIGURE IB, corresponding to G12-3 binding only to GaAs.
• the Si0 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 (FIGURE 1A) . The same result was obtained using non-phage bound G12-3 peptide.
• the GaAs clone G12-3 was observed to be substrate- specific for GaAs over AlGaAs (FIGURE IC) .
• AlAs and GaAs have essentially identical lattice constraints at room temperature, 5.66 A° and 5.65 A°, respectively, and thus ternary alloys of AlxGal-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 0 . 98 Ga 0 . 02 As . The substrate material was cleaved and subsequently reacted with the G12-3 clone.
• 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 0 . 98 Gao . o 2 As within the heterostructure (FIGURE IC) .
• 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.
• FIGURE ID a model is depicted for the discrimination of phage for semiconductor heterostructures, as seen in the fluorescence and SEM images (FIGURES 1A-C) .
• 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 3 0 4 , Fe 2 0 3 , CdSe, ZnSe and CaC0 3 using peptide libraries.
• 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) .
• 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.
• 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 .
• 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.
• 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.
• 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.
• 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.
• 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.
• peptides 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.
• 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.
• 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.
• 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.
• 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.
• 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 .
• magnétique separation examples include classical affinity based separations in-vitro and localization of reagents in-vivo.
• 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.
• 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.
• 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 ( ⁇ -Co, CoPt, FePt) .
• combinatorial peptide phage display libraries i.e., large collections of bacterial phage that express millions of different peptide sequences on their surfaces
• biopanning techniques to select specific peptide sequences that tightly bind directly to magnetic materials ( ⁇ -Co, CoPt, FePt) .
• 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.
• Co nanoparticles have been prepared of hexagonally close packed Co
• 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.
• memory devices are prepared using a CoCr alloy with a density of 16.3 Gb/in2.
• Nanoparticles prepared that possess HCP P6/mm crystallinity are prepared that possess HCP P6/mm crystallinity.
• 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 .
• 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.
• 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-HCI (pH 9.1) .
• the eluted phage were titered and binding efficiency was compared.
• the phage eluted after third-round substrate exposure were mixed with their Escherichia coli ⁇ E. coli ) ER2537 host and plated on LB XGal/lPTG plates. Since the library phage were derived from the vector M13mpl9, which carries the lacZ ⁇ gene, phage plaques were blue in color when plated on media containing Xgal (5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside) and IPTG (isopropyl- ⁇ -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 .
• 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 4 0H:H 2 0 (1:10), HC1:H 2 0 (1:10), H 3 P0 4 :H 2 ⁇ 2:H2 ⁇ (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 2 0 for 1 minute followed by a deionized water rinse for 1 minute.
• XPS X-ray Photoelectron Spectroscopy
• the XPS instrument used was a Physical Electronics Phi ESCA 5700 with an aluminum anode producing monochromatic
• TEM Transmission Electron Microscopy
• SEM Scanning Electron Microscopy
• 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.
• 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.
• 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.
• 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.
• 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 .
• inventions include electrical and magnetic interfaces, or even the organization of 3D electronic nanostructures for high-density storage, e.g., for use in quantum computing.
• 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.
• 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.
• 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.
• 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.
• 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.
• the filamentous bacteriophage, Fd has a long rod shape (length: 880 nm; diameter: 6.6 nm) and monodisperse molecular weight (molecular weight: 1.64 x 10 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.
• M13 a similar filamentous bacteriophage, was genetically modified to bind nanoparticles such as zinc sulfide, cadmium sulfide and iron sulfide.
• Nanoscale 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.
• 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.
• 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 "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.
• FIGURES 2A and 2B are schematic diagrams of the smectic alignment of M13 phages observed using AFM.
• TEM TEM images were taken using a Philips EM208 at 60 kV.
• the Gl-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.
• 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
• M13 bacteriophage that had specific binding properties to semiconductor surfaces was amplified and purified using standard molecular biological techniques.
• the suspensions were prepared by adding Na 2 S solutions to ZnCl 2 doped A7 phage suspensions at room temperature.
• the highest concentration of A7-phage suspension was prepared by adding 20 ⁇ L of 1 mM ZnCl 2 and Na 2 S solutions, respectively into the -30 mg of phage pellet . The concentration was measured using extinction coefficient of 3.84 mg/mL at 269 nm.
• nemetic phase that has directional order
• cholesteric phase that has twisted nemetic structure
• smectic phase that has directional and positional orders as well
• 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
• the cholesteric pitches in FIGURE 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.
• M13 bacteriophage suspension (concentration: 30 mg/mL) was dried for 24 hours on the 8 mm x 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 FIGURE 3C, M13 phage lie in the plane of the photo and form smectic alignment.
• Nanoscale bacteriophage alignment of the A7-ZnS film were observed using SEM.
• the film was cut then coated via vacuum deposition with 2 nm of chromium in an argon atmosphere. Highly close-packed structures, FIGURE 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 ⁇ 20nm analogous to the TEM observation of individual particles.
• the SAED patterns of the aligned particles showed that the ZnS particles have the wurzite hexagonal structure.
• FIGURE 4C The surface orientation of the viral film was investigated using AFM.
• FIGURE 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.
• 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 .
• a phage display technique was used to discover novel peptides that bind selectively to magnetic materials.
• 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 2 to generate the desired crystal structure. Phage display was then performed on these films ( ⁇ -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.
• Ferromagnets 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.
• 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 IO 7 ergs/cm 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.
• 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.
• XRD x-ray diffraction
• the XRD pattern obtained for ⁇ -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 ⁇ -Co.
• the FePt and CoPt patterns also agreed with the literature spectra for FePtll 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.
• FIGURE 5A is a high resolution TEM image of a SmCo5 nanoparticle and FIGURE 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 (FIGURE 51B) .
• FIGURE 5C is a STEM image of the annealed SmCo5 nanoparticles and illustrates their size, shape, and overall morphology. Sequence Analysis and Binding Assays of Binding Phage
• 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 .
• 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.
• FIGURE 7 High resolution TEM images of CoPt nanoparticles grown using the 7-constrained sequence from Table 1 are shown in FIGURE 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 (FIGURE 7A) and with the lattice spacing of Ll 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.
• nanoparticles In control experiments with either wild-type phage (FIGURE 7C) , nonspecific phage (FIGURE 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.
• FIGURE 8 shows high resolution TEM images of Co nanoparticles grown using the phage that expressed the 12mer peptide that binds specifically to Co (FIGURE 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 (FIGURE 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.
• FIGURE 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 Ll 0 FePt.
• FIGURE 9B is the corresponding electron diffraction pattern
• FIGURE 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.
• Magnetic Force Microscopy 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 (FIGURE 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 (FIGURE 11B) ) .
• 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.
• 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.
• a room temperature hysteresis loop for FePt nanoparticles grown using the 12mer peptide expressed on phage was taken (FIGURE 12A) .
• a high- resolution hysteresis loop of the central portion of the scan was also taken to clarify the presence of the coercivity (FIGURE 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 5 nanoparticles (FIGURE 13) . The hystersis was much larger for these nanoparticles (400 Oe) . This result was expected since macroscopic samples of SmCo 5 typically display higher coercivity values than FePt.
• 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) .
• 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.
• the peptides 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°C or 1,000°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.
• Co nanoparticle Synthesis of ⁇ -Co was prepared by first dissolving 0.6 g of Co 2 (CO) 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 L 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 0 2 and H 2 0. 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.
• Nanoparticle Synthesis of CoPt Preparation was identical to FePt, except 0.16 g of Co 2 (CO) 8 was substituted for 0.13 mL of Fe(CO) 5 .
• 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 2 was mixed with 16.0 mg of SmCl 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 0 2 or H 2 0 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
• Peptide Selection The use of a phage display library technique was used to find peptides that bind exclusively to ⁇ -Co, and the LlO-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 ⁇ L (or an initial amount) of phage display library to initiate selection against the magnetic substrates (in 1 mL of TBS) . For ⁇ -Co, selections were performed in a 10 mM solution of NaBH 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.
• 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.
• Binding Affinity To determine that the peptides bind specifically to ⁇ -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 ⁇ -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 .
• FePt was prepared in a similar fashion to CoPt, except a FeCl 2 solution was used in place of CoCl 2 -
• 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 5mM CoCl 2 and 5mM H 2 PtC ⁇ 6 is added to 1 L 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 lOOmM NaBH4 is added as a reducing agent.
• FIGURE 14A depicts the TEM image of the nanoparticles
• FIGURE 14B the resolution image with the selected area electron diffraction pattern (FIGURE 14C) showing many bands corresponding to the expected values for the CoPt facets.
• FIGURE 14D The STEM image of one of these phage with CoPt nanoparticles grown along its P8 proteins is shown in FIGURE 14D.
• the EDS mapping for Pt (FIGURE 14E) and Co (FIGURE 14F) indicate that Co and Pt are both found along the length of the structure in equal concentrations.
• 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.
• 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 .

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