JP4250481B2 - Porous structure - Google Patents

Description

  The present invention relates to a porous structure in which a functional material is supported in pores. The porous structure is a silicon-biopolymer composite in which a biopolymer is supported in pores, and is applied to a biosensor device and a bioreactor device as a module.

  In recent years, development of apparatuses that industrially utilize the selectivity of biopolymers such as nucleic acids and proteins has become active. Among them, DNA sensor chips that use sequence-dependent complementary hydrogen bonding of deoxyribonucleic acid (hereinafter referred to as DNA) and ribonucleic acid (hereinafter referred to as RNA), receptors for disease markers that elute in blood, and antibody molecules are used. Research and development of biosensors such as enzyme sensors using oxidoreductases and hydrolases, such as protein sensors and glucose sensors for diabetic patients, have been promoted.

  Furthermore, in the bioreactor field using the selective substance conversion ability of enzymes, in addition to the synthesis of conventional amino acids and isomerized sugars, the synthesis of pharmaceutical intermediates, commodity chemicals such as acrylamide synthesis (chemical raw materials) Research and development for its application is also underway in the field of synthesis and in the field of decomposition and detoxification of environmental pollutants.

  One of the major problems common to the product areas to which biopolymers are applied is the stability of the biopolymer supported on the device. In particular, proteins are very unstable with respect to the external environment, and various stabilization techniques have been tried.

  As a method for improving the stability of a protein such as an enzyme, a method for immobilizing the protein on a supporting solid and a method for modifying the protein surface with polyethylene glycol or glycolipid are known.

  As a conventional immobilization method, a physical adsorption method in which a substrate is immersed in a protein solution or dried by applying a protein solution onto a substrate and then drying, a method using an alkanethiol used in the case of a gold substrate, a substrate Alternatively, there is a method of immobilizing by chemical bonding using a crosslinking agent such as glutaraldehyde after treating the carrier surface with a silane coupling agent. However, such chemical bonding by cross-linking reaction has low selectivity with respect to the binding site, and since it occurs randomly, the active site of a protein such as an enzyme or a site indirectly related to the activity is considered to be a binding site with a carrier. There is a case. In such a case, there is a concern that the activity may be greatly affected, the protein's higher order structure may be disrupted by random chemical bonding, or the structural change necessary for the reaction may be hindered.

  For the immobilization technique as described above, for example, an organic polymer resin solution such as acrylamide or polyvinyl alcohol, or a sol or colloidal suspension of an organic modifying group such as silane or the like is subjected to a gelation reaction in the enzyme. There is a method of immobilization by inclusion.

  In general, a technique is known in which an enzyme is included in a polymer “gel” having a network such as poly (hydroxyethyl methacrylate).

  This is done by physically immobilizing the enzyme by means of a gel polymer disposed all around the enzyme, which stabilizes the higher-order structure of the enzyme and is expected to have long-term stability. However, on the other hand, the inclusion of the enzyme by the gel always impairs the flexibility of the higher-order structure of the enzyme necessary for the enzyme activity by the interaction between the enzyme and the gel substance from all angles and the cross-linking formation by the gelling agent. There is concern about the reduction in enzyme reaction activity.

  Also, when considering application to biosensors or bioreactor devices, proteins such as enzymes that serve as biosensor device recognition elements or bioreactor device reaction catalysts should be detected or reacted in a state of being included in the gel. There are several problems to achieve high sensitivity, such as whether sufficient contact with the target molecule is possible.

  In the following Patent Document 1, a technique for immobilizing an enzyme on a structural unit having structural stability is disclosed. As the structural unit, mesoporous silica, porous polymer and the like are disclosed. According to this, since the structural unit is stable, it is described that the change in the three-dimensional structure of the immobilized enzyme is suppressed even when the external environment changes. As the enzyme immobilization by such a structural unit, a fuel reforming stabilizing enzyme applied to the above method for at least one enzyme selected from peroxidase, sulfinase, monooxygenase, dioxygenase, and isomerase is known. In addition, an enzymatic degradation method of lignin using peroxidase, an immobilized lipase applied to a lipase exhibiting substrate specificity for optical isomers, and a method for improving the substrate specificity of lipase are known. Patent Document 2 below discloses an enzyme immobilization method and an immobilized enzyme characterized in that a network structure of a gelling substance is formed in the opening and / or internal voids of the mesoporous silica. ing.

  Non-Patent Document 1 below shows the results of immobilizing horse heart cytochrome c having a diameter of 300 nm in silica or aluminosilicate mesoporous molecular sieves having pores having a diameter of 320 to 340 nm and evaluating the oxidation-reduction reaction. It has been reported. In addition, there are reports that trypsin was supported in mesoporous silica, and that α-chymotrypsin was supported in the pores of nanoporous sol-gel glass with trimethoxysilylpropanal as a ligand to improve thermal stability and activity in methanol. is there.

However, in any of the immobilized enzymes using mesoporous silica or polymer gel, the pores of the structural unit are randomly formed randomly, and biological macromolecules such as proteins are quantitatively immobilized in the pores. It ’s difficult. For this reason, there are many problems in realizing “high sensitivity and downsizing” which is the required quality of recent sensor devices. In addition, since the reaction liquid hardly penetrates into the structural unit inside the enzyme-immobilized member as the reactor device, it is necessary to increase the area of the enzyme-immobilized member and extend the reaction time in order to increase the reaction rate.
JP 2000-139459 A JP 2001-178457 A JOURNAL of Molecular Catalysis. B, Enzymatic, Vol. 2 (2-3), 1996

  As can be seen from a number of reports as described above, the method of supporting a protein on a conventionally known inorganic porous material typified by silica is very excellent in terms of stabilizing the protein against the external environment.

  On the other hand, in the biosensor field and bioreactor field supporting biopolymers, downsizing of the apparatus and high-throughput processing are indispensable in consideration of future needs. Correspondingly, there is a demand for further improvement in detection sensitivity, reaction time, and reaction efficiency in a microscopic region, and many development elements for achieving these technical elements remain. In contrast to such “miniaturization / high sensitivity”, the technology disclosed above achieves further improvements in sensitivity, reaction time, and efficiency because the direction of the pores of the porous material is not necessarily constant. Therefore, it is not necessarily a preferable form and a problem remains.

  Therefore, in order to achieve “miniaturization / high sensitivity” in the biosensor and the bioreactor device, it is necessary to overcome the above-described problems.

  Due to such technical background, the present inventors have made various studies, and as a result, have at least one or more columnar pores on one plane of the structure, and silicon is surrounded by the pores. A silicon structure having a silicon region as a main metal element, a method for forming a biopolymer in the pores of the structure in which the pores are perpendicular or nearly perpendicular to the plane, has been found, leading to the present invention. It was.

  That is, this invention is providing the porous structure which carry | supported the functional material in the hole which has a novel structure which can expand the application range to the various devices which combined the inorganic material and the biomaterial.

  Moreover, this invention is providing the novel biosensor apparatus and bioreactor apparatus which contain the said porous structure as a module.

In order to solve the above problem, the present invention provides a porous structure supporting the functional material in the hole, before Ki構 Zotai contains silicon and aluminum as a component for forming a eutectic, multiple And a biopolymer is supported on the wall surface of the hole as a functional material.

  In particular, the average diameter of the holes is 10 nm or less.

  The biopolymer may be a protein, a nucleic acid, or a protein and a nucleic acid.

  The nucleic acid is any one of deoxyribonucleic acid and ribonucleic acid.

  Further, the porous structure contains an oxide.

  Further, the porous structure contains 80 atomic% or more of silicon with respect to the total amount of elements excluding oxygen.

  Furthermore, the present invention is a biosensor device provided with the porous structure.

  Moreover, this invention is a bioreactor apparatus provided with the said porous structure.

According to the present invention, there is provided a porous structure in which a functional material is supported in pores, wherein the structure contains silicon and aluminum as components for forming a eutectic and has a plurality of columnar pores. In addition, since a biopolymer is supported on the wall surface of the hole as a functional material, when applied as a biosensor device or a bioreactor device, downsizing and high sensitivity can be achieved.

  First, an outline of an embodiment of the present invention will be described.

  The porous structure in which the functional material is supported in the pores of the present invention is a nanostructure, has at least one or more pores on one plane of the structure, and has columnar pores and silicon surrounding the pores. A silicon-biopolymer composite in which a biopolymer is supported in pores of a silicon structure having a silicon region in which the main metal element is silicon, wherein the pores are perpendicular or nearly perpendicular to the plane It is formed in a columnar shape in the direction, and a biopolymer is supported on either the wall surface or the bottom surface of the pore.

  In such a structure, the direction of the columnar shape of the pores is perpendicular or almost perpendicular to one plane of the structure, that is, a unidirectional porous body. The protein can be immobilized quantitatively. In addition, it can be expected that the surface area of the inner surface of the pores in contact with the external environment of the structure, for example, the enzyme immobilization reaction solution and the measurement solution is increased. When such a structural material is actually used as a bioreactor or biosensor, further improvement in sensitivity, reaction time, and efficiency can be expected.

  In addition, the present invention is preferably a silicon-biopolymer composite characterized in that the average pore diameter of the pores is 10 nm or less and the average interval between the pores is 15 nm or less. Thus, by setting the average pore diameter of the pores to 10 nm or less, proteins such as enzymes having a diameter of 10 nm or less and nucleic acid molecules such as DNA can be retained in the pores. In particular, in an enzyme having a diameter of 10 nm or less, the three-dimensional structure is stable, and at least the pores of the present invention are open to at least one direction outside the structure. Is expected to have little impact on activity.

  Moreover, it is preferable that the said structure is a film | membrane form. By being in the form of a film, it is also possible to transfer onto an electrode having an arbitrary area.

  The film-like structure is preferably formed on a substrate. By providing the structure on the substrate, the structure can be stabilized. It is also possible to use a deposition substrate as an electrode.

  In addition, it is desirable that at least one kind of the biopolymer is selected from the nucleic acid molecule or the protein molecule.

  The silicon structure may be oxidized. By using the entire structure as an oxide, module control utilizing the function as a silicon oxide semiconductor can be performed.

  Further, the silicon region preferably contains 80 atomic% or more of silicon, more preferably 90 atomic% or more with respect to the total amount of all elements except oxygen. In such a structure, the structure around the pores is stable and exhibits physical properties as silicon or silicon oxide and is easy to control.

  The biosensor device of the present invention includes a silicon structure having at least one or more pores on one plane of the structure, and a silicon region having silicon as a main metal element so as to surround the pores. The module includes a silicon-biopolymer complex in which pores are formed in a columnar shape in a direction perpendicular or substantially perpendicular to the plane, and a biopolymer is supported on the wall surface of the pores.

  Moreover, the silicon oxide-biopolymer complex in which the silicon structure is oxidized is included as a module.

  The bioreactor device of the present invention is a silicon structure having at least one or more pores on one plane of the structure and having a silicon region having silicon as a main metal element so as to surround the pores. The module includes a silicon-biopolymer complex in which pores are formed in a columnar shape in a direction perpendicular or substantially perpendicular to the plane, and a biopolymer is supported on the wall surface of the pores.

  Moreover, the silicon oxide-biopolymer complex in which the silicon structure is oxidized is included as a module.

  Next, embodiments of the present invention will be described with reference to the drawings.

<Configuration of silicon structure>
FIG. 1 is a schematic diagram illustrating a silicon or silicon oxide structure of the present invention.

  FIG. 1 (a) shows that the average pore diameter of pores is 10 nm or less, the average interval between adjacent pores is 15 nm or less, the pores are independent of each other, and the membrane surface (one or more fine pores). FIG. 3 is a schematic plan view of a silicon structure made of a material whose main component is silicon in which the silicon region of the wall material separating the pores is perpendicular or substantially perpendicular to the plane of the structure having holes). FIG. 1B is a schematic cross-sectional view when the silicon structure is cut along the chain line AA in FIG. In FIG. 1, 1 is a pore (nanohole), 2 is a silicon region, and 3 is a substrate.

  The silicon structure of the present invention is characterized by comprising a pore 1 and a silicon region 2 (wall material) having a composition containing silicon as a main metal component. Further, as shown in FIG. 1B, the pores are separated from each other by a silicon region, and are formed perpendicular or nearly perpendicular to the substrate.

  Further, the shape of the pores constituting the silicon structure of the present invention is a columnar shape as shown in FIG. The pore diameter (indicating average pore diameter) 2r of the pores is 10 nm or less, and the pore spacing (indicating average spacing) 2R is 15 nm or less. Preferably, the pore diameter 2r is 1 to 9 nm, and the interval 2R is 3 to 10 nm. The length L is in the range of 5 nm to several μm, preferably 2 nm to 1000 nm. Here, the average pore diameter is a value derived, for example, by performing image processing with a computer on a pore portion observed in an actual SEM photograph (a range of about 100 nm × 70 nm).

  In addition, as shown in FIG. 1 (b), the pores in the silicon structure of the present invention can directly connect the pores and the substrate. However, the present invention is not limited to this. It is not necessary to connect.

  The composition of the silicon region constituting the silicon structure of the present invention is mainly composed of silicon (Si), but in addition to oxides such as aluminum oxide (AlOx), argon (Ar), nitrogen ( Various elements such as N) may be contained. The silicon (Si) content in the silicon oxide region is 80 atomic% or more, preferably 85 to 99 atomic%, with respect to all elements except oxygen.

  The aluminum content is in the range of 0.01 to 20 atomic%, preferably in the range of 0.1 to 10 atomic%, with respect to all elements except oxygen.

  The silicon structure constituting the silicon structure of the present invention is preferably amorphous silicon, but may be crystallized to contain crystalline silicon oxide.

  Further, the silicon structure of the present invention may be oxidized to form a silicon oxide structure. The silicon oxide structure of the present invention obtained by oxidation is obtained by substituting silicon of the silicon structure with silicon oxide, and can obtain the same characteristics.

  Further, the shape of the pore portion constituting the silicon structure of the present invention viewed from the upper surface of the substrate may be substantially circular as shown in FIG. 1A, or any shape such as an ellipse is possible. It is. Further, the shape of the pore portion constituting the silicon structure of the present invention viewed from the cross section of the substrate may be a rectangular shape as shown in FIG. 1B, or an arbitrary shape such as a square or a trapezoid. The columnar shape of the pore includes a shape having an arbitrary aspect ratio (length L / pore diameter 2r) as long as the size is satisfied. The aspect ratio (length L / hole diameter 2r) is preferably in the range of 0.5 to 1000.

  Next, the manufacturing method of the silicon or silicon oxide structure of the present invention will be described.

  FIG. 2 is an explanatory view showing an example of a method for producing a silicon or silicon oxide structure of the present invention. FIG. 3 is an explanatory view showing another example of the manufacturing method.

(A) Process: The process of preparing aluminum and silicon The silicon | silicone chip 13 is arrange | positioned on the target 12 of aluminum as shown in FIG.

(B) Process: Formation process of aluminum silicon mixed film Next, an aluminum silicon mixed film is formed on a substrate. Here, an example in which a sputtering method is used as a film formation method for forming a substance in a non-equilibrium state is shown.

  An aluminum silicon mixed film 23 is formed on the substrate 22 by magnetron sputtering, which is a film forming method for forming a substance in a non-equilibrium state. The aluminum silicon mixed film 23 includes an aluminum columnar structure 21 made of a composition containing aluminum as a main component, and a silicon region 24 containing silicon around it as a main component.

  FIG. 4 is a schematic view showing an example of a method for forming an aluminum silicon mixed film in the present invention.

  With reference to FIG. 4, a method for forming an aluminum silicon mixture by sputtering will be described as a method for forming a film in a non-equilibrium state. In FIG. 4, 11 is a substrate and 12 is an aluminum sputtering target. When the sputtering method is used, the ratio of aluminum and silicon can be easily changed.

  An aluminum silicon mixed film is formed on the substrate 11 by a magnetron sputtering method which is a film forming method for forming a substance in a non-equilibrium state.

  Silicon and aluminum as raw materials are achieved, for example, by disposing a silicon chip 13 on an aluminum target 12 as shown in FIG. In FIG. 4, the silicon chips are divided into a plurality of parts, but of course, the present invention is not limited to this, and one silicon chip may be used as long as desired film formation is possible. However, in order to uniformly disperse the columnar structure containing aluminum in the silicon region, it is preferable to dispose the columnar structure on the substrate 11 (for example, concentrically).

  Alternatively, a fired aluminum silicon product obtained by firing a predetermined amount of aluminum and silicon powder can be used as a target material for film formation.

  Alternatively, a method of separately preparing an aluminum target and a silicon target and simultaneously sputtering both targets may be used.

  The amount of silicon in the formed film is 20 to 70 atomic%, preferably 25 to 65 atomic%, more preferably 30 to 60 atomic%, based on the total amount of aluminum and silicon. If the amount of silicon is within such a range, an aluminum silicon mixed film in which aluminum columnar structures are dispersed in the silicon region can be obtained.

  In the present invention, atomic% indicating the ratio of silicon and aluminum, for example, indicates the ratio of the number of atoms with respect to a single atom such as silicon and aluminum, and is also described as atom% or at%. For example, inductively coupled plasma emission It is a value when the amount of silicon and aluminum in the aluminum silicon mixed film is quantitatively analyzed by the analysis method.

  In the above ratio, atomic% is used as a unit. However, when wt% is used as a unit, for example, when silicon is 20 atomic% or more and 70 atomic% or less, it is 20.65 wt% or more and 70.84 wt% or less. (Conversion from atomic% to wt% is performed by calculating the weight ratio of Al and Si by assuming that the atomic weight of Al is 26.982 and the atomic weight of Si is 28.086, and the weight ratio (wt. Ratio) × (atomic%) is wt. % Can be converted.

  The substrate temperature is 200 ° C. or lower, preferably 100 ° C. or lower, more preferably room temperature (25 ° C.) or lower.

  In addition, when an aluminum silicon mixed film is formed by such a method, aluminum and silicon become a metastable eutectic structure, and aluminum forms a nanostructure (columnar structure) of several nanometers in the silicon matrix. Aluminum and silicon are separated in a self-organizing manner. The aluminum at that time has a substantially cylindrical shape, the pore diameter is 1 to 10 nm, and the interval is 3 to 15 nm.

  The amount of silicon in the aluminum silicon mixed film can be controlled, for example, by changing the amount of silicon chips placed on the aluminum target.

  When film formation is performed in a non-equilibrium state, particularly in the case of sputtering, the pressure in the reaction apparatus when argon gas is flowed is preferably about 0.2 to 1 Pa, and the output for forming plasma is 4 inches. In the case of a target, about 150 to 1000 W is preferable. However, the present invention is not particularly limited to this, and any pressure and output may be used as long as argon plasma is stably formed.

  As a substrate, for example, an insulating substrate such as quartz glass or plastic, a semiconductor substrate such as silicon or gallium arsenide, a substrate such as a metal substrate, or one or more films formed on these substrates Is mentioned. If there is no problem in forming the aluminum silicon mixed film, the material, thickness, mechanical strength, etc. of the substrate are not particularly limited.

  In addition, the shape of the substrate is not limited to a flat plate shape, but may include a curved surface, a surface having a certain degree of unevenness or a step, etc. Is not to be done.

  A sputtering method is preferable as a film forming method for forming a substance in a non-equilibrium state, but a film forming method for forming a substance in any non-equilibrium state such as resistance heating vapor deposition and electron beam vapor deposition (EB vapor deposition) can be applied. is there.

  As a method for forming a film, a simultaneous film formation process in which silicon and aluminum are formed at the same time may be used, or a stacked film formation process in which silicon and aluminum are stacked in several atomic layers may be used.

  The aluminum silicon mixed film 23 formed as described above includes an aluminum columnar structure 21 made of a composition containing aluminum as a main component, and a silicon region 24 containing silicon as its main component.

  The composition of the columnar structure 21 containing aluminum is mainly composed of aluminum. However, if a fine structure having a columnar structure is obtained, it contains other elements such as silicon, oxygen, argon, and nitrogen. May be. Note that the main component means that the proportion of aluminum is 80 atomic% or more, preferably 90 atomic% or more in the component composition ratio of the columnar structure portion.

  The composition of the silicon region 24 surrounding the aluminum columnar structure is mainly composed of silicon. If a microstructure having a columnar structure is obtained, aluminum, oxygen, argon, nitrogen, hydrogen, etc. These various elements may be contained. Note that the main component is preferably a silicon ratio of 80 atomic% or more, and preferably 90 atomic% or more in the component composition ratio of the silicon region.

(C) Process: Pore formation process Only the aluminum area | region (columnar structure body area | region containing aluminum) in said aluminum silicon mixed film is selectively etched. As this etching method, wet etching using an acid or alkali that selectively dissolves only aluminum is preferable. As a result, only the silicon region having pores remains in the aluminum silicon mixed film, and the silicon structure 25 is formed. The pores 26 in the silicon structure 25 have an interval 2R of 15 nm or less and a pore diameter 2r of 10 nm or less. Preferably, the pore diameter 2r is 1 to 9 nm, and the center distance 2R is 3 nm. -10 nm. The length L is in the range of 1 nm to several μm. Note that selective etching of only the aluminum region means that the aluminum portion is substantially removed.

  Examples of the solution used for wet etching include acids such as phosphoric acid, sulfuric acid, hydrochloric acid, and chromic acid solution in which aluminum is dissolved but silicon is hardly dissolved. The alkali can be used, and is not particularly limited to the type of acid or the type of alkali. Moreover, you may use what mixed several types of acid solutions or several types of alkali solutions. Etching conditions can be set as appropriate according to the silicon structure to be manufactured, for example, solution temperature, concentration, time, and the like.

  Furthermore, a silicon oxide structure can be obtained by adding the following step (d).

Step (d): Oxidation step of silicon structure As a method for oxidizing the silicon structure produced in step (c), in addition to a method of heating in an oxygen atmosphere, heating in water vapor or air, anodization, Any silicon oxidation method such as exposure to oxygen plasma can be applied. The silicon in the silicon region is oxidized into a silicon oxide region 29, and a silicon oxide structure 28 is obtained.

  In addition, as shown in the step (e ′) of FIG. 3, the present invention heats the silicon structure in which the pores of the silicon structure produced in the step (c) are enlarged in an oxygen atmosphere, A method of obtaining a silicon oxide structure may be used. Moreover, when it is not necessary to make all the hole walls into an oxide, the time of the oxidation process may be shortened.

Steps (e) and (e ′): pore diameter expansion step As shown in FIG. 2, the pore diameter expansion step is performed by expanding the pores of the silicon oxide structure produced in the step (d) [(e ) Step]. Alternatively, as shown in FIG. 3, enlargement of pores of the silicon structure produced in the step (c) [(e ′) step] is performed. Reference numeral 27 denotes enlarged pores.

  The enlargement of the pore diameter can be achieved by an acid solution (for example, a solution obtained by diluting hydrogen fluoride) or an alkaline solution (for example, sodium hydroxide) that dissolves silicon or silicon oxide in the silicon structure or silicon oxide structure. The pore diameter can be appropriately expanded by pore wide treatment (pore diameter enlargement treatment) immersed in the inside.

  Any acid and alkali may be used for this solution as long as there is no problem in pore enlargement. Moreover, you may use what mixed several types of acid solutions or several types of alkali solutions.

  In addition, for pore diameter expansion (pore wide treatment) conditions, for example, solution temperature, concentration, time, and the like can be appropriately set according to the size of the pores to be produced.

  As described above, the method for producing a silicon oxide structure according to the present invention is a method in which the pore diameter of pores is increased after the production of the silicon structure, or the pore diameter of pores after the production of the silicon oxide structure. A method for performing enlargement processing is included. At this time, any acid or alkali solution capable of dissolving silicon oxide can be used.

  Next, a method for supporting a biopolymer in the pores of the silicon oxide structure obtained by the above method will be described.

  In the present invention, the biopolymer supported on the silicon oxide structure is selected from at least one nucleic acid molecule or protein. In particular, it is desirable that a protein molecule having a large molecular weight has a diameter not larger than the pore diameter of the silicon oxide structure.

  The simplest method for supporting the biopolymer in the pores of the silicon oxide structure is to immerse the structure in an aqueous solution containing the biopolymer. At this time, the reaction conditions such as the biopolymer concentration of the aqueous solution containing the biopolymer, the other salt concentrations, and the temperature are determined by the surface charge characteristics of the biopolymer, the diameter, and the shape and size of the pores of the silicon oxide. It chooses suitably in consideration.

  The surface of the silicon oxide structure obtained as described above is washed, the biopolymer adsorbed on the surface of the structure other than the pores is removed, and the structure is dried under the desired conditions to remove the biopolymer. A supported porous structure of the present invention is obtained.

  Hereinafter, the present invention will be specifically described with reference to examples.

[Example 1]
FIG. 5 is a schematic view showing an example of the silicon or silicon oxide structure of the present invention.

  31 is a substrate, 32 is a pore, and 33 is a silicon oxide region.

  This example shows an example in which a silicon oxide structure having pores having an average pore interval 2R of 8 nm, an average pore diameter 2r of 5 nm, and a height L of 200 nm is formed.

  As shown in FIG. 2B, an aluminum-silicon mixed film containing silicon at 37 atomic% with respect to the total amount of aluminum and silicon was formed on a silicon substrate by a magnetron sputtering method to a thickness of about 200 nm. The target used was a 6-inch silicon chip of 15 mm square placed on a circular aluminum target having a diameter of 4 inches (101.6 mm). Sputtering conditions were as follows: using an RF power source, Ar flow rate: 50 sccm, discharge pressure: 0.7 Pa, and input power: 1 kW. The substrate temperature was room temperature (25 ° C.).

  In this case, the target used was six silicon chips placed on an aluminum target. However, the number of silicon chips is not limited to this, and the composition of the aluminum silicon mixed film varies depending on sputtering conditions. Should be around 37 atomic%. The target is not limited to a silicon chip placed on an aluminum target, and an aluminum chip placed on a silicon target may be used, or a target obtained by sintering silicon and aluminum may be used.

  When the aluminum silicon mixed film was observed with an FE-SEM (field emission scanning electron microscope), a circular aluminum columnar structure surrounded by a silicon region was two-dimensionally shown in FIG. Was arranged. Moreover, the hole diameter of the aluminum columnar structure was 6 nm, and the average center-to-center distance was 8 nm. The height was 200 nm, and the respective aluminum columnar structure portions were separated from each other by the silicon region.

  An aluminum / silicon mixed film containing 37 atomic% of silicon and aluminum based on the total amount of silicon is immersed in 5 wt% of phosphoric acid for 4 hours, and only the aluminum columnar structure is selectively etched to form pores. did. As a result, a silicon structure was produced.

  Next, the phospho-etched aluminum silicon mixed film (silicon structure) was observed with FE-SEM. The shape of the surface viewed from directly above the substrate was a two-dimensional array of pores surrounded by silicon regions. The pore diameter of the pores was 6 nm, and the average interval was about 8 nm. Moreover, when the cross section was observed with FE-SEM, the height was 200 nm, and each pore part was separated by the silicon | silicone area | region, and was mutually independent. Further, no film was formed between the pore and the substrate, and the substrate and the bottom of the pore were directly connected.

  In addition, the produced silicon oxide structure was observed with FE-SEM. The shape of the surface viewed from directly above the substrate was a two-dimensional array of pores surrounded by silicon. The pore diameter of the pores was 5 nm, and the average interval was about 8 nm. Moreover, when the cross section was observed with FE-SEM, the height was 200 nm and each pore part was separated by the silicon | silicone area | region and was mutually independent. Moreover, there was no film formation between the pores and the substrate, and they were directly connected.

  As a result, a silicon structure as shown in FIG. 4 was produced.

  The silicon content in the silicon region was about 90 atomic% for all atoms except oxygen.

  Next, a method for immobilizing horseradish-derived peroxidase (HRP) in the pores of the obtained silicon structure will be described below.

  To an aqueous phosphoric acid solution (pH 7.4), 0.5 g of HRP and potassium iodide are added to 30 mM. The silicon oxide structure is immersed in the resulting phosphoric acid aqueous solution and stirred overnight at room temperature. Thereafter, the silicon oxide structure is pulled up, the front and back surfaces are washed with ion exchange water, and left to stand at room temperature for one day to dry. Thereby, an HRP-immobilized silicon oxide structure is obtained.

[Example 2]
Next, a method for producing a porous structure obtained by the present invention will be described below.

In Example 1, a silicon structure is formed on a substrate by the same method except that the substrate is a platinum oxide substrate. The resulting silicon oxide structure has an average pore spacing 2R of 8 nm and an average pore diameter of 2r. Is 5 nm, and 10 nm is formed at the height L.

  Note that, when measured by Raman spectroscopy as in Example 1, it was confirmed that the silicon structure was amorphous silicon.

  The silicon structure was heated in an oxygen atmosphere. Here, heating was performed at 800 ° C. for 2 hours while flowing oxygen at 50 sccm at atmospheric pressure. As a result, a silicon oxide structure was produced.

  Further, when a sample obtained by oxidizing a silicon structure was measured by wide area electron energy loss structure analysis (EELS), it was confirmed that oxygen and silicon were bonded to this sample, and this sample was silicon oxide.

  In addition, the produced silicon oxide structure was observed by FE-SEM as in Example 1. The shape of the surface viewed from directly above the substrate was a two-dimensional array of pores surrounded by silicon oxide. The pore diameter of the pores was 5 nm, and the average interval was about 8 nm. Moreover, when the cross section was observed with FE-SEM, the height was 200 nm, and each pore part was separated by the silicon oxide region and was mutually independent. Moreover, there was no film formation between the pores and the substrate, and they were directly connected.

  The silicon content in the silicon oxide region was about 90 atomic% for all atoms except oxygen.

  Next, horseradish-derived peroxidase (HRP) is immobilized in the pores of the silicon oxide structure obtained in the same manner as in Example 1 by the following method.

  To an aqueous phosphoric acid solution (pH 7.4), 0.5 g of HRP and potassium iodide are added to 30 mM. The silicon oxide structure is immersed in the resulting phosphoric acid aqueous solution and stirred overnight at room temperature. Thereafter, the silicon oxide structure is pulled up, the front and back surfaces are washed with ion exchange water, and left to stand at room temperature for one day to dry. Thereby, an HRP-immobilized silicon oxide structure is obtained.

[Example 3]
A biosensor using the silicon oxide structure obtained above will be described below.

  The HRP-immobilized electrode obtained in Example 2 was immersed in a container filled with a working electrode, platinum as a counter electrode, and a silver / silver chloride electrode as a reference electrode filled with 30 mM potassium iodide / phosphoric acid aqueous solution (pH 7.4). An electrode measurement system is prepared.

  Next, with the potential of the working electrode HRP immobilized electrode fixed at −100 mV, a hydrogen peroxide solution was added to the measurement system to a final concentration of 10 to 500 μM. Measure the current value.

As a result, an increase in HRP-immobilized electrode current proportional to the amount of hydrogen peroxide added is observed.
The hydrogen peroxide electrode is expected to function as a hydrogen peroxide sensor.

[Example 4]
A bioreactor using the silicon oxide structure obtained in Example 2 is shown below.

  The HRP-immobilized silicon oxide structure obtained above is weighed into a 50 ml beaker so as to be about 1.0 mg in terms of HRP. Next, 20 mL of 50 mM Tris-sodium acetate solution is added and gently stirred. Further, the microtube is bathed at 60 ° C. for 1 hour. The HRP-immobilized silicon oxide structure is taken out and cooled while being left at room temperature. The cooled HRP-immobilized silicon oxide structure is immersed in a 50 mM Tris-sodium acetate solution and washed with stirring.

  The enzyme activity of the HRP-immobilized silicon oxide structure thus obtained is measured by the following procedure.

  10 mL of 50 mM Tris-sodium acetate aqueous solution, 400 μL of 3000 ppm phenol aqueous solution and 100 μL of 30% aqueous hydrogen peroxide are added, and the mixture is reacted at 30 ° C. for 1 hour with slow stirring.

  Next, 1% 4-aminoantipyrine / 1M glycine solution (pH 9.6) is added to 100 μL of the reaction solution, and the following enzyme activity decrease is evaluated from the absorbance at 490 nm.

The evaluation of enzyme activity is a relative comparison to the absorbance of HRP that has not been heat-treated.
Further, HRP / silica obtained by immobilizing HRP with glutaraldehyde on silica subjected to aminosilane treatment is also subjected to the same heat treatment as described above to evaluate the decrease in enzyme activity.

  In the HRP immobilized on the silicon oxide structure of this example, an enzyme activity of about 15% was observed. On the other hand, about 70% of the activity is reduced in HRP / silica.

  From the above results, HRP immobilized on the silicon oxide structure is expected to be excellent in thermal stability and function as a stable bioreactor.

It is the schematic which shows the silicon | silicone or silicon oxide structure of this invention. It is explanatory drawing which shows an example of the manufacturing method of the silicon | silicone or silicon oxide structure of this invention. It is explanatory drawing which similarly shows the other example of a manufacturing method. It is the schematic which shows an example of the film-forming method of the aluminum silicon mixed film in this invention. It is the schematic which shows an example of the silicon | silicone or silicon oxide structure of this invention.

Explanation of symbols

1,26,32 Pore 2,29,33 Silicon or silicon oxide region 3,22,31 Substrate 11 Substrate 12 Sputtering target 13 Silicon chip 14 Ar plasma 21 Aluminum columnar structure 23 Aluminum silicon mixed film 24 Silicon region 25 Silicon Structure 27 Expanded pores 28 Silicon oxide structure

Claims (8)

A porous structure supporting the functional material in the hole,
The structure contains silicon and aluminum as components for forming a eutectic, has a plurality of columnar holes, and a biopolymer is supported on the wall surface of the holes as a functional material. A porous structure.   The porous structure according to claim 1, wherein an average diameter of the pores is 10 nm or less. The porous structure according to claim 1 , wherein the biopolymer is a protein, a nucleic acid, or a protein and a nucleic acid. The porous structure according to claim 3 , wherein the nucleic acid is either deoxyribonucleic acid or ribonucleic acid. The porous structure according to any one of claims 1 to 4 , comprising an oxide. The porous structure according to any one of claims 1 to 5 , wherein silicon is contained in an amount of 80 atomic% or more based on a total amount of elements excluding oxygen. The biosensor apparatus provided with the porous structure in any one of Claims 1-6 . The bioreactor apparatus provided with the porous structure in any one of Claims 1-6 .

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