JP6628299B2 - Particle forming method and particles - Google Patents

Description

  The present invention relates to a method of forming a micrometer-sized structure composed of bio-based molecules by creating local supersaturation of a solution by local heating of an electric current, and to a structure formed by the method.

  Molecular self-assembly is widespread in nature, and a variety of supramolecular nano / micro self-assembled structures with well-defined morphologies form biological structures such as proteins and peptides. They have been formed by appropriately arranging them by non-covalent interaction between elements (Non-Patent Documents 1 to 7). Although the molecular structure of such a self-assembled structure is confirmed by X-ray diffraction (Non-Patent Documents 8 and 9), there are few experimental reports on the process of growing into a microstructure. Previously reported growth models for peptide-based microtubes were presented primarily based solely on scanning electron microscopy of the final structure (Non-Patent Documents 10 to 12). There have been few reports on the experimental verification of the mechanism by which microstructures grow from biomolecules in a self-organizing manner, and there is a large gap between the experimental results and the proposed model. Therefore, in order to control and manufacture a micro-sized structure that resembles or mimics the form of an organism, a deep understanding of the self-assembly process composed of molecules is strongly required.

  On the contrary, the examples of artificially producing the structures of these peptide aggregates are mostly simple, and techniques for constructing complex and functional morphologies have attracted much interest. In a complex self-assembled architecture, the functionality of the molecule itself is enhanced by the hierarchical cooperation between the nanoscale components and the entire microscale size (Non-Patent Documents 13 to 16). However, most of the hierarchical architectures reported to date have been made using inorganic materials (13, 17). In the field of catalysts, sensors and cell cultures, there is a need to create an architecture based solely on organic molecules, especially biomolecules, to achieve cost-effective and environmentally friendly applications (Non-Patent Document 14). ~ 16). Designing, controlling and fabricating molecular self-assembly architectures has led to significant advances in nanopharmaceuticals (Non-Patent Document 18) and nanophotonics (Non-Patent Document 19).

  An object of the present invention is to create a partially supersaturated state by rapidly heating a part of a solution in which a peptide molecule is dissolved. In a supersaturated solution, aggregation of molecules is likely to occur, and as a result, nuclei for self-organization of peptide molecules are formed. In other words, rapid heating of a part of the solution is to realize a new condition for forming a microstructure. It is another object of the present invention to provide a microstructure of a peptide having a structure and shape that has not been realized until now under the novel conditions.

According to one aspect of the present invention, a solution in which a peptide is dispersed in a solvent that evaporates by increasing the temperature is prepared, and the solution is locally heated by Joule heating by applying an electric current to form crystal nuclei of the peptide. A particle forming method for forming particles of the peptide by self-growth around the crystal nucleus.
Here, the supersaturated state of the peptide may be expressed in the solution by the local temperature increase, thereby forming a crystal nucleus of the peptide.
Further, the current application may be performed by causing two electrodes to face each other and applying a voltage between the two electrodes, thereby causing a current to flow through the solution existing in a gap between the electrodes.
Further, the two electrodes may have a tapered shape, and the voltage may be applied with their tapered tips facing each other.
Further, the current application may be performed by applying a voltage to at least one conductive path to locally heat the solution on the conductive path.
Further, the temperature of the solution may be locally raised to the boiling point of the solvent.
After the temperature rise, the application of the current may be stopped.
Further, the peptide may be a peptide having less than 10 amino acid residues (hereinafter, referred to as a short peptide).
The particles may be spherical particles having a particle size of 1 μm to 20 μm and having a porous body on the surface.
The average pore diameter of the porous body may be 20 nm to 500 nm.
Further, the porous body may have a network structure including a plurality of nanowires having an average diameter of 20 nm to 300 nm.
According to another aspect of the present invention, particles having a particle size of 1 μm to 20 μm, which are made of a peptide and have a porous body having an average pore diameter of 20 nm to 500 nm on the surface, are provided.
Here, the porous body may have a network structure including a plurality of nanowires having an average diameter of 20 nm to 300 nm.
Further, the peptide may be a short peptide.
Further, the short peptide may be selected from the group consisting of a diphenylalanine peptide and a phenylalanine peptide.

  According to the present invention, a new growth mode can be realized by creating a region where molecules are supersaturated locally and rapidly in a solution. Also, a novel microstructure having a porous shell around the core was obtained.

Schematic diagram of an electrode and a solution used for controlling self-assembly of molecules. The distance between the two gold electrodes is about 120 μm. A photograph of the electrode after the solution was dropped. The graph which shows the time change of an electric current after dripping 4 microliters of 1 mg / mL solution (solvent is ethanol) between the electrodes to which the voltage of 100V was applied. The voltage application time is 25 seconds. The figure which shows the temperature distribution measured by thermography when the solution is dripped at the gap which applied the voltage of 100V. The scanning electron beam microscope image of the porous microstructure produced using the methanol solution of the diphenylalanine peptide molecule of 1 mg / microliter concentration. Scanning electron microscope image of a microsphere generated between electrodes after voltage application. FIG. 2B is a scanning electron microscope image of an enlarged view of the microsphere shown by an arrow in FIG. 2A. Scanning electron microscope image of a microtube created by applying a voltage immediately after dropping. Scanning electron microscope image of a molecular sphere created without applying a voltage. The figure which shows the spectrum obtained by performing time-of-flight secondary ion mass spectrometry (TOF-SIMS) with respect to the created microstructure. FIG. 4 is a conceptual diagram of an electrode for applying an electric field of 10 ⁷ to 10 ⁵ V / m and a solution dripped by forming an arrangement in which no current flows between two electrodes or a probe with a cover glass (about 100 μm in thickness) interposed therebetween. FIG. 4 is a conceptual diagram of an electrode on a glass substrate in which two electrodes are connected by a thin gold wire (thickness: 100 nm, width: 300 nm, length: 600 μm). Scanning electron microscope image of a porous microstructure created by applying a voltage of 0.5 V between electrodes for 50 seconds and controlling self-organization by local Joule heat. Phenylalanine (FY) peptide molecule (inset shows molecular structure) was dropped on a glass substrate without applying voltage to a solution (concentration: 1 mg / 1 mL) dissolved in methanol and formed by self-assembly. Scanning electron microscope image of a wire-like structure. Scanning electron microscope image of a microstructure grown after a similar solution was dropped under an electrode and a voltage of 100 V was applied between the electrodes for 30 seconds.

  In the method of the present invention, supersaturation of a peptide molecule is expressed in a heated portion by a local rapid rise in temperature caused by locally heating a solution in which a peptide molecule is dispersed using Joule heating by applying a voltage. Molecular collisions frequently occur in a supersaturated solution, and crystal nuclei are formed in a region where the supersaturation occurs. Microparticles are formed by self-organizing growth centering on this crystal nucleus. Here, the microparticle refers to a particle having a size of 1 μm to 20 μm. The present invention is the first invention that artificially creates a locally supersaturated region to control the self-assembly of molecules. When an electric current is applied to a very small area of the solution, Joule heating can partially and rapidly heat the solution. Therefore, a condition for generating a unique microstructure is given from a balance between nucleation and sufficient supply of molecules from a nearby region due to convection and other flows of the solution. Under the conditions for generating the microstructure, the surface of the microparticle is formed by entanglement of nanowires having an average diameter of 20 nm to 300 nm (in the embodiment, 50 nm to 100 nm). Structure) is formed. The present invention also relates to microparticles that can be formed by such a method.

  In this application, one method to achieve controlled production using a low molecular weight diphenylalanine (FF) peptide is shown. The FF peptide is an important molecule constituting a protein, and is known as a core recognition motif of Alzheimer's β-amyloid polypeptide (Non-Patent Documents 8, 20 to 23). Self-assembled peptides are very attractive components due to their chemical diversity, specific molecular detection capabilities, availability and functional flexibility (24-27). In the present application, a unique peptide-based flower-shaped microstructure, similar in shape and size to the marine algae diatom, is created using early nucleation under artificial supersaturation. The microstructure formation technique by local supersaturation formation in the present application is a self-organization control using commonly used external stimuli such as solvent polarity, concentration, pH or enzyme to control the self-assembly of molecules. It is different (Non-Patent Documents 5, 6, 28).

<Method for fabricating microstructure>
[Preparation of FF peptide solution and gold electrode]
FF peptide (Bachem) powder at a concentration of 100 mg / mL in 1,1,1,3,3,3-hexafluoro-2-propanol (1,1,1,3,3,3-hexafluoro-2-propanol) , HFIP). This FF peptide solution was diluted with methanol to a final concentration of 1 mg / mL. To avoid aggregation and self-assembly in advance, a new FF peptide solution was prepared for each experiment. A gold electrode was formed on a glass substrate by depositing chromium (10 nm) and gold (50 nm) through a mask pattern by an electron gun (e-gun) deposition method. This electrode structure has a shape in which two triangular electrodes face each other with their vertexes close to each other, and a gap between these vertices is called a gap.

[Preparation and measurement of microstructure]
To study the growth of the microstructure, a 4 mL droplet of the FF peptide solution was dropped into the gap between the electrodes, as conceptually shown in FIG. 1a, and a voltage range V (10 １００100 V) was applied for 25 to 85 seconds. FIG. 1B shows a photograph near the gap immediately after the dropping. A voltage was applied, and the current was monitored by a measuring instrument SourceMeter2400 (manufactured by Keithley). The result is shown in FIG. 1c. The microstructure growth was recorded using an optical microscope fitted with a CCD camera. After the solvent air dried (3-20 minutes), porous, unique microspheres and smooth microtubes grew. The TOF-SIMS positive ion spectrum of the prepared microstructure measured using PHI TRIFT V nano TOF manufactured by ULVAC-PHI was, as shown in FIG. At 313 m / z (mass-charge ratio), corresponding to singularly charged ions. This indicates the structural integrity of the FF peptide molecule under the experimental conditions applied in this example. As the applied voltage was reduced, the ratio of the fully grown "diatom-like" porous structure to the partially grown porous structure was reduced. When the applied voltage is reduced, a long application time is required to create a completely porous structure.

[Temperature measurement]
The temperature distribution of the sample during the application of the voltage was measured using a thermograph (ViewOhre Imaging, AIR32 Micro3x). In the initial stage, the substrate is covered with a thick solution layer having a thickness of 100 to 400 μm. Current flows mainly between the electrodes located at the bottom of the solution. Several seconds after the start of the voltage application, foaming was observed in a part of a slightly darker region in the center of the gap in FIG. 1D. This suggests that the local temperature of the region has reached the boiling point of methanol (about 64.7 ° C.). When the voltage application time is short (about 60 seconds or less), the volume of the local region where the temperature rise is greatly increased by Joule heating is considerably smaller than the total volume of the solution. Therefore, when the heated solution moves far by convection, it is cooled to room temperature. The slightly darker part in the center and the surrounding part in FIG. 1d correspond to about 60 ° C. and about 24 ° C., respectively. In other words, it can be seen that the temperature directly above the gap between the electrodes and in the vicinity of the gap is greatly increased locally.

[Formation of diatom-like porous microstructure]
FIG. 2a shows a scanning electron microscope image (SEM image) of a porous microstructure (average particle size of 5 to 10 μm) prepared using a diphenylalanine peptide solution (solvent: methanol) having a concentration of 1 mg / μL. In a typical configuration for creating microstructures as shown in FIG. 1a, one drop of methanol solution of FF peptide is dropped between two Au electrodes with a gap of 120 μm, as shown in FIG. 1b. did. When a voltage (eg, 100 V) is applied between the electrodes, a current on the order of 100 μA flows through the solution. This current caused the temperature of the gap region to increase due to the Joule heating effect. FIG. 1d shows thermographic mapping of a region centered on the gap when 4 μL of the above solution is dropped into the gap and a voltage of 100 V is applied between the two electrodes for 25 seconds. It was found that the slightly dark portion at the center was about 60 ° C, and the brightest part slightly deviated therefrom was about 40 ° C. After drying of the solvent (3-6 minutes), several microspheres and microtubes were observed on the substrate, as shown in FIGS. 2b-2d. The shape of these microspheres closely resembles the micrometer-sized diatom (marine algae diatom) shown in the inset in the lower right part of FIG. 2a. The surface morphology can be seen from the high magnification SEM image of the microsphere shown in the upper right inset of FIG. 2a. Specifically, a plurality of wires having a large number of protrusions (average diameter of 50 to 100 nm) are entangled to form a three-dimensional network structure as a whole, which is very similar to a diatom frustule. I have. When a typical voltage of 100 V is applied, the initial growth stage for forming the microstructure requires an application time of about 25 to 30 seconds after 4 μL of the solution is dropped. When the methanol solution of the FF peptide is naturally evaporated, a spherical dot-like structure having a diameter of 100 to 400 nm shown in FIG. Our results showed a clear morphological transition from nanoscale dot-like structures to micron-scale spheres and tubes. The structural integrity of FF peptide molecules in porous microspheres and microtubes was determined by time-of-flight secondary ion mass spectroscopy (TOF-SIMS) (Non-Patent Document 22). It was confirmed by using (FIG. 3).

Several systematic experiments were performed to confirm the effects of temperature, electric field and current on the formation of the microstructure. ¹⁰ 5 ~10 7 V / ^m electric field comparable which is realized between the electrodes of the present experiment is maintained was prepared three different electrode patterns, no current flows (see FIG. 4). When the FF peptide solution was dropped on these electrode patterns, a porous microstructure could not be formed at all, and a typical dot-like sphere similar to the case where no voltage was applied was observed. Furthermore, even when the temperature of the solution was uniformly raised to 30 to 65 ° C. using a heater, the microstructure according to the present invention could not be produced at all. From these results, the electric field and temperature could be excluded from the list of factors that could affect the microstructure formation according to the present invention. The temperature distribution shown in FIG. 1d also has a low current density (about 1 × 10 ⁻¹⁰ A / μm ² ) due to the wide current path between the electrodes. Therefore, the influence of the current could be removed from the above list. Further, instead of passing a current through the solution, a narrow metal wire was provided between the electrodes as shown in FIG. 5A, and a voltage as low as 0.5 V was applied between the electrodes. Another type of artificial supersaturation caused by Joule heating in the metal wire also formed the microstructure according to the present invention as shown in FIG. The possible involvement of the reaction was also ruled out.

  For the purpose of confirming whether the present invention can be applied only to a specific molecule or extend to other peptide molecules, a phenylalanine (FY) peptide molecule (the molecular structure is shown in the inset of FIG. 6a) is dissolved in methanol. An experiment was performed using the solution (concentration 1 mg / 1 mL). When the FY peptide solution was dropped on the glass substrate without applying a voltage, a wire-like structure was formed by self-organization (see FIG. 6a). When a similar solution is dropped under the electrodes and a voltage of 100 V is applied between the electrodes for 30 seconds, the diameter of the network-like porous structure in which a large number of nanowires are entangled on the surface can be seen in the scanning electron microscope image of FIG. 6b. However, a microstructure of about 2 to 10 μm was realized. That is, it was confirmed that the method was a microstructure preparation method having generality even for small peptide-based molecules.

[Conclusion]
The present application has presented, for the first time, a simple production method for producing a “diatom-like” composite structure from a small dipeptide and its growth mechanism. The formation of small nuclei induced by artificial supersaturation causes morphological transitions by self-assembly and the creation of unique microstructures. Microspheres have a porous structure similar to diatoms, but the structure of diatoms is known as a high-sensitivity biosensor (Non-Patent Documents 32-34). Therefore, even if a short peptide (a peptide having less than 10 amino acid residues) is replaced with another functional biomolecule such as a fluorescent protein, polypeptide or enzyme, a hierarchical functional biomaterial may still be produced. However, this would result in low cost and environmentally friendly biosensors, biophotonic devices and biocatalysts.

  Since a porous and hydrophilic structure was produced, applications such as sensors and catalysts can be expected. In addition, the method of production is general because it has been realized with other peptide molecules, and when creating a desired microstructure from biomaterials related to medicine, create a microstructure containing functional molecules required for photonics May be adaptable in some cases.

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Claims (12)

Prepare a solution in which a peptide selected from the group consisting of diphenylalanine peptide and phenylalanine peptide is dispersed in a solvent that evaporates by raising the temperature,
The solution is locally heated by Joule heating by applying an electric current to form crystal nuclei of the peptide,
Forming particles of the peptide by self-growth around the crystal nucleus,
Particle formation method. Expressing the supersaturated state of the peptide in the solution by the local temperature increase, thereby forming a crystal nucleus of the peptide,
The method for forming particles according to claim 1.   3. The method according to claim 1, wherein the current application is performed by causing two electrodes to face each other and applying a voltage between the two electrodes to cause a current to flow through the solution existing in a gap between the electrodes. 4. The method for forming particles according to the above.   The particle forming method according to claim 3, wherein the two electrodes have a tapered shape, and the voltage is applied with the tapered tips facing each other. The current application is performed by applying a voltage to at least one conductive path,
Locally heating the solution on the conductive path,
The method for forming particles according to claim 1.   The particle forming method according to claim 1, wherein the temperature of the solution is locally increased to a boiling point of the solvent.   The method according to claim 1, wherein the application of the current is stopped after the temperature is increased. The particles are particle size 1 m to 20 m, a spherical particle with a porous material to the surface, the particles forming method according to any one of claims 1 to 7. The particle forming method according to claim 8 , wherein the porous body has an average pore diameter of 20 nm to 500 nm. The porous body having a network structure in which the average diameter comprising a plurality of nanowires 20 nm to 300 nm, the particles forming method according to claim 8 or 9. Consisting of a peptide selected from the group consisting of diphenylalanine peptide and phenylalanine peptide ,
Having a core and a porous shell around the core,
The porous body has an average pore diameter of 20 nm to 500 nm,
Particles having a particle size of 1 μm to 20 μm. The porous body having a network structure in which the average diameter comprising a plurality of nanowires 20 nm to 300 nm, the particles of claim 1 1.