JP3146985B2 - Crystal growth method and solid state element for crystal growth - 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 polymer compound and a solid-state device used for the method, and more particularly to a method for crystallizing various kinds of proteins such as proteins using a semiconductor substrate or the like in which valence electrons are controlled. The present invention relates to a technique for crystallization of a biopolymer.

[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 disadvantage of the batch method is improved. For example, as shown in FIG.
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. The gas-liquid interphase diffusion method uses, for example, a sample stage 111 such as a cover glass as shown in FIG.
A container 11 on which a droplet 112 of the sample solution is placed
In this method, the droplets and the precipitant solution 114 are put into the liquid crystal 3 to slowly establish an equilibrium by evaporating 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 crystallization conditions. Biological macromolecules are stabilized in a solvent by the interaction between individual molecular surfaces, while the charge distribution on the molecular surface, especially the conformation of amino acids near the molecular surface, depends on the environment, that is, the pH of the solution, 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.

As described above, crystallization of proteins and other biomacromolecules and their complexes is an important process in science and industry, but has been carried out through trial and error until now. 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.

[0012]

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.

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

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

[0015]

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. A step of providing a solid element 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; And holding the solution in the grooves or holes by contacting the solution with the solution. In the solid-state device, valence electrons are controlled such 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.

A semiconductor substrate doped with impurities can be used as the solid-state element. 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 is different between the inside and the outside of the groove or the hole in order to promote the crystallization of the polymer compound inside the outside of the groove or the hole. Can be closed.

The present invention provides a solid state device for controlling crystal growth. The solid state device has two or more grooves or holes having different depths and / or opening widths. In this solid-state device, the valence electrons are controlled so that the concentration of holes or electrons in the surface portion can be controlled according to the environment of the solution containing the substance to be crystallized, and the valence is different between inside and outside the groove or hole. The electronics are controlled.

As such a solid-state element, a semiconductor substrate doped with impurities as described above can be used.
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.

[0020]

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 unclear 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 crystal nuclei, 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 substantially isotropically exerted on molecules 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.

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.

[0026] 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 and 3 schematically show the structure of the solid state element for crystal growth according to the present invention. FIG. 2 is directed to crystallization of a polymer compound having a negative effective surface charge after dissociation in a solution. In the device, an N-type silicon layer 11 is formed on a surface of a P-type silicon substrate 10. Further, a plurality of V-shaped grooves (V grooves) 12 are provided on the surface.
a, 12b, 12c and 12d are formed. The depth and the width of the opening in each V-groove are different. FIG. 3 also targets crystallization of a polymer compound having similar dissociation characteristics. The surface of the P-type silicon substrate 20 has N
A mold silicon layer 21 is formed. A plurality of concave grooves 22a, 22b and 22c are formed on the surface. The width of the opening in each groove is different. The groove 22a is a well-shaped or prism-shaped groove whose depth is significantly longer than the width of the opening. Grooves 22b and 22
c has a stepped inner wall. The width of the opening becomes narrower toward the depth of the groove. As described above, it is more preferable that the width of the opening becomes narrower toward the deeper part of the groove. Note that for a polymer compound having dissociation characteristics opposite to the above, the polarity of the silicon substrate may be reversed from that shown in FIG. That is, when the purpose is to crystallize a polymer compound having a positive effective charge in a solution, an element in which a P-type silicon layer is formed on an N-type silicon substrate can be used. In any of the devices, the substrate surface is exposed inside the groove. That is, in the case of the element shown in FIG. 2 or FIG. 3, an N-type silicon layer is formed at a portion outside the groove and in contact with the solution, while an inner wall of the groove is formed by a P-type silicon substrate. Similarly, when a P-type silicon layer is formed on an N-type silicon substrate, the inner wall of the groove is formed of the N-type silicon substrate, and the P-type silicon layer is provided on a portion outside the groove that comes into contact with the solution.

FIG. 4 schematically shows a manufacturing process of the device shown in FIG. First, a P-type silicon substrate 30 whose surface is cleaned and, for example, mirror-polished is prepared (FIG. 4A). Next, an N-type silicon layer 31 is formed on the surface of the P-type silicon substrate 30 by a method such as ion implantation (FIG. 4B). Thereafter, a silicon oxide film (SiO ₂ ) 33 is formed by a method such as CVD.
(C)). Next, after performing an appropriate method such as lithography, the silicon oxide film in the region where the groove is to be formed is removed (FIG. 4D). If the portions not covered with the oxide film are etched, the grooves 32a, 31b,
32c and 32d are formed (FIG. 4E). Next, by removing the silicon oxide film, a necessary solid-state element is obtained (FIG. 4F). 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.

FIG. 5 schematically shows a manufacturing process of the solid-state device shown in FIG. First, a P-type silicon substrate 40 is prepared (FIG. 5A). Next, an N-type silicon layer 41 is formed on the substrate 40 by a method such as ion implantation.
(B)). After performing appropriate lithography and the like, a groove 42a is formed in a predetermined portion by dry etching (FIG. 5C). The portion where the opening of the groove is desired to be widened is further subjected to dry etching to form a plurality of grooves 42 b and 42 b that are anisotropically deep and have different opening areas.
c is formed (FIGS. 5D and 5E).

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 impurities on the silicon surface. 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 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 which can easily control valence electrons has been described above, other materials having the same function can be appropriately used 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 V-groove, and the device shown in FIG. 3 has a concave groove. Instead of the grooves, for example, pyramidal or conical holes may be provided on the surface of the solid-state device. It is more preferable that the width of the opening of these grooves or holes 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.

The solid-state device used for crystallization is desirably applicable 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.

The size of the opening of the groove or the hole formed on the substrate surface and the depth of the groove or the hole are desirably set as appropriate in accordance with the kind of the target polymer. Generally, the width of the opening of the groove or hole is 0.01
１００100 μm is preferable, and the groove length is 1-10 m
The range of m is preferred. In addition, the plurality of grooves or holes are 1 μm.
It can be appropriately prepared at intervals of m to 1 mm.
Preferably, the depth of the groove or hole is adjusted, for example, in the range of 0.01 to 200 μm. 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 and 7 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 immersed in an electrolyte solution containing molecules to be crystallized, the V-groove portion 52 made of P or P ^- silicon is compared with the surface portion 51 made of an N or N ⁺ silicon layer. Therefore, it is expected that the range (which may be considered as the width of the electric double layer) that the electrostatic interaction with the dissociated polymer reaches becomes wide. In the figure, an area 54 indicated by a dotted line
Is the range of electrical interaction. That is, the region 54 is thicker in the V-groove portion 52 than on the surface portion 51. In particular, it is expected that the width of the region 54 will be the widest at the deepest central portion of the V-groove portion 52 due to the overlapping of the regions to which this effect is exerted.

Therefore, at the deepest part of the V-groove portion 52 made of P or P ^- silicon, the crystal nucleus or the molecular aggregate 55 serving as a crystal nucleus receives an electrostatic attraction from the V-groove surface almost isotropically, Within. V
Regarding the molecular aggregate 55 in the deep part of the groove, the influence of convection in the solution due to gravity is suppressed by electrostatic attraction,
It is expected that generation of crystal nuclei and crystal growth can occur stably. On the other hand, on the surface portion 51 made of N or N ⁺ silicon, the formation of crystal nuclei is suppressed as described above. Also, even if crystal nuclei are formed on this surface, the width of the diffusion supply layer near the crystal nuclei fluctuates under the influence of convection in the solution, resulting in a decrease in crystallinity or a decrease in growth rate. It is considered to be. Therefore, crystal growth proceeds selectively in the groove 52, and a large crystal is obtained.

It is considered that stable crystal growth occurs on the solid-state device shown in FIG. 7 by the same mechanism. That is, in the portion where the opening width of the groove 62 is the narrowest, the crystal nucleus 65 is generated and is restrained by the electrostatic attraction. The region 64 to which the electrostatic interaction is exerted has the largest width at the deepest part of the groove 62. On the other hand, the substrate 60
On the N or N ⁺ silicon layer 61 formed thereon, the generation of crystals is suppressed as described above. Therefore, a crystal nucleus is generated in the deep portion of the groove 62, and the crystal grows.

FIG. 8 shows a more specific apparatus for carrying out the present invention. In the apparatus shown in FIG. 8, a buffer solution 72 is contained in a container 71, and a dialysis membrane tube 73 is provided therein. In the dialysis membrane tube 73, together with a mother liquor 72 containing a polymer compound, a solid element for crystal growth, for example, a silicon semiconductor substrate 75 as described above is accommodated. The dialysis membrane tube 73 is sealed with the closers 75 a and 75 b and immersed in the buffer solution 72. Container 7
The opening 4 is covered by a lid 76. In this device, dialysis is advanced and the silicon semiconductor substrate 7
Polymer crystals are precipitated from the mother liquor 74 on the surface 5. 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.

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.

[0044]

[Example] Lysozyme made from chicken egg white (Lysozyme, From
Chicken Egg White) was dissolved in a standard buffer solution at pH = 6.8 to a concentration of 20 mg / ml. 5m of the solution
was sealed together with silicon crystals in a dialysis tube that had been sufficiently boiled and washed. The following two types of silicon crystals were used, and lysozyme was crystallized in an apparatus as shown in FIG.

(1) Sample-1 A sample in which a low-resistance N-type silicon layer is entirely formed on the surface of a P-type silicon substrate having a specific resistance of about 20 Ωcm by ion implantation of phosphorus. The specific resistance of the formed N-type silicon layer was about 0.1 Ωcm, and its thickness was about 0.5 μm. This silicon substrate was cut into a size of 5 mm long and 10 mm wide and used for crystallization.

(2) Sample-2 Using a silicon substrate on which an N-type silicon layer was formed in the same manner as in Sample-1, processing was performed by the method shown in FIG. 4 to form a V-groove. 1. 0.8 and 2.
Five types of V-grooves having the same width of 0, 10, 50, 100 μm, length 5 mm, and depth were formed on the silicon substrate at a pitch of 0.5 mm. The silicon substrate on which the V-groove was formed was cut into a size of 5 mm in length and 10 mm in width, and used as a solid state element for crystallization. In this solid-state element, the P-type silicon is exposed in the portion where the V-groove is formed as described above.

The silicon crystals (1) and (2) were placed in a dialysis tube containing lysozyme, and in a device as shown in FIG. 8, the dialysis tube was filled with 200 ml of a standard buffer solution of pH = 4.5 and 1M of 1M. It was immersed in an aqueous solution mixed with 20 ml of NaCl, and stored in a cold and dark place at 10 ° C. After being stored in a cool and dark place for 96 hours, a sample was taken out, and lysozyme crystals formed on the silicon crystal were observed with a microscope.

On sample-1, as shown in FIG.
A large amount of microcrystals or twins of lysozyme were randomly deposited on the entire surface of the silicon substrate. The average size of the precipitated crystals was 0.05 mm.

On the other hand, on Sample-2, the size of the precipitated crystal was different depending on the size of the groove. The width is 0.
In the V-groove portions having the sizes of 8, 2.0 μm, as shown in FIG. 10, crystals of the same form as Sample-1 were randomly deposited. On the other hand, in a V-groove having a width of 10, 50, or 100 μm, as shown in FIG.
Large single crystals were regularly deposited along the grooves. The formation of large single crystals in the V-groove portion as described above suppresses the generation of crystals on the N-type silicon layer, while selectively depositing crystal nuclei in the V-groove portion of P-type silicon. -It is considered that they aggregated and this grew to a large crystal.

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.

[0051]

As described above, according to the present invention,
It is possible to overcome 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.

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 purification 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.

[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 schematic sectional view showing a specific example of the solid-state device of the present invention.

FIG. 3 is a schematic cross-sectional view showing another specific example of the solid-state device of the present invention.

FIG. 4 is a schematic sectional view showing a process for manufacturing the solid-state device of FIG.

FIG. 5 is a schematic cross-sectional view showing a process for manufacturing the solid-state device of FIG.

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 for explaining how crystal growth proceeds in a groove formed in the solid-state element.

FIG. 8 is a schematic diagram showing one specific example of an apparatus for performing the crystal growth method of the present invention.

FIG. 9 is a micrograph of a crystal structure generated in an example.

FIG. 10 is a micrograph of a crystal structure generated in an example.

FIG. 11 is a micrograph of a crystal structure generated in an example.

FIG. 12 is a schematic diagram illustrating an example of an apparatus used in a conventional method.

FIG. 13 is a schematic view showing another example of the device used in the conventional method.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Solid element 2 Crystal nucleus 10, 20, 30, 40 P-type silicon substrate 11, 21, 31, 41 N-type silicon layer 12a, 12b, 12c, 12d, 32a, 32b, 3
2c, 32d V-groove 22a, 22b, 22c, 42a, 42b, 42c Recessed groove

──────────────────────────────────────────────────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 (4)

(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 solid device having two or more grooves or holes having different controlled depths and / or opening widths; and contacting the solid device with the solution containing the polymer compound, Holding the solution in a groove or a hole, wherein in the solid-state device, the valence electrons are controlled so that crystallization of the polymer compound is promoted within the outside of the groove or the hole, In the groove or hole holding the solution, growing a crystal of the polymer compound under an electric state brought to the surface of the solid state device by the controlled valence electrons, Crystal growth method. 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. A solid-state element for crystal growth used in the method according to claim 1, comprising at least two grooves or holes having different depths and / or opening widths, and a solution containing a polymer compound. Valence electrons are controlled so that the concentration of holes or electrons on the surface can be controlled according to the environment of the
A solid state element for crystal growth, wherein the valence electrons are controlled differently inside and outside the groove or hole. 4. The solid-state element is a semiconductor substrate to which an impurity is added, and the kind and / or concentration of the impurity in the semiconductor substrate is different between inside and outside the groove or the hole. The solid-state device for crystal growth according to claim 3.