JP2001213699A - Crystal preparation equipment, crystal preparation method, and its equipment kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the screening equipment which enables to find the condition in a short time suitable to various high polymer crystallization. SOLUTION: The crystal preparation equipment 70 is equipped with No.1 substrate 71 having multiple penetration apertures 73 detached each other and multiple No.2 substrates 72 having multiple kinds of surface showing surface electric potential or zeta electric potential different each other in a high polymer contained solution. No.2 substrates 72 are set to cover multiple penetration apertures 73 in No.1 substrate 71. The multiple portions 74, retaining the solution, are set in order that multiple kinds of surface, showing surface electric potential or zeta electric potential different each other, can be touched with the solution at the same time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an apparatus and a method for preparing a polymer crystal, and a kit for the apparatus.
In particular, the present invention relates to an apparatus, a method, and a kit applied to crystallization of various biopolymers such as proteins and enzymes, and polymers including a complex thereof.

[0002]

2. Description of the Related Art In the crystallization of biological macromolecules such as proteins, supersaturation is achieved by removing the solvent from aqueous or non-aqueous solutions containing macromolecules, as in the case of low molecular weight compounds such as ordinary inorganic salts. Then, it is fundamental to grow a crystal. Representative methods for this include a batch method, a dialysis method, and a gas-liquid interphase diffusion method, which are properly used depending on the type, amount, and property of the sample.

FIGS. 1A and 1B show an apparatus for attempting crystallization under various conditions by a vapor diffusion method. The apparatus 1 has a number of wells 2, where each well 2 and each plate 3 constitute a cell 4 for crystallization. A precipitant 5 is held in each well 2, while a mother liquor 6 containing a biopolymer to be crystallized is dripped on each plate 3 covering the opening of the well 2. In this apparatus, an equilibrium is established by the evaporation of the volatile components in the precipitant 5 and the mother liquor 6. Screening using this apparatus is usually performed by changing the concentration and type of a precipitant, the concentration of a protein solution, the pH and type of a buffer solution, and the like. However, preparing many conditions in this device is a laborious task, and there are many conditions to try.

[0004]

In order to determine the three-dimensional structure of a biopolymer by X-ray crystal structure analysis, it is essential to extract and purify the target substance and then crystallize it. However, at present, there is no method or device that can be applied to any substance to ensure crystallization, so the reality is that crystallization is being promoted by repeating trial and error based on intuition and experience. . In order to obtain biopolymer crystals, it is necessary to search under a great number of experimental conditions, and crystal growth is the biggest bottleneck in the field of X-ray crystal analysis. In particular, depending on the pH of the solution, ionic strength, temperature, type of buffer, relative permittivity, etc.
Since the charge distribution on the surface of the biopolymer and the conformation of the molecule change remarkably, finding suitable conditions for crystallization has been a very laborious task.

[0005] An object of the present invention is to solve the above problems.
An object of the present invention is to provide a technique that can easily find conditions suitable for crystallization.

In particular, it is an object of the present invention to provide a novel screening apparatus and a novel screening method which can provide conditions suitable for crystallization or aggregation of various polymers in a shorter time.

[0007] It is a further object of the present invention to provide an apparatus and method that can provide large crystals that can enable X-ray structural analysis.

A further object of the present invention is to provide a technique for enabling crystallization with a small amount of a biopolymer solution.

[0009]

According to the present invention, there is provided an apparatus for preparing a polymer crystal contained in a solution, the apparatus comprising a first apparatus having a plurality of through holes spaced apart from each other. And a plurality of second members each having a plurality of types of surfaces exhibiting different surface potentials or zeta potentials in the solution. In the device, the first member and the plurality of second members are combined so as to cover the plurality of through holes of the first member, and the plurality of through holes and the plurality of second members form: A plurality of portions for holding the solution are formed. In a plurality of portions for holding the solution, a plurality of types of surfaces exhibiting mutually different surface potentials or zeta potentials are arranged so as to be able to simultaneously contact the solution.

[0010] In the device according to the present invention, a combination of a plurality of types of surfaces exhibiting different surface potentials or zeta potentials among the plurality of second members may be different. In a preferred embodiment of the present invention, the polymer is more strongly electrostatically adsorbed to any of the plurality of surfaces, and the plurality of surfaces are arranged adjacent to each other in a predetermined region, In addition, in a predetermined area, the area occupied by the surface that strongly electrostatically adsorbs the polymer is equal to or less than the area occupied by the remaining surface. The surface on which the polymer is more strongly electrostatically adsorbed can be made of at least one material selected from the group consisting of doped silicon, metal oxide and metal hydroxide. Meanwhile, the remaining surface may be made of at least one material selected from the group consisting of silicon, doped silicon and silicon oxide. The device according to the present invention is particularly useful for crystallization of biopolymers such as proteins.

According to the present invention, there is provided another apparatus for preparing a crystal of a polymer contained in a solution, the apparatus comprising a first apparatus having a plurality of through holes spaced apart from each other.
And a second member having a plurality of types of regions combining a plurality of types of surfaces exhibiting different surface potentials or zeta potentials in the solution. In the apparatus, the first member and the second member are combined so as to close the plurality of through holes of the first member, and the plurality of through holes and the second
A plurality of portions for holding the solution are formed by the member. In a plurality of portions for holding the solution, a plurality of types of surfaces exhibiting mutually different surface potentials or zeta potentials are arranged so as to be able to simultaneously contact the solution. A plurality of types of surfaces exhibiting different surface potentials or zeta potentials among the plurality of parts for holding the solution are different. That is, in the second member, there are a plurality of types of regions in which a plurality of types of surfaces are combined.

In one embodiment of the present invention, a plurality of regions in which a plurality of types of surfaces are combined are provided on one main surface of a substrate so as to be separated from each other, and are arranged so as to correspond to the arrangement of the plurality of regions. Are disposed in the first member. In a preferred embodiment of the present invention, the polymer is more strongly electrostatically adsorbed to any of the plurality of surfaces, and the plurality of surfaces are arranged so as to be adjacent to each other in the region, and In this region, the area occupied by the surface that strongly electrostatically adsorbs the polymer is equal to or less than the area occupied by the remaining surface. The surface on which the polymer is more strongly electrostatically adsorbed can be made of at least one material selected from the group consisting of doped silicon, metal oxide and metal hydroxide. Meanwhile, the remaining surface may be made of at least one material selected from the group consisting of silicon, doped silicon and silicon oxide. The device according to the present invention is particularly useful for crystallization of biopolymers such as proteins.

According to the present invention, there is provided a method of preparing charged polymer crystals contained in a solution from the solution,
The method comprises the steps of: containing a polymer and having a pH other than the isoelectric point of the polymer.
Placing the solution having a plurality of parts in a plurality of portions for holding the solution of the above-described apparatus, and contacting the plurality of types of surfaces, and growing a polymer crystal on any of the plurality of types of surfaces. , Maintaining the contact. In this method, the device holding the solution may be placed in an enclosed place, optionally with a precipitant. Preferably, in at least one of the plurality of portions for holding the solution, the pH of the solution containing the polymer provides a surface potential or zeta potential of the opposite polarity to the polymer on one of the surfaces. And a surface potential or zeta potential of the same polarity as that of the polymer on the other surface.

There is further provided a kit for the device according to the invention, the kit comprising a first member having a plurality of through-holes spaced apart from each other, and a different surface potential or zeta in the solution. A plurality of second members each having a plurality of types of surfaces exhibiting potentials are provided.

There is also provided another kit for the device according to the invention, the kit comprising a first member having a plurality of through-holes spaced apart from each other, and a different surface potential or different potential in solution. A second member having a plurality of types of regions in which a plurality of types of surfaces exhibiting zeta potential are combined is provided.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Most of biomolecules such as proteins are geometrically specific in a solution and have an electrostatic interaction (electrostatic repulsion / attractive force, van der Waals force). Recognition between molecules is performed. In the interaction between molecules based on electrostatic energy, the slight difference in the spatial charge distribution on the outermost surface of each molecule is crucial to the degree of recognition between molecules and the ease of forming molecular aggregates. Expected to have an effect. Therefore, it is considered that nuclei of a molecular aggregate having a periodic and regular structure are extremely unlikely to be formed in individual molecules that repeatedly collide while performing Brownian motion in a solution. Furthermore, even when a crystal nucleus is formed, if the surface structure and charge distribution of each molecule are not the same and are redundant, the molecules gathering around the nucleus are loosely bonded to each other, resulting in a decrease in crystallinity. obtain.

It has been reported that the initial step of nucleation is important for crystal formation of protein molecules. If some conditions for two-dimensionally arranging nuclei molecules in the initial stage of crystallization are established, subsequent crystallization is considered to proceed epitaxially using the nuclei as nuclei.

The apparatus and method according to the present invention perform selective adsorption of a polymer based on the mechanism of action described below, and as a result, crystal nuclei are formed in a specific region, resulting in favorable crystal growth. it can.

FIG. 2 shows an example of a portion used in a crystallization operation in the apparatus according to the present invention. Crystallizing member 10
Has a first solid 11 having a first surface 11a and a second solid 12 having a second surface 12a. As shown, the second solid 12 can be a protrusion or an island formed on the first solid 11.
The first solid 11 and the second solid 12 may be made of substantially different materials, or may have a common main material. Here, “substantially different” means that the main materials constituting the solid are different. When the two solids include a common main material, generally, the amounts or types of sub-materials such as impurities and trace components are different from each other. The material constituting the solid is, for example, a metal, a semiconductor, or a compound thereof, for example, an oxide, a hydroxide, or a nitride. The member 10 is a polymer 1 to be crystallized, such as a protein.
3 is brought into contact with a solution 14 containing. When the entire molecule is macroscopically viewed, the surface of the polymer 13 such as a protein is usually positively or negatively charged in a solution having a pH other than the isoelectric point of the molecule. On the other hand, in the member 10, the surfaces 11 a and 12 a of
Charged in 4. At this time, the magnitude and polarity of the potential on the surface depend on the material of the solid and the pH of the solution. For example, in a solution at a certain pH, the solid surface 11a is negatively charged and the solid surface 12a is positively charged. On the other hand, the polymer 13 is negatively charged in the solution having the pH. In this case, the polymer 13 in the solution 14 is selectively adsorbed on the solid surface 12a charged with the polarity opposite to that of the polymer 13 according to the electrostatic attraction. Adsorption to the solid surface 11a charged with the same polarity as the polymer 13 is inhibited by the electrostatic action. Thus, crystal nuclei of the polymer 13 are formed on the solid surface 12a, and crystallization proceeds. However, it is not necessary that the first surface and the second surface show opposite polarities in the solution. Even if the first and second surfaces are charged with the same polarity, if the charge amounts are different, the polymer can be more strongly attracted and adsorbed to either of them. As described above, by controlling the electrostatic property of the solid surface, a surface capable of specifically or preferentially adsorbing or aggregating a macromolecule such as a protein is provided. Further, by the electrostatic adsorption on the solid surface, the influence of convection in the solution can be reduced, and stable crystal growth can be achieved.

As described above, in a specific embodiment of the present invention, one solution contact area formed on the substrate surface includes:
A plurality of types of surfaces having different surface potentials (zeta potentials) are formed in the solution. In this solution contact area,
Desirably, the seed surface occupies most of the area and the other surface occupies a small percentage of the area. For example,
When two types of surfaces, a first surface and a second surface, are formed in the solution contact region, the area occupied by the first surface is the second surface.
It is desirable that the area is sufficiently large as compared with the area occupied by the surface.

In the device according to the invention, any material which has the desired surface potential and is stable in solution can be used for the crystallization element. Such materials include, in addition to semiconductor materials such as silicon, whose surface potential can be controlled by doping, surface hydroxyl groups are generated in an electrolyte solution, and oxidization of metals or semiconductors that can provide a desired surface charge by dissociation of the hydroxyl groups. Substances or hydroxides, as well as other inorganic compounds that can provide a surface charge, and organic compounds such as organic macromolecules. In the present invention, the material constituting the crystallization member can be selected in consideration of the mechanism described below.

Generally, protein molecules, colloid particles,
And metals, semiconductors and their oxides, hydroxylated
Surfaces of compounds such as nitrides or nitrides
Surface potential (generally zeta
(Which can be measured as a potential). This surface potential is
The pH value of the solution at the time when it is virtually zero is the isoelectric point.
You. The isoelectric point differs depending on the substance, but is lower than this isoelectric point.
At positive pH the substance is positively charged and has a pH above the isoelectric point.
At, the material is negatively charged. The present invention relates to such an object.
The selective crystallization of macromolecules is carried out by utilizing the properties of quality.
For example, the relationship shown in FIG.
It is assumed that it is established between proteins. Curve S ₁Is the first
Represents the relationship between surface potential and pH for solid surfaces,
Line S_TwoIs the surface of the second solid surface (convex surface)
The relationship between the potential and the pH is shown.
It shows the relationship between surface potential and pH. Isoelectric point of the first solid surface
Is 3, the isoelectric point of the protein is 6, the isoelectric point of the second solid surface
The point is 9. Therefore, the pH (ta
P between the isoelectric point of the protein and the isoelectric point of the second solid surface
H), the first solid surface and the tan
Parkin is negatively charged and the second solid surface is positively charged.
In this pH range, the protein is on the second solid surface
Is selectively attracted or fixed to the surface by electrostatic attraction.
As a result, protein crystal growth is promoted on the second solid surface.
obtain. On the other hand, between the protein and the first solid surface,
An electrostatic repulsion works. On the other hand, the relationship as shown in FIG.
And In this case, the isoelectric point of the first solid surface is 9,
The isoelectric point of the protein is 6, the second solid surface (convex surface)
The isoelectric point is 3. And the isoelectric point of the second solid surface
In a solution having a pH between the isoelectric points of the proteins,
The first solid surface and the protein are positively charged and the second
The solid surface is negatively charged. Therefore, the shaded area
At a pH of, the electrostatic attraction forces the protein to a second
It can be selectively adsorbed on a solid surface. like this
By setting the pH of the mother liquor to an appropriate value,
Molecules are selectively electrostatically adsorbed on the surface of the
Crystal nuclei are formed on the surface, causing crystal growth of molecules.
Can be

For example, the isoelectric point of silicon oxide (SiO ₂ ) is 1.8-2.8, so that at lower pH, SiO ₂ is positively charged and higher p
At H, it is negatively charged. On the other hand, alumina (α-Al ₂
The isoelectric point of O ₃ ) is around 9 (the isoelectric point of γ-Al ₂ O ₃ is about 7.4 to 8.6). Most proteins also have an isoelectric point of 4-7 (e.g., human serum albumin 4.7-5.2, bovine insulin 5.0.
3 to 5.8, interferon (chick embryo) 7 to 8,
Human growth hormone 4.9-5.2). Therefore, for example, in the member shown in FIG.
Assuming that O ₂ is used and alumina is used as the second solid 12, in the solution 14 containing protein and having a pH of 6 to 8 (a value between the isoelectric point of protein and the isoelectric point of alumina),
The iO ₂ surface is negatively charged and the alumina surface is positively charged. On the other hand, proteins having an isoelectric point of 4 to 7 are usually negatively charged. Thus, proteins in solution can be selectively adsorbed on positively charged alumina surfaces. On the other hand, adsorption of proteins on the SiO ₂ surface can be inhibited. Thus, the combination of SiO ₂ and alumina is suitable for selective adsorption.

The isoelectric point of silicon (Si) differs depending on the type and concentration of the added impurity. For example, the isoelectric point of general n-type Si is about 3.5 to 4, At lower pH the n-type Si surface is positively charged and at higher pH it is negatively charged. Further, the isoelectric point of general p-type Si is about 5 to 6. Therefore, in the member shown in FIG.
If the second solid 12 is alumina formed on a Si substrate, the second solid 12 contains protein and has a p-value of 6-8.
H (value between isoelectric point of protein and isoelectric point of alumina)
, The n-type Si surface and the protein are negatively charged, and the alumina surface is positively charged. Thus, proteins in solution can selectively adsorb to positively charged alumina surfaces, while inhibiting adsorption on negatively charged Si surfaces. Thus, the combination of silicon and alumina is also suitable for the selective adsorption of protein molecules.

In the apparatus according to the present invention, the combination of the first solid surface and the second solid surface is arbitrary, but the isoelectric point of the molecule to be crystallized is the same as that of the first solid surface. It is desirable to select the combination so as to be between the isoelectric point of the second solid surface. That is, as shown in FIGS. 3 and 4, it is desirable that the pH-surface potential curve of the molecule to be crystallized falls between the pH-surface potential curves of the first solid surface and the second solid surface.

Preferred semiconductors for use in the device according to the invention include silicon, gallium arsenide (GaAs), gallium phosphorus (GaP) and the like. Preferred semiconductor compounds include silicon oxide and silicon nitride, and preferred metal compounds include aluminum oxide (α-Al ₂
O ₃ , γ-Al ₂ O ₃ ), metal oxides such as titanium oxide and copper oxide, metal nitrides such as aluminum nitride, titanium nitride, tungsten nitride, tantalum nitride, TaSiN, WSiN, aluminum hydroxide, water There are metal hydroxides such as magnesium oxide. Preferred combinations include silicon-alumina, silicon oxide-alumina, silicon nitride (isoelectric point is about 4 to 5) -alumina, silicon oxide-titanium oxide (isoelectric point is about 5 to 6.5), and silicon-alumina. There are titanium oxide and titanium oxide-alumina.
For reference, FIG. 5 shows the pH dependency of the surface potential (measured value of zeta potential) of alumina, p-type silicon, silicon nitride, n-type silicon and silicon oxide.

The first surface and the second surface are preferably formed on the same substrate, more preferably formed on a semiconductor substrate, and particularly preferably formed on a silicon substrate. For example, a combination of silicon and alumina can be formed by forming alumina only in a predetermined region on the surface of a silicon substrate. Further, a silicon oxide-alumina combination can be formed by forming a silicon oxide film (SiO ₂ film) on the entire surface of the silicon substrate and forming alumina only in a predetermined region on the surface of the SiO ₂ film. Silicon nitride film (Si ₃ N ₄ film) instead of SiO ₂ film
Is formed, a combination of silicon nitride-alumina can be formed in the same manner. If titanium oxide is formed instead of alumina, a combination of silicon-titanium oxide or silicon oxide-titanium oxide can be formed.

By using a semiconductor substrate, more preferably, a silicon substrate, a device having a plurality of types of surfaces can be easily manufactured by a method similar to a method of manufacturing a normal semiconductor integrated circuit such as CVD, photolithography, and etching.
That is, a film of a desired material is formed on a silicon substrate using a CVD technique, and a film of a different material is formed thereon as needed to form a multilayer structure, and a desired shape is formed using a photolithography technique. By forming the mask described above and exposing the base by removing the area other than the masked area using an etching technique, a device having a plurality of types of solid surfaces in various combinations can be manufactured. For example, a silicon-alumina combination can be formed by forming an alumina film on the surface of a silicon substrate and removing the alumina film by etching while leaving only a predetermined region to expose the surface of the silicon substrate. Also, if a silicon oxide film is formed on the surface of the silicon substrate, an alumina film is further formed thereon, and the alumina film is removed by etching while leaving only a predetermined region to expose the silicon oxide film, the silicon oxide film is formed. -A combination of aluminas can be formed. Thus, various combinations can be easily formed by changing the material of the film formed on the silicon substrate.

The surface of a metal or semiconductor oxide or hydroxide hydrates upon contact with water to generate hydroxyl groups. Due to the dissociation of the hydroxyl groups, the surface of the oxide or hydroxide generates a surface potential (zeta potential) according to the pH of the aqueous solution. For example, the following dissociation occurs in SiO ₂ . Solid surface-SiOH + H ⁺ → Solid surface-SiOH ₂ ⁺ +
OH ^- solid surface -Si · OH + OH ^- → solid surface -SiO ^- + H
₂ O + H ⁺ The oxide or hydroxide surface therefore has a low pH
At a positive potential by protonation, at high pH,
A negative potential is obtained by the extraction of a proton from the OH group. In general, oxides or hydroxides have a point at which the apparent potential becomes zero (isopotential point), and a higher p than this point
At H, it has a negative surface potential, and at pH below this point it has a positive surface potential. Therefore, a combination of oxides or hydroxides having different equipotential points can be selected and preferably used in the present invention.

In the device according to the invention, one surface can be provided on the island (convex). This island can be of any shape. For example, as shown in FIG. 6 (a) or (b), the island portion may have a shape of an elongated rectangular parallelepiped having a substantially rectangular cross section or an elongated truncated pyramid having a substantially trapezoidal cross section. 6) may be a substantially cylindrical shape or a prismatic shape as shown in FIG. 6 (d). The surfaces 32a to 32d on which the polymer should be adsorbed in the island portion preferably have a width d such that the polymer crystal protrudes and grows thereon.
That is, the width d is preferably smaller than the diameter of the crystal to be obtained. Further, a plurality of islands having the same shape may be provided as shown in FIG. 7A, or a plurality of types of islands having different widths within a limited range may be provided as shown in FIG. 7B. . Depending on the properties of the desired polymer,
Also, depending on the pH, concentration, temperature, etc. of the solution, the appropriate island width may differ depending on the balance between the adsorption power required for crystallization and the ease of taking out the grown crystals.
By preparing multiple types of islands with different widths,
Crystal growth is favorably performed on any of the islands,
It can be expected that the grown crystal can be easily taken out.

Further, in the apparatus according to the present invention, the arrangement pattern of the solid surface (supposed to be the first surface) for suppressing crystallization and the island surface (supposed to be the second surface) for promoting crystallization is arbitrary. It is. For example, as shown in FIG. 8A, an arrangement in which the second surface 42a is surrounded by the first surface 41a is preferably used. In this case, the first surface is significantly wider than the second surface. In addition to the one shown in FIG. 8A, as shown in FIG.
8B, a plurality of second surfaces 42b having a predetermined width may be arranged at predetermined intervals, or as shown in FIG. The second surfaces 42c having a shape and an area may be arranged in a matrix at predetermined intervals.

As shown in FIG. 9, a plurality of types of second surfaces 62 and 63 different from the first surface 61 are provided.
Can be arranged. Surfaces 62 and 63 have different potentials for a solution having a predetermined pH. For example, surface 61 can be silicon oxide, surface 62 can be alumina, and surface 63 can be titanium oxide. The particular polymer to be crystallized can be strongly adsorbed by either of the surfaces 62 and 63. The optimum material for the surface capable of adsorbing the polymer more strongly may vary depending on the intended polymer. By forming a plurality of types of second surfaces as shown in FIG. 9, an apparatus that can be used for crystallization of various polymers with one apparatus can be provided. In the apparatus of FIG. 9, there are two types of second surfaces, but three or more types can be formed. Such a device can also be easily manufactured using a general method for manufacturing a semiconductor integrated circuit. For example, an SiO ₂ film, a TiO ₂ film, an Al ₂ film
O ₃ film sequentially formed, by laminating, leaving a region of the surface 62 and 63 to remove the Al ₂ O ₃ film to expose the TiO ₂ film underlying, then the surface of the exposed TiO ₂ film 63 it is sufficient to expose the SiO ₂ film by removing the TiO ₂ film to leave the area.

In many cases, important information for crystallization, such as the isoelectric point of the target polymer, the relationship between the pH of the solution and the surface potential (zeta potential) of the polymer, is not clear. In such a case, it is not clear what the pH of the solution should be, and what combination of materials should be used to form the first surface and the second surface. Therefore, the present invention provides an apparatus suitable for screening that enables a crystal preparation experiment to be performed under a plurality of conditions at the same time and searches for optimal conditions for crystallization. In order to set a plurality of conditions simultaneously, the apparatus according to the present invention has a plurality of members having a plurality of types of surfaces as described above, or has a plurality of combinations of a plurality of types of surfaces in a crystallization member. The present invention combines a member having a plurality of crystallization members or a plurality of types of crystallization regions with a member having a plurality of through-holes, thereby enabling an apparatus capable of simultaneously screening a large number of conditions with a simple structure. I will provide a. In the device according to the invention, favorable crystal growth can occur under any of a number of conditions.

FIGS. 10A and 10B show a preferred embodiment of the apparatus according to the present invention. Crystal preparation equipment 7
Reference numeral 0 denotes an assembly of the first substrate 71 and five second substrates 72. The first substrate 71 has 25 cylindrical through-holes 73 arranged apart from each other. The substrate 72 is arranged at each position where the through holes 73 are arranged. One opening of the through hole 73 is closed by the substrate 72, and the other opening is left open. The through-hole 73 closed by the substrate 72 forms a part 74 for holding a solution containing the material to be crystallized. Each solution holding section 74 becomes a well for a crystallization test. The substrate 72 is formed as shown in FIG.
And (b). The substrate 72 is
One protrusion 75 has five regions 77 sandwiched between two recesses 76. The regions 77 are arranged corresponding to the arrangement of the through holes 73. As will be described later, the material of the surface of the convex portion 75 and the surface of the concave portion 76 are different, so that both surfaces have different surface potentials (zeta potential) with respect to the solution to be held in the portion 74. In addition, the surface of the tip of the convex portion 75 is significantly narrower than the surfaces of the two concave portions 76 sandwiching the convex portion. As shown in FIG. 12, the solution 78 containing the polymer to be crystallized is held in the part 74. At this time, the surface of the convex portion 75 and the surface of the concave portion 76 are simultaneously in contact with the solution 78, and both surfaces show different zeta potentials in the solution 78, enabling selective adsorption of the polymer as described above. To That is, the surface on which the polymer is more strongly electrostatically adsorbed and the crystal of the polymer is grown is called a second surface, and the surface that inhibits the adsorption of the polymer and suppresses crystallization is referred to as a first surface. In terms of the surface, the surface of the convex portion 75 functions as a second surface, and the concave portion 76 functions as a first surface.

In the apparatus shown in FIG.
2 may be all the same, some of them may be different, or all may be different. For example, the five substrates can have any of the structures as shown in FIGS. FIG.
9A, an n ⁺ doped layer 8 is formed on a p-type silicon substrate 81.
2, so that the surface of the top of the protrusion is made of n ⁺ -doped silicon, and the surface of the recess is made of p-type silicon. In FIG. 13B, ap ⁺ -doped layer 84 is formed on an n-type silicon substrate 83, so that the top surface of the convex portion is made of p ⁺ -doped silicon, and the surface of the concave portion is n-type silicon. Consists of FIG.
In (c), an n ⁺ doped layer 8 is formed on a p-type silicon substrate 81.
2 is formed thereon, and a silicon oxide layer 8 is further formed thereon.
5, the surface of the top of the projection is made of silicon oxide, and the surface of the recess is made of p-type silicon. In this case, the n ⁺ doped layer 82 may not be provided. In FIG. 13 (d), a silicon oxide layer 85 is formed on a p-type silicon substrate 81, and a silicon nitride layer 86 is further formed thereon. And the surface of the recess is made of p-type silicon. When a silicon nitride layer is formed directly on a silicon substrate, the silicon nitride layer is easily peeled off. Therefore, a silicon oxide layer is formed to improve the adhesiveness of the layer. In FIG. 13 (e), a silicon oxide layer 85 is formed on a p-type silicon substrate 81, and an alumina layer 87 is further formed thereon. Therefore, the surface of the top of the projection is made of alumina, The surface of the recess is made of p-type silicon. Also in this case, a silicon oxide layer is provided as a base to improve the adhesion of the alumina layer. When the alumina layer 87 is formed by the anodic oxidation method, the silicon oxide layer 85
Also plays the role of insulating the aluminum film remaining at the bottom from the silicon substrate. The structure as shown in FIGS. 13A to 13E can be formed according to the steps as shown in FIGS. First, as shown in FIG. 14A, necessary layers 92 such as a layer having a different conductivity type from the substrate 91, a silicon oxide layer, a silicon nitride layer, and an alumina layer are formed on the substrate 91. Next, as shown in FIG. 14B, a resist pattern 93 is formed on the layer 92 by photolithography using an appropriate mask. Next, a structure as shown in FIG. 14C is obtained through etching, ashing, and the like. As described above, a region where different surface potentials are combined can be easily obtained by forming a necessary layer on the surface of the silicon substrate and etching the layer.

A crystallization substrate (second substrate) to be combined with a substrate having a plurality of through holes (first substrate)
May have any of the structures shown in FIGS. 15A to 15E in addition to the above-described example. In FIG. 15A, the substrate 1
A protrusion or island 122 is formed on 21. For example, the substrate 121 is made of silicon, and the projection 122 is made of silicon oxide. Further, the entire convex portion need not be formed of the same material. For example, in FIG.
The protrusion or island 123 formed on the substrate 1 has a base layer 123a and an upper layer 123b. For example, substrate 1
Reference numeral 21 denotes silicon, the base layer 123a is silicon oxide, and the upper layer 123 (b) is alumina, silicon nitride, or the like. Materials having poor adhesion to the substrate are preferably laminated via an intermediate layer having good adhesion to the substrate. FIG.
In (c), a layer 124 of another material is formed on the substrate 121, and a convex portion or an island 125 is formed thereon. In this case, the surface (first surface) of the layer 124 and the top surface (second surface) of the protrusion 125 contribute to selective crystallization. For example, substrate 121 is silicon and layer 1
Reference numeral 24 denotes silicon oxide, and the upper layer 126 of the projection 125 is made of alumina, silicon nitride, or the like. It is not always necessary to form the projection on the substrate. In FIG. 15D, the substrate 12
A layer 124 of another material is formed on 1, and a different layer 127 is formed in the layer 124. The surface of layer 124 (first surface) and the surface of layer 127 (second surface)
They are on the same plane. For example, the substrate 121 is silicon,
The layer 124 is made of silicon oxide, and the layer 127 is made of alumina, silicon nitride, or the like. In FIG. 15E, a groove or hole 128 is formed in the substrate 121, and another material layer 129 is formed on the bottom thereof.
Are formed. As described above, a region for crystallization may be provided in the concave portion. However, a specific surface (second surface)
In order to easily take out the crystal grown on it without breaking it, it is better that the area where the crystal contacts the surface is small,
Considering this point, a projection or an island having a surface with a width smaller than the size of the crystal is preferable. When the cross section of the second substrate has a structure as shown in FIGS. 15A to 15E, similarly to the above-described example, FIG.
As shown in the plan view of FIG. 16, a plurality of second surfaces (five in FIG. 16A) can be formed separately from each other. Then, by bonding to the first substrate as shown in FIG. 16B, a crystal preparation apparatus is manufactured in the same manner as in the above-described example. Note that, as shown in FIG. 15D, the second surface of the structure in which the first surface and the second surface are on the same plane.
In the case of the substrate described above, the plurality of second surfaces need not always be formed separately from each other, and may be formed in a shape continuous in the longitudinal direction. Even in the case of a continuous shape, it is automatically isolated by the through hole when it is bonded to the first substrate.

As the five crystallization substrates to be combined with the substrate having the through holes, the following ones may be used. (1) The first surface is p-type silicon and the second surface is
A substrate whose surface is alumina, (2) a substrate whose first surface is p-type silicon and whose second surface is silicon oxide,
(3) a substrate whose first surface is p-type silicon and whose second surface is silicon nitride; (4) a substrate whose first surface is silicon oxide and whose second surface is silicon nitride; and (5) A) a substrate wherein the first surface is silicon oxide and the second surface is alumina. In these substrates, the upper surface of the protrusion is the second surface, and the other surface adjacent to the protrusion is the first surface.
Surface.

According to the present invention, by changing the combination of a substrate having a through hole (first substrate) and a crystallization substrate (second substrate), various types of crystal preparation apparatuses can be used according to the purpose. It can be easily manufactured. For example, in the apparatus as shown in FIG. 10, the above-described five types of second substrates may be used one by one, or any one of the five types of second substrates may be used. The number may differ depending on the type. For example, two types may be used by several sheets, or one type may be used by plural sheets. Although a plurality of second substrates are used, it is not necessary to cover all the through holes of the first substrate. By appropriately selecting the second substrate to be combined with the first substrate, a crystal preparation apparatus suitable for the purpose can be easily manufactured.

In the present invention, one second substrate includes:
Preferably, a plurality of crystallization regions are formed. For example, in the example shown in FIG. 11, five crystallization regions are formed on the substrate. The five crystallized regions of the substrate shown in FIG. 11 have the same structure. However, the plurality of crystallization regions formed on the second substrate may have different structures. That is, in the present invention, the combination of a plurality of types of surfaces may be the same or different between a plurality of crystallization regions. The apparatus shown in FIG. 10 provides 25 crystallization wells. Therefore, in the case of this apparatus, a maximum of 25 types (for example, the structure of five types of crystallized regions (five types of second substrates having different types of surface combinations) × the conditions of five types of protein solutions) are different. Crystallization can be performed simultaneously under the conditions. However, not all wells need to be under different conditions. Of the 25 wells, in a plurality of wells having the same crystallization region structure (wells on the second substrate of the same or the same type), the conditions such as the pH and the concentration of the soluble protein were changed to change the conditions under different conditions. Crystallization can be performed. Further, a plurality of types of protein solutions may be supplied to a plurality of wells having the same crystallization region structure. For example, five types of protein solutions with different conditions can be supplied. On the other hand, a plurality of wells having different structures of crystallization regions (wells of different types on the second substrate) provide different crystallization conditions from the beginning. Multiple wells with different structures can be supplied with the same pH or the same concentration of protein.

As in the substrate shown in FIG. 11, one crystallization region may be provided with one projection surface (second surface), and FIGS. 17A and 17B and FIG. 18A ) And (b), one or more crystallization regions may be provided with a plurality or a plurality of types of convex surfaces (second surfaces). In FIGS. 17A and 17B, one separated crystallization region 130 is provided with four separate convex surfaces 132a to 132d. The four surfaces 132a-132d may be the same or different. FIG.
In (a) and (b), two crystallization regions 140
Two separate convex surfaces 142a and 142b are provided. The two surfaces 142a and 142b may be the same or different. Surface 142a
And 142b are different from each other, three types of surfaces are provided for the crystallized region in addition to the surface of the substrate 141.

When preparing a crystal of an unknown biopolymer,
Initially, the type of the second substrate is increased as much as possible, and the crystallization experiment can be performed in an apparatus having as many physical conditions as possible. In the experiment, if crystal growth was confirmed only on a certain second substrate, then, using a device using only a plurality of the second substrates, a large number of solutions in which the pH and concentration of the solution were more finely changed. A crystallization experiment can be performed under the following conditions. With this procedure, more appropriate conditions can be searched.

For example, when the apparatus shown in FIG. 10 is used, first, as a second substrate, FIG.
(E) or use the above (1) to (5) one by one.
In the experiment, if crystal growth was observed only on the substrate 13 (d) or the substrate (3), then, using only five substrates, the solution conditions such as pH and concentration were changed more finely. Crystallization experiments can be performed.

FIGS. 19A and 19B show another preferred embodiment of the device according to the present invention. The crystal preparation apparatus 170 is an assembly of a first substrate 171 and a second substrate 172. The first substrate 171 has 32 cylindrical through holes 173 spaced apart from each other. Through hole 1
One opening of 73 is closed by the substrate 172 and the other opening remains open. The through hole 173 closed by the substrate 172 forms a part 174 for holding a solution containing the material to be crystallized. Each solution holding unit 17
4 is a well for a crystallization test. Substrate 172
Has a shape as shown in FIG. The substrate 172 is
Region 1 in which one convex portion 175 is sandwiched between two concave portions 176
There are 32 with 77. The regions 177 are arranged corresponding to the arrangement of the through holes 173. The material of the surface of the convex portion 175 is different from that of the surface of the concave portion 176. Therefore, both surfaces have different surface potentials (zeta potential) with respect to the solution to be held in the portion 174. Further, the surface of the tip of the convex portion 175 is significantly narrower than the surfaces of the two concave portions 176 sandwiching the convex portion 175. The 32 regions 177 are classified into two types or three types or more. That is, the protrusion 17
There are two, three or more combinations of the surface of No. 5 and the surface of the recess 176 in the 32 regions 177. For example, the combination of surfaces can be different for each row of regions. In this case, there are the number of columns, that is, eight combinations. On the other hand, the combination of surfaces may be different for each row, and in this case, there are four types of combinations. Also,
It may be classified into two types, left 16 and right 16, or all 32 regions may be different from each other. in this way,
Different combinations can be arranged in any manner depending on the application. The combination of the surfaces is, for example, as shown in FIGS.
(E), FIGS. 15 (a)-(e) and the above (1)-
(5) Can be selected. The second substrate shown in FIG. 20 can also be manufactured through film formation, photolithography, etching, and the like as shown in FIGS.

As shown in FIG. 21, in an apparatus 170, a solution 178 containing a polymer to be crystallized
4 is held. At this time, the surface of the convex portion 175 and the concave portion 17
The surfaces of 6 simultaneously contact solution 178, and both surfaces exhibit different zeta potentials in solution 178, allowing selective adsorption of macromolecules as described above.

The device shown in FIGS. 19A and 19B also
Since a plurality of crystallization regions having different structures are provided, a plurality of crystallization conditions are simultaneously provided. This device provides 32 crystallization wells. Therefore, in the case of this apparatus, crystallization can be performed simultaneously under a maximum of 32 different conditions. However, not all wells need to be under different conditions. Of the 32 wells, in a plurality of wells having the same crystallization region structure, crystallization can be performed under different conditions by changing conditions such as the pH and the concentration of the soluble protein. Further, a plurality of types of protein solutions may be supplied to a plurality of wells having the same crystallization region structure. On the other hand, a plurality of wells having different crystallization regions provide different crystallization conditions from the beginning. Multiple wells with different structures can be supplied with the same pH or the same concentration of protein. This apparatus is also suitable for screening of optimal conditions when crystallization of a biopolymer whose properties are unknown.

However, it is preferable to combine a plurality of second substrates and the first substrate as shown in FIG. 10 in terms of versatility and flexibility. Further, a substrate having a smaller number of crystallization regions can be manufactured at lower cost. By manufacturing a large number of second substrates of the same type on one wafer, the number of steps such as film formation, photolithography, and etching necessary for manufacturing a large number of substrates can be reduced.

In the apparatus described above, the substrate (first substrate) having a plurality of through holes is made of glass, plastics such as polycarbonate resin and acrylic resin, and metals such as stainless steel. The first substrate is preferably formed of a material that can inhibit adsorption of a polymer in a solution, such as glass having a low isoelectric point. However, if the distance between the solid surface on which the polymer is adsorbed and the surrounding wall is sufficiently longer than the diffusion distance of the polymer in the solution (the distance over which the polymer can move in the solution), this does not apply. No problem with the material. The through hole can be formed in the substrate by etching using a resist pattern, machining, or the like. Also,
A substrate having a through hole can be obtained from the beginning by molding the raw material. The substrate having the crystallization region (the second substrate) is preferably manufactured using a semiconductor substrate, particularly, a silicon wafer. When a silicon wafer is used, a substrate having a plurality of types of surfaces can be easily manufactured by using a normal semiconductor integrated circuit manufacturing technique. That is, the second layer can be easily formed by using a film forming technique, photolithography, etching and, if necessary, chemical mechanical polishing (CMP).
Substrate can be manufactured. A metal oxide formed on a substrate,
The layer of metal nitride or the like can be formed using an anodizing method, vapor deposition such as CVD, or the like.

The first substrate and the second substrate may be joined in a separable manner or inseparably joined. In the case of releasable bonding, a specific second substrate can be detached from the first substrate and replaced with another second substrate. In this case, more second substrates can be tried for one first substrate. The first substrate and the second substrate can be joined and fixed by an anodic bonding method or an adhesive. When bonding a glass substrate and a semiconductor substrate, an anodic bonding method is preferably used. On the other hand, the first substrate and the second substrate may be combined so that they can be separated later by using a sealant or an adhesive having a low adhesive strength. In addition, the first substrate and the second substrate may be combined with each other without using another material depending on a fitting structure.

According to the present invention, a kit including a first substrate and a second substrate is also provided. Various kits can be provided by combining a first substrate with an appropriate type and / or number of second substrates. The user of the kit can appropriately prepare an appropriate combination for preparing crystals or screening for crystallization. In this case, 1
The number of second substrates combined with one first substrate is arbitrary. Regardless of the number of through holes in the first substrate, a large number of second substrates may be combined. That is, the number of the second substrates that is larger than the number sufficient to cover all the through holes of the first substrate is 1
One first substrate may be combined.

The device according to the invention can include means for measuring the pH of the solution containing the polymer to be crystallized. As described above, the surface potential or effective surface charge of the solid surface and the molecule to be crystallized depends on the pH of the solution, and therefore, it is significant to monitor the pH of the solution during the crystallization operation. As the pH measuring means, a normal pH meter, a conventional pH sensor in which an ion-sensitive field effect transistor (ISFET) and a reference electrode are combined, and the like can be used.

On the other hand, an apparatus as shown in FIG. 22 may be used as the pH measuring means. The pH measuring means includes a semiconductor layer, an insulating layer formed on the semiconductor layer, an enclosing wall for holding the solution on the insulating layer, and a metal electrode provided on the enclosing wall so as to contact the solution. Can be prepared.
In the pH measuring device 100, an n-type silicon substrate 101
An SiO ₂ film 102 is formed thereon. Substrate 101
A solution holding unit 110 is provided above. Solution holding unit 11
Reference numeral 0 denotes an enclosure wall 106 for blocking the flow of the solution 105 and the SiO ₂ film 102. On the surrounding wall 106, a metal electrode 107 is provided. The metal electrode 107 is
It extends toward the iO ₂ film 102 and is arranged to be in contact with the solution held in the solution holding unit 110. A terminal electrode 108 is provided on the back surface of the silicon substrate 101 (the surface facing the surface on which the SiO ₂ film 102 is provided).

The surface of the oxide undergoes a hydration reaction as described above to generate a hydroxyl group. An electric charge is generated on the oxide surface by the dissociation of the hydroxyl group. Therefore, a surface potential corresponding to the pH of the solution is generated on the surface of the oxide.
For example, in the case of SiO ₂ , the following dissociation occurs, and the surface potential changes depending on pH. Surface potential is generated by the same mechanism in other oxides, and the isoelectric point and the value of the generated potential are different depending on the type of the oxide.
The isoelectric point of SiO ₂ is approximately 1.8 to 2.8. Although the detailed mechanism is unknown, a potential corresponding to the pH of the aqueous solution is generated in the aqueous solution as well as the oxide on the surface of the nitride. For example, Si ₃ N ₄ has an isoelectric point of about 4 to 5, and has a positive surface potential at a lower pH and a negative surface potential at a higher pH.
For this reason, instead of the SiO ₂ film, Si ₃ N ₄
A nitride film such as a film may be used.

Therefore, in the apparatus 100 shown in FIG. 22, when an aqueous solution is put into the solution holding section 110 where the SiO ₂ film 102 is exposed, a potential corresponding to the pH of the aqueous solution is generated on the surface of the SiO ₂ film 102. With this potential,
The carrier concentration on the surface of the silicon substrate provided via the oxide film changes. Therefore, the capacitance of the depletion layer 109 formed near the solution 105 of the silicon substrate 101 changes (the width of the depletion layer changes). Therefore, MO
In the device 100 having a structure corresponding to S (MIS), the capacitance-voltage characteristics (high-frequency characteristics) between the metal electrode 107 and the terminal electrode 108 change according to the pH of the solution 105. This change is shown in FIG. FIG. 23 shows the capacity-voltage characteristics of two kinds of solutions having different pHs. The capacitance-voltage characteristics change in the voltage axis direction according to the pH as shown in the figure.

Using a pH measuring apparatus shown in FIG. 22, the capacitance-voltage characteristics of various solutions whose pH is known are measured at a measurement frequency of about 1 MHz, and the pH value and the flat band potential (V _FB ) are measured. You can get a relationship. p
The H value and the flat band potential (V _FB ) are, for example, as shown in FIG.
Has the relationship shown in FIG. Based on this relationship, the pH of the unknown solution is determined. That is, the pH measuring device 1
00 is connected to a CV meter and a CV recorder. Next, the solution to be measured is placed in the solution holding unit 110, and the CV characteristics between the electrodes 107 and 108 are measured.
_Find V _FB . And V _FB obtained from the relationship between the previously obtained pH values and the flat band potential (V _FB), pH of the solution is determined.

In this pH measuring apparatus, a p-type Si substrate may be used instead of the n-type Si substrate, or another semiconductor substrate, for example, a compound semiconductor substrate such as a Ge substrate or GaAs may be used. Further, instead of the SiO ₂ film, another oxide film such as an Al ₂ O ₃ film or a TiO ₂ film may be used, or a nitride film such as Si ₃ N ₄ may be used. The thickness of the insulating layer is, for example, 100 ° to 1 μm, and preferably 500 ° to 3000 °. The material for the electrode is P
t, Pd, Au or the like can be used.

This pH measuring device has a very simple structure (MOS (MIS) structure) and can be easily manufactured using ordinary semiconductor processing techniques (lithography, CVD, etching, etc.). The solution can be dropped on the solution holding section of the apparatus by a pipette or the like, and the pH can be measured for a very small amount of several μl to several tens μl of the solution. This device can be made on a silicon substrate and can therefore be built on the same substrate as the crystal preparation device according to the invention. For example, in the case of the device as shown in FIG. 10, this pH measuring device can be built in one of the five second substrates 72.

As shown in FIG. 25, the well of the device 230 can be sealed with a transparent glass lid 200 and stored in a cool and dark place. The state of crystal growth in each well is observed with a microscope from above the transparent lid. At that time, as shown in FIG.
01 is connected, and the XY recorder 202 measures CV characteristics. This monitors the pH of the solution during crystal growth.

The device of the present invention may be sealed with a precipitant. The precipitant may be placed in another container and placed side by side with the crystal growing apparatus. Although a pH monitor is not always necessary, it is preferable to prepare a crystal growth well, a pH monitor cell, and a precipitant cell on the same substrate and use them as one chip, because it is convenient and convenient. Such a one-chip device can be easily manufactured by using a general semiconductor device manufacturing process as described above.

[0059]

EXAMPLE Crystallization was performed using the apparatus shown in FIGS. 10 (a) and 10 (b). In the apparatus, the first substrate comprises about 5
It was a Pyrex glass plate having a size of cm × about 3 cm and having 25 through-holes having a diameter of about 4 mm and a thickness of about 1 mm. The five second substrates are (A) to (E) below, respectively, and are respectively shown in FIGS.
It has the structure shown in FIG. The first substrate and the five second substrates were anodically bonded. (A) A substrate in which an n ⁺ -doped layer is formed at a depth of about 5 μm on a p-type silicon surface having a specific resistance of about 100 Ωcm. (B) A substrate having a p ⁺ -doped layer formed at a depth of about 5 μm on the surface of n-type silicon having a specific resistance of about 10 Ωcm. (C) A substrate in which an n ⁺ -doped layer is formed at a depth of about 5 μm on a p-type silicon surface having a specific resistance of about 100 Ωcm, and a SiO ₂ layer having a thickness of about 100 nm is further formed thereon. (D) A substrate in which a SiO ₂ layer having a thickness of about 100 nm is formed on a p-type silicon surface having a specific resistance of about 100 Ωcm, and a Si ₃ N ₄ layer having a thickness of about 100 nm is further formed thereon. (E) A substrate in which a SiO ₂ layer having a thickness of about 100 nm is formed on a p-type silicon surface having a specific resistance of about 100 Ωcm, and an alumina layer having a thickness of about 10 μm is further formed thereon by anodization.

(A)-(E) In the substrate, the depth of the recess formed by etching is about 100 μm, and the area of the silicon surface in contact with the protein solution is about 15 m.
m ² , and the area of the upper surface of the convex portion was about 0.3 mm ² . As shown in the figure, five crystallization regions are provided on each substrate.

Bovine liver catalase was used as the protein to be crystallized. The following three buffer solutions were used. (1) pH 5.2: 0.1 M citric acid-0.1 M Na
₂ HPO ₄ (2) pH 8.0: 0.1 M Na ₂ HPO ₄ -0.1 M
KH ₂ PO ₄ (3) pH 11.0: 0.05M boric acid-0.05MK
Cl-0.05 M Na ₂ CO ₃ A catalase solution having a concentration of 30 mg / ml was prepared with these buffer solutions, and 10 μl of the obtained solution was dropped into each well of the apparatus. Further, NaCl was added to each well,
The concentration was 0.1M. Cover the device with a cover and seal each well with a precipitant containing 0.5 M NaCl,
It was left at a temperature of 4 ° C. for 10 days. As a result, pH 8.0
Only about 0.5 mm on the surface of the substrate (E)
It was confirmed that crystals having a size of?

[0062]

According to the present invention, more conditions for crystallization can be made in an apparatus having a simple structure. According to the present invention, a crystal of a polymer such as a protein can be selectively grown on a solid surface. Further, according to the present invention, it is possible to obtain a large crystal that enables X-ray structure analysis. Further, according to the present invention, by using a large number of solid surfaces for crystallization, it is possible to provide an apparatus corresponding to crystallization of many types of polymers. In the present invention, crystallization can be performed on a small amount of a sample.

The present invention can be used for purifying or crystallizing various polymer compounds, particularly polymer electrolytes, in the pharmaceutical industry, the food industry, and the like. The present invention is particularly preferably applied to purify or crystallize proteins such as enzymes and membrane proteins, polypeptides, peptides, polysaccharides, nucleic acids, and complexes and derivatives thereof. In particular, the present invention is preferably applied for purification or crystallization of a biopolymer. Further, since the device of the present invention can selectively adsorb and immobilize a biopolymer or the like, it can be applied to a biosensor, a device for measuring various living tissues and biomaterials by the biosensor, and the like.

[Brief description of the drawings]

FIGS. 1A and 1B are a plan view and an X′-X ′ cross-sectional view showing an example of a crystallization apparatus using a vapor diffusion method.

FIG. 2 is a schematic sectional view showing a crystallization region of the device according to the present invention.

FIG. 3 is a diagram showing how two types of solid surfaces in a solution and the surface charge of a protein change with the pH of the solution.

FIG. 4 is a diagram showing how two kinds of solid surfaces in a solution and the surface charge of a protein change depending on the pH of the solution.

FIG. 5 is a diagram showing pH dependence of zeta potential of some substances.

FIGS. 6A to 6D are perspective views schematically showing islands of various shapes in a crystallization region.

FIGS. 7A and 7B are perspective views showing a crystallization region provided with a plurality of islands.

FIGS. 8A to 8C are plan views showing examples of patterns in which a plurality of surfaces are arranged in a crystallization region.

FIG. 9 is a plan view showing an example of a crystallization region having a plurality of preferable adsorption surfaces in the apparatus according to the present invention.

FIG. 10 (a) is a plan view showing a specific example of an apparatus for preparing crystals according to the present invention, and FIG. 10 (b) is a sectional view taken along line XX.

11A is a plan view showing the second substrate shown in FIGS. 10A and 10B, and FIG. 11B is a cross-sectional view taken along the line YY.

FIG. 12 is a cross-sectional view showing a state in which a solution containing a polymer is held in the devices shown in FIGS. 10 (a) and 10 (b).

FIGS. 13A to 13E are cross-sectional views schematically showing the structure of a second substrate used in the device shown in FIGS. 10A and 10B, respectively.

FIG. 14 is a schematic cross-sectional view showing a step for manufacturing the substrate shown in FIGS. 13 (a) to 13 (e).

15 (a) to (e) are cross-sectional views schematically showing specific crystallization members used in the device according to the present invention.

FIG. 16A is a plan view illustrating an example of a second substrate according to the present invention, and FIG. 16B is an example of an apparatus combining the first substrate and the second substrate according to the present invention. It is sectional drawing.

FIG. 17A is a plan view showing an example of the arrangement of surfaces in a crystallization region, and FIG. 17B is a cross-sectional view thereof.

18A is a plan view showing another example of the arrangement of the surface in the crystallization region, and FIG. 18B is a cross-sectional view thereof.

FIG. 19 (a) is a plan view showing another specific example of the device according to the present invention, and FIG. 19 (b) is a ZZ sectional view thereof.

FIG. 20 is a plan view of the second substrate shown in FIGS. 18 (a) and (b).

FIG. 21 is a cross-sectional view showing a state in which a solution containing a polymer is held in the devices shown in FIGS. 18 (a) and (b).

FIG. 22 is a schematic sectional view showing an example of a pH sensor provided in the device of the present invention.

23 is a diagram showing an example of a capacitance-voltage characteristic measured by the pH sensor shown in FIG.

FIG. 24 shows a flat band value obtained from the capacitance-voltage characteristic measured by the pH sensor shown in FIG.
It is a figure showing the relation with H.

FIG. 25 is a schematic diagram showing a flow for measuring pH in the device according to the present invention.

[Explanation of symbols]

11 first solid, 11a first surface, 12 second solid, 12a second surface, 13 polymer, 14 solution, 70,170 crystal preparation equipment, 71,171 first substrate, 72,172 Second substrate, 73, 173
Through holes.

Claims (14)

[Claims] 1. An apparatus for preparing a polymer crystal contained in a solution, comprising: a first member having a plurality of through holes provided apart from each other; and a surface potential different from each other in the solution. Or a plurality of second members each having a plurality of types of surfaces exhibiting a zeta potential, wherein the first member and the plurality of second members are arranged so as to cover a plurality of through holes of the first member. Are combined, and by the plurality of through holes and the plurality of second members,
A plurality of portions for holding the solution are formed,
And in each of a plurality of portions for holding the solution, characterized in that a plurality of types of surfaces showing different surface potentials or zeta potentials are arranged so as to be able to simultaneously contact the solution, for preparing crystals. apparatus. 2. The apparatus according to claim 1, wherein a combination of a plurality of types of surfaces exhibiting different surface potentials or zeta potentials differs between the plurality of second members. 3. The method according to claim 2, wherein the polymer is more strongly electrostatically adsorbed to any of the plurality of types of surfaces, and the plurality of types of surfaces are arranged adjacent to each other in a predetermined region. And, in the predetermined region, the area occupied by the surface for more strongly electrostatically adsorbing the polymer,
Characterized by being less than the area occupied by the remaining surface,
An apparatus according to claim 1. 4. The surface on which the polymer is more strongly electrostatically adsorbed is made of at least one material selected from the group consisting of doped silicon, metal oxide and metal hydroxide, and 4. The device of claim 3, wherein the remaining surface comprises at least one material selected from the group consisting of silicon, doped silicon, and silicon oxide. 5. The device according to claim 1, wherein the polymer is a protein. 6. An apparatus for preparing a polymer crystal contained in a solution, comprising: a first member provided with a plurality of through holes separated from each other; and a surface potential different from each other in the solution. Or a second member having a plurality of regions in which a plurality of types of surfaces exhibiting a zeta potential are combined, wherein the first member and the second member are closed so as to cover a plurality of through holes of the first member. A plurality of portions for holding the solution are formed by the plurality of through-holes and the second member, and the plurality of portions for holding the solution each A plurality of types of surfaces exhibiting different surface potentials or zeta potentials are arranged so as to be able to simultaneously contact the solution, and between a plurality of parts for holding the solution, the different surface potentials or Wherein the combination of plural kinds of surface showing the over data potential are different, crystal preparation devices. 7. A plurality of regions in which the plurality of types of surfaces are combined are provided on one main surface of the substrate so as to be separated from each other, and the plurality of through holes are formed so as to correspond to the arrangement of the plurality of regions. 7. The device according to claim 6, wherein the device is arranged on the first member. 8. The method according to claim 8, wherein the polymer is more strongly electrostatically adsorbed to any of the plurality of types of surfaces, and the plurality of types of surfaces are arranged adjacent to each other in the region, 7. An area occupied by a surface which more strongly electrostatically adsorbs the polymer in the region is smaller than an area occupied by the remaining surface.
Or the apparatus according to 7. 9. A surface for strongly electrostatically adsorbing the polymer is made of at least one material selected from the group consisting of doped silicon, metal oxide and metal hydroxide, and 9. The apparatus of claim 8, wherein the remaining surface comprises at least one material selected from the group consisting of silicon, doped silicon, and silicon oxide. 10. The device according to claim 6, wherein the polymer is a protein. 11. A method for preparing charged polymer crystals contained in a solution from the solution, comprising: a solution containing the polymer and having a pH other than the isoelectric point of the polymer. 11. A step of contacting the plurality of types of surfaces with the plurality of portions for holding the solution of the apparatus according to any one of 1 to 10, respectively, and on any of the plurality of types of surfaces. A method for preparing a crystal, comprising: maintaining the contact so that the polymer crystal grows. 12. The method according to claim 12, wherein at least one of the plurality of portions for holding the solution includes a polymer solution containing the polymer.
H provides a surface potential or zeta potential of the opposite polarity to the polymer on one of the plurality of types of surfaces, and provides a surface potential or zeta potential of the same polarity as the polymer on the other surface The method of claim 11, wherein the method is: 13. The kit for an apparatus for preparing a crystal according to claim 1, wherein the first member has a plurality of through holes provided apart from each other, and surfaces different from each other in the solution. A kit comprising: a plurality of second members each having a plurality of types of surfaces exhibiting a potential or a zeta potential. 14. The kit for a crystal preparation device according to claim 6, wherein the first member has a plurality of through-holes spaced apart from each other, and surfaces different from each other in the solution. A kit comprising a second member having a plurality of types of regions in which a plurality of types of surfaces exhibiting a potential or a zeta potential are combined.