JP2001240499A - Crystal preparation equipment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject equipment which can spatially control the formation of crystal nucleus when crystallizing various kinds of biopolymers. SOLUTION: The equipment, which prepares crystal of biopolymers contained in a solution, is equipped with a pair of convex parts 1 and 2 being positioned face to face to a substrate and on the top of the substrate. The space is formed between a pair of the convex parts 1 and 2 to crystallize biopolymers 3. The surface of the convex parts 1a and 2a, being positioned face to face in the space for crystallization, are characterized to orient the biopolymers 3 toward a certain direction in the solution. For instance, the surface of the convex parts 1a and 2a, which have minus and plus in zeta potential, respectively, are able to orient the biopolymers 3 by bipolar in the static electrical field.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for preparing crystals of organic molecules, and more particularly, to an apparatus for crystallization of various biological macromolecules such as proteins and enzymes, and macromolecules including complexes thereof. Related devices.

[0002]

2. Description of the Related Art Crystallization of biopolymers such as proteins
As in the case of ordinary low molecular weight compounds such as inorganic salts, it is fundamental to grow the crystals in a supersaturated state by performing a treatment for removing the solvent from water or a non-aqueous solution containing a polymer. 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 schematically show a hanging drop method and a sitting drop method included in the gas-liquid interphase diffusion method. In the hanging drop method shown in FIG. 1A, a mother liquor 221 containing a biopolymer to be crystallized is dropped in a closed container 220 containing a precipitant 222. In the sitting drop method shown in FIG. 1B, a mother liquor 221 containing a biopolymer to be crystallized is placed on a plate 233 in a closed container 230. The precipitant 222 is accommodated in another container 231 in the closed container 230. With these methods,
Evaporation of the precipitant and volatile components in the mother liquor slowly establishes equilibrium.

[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.

It is an object of the present invention to technically solve the drawbacks of the conventional crystallization process which has been carried out through trial and error due to having various characteristics as described above.

Specifically, an object of the present invention is to provide an apparatus capable of spatially controlling the formation of crystal nuclei in crystallization of various biopolymers and a biological tissue mainly composed of biopolymers. It is.

It is a further object of the present invention to provide an apparatus that can facilitate the preparation of large crystals.

[0008] It is a further object of the present invention to provide an apparatus which can enable crystallization with a small amount of a biopolymer solution.

[0009]

According to the present invention, there is provided an apparatus for preparing a crystal of an organic molecule contained in a solution, the apparatus comprising a substrate, and a pair of opposed convexes formed on the substrate. It has a unit. The device is used by forming a space for crystallization of organic molecules between a pair of opposed convex portions and allowing a solution containing the organic molecules to exist in the space. In this apparatus, a pair of convex surfaces sandwiching a space for crystallization has the property of orienting organic molecules in a specific direction between the surfaces in a solution.

Preferably, in the device according to the invention,
The pair of convex surfaces have different surface potentials or zeta potentials from each other in the solution,
The property of orienting organic molecules in a specific direction by electrostatic action between the surfaces is provided. Preferably, the pair of convex surfaces are made of materials having different isoelectric points from each other, and the surface of the substrate existing between the pair of convex portions is between the isoelectric points of the pair of convex surfaces. It is made of a material having an isoelectric point. Further, it is preferable that the surfaces existing on the tops of the pair of projections also be made of a material having an isoelectric point between the isoelectric points of the surfaces of the pair of projections.

In the apparatus according to the present invention, it is preferable that a wall is provided on the substrate so as to surround the pair of projections, and the solution is held inside the wall.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The surface of a biopolymer such as a protein or an enzyme is usually dissociated in a solution and has a pH-dependent surface charge. For example, as shown in FIG.
FIG. 2 (b) shows a macroscopic view of a biopolymer having many positive charges on one surface and many negative charges on the other surface.
Can be regarded as having a dipole structure as shown in FIG. Regarding the crystallization of a biopolymer in the form of such a dipole, a crystal nucleus can be formed by orienting a large number of such molecules in a certain direction (for example, the direction of a dipole moment) and assembling them. Thus, crystal growth centering on this nucleus can be expected.

Therefore, according to the present invention, for example, as shown in FIG. 3, a first solid material 1 whose surface 1a is negatively charged in a solution, and a second solid material 2 whose surface 2a is positively charged, as shown in FIG. And are arranged facing each other. As shown in the figure, between them, a large number of biopolymers 3 in the form of dipoles are dipolar with respect to the direction from the surface 1a to the surface 2a indicated by the arrow (a direction substantially perpendicular to these surfaces). The child moments can be oriented to be substantially parallel. The positive surface of the biopolymer 3 can be adsorbed to the negatively charged surface 1a, while the negative surface of the biopolymer 3 can be adsorbed to the positively charged surface 2a. In this way, a large number of molecules 3 can be arranged between the surface 1a and the surface 2a to form a crystal nucleus, thereby causing crystallization. The solid material arranged for crystallization may be arranged as shown in FIG. In this case, the positively charged first solid material 1
And a pair of combined bodies 5 in which the second solid material 2 negatively charged and the second solid material 2 are stacked back to back.

Thus, the present invention provides a pair of different solid surfaces that have the property of providing the orientation of the molecules to be crystallized, creating a space between the surfaces for crystallization. In the present invention, the solid material providing such a surface forms a protrusion on the substrate. In the present invention, the distance between the surface of a pair of opposing convex portions (d1, d shown in FIGS. 3 and 4)
In 2), an organic molecule to be crystallized may be oriented and fixed in a certain direction by electrostatic action in the meantime, and an interval may be set such that crystal nuclei are formed there. Therefore, it may be adjusted according to the size of the target organic molecule. For example, in the case of protein molecules, the spacing can be between 5 nm and 2 μm, and between 30 nm and 50 μm.
0 nm is preferred. Similarly, the height of the convex portion is 5 nm to 2 μm.
And it is preferably 30 nm to 500 nm.

Particularly, in the present invention, a pair of convex surfaces sandwiching a space for crystallization have different surface potentials or zeta potentials or surface charges in a solution, and form the above-mentioned electrostatic field. Is preferred. Such a structure can be obtained by making a substance having a high isoelectric point face a substance having a low isoelectric point. If solid surfaces with different isoelectric points are opposed to each other and the pH of the solution contacting both surfaces is adjusted between those isoelectric points, a solid surface with a high isoelectric point is positive and a solid surface with a low isoelectric point is It can be negatively charged. For example, in FIG. 3 and FIG.
When iO ₂ (isoelectric point: about 2) and the second solid 1 were Al ₂ O ₃ (isoelectric point: about 9) and the pH of the solution was adjusted to 5, the surface 1a
Is negatively charged and the surface 2a is positively charged.

In general, 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. For example, metal or
When the surface of the oxide or hydroxide of the semiconductor comes in contact with water,
Causes hydration to generate hydroxyl groups. Dissociation of this hydroxyl group
Due to the oxide or hydroxide surface, the pH of the aqueous solution
A surface potential (zeta potential) is generated in accordance with. for example
For example, SiO _TwoThen, the following dissociation occurs. Solid surface -SiOH + H⁺→ Solid surface-SiOH_Two ⁺+
OH^- Solid surface-Si OH + OH^-→ Solid surface-SiO^-+ H
_TwoO + H⁺ Therefore, the surface of the oxide or hydroxide has a low pH
At a positive potential by protonation, at high pH,
Negative potential due to proton abstraction from OH group
You.

For reference, FIG. 5 shows the pH dependence of the zeta potential of alumina, p-type silicon, silicon nitride, n-type silicon and silicon oxide. As described above, the zeta potential at a predetermined pH differs depending on the type of the substance. In the present invention, a space for crystallization is formed by a combination of different materials utilizing the properties of such substances. For example, from FIG. 5, when the pH of the solution is between 6 and 8, the alumina-silicon nitride, alumina-n-type silicon, and alumina-silicon oxide combinations can each provide a significant positive-negative surface combination. In particular, alumina
Combinations of silicon oxides can produce strong electrostatic fields in the space for crystallization over a wide range of pH.

In the device according to the invention, the material of the solid surface for crystallization may be a metal, semiconductor,
It can be appropriately selected from metal compounds, semiconductor compounds, polymers such as resins, and the like. The combination of the pair of solid surfaces is optional, but it is preferable that the zeta potentials of both surfaces are significantly different. Preferred semiconductors include silicon, gallium arsenide (GaAs), gallium phosphorus (GaP), and the like. Preferred semiconductor compounds include silicon oxide,
Preferred examples of the metal compound include metal oxides such as aluminum oxide (α-Al ₂ O ₃ , γ-Al ₂ O ₃ ), titanium oxide, and copper oxide; aluminum nitride, titanium nitride, and tungsten nitride. , Tantalum nitride, T
There are metal nitrides such as aSiN and WSiN, and metal hydroxides such as aluminum hydroxide and magnesium hydroxide. Preferred combinations include silicon-alumina, silicon oxide-alumina, silicon nitride-alumina, silicon oxide-titanium oxide, silicon-titanium oxide, titanium oxide-alumina, and the like.

In the device according to the present invention, the surfaces other than the pair of surfaces facing each other to orient the biopolymer may have a low surface potential or zeta potential (for example, the surface potential or zeta potential of a pair of surfaces) in a solution. It is preferably made of a material having an intermediate potential). As shown in FIGS. 6A and 6B, it is preferable that the surface 10a of the substrate 10 between the opposing projections 11 and 12 is made of a material having such a potential. As shown in FIGS. 7A and 7B, the potential of the surface 10a is changed to the positive side or the negative side.
If it is higher on the side, the molecules 13 will be affected by the surface 10a and the preferred orientation may be impaired. FIG. 6 (a)
As shown in (b) and (b), the upper surfaces (surfaces constituting the apexes) 11b and 12b of the protrusions 11 and 12 and the other substrate surface 10b are also preferably formed of a material exhibiting a small potential in a solution. For example, the surfaces 10a, 10b, 1
Preferably, 1b or 12b is made of a material having an isoelectric point between the isoelectric points of the pair of surfaces 11a and 12a. That is, when the convex portion 11 is made of a material having an isoelectric point of about 2 and the convex portion 12 is made of a material having an isoelectric point of about 9, the surface 10a, 10b, 11b or 12b has an isoelectric point of about 2 5 of the material. In this case, assuming that the pH of the solution is about 5, the surface potential of the convex portion 11 becomes negative,
Although the surface potential of the projection 12 becomes positive, the surface 10a, 10
The potential of b, 11b or 12b approaches zero. Thus, the dipole-structured biomolecule can be well oriented between the pair of protrusions without being affected by the potential of the upper surface of the protrusions or the substrate surface.

Specifically, the protrusion 11 is made of SiO ₂ , and the protrusion 1
2 is Al ₂ O ₃ , surface 10a, 10b, 11b or 12
When the layer constituting b is formed of Si ₃ N ₄ (isoelectric point of about 5) and the pH of the solution is adjusted to about 5, the surface potential of the projection 11 is negative, the surface potential of the projection 12 is positive, and the others. Can be made substantially zero.

By using a semiconductor substrate, more preferably, a silicon substrate, the projections can be formed on the substrate relatively easily by the same method as that for manufacturing a normal semiconductor integrated circuit such as CVD, photolithography, and etching. Structure can be made. That is, a film of a desired material is formed on a silicon substrate using a CVD technique, a mask having a desired shape is formed using a photolithography technique, and an area other than the masked area is removed using an etching technique. As a result, a projection can be formed on the substrate.

[0022] For example, as shown in FIG. 8 (a), providing a silicon substrate 20, then as shown in FIG. 8 (b), SiO ₂ film by CVD on the silicon substrate 20 2
1 and a Si ₃ N ₄ film 22 are sequentially deposited. Further FIG.
As shown in (c), Al ₂ O ₃ by sputtering
A film 23 is formed. Then, as shown in FIG.
The resist mask 24 is formed by using photolithography.
To form After etching and removing the mask,
A projection 31 made of Al ₂ O ₃ is formed as shown in FIG. Next, as shown in FIG. 8F, a SiO ₂ film 25 is formed by CVD to cover the convex portions 31. At this time, irregularities are formed in the SiO ₂ film 25 according to the shape of the convex portion 31. Therefore, the SiO ₂ film 25 is subjected to chemical mechanical polishing,
As shown in FIG. 8G, Al ₂ O ₃ is exposed to obtain a flat surface. Next, as shown in FIG. 8 (h), Si ₃ N ₄
A film 26 is formed, and a resist mask 27 is formed thereon. After etching and removing the mask, FIG.
The structure as shown in (i) is obtained. The upper surface of the projection 31 made of Al ₂ O ₃ is covered with the Si ₃ N ₄ film 26. Si
The upper surface of the convex portion 32 made of O _{2 is} also covered with the Si ₃ N ₄ film 26. The space between the opposing convex portions 31 and 32 is a space for crystallization. The space is sandwiched between the Al ₂ O ₃ surface 31a and the SiO ₂ surface 32a. The surface of the substrate 20 is also covered with the Si ₃ N ₄ film 22. Si ₃ N ₄ is composed of Al ₂ O ₃ and S
Since the material has an isoelectric point between iO _2, the influence of the substrate surface and the upper surface of the convex portion on crystallization is suppressed.

Another projection forming process is shown in FIGS.
It is shown in (i). As shown in FIG. 9A, a silicon substrate
40, and then, as shown in FIG.
SiO on the substrate 30 by CVD_TwoMembrane 41 and S
i_ThreeN_FourThe films 42 are sequentially deposited. SiO_TwoThe membrane 41 is
Si for plate_ThreeN_FourFormed to enhance the bonding of membrane 42
You. Further, as shown in FIG.
Therefore Al_TwoO_ThreeA film 43 is formed. Then, FIG.
As shown in the figure,
A mask 44 is formed. Perform dry etching,
When the mask is removed, Al as shown in FIG._TwoO_Three
Is formed. Next, FIG.
As shown, the SiO_TwoForming a film 45,
The projection 51 is covered. At this time, according to the shape of the convex portion 51, S
iO_TwoThe film 45 has irregularities. Therefore, SiO_TwoMembrane 45
Is chemically and mechanically polished, and as shown in FIG._TwoO_ThreeTo
Exposure to get a flat surface. Then, as shown in FIG.
As shown in FIG. _ThreeN_FourForming a film 46;
Resist mask using photolithography on top
47 is formed. Perform etching and remove the mask
Then, a structure as shown in FIG. 9 (i) is obtained. Al_TwoO_Three
Convex portion 51 made of SiO and SiO_TwoThe convex part 52 consisting of
Combined convex portions 53a and 53b are formed in parallel.
Is done. The upper surfaces of the projections 53a and 53b are Si_ThreeN_FourMembrane 4
6 is covered. Opposing convex portions 53a and 53b
Is a space for crystallization. The space is Al_Two
O_ThreeSurface 51a and SiO_TwoIt is sandwiched between the surface 52a. Base
The surface of the plate 40 is also Si_ThreeN_FourCovered with membrane 42. Si_ThreeN_FourTo
Therefore, the effect of the substrate surface and the upper surface of the projection on crystallization
It is suppressed.

Further, the method of forming the convex portion may be a method other than the above-described method, and for example, an electrochemical method such as anodic oxidation may be used.

The device shown in FIGS. 10A and 10B can be provided by the present invention. Nine pairs of convex portions 220 are formed on a substrate 210 constituting the crystal preparation apparatus 200. In each convex portion pair 220, the convex portions 22
1 and the convex portion 222 face each other at a predetermined interval. Convex part 2
The space between the projection 21 and the projection 222 is a space for crystallization. The distance between adjacent pairs of convex portions is determined by the convex portions 221 and 222.
Significantly longer than the distance between. Therefore, each pair of protrusions can proceed with crystallization independently of each other. The structure of the substrate and the pair of projections is, for example, as shown in FIG.
It can be as shown in (i). In the nine pairs of convex portions 220, the combination of the material of the convex portions 221 and the material of the convex portions 222 may be all the same, may be partially different, or may be a combination in which all the pairs of convex portions are different from each other. May be provided. Further, the intervals between the convex portions 221 and the convex portions 222 may be the same for each pair of convex portions, or may be partially different or different for all the pairs of convex portions.

Further, on the substrate 210, a wall 230 surrounding the nine sets of convex portions 220 is provided. As shown in FIG. 11, a solution 240 containing organic molecules to be crystallized can be held inside the wall 230. The protrusions 221 and 222 come into contact with the held solution 240. Due to the surrounding wall, a portion for holding the solution can be easily formed on the substrate. The surrounding wall is made of glass, ceramics, plastics such as polycarbonate resin and acrylic resin, and metals such as stainless steel. The enclosure wall is preferably formed of a material that can inhibit the adsorption of organic molecules in the solution, that is, a material having a small potential in the solution. However,
If the distance between the space for crystallization (each pair of projections 220) and the surrounding wall is sufficiently longer than the diffusion distance of the organic molecules in the solution (the distance that the organic molecules can move in the solution), However, there is no problem if the surrounding wall is formed of another material.

The surrounding wall can be formed by joining another substrate having a through hole, for example, a Pyrex glass substrate, to the substrate 210 having the pair of convex portions 220. The substrate having the through hole forms a wall, and the solution can be held in the portion of the through hole. The through hole can be formed in a substrate such as glass by etching using a resist pattern, machining, or the like.
In addition, a substrate having a through hole from the beginning can be obtained by molding a raw material or the like. The substrate having the through hole and the substrate having the pair of projections may be joined in a separable manner or inseparable. For example, they 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, a sealant or an adhesive having a low adhesive strength may be used so as to be joined so as to be separated later. In addition, depending on the structure of the fitting, the bonding may be performed without using any other material. Alternatively, the surrounding wall may be formed by forming a relatively thick film on a substrate having a pair of convex portions and integrally forming the film using photolithography and etching.

Further, FIGS. 12 (a) and 12 (b)
The present invention can provide the apparatus shown in the following. The apparatus for crystal preparation 270 includes a first substrate 271 for forming an enclosure wall.
And an assembly in which three second substrates 272 each having a pair of convex portions are joined. The first substrate 271 has nine cylindrical through holes 273 arranged apart from each other. Through hole 27
The substrate 272 is arranged at each position where 3 is arranged. One opening (the lower opening in FIG. 12B) of the through hole 273 is closed by the substrate 272, and the other opening (the upper opening in FIG. 12B) remains open. The through hole 273 closed by the substrate 272 is
A portion 274 is formed that holds a solution containing the organic molecules (eg, protein molecules) to be crystallized. Each solution holding section 274 becomes a well for crystallization. Each substrate 272
Are formed with three pairs of convex portions corresponding to the arrangement of the through holes of the substrate 271. In the convex part pair, the convex part 27
5 and the convex portion 276 face each other. The three substrates 272 may all be the same, some of them may be different, or all may be different. The combination and spacing of the materials of the pair of protrusions may be different for each substrate, or may be the same for all. By combining a substrate having a plurality of through holes and a plurality of substrates each having a pair of projections, an apparatus for simultaneously performing crystallization under a plurality of conditions can be easily provided.

Further, an apparatus as shown in FIGS. 13A and 13B can be provided by the present invention. The crystal preparation apparatus 370 includes a first substrate 371 and a second substrate 37.
2 is an assembly. The first substrate 371 has nine cylindrical through holes 373 arranged apart from each other. One opening of the through hole 373 is closed by the substrate 372,
Other openings remain open. The through hole 373 closed by the substrate 372 forms a portion 374 that holds a solution containing the organic molecules to be crystallized. Each solution holding section 374 becomes a well for crystallization. Substrate 372
On the upper side, nine pairs of convex portions are arranged corresponding to the arrangement of the through holes of the substrate 371. In the convex portion pair, the convex portion 375
And the convex portion 376 are opposed to each other. The combinations and intervals of the materials of the nine pairs of convex portions may be the same, may be partially different, or may be all different. Also in this apparatus, crystallization can be performed simultaneously under a plurality of conditions.

The second substrate 272 shown in FIG.
The second substrate 372 shown in FIG. 3 can be manufactured by a method as shown in FIG. 8 or FIG.

[0031] The device according to the invention may include means for measuring the pH of the solution. As described above, the surface potential or the effective surface charge of the solid surface and the organic molecule to be crystallized depends on the pH of the solution, and therefore, it is significant to monitor the pH of the solution in 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. 14 may be used as the pH measuring means. In the pH measuring device 100, an SiO ₂ film 102 is formed on an n-type silicon substrate 101. A solution holding unit 110 is provided on the substrate 101. The solution holding unit 110 includes 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 extends toward the SiO ₂ film 102 and is arranged so as to be in contact with the solution held in the solution holding unit 110. Back surface of silicon substrate 101 (Si
A terminal electrode 108 is provided on the surface opposite to the surface on which the O ₂ film 102 is provided).

As described above, the surface of the oxide undergoes a hydration reaction 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. 14, 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. 15 shows the capacitance-voltage characteristics of two 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 device shown in FIG. 14 at a measuring frequency of about 1 MHz, the capacitance-voltage characteristics of various solutions whose pH is known are measured, 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 device for crystal growth according to the invention.

The apparatus 430 shown in FIG. 17 has the above-mentioned pH measuring apparatus built in one of the nine solution holding sections (wells for crystallization) of the apparatus for preparing crystals as shown in FIG. This is an application example, and is a crystal growth apparatus in which a pH monitor is integrated. In this example, one of the nine solution holding units can be used for measuring the pH of the solution, and the remaining eight can be used for preparing crystals of organic molecules. Further, a precipitant may be retained in some of the remaining eight.

As shown in FIG. 17, a crystal can be prepared (prepared) by sealing the well of the apparatus 430 with a transparent glass lid 500 and storing it 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 this time, as shown in the figure, a CV meter 401 is connected to the electrode of the pH monitor cell, and an XY recorder 402 measures CV characteristics. This monitors the pH of the solution during crystal growth.

In order to prepare (prepare) a crystal of an organic molecule using the apparatus of the present invention, first, a solution containing the organic molecule to be crystallized is formed in the region where the pair of convex portions of the apparatus of the present invention is formed. (In the case of an apparatus provided with an enclosure wall, the solution is supplied to the inside of the enclosure wall and held therein. In the case of an apparatus having no enclosure wall, a pair of convex portions on the substrate surface is provided. The solution is dropped on the portion where is formed, and held by surface tension). Then, it is sealed with a precipitant and left to stand until crystals grow, whereby crystals of organic molecules can be produced. The precipitant may be put in a separate container, placed side by side with the crystal preparation device of the present invention, and sealed with a glass lid or the like. Further, a precipitant holding section may be formed integrally with the crystal growth apparatus, and the precipitant may be put therein. That is, the apparatus may be configured to have a plurality of solution holding units (wells), some of which may be used as wells for holding the precipitant, and into which the precipitant may be charged. The volumes of the crystallization well and the precipitant holding well may be changed. Note that a pH monitor is not always necessary.

[0041]

According to the present invention, the organic molecules are selectively arranged in the space formed by the pair of convex portions, thereby reducing the influence of convection on the organic molecules and forming the crystal nuclei of the organic molecules. Can be stabilized. According to the present invention, it is possible to obtain a large crystal capable of performing X-ray structural analysis. Further, according to the present invention, it is possible to cope with crystallization of many types of organic molecules by using a large number of pairs of convex portions for crystallization. In the present invention, crystallization can be performed on a very small amount of sample.

The present invention can be used to purify or crystallize 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.

[Brief description of the drawings]

FIGS. 1A and 1B are schematic diagrams showing a conventional crystal growth method.

2A is a schematic diagram showing a polymer having a surface charge distribution, and FIG. 2B is a schematic diagram showing that the molecule shown in FIG. 2A can be regarded as a dipole.

FIG. 3 is a schematic sectional view showing a specific example of the device according to the present invention.

FIG. 4 is a schematic sectional view showing another embodiment of the device according to the present invention.

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

6 (a) and 6 (b) are schematic cross-sectional views showing another specific example of the device according to the present invention.

FIGS. 7A and 7B are schematic diagrams showing a state in which the substrate surface inhibits the orientation of molecules.

8 (a) to (i) are schematic sectional views showing a method for manufacturing a device according to the present invention.

9 (a) to (i) are schematic sectional views showing another method for manufacturing a device according to the present invention.

FIG. 10 (a) is a plan view showing a specific example of an apparatus having an enclosure for holding a solution according to the present invention,
(B) is a sectional view thereof.

FIG. 11 is a cross-sectional view showing how the apparatus shown in FIGS. 10A and 10B holds a solution.

FIG. 12A is a plan view showing an example of an apparatus capable of simultaneously performing crystallization under a plurality of conditions according to the present invention, and FIG. 12B is a cross-sectional view thereof.

FIG. 13A is a plan view showing another example of an apparatus capable of simultaneously performing crystallization under a plurality of conditions according to the present invention, and FIG. 13B is a cross-sectional view thereof.

FIG. 14 is a schematic sectional view showing an example of a pH sensor provided in the device of the present invention.

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

FIG. 16 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.

17 is a schematic diagram showing a flow of measuring pH in a pH monitor-integrated device in which the device shown in FIG. 14 is incorporated in the crystal growth device shown in FIG.

[Explanation of symbols]

1, 2, 11, 12, 31, 32, 51, 52 convex portion,
10, 20, 40 substrates.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) B01D 9/02 625 B01D 9/02 625Z C30B 7/06 C30B 7/06 // C12M 1/42 C12M 1 / 42

Claims (5)

[Claims] 1. An apparatus for preparing a crystal of an organic molecule contained in a solution, comprising: a substrate; and a pair of opposed convex portions formed on the substrate, wherein the pair of opposed convex portions are provided. A space for crystallization of the organic molecule is formed between them, a solution containing the organic molecule is used in the space, and a pair of protrusions sandwiching the space for crystallization are used. An apparatus for preparing a crystal, wherein the surface of the part has a property of orienting the organic molecules in a specific direction between the surfaces in the solution. 2. The surface of the pair of convex portions has a surface potential or a zeta potential different from each other in the solution, whereby a property of orienting the organic molecules in a specific direction between the surfaces is provided. The device according to claim 1, wherein the device is provided. 3. The surface of the pair of protrusions is made of a material having a different isoelectric point from each other, and the surface of the substrate existing between the pair of protrusions has the surface of the pair of protrusions. 3. The device according to claim 2, wherein the device comprises a material having an isoelectric point between the isoelectric points. 4. The surface of the pair of protrusions is made of a material having a different isoelectric point, and the surface existing on the top of the pair of protrusions is the isoelectric point of the pair of protrusions. Device according to claim 2 or 3, characterized in that it consists of a material having an isoelectric point between: 5. The device according to claim 1, wherein a wall is provided on the substrate so as to surround the pair of protrusions, and the solution is held inside the wall. An apparatus according to claim 1.