Crystal manufacturing method and apparatus using a microchannel device

Abstract

To provide a crystal production method and a crystal production device, capable of controlling production of a crystal nucleus of protein.SOLUTION: A method for producing a biopolymer crystal includes steps of: producing a crystal nucleus of a biopolymer by introducing supersaturated solution of the biopolymer containing a crystallization agent into a micro flow path having a cross sectional area of 10,000 μm2 or less, and by placing it calmly; and growing the crystal by adding thereafter the solution containing the produced crystal nucleus into a solution in a metastable region containing the biopolymer, as the need arises: and to provide a biopolymer crystal production device utilizable for the method.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a crystal production method and apparatus using a microchannel device. [Background technology]

[0002] Structural biology of biopolymers such as proteins using quantum beams, including cryo-electron microscopy (cryoEM) and X-ray free electron lasers (XFEL), has developed into a very powerful analytical method in recent years. The main reason for this progress is that the molecular structure of proteins has been determined at the atomic level using X-rays and electron beams, and their functions have been elucidated. However, crystallization, which is performed when analyzing the structure of biopolymers such as proteins, is still extremely labor-intensive, and obtaining high-quality crystals is difficult.

[0003] When analyzing the crystal structure of biopolymers using X-rays or electron beams, it is difficult to observe hydrogen atoms and electrons, which are closely related to protein function. Furthermore, ionizing radiation can strip away electrons, damaging the sample. Neutron diffraction, on the other hand, allows for easy observation of hydrogen (especially the hydrogen and protonated states of water molecules) without damaging the sample. However, the weak incident light intensity requires high-quality, larger crystals as samples. While various techniques for biopolymer crystallization are known, they all rely heavily on trial and error when determining crystallization conditions, which poses a significant bottleneck in research. Therefore, there is still a need for improved methods for producing biopolymer crystals. In particular, few techniques have been reported that can control the generation of crystal nuclei.

[0004] Patent Document 1 discloses a device for promoting protein crystal growth, in which a protein solution and a solvent solution are flowed in parallel in a thin layer in a microfluidic channel, causing diffusion and forming a concentration gradient, thereby promoting crystallization. However, this method is aimed at simply promoting crystallization, rather than controlling the generation of crystal nuclei, and the structure of the microchannel does not allow for large crystal growth.

[0005] Patent Document 2 discloses a method for promoting crystallization by changing the concentration of a biomolecule solution through dialysis through a membrane in a channel, and a microfluidic chip for this purpose. This method is a more complex system in that it changes solution conditions such as concentration through dialysis in the channel, and it is thought that controlling the generation of crystal nuclei is difficult. It is also difficult to grow large crystals using this method.

[0006] Patent Document 3 discloses a protein crystallization method that improves the crystallization success rate and shortens the crystal growth time by applying a voltage to a supersaturated solution of protein introduced into the channel of a microfluidic device. However, Patent Document 3 does not disclose how to control the generation of crystal nuclei, and there are concerns about the effect of voltage application on the sample.

[0007] Patent Document 4 discloses a method in which a microchannel is filled with a medium such as fluorinated oil, microdroplets of a protein solution are formed in the medium with a controlled size, and one to three small crystals are generated in each microdroplet. However, with this method, the size of the microdroplets is a constraint, making it difficult to grow large crystals. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 2004-500241 [Patent Document 2] Special Publication No. 2005-538163 [Patent Document 3] Japanese Patent Application Laid-Open No. 2007-061672 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-112545 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a crystal production method and a crystal production apparatus that enable control of protein crystal nucleation. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to solve the above problems, and have found that 2 The present inventors have found that by using a microchannel having the cross-sectional area specified below, it is possible to discretely generate crystal nuclei of biopolymers in a supersaturated solution of biopolymers such as proteins that fills the microchannel, and that the number and density of the generated crystal nuclei can be controlled, thereby enabling a controlled number of crystal nuclei to be obtained and used for crystal growth, and have completed the present invention.

[0011] That is, the present invention includes the following. [1] A supersaturated solution of biopolymers containing a crystallizing agent is placed in a 10,000 μm 2 A method for producing biopolymer crystals, comprising introducing a biopolymer into a microchannel having the following cross-sectional area and allowing it to stand to generate crystal nuclei of the biopolymer. [2] The method according to [1] above, wherein the microchannel has a depth of 10 μm to 100 μm and a width of 10 μm to 100 μm. [3] The method according to [1] or [2] above, wherein the microchannel has a length of 10 mm or more. [4] The method according to any one of [1] to [3] above, comprising adding the generated crystal nuclei to a solution in a metastable region containing the biopolymer to grow crystals. [5] The method according to [4] above, wherein the supersaturated solution of the biopolymer containing the generated crystal nuclei is added to the solution in the metastable region in a volume containing 1 to 30 crystal nuclei. [6] The method according to [4] above, wherein the supersaturated solution of the biopolymer containing the generated crystal nuclei is added to the solution in the metastable region in a volume containing 1 to 10 crystal nuclei. [7] The method according to any one of [4] to [6] above, wherein the volume of the solution containing the generated crystal nuclei to be added is determined according to a prediction of the number of generated crystal nuclei by regression analysis using the initial concentration conditions of the supersaturated solution. [8] The method according to any one of the above [1] to [7], wherein the biopolymer is a protein. [9] The method according to any one of the above [1] to [8], wherein the crystallizing agent is sodium chloride or ammonium sulfate.

[10] The method according to any one of [1] to [9] above, wherein the standing is carried out for 1 hour to 26 hours.

[11] The method according to any one of [1] to [9] above, wherein the standing is carried out for 12 hours to 2 weeks.

[12] 10,000 μm for generating crystal nuclei of biopolymers 2 An apparatus for producing biopolymer crystals, comprising a microchannel device having a microchannel having the following cross-sectional area, a fluid inlet for introducing a supersaturated solution of a biopolymer containing a crystallization agent into the microchannel, and a fluid outlet from the microchannel.

[13] The device according to

[12] above, wherein the microchannel has a depth of 10 μm to 100 μm and a width of 10 μm to 100 μm.

[14] The device according to

[12] or

[13] above, wherein the microchannel has a length of 10 mm or more.

[15] The apparatus according to any one of the above

[12] to

[14] , wherein the fluid inlet is connected to a fluid injection means for injecting the supersaturated solution.

[16] The device according to

[15] above, wherein the fluid injection means is configured to be able to push out the supersaturated solution introduced into the microchannel from a fluid outlet of the microchannel.

[17] The apparatus according to any one of the above

[12] to

[16] , further comprising a reservoir for crystal growth.

[18] The apparatus described in

[17] above, wherein the supersaturated solution of the biopolymer containing the generated crystal nuclei is introduced into a crystal growth reservoir in a volume containing a controlled number of crystal nuclei.

[19] The device according to any one of the above

[12] to

[18] , further comprising: (i) a memory unit that stores the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallization agent; (ii) a calculation unit that predicts the number of crystal nuclei generated in a microchannel by regression analysis using the initial concentration conditions of the supersaturated solution; and (iii) a control unit that introduces the supersaturated solution of the biopolymer containing the generated crystal nuclei from the microchannel into a crystal growth reservoir in a volume that contains a controlled number of crystal nuclei.

[20] The method according to any one of [1] to

[11] above, which is carried out using the device according to any one of

[12] to

[19] above.

[0012] This invention is based in part on the results of the "FY2019 Ibaraki Prefecture, Ibaraki Prefecture Leading Research Commission on Neutron Structure Analysis Utilizing the Characteristics of the Life Material Structure Analysis Device." [Effects of the Invention]

[0013] According to the present invention, crystal nuclei of biopolymers such as proteins can be generated discretely, and the production of biopolymer crystals can be controlled. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram of an example of a crystal manufacturing apparatus using a microchannel device. [Figure 2] Figure 2 shows the crystallization phase diagram of hen egg white lysozyme (HEWL) prepared using HEWL and a crystallization agent (NaCl). The dotted line indicates the boundary between the supersaturated and metastable regions. [Figure 3] Figure 3 shows photographs of the formation of lysozyme crystals in the microchannels. A: 67 μm deep x 63 μm wide microchannel (lysozyme 40 mg / ml), B: 67 μm deep x 100 μm wide microchannel (lysozyme 20 mg / ml), C: 67 μm deep x 100 μm wide microchannel (lysozyme 15 mg / ml), D: 30 μm deep x 38 μm wide microchannel (lysozyme 40 mg / ml), E: 95 μm deep x 94 μm wide microchannel (lysozyme 40 mg / ml). [Figure 4] Figure 4 shows an example of the inter-crystal distance frequency distribution when a crystallization test was conducted in a microchannel. A: Microchannel with a channel depth of 95 μm and a channel width of 94 μm, B: Microchannel with a channel depth of 49 μm and a channel width of 60 μm. The horizontal axis shows the division of the inter-crystal distance between adjacent crystals (in 0.1 mm units), and the vertical axis shows the number of generated crystals (observation frequency) classified by the inter-crystal distance. [Figure 5] Figure 5 is a graph showing the relationship between the total solution volume (channel volume) of the microchannel and the number of lysozyme crystals formed in the entire microchannel. The lines and equations represent the approximate lines and equations for each lysozyme concentration obtained by regression analysis using least-squares fitting. Black circles: 40 mg / ml lysozyme solution + 0.8 M NaCl solution, black squares: 20 mg / ml lysozyme solution + 0.8 M NaCl solution, black triangles: 15 mg / ml lysozyme solution + 0.8 M NaCl solution. [Figure 6] Figure 6 is a graph showing the relationship between the channel volume per crystal produced and the lysozyme concentration. The approximation line is a logarithmic approximation curve. [Figure 7] FIG. 7 shows photographs showing observation images (A, B) of lysozyme crystals formed in a microfluidic device and their X-ray diffraction image (C). [Figure 8] Figure 8 shows photographs showing the time-dependent crystal growth of lysozyme crystal nuclei in a reservoir generated in a microfluidic device: A: Control (reservoir without lysozyme crystal nuclei left undisturbed for 6 days), B: reservoir with lysozyme crystal nuclei added left undisturbed for 5 days, C: reservoir with lysozyme crystal nuclei added left undisturbed for 6 days, D: reservoir with lysozyme crystal nuclei added left undisturbed for 8 days, E: reservoir with lysozyme crystal nuclei added left undisturbed for 12 days. [Figure 9] Figure 9 shows photographs showing the results of crystal growth in a reservoir of lysozyme nuclei generated in a microfluidic device. A: The state after 7 days of standing in a reservoir containing lysozyme nuclei (a total of approximately 15 crystals were confirmed). B: The state after 7 days of standing in a control reservoir (no lysozyme nuclei added) (no crystals). The presence of crystals is indicated by a white dashed line in Figure 9A. [Figure 10]Figure 10 shows the crystallization phase diagram of glucose isomerase prepared using glucose isomerase and a crystallization agent (ammonium sulfate). The dotted line indicates the boundary between the supersaturated region and the metastable region. [Figure 11] Figure 11 shows photographs (A to C) of observation images of glucose isomerase crystals formed in a microchannel device. A: Microchannel with a channel depth of 30 μm and a channel width of 38 μm, B: Microchannel with a channel depth of 30 μm and a channel width of 63 μm, C: Microchannel with a channel depth of 30 μm and a channel width of 100 μm. [Figure 12] Figure 12 shows photographs (A, B) of glucose isomerase crystals formed in a microchannel device. A: Microchannel with a channel depth of 49 μm and a channel width of 63 μm. B: Microchannel with a channel depth of 49 μm and a channel width of 100 μm. The arrows indicate the crystals. [Figure 13] Figure 13 is a graph showing the relationship between the amount of solution in the entire microchannel (channel capacity (channel volume)) and the number of glucose isomerase crystals formed in the entire microchannel. GI20 indicates a sample solution containing 20 mg / ml of glucose isomerase, and GI30 indicates a sample solution containing 30 mg / ml of glucose isomerase. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below.

[0016] The present invention relates to a method for producing biopolymer crystals, which is based on the finding that crystal nuclei of biopolymers can be generated discretely by crystallizing the biopolymers in a microchannel of a predetermined size (thinness) using a supersaturated solution of the biopolymer containing a crystallizing agent. 2We provide a method for producing biopolymer crystals, which comprises discretely generating crystal nuclei of the biopolymer by placing a supersaturated solution of the biopolymer containing a crystallization agent, filled in a microchannel having the following cross-sectional area, under conditions suitable for crystallization of the biopolymer (e.g., leaving it to stand). The method of the present invention may further comprise adding the crystal nuclei thus generated in the microchannel to a solution in the metastable region of the same biopolymer as the biopolymer in the supersaturated solution used, and growing crystals of the biopolymer.

[0017] In the present invention, a supersaturated solution of a biopolymer containing a crystallization agent is added to a 10,000 μm 2 By introducing the biopolymer into a microchannel having a cross-sectional area of ​​the following size and inducing crystal nucleation of the biopolymer, crystal nuclei of the biopolymer can be generated discretely within the microchannel.

[0018] In a preferred embodiment, the nucleation of biopolymer crystals in a microchannel can be induced using a batch method. The "batch method" is a technique for inducing crystallization of a crystalline substance (e.g., a biopolymer) by allowing a supersaturated solution of the crystalline substance (e.g., a biopolymer) containing a crystallization agent to stand in a sealed environment to prevent evaporation. Alternatively, the nucleation of biopolymer crystals in a microchannel can be induced using other crystallization methods, such as dialysis.

[0019] More specifically, the present invention provides a method for preparing a supersaturated solution of a biopolymer containing a crystallization agent by dissolving the solution in a 10,000 μm solution. 2 The present invention provides a method for producing biopolymer crystals, comprising: introducing a supersaturated solution of a biopolymer containing a crystallizing agent into a microchannel having a cross-sectional area of ​​10,000 μm or less, allowing the microchannel to stand to discretely generate crystal nuclei of the biopolymer, and then adding the generated crystal nuclei to a solution in a metastable region containing the biopolymer to grow crystals. 2 It is preferable to introduce the supersaturated solution into a microchannel having a cross-sectional area as follows and fill (pack) the microchannel with the supersaturated solution.

[0020] In the present invention, a "microchannel" refers to a channel whose depth and width (both internal dimensions) are within the range of 1 μm or more and less than 1 mm. The microchannel in the present invention preferably has a constant or nearly constant depth and width over its entire length or nearly its entire length in the flow direction. Here, "nearly" means that an error of ±1% is allowed.

[0021] In the present invention, 10,000 μm 2 By using a microchannel having the following cross-sectional area, crystal nuclei of biopolymers can be generated discretely. Here, the "cross-sectional area" of the microchannel refers to the area of ​​the cross section of the microchannel perpendicular to the bottom surface and central axis (flow direction) of the microchannel. In one embodiment, the microchannel used in the present invention has a cross-sectional area of ​​10 μm 2 ~10,000μm 2 and in a more preferred embodiment, a cross-sectional area of ​​100 μm 2 ~10,000μm 2 For example, 400 μm 2 ~10,000μm 2 , 500 μm 2 ~10,000μm 2 , 1000 μm 2 ~10,000μm 2 , 2500 μm 2 ~10,000μm 2 , 4000 μm 2 ~10,000μm 2 , 500 μm 2 ~9,000μm 2 , 1000 μm 2 ~9,000μm 2 , 2500 μm 2 ~9,000μm 2 , 4000 μm 2 ~9,000μm 2 , 1000 μm 2 ~7,000μm 2 , 2500 μm 2 ~7,000μm 2 , or 4000 μm 2 ~7,000μm 2 The cross-sectional area may be

[0022] The microchannel used in the present invention may have a depth and width of 200 μm or less, but is not limited thereto. In a preferred embodiment, the microchannel used in the present invention has a depth and width of 10 μm to 100 μm. The microchannel used in the present invention may have a depth and width independently of, for example, 10 μm to 100 μm, 14 μm to 100 μm, 20 μm to 100 μm, 30 μm to 100 μm, 40 μm to 100 μm, 60 μm to 100 μm, 80 μm to 100 μm, 10 μm to 80 μm, 20 μm to 80 μm, 30 μm to 80 μm, 40 μm to 80 μm, 60 μm to 80 μm, 10 μm to 70 μm, 20 μm to 70 μm, 30 μm to 70 μm, 40 μm to 70 μm, 60 μm to 70 μm, 10 μm to 50 μm, 20 μm to 50 μm, 30 μm to 50 μm, or 40 μm to 50 μm. In one embodiment, the microchannel used in the present invention may have a depth of, for example, 10 μm to 100 μm (e.g., 30 μm to 100 μm) and a width of 10 μm to 100 μm, 30 μm to 100 μm, 30 μm to 70 μm, or 60 μm to 100 μm.

[0023] In one embodiment, the microchannel used in the present invention may have a depth:width ratio of 1:4 to 4:1, preferably 1:2 to 2:1, for example, a depth:width ratio of 1:1.8 to 1.8:1, 1:1.5 to 1.5:1, 1:1.3 to 1.3:1, or 1:1.1 to 1.1:1.

[0024] The microchannel used in the present invention may have any length, but typically has a length of 1 mm or more. In a preferred embodiment, the microchannel may have a length of 10 mm or more, for example, 10 mm to 100 mm, 10 mm to 80 mm, 10 mm to 70 mm, 20 mm to 100 mm, 20 mm to 80 mm, 20 mm to 70 mm, 30 mm to 100 mm, 30 mm to 80 mm, 30 mm to 70 mm, 40 mm to 100 mm, 40 mm to 80 mm, 40 mm to 70 mm, 50 mm to 100 mm, 50 mm to 80 mm, or 50 mm to 70 mm.

[0025] The microchannel used in the present invention may be formed in a microchannel device (also referred to as a microfluidic device). The microchannel used in the present invention may have any shape. The microchannel used in the present invention may be linear, curved, bent, or serpentine when viewed from above in a state where it is installed on a horizontal plane (a plane perpendicular to the direction of gravity). In a more preferred embodiment, the microchannel used in the present invention is linear when viewed from above in a state where it is installed on a horizontal plane. The interior of the microchannel used in the present invention is preferably a closed space except for the fluid inlet to the microchannel and the fluid outlet from the microchannel. The microchannel device used in the present invention is used for producing biopolymer crystals and is preferably configured to be suitable for that application.

[0026] The microchannel used in the present invention does not need to have a means for actively generating fluid non-uniformity such as a concentration gradient, a concentration difference, or two or more fluid layers within a single microchannel (for example, a dialysis means, a sub-channel for forming a concentration gradient, etc.).

[0027] The microchannel and microchannel device used in the present invention may be made of any material suitable for microchannels and crystal production, and may be made of, for example, one or more materials selected from resins such as silicone resins such as polydimethylsiloxane (PDMS), cyclic olefin resins such as cycloolefin polymers (COP), and acrylic resins such as polymethyl methacrylate (PMMA), glass, quartz, and the like.

[0028] The biopolymers used in the methods of the present invention may be, for example, polypeptides such as proteins and oligopeptides, nucleic acids such as DNA or RNA, or crystal-forming polymers such as sugar chains. The biopolymers used in the methods of the present invention may be naturally occurring, or may not exist in nature but be artificially created (for example, by genetic engineering, or chemical synthesis or chemical modification). The biopolymers used in the methods of the present invention may be isolated from a living organism or synthesized.

[0029] In a preferred embodiment, the biopolymer used in the method of the present invention may be a protein. The protein may be a natural protein or a synthetic protein. The protein may be modified by the addition of a sugar chain, lipid, or the like, may be labeled with a radioisotope, a dye such as a fluorescent dye, or may be complexed with any compound, such as a low-molecular-weight compound such as a nucleic acid or a peptide. The protein may be a monomer or a polymer. The protein may also be a complex of two or more proteins. Examples of proteins include enzymes such as lysozyme and glucose isomerase.

[0030] In a preferred embodiment of the present invention, a supersaturated solution of a biopolymer containing a crystallization agent is introduced into a microchannel. The crystallization agent (precipitant) may be any additive used to promote crystallization, such as salts, amino acids and amino acid derivatives, organic solvents, or water-soluble polymers. Examples of crystallization agents include chlorides such as sodium chloride, potassium chloride, magnesium chloride, and calcium chloride; sulfates such as sodium sulfate, ammonium sulfate, magnesium sulfate, lithium sulfate, and cadmium sulfate; phosphates such as sodium phosphate, potassium phosphate, anhydrous ammonium phosphate, and anhydrous potassium phosphate; hydrogen phosphates such as sodium dihydrogen phosphate and potassium dihydrogen phosphate; nitrates such as sodium nitrate; acetates such as sodium acetate, potassium acetate, magnesium acetate, calcium acetate, ammonium acetate, and zinc acetate; citrates such as sodium citrate; formates such as sodium formate and potassium formate; potassium sodium tartrate; Examples of suitable crystallizing agents include, but are not limited to, organic acid salts such as sodium malonate and their solvates, amino acids such as arginine, glycine, lysine, aspartic acid, glutamic acid, and proline and their salts, amino acid derivatives such as glycine ethyl ester, glycinamide, and prolinamide and their salts, organic solvents such as 2-methyl-2,4-pentanediol, ethanol, methanol, isopropanol, n-propanol, tert-butanol, and dioxane, and water-soluble polymers such as polyethylene glycols and their derivatives having a molecular weight of approximately 400 to 20,000, polyethylene glycol monoalkyl ethers, polyethyleneimine, and glycerol. It is also preferable to select the crystallizing agent according to the type of biopolymer to be crystallized.

[0031] The supersaturated solution of biopolymer containing a crystallization agent is preferably a buffer solution containing the crystallization agent and the biopolymer. The buffer may be any buffer solution used for crystallization samples, including, but not limited to, acetic acid-sodium acetate buffer (e.g., 50 mM sodium acetate buffer; pH 3.7 to 5.6), citric acid-sodium citrate buffer (pH 3.0 to 6.2), sodium succinate-NaOH buffer (pH 3.8 to 6.0), sodium cadicholate-HCl buffer (pH 5.0 to 7.4), MES-NaOH buffer (pH 5.4 to 6.8), PIPES-NaOH buffer (pH 6.2 to 7.3), MOPS-NaOH buffer (pH 6.4 to 7.8), HEPES-NaOH buffer (pH 7.2 to 8.2), and Tris-HCl buffer (pH 7.1 to 8.9).

[0032] Depending on the solution conditions, biopolymer solutions containing crystallization agents are generally classified into (i) an undersaturated region (undersaturated phase) in which neither nucleation nor growth of biopolymer crystals occurs, (ii) a metastable region (metastable phase) in which only crystal growth of biopolymer crystals occurs, and (iii) a supersaturated region (supersaturated phase) in which nucleation and growth of biopolymer crystals occur. For example, those skilled in the art can easily create a crystallization phase diagram showing the undersaturated, metastable, and supersaturated regions by plotting the biopolymer concentration of the solution on the vertical axis and the crystallization agent concentration on the horizontal axis. A solution in the supersaturated region of the biopolymer is called a supersaturated solution of the biopolymer. As an example, a solution of 15 to 40 mg / mL of lysozyme in 50 mM sodium acetate buffer (pH 4.5) containing 0.8 M sodium chloride as a crystallization agent is a supersaturated solution of lysozyme. Furthermore, a solution of glucose isomerase at a concentration of 20 mg / mL to 40 mg / mL in 10 mM HEPES buffer (pH 7.7) containing 10 to 45% (w / v) ammonium sulfate as a crystallization agent is a supersaturated solution of glucose isomerase. Whether a biopolymer solution is in the supersaturated region (supersaturated solution), metastable region, or unsaturated region can be easily determined by those skilled in the art based on a crystallization phase diagram, for example, a crystallization phase diagram in which the biopolymer concentration is plotted on the vertical axis and the crystallization agent concentration is plotted on the horizontal axis.

[0033] In one embodiment of the present invention, it is preferable to introduce a supersaturated solution of a biopolymer containing a crystallization agent into the microchannel and fill the microchannel with the supersaturated solution. The supersaturated solution of a biopolymer containing a crystallization agent does not form microdroplets in the microchannel. The supersaturated solution of a biopolymer containing a crystallization agent may be a homogeneously mixed solution when introduced into the microchannel.

[0034] A supersaturated solution of a biopolymer containing a crystallization agent can be left standing in a microchannel to generate biopolymer crystal nuclei. In the present invention, "crystal nuclei" encompasses microcrystals, which are crystal nuclei in the early stages of growth. In one embodiment of the present invention, biopolymer crystals (nuclei) with lengths of 50 μm or less, 500 μm or less, 5 mm or less, or longer than 5 mm may be generated in the microchannel. When introduced into a reservoir as described below, the crystals (nuclei) generated in the microchannel may be, but are not limited to, shorter crystal nuclei (e.g., 50 μm or less in length). The time for leaving the solution in the microchannel may be any time that allows for the generation of biopolymer crystal nuclei. The time is not limited to the following, and may typically be 1 to 26 hours, e.g., 1 to 24 hours, 1 to 12 hours, 1 to 5 hours, or 1 to 3 hours. Alternatively, the standing time in the microchannel may be longer, such as 12 hours or more, 24 hours or more, 26 hours or more, or 48 hours or more, for example, 1 hour to 1 week, 1 hour to 10 days, 1 hour to 2 weeks, 1 hour to 1 month, 1 hour to 2 months, 12 hours to 1 week, 12 hours to 10 days, 12 hours to 2 weeks, 12 hours to 1 month, or 12 hours to 2 months, 24 hours to 1 week, 24 hours to 10 days, 24 hours to 2 weeks, 24 hours to 1 month, or 24 hours to 2 months. In the present invention, the standing time in the microchannel for crystal nucleation may be shorter than the time required for the crystal nuclei to grow into larger crystals, but is not limited thereto.

[0035] The supersaturated solution of biopolymer containing a crystallization agent may be left standing in a microchannel under temperature conditions suitable for crystallization. Such temperature conditions can be selected depending on the type of crystallization agent and biopolymer, but may typically be 4 to 25°C or 4 to 39°C, for example, 4 to 25°C, 5 to 30°C, 15 to 25°C, or 18 to 22°C.

[0036] In a microchannel into which a supersaturated solution of a biopolymer containing a crystallization agent is introduced, preferably filled with the solution, it is preferable to take measures to prevent evaporation of the solution when the microchannel is left standing, such as sealing the openings of the fluid inlet and outlet ports or connecting means such as tubes connected thereto with a sealing material (e.g., seal, grease) or covering the material surface.

[0037] As described above, crystallization of biopolymers occurs within the microchannel. By using the microchannel of the present invention, biopolymer crystal nuclei or single crystals grown from the crystal nuclei can be discretely generated. In the present invention, "discrete" refers to the overall tendency of each crystal nuclei or single crystal to form at dispersed locations within the microchannel without concentrating or agglomerating at a specific location. By using the microchannel of the present invention, biopolymer crystal nuclei or single crystals can be generated at intervals within a certain range, rather than at random intervals. This means that the number of crystal nuclei or single crystals generated can be controlled depending on the size of the microchannel used. Therefore, in the present invention, a solution containing the discretely generated biopolymer crystal nuclei or single crystals can be extruded through the microchannel to obtain a controlled number of crystal nuclei or single crystals. The present invention also provides a method for discretely generating biopolymer crystal nuclei or single crystals.

[0038] In the present invention, there is a correlation between the initial concentration of the supersaturated solution of biopolymer containing a crystallization agent, the volume of the microchannel used, and the number of biopolymer crystals formed throughout the entire microchannel. For example, the number of biopolymer crystals formed throughout the entire microchannel for each initial biopolymer concentration (typically the concentration upon introduction into the microchannel) of the supersaturated solution of biopolymer containing a crystallization agent is positively correlated with the volume of the microchannel (see, for example, Figure 5). In a preferred embodiment, this correlation can be expressed by a linear regression equation passing through the origin using the least squares method. The initial biopolymer concentration (typically the concentration upon introduction into the microchannel) of the supersaturated solution of biopolymer containing a crystallization agent is positively correlated with the number of biopolymer crystals formed per unit volume of the microchannel; in other words, it is negatively correlated with the volume of the microchannel per biopolymer crystal formed (the volume of the microchannel occupied by one crystal) (see, for example, Figure 6). Based on this correlation, it is possible to predict the number of crystal nuclei or single crystals (absolute number or density, etc.) generated for a given microchannel capacity using initial concentration conditions, such as the initial biopolymer concentration, for the supersaturated solution. Furthermore, based on the predicted number of crystal nuclei or single crystals generated, it is possible to predict the volume (volume or weight) of a solution (a supersaturated solution of a biopolymer containing crystal nuclei or single crystals, such as a supersaturated solution after standing or a diluted solution thereof) containing a controlled number of crystal nuclei or single crystals to be generated, preferably a desired number (e.g., but not limited to, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5). In the present invention, the term "controlled number" for crystals or crystal nuclei refers to a number within a specific intended range.

[0039] The number of crystal nuclei or single crystals to be produced, or the volume of a solution containing a desired number of crystal nuclei or single crystals to be produced, can be predicted by regression analysis based on the correlation between the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallization agent (such as the initial biopolymer concentration, initial crystallization agent concentration, initial buffer concentration, and / or initial hydrogen ion concentration (pH)) and the number of biopolymer crystals produced per unit volume of a microchannel or the volume of a microchannel per biopolymer crystal to be produced. Regression analysis can be performed, for example, by the least squares method, but is not limited to this. Regression analysis may be simple or multiple regression analysis. In regression analysis, the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallization agent, such as the initial biopolymer concentration and, optionally, the initial crystallization agent concentration, can be used to predict the number of crystal nuclei or single crystals to be produced, or the volume of a solution containing a controlled number, preferably a desired number (e.g., 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5), of crystal nuclei or single crystals from a regression equation.

[0040] According to such predicted values, a solution that will contain the predicted or controlled number, preferably the desired number (e.g., 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5) of crystal nuclei or single crystals can be removed from the microchannel after standing. The method for removing the solution containing crystal nuclei or single crystals from the microchannel is not particularly limited, but may be performed by additionally injecting a supersaturated solution of a biopolymer containing a crystallization agent or other liquid medium into the microchannel from a fluid inlet, thereby pushing the supersaturated solution in the microchannel and the generated crystal nuclei or single crystals contained therein out of the fluid outlet of the microchannel.

[0041] To promote the growth of crystal nuclei, the solution containing the generated crystal nuclei or single crystals removed from the microchannel, particularly the crystal nuclei (e.g., a supersaturated solution of the biopolymer containing the generated crystal nuclei), can be added to a metastable region solution containing a biopolymer. The metastable region solution containing a biopolymer may be contained in a crystal growth reservoir. The metastable region solution typically contains the same biopolymer as the biopolymer in the supersaturated solution used. The metastable region solution containing a biopolymer may contain a crystallization agent. Preferably, the metastable region solution contains the same crystallization agent as the crystallization agent in the added supersaturated solution, but this is not a limitation. The metastable region solution may contain the crystallization agent at a concentration that is the same as or approximately the same (±20%) as the initial crystallization agent concentration in the supersaturated solution used for crystal nucleation in the microchannel, but this is not a limitation.

[0042] The solution containing the generated crystal nuclei removed from the microchannel may be added to the solution in the metastable region at a volume containing a controlled number of crystal nuclei, preferably a relatively small number, preferably 1 to 100, more preferably 1 to 30 or 1 to 20, even more preferably 1 to 15 or 1 to 10, particularly preferably 1 to 5, and most preferably 1 crystal nuclei. Such a volume can be determined, as described above, according to the initial concentration conditions of the supersaturated solution of biopolymer containing a crystallization agent, for example, the number of crystal nuclei generated by regression analysis using the initial biopolymer concentration and, optionally, the initial crystallization agent concentration. Adding a relatively small number of crystal nuclei to the solution in the metastable region can further promote the growth of the crystal nuclei, allowing them to grow into larger crystals.

[0043] The solution containing the generated crystal nuclei (crystal nuclei) extracted from the microchannel is added to a solution in the metastable region, and the crystal nuclei can be grown by a conventional method, such as a batch method. For example, crystal growth can be promoted by allowing the solution in the metastable region to which the crystal nuclei have been added to stand, preferably in a sealed state. In this way, it becomes possible to produce high-quality crystals of a size suitable for structural analysis of biopolymer crystals.

[0044] In a preferred embodiment, the method for producing biopolymer crystals of the present invention can be suitably carried out using the above-mentioned microchannel device or an apparatus for producing biopolymer crystals suitable for the method. The present invention also provides such an apparatus for producing biopolymer crystals, preferably for use in the method of the present invention.

[0045] The biopolymer crystal manufacturing apparatus usable in the biopolymer crystal manufacturing method of the present invention is a 10,000 μm 2 The present invention may include a microchannel device having a microchannel having the following cross-sectional area, a fluid inlet to the microchannel, and a fluid outlet from the microchannel. The cross-sectional area, depth, width, length, shape, and other structures and materials of the microchannel are as described above. The microchannel of the present invention may not have branching channels and may be one-dimensional. The microchannel of the present invention can discretely generate crystal nuclei of a biopolymer. The fluid inlet is for introducing a supersaturated solution of a biopolymer containing a crystallization agent into the microchannel, but can also be used to introduce other fluids into the microchannel. The microchannel device of the present invention does not need to further include an inlet and / or channel for introducing a crystallization agent into the microchannel separately from the biopolymer. The microchannel device of the present invention may have one or more microchannels.

[0046] The biopolymer crystal production apparatus according to the present invention may further include a crystal growth reservoir. The crystal growth reservoir may be connected to the fluid outlet of the microchannel device. The crystal growth reservoir is a container that contains a biopolymer solution in a metastable region and into which a solution containing crystal nuclei removed from the microchannel is added to grow crystals. The fluid outlet of the microchannel device and the crystal growth reservoir may be connected by any connecting means (such as a tube, a channel, and / or a connector) so that the solution containing crystal nuclei can be introduced into the crystal growth reservoir.

[0047] In one embodiment, the fluid inlet is connected to a fluid injection means for injecting a supersaturated solution of a biopolymer containing a crystallization agent. Such a fluid injection means may be, for example, a syringe, a syringe pump, a dispenser, etc. The fluid inlet may be connected to the fluid injection means via any connecting means, for example, a tube. The fluid injection means may also be used to inject a fluid other than a supersaturated solution of a biopolymer containing a crystallization agent into the fluid inlet.

[0048] The microchannel device (also referred to as a microfluidic device) may be, for example, a microchannel chip.

[0049] The fluid injection means may be configured to extrude a solution introduced into the microchannel, typically a supersaturated solution of a biopolymer containing a crystallization agent, from the fluid outlet of the microchannel. Specifically, the fluid injection means is preferably configured to apply pressure to send a liquid so that an additional solution can be injected into the microchannel from the fluid inlet while the solution introduced into the microchannel is extruded from the fluid outlet.

[0050] The microchannel device of the present invention may also be configured so that a solution containing crystal nuclei generated in the microchannel (typically, a supersaturated solution of the biopolymer containing the generated crystal nuclei) is introduced into a crystal growth reservoir at a volume containing a controlled number of crystal nuclei, preferably a desired number, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5. Such a volume can be determined according to the initial concentration conditions of the supersaturated solution of the biopolymer containing a crystallization agent, for example, a prediction of the number of crystal nuclei generated by regression analysis using the initial biopolymer concentration and, optionally, the initial crystallization agent concentration.

[0051] FIG. 1 illustrates the structure of a biopolymer crystal production apparatus. As shown in FIG. 1, a microchannel device 6 having a microchannel 1 is composed of a substrate 5 and a glass slide 4. The microchannel 1 is formed by a groove formed on the substrate 5 and the glass slide 4 attached to the substrate 5 to cover the groove. At one end of the microchannel 1, a fluid inlet 2 is provided on the substrate 5 to allow fluid to be introduced into the microchannel from outside the microchannel device. At the other end of the microchannel 1, a fluid outlet 3 is provided on the substrate 5 to allow fluid to be discharged from the microchannel to outside the microchannel device. The fluid inlet 2 is connected to a fluid injection means 7, which injects a supersaturated solution of a biopolymer containing a crystallization agent (crystallization agent-biopolymer supersaturated solution) into the fluid inlet 2 and then introduces it into the microchannel. A crystal growth reservoir 8 is connected to the fluid outlet 3 from the microchannel of the microchannel device, preferably via a tube or the like. After a settling time, the solution introduced into the microchannel is removed from the microchannel by, for example, being pushed out or collected through the fluid outlet 3, and then introduced into the crystal growth reservoir 8. The fluid outlet 3 and the crystal growth reservoir 8 are preferably connected so that the solution removed from the microchannel is added to the solution in the metastable region in the crystal growth reservoir 8. The microchannel device 6 and its components, such as the substrate 5, may be made of any material suitable for microchannels and crystal production, and may be made of one or more materials selected from the group consisting of silicone resins such as polydimethylsiloxane (PDMS), cyclic olefin resins such as cycloolefin polymer (COP), and acrylic resins such as polymethyl methacrylate (PMMA), as well as glass, quartz, and the like.

[0052] In one embodiment, the biopolymer crystal production apparatus according to the present invention may further comprise a memory unit, a calculation unit, and / or a control unit. The biopolymer crystal production apparatus according to the present invention may further comprise: (i) a memory unit that stores the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallization agent; (ii) a calculation unit that predicts the number of crystal nuclei generated in the microchannel by regression analysis using the initial concentration conditions of the supersaturated solution (such as the initial biopolymer concentration, for example, the initial biopolymer concentration and the initial crystallization agent concentration); and (iii) a control unit that introduces a solution containing the generated crystal nuclei (typically, a supersaturated solution of the biopolymer containing the generated crystal nuclei) from the microchannel into a crystal growth reservoir at a volume containing a controlled number of crystal nuclei, preferably a desired number, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 crystal nuclei.

[0053] The memory unit may store data on the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallization agent (initial biopolymer concentration, initial crystallization agent concentration, initial buffer concentration, and / or initial hydrogen ion concentration (pH), etc.). The memory unit may also store experimental data or analytical data thereof, such as the type of biopolymer, the type of crystallization agent, the type of buffer solution, the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallization agent (initial biopolymer concentration, initial crystallization agent concentration, initial buffer concentration, and / or initial hydrogen ion concentration (pH)), crystallization phase diagram data (e.g., data such as those shown in Figures 2 or 10), microchannel conditions (e.g., data on channel volume or channel depth and channel width), data on the number of biopolymer crystals generated associated with the channel volume of the microchannel, data indicating the number of biopolymer crystals generated per unit volume of the microchannel (e.g., data such as those shown in Figures 5 or 13), and / or data on the microchannel volume per generated biopolymer crystal (e.g., data such as those shown in Figure 6), data on the results of correlation analysis and / or regression analysis thereof, or a database or dataset containing such data. The memory unit may also store programs for performing such correlation analysis and / or regression analysis, or analyses such as predictions based thereon. The data such as the initial concentration conditions of the supersaturated solution stored in the storage unit may be data (input data) inputted by an input unit described later.

[0054] The calculation unit may perform calculation processing to predict the number of crystal nuclei generated in the microchannel used by regression analysis using the initial concentration conditions of the supersaturated solution used, such as the initial biopolymer concentration and, optionally, the initial crystallization agent concentration. This regression analysis may be performed using the initial concentration conditions of the supersaturated solution used, such as the initial biopolymer concentration and, optionally, the initial crystallization agent concentration, with reference to experimental data or analytical data thereof, such as crystallization phase diagram data (e.g., data such as those in Figures 2 or 10), microchannel conditions (e.g., data on channel volume or channel depth and channel width), data on the number of biopolymer crystals generated associated with the channel volume of the microchannel, data showing the number of biopolymer crystals generated per unit volume of the microchannel (e.g., data such as those in Figures 5 or 13), and / or data on the microchannel volume per generated biopolymer crystal (e.g., data such as those in Figure 6), or a database or dataset containing such data. For example, the initial biopolymer concentration of a supersaturated solution of biopolymer containing a crystallization agent shows a positive correlation with the number of biopolymer crystals produced per unit volume of the microchannel, and a negative correlation with the microchannel volume per biopolymer crystal produced. Therefore, a predicted value for the number of crystal nuclei produced in the entire microchannel can be obtained by regression analysis from the initial biopolymer concentration of the supersaturated solution of biopolymer containing a crystallization agent and, in some cases, the initial crystallization agent concentration.

[0055] The calculation unit may also perform calculations to predict the volume of a solution containing a controlled number of crystal nuclei generated in the microchannel, preferably a desired number, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 (typically, a supersaturated solution of a biopolymer containing the generated crystal nuclei, which may contain a crystallization agent) by regression analysis using the initial biopolymer concentration of the supersaturated solution and, optionally, the concentration of the crystallization agent. For example, the initial biopolymer concentration of a supersaturated solution of biopolymer containing a crystallization agent shows a positive correlation with the number of biopolymer crystals generated per unit volume of the microchannel, and a negative correlation with the microchannel volume per generated biopolymer crystal. Therefore, a predicted value for the number of crystal nuclei generated in the entire microchannel can be obtained by regression analysis from the initial biopolymer concentration of a supersaturated solution of biopolymer containing a crystallization agent and, in some cases, the initial crystallization agent concentration. Based on this predicted value, a predicted value for the volume of a solution containing a controlled number of generated crystal nuclei, preferably a desired number, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 (typically, a supersaturated solution of biopolymer containing generated crystal nuclei, which may contain a crystallization agent) can be calculated (determined).

[0056] More specifically, for example, by referring to the initial concentration conditions of the supersaturated solution of the biopolymer containing the crystallizing agent stored in the memory unit, and reading and executing a program, database, or data set for performing data analysis such as correlation analysis and / or regression analysis stored in the memory unit, etc., the predicted volume of the supersaturated solution of the biopolymer containing the crystallizing agent containing a controlled number of crystal nuclei to be generated, preferably a desired number, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, can be calculated. The calculation unit may also perform further calculations to calculate the predicted number of crystal nuclei generated in the entire microchannel (e.g., absolute number or generation density), the predicted number of generated crystal nuclei to be added to the biopolymer solution in the metastable region, or the predicted volume of the solution containing the crystal nuclei to be added, by referring to the initial concentration conditions of the supersaturated solution of the biopolymer containing the crystallizing agent stored in the memory unit, and reading and executing a program, database, or data set for performing data analysis such as correlation analysis and / or regression analysis stored in the memory unit.

[0057] The control unit may control the microchannel device (e.g., its fluid injection means, fluid outlet, or crystal growth reservoir, means for connecting the fluid outlet and the crystal growth reservoir, etc.) so that a solution containing crystal nuclei generated in the microchannel is introduced from the microchannel to the crystal growth reservoir in a volume containing a controlled number of crystal nuclei, preferably a desired number, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 crystal nuclei.

[0058] The biopolymer crystal production apparatus according to the present invention may further comprise an input unit for inputting data such as the initial concentration conditions of the supersaturated solution of biopolymer containing a crystallization agent. The biopolymer crystal production apparatus according to the present invention may further comprise an output unit for outputting a predicted value of the number of crystal nuclei generated in the microchannel, and a display unit for displaying the predicted value. The biopolymer crystal production apparatus according to the present invention may also further comprise an output unit for outputting a predicted value of the volume of solution containing a controlled number of generated crystal nuclei, preferably a target number, for example, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 nuclei, to be added to the biopolymer solution in the metastable region, and / or a display unit for displaying the predicted value.

[0059] The biopolymer crystal manufacturing apparatus according to the present invention may be configured to be connectable to a communications network. Examples of communications networks include, but are not limited to, the Internet, intranets, LANs, VANs, CATV communications networks, virtual communications networks, and satellite communications networks. When connected to a communications network, the biopolymer crystal manufacturing apparatus according to the present invention may have a control unit that, using the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallizing agent, performs a calculation process on the communications network to predict the number of crystal nuclei generated in a microchannel and / or the volume of a solution containing a controlled number of generated crystal nuclei, preferably a target number, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, by regression analysis, and introduces the predicted volume of solution from the microchannel into a crystal growth reservoir.

[0060] The method and apparatus for producing biopolymer crystals according to the present invention as described above can control the number or density of biopolymer crystal nuclei generated, thereby allowing a controlled number of crystal nuclei to grow. It is believed that the method and apparatus for producing biopolymer crystals according to the present invention can also be advantageously used to grow larger, higher-quality crystals of biopolymers such as proteins. For example, the ability to easily produce large, high-quality crystals is extremely useful for crystal structure analysis using neutron diffraction, which requires larger crystals than X-ray diffraction. [Example]

[0061] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0062] [Example 1] Creation of crystallization phase diagram In this example, a crystallization phase diagram for the protein hen egg white lysozyme (HEWL; Sigma-Aldrich) was created.

[0063] First, a 100 mM acetic acid solution and a 100 mM sodium acetate solution were mixed and adjusted to pH 4.5, and then an equal volume of distilled water was added to prepare a 50 mM sodium acetate buffer solution (pH 4.5).

[0064] Lysozyme solutions and NaCl solutions were prepared by adding and dissolving uncrystallized HEWL or sodium chloride (NaCl) as a crystallization agent to the above-prepared buffer solution to a concentration twice the desired concentration (HEWL: 5, 10, 15, 20, 25, 30, 35, or 40 mg / mL, NaCl: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 M). The lysozyme (HEWL) concentration (C [mg / mL] (wt / v)) was determined by measuring the absorbance A at 280 nm using a spectrophotometer (Ultrospec 2100 pro; Amersham Bioscience). The following lysozyme absorbance coefficient (ε) and equation were used to calculate the concentration. L represents the optical pathlength.

[0065] Absorbance A = Lysozyme absorbance coefficient (ε) 2.69 × C [mg / mL] × L [cm]

[0066] 3 μl of each of the prepared lysozyme solution and NaCl solution was added to the wells of a plate in a microbatch kit (Greiner Bio-One) and mixed to prepare a sample. This was then covered with 5 μl of liquid paraffin to prevent evaporation. The plate was left in a constant temperature bath (incubator) set at 20°C for 4 days to allow crystallization. Each sample was tested in quadruplicate on 4 plates. The crystallization conditions were as follows:

[0067] [Table 1]

[0068] After 4 days of standing, the droplets in the wells of the plate were observed using a stereomicroscope to determine whether or not crystals formed. A crystallization phase diagram was created based on the lysozyme and NaCl concentrations of each sample and the presence or absence of crystal formation (Figure 2). In Figure 2, a star indicates no crystal precipitation (0 / 4), an × indicates crystal precipitation in one of four plates (1 / 4), a triangle indicates crystal precipitation in two of four plates (2 / 4 = 1 / 2), a square indicates crystal precipitation in three of four plates (3 / 4), and a circle indicates crystal precipitation in four of four plates (4 / 4). Furthermore, for each marker (star, ×, triangle, square, and circle), open circles represent samples with lysozyme concentrations of 35-40 mg / ml and NaCl concentrations of 0.3-1.0 M, shaded circles represent samples with lysozyme concentrations of 20-30 mg / ml and NaCl concentrations of 0.3-1.0 M, and filled circles represent samples with lysozyme concentrations of 5-30 mg / ml and NaCl concentrations of 0.1-0.2 M, or lysozyme concentrations of 5-15 mg / ml and NaCl concentrations of 0.3-1.0 M.

[0069] The boundary between the supersaturated and metastable regions (dotted line in Figure 2; crystal nucleation boundary) is shown for each lysozyme concentration and NaCl concentration. In Figure 2, the supersaturated region is above the dotted line, and the metastable region is below the dotted line.

[0070] [Example 2] Crystal formation using a microfluidic device In this example, to investigate the conditions for discrete formation of protein crystals in microchannels, crystallization tests were conducted using a microchannel device (also called a microfluidic device) with protein solutions of different concentrations (all within the supersaturated region) and microchannels of different widths and depths (both internal dimensions).

[0071] The test conditions were as follows:

[0072] [Table 2]

[0073] The microchannel device used was a microchip that had a microchannel (length 60 mm, depth and width of the channel as shown in Table 2) formed by adhering a glass slide to a substrate made of polydimethylsiloxane (PDMS) that had a groove that constituted the microchannel, a fluid inlet to the microchannel, and a fluid outlet from the microchannel, to cover the groove.

[0074] A 100 mM acetic acid solution and a 100 mM sodium acetate solution were mixed and adjusted to pH 4.5, and then an equal volume of distilled water was added to prepare a 50 mM sodium acetate buffer solution (pH 4.5).

[0075] Lysozyme solutions and NaCl solutions were prepared by adding and dissolving uncrystallized lysozyme (HEWL) or NaCl as a crystallization agent to the buffer solution to a concentration twice the target concentration (HEWL: 15, 20, or 40 mg / ml, NaCl: 0.8 M). The lysozyme concentration was measured using a spectrophotometer (Ultrospec 2100 pro; Amersham Bioscience) in the same manner as above.

[0076] The prepared lysozyme solution and NaCl solution were mixed in a 1:1 volume ratio to prepare a sample solution. The prepared sample solution was introduced into the microchannel through the fluid inlet of the microchannel device, filling the microchannel with the sample solution. After the sample solution was introduced, the fluid inlet was sealed with vacuum grease, and a clear seal was attached to the surface of the microchannel device to prevent evaporation of the sample solution. Next, the microchannel device, into which the sample solution had been introduced and filled, was left at 293 K (20°C) for the time listed in Table 2, allowing crystals (crystal nuclei) to form within the microchannel. After the standing time had elapsed, the microchannel was observed using a stereomicroscope, and the number of crystals formed and the spacing between each crystal were measured.

[0077] Observation results showed that lysozyme (HEWL) crystals were formed discretely (dispersed) within all microchannels tested. The crystals that formed were single crystals. Figure 3 shows an example of a photograph showing the discrete formation of lysozyme crystals within a microchannel. This is the first time that discrete crystal formation has been observed in such a one-dimensional microchannel.

[0078] Furthermore, the channel depth x channel width (both internal dimensions) = 14 μm x 31 μm (HEWL 30 mg / ml, NaCl 0.8 M), 14 μm x 50 μm (HEWL 30 mg / ml, NaCl 0.8 M), 30 μm x 19 μm (HEWL 40 mg / ml, NaCl 0.8 M), 30 μm x 38 μm (HEWL 17.5 mg / ml, NaCl 0.4 M), 30 μm x 63 μm (HEWL 40 mg / ml, NaCl 0.8 M), 30 μm x 100 μm (HEWL 40 mg / ml, NaCl 0.8 M), 49 μm x 60 μm (HEWL 17.5 mg / ml, NaCl 0.4 M), 49 μm x 63 μm (HEWL 40 mg / ml, NaCl In experiments conducted in the same manner as above, except that microchannels with dimensions of 67 μm x 63 μm (HEWL 17.5 mg / ml, NaCl 0.8 M), 67 μm x 63 μm (HEWL 17.5 mg / ml, NaCl 0.4 M), and 95 μm x 94 μm (HEWL 17.5 mg / ml, NaCl 0.4 M) were used (the lysozyme concentration and NaCl concentration conditions are in parentheses), discrete formation of lysozyme crystals was observed in each microchannel. In experiments using microchannels with channel depth x channel width = 14 μm x 31 μm (HEWL 30 mg / ml, NaCl 0.8 M), 30 μm x 38 μm (HEWL 17.5 mg / ml, NaCl 0.4 M), 49 μm x 60 μm (HEWL 17.5 mg / ml, NaCl 0.4 M), 67 μm x 63 μm (HEWL 17.5 mg / ml, NaCl 0.4 M), and 95 μm x 94 μm (HEWL 17.5 mg / ml, NaCl 0.4 M), 13, 9, 3, 7, and 4 crystals were observed, respectively.

[0079] The crystal spacing measurements showed that many crystals formed within a certain range of spacing within the microchannel. Figure 4 shows the frequency distribution of intercrystal distances for (A) a crystallization test using a 40 mg / ml lysozyme solution (NaCl 0.8 M) in a microchannel with a channel depth of 95 μm and a channel width of 94 μm, and (B) a crystallization test using a 40 mg / ml lysozyme solution (NaCl 0.8 M) in a microchannel with a channel depth of 49 μm and a channel width of 60 μm.

[0080] Figure 5 shows the relationship between channel volume and the number of crystals generated in the entire channel. A positive correlation was observed between channel volume and the number of crystals generated in the entire channel at all lysozyme concentrations. Furthermore, Figure 6 shows the relationship between the channel volume per generated crystal and the lysozyme concentration, using the coefficient (slope) of the approximation line for each lysozyme concentration shown in Figure 5, as a function of the lysozyme concentration. As shown in Figure 6, a negative correlation was observed between the lysozyme concentration and the channel volume per generated crystal at a given crystallization agent concentration. Therefore, for example, based on the graph in Figure 5 and the approximation line shown in Figure 6, it can be said that the number of protein crystals (crystal nuclei) generated can be predicted from the lysozyme concentration used and the volume of the microchannel.

[0081] The results shown in Figures 4 to 6 demonstrate that the number of protein crystal nuclei and single crystals generated and their distribution within the microchannel can be controlled with high reproducibility by adjusting the protein concentration of the supersaturated solution introduced into the microchannel and the volume of the microchannel. In other words, it is possible to calculate the protein concentration and the volume (size) of the microchannel that allow one to a few protein crystal nuclei to be discretely formed within the microchannel.

[0082] [Example 3] X-ray diffraction measurement of crystals In this example, X-ray diffraction measurements were carried out on lysozyme crystals formed in a microfluidic device.

[0083] A sample solution containing 40 mg / mL of lysozyme (HEWL) and 0.8 M NaCl, prepared as described above, was introduced into the fluid inlet of the microchannel described above, which had a depth of 30 μm, a width of 63 μm, and a length of 60 mm. The microchannel device was then placed in an incubator at 20°C and allowed to stand for one week to allow crystal formation. To prevent evaporation of the solution through the PDMS during this time, the microchannel device was placed in a sealed container with a volume of approximately 1 L, along with approximately 1 mL of 50 mM sodium acetate buffer (pH 4.5) containing 0.8 M NaCl. Images of crystals formed in the microchannel are shown in Figures 7A and B. Figure 7B is a magnified image of the crystal shown in Figure 7A.

[0084] X-ray diffraction measurements of approximately 300 μm long crystals grown in the microchannel were performed using beamline BL-5A at the Photon Factory (PF) of the High Energy Accelerator Research Organization (KEK). The solution containing the crystals in the microchannel was not pretreated. The microchannel device was mounted on an X-ray diffractometer with the PDMS substrate on the upstream side and the glass slide on the downstream side. Diffraction data were collected at a wavelength of 1 Å, an oscillation angle of 1.0°, five images from 90.0° to 95.0°, an exposure time of 20.0 seconds, a camera distance of 382.81 mm, and room temperature. An example of a diffraction image obtained from the X-ray diffraction measurement is shown in Figure 7C.

[0085] The diffraction data analysis results are shown in Table 3.

[0086] [Table 3]

[0087] The lattice constant of this crystal, shown in Table 3, indicates that it is a tetragonal crystal (P43212), which is commonly seen in lysozyme.These X-ray diffraction results confirmed that lysozyme single crystals were formed within the microchannel.

[0088] [Example 4] Protein crystal nucleation and crystal growth in the metastable phase in the reservoir (1) A 100 mM acetic acid solution and a 100 mM sodium acetate solution were mixed and adjusted to pH 4.5, and then an equal volume of distilled water was added to prepare a 50 mM sodium acetate buffer solution (pH 4.5).

[0089] Lysozyme and NaCl solutions were prepared by adding and dissolving uncrystallized lysozyme (HEWL) or NaCl as a crystallization agent to the buffer solution to a concentration twice the target concentration (HEWL 20 mg / mL, NaCl 0.8 M). The lysozyme concentration (C [mg / mL] (wt / v)) was determined as described above using a spectrophotometer (Ultrospec 2100 pro; Amersham Bioscience).

[0090] The sample solution was prepared by mixing the prepared lysozyme solution and NaCl solution in a 1:1 ratio (volume). The prepared sample solution (a supersaturated solution of 20 mg / ml lysozyme and 0.8 M NaCl) was introduced into a 95 μm deep, 63 μm wide, and 60 mm long microchannel through the fluid inlet of the microchannel device (Figure 1) using a PEEK tubing and microsyringe connected to the fluid inlet, filling the microchannel with the sample solution. The microchannel device was then left at 293 K (20 °C) for 1 h. After the leave-and-stand period, an additional 5 μL of sample solution (a 1:1 mixture of lysozyme and NaCl solutions) was injected into the PEEK tubing and microsyringe connected to the fluid inlet of the microchannel device, pushing the sample solution through the fluid outlet and ensuring the removal of crystal nuclei. 0.5 μl of the 5 μl of fluid discharged from the microchannel by extrusion through the fluid outlet was quickly added to 40 μl of metastable lysozyme solution in a sitting drop plate as a reservoir. The metastable lysozyme solution was prepared using the above-mentioned 50 mM sodium acetate buffer (pH 4.5) containing 5 mg / ml lysozyme (HEWL) and 0.8 M NaCl.

[0091] The sitting drop plate containing the sample solution was sealed with an OPP (Oriented Polypropylene) tape and placed in an incubator at 20°C. This allowed for crystal growth using the so-called batch method. As a control experiment, 40 μl of metastable lysozyme solution in the reservoir was prepared without adding the sample solution removed from the microchannel and similarly placed in an incubator at 20°C. The solutions in these reservoirs were observed over time for up to 12 days.

[0092] In the control reservoir under metastable conditions, to which no sample solution (lysozyme crystal nuclei) had been added after removal from the microchannel, no visible crystals were observed growing (Figure 8A shows the state after 6 days of standing).

[0093] On the other hand, in the reservoir to which the sample solution (lysozyme crystal nuclei) removed from the microchannel was added, no crystals were observed by visual observation immediately after addition. However, after 5 days of standing, numerous crystals were observed to have grown (Figure 8B). These crystals grew larger each day, and the formation of multiple crystals approximately 0.4 mm in length was observed (Figures 8C-E). This indicates that even when a solution containing a small amount of crystal nuclei grown under supersaturated conditions was poured into a large amount of reservoir solution under metastable conditions, the crystal nuclei did not break and crystals continued to grow.

[0094] These results indicate that crystal growth can be promoted by injecting protein crystal nuclei generated in the microchannel into a metastable region protein solution. Crystal growth can be promoted more effectively by injecting one or a small number of protein crystal nuclei formed in the microchannel into a metastable region lysozyme solution in the reservoir.

[0095] [Example 5] Protein crystal nucleation and crystal growth in the metastable phase in the reservoir (2) A 100 mM acetic acid solution and a 100 mM sodium acetate solution were mixed and adjusted to pH 4.5, and then an equal volume of pure water was added to prepare a 50 mM sodium acetate buffer solution (pH 4.5).

[0096] Lysozyme and NaCl solutions were prepared by adding uncrystallized HEWL or NaCl as a crystallization agent to the buffer solution to a concentration twice the target concentration (HEWL 15 mg / mL, NaCl 0.8 M). The lysozyme concentration (C [mg / mL] (wt / v)) was determined as described above using a spectrophotometer (Ultrospec 2100 pro; Amersham Bioscience).

[0097] The prepared lysozyme solution and NaCl solution were mixed in a 1:1 ratio to prepare a sample solution. 294 nL of the sample solution (supersaturated solution of 15 mg / mL lysozyme and 0.8 M NaCl) immediately after preparation was introduced into the microchannel (depth 49 μm, width 100 μm, length 60 mm) of the microchannel device. Specifically, a microsyringe (Hamilton, capacity 10 μL) filled with the sample solution was connected to a PEEK tube connected to the fluid inlet of the microchannel of the microchannel device, and the sample solution was injected from the microsyringe to fill the microchannel. The syringe was removed, and the end of the PEEK tube leading to the microchannel was sealed with grease. As a result, the microchannel was filled with the sample solution, while the sample solution was not present in the adjacent PEEK tube, and the sample solution in the microchannel was sandwiched between the air phase inside the PEEK tube. Furthermore, a clear seal (FastGene) was attached to the microchannel device. TM Evaporation of the sample solution was suppressed by applying a qPCR pressure-bonded clear seal (Nihon Genetics). The microfluidic device was placed in a Tupperware-type storage container together with a weighing dish filled with a 0.8 M NaCl solution to maintain a constant vapor pressure.

[0098] The microfluidic device was left standing at 293 K (20 °C) for 6 hours for crystallization. After the standing time, an empty syringe was connected to the PEEK tubing connected to the fluid inlet of the microfluidic device, and 294 nL of the sample solution in the microchannel was pushed out through the fluid outlet and added to the reservoir. The reservoir was a sitting drop plate containing 30 μL of metastable lysozyme solution (5 mg / mL lysozyme (HEWL) in the above-mentioned 50 mM sodium acetate buffer (pH 4.5), 0.8 M NaCl) in each well.

[0099] The reservoir (sitting drop plate) containing the sample solution removed from the microchannel was sealed with a lid using OPP tape and placed in an incubator at 20°C. This allowed crystal growth using the so-called batch method. As a control experiment, 30 μl of metastable lysozyme solution (HEWL 5 mg / ml in the above-mentioned 50 mM sodium acetate buffer (pH 4.5) and NaCl 0.8 M) was prepared in a reservoir without the sample solution removed from the microchannel and similarly placed in an incubator at 20°C. The solutions in these reservoirs were observed over time for up to 7 days.

[0100] As a result, in the reservoir to which the sample solution removed from the microchannel was added, no crystals were observed by visual observation immediately after addition to the reservoir, but approximately 15 lysozyme crystals (average side length of approximately 0.1 mm, maximum length of approximately 0.3 mm) were confirmed seven days later. On the other hand, no crystals were confirmed in the reservoir to which the sample solution removed from the microchannel was not added. Figure 9 shows an example of these results.

[0101] Applying the lysozyme and NaCl concentrations of the sample solution used in this example and the volume of the solution (294 nl) filled into the microchannel to the graph in Figure 5, the number of crystal nuclei generated in the microchannel is predicted to be around 10. The number of crystals grown in the reservoir from the sample solution was 15, a value quite close to the number of crystal nuclei predicted from the crystallization phase diagram. This demonstrates that the generation of protein crystals (nuclei) can be controlled using the method and microchannel device of the present invention.

[0102] [Example 6] Preparation of a glucose isomerase crystallization phase diagram In the following examples, crystals of glucose isomerase were produced using the method of the present invention. In these examples, in order to grow crystals of glucose isomerase using a microfluidic device, a crystallization phase diagram of glucose isomerase was first created.

[0103] To prepare a crystallization phase diagram, a glucose isomerase protein solution derived from Streptomyces rubiginosus (SPEZYME GIpf, Nagase ChemteX Corporation) was subjected to solvent exchange in advance. The exchange solution was 10 mM hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer (pH 7.7), and the solution was filtered using a centrifugal filter device (Amicon). (R) The HEPES buffer solution was prepared by ultrafiltration (8500 rpm, 277 K) using an Ultra filter (MWCO (molecular weight cutoff): 10,000). The HEPES buffer solution was prepared by dissolving 0.2384 g of HEPES (238.31 g / mol) in pure water, adjusting the pH to 7.7 with an aqueous NaOH solution, and then adding pure water to make a final volume of 100 ml.

[0104] After solvent exchange, the glucose isomerase concentration was determined and diluted to the required concentration with 10 mM HEPES buffer (pH 7.7). The glucose isomerase concentration (C' [mg / mL] (wt / v)) was calculated using the absorbance A' of the glucose isomerase solution measured at 280 nm and the molar extinction coefficient ε' (= 1.06) of glucose isomerase at 280 nm using the following formula: absorbance A' = ε' × C' [mg / mL] × d [cm], where d represents the optical path length. Crystallization tests were performed using the glucose isomerase solution prepared in this manner under the conditions shown in Table 4.

[0105] [Table 4]

[0106] For the crystallization test, 3 μl of each of the prepared glucose isomerase solution and crystallization agent solution was added to the wells of a plate in a microbatch kit (Greiner Bio-One) and mixed to prepare a sample. This was then covered with 5 μl of liquid paraffin to prevent evaporation. The plate was then covered and placed in an incubator set at 6°C for 2 weeks to crystallize the glucose isomerase.

[0107] After two weeks of standing, the lid was removed, and the droplets in the wells of the plate were observed using a stereomicroscope to determine whether or not crystals were present. The presence or absence of crystal formation was plotted against the glucose isomerase concentration and crystallization agent (ammonium sulfate) concentration for each sample, creating a crystallization phase diagram (Figure 10). In Figure 10, × indicates no crystal precipitation, and black circles indicate the presence of crystal precipitation.

[0108] Under these test conditions, a boundary line (crystal nucleation boundary line; dotted line in Figure 10) between the supersaturated and metastable regions was observed between glucose isomerase concentrations of 10 mg / mL and 15 mg / mL. In Figure 10, the supersaturated region is located above the dotted line, and the metastable region is located below the dotted line.

[0109] Based on the results obtained, it was decided that in the crystal formation test using the microchannel device described below, two types of solutions would be introduced into the microchannel: (i) a glucose isomerase concentration of 30 mg / ml and a crystallization agent concentration of 10% (w / v), or (ii) a glucose isomerase concentration of 20 mg / ml and a crystallization agent concentration of 10% (w / v).

[0110] [Example 7] Fabrication of a microfluidic device Polydimethylsiloxane (PDMS) and its polymerization agent were mixed in a 10:1 ratio, placed in a desiccator, and the pressure was reduced to remove any air bubbles. The mixture was then added to a Petri dish containing a microchannel mold, taking care to avoid introducing air bubbles. The Petri dish was then placed in an oven and heated at 70°C for 1 hour. Once solidified, the PDMS was cut out and washed and rinsed with acetone and 2-propanol. A 0.5 mm hole was punched into the solidified PDMS after removal from the mold, creating a fluid inlet for introducing sample solution into the microchannel. One side of the solidified PDMS and a glass slide were then surface-treated using a plasma cleaner, and the two were then bonded together, taking care to avoid air bubbles. A PEEK tube was inserted into the hole punched with the Seiken punch and secured in place with adhesive. The microchannel devices fabricated in this way had linear microchannels with channel depths of 30 μm or 49 μm and channel widths of 38, 63, or 100 μm (all internal dimensions).

[0111] [Example 8] Crystal formation using a microfluidic device A crystal growth test of glucose isomerase was carried out using the microfluidic device prepared in Example 7. The test conditions were as follows.

[0112] [Table 5]

[0113] Specifically, first, glucose isomerase solution and crystallization agent solution were prepared by adding and dissolving non-crystallized glucose isomerase or ammonium sulfate as a crystallization agent in 10 mM HEPES buffer (pH 7.7) to a concentration twice the target concentration (Table 5). Then, the glucose isomerase solution and the crystallization agent solution were mixed in a 1:1 ratio (volume ratio) to prepare a sample solution.

[0114] The prepared sample solution was introduced into the microchannel from the fluid inlet of the microchannel device through the PEEK tube, filling the microchannel with the sample solution. After the sample solution was introduced, the end of the PEEK tube connected to the fluid inlet was sealed with grease, and a clear seal was attached to the surface of the microchannel device to prevent evaporation of the sample solution. Next, the microchannel device, which had been filled with the sample solution introduced into the microchannel in this way, was placed in a sealed container with Kimwipes soaked in the crystallization agent solution. (R) The microchannel was then observed using a stereomicroscope and the number of crystals that had formed was counted.

[0115] The results are shown in Table 6 and Figures 11 and 12. It was shown that glucose isomerase crystals were formed discretely within the microchannel.

[0116] [Table 6]

[0117] Figure 13 shows the relationship between the channel volume and the number of crystals produced in the entire channel. It was found that there was a positive correlation between the channel volume and the number of crystals produced in the entire channel at any glucose isomerase concentration. It can be said that the number of protein crystals (crystal nuclei) produced can be predicted from the glucose isomerase concentration used and the volume of the microchannel. Note that the microchannels used in this example had the same channel length and each microchannel had a uniform cross-sectional area overall, so the channel volume was determined as a function of the cross-sectional area of ​​the microchannel (μm 2) is proportional to

[0118] [Example 9] X-ray diffraction measurement of crystals The glucose isomerase crystals formed in the microchannel in Example 8 were subjected to X-ray diffraction measurement.

[0119] Glucose isomerase crystals (approximately 1.5 mm long, Figure 12A) were fabricated as described above using a microfluidic device with a microchannel measuring 49 μm deep, 63 μm wide, and 60 mm long. X-ray diffraction measurements were performed using beamline BL-5A at the Photon Factory (PF) of the High Energy Accelerator Research Organization (KEK). The solution containing the crystals in the microchannel was not pretreated. The microfluidic device was mounted on the X-ray diffractometer with the PDMS substrate on the downstream side and the glass slide on the upstream side. Diffraction data were collected at a wavelength of 1 Å, an oscillation angle of 1.0°, five images ranging from 0.0° to 5.0°, an exposure time of 30.0 s, a camera distance of 382.81 mm, and room temperature.

[0120] The results of the diffraction data analysis are shown in Table 7.

[0121] [Table 7]

[0122] The lattice constants of this crystal, shown in Table 7, indicate that the crystal is an orthorhombic crystal (I222), which is commonly found in glucose isomerase. The X-ray diffraction results confirmed that a single crystal of glucose isomerase was formed in the microchannel. [Explanation of symbols]

[0123] 1...microchannel, 2...fluid inlet, 3...fluid outlet, 4...slide glass, 5...substrate, 6...microchannel device, 7...fluid injection means, 8...crystal growth reservoir

Claims

1. A supersaturated solution of biopolymer containing a crystallization agent was added to a 10,000 μm ２ A method for producing biopolymer crystals, comprising: introducing a biopolymer into a microchannel having a cross-sectional area of ​​100 nm or less and allowing the microchannel to stand to generate a crystal nucleus of the biopolymer; and adding the generated crystal nucleus to a solution in a metastable region containing the biopolymer to grow a crystal. 1) adding a supersaturated solution of the biopolymer containing the generated crystal nuclei to the solution in the metastable region in a volume containing 1 to 30 crystal nuclei; and / or 2) determining the volume of the solution containing the generated crystal nuclei to be added according to the prediction of the number of generated crystal nuclei by regression analysis using the initial concentration conditions of the supersaturated solution; method.

2. The method of claim 1, wherein the microchannel has a depth of 10 μm to 100 μm and a width of 10 μm to 100 μm.

3. The method according to claim 1 or 2, wherein the microchannel has a length of 10 mm or more.

4. The method according to any one of claims 1 to 3, wherein the supersaturated solution of the biopolymer containing the generated crystal nuclei is added to the solution in the metastable region in a volume containing 1 to 10 crystal nuclei.

5. The method according to any one of claims 1 to 4, wherein the biopolymer is a protein.

6. The method according to any one of claims 1 to 5, wherein the crystallization agent is sodium chloride or ammonium sulfate.

7. The method according to any one of claims 1 to 6, wherein the standing is carried out for 1 hour to 26 hours.

8. The method according to any one of claims 1 to 6, wherein the standing is carried out for 12 hours to 2 weeks.

9. 10,000 μm for generating crystal nuclei of biopolymers ２ 1. A biopolymer crystal production apparatus comprising a microchannel device having a microchannel having a cross-sectional area as follows: a fluid inlet for introducing a supersaturated solution of a biopolymer containing a crystallization agent into the microchannel; and a fluid outlet from the microchannel, 1) a supersaturated solution of the biopolymer containing the generated crystal nuclei is introduced into a crystal growth reservoir in a volume containing a controlled number of crystal nuclei; and / or 2) (i) a memory unit that stores the initial concentration conditions of a supersaturated solution of a biopolymer containing a crystallization agent; (ii) a calculation unit that predicts the number of crystal nuclei generated in the microchannel by regression analysis using the initial concentration conditions of the supersaturated solution; and (iii) a control unit that introduces the supersaturated solution of the biopolymer containing the generated crystal nuclei from the microchannel into a crystal growth reservoir in a volume that contains a controlled number of crystal nuclei. Device.

10. 10. The device of claim 9, wherein the microchannel has a depth of 10 μm to 100 μm and a width of 10 μm to 100 μm.

11. 11. The device according to claim 9 or 10, wherein the microchannel has a length of 10 mm or more.

12. 12. The apparatus according to any one of claims 9 to 11, wherein the fluid inlet is connected to a fluid injection means for injecting the supersaturated solution.

13. The device according to claim 12, wherein the fluid injection means is configured to be able to push the supersaturated solution introduced into the microchannel out of a fluid outlet of the microchannel.

14. The apparatus of any one of claims 9 to 13, further comprising a reservoir for crystal growth.

15. A method according to any one of claims 1 to 8, carried out using an apparatus according to any one of claims 9 to 14.

Images