JP6598234B2 - Sericin-copper phosphate hybrid structure, production method thereof, and heavy metal ion adsorbent - Google Patents

Description

  The present invention relates to an organic-inorganic hybrid structure, and more particularly to a sericin-copper phosphate hybrid structure that can be prepared from sericin and copper sulfate, a method for producing the same, and an application of heavy metal ion removal using the same.

  The structural complexity, ease of creation and potential application of the myriad natural patterns of a functional hybrid structure combining inorganic and organic components has always fascinated researchers and assembled them It has continued to inspire efforts to understand and control the process (Non-Patent Documents 1 to 3). The approach of synthesizing a hierarchical hybrid structure, which has a micron-scale size as a whole and is composed of complex parts of nanoscale on a laboratory basis, is used to produce a well-defined structure. It cannot be controlled at the same level of precision (Non-Patent Documents 4 to 8). Organic-inorganic hybrid “flower-like structures” (also referred to as bioinorganic hybrids) produced from biomolecules are widely used in biocatalysis, drug delivery, biosensing, etc. In order to have patent documents 9 to 12), there has been an increasing interest recently. The interaction between the biomolecule and the inorganic material plays a role of further increasing the functionality of the hybrid material itself. Compared to compact structures such as dense spheres or polyhedrons, hierarchical flower-like structures have a much larger surface area, and if porous, their functionality is further expanded ( Non-patent document 9). By immobilizing and taking in biomolecules in the core of the nanoscale flower-like structure, it shows greater activity, durability and stability than molecules that move around freely in the solution (Non-Patent Documents 9 to 13). ). In addition, if the biomolecules contained in the hybrid structure can be removed without breaking the hierarchical morphology that constitutes the structure, the place where the biomolecules were contained becomes an empty area, and the porosity and surface area increase. A structure produced by such a method can be an excellent catalyst (Non-Patent Document 14).

  Biomolecular hybrids are not only due to the application of their combined functions, but are also chemically diverse, flexible in composition, and have good biocompatibility, making great efforts to produce them. Has been poured. However, as reported so far, most of the hierarchical bio-inorganic hybrids have been made using enzyme molecules. Therefore, in order to explore possibilities in various aspects, it is strongly required to realize other functional biomolecules.

  Silk, a natural polymer, is one of the most promising biopolymers, but it has a rich history of traditional uses in textile and medical applications for thousands of years. (Non-Patent Documents 15 and 16). Silk is a promising candidate for multifunctional advanced materials because it is readily available, has good mechanical strength, optical properties and biocompatibility, and is capable of managing degradation (non-patented) References 15-17).

  Recent major developments include photonics (Non-Patent Document 18), lithographic resists (Non-Patent Document 19), microstructure patterns (Non-Patent Document 20), biomedical devices (Non-Patent Document 21) and drug delivery (Non-Patent Documents). 22) A new hopeful possibility for the application of silk to ref. 22) has been clarified, which further provides an innovative method for producing silk-based materials on multiple scales for use as a host for silk applications. It became necessary to develop. Silk obtained from silkworms is mainly composed of two types of proteins, namely, fibroin, which is a protein in the core of the fiber, and glue protein called sericin. Sericin is coated as a continuous adhesive layer on fibroin fibers, and silk fibers are bonded together to produce wrinkles. Unlike insoluble fibroin, sericin has unique properties such as good hydrophilicity, biodegradability, and resistance to oxidation, bacteria and ultraviolet light (Non-Patent Documents 23 to 26). Sericin also exhibits various biological activities and pharmacological functions, while promoting cell attachment, growth and proliferation by addition to culture media (Non-patent Documents 28 and 29).

  Despite these various advantageous properties, the use of sericin for the creation of functional structures has hitherto been difficult because it is difficult to process sericin to obtain useful materials. Compared to fibroin, which has been well studied, its scope is still limited. However, several recent studies have explored interesting applications of silk sericin and opened up new perspectives on the usefulness of sericin (29, 30).

  Apart from this possibility, there are two main reasons that strongly motivate the selection of sericin to create hierarchical hybrid floral structures. Firstly, most of the amino acids possessed by sericin have molecular chains having strong polarities such as hydroxyl groups, carboxyl groups and amino groups. As a result, sericin is water-soluble and has a characteristic of strongly binding to metal (Non-patent Documents 17 and 31). Secondly, sericin is a very cheap and abundant material that has usually been discarded as a by-product during the degumming process during raw silk production (Non-Patent Document 17). .

   The present invention solves the above-mentioned problems of the prior art, and uses sericin that has not been sufficiently utilized as a by-product in raw silk production, and uses a sericin-copper phosphate hybrid structure having a complex nanostructure. It is an object of the present invention to provide a body (hereinafter referred to as a sericin-copper phosphate hybrid floral structure or simply a hybrid floral structure). Another object is to use this hybrid structure to remove heavy metal ions dissolved in water.

According to an aspect of the present invention, the center portion includes a plurality of sheets in which one or a plurality of sericin-copper phosphate hybrid nanosheets are stacked on each other, and the plurality of sheets extend in different directions from the center portion. A bound sericin-copper phosphate hybrid structure is provided.
Here, the hybrid structure may have a substantially spherical shape.
The size of the hybrid structure may be in the range of 2 to 20 μm.
The average thickness of the sheet may be in the range of 200 to 600 nm.
The average thickness of the sericin-copper phosphate composite nanosheet may be in the range of 15 to 50 nm.
The sheet may have pores.
According to another aspect of the present invention, the sericin-copper phosphate hybrid structure has a plurality of sheets in which one or a plurality of sericin-copper phosphate hybrid nanosheets are stacked, and the plurality of sheets Provided with a copper phosphate structure having a flower- like morphology of structures connected at the center so as to extend in different directions from the center , and having sericin removed from the sericin-copper phosphate hybrid structure. It is done.
According to yet another aspect of the present invention, a method is provided for producing a sericin-copper phosphate hybrid structure as a precipitate from a phosphate buffer containing sericin and copper ions.
Here, the phosphate buffer may further contain chloride ions.
The copper ions may be provided by adding copper sulfate to the phosphate buffer.
Moreover, the density | concentration of sericin may be 0.1-0.3 mg / mL, and the density | concentration of the copper sulfate which gives the said copper ion may be 1-3 mM.
Or the density | concentration of sericin may be 0.01-0.05 mg / mL, and the density | concentration of the copper sulfate which gives the said copper ion may be 0.1-0.5 mM.
Or the density | concentration of sericin may be 0.4-1 mg / mL, and the density | concentration of the copper sulfate which gives the said copper ion may be 4-10 mM.
According to still another aspect of the present invention , one or a plurality of sericin-containing sericin-copper phosphate hybrid structures are obtained by heat-treating the sericin-copper phosphate hybrid structures manufactured by any of the above methods. The sericin has a plurality of sheets in which copper phosphate hybrid nanosheets are stacked on each other, and maintains the flower- like morphology of the structure in which the plurality of sheets extend from the central part in different directions. A method of manufacturing a copper phosphate structure is provided that removes.
According to still another aspect of the present invention, there is provided a heavy metal ion adsorbent comprising any one of the sericin-copper phosphate hybrid structures.
Here, the heavy metal ion may be an ion of one or more elements selected from the group consisting of lead, mercury and cadmium.

  According to the present invention, a hybrid structure having a larger surface area than a conventional similar material can be produced in a shorter time. Further, by annealing the hybrid structure at a high temperature, it is possible to produce a structure having a further increased porosity while maintaining the overall structure.

The figure which shows notionally the synthetic | combination process of the organic-inorganic hybrid floral structure of one Example of this invention incorporating the sericin which is silk protein. (A) SEM image of a single hybrid flower-like structure of one embodiment of the present invention. The inset is a picture of the natural flower that made this metaphor. (B) Scanning electron microscope (SEM) image showing the hierarchical structure of porous petals corresponding to (a). (C) High-resolution SEM image of the stacked nanosheets constituting one petal. (D) A high-resolution SEM image of the surface of a single porous petal. (E) Transmission electron microscope (TEM) image of the hybrid flower-like structure of one example of the present invention. (F) High-resolution TEM image corresponding to (e), showing the crystal lattice structure of the petals. The inset shows the plaid of the selected area. (G) The laser scanning microscope (LSM) image of the hybrid flower-like structure of one Example of this invention. The figure which shows the elemental-analysis result of the hybrid floral structure of one Example of this invention performed with the energy dispersive X-ray analysis (EDX) measuring apparatus. (A) is the result obtained using the device attached to the SEM, and (b) is the result obtained using the device attached to the TEM. Shows sericin silk protein amorphous, copper phosphate crystals (CuSO ₄ and sericin what was synthesized from non with PBS added) and the XRD pattern of an embodiment crystalline hybrid flower-like structures of the present invention, respectively . Furthermore, the XRD pattern (Cu ₃ (PO ₄ ) ₂ .3H ₂ O, (JCPDS 00-022-0548)) of a standard copper phosphate is shown at the bottom of the figure, and this pattern is a hybrid flower-like structure. It perfectly matches the body's XRD pattern. Hybrid flower-like structure of an embodiment of the present invention, shows an XPS spectrum data of sericin and copper phosphate (which was synthesized from a PBS without the addition of CuSO ₄ and sericin). The figure which shows the XPS binding energy curve of Cu, P, and O from the hybrid flower-like structure of one Example of this invention, respectively. SEM images showing various morphologies of hybrid floral structures of one example of the present invention made by varying the sericin to Cu ⁺² concentration ratio in 1X PBS. (A) and (b) are SEM images of a hybrid flower-like structure of one example of the present invention obtained from 0.01 mg / mL and 0.5 mg / mL sericin and 1 mM Cu ⁺² , respectively (c ) And (d) are SEM images of the hybrid floral structure of one example of the present invention obtained from 0.5 mM and 2 mM Cu ⁺² and 0.1 mg / mL sericin, respectively. Further, (a ′) to (d ′) are enlarged views of one hybrid flower-like structure indicated by a white broken arrow in (a) to (d), respectively. The SEM image of the structure produced by changing the density | concentration of PBS. (A) is prepared using 2X PBS, and (b) and (c) are prepared using 05X PBS. Also, these insets show the respective high magnification images. The figure which shows the diversity of the morphology change of the hybrid floral structure of one Example of this invention when the density | concentration of sericin is changed. (A) is 1 mg / mL, (b) is 0.5 mg / mL (c) is 0.1 mg / mL, and (d) is the case of sericin concentration of 0.01 mg / mL. The insets of (a) to (d) are a corresponding single flower-like structure (upper side) and an SEM image (lower side) in a further enlarged state. The figure which shows the time change (0.1 mg / mL sericin is used) of the growth of the hybrid flower-like structure of one Example of this invention. (E) shows SEM images for 10 minutes, (f) for 2 hours, (g) for 6 hours, and (h) for 12 hours of growth time. The insets of (f) to (h) are enlarged views of a rectangular region indicated by a black broken line in (f) to (h). The SEM image which expands and shows one hybrid floral structure of one Example of this invention when changing a sericin density | concentration, and its petal. The sericin concentrations were 0.01 mg / mL for (a), 0.05 mg / mL for (b), 0.1 mg / mL for (c) and 0.25 mg / mL for (d). Further, (a ′) to (d ′) are enlarged images of white square regions in the vicinity of the center in (a) to (d), respectively. The petal thickness increased with increasing sericin concentration. The SEM image of the spherical hybrid floral structure of one Example of this invention which has a dense petal which has a mesh structure. The sericin concentration is 1 mg / mL in (a) and 0.5 mg / mL in (c), and (b) and (d) are enlarged SEM images of the surfaces of (a) and (c), respectively. The SEM image of the hybrid flower-like structure of one Example of this invention when changing sericin density | concentration to 0.01 mg / mL and growth time. The growth time is 10 minutes for (a), 2 hours for (b), 6 hours for (c) and (d), and 12 hours for (e) and (f), respectively. The insets of (b), (d), and (f) are high-resolution images of the respective drawings. (A) And (b) is the SEM image of the hybrid floral structure of one Example of this invention processed with glutaraldehyde and EDTA, respectively. (C) is an ATR-FTIR spectrum of the hybrid flower-like structure of one example of the present invention. (D) is a nitrogen adsorption-desorption isotherm of the hybrid flower-like structure and copper phosphate of one example of the present invention. The inset shows the pore size distribution. (E) is an SEM image showing the thermal stability of the hybrid flower-like structure of one example of the present invention after annealing at 400 ° C. for 2 hours. The inset shows a single flower-like structure. (F) is an SEM image of the petals in the hybrid flower-like structure after calcination shown in (e). The inset shows the magnified image. (A) And (b) is the low-magnification SEM image of the flower-like structure of one Example of this invention which deform | transformed and destroyed after adding glutaraldehyde and EDTA, respectively. SEM image of a precipitate formed from 1X (10 mM) phosphate buffer containing 0.1 mg / mL sericin and 1 mM CuSO ₄ but no chloride ions (ie, no NaCl or KCl). It can be seen that no hybrid flower-like structures are found. Formed from 1X (10 mM) phosphate buffer containing 0.1 mg / mL sericin and 1 mM CuSO ₄ , further (a) containing bromide ions (KBr) and (b) iodine ions (KI). SEM image of the sediment. It can be seen that no hybrid flower-like structures are found. The inset is an enlarged image of each. ATR-FT IR spectrum of silk proteins sericin and copper phosphate (synthesized without adding sericin from CuSO ₄ and PBS). The figure which shows the EDX and ATR-FT IR measurement result after heat-processing the hybrid floral structure of one Example of this invention at 400 degreeC for 2 hours. (A) The figure which shows an EDX elemental analysis result. Carbon peaks are not shown. (B) ATR-FT IF spectrum. The characteristic amide band of the silk protein is not shown (only the phosphate band is present). The figure which shows the thermal-analysis result ((a) is TG, (b) is DSC) of the hybrid flower-like structure of one Example of this invention. (A) and (b) are TG and DSC curves for sericin, respectively, and (c) and (d) are TG and DSC curves for copper phosphate (CuSO ₄ and PBS without sericin added), respectively. . The figure which shows a mode that the density | concentration of the lead ion in the aqueous solution which disperse | distributed the hybrid flower-like structure of one Example of this invention reduces with progress of time. The figure which shows a mode that the density | concentration of the mercury ion and cadmium ion in the aqueous solution which disperse | distributed the hybrid floral structure of one Example of this invention reduces with progress of time.

  According to one aspect of the present invention, a complex one-pot coprecipitation method that uses silk protein sericin as an organic component and copper (II) phosphate as an inorganic component and does not modify the original protein structure. An organic-inorganic hybrid floral structure is produced in a controlled manner. This organic-inorganic hybrid floral structure can be fully characterized by various microscopic and spectroscopic techniques. The inventor has previously optimized the growth conditions of biomolecules and metal ions for the formation of biohybrid floral structures in order to adapt to growth scalability. The floral morphology of the synthesized hybrid structure is highly dependent on the concentration of sericin and the strength of the phosphate buffer solution. Furthermore, it was observed that the surface area of this porous hybrid structure was significantly increased compared to the corresponding metal phosphate. This is very important from the viewpoint of various applications.

  The size of the hybrid floral structure is preferably 2 to 20 μm. The “petal” portion of the hybrid flower-like structure has a structure in which sericin-copper phosphate hybrid nanosheets are stacked on each other, but the average thickness of each nanosheet is preferably 15 to 50 nm, and the total petal thickness Is preferably 200 to 600 nm.

  Furthermore, when the hybrid flower-like structure in an as-grown state completely dissipates the sericin molecules by calcination, the porosity of the petal portion of the flower-like structure is significantly increased, but good thermal stability is achieved. Indicates. In such a state after calcination, the flower-like structure loses the characteristics of an organic-inorganic hybrid, but the porosity becomes more remarkable while maintaining the morphology of the structure before calcination, and the organic matter It has useful characteristics such as being able to withstand high temperature use environment because it does not contain.

  As an example of utilization of the structure of the present invention, in the examples described below, it is shown that the hybrid flower-like structures produced in the examples have the ability to adsorb lead ions from water.

  Hereinafter, the present invention will be described in more detail based on examples. Here, it should be noted that the following examples are intended to assist the understanding of the present invention and are not intended to limit the present invention.

A general procedure according to one embodiment of the present invention for the synthesis of a silk protein sericin-inorganic hybrid floral structure is shown in FIG. In a typical experiment, an aqueous CuSO ₄ solution (1 mM) was added to 10 mM phosphate buffered saline (1 × PBS, pH 7.4) containing a clear solution of silk protein sericin (0.1 mg / mL). Added. The resulting mixture was gently shaken for a few minutes and left at room temperature (about 25 ° C.). After 12 hours, a large amount of blue precipitate appeared, having a porous and uniformly grown flower-like structure. In contrast, in sericin alone 1X PBS solution, sericin self-assembles to form nanoparticles of various diameters, and from copper sulfate alone 1X PBS solution precipitates as large crystals of copper sulfate, In these, no traces of flower morphology were seen.

  FIG. 2A shows a scanning electron microscope (SEM) image of one hybrid flower-like structure (average size 6 to 8 μm). This SEM image shows that petals (FIG. 2 (b)) having a thickness in the range of 200 to 600 nm have a clear hierarchical structure. When observed in detail, each petal is actually a combination of several tens of nanosheets (average thickness 15-30 nm) attached to each other, and there are numerous pores or cracks on these surfaces ( 2 (c) and (d)) were found.

A transmission electron microscope (TEM) image and a laser scanning microscope (LSM) image of the hybrid flower-like structure are shown in FIG. 2 (c) and FIG. 2 (g), respectively. Here, one crystal lattice fringe of the nanosheet petal is clearly observed by a high-resolution TEM image (inset of FIG. 2 (c)). Energy dispersive X-ray (EDX) analysis of the hybrid floral structure showed the presence of C, O, P, Cl and Cu (FIG. 3). The crystal structure of the hybrid floral structure was examined using X-ray diffraction (XRD). The sharp and strong diffraction peaks confirm the highly crystallized characteristics of the hybrid flower-like structure different from amorphous silk sericin, and these peaks are Cu ₃ (PO ₄ ) _2. Fits well for 3H ₂ O. Furthermore, by performing X-ray photoelectron analysis (XPS), individual components of the hybrid material and their atomic concentrations were examined. The results are shown in Table 1 and FIGS. The characteristic binding energy peak clearly shows the presence of protein molecules and copper phosphate in the hybrid structure.

In order to optimize the growth conditions for the formation of flower-like hierarchical hybrid structures, several synthetic experiments were performed with varying reaction conditions. The morphology of the as-grown hybrid material depends mainly on the concentration of sericin, Cu ⁺² , PBS and the reaction time. As shown in FIG. 7, various types of flower-like structures were generated by combining the range of concentration ratios of sericin and Cu ^{+2 in} 1 × PBS. In contrast, when the PBS concentration was doubled (2X or 20 mM) without changing other parameters, it was observed that a somewhat ambiguous morphological hybrid structure precipitated relatively quickly, At half concentrations of PBS (0.5X or 5 mM), only a few microstructures in solution were possible, which were slightly lumpy solid precipitates. These results are shown in FIG. Based on the above experimental results, for the subsequent experiments, the condition of creating a hybrid flower-like structure using 1 × PBS containing 0.1 mg / mL sericin and 1 mG Cu ⁺² was selected.

  Here, the effect of the sericin concentration on the formation of a hybrid flower-like structure was examined while the above-mentioned optimum ratios of the individual components were fixed. By changing the sericin concentration within a wide range (0.01 to 1 mg / mL), a large variation was observed in the morphology of the synthesized hybrid floral structure. The results of this experiment showed that the hybrid flower-like structures made from 1 mg / mL and 0.5 mg / mL sericin were smaller and uniformly grown spherical objects (FIGS. 9A and 9B). . As the concentration of sericin is gradually reduced (0.1 mg / mL and 0.01 mg / mL), the morphology of the hybrid flower-like structure changes, mimicking larger, full bloom nature flowers. (Fig. 9A (c), (d)). At very low sericin concentrations (0.01 mg / mL), the individual petals appeared to be very thin, but as the concentration increased to 0.25 mg / mL, they integrated as shown in FIG. Much thicker and smaller petals. When the concentration was further increased, the morphology changed to a spherical shape, as shown in FIG. 11, where the surface appeared to form a dense petal mesh structure and the petals were curved. There are a number of voids between the petals as well as the undulating edges on the wave (FIG. 11). This result indicates that the formation and morphology of hybrid floral structures are strongly dependent on sericin concentration. This result is also significantly different from previous reports (Non-Patent Documents 9 and 11) on enzyme hybrids in which the dramatic change in morphology as described above was not observed depending on changes in protein concentration.

Furthermore, by changing the conditions in various ways, when the petals have a multi-layer structure and a single-layer structure, and as a whole, the structure of a high-density petal mesh on the surface (that is, a large number of holes) as shown in FIG. As a result of obtaining the production conditions in the case of taking a spherical structure with the following, the following results were obtained.
-Preparation conditions of the flower-like structure of a multilayer structure: Sericin concentration is 0.1-0.3 mg / mL. CuSO ₄ concentration is 1 to 3 mM.
-Preparation conditions of the flower-like structure of a single layer structure: Sericin concentration is 0.01-0.05 mg / mL. CuSO ₄ concentration is 0.1-0.5 mM.
Spherical structure production conditions: Sericin concentration is 0.4 to 1 mg / mL. CuSO ₄ concentration is 4-10 mM.

  The peak position in the XRD pattern of the hybrid flower-like structure of the present invention is the same as the peak position of the copper phosphate crystal (FIG. 4), and a large regularly arranged lattice structure is also observed in the TEM image of the petal. (FIG. 2 (f)), it may be concluded that the petals in the hybrid floral structure are formed by regular arrangement of copper phosphate crystals. In order to understand the formation mechanism, the growth of hybrid floral structures was observed over time using two different sericin concentration settings (0.1 mg / mL and 0.01 mg / mL). Here, the individual concentrations at various growth times were kept constant. FIGS. 9B (e) to 9 (h) and FIG. 12 show how the morphology gradually changes with time. These changes suggest that there are at least two stages of growth. In the initial stage within 2 hours from the start of growth, a flower-like morphology composed of very thin (about 10 nm) curled nanosheets of copper phosphate grows (FIGS. 9B (e) and (f) and FIG. 12 (a) ) And (b)). In the second stage (6 hours), nucleation and subsequent growth of copper phosphate nanocrystals is initiated on the surface of the thin nanosheet petals to form a relatively thick and layered petal (FIG. 9B (g) FIG. 12 (c) and (d)). In the final stage (12 hours), the growth process continues and a fully grown multilayered flower-like structure is formed (FIGS. 9B (h), 12 (e) and 12 (f)).

Throughout this process, the role of sericin may be similar to the role of protein in protein (enzyme) -inorganic hybrid nanofloral structures (Non-Patent Document 9). In a protein (enzyme) -inorganic hybrid nanofloral structure, the interaction between protein molecules and copper ions results in complex formation and subsequent growth of the hybrid floral structure. However, a great difference was found between the two at the initial growth stage. This is inherently “adhesive” in that a very strong complex is formed with a metal mainly by coordination mechanism elements (Non-Patent Documents 32-34) for bonding with various metals in silk protein. "It depends on the ability of the protein sericin. In addition to the amide skeleton, various polar side chains of sericin such as hydroxyl, carboxyl and amino groups also form complexes with Cu ⁺² to form large folded nanosheets of copper phosphate in the initial growth mode The formation of petals (FIGS. 9B (e) and (f)) can be promoted. The role of sericin is also the result of the above, i.e. lowering sericin concentration correspondingly reduces the number of nucleation sites and "adhesive" binder, resulting in thinner petals and wider opening. This is consistent with the fact that the structure is obtained (FIGS. 9A (a) to 9 (d)). Furthermore, the time required for forming a sericin flower-like structure in full bloom (12 hours) is conspicuous compared to the formation time of the enzyme hybrid flower-like structure (72 hours) (Non-Patent Documents 9 and 11). short. This may be due to the rapid coprecipitation reaction due to the dynamic interaction between the Cu ⁺² ion and the binding site of the sericin protein, which is the main driving force for the formation of hierarchical hybrid structures.

In the absence of sericin molecules, large crystals were formed, but no flower-like structures were formed. Furthermore, by applying a controlled treatment to the hybrid flower-like structure with glutaraldehyde (Non-patent Document 37), which is known as an excellent cross-linking agent for silk sericin by blocking the binding site, flower-like morphology and petals are obtained. This leads to a total deformation of the aggregates (FIGS. 13 (a) and 14 (a)). On the other hand, by adding ethylenediaminetetraacetic acid (EDTA), which is a strong chelating agent of Cu ⁺² ions, the hybrid floral structure was disordered and the petals were scattered (FIG. 13 (b)). And FIG. 14B). This result further shows that a strong protein-metal ion interaction is required as a pillar to stabilize such a hierarchical structure.

Furthermore, it has been found that chloride ions (Cl ⁻ ) play an important role in the growth of hierarchical hybrid structures. EDX and XPS spectra also showed the presence of chlorine (FIGS. 3 and 5). As shown in FIG. 15, from the phosphate buffer without the Cl ⁻ ion, the hybrid flower-like structure is nothing even though the ratio of Cu ⁺² to sericin was kept the same as before. Not formed. Even when an alkali metal salt of another halogen element (Br ⁻ , I ⁻ ) having a similar concentration is added under the same experimental conditions as above, no flower-like morphology is generated, and some solid as shown in FIG. A precipitate was obtained. Thus, Cl ⁻ ions influence the copper phosphate crystallization process and assist in the hierarchical growth of hybrid floral structures by helping to direct protein-induced nucleation in a favorable orientation ( Non-patent literature 38, 39).

Infrared spectroscopy is an important tool for determining the secondary structure of a protein mainly by analyzing the characteristic amide bond of the protein (Non-patent Document 40). Examination of attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy not only provides direct proof for the formation of hybrid floral structures, but also conformational constancy of sericin proteins. ) Is also provided with valuable information. As shown in FIG. 13 (c) and FIG. 17, the ATR-FTIR spectrum showed no significant change in the major amide bond of sericin protein before and after hybrid formation (amide I: 1636 cm ⁻¹ and 1634 cm ^-1, amide II: 1515cm ^-1 and 1514cm ^-1 and amide III: both 1238cm ^-1) (non-Patent Document 41). This clearly shows that the true conformation of the protein remains intact in the hybrid flower-like structure and no covalent bond has formed between the protein molecule and its partner inorganic. Yes. In contrast, the newly formed strong IR band at 1048 cm ⁻¹ was associated with the phosphate group of the hybrid floral structure.

Furthermore, it can be shown that these synthesized biological hybrid floral structures have a large surface area. This was confirmed by the standard Brunauer-Emmett-Teller (BET) method. FIG. 13 (d) shows a typical nitrogen adsorption-desorption isotherm and a BJH (Barrett-Joyner-Halenda) pore size distribution curve for each of the hybrid flower-like structure and copper phosphate. The BET surface area of the hybrid flower-like structure was calculated to be 171 m ² g ⁻¹ , which was about 5 times larger than the BET surface area of the as-synthesized copper phosphate (34 m ² g ⁻¹ ). This remarkable increase in characteristic surface area is due to the nanosheet petals of hybrid floral structures that have characteristic surface porosity and are interconnected and stacked. In addition, the observed surface area is very promising when compared to various floral hybrid materials (Non-Patent Documents 42 and 43) as well as other hierarchical metal phosphates. A typical hysteresis loop in the low pressure region indicates the presence of mesopores in the flower-like structure with a corresponding narrow pore size distribution, as shown in the inset of FIG. 13 (d). is doing. The average pore diameter and cumulative pore volume of the hybrid flower-like structure are 3.08 nm and 0.363 cm ³ g ⁻¹ , respectively. These selectively grown relatively small mesopores (3-10 nm) reflect the pores in the nanosheets, while the relatively large few pores (> 10 nm) were stacked at intervals. Can be associated with petal nanosheets. For as-synthesized copper phosphate, the average pore diameter and the corresponding cumulative pore volume are 3.09 nm and 0.085 cm ³ g ⁻¹ , respectively.

  In contrast to previous reports of enzyme hybrids, hybrid sericin structures incorporating these sericins have been allowed to stand at high temperatures even after the sericin molecules have been completely dissipated (Non-Patent Document 9). 11), which showed exceptionally good thermal stability. By calcining the hybrid flower-like structure at 400 ° C. for 2 hours, as shown in FIGS. 13 (e) and (f), the protein molecules are completely formed without affecting the overall flower-like morphology of the hybrid structure. Was broken down. However, the porosity of the microstructure was greatly increased during this process. The most probable reason is that the site occupied by the sericin molecule is empty. EDX analysis and ATR-FTIR spectrum confirmed that no protein molecules were present in the hybrid flower-like structure after heat treatment, as shown in FIG. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were also performed to investigate the thermal behavior of the hybrid flower-like structure, and the results shown in FIG. 19 were obtained. As can be seen from the TG curve in FIG. 19 (a), the first weight loss (about 6%) (DSC endothermic peak at 125 ° C.) in the low temperature region of 25 to 135 ° C. was due to the loss of water. . The second weight loss occurred in the 150-450 ° C region (broad DSC endothermic peaks at 230 ° C and 355 ° C), which is associated with the complete degradation of the sericin molecule of the hybrid flower-like structure. (Non-patent document 44). The amount of weight loss within this temperature region was about 2.5%, which should be attributed to the amount of sericin in the hybrid flower-like structure. Therefore, the mass ratio of the inorganic component in the hybrid flower-like structure calculated from the mass remaining after heating at 450 ° C. was about 91.5%. This phenomenon was further confirmed by thermal analysis of sericin and copper phosphate, which are precursor materials shown in FIG.

  In summary, by using sericin, a silk protein, and extracting the desirable properties of silk sericin and copper phosphate, an organic-inorganic hybrid floral structure was produced using an environmentally friendly and simple coprecipitation method. . With only a few percent of the total mass, silk sericin helps to form well-ordered flower-like structures with high precision and high manufacturing scalability. Moreover, the inherent structural integrity of silk proteins is maintained even in hybrid structures, thereby opening up the possibility of the hybrid structure exerting various biological functions. Silk sericin having biocompatibility is well known for its application to culture media (Non-patent Documents 27 and 28), and copper-based materials act as promising antibacterial agents (Non-patent Document 45, 46). Thus, the combination of both forming a hierarchical porous structure would be a very effective scaffold for 3D cell culture. Furthermore, the porous structure functionalized with these proteins can be an excellent adsorbent for heavy metals and other toxic substances (Non-patent Documents 42 and 43 to 47). Apart from that, transition metal phosphates, especially copper phosphate, are well known for a variety of catalytic reactions and have many special properties, due to the spatial arrangement of the transition metal's d-orbital electrons. (Nonpatent literature 14, 48-50). On the other hand, the improved porosity provided by calcining facilitates mass transport, bringing the hybrid flower-like structure to a wide range of new and interesting applications in the catalytic field.

  Here, the hybrid flower-like structure according to one embodiment of the present invention has not only the possibility of being used as a catalyst but also the ability to adsorb various substances as described above due to the presence of a wide surface and a large number of pores. Was confirmed by an experiment of lead ion adsorption ability.

Lead nitrate (Pb) in an aqueous solution (5 mg / 20 mL) in which the hybrid flower-like structure shown in FIG. ₂ (NO ₃ ) ₂ ) was added, and the change in the lead concentration in this mixed solution was measured using inductively coupled plasma atomic emission spectrometry (ICP) to reduce the lead concentration in the solution due to adsorption. Observed. Thereby, the high adsorption capability of the hybrid floral structure of the present invention was confirmed. The result is shown in FIG. In this analysis, measurements were made for two cases of 50 mg / L and 25 mg / L as the initial concentration of lead nitrate, and the measurement results were plotted on the graph with ● and Δ, respectively.

In addition, for the purpose of investigating the ability to remove other heavy metal ions, solutions of mercury nitrate (Hg ₂ (NO ₃ ) ₂ ) and cadmium nitrate (Cd ₂ (NO ₃ ) ₂ ) were prepared, and a lead nitrate solution was added to 20 mL of each aqueous solution. A graph in which 10 mg of the same hybrid flower-like structure as used for the measurement of adsorption from the sample is added, and the behavior in which the heavy metal ions are adsorbed on the structure with time and the concentration of heavy metal ions in the aqueous solution decreases is plotted with time. Is shown in FIG. In FIG. 22, the measurement results for lead nitrate measured under the same conditions are also plotted for comparison. In the present application, heavy metal means a metal having a specific gravity of iron or more.

[Experiment]
The materials, specific synthesis and measurement procedures / devices used in the above examples are as follows.

<Material>
Pure sericin (Wako Pure Chemical Industries, Ltd.), anhydrous copper (II) sulfate (Sigma-Aldrich) and phosphate buffered saline (Lonza, USA) were used as precursor reagents for the coprecipitation reaction. A phosphate buffer solution containing no salt was purchased from Nacalai Tesque. A stock solution was made using Milli-Q purified deionized water.

<Details of synthesis of hybrid floral structure>
First, a stock solution of sericin, a silk protein, was made by dissolving 20 mg of sericin powder in 1 mL of Milli-Q purified deionized water. Next, this stock solution was diluted with 1 × (10 mM) phosphate buffered saline (PBS, pH 7.4) to prepare solutions of various concentrations for coprecipitation reaction. A 100 mM CuSO ₄ stock solution was also made using deionized water. In a typical experiment, 40 μL of CuSO ₄ aqueous solution (100 mM) was added to 4 mL of sericin 1 × PBS solution (0.1 mg / mL) and the resulting mixture was gently shaken for several minutes. Then, it left still at 25 degreeC for 12 hours. A light blue precipitate was collected from this mixture, washed with deionized water, and dried at room temperature. These hybrid flower-like structures in the as-grown state were evaluated by various microscopic observations and measurements using the experimental techniques described in this specification.

In accordance with the same experimental procedure, controlled experiments were performed for several changes in the ratio of sericin to CuSO ₄ and also for phosphate buffer without salt (1 ×, pH 7.4).

  Dissolve 16.7 mg KBr and 23.2 mg KI in 100 mL of 1X phosphate buffer (pH 7.4, not including NaCl, KCl) to obtain a phosphate buffer containing bromine and iodine, respectively. Produced. The sericin stock was diluted with this newly prepared phosphate buffer containing bromine or iodine (1X, pH 7.4) and the same manufacturing procedure as described above was performed.

  In order to examine the change over time of the growth of the hybrid flower-like structure, the precipitates were collected at various points during the reaction, washed with deionized water, and then observed with a microscope.

  In order to investigate glutaraldehyde and EDTA treatment, a dispersion of water in a hybrid flower-like structure incorporating sericin in an as-grown state (synthesized from 0.1 mg / mL sericin) was mixed with glutaraldehyde (0. 8 wt%) solution and EDTA (1 wt%) solution were treated separately and allowed to stand for 12 hours.

  For calcination, the as-grown hybrid floral structure was dried on a Si wafer and heated at 400 ° C. for 2 hours.

<Characteristic evaluation>
-About SEM analysis The dispersion liquid of the hybrid flower-like structure as it was produced was washed completely and placed on a glass substrate, and then coated with platinum. SEM measurements were performed with Hitachi S4800 and SU8000 field emission microscopes. Elemental analysis was performed using an EDX measuring device attached to the SEM.

-About TEM analysis The 2 microliters diluted dispersion liquid of the hybrid flower-like structure was applied to the TEM grid, and it was made to dry at room temperature, Then, the image was acquired with the JOEL JEM-2100F high resolution electron microscope with the operating voltage of 200 kV.

-About LSM imaging LSM imaging of the hybrid flower-like structure was performed by KEYENCE VK-9710 (purple laser).

-About XRD analysis The XRD analysis of the dried hybrid floral structure was performed by Rigaku Rint 2000 Ultima III X-ray diffractometer using Cu Ka radiation ((lambda) = 1.5406 *, m40kV / 40mA). The 2θ scanning range was 1 to 80 ° in steps of 0.02 °, and the scanning speed was 1 ° / min.

-About XPS analysis The surface characteristic of the hybrid flower-like structure deposited on Si substrate was evaluated by XPS using Thermo Fisher Science, Theta Probe system. FIG. 5 shows the XPS profile of the sample scanned in the range of 0-1400 eV. Here, the presence of C, O, P, Cl and Cu elements is shown. Table 1 and FIG. 6 show the binding energies of the C1s, N1s, O1s, P2p, Cl2p and Cu2p peaks together with the atomic concentrations of the respective elements. The high-resolution XPS spectra of C1s and N1s clearly show the presence of characteristic functional groups of silk protein sericin (Non-Patent Documents 51 and 52). On the other hand, the presence of copper phosphate is confirmed by the binding energy data of Cu (Cu2p _3/2 : 934.7 eV). In this regard, the main two components, the O1s peak (531.3 eV) and the P2p peak (133.5 eV), are associated with the PO ₄ ^-3 group (FIG. 6) (Non-patent Documents 53 and 54).

・ ATR-FTIR measurement
Each sample was placed on a silicon wafer and measured with a NICOLET 4700 FTIR measuring device.

<Thermal analysis>
For measurement of thermal stability, the as-grown hybrid flower-like structure was dried on a Si wafer, and this Si wafer was subjected to forced airflow programmable heating furnace (Yamato Muffle Furnace FO 610, accuracy: ± 2 at 1150 ° C) C.) at 400 ° C. for 2 hours, and then SEM analysis was performed.

・ About TG analysis
It was carried out on a SII Exstar TG / DTA 6200 thermal analyzer with N ₂ dynamic atmosphere of dinitrogen (flow rate = 30 cm ³ / min). The sample was heated from 25 ° C. to 500 ° C. at a rate of 5 ° C./min in an alumina crucible. DSC measurement was performed with Exstar X-DSC 7000 high sensitivity thermal analyzer.

-Measurement of surface area and pore size After the sample was vacuum dried at 100 ° C for 20 hours, the specific surface area and pore size distribution were tested with a Quantachrome Autosorb iQ2 automated gas sorption analyzer.

  The hybrid flower-like structure according to the present invention has a large surface area due to its complicated nanostructure, and is expected to be applied to a wide range of fields such as adsorption of catalysts and various substances.

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Claims (16)

A plurality of sheets in which one or more sericin-copper phosphate hybrid nanosheets are stacked on each other;
A sericin-copper phosphate hybrid structure bonded at the center so that the plurality of sheets extend from the center in different directions.   The sericin-copper phosphate hybrid structure according to claim 1, which has a substantially spherical shape.   The sericin-copper phosphate hybrid structure according to claim 1 or 2, wherein the size is in the range of 2 to 20 µm.   The sericin-copper phosphate hybrid structure according to any one of claims 1 to 3, wherein an average thickness of the sheet is in a range of 200 to 600 nm.   The sericin-copper phosphate hybrid structure according to any one of claims 1 to 4, wherein an average thickness of the sericin-copper phosphate composite nanosheet is in a range of 15 to 50 nm.   The sericin-copper phosphate hybrid structure according to any one of claims 1 to 5, wherein the sheet has pores. The sericin-copper phosphate hybrid structure according to any one of claims 1 to 6, wherein the sericin-copper phosphate hybrid structure has a plurality of sheets in which one or a plurality of sericin-copper phosphate hybrid nanosheets are stacked on each other, and the plurality of sheets are in a central portion. A copper phosphate structure having a flower- like morphology of structures bonded at the center so as to extend in different directions from each other , and sericin removed from the sericin-copper phosphate hybrid structure.   A method for producing a sericin-copper phosphate hybrid structure as a precipitate from a phosphate buffer containing sericin and copper ions.   The method for producing a sericin-copper phosphate hybrid structure according to claim 8, wherein the phosphate buffer further contains chloride ions.   The method for producing a sericin-copper phosphate hybrid structure according to claim 8 or 9, wherein the copper ions are provided by addition of copper sulfate to the phosphate buffer.   The method for producing a sericin-copper phosphate hybrid structure according to claim 10, wherein the sericin concentration is 0.1 to 0.3 mg / mL, and the copper sulfate concentration is 1 to 3 mM.   The sericin-copper phosphate hybrid structure according to claim 10, wherein the sericin concentration is 0.01 to 0.05 mg / mL, and the concentration of the copper sulfate concentration is 0.1 to 0.5 mM. How to manufacture.   The method for producing a sericin-copper phosphate hybrid structure according to claim 10, wherein the sericin concentration is 0.4 to 1 mg / mL and the copper sulfate concentration is 4 to 10 mM. One or a plurality of sericin-copper phosphate hybrid nanosheets of the sericin-copper phosphate hybrid structure are obtained by heat-treating the sericin-copper phosphate hybrid structure manufactured according to any one of claims 8 to 13. Phosphoric acid having a plurality of stacked sheets, and removing the sericin while maintaining the flower- like morphology of the structure in which the plurality of sheets extend from the central portion in different directions. A method for producing a copper structure.   A heavy metal ion adsorbent comprising the sericin-copper phosphate hybrid structure according to any one of claims 1 to 6.   The heavy metal ion adsorbent according to claim 15, wherein the heavy metal ions are ions of one or more elements selected from the group consisting of lead, mercury, and cadmium.