JP3146983B2 - Crystal growth method and crystal growth apparatus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for crystallizing a high molecular compound and an apparatus used for the method, and more particularly, to a method using a semiconductor substrate or the like in which valence electrons are controlled.
The present invention relates to a technique for crystallization of various biopolymers including proteins.

[0002]

2. Description of the Related Art In order to understand the specific properties and functions of various biomacromolecules such as proteins and their complexes, their detailed three-dimensional structures are indispensable information. For example, from the viewpoint of basic chemistry, information on the three-dimensional structure of a protein or the like is a basis for understanding the mechanism of function expression in a biochemical system by enzymes, hormones, and the like. In addition, in the fields of pharmacy, genetic engineering, and chemical engineering, especially in the fields of industry, the three-dimensional structure provides information essential for rational molecular design in advancing drug design, protein engineering, biochemical synthesis, etc. provide.

Atomic level 3 of such biopolymers
As a method of obtaining three-dimensional structure information, at present X
Line crystal structure analysis is the most powerful and accurate means. In addition to the shortening of the measurement time and the improvement of the measurement accuracy due to the recent hardware improvement of the X-ray light source / diffraction apparatus, the analysis speed has been greatly improved due to the dramatic improvement in the calculation processing speed of the computer. It is expected that the three-dimensional structure will be clarified by using this method as the mainstream in the future.

On the other hand, in order to determine the three-dimensional structure of a biopolymer by X-ray crystal structure analysis, it is necessary to extract and purify a 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.

[0005] Crystallization of biological macromolecules such as proteins can be carried out in a supersaturated state by performing a treatment for removing a solvent from water or a non-aqueous solution containing macromolecules, as in the case of ordinary low molecular weight compounds such as inorganic salts. Basically, growing a crystal. Representative methods for this include (1) batch method, (2) dialysis method, and (3) gas-liquid interphase diffusion method, which are used properly depending on the type, amount, and property of the sample.

[0006] The batch method is a method in which a precipitant for depriving water of hydration is directly added to a solution containing a biopolymer to reduce the solubility of the biopolymer and convert the biopolymer to a solid phase. In this method, for example, solid ammonium sulfate (ammonium sulfate) is often used. This method has the drawbacks that it requires a large amount of a solution sample, that it is difficult to finely adjust the salt concentration and the pH, that the operation requires skill, and that the reproducibility is low. The dialysis method is a method in which the drawback of the batch method is improved. For example, as shown in FIG. 16, a solution 162 containing a biopolymer is sealed inside a dialysis tube 161 and a dialysis tube external solution 16 is sealed.
In this method, the crystallization is performed by continuously changing the pH or the like of the buffer 3 (for example, a buffer solution). According to this method, since the salt concentration and the pH difference between the inner and outer liquids can be adjusted at an arbitrary speed, it is easy to find the conditions for crystallization. In the gas-liquid interphase diffusion method, for example, as shown in FIG.
A container 17 in which a droplet 172 of the sample solution is placed
In this method, the droplets and the precipitant solution 174 are put into the liquid crystal 3 so as to gradually establish an equilibrium by evaporation of volatile components between the two.

However, there are various problems in the crystallization of biological macromolecules such as proteins as described above.

First, it has been difficult to obtain a crystal having good crystallinity or a large single crystal. This is thought to be due to the fact that biopolymers are generally susceptible to gravity due to their high molecular weight, causing convection in solution (eg, F. Rosenberger, J. Cryst. Growt
h, 76, 618 (1986)). That is, the biological macromolecules and the generated fine crystal nuclei settle under their own weight, thereby causing convection of the solution around the molecules and the nuclei. Furthermore, local convection of the solution occurs also at the generated crystal surface portion because the concentration of the molecule is reduced. Due to the convection in the solution generated as described above, generated crystals move in the solution, and the supply layer of molecules due to peripheral diffusion is significantly reduced. For this reason, it is considered that the crystal growth rate is reduced or the growth anisotropy on the crystal plane is generated, thereby preventing crystallization.

Also, unlike the crystals of other substances, biopolymer crystals contain a large amount of solvent (mainly water) (≧ 50% by volume). This solvent can easily move in the disordered and intermolecular voids in the crystal. Also, despite the large size of the molecule, there are few packing contacts between the molecules in a wide range in the crystal, and only few molecule-to-molecule contacts or contacts by hydrogen bonding through water. Such a state is also a factor preventing crystallization.

In addition, biopolymers are very sensitive to the conditions used for crystallization. Biomacromolecules are stabilized in solvents by the interaction between individual molecular surfaces,
The charge distribution on the molecular surface, especially the conformation of the amino acid near the molecular surface, is dependent on the environment,
It varies greatly depending on ionic strength, temperature, type of buffer solution, dielectric constant, and the like. Therefore, the crystallization process is a multi-parameter process in which various complicated conditions are intertwined, and a unified method applicable to any substance has not been established. Regarding proteins, it is very difficult to crystallize hydrophobic membrane proteins at present, although they are very important biochemically compared to water-soluble proteins. So far only two cases have been successfully analyzed.

[0011] In addition, the obtained biopolymer is often very small. For example, proteins such as enzymes are generally extracted from cells and the like and purified.
Very few samples are ultimately obtained for crystallization. It is said that when crystallization is performed, the concentration of the biopolymer in the solution needs to be about 50 mg / ml. Therefore, it is necessary to repeat the crystallization experiment under various conditions for the minimum amount of solution (screening).
Need to be done.

As described above, crystallization of proteins and other biological macromolecules and their complexes is an important process in academic and industrial fields, but has been carried out through trial and error. This has been the biggest bottleneck in X-ray crystal structure analysis. Therefore, it is necessary to understand the basic principle of crystallization in the future and to develop a crystallization technique applicable to any molecule.

[0013]

SUMMARY OF THE INVENTION An object of the present invention is to provide a conventional crystal which has been developed by repeating trial and error because there is no method applicable to any substance because of its various characteristics as described above. The technical disadvantages of the conversion process are to be eliminated.

More specifically, the present invention reduces the influence of convection in a solution due to the effect of gravity and controls nucleation in the crystallization of various biopolymers and biological tissues mainly composed of biopolymers. The purpose is to do.

A further object of the present invention is to provide a technique for suppressing or controlling the mass production of microcrystals and obtaining a large crystal capable of X-ray structural analysis.

It is a further object of the present invention to provide a method and apparatus for enabling crystallization with a small amount of solution.

[0017]

The crystal growth method of the present invention is a method for growing crystals of a polymer compound contained in a solution, wherein the surface portion of the crystal is grown in accordance with the environment of the solution containing the polymer compound. Providing a plurality of solid devices having two or more grooves or holes in which valence electrons are controlled so that the concentration of holes or electrons can be controlled and depth and / or opening width are different; A step of holding the plurality of solid-state elements with a predetermined gap opened and holding a solution containing a polymer compound in the gap to form a groove or a hole. Contacting the solution with the surfaces of the plurality of solid devices. In the solid-state device, valence electrons are controlled so that crystallization of the polymer compound is promoted inside the groove or the hole rather than outside. In this method, a crystal of a polymer compound is grown in a groove or a hole for holding a solution under an electric state brought to a surface of a solid-state device by a controlled valence electron.

As the solid-state element, a semiconductor substrate doped with impurities can be used. Two or more grooves or holes having different depths and / or different widths of the openings are formed in the semiconductor substrate. In this semiconductor substrate, the type and / or concentration of the added impurity may be different between inside and outside the groove or hole in order to promote crystallization of the polymer compound inside the groove or hole. it can.

In the step of holding a plurality of solid elements with a gap therebetween, the solid elements can be held so that the distance between the opposed solid elements continuously decreases or increases. .

In the apparatus of the present invention, the valence electrons are controlled so that the concentration of holes or electrons on the surface can be controlled according to the environment of the solution containing the polymer compound, and the depth and / or the width of the opening are controlled. A plurality of solid devices having two or more different grooves or holes, and means for facing the plurality of solid devices and holding a predetermined gap therebetween are provided. Valence electrons are controlled differently inside and outside the grooves or holes of the solid state device.

In the device of the present invention, the semiconductor substrate doped with impurities as described above can be used as the solid-state element. The type and / or concentration of the added impurity can be different between inside and outside of the groove or hole formed in the semiconductor substrate.

The apparatus of the present invention comprises a container capable of holding the above-mentioned plurality of solid elements and means for holding the same in a hermetically sealed state together with a desiccant or a buffer solution, and means for supporting the solid elements in the container. May be further provided.

In the device of the present invention, the solid state elements can be arranged such that the distance between the plurality of opposed solid state elements continuously decreases or increases.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Most of biomolecules, including 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 if a crystal nucleus is formed, if the molecular structure and charge distribution on the surface of each molecule are not exactly the same and have redundancy, the molecules assembled around the nucleus will loosely bind to each other. Therefore, it is considered that the crystallinity is reduced.

It has been reported that the initial process of nucleation is important for the crystal formation of protein molecules.
Yonath et al. Use an electron microscope to observe the initial crystallization process of a large ribosome subunit extracted from Bacillus Stearothermophilus. According to this, in order for crystallization to proceed, it is essential that each molecule take a two-dimensional regular structure (a stitch, a star, a staggered lattice, etc.) and aggregate as an initial process. (Biochemist
ry International, Vol. 5, 629-636 (1982)).

It is unknown whether this is essential for all substances. However, protein molecules generally have weak intermolecular interactions, and because the molecular surface is locally charged, it is difficult to aggregate. Considering that protein molecules are two-dimensionally arranged in the initial stage of crystallization, If some conditions are satisfied, the subsequent crystallization is considered to proceed epitaxially with this as a nucleus.

In the present invention, in order to stably generate a crystal nucleus, a solid-state element whose valence electrons are controlled is brought into contact with a liquid containing a substance to be crystallized. In the solid-state device, the concentration of electrons and holes can be controlled by valence electron control from the surface in contact with the liquid toward the inside or within the cross-section of the solid-state device, whereby the electric surface of the solid-state device can be electrically controlled. The state can be controlled. For example, FIG. 1 schematically shows a state in which a crystal nucleus is fixed on the surface of a solid device and a crystal grows according to the present invention. As shown in FIG. 1A, the crystal nucleus 2 is fixed by electrostatic action to the surface of the solid-state element 1 which is brought into a predetermined electrical state by valence electron control. Then, as shown in FIG. 1B, the compound such as a protein is aggregated on the surface of the solid-state element by electrostatic interaction, the generation of crystal nuclei is promoted, and the crystal grows. Therefore, crystallization can be controlled by controlling the electrical characteristics of the surface of the solid-state element. For example, the type, amount, arrangement density, and the like of crystal nuclei fixed on the surface of the solid-state element can be adjusted by valence electron control, whereby crystallization can be controlled. In addition, since the generated crystal nuclei are fixed to the surface of the solid-state element, minute fluctuations of the nuclei due to convection in the solution and the like are suppressed, molecules are regularly assembled according to the nucleation, and the crystallinity is improved. Is also expected. Even if the charge distribution on the surface of the molecule to be crystallized is slightly changed due to the pH of the solution or the denaturation of the molecule, a space charge is always induced on the solid device surface to compensate for the effective surface charge of the molecule. Is expected to be easily and preferentially generated.

Further, as will be described in more detail later, at the bottom of the groove formed in the solid-state element, an electrostatic interaction can be exerted almost isotropically on the molecule to be crystallized. When a crystal nucleus is formed at the bottom of the groove, the crystal nucleus can be protected from convection due to the effect of gravity. That is, the crystal nucleus can be stopped at the bottom of the groove by the electrostatic interaction. If crystals grow on the basis of substantially stationary nuclei, generation of excessive microcrystals is suppressed, and it is expected that large crystals in which molecules are regularly assembled on the surface of the crystal nuclei can be obtained.

Further, in the present invention, a plurality of solid-state elements are opposed to each other with a predetermined gap therebetween, and a solution containing a molecule to be crystallized is held in the gap, whereby crystallization can be performed with a smaller amount of solution. . The solution held in the gap has a surface tension that causes convection, but the effect of the surface tension can be suppressed by the action of electrostatic attraction brought on the surface of the solid-state element as described later.

In general, the cohesiveness of a charged substance or molecule in an electrolyte solution depends on the sum of the electric double layer repulsion and Van der Waals force between them. It is very important to control the salt concentration for adjusting the surface potential added to the solution. However, according to the present invention, since the electrostatic characteristics of the surface of the solid element can be adjusted in advance by valence electron control, there is an advantage that the adjustment of the salt concentration is easy or unnecessary.

The solid used for such a purpose includes:
Any substance may be used as long as it is a substance having the above-mentioned electrostatic characteristics and capable of controlling the amount of charge and the polarity, and is further chemically stable in a solution. One of the most suitable materials for achieving this purpose is a silicon crystal. The crystallization mechanism expected when a silicon crystal is used will be described below. However, the mechanisms described below can be applied to other solid state devices used in accordance with the present invention.

FIGS. 2, 3 and 4 show one embodiment of the device according to the invention. As shown in FIG. 2, in the crystal growth apparatus, two solid-state elements 10 and 20 are arranged via a predetermined gap 25. A plurality of V-shaped grooves (V-grooves) 11a, 11b, 11c having different depths and / or opening widths are formed on the surface of the solid-state element 10.
A plurality of V-grooves 2 of different sizes are also provided on the surface of the solid-state device 20.
1a, 21b, 21c and 21d are formed. The two solid-state elements 10 and 20 are held with the surfaces on which the V-grooves are formed facing each other. In this device, two solid-state elements are arranged such that the longitudinal direction of each groove is substantially vertical. In FIG. 2, a spacer for maintaining the gap 25 is omitted.

FIG. 3 shows a state in which a gap between two solid-state elements is maintained by a spacer in the apparatus of FIG. Spacers 15a and 15b are provided between the solid state device 10 and the solid state device 20, and a predetermined gap 25 is maintained. A mother liquor 16 containing molecules for crystallization is injected into a gap between the two solid-state elements formed by the spacers. If the gap between the solid elements is made very small by a spacer of a predetermined size, the injected mother liquor is retained in this gap by capillary action. The held mother liquor 16 is in contact with the surface of the solid element where the V-groove is formed. The amount of mother liquor held in these small gaps is very small.

FIG. 4 shows a more detailed structure of one of the solid-state devices. In the solid state device 20,
An N-type silicon layer 2 is formed on a substrate 22 made of P-type silicon.
3 are formed. A plurality of V-grooves 2 are provided on the surface of the substrate 22.
1a, 21b, 21c and 21d are formed. P-type silicon is exposed in the V groove. On the other hand, the surface that comes into contact with the solution outside the V groove is covered with the N-type silicon layer 23. On the surface of the substrate 22, a water-repellent layer 24 made of, for example, a water-repellent resin is further provided so as to surround the V groove. If the spacers 15a and 15b are provided via the water-repellent layer, the mother liquor containing the molecules to be crystallized in the gap can be more stably held. An element in which an N-type silicon layer is formed on P-type silicon as shown in FIG. 4 can be preferably used for crystallization of a polymer compound having a negative effective surface charge after dissociation in a solution. on the other hand,
For a polymer compound which dissociates in a solution and has a positive effective surface charge, a solid-state element using silicon having a polarity opposite to that shown in FIG. 4 can be preferably used. That is, a solid-state element in which a P-type silicon layer is formed on an N-type silicon substrate and N-type silicon is exposed in a groove can be used for crystallization of a polymer compound having a positive effective surface charge.

FIG. 5 schematically shows a manufacturing process of the solid-state device shown in FIGS. First, a P-type silicon substrate 32 whose surface is cleaned, for example, mirror-polished
Is prepared (FIG. 5A). Next, an N-type silicon layer 33 is formed on the surface of the P-type silicon substrate 32 by a method such as ion implantation (FIG. 4B). Thereafter, a silicon oxide film (SiO ₂ ) 39 is formed by a method such as CVD (FIG. 5C). Next, after performing an appropriate method such as photolithography, the silicon oxide film in the region where the groove is to be formed is removed (FIG. 5D). If the portion not covered with the oxide film is etched, the groove 3
1a, 31b, 31c and 31d are formed.
(E)). Next, after removing the silicon oxide film, the groove 31 is removed.
A water-repellent resin layer 34 is formed so as to surround a to d (FIG. 5F). Although not shown, after removing the silicon oxide film, a silicon oxide film may be formed again on the entire surface of the solid-state device. The substrates 32 and 32 'on which the N-type silicon layer and the groove have been formed as described above are overlapped via the glass spacers 35a and 35b (FIG. 5 (g)). The spacers 35a and 35b are sandwiched between the water-repellent resin layers 34 and 34 'formed on the substrate. If the two substrates are fixed with an adhesive or the like, an apparatus for crystallization can be obtained.

The mechanism by which crystallization is controlled using the solid-state device of the present invention will be described below. When an aqueous electrolyte solution containing a polymer compound having a dissociated negative effective surface charge is brought into contact with a valence-controlled N or P-type silicon crystal, a Schottky barrier is formed on the N-type silicon surface. On the other hand, an ohmic contact is obtained on the P-type silicon surface. On the P-type silicon surface, holes are always supplied from the bulk silicon side to the polymer electrolyte having a negative charge (ohmic characteristic), so that the polymer is expected to always aggregate on the silicon surface. . On the other hand, a surface potential depending on the electrolyte concentration of the aqueous solution is generated on the surface of the N-type silicon, and a space charge layer region is formed inside. This space charge amount also depends on the dopant concentration of N-type silicon. Therefore, a polymer having a negative charge in the electrolyte solution is expected to continue to aggregate on the silicon surface until at least compensating for the positive space charge of the N-type silicon. Therefore, the aggregation and crystallization of the polymer compound are restricted on the silicon surface where the space charge layer region is formed, while the polymer surface is not polymerized on the silicon surface where the ohmic contact is formed. It is expected that aggregation of the compound will proceed indefinitely.

Also, for example, when two or more regions having different impurity concentrations are formed in N-type silicon, crystallization is expected to proceed in a different manner depending on those regions. For the case where the impurity concentration of N-type silicon is low and high resistance, and the case where the impurity concentration is high and low resistance,
The difference between the effects will be described. For N-type silicon,
In a low-impurity-concentration (or high-resistance) substrate, the space charge layer formed near the surface is widened due to the low dopant concentration, so that the capacitance of the depletion layer is small and the surface potential induced on the silicon surface is high. It is expected to be larger than in the case of an impurity concentration (or low resistance) substrate. Since this surface potential is opposite in polarity to the effective surface potential of the polymer compound, aggregation is expected to be promoted by the action of electrostatic attraction. That is, it is expected that an N-type silicon substrate having a low impurity concentration and a high resistance can deposit more crystals on the substrate surface than an N-type silicon substrate having a high impurity concentration and a low resistance.

As described above, the formation of regions having spatially different resistances can be easily achieved by selectively doping the silicon surface with impurities. As another method, it is possible to expose surfaces having different resistance values by etching the silicon surface.

The N-type and P-type silicon crystals used in the present invention may have characteristics equivalent to those of a silicon wafer used in a normal LSI process. The specific resistance of the silicon crystal may be in the range of about 0.0001 to 1000 Ωcm, and more preferably 0.001 to 100 Ωc.
m can be used. Various methods are available for preparing silicon whose valence electrons are controlled to be N-type and P-type, and any method may be used. However, the simplest and most accurate method for controlling impurity concentration is ion-implantation. An injection method may be used. In this case, P-type and N-type valence electron control can be easily performed by implanting ions of elements belonging to Group III and Group V of the periodic table into silicon and annealing the silicon. P
B, Al, Ga, I
n and Tl. In particular, B is common. N, P, As, as a Group V element for making an N type
Sb, Bi, etc. can be mentioned, and especially P, As, Sb
Is common. Further, the surface of the crystal is preferably mirror polished in order to control the crystal nuclei to be precipitated.

In the present invention, when forming the impurity layer on the surface of the silicon substrate, the thickness thereof is preferably 0.1 to 200 μm, more preferably 1 to 50 μm. Outside of this range, the fabrication is not easy or the effect is lost, which is not desirable.

Although an example using semiconductor crystalline silicon that can easily control valence electrons has been described above, other materials having the same function can be used as appropriate to achieve the object. For example, a semiconductor crystal other than silicon can be preferably used, and further, a material other than the semiconductor crystal, for example, an inorganic compound, an organic compound, a polymer compound, and a composite thereof having a controlled charge distribution may be mentioned as a candidate. Can be.

In the present invention, a plurality of grooves or holes are formed in the solid-state device. The device shown in FIG. 2 has a plurality of V grooves. Instead of the grooves, for example, pyramidal or conical holes may be provided on the surface of the solid-state device. Grooves or holes
It is more preferable that the width of the opening becomes narrower as going deeper. In actual crystal growth, it is advantageous to provide a large number of grooves or holes of several sizes on one element surface.

It is desirable that the solid-state element used for crystallization can be applied to any polymer compound. On the other hand, it is considered that the required characteristics of the solid-state device also vary depending on the size of the molecules to be crystallized, the charging characteristics, and the like. It is considered that the size of the groove or hole also needs to be changed according to the size of the molecule to be crystallized, the charging characteristics, and the like. However, if a solid-state device having a groove for each target polymer compound was prepared, it would be costly and time-consuming, and would not be efficient.
Therefore, if a plurality of grooves having different sizes are formed on the solid-state element in advance, even if the target molecular species is changed, more preferable crystallization conditions can be provided in any one of the grooves. Therefore, various molecules can be crystallized with one solid-state device. Thereby, the labor and cost for manufacturing the solid-state device are also reduced.

It is desirable that the size of the opening of the groove or hole and the depth of the groove or hole formed on the substrate surface be appropriately set in a suitable range depending on the kind of the polymer to be treated. Generally, the width of the opening of the groove or hole is 0.0
The range of 1 to 100 μm is preferable, and the length of the groove is 1 to 10 μm.
The range of mm is preferred. Also, the plurality of grooves or holes are
It can be produced as appropriate at intervals in the range of μm to 1 mm. The depth of the groove or hole is, for example, 0.01 to 200 μm.
It is preferable to adjust in the range of m. However, the size described above mainly comes from restrictions on the fabrication of the solid-state device. Even if the size is other than this, the performance of the solid-state device, that is, the one that has a detrimental effect on crystallization is not considered. Absent.

FIGS. 6 to 8 are for explaining the effect of the grooves or holes on the crystal growth. As shown in FIG. 6, when the solid-state device is brought into contact with an electrolyte solution containing a molecule to be crystallized, V or P ^- silicon is formed.
The grooves 41 and 51 have a wider range (which may be considered as the width of the electric double layer) of the electrostatic interaction with the dissociated polymer than the surface portions 43 and 53 made of the N or N ⁺ silicon layer. It is expected to be wider. In the figure, regions 58 and 58 'shown by dotted lines are the ranges that the electric interaction can reach. That is, the regions 58 and 58 ′ are thicker in the V-groove portions 41 and 51 than on the surface portions 43 and 53. In particular, the deepest central portion of the V-groove overlaps with the region where this effect is exerted, so that regions 58 and 58 'are formed.
Is expected to be the widest.

Therefore, at the deepest part of the V-groove portion made of P or P ^- silicon, the crystal nucleus or the molecular aggregate 47 serving as the crystal nucleus receives an electrostatic attraction 49 from the V-groove surface almost isotropically, Within. V
Regarding the molecular aggregate 47 in the deep part of the groove, the influence of the convection in the solution based on the gravity and the electrostatic attraction are offset, and it is expected that the generation of crystal nuclei and the growth of the crystal can occur stably.

On the other hand, as shown in FIG. 7, on surface portions 43 and 53 made of N or N.sup. ⁺ Silicon, it is expected that convection 59 of the solution is caused inward in the gap by surface tension. . However, in the narrow gap, the convection is suppressed as a result by the electrostatic attractive force 49 acting in a direction substantially perpendicular to the direction of the surface tension. This electrostatic attraction comes from the silicon substrate. In a groove where a larger electrostatic attraction acts, the effect of convection is almost suppressed, whereas outside a groove where only a small electrostatic attraction acts, molecules to be crystallized are slightly affected by convection. Therefore, it is considered that the diffusion supply layer width in the vicinity of the crystal nucleus fluctuates outside the groove, resulting in a decrease in crystallinity or a decrease in growth rate.

As shown in FIG. 8A, the crystal nuclei 80 formed in a state where there is no convection are supplied to the molecule supply layer 8.
If 2 is uniform and isotropic, the crystal grows uniformly and isotropically, and its growth rate increases. On the other hand, FIG.
As shown in (2), when the convection 89 occurs in the solution, the molecular supply layer 82 ′ of the crystal nucleus 81 is narrowed by the convection. As a result, crystals grow non-uniformly, and twins are easily formed. In addition, the growth rate becomes slow. In order to suppress the influence of convection generated by gravity, buoyancy, etc., in the present invention, the molecules to be crystallized are restrained in the grooves by electrostatic attraction acting on the surface of the solid element, and the movement is stopped. .
For example, when a solution is sandwiched between two solid devices, molecules in the solution receive electrostatic attraction from each device. That is, the electrostatic attraction works from up and down or left and right. In this way, the electrostatic effect on molecules is superimposed on the surface of the solid-state device. Therefore, molecules are more likely to be restrained by electrostatic attraction. Even if the molecules try to move in the gap under the influence of surface tension, gravity, or the like, the molecules are eventually restrained in the groove by the electrostatic effect, and the crystal grows there.

As described above, since the molecules to be crystallized are restricted in the grooves, it is preferable that the grooves sandwiching the solution overlap as much as possible. On the other hand, crystallization in a flat portion without a groove is suppressed. Therefore, it is considered more preferable that the grooves are opposed to each other with the solution interposed therebetween so that the longitudinal directions coincide with each other. However, since the size, for example, the width of the groove is very small, it is considered that it is quite difficult to arrange the solid-state elements so that the positions of the grooves overlap well. Therefore, as shown in FIG.
It is more practical to arrange a plurality of solid-state elements so that the longitudinal directions of the opposing grooves intersect substantially vertically. In this case, the overlapping of the opposing grooves can be maximized.

Further, as shown in FIG. 9, the distance between a plurality of solid state elements can be varied depending on the position. In the device shown in FIG. 9, the spacer 95 is inserted only at one end between the solid state devices 90 and 90 '. In this structure, the distance between the elements on the solid state element decreases as the distance from the spacer 95 increases. That is, the spacer 9
As one moves away from 5, the gap decreases continuously.
Although this apparatus uses one spacer, it is also possible to continuously reduce or increase the gap by using two spacers having different sizes. As shown in FIG. 3, when two elements are held at a constant interval, the predetermined gap may not be optimal depending on the material to be crystallized. This is because the gap reflects a region where electrostatic attraction is exerted between elements and a distribution of surface potential. Therefore, as shown in FIG. 9, it is considered that the optimum conditions for crystallization can be provided at any position by changing the interval between elements in one device. For example, as shown in the figure, the optimum conditions for the substance A can be provided in the region X, while the optimum crystallization conditions can be provided for the material B different from A in the region Y. Therefore, it is possible to crystallize various molecules in one apparatus. This allows
The labor and cost for device fabrication are also reduced.

As described above, the solid-state device having the V groove has been described.
Various shapes can be used for the shape of the groove, for example,
A concave groove as shown in FIG. 10 may be formed. In the solid-state device 100, an N-type silicon layer 113 is formed on a P-type silicon substrate 112, and the concave grooves 10 having different openings are formed.
1a, 101b and 101c are provided.

As described above, a spacer is used to oppose a solid-state element such as a silicon substrate with a gap therebetween. The material of the spacer is not particularly limited,
For example, glass can be preferably used. As the spacer, for example, a cylindrical or prismatic spacer can be used. The distance between the elements is, for example, 0.1
It is preferably in the range of 10 to 10 mm. Therefore,
When a cylindrical spacer is used, the diameter of the spacer can be set in the range of 0.1 to 10 mm. For example, a plurality of solid state elements sandwiching the spacer can be fixed by bonding or welding.

As shown in FIG. 3, a water-repellent layer is preferably formed on the surface of a solid element such as a silicon substrate so as to surround the groove. This layer can effectively prevent the solution from flowing to the surroundings when the solution is held between the devices. For example, a silicon surface from which an oxide film has been removed is generally water-repellent to water or pure water containing only acids and alkalis, but not to an aqueous solution containing a salt such as a buffer solution. . Therefore, when a buffer solution is used, it is necessary to form a layer made of a water-repellent substance around the silicon substrate. The water-repellent layer can be formed of, for example, an organic resin, and a polyimide resin is one of the materials that can form the water-repellent layer most easily. In the case of forming a water-repellent layer made of polyimide, for example, after coating and curing a photosensitive or non-photosensitive polyimide resin, unnecessary portions can be removed by etching or development to obtain a desired pattern. .

The thickness of the water-repellent layer used in the present invention does not need to be particularly limited functionally, but a layer having a thickness in the range of 0.1 to 100 μm is relatively easy to manufacture. Also, various materials that exhibit water repellency and are chemically stable in solution can be used for this layer. Further, when a solution having water repellency to a solid element such as silicon is used, such a water repellent layer is unnecessary.

FIG. 11 shows a more specific apparatus for carrying out the present invention. In the apparatus shown in FIG. 11, a buffer solution 112 is contained in a container 111, and a dialysis membrane tube 113 is provided therein. In a dialysis membrane tube 113, together with a mother liquor 112 containing a polymer compound, a crystal growth apparatus 110 in which two solid-state elements for crystallization control, for example, two silicon semiconductor substrates as described above are opposed to each other at a predetermined interval. Is housed. Dialysis membrane tube 11
3 is sealed with closers 115a and 115b and immersed in buffer solution 112. The opening of the container 114 is
Covered by 16. In this apparatus, dialysis proceeds and mother liquor 112 is placed on crystal growing apparatus 110.
From which polymer crystals are precipitated. In addition, other than this apparatus, for example, a method and an apparatus for performing crystallization by holding a mother liquor in the form of droplets on a silicon substrate without using a dialysis tube are also applicable.

FIG. 12 shows an example of an apparatus which enables crystal growth with a very small amount of mother liquor. In this device, a substrate in which an upper substrate 120 and a lower substrate 130, which are solid-state elements, are stacked with a spacer 125 interposed therebetween is held above a container 140 by a support 143. A small amount of a solution (mother liquor) 126 containing a substance to be crystallized, such as a protein, is injected between the substrate 120 and the substrate 130 from the side thereof by a pipette or the like. The injection of the mother liquor is stopped when the mother liquor drops are stably held without dropping from the gap between the substrates. The bottom of the container 140 is filled with a buffer solution 146 in advance. Buffer solution 146 contains mother liquor 1
26 is used to maintain gas-liquid equilibrium. The container 140 containing the substrate holding the mother liquor 126 together with the buffer solution 146 can be sealed by the cap 141. If the mother liquor 126 is left standing between the substrates in a sealed state, crystals grow in the grooves formed in the substrates. In the apparatus shown in FIG. 12, a desiccant for absorbing a solvent (eg, water) in the mother liquor may be used instead of the buffer solution.

The present invention can be used to crystallize various polymer compounds, especially polymer electrolytes. The present invention is particularly preferably applied for crystallizing proteins such as enzymes and membrane proteins, polypeptides, peptides, polysaccharides, nucleic acids, and complexes and derivatives thereof. The present invention is preferably applied for crystallization of biopolymers.

[0058]

[Example] Lysozyme made from chicken egg white (Lysozyme, From
Chicken Egg White) was dissolved in a solution in which a standard buffer solution at pH = 4.5 and 0.1 M NaCl were mixed at a volume ratio of 10: 1 to give a concentration of 50 mg / ml. The following two types of silicon crystals were used as solid elements for crystallization.

(1) Sample-1 A low-resistance N-type silicon layer was entirely formed on the surface of a P-type silicon substrate having a specific resistance of about 20 Ωcm by ion implantation of phosphorus. Thereafter, according to the process shown in FIG. 5, six types of V-grooves having different sizes were formed. The widths of the V-grooves are 0.1, 0.5, 2.0, 10, 50, 10 respectively.
It was 0 μm. The depth of each V groove was almost the same as its width. The lengths of the V-grooves were all 10 mm, and six types of V-grooves were produced at a pitch of 1.0 mm. further,
A polyimide layer having a thickness of 10 μm was formed so as to surround the six types of V grooves. The obtained silicon substrate was cut into a size of 15 mm in length and 15 mm in width, and used as a solid device. The specific resistance of the N-type silicon layer formed on the surface is about 0.2Ω.
cm and its thickness was about 0.7 μm.

(2) Sample-2 Ion implantation was performed in the same manner as in Sample-1 to form an N-type silicon layer on a P-type silicon substrate. The specific resistance and the thickness of the N-type layer are the same as in Sample-1. on the other hand,
No V-groove was formed on the substrate.

Two sets of silicon crystals having grooves of Sample-1 were prepared, and they were stacked with a glass rod having a diameter of 2 mm and a length of about 5 mm interposed therebetween. A glass rod was placed over the polyimide layer formed on each silicon substrate. The two sets of silicon substrates were arranged such that the longitudinal directions of the opposing grooves intersect vertically.

A stack of silicon substrates via glass rods was accommodated in a container as shown in FIG. A reactive vial having a diameter of about 20 mm was used as a container.
At the top of this vial, the substrate was held on a support so that the substrate surface was horizontal, and about 0.5 ml of the lysozyme mother liquor was injected between the substrates. In addition, about 10 ml of a standard buffer solution of pH = 4.5 was previously injected into the bottom of the vial. Seal vial with cap, 10 ° C
Was stored in a cool dark place.

Sample-2 under the same conditions as Sample-1
The two silicon substrates were opposed to each other to prepare an apparatus for crystallization. The substrate overlapped with the glass rod was held on the top of the reactive vial as in the case of Sample-1, and the mother liquor of lysozyme described above was placed about 0.5 m between the substrates.
1 was injected. In addition, pH = 4.
Five standard buffer solutions had been injected. After the vial was sealed with a cap, it was stored in a cool dark place at 10 ° C.

Each sample was prepared in a cool dark place for 72 hours.
After storage for a time, the stacked silicon substrates were carefully opened, and the lysozyme crystals formed on the surface were observed under a microscope. On the substrate of Sample-1, the size of the precipitated crystal was different depending on the size of the groove. Microcrystals and twins were randomly deposited on the surfaces of the V-grooves having widths of 0.1, 0.5, and 2.0 μm and the N-type silicon layer, as shown in FIG. On the other hand, when the width is 10, 50, 10
In the 0 μm V groove, a large single crystal having a size of about 0.3 mm as shown in FIG. 14 was regularly deposited along the groove.

On the substrate of sample-2, as shown in FIG. 15, a large amount of lysozyme microcrystals or twins was randomly and randomly deposited on the entire surface of the silicon substrate. The average size of the precipitated crystals was about 0.05 mm.

Thus, according to the present invention, it was found that a large-sized and high-quality single crystal can be produced with a small amount of sample by using the apparatus as shown in FIG.

In the above embodiment, an element in which an N-type silicon layer is formed on P-type silicon is used. However, a low-resistance N-type layer is formed on a high-resistance N-type substrate. It is expected that the same result will be obtained even if the groove is formed to expose the high-resistance N-type substrate. That is, it can be expected that precipitates serving as crystal nuclei are selectively aggregated and aggregated in the high-resistance N-type region exposed in the groove, and grow into large crystals.

[0068]

As described above, according to the present invention,
Can either be solved the drawbacks of the conventional crystallization process method can be applied to any materials because it has a variety of characteristics as described above have been conducted by trial and error without. In particular, according to the present invention, the influence of convection of the solution due to gravity can be suppressed, and nuclei can be formed stably in the initial stage of crystallization. Further, according to the present invention, it is possible to suppress or control the mass production of microcrystals, and to obtain a large crystal capable of performing X-ray structural analysis. Further, according to the present invention, by using a solid element provided with a plurality of grooves having different sizes for crystallization,
It can cope with crystallization of all kinds of polymers. By forming a plurality of grooves, it is not necessary to individually process substrates according to the type of polymer to be crystallized, and crystallization can be performed at low cost. Further, by stacking a plurality of solid-state elements and holding a solution containing a molecule to be crystallized in a gap between the solid-state elements, crystallization can be performed on a very small amount of sample.

The present invention is applied to the research, development and production of useful substances, particularly biopolymers such as proteins and nucleic acids, in the pharmaceutical and food industries. According to the present invention, X
Crystals with good crystallinity that enable line structure analysis can be grown. The information obtained as a result of the crystal analysis on the molecular structure and the mechanism of the activity is used for designing and manufacturing a drug. The invention also applies to the production or crystallization of the molecule of interest. Further, the present invention is expected to be applied to the production of electronic devices using biopolymers such as proteins. Also, the device of the present invention can selectively adsorb and immobilize biopolymers and the like,
The present invention can be applied to a biosensor, an apparatus for measuring various biological tissues and biological substances using the biosensor, and the like.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a state in which crystal nuclei are fixed on the surface of a solid-state device and crystal growth proceeds according to the present invention.

FIG. 2 is a perspective view showing a specific example of the crystal growth apparatus of the present invention.

FIG. 3 is a schematic cross-sectional view showing a state in which a space between two solid elements is maintained by a spacer in the apparatus shown in FIG. 2;

FIG. 4 is a sectional view showing a detailed structure of a main part of the device shown in FIG. 3;

FIG. 5 is a schematic cross-sectional view showing a process for manufacturing the crystal growth apparatus of the present invention.

FIG. 6 is a schematic cross-sectional view for explaining how crystal growth proceeds in a groove formed in the solid-state element.

FIG. 7 is a schematic cross-sectional view showing the direction of surface tension and the direction of electrostatic attraction in a solution held between solid elements.

FIG. 8 is a diagram schematically showing a state of crystal growth with and without convection in a solution.

FIG. 9 is a schematic sectional view showing another example of the crystal growth apparatus of the present invention.

FIG. 10 is a schematic sectional view showing another example of the solid-state element used in the present invention.

FIG. 11 is a schematic view showing a specific example of an apparatus for performing the crystal growth method of the present invention.

FIG. 12 is a schematic sectional view showing a specific example of a crystal growth apparatus of the present invention.

FIG. 13 is a micrograph of a crystal structure generated in an example.

FIG. 14 is a micrograph of a crystal structure generated in an example.

FIG. 15 is a micrograph of a crystal structure generated in an example.

FIG. 16 is a schematic view showing an example of an apparatus used in a conventional method.

FIG. 17 is a schematic view showing another example of the device used in the conventional method.

[Description of Signs] 1 solid element 2 crystal nucleus 10, 20 solid element 11a to c, 21a to d V groove 15a, 15b spacer 16 mother liquor 24 water repellent layer

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI B01D 9/02 620 B01D 9/02 620 621 621 625 625 625E 625Z C30B 7/00 C30B 7/00 30/02 30/02 (56) References JP-A-2-18373 (JP, A) JP-A-1-111799 (JP, A) JP-A 8-294601 (JP, A) JP-A 8-34699 (JP, A) JP-A-6 -116098 (JP, A) JP-A-5-319999 (JP, A) Rosenberger, "IN ORGANIC AND PROTEIN CRYSTAR GROWTH-S IMILORITES AND DI FFERENCES", Journal of Crystal Growth, Vol. 76, No. 3, 1986, p. 618-636 (58) Fields investigated (Int. Cl. ⁷ , DB name) C30B 1/00-35/00 B01D 9/02 C07B 63/00 C07H 21/00 C07K 1/14 C08H 1/00 C12N 9/00 CA (STN) JICST file (JOIS)

Claims (7)

(57) [Claims] 1. A method for growing a crystal of a polymer compound contained in a solution, comprising the steps of: controlling the concentration of holes or electrons at a surface portion according to the environment of the solution containing the polymer compound. Providing a plurality of solid elements having two or more grooves or holes, each of which has a controlled depth and / or a different opening width, and surfaces of the plurality of solid elements on which the grooves or holes are formed. Facing each other, a step of holding the plurality of solid state elements by opening a predetermined gap, and holding the solution containing the polymer compound in the gap,
Contacting the solution with the surface of the plurality of solid devices in which the grooves or holes are formed, and in the solid devices, crystallization of the polymer compound is promoted within the outside of the grooves or holes. The valence electrons are controlled such that the crystal of the polymer compound is formed in the groove or the hole holding the solution under an electric state brought to the surface of the solid-state device by the controlled valence electrons. Growing a crystal. 2. The semiconductor device according to claim 1, wherein the solid-state element is a semiconductor substrate to which impurities are added, and a kind and / or a concentration of the impurities in the semiconductor substrate are different between inside and outside of the groove or the hole. The method of claim 1. 3. The method according to claim 1, wherein a distance between the plurality of opposed solid-state devices is continuously reduced or increased.
2. The solid state device is held.
Or the second method. 4. A crystal growth apparatus used in the method according to claim 1, wherein valence electrons are controlled so that the concentration of holes or electrons on a surface portion can be controlled according to the environment of a solution containing the polymer compound.
And a plurality of solid elements having two or more grooves or holes having different depths and / or opening widths, and means for holding the plurality of solid elements facing each other with a predetermined gap therebetween. An apparatus for crystal growth, wherein valence electrons are controlled differently inside and outside the groove or hole. 5. The semiconductor device according to claim 1, wherein the solid-state element is a semiconductor substrate to which impurities are added, and a kind and / or a concentration of the impurities in the semiconductor substrate are different between inside and outside of the groove or the hole. The device of claim 4. 6. A container capable of holding the plurality of solid elements and the means for holding in a sealed state together with a desiccant or a buffer solution, and further comprising means for supporting the solid elements in the container. Apparatus according to claim 4 or 5, characterized in that: 7. The solid state device according to claim 4, wherein the distance between the plurality of solid state devices facing each other is continuously reduced or increased.
The device according to any one of claims 6 to 10.