JP3146984B2 - Crystal growth method, crystal growth solid state element 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 technique for crystallizing a high molecular compound, and more particularly, to a method for crystallizing various high molecular compounds such as proteins using a semiconductor substrate or the like in which valence electrons are controlled. The present invention relates to a technique for performing crystallization.

[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 a basic biochemical point of view,
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 in the future mainly by X-ray crystallography.

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 and apparatus that can be applied to any substance to ensure crystallization. Therefore, the reality is that crystallization is 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 subjecting water or a non-aqueous solution containing the macromolecules to a solvent as in the case of ordinary low molecular weight compounds such as inorganic salts. Basically, growing a crystal. Representative methods for this are (1) batch method, (2) dialysis method, and (3) gas-liquid correlation diffusion method,
They are used differently depending on the type, amount, and properties 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, ammonium sulfate (ammonium sulfate) is often used. This method requires a large amount of solution sample, salt concentration,
It has the drawback that it is difficult to finely adjust the pH, and that the operation requires skill and the reproducibility is low. The dialysis method is
In a method in which the disadvantages of the batch method are improved, for example, as shown in FIG. 14, a solution 102 containing a biopolymer is sealed inside a dialysis tube 101, and the pH of an external solution 103 (eg, a buffer solution) of the dialysis tube is continuously adjusted. This is a method in which crystallization is performed by changing the temperature. According to this method, the salt concentration of the inner and outer liquids,
Since the difference in pH can be adjusted at an arbitrary rate, it is easy to find the conditions for crystallization. The gas-liquid interphase diffusion method is, for example, shown in FIG.
As shown in FIG. 5, on a sample stage 111 such as a cover glass,
In this method, a droplet 112 of a sample solution is placed, and the droplet and the precipitant solution 114 are placed in a closed container 113, whereby a gradual equilibrium is established by evaporation of volatile components between the two.

However, crystallization of biological macromolecules such as proteins has various problems as described above.

First, according to the conventional method, the obtained substance has poor crystallinity, and it is difficult to obtain a large single crystal.
This is because biopolymers generally have a large molecular weight,
It is susceptible to gravity and is believed to be caused by convection in solution (eg, F. Rosenb
erger, J. Cryst. Growth, 76, 618 (1986)). That is, the biopolymer and the generated fine crystal nuclei settle under their own weight, which causes convection of the solution around the molecules and nuclei. In addition, local convection of the solution occurs at the surface of the generated crystal because the concentration of the molecule is reduced. Due to the convection of the solution thus generated, the 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 growth rate of the crystal is reduced or the anisotropy of the growth on the crystal plane is generated, so that the crystallization is prevented.

Also, unlike the crystals of other substances, the biopolymer crystal contains a large amount of solvent (mainly water) (≧ 50% by volume). This solvent can easily move in the disordered and intermolecular voids in the crystal. In addition, despite the large size of the molecule, there is almost no packing contact between a wide range of molecules in the crystal, and there are only a few molecule-to-molecule contacts or contacts by hydrogen bonding via water molecules. These are factors that hinder crystallization.

Furthermore, 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 involving complicated various conditions, and a unified method applicable to any substance has not been established.
For proteins, compared to water-soluble proteins,
Despite its very important biochemistry, crystallization of hydrophobic membrane proteins is currently very difficult, and only two cases have been successfully crystallized and analyzed at higher resolution. It is.

Furthermore, the biopolymer obtained is often very small. Proteins such as enzymes are generally extracted and purified from cells and the like, but the content of the proteins is small, so that the final sample is often very small. When performing crystallization, the concentration of the solution containing the biopolymer is 50 mg.
It is said that about / ml is required. For this reason, it is necessary to prepare the solution so that the solution becomes as small as possible, and to repeat the crystallization experiment (screening) under various conditions.

As described above, crystallization of proteins and other biomacromolecules is an important process in academic and industrial fields, but has been carried out through trial and error until now. For this reason, the crystallization process is 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 disadvantage of the chemical process is to eliminate it.

Specifically, an object of the present invention is to reduce the influence of convection in a solution due to the influence of gravity on the crystallization of a biological tissue mainly composed of various biopolymers and biopolymers. It is to provide a technique for controlling the formation.

Still another object of the present invention is to provide a technique capable of 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 technique for enabling crystallization with a small amount of a biopolymer solution.

[0017]

A crystal growth method according to the present invention is a method for growing a crystal of a polymer compound contained in a solution. The method for growing a crystal of a surface portion according to the environment of a solution containing a polymer compound. The method includes a step of providing a solid element in which valence electrons are controlled so that the concentration of holes or electrons can be controlled, and a step of bringing the solid element into contact with a solution containing a polymer compound. The solid state element has different depths and / or different widths of openings formed on one surface side of two opposing main surfaces.
It has one or more grooves or holes and a through hole for supplying a solution containing a polymer compound to the grooves or holes from the other surface side of the two main surfaces. The valence electrons are controlled so that the crystallization of the polymer compound is promoted in the portion of the solid device that comes into contact with the solution, outside the groove or the hole. Using such a solid-state element, a solution containing a polymer compound is supplied to a downward groove or hole through a through hole. As a result, the solution is held in the groove or the hole in a state where the droplet of the solution hangs down in the direction of gravity on the solid device. In this way, in the groove or hole holding the solution, the crystal of the polymer compound is grown under the electric state brought to the surface of the solid-state device by the controlled valence electrons.

In the present invention, the solid state element may be a semiconductor substrate doped with impurities. The type and / or concentration of impurities in the semiconductor substrate can be different between inside and outside of a groove or hole formed in the semiconductor substrate.

Further, a water-repellent layer can be provided on the solid element so as to surround the plurality of formed grooves or holes.

In the present invention, the growth of crystals in the grooves or pores holding the solution depends on the presence of a precipitant absorbing the vapor of the solvent contained in the solution or of a buffer solution for maintaining the gas-liquid equilibrium of the solution. Can be done below.

The present invention provides a solid-state device for use in crystal growth. In this solid-state device, valence electrons are controlled so that the concentration of holes or electrons on the surface portion can be controlled according to the environment of the solution containing the substance to be crystallized.
The solid state device has two opposing main surfaces, and two or more grooves or holes formed on one surface side of the two main surfaces and having different depths and / or opening widths; Said 2
And a through hole for supplying a solution containing a polymer compound to the groove or hole from the other surface side of the one main surface. The valence electrons are controlled so that the crystallization of the polymer compound is promoted in the portion of the solid device to be brought into contact with the solution, rather than outside the groove or hole.

Such a solid-state device can be a semiconductor substrate doped with impurities. A plurality of grooves or holes having different sizes are formed in a semiconductor substrate, and the type and / or concentration of impurities in the semiconductor substrate differs between inside and outside of the grooves or holes.

Further, a water-repellent layer can be provided on such a solid element so as to surround a plurality of grooves or holes.

The present invention further provides an apparatus for growing a crystal, the apparatus comprising a solid element as described above, means for supporting the solid element, and the solid element and the means contained in a sealed state together with a precipitant or a buffer solution. And a container that can be used.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION Most of biomolecules such as proteins are geometrically specific in a solution and have an electrostatic interaction (electrostatic repulsion / attractive force, van der Waals force). Recognition between molecules is performed. In the interaction between molecules based on electrostatic energy, a 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. Is expected to have a significant effect. It is considered that individual biopolymers that repeatedly collide while performing Brownian motion in a solution are unlikely to become nuclei of a molecular assembly having a periodic regular structure. Furthermore, even if a crystal nucleus is formed, if the structure and charge distribution of each molecule surface are not exactly the same and have redundancy, the molecules assembled around the nucleus will loosely bind to each other, It is considered that the crystallinity decreases.

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 the report, in order for crystallization to proceed, it is essential that each molecule take a two-dimensional regular structure (mesh, star, staggered, 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. In general, protein molecules have weak intermolecular interaction and are hardly aggregated because the molecular surface is locally charged. In view of this, it is considered that, if some conditions are provided for arranging the molecules serving as nuclei in a two-dimensional manner in the initial stage of crystallization, the subsequent crystallization proceeds epitaxially using the nuclei as nuclei.

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 compound 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. Properties 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. In the present invention, FIG.
As shown in (a), 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. 1 (b), the compound such as a protein aggregates on the surface of the solid-state device by electrostatic interaction, and the generation of crystal nuclei is promoted.
Crystal growth results. 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.
Further, since the generated crystal nuclei are fixed on the surface of the solid-state element, minute fluctuations of the nuclei due to convection in the solution are suppressed,
It is also expected that the molecules are regularly assembled according to the nucleus formation and the crystallinity is improved. 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.

In general, the cohesiveness of a charged substance or molecule in an electrolyte solution depends on the sum of an electric double layer repulsive force and a Van der Waals force between them. It is very important to control the salt concentration for adjusting the surface potential added to the solution. On the other hand, according to the present invention, the surface potential based on the space charge layer induced on the surface of the solid-state device is:
For example, it changes in proportion to the concentration of an impurity added for controlling valence electrons. Therefore, for example, by adjusting the impurity concentration in the solid state element, it is possible to previously adjust the electrostatic characteristics brought to the element surface. Therefore, in the present invention, it is not necessary to adjust the salt concentration in the electrolyte solution, or even if it is adjusted, simple adjustment is sufficient.

As will be described later, a region where the electrostatic interaction is isotropic can be provided at the bottom of the groove formed in the solid-state device. In such a region, the crystal nucleus can be fixed by electrostatic interaction, and the movement of the crystal nucleus by convection based on the influence of gravity can be suppressed. As a result, the crystal can grow stationary at the bottom of the trench. Therefore, it is expected that generation of excessive microcrystals is suppressed, and large crystals having good crystallinity in which molecules are regularly gathered on the surface of crystal nuclei can be obtained.

Further, according to the present invention, the width of the opening and / or
Alternatively, a plurality of grooves or holes having different depths are formed in the solid-state element. It is believed that the appropriate groove or pore size will depend on the size of the molecule to be crystallized, charging characteristics, and the like. Therefore, if a plurality of grooves or holes having different sizes are previously formed on the solid-state element, it is considered that more appropriate crystallization conditions can be realized in any of the grooves or holes. Even if the molecular species to be crystallized is changed, grooves or holes suitable for the crystallization conditions must exist, and it is considered that one solid-state device can be applied to crystallization of various molecules.

In the present invention, the through holes are formed in the solid state device. Assuming that the surface of the solid element on which the grooves or holes are formed is the front surface, and the surface opposite thereto is the back surface, the front surface and the back surface of the solid device communicate with each other by through holes. FIG. 2A shows an example of a solid state device in which a through hole is formed. When the mother liquor containing the compound to be crystallized is injected into the through-hole 6 from the back surface 5b side of the solid-state element 5 having the through-hole 6, droplets 7 of the mother liquor are easily formed on the surface 5a by the action of surface tension. Can be. Through the through holes, a small amount of solution can be held on the surface of the solid-state element. As will be described later, if crystallization is performed in such a droplet, a very small amount of sample is required for crystallization. In this manner, if the liquid is suspended in the direction of gravity, a small amount of droplets can be easily held. On the other hand, as shown in FIG. 2B, when the mother liquor is dropped onto the solid element 5 'to form a droplet, an appropriate droplet is formed due to the wettability of the surface of the solid element with the mother liquor. Or not. Droplets are less likely to form on highly wettable surfaces.
Also, it is difficult to form a droplet that hangs in the direction of gravity without passing through the through hole. For example, as shown in FIG. 2C, first, the droplet 7 is placed on the surface of the solid-state element 5 '.
It is conceivable that the liquid droplet 7 is made to hang down in the direction of gravity by turning over this. However, in this case, the liquid often flows on the solid-state element 5 ', and cannot hold the droplet in many cases. Further, as shown in FIG. 2D, it is conceivable to form droplets on the surface of the turned-down solid-state element 5 ′ by using the spoiler 9. However, in this case, the liquid often falls down, and it is difficult to hold a prescribed amount of droplets on the substrate. As mentioned above, the through holes facilitate the formation of droplets that hang in the direction of gravity.

The solid used for crystallization in the present invention is a substance having the above-mentioned electrostatic properties and capable of controlling the amount of charge and polarity, and a substance which is chemically stable in a solution. Anything is fine,
One of the preferable materials is silicon which is a semiconductor 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.

FIG. 3 shows a specific example of the solid-state device of the present invention. FIG. 3A is a schematic sectional view, and FIG. 3B is a schematic plan view. The purpose of this solid-state device is to crystallize a polymer compound having a negative effective surface charge after dissociation in a solution. In the solid-state device, an N-type silicon layer 11 is formed on a surface of a P-type silicon substrate 10. A plurality of V-shaped grooves (V-grooves) having different widths and depths of the openings are formed on the front side 10 a of the P-type silicon substrate 10.
12a, 12b, 12c, 12d and 12e are formed. V-grooves 12c are formed at substantially the center of the silicon substrate 10.
A through-hole 16 is formed between the substrate and the substrate 12d so that the liquid can be supplied from the back surface 10b to the front surface 10a of the substrate. The opening on the back side of the through hole 16 is wider than the opening on the front side. Further, a water-repellent layer 17 made of a water-repellent resin such as polyimide is formed on the front side 10a of the silicon substrate 10 so as to surround the V grooves 12a to 12e.

FIG. 4 shows another example of the solid-state device of the present invention. This solid-state element is also intended to crystallize a polymer compound having a negative effective surface charge after dissociation in a solution. An N-type silicon layer 21 is formed on the surface of the P-type silicon substrate 20. Further, a plurality of concave grooves 22a, 22b, 22c, 22d and 22e are formed on the front side 20a. The groove 22a is a well-shaped or prism-shaped groove whose depth is longer than the width of the opening. on the other hand,
The grooves 22b to 22e have stepped inner walls. In these grooves, the width of the opening becomes smaller as going deeper. As described above, it is more preferable that the opening of the groove or the hole formed in the solid element becomes narrower as it goes deeper. Groove 2 at the center of silicon substrate 20
A through hole 26 is formed between 2c and 22d. The opening on the back side 20b in the through hole 26 is the front side 20a.
It is wider than the opening. On the front side 20a of the silicon substrate 20, a water-repellent layer 27 made of a water-repellent resin is formed so as to surround five more grooves.

In the solid-state device described above, P
While the mold silicon is exposed, the surface outside the trench is covered by an N-type silicon layer. In such a solid-state device, a solution sample is supplied to a surface region where a plurality of grooves are formed and covered with a water-repellent resin. Note that when dissociating in a solution to crystallize a compound having a positive effective charge, it is preferable to reverse the polarities of the silicon substrate and the silicon layer in the above-described solid-state element. That is, it is desirable to form a P-type silicon layer on an N-type silicon substrate. In this case, the N-type silicon substrate is exposed in the groove, and the surface outside the groove is covered with the P-type silicon layer.

FIG. 5 is a schematic sectional view showing a process for manufacturing the solid-state device shown in FIG. First, for example, a mirror-polished P-type silicon substrate 30 is prepared (FIG. 5).
(A)). Next, an N-type silicon layer 31 is formed on the P-type silicon substrate 30 by a method such as ion implantation (FIG. 5B). Thereafter, a silicon oxide film (SiO ₂ ) 33 is formed by a method such as CVD (FIG. 5C). After an appropriate photolithography step or the like, the silicon oxide film in the region where the groove is to be formed is removed (FIG. 5D). Next, the N-type silicon layer and the P-type silicon substrate in the portion where the silicon oxide film has been removed are etched to form V-grooves 32a, 32b, 32c, 32d and 32e having different sizes (FIG. 5E). Next, the silicon oxide film is removed (FIG. 5F). Thereafter, through holes 36 are provided in the substrate 30 by anisotropic etching using a KOH solution or the like (FIG. 5G). Next, a water-repellent layer 37 made of a water-repellent resin such as polyimide is formed so as to surround the groove.
(H)). Although not shown, after removing the silicon oxide film, a silicon oxide film may be formed again on the surface of the solid-state device. Further, the through holes may be formed in advance at the beginning of the process.

FIG. 6 shows a process for manufacturing the solid-state device shown in FIG. First, a P-type silicon substrate 40
Is prepared (FIG. 6A), and an N-type silicon layer 41 is formed by a method such as ion implantation (FIG. 6B). After an appropriate photolithography step or the like, a groove 42a having a high aspect ratio is formed by dry etching (FIG. 6).
(C)). Further, by repeating the dry etching, the concave grooves 42 which are anisotropically deep and have different opening widths are formed.
b, 42c, 42d and 42e (FIG. 6)
(D) and (e)). Next, through holes 46 are formed in the substrate 40 by anisotropic etching using a KOH solution (FIG. 6F). Thereafter, a water-repellent layer 47 made of a water-repellent resin such as polyimide is formed so as to surround the groove (FIG. 6G).

Hereinafter, the mechanism of crystal growth in the solid-state device of the present invention will be described in more detail. When an electrolyte solution containing a polymer compound having dissociated and having a negative effective surface charge is brought into contact with a valence-controlled N-type 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 to the negatively charged molecules from the bulk silicon side (ohmic characteristics), and thus it is expected that the polymer compound will continue to aggregate on the silicon surface. On the other hand, a surface potential depending on the electrolyte concentration of the 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 impurity concentration contained in the N-type silicon. Therefore, on the N-type silicon surface, it is expected that the polymer compound having a negative charge in the solution will continue to aggregate on the silicon surface until at least compensating for the positive space charge of the N-type silicon. Therefore, while aggregation and crystallization of molecules are restricted on the N-type silicon surface where the space charge layer region is formed, the molecular aggregation and crystallization are restricted on the P-type silicon surface where the ohmic contact is formed. Is expected to proceed indefinitely.

On the other hand, if the impurity concentration in N-type silicon is different, the degree of crystallization is considered to be different as described below. The difference between N-type silicon having a low impurity concentration and high resistance and N-type silicon having a high impurity concentration and low resistance will be described below. In N-type silicon with a low impurity concentration (high resistance), the width of the space charge layer formed near the surface is increased, so that the capacity of the depletion layer is small, and therefore, compared to the case where the impurity concentration is high (low resistance). It is expected that a large surface potential will be induced. Since the polarity of this surface potential is opposite to the effective surface potential of the polymer compound, aggregation of molecules is promoted by the action of electrostatic attraction. In other words, the N-type silicon substrate having a low impurity concentration and a high resistance causes more crystals to precipitate for molecules having a negative effective charge in the solution than the N-type silicon substrate having a high impurity concentration and a low resistance. Expected to have an effect.

Although the above description has been made with respect to molecules having a negative effective surface charge in a solution, similar effects can be expected with respect to molecules having a positive effective surface charge with respect to silicon having a polarity opposite to that described above. Can be.

As described above, regions having different resistances can be easily formed by selectively doping the silicon surface with impurities. Further, by etching the silicon surface, the silicon surfaces having different resistance values can be exposed.

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 an ordinary LSI process. The specific resistance of the silicon crystal may be in the range of about 0.0001 to 1000 Ωcm, and more preferably in the range of 0.001 to 100 Ωcm. The surface of the crystal is preferably mirror-polished for controlling crystal nuclei to be precipitated.

In the present invention, when a layer containing impurities is formed on the surface of the silicon substrate, the thickness of the layer is preferably 0.1 to 200 μm, more preferably 1 to 200 μm.
The range is 50 μm. Outside of this range, the fabrication is not easy or ineffective, which is not desirable.

Various methods are available for preparing N-type and P-type valence-controlled silicon used in the present invention, and any method may be used.
An ion implantation method is the simplest and most accurate method of controlling the impurity concentration. In the case of this method, P-type and N-type valence electron control are respectively performed in the periodic table III.
This can be easily performed by implanting ions of elements belonging to the group V and group V into silicon and annealing them. B, Al, as a group III element for forming a P-type
Ga, In, Tl, etc. can be mentioned, and B is particularly common. N, P, as Group V elements for N-type
As, Sb, Bi and the like can be mentioned, and especially P, A
s and Sb are common.

In the above, an example using semiconductor crystal silicon which can easily control valence electrons has been described. However, in order to achieve the object, another semiconductor or another substance having the same function can be used as appropriate. In addition, a substance other than a semiconductor crystal can be used as long as the substance is stable in a solution. For example, an inorganic compound, an organic compound, an organic polymer, or the like whose charge distribution is controlled are cited as candidates for a solid-state element for crystallization. be able to.

In the present invention, a plurality of grooves or holes are formed in the solid-state device. The element shown in FIG. 3 has a V groove, and the element shown in FIG. 4 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 element having a groove or a hole for each target polymer compound is prepared, it is costly and time-consuming, and is not efficient. Therefore, if a plurality of grooves or holes having different sizes are previously formed on the solid-state element, even if the target molecular species changes, more preferable crystallization conditions should 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 and depth of the opening of the groove or hole formed on the surface of the solid element can be appropriately set in a suitable range depending on the kind of the polymer to be treated. Generally, the width of the opening is preferably in the range of 0.01 to 100 μm, and the length is preferably in the range of 1 to 10 mm. Also,
The plurality of grooves or holes can be appropriately formed at intervals of 1 μm to 1 mm. The depth of the groove or hole is preferably 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. 7 and 8 are for explaining the effect of the grooves or holes on the crystal growth. As shown in FIG. 7, when the solid-state device is brought into contact with an electrolyte solution containing a molecule 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 on 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 the crystal nucleus receives an electrostatic attraction 58 from the V-groove surface almost isotropically. It will be restrained in the groove. Regarding the molecular aggregate 55 in the deep part of the V-groove, the influence of the convection 59 in the solution due to gravity and the electrostatic attraction 58 cancel each other, so that it is expected that the generation of crystal nuclei and the growth of crystals can occur stably. You. 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 due to the influence of convection 59 in the solution. It is thought to be brought. 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. 8 by the same mechanism. That is, the crystal nucleus 65 is generated at the portion where the opening width of the groove 62 is the narrowest, and is restrained by the electrostatic attraction 68. The region 64 to which the electrostatic interaction reaches is formed by the groove 62.
Is the widest at the deepest part. On the other hand, on the N or N ⁺ silicon layer 61 formed on the substrate 60, 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 stably.

In the present invention, a water-repellent layer is formed on the surface of the solid element so as to surround a plurality of grooves or holes. This layer is for preventing the droplets of the solution to be held on the solid state element from flowing out to the surroundings. For example, a silicon surface from which an oxide film on the surface has been removed is generally water-repellent to water or pure water containing only an acid or alkali, but not to an aqueous solution containing a salt such as a buffer solution. , Loses water repellency. Therefore, in order to stably hold the droplet of the aqueous solution containing the salt, it is desirable to form a layer made of a water-repellent substance around the region where the droplet is to be held. In order to achieve such an object, an organic resin can be used as the water-repellent material. One of the materials that can be used most easily is, for example, polyimide. For example, after coating a photosensitive or non-photosensitive polyimide resin and curing, an unnecessary coating portion is removed by etching or development to form a desired pattern to form a water-repellent layer on the solid-state device. be able to.

The thickness of the water-repellent layer used in the present invention does not need to be particularly limited in terms of function, but it can be set to a range of 0.1 to 100 μm as a thickness that can be easily produced.
The material is not limited to polyimide as long as it shows water repellency and is chemically stable to the solution. Further, when a solution having water repellency to a solid element such as silicon is used, such a water repellent layer is unnecessary.

FIG. 9 shows a specific example of the crystal growth apparatus of the present invention. In the device shown in FIG. 9, the solid state device 100 is supported by a support base 90 and housed in a container 91. The opening of the container 91 can be closed by a lid 92. In such an apparatus, a mother liquor droplet 88 containing molecules to be crystallized is injected from a through-hole 86 and held on the solid-state element 100 so as to hang in the direction of gravity. At the bottom of the container 91 is a precipitant capable of absorbing the solvent vapor in the droplet 88, for example, water in the case of an aqueous solution, to supersaturate the molecules in the droplet, or the vapor-liquid equilibrium for the droplet. A buffer solution 93 for holding is stored. When the container 91 containing the precipitant or the buffer solution is closed by the lid 92, crystallization in the droplet 88 is promoted.

FIG. 10 is an enlarged view showing how the droplet 88 is held on the solid-state device 100. Above the precipitant or buffer solution 93 contained in the container, the solid state element 100 is held by the support base 90. Solid state device 10
A silicon layer (for example, an N-type silicon layer) 81 having a different type or concentration of impurities is formed on a substrate (for example, a P-type silicon substrate) 80 constituting 0. Also,
On the substrate 80, a plurality of V grooves 82a, 82b, 82c,
82d, 82e and 82f are formed. A water-repellent layer (for example, a layer made of a water-repellent resin) 87 is formed so as to surround these grooves. At a substantially central portion of the substrate 80, a through hole 86 for injecting a mother liquor is provided. The solid state element 100 is held on the support base 90 with the V-groove facing downward. When a small amount of a solution (mother liquor) containing molecules to be crystallized, such as protein, is injected into the through-hole 86 from the back side of the solid-state device by a pipette or the like, the mother liquor gradually moves to the surface on which the groove is formed. Drops are formed. The droplet of the mother liquor does not fall on the surface of the solid state element 100,
The injection of the mother liquor is terminated when the liquid has spread in all the grooves and has been stably held. In this manner, as shown in FIG.
7, the droplet 88 is stably held in a state of hanging in the direction of gravity. If the droplets are held in this manner, the amount of the solution required for crystallization is small. In such an apparatus, a crystal is grown inside the groove as described above.

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, polyzacarides, nucleic acids, and complexes and dielectrics 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 standard buffer solution at pH = 6.8 to a concentration of 50 mg / ml. The following two types of silicon crystals were used as solid devices, and lysozyme was crystallized in an apparatus as shown in FIGS.

(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, processing was performed based on the process shown in FIG. 5 to form a silicon substrate having a plurality of V grooves. The widths of the V-grooves having different sizes are each 0.
8, 2.0, 10, 50, and 100 μm, and the depth was almost the same as the width of each groove. The lengths of the grooves were all 7 mm, and the five types of V grooves were formed at a pitch of 1.0 mm. The through hole has a diameter of about 200 μm at the center of the substrate.
The size was formed. The thickness of the polyimide layer formed so as to surround the five types of grooves was 10 μm. The obtained silicon substrate was cut into a size of 10 mm in length and 10 mm in width, and provided as a solid device for crystallization. The specific resistance of the N-type silicon layer formed on the surface was about 0.1 Ωcm, and the thickness was about 0.5 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. Thereafter, a through hole having a diameter of about 200 μm and a polyimide layer were formed at the center of the substrate under the same conditions as in Sample-1. The obtained silicon substrate was cut into a size of 10 mm in length and 10 mm in width, and provided as a solid device for crystallization. Therefore, Sample-2 has the same structure as Sample-1 except that there are no plurality of V-grooves.

The substrates of Sample-1 and Sample-2 were supplied to the apparatus shown in FIGS. 9 and 10, respectively. In this apparatus, a silicon substrate was held on a reactive vial having a diameter of about 15 mm such that the surface on which an N-type silicon layer was formed was down. About 0.1 lysozyme mother liquor
ml was injected into the through-hole from the back surface to form a droplet that hangs in the direction of gravity. Note that p
H = 4.6 standard buffer solution and 1 M NaCl
About 3 ml of the buffer solution mixed at a volume ratio of 0: 1 was injected,
It was held at the bottom. The apparatus for crystallization was stored in a cool dark place at 10 ° C. and allowed to stand.

After standing in a cool and dark place for 96 hours, each sample was taken out, and lysozyme crystals formed on the silicon substrate were observed with a microscope.

On the substrate of Sample-1, the size of the precipitated crystal was different depending on the size of the groove. The width is 0.
Microcrystals and twins were randomly deposited on the V-grooves having a size of 8, 2.0 μm and on the surface of the N-type silicon layer, as shown in FIG. On the other hand, the width is 10, 50, 100 μ
In the V-groove having a size of m, a large single crystal having a size of about 0.3 mm was regularly deposited along the groove as shown in FIG. It is considered that the precipitate serving as the nucleus of the crystal selectively aggregated and aggregated in the specific V-groove made of P-type silicon, which grew to a large single crystal.

On the other hand, on the sample-2 substrate, FIG.
As shown in (1), 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.

From the above results, it was proved that the present invention could provide a large-sized and high-quality single crystal for a small amount of sample.

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.

[0067]

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. Further according to the invention,
Effective crystallization of a small amount of sample by supplying droplets of a solution containing molecules to be crystallized onto the solid-state device through through holes and holding the droplets so that they hang down in the direction of gravity Can be.

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. ADVANTAGE OF THE INVENTION According to this invention, the crystal with favorable crystallinity which enables X-ray 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. The present invention is also 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 cross-sectional view showing a state in which a mother liquor is injected from a through hole in the solid-state device of the present invention.

FIGS. 3A and 3B are a schematic sectional view and a plan view showing a specific example of the solid-state device of the present invention. FIGS.

FIG. 4 is a schematic sectional view showing another specific example of the solid-state device of the present invention.

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 showing a process for manufacturing the solid-state device shown in FIG.

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

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

10 is an enlarged schematic cross-sectional view of a main part of the device shown in FIG.

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

FIG. 12 is a micrograph of a crystal structure generated in an example.

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

FIG. 14 is a schematic view showing an example of an apparatus used in a conventional method.

FIG. 15 is a schematic view showing another example of the device used in the conventional method.

[Description of Signs] 1 Solid-state element 2 Crystal nucleus 10, 20, 30, 40 P-type silicon substrate 11, 21, 31, 41 N-type silicon layer 12a-e, 32a-e V groove 22a-e, 42a-e concave shape groove

────────────────────────────────────────────────── ─── front page continued (51) Int.Cl. ⁷ identifications FI B01D 9/02 605 B01D 9/02 605 620 620 625 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 (8)

(57) [Claims] 1. A method for growing crystals of a polymer compound contained in a solution, the method comprising: controlling the concentration of holes or electrons at a surface portion according to the environment of the solution containing the polymer compound. A step of providing a solid-state element in which electrons are controlled; and a step of bringing the solid-state element into contact with the solution containing the polymer compound, wherein the solid-state element is formed on one of two opposing main surfaces. And / or two or more grooves or holes having different depths and / or opening widths, and a solution containing the polymer compound supplied to the grooves or holes from the other surface side of the two main surfaces. Having a through-hole, in the portion of the solid-state element that is in contact with the solution, the valence electrons are controlled such that crystallization of the polymer compound is promoted within the groove or hole, The groove facing down By supplying the solution containing the polymer compound to the hole through the through hole, the solution is held in the groove or the hole in a state where the droplet of the solution hangs down in the direction of gravity on the solid-state device. And 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 in the groove or the hole holding the solution, 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. The method according to claim 1, wherein a water-repellent layer is provided on the solid device so as to surround the two or more grooves or holes. 4. The presence of a precipitant absorbing the vapor of a solvent contained in the solution or a buffer solution for maintaining a vapor-liquid equilibrium for the solution, wherein the growth of crystals in the grooves or holes holding the solution is carried out. Claim 1 which is performed under
3. The method according to any one of 3. 5. A solid-state element for crystal growth used in the method according to claim 1, wherein two of the two main faces opposing each other have different depths and / or different widths of the openings. Having the above-mentioned groove or hole, and a through hole for supplying a solution containing a polymer compound to the groove or hole from the other surface side of the two main surfaces; Valence electrons are controlled so that the concentration of holes or electrons on the surface can be controlled accordingly,
A solid state element for crystal growth, wherein the valence electrons are controlled differently inside and outside the groove or hole. 6. The semiconductor device according to claim 6, wherein 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 hole. The solid-state element for crystal growth according to claim 5. 7. The solid-state device for crystal growth according to claim 5, wherein a water-repellent layer is provided on the solid-state device so as to surround the two or more grooves or holes. 8. The solid-state device according to claim 5, a means for supporting the solid-state device, and the solid-state device and the means can be housed in a sealed state together with a precipitant or a buffer solution. An apparatus for growing a crystal, comprising: a container;