JP5800195B2 - Crystal growth container, droplet preparation device, and crystal acquisition method - Google Patents

Description

  The present invention relates to a crystal growth container that acquires at least one crystal in a microdroplet, a droplet preparation device, and a crystal acquisition method.

  In recent years, in the single crystallization of a substance typified by protein and the determination of the three-dimensional structure by the structural analysis, at least one crystal is generated in a droplet, or each crystal is generated at a remote location. It is requested to do. That is, it is required that the individual crystals are separated without being combined. This is to improve the performance of a structural analysis apparatus using X-rays (for example, to increase the output of X-rays irradiated on the crystal, and to improve the performance of a detector that detects X-rays scattered by the crystal). Is attributed. In this analysis, it is not necessarily required that the target crystal is large.

  For example, Non-Patent Document 1 reports a crystallization method under microgravity in space or the like as an environment for achieving such crystallization. In a microgravity environment, protein crystals grow as a result of exquisite control of kinetic conditions and material transport, and as a result, crystal growth by diffusion rate control is possible. Therefore, in general, a single crystal obtained in the universe is large and the integrity of the crystal lattice is high. That is, it can be said that a microgravity environment such as the universe is convenient for crystal growth.

  Moreover, as mentioned above, as a technique for obtaining at least one crystal, for example, Non-Patent Documents 2 to 4 can be cited. Non-patent document 2 reports that at least one nucleation was achieved by theoretical control, and non-patent document 3 by kinetic control. Further, in Non-Patent Document 4, at least one crystal is obtained in a droplet by using a microfluidic technology (for example, a microreactor) to separate and control nucleation and growth. It has been reported.

F. Rosenberger, P. G. Vekilov, M. Muschol and B. R. Thomas, J. Crystal Growth, 1996, 168, 1-27 D. Kashchiev, D. Clausse, C. Jolivet-Dalmazzone, Journal Colloid and Interface Science 1994, 165, 148-153 P. Laval, J. Salmon, M. Joanicot, Journal of Crystal Growth 2007, 303, 622-628. J. Shim, G. Cristobal, D. R. Link, T. Thorsen, S. Fraden, Crystal Growth & Design 2007, 7, 2192-2194.

  Non-Patent Document 1 discloses that it is possible to achieve crystal growth by diffusion-controlled by performing experiments in space such as a microgravity environment. However, it is not easy to go to space and perform the experiment because of problems such as cost. Moreover, even if the experiment can be performed in space, the time for the experiment is limited, and there is a possibility that a sufficient experiment cannot be performed. Therefore, there is a need for a technique for achieving crystal growth based on the diffusion-limited method at a low cost and easily, such as a simulation or an experiment in an alternative environment of microgravity.

  Further, the methods or techniques of Non-Patent Documents 2 to 4 disclose that at least one crystal is grown in a droplet, but do not disclose any crystal growth based on diffusion rate control. Furthermore, in these methods or techniques, there are limitations on substances to which the methods or techniques can be applied, and complicated processing is required.

  Further, the techniques of Non-Patent Documents 1 to 4 do not aim to achieve such crystal growth in a gravitational environment such as the ground. If the crystal growth is not diffusion-controlled, the size of the microdroplet when at least one crystal is grown in the microdroplet cannot be defined (estimated).

  On the other hand, the inventor of the present invention pays attention to the similarity between the behavior of a substance inside a microdroplet (internal fluid behavior) under a gravitational environment and the internal fluid behavior of the droplet under microgravity. Crystal growth by diffusion-controlled using droplets has been achieved. Naturally, for the first time, the inventor of the present invention has found a technique for achieving crystal growth by the above-mentioned diffusion-controlled method after defining the size of the microdroplet.

  The present invention has been made to solve the above-described problems, and its object is to provide a crystal growth container, a droplet preparation device, and a container capable of efficiently acquiring at least one crystal in a microdroplet. It is to provide a crystal acquisition method.

  A crystal growth container according to the present invention is a crystal growth container for growing a crystal of a substance in order to solve the above-mentioned problems, and a substance to be crystallized is placed inside the crystal growth container. A microdroplet of a solution containing the microdroplet, the length of the longest part of the microdroplet being less than the maximum movable distance indicating the maximum distance that can be moved by natural diffusion of the substance to be crystallized It is characterized by being prepared in such a size as to become.

  According to the above configuration, since the microdroplet is arranged in the crystal growth container, the convection driven by the density difference in the microdroplet is suppressed. Thereby, the crystal growth by diffusion control can be achieved. The achievement of this crystal growth provides an environment in which the substance moves only by natural diffusion in the fine droplets arranged inside the crystal growth vessel. Therefore, the crystal of the substance can be generated in the fine droplets by diffusion rate control.

  In addition, the microdroplet placed inside the crystal growth container is prepared in such a size that the length of the longest portion thereof is not more than the maximum possible moving distance. Can be reliably grown and the number thereof can be suppressed to at least one. That is, in the crystal growth container of the present invention, at least one substance crystal can be efficiently grown in a microdroplet.

  Furthermore, in the crystal growth container according to the present invention, it is preferable that the periphery of the microdroplet is filled with a medium substance that is not compatible with the microdroplet.

  According to the above configuration, the microdroplet is in an isolated state where no substance enters or exits from the outside. Therefore, unless the crystal growth container is handled roughly, the crystal growing in the microdroplet is in the form of other microscopic droplets. Does not coalesce with droplets or other substances. Therefore, the crystal can be easily analyzed.

  Furthermore, in the crystal growth container according to the present invention, the size of the microdroplet is preferably such that convection driven by a density difference in the microdroplet is suppressed.

  According to the above configuration, the microdroplet is adjusted to a size that suppresses the convection driven by the density difference, so that crystal growth can be caused by diffusion rate control.

  Furthermore, in the crystal growth container according to the present invention, it is preferable that the length of the shortest portion inside the microdroplet is the same as the length of the longest portion of the microdroplet.

  According to the above configuration, the fine droplets can be reliably arranged inside the crystal growth container.

  Furthermore, in the crystal growth container according to the present invention, the substance to be crystallized is preferably a protein.

  According to the said structure, the maximum movable distance calculated | required using the diffusion constant of protein is close to the value which can suppress the convection in a microdroplet efficiently. In other words, when the maximum movable distance is calculated using a diffusion constant, the convection can be efficiently suppressed by using a protein as a substance to be crystallized in a microdroplet, and it is easy to achieve crystal growth by diffusion rate control. be able to.

  Furthermore, the crystal growth vessel according to the present invention is preferably a cylindrical capillary.

  According to the above configuration, since the crystal growth container is realized by the capillary having a high affinity with the crystal structure analysis apparatus that is generally used, it is possible to provide a highly versatile container.

  Furthermore, in the crystal growth container according to the present invention, it is preferable that a crystal of the substance to be crystallized grown in a microdroplet is an analysis target of a crystal structure analysis apparatus for analyzing the crystal structure.

  According to the above configuration, the crystal of the substance crystallized in the crystal growth instrument is the analysis target of the crystal structure analysis apparatus. That is, the crystal growth container in which the crystal of the substance is grown can be set as it is on the sample stage provided in the crystal analysis structure apparatus. Therefore, it is possible to save the trouble of taking out from the instrument on which the crystal has been grown and setting it on the sample stage of the crystal analysis structure apparatus as in the prior art.

  Furthermore, it is preferable that the droplet preparation device according to the present invention is connected to the crystal growth container described above, prepares the microdroplets, and sends the liquid to the crystal growth container.

  According to the above configuration, the droplet preparation device prepares a microdroplet capable of growing the substance to be crystallized as a crystal therein, and sends the prepared microdroplet to the crystal growth container. Liquid. Therefore, microdroplets containing a substance to be crystallized and limited in size can be arranged in the crystal growth container.

  Furthermore, the crystal acquisition method according to the present invention is a crystal acquisition method for acquiring a crystal of a substance, wherein the substance has a maximum movable distance indicating a maximum distance that the substance can move in a microdroplet by natural diffusion, (1) The rate at which the substance in the solution containing the substance is consumed as a result of the crystal growth of the substance growing from the parameters shown in (1), (2), and (3) below (2) the initial concentration of the substance, which is the concentration of the substance with respect to the microdroplet when the diffusion of the substance is started; (3) the diffusion constant of the substance; estimated by the estimation step A preparation step for preparing the microdroplet so as to have a size based on the maximum movable distance, and growth for growing at least one crystal of the substance in the microdroplet prepared by the preparation step. It is characterized in that it comprises a degree, the.

  According to the said structure, the convection driven by the density difference in the micro droplet is suppressed. Thereby, the crystal growth by diffusion control can be achieved. And, by achieving this crystal growth, an environment in which the substance moves only by natural diffusion is prepared, so that the maximum movable distance of the substance as described above can be estimated.

  In addition, the above-described estimation process makes it possible to estimate the above-mentioned maximum movable distance for each substance that is the target of crystal growth by diffusion-limited (without any substance restrictions), so that the above-mentioned maximum distance can be obtained. Can be easily performed.

  Then, a micro droplet having a size based on the maximum movable distance obtained by the above estimation step is prepared, and at least one crystal is grown in the micro droplet, whereby the crystal is formed in the micro droplet. It can be grown efficiently.

  In the crystal growth container according to the present invention, as described above, microdroplets of a solution containing a substance to be crystallized are arranged inside the crystal growth container. In this configuration, the length is adjusted to be not more than the maximum movable distance indicating the maximum distance that can be moved by natural diffusion of the substance to be crystallized.

  Further, in the crystal growth method according to the present invention, as described above, the maximum movable distance indicating the maximum distance that the substance can move in the microdroplet by natural diffusion is represented by the following (1), (2), and (3) an estimation step that is estimated from the parameters shown in (3), and (1) a substance consumption rate that is a rate at which the substance in the solution containing the substance is consumed by growing the crystal of the substance; The initial concentration of the substance, which is the concentration of the substance relative to the microdroplets when the diffusion of the substance is started; (3) the diffusion constant of the substance; the size based on the maximum movable distance estimated by the estimation step; The method includes a preparation step of preparing the microdroplet, and a growth step of growing at least one crystal of the substance in the microdroplet prepared by the preparation step.

  Therefore, in the crystal growth container and the crystal acquisition method of the present invention, crystals of at least one substance can be efficiently grown in the microdroplets.

FIG. 1 is a view showing an example of a crystal growth container according to an embodiment of the present invention, and shows a state in which micro droplets containing crystals of a grown substance are arranged therein. It is a figure which shows an example of the droplet preparation instrument connected to the said container for crystal growth. It is a schematic diagram which shows an example of the structure of the main channel and subchannel of the said droplet preparation instrument. FIG. 2 shows an example of an experimental result on the number of crystals when thaumatin crystals are grown in microdroplets arranged in the crystal growth vessel, wherein (a) shows the crystal growth vessel. Shows an example of the experimental result when the inner diameter of the crystal growth is 360 μm, (b) shows an example of the experimental result when the inner diameter of the crystal growth vessel is 200 μm, and (c) An example of the experimental results when the inner diameter of the crystal growth vessel is 130 μm is shown. FIG. 2 shows an example of an experimental result regarding the number of crystals when a lysozyme crystal is grown in a microdroplet arranged in the crystal growth vessel; FIG. 2 shows an example of an experimental result when the inner diameter of the crystal growth vessel is 360 μm, and FIG. 4B shows an example of the experimental result when the inner diameter of the crystal growth vessel is 200 μm. It is a block diagram which shows an example of a structure of the crystal acquisition apparatus which concerns on this embodiment. It is a figure which shows an example of the microsphere shell used as the model when estimating the magnitude | size of a microdroplet in the crystal | crystallization acquisition method which concerns on this embodiment. It is a figure which shows an example of the crystal acquisition method including the process in the crystal acquisition apparatus which concerns on this embodiment.

  The following describes one embodiment of the present invention with reference to FIGS. For convenience of explanation, members having the same functions as those shown in the drawings are denoted by the same reference numerals, and description thereof is omitted.

[Outline of the Invention]
<Background to the Present Invention>
The microdroplets arranged in the crystal growth container of this embodiment are utilized for various applications. In particular, it is used in drug delivery and other biomedical fields. This is because, for example, by using microdroplets as a template, liposomes with high uniformity in size can be prepared, and liposomes can be highly controlled, such as achieving high encapsulation efficiency. is there. In such a field, such highly controlled liposomes are essential. In addition, since microdroplets have the characteristics of a small diffusion distance and a large specific surface area, the fact that chemical reactions of substances can be performed efficiently in the microdroplets is also used for various applications. It is a cause.

  In addition, in order to use microdroplets for the above applications, it is necessary to prepare a large amount of highly controlled microdroplets. For such preparation, a microfluidic technique (for example, a microreactor) is useful as a tool for producing a large amount of monodispersed and controlled-size microdroplets in a reproducible amount.

  In addition, due to its strong surface tension, the microdroplet is not a convection driven by density difference (density difference driven convection), but a characteristic internal fluid behavior in which Marangoni convection dominates. Indicates.

  In a microgravity environment, density difference driven convection does not occur, so only Marangoni convection occurs inside the microdroplet. Therefore, in a microgravity environment, an environment in which the kinetic conditions and the transport of the material are exquisitely controlled is brought about in the crystal growth of the material, and as a result, the crystal growth by diffusion rate control becomes possible. Therefore, in general, in a microgravity environment such as the universe, it is possible to grow a large crystal (single crystal) with high crystal lattice integrity.

  However, although the microgravity environment is favorable for crystal growth, it is not possible to easily go to the environment, and in this environment, the experiment time for crystal growth is also limited. Therefore, there has been a demand for a technique for achieving crystal growth by the diffusion-controlled method at a low cost and easily, such as a simulation or an experiment in an alternative environment of microgravity.

<Outline of the present invention>
Therefore, the inventor of the present invention has similarities in internal fluid behavior between microdroplets under a gravitational environment such as the ground and microgravities such as space (not necessarily “micro”) (that is, microdroplets). Focusing on the point that the convection driven by the density difference in the droplet is suppressed), we studied the possibility of the technology to reproduce the microgravity environment under the gravity environment. As a result, we have succeeded in diffusion-controlled crystal growth that can obtain large crystals with high lattice integrity by using microdroplets in a gravitational environment.

  In addition, as a crystallization method using micro droplets, for example, surface diffusion method, two-layer fluid, diffusion method in a container, etc. have been reported. The goal was not to achieve diffusion-controlled crystal growth.

  Based on these studies, the present inventor has found that the length of the longest part of the microdroplet is a microfluid prepared so as to be not more than the maximum movable distance indicating the maximum distance that can be moved by natural diffusion of the substance to be crystallized. It came to realize the container for crystal growth which arranged the drop.

  As a result, microdroplets whose longest part is less than or equal to the maximum possible movement distance are arranged inside the crystal growth container, so that a substance crystal is efficiently grown in the microdroplets. And the number thereof can be suppressed to at least one (for example, about 1 to 3).

  Further, in the crystal growth vessel, in order to achieve crystal growth by diffusion rate control, the size of the microdroplet is such that convection driven by the density difference in the microdroplet is suppressed. It is prepared as follows.

  In addition, the present inventor has conducted theoretical research on the background of crystallization in “isolated” microdroplets that are not exposed to or from the outside. That is, the present inventor obtains at least one crystal inside an “isolated” microdroplet that does not exchange substances with the outside by the theoretical consideration of the internal fluid behavior of the microdroplet described above. I paid attention to. In order to achieve this, in the crystal growth container according to the present embodiment, the microdroplets are arranged so that the periphery of the microdroplets is filled with a medium material that is not compatible with the microdroplets. Yes.

  In addition, the present inventor estimates (estimates) the size of the crystal to obtain one crystal in one minute droplet, and as a result, obtains the estimated size and the actual experiment. It was confirmed that the size of the obtained microdroplets almost coincided.

Based on this examination, the present inventor has determined the maximum movable distance as described above.
(1) Substance consumption rate, which is the rate at which the substance in the solution containing the substance is consumed as the crystal of the substance grows. (2) The microdroplet when the diffusion of the substance is started. The initial concentration of the substance, which is the concentration of the substance with respect to (3), is estimated from the parameter indicated by the diffusion constant of the substance, and a microdroplet is prepared so as to have a size based on the estimated maximum movable distance. A crystal acquisition method has been realized in which at least one substance crystal is grown in the droplet. As a result, the maximum movable distance can be estimated simply and universally, so that the crystal can be easily obtained. Then, a microdroplet having a size based on the estimated maximum movable distance is prepared, and at least one crystal is grown in the microdroplet, thereby efficiently growing the crystal in the microdroplet. be able to.

  In addition, the present inventor has developed a crystal structure analysis apparatus for analyzing a crystal structure of a crystal of a substance to be crystallized, which has been grown in a microdroplet disposed in the crystal growth container. The container was realized.

  In general, the three-dimensional structure of a protein determined by a crystal structure analyzer is the most basic and important information source in the field of molecular biology or drug discovery. As a step before the determination of the three-dimensional structure, a step of growing a crystal is required. Conventionally, after the crystal has been grown, manual operation has occurred in which the crystal is taken out from the grown container and placed on the sample stage (gonio stage) of the crystal structure analyzer.

  However, in the present embodiment, the crystal growth container including the crystal grown as described above is set as an analysis target of the crystal structure analysis apparatus, so that a final analysis can be performed without causing a step of taking out the crystal. The target crystal can be provided to the crystal structure analysis apparatus. That is, it is possible to grow at least one crystal in a microdroplet and provide it as it is to the crystal structure analysis apparatus without taking it out of the crystal growth container.

  In addition, in the crystal structure analysis, it is not necessary to take out the crystal from the crystal growth container in which the microdroplet on which at least one crystal has grown is arranged. It can be used as a compatible “crystal structure analysis support instrument”.

  Note that the microdroplet with the adjusted size disposed in the crystal growth container of the present embodiment provides an environment in which the substance moves only by natural diffusion. Therefore, after the first generation of one nucleus inside the microdroplet, a concentration gradient of the substance can be generated, so that the “controlled” supersaturation can be eliminated. Therefore, when a crystal is grown in a microdroplet, it is possible to grow “only one” crystal in the microdroplet by diffusion-controlled.

[Experimental system]
<Droplet preparation device>
Next, with reference to FIG. 2 and FIG. 3, the droplet preparation device 1 for preparing the micro droplets 10 arranged in the crystal growth container 2 according to the present embodiment will be described. FIG. 2 is a view showing an example of the droplet preparation device 1 connected to the crystal growth vessel 2. FIG. 3 is a schematic diagram showing an example of the configuration of the main flow path 1 a and the sub flow path 1 b of the droplet preparation device 1.

As shown in FIG. 2, the droplet preparation device 1 is connected to, for example, a crystal growth container 2 (capillary) to prepare a microdroplet 10 (see FIG. 1) and send it to the crystal growth container 2. It ’s liquid. In the droplet preparation device 1, in order to prepare the micro droplet 10 to a predetermined size (a size that is not more than the maximum movable distance R _c ), the type, concentration, The flow rate is controlled.

  The droplet preparation instrument 1 is realized by, for example, a microchannel device. In the present embodiment, for example, a microchannel device made of polydimethylsiloxane (PDMS) is used. Since the structure is the same as that of a conventionally used microchannel device, a specific description thereof is omitted here.

  Moreover, as shown in FIG. 3, the droplet preparation device 1 includes at least a main channel 1a and a sub channel 1b. In order to prepare a microdroplet 10 in which at least one crystal 11 of the substance (see FIG. 1) can be grown, a solution containing the substance to be crystallized, a precipitant solution, and Fluoride oil is fed. The solution containing the substance to be crystallized and the precipitant solution are compatible with each other, and the microdroplets 10 are prepared by combining. Further, the fluoride-based oil functions as a medium substance that is not compatible with the microdroplets 10. For this reason, the fluoride-based oil can surround the periphery of the prepared microdroplet 10 and enables the microdroplet 10 to be placed inside the crystal growth container 2 in an “isolated” state. .

  Specifically, among the plurality of sub-channels 1b, fluoride-based oil is fed from the sub-channel 1b on the side connected to the crystal growth vessel 2, and from the side connected to the crystal growth vessel 2 The solution to be crystallized and the precipitant solution are sent from the sub-flow channel 1b provided at a distant position. In the example shown in FIG. 3, a protein solution is fed as the solution.

  Note that the medium substance that is not compatible with the microdroplet 10 is not limited to fluoride-based oil as long as it can realize the “isolated” state of the microdroplet 10, but the crystallized solution and It is preferred that there is no extremely large density difference from the precipitant solution.

  The plurality of sub-channels 1b are connected to the main channel 1a, and one end of the main channel 1a is connected to the crystal growth vessel 2. As a result, various materials (various solutions) are fed from the sub-channel 1b, whereby the micro droplet 10 is prepared in the main channel 1a. In the present embodiment, it is assumed that the micro droplet 10 is adjusted to have a predetermined size inside the droplet preparation instrument 1 and is sent to the crystal growth container 2, but not limited thereto. In the crystal growth vessel 2 connected to the droplet preparation device 1, the microdroplet 10 may be prepared until it reaches a predetermined size.

  In the example shown in FIG. 3, the cross section of the main channel 1a (the plane perpendicular to the direction in which various materials flow) is a rectangle whose one end connected to the crystal growth vessel 2 is a rectangle of 100 μm × 200 μm, and other portions. Is a square of 100 μm × 100 μm. The cross section of the sub-channel 1b is also a square of 100 μm × 100 μm. The reason why the cross section of one end connected to the crystal growth container 2 is larger than the cross section of the other part is to facilitate feeding of various materials to the crystal growth container 2.

  In addition, how to provide the sub-channel 1b, the number of sub-channels 1b, the relationship between the sub-channel 1b and the material to be sent (which sub-channel 1b sends which material), and the mainstream The dimensions of the channel 1a and the subchannel 1b are merely examples, and are not limited to these as long as the microdroplet 10 capable of growing at least one substance crystal 11 therein can be prepared. .

<Crystal growth container>
Next, the crystal growth container 2 according to the present embodiment will be described with reference to FIG. FIG. 1 shows an example of a crystal growth container 2, and shows a state in which minute droplets 10 containing a crystal 11 of a grown substance are arranged therein.

The crystal growth container 2 is used for growing a crystal 11 of a substance, and a microdroplet 10 of a solution containing the substance to be crystallized is arranged (arranged) therein. The length of the longest portion of the deployed microdroplets 10 are prepared on a movable maximum distance R _c below become such magnitude.

  In the present embodiment, the crystal growth container 2 is a capillary (Teflon capillary tube) made of Teflon (registered trademark), and in the example shown in FIG. 1, its inner diameter is 200 μm. In the following experimental examples, a cylindrical capillary having four different inner diameters (130, 200, 360, and 500 μm) is used as the crystal growth vessel 2. Note that, regardless of the inner diameter of the crystal growth container 2, the size of the droplet preparation device 1 to be connected is the same.

  In the example shown in FIG. 1, three micro droplets 10 are arranged. Further, the shape is substantially “spherical” and has a diameter substantially the same as the inner diameter of the crystal growth vessel 2. That is, the length of the shortest portion inside the crystal growth container 2 where the microdroplet 10 is disposed is the same as the length of the longest portion of the microdroplet 10. A crystal 11 grows inside the microdroplet 10.

The shape is not limited to a true sphere, and may be an elongated shape. Note that the “maximum movable distance R _c ” according to the present embodiment refers to the “radius”, not the “diameter”, when defined for the true micro droplet 10.

<Example>
Next, an example (experimental example) when the microdroplet 10 is prepared using the above-described droplet preparation device 1 and the crystal growth container 2 and the crystal 11 is grown therein will be described. In addition, the Example described below is only an example, and does not cause the droplet preparation instrument 1, the crystal growth container 2, and the crystal acquisition method using them to be interpreted in a limited manner. . In the following description, it is assumed that the droplet preparation device 1 and the crystal growth container 2 are connected.

In this embodiment, protein is used as the substance to be crystallized. The maximum movable distance R _c estimated using the protein diffusion constant D (that is, using the equation (6) described later) is close to a value that can efficiently suppress density difference-driven convection in the microdroplet 10. . Therefore, when protein is used as the substance to be crystallized, the microdroplet 10 can be prepared from the estimated value. Therefore, the convection of the protein in the microdroplet 10 can be efficiently suppressed, and the diffusion rate is controlled. The crystal growth due to can be easily achieved. Many experiments on proteins have been carried out especially in the field of biomedical. Therefore, the crystal growth container 2 can be effectively applied particularly in the biomedical field.

  This experiment uses a protein called thaumatin as a model. This is because thaumatin is typically used extensively as the model and is very well studied in crystallization on a macro scale, kinetic analysis, and the like. Further, a microfluidic device made of PDMS was used as the droplet preparation instrument 1, and a Teflon capillary tube was used as the crystal growth container 2. By preparing Teflon capillary tubes having four different inner diameters (130, 200, 360 and 500 μm), “spherical” microdroplets 10 were prepared for the four different inner diameters.

  Further, the protein solution and the precipitant solution are sent from the sub-flow channel 1b of the droplet preparation device 1, and the fluoride-based oil is sent from the main flow channel 1a. The protein solution is prepared by dissolving thaumatin having a concentration of 20 mg / mL in 100 mM N- (2-acetamido) iminodiacetic acid (ADA) buffer solution (pH 6.5). The precipitant solution is a solution of 1.6 M potassium sodium tartrate dissolved in 50 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer solution (pH 7.0). Fluoride-based oil is a mixture of fluorine-based inert liquid (Fluorinert (registered trademark) manufactured by Sumitomo 3M) and 1H, 1H, 2H, 2H-perfluoro-1-octanol in a volume ratio of 10: 1. It is a thing.

  The protein solution and the precipitant solution sent from the sub-channel 1b merge in the main channel 1a and mix with each other to form one micro droplet 10 in the main channel 1a. The protein solution and the precipitant solution are controlled to have substantially the same flow rate. In addition, the fluoride oil surrounds the microdroplet 10 to be prepared, and in order to realize the “isolated” state of the microdroplet 10, a side stream to which the protein solution and the precipitant solution are fed Liquid is fed from two sub-channels 1b different from the channel 1b. The fluoride oil is also controlled to have a flow rate substantially the same as the above flow rate. Then, by finely adjusting these flow velocities, true spherical microdroplets 10 were prepared.

  Crystal droplet growth is carried out by supplying a microdroplet 10 having a predetermined size to the crystal growth vessel 2 or by preparing a microdroplet 10 having a predetermined size in the crystal growth vessel 2. It is arranged inside the container 2 for use. After the minute droplets 10 of a predetermined size were placed, the liquid feeding of the material was stopped, the crystal growth container 2 was removed from the droplet preparation device 1, and both ends thereof were sealed with wax. Then, the crystal growth container 2 in which the microdroplets 10 are arranged is stored at about 4 ° C., and imaging by the imaging device is performed until the protein crystal growth in the microdroplets 10 is completed. The crystal growth process was observed.

<Experimental result>
The microdroplets 10 prepared in the above experiment were not naturally distorted and crushed by the strong surface tension. Also, it took approximately several hours from the time when the microdroplet 10 was placed inside the crystal growth vessel 2 and the crystal 11 began to grow until the growth stopped.

  Here, the experiment was performed on the four crystal growth containers 2. 100 to 200 microdroplets 10 arranged in each crystal growth container 2 were observed, and the number of crystals 11 grown in the microdroplets 10 was counted. The results are shown in Table 1. In Table 1, the number of crystals 11 is shown as an average value ± standard deviation of the numbers observed in the plurality of microdroplets 10.

  As shown in Table 1, in the microdroplet 10 disposed in each of the crystal growth containers 2 having an inner diameter of 360 μm, 200 μm, and 130 μm (in this experiment, the inner diameter and the diameter of the microdroplet 10 are substantially the same. ), About one crystal 11 was confirmed. On the other hand, approximately two crystals 11 were confirmed in the microdroplet 10 disposed in the crystal growth vessel 2 having an inner diameter of 500 μm.

<Investigation of crystal growth by Abram's formula>
Here, in order to examine the mechanism of crystal growth, analysis was performed using the Arami equation used for kinetic analysis of crystal growth. Here, in the formula (1), α is the growth rate of the crystal 11 (α = 1 is the end of crystal growth, α = 0 is before the crystal 11 is formed, that is, all the proteins are dissolved in the solution), k Is the rate constant of crystallization, and t is the time taken for crystal growth. Abram index m reflects basic information on nucleation and crystal growth.

  In this study, the crystal growth of about 10 microdroplets 10 each of a microdroplet 10 having a diameter of 360 μm, a microdroplet 10 having a diameter of 200 μm, and a microdroplet 10 having a diameter of 130 μm. The process was observed. Then, the Abram index m was determined from the time taken for crystal growth and the size of the crystal 11. As a result, the Abram's index m was about 1.5 regardless of the diameter of the micro droplet 10.

  In the experiment using thaumatin as described above, the crystal 11 grown in the microdroplet 10 was apparently three-dimensional, so m≈1.5 indicates that the nucleus generation rate is It means infinite and crystal growth by diffusion rate control. The result that “the nucleation rate is infinite” is consistent with the result that one crystal 11 is generated and grown in one microdroplet 10. Further, the result of “crystal growth by diffusion rate control” means that convection due to the density difference in the microdroplets 10 is sufficiently suppressed.

<Further experimental examples>
(In the case of thaumatin)
Next, an example of further experimental results when thaumatin is used as the substance to be crystallized will be described with reference to FIG. 4A shows an example of an experimental result when the inner diameter of the crystal growth container 2 is 360 μm, and FIG. 4B shows an example of an experimental result when the inner diameter of the crystal growth container 2 is 200 μm. c) shows an example of each experimental result when the inner diameter of the crystal growth vessel 2 is 130 μm.

  FIG. 4 shows the number of crystals 11 grown in one microdroplet 10 and the value related to the size of the microdroplet 10 as in Table 1 above. For the further experimental example, the experiment was performed in the same procedure as the above-described experimental example. However, the shape of the microdroplet 10 is not limited to a true spherical shape, and is prepared to be a non-spherical shape (elongate shape). Also included.

  For example, in FIG. 4A, experimental results (a) to (e) were obtained. In the case of the experimental result (A), the volume of the microdroplet 10 was the largest, and a horizontally long (elongated shape) microdroplet 10 was confirmed. The number of crystals 11 in the microdroplet 10 was about three. On the other hand, in the case of the experimental result (e), the volume of the micro droplet 10 was the smallest, and a substantially spherical micro droplet 10 was confirmed. The number of crystals 11 in the microdroplet 10 was about one.

That is, the smaller the droplet 10 is, the smaller the volume is, that is, the smaller the movable maximum distance R _c , the smaller the droplet 10 having a shape close to a true sphere. Therefore, one crystal 11 is grown in the droplet 10. It can be said that it is easy. Since each of the microdroplets 10 is arranged in the crystal growth container 2 in an “isolated” state, if only one crystal 11 is growing on the microdroplet 10, the crystal 11 is increased accordingly. Can be easily analyzed. On the other hand, if there is no problem in the analysis of the crystal 11 even if the microdroplet 10 includes, for example, two to three (that is, a plurality of) crystals 11, the volume of the microdroplet 10 is increased (that is, , In a non-spherical shape).

  In addition, the results in Table 1 above show that when the microdroplet 10 was prepared in accordance with the inner diameter of the crystal growth container 2 having an inner diameter of 500 μm, a true microdroplet 10 could not be obtained. ing. This is because a droplet with a short axis exceeding 500 μm has a relatively small surface tension due to a decrease in specific surface area, and the droplet is distorted by its own weight, so that a true spherical droplet can be obtained. It is thought that there was not. Therefore, this result means that a true micro droplet 10 cannot be obtained with a micro droplet 10 having a length exceeding 500 μm.

  Further, as shown in FIGS. 4B and 4C, each of the crystal growth containers 2 having an inner diameter of 200 μm and 130 μm also has a volume of the microdroplet 10 disposed therein as described above. If it is larger, the number of crystals 11 that grow inside increases (see (b) and (c) of each experimental result (a) in FIG. 4)). On the other hand, the smaller the volume of the microdroplet 10 is, the fewer the number of crystals that grow inside it is. When the shape is close to a true sphere, the number of crystals 11 that grow inside it can be controlled to about one. (See the experimental result (co) in FIG. 4B and the experimental result (b) in (c)).

(In the case of lysozyme)
Next, an example of further experimental results when lysozyme is used as a substance to be crystallized will be described with reference to FIG.

  FIG. 5 shows the number of crystals 11 grown in one microdroplet 10 and the value related to the size of the microdroplet 10 as in the case of thaumatin. Moreover, FIG. 5 has shown the experimental result at the time of experimenting in the procedure similar to thaumatin.

  For example, in FIG. 5A (when the inner diameter is 360 μm), experimental results (a) and (b) were obtained. In both results, the volume of the microdroplet 10 was small, and a substantially spherical microdroplet 10 was confirmed. The number of crystals 11 in the microdroplet 10 was about 1 to 2. In FIG. 5B (when the inner diameter is 200 μm), experimental results (a) and (b) were obtained. In the experimental result (a), the number of the crystals 11 in the microdroplet 10 is about 1 to 2, and in the experimental result (a), the number of the crystals 11 in the microdroplet 10 is about one. There wasn't.

  From this result, it can be seen that, like thaumatin, about one crystal 11 can be grown in a microdroplet 10 having a predetermined size (at least 200 to 360 μm in diameter). That is, the crystal growth container 2 of the present embodiment is the result of density difference-driven convection being suppressed by arranging the microdroplets 10 having a predetermined size even in the crystallization of substances other than thaumatin. Crystal growth by diffusion rate control can be realized, and a crystal of at least one substance can be grown in the microdroplet 10.

[Crystal acquisition device and crystal acquisition method]
Next, the configuration of the crystal acquisition device 3 will be described with reference to FIGS. 6 and 7. FIG. 6 is a block diagram showing an example of the configuration of the crystal acquisition device 3 according to this embodiment. FIG. 7 is a diagram illustrating an example of a microspherical shell that serves as a model for estimating the size of the microdroplet 10.

  As illustrated in FIG. 6, the crystal acquisition device 3 is a device for acquiring a crystal 11 of a substance, and includes, for example, a control unit 4, a storage unit 5, and an operation unit 6. The crystal acquisition device 3 is included in the crystal acquisition system 20 for acquiring the substance crystal 11 together with the droplet preparation device 1 and the crystal growth container 2, and is connected to the droplet preparation device 1, and the flow rates of various solutions, etc. Is adjusted to control liquid feeding of the droplet preparation device 1 to the solution.

The control part 4 controls the member which comprises the crystal acquisition apparatus 3, for example by running a control program. The control unit 4 reads the program stored in the storage unit 5 into a primary storage unit (not shown) configured by, for example, a RAM (Random Access Memory), for example, and executes the program, for example, It performs various processing such as calculation processing movable maximum distance R _c. Note that the maximum movable distance R _c is a value estimated by the maximum distance estimation unit 42.

The storage unit 5 records (1) a control program for each unit executed by the control unit 4, (2) an OS program, (3) an application program, and (4) various data read when these programs are executed. It is. The control unit 4 is configured by a non-volatile storage device such as a ROM (Read Only Memory) flash memory. The primary storage unit described above is configured by a volatile storage device such as a RAM. However, in the present embodiment, the storage unit 5 may be described as having the function of a primary storage unit. Storage unit 5 stores, for example, various data such as a formula that used when calculating the movable maximum distance R _c.

  The operation unit 6 allows an operator operating the crystal acquisition device 3 to acquire data necessary for the crystal acquisition device 3 in order to acquire the crystal 11 of the substance in the microdroplet 10 disposed in the crystal growth container 2. It is used for input, and is realized by, for example, a keyboard or a mouse. Examples of the necessary data include values of various parameters used in the maximum distance estimation unit 42.

  The operation unit 6 may be realized by an external device different from the crystal acquisition device 3. In this case, the parameter acquisition unit 41 of the crystal acquisition device 3 may acquire the values of various parameters acquired via the operation unit 6 via a wireless or wired communication medium.

  Next, the configuration of the control unit 4 will be described. The control unit 4 mainly includes a parameter acquisition unit 41, a maximum distance estimation unit 42, and a droplet preparation control unit 43.

Parameter acquisition unit 41 is for acquiring various parameters used when the maximum distance estimation unit 42 estimates the movable maximum distance R _c (calculated). For example, the values of various parameters acquired by the parameter acquisition unit 41 are displayed on the display unit (not shown) that prompts the control unit 4 to input various parameters necessary for the estimation, and the input screen is operated. Entered by the user. The parameter acquisition unit 41 temporarily stores these values in the storage unit 5.

As various parameters acquired by the parameter acquisition unit 41,
(1) Substance consumption rate q, which is the rate at which the substance is consumed in the solution containing the substance by the growth of the substance crystal 11
(2) Material initial concentration C ₀ which is the concentration of the substance with respect to the microdroplet 10 when the substance diffusion is started
(3) Diffusion constant D of the substance
There are three. The parameters (1) to (3) are merely examples. For example, when the maximum distance estimation unit 42 estimates the maximum movable distance _Rc by a method other than the method described below, The parameters may be acquired. For example, the parameter acquisition unit 41 does not depend on the substance consumption rate as a constant value (“q” in the following formula (6)) but the actual substance consumption rate −r _p and the crystal growth as the substance consumption rate. You may get time.

The maximum distance estimation unit 42 reads various parameters acquired by the parameter acquisition unit 41 from the storage unit 5 and estimates the maximum movable distance R _c from the various parameters. Specifically, the maximum movable distance R _c is estimated using the following equation (6) obtained. In this estimation, it is assumed that one crystal 11 is obtained in one minute droplet 10. Further, in this estimation, a crystal growth process is assumed in which a crystal nucleus is generated at the center of a true spherical droplet 10 and the nucleus grows. In this case, the concentration of the substance in the immediate vicinity of the crystal 11 is lower than the concentration of the substance in the periphery. For this reason, the molecule | numerator of the substance in a solution moves to the surface of the crystal | crystallization 11 by natural diffusion, and is absorbed by the crystal | crystallization 11 after that.

In the following description, it is assumed that the substance is a protein. This is because the maximum movable distance R _c obtained using the protein diffusion constant D is close to a value that can efficiently suppress density difference driven convection in the microdroplet 10 as described above. If this point is not taken into consideration, the substance to be crystallized need not necessarily be a protein.

  Here, it is assumed that a microspherical shell surrounded by a circle having a radius r and a circle having a radius (r + dr) in a micro droplet 10 having a radius R as shown in FIG. Fick's first law is expressed by the following equation (2). Here, in Equation (2), N is the diffusion flux of the protein, D is the diffusion coefficient of the protein, and C is the concentration of the protein with respect to the microdroplet 10. The size of the crystal nucleus and the crystal 11 in the initial stage of crystal growth are sufficiently small and negligible with respect to the size of the microdroplet 10 on which the crystal 11 is grown.

Moreover, the mass balance formula about protein is represented by following Formula (3). Here, in the formula (3), the consumption rate of the protein, i.e., by the crystal 11 of protein grows, the speed at which the protein in a solution containing a protein is consumed with -r _p.

  This equation (3) is rearranged as shown in the following equation (4).

Here, the following boundary conditions are considered. Here, C ₀ is the initial concentration of protein.

In the protein crystal growth in the present embodiment, the protein concentration in the vicinity of the protein crystal 11 with respect to the microdroplet 10 is sufficiently low, and protein crystallization proceeds by diffusion rate control (using the above Arami equation (1)). Therefore, this crystallization can be regarded as a zero-order reaction. That is, consumption concentration -r _p proteins are the values that do not depend on the radius r, can be placed a constant value q. Therefore, equation (4) can be rewritten as shown in the following equation (5).

The radius R of the microdroplet 10 and the initial protein concentration C ₀ are positive values. Therefore, when this equation (5) is solved, the maximum movable distance (the “critical” size of the microdroplet 10) R _c for obtaining one crystal 11 in one microdroplet 10 is given by (6).

That is, the maximum distance estimation unit 42 estimates the maximum movable distance R _c from the parameters of the protein substance consumption rate q, the substance initial concentration C ₀ , and the diffusion constant D (parameters acquired by the parameter acquisition unit 41). Then, the maximum distance estimation unit 42 temporarily stores the maximum distance estimation result, which is the estimated result, in the storage unit 5.

When the parameter acquisition unit 41 acquires the actual substance consumption rate −r _p and the time taken for crystal growth as the substance consumption rate, the maximum distance estimation unit 42 determines that these two parameters From the above, the substance consumption rate q used in the above equation (6) may be obtained.

Further, in the above, although the movable maximum distance R _c using Equation (6) is estimated, not limited to this, for example, to the different conditions of the above boundary conditions, material consumption concentration -r _p constant The above estimation may be performed without replacing with the value q. In this case, the size of the microdroplets 10 for growing the plurality of crystals 11 in the microdroplets 10 can be estimated.

  Furthermore, the maximum distance estimation unit 42 performs estimation using the various parameters acquired by the parameter acquisition unit 41 and the above equation (6), but is not limited thereto. For example, from the above equation (2), The maximum distance estimation unit 42 may perform the processing until Expression (6) is obtained.

Droplet preparation control unit 43 is mainly maximum distance estimation unit 42 reads the movable maximum distance R _c estimated from the storage unit 5, the movable maximum distance identical to R _c, or it from smaller microdroplets 10 The flow rate of various solutions (a solution containing a substance to be crystallized, a precipitant solution, a fluoride oil, etc.) sent to the droplet preparation device 1 is determined. Then, according to the determined flow rate, the liquid preparation device 1 is controlled to feed various solutions.

Whether prepared microdroplets 10 to the size of the extent, for example, (1) preparing to movable maximum distance R _c, (2) several tens of percent of the movable maximum distance R _c (for example, 80% (set (3) The maximum distance _Rc that can be moved is set as an upper limit, and the operator of the crystal acquisition device 3 inputs the value.

  In addition, the droplet preparation control unit 43 determines the shape of the micro droplet 10 so as to be a desired shape (for example, a true spherical shape) by monitoring the process of preparing the micro droplet 10. Fine adjustment of the flow rate of the various solutions may be performed. In this case, for example, the droplet preparation control unit 43 captures an image of the minute droplet 10 in the preparation process with an imaging device (not shown) and analyzes the captured image to thereby adjust the minute droplet in the adjustment process. Recognize 10 shapes. After that, by comparing with data indicating the desired shape stored in the storage unit 5 (for example, image data as a model), it is determined whether or not the micro droplet 10 has the desired shape, and the desired shape is obtained. If it is determined that the shape is not, the flow velocity is re-determined from the recognized shape.

  In addition, when the shape of the micro droplet 10 in a preparation process is not a desired shape, the flow velocity for obtaining a desired shape from the shape is grasped from an empirical rule. Therefore, for example, the shape and the flow rate change rate are associated with each other and stored in the storage unit 5 so that it can be determined how much the determined flow velocity should be increased or decreased based on the empirical rule. Has been.

The droplet preparation control unit 43, if it is determined that the microdroplets 10 are prepared on a movable maximum distance R _c below a predetermined size, to stop the liquid feed of various solutions. Thereafter, the microdroplet 10 is fed from the droplet preparation device 1 to the crystal growth container 2, and at least one crystal 11 of the substance grows in the microdroplet 10. This crystal growth is realized by leaving the crystal growth vessel 2 stationary.

  That is, the acquisition of the substance crystal 11 is realized through the flow shown in FIG. FIG. 8 is a diagram illustrating an example of a crystal acquisition method including processing in the crystal acquisition device 3 according to the present embodiment.

In the crystal acquisition device 3, after the parameter acquisition unit 41 acquires values of various parameters (material consumption rate q, substance initial concentration C ₀ and diffusion constant D of the substance to be crystallized) (S 1), the maximum distance estimation unit 42 Then, the maximum movable distance R _c is estimated from the value of the parameter (S2: estimation step).

Then, as the droplet preparation control unit 43, a size based on the movable maximum distance R _c, to prepare the microdroplets 10 (S3: Preparation Step), then, the microdroplets 10 therein When the arranged crystal growth container 2 is left still, at least one crystal 11 grows in the microdroplet 10 (S4: growth step).

In S3, the droplet preparation control unit 43 controls the flow rates of various solutions so that the micro droplets 10 have a predetermined size. However, the present invention is not limited to this. For example, the maximum movable distance R _c estimated by the maximum distance estimation unit 42 is displayed on a display unit (not shown), and the operator who has confirmed the maximum movable distance R _c sends the liquid to the droplet preparation device 1. The flow rate of the various solutions to be performed may be determined. In addition, for example, the operator may finely adjust the flow velocity by monitoring the shape of the microdroplet 10 in the preparation process as needed using the above image or microscope.

[Comparison between estimated maximum movable distance and experimental micro droplet size]
Next, the estimated value of the movable maximum distance R _c, a comparison between the size of the microdroplets 10 obtained by the above experiment.

In the above, substances initial concentration _{C 0} of the protein (thaumatin) is 10 mg / mL. The protein consumption rate q when the time taken for crystal growth is 6000 seconds is calculated as q≈1 / 6 × 10 ⁻² mg / mL · s. The diffusion constant D of thaumatin is about 10 ^-11 m ² / s. The maximum distance estimation unit 42 calculates the maximum movable distance R _c ≈600 μm by substituting the values of these parameters into Equation (6).

This value is a theoretical value, and the actual value is considered to be smaller than this. This is because the nucleation of the crystal 11 does not always occur at the center of the microdroplet 10, and the size of the crystal 11 itself accompanying crystal growth cannot be ignored. Considering this, it can be said that this value roughly matches the actual experimental result (Table 1). Therefore, the estimation of the maximum movable distance R _c by the crystal acquisition device 3 makes it possible to efficiently generate the crystal 11 in the microdroplet 10. In particular, when preparing a true microdroplet 10 having a radius smaller than the estimated value, only one crystal 11 can be grown in the microdroplet 10. That is, when it is desired to grow one crystal 11 in the micro droplet 10, the estimated value is particularly effective.

  It should be noted that the nucleation of the crystal 11 described above does not necessarily occur at the center of the microdroplet 10 and the size of the crystal 11 itself accompanying crystal growth cannot be ignored, as shown in Table 1, 360 μm. In addition, in the 200 μm microdroplets 10, the average number of crystals obtained in one microdroplet 10 is slightly larger than one. This is because the high concentration portion of the protein is maintained at a location away from the growing crystal 11, resulting in a second nucleation and opportunity for its growth.

[Example of software implementation]
Finally, each block of the crystal acquisition device 3, in particular, the parameter acquisition unit 41, the maximum distance estimation unit 42, and the droplet preparation control unit 43 of the control unit 4 may be configured by hardware logic, as follows: You may implement | achieve by software using CPU.

  That is, the crystal acquisition device 3 includes a CPU (central processing unit) that executes a command of a control program that realizes each function, a ROM (read only memory) that stores the program, and a RAM (random access memory) that expands the program And a storage device (recording medium) such as a memory for storing the program and various data. An object of the present invention is to provide a recording medium in which a program code (execution format program, intermediate code program, source program) of a control program of the crystal acquisition device 3 which is software that realizes the above-described functions is recorded in a computer-readable manner. This can also be achieved by supplying the crystal acquisition apparatus 3 and reading and executing the program code recorded on the recording medium by the computer (or CPU or MPU).

  Examples of the recording medium include a tape system such as a magnetic tape and a cassette tape, a magnetic disk such as a floppy (registered trademark) disk / hard disk, and a compact disk-ROM / MO / MD / digital video disk / compact disk-R. A disk system including an optical disk, a card system such as an IC card (including a memory card) / optical card, or a semiconductor memory system such as a mask ROM / EPROM / EEPROM / flash ROM can be used.

  Further, the crystal acquisition device 3 may be configured to be connectable to a communication network, and the program code may be supplied via the communication network. The communication network is not particularly limited. For example, the Internet, intranet, extranet, LAN, ISDN, VAN, CATV communication network, virtual private network, telephone line network, mobile communication network, satellite communication. A net or the like is available. Further, the transmission medium constituting the communication network is not particularly limited. For example, even in the case of wired such as IEEE 1394, USB, power line carrier, cable TV line, telephone line, ADSL line, etc., infrared rays such as IrDA and remote control, Bluetooth ( (Registered trademark), 802.11 wireless, HDR, mobile phone network, satellite line, terrestrial digital network, and the like can also be used. The present invention can also be realized in the form of a computer data signal embedded in a carrier wave in which the program code is embodied by electronic transmission.

[Another expression of the present invention]
The present invention can also be expressed as follows.

  The method of the present invention is used as a microgravity alternative experimental environment on the ground by utilizing the fact that the internal fluid behavior of microdroplets is similar to that under microgravity.

  The method of the present invention is a method for obtaining a single crystal by utilizing the properties of microdroplets as such a microgravity alternative experimental environment.

  Further, the method of the present invention is a method for growing a single crystal, in which the growth of the single crystal in the microdroplet is diffusion-controlled.

  Further, the method of the present invention is a method in which only one or a small number of single crystals are produced in one droplet by utilizing the special characteristics of the internal fluid behavior of the micro droplet.

  In addition, the method of the present invention can be applied to a substance consumption rate in a solution, a substance for a substance that is desired to be a single crystal having a radius of a minute droplet that can obtain only one or a small number of single crystals in one droplet. It is obtained from the initial concentration and the diffusion constant of the substance.

  In addition, the instrument of the present invention is an instrument that makes it possible to obtain one or a small number of single crystals in one droplet by using the above-mentioned micro droplets.

  The instrument of the present invention is a capillary-like cylindrical tube.

  In the method and instrument of the present invention, the target substance to be crystallized is protein.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

  The present invention is a technique for obtaining at least one crystal in a microdroplet by paying attention to the similarity in internal fluid behavior between a microdroplet under a gravity environment and a droplet under microgravity. This technique is particularly suitable for crystal structure analysis by X-rays. In addition, at present, since the search for crystallization conditions is performed in a situation where a large number of crystals are generated by the batch method, the above crystal acquisition apparatus and crystal acquisition method are incorporated into a robot that automatically searches for the crystallization conditions. Is also possible.

1 Droplet preparation device 2 Crystal growth vessel (capillary)
10 Micro droplet 11 Crystal q Material consumption rate C ₀ Material initial concentration D Diffusion constant R _c Maximum movable distance

Claims (9)

A crystal growth vessel for growing a crystal of a substance,
Inside the crystal growth vessel, microdroplets of a solution containing a substance to be crystallized are arranged,
The microdroplet is prepared in such a size that the length of the longest part of the microdroplet is not more than the maximum movable distance indicating the maximum distance that can be moved by natural diffusion of the substance to be crystallized ,
The maximum movable distance is
(1) a substance consumption rate, which is a rate at which the substance is consumed in a solution containing the substance by growing crystals of the substance;
(2) a substance initial concentration which is a concentration of the substance with respect to the microdroplet when the diffusion of the substance is started; and
(3) Diffusion constant of the above substances
Crystal growth vessel, wherein Rukoto estimated from the parameters shown in.   2. The crystal growth container according to claim 1, wherein the periphery of the microdroplet is filled with a medium substance that is not compatible with the microdroplet.   3. The crystal growth container according to claim 1, wherein the size of the microdroplet is such a size that convection driven by a density difference in the microdroplet is suppressed.   4. The crystal according to claim 1, wherein a length of the shortest inner portion where the microdroplet is disposed is the same as a length of the longest portion of the microdroplet. 5. Growth container.   The crystal growth container according to any one of claims 1 to 4, wherein the substance to be crystallized is a protein.   The container for crystal growth according to any one of claims 1 to 5, wherein the container is a cylindrical capillary.   The crystal of the substance to be crystallized grown in a microdroplet is set as an analysis target of a crystal structure analyzing apparatus for analyzing the crystal structure. Crystal growth container.   A droplet preparation device connected to the crystal growth vessel according to any one of claims 1 to 7, wherein the microdroplet is prepared and fed to the crystal growth vessel. A method for obtaining a crystal for obtaining a crystal of a substance,
An estimation step of estimating the maximum movable distance indicating the maximum distance that the substance can move in the microdroplet by natural diffusion from the parameters shown in the following (1), (2), and (3):
(1) A substance consumption rate, which is a rate at which the substance is consumed in a solution containing the substance by growing crystals of the substance;
(2) Initial substance concentration which is the concentration of the substance with respect to the microdroplet when the diffusion of the substance is started;
(3) Diffusion constant of the above substance;
A preparation step of preparing the microdroplet so as to have a size based on the maximum movable distance estimated by the estimation step;
And a growth step of growing at least one crystal of the substance in the microdroplet prepared by the preparation step.