JP2006520317A - Peptide-mediated synthesis of metal and magnetic materials - Google Patents

Abstract

The present invention includes a method of producing magnetic nanocrystals by using biomolecules modified to include amino acid oligomers that can specifically bind to magnetic materials.

Description

This application claims the right to Belcher et al., US Provisional Patent Application No. 60 / 411,804 (filed September 18, 2002), which is hereby incorporated herein by reference, It is incorporated into this application.

Description of Government Assistance Part of the research carried out in this application was supported by Grant No. DADD19-99-0155 from the Army Research Office. May have.

  Further, the sequence listing of the base sequence and / or amino acid sequence is incorporated into the present application by referring to the computer-readable sequence listing.

TECHNICAL FIELD OF THE INVENTION The present invention relates to organic materials that can bind to inorganic materials, and more particularly to specific peptide sequences that bind tightly and directly to metallic materials, such as magnetic materials.

BACKGROUND OF THE INVENTION In biological systems, organic molecules largely control the nucleation and mineral phases of inorganic materials, such as calcium carbonate and silica, and the process by which building blocks are assembled into complex structures required for biological functions. is doing.

  Materials formed by biological processes are usually flexible, and molecular structural units (ie, lipids, peptides, nucleic acids) are assembled in a very simple form and arranged in a surprisingly complex structure. Unlike relying on a series of lithographic process approaches to constructing integrated circuit microstructures, as in the semiconductor industry, organisms are primarily non-covalent forces that act on many constituent molecules simultaneously. To achieve a structural “design drawing”. Moreover, these structures are often rearranged in a refined manner between two or more useful forms without any change in their constituent molecules.

  Processing “next generation” microelectronic devices using “biological” materials can be a way to overcome the limitations of traditional process methods. In this approach, it is important to identify the appropriate compatibility and combination of biomaterial and inorganic material and synthesize the appropriate building blocks.

SUMMARY OF THE INVENTION The inventors have designed a construct and implemented and controlled a collection of inorganic materials, including metals and magnetic materials, to produce biomaterials with controlled and elaborate structures. Of particular importance are ferromagnetic materials and particulate materials, such as nanoparticle materials. When biomaterials can be used to create and design materials with interesting electrical, magnetic, or optical properties, structures can be miniaturized, the photoelectric properties of materials can be better controlled, The manufacturing process may be better controlled. For example, in the present invention, a manufacturing method at room temperature has been developed, but this method enables manufacturing of a material at room temperature that has been conventionally manufactured at high temperature.

  Combinatorial peptide phage display libraries with a large number of bacteriophages expressing millions of different peptide sequences on the surface are combined with biopanning methods to produce metallic materials such as magnetic materials (e.g. , Co, CoPt, SmCo5, or FePt) were selected for specific peptide sequences that bind tightly and directly. We have nucleated inorganic materials by using molecules that are binding to these metals and magnetic materials, such as peptides, as exemplified for the natural state and II-VI semiconductors. I found that I can control. If proteins can be used to control the nucleation of metals, including magnetic materials, magnetic nanoparticles and their applications can be manufactured much cheaper and easier than using conventional methods. Nanomolecular metals, including magnets and magnetic materials, can be used in any application possessed by magnetic or magnetizable materials, including micro or nanomachines, dynamos, generators, magnetic storage devices. Another use of these materials is to modify the surface of metals, including magnetic materials. Peptides can function as linkers when binding materials to the surface of magnetic materials, allowing for the self-assembly of complex nanostructures that can be the basis for new electronic devices.

  These approaches to selecting binding peptides (using combinatorial peptide libraries and panning methods) allow us to form metallic materials, including magnetic materials, and control their nucleation. Also noticed that it could be used. Other technologies being studied for the purpose of synthesizing metal particles, including magnetic particles, are based on synthesis at high temperatures using expensive materials in an inert atmosphere and have the desired shape and crystallinity. In order to produce particles including nanoparticles, further processing and purification are required after synthesis. For this reason, the production of magnetic nanoparticles by conventional methods is not suitable for large-scale production and / or mass production. The approach proposed in the present invention is carried out at room temperature using inexpensive materials, and as a result, nanoparticles with controlled crystallinity are obtained, so that metal particles including magnetic nanoparticles are synthesized. Cost is reduced, and the crystal structure and orientation are also controlled.

  When metal particles such as magnetic nanoparticles are synthesized using a peptide as a medium, metal materials such as magnetic nanoparticles can be synthesized at a much lower cost and with less environmental impact. Currently used protocols for synthesizing metal nanoparticles, including magnetic nanoparticles, are time consuming, expensive and result in nanoparticles coated with an organic surfactant. Such surfactants are unsuitable for further modification of the nanoparticles. Due to the progress of molecular biology, functional groups can be introduced into peptides, and therefore functional groups can be easily introduced into particles and nanoparticles formed from peptides. Giving a peptide a functional group / function allows the peptide to be incorporated into an electronic element or integrated into a magnetic memory element.

  One aspect of the present invention is a self-assembled biomolecule that has been genetically modified to bind a variety of materials, including metals, nanoparticles, and magnetic materials, and to organize highly ordered structures, such as bacteriophages. Is the way to use. Such structures are, for example, nanoscale arrays of particles and nanoparticles. Taking bacteriophage as an example, self-assembling biomaterials can be selected for specific binding properties to a particular surface (eg, a semiconductor), and thus using the modified bacteriophages and methods taught in the present invention, A highly regular structure of the selected substance can be created.

  More particularly, the present invention includes compositions and methods for producing metallic materials, including magnetic materials, particles, and nanoparticles. In one aspect, a method for producing metal particles such as magnetic particles, wherein a molecule including a moiety that specifically binds to a metal surface such as a magnetic surface is produced, and a metal such as a magnetic material is produced. Examples of the method include a step of bringing a precursor of a metal material including a precursor of one or more kinds of magnetic materials into contact with the molecule under such a condition that the material is formed. The molecule can be, for example, a biomolecule such as an amino acid oligomer or peptide. The oligomer can be, for example, from about 7 to about 100 amino acids in length, more preferably from about 7 to about 30 amino acids in length, and even more preferably from about 7 to about 20 amino acids in length. It may form part of a library and / or contain a chimeric molecule.

  Metal materials including magnetic particles of the type disclosed in the present invention can be formed from, for example, Co, CoPt, SmCo5, and / or FePt. Another method of the present invention is a method for identifying a molecule that binds to a magnetic material by non-magnetic interaction, wherein an oligomer that specifically binds to a magnetic material by contacting an amino acid oligomer library with the magnetic material. And eluting an oligomer that specifically binds to the magnetic material. The oligomer library can be a library of self-assembling molecules, for example a phage library such as the M13 phage library. Libraries can be encapsulated in bacteria or collected externally.

  The method for producing magnetic particles can also include a step of bringing a molecule that initiates the formation of a magnetic molecule into contact with a precursor of a magnetic material and a reducing agent. Molecules that initiate the formation of magnetic molecules with the precursor of the magnetic material can be contacted, for example, at room temperature or at temperatures below 100 ° C, below 200 ° C, and in some cases below 300 ° C. The molecule can be an amino acid oligomer, such as an amino acid oligomer that is about 7-20 amino acids in length. The magnetic particles can be magnetic quantum dots or, optionally, film-shaped Co, CoPt, SmCo5, or FePt magnetic particles. A person skilled in the art can arrange one or more of the magnetic particles disclosed in the present specification in various one-dimensional, two-dimensional, or three-dimensional positions, shapes, etc. according to a specific application. You should realize that you can.

  The present invention also includes magnetic particles, such as nanoparticles, produced by the method disclosed in the present invention. These magnetic particles have a magnetic material-binding peptide immobilized on a substrate, and one or more magnetic material precursors are brought into contact with the magnetic material-binding peptide under conditions such that the magnetic particles are formed. A part of an integrated circuit manufactured by forming a magnetic crystal can be formed. The magnetic material binding peptide can be chemically bound to a substrate, such as silicon or other semiconductor substrate. The magnetic particles of the present invention can be used in memory, short or long term memory, ID systems, or any application that one skilled in the art would think can be made using such particles. Examples of other uses of the magnetic micro, nano, and femto particles of the present invention include micro or nano motors, generators, and the like.

  Another aspect of the present invention is a method for forming nanoparticles having specific alignment characteristics. This method creates, for example, an M13 bacteriophage with specific binding properties and amplifies this bacteriophage to a high concentration (e.g., incubating a phage library with a bacterial host culture to infect, Amplification by replication and subsequent virus purification) and by resuspending the phage.

  This same method is used to create a bacteriophage with three liquid crystal phases: nematic phase directional regularity, cholesteric phase twisted nematic structure, and smectic phase directional and positional regularity. can do. One aspect of the present invention relates to a method for producing a polymer, such as a film, which amplifies a self-assembling biomolecule comprising a moiety that binds to a specific semiconductor surface at a high concentration, The method includes a step of bringing a semiconductor material precursor into contact with a self-assembling biomolecule to induce crystal formation or crystal formation induction.

  Another aspect of the invention is a method of creating nanoparticles with different cholesteric pitches, which uses, for example, M13 bacteriophage selected to bind to a semiconductor surface, and the phages at various concentrations. Perform by resuspending. Another aspect of the present invention is a method of producing a cast film in which nanoparticles are arranged, the method comprising, for example, using genetically modified M13 bacteriophage and resuspending the bacteriophage. carry out.

  Yet another aspect of the present invention is a method for producing a nanoparticle film, the method comprising adding a nanoparticle solution to a surface, evaporating the nanoparticle solution on the surface, and annealing the nanoparticles to the surface. In which the nanoparticle is a magnetic molecule. The surface can be a micro-manufactured solid surface to which molecules can attach via covalent or non-covalent bonds, such as Langmuir-Bodgett film, glass, functionalized / functionalized glass, germanium, silicon, PTFE, It may include polystyrene, gallium arsenide, gold, silver, or any material having amino, carboxyl, thiol, or hydroxyl functional groups introduced on the surface. Annealing is usually performed at an elevated temperature in the presence of an inert gas (eg, nitrogen). Another aspect of the present invention is a film of nanoparticles produced by the method described above.

DETAILED DESCRIPTION OF THE INVENTION This application claims rights to Belcher et al. Provisional patent application No. 60 / 411,804 (filed Sep. 18, 2002), which is hereby incorporated by reference herein, The entire application, including drawings, disclosure, detailed description, examples, claims, and sequence listing, is incorporated into this application.

  In the following, the manufacture and use of the various aspects of the present invention will be discussed in detail, but it will be understood that the present invention provides a number of applicable inventive concepts that can be implemented in a wide variety of ways in a specific context. I want. The specific embodiments described herein are merely illustrative for the specific production and use of the present invention, and the scope of the present invention is not limited by these embodiments.

  In order to facilitate understanding of the present invention, some terms are further described below. As used herein, a “metal material” may be, for example, magnetic and / or ferromagnetic, may or may not be crystalline, polycrystalline or amorphous. The metal material may be a metal alloy, metal oxide, or a substance containing a pure metal, but the metal material is not limited thereto. The metallic material can also exist in several spatial forms, such as particles, patterned surfaces, layered films. The term “particle” can describe the size and shape of the material, and is not described herein, such as a micron scale particle, a nanoscale particle (referred to as a nanoparticle), a metal material single molecule, or not described herein. However, it includes, but is not limited to, various sizes and shapes controlled by the biological methods described herein.

  The term binding molecule is defined herein as a molecule that binds to, recognizes, or induces the growth of a metallic material. Examples of binding molecules include, but are not limited to, peptides, amino acid oligomers, and nucleic acid oligomers. These binding molecules can be selected by screening combinatorial libraries, or synthesized, bound, or formulated independently of such libraries. These binding molecules can be bound to a substrate, i.e. bound to a surface or scaffold, e.g. M13 virus with binding molecules displayed on the viral envelope, or to various binding molecular junction structures. it can.

  We have already shown that peptides can bind to semiconductor materials. In the present invention, we demonstrate that binding molecules, such as peptides, can specifically bind to metallic materials, such as magnetic materials. These peptides are further developed to nucleate nanoparticles and induce self-assembly of nanoparticles. The main feature of this peptide is that it recognizes and binds technically important materials with surface specificity, nucleates crystalline semiconductor materials of limited size, and controls the crystalline phase of the nucleated nanoparticles. It has the ability to be able to. This peptide can also control the aspect ratio of the nanoparticles and thus the optical properties.

  Briefly, biological systems have a mechanism for assembling very complex structures on a very small scale, and such mechanisms have nurtured the motivation to identify non-biological systems that can work in the same way. Also, despite the fact that it can be applied to materials with interesting electronic or optical properties, depending on the evolution of nature, methods that have not been chosen to interact with biomolecules and such materials. If it can be found, the method should be of particular value.

  The present invention enables biological systems to efficiently and accurately assemble nanoscale building blocks into complex and highly functional structures that exhibit high completeness and controlled size and composition uniformity. It is based on the recognition.

Selection of Peptide Sequences One method of producing a pool of random organic polymers is to generate a phage library based on a combinatorial library of random peptides containing 7-12 amino acids fused to the pIII coat protein of M13 bacteriophage. A display library is used to obtain various peptides that have reacted with crystalline semiconductor structures. Five copies of pIII coat protein were located at one end of the phage particle, making up 10-16 nm of the particle. This phage display approach has provided a physical link between the peptide-substrate interaction and the DNA encoding this interaction. In the examples described herein, by way of example, five different single crystal semiconductors, namely GaAs (100), GaAs (111) A, GaAs (lll) B, InP (100), Si (100) It was used. When such a substrate is used, it is possible to systematically evaluate the interaction between the peptide and the substrate and confirm the general usefulness of the method of the present invention for various crystal structures.

  The protein sequence that successfully binds to a specific crystal is eluted from the surface, amplified up to 1 million times, and reacted with the substrate under strict conditions, and this procedure is repeated 5 times in the library. The phage showing the most specific binding was selected. After phage selection such as the third, fourth, and fifth rounds, the phage that was specific for the crystal was isolated and its DNA sequence was analyzed. Peptide bonds are selective for crystal composition (for example, binding to GaAs but not to Si) and for crystal planes (for example, binding to (100) GaAs but (111) B Specified that it does not bond to GaAs).

  Twenty clones selected with GaAs (100) were analyzed to determine the epitope binding domain for the GaAs surface. Table 1 shows partial peptide sequences of the modified pIII protein or pVIII protein. It has been shown that similar amino acid sequences exist between peptides contacted with GaAs.

(Table 1) Partial peptide sequence of modified pIII or pVIII protein

  As the number of contacts with the GaAs surface increased, the number of uncharged polar functional groups and Lewis base functional groups increased. Phage clones obtained by the 3rd, 4th and 5th round sequence analysis contained 30%, 40% and 44% polar functional groups on average, respectively, while the proportion of Lewis base functional groups Increased from 41% to 48% -55%. The observed increase in Lewis bases, which should constitute only 34% of the functional groups of random 12-mer peptides in our library, suggests that This suggests that interactions with Lewis acid sites on the GaAs surface may mediate the selective binding exhibited by these clones.

  The predicted structure of the modified 12mer selected from the library may be in an extended conformation, as is often seen with small peptides, so that the peptide is a unit of GaAs. It may be much longer than the lattice (5.65 cm). Therefore, only a small binding domain is needed for the peptide to recognize GaAs crystals. These short peptide domains highlighted in Table 1 contain regions of high serine and threonine content in addition to the presence of amine Lewis bases such as asparagine and glutamine. To determine the correct binding sequence, the surface was screened with a shorter library including a 7-mer library and a disulfide-constrained 7-mer library. Using these shorter libraries to reduce the size and flexibility of the binding domain reduces the interaction that occurs between the peptide and the surface and can enhance the interaction as expected between selected generations. .

  For quantitative analysis of specific binding, 20-nm colloidal gold particles labeled with streptavidin were bound to the phage via a biotin-labeled anti-M13 coat protein antibody, and a tagged phage was used. Elemental composition analysis using X-ray photoelectron spectroscopy (XPS) was performed, and the interaction between the phage and the substrate was monitored by the intensity of the gold 4f-electron signal (FIGS. 1A-C). In the absence of G1-3 phage, antibody and gold-streptavidin did not bind to the GaAs (100) substrate. Therefore, gold-streptavidin binding was specific for the phage and was indicative of the binding of the phage to the substrate. It was also found that by using XPS, the G1-3 clone isolated from GaAs (100) specifically bound to GaAs (100) and not to Si (100) (see FIG. 1A). Complementarily, the S1 clone screened on the (100) Si surface showed low binding to the (100) GaAs surface.

  Some of the GaAs clones also bound to the surface of another sphalerite structure, InP (100). The reason why such selective bonding occurs, whether chemical, structural or electronic, is still under investigation. In addition, the selectivity of peptide binding may change due to the presence of oxides originally present on the substrate surface.

  The G1-3 clone has been shown to bind selectively and specifically to GaAs (100) compared to the (111) A (gallium terminated) or (111) B (arsenic terminated) surface of GaAs. (FIGS. 1B and C). The surface concentration of the G1-3 clone was higher on the (100) surface used for selection than on the (111) A surface with more gallium or the (111) B surface with more arsenic. These various surfaces are known to exhibit different chemical reactivities, and it is not surprising that phage show selectivity for various crystal surfaces. Although the bulk of both 111 surfaces terminates with the same geometric structure, the difference between whether Ga atoms are located on the outermost surface of the double layer or whether As atoms are located is compared with the surface reconstruction. Then it becomes clearer. The composition of the oxides on the various GaAs surfaces is also expected to be different, and as a result, the binding properties of the peptides may be affected.

  The intensity of Ga 2p electrons versus the binding energy of the substrate in contact with the G1-3 phage clone is plotted in FIG. 1C. As predicted from the results in FIG. 1B, the 2p intensity of Ga observed on the GaAs (100), (111) A and (111) B planes is inversely proportional to the gold concentration. The reason why the Ga 2p intensity is lower at the surface where the gold-streptavidin concentration is higher is that the surface coverage by the phage is increased. XPS is a surface technology with a sampling depth of about 30 angstroms, so the signal from the inorganic substrate decreases as the organic layer thickness increases. Using this observation result, it was confirmed that the intensity of gold-streptavidin was actually generated due to the presence of a phage containing a crystal-specific binding sequence on the GaAs surface. A test for binding, correlated with XPS data, was performed in which an equal number of specific phage clones were contacted with various semiconductor substrates of equal surface area. Wild type clones (without random peptide inserts) did not bind to GaAs (no plaque detected). For the G1-3 clone, the eluted phage population was 12 times greater for those eluted from GaAs (100) than those eluted from the GaAs (111) A surface.

  G1-3, G12-3, and G7-4 clones bound to GaAs (100) and InP (100) were imaged by atomic force microscopy (AFM). InP crystals have a zinc blende structure, and the In—P bond is isomorphic to GaAs even though it is more ionic than the Ga—As bond. The width (10 nm) and length (900 nm) of the phage observed with AFM match the size of the M13 phage observed with a transmission electron microscope (TEM), and the gold sphere bound to the M13 antibody is Binding to the phage was observed (data not shown). A high concentration of phage was present on the surface of InP. These data suggest that many factors are involved in substrate recognition, including atomic size, charge, polarity, and crystal structure.

  In a TEM image (not shown), it was observed that the (negatively stained) G1-3 clone was bound to a GaAs crystalline wafer. This data confirms that it is generated by the modified pIII protein of G1-3 and not by non-specific interaction with the large coat protein. Thus, the peptides of the present invention can be used to generate specific interactions between peptides and semiconductors when assembling nanostructures and heterostructures (FIG. 1E).

Using observations with X-ray fluorescence microscopy, it was demonstrated that phage selectively attached to sphalerite surfaces in close proximity to surfaces with different chemical and structural compositions. The square pattern on the nest was etched in a GaAs wafer. This pattern included a 1 μm GaAs line and a 4 μm SiO ₂ gap between the lines (FIGS. 1A-1B). The G12-3 clone was interacted with a patterned substrate of GaAs / SiO ₂ and washed to reduce non-specific binding and tagged with an immunofluorescent probe, tetramethylrhodamine (TMR). The tagged phage is found in Figure 1B as three light lines (red in the color picture) and a central dot, consistent with G12-3 binding only to GaAs. Was. No phage binds to the SiO ₂ region of the pattern and this region remains dark. These results were not observed in controls that were not contacted with phage and contacted with primary antibody and TMR (FIG. 1A). The same results were obtained when using non-phage bound G12-3 peptide.

The GaAs clone G12-3 was observed to have substrate specificity for GaAs rather than AlGaAs (FIG. 1C). AlAs and GaAs have approximately the same lattice constants of 5.66% and 5.65% at room temperature, so that the ternary alloy AlxGal-xAs can be grown epitaxially on a GaAs substrate. GaAs and AlGaAs have a zinc blende crystal structure, but the G12-3 clone showed selectivity to bind only to GaAs. A multilayer substrate in which GaAs layers and Al _0.98 Ga _0.02 As layers were alternately stacked was used. The substrate material was cut and then reacted with the G12-3 clone.

G12-3 clones were labeled with 20-nm gold-streptavidin nanoparticles. When observed and examined with a scanning electron microscope (SEM), GaAs and Al _0.98 Ga _0.02 As were alternately stacked in the heterostructure (FIG. 1C). Using X-ray elemental analysis of gallium and aluminum, the gold-streptavidin particles were exclusively mapped to heterostructure GaAs layers, which showed a high degree of binding specificity for chemical composition. FIG. 1D shows a model for phage differentiation with respect to semiconductor heterostructures, as shown in fluorescence and SEM images (FIGS. 1A-C).

The present invention illustrates the powerful use of phage display libraries in identifying, growing and amplifying binding between organic peptide sequences and inorganic semiconductor substrates. This recognition by peptides and the specificity of inorganic crystals are also found in other substrates using peptide libraries, such as GaN, ZnS, CdS, Fe ₃ O ₄ , Fe ₂ O ₃ , CdSe, ZnSe, and CaCO ₃ Could be expanded.

  A bivalent synthetic peptide (FIGS. 1E-F) is currently designed with an attachment that recognizes the two components. Such peptides may have the ability to direct nanoparticles to specific locations on the semiconductor structure. These organic / inorganic pairs should be powerful building blocks in the production of new generations of complex and sophisticated electronic structures.

Metals and Magnetic Materials In the present invention, specific binding and recognition by binding molecules has been unpredictably extended to metallic materials, including but not limited to magnetic and ferromagnetic materials, particles and nanoparticles. Combinatorial peptide phage display libraries that express large numbers of bacteriophages that express millions of different peptide sequences on the surface, combined with biopanning techniques, can be combined with magnetic materials (e.g., Co , SmCo5, CoPt, FePt) and other specific peptide sequences that bind firmly and directly to metal materials were recognized. The inventor is able to control the nucleation of inorganic materials using these magnetic material binding peptides, as shown in the native state and in III-V and II-VI semiconductors. I found. If proteins can be used to control the nucleation of magnetic materials, it will be possible to produce magnetic nanoparticles much cheaper and easier than using conventional methods. Nanomolecular magnets and magnetic materials can be used for any application possessed by magnetic or magnetizable materials including, for example, micro or nanomachines, dynamos, generators, magnetic memory. Other uses for these materials include modifying the surface of magnetic materials, where peptides act as linkers to attach other materials to the surface of magnetic materials, thus creating the basis for new electronic devices. Enables self-assembly of complex nanostructures that can be

  We have not used this approach of selecting binding peptides (using combinatorial peptide libraries and panning techniques) to control nucleation of magnetic materials. Above I noticed that the peptide has never been used. Many other techniques are still being studied for the purpose of synthesizing magnetic nanoparticles. Both of these attempts are based on high temperature synthesis that needs to be carried out in an inert atmosphere using expensive materials to produce nanoparticles with the desired shape and crystallinity. Often requires further processing and purification. As a result, when magnetic nanoparticles are produced by a conventional method, the cost is extremely high and it is difficult to expand the production scale. The approach presented here can be performed at room temperature using inexpensive materials to obtain nanoparticles with controlled crystallinity, and thus a much cheaper approach for synthesizing magnetic nanoparticles. Become. This approach can also be used in controlling crystal structure and crystal orientation.

  When peptide-mediated magnetic materials are synthesized, it is possible to take a much cheaper and less environmentally friendly approach for the synthesis of magnetic nanoparticles. Current magnetic nanoparticle production protocols are time consuming and expensive. Also, current protocols result in nanoparticles coated with organic surfactants, which are not suitable for further modifying the nanoparticles. With the progress of molecular biology, it is possible to add functions / functional groups to peptides, and it is also possible to easily add functions / functional groups to nanoparticles grown from peptides for incorporation into electronic devices. This suggests that integration into magnetic memory elements can be facilitated.

  Current technology for producing magnetic nanoparticles is expensive, time consuming, requires high temperatures, inert atmospheres, expensive materials, cumbersome purification, and post-synthesis modification. This new technology, which uses peptides to mediate particle formation, alleviates all of these problems and enables much faster and cheaper synthesis of particles compared to the prior art. The crystal structure and orientation control are also improved.

  By using known techniques, sufficient peptides can be produced to produce large quantities of nanoparticles. A genetically designed organism can be used to produce one or more peptides of interest. The peptide can be produced, for example, with one of the coat proteins of M13 bacteriophage. The bacteriophage can be further designed or modified to express the protein in another coat protein. In addition, bacteria, such as E. coli, can be modified to express the target peptide at one or more designs or target positions. One obvious advantage of using peptides in the localization or positioning of magnetic materials produced in the present invention is that they include semiconductor processing, which is generally limited to two dimensions, such as by the use of photolithography. There are no various limitations inherent to. The peptides of the present invention can be used in or around a matrix that allows three-dimensional positioning or synthesis of the peptides. By doing so, these peptides can be formed on a film, line or stripe, layer, dot, groove, on the surface, on the side or bottom of the opening, and the like.

  Magnetic nanoparticles have a variety of uses, including memory elements, sensors, and ferrofluids. The materials, particles, and nanoparticles described herein are applicable to all of these fields.

  Moreover, the metal and magnetic material of this invention can be used with the utilization method in various uses including the following uses. Further applications include therapeutic, diagnostic, engineering, chemical engineering treatment of reactions, cellular and environmental applications. For example, separation using magnetism can be performed (including bulk separation in a large-scale process of a reaction process). Other applications can include purification, therapy, biocompatibility, drug delivery, imaging contrast agents, and use of externally addressable magnetic materials for localization (in vivo). For drug delivery, the particles can be combined with a drug or chemotherapeutic agent and then localized by a magnetic field. If the particles are properly designed, they can also penetrate cells. Another application is blood-urine detection. In engineering applications, display elements can be manufactured by bonding magnetic particles with controlled aspect ratios to optically active materials such as fluorescent and antirefractive materials. It is also possible to manufacture a sensor element in which the moment of inertia of the magnetic particles combined with the binding element changes when the binding occurs. Changes in the moment of inertia can be detected through polarization decay by using a bound optically active material. Another use is for use in memory. For example, it is possible to manufacture a memory that produces a response to a magnetic field that changes with time during reading. In the writing process, a specific part can be bound to a specific address. Cell uses include cell modification and cell triggering. In cell modification, the size of the magnetic particles can be adjusted so that the magnetic particles penetrate into the cells and the particles bind to the substance. A magnetic field can be used as power for penetration. Cell modifications may be useful during the transfection process. In cell triggering, a substance combined with magnetic particles can be placed in the cell and then a time-varying magnetic field can be used to trigger the cell response.

  Examples of magnetic separations include classical separations using in vitro affinity and localization of substances in vivo. In the case of separation utilizing affinity, magnetic nanoparticles may be advantageous in that they are small in size, have a high aspect ratio, and are well controlled in size and shape distribution. There is a further advantage when the magnetic permittivity of the particles is high. That is, the particles can be elongated and can be rotated in a magnetic field, in which case additional forces due to shape effects can be generated. As a result, the separation power per mg of substance increases. In the case of in vivo substance localization, the magnetic particles can be injected or ingested while bound to the substance. When a magnetic field that varies spatially from the outside is applied to the object, particles can be collected in the region of the maximum gradient B. Because the combined size of the particles and the substance is small, the substance can approach the tissue and possibly penetrate the cell.

  More particularly, the inventors have described combinatorial peptide phage display libraries (i.e., large collections of bacteriophages that express millions of different peptide sequences on their surface) and biopanning methods. Used to select specific peptide sequences that bind directly and tightly to magnetic materials (ε-Co, CoPt, FePt). By selecting and identifying specific peptide sequences that bind to magnetic materials with high affinity, it is possible to quickly and easily identify peptides that may be used to control nucleation of peptide magnetic nanostructures. It becomes possible. The use of peptides in controlling the nucleation of magnetic nanoparticles enables the synthesis of magnetic nanostructures at ambient conditions. Traditional manufacturing protocols for magnetic nanoparticles often require elaborate synthesis schemes and extensive purification, ie peptide-mediated nucleation is a much cheaper alternative for nanoparticle synthesis Should be.

One particular advantage of the present invention is that the peptides selected by this approach have been selected as peptides that are specific and direct to the magnetic material. Such peptides have been demonstrated to selectively form nuclei of magnetic nanostructures with controlled crystallinity. So far, Co nanoparticles have been manufactured in a state where Co is packed in a hexagonal close-packed state, and CoPt and FePt nanoparticles have been manufactured in a layered crystalline state usually accompanied by an Invar alloy. . Such a crystal structure is maximally easy to be magnetized for each material, and these materials retain desired magnetic properties even at a nanometer scale. Because of these characteristics, these materials are excellent candidates for producing next-generation magnetic memory elements. Currently, memory elements are manufactured using a CoCr alloy with a density of 16.3 Gb / in ² . Since these nanoparticles are smaller in size, it is considered that a memory element having a density of terabit / in ² can be constructed. In the present invention, SmCo5 nanoparticles having a crystallinity of HCP P6 / mm are produced.

  Controlling nanoparticle nucleation using peptides also allows the nanoparticle to be further functionalized / functionalized. Nanoparticles produced by conventional methods are often coated with a hydrophobic surfactant, and it is troublesome to add a function / functional group (provide an activity or add an active group). It was. Nanoparticles produced as disclosed herein are coated with peptides, compared to imparting functionality / functionality using various chemical and biological techniques known to those skilled in the art. Easy. Adding further functionality / functional groups to these nanoparticles allows complex architectures and memory elements to be formed by self-assembly of these nanoparticles.

  Particles and nanoparticles produced using peptides to control crystallinity have the potential to revolutionize the magnetic recording industry due to their small size, high degree of magnetization, and ease of manufacture Yes.

Example I: Peptide Production, Isolation, Selection, and Characterization Peptide Selection: A phage display or peptide library is prepared from a semiconductor or other crystal and a Tris buffered saline solution containing 0.1% TWEEN-20 ( In TBS) to reduce the interaction between phages on the surface. After rocking for 1 hour at room temperature, the surface was washed by contacting 10 times with Tris buffered saline (pH 7.5) and increasing the concentration of TWEEN-20 from 0.1% to 0.5% (v / v). Elution from the surface of the phage was performed by adding glycine-HCl (pH 2.2) for 10 minutes, transferring to a new tube, and then neutralizing with Tris-HCl (pH 9.1). The titer of the eluted phage was measured and the binding efficiency was compared.

  Phages eluted after the third round of substrate contact were mixed with E. coli ER2537 host and plated on LB XGal / IPTG. Since the library phage is derived from the vector M13mpl9 having the lacZα gene, Xgal (5-bromo-4-chloro-3-indoyl-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactoside) When phage plaques were cultured in the containing medium, the phage plaques turned blue. Blue / white screening was used to select phage plaques with random peptide inserts. Plaques were picked from these plate cultures and analyzed for DNA sequences.

Production of substrate: The orientation of the substrate was confirmed by X-ray diffraction, and the oxides originally present were removed by appropriate chemical specific etching. The following etchants: NH ₄ OH: H ₂ O (1:10), HCl: H ₂ O (1:10), H ₃ PO ₄ : H ₂ O ₂ : H ₂ O (3: 1: 50) Were examined on GaAs and InP surfaces for etching times of 1 and 10 minutes. For the etched surfaces of GaAs and InP, the element ratio was the best and the oxide formation was minimal (using XPS), using HCl: H ₂ O for 1 minute, then washed with deionized water for 1 minute Was the case. However, since the first screening of the library used an ammonium hydroxide etchant for GaAs, this etchant was also used for all other GaAs substrate examples. A Si (100) wafer was etched in a solution of HF: H ₂ O (1:40) for 1 minute and then washed with deionized water. All surfaces were removed directly from the wash and immediately inserted into the phage library. The surface of the control substrate not in contact with the phage was also analyzed and mapped for the effectiveness of the etching process and the surface shape by AFM and XPS.

A multilayer substrate of GaAs and Al _0.98 Ga _0.02 As was grown on GaAs (100) by molecular beam epitaxy. The layer grown by epitaxy was doped (n-type) with Si at a level of 5 × 10 ¹⁷ cm ⁻³ .

  Antibody and gold labeling: For XPS, SEM, AFM examples, the substrate is contacted with phage in Tris buffered saline for 1 hour and then an anti-fd bacteriophage that is an antibody against the fIII phage pIII protein -Introduced into biotin conjugate (1: 500 solution in phosphate buffer from Sigma) for 30 minutes and washed in phosphate buffer. A solution of streptavidin-20-nm colloidal gold label (1: 200) in phosphate buffer (PBS, Sigma) is bound to biotin-binding phage by the interaction of biotin and streptavidin, and the surface is labeled. Contacted for 30 minutes and then washed several times with PBS.

  X-ray Photoelectron Spectroscopy (XPS): For the XPS example, the following controls were performed, where the gold signal observed with XPS was derived from gold bound to phage, and the non-specific antibody GaAs It was confirmed that it was not due to interaction with the surface. The manufactured GaAs (100) surface was subjected to three conditions. (1) Contact with antibody and streptavidin-gold label but not with phage, (2) Contact with G1-3 phage and streptavidin-gold label but not with antibody , (3) Streptavidin-Gold label, but not G1-3 phage or antibody.

  The XPS apparatus used was a Phi ESCA 5700 equipped with an aluminum anode that produces a monochromatic 1,487 eV X-ray from Physical Electronics. All samples were introduced into the chamber immediately after gold was bound to the phage (as described above) to inhibit oxidation of the GaAs surface, and then evacuated in high vacuum overnight to produce an XPS chamber. The outgassing of the sample in the inside was reduced.

Atomic Force Microscope (AFM) Observation: The AFM used was a Digital Instruments Bioscope mounted on a Zeiss Axiovert 100s-2tv, which was used with a G scanner in chip scan mode. Images were taken in the air using tapping mode. The AFM probe was made of etched silicon with a 125 mm cantilever and a spring constant of 20 ± 100 Nm ⁻¹ and was driven at a resonance frequency of around 200 ± 400 kHz. The scanning rate was about 1 ± 5 mms ⁻¹ . Images were leveled using a primary plane to remove sample tilt.

  Observation with Transmission Electron Microscope (TEM): TEM images were taken using Philips EM208 at 60 kV. Gl-3 phage (diluted in TBS1: 100) was incubated with GaAs strips (500 mm) for 30 minutes, centrifuged to separate particles that did not bind, washed with TBS, and resuspended in TBS. Samples were stained with 2% uranyl acetate.

  Observation with Scanning Electron Microscope (SEM): G12-3 phage (diluted 1: 100 with TBS) was incubated with the freshly cut heterostructure surface for 30 minutes and washed with TBS. G12-3 phage was bound with 20 nm colloidal gold. SEM and elemental mapping images were taken using a Norian detection system mounted on a Hitachi 4700 field emission scanning electron microscope at 5 kV.

Example II: Biofilm The inventors have realized that organic and inorganic hybrid materials provide a new path to new materials and devices. Size-controlled nanostructures impart optically and electrically tunable properties to semiconductor materials, and organic additives modify inorganic shapes, phases, and nucleation directions. Since biomaterials have a monodisperse nature, the system is suitable for highly ordered smectic ordered structures. By using the method of the present invention, genetically modified self-assembling biomolecules, such as M13 bacteriophage with a portion that recognizes a specific semiconductor surface, are highly ordered and nanometer An array of scale and multi-length scale II-VI semiconductor materials was created.

  By using the semiconductor recognition and self-ordering system described in the present invention, nanoscale and multi-length scale arrays of semiconductor materials were realized by using the compositions and methods of the present invention. Utilizing semiconductor recognition and self-ordering can enable micro-micro fabrication of electronic devices that surpass the characteristics of current photolithography. Applications of such materials include light emitting displays, optical detectors, photoelectric elements such as lasers, robust wiring, nanometer scale computer components and biosensors. Other uses for biofilms formed using the present invention include highly ordered liquid crystal display and organic-inorganic display technologies.

  Various structures including films and fibers can include high-density sensors for detecting small molecules including biological toxins. Other uses can include optical coatings and optical switches. By using one or more materials disclosed in the present invention as a single layer or multiple layers, or in some cases as stripes, and as any combination thereof as will be apparent to those skilled in the art, Scaffolding materials for medical implants or possibly bone implants can also be constructed.

  Other uses of the present invention may include the organization of 3D electronic nanostructures used for electrical and magnetic interfaces, or in some cases, for high density storage, eg, quantum computing. Also, high density and stable storage of reconfigurable medical viruses such as biocompatible vaccines, adjuvants, and vaccine containers can be performed using the films and / or matrices produced in the present invention. It is also possible to use information storage based on quantum dot patterns for identification, such as equipment for identifying enemy ally of the Ministry of Defense or cloth for coding. The nanofibers of the present invention can also be used for currency coding and identification in some cases.

  Producing highly ordered, well-controlled two-dimensional or three-dimensional structures at the nanoscale is a major goal in producing the next generation of optical, electronic, and magnetic materials and devices. Current methods of producing specific nanoparticles are limited in terms of length scale and material type. The present invention takes advantage of the properties of self-assembling organic or biological molecules or particles, such as M13 bacteriophage, to extend the range of arrays, sizes, nanoparticle scales, and usable semiconductor materials To do.

  The inventors have realized that monodisperse biomaterials with anisotropic shapes are an alternative method for producing highly ordered structures. By using genetically modified M13 bacteriophages with sites (peptides or amino acid oligomers) that recognize specific semiconductor surfaces, II-VI semiconductor materials were successfully aligned on the nanoscale and multi-length scales .

  Seth et al. Described a smectic ordered structure of the Fd virus with both positional and directional regularity. The smectic structure of the Fd virus has the potential to be used in applications where the structure is ordered on multiple and nanoscales to form two- and three-dimensional arrays of nanoparticles. The bacteriophage M13 was used because the bacteriophage M13 is genetically modifiable and has been successfully selected to have the same shape as the Fd virus and is specific for II-VI semiconductor surfaces. This is because it has binding affinity. Thus, M13 is an ideal source of smectic structures that can be used in ordering nanoparticles to be ordered on multiple or nanoscales.

  The inventors have used a combinatorial screening method to find M13 bacteriophages containing peptide inserts that can bind to semiconductor surfaces. Examples of these semiconductor surfaces include materials such as zinc sulfide, cadmium sulfide, and iron sulfide. By using molecular biology techniques, clones of specific semiconductor materials and bacteriophage combinatorial libraries that bind to the material surface were cloned and amplified to a concentration sufficient to form liquid crystals.

Filamentous bacteriophage Fd has an elongated rod shape (length: 880 nm, diameter: 6.6 nm) and a monodisperse molecular weight (molecular weight: 1.64 × 10 ⁷ ). As a result of these properties, this bacteriophage behaves like a lyotropic liquid crystal in high concentration solutions. These anisotropic shapes of bacteriophages were utilized as a method for forming highly ordered layers of nanoparticles using biological selectivity and self-assembly. In the present invention, where monodispersed bacteriophages were produced by standard amplification methods, a similar filamentous bacteriophage, M13, was inherited to bind to nanoparticles such as zinc sulfide, cadmium sulfide, and iron sulfide. Modified.

  It was shown that nanoscale arrays of nanoparticles were formed by the bacteriophage ordering at the mesoscale. These nanoparticles are further ordered into micron-level domains and are further centimeter scale long. Semiconductor nanoparticles exhibit a quantum confinement effect and can be synthesized and ordered in liquid crystals.

  Suspensions of bacteriophage M13 containing specific peptide inserts were prepared and examined by AFM, TEM, and SEM. Throughout the sample, nanoparticles were observed to be uniformly ordered in 2D and 3D.

AFM: A Digital Instruments Bioscope mounted on Zeiss' Axiovert 100s-2tv was used and was used in chip scan mode with a G scanner. Images were taken in the air using tapping mode. The AFM probe was made of etched silicon with a 125 mm cantilever and a spring constant of 20 ± 100 Nm ⁻¹ and was driven at a resonance frequency of around 200 ± 400 kHz. The scanning rate was about 1 ± 5 mms ⁻¹ . Images were leveled using the primary plane to remove sample tilt. 2A and 2B are schematic diagrams of the smectic sequence of M13 phage observed by using AFM.

  TEM: TEM images were taken using Philips EM208 at 60 kV. Gl-3 phage (diluted in TBS1: 100) was incubated with semiconductor material for 30 minutes and centrifuged to separate particles from unbound phage, washed with TBS, and resuspended in TBS. Samples were stained with 2% uranyl acetate.

  SEM: Phages (diluted 1: 100 with TBS) were incubated with the newly cleaved heterostructure surface for 30 minutes and washed with TBS. G12-3 phage was bound with 20 nm colloidal gold. SEM and elemental mapping images were taken using a Norian detection system mounted on a Hitachi 4700 field emission scanning electron microscope at 5 kV.

A genetically modified M13 bacteriophage with specific binding properties to the semiconductor surface was amplified and purified by standard molecular biology techniques. 4 mL of an overnight culture with 3.2 mL of bacteriophage suspension (concentration: about 10 ⁷ phage / μL) was added to 400 mL of LB medium to amplify the amount. After amplification, approximately 30 mg of pellet was precipitated. The suspension was prepared by adding the Na ₂ S solution to the ZnCl ₂ doped A7 phage suspension at room temperature. The highest concentration A7 phage suspension was prepared by adding 20 μL of 1 mM ZnCl ₂ and Na ₂ S solutions to approximately 30 mg of phage pellet, respectively. The concentration was measured by using 3.84 mg / mL (269 nm) as extinction coefficient.

  As the concentration of the isotropic suspension increased, a nematic phase with directional regularity, a cholesteric phase with a twisted nematic structure, and a smectic phase with directional and positional regularity were observed. . These phases have been observed with Fd viruses without nanoparticles.

  Observation with a polarizing microscope (POM): The suspension of the M13 phage was examined with a polarizing microscope. Each suspension was filled into a 0.7 mm diameter glass capillary tube. The highly concentrated suspension (127 mg / ml) showed iridescence under collimated polarization [5] and a smectic structure under orthogonal polarization (FIG. 3A). As shown in Table 2, the cholesteric pitch of FIG. 3B can be controlled by changing the concentration of the suspension. The pitch length was measured and micrographs were taken 24 hours after sample preparation.

(Table 2) Relationship between cholesteric pitch and concentration

  AFM: As observed by AFM, 5 μL of M13 bacteriophage suspension (concentration: 30 mg / ml) was silanized with 3-aminopropyltriethylsilane for 4 hours in a desiccator for 8 hours x 8 mm Dried on a mica substrate. Images were taken in the air using tapping mode. An ordered structure was observed by self-assembly due to the anisotropic shape of M13 bacteriophage (length 880 nm, width 6.6 nm). In FIG. 3C, the M13 phage extends in the plane of the photograph to form a smectic sequence.

  SEM: For SEM observation, a sample was prepared by drying a smectic suspension of bacteriophage and ZnS nanoparticles (concentration of bacteriophage suspension: 127 mg / ml) to the critical point. In FIG. 3D, a region rich in nanoparticles and a region rich in bacteriophages were observed. The spacing between the nanoparticles and the bacteriophage corresponds to the length of the bacteriophage. Electron diffraction patterns using diluted samples of smectic suspensions with TEM confirmed the ZnS fiber zinc ore crystal structure (FIGS. 3E and 3F).

Biofilm production: Bacteriophage pellets were suspended in 400 μL Tris buffered saline (TBS, pH 7.5) and 200 μL 1 mM ZnCl ₂ and 1 mM Na ₂ S was added. After rocking for 24 hours at room temperature, the suspension (enclosed in an Eppendorf tube containing 1 ml) was gradually dried in a desiccator for 1 week. A translucent film about 15 μm thick was formed in the tube. The film shown in FIG. 4A was carefully taken using tweezers. A schematic diagram of the biofilm is shown in FIG. 4B.

  Biofilm SEM observation: SEM was used to observe the nanoscale bacteriophage sequence of A7-ZnS film. For analysis by SEM, the film was cut and then coated with 2 nm chromium by vacuum evaporation in an argon atmosphere. A densely packed structure (FIG. 4D) was observed throughout the sample. The average length of individual phage, 895 nm, had reasonable similarity to the phage length of 880 nm. The film exhibited a smectic A or C-like lamellar morphology, with periodicity observed between the nanoparticles and the bacteriophage phase. The length of the periodicity corresponded to the length of the bacteriophage. The average particle size of the nanoparticles was about 20 nm, similar to the observation of individual particles with TEM.

  Observation of biofilm by TEM: In order to examine the arrangement of ZnS nanoparticles, the film was embedded in an epoxy resin (LR white) for one day and polymerized by adding 10 μl of a curing accelerator. After curing, resin sections were made using a Leica ultramicrotome. These approximately 50 nm sections were floated in distilled water and picked up on a blank gold grid. Parallel aligned nanoparticles corresponding to the xz plane in the schematic diagram were observed (FIGS. 4E-F). Since each bacteriophage has 5 copies of the A7 portion, each A7 recognizes one nanoparticle (size, 2-3 nm), is arranged approximately 20 nm wide, and extends over a length of more than 2 μm. Existed. Bands of 2 μm × 20 nm were formed in parallel, and the interval between each band was about 700 nm. This discrepancy may be caused by the smectic arrangement of the phage layer being tilted with respect to the TEM observation, as reported by Marvin's group. A plane of a nanoparticle layer similar to that shown in FIG. 1F was also observed. The SAED pattern of the arrayed particles showed that the ZnS particles have a hexagonal structure of fibrite.

  Observation of biofilm by AFM: The surface orientation of the virus film was examined using AFM. In FIG. 4C, the phage forms a pattern of herringbones arranged in parallel such that for most of the surface called Smectic O, the normals of adjacent directors (bacteriophage axis) are approximately perpendicular. It was shown that The film exhibited a long range ordered normal director lasting tens of microns. In the part of the region where the two magnetic domain layers meet each other, two or three multi-length scale bacteriophages were parallel and arranged with a smectic C-ordered structure.

  Nanoscale and multi-length scale arrangements of semiconductor materials obtained by using recognition and self-ordering systems will be useful in the future for microfabrication of electronic devices. These elements have the potential to surpass the characteristics of current photolithography. Other potential uses for these materials include light emitting displays, optical detectors, photoelectric elements such as lasers, robust wiring, nanometer scale computer components and biosensors.

Example III: Formation of metal and magnetic materials Phage display technology was used to find novel peptides that selectively bind to magnetic materials. In these specific studies, the production of magnetic material films was performed by first synthesizing a colloidal dispersion of magnetic material. These colloidal solutions were then drop coated onto Si wafers and annealed in N ₂ to produce the desired crystal structure. Next, phage display was performed on these films (ε-Co, CoPt, and FePt) to find peptides that selectively bind to each substrate. Next, nucleation of unique nanoparticles using these peptides was performed by mixing phage expressing the peptide of interest, a metal salt, and a reducing agent.

  The synthesis of nanoparticles with controlled size and composition is a fundamental and important technique. In the last few years, many papers have been published describing synthetic methods that can produce nanoparticles of metal and semiconductors with well-controlled size and shape. Recently, it has been found that peptides identified using phage display can be selectively bound to inorganic surfaces and can be used to control nucleation of semiconductor nanoparticles. In this case, the peptide can control the size, shape, composition, and possibly even crystallinity of the resulting nanoparticles. Because of the successful control of the synthesis of semiconductor nanoparticles with peptides, there is great interest in applying this technology to other important materials.

One particularly important and industrially useful group of materials are ferromagnets, including those of particles and nanoparticles. Ferromagnetic materials are the foundation of the billions of years of the magnetic recording industry. Current devices use CoCr alloys for data storage because they are easy to magnetize and easy to manufacture. Other materials are currently under development, and one such material is metallic Co with a magnetic anisotropy level of 10 ⁷ erg / cm ³ . Such high magnetic anisotropy suggests that even small particles with a diameter of 10 nm can act as a single magnetic domain and function as a memory element. Current technology uses storage elements whose domain size is on the order of hundreds of nanometers, so the generation of Co nanoparticles with a size of 10 nm is the production of much higher density storage elements. It is also a dramatic advancement that makes possible. Further interesting ferromagnetic materials are Pt magnetic alloys, in particular FePt and CoPt magnetic alloys. These materials have extremely high magnetic anisotropy (10 ⁸ erg / cm ³ ). This is because of the invar effect that Pt becomes magnetic due to the disturbance of the lattice constant caused by superposition of Fe and Pt atoms. This is because it has occurred. Because these systems have high anisotropy, small nanoparticles as small as 2 nm can also act as ferromagnetic materials at room temperature, and these nanoparticles will develop extremely high density memory elements. It can be seen that it can be used above.

  Since such systems have high magnetic anisotropy, great efforts have been devoted to the synthesis of particles and nanoparticles composed of these materials. Several different synthetic protocols have been developed for ε-Co, FePt, and CoPt, all of which have the same fundamental weaknesses. All of these synthetic strategies rely on the limited precipitation of nanoparticles at high temperatures in the presence of surfactants. That is, any such production of nanoparticles is costly due to the need to use expensive materials in an inert atmosphere and cannot be scaled up. Also, these productions often require further modification of the particles, such as annealing at high temperatures to achieve the desired crystallinity, and size selective precipitation to form a monodisperse population. Because of these extra synthetic steps, these synthetic strategies are costly.

Because these materials are of great industrial importance, new synthetic strategies have been required. Such an alternative strategy is possible using the principle of peptide-mediated magnetic material synthesis. In these studies, selection using phage display is performed on key magnetic materials (Co, CoPt, SmCo5, and FePt) to identify peptides that specifically bind to magnetic materials with high affinity. . Such peptides are used after characterization to control nucleation of magnetic nanoparticles. In such studies, the phage expressing the peptide was mixed with the metal salt of the metal, and then a reducing agent (NaBH ₄ ) was added to produce nanoparticles. Nanoparticles were formed and the properties of the nanoparticles were analyzed by TEM. The synthesis of the present invention is performed at ambient conditions and provides an alternative strategy that is much cheaper than existing synthetic strategies to produce magnetic nanoparticles.

Analysis of magnetic nanoparticles by X-ray diffraction It was necessary to generate a magnetic surface for use as a substrate for phage display. For this purpose, magnetic nanoparticles were produced by conventional methods and applied dropwise to a Si wafer. Before starting the phage display study, surface properties were analyzed by X-ray diffraction (XRD) to confirm that the crystallinity of this material was appropriate.

  The XRD pattern obtained for ε-Co correlates well with the pattern obtained in the literature, showing a triplet peak between 45 and 50 degrees, these peaks being the (221) of ε-Co , (310), and (311) because they correspond to the crystal planes. The pattern of FePt and CoPt is also consistent with the literature spectrum for FePtll, (001), (110), (111), (200), (002), (210), (112) of FePt and CoPt And peaks corresponding to the (202) plane. The XRD for SmCo5 also agreed with the literature values for HCP SmCo5 and showed peaks representing the (101), (110), and (111) planes. This is the first reported synthesis for HCP SmCo5 nanoparticles. FIG. 5A shows a high resolution TEM image for SmCo5 nanoparticles, and FIG. 5B shows an electron diffraction pattern in selected regions of the TEM image. Several spots in the diffraction pattern correlated well with the known surface of HCP SmCo5 (FIG. 51B). FIG. 5C is a STEM image of the annealed SmCo5 nanoparticles, illustrating the size, shape, and overall shape of the nanoparticles.

Binding Phage Sequence Analysis and Binding Assay Table 3 lists and lists all peptides selected using phage display for binding to the magnetic material of interest.

*ε-Co上の7量体の拘束ライブラリーについては、コンセンサス配列は得られたかった。 Table 3 Selected clones with magnetic binding properties

* Consensus sequences were not obtained for the 7-mer constrained library on ε-Co.

  Any of the sequences selected are likely to be effective sequences with a high degree of affinity for the metal surface. In some of the sequences, histidine residues appear. Since histidine has an imidazole side group, it is an excellent ligand for metals, and therefore the presence of histidine in these sequences is an expected situation. Except for the 7-mer constrained sequence on CoPt, the entire sequence isolated for the Pt alloy contains lysine residues. The interaction between lysine and Pt is thought to be important in the action of cisplatin, which is important as an anticancer agent. The interaction of lysine and Pt suggests that these sequences bind selectively to such materials. Nevertheless, the present invention is not limited to the interaction of a specific mechanism, whether known or unknown.

  Specific binding assay: Two tests were performed to determine the affinity of the isolated phage for the magnetic substrate. In the first test, several different peptide-containing phages, including our Co-specific phage, random phage, and wild-type phage, were contacted with the Co surface. In addition, Co-specific phage were contacted with several different material surfaces. The results are shown in FIG. Co-specific phage showed higher affinity for Co than wild-type phage or random phage library sequences (FIG. 6A). The Co-specific phage showed higher affinity for Co than Si, suggesting that this phage selectively binds to the Co surface.

In the second test, the Co surface was immersed in a solution of Co specific phage. This test was repeated at several different phage concentrations. When the amount of adsorbed phage was plotted against the concentration of phage (FIG. 6B), it was suggested that the adsorption of the phage to the Co surface follows the Langmuir model for the adsorption of the analyte to the surface. Since the adsorption follows the Langmuir model, generating an inverse plot showed a linear correlation between the adsorbed phage and the concentration (not shown). The slope of this line is equal to the binding constant, and in the case of Co, the phage k _ads was 2 × 10 ⁻¹² M. Although this measurement is the first measurement of the thermodynamic properties associated with the binding between the phage and the inorganic surface, it is difficult to interpret, but the magnitude of this binding constant is comparable to some other biological interactions. To do. This approach can be used for CoPt and FePt systems.

  These studies show that peptides selected using phage display screening have specific binding properties for Co but not such binding properties for other materials. . It is this specificity that can be used to form metallic materials, including magnetic materials.

Analysis by TEM of Nanoparticles Produced by Peptide-Mediated Nucleation In one aspect of the invention, peptides were used to produce nanoparticles to control modification and / or crystallinity. High resolution TEM images of CoPt nanoparticles grown using the 7-mer constrained array shown in Table 3 were also taken (not shown). These nanoparticles have a lattice spacing of 0.19 and 0.22 nm, which correlates with the lattice spacing of L10 CoPt.

  High resolution TEM images of nanoparticles grown using wild-type phage were also taken with images of CoPt nanoparticles grown using phage containing random peptide inserts (not shown). In both of these control studies, although nanoparticles were formed, they did not have the crystallinity that particles grown using CoPt selective peptides had. Nanoparticles grown in the absence of phage aggregate and precipitate out of solution, making it almost impossible to take images with TEM.

  High resolution TEM images were also taken of FePt nanoparticles grown using phage expressing a 12-mer peptide selective for FePt (not shown). These nanoparticles showed lattice spacing similar to CoPt nanoparticles, suggesting that these nanoparticles are composed of L10 FePt. An electron diffraction pattern was photographed (not shown) for this same particle, eg, FePt nanoparticles grown in the presence of wild-type phage. These nanoparticles also do not have the crystallinity that particles grown using FePt-selective phage have. Nanoparticles grown in the absence of phage aggregate and precipitate out of solution, making it impossible to take an image.

  A high resolution TEM image of CoPt nanoparticles grown using the 7-mer constrained array shown in Table 1 is shown in FIG. The lattice spacing of these nanoparticles is about 0.22 nm, which correlates well with the literature value for HCP Co of about 0.19 nm and the lattice spacing of Llo CoPt (FIG. 7A). In addition, the selected region was used to observe the electron diffraction pattern of the nanoparticles (not shown). In this diffraction pattern, there are some bands that correlate with the surface of HCP Co, indicating that the nanoparticles are actually composed of HCP Co. In control experiments with wild-type phage (Figure 7C) or non-specific phage (Figure 7B), nanoparticles were formed once, but these nanoparticles were grown using CoPt-selective peptides. The crystallinity was not provided. Since nanoparticles grown in the absence of phage aggregate and precipitate from solution, it is almost impossible to take an image with a TEM.

  FIG. 8 is a high resolution TEM image of Co nanoparticles grown using phage expressing a 12-mer peptide that specifically binds to Co (FIG. 8A). The lattice spacing of these nanoparticles is 0.2 nm, which correlates well with the literature value for HCP Co (0.19 nm). In order to observe the electron diffraction pattern of these nanoparticles, a specific region was selected (Fig. 8B), and the diffraction pattern had several bands correlated with the HCP Co surface. Was composed of HCP Co. In control experiments using wild-type phage or non-specific phage or no phage, Co particles aggregate and precipitate out of solution (not shown).

  FIG. 9A is a high resolution TEM image of FePt nanoparticles grown using phage expressing a 12-mer peptide selective for FePt. These nanoparticles showed a lattice spacing similar to CoPt nanoparticles and were likely composed of Llo FePt. FIG. 9B is the corresponding electron diffraction pattern and FIG. 9C is an image of FePt nanoparticles grown in the presence of wild type phage. In the absence of wild-type phage, the nanoparticles did not have the crystallinity as provided by nanoparticles grown using phage selective for FePt. Further, since the nanoparticles grown in the absence of phage aggregate and precipitate from the solution, it is impossible to take an image.

  High resolution TEM images were also taken of SmCo5 nanoparticles grown using phage expressing a 12-mer peptide specific for SmCo5 (FIG. 10A). In addition, using the selected region, the electron diffraction pattern of the nanoparticles was observed (FIG. 10B). Also in this case, there were some bands correlated with the surface of HCP SmCo5 in the diffraction pattern. Control experiments performed using the SmCo5 system yielded results similar to those observed in the Co system where nanoparticles were aggregated and / or precipitated from solution using non-specific phage. TEM images of these particles showed some crystalline domains, but most of the material was amorphous.

Characterization of nanoparticles by MFM The magnetic properties of the nanoparticles were analyzed using magnetic force microscopy (MFM). An image of the phage used for nucleation of Co nanoparticles was first taken with an atomic force microscope (FIG. 11A). The large aggregation of nanoparticles at the phage ends was clearly observed, suggesting that, as expected, the P3 protein controls the nucleation of the nanoparticles. Corresponding MFM images were taken to confirm these results (FIG. 11B). In this case, the phage could not be seen because it was non-magnetic, but the appearance of the aggregated nanoparticles was clearly visible, suggesting that the nanoparticles had high magnetic anisotropy. It was.

  SQUID: In one embodiment of the present invention, the magnetic properties of the nanoparticles can be measured quantitatively using a superconducting quantum interference device (SQUID) magnetometer. The particles were further investigated using SQUID magnetometry. SQUID was used to measure the hysteresis loop at room temperature for FePt nanoparticles grown using 12-mer peptides expressed by phage (FIG. 12A). A high resolution hysteresis loop in the center of the scan was also measured to clarify the presence of coercivity (FIG. 12B). The coercivity of these samples was relatively low (about 50 Oe). This data is the first case for ferromagnetic nanoparticles grown at ambient conditions. Hysteresis loops were also measured for biologically produced SmCos nanoparticles (FIG. 13). Hysteresis was much greater for these nanoparticles (400 Oe). This result is as expected because the macroscopic sample of SmCos usually has a higher retention value than FePt.

Magnetic Specific Peptides on P8 Coat Protein In one embodiment of the invention, material-specific phage expressed in the M13 bacteriophage p3 protein were used to produce nanoparticles that displayed magnetic behavior. The p3 protein is present only at one end of the rod-shaped phage, and there is a limited number (3-5 per phage). On the other hand, p8 coat protein is expressed along the length of the phage, and hundreds of copies are expressed per phage. For this reason, the p8 protein was modified to express a CoPt-specific peptide to form the nucleus of CoPt nanoparticles along the length of the phage. An example of manufacturing the material is shown below. Other methods obvious to those skilled in the materials and biological sciences can also be used without undue experimental validation.

  In nucleation of magnetic materials such as magnetic particles and nanoparticles, the peptide is heated to a sufficiently high temperature with or without phages, and the binding molecules associated with the layers are incinerated during the high-temperature annealing process. Can be removed. For example, heating to 500 ° C. or 1,000 ° C. can be performed for a time such that incineration and removal occurs optimally. By setting this temperature within the range of the annealing temperature of the metal, the polycrystalline domains can be fused to form a single crystal domain.

Method Materials: Samarium (III) chloride, platinum (II) acetylacetate (Pt (Acac) ₂ ), dihydrogen hexachloroplatinate (H ₂ PtCl ₆ ), octacarbonylcobalt (Co ₂ (CO) ₈ ), Alfa Aesar Purchased from. Iron pentacarbonyl (Fe (CO) ₅ ), cobalt chloride (II) (CoCl ₂ ), iron chloride (II) (FeCl ₂ ), trioctylphosphine oxide (TOPO), sodium borohydride (NaBH ₄ ), oleylamine Oleic acid was purchased from Aldrich.

Synthesis of ε-Co nanoparticles: Co nanoparticles were first prepared by dissolving 0.6 g Co ₂ (CO) ₈ in 5 ml o-dichlorobenzene. The mixture was stirred for 1 hour to dissolve Co, and 20 ml of o-dichlorobenzene, 0.416 g of TOPO, 0.2 ml of oleic acid were mixed in a three-necked reaction vessel containing 500 ml in an Ar atmosphere. . The mixture was then heated to 100 ° C. The mixture was then depressurized for 5 minutes to remove all dissolved O ₂ and H ₂ O. Next, this mixture was heated to boiling (180 ° C.), and a Co solution was added. As a result, the mixture turned black and CO gas was generated in a cloud shape. After refluxing for 20 minutes, the reaction system was cooled to room temperature. To purify the particles, 3 ml Co nanoparticle solution was mixed with 3 ml ethanol. After 1 hour, the mixture was centrifuged at 10,000 rpm for 5 minutes. The precipitate was resuspended in 3 ml CH ₂ Cl ₂ and another 3 ml ethanol and the centrifugation step was repeated. The precipitate was then resuspended in 3 ml CH ₂ Cl ₂ .

Synthesis of FePt nanoparticles: Mix 20 ml phenyl ether, 0.205 g Pt (Acac) ₂ , 0.358 g 1,2-tetradecanediol, heat to 100 ° C. in Ar atmosphere, then 0.16 ml oleic acid 0.17 ml oleylamine, 0.13 ml Fe (CO) ₅ was added. The mixture was heated to 300 ° C., refluxed for 30 minutes, and cooled to room temperature. FePt nanoparticles were purified in the same way as Co nanoparticles.

Synthesis of CoPt nanoparticles: The production was carried out in the same manner as FePt, except that 0.16 g CO ₂ (CO) ₈ was used instead of 0.13 ml Fe (CO) ₅ .

Synthesis of SmCo5 Nanoparticles: SmCo5 nanoparticles were produced using an arrested precipitation method. This technology takes advantage of previous nanoparticle production attempts. 38.75 mg CoCl ₂ was mixed with 16.0 mg SmCl ₃ and mixed with 20 ml phenyl ether. Next, 0.357 ml of oleic acid was added to the mixture and further heated to 100 ° C. in an Ar atmosphere. Next, 1.35 ml of trioctylphosphine was added. The mixture was then depressurized for 10 minutes to remove any O ₂ and H ₂ O dissolved in the solution. After vacuum purging of the solution, the solution was heated to 290 ° C. to boil the phenyl ether. Then 1 ml of the superhydride solution was added and the solution immediately changed from blue to black. The black mixture was then refluxed for 20 minutes and cooled to room temperature.

  Film formation: To produce a film for selection by phage display, a colloidal solution of nanoparticles was coated dropwise on a Si slide and the solvent was evaporated.

In the case of FePt and CoPt, the slide was then annealed at 700 ° C. for 30 minutes in a N ₂ atmosphere to form the L10 phase. XRD analysis was performed on all of these slides to confirm proper material.

Peptide selection: The phage display library method was used to find peptides that bind exclusively to the L10 phase of ε-Co and CoPt and FePt. More specifically, the Ph.D.-12 (tm) and Ph.D.-7 CTM phage display peptide library kits start with 1 μL (or initial amount) of phage display library Used to initiate selection for magnetic substrates (in 1 ml TBS). For ε-Co, selection was performed in a 10 mM solution of NaBH ₄ in TBST. After five rounds of panning, peptides and peptide DNA were isolated and sequences were obtained from the University of Texas DNA Core Facility. These sequences corresponding to the peptides presented by the bacteriophage were analyzed to determine the consensus sequence. In the analysis result of the DNA sequence, the ratio of amino acids was excessive with respect to the position. The analysis was performed only for the last three rounds of panning, as non-specific binding may have occurred in the first two rounds.

  Binding affinity: Binding affinity was measured to confirm that the peptide specifically bound to ε-Co, CoPt, FePt. The consensus peptide was examined by panning to obtain titer values that were compared to those of wild-type and random peptides that did not bind to ε-CO, CoPt, FePt. Next, using various concentrations of phage, it was examined by panning to determine the binding constant of the phage to the target metal surface.

Co peptide-mediated nucleation: Approximately 880 μl of H ₂ O was mixed with 100 μL of 1 mM CoCl ₂ and 20 μL of phage solution (pfu = 1011). The mixture was gently stirred for 30 minutes, then 100 μL of 100 mM NaBH ₄ was added. The solution was vortexed and incubated for an additional 5 minutes. Next, 100 ml of a solution of TOPO and oleic acid in CH ₂ Cl ₂ was added. The mixture was vortexed and stirred gently for 1 hour. During this time, the CH ₂ Cl ₂ layer turned dark gray. This procedure was repeated with Co-1, Co-2, several different phages including wild type phage, and a TBS solution containing no phage.

CoPt peptide-mediated nucleation: For nucleation, 50 μL of 1 mM CoCl ₂ solution was mixed with 50 μL of 1 mM H ₂ PtCl ₆ solution. Next, 10 ml of phage solution was added (pfu = 1011). The mixture was gently stirred for 30 minutes and then 20 μL of 100 mM NaBH ₄ was added. This solution was immediately vortexed and placed in a tumbler for 30 minutes. The final solution was yellow.

FePt peptide-mediated nucleation: FePt was prepared in the same manner as CoPt, except that FeCl ₂ solution was used instead of CoCl ₂ solution.

Pm mediated nucleation of SmCo5: Same as Co synthesis except that 16.7 μL of 1 mM SmCl ₃ and 83 ul of 1 mM CoCl ₂ were used instead of 100 μL of 1 mM CoCl ₂ .

Peptide expression by P8: Genetically modified E. coli was grown overnight in 20 ml LB broth, diluted 1: 100 and then grown to OD = 0.6. Tetracycline-HCl (1000 ×) and 100 mM IPTG were added to a final concentration of lMM. IPTG initiates the production of modified p8 protein in the cell, and the modified p8 protein is incorporated into the viral coat during assembly. The mixture was left for 1 hour without stirring. One hour later, helper phage was infected and then shaken overnight at 39 ° C. Next, the phages were separated and purified by centrifugation and PEG precipitation. The amplified phage pellet was resuspended in 10 ml TBS (pH 7.5) and dialyzed against 18 MW water. Both 0.5 ml of 5 mM CoCl ₂ and 5 mM H ₂ PtCl ₆ were added to 1 ml of amplified phage stock that had been spun down and the supernatant removed. A further 60 minutes of shaking was performed, after which 0.5 ml of 100 mM NaBH ₄ was added as a reducing agent.

  TEM images of nanoparticles were taken with selected region electron diffraction patterns showing many bands corresponding to the expected values for CoPt facets. STEM images were also taken of one of these phages where CoPt nanoparticles were grown along the P8 protein. The length of this structure correlates with the length of the phage (800 nm). Figure 14A is a TEM image of a nanoparticle, Figure 14B is this high-resolution image, and Figure 14C is an electron in a selected region showing many bands corresponding to the expected values for CoPt facets. It is a diffraction pattern. An STEM image of one of these phage with CoPt nanoparticles grown along with its P8 protein is shown in FIG. 14D. The EDS mapping of Pt (FIG. 14E) and Co (FIG. 14F) shows that both Co and Pt are found at equal concentrations along the length of the structure.

  The present invention demonstrates that phage display can be used in identifying peptides that bind to magnetic materials. This identification is quick, cost effective, and requires less material. The obtained peptide can be used in controlling the nucleation of magnetic nanoparticles, and the user can control the size, composition, and crystallinity of the generated nanoparticles. The use of these peptides makes it possible to synthesize nanoparticles under ambient conditions, thus making it possible to replace the currently used nanoparticle synthesis strategy in a favorable manner.

  Phage display libraries and experimental methods for using such libraries for biopanning are described, for example, in the following Belcher et al. US patent publication. (1) “Biological Control of Nanoparticle Nucleation, Shape, and Crystal Phase”, 2003/0068900 (published on April 10, 2003), (2) “Nanoscale Ordering of Hybrid Materials Using Genetically Engineered Mesoscale Virus”, 2003/0073104 ( (Published April 17, 2003), (3) "Biological Control of Nanoparticles", 2003/0113714 (published June 19, 2003), and (4) "Molecular Recognition of Materials", 2003/0148380 (2003 2003) (Released on March 7).

  Applications of the present invention are described in the following references, including how to use them. The use of superparamagnetic materials in magnetic resonance imaging is described, for example, in Palmacci et al. US Pat. No. 5,262,176 (November 16, 1993), which is coated with colloids and polymers. The use of superparamagnetic metal oxides is also described. This reference is hereby incorporated by reference in its entirety into the present invention. Superparamagnetic materials are described, for example, in Lee Josephson et al., Bioconjugate Chem., 1999, 10, 186-191, which includes biocompatible dextran-coated superparaffins derivatized with peptide sequences. Magnetic iron oxide particles are described. This reference is hereby incorporated by reference in its entirety into the present invention. Applications include separation using magnetic resonance imaging and magnetism. J. Manuel Perez et al., J. Am. Chem. Soc., 2003, 125, 10192-10193 are used for various magnetic nanosensors such as MRI that can detect various targets including nucleic acids and proteins. Magnetic nanoparticles that self-assemble by virus induction are described. This reference is hereby incorporated by reference in its entirety into the present invention.

  Finally, the surface can be made using various methods known in the art, such as microlithography and nanolithography, and also the use of resist and self-assembled monolayers, such as self-assembled monolayers with functional groups introduced. Can be patterned.

  In the following, the manufacture and use of various aspects of the present invention will be described, but the present invention provides many applicable inventive concepts that can be embodied in various specific situations. I want you to understand. The specific embodiments described herein are merely illustrative of specific ways to make and utilize the invention, and the scope of the invention is not limited by these embodiments.

For a more complete understanding of the features and advantages of the present invention, reference is now made to the following detailed description of the present invention, taken in conjunction with the accompanying drawings. In these drawings, the same number corresponds to the same component even in different drawings.
Analysis of elemental composition analysis using phage X-ray photoelectron spectroscopy (XPS) based on gold 4f-electron signal intensity (XPS), model for discriminating semiconductor heterostructures with phage (D), two components An example of a bivalent synthetic peptide (EF) having an attachment to recognize is shown. It is a schematic diagram about the smectic arrangement | sequence of M13 phage of this invention. A7-ZnS suspension image taken using (AB) POM, (C) AFM, (D) SEM, (E) TEM, and (F) TEM with electron diffraction insert. is there. It includes images of M13 bacteriophage nanoparticles, (A) is a photograph of the film, (B) is a schematic diagram of the structure of the film, (C) is an AFM image, (D) is an SEM image, (E ~ F) is a TEM image viewed along the xz and zy planes. Fig. 5 (A) is a TEM image of annealed SmCo5 nanoparticles, Fig. 5 (B) is a TEM image showing an electron diffraction pattern of a selected region, and Fig. 5 (C) is an STEM of annealed SmCo5 nanoparticles. It is an image. An example of a binding assay is shown. (A) shows the specificity of Co-specific phage for Co, and (B) shows the isotherm of Co-specific phage of the present invention for Co. A series of CoPt nanoparticles produced by using (A) a phage expressing a 7-mer constrained peptide that selectively binds to CoPt, (B) a phage expressing a random peptide, and (C) a wild-type phage. Including high-resolution TEM images. Figure 8 (A) is a high resolution TEM image of Co nanoparticles grown using a 12-mer peptide that selectively binds to Co, and Figure 8 (B) shows the corresponding electrons. It is a diffraction pattern. Figure 9 (A) is a high resolution TEM image of FePt nanoparticles grown using phage expressing 12-mer peptides, and Figure 9 (B) is an electron diffraction pattern, which is FIG. 9 (C) was compared with FePt nanoparticles grown using wild-type phage. FIG. 10 (A) is a high resolution TEM image of SmCo5 nanoparticles grown by using a 12-mer peptide that selectively binds to SmCo5 as a template, and FIG. ) Electron diffraction patterns for selected regions, FIG. 10 (C), are SmCo5 nanoparticles grown by using wild type phage as a control. FIG. 11 (A) is an AFM image of Co-specific phage in which Co nanoparticles are bound to P3 protein, and FIG. 11 (B) is a corresponding MFM image. Figure 12 (A) shows the hysteresis loop of biologically produced FePt nanoparticles, and Figure 12 (B) shows a higher resolution scan of the center of the loop to reveal retention. is there. Figure 13 (A) is a hysteresis loop of biologically produced SmCo5 nanoparticles, and Figure 13 (B) is a plot of the center of the loop on a smaller axis to reveal retention. is there. (A) TEM of CoPt nanoparticles grown using phage genetically modified to express a CoPt-specific 12-mer sequence of the present invention on its P8 protein, (B) Same CoPt Higher resolution TEM image of nanoparticles, (C) corresponding electron diffraction pattern, (D) STEM image of particles produced in the same manner, STEM mapping for (E) Pt, STEM mapping for (F) Co Show.

Claims (32)

A method for producing a magnetic material comprising the following steps:
Providing a molecule comprising a moiety that specifically binds to the surface of the magnetic material;
The step of bringing one or more kinds of magnetic material precursors into contact with molecules under such conditions that a magnetic material is formed.   A method for producing a magnetic material, comprising contacting a molecule that initiates formation of the magnetic material with a magnetic material precursor and a reducing agent. A method for producing a magnetic material comprising the following steps:
Binding a magnetic material binding molecule to a substrate;
Contacting one or more magnetic material precursors with a magnetic material binding molecule under conditions such that the magnetic material is formed;
Forming a magnetic material;   4. The method of claim 1, 2, or 3, wherein the molecule comprises an amino acid oligomer as a moiety that specifically binds to the surface of the magnetic material.   4. The method of claim 1, 2 or 3, wherein the molecule is selected by screening a combinatorial library.   4. The method of claim 1, 2 or 3, further comprising the step of isolating the magnetic material.   4. The method of claim 1, 2 or 3, wherein the molecule is further defined as being a peptide comprising a portion of a self-assembling molecule.   8. The method of claim 7, wherein the self-assembling molecule is a phage.   4. A method according to claim 1, 2 or 3, wherein the magnetic material is nanoparticles.   4. The method of claim 1, 2 or 3, wherein the molecule comprises a chimeric protein that expresses one or more magnetic material binding amino acid oligomers on the surface.   A magnetic material formed by using binding molecules and one or more magnetic material precursors.   A magnetic material formed by using a binding molecule specific to a magnetic material in the presence of a metal salt and a reducing agent.   13. A magnetic material according to claim 11 or 12, comprising nanoparticles.   A composition comprising a peptide that specifically binds to ε-Co.   A composition comprising a peptide that specifically binds to CoPt.   A composition comprising a peptide that specifically binds to FePt.   A composition comprising a peptide that specifically binds to SmCo5.   A composition comprising a peptide that specifically binds to a ferromagnetic surface. A method of isolating a molecule that specifically binds to a magnetic material, comprising the following steps:
Contacting a library of molecules with a magnetic material;
Removing unbound molecules from the library;
Eluting bound molecules from the magnetic material.   20. The method of claim 19, wherein the library of molecules is further defined as comprising a phage display library.   20. The method of claim 19, wherein the library of molecules is further defined as comprising a combinatorial chemistry library.   20. The method of claim 19, wherein the molecule comprises an amino acid oligomer that specifically binds to the magnetic material. A method of preparing a particle film comprising the following steps:
Adding a solution of particles to the surface, wherein the particles are synthesized by using binding molecules;
Evaporating a solution of nanoparticles on the surface;
The process of creating a film of particles by annealing the particles to the surface.   24. The method of claim 23, wherein the particles are nanoparticles. A method for producing a metal material including the following steps:
Providing a molecule comprising a moiety that specifically binds to a metal surface;
Contacting one or more metal material precursors with molecules under conditions such that a metal material is formed;   A method for producing a magnetic material, comprising a step of bringing a molecule that specifically binds to a magnetic material into contact with a magnetic material precursor at a temperature of 300 ° C. or lower to form the magnetic material.   A method for producing a metal material, comprising a step of contacting a molecule that specifically binds to a metal material with a metal material precursor at a temperature of 300 ° C. or lower to form the metal material.   A composition comprising a magnetic material and an oligopeptide that specifically binds to the magnetic material.   A composition comprising SmCo5 nanoparticles.   Metal materials containing metal particles synthesized by binding molecules.   A metal material comprising a collection of metal nanoparticles synthesized by binding molecules, wherein the metal nanoparticles are annealed from a polycrystalline material to a single crystal material.   A metal material comprising a collection of metal nanoparticles synthesized by binding molecules, wherein the metal nanoparticles are each independently synthesized from a virus.

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