JP3146990B2 - Solid state element for crystal growth, crystal growth apparatus and crystal growth method - 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 biopolymers 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 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. 18, a solution 172 containing a biopolymer is sealed inside a dialysis tube 171 and the dialysis tube external solution 17 is sealed.
In this method, the crystallization is performed by continuously changing the pH or the like of the buffer 3 (for example, a buffer solution). According to this method, since the salt concentration and the pH difference between the inner and outer liquids can be adjusted at an arbitrary speed, it is easy to find the conditions for crystallization. In the gas-liquid interphase diffusion method, for example, as shown in FIG.
A sample solution droplet 182 is placed on the
In this method, the liquid droplets and the precipitant solution 184 are put into the liquid crystal 3 so that a volatile component between the liquid and the liquid evaporates to gradually establish equilibrium. In the inter-liquid diffusion method among the diffusion methods, as shown in FIG. 20, a droplet 192 of a mother liquor containing a target substance and a droplet 191 of a precipitant are formed on a substrate 190.
Are placed about 5 mm apart from each other, and a thin liquid flow 193 is formed between them by a tip of a needle or the like. This liquid flow 19
Interdiffusion is carried out via 3 to promote crystallization.
This diffusion method has an advantage that the amount of the solution is very small as compared with the batch method or the like.

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

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

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

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

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

As described above, the diffusion method requires a small amount of sample, but in order to obtain good-quality crystals, the optimum conditions for crystallization have been found by changing the salt concentration and pH of the precipitant over a wide range. I have to work. In this case, the condition can only be found by try and error. Further, a glass substrate on which a sample droplet is formed tends to generate a large amount of unnecessary crystal nuclei. In order to suppress this, surface treatment such as surface polishing and water repellent treatment needs to be performed in advance.

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

[0014]

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

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

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

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

[0018]

The present invention first provides a solid-state device used for growing a crystal of a polymer compound contained in a solution. This solid-state device is composed of a substrate whose valence electrons are controlled so that the concentration of holes or electrons on the surface can be controlled according to the environment of the solution containing the polymer compound.
A surface of the substrate, a plurality of first solution reservoirs for holding two or more types of solutions, a plurality of second solution reservoirs for retaining a solution containing a polymer compound for growing crystals, And a plurality of flow paths that connect the first solution storage section and the plurality of second solution storage sections to allow the solution to flow. In at least the second solution storage section of the substrate, 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 polymer compound.

[0019] The solid-state device of the present invention may further include means for heating the solution in the first solution storage section.

Further, the solid-state device of the present invention may further have an electrode for applying a voltage to at least the second solution storage part.

In the plurality of flow paths formed in the solid-state element, it is preferable that the width and / or the depth are different.

[0022] The second solution storage section formed in the solid state element includes:
It is preferable to have a groove or a hole formed by fine processing.

The solid-state device of the present invention is preferably made of, for example, a semiconductor substrate doped with impurities. Valence electron control in the solid-state device can be performed by controlling the concentration and / or type of impurities.

As a preferred semiconductor substrate, a silicon crystal substrate can be used. In a solid-state device formed of a semiconductor substrate, a groove or a hole can be formed in the second solution storage section. In this case, the concentration and / or type of impurities can be different between inside and outside of the groove or hole.

In the solid-state device of the present invention, a coating made of an oxide can be provided on the surface to be brought into contact with the solution.
Such a coating imparts hydrophilicity to the surface.

The present invention provides an apparatus for growing a crystal, which comprises a solid element for crystal growth as described above, a container capable of holding the solid element together with a precipitant or a buffer solution, and a solid element in the container. Means for supporting the

The present invention provides a method for growing crystals of a polymer compound contained in a solution. In this method, the step of providing the above-described solid element for crystal growth and the step of holding the first solution containing the polymer compound and the second solution different from the first solution in the plurality of first solution reservoirs provided in the solid element, respectively. And transferring the first solution and the second solution to a plurality of second solution reservoirs provided in the solid-state device through a plurality of flow paths, and the first solution is stored in the plurality of second solution reservoirs. For storing a plurality of types of mixed liquids in which the mixed solution and the second solution are mixed at different ratios, and in a plurality of second solution storing sections for storing the mixed liquids respectively, the surface of the solid element is controlled by controlled valence electrons. Growing a crystal of the polymer compound under the electrical state brought to the above.

[0028] The method of the present invention may further comprise the step of heating the solution in the first solution storage, whereby
The transfer of the solution from the first solution storage section through the flow path can be promoted.

[0029] The method of the present invention can further include a step of applying a voltage to at least the second solution storage section in the solid-state device, whereby the electric state brought to the surface of the solid-state device can be controlled. .

In the method of the present invention, when a solid element having a groove or a hole formed by microfabrication in the second solution storage portion is used, crystallization of the polymer compound in the groove or the hole can be promoted.

In the method of the present invention, a buffer solution and / or a salt solution for changing the pH and / or the salt concentration of the first solution can be used as the second solution.

[0032]

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

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

In the present invention, in order to stably generate crystal nuclei, a solid-state element having controlled valence electrons 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.

According to the present invention, a plurality of first solution reservoirs for holding a mother liquor, a precipitant, a buffer solution, and the like on the surface of a substrate constituting a solid-state device, and a plurality of liquids from the plurality of first solution reservoirs, respectively. And a plurality of second solution reservoirs for receiving and growing crystals. The flow of the liquid between the first solution storage part and the second solution storage part is performed by a flow path provided on the surface of the substrate. The flow path is provided between the first solution storage section and the second solution storage section, but may be further provided between the plurality of second solution storage sections. By circulating the liquid through the flow path, different types of solutions can be supplied from the plurality of first solution storage units to the second solution storage unit, and a mixed solution can be obtained. The mixed liquid obtained in each of the plurality of second solution reservoirs can have a different composition depending on the position of each of the second solution reservoirs and the arrangement of the flow paths or the size and shape thereof. That is, a plurality of types of solutions can be mixed at different ratios in the plurality of second solution storage units. In a system in which the solution storage units are connected by a flow path, the mutual diffusion of a plurality of solutions can be changed continuously and spatially and temporally, and different crystals are formed in a plurality of second solution storage units to be crystallized. Conditions can be obtained with good reproducibility. If many second solution reservoirs are provided on the solid-state device, slightly different conditions can be adjusted. In this manner, a plurality of different crystallization conditions can be obtained on one solid-state device, and it can be expected that any of them is optimal for crystallization of a necessary compound.

In a plurality of flow paths formed on the substrate,
Preferably, they have different widths and / or depths. By changing the size of the channel, the supply amount of the solution can be changed. By changing the flow rate of the solution supplied to the second solution reservoir for performing crystallization,
The mixing ratio of a plurality of types of solutions can be controlled. If the flow rate is adjusted for each of the plurality of flow paths, more various mixing ratios can be obtained, and thus more conditions for crystallization can be prepared.

Further, in the present invention, means for heating the solution stored in the first solution storage section can be formed in the solid state device. Such a means can be, for example, a heating electrode formed on the substrate of the solid state device. Each heated solution is extruded into the flow channel by using the expansion of the volume as a driving force. In addition, the heated solution becomes easier to flow because the viscosity decreases. Therefore, the transfer of the solution is promoted by heating. Other ways to facilitate solution transfer include:
A height difference can be provided between the solution storage portions connected by the flow path. Further, a groove having a fine width may be formed as a flow path. When the gap between the grooves is small, the liquid can be moved by the surface tension.

When the solid state element is partially heated by the heating means, a temperature gradient can be provided in the solid state element. According to the temperature gradient, a temperature difference occurs in the plurality of second solution storage units. In solutions at different temperatures, the solubility of the substance to be crystallized also differs. Therefore, more conditions for crystallization can be obtained in one solid state device due to the temperature difference.

Further, in the present invention, a voltage can be applied to the solid-state device. The voltage can be applied, for example, via an electrode formed on the substrate surface opposite to the surface on which the solution storage section is formed. Such an electrode has at least a second
It is preferable to provide the solution storage unit so that a voltage can be applied. By applying a bias voltage through the electrodes,
As will be described in detail later, the electrostatic effect acting on the surface of the solid state device can be increased or decreased. In particular, crystal growth can be promoted by increasing the surface potential of the solid state element in the second solution storage section.
By the application of the voltage, the selective reaction of the molecules to be crystallized on the solid element surface and the aggregating action can be improved.

Further, by forming a groove or a hole in the second solution storage section by micromachining, the effect of suppressing the convection of electrostatic attraction acting on the solid-state element can be improved. In particular, at the bottom of the groove formed in the solid-state device, an electrostatic interaction can be exerted almost isotropically on the molecule to be crystallized. When a crystal nucleus is formed at the bottom of a groove, the crystal nucleus can be stopped at the bottom of the groove by electrostatic interaction, and the crystal nucleus can be protected from convection due to the influence of gravity. 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.

The solid-state device provided for such a purpose is a material having the above-mentioned electrostatic characteristics and capable of controlling the amount of charge and polarity, and a material which is chemically stable in a solution. Anything is acceptable. One of the most suitable materials for achieving this purpose is a silicon crystal. The crystallization mechanism expected when a silicon crystal is used will be described below. However, the mechanisms described below can be applied to other solid state devices used in accordance with the present invention.

FIGS. 2, 3 and 4 show one specific example of the solid state element for crystal growth of the present invention. The structure shown in the figure is obtained by processing a silicon substrate. FIG.
1 schematically shows a structure formed on a surface of a silicon substrate. 3 and 4 show XX, Y of the device shown in FIG.
It is -Y sectional drawing.

As shown in FIG. 2, a plurality of solution reservoirs 12a, 12b, 12c, 12d,
14 are formed. The solution storage units 12a to 12d
It corresponds to a first solution storage section according to the present invention, and holds a solution containing a polymer compound and other necessary solutions, respectively. The large number of solution storage sections 14 correspond to the second solution storage section of the present invention, and are for receiving a plurality of types of solutions supplied from the solution storage sections 12a to 12d to prepare a mixed solution. In the solution storage section 14, the solution containing the polymer compound meets another solution, and the conditions for crystallization are adjusted. Then, in any one of the large number of solution storage sections 14, optimal crystallization conditions are created, and crystallization is promoted. The solution storage section 12a and the solution storage section 14 are connected by a flow path 16a. Similarly, the storage unit 12
b, 12c and 12d are flow paths 16b, 16c, 16
Reservoir 1 corresponding to d ₁ , 16d ₂ and 16d ₃
4. In addition, a flow path 26 is provided between a number of solution storage sections 14. The flow path 26 connects the adjacent storage units 14.

A heating electrode 18 is formed around the solution storage sections 12a, 12b and 12c. Electrode 18
Is connected to the pad 21. A heating electrode 28 is also provided around the solution storage section 12 d, and the electrode 28 is connected to the pad 31. When the electrodes 18 and 28 are energized via the pads 21 and 31, respectively, the solutions held in the reservoirs 12a to 12d are heated.
As described above, the transfer of the solution is promoted by heating, but these heating means may be omitted.

FIG. 3 shows a cross section of a substrate on which a solid-state element is formed. V-grooves having different sizes are formed on the silicon substrate 10, and these portions serve as solution storage portions 12b, 14 and 12d, respectively. Although not shown, V-grooves are also formed in the solution storage sections 12a and 12c shown in FIG. On the substrate 10, a water-repellent layer 17 made of, for example, a water-repellent resin is formed around the portion where the V-groove is formed. This layer prevents the solution from spreading on the substrate and ensures that a given solution is retained in each solution reservoir. The solutions 15b, 15d, 25a, 25b, and 25c are held in spaces formed by the V-grooves and the water-repellent layer 17. An electrode 19 is provided on the back surface of the silicon substrate 10. In the silicon substrate, the portions where the solution storage portions 12b and 12d are formed and the portion where the solution storage portion 14 is formed have different thicknesses. In such a structure, when a voltage is applied to the electrode 19, the electrical state (surface potential) brought to the substrate surface differs between the storage portions 12b and 12d and the storage portion 14. If the distance between the back electrode and the surface of the substrate is reduced, the strength of the field applied to the surface of the substrate when a voltage is applied can be increased. Therefore, a high bias voltage can be selectively applied to the solution storage section 14 where crystallization is to be performed, and the surface potential can be increased at this portion to promote crystal growth.

FIG. 4 shows a structure in which a flow path is formed on a silicon substrate. V on the silicon substrate 10
Channels 16a, 16b, 16c having grooves are formed.
A water-repellent layer 17 is also provided around the flow paths 16a to 16c. The solution 25 is placed in the space defined by the V-groove and the water-repellent layer 17.
Is held.

In the solid-state device shown in FIGS. 2, 3 and 4, for example, p
A buffer solution in which H is adjusted, an aqueous solution of a predetermined salt in the storage unit 12b, and an aqueous solution (mother liquor) containing a molecule (eg, protein) to be crystallized in the storage unit 12d can be held as droplets. . Further, a buffer solution or an aqueous solution of a salt may be held in various parts of the storage unit 14 as needed. Next, the entire substrate or the solution storage section is heated by energization. The heating promotes the transfer of the solution from each reservoir. As shown in the figure, each flow path connects the reservoirs 14 in a complicated manner, and thus the mother liquor, the buffer solution, and the salt solution are gradually mixed in the respective reservoirs at various concentration ratios. At this time, the inflow and outflow of the solution are repeated through the flow path, and the mixing ratio changes temporally and spatially little by little. That is, many storage units 14
In, various mixtures are prepared with slightly different concentrations of the molecules to be crystallized, the buffer solution and the salt. An optimum mixing ratio for crystallization is provided in any of these mixtures, where crystallization is promoted. Further, as described later, if a groove is formed in a solution storage portion where a crystal is to be grown, it is possible to control the formation of crystal nuclei and promote the crystal growth.

In the solid-state device shown in FIGS. 2 to 4, a solution storage section having a V-groove and a flow path are used, but their shapes may be various. FIGS. 5, 6, and 7 further show specific examples of the first solution storage section, the flow path, and the second solution storage section.

The first solution storage section formed in the solid-state device of the present invention can have, for example, a shape as shown in FIGS. 5 (a) to 5 (e). As shown in FIGS. 5A to 5D, a groove is preferably formed in the first solution storage section in order to hold a necessary and sufficient amount of droplets. FIG.
In the storage section shown in FIG.
A U-shaped groove 32a is formed. The groove 32a is surrounded by a water-repellent layer 37 made of, for example, a water-repellent resin. Groove 3
The storage section 32 that holds the solution 35 is formed by the 2a and the water-repellent layer 37. In the storage section shown in FIG. 5B, a silicon oxide film 33 is formed on a silicon substrate 30. This oxide film imparts hydrophilicity to the silicon substrate.
Other structures are the same as those in FIG. FIG. 5 (c)
In the storage section shown in FIG. 5, a V-groove 42 a is formed on a silicon substrate 40. A water-repellent layer 47 made of, for example, a water-repellent resin is formed around the groove 42a. The solution 45 is held in the storage part 42 composed of the groove 42 a and the water-repellent layer 47. In the storage section shown in FIG. 5D, a silicon oxide film 43 is formed on a silicon substrate 40. Other structures are the same as those shown in FIG. In these structures, the U-shaped groove and the V-shaped groove can be formed by etching the silicon substrate. The water-repellent layer can be formed, for example, by coating a silicon substrate with a water-repellent resin and performing patterning using photolithography or the like. Also, as shown in FIG.
The solution storage section can be configured without forming the groove. In this case, the water-repellent layer 47 'can be formed in a predetermined pattern on the silicon substrate 40', and the portion where the surface of the silicon substrate 40 'is exposed can be used as a solution storage section. When a water-soluble solution is used, by forming an oxide film on the silicon surface where the groove is formed, the hydrophilicity of that portion can be increased, and the flow of the solution can be improved.

FIGS. 6A to 6E show specific examples of the flow path. As shown in FIG. 6A, a U-shaped groove 56a is formed on a silicon substrate 50, and a water-repellent layer 57 made of a water-repellent resin can be formed therearound. The channel 56 is constituted by the groove 56a and the water repellent layer 57, and the solution 55 is passed therethrough. The structure shown in FIG. 6B is the same as the structure shown in FIG. 6A except that a silicon oxide film 53 is formed on a silicon substrate 50. The channel 66 shown in FIG. 6C has a V groove 66 a formed on the silicon substrate 60. The solution 65 is passed through a flow path 66 composed of the groove 66a and the water-repellent layer 67 surrounding the groove 66a. 6D except that a silicon oxide film 63 is formed on a silicon substrate 60. The structure shown in FIG.
It is the same as the structure of FIG. In the structure having such a groove, the U groove and the V groove can be formed by etching the silicon substrate. The water-repellent layer is obtained, for example, by coating a silicon substrate with a water-repellent resin and patterning the same using photolithography or the like. Further, as shown in FIG. 6E, a structure in which no groove is formed can be obtained more easily. In this case, the water-repellent layer 77 is formed on the silicon substrate 70 except for the portion where the flow path is to be formed.
Is provided. The solution 75 is passed through a channel 76 surrounded by a water-repellent layer 77. When a water-soluble solution is used, a silicon oxide film is formed on the surface of the silicon where the grooves are formed, whereby the hydrophilicity can be increased and the flow of the solution can be improved.

FIG. 7 shows a specific example of the structure of the second solution storage section. In the structure shown in FIG. 7A, a water-repellent layer 87 made of, for example, a water-repellent resin is formed on a silicon substrate 80 in a predetermined pattern. A solution storage portion 84 for holding the solution 85 is formed in a portion surrounded by the water-repellent layer 87 and exposing the silicon substrate 80. Since the surface of a silicon substrate usually has very few crystal defects and fixed charges, it has the advantage that it is difficult to generate extra crystal nuclei for molecules to be crystallized. Therefore, FIG.
Even with the structure as shown in (a), the space for crystallization can be prepared. In the structure shown in FIG.
A silicon oxide film 83 is formed on a silicon substrate 80. Other parts are the same as the structure shown in FIG. An oxide film may be formed on the silicon surface according to the characteristics of the molecule to be crystallized (for example, a protein molecule). The oxide film improves the hydrophilicity of the silicon surface. FIG. 7 (c)
In the structure shown in FIG. 5, a second silicon layer 80b is formed on a first silicon layer 80a. Such different silicon layers may have different concentrations of impurities added to silicon and / or
Alternatively, it can be formed by changing the type.
A water-repellent layer 87 is formed in a predetermined pattern on silicon having a laminated structure, and a solution storage section 84 is formed in a portion surrounded by the water-repellent layer 87. In the structure shown in FIG.
A second silicon layer 90b is formed on the silicon layer 90a, and a V-groove 94a is formed in the stacked body. The water-repellent layer 97 formed in a predetermined pattern and the V-shaped groove 94a constitute a reservoir 94 for holding the solution 95.
The first silicon layer 90a is exposed inside the V groove 94a. On the other hand, outside the V groove 94a, the first silicon layer 90a is covered with the second silicon layer 90b. In the structure shown in FIG. 7E, a trench 104a is formed in the solution storage section 104. Trench 104a
Has a shape with a depth greater than the width of the opening and a high aspect ratio. As shown in the figure, it is preferable to provide a plurality of trenches. The solution storage section 104 is provided on a substrate in which the second silicon layer 100b is formed on the first silicon layer 100a. A water-repellent layer 107 is formed around the solution storage section 104. In the structure shown in FIG.
A U-shaped groove 114a is formed in the solution storage section 114. First
A second silicon layer 110b on the first silicon layer 110a.
Is formed, and a water-repellent layer 117 is formed around the solution storage portion 114.
Is provided.

In the structure shown in FIGS. 7D to 7F,
The V-groove, trench and U-groove can be formed by, for example, chemically etching the silicon surface. By forming a groove in the solution storage section, convection of the solution in the storage section can be effectively suppressed, and crystal nuclei can be generated and large crystals can be grown more stably. In the structure shown in FIGS. 7D to 7F, a silicon oxide film may be formed on the surface. The oxide film
Improves the hydrophilicity of the silicon surface. In the storage section shown in FIG. 7, the water-repellent layer can be formed by, for example, coating a water-repellent resin and then performing patterning using photolithography or the like.

In the solution storage for crystallization shown in FIG. 7, silicon whose valence electrons are controlled can be used. Due to the controlled valence electrons, an electrical state corresponding to the state of the solution is provided on the silicon surface in contact with the solution. In order to control valence electrons, it is particularly preferable to form a plurality of silicon layers having different impurity concentrations and / or types in the solution storage section. The structures shown in FIGS. 7C to 7F are examples in which a plurality of silicon layers having different valence electron control states are formed. In these structures, for example, P-type silicon can be used as the first silicon layer and N-type silicon can be used as the second silicon layer. In this case, it is considered that molecules having a negative effective surface charge can be more effectively crystallized particularly in the groove where the P-type silicon layer is exposed. Further, N-type silicon having a low impurity concentration and high resistance can be used as the first silicon layer, and N-type silicon having a high impurity concentration and low resistance can be used as the second silicon layer. Also in this case, it is considered that molecules having a particularly negative effective surface charge can be effectively crystallized in the groove where the high-resistance N-type silicon is exposed. on the other hand,
It is considered that the polarity of silicon described above should be reversed for crystallization of molecules having a positive effective surface charge.
That is, N-type silicon may be used as the first silicon layer, and P-type silicon may be used as the second silicon.
It is also considered effective to form layers or regions having different impurity concentrations for P-type silicon.
Hereinafter, a mechanism in which crystallization is controlled by valence electron control in a solid-state device will be described. An electrolyte aqueous solution containing a polymer compound having dissociated and having a negative effective surface charge,
Contact with a valence-controlled N or P-type silicon crystal forms a Schottky barrier on the N-type silicon surface while providing an ohmic contact 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, the polymer having a negative charge in the electrolyte solution is at least compensated for the positive space charge of the N-type silicon.
It is expected that it will continue to aggregate on the silicon surface. 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.

By utilizing the characteristics described above, the formation of crystal nuclei in a specific region of the solid-state device can be suppressed, and the generation of crystal nuclei in a specific region can be promoted. For example, FIG.
In the structure shown in (c), a high-resistance N-type silicon is used as the first silicon, and a low-resistance N-type silicon is used as the second silicon. Crystals can be prevented from depositing in the flow path. Further, by forming a region suitable for crystallization by valence electron control in a specific solution storage portion, it is possible to control a position where crystal growth is to be performed on the solid-state device.

As described above, 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 the same characteristics as those on silicon 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 Ωcm.
Can be used. Various methods can be considered as a method of adjusting silicon whose valence electrons are controlled to N-type and P-type, and any method may be used. However, the simplest method for accurately controlling impurity concentration is as follows.
An ion implantation 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. B, Al, Ga, In,
Tl and the like can be mentioned. In particular, B is common.
N, P, As, S as Group V elements for making N-type
b, Bi and the like can be mentioned, and P, As, and Sb are particularly common. Further, the surface of the crystal is preferably mirror polished in order to control the crystal nuclei to be precipitated.

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

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

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

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.

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

Further, it is preferable to form a water-repellent layer on the surface of the solid element such as a silicon substrate so as to surround the solution storage section and the flow path. This layer can effectively prevent the solution from flowing to the surroundings when holding the solution. For example, a silicon surface from which an oxide film has been removed is generally sufficiently water-repellent to water containing only acids and alkalis or pure water, but is water-repellent to aqueous solutions containing salts such as buffer solutions. Drops. Therefore, when a buffer solution is used, it is necessary to form a layer made of a water-repellent substance around the silicon substrate. The water-repellent layer can be formed of, for example, an organic resin, and a polyimide resin is one of the materials that can form the water-repellent layer most easily. In the case of forming a water-repellent layer made of polyimide, for example, after coating and curing a photosensitive or non-photosensitive polyimide resin, unnecessary portions can be removed by etching or development to obtain a desired pattern. .

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

In the present invention, the distribution of the surface potential can be controlled by applying a voltage to the solid state device. FIG.
The principle of operation when a bias voltage is applied to the substrate constituting the solid-state device of the present invention will be described with reference to FIG. In the solid-state element shown in the drawing, a second silicon layer 150b is formed on a first silicon layer 150a. The second silicon layer is not formed in the portion where the solution is stored, and the first silicon layer is exposed. An electrode layer 159 is formed on the back side of the first silicon layer 150a so that a bias voltage can be applied. For example, high-resistance silicon (N ^- silicon) can be used as the first silicon, and low-resistance silicon (N ⁺ silicon) can be used as the second silicon. In this case, when no bias voltage is applied, the distribution of the surface potential on the silicon surface is as shown in the chart indicated by A. When a positive bias voltage is applied to the solid state device, the distribution of the surface potential shifts in the positive direction, and the potential increases as a whole (see Chart B). On the other hand, when a negative bias voltage is applied, the silicon surface becomes electrically ohmic to the solution,
The distribution of the surface potential becomes substantially flat, and the potential becomes substantially zero as a whole (Chart C). Thus, by applying a voltage, the potential distribution on the surface of the silicon substrate can be changed arbitrarily. By controlling the surface potential by applying a voltage, aggregation and crystallization of the polymer compound on the device surface can be more positively controlled. The electrode for applying a voltage may be provided on the entire back surface of the solid-state element, or may be formed only on a portion where crystal growth is desired. For example, as shown in FIG. 9, an electrode 90 is formed in a portion corresponding to a solution storage portion for crystal growth, and a pad 91 is formed.
A voltage may be applied via the.

In the present invention, the effect of convection is suppressed by forming a groove in the solution storage portion where the crystal is to be grown.
Crystal growth can be performed more stably. FIG. 10 illustrates the effect of the groove. For example, in a portion where a groove is formed by etching, a range where an electrostatic interaction between a molecule dissociated in a solution and a surface of a solid-state element reaches (a width of an electric double layer) as compared with a portion where a groove is not formed. Can be expected to be wider. For example, as shown in FIG.
When the solution 125 is held in the storage part 126 in which the V groove 126a is formed, the deepest central portion of the V groove 126a
It is expected that the areas that this interaction will reach overlap and their width will be wider. In the drawing, a region 121 to which the electrostatic interaction is exerted is indicated by a dotted line. Therefore, in the central part of the V-groove, the crystal nucleus or the molecular aggregate serving as the crystal nucleus is V V
Electrostatic attraction can be received almost isotropically from the groove surface,
It can be more constrained within the space of the V-groove. Above the solution 125, convection 129 is generated due to the influence of gravity or the like.
On the other hand, in the V-groove, molecules are restrained by electrostatic attraction, so that the influence of convection is suppressed. Therefore,
In the V groove, crystal nuclei are generated stably, and it can be expected that crystal growth is performed stably. This is illustrated in FIG.
It is considered that this also applies to a solid-state device having a trench as shown in FIG. In the trench 136a formed in the silicon substrate 130, the region 131 to which the electrostatic attraction is exerted overlaps, and it is expected that the influence of the convection 135 generated above the solution 136 does not extend to the deep portion of the trench 136a. In the trench 136a, molecules to be crystallized are restrained by the electrostatic attractive force 133, and it can be expected that the crystal growth proceeds stably. Generally, the width of the diffusion supply layer in the vicinity of the crystal nucleus fluctuates due to the influence of convection in the solution, so that it is considered that the crystallinity and the growth rate decrease. Therefore, convection should be suppressed as much as possible.

In the case of the structure shown in FIG. 10C, it is considered that the molecules to be crystallized are relatively easily affected by convection. On the other hand, the region 121 to which the electrostatic attraction 123 is applied is spatially uniform, and no electric field gradient is formed in the plane of the substrate 140. For this reason, depending on the type of the molecule to be crystallized, it is considered that the molecules are easily arranged two-dimensionally during aggregation, which may be advantageous in some cases.

FIG. 11 shows a specific example of an apparatus for growing a crystal. In this apparatus, a substrate 10 constituting a solid element for crystal growth is supported on a support 162 in a container 161.
a and 162b. An electrode 19 is formed on the back surface of the substrate 10. An electrode 163b is also formed on the support 162b, and a voltage can be applied to the electrode 19 via the terminal 164b. The electrode 163a is also provided on the support 162a, and the electrode 1
63 a is electrically connected to the pad 31 formed on the substrate 10. Electric power is supplied to the pad 31 via the terminal 164a, so that the solution storage section formed on the substrate 10 can be heated. A buffer solution 166 is accommodated in the bottom of the container 161, and the opening thereof can be sealed with an upper lid 165. The supports 162a and 162b of the container 161 capable of preventing the solution from evaporating in this way.
The substrate 10 is placed thereon, and necessary solutions such as the solutions 15 b and 15 d are dropped on the substrate 10. The substrate 10 may be either upward or downward. After the buffer solution 166 or the like is added to the bottom of the container 161, the opening is sealed with the upper lid 165, and crystallization can be started. At the start of crystallization, a current can be applied from the terminal 164a to heat the solution. Further, a bias voltage can be applied to the back surface of the substrate 10 from the terminal 164b. Heating of the solution and application of the bias voltage may be performed continuously or intermittently. In this state, if the substrate 10 holding the droplets is allowed to stand still, crystal growth can be performed on a predetermined portion of the substrate.

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.

[0072]

[Example] Lysozyme made from chicken egg white (Lysozyme, From
Chicken Egg White) was dissolved in a standard buffer solution of pH = 4.5 to a concentration of 50 mg / ml. This solution was crystallized using the following two types of silicon crystals.

(1) Sample-1 A low-resistance N-type silicon layer (specific resistance: about 0.01 Ωcm, thickness: about 0.5 μm) was implanted on the surface of an N-type silicon substrate having a specific resistance of about 30 Ωcm by ion implantation of phosphorus element. ) Was formed over the entire surface. Then, the surface is heated to 200 酸化 by thermal oxidation.
Was formed. Next, a solution storage section, a flow path, an electrode pattern, and a pad were formed on the surface with the configuration shown in FIG. These could be formed using a photolithography method, an etching technique, and the like that are usually used in an LSI manufacturing process. The solution storage units 12a, 12b and 12c shown in FIG.
The size of the solution storage unit 12d is 3 mm × 10 mm.
mm. V-grooves were formed in each solution storage section by anisotropic etching of silicon. All V grooves had a depth of 200 μm. On the other hand, all the solution storage sections 14 for crystal growth shown in FIG.
It was the size of. Also for these, V grooves were formed by etching. The depth of the groove was about 5 μm. The flow path connecting the respective solution storage sections to each other had a V-groove shape.
Among the flow paths shown in FIG. 2, the flow path arranged vertically in the drawing was a V-shaped groove having a width and a depth of 50 μm.
The flow path arranged in the left-right direction as viewed in the drawing was a V-shaped groove having a width and a depth of 100 μm. The flow path connecting the crystal growth solution storage portions at oblique positions to each other was a V-groove having a width and depth of 20 μm. All V grooves were formed by anisotropic etching of the silicon surface.

The heating electrodes 18 and 28 shown in FIG.
It was obtained by forming a Cr thin film on a silicon oxide film and patterning it. Its film thickness was 0.2 μm. Also, as the pads 21 and 31, an Al layer having a thickness of 1 μm was formed on the Cr thin film. After the silicon oxide film is removed on the back surface of the silicon substrate, thin films of Ti, Ni and Au are continuously formed at 0.05 μm and 0.2 μm, respectively.
A back electrode as shown in FIG. 3 was prepared by forming the electrodes with a thickness of μm and 0.2 μm. Thereafter, a photosensitive polyimide was applied to the surface of the silicon substrate, and was patterned using photolithography to form a polyimide layer having a thickness of 10 μm on the surface other than the solution storage part and the flow path.
The obtained structure was used for a solid-state device for crystallization.

(2) Sample-2 Ion implantation was performed in the same manner as in Sample-1 to form a low-resistance N-type silicon layer on an N-type silicon substrate.
The specific resistance and the thickness of the N-type layer are the same as in Sample-1. Thereafter, without forming a V-groove, the device was used as it was as a solid device for crystallization.

An experiment of crystallization was carried out in an apparatus as shown in FIG. The silicon substrate of Sample-1 was held in a cell plate with a lid having a diameter of about 40 mm such that the surface on which the V-groove was formed faced up. About 5 ml of a pH 4.5 buffer solution was injected into the bottom of the cell plate. After the cell plate holding the silicon substrate was allowed to stand in a cool and dark place at 10 ° C., the above-mentioned lysozyme solution was dropped into the solution storage section 12 d shown in FIG. 2 so as not to overflow. Further, a buffer solution having a pH of 4.5 was dropped into the solution storage portions 12a and 12c, and a 1.0M NaCl aqueous solution was dropped into the solution storage portion 12b so as not to overflow. After that, close the lid of the cell plate,
A current was passed through the heating electrode, and the solutions were heated until the temperature of each solution rose to around 50 ° C. When each solution started to reach the storage for crystal growth along the flow path, heating was stopped. Crystallization experiments were performed on two types of cases: a case where a voltage of +1.0 V was applied from the outside via a back electrode, and a case where a floating state was applied without applying a voltage. In the case of applying a voltage, the application of voltage was started after each solution reached the storage part for crystal growth through the flow path by heating, and then the voltage was continuously applied.

With respect to the silicon substrate of Sample-2,
It was stored in a cell plate holding a buffer solution of pH 4.5 at the bottom as it was. Next, the same lysozyme solution as in the case of Sample-1 was dropped on the silicon substrate to form a droplet having a diameter of about 10 mm. Further, at a position about 5 mm away from the droplet on the silicon substrate, 1.0 M NaC
A solution obtained by mixing an aqueous solution and a buffer solution having a pH of 4.5 at a volume ratio of 1: 1 was dropped, and a droplet having a diameter of about 10 mm was similarly formed. Thereafter, a thin channel was formed between the droplets by drawing a line from one droplet to the other with a needle, and they were connected. Close the lid of the cell plate and put the silicon substrate
Stored in a cool, dark place at 0 ° C.

After the silicon substrates of Sample-1 and Sample-2 were stored in a cool and dark place for 50 hours, respectively, each sample was taken out and lysozyme crystals were observed with a microscope. The results are shown in FIGS. The forms and states of the crystals shown in FIGS. 12, 13 and 14 are schematically shown in FIGS. 15, 16 and 17, respectively. As shown in FIGS. 12 and 15, relatively large and well-grown crystals were obtained on the V-groove of the solution storage portion on the silicon substrate of Sample 1, even when no voltage was applied. The obtained crystal had good crystallinity on the crystal plane and was in a single crystal state. When a voltage was further applied, as shown in FIGS. 13 and 16, larger crystals having better crystallinity were obtained on the V-grooves in the solution storage portion. On the other hand, as shown in FIGS. 14 and 17, large crystals of about 1 mm grew on the silicon substrate of Sample-2, but all of them were twin crystals or the grown crystals had a poor surface condition, which was very good. No crystals were obtained. From this result, it can be seen that according to the present invention, a large single crystal with good quality can be produced even with a small amount of sample.

[0079]

As described above, according to the present invention,
Can either be solved the drawbacks of the conventional crystallization process method can be applied to any materials because it has a variety of characteristics as described above have been conducted by trial and error without. In particular, according to the present invention, the influence of convection of the solution due to gravity can be suppressed, and nuclei can be formed stably in the initial stage of crystallization. Further, according to the present invention, it is possible to suppress or control the mass production of microcrystals, and to obtain a large crystal capable of performing X-ray structural analysis. Further, according to the present invention, it is possible to prepare a number of different crystallization conditions for a small amount of a solution on one substrate. As a result, optimal conditions for crystallization of a specific molecule can be created. Further, in the present invention, a more appropriate electrical state for agglomeration and crystallization of molecules can be provided on the surface of the substrate by applying a voltage. Crystal growth can be promoted by applying a voltage. Further, according to the present invention, the influence of the convection of the solution in the groove can be suppressed, and the crystal can be stably grown. According to the present invention, even if the amount of the sample is very small, it is possible to create more appropriate conditions in a short time for growing a large crystal.

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

[Brief description of the drawings]

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

FIG. 2 is a schematic view showing a specific example of the solid-state element for crystal growth of the present invention.

FIG. 3 is a cross-sectional view of the solid state device shown in FIG.

FIG. 4 is a sectional view of the solid-state element shown in FIG.

FIG. 5 is a schematic sectional view showing a specific example of a first solution storage section in the solid-state element for crystal growth of the present invention.

FIG. 6 is a schematic cross-sectional view showing a specific example of a flow channel in the solid-state element for crystal growth of the present invention.

FIG. 7 is a schematic sectional view showing a specific example of a second solution storage section in the solid-state element for crystal growth of the present invention.

FIG. 8 is a schematic diagram showing a surface potential generated when a bias charge is applied to a solid-state element for crystal growth.

FIG. 9 is a plan view showing a specific example of an arrangement of a back surface electrode in the solid-state element for crystal growth of the present invention.

FIG. 10 is a schematic cross-sectional view for explaining the function and effect of a groove formed in the solid-state element for crystal growth.

FIG. 11 is a schematic sectional view showing a specific example of a crystal growth apparatus of the present invention.

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 micrograph of a crystal structure generated in an example.

FIG. 15 is a perspective view schematically showing the crystal structure shown in FIG.

FIG. 16 is a perspective view schematically showing the crystal structure shown in FIG.

FIG. 17 is a perspective view schematically showing the crystal structure shown in FIG.

FIG. 18 is a schematic view showing an example of an apparatus used in a conventional method.

FIG. 19 is a schematic view showing another example of the device used in the conventional method.

FIG. 20 is a schematic view showing another example of the device used in the conventional method.

[Explanation of symbols]

1 solid element 2 crystal nuclei 10 silicon substrate 12a, 12b, 12c, 12d first solution storage part 14 and the second solution storage section _{16a, 16b, 16c, 16d 1} , 16d 2, 16d
₃ , 26 flow path 18, 28 heating electrode 21, 31 pad 17 water-repellent layer 19 back electrode

────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification symbol FI C07K 1/30 C07K 1/30 C30B 7/00 C30B 7/00 (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 H01L 51/00 CA (STN) JICST file (JOIS)

Claims (15)

(57) [Claims] 1. A solid-state device used for growing a crystal of a polymer compound contained in a solution, wherein the concentration of holes or electrons on a surface portion is controlled according to the environment of the solution containing the polymer compound. The surface of the substrate has a plurality of first and second solutions for holding two or more types of solutions, respectively.
A solution storage unit, a plurality of second solution storage units for retaining a solution containing the polymer compound for growing the crystals, and a connection between the plurality of first solution storage units and the plurality of second solution storage units And a plurality of flow paths for allowing a solution to flow therethrough, and at least in the second solution storage unit, controlling the concentration of holes or electrons on the surface according to the environment of the solution containing the polymer compound. A solid-state device for crystal growth, characterized in that valence electrons are controlled so as to be able to. 2. The solid state device for crystal growth according to claim 1, further comprising means for heating the solution in said first solution storage section. 3. An electrode for applying a voltage to at least the second solution storage unit.
The solid state element for crystal growth according to claim 1. 4. The solid state element for crystal growth according to claim 1, wherein the plurality of flow paths have different widths and / or depths. 5. The solid element for crystal growth according to claim 1, wherein said second solution storage section has a groove or a hole formed by fine processing. 6. The solid-state device according to claim 1, wherein the solid-state element is formed of a semiconductor substrate doped with an impurity, and the valence electron control is performed by controlling the concentration and / or type of the impurity. The solid-state device for crystal growth according to any one of claims 1 to 7. 7. The solid state element for crystal growth according to claim 6, wherein said semiconductor substrate is made of silicon crystal. 8. The method according to claim 6, wherein the second solution storage section has a groove or a hole, and the concentration and / or type of the impurity is different between inside and outside of the groove or the hole. Or the solid state element for crystal growth of 7. 9. The solid state element for crystal growth according to claim 1, further comprising a coating made of an oxide on a surface to be brought into contact with the solution. 10. A solid-state element for crystal growth according to claim 1, a container capable of holding the solid-state element in a sealed state together with a precipitant or a buffer solution, and supporting the solid-state element in the container. Means for growing a crystal. 11. A method for growing a crystal of a polymer compound contained in a solution, a step of providing the solid-state element for crystal growth according to claim 1, wherein the plurality of first solutions are provided. A step of holding a first solution containing the polymer compound and a second solution different from the first solution in the storage unit, and the plurality of the first solution and the second solution are passed through the plurality of flow paths. The first solution is transferred to the second solution storage section, and the first solution and the second solution are stored in the plurality of second solution storage sections.
And a step of storing a plurality of mixed liquids mixed at different ratios with each other, and in the plurality of second solution storing sections respectively storing the plurality of mixed liquids, the solid element is controlled by the controlled valence electrons. Growing a crystal of the polymer compound under an electrical state brought to the surface of the crystal. 12. The method according to claim 11, further comprising a step of heating the solution in the first solution storage part, whereby the transfer of the solution from the first solution storage part through the flow path is promoted. The method for growing a crystal according to claim 11. 13. The solid state device further comprising a step of applying a voltage to at least the second solution storage part, whereby an electric state provided on a surface of the solid state device is controlled. Item 11 or 12
Crystal growth method. 14. The method according to claim 11, wherein a groove or a hole is formed in the second solution storage portion by micromachining, and crystallization of the polymer compound is promoted in the groove or the hole. 14. The method for growing a crystal according to any one of items 13 to 13. 15. The method according to claim 11, wherein the second solution is a buffer solution and / or a salt solution for changing a pH and / or a salt concentration of the first solution. The crystal growth method according to any one of claims 1 to 7.