WO2001086037A1

WO2001086037A1 - Crystal growth apparatus and crystal growth method

Info

Publication number: WO2001086037A1
Application number: PCT/JP2001/003782
Authority: WO
Inventors: Shigeru Inoue; Akira Sanjoh; Koji Akioka
Original assignee: Sumitomo Metal Industries, Ltd.
Priority date: 2000-05-12
Filing date: 2001-05-01
Publication date: 2001-11-15
Also published as: JP2001322900A

Abstract

An apparatus for promoting crystallization of biopolymer. An apparatus (10) for growing a crystal comprises a solid material (12) brought into contact with a solution (14) containing a biopolymer and electrodes (11a, 11b) for producing an electric field the direction of which is from the solution (14) to the solid material (12) or from the solid material (12) to the solution (14). The solid material (12) has at least first and second regions (12a, 12b) brought into contact with the solution (14) through a dielectric body. The density of charge in the dielectric body over the first region (12a) is different from that over the second region (12b) when a predetermined electric field is produced through the electrodes (11a, 11b). Therefore if more charge is created in either the first or second region (12a, 12b) when a voltage is applied, charged biopolymer in the solution (14) is selectively adsorbed to the region having the higher charge density and crystallized.

Description

Description Crystal growth equipment and crystal growth method ¾ Technical field

The present invention relates to an apparatus for growing crystals of a necessary substance, and more particularly to an apparatus applied to crystallization of various biopolymers such as proteins and enzymes.

Background art

Crystallization of biological macromolecules such as proteins is carried out in the same manner as in the case of low molecular weight compounds such as ordinary inorganic salts, by removing the solvent from water or a non-aqueous solution containing the macromolecules so that they become supersaturated and crystallized. It is fundamental to grow. Typical methods for this include a batch method, a dialysis method, and a gas-liquid phase diffusion method, which are properly used depending on the type, amount, and properties of the sample.

1A and 1B schematically show the hanging drop method and the sitting drop method included in the gas-liquid phase diffusion method. In the hanging-drop method shown in FIG. 1A, a mother liquor 221 containing a biomolecule to be crystallized is dripped in a closed container 220 containing a precipitant 222. In the sitting drop method shown in FIG. 1B, a mother liquor 221 containing a biopolymer to be crystallized is placed on a plate 233 in a closed container 230. The precipitant 222 is contained in another container 231 in the closed container 230. In these methods, equilibrium is slowly established by evaporation of the precipitant and volatile components in the mother liquor.

In order to determine the three-dimensional structure of a biopolymer by X-ray crystal structure analysis, it is essential to extract and purify the target substance and then crystallize it. In order to obtain biopolymer crystals, it is necessary to search under a large number of experimental conditions, and crystal growth is a bottleneck in the field of X-ray crystallography.

Disclosure of the invention

An object of the present invention is to provide an apparatus capable of promoting crystallization of a biopolymer. Specifically, an object of the present invention is to reduce the influence of convection in a solution due to the effect of gravity on the crystallization of various biopolymers and biological tissues mainly composed of biopolymers. And a device capable of controlling nucleation.

A further object of the present invention is to provide an apparatus capable of suppressing or controlling the mass production of microcrystals and obtaining a large crystal capable of X-ray structure analysis. It is a further object of the present invention to provide an apparatus for enabling crystallization with a small amount of a biopolymer solution.

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

It is a further object of the present invention to provide a method for preparing biopolymer crystals.

According to the present invention, there is provided an apparatus for growing crystals of a biopolymer contained in a solution. The apparatus comprises: a solid member having a surface for growing crystals in contact with a solution; a solution and a solid member in contact with the solution; and a direction from the solution to the solid member or from the solid member. Means for applying an electric field in a direction to the solution. In the device according to the present invention, at least a portion of the solid member that contacts the solution is formed of a dielectric. The solid member has at least a first region and a second region that are in contact with the solution via the dielectric and are adjacent to each other, and when a predetermined electric field is applied through a means for applying an electric field. The capacitance per unit area of the first region is different from the capacitance per unit area of the second region, so that either the surface of the first region or the surface of the second region is different. It promotes crystal growth more than the other.

In the device according to the present invention, the solid member can be composed of a plurality of types of dielectrics, and the dielectric in the first region and the dielectric in the second region can have different dielectric constants from each other.

In the device according to the present invention, the solid member may be a combination of a semiconductor and one or more dielectrics, and the dielectric may be laminated on the semiconductor. In this case, two or more kinds of dielectrics may be laminated on one kind of semiconductor, and the dielectric in the first region and the dielectric in the second region have different relative dielectric constants from each other. Can be.

Furthermore, in the device according to the present invention, the solid member is composed of a semiconductor having a different conductivity type and a 1 ' Alternatively, it can be a combination with two or more kinds of dielectrics, and the dielectrics can be laminated on a semiconductor. In this case, the semiconductor forming the first region and the semiconductor forming the cable 2 region can be of different conductivity types.

In a preferred embodiment, the device according to the invention further comprises an electrically insulating enclosure formed on the solid member for retaining the solution on the first and second regions. Further, the device according to the present invention can have a structure in which one of the first region and the second region protrudes from the solution more than the other.

In the device according to the invention, typically the means for applying an electric field consists of a pair of electrodes which respectively contact the solution and the solid member.

In the device according to the present invention, one of the surface of the first region and the surface of the second region can be smaller than the other. In that case, crystal growth can be promoted on the surface of the region having the smaller area.

In a preferred embodiment, the device according to the present invention is a device for growing a crystal of a biopolymer contained in a solution, comprising: a solid member having a surface on which a crystal is grown by contacting the solution; and a solid member. An enclosure wall provided on the solid member to hold the solution on the surface of the solid member, and an electrode attached to the enclosure wall, at least a portion of the solid member surrounded by the enclosure wall is formed of a dielectric. The dielectric has at least a first dielectric and a second dielectric, and the first dielectric and the second dielectric have different dielectric constants from each other. Features.

According to the present invention, there is provided a method for growing a crystal of a biopolymer contained in a solution using the above-described apparatus. The method comprises the steps of: holding a solution containing a biopolymer on a solid member; and applying an electric field to the solution and the solid member in contact with the solution in a direction from the solution to the solid member or from the solid member to the solution. Generating a charge on the surface of the first region and the surface of the second region; and maintaining the contact between the surface of the first region and the surface of the second region having the charge and the solution. In this case, the growth of biomolecular crystals is promoted more than either one of the surface of the first region and the surface of the second region.

The present invention also provides another method for growing crystals of a biopolymer contained in a solution using the above-described apparatus. The method comprises the steps of: A step of holding a solution containing a biopolymer on a member, a step of applying a voltage to the electrodes of the device to generate electric charges on the surfaces of the first dielectric and the second dielectric, Maintaining contact between the surface of the charged first dielectric and the surface of the second dielectric and the solution, wherein the surface of the first dielectric or the surface of the second dielectric is provided. In either case, the growth of the crystal of the biopolymer is promoted more than the other.

BRIEF DESCRIPTION OF THE FIGURES

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

FIG. 2 is a schematic sectional view showing a specific example of the device according to the present invention. · FIG. 3 is a schematic diagram showing a state in which biopolymers to be crystallized gather in the first region in the apparatus shown in FIG.

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

FIG. 5 is a diagram showing the relationship between capacitance and voltage for the device shown in FIG.

FIG. 6 is a schematic sectional view showing a solid member of the device according to the present invention.

FIG. 7 is a schematic sectional view showing another solid member of the device according to the present invention. FIG. 8 is a schematic sectional view showing a further solid component of the device according to the invention.

FIG. 9 is a schematic sectional view showing another solid member of the device according to the present invention.

FIG. 10 is a schematic diagram showing a state in which the biopolymer to be crystallized in the device according to the present invention gathers in the protruding first region.

FIG. 11 is a schematic diagram showing how a biopolymer to be crystallized is attracted to an electrode in the device according to the present invention.

FIG. 12A is a schematic sectional view showing another specific example of the device according to the present invention, and FIGS. 12B and 12C are schematic sectional views of electrodes of the device.

FIG. 13 is a schematic view showing a state in which a crystal grows in the narrowed first region in the device according to the present invention.

FIG. 14 is a schematic diagram showing patterns of the first and second regions used in the device according to the present invention.

FIG. 15 is a schematic diagram showing another pattern of the first and second regions used in the device according to the present invention.

FIG. 16 shows another pattern of the first and second regions used in the device according to the invention. It is a schematic diagram which shows a turn.

FIG. 17 is a schematic plan view showing a further specific example of the device according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

FIG. 2 shows an example of the device according to the present invention. The crystal growth apparatus 10 has a pair of opposing electrodes 11a and 11b, and a solid member 12 provided on the electrode 11a. If necessary, the device 10 has a structure 15 for securely damping the flow of the solution 14 containing the biopolymer to be crystallized on the solid member 12, and the structure 15 is an electric device. Made of insulating material. The structure 15 is typically an electrically insulating enclosure formed on the solid member 12. The electrode 11 b contacts the solution 14 held on the solid member 12. An electric field is generated by applying a voltage between the electrodes 11a and 11b. The direction of the electric field is the direction from the solution 14 to the solid member 12 or the opposite direction.

In the device 10, the solid member 12 has a first region 12a and a second region 12b adjacent thereto. The first region 12a has a surface area in contact with the solution 14 smaller than that of the second region 12b, and is arranged so as to be surrounded by the second region 12b. The first region 12a is made of a first dielectric, and the second region 12b is made of a second dielectric having a dielectric constant different from that of the first dielectric. Therefore, the capacitance per unit area in the first region 12a is different from the capacitance per unit area in the second region 12b. In the device 10, the surface of the first region 12a agglutinates biomolecules such as proteins on the surface of the solid member 12 in contact with the solution 14 by stronger electric action, and more strongly adsorbs them. It is an area to be made. The surface of the second region 12b is a region that does not adsorb much biomolecules such as proteins. This is achieved by making the capacitance per unit area of the first region larger than the capacitance per unit area of the second region, as described below. This is realized by making the relative permittivity of the second dielectric material larger than that of the second dielectric.

The capacitance per unit area of the first dielectric is C 1 and its relative permittivity is ε1, and the capacitance per unit area of the second dielectric is C 2 and its relative permittivity is ε 2 And Further, the first dielectric and the second dielectric have the same thickness. At this time, the following relationship holds regardless of the value of the applied voltage V g. C 1 = ( _£ 1 / ^/ _£ 2) C2

If the capacitance per unit area is different in each region, the applied charge voltage will cause a different charge density in each region. In the case of the apparatus 10 shown in FIG. 2, when the materials are selected so that _ε 1 is sufficiently larger than ε 2 to form the first dielectric and the second dielectric, C 1 is sufficiently larger than C 2 growing. Therefore, when a predetermined voltage Vg is applied between the electrode 11a and lib in the device 10 shown in FIG. 2, for example, as schematically shown in FIG. A high density of charges can be generated on the surface of the first region 12a. As shown schematically in FIG. 3, when the biopolymer 16 to be crystallized (for example, a protein) contained in the solution 14 is negatively charged, the electrode 11 a is positive and the electrode 11 b is negative. It is considered that by selecting the voltage Vg as described above, a high-density positive charge can be generated on the surface of the first region 12a, and the molecule 16 can be selectively adsorbed thereon. By this selective adsorption, crystal nuclei can be formed on the first region 12a, and crystal growth can be advanced. If the biopolymer to be crystallized is positively charged, a high-density negative charge may be generated in the first region 12a by applying a reverse electric field. As described above, in the device according to the present invention, by applying a voltage, the charge density generated in the first region and the charge density generated in the second region are positively differentiated, and the charged biopolymer is converted into a specific region. Can be absorbed and assembled.

In the present invention, the first region and the second region may each be formed from a plurality of materials. Also in this case, by making the electric capacity per unit area of the first region different from the electric capacity per unit area of the second region, the charge density generated in each region can be made different.

For example, if each region is composed of a combination of a dielectric and a semiconductor, the capacitance C 1 of the first region is the capacitance C d 1 of the dielectric and the capacitance of the semiconductor near the dielectric (capacity of the depletion layer) C s 1 Can be obtained as a combined capacitance connected in series. Similarly, the capacitance C 2 of the second region can be obtained as a combined capacitance of the capacitance C d 2 of the dielectric and the capacitance C s 2 of the semiconductor.

C 1 = 1 / (1 / C d 1 + 1 / C s 1)

C 2 = 1 / (1 / C d 2+ 1 / C s 2) While the same semiconductor is used for the first and second regions, if the dielectric of the first region and the dielectric of the second region are formed of different materials, the combined capacitance of each region is different. Thus, the C-V characteristics in the first region and the C-V characteristics in the second region when a voltage is applied can be made different. By appropriately adjusting the applied voltage, the capacitance C1 per unit area of the first region can be made different from the capacitance C2 per unit area of the second region. Different degrees can also be adjusted.

FIG. 4 shows a specific example of the device according to the present invention using a semiconductor. The crystal growth apparatus 40 includes a semiconductor substrate 42 such as a silicon substrate, a first dielectric layer 43 a and a second dielectric layer 43 b formed thereon, and both dielectric layers 43. It has an electrode 41 that contacts the solution 44 held on a and 43b. Adjacent first dielectric layers 43a 'and second dielectric layers 43b are provided on the same substrate 42 at substantially the same height. If necessary, the device 40 may be used to reliably block the flow of the solution 44 containing the biopolymer to be crystallized on the first dielectric layer 43a and the second dielectric layer 43b. It has a structure 45, and the structure 45 is made of an electrically insulating material. Structure 45 is typically an electrically insulating enclosure. The semiconductor substrate 42 also has a function as an electrode, and an electric field is generated by applying a voltage between the semiconductor 42 and the electrode 41. The direction of the electric field is the direction from the solution 44 to the semiconductor substrate 42 or the opposite direction.

In the device 40, the first region 46a is formed by the first dielectric layer 43a and the portion of the semiconductor substrate 42 thereunder, and the second dielectric layer 43b and the lower half thereof are formed. The second region 46 b is formed by the portion of the conductive substrate 42. The first region 46a is arranged such that the area of the surface in contact with the solution 44 is smaller than that of the second region 46b, and is surrounded by the second region 46b. Since the first dielectric layer 43a and the second dielectric layer 43b have different relative dielectric constants, when a predetermined voltage is applied, as described later, a unit of the first region is used. The capacitance C 1 per area is different from the capacitance C 2 per unit area of the second region, and the values of C 1 and C 2 change depending on the applied voltage. In the device 40, the surface of the first region 46a is a region in which biopolymers such as proteins are aggregated by stronger electric action and are more strongly adsorbed. The surface of the second region 46b is a region that does not adsorb much biomolecules such as proteins. this is, This is realized by making the relative permittivity of the first dielectric layer 43a larger than the relative permittivity of the second dielectric layer 43b. '

For example, a protein solution (electrolytic solution) is held on the device 40, and a voltage Vg is applied between the solution and the back surface of the substrate. The capacitance per unit area of the first dielectric layer is C d 1, its relative permittivity is ε 1, the capacitance per unit area of the second dielectric layer is C d 2, its relative permittivity If the rate is ε 2, then C d 2 = (ε 2 / ε 1) C d 1. If the semiconductor substrate is an n-type silicon substrate, the C 1 -V characteristics of the first region and the C 2 -V characteristics of the second region are as shown in FIG. 5, for example. In this case, C 1 is the combined capacitance of the capacitance C d 1 of the dielectric layer in the first region and the capacitance C s 1 of the semiconductor present in the first region, and C 2 is the capacitance of the dielectric layer in the second region. This is a combined capacitance of the capacitance C d 2 and the capacitance C s 2 of the semiconductor present in the second region. The capacitance of a semiconductor changes according to the applied voltage because the state of the depletion layer (width of the depletion layer) generated near the dielectric of the semiconductor changes according to the value of the applied voltage. Therefore, as shown in FIG. 5, the combined capacitance of the first region and the combined capacitance of the second region are not constant but change with various voltages. In this way, by combining the dielectric and the semiconductor, a region where the capacitance can be changed according to the value of the voltage can be formed.

In FIG. 5, at Vg く VI or V3 く Vg, the combined capacitance C1 of the first region is approximately equal to the dielectric layer capacitance Cd1 of the first region. Similarly, when Vg <VO or V2 <Vg, the combined capacitance C2 in the second region is approximately equal to the dielectric layer capacitance Cd2 in the second region. Therefore, in the case of Vg * VO or V3 * Vg, the capacitance difference between both regions is equal to the capacitance difference between the dielectric layers. On the other hand, when VO≤Vg≤V3, the capacitance difference between the two regions can be adjusted according to the value of Vg. For example, when Vg = Va, the capacitance difference between the two regions is maximum, and when Vg = Vb, the capacitance difference between the two regions is minimum. In this way, the charge density generated on the surface of each region can be made different, and the charge density between the first region and the second region can be adjusted by adjusting the applied voltage within an appropriate range. The difference can be adjusted. When a semiconductor and a dielectric are combined, the capacitance can be changed by changing the voltage, and the difference in charge density between the two regions can be changed. This means that the same equipment can be used to change the crystallization conditions and select more appropriate crystallization conditions. In the device according to the present invention, different dielectric layers 61 and 62 may be formed at the same height on a substrate 60 such as a semiconductor substrate as shown in FIG. 6, or as shown in FIG. The dielectric layer 72 forming one region may be formed so as to protrude from the dielectric layer 71 forming the second region. However, since the capacitance is proportional to the relative permittivity of the dielectric and is inversely proportional to the thickness of the dielectric, the capacitance per unit area of the first region is sufficiently larger than that of the second region. In order to achieve this, not only the permittivity but also the thickness must be appropriately selected.

Further, in the device according to the present invention, as shown in FIG. 8 or FIG. 9, the conductivity type of the semiconductor in the first region may be different from the conductivity type of the semiconductor in the second region. In the device 80, a region 83 made of p-type silicon is formed on a part of the n-type silicon substrate 81, and a dielectric layer 82 is formed thereon. The first region 86a includes a p-type Si, a portion of the dielectric layer 82 thereon, and a portion of the n-type Si below the p-type Si, and the second region 86b includes The remaining n-type Si portion and the portion of the dielectric layer 82 thereon are formed. The same dielectric is used for the first and second regions. In the device 80, regions of both conductivity types are formed in the substrate 81, and they are covered with the dielectric layer 82. On the other hand, in the device 90, the first region 96a has a protruding shape. On the n-type silicon substrate 91, projections 93 made of p-type silicon are formed, which are covered with a dielectric layer 92. The first region 96 a is composed of a p-type Si and a portion of the dielectric layer 92 thereon and an n-type Si below the p-type Si, and the second region 96 b The remaining n-type Si portion and the portion of the dielectric layer 92 thereon are formed. The same dielectric is used for the first and second regions. The first region has a structure protruding from the second region. These structures can be easily formed by ordinary techniques used in semiconductor integrated circuit manufacturing processes, such as ion implantation and lithography.

8 and 9, the CI-V characteristics in the first region are different from the C2-V characteristics in the second region. This is because the conductivity type of the semiconductor is different between the two regions. Therefore, when a voltage is applied between the solution and the back surface of the substrate, as in the case where the material of the dielectric is changed as described above, the value of the voltage per unit area of the first region can be selected by appropriately selecting the value of the voltage. The capacitance and the capacitance per unit area of the second region can be made different, so that the charge density generated on the surface of the first region and the capacitance of the second region The charge density generated on the surface can be different.

The voltage applied to the device according to the present invention is preferably a DC voltage such that the polarity of the electrode in contact with the solution is the same as the charge polarity of the biopolymer to be crystallized contained in the solution. For example, if a protein molecule to be crystallized in a solution is negatively charged, a negative voltage is applied to the electrode that contacts the solution. Then, the type, thickness, and applied voltage of the dielectric in each region are selected such that the capacitance per unit area of the first region is larger than the capacitance per unit area of the second region. With this selection, as schematically shown in FIG. 10, the charge density generated on the surface of the first region 102 becomes larger than the charge density generated on the surface of the second region 102. It is thought that the protein molecules 104 can be more strongly aggregated and adsorbed on the surface of the first region. In the case of a device having a semiconductor substrate, the magnitude of the voltage is desirably selected, for example, in the range of V0 to V3 shown in FIG. For example, it can be selected in the range of 15V to 0V. Conversely, when the polarity of the voltage applied to the electrode 101 is different from the polarity of the charge of the protein in the solution, the protein molecule 104 is connected to the electrode 101 as schematically shown in FIG. It is thought that the protein molecule 104 cannot move to the first region and cannot aggregate into the first region 102.

Further, the applied voltage may be an AC voltage obtained by biasing a positive or negative DC voltage.

The voltage can be applied at the appropriate intensity and for the required time. The voltage may be applied until crystal nuclei are formed, and the voltage application may be stopped after the nuclei are formed.

In order to hold the biopolymer solution on the substrate and bring the electrodes into contact with the solution, a structure (dam) that blocks the flow of the solution is created as shown in Fig. 2 or Fig. 4, and the inside of the structure After filling with a solution, the electrodes can be arranged to cover the structure. Instead, a hole may be provided in a part of the electrode, and after the electrode is placed on the structure, the biopolymer solution may be supplied from the hole until it comes into contact with the electrode. On the other hand, the biopolymer solution may be held on the substrate by surface tension, and the electrode may be held in contact with the solution.

In consideration of workability, as shown in Fig. 12A, an electrically insulating enclosure wall 1 25 is provided so as to surround the surface of the first area 1 26a and the surface of the second area 1 26b, Fence Preferably, the electrode 125 is attached to the inner surface of the electrode 125 in contact with the solution 124. In the device 120, a second dielectric film 123b is formed on a large part of the semiconductor substrate 122, and the first dielectric film 123a is formed only in a part thereof in a predetermined pattern. Have been. The first dielectric film 123a is formed so as to protrude from the surface of the second dielectric film 123b (to have a convex part). An enclosure wall 125 made of an electrically insulating material is formed on the second dielectric film 123 b, and an electrode 121 in contact with the solution 124 is provided on the enclosure wall 125. Further, an electrode 127 for connection to a power supply is arranged also on the back surface of the semiconductor substrate 122. These H poles 1 2 1 and

127 can be formed by using a general semiconductor integrated circuit manufacturing technique such as a sputtering method. The electrode 121 has, for example, a 〇r layer 121 a with a thickness of about 1 001 111 and a thickness of about 20011111 as shown in FIG. It can have a two-layer structure consisting of a t-layer 12 1 b force. Also, as shown in FIG. 12C, for example, the electrode 127 has a layer 501 a having a thickness of about 501 111, a layer 127 b having a thickness of about 30011111, and a layer 127 b having a thickness of about 50! ! ! ! ! It can have a three-layer structure consisting of the layer 丄 27 c. In addition, from the viewpoint of easy removal of the grown biopolymer crystal, the first region preferably has a convex portion as shown in FIG. 7, FIG. 9 or FIG. If crystals are grown on the projections, the crystals can be easily separated and collected from the upper surface of the projections having a relatively small area. In particular, it is desirable that the projection has a width such that the biopolymer crystal protrudes and grows. That is, it is desirable that the width of the projection is smaller than the diameter of the crystal to be obtained.

For example, as shown in FIG. 13, in the device according to the present invention, it is preferable that the surface 132a on which the biopolymer is to be strongly electrostatically adsorbed is provided on the protruding portion. As shown in FIG. 13, the protrusions are provided so as to protrude from the surface 132 b where the biopolymer is not easily adsorbed. The surface on which the organic molecules are to be adsorbed is provided on the projection. Usually, this surface is provided on top of the protrusion. In addition, as shown in Figure 13

Preferably, 132a has a width d such that the biopolymer crystal 134 can grow out of the surface 132a. That is, the width is smaller than the size (diameter) of the crystal to be formed. By setting the width of the projection surface in this way, the contact area between the grown crystal and the projection surface is limited. Thus, the convex part table The surface has limited adsorption power to crystals, making it easier to take out grown crystals. The width is set in an appropriate range according to the molecular species to be crystallized. For example, in the case of protein, crystals having a diameter of about 0.2 to about 0.5 mm are generally suitable for X-ray crystal structure analysis, so a width smaller than that diameter, for example, a width of 10 to 200 in Preferably, 10 to: The width of L 0 Ομπι is more preferable.

In the apparatus according to the present invention, the arrangement pattern of the region where crystallization is to be suppressed (second region) and the region where crystallization is to be promoted (first region) is arbitrary. For example, as shown in FIG. 14, an arrangement in which the first region 141 is surrounded by the second region 142 is preferably used. In addition, as shown in FIG. 15, a plurality of first regions 151 having a predetermined width may be arranged at predetermined intervals with respect to the second region 152, as shown in FIG. The first region 161 having a predetermined shape and area may be arranged in a matrix at a predetermined distance from the second region 162. In each case, the surface of the second regions 142, 152, 162 is significantly wider than the surface of the first regions 141, 151, 161.

These structures can be formed using general semiconductor integrated circuit manufacturing techniques such as a film forming technique, a photolithography technique, and an etching technique.

The dielectric used in the present invention include, for example, aluminum oxide (specific one _{_{Α 1 2 0 3, yA 1}} 2 0 3), titanium oxide, metal acid I arsenide materials such as copper oxide, aluminum nitride, nitride titanium , Tungsten nitride, tantalum nitride, metal nitrides such as TaSiN and WSin, and semiconductor compounds (oxides and nitrides) such as silicon oxide and silicon nitride. Preferred semiconductors for use in the present invention include silicon, gallium, arsenic (GaAs), gallium phosphorus (GaP), and the like.

For example, in the apparatus shown in FIG. 1 2, the second dielectric film which covers the majority of the substrate surface can be used S i 0 _2, S i ₃ in the first dielectric film to form a protrusion N ₄ and A 1 ₂ 〇 ₃ can be used. The dielectric constant of the S i 0 ₂ is the relative dielectric constant of about 3. 9, S i ₃ N ₄ is a dielectric constant of about 7. 5, A 1 ₂ 0 ₃ is about 9.5.

In order to grow protein crystals using the apparatus according to the present invention as shown in FIGS. 2, 4, and 12, a solution in which the target protein is dissolved is placed at a predetermined position (dam or enclosure wall) of the apparatus. Supply and store the solution and keep the electrode in contact with the solution. That is, the entire apparatus is sealed together with a precipitant in a glass container or the like, and stored in a cool place until crystals grow to a sufficient size. For example, keep it for about 100 hours. At this time, an appropriate voltage is applied between the electrodes of the device for the required time. For example, a voltage is applied until crystal nuclei are formed. The state of crystal growth can be observed on a closed glass container or with a microscope.

The precipitant may be placed in a separate container and sealed with the apparatus of the present invention, or a storage section 172 for storing the precipitant in the semiconductor substrate 17 1 as shown in FIG. 17 may be formed. However, the precipitant 173 may be stored therein and the entire surface may be covered with a glass lid 174. Such a reservoir can be easily prepared by anisotropically wet-etching the silicon substrate with a K〇H etching solution or the like.

Industrial applicability

ADVANTAGE OF THE INVENTION According to this invention, a biopolymer is selectively adsorbed to the surface of a specific area of a solid member by electric action, thereby reducing the influence of convection on the biopolymer. Nucleation can be stabilized. Further, according to the present invention, the crystallization conditions can be easily changed by changing the applied voltage. INDUSTRIAL APPLICABILITY The present invention can be used for purifying or crystallizing various biopolymers, particularly biopolymer electrolytes, in the pharmaceutical industry, the food industry, and the like. The present invention is particularly preferably applied to purify or crystallize proteins such as enzymes and membrane proteins, polypeptides, peptides, polysaccharides, nucleic acids, and complexes and derivatives thereof.

Claims

The scope of the claims

1. An apparatus (10, 40, 120) for growing a crystal of a biopolymer contained in a solution (14, 44, 124),

A solid member (1 2, 43 a, 43 b, 123 b, 123 b, 123 b) having a surface for growing a crystal by contacting the solution (14, 44, 124);

The solution (14,44,124) is added to the solution (14,44,124) and the solid member (12,43a, 43b, 123a, 123b) in contact therewith. Direction to the solid member (12, 43a, 43b, 123a, 123b) or the solid member (12, 43a, 43b, 123a, 123b) Means (11a, lib, 41, 121) for applying an electric field in the direction from the solution to the solution (14, 44, 124),

At least a portion of the solid member (12, 43a, '43b, 123a, 123b) that comes into contact with the solution (14, 44, 124) is formed of a dielectric. A first region (1 2a, 1 2a, 43 b, 123 b, 123 b, 123 b) that contacts the solution via the dielectric and is adjacent to each other; 43 a, 1 26 b) and at least a second region (1 2 b, 43 b, 1 26 b).

When a predetermined electric field is applied through the means (11a, lib, 41, 121) for applying the electric field, the first region (12a, 43a, 126a) The capacitance per unit area is different from the capacitance per unit area of the second region (12b, 43b, 126b), whereby the first region (1 2a, 43a, 126a) or the surface of the second region (12b, 43b, 126b), wherein the growth of the crystal is promoted more than the other. Growth equipment (10, 40, 120).

2. The solid member (12, 43a, 43b, 123a, 123b) is composed of a plurality of dielectrics,

The dielectric of the first region (12a, 43a, 126a) and the dielectric of the second region (12b, 43b, 126b) have different dielectric constants from each other. The crystal growth apparatus according to claim 1.

3. The solid member includes a semiconductor (60, 70, 81, 83, 91, 93, 122) and one or more dielectrics (61, 62, 71, 7). 2, 8 2, 9 2, 1 2 3 a, 1 23 b)

The dielectric (61, 62, 71, 72, 82, 92, 123 a, 123 b) is formed of the semiconductor (60, 70, 81, 83, 91, 93). The crystal growth apparatus according to claim 1, wherein the crystal growth apparatus is stacked on the crystal growth apparatus.

4. Two or more dielectrics (61, 62, 71, 72, 123a, 123b) are laminated on one type of semiconductor (60, 70, 122).

The dielectric (61, 72, 123a) of the first region and the dielectric (62, 71, 123b) of the second region have different dielectric constants from each other. Item 3

5. The solid member is a combination of a semiconductor (81, 83, 91, 93) having a different conductivity type and one or more dielectrics (82, 92).

The dielectric (82, 92) is laminated on the semiconductor (81, 83, 91, 93);

The semiconductor (83, 93) forming the first region (86a, 96a) and the semiconductor (81, 91) forming the second region (86b, 96b) are mutually 2. The crystal growth apparatus according to claim 1, wherein the apparatus is of a different conductivity type.

6. Place the solution (14, 44, 124) on the first area (12a, 43a, 126a) and the second area (12b, 43b, 126b). An electrically insulating enclosure (15, 45, 125) formed on the solid member (12, 43a, 43b, 123a, 123b) for holding. The crystal growth apparatus according to any one of claims 1 to 5.

7. Either of the first region (72, 96a, 126a) or the second region (71, 96b, 126b) is higher than the other with respect to the solution (124). The crystal growth apparatus according to claim 1, wherein the crystal growth apparatus has a protruding structure.

8. Means for applying the electric field (11a, 11b) Consisting of a pair of electrodes (11a, lib) that respectively contact the solution (14) and the solid member (12) The crystal growth apparatus according to any one of claims 1 to 7.

9. The surface of the first region (1 2a, 43a, 61, 72, 86a, 96a, 126a, 141, 1, 51, 161) and the second region Area (1 2 b, 4 3 b, 62, 71, 86 b, 96 b, 126 b, 142, 152, 16 2) Smaller than the area of

The crystal growth apparatus according to any one of claims 1 to 8, wherein the growth of the crystal is promoted on a surface of a region having a smaller area.

10. An apparatus (10, 40, 120) for growing a crystal of a biopolymer contained in a solution (14, 44, 124),

A solid member (12, 43a, 43b, 123a, 123b) having a surface for growing a crystal by contacting the solution (14, 44, 124);

In order to hold the solution (14, 44, 124) on the surface of the solid member (12, 43a, 43b, 123a, 123b), the solid member (12) , 43a, 43b, 123a, 123b) and the enclosure wall (15, 45, 125) provided above and attached to the enclosure wall (15, 45, 125). Electrodes (1 1 a, lib, 4 1, 1 2 1)

At least a part of the solid member (12, 43a, 43b, 123a, 123b) surrounded by the enclosing wall (15, 45, 125) is formed of a dielectric material. Wherein the dielectric has at least a first dielectric (12a, 43a, 123a) and a second dielectric (12b, 43b, 123b),

The first dielectric (12a, 43a, 123a) and the second dielectric (12b,

Crystal growth apparatus (10, 40, 120), characterized in that 43b, 123b) have different dielectric constants from each other.

1 1. Using the device (10, 40, 120) according to any one of claims 1 to 9, crystallizing a biopolymer crystal contained in the solution (14, 44, 124). A way to grow,

Holding a solution (14,44,124) containing a biopolymer on the solid member (12,43a, 43b, 123a, 123b);

The solution (14,44,124) is applied to the solution (14,44,124) and the solid member (12,43a, 43b, 123a, 123b) in contact with the solution (14,44,124). From the solid member (12, 43a, 43b, 123a, 123b) or from the solid member (12, 43a, 43b, 123a, 123b) An electric field in the direction toward the solution (14, 44, 124) is applied to the surface of the first region (12a, 43a, 126a) and the surface of the second region (12b, Generating a charge on the surface of 43b, 126b),

The surface of the first region (12a, 43a, 126a) having the charge and the surface of the second region (12b, 43b, 126b) and the solution (14, 44) , 1 2 4) to maintain contact with

Either one of the surface of the first region (12a, 43a, 126a) or the surface of the second region (12b, 43b, 126b) is more effective than the other. A crystal growth method that promotes crystal growth.

1 2. A method for growing crystals of a biopolymer contained in a solution (14, 44, 124) using the apparatus (10, 40, 120) according to claim 10. Holding a solution (14, 44, 124) containing a biopolymer on the solid member (12, 43a, 43b, 123a, 123b);

A voltage is applied to the electrodes (11a, lib, 41, 121) to apply a voltage to the surface of the first dielectric (12a, 43a, 123a) and the second dielectric. Generating a charge on the surface of the body (1 2 b, 4 3 b, 123 b);

The surface of the first dielectric (12a, 43a, 123a) having the charge and the surface of the second dielectric (12b, 43b, 123b) Maintaining contact with said solution (14, 44, 124);

Either the surface of the first dielectric (12a, 43a, 123a) or the surface of the second dielectric (12b, 43b, 123b) The method of growing a crystal according to any one of the preceding claims, wherein the growth of the crystal of the biopolymer is promoted.