WO2007063898A1

WO2007063898A1 - Method and apparatus for crystallization of protein, and apparatus for protein crystallization treatment

Info

Publication number: WO2007063898A1
Application number: PCT/JP2006/323812
Authority: WO
Inventors: Toru Torii; Muhammad Imran Al-Haq; Eric Lebrasseur; Hiroki Yamazaki; Koji Nagata; Masaru Tanokura
Original assignee: The University Of Tokyo; Techno Medica Co., Ltd.
Priority date: 2005-11-29
Filing date: 2006-11-29
Publication date: 2007-06-07
Also published as: JPWO2007063898A1

Abstract

Disclosed is a method for crystallization of a protein by a microbatch process comprising directly adding a droplet of a reagent required for the crystallization of the protein to a trace amount of a droplet of a solution of the protein to yield a sample droplet and placing the sample droplet under a predetermined environment to cause the crystallization of the protein in the sample droplet. The method is characterized in that, in the microbatch process, a pair of electrodes are arranged side by side over or below the sample droplet and an electric field is applied to the sample droplet from the upper or lower side thereof by means of the electrodes to cause the crystallization of the protein.

Description

Protein crystallization method and apparatus, and protein crystallization treatment apparatus

[0001] The present invention relates to a method and apparatus for crystallizing a protein and a protein crystallization treatment apparatus to which the method for crystallizing the protein is applied.

Background art

[0002] In order to know the structure and function of proteins from the viewpoint of biology and medicine, protein structure analysis has been actively studied.

The most common method for analyzing the three-dimensional structure of proteins is the X-ray structure analysis method. In order to analyze the three-dimensional structure of a protein by this X-ray structure analysis method, a high-quality single crystal of the protein is required.

For this reason, protein crystallization is indispensable for protein structural analysis. Protein crystals are precipitated and grown by mixing reagents necessary for protein crystallization and a protein solution at an appropriate concentration and placing the mixed solution in an appropriate environment.

However, very few proteins have confirmed crystallization conditions. In order to confirm the crystallization conditions, and in order to confirm the protein crystallization conditions, the types of reagents, reagent concentrations and temperature conditions are changed by trial and error, and more than several thousand crystallization conditions are set. Need to be screened.

To screen protein crystallization conditions for a very large variety of crystallization conditions, a very large number of sample solutions are required. For this reason, it is necessary to secure and store a very large amount of sample solution. For the same reason, it is necessary to store many reagents necessary for protein crystallization. As a result, very large storage facilities are required.

The inventors have already proposed a microbatch method using the fine droplets in the sample solution to solve the above-described problems (see Patent Documents 1 to 5). As a result, a large number of sample solutions can be screened without requiring large storage facilities. On the other hand, biopolymers such as proteins have a very large molecular weight and a small self-diffusion coefficient in the sample solution, which makes it difficult for the molecules to aggregate or associate to form crystal nuclei. There are many sample solutions that cannot be crystallized even if sample solutions are prepared.

In order to promote the crystallization of biopolymers such as proteins that are difficult to crystallize, it has already been proposed to apply a voltage to the sample solution (see Patent Document 6).

[0003] Patent Document 1: Japanese Patent Application 2005— 176339

Patent Document 2: Japanese Patent Application 2005— 176344

Patent Document 3: Japanese Patent Application 2005— 176351

Patent Document 4: # 112005-176354

Patent Document 5: Japanese Patent Application 2005— 176359

Patent Document 6: JP 2000-63199 A

Disclosure of the invention

Problems to be solved by the invention

[0004] As described above, by applying a voltage to the sample solution, the molecules to be crystallized in the solution can be electrophoresed, the amount of movement of the molecules increases, and the molecules collide with each other in the solution. Is likely to occur, and the formation of crystal nuclei is promoted.

However, since the conventional crystallization method described above is configured to apply a voltage to the sample solution with the sample solution sandwiched between a pair of electrodes, the apparatus itself has a very special and complicated structure.

In particular, when this conventional crystallization method is applied to the micronotch method using microdroplets proposed by the inventors, the generated microsample droplets are electrostatically transported to a predetermined storage position. Since a small sample droplet must be moved to a crystallization apparatus having a pair of electrodes and a voltage must be applied, if the structure of the apparatus becomes complicated, the entire structure of the apparatus will naturally increase in size. It will be against the purpose.

In addition, when performing X-ray structural analysis, the obtained crystal is irradiated with X-rays, and an X-ray reflection image or transmission image is taken. However, if the quality of the obtained crystal is not good, the resolution of the photographed crystal will be low, and an accurate three-dimensional structure analysis will not be possible. The present invention solves the above-mentioned conventional problems, and can promote crystallization without complicating the structure of the apparatus and enlarging the apparatus itself in the microbatch method using microdroplets. In addition, an object of the present invention is to provide a crystallization method and apparatus capable of obtaining high-quality crystals with high X-ray diffraction resolution, and a protein crystallization treatment apparatus to which the crystallization method is applied! .

Means for solving the problem

In order to achieve the above-mentioned object, the crystallization method in the microbatch method according to the present invention is a sample droplet obtained by directly adding a droplet of a reagent necessary for protein crystallization to a small amount of a protein solution droplet. A microbatch method in which the sample droplet is crystallized in a predetermined environment to crystallize the protein in the sample droplet, and a pair of electrodes are arranged side by side above or below each sample droplet. In addition, an electric field for crystallization is applied to the sample droplet by the electrode with an upward or downward force.

The crystallization apparatus used in the microbatch method according to the present invention is a sample droplet generated by directly adding a droplet of a reagent necessary for protein crystallization to a small amount of a protein solution droplet. And a pair of electrodes arranged above or below the holding means and above or below each sample droplet, and the sample held by the holding means by the electrodes It is characterized in that an electric field for crystallization is applied to the droplet from the upper or lower side.

Furthermore, the protein crystallization treatment apparatus according to the present invention comprises a traveling wave electric field generating electrode and a liquid having an unresolved solution layer mixed with sample droplets disposed on the traveling wave electric field generating electrode. A droplet holding means, and applying a voltage at a predetermined frequency to the traveling wave electric field generating electrode, thereby providing a droplet of a reagent necessary for crystallization of the protein supplied in the droplet holding means. A solution processing means configured to move and combine the droplets of the protein solution to generate a sample droplet, and then hold the sample droplet in the droplet holding means; and the solution A droplet supply means for supplying the droplet to the droplet holding means in the processing means, and an upward or downward force on each sample droplet in the droplet holding means for holding the sample droplet. Electric field for crystallization So that you can give There the arrangement crystallisation unit, Ekishizukuho crystallization have been made in the crystallization unit Crystallization growth monitoring means for monitoring the crystallization growth of the sample droplet in the holding means, and the droplet holding means is transferred to the crystallization means after holding the sample droplet, and the crystallization means and the crystallization growth monitoring And a transfer means capable of transferring the droplet holding means to and from the means.

The invention's effect

In the crystallization method according to the present invention, a pair of electrodes are arranged side by side above or below each sample droplet in the microbatch method, and an upward or downward force is applied to the sample droplet by the electrodes for crystallization. By applying an electric field, crystallization in the sample droplet can be promoted, and high-quality crystals with high z or X-ray diffraction resolution can be obtained. Compared to the conventional accelerating method that promotes crystallization by interposing a droplet, the crystallization is promoted and the resolution of z or X-ray diffraction becomes higher with a simple structure. , Has an effect.

The electric field applied for the crystallization can be 200 V / mm or more. By increasing the applied electric field to 200 V / mm or more, crystallization in the sample droplet is promoted.

In addition, by setting the electric field applied for the crystallization to lOOOV / mm or more, high quality crystals with high X-ray diffraction resolution are precipitated in the sample solution.

The crystallization apparatus according to the present invention includes a holding means for holding a sample droplet generated by directly adding a reagent droplet necessary for protein crystallization to a minute amount of a protein solution droplet; A pair of electrodes arranged above or below the holding means and above or below each of the sample droplets, and by the electrodes, an upward or downward force is exerted on the sample droplet held by the holding means. Since the electric field for crystallization is configured to be applied, the crystallization is promoted and Z or X is simply configured so that the sample droplet can be disposed above or below the electrodes arranged side by side. High-quality crystals can be precipitated with higher resolution of line diffraction. In the case of a conventional device that promotes crystallization by sandwiching a sample droplet between a pair of electrodes, the sample droplet must be placed between the pair of electrodes, which complicates the structure and each sample. Although there is a problem that the apparatus becomes large because the droplet is sandwiched between the pair of electrodes, the crystallization apparatus according to the present invention can arrange the sample droplet above or below the electrodes arranged side by side. Configure Therefore, the structure of the apparatus is simple, and the apparatus itself can be made smaller than the conventional apparatus.

Furthermore, the protein crystallization apparatus according to the present invention comprises a traveling wave electric field generating electrode and a droplet holding means having a solution layer that does not mix with the sample droplet disposed on the traveling wave electric field generating electrode. And applying a voltage at a predetermined frequency to the traveling-wave electric field generating electrode, thereby supplying a droplet of a reagent necessary for crystallization of the protein supplied in the droplet holding means and a liquid of a protein solution. A liquid processing unit configured to move and combine the droplets to form a sample liquid droplet, and then hold the sample droplet in the droplet holding unit; and a droplet in the solution processing unit Electrodes are provided so that an electric field for crystallization can be applied to each sample droplet in the droplet supply means for supplying the droplet to the holding means and each sample droplet in the droplet holding means for holding the sample droplet. Arrangement The crystallization growth monitoring means for monitoring the crystallization growth of the sample droplet in the crystallization means placed in the crystallization means and the crystallization growth monitoring means for monitoring the crystallization growth of the sample droplet in the crystallization means; Since it is transported to the crystallization means after holding the droplet, and the transfer means capable of transferring the droplet holding means between the crystallization means and the crystallization growth monitoring means, the generation force of the sample droplet is crystallized. It is possible to automatically perform a series of operations up to observation. In addition, sample droplet generation, crystallization, and crystallization observation can be performed using common droplet holding means, so sample droplets can be moved very easily. Become.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of a crystallization method and apparatus according to the present invention and a crystallization processing apparatus to which the crystallization method is applied will be described with reference to one embodiment shown in the accompanying drawings.

[0008] FIGS. 1A and 1B are a schematic perspective view and a schematic cross-sectional view of a crystallization apparatus for explaining the principle of a crystallization method according to the present invention.

As shown in the drawing, this crystallization apparatus is

An electrode plate 6 in which a plurality of rows of a pair of electrodes 2a and 2b are arranged on the substrate 1,

A solution that does not mix with the sample droplet to hold the sample droplet 5. Have The electrodes 2a and 2b are covered with an insulator 3.

Sample droplets 5 consisting of a mixture of various protein crystallization reagents and protein solutions such as precipitants, salts and surfactants are placed on insulator 3 and each sample solution The drop 5 is placed in the middle of the pair of electrodes.

The sample droplet 5 is surrounded by a solution 4 that does not mix with sample droplets such as oil to prevent evaporation. The setup that surrounds the sample droplet 5 with the solution 4 in combination with the sample droplet corresponds to the so-called microbatch method.

When a potential difference is applied between the electrodes 2a and 2b, an electric field is generated on the electrodes 2a and 2b on which the sample droplet 5 is placed. A high-voltage power supply (not shown) is used to create a continuous electric field. Although not shown, it is also possible to generate an AC electric field by using a signal generator connected to an amplifier, for example.

The sample droplet 5 can be positioned, for example, by creating a hydrophilic spot on a hydrophobic surface. The droplet positioned by the hydrophilic spot is fixed with a tenacity.

2 and 3 show the results of observing the crystallization of protein (egg white lysozyme) over time by actually applying a predetermined electric field to the sample droplet 5 using the protein crystallization apparatus described above. It is a graph.

In this experiment, egg white lysozyme was dissolved in 0.2 M phosphate buffer (pH 4.6), and the solution prepared at 40 mg / mL was used as the protein solution, and NaCl and 0.2 M phosphate buffer (pH 4.6) were used as the precipitant. Using 60 sample droplets (1 1 and 1.2 mm in diameter) each mixed with 0.5 1 (diameter 1 mm), and mixed with the sample droplets made of paraffin oil force, the sample droplets in solution Prepare ^three sample holders that hold

No electric field is applied to the first sample holder,

In the second sample holder, an electric field of DC 200 V / mm (60 V) is applied to each sample droplet by an electrode arranged below,

To the third sample holder, an electric field of DC 400 V / mm (120 V) is applied to each sample droplet by an electrode arranged below,

The fourth sample holder has a DC 6 electrode connected to each sample drop. Apply an electric field of 00V / mm (180V),

In the fifth sample holder, an electric field of DC 800 V / mm (240 V) is applied to each sample droplet by an electrode disposed below,

The state of crystallization was observed for 1 week.

In the protein crystallization apparatus used in the experiment, the distance between the electrodes of the electrode plate was 0.3 mm, and the voltage described in the parenthesis indicates the actually applied voltage value. FIG. 2 is a graph showing the relationship between the size of the largest crystal finally produced in each sample droplet of each sample and the number of crystals.

FIG. 3 is a graph showing the number of crystals finally formed in each sample droplet of each sample and the ratio of the crystals in the droplet.

FIG. 4 is a schematic top view of sample droplets picked up from the first to fifth samples and photographed from above.

As is clear from the experimental results in Figs. 2 to 4, when an electric field is applied compared to the first sample that does not apply an electric field at all, the number of crystals generated in one droplet increases as the electric field strength increases. This means that the ratio of -3 increases. On the other hand, in terms of crystal size, when an electric field of 400 V / mm was applied, the force with which a crystal of 0.3 mm or more was obtained was all less than 0.3 mm under other conditions. This shows that when an electric field is applied, crystallization is promoted and the size of precipitated crystals increases. In particular, in egg white lysozyme, it can be seen that when an electric field of 200 V / mm to 600 V / mm is applied, crystallization is promoted and the effect of increasing the crystal size appears prominently.

Without applying an electric field, if the number of crystals generated in each sample droplet is large as in the first sample, it will not grow into a large crystal and the necessary crystals will not be obtained. If given, the number of crystals generated in each sample droplet is reduced, and the crystal size is increased accordingly, so that necessary crystals can be obtained.

Since the diameter of the droplet is typically 1 mm or less (volume 1 μ1 or less), a very large number of droplets can be set on a small surface. For example, if the volume is: L 1 droplets, 96 droplets can be easily placed in a 8 x 12 row on a 3 x 4.5 cm surface. FIG. 5 is a graph showing the results of observing the crystallization of another protein (thaumatin) by actually applying a predetermined electric field to the sample droplet 5 using the protein crystallization apparatus described above.

In this experiment, thaumatin was dissolved in 0.1 M phosphate buffer (pH 6.25) and adjusted to 100 mg / mL as the protein solution, and 1.0 M sodium tartrate solution as the precipitating agent in 0.1 \ 1 phosphate buffer. Using 60 samples dissolved in (pH 6.25), 60 sample droplets (2 μL) each mixed with 1.0 μL (diameter 1.2 mm) were generated, and the sample was prepared in an inert solution of paraffin oil. Prepare four sample holders holding droplets,

No electric field is applied to the first sample holder,

To the second sample holder, an electric field of DC 500 V / mm (100 V) is applied to each sample droplet by an electrode arranged below,

The third sample holder is given an electric field of DC 1 000 V / mm (200 V) by the electrode placed below each sample droplet,

In the fourth sample holder, an electric field of DC 1 350 V / mm (270 V) was applied to each sample droplet by an electrode disposed below, and the state of crystallization was observed.

In the protein crystallization apparatus used in the experiment, the distance between the electrodes of the electrode plate is 0.2 mm, and the voltage described in the parenthesis indicates the actually applied voltage value. FIG. 5 is a graph showing an average value of the number of crystals finally generated in each sample droplet of each sample.

As can be seen from the experimental results in Fig. 5, when an electric field is applied compared to the first sample that does not apply an electric field at all, the number of crystals generated in one droplet decreases as the electric field strength increases. Represents. This shows that when an electric field is applied, crystallization is promoted and the size of the precipitated crystals increases. In particular, in the case of thaumatin, when an electric field of 1000 V / mm to 1350 V / mm is applied, the effect of reducing the number of crystals due to the electric field is noticeable.

Without applying an electric field, if the number of crystals generated in each sample droplet is large as in the first sample, it will not grow into a large crystal and the necessary crystals will not be obtained. If given, the number of crystals generated in each sample droplet is reduced, and the crystal As the size of the crystal increases, the crystals necessary to obtain sufficient X-ray diffraction resolution can be obtained.

Fig. 6 shows X-ray diffracting equipment (super high brightness X-ray generator FR-E SuperBright (Rigaku)) for crystals picked up from samples 1 to 4 one by one. This is a diffraction image detected by a detector (high-speed imaging plate X-ray detector R-AXIS VII (Rigaku)). Table 1 shows the resolution.

[table 1]

From Fig. 6 and Table 1, the electric field is not applied! The resolution when the electric field is 500 V / mm and the resolution when the electric field is 500 V / mm are both 1.54 A. When the electric field of lOOOV / mm is applied, the resolution is 1.3 7 A. It was confirmed that the resolution improved to 1.35A when an electric field of 1350 V / mm was applied. Thus, it was confirmed that the quality of crystals obtained by the electric field was improved.

Next, a more preferred embodiment of the crystallization apparatus according to the present invention will be described with reference to FIG.

FIGS. 7 (a) and 7 (b) show schematic cross-sectional views of the second embodiment of the crystallization apparatus, respectively. (A) shows that the sample droplet holding means is set on the substrate 1 having the electrode 2. (B) shows a state in which the sample droplet holding means is separated from the substrate 1 having the electrode 2.

In the present embodiment, the same or equivalent elements as those shown in FIG. 1 are denoted by the same reference numerals used in FIG. 1, and detailed description thereof is omitted.

The electrode plate 6 is formed by arranging a plurality of pairs of electrodes 2 a and 2 b on a substrate 1. The material of the substrate 1 can be any non-conductive material such as glass or plastic plate. For the processing of the electrode 2, for example, any conductive material such as copper, gold, ITO (isodium stannate) can be used.

A sample droplet holding means 9 is detachably provided on the electrode plate 6 described above. The separable sample droplet holding means 9 is formed by fixing a plastic film 7 to a holder 8 and contains therein a solution 4 that does not mix with sample droplets. The sample droplet 5 is disposed in the solution 4 that does not mix with the sample droplet in the sample droplet holding means 9 described above. In this embodiment, since the plastic film 7 functions as an insulator, it is not necessary to provide the electrode plate 6 with an insulator (the insulator 3 in the embodiment of FIG. 1).

The thickness of the plastic film 7 is in the range between 5 μm and 100 μm, preferably in the range between 5 μm and 40 μm. The film material can be any insulating material, preferably a material that is impermeable to oil when the soot solution mixed with the sample droplets is oil.

The protein crystallization apparatus configured as described above generates an electric field on the electrodes 2a and 2b on which the sample droplet 5 is placed by applying a potential difference between the electrodes 2a and 2b.

[0012] Finally, an example of a protein crystallization treatment apparatus including the crystallization apparatus configured as described above will be described.

FIG. 8 is a schematic block diagram of a protein crystallization treatment apparatus (hereinafter simply referred to as a crystallization treatment apparatus) provided with a protein crystallization apparatus according to the present invention.

As shown in the drawing, this protein crystallization treatment apparatus is

A liquid particle supply unit A for supplying liquid fine particles of a reagent and a protein solution, a solution processing unit B for mixing the reagent and protein solution supplied from the liquid particle supply unit A and holding them on the sample droplet holding means 9;

A protein crystallization apparatus C for applying an electric field to the sample droplet 5 held on the sample droplet holding means 9 to promote protein crystallization; and

Sample droplets that are promoted by crystallization in the protein crystallization apparatus C The crystallization growth monitoring unit D that periodically monitors the crystallization growth of proteins in the sample and the sample droplet holding means 9 are transferred from the solution processing unit B to the crystallization device C and, if necessary, the crystallization device. Transfer means E for transferring from C to the crystallization growth monitoring section D, and from the crystallization growth monitoring section D to the crystallization apparatus C.

Have

First, the solution processing unit B will be described.

FIG. 9 is a schematic diagram showing a circuit configuration of the solution processing unit B, and FIG. 10 is a schematic cross-sectional view corresponding to the AA cross section of the solution processing unit B in FIG.

The solution processing unit B includes a circuit unit 13 in which a plurality of electrode lines for generating static electricity are arranged on a substrate 11, and a sample droplet holding unit 9 configured to be detachable from the circuit unit 13.

As shown in FIG.

A first electrode group 13a in which 18 electrode wires are concentrically arranged in a fan shape of 90 degrees;

A second electrode group 13b composed of 26 electrode wires arranged in parallel with the electrode line located in the center of the first electrode group 13a;

A third electrode group 13c that is arranged adjacent to the second electrode group 13b and that also includes five electrode line forces arranged in parallel to the direction of each electrode line of the second electrode group 13b; and

A fourth electrode group 13d which is arranged adjacent to the third electrode group 13c, and which also has 26 electrode line forces arranged in parallel to the direction of each electrode line of the third electrode group 13c,

A fifth electrode group 13e that is arranged adjacent to the fourth electrode group 13d and that also includes five electrode line forces arranged in parallel to the direction of each electrode line of the fourth electrode group 13d; and

A sixth electrode group 13f that is arranged adjacent to the fifth electrode group 13e and that also has 26 electrode line forces arranged in parallel to the direction of each electrode line of the fifth electrode group 13e; and

A seventh electrode group 13g consisting of five electrode line forces arranged adjacent to the sixth electrode group 13f and parallel to the direction of each electrode line of the sixth electrode group 13f;

An 8th electrode group 13h that is arranged adjacent to the 7th electrode group 13g, and that also has 26 electrode line forces arranged in parallel to the direction of each electrode line of the 7th electrode group 13g;

A ninth electrode group 13i that is arranged adjacent to the eighth electrode group 13h and that also has five electrode line forces arranged in parallel to the direction of each electrode line of the eighth electrode group 13h; A tenth electrode group 13j, which is arranged adjacent to the ninth electrode group 13i, and also includes 26 electrode line forces arranged in parallel to the direction orthogonal to the direction of each electrode line of the ninth electrode group 13i;

The eleventh electrode group 13k, which is also arranged adjacent to the tenth electrode group 1¾, and also has five electrode line forces arranged in parallel to the direction orthogonal to the direction of each electrode line of the tenth electrode group 1¾,

Have

The solution processing unit B has one or more control devices (not shown) that control the voltage of the electrode lines of the electrode groups 13a to 13k.

As shown in FIG. 10, the sample droplet holding means 9 includes a base portion 16 made of an electrically insulating material. The base portion 16 includes a lower portion 16a configured to be detachable from the circuit portion 13 and an upper portion 16b that contains a solution (for example, oil) mixed with the sample droplet. 10 shows a state in which the sample droplet holding means 9 is attached to the circuit unit 13, and FIG. 11 shows a state in which the sample droplet holding unit 9 is removed from the circuit unit 13. A hydrophilic film 17 is formed on the bottom surface inside the upper portion 16b, and a hydrophobic film 8 is further formed on the upper surface of the hydrophilic film 7.

A plurality of hydrophilization spots 19 are formed at positions corresponding to the fourth electrode group 13d, the sixth electrode group 13f, the eighth electrode group 13h, and the tenth electrode group 1¾ in the hydrophobic membrane 8. . These hydrophilized spots 19 are formed by, for example, forming a resist in a predetermined pattern in advance before depositing the hydrophobic film 18 on the hydrophilic film 17, and then depositing the hydrophobic film 18 on the resist portion. And the hydrophilic film 17 is partially exposed. Further, at the position corresponding to the first electrode group 13a on the upper wall of the upper portion 16b of the base portion 16, a necessary number of openings 16c for introducing the liquid fine particles from the liquid fine particle supply portion 1 are provided (( Two in this example).

FIG. 12 is a diagram showing a positional relationship between the electrode group of the circuit unit 13 and the hydrophilic spot 19 and the opening 16c of the sample droplet holding means 9.

The sample droplet holding means 9 configured as described above is used by being attached to the circuit unit 13 when the sample droplet is generated, and is removed from the circuit unit 13 after the required number of sample droplets are generated and held. Then, it is transferred to the crystallization apparatus C and the crystallization growth monitoring unit D by the transfer means E. Here, the principle of moving a droplet by applying a voltage to a plurality of electrode lines as described above will be described.

First, the relationship between the voltage applied to the electrode lines arranged in the same direction and the movement of the droplet will be described with reference to FIG. This is the principle of droplet movement within each electrode group.

As shown in Figs. 13 (a) to (d), six-phase voltages (++) are sequentially applied to the electrode lines so that the electrode lines are moved one by one at an appropriate cycle time. The direction in which the voltage is moved is the direction in which the droplet travels (note that the voltage application pattern is not limited to this example).

As described above, the voltage of 6 phases (+ + +) is moving at an appropriate cycle time. When a droplet is placed on the electrode wire, a negative voltage is applied to the negatively charged droplet. The electrode repels and is attracted to the electrode to which a positive voltage is applied, so that the voltage moves in the moving direction. When the voltage stops moving, the droplet stops at that position.

In each electrode group, the droplet moves according to the principle described above.

Next, the relationship between the voltage applied to the electrode lines arranged in different directions and the movement of the droplet will be described with reference to FIGS. 14 (a) and 14 (b). This is the principle of moving droplets between electrode groups with different electrode wire directions.

In FIG. 14, electrode group X and electrode group Y have different electrode line directions. Figure 1

4 In (a), a six-phase voltage (+++) is applied to the electrode group X. However, since there is no voltage movement in the electrode group X, a positive voltage is applied to the droplet. Between the active electrode and the voltage to which a negative voltage is applied.

Then, from the state shown in FIG. 14 (a), as shown in FIG. 14 (b), a negative voltage is applied to all electrode lines of the electrode group X, and the electrode group Y is adjacent to the electrode group X. By applying a positive voltage to the electrode wire (or all electrode wires), the negatively charged droplets repel the electrode wire of electrode part X and attract to the electrode line of electrode part Y. Therefore, the electrode part X changes to the electrode part Y.

The droplet moves between the electrode portions having different electrode line directions on the principle described above. [0016] The droplet 5a of the protein solution supplied to the first electrode group 13a through the opening 16c and the droplet 5b of the reagent necessary for crystallization are formed in the first electrode group 13a according to the principle described above. As a result of the electrostatic transport, they collide at the center of the first electrode group 13a and coalesce into a sample droplet 5 (see FIG. 15).

Next, the sample droplet 5 is electrostatically conveyed from the second electrode group 13b to the hydrophilic spot 19 of the predetermined electrode group according to the principle described above.

Since the hydrophilic film is exposed in the hydrophilization spot 19, the sample droplet contacts the exposed hydrophilic film when it reaches the hydrophilization spot. When the sample droplet comes into contact with the hydrophilic film, its spherical shape collapses, so that the sample droplet does not move any more due to the action of the voltage applied to the electrode wire, and as a result is held on the hydrophilization spot. Therefore, once held on the lyophilic spot, the sample droplet 5 does not move even if no voltage is applied to the electrode wire.

FIG. 16 is a diagram conceptually showing a state where the sample droplet is held in the hydrophilization spot.

FIG. 17 shows a state in which the necessary number of sample liquid droplets are held on the hydrophilized spot by repeating the above-described process continuously the required number of times.

As described above, once the sample droplet is held on the hydrophilic spot, the sample droplet does not move even if a voltage is not applied to the electrode. (See FIG. 11) and transported to the protein crystallizer C by the transfer means 40.

In the above-described embodiment, when a droplet is moved from one electrode group to another electrode group in which the direction of the electrode line is different from that of the electrode group, all the electrode lines of the electrode group before the transfer are transferred. Force to apply a positive voltage and apply a negative voltage to all electrode wires of the electrode group after replacement. This is not limited to this example. Apply reverse-phase voltage to the later electrode group.

The protein crystallization apparatus C includes an electrode plate 6 on which a sample droplet holding means 9 can be attached.

Fig. 18 shows the sample droplet holding means 9 on the electrode plate 6 in the protein crystallizer C. FIG. 19 is a schematic cross-sectional view showing a state in which the sample droplet holding means 9 is removed from the electrode plate 6.

As shown in the drawing, the electrode plate 6 is formed by arranging a plurality of rows of a pair of electrodes 2 on the substrate 1, and is held on the hydrophilization spot 19 in a state where the sample droplet holding means 9 is mounted on the electrode plate 6. The sample droplet 5 is configured to be positioned between the pair of electrodes 2a and 2b.

In this state, a potential difference is applied between the electrodes 2a and 2b by using a control device (not shown), and the sample droplet 5 is placed! /, And an electric field is generated on the electrodes 2a and 2b. And promotes the precipitation of high quality crystals that increase the resolution of Z or X-ray diffraction.

Here, the droplet supply unit A that supplies droplets to the solution processing unit B configured as described above will be briefly described.

The droplet supply unit A is configured to supply the solution processing unit B with a protein solution droplet and a reagent droplet.

Preferably, the droplets supplied from the droplet supply unit A are adjusted so that the combined sample droplets are lm m or less (volume 1 μ 1 or less).

[0019] Finally, the configuration of the crystallization growth monitoring unit D will be described.

The transfer means 定期 periodically takes out the sample droplet holding means 9 from the crystallization part C and transfers it to the crystallization growth monitoring part D.

The crystallization growth monitoring unit D optically or electrically monitors the crystallization of the sample droplets of the sample droplet holding means 9 transferred by the transfer means 記録, and records and displays or displays the result.

Various configurations are possible for the crystallization growth monitoring unit D. Here, some examples will be described.

For example, the crystallization growth monitoring unit D includes a light source and a photomultiplier tube. The light source irradiates the sample droplet on the sample droplet holding means with light, and the photomultiplier tube irradiates the refractive index of the sample droplet. And the degree of crystallization growth of the protein in the sample droplet can be determined based on the detected change in the refractive index. In this case, for example, an increase in the detected refractive index. In addition, it can be determined that the crystallization of the protein in the sample droplet is progressing. The detection results can be recorded on a suitable recording device and, if necessary, visually and Z or audibly by a display, printer, lamp and Z or speaker etc.

The degree of crystallization growth of the protein can be displayed to the user.

As another example, for example, the crystallization growth monitoring apparatus D includes an optical measurement unit having an automatic focusing function, and the focal position in the sample droplet on the sample droplet holding unit by the automatic focusing function. And the degree of crystallization growth of the protein can be determined by detecting the focal position. In this case, for example, when the focal position cannot be detected, crystallization growth has not progressed, but when the focal position can be detected, it can be determined that crystallization growth has progressed. The detection results can be recorded on an appropriate recording device, and if necessary, the crystallized growth of the protein visually and Z or audibly with a display, printer, lamp and / or speaker. The degree can be displayed to the user.

As yet another example, for example, the crystallization growth monitoring apparatus D includes a light source and a light receiving unit, and after irradiating the sample droplet on the sample droplet holding unit with the light source, the light irradiated by the light receiving unit is emitted. Based on the amount of light received and received by the light receiving means, the absorbance of the sample droplet can be detected, and the degree of crystallization growth of the protein can be determined based on the detected absorbance. In this case, for example, it can be determined that the crystallization of the protein in the sample droplet is progressing as the amount of light absorption increases. The detection results can be recorded in an appropriate recording device, and the degree of protein crystallization growth can be visually and Z or audible visually, using a display, printer, lamp and z or speaker, etc., if necessary. Can be displayed to the user.

In the embodiment of the crystallization processing apparatus described above, an example is shown in which eight sample droplets are held in one sample droplet holding means so that the configuration can be easily divided. The number of droplets to be held in is not limited to this embodiment.

As described above, the diameter of the droplets is typically 3 mm or less (volume 10 1 or less), allowing a very large number of droplets to be set on a small surface. For example, for a droplet with a volume of 101, there are 96 liquids placed in 8 x 12 rows on a 3 x 4.5 cm surface. Drops can be placed easily.

Further, in the above-described embodiment, an electrode is arranged below the droplet holding means, and a force for applying an electric field for crystallization of the downward force to the sample droplet is limited to this embodiment. For example, the electrode may be arranged above the droplet holding means so that the upper force applies an electric field to the sample droplet.

Furthermore, in the embodiment of the protein crystallization apparatus described above, the solution processing section B and the protein crystallization apparatus C are configured to have different electrodes. The electrode for generating the traveling wave electric field of the solution processing unit B and the electrode for generating the electric field for crystallization of the protein crystallization apparatus C are used in common, and the electrode of the solution processing unit B is used. Configure the sample droplet to apply a voltage for crystallization.

In the above-described embodiment, the force for applying the voltage for crystallization from the beginning to the sample droplet in the crystallization apparatus. Preferably, before applying the voltage for crystallization, the sample droplet is applied to the sample droplet. And can be configured to apply an electric field of at least 800 V / mm instantaneously (preferably less than 1 second). By applying a high electric field instantaneously to the sample droplet in this way, the sample droplet is strongly attracted to the electrode and deforms under a high electric field, and the area where the droplet contacts the electrode surface increases. It is possible to increase the crystallization promotion effect by applying an electric field and increase the precipitation of high-quality crystals that increase the resolution of Z or X-ray diffraction. Also, by deforming the droplets so as to come into contact with the electrode surface, the shadow (black portion) around the droplets is reduced. When observing crystallization in a sample droplet by image processing, if there are many shadow portions around the droplet, the force that makes it difficult to determine whether the crystal overlapping the shadow portion is a shadow or a crystal. By deforming the droplets so as to be in contact with the electrode surface, shadows around the droplets can be reduced, and as a result, crystallization can be easily observed.

Figure 20 (a) and (b) shows the state of the sample droplet after applying a voltage of 800 V / mm to a sample droplet under the same conditions for one week and after applying a voltage of 880 V / mm for 1 second. It is a photograph which shows the state of a sample droplet. From the results of this experiment, the shape of the sample droplet was deformed even when applied at a voltage of 800 V / mm for one week, whereas a voltage of 880 V / mm was applied. It can be seen that the sample droplet can be deformed in 1 second. Therefore, it can be seen that the sample droplet can be deformed by instantaneously applying a voltage higher than 800 V / mm (preferably a voltage of 880 V / mm or more).

Furthermore, as described above, for the electrode, any conductive material such as copper, gold, or ITO (isodium stannate) can be used. Note that the above-described photograph of FIG. 4 is a top view photograph of a sample droplet as a result of an experiment using a copper electrode. In the case of using a copper electrode, only a photograph using reflected light can be taken because copper is not translucent. For this reason, as shown in Fig. 4, the electrode appears in the photograph. However, when an ITO film is used for the electrode, the ITO film is transparent, so as shown in FIG. 21, the sample droplet holding means 35 is mounted on the electrode plate 30, and the microscope is used with the polarized light of the transmitted light. Crystals can be observed at 40 mag.

FIG. 21 is a schematic cross-sectional view of the crystallization apparatus when the crystal is observed with a microscope 40 or the like while the sample droplet holding means 35 is mounted on the electrode plate 30. In FIG. ITO electrode, symbol 32 is a glass substrate with ITO electrode, symbol 36 is an insulating film such as plastic film, symbol 37 is a holder, symbol 38 is a solution that does not mix with sample droplets (eg oil), symbol Reference numeral 39 denotes a droplet, reference numerals 41 and 42 denote deflecting plates, and reference numeral 43 denotes a light source.

FIG. 22 is a photograph of a crystal actually crystallized using the crystallization apparatus of FIG. 21 using an ITO film, using the polarization of transmitted light.

Brief Description of Drawings

FIG. 1 (a) and (b) are a schematic perspective view and a schematic cross-sectional view of a crystallization apparatus for explaining the principle of a crystallization method according to the present invention.

FIG. 2 is a graph showing the relationship between the maximum crystal size generated in each sample droplet of each sample using lysozyme and the number of crystals.

FIG. 3 is a graph showing the number of crystals generated in each sample droplet of each sample.

[FIG. 4 (a)] A schematic top view of a sample droplet in which one first sample force is also picked up.

FIG. 4 (b) is a schematic top view photograph of a sample droplet picked up from the second sample.

[FIG. 4 (c)] A schematic top view of a sample droplet picked up by one third sample force. FIG. 4 (d) is a schematic top view photograph of a sample droplet picked up from the fourth sample.

[FIG. 4 (e)] A schematic top view of a sample droplet in which a fifth sample force is also picked up.

FIG. 5 is a graph showing the average number of crystals generated in each sample droplet of each sample finally using thaumatin.

[Fig. 6] (a) to (d) are X-ray diffractometers (super-high brightness X-ray generators) for crystals picked up one by one from each sample applied with an electric field at 0, 500, 1000, 1350 V / mm FR-E SuperBright (Rigaku)) is used to irradiate X-rays, and is a diffraction image detected by a detector (high-speed imaging plate X-ray detector R-AXIS VII (Rigaku)).

7] (a) and (b) show schematic cross-sectional views of the second embodiment of the crystallization apparatus, respectively. (A) shows that the sample droplet holding means is set on the substrate 1 having the electrode 2. (B) shows a state where the sample droplet holding means is separated from the substrate 1 having the electrode 2.

[8] FIG. 8 is a schematic block diagram of a protein crystallization treatment apparatus including the crystallization apparatus according to the present invention.

FIG. 9 is a schematic diagram showing a circuit configuration of a solution processing unit B.

FIG. 10 is a schematic cross-sectional view of the solution processing section B corresponding to the AA cross section in FIG.

FIG. 11 is a schematic cross-sectional view corresponding to FIG. 10, showing a state where the sample droplet holding means is removed from the circuit section 13.

FIG. 12 is a diagram showing the positional relationship between the electrode group of the circuit section 13 and the hydrophilization spot 19 and opening 16c of the sample droplet holding means 9.

FIG. 13 is a diagram for explaining the relationship between the voltage applied to the electrode lines arranged in the same direction and the movement of the droplet.

FIG. 14 is a diagram for explaining the relationship between the voltage applied to electrode lines arranged in different directions and the movement of a droplet.

FIG. 15 is a diagram illustrating a process of generating a sample droplet 5 by combining a droplet 5a of a protein solution and a droplet 5b of a reagent necessary for crystallization.

FIG. 16 is a diagram conceptually showing a state in which a sample droplet is held in a hydrophilic spot. FIG. 17 is a view showing a state in which a necessary number of sample droplets are held on a hydrophilization spot. FIG. 18 is a schematic cross-sectional view showing a state where the sample droplet holding means 9 is attached to the electrode plate 6 in the protein crystallization apparatus C.

FIG. 19 is a schematic cross-sectional view showing a state where the sample droplet holding means 9 is removed from the electrode plate 6 in the protein crystallization apparatus C.

FIG. 20 (a) is a photograph showing the state of the sample droplet after applying a voltage of 800 V / mm to the sample droplet for one week.

FIG. 20 (b) is a photograph showing the state of the sample droplet after a voltage of 880 V / mm is applied to the sample droplet under the same conditions as in FIG. 20 (a) for 1 second.

FIG. 21 is a schematic cross-sectional view of a crystallization apparatus when a crystal is observed with a microscope 40 or the like while the sample droplet holding means 35 is mounted on the electrode plate 30.

FIG. 22 is a photograph of a crystal crystallized using an ITO film electrode, taken using polarized light of transmitted light.

Explanation of symbols

1 Board

2 electrodes

3 Insulator

4 Solution that does not mix with sample droplets

5 Sample droplet

6 Electrode plate

7 Plastic film

8 Holder

9 Sample droplet holding means

液体 Liquid particulate supply unit

B Solution processing section

11 Board

13 Circuit part

13a-13k electrode county 16 base

16a Lower part

16b Upper part

16c Opening (droplet introduction part) 17 Hydrophilic membrane

18 Hydrophobic membrane

19 Hydrophilic spot

C Protein crystallization accelerating device D Crystallization growth monitoring unit E Transfer means

Claims

The scope of the claims

[1] A sample droplet is generated by directly adding a droplet of a reagent necessary for protein crystallization to a small amount of a protein solution droplet, and the sample droplet is placed in a predetermined environment. In the microbatch method to crystallize the protein in the drop,

A pair of electrodes are placed side by side above or below each sample droplet, and an electric field for crystallization is applied to the sample droplet by the above-mentioned electrodes.

The crystallization method in the microbatch method characterized by the above-mentioned.

[2] Before applying the electric field for crystallization, an electric field higher than 800 V / mm is instantaneously applied to the sample droplet by the electrode to enlarge the area where the droplet contacts the electrode surface. The crystallization method according to claim 1, wherein

[3] The electric field is a DC electric field or an AC electric field

The crystallization method according to claim 1 or 2, wherein:

[4] The electrode is made of a transparent material so that the sample droplet can be observed with the electrode placed above or below the sample droplet.

The crystallization method according to any one of claims 1 to 3, wherein:

[5] holding means for holding a sample droplet generated by directly adding a droplet of a reagent necessary for protein crystallization to a minute amount of a protein solution droplet;

A pair of electrodes arranged above or below the holding means and above or below each Sampnore droplet;

Have

The electrode is configured to apply an electric field for crystallization from the upper or lower side to the sample droplet held by the holding means.

A crystallization apparatus for use in a microbatch method.

[6] An electrically insulating hydrophobic layer is provided above or below the electrode,

A holding means consisting of a soot solution layer mixed with sample droplets is arranged adjacent to the electrically insulating hydrophobic layer,

6. The crystallization apparatus according to claim 5, wherein the sample droplet is held in the solution layer when mixed with the sample droplet.

[7] Before applying the electric field for crystallization, an electric field higher than 800 V / mm is instantaneously applied to the sample droplet by the electrode to enlarge the area where the droplet contacts the electrode surface. The crystallization apparatus according to claim 5 or 6, characterized in that

[8] The electric field is a DC electric field or an AC electric field

The crystallization apparatus according to any one of claims 5 to 7, wherein

[9] The electrode is made of a transparent material so that the sample droplet can be observed with the electrode placed above or below the sample droplet.

The crystallization apparatus according to any one of claims 5 to 8, wherein

[10] a traveling wave electric field generating electrode;

A droplet holding means having a solution layer that does not mix with the sample droplets disposed on the traveling wave electric field generating electrode;

By applying a voltage at a predetermined frequency to the traveling wave electric field generating electrode, a droplet of a reagent and a droplet of a protein solution necessary for crystallization of the protein supplied into the droplet holding means are used. Are combined to generate sample droplets, and then a solution processing means configured to hold the sample droplets in the droplet holding means, and a droplet holding in the solution processing means Droplet supply means for supplying the droplets to the means;

Crystallization means in which an electrode is arranged so that an electric field for crystallization can be applied to each sample droplet in the droplet holding means holding the sample droplet, and the crystallization means has a crystal. A crystallization growth monitoring means for monitoring the crystallization growth of the sample droplet in the droplet holding means that has been converted into a liquid;

The droplet holding means is transferred to the crystallization means after holding the sample droplet, and has a transfer means capable of transferring the droplet holding means between the crystallization means and the crystallization growth monitoring means. A protein crystallization treatment apparatus.

[11] The traveling wave electric field generating electrode of the solution processing means and the electrode of the crystallization means are separate electrodes.

The protein crystallization treatment apparatus according to claim 10, wherein:

[12] The traveling wave electric field generating electrode of the solution processing means and the electrode of the crystallization means are the same. Electrode

The protein crystallization treatment apparatus according to claim 10, wherein: