JP4407416B2 - Precipitation method - Google Patents

Description

  The present invention relates to a method for depositing a deposited substance at a predetermined position in a medium.

  Calcium phosphates are known to have a high affinity for biological materials (for example, DNA, protein, bone, etc.), cells, microorganisms and the like. Among the calcium phosphates, apatites have biocompatibility, and particularly hydroxyapatites have high affinity. Furthermore, hydroxyapatite is known as an inorganic component that forms bones of living bodies together with collagen. Hydroxyapatites are attracting attention as functional materials having biocompatibility (for example, materials such as artificial teeth and artificial bones) because they are biological components and have high affinity with living bodies.

  For example, cells such as cells and microorganisms are supported in a structure containing hydroxyapatite, which is a calcium phosphate, and biosensors, bioreactors, artificial organs, etc. that utilize the sensing functions, substance production functions, and physiological functions of these living bodies It is thought that it can contribute to creation.

As a method for producing such a structure containing hydroxyapatite, (1) a region where a hydroxyapatite layer is formed on a substrate and a region where a hydroxyapatite layer is not formed are divided, and a region where a hydroxyapatite layer is not formed is divided. A method of obtaining an electronic device or a biocompatible material by coating with a coating material, (2) forming a hydroxyapatite layer, and then (3) removing the coating material has been reported (see Patent Document 1). This method is an excellent method for forming a pattern having an arbitrary shape on a substrate. However, in this method, a hydroxyapatite structure that needs to be formed on a base material and is arranged in a three-dimensional manner by placing another substance that may have some function in the center is formed. It is difficult.
JP 2001-294411 A

  An object of this invention is to provide the method of depositing the various desired substances which are not limited to the above calcium phosphates at a predetermined position three-dimensionally.

  The present invention provides a method for precipitating a precipitation substance at a predetermined position in a medium, and the method includes surrounding the precipitation-inducing substance in a state where an aggregate of precipitation induction substances is held at a predetermined position in the medium. A step of precipitating a depositing substance.

  The present invention also provides a method for precipitating a deposited substance at a predetermined position in a medium, the method comprising collecting the dispersed precipitation-inducing substance at a predetermined position in the medium, and A step of depositing a deposition substance around the deposition induction substance in a state where the deposition induction substance is held at the predetermined position in the medium.

  In a preferred embodiment, before the step of depositing the deposition material, a step of collecting a desired material at the predetermined position in the medium is included.

  In a more preferred embodiment, the assembling step is performed by dielectrophoresis.

  In a further preferred embodiment, in the assembling step, the medium is a fluid, and in the precipitating step, the medium is a gel or a solid.

  In another preferred embodiment, the medium is a colloidal dispersion.

  The present invention also provides a gel containing a collection of precipitation-inducing substances therein.

  The present invention further provides a gel containing a desired substance and a precipitation inducing substance present in the vicinity of the desired substance.

  In a preferred embodiment, the desired substance and the precipitation inducing substance form an aggregate.

  The present invention further provides a method for producing a complex of a desired substance and a precipitated substance, the method comprising: (i) assembling the desired substance in a medium; and (ii) inducing precipitation in the medium. Collecting the substance in the vicinity of the desired substance, and (iii) depositing the deposited substance around the deposition-inducing substance in the medium.

  In a preferred embodiment, steps (i) and (ii) above are performed by dielectrophoresis.

  In a more preferred embodiment, in the step (iii), the desired substance and the precipitation inducing substance existing in the vicinity of the desired substance are fixed with a gel, and the precipitated substance is precipitated around the precipitation inducing substance. It is a process to make.

  In a more preferred embodiment, in steps (i) and (ii) above, the medium is a fluid, and in step (iii), the medium is a gel or a solid.

  The present invention also provides a complex of a desired substance and a deposited substance, which is obtained by depositing the deposited substance around the deposition-inducing substance in the gel.

  The present invention further provides a complex of a desired substance and a precipitated substance obtained by any of the above methods.

  According to the method of the present invention, it is possible to easily obtain a capsule-like material in which a desired substance is coated with a deposited substance. For example, what was coat | covered with calcium phosphates demonstrated below has various uses. Alternatively, according to the present invention, since the substance can be deposited at a predetermined position in the solid, for example, as described below, glass containing the deposited substance at a predetermined position can be obtained.

(Deposition of calcium phosphates)
Since calcium phosphates are biological components, they are excellent in biocompatibility and are preferably used as harmless drug delivery systems (DDS). Calcium phosphates can cross the cell membrane and can be lysed in the cell, so that they can be used for the purpose of introducing a desired substance into the cell. It can also be used for food applications or architectural applications such as preventing concrete collapse.

  In this case, examples of desired substances include drugs (for example, anticancer drugs, anticancer drugs, etc.), radioactive substances, antibodies, antioxidants, magnetic substances for therapeutic purposes, osteogenesis promoters, proteins, nucleic acids (DNA, RNA), enzymes, nutrients, magnetic substances, metals, semiconductors, glasses, ceramics, carbon materials (for example, amorphous carbon), diamond, gypsum salts, organic polymers, natural products, poisons, deleterious substances, and the like. These substances coated with calcium phosphates can be placed at any place (for example, a place requiring treatment) to exert the function of the substance.

  Examples of food applications include antioxidants (vitamin C, vitamin E, sesame oil, etc.), antibacterial agents (silver, tannin, ginger extract, etc.), preservatives, and the like. These substances coated with calcium phosphates have an effect of gradually releasing encapsulated substances in food over a long period of time and suppressing deterioration of the food over a long period of time.

  Alternatively, if an alkali agent, fungicide, bactericidal agent, etc. is coated, knead it into a concrete material and release it gradually, thereby causing deterioration due to neutralization, rusting of reinforcing bars, sulfur-oxidizing bacteria, mold, etc. And can extend the life of concrete structures.

(Glass containing precipitated substances)
For example, crystals can be precipitated in glass, and can be a photonic crystal, which can be applied to optical elements (super prisms, optical wavelength filters, polarization separators, optical branching elements, optical switches, etc.). Alternatively, it can be used as an ornament.

  The present invention includes a step of precipitating a deposited substance at a predetermined position in the medium, including a step of depositing the deposited substance around the precipitation-inducing substance in a state where the aggregate of the precipitation-inducing substance is held at the predetermined position in the medium. On how to do.

<Precipitated material>
In the present invention, the deposited substance refers to a solid substance that can be deposited from a deposition fluid (liquid, gas, etc.) defined below, and is not particularly limited. For example, a solute that can be dissolved in a liquid, a substance that forms a hardly soluble salt, and the like can be given. Specifically, in the present invention, salts such as calcium phosphates, metal oxides, metals, semiconductors, glass, ceramics, carbon materials (for example, amorphous carbon), diamond, organic polymers, and the like are particularly suitable. The state of the deposited substance is not particularly limited, and may be, for example, a crystalline state or an amorphous state.

(Calcium phosphates)
In the present invention, calcium phosphates refer to primary calcium phosphate (Ca (H ₂ PO ₄ ) ₂ ), dibasic calcium phosphate (CaHPO ₄ ), tertiary calcium phosphate (Ca ₃ (PO ₄ ) ₂ ), tetracalcium phosphate (Ca ₄ (PO ₄ ) ₂ O), octacalcium phosphate (Ca ₈ H ₂ (PO ₄ ) ₆ ), apatites including hydroxyapatites, amorphous calcium phosphate, and the like, including those having crystal water.

(Hydroxyapatite)
In the present invention, hydroxyapatite refers to a compound represented by the chemical formula Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ . Hydroxyapatites are those in which hydroxyapatite or its constituent elements are substituted and / or missing. Hydroxyapatites are, for example, elements of the hydroxyapatite or a part of the group are elements of Group I of the periodic table such as Na and K, elements of Group II of the periodic table such as Mg and Zn, F, Substituted with a group such as Cl; group VII elements such as Cl; CO ₃ ²⁻ , HPO ₄ ²⁻ , SO ₄ ²⁻ . Further, it may be substituted with a rare earth. Such hydroxyapatites are derived from various elements or groups contained in a precipitation fluid (calcium phosphates forming solution) for forming calcium phosphates.

(Metal oxide)
In the present invention, the metal oxide is not particularly limited, and preferably a high dielectric constant oxide such as TiO ₂ , Ta ₂ O ₅ , ZrO ₂ , HfO ₂ , or a magnetic oxide such as Fe ₃ O ₄ is used. Can be mentioned.

(metal)
In the present invention, examples of the metal include cobalt, nickel, iron, Fe—Pt compounds, Sm—Co compounds, Nd—Fe—B compounds, and various compounds containing other iron group or rare earth elements. Can be mentioned.

  In the present invention, examples of the semiconductor include silicon, germanium, GaN, GaAs, and InP.

  In the present invention, examples of the ceramic include aluminum oxide, silicon oxide, boron nitride, and silicon carbide.

  In the present invention, the organic polymer is not particularly limited. For example, collagen, chitin, polylactic acid, silicone, polyvinyl chloride, polyethylene terephthalate, polyether sulfone, polypropylene, polyethylene, nylon 6, polymethyl methacrylate, polyurethane, synthesis For example, rubber.

<Precipitation inducer>
In the present invention, the precipitation-inducing substance refers to a substance that can promote the precipitation or crystallization of the above-mentioned precipitation substance. The precipitation inducing substance is not limited to one type, and two or more types may be mixed. Moreover, the substance which induces precipitation of a different kind of precipitation substance may be mixed. It also includes substances that are converted into substances that cause chemical or physical changes at predetermined positions in the medium to induce precipitation of the precipitated substances. The precipitation inducing substance may be in the form of fine particles such as seed crystals.

  The size of the fine particles of the precipitation inducing substance is not particularly limited, and is generally 1 nm to 1 mm, preferably 1 nm to 100 μm, more preferably 10 nm to 10 μm.

(Seed crystal)
As a precipitation inducing substance, a seed crystal of the substance to be precipitated can be used. The seed crystal may have a different chemical composition from the target phase as long as it has the same or similar crystal form or a crystal form with similar atomic spacing. The seed crystal is usually a fine one of 1 nm to 500 nm, preferably 1 nm to 100 nm, and the amount used is arbitrary. As such a seed crystal, the fine crystal in the filtrate that has been filtered through a microfilter may be used as it is.

For example, in the case of calcium phosphates, the seed crystal can be obtained as follows. When CaO-SiO ₂ glass powder and a resin film are brought into contact with each other in a precipitation fluid (calcium phosphates forming solution) and left for several days, for example, calcium phosphates microcrystals are deposited on the film surface, and calcium phosphates microcrystals are deposited. The obtained resin film is dissolved in a solvent such as acetone. The calcium phosphates microcrystals remaining in the solvent are recovered and used as seed crystals. When using a resin film (for example, fluororesin) having poor adhesion with calcium phosphates microcrystals, the calcium phosphates microcrystals may be peeled off from the resin film by applying ultrasonic vibration in a solvent such as distilled water.

  As described above, the microcrystals obtained by once taking out the precipitation fluid or performing a solvent treatment with acetone or the like act as seed crystals by being immersed again in the precipitation fluid.

  In the present invention, the seed crystal is not limited to the fine crystal as described above, but may be a fine powder obtained by grinding a solid block or a fine powder obtained by grinding a crystal. For example, a metal or metal oxide powder can be used.

As other precipitation substances, for example, precipitation inducers for calcium phosphates, for example, phosphate glass, Na ₂ O—CaO—SiO ₂ —P ₂ O ₅ glass, Na ₂ O—K ₂ O—MgO—CaO Glass of specific composition such as —SiO ₂ —P ₂ O ₅ —CaF ₂ glass, MgO—CaO—SiO ₂ —P ₂ O ₅ —CaF ₂ crystallized glass, and / or crystallized glass, and wollastonite are preferable. Used. A substance having a Si—OH group, a Ti—OH group, a Ta—OH group, a Zr—OH group, a PO ₃ group, a COOH group, or the like on the surface can also be used. These precipitation inducing substances are used in the form of fine particles.

<Precipitated fluid>
In the present invention, the deposition fluid refers to a fluid used for depositing a deposited substance. Examples of the fluid include liquid (liquid, dispersion liquid, etc.), gas, supercritical fluid, plasma, sol, aerosol containing fine particles, colloidal solution, and the like. This deposition fluid contains a deposition material and / or a component of the deposition material that is deposited by contacting with a deposition inducing material disposed at a predetermined position in the medium defined below. The precipitation inducing substance may be dispersed in the precipitation fluid and used as a medium. The composition, concentration, and the like of the deposition fluid can be appropriately determined according to the deposited substance.

  The precipitation fluid may contain various substances other than the precipitation substance and / or the components of the precipitation substance. For example, a substance (for example, a pH adjuster) for adjusting the precipitation conditions and the above-described precipitation inducing substance can be used. Specifically, the concentration of sodium ions and chloride ions in the calcium phosphates forming solution affects the precipitation rate of calcium phosphates.

Specific examples of the deposition fluid suitably used in the present invention include calcium phosphates forming solution for depositing calcium phosphates, metal oxide deposition solution for depositing metal oxide, and electroless plating for depositing metal. Examples thereof include a liquid and a Ga—As ₂ molecular beam or nitrogen plasma for depositing a semiconductor film.

(Calcium phosphates forming solution)
In the present invention, the calcium phosphates forming solution refers to a solution used for precipitating calcium phosphates. The calcium phosphates forming solution preferably contains 0.02 to 25 mM of calcium ions (Ca ²⁺ ), 0.01 to 10 mM of hydrogen phosphate ions (HPO ₄ ²⁻ ), and has a pH of 6 to 8. More preferably, the calcium ion is 0.2 to 20 mM, the hydrogen phosphate ion is 0.1 to 8 mM, and the pH is 6.8 to 7.6. More preferably, the calcium ion is 1.2 to 5 mM, the hydrogen phosphate ion is 0.5 to 2 mM, and the pH is 7.2 to 7.5.

In the present invention, hydrogen phosphate ion means a general term for phosphoric acid capable of producing PO ₄ ³⁻ in an aqueous solution. Phosphoric acid (H ₃ PO ₄ ) has the following formula in aqueous solution:

As shown in FIG. 2, the compound is in a dissociation equilibrium state, and is phosphate (H ₃ PO ₄ ), dihydrogen phosphate ion (H ₂ PO ₄ ⁻ ), hydrogen phosphate ion (HPO ₄ ²⁻ ), and phosphate ion ( PO ₄ ³⁻ ) coexists. Each abundance varies depending on the pH, and in an aqueous solution having a pH of about 7, there are many dihydrogen phosphate ions and hydrogen phosphate ions. Since phosphoric acid, dihydrogen phosphate ion, hydrogen phosphate ion, and phosphate ion all have the same amount of PO ₄ ³⁻ , in the present invention, these are collectively described as “hydrogen phosphate ion”. . Note that condensed phosphoric acid and the like are also hydrolyzed in an aqueous solution to produce PO ₄ ^3−, and are therefore collectively described as hydrogen phosphate ions having an equivalent amount of PO ₄ ³⁻ .

For the preparation of the calcium phosphates forming solution, it is preferable to use dipotassium hydrogen phosphate trihydrate and calcium chloride. It is preferable to adjust the pH of the calcium phosphates forming solution by using an appropriate buffer solution such as NH ₂ C (CH ₂ OH) ₃ and further adding an acid such as hydrochloric acid.

In order to form calcium phosphates with excellent biocompatibility, in addition to calcium ions and hydrogen phosphate ions, for example, sodium chloride, sodium bicarbonate, potassium chloride, magnesium chloride / hexahydrate, sodium sulfate, etc. Furthermore, it is preferable to include a so-called simulated body fluid. When preparing a simulated body fluid, sodium ion (Na ⁺ ) is 1.4 to 1420 mM, potassium ion (K ⁺ ) is 0.05 to 50 mM, magnesium ion (Mg ²⁺ ) is 0.01 to 15 mM, chloride ion (Cl ^- ) May contain 1.4 to 1500 mM, hydrogen carbonate ion (HCO ₃ ⁻ ) 0.04 to 45 mM, and sulfate ion (SO ₄ ²⁻ ) 5.0 × 10 ⁻³ to 5 mM. Preferably, sodium ion is 14 to 1140 mM, potassium ion is 0.5 to 40 mM, magnesium ion is 0.1 to 12 mM, chlorine ion is 14.5 to 1200 mM, bicarbonate ion is 0.4 to 36 mM, and sulfate ion is Contains 0.05-4 mM. More preferably, sodium ion is 70 to 290 mM, potassium ion is 2.5 to 10 mM, magnesium ion is 0.7 to 3.0 mM, chlorine ion is 70 to 300 mM, bicarbonate ion is 2.0 to 9.0 mM, sulfuric acid It may contain 0.2-1.0 mM of ions.

  The most preferable calcium phosphates formation solution (simulated body fluid) has a composition of inorganic components similar to those in the human plasma, 142.0 mM sodium ions, 5.0 mM potassium ions, 1.5 mM magnesium ions, It contains 2.5 mM calcium ions, 148.8 mM chloride ions, 4.2 mM bicarbonate ions, 1.0 mM hydrogen phosphate ions, and 0.5 mM sulfate ions.

  An organic polymer may be dissolved in the calcium phosphates forming solution. As the organic polymer, for example, collagen, chitin, polylactic acid, polyethylene, polyethylene terephthalate, polymethyl methacrylate, polypropylene, polystyrene, silicone and the like are used, and biopolymers such as collagen and chitin are particularly preferably used. For example, when collagen is used, it is used as an artificial bone material.

(Metal oxide deposition solution)
In the present invention, the metal oxide deposition solution refers to a solution used for depositing a metal oxide. For example, the titanium oxide precipitation solution is preferably a solution obtained by mixing an aqueous ammonium hexafluorotitanate solution and an aqueous boron oxide solution.

(Electroless plating solution)
In the present invention, the electroless plating solution refers to a solution used for depositing a metal. As the electroless plating solution, a commonly used electroless plating solution is suitable.

<Medium>
In the present invention, the medium is a fluid, gel, or solid used to hold the precipitation inducing substance, and the substance in which the precipitation substance and / or the components of the precipitation substance can move in the medium. It may be sufficient, and may be appropriately selected according to the precipitation inducing substance, the precipitated substance and / or the component of the precipitated substance. For example, a colloidal dispersion system may be used. Alternatively, the medium may contain a precipitated substance and / or a component of the precipitated substance in a dissolved state. As the medium, a substance that can easily change from a fluid state to a gel or a solid depending on various conditions such as temperature is suitable. For example, if the medium is a substance that gels with temperature, the precipitation-inducing substance is dispersed in the medium in a fluid state, gathered at a predetermined position in the medium by appropriate means, and then cooled to remove the medium. When gelled, the aggregated precipitation-inducing substances can be held at predetermined positions in the medium. Examples of the gel include agar, gelatin, agarose gel, polyacrylamide gel, and silica gel. Examples of the solid include amorphous substances such as glass; molten salts, metals, ceramics, and the like. In addition to temperature, other chemical substances such as gelling agents may work effectively for gelation.

<Gathering>
In the present invention, the term “aggregation” means that the precipitation inducing substances and / or desired substances dispersed in the medium are collected at a predetermined position by an appropriate means. When assembling a desired substance and a precipitation inducing substance, after assembling the desired substance, the precipitation inducing substance is assembled in the vicinity thereof, or after the precipitation inducing substance is assembled, the desired substance is placed in the vicinity thereof. They may be assembled, or both substances may be assembled simultaneously.

  Examples of means for assembling include dielectrophoresis, electrophoresis, chromatography, centrifugal force, and magnetic force.

For example, when using dielectrophoresis, a deposition-inducing substance or a desired substance in a fluid medium can be electrically collected and collected in one place. Here, the dielectrophoretic force is a force caused by the interaction between the polarization charges generated as a result of the polarization of the particles present in the non-uniform electrolysis and the non-uniform electrolysis, and the magnitude F _DEP thereof. Is
F _DEP = 2πa ³ ε _m Re [K ^* (ω)] ▽ (E ² ) (1)
Given in. Here, a represents the particle radius. K ^* (ω) is the frequency ω of the applied voltage,
_{^{K * (ω) = ε p}} * -ε m * / ε p * + 2ε m * (2)
Ε _p ^* and ε _m ^* in the equation (2) are respectively expressed as ε _p ^* = ε _p −jσ _p / ω (3)
ε _m ^* = ε _m −jσ _m / ω (4)
It is. However, ε _p , ε _m , σ _p , and σ _m are the dielectric constant and conductivity of the target substance and medium to be assembled, respectively, and in the above formula, ^* is added to the complex quantity. In the above formula (1),
Re [K ^* (ω)]> 0 (5)
If so, the target substance moves to the one where the electric field strength is strong (positive dielectrophoresis),
Re [K ^* (ω)] <0 (6)
If so, the target substance moves toward the weaker electric field strength (negative dielectrophoresis). As can be seen from these equations, whether positive dielectrophoresis or negative dielectrophoresis occurs in the target substance depends on the frequency of the applied voltage, the conductivity and dielectric constant of the fluid medium, and the conductivity of the target substance. Determined by the parameters of the dielectric constant and dielectric constant. Therefore, by adjusting the parameters so that a negative dielectrophoretic force acts on the medium, the target substance can be captured and collected in one place.

  In the present invention, dielectrophoresis is performed regardless of whether or not a substance in a medium has a charge, for example, a virus, a microorganism, a unicellular organism, an animal cell, a plant cell, and a biological fine particle such as DNA or protein derived therefrom, a metal, The present invention can be applied to all substances including ceramics, organic substances, inorganic substances and the like. These substances can be used as desired substances in the present invention.

  For example, first, a precipitation-inducing substance (for example, a calcium phosphate seed crystal) is aggregated in a fluid medium by gel electrophoresis, the medium is gelled, and then a desired substance such as DNA is precipitated by electrophoresis. It is also possible to gather at the position where the inducer is gathered, and then immerse this gel in the deposition fluid to deposit the deposited substance.

  In the present invention, the vicinity of the desired substance means the vicinity of the position of the desired substance. For example, it is preferable that the precipitation inducing substance exists in the vicinity of the desired substance. The precipitation inducing substance is preferably present at a position surrounding the desired substance or mixed with the desired substance. In the latter case, the precipitation-inducing substance and the desired substance may form an aggregate.

  In the present invention, the periphery of the precipitation-inducing substance refers to a position that directly surrounds the precipitation-inducing substance. For example, the precipitated substance is deposited around the precipitation inducing substance.

<Retention>
In the present invention, holding refers to maintaining a position where the substance is fixed three-dimensionally in the medium. Examples of the holding means include gelling or solidifying the fluid medium; continuing to collect substances at predetermined positions in the fluid medium by dielectrophoresis, centrifugal force, or magnetic force.

  For example, in the case of a gelable medium, the substance can be held at a predetermined position by cooling and gelling a fluid medium in which the substance is gathered. For example, a gel containing an aggregate of precipitation-inducing substances therein; a gel containing a desired substance and a precipitation-inducing substance present in the vicinity of the desired substance; containing aggregates of the desired substance and the precipitation-inducing substance Gel can be obtained.

<Deposition>
When the deposited substance comes into contact with the deposition-inducing substance held in the medium, the deposited substance is deposited around it. Alternatively, the components of the deposited substance are retained in the medium and form a deposited substance around the precipitate inducing contact with the deposition inducing substance. The deposited substance and / or the components of the deposited substance are supplied as the deposition fluid or previously dissolved in the medium. The amount of the deposited substance can be appropriately adjusted depending on the concentration of the deposited substance to be supplied, the time required for deposition, and the like.

  For example, in the case of a gel, the deposition fluid can be supplied from the outside of the gel. Specifically, by immersing a gel containing a collection of precipitation-inducing substances in the precipitation fluid, the precipitation substance penetrates into the gel and comes into contact with the precipitation-inducing substance present at a predetermined position. Material precipitates out. In addition, for example, when a desired substance and a precipitation-inducing substance existing in the vicinity of the desired substance are held in the medium, the precipitated substance is deposited so as to surround these, and as a result, the precipitated substance is a desired substance. A deposited substance shaped to cover the surface is obtained. If necessary, the gel can be removed by suitable means to obtain a mass of deposited material or a desired material coated with deposited material.

  Or, for example, in the case of glass, the precipitation inducing substance is placed in a predetermined position in a high-temperature molten state, and then cooled and solidified, and the precipitation substance and / or the precipitation substance previously mixed in the glass is removed. Precipitating substances from the components can be deposited around the precipitation inducing substance.

  In this way, the deposited substance can be deposited at a predetermined position three-dimensionally in the medium.

(Preparation of calcium phosphates forming solution (SBF1.0))
In 700 mL of distilled water, 7.996 g of NaCl, 0.350 g of NaHCO ₃ , 0.224 g of KCl, 0.228 g of K ₂ HPO ₄ .3H ₂ O, 0.305 g of MgCl ₂ .6H ₂ O, 1M hydrochloric acid 40 mL, CaCl ₂ 0.278 g, Na ₂ SO ₄ 0.071 g, and trishydroxymethylaminomethane 6.057 g are dissolved in 1M hydrochloric acid so that the pH is 7.40 at 36.5 ° C. It was adjusted. Then, distilled water was added to make the whole solution 1 L, and a calcium phosphates forming solution (SBF1.0) was prepared.

(Preparation of calcium phosphates forming solution (SBF1.5))
In 700 mL of distilled water, 11.999 g of NaCl, 0.525 g of NaHCO ₃ , 0.336 g of KCl, 0.342 g of K ₂ HPO ₄ .3H ₂ O, 0.458 g of MgCl ₂ .6H ₂ O, 1M hydrochloric acid 52.5 mL, CaCl ₂ 0.417 g, Na ₂ SO ₄ 0.107 g, and trishydroxymethylaminomethane 9.086 g are dissolved in 1M so that the pH is 7.40 at 36.5 ° C. Adjusted with hydrochloric acid. Then, distilled water was added to make the whole solution 1 L, and a calcium phosphates formation solution (SBF1.5) was prepared.

(Preparation of aqueous solution containing calcium phosphate seed crystals)
CaF ₂ : MgO: P ₂ O ₅ : SiO ₂ : CaO was weighed so as to be 0.5: 4.6: 16.2: 34.0: 44.7% by mass, mixed, and then 1450 ° C. For 2 hours. The melt was quenched to obtain a glass solid. The obtained glass was pulverized by a ball mill and classified by a sieve to obtain glass fine particles having a particle size of 150 μm or less.

  Next, 30 mL of the calcium phosphate forming solution (SBF1.0) prepared as described above was measured in a plastic container, and 0.2 g of the glass fine particles were added and dispersed. In this dispersion, a 1 cm × 1 cm polyethylene sulfone base material was immersed in a clip and allowed to stand for 1 day to precipitate calcium phosphates microcrystals on the surface of the base material. Subsequently, this base material was taken out and immersed in distilled water in a state of being hung with a clip to remove water-soluble salts derived from the calcium phosphates forming solution. Then, this base material was immersed in a screw bottle containing 15 mL of distilled water, applied with ultrasonic waves, and microcrystals of calcium phosphates precipitated on the surface of the base material were dispersed in distilled water to obtain an aqueous solution containing seed crystals. .

(Create agarose gel with calcium phosphate seed crystals)
To the aqueous solution containing calcium phosphate seed crystals prepared as described above, 0.3 g of Agarose H (manufactured by Nippon Gene Co., Ltd.) was added, stirred at room temperature for 30 minutes, and then stirred at 80 ° C. for 20 minutes. This solution was cooled to room temperature to prepare an agarose gel containing calcium phosphate seed crystals.

(Growth of calcium phosphates from aggregated calcium phosphate seed crystals)
A gel containing calcium phosphates seed crystals assembled by dielectrophoresis was immersed in a calcium phosphates formation solution (SBF1.5) prepared as described above at 36.5 ° C. for 4 days. This solution was replaced with a fresh one every two days. Next, the gel was observed with a scanning electron microscope, and it was confirmed that spherical particles made of calcium phosphates having a diameter of about 5 μm were formed at predetermined positions in the gel.

(Model experiment-1)
Using a polystyrene standard sphere as a model of the precipitation-inducing substance, it was confirmed that the precipitation-inducing substance can be held at a predetermined position by dielectrophoresis. FIG. 1 shows a cross-sectional view of the dielectrophoresis apparatus used in this example. This apparatus 1 is composed of a quadrupole electrode 2, an insulating rubber 3, and a transparent acrylic plate 4, and a sample 6 supplied from a sample inlet 5 passes through the quadrupole electrode 2 and is a polyimide tube 7. It is configured to be recovered from. The quadrupole electrode 2 is configured so that a voltage is applied from an AC power source (WAVEFACTORY WF1946), and the behavior of the sample at the quadrupole electrode of the sample 6 from the sample inlet 5 is measured by a microscope 8 (attached to the lower part of the apparatus. Nikon inverted microscope ECLIPSE TE-2000U) was observed continuously.

  The quadrupole electrode 2 used was a three-dimensional electrode formed by depositing gold on a polyester resin sheet and bonding the three.

A 10 μm polystyrene standard sphere (manufactured by MAGSPHER inc, Monosized PS Microsphere: 1.05 g / cm ³ aqueous solution) was diluted with about 20 times the amount of 0.02 mass% potassium chloride solution and used as a sample. A sample was dropped into the chamber, and an AC voltage of 10 V and 1 MHz was applied to the quadrupole electrode. The results are shown in FIG. As shown in FIG. 2, it can be seen that polystyrene standard spheres are gathered at the center of the electrode due to the three-dimensional quadrupole electrode.

(Model experiment-2)
In this model experiment 2, a single-layer three-dimensional electrode was used instead of the three-dimensional electrode of Example 2, and a syringe pump (syringe pump mute mini (manufactured by Senshu Kagaku)) was attached to the tip of the polyimide tube of the apparatus of FIG. (Not shown). As the polystyrene standard sphere, a mixture of the above 10 μm polystyrene standard sphere and 1 μm polystyrene standard sphere (Duke Scientific Corporation, Latex Microspher Suspension-5100A) in a quantity ratio of about 1000: 1 is used, and 10 nl / min with a syringe pump. The behavior of polystyrene standard spheres was observed in the same manner as in Example 2 except that the sample was sucked at a speed of and a flow was formed.

  Changes with time when a voltage of 12 V and 100 kHz is applied are shown in FIGS. As shown in FIG. 2 and FIG. 3, it can be seen that 1 μm or 10 μm polystyrene standard spheres carried by the flow gather around the 10 μm polystyrene standard spheres migrated in the center of the quadrupole electrode.

  The results of the above model experiment indicate that the target sample can be migrated to the center by the dielectrophoresis method. That is, when a precipitation inducing substance is used as the target sample, the precipitation inducing substance is held at a predetermined position, and the precipitation substance can be deposited around the precipitation inducing substance. Furthermore, the results of this model experiment show that the target sample can be migrated to the center by dielectrophoresis, and then another sample can be collected around the target sample to surround the target sample. That is, when a desired substance and a precipitation-inducing substance are used as the target sample and another sample, respectively, the desired substance is held at a predetermined position, surrounded by the precipitation-inducing substance, and the precipitation substance is surrounded around the precipitation-inducing substance. It shows that it can be deposited and coated with the desired material.

  The method of the present invention is widely used in various fields. For example, pharmaceutical manufacturing field (drug delivery system (DDS), etc.), food field (antioxidation, food deterioration prevention, etc.), building field (concrete prevention, etc.), or optical field (photonic crystal, optical element ( Super prisms, optical wavelength filters, polarization separators, optical branching elements, optical switches, etc.).

It is sectional drawing of the dielectrophoresis apparatus used in the Example. It is a microscope picture which shows that a 10 micrometer polystyrene standard sphere gathers in the center part of a quadrupole electrode when a three-dimensional quadrupole electrode is used. It is a microscope picture which shows that a polystyrene standard sphere gathers toward the center part of a quadrupole electrode. It is a microscope picture of the time after FIG. 3 which shows that a polystyrene standard sphere gathers toward the center part of a quadrupole electrode.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Dielectrophoresis apparatus 2 Quadrupole electrode 3 Insulation rubber 4 Acrylic board 5 Sample inlet 6 Sample 7 Polyimide tube 8 Microscope

Claims (8)

A method of depositing a deposited substance at a predetermined position in a medium,
Including the step of precipitating the precipitation substance around the precipitation-inducing substance in a state where the aggregate of the precipitation-inducing substance is held at a predetermined position in the medium,
The medium is a gel, and the precipitate containing the aggregate of the precipitation-inducing substance is immersed in a precipitation fluid, so that the precipitate penetrates into the gel, and the precipitate is present at the predetermined position. A method wherein the deposited material precipitates upon contact with the inducing material. A method of depositing a deposited substance at a predetermined position in a medium,
A step of collecting the dispersed precipitation-inducing substance at a predetermined position in the medium, and a precipitation around the precipitation-inducing substance in a state where the aggregated precipitation-inducing substance is held at the predetermined position in the medium. Including the step of precipitating the substance,
In the step of assembling, the medium is a fluid that can be gelled. After the step of assembling, the medium in the step of precipitating by gelling the medium is used as a gel, and the aggregate of the precipitation-inducing substances is contained inside. By immersing the gel contained in the precipitation fluid, the deposited substance penetrates into the gel, contacts with the deposition-inducing substance present at the predetermined position, and the deposited substance is deposited.
Method.   The method according to claim 2, further comprising the step of collecting a desired substance at the predetermined position in the medium before the step of depositing the deposition substance.   The method according to claim 2 or 3, wherein the assembling step is performed by dielectrophoresis.   The method according to claim 1, wherein the medium is a colloidal dispersion. A method for producing a composite of a desired substance and a precipitated substance,
(I) assembling the desired substance in a medium;
(Ii) a step of causing a precipitation-inducing substance to gather in the vicinity of the desired substance in the medium; and (iii) a step of depositing the precipitation substance around the precipitation-inducing substance in the medium.
Including
In step (i) and (ii), the medium is a gelable fluid, and after step (ii), the medium in step (iii) is gelled by gelling the medium, and the desired By immersing the gel containing the substance and the aggregate of the precipitation-inducing substance in the precipitation fluid, the precipitation substance penetrates into the gel and comes into contact with the precipitation-inducing substance. To precipitate,
Method.   The method according to claim 6, wherein the steps (i) and (ii) are performed by dielectrophoresis.   The method according to any one of claims 1 to 7, wherein the precipitation inducing substance is calcium phosphates.

Images