Cylindrical device, method for manufacturing a cylindrical device, and method for crystallizing a protein

Abstract

Provided are a cylindrical device that facilitates crystallization by the counterdiffusion method, a method for producing the cylindrical device, and a method for crystallizing a protein using the cylindrical device. [Solution] The tubular device 100 comprises a long tubular body 1 and a metal structure 2 arranged on the inner surface of the tubular body 1, the metal structure 2 having metal particles 3 arranged on the inner surface of the tubular body 1, the metal particles 3 having an average diameter of 5 nm or more and 50 nm or less when viewed in a direction perpendicular to the inner surface of the tubular body 1, and the average particle surface distance between adjacent metal particles 3, 3 being 3 nm or more and 90 nm or less.

Description

[Technical Field]

[0001] The present disclosure relates to a tubular device, a method for manufacturing a tubular device, and a method for crystallizing a protein. [Background technology]

[0002] Patent Document 1 discloses a nanostructure substrate. This nanostructure substrate is composed of a metal structure made of a composite particle group, a substrate made of a resin base, and a support, and has a front and back surface. In this nanostructure substrate, the geometric surface area of ​​the front side of the composite particle group is larger than the geometric surface area of ​​the back side. The composite particles are composed of fine particles of metal or the like and a coating layer of a metal or a precipitant deposited on the upper part by reduction, and the lower parts of the fine particles of metal or the like are embedded in the resin base, and the embedded fine particles of metal or the like are separated from each other. It is disclosed that in this nanostructure substrate, the composite particle group exhibits plasmon properties.

[0003] Patent Document 2 discloses a method for concentrating and crystallizing biopolymers, and a nanostructure substrate. As an example of a method for crystallizing biopolymers, Patent Document 2 discloses that reduced gold microparticles (average particle size 20 nm) are self-assembled on a transparent polyester resin film, half-submerged and fixed, the substrate is repeatedly immersed in an electroless gold plating solution to precipitate gold particles on the gold microparticles, 10 microliters of protein solution is dropped onto the nanostructure substrate, and crystallization is carried out by the hanging drop vapor diffusion method.

[0004] Non-Patent Document 1 discloses a crystallization method called the counter-diffusion method.

[0005] The counterdiffusion method disclosed in Non-Patent Document 1 is used for crystallization of a crystallization target (a substance to be crystallized) such as a protein.

[0006] Taking the case where the object to be crystallized is a protein as an example, crystallization can be attempted using the counterdiffusion method as follows: First, a protein solution (solution of the object to be crystallized) is filled into a capillary (cylindrical device) such as a thin glass tube, and the lower end of the capillary is closed with a porous body (a gel-like substance, hereinafter referred to as a gel layer).Then, the lower end of the capillary is immersed in a precipitant solution (a solution of a substance that promotes crystallization of the object to be crystallized).

[0007] The protein solution then diffuses outward from the capillary through the gel layer, and the precipitant solution diffuses through the gel layer into the capillary. When the protein solution and precipitant solution interdiffuse in this way, convection in the liquid is suppressed within the narrow capillary. This results in various gradients of protein and precipitant concentration within the capillary, which also change over time. This allows the counterdiffusion method to simultaneously experiment under a wide range of conditions within a single capillary, making it more likely to successfully crystallize substances that are generally considered difficult to crystallize.

[0008] In this way, the counterdiffusion method allows experiments under a wide range of conditions to be conducted simultaneously within a single capillary, and has the advantage of being more likely to successfully crystallize substances that are generally considered difficult to crystallize. Therefore, it is suitable for crystallization experiments on substances such as proteins, which are generally known to be difficult to crystallize.

[0009] Furthermore, while taking advantage of the characteristic that convection in liquids is suppressed within thin capillaries, they are also sometimes used to attempt crystallization in microgravity environments such as those found in outer space, in order to eliminate the effects of gravity.

[0010] Non-Patent Document 2 discloses a general explanation of Voronoi diagrams and the generating points and Voronoi regions in the Voronoi diagrams. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Publication No. 2020-180091 [Patent Document 2] Japanese Patent Application Publication No. 2020-180356 [Non-patent literature]

[0012] [Non-Patent Document 1] Sano et al., "Technology for successful protein crystallization experiments in space," Journal of the Japan Society for Microgravity Applications, Vol. 25, No. 2, 2008, pp. 157-160 [Non-patent document 2] “Voronoi diagram”, Wikipedia, [Retrieved January 6, 2025], Internet<https: / / en.wikipedia.org / wiki / Voronoi_diagram> Summary of the Invention [Problem to be solved by the invention]

[0013] As mentioned above, the counterdiffusion method is often used for substances that are difficult to crystallize, and is often used in costly environments such as outer space. Therefore, there is a need for innovations that make it easier to achieve crystallization (increasing the probability of successful crystallization) using the counterdiffusion method.

[0014] The present disclosure has been made in consideration of the above-mentioned circumstances, and its purpose is to provide a tubular device that facilitates crystallization by the counterdiffusion method, a method for manufacturing this tubular device, and a method for crystallizing proteins using this tubular device. [Means for solving the problem]

[0015] In order to achieve the above object, the tubular device according to the present disclosure comprises: A long cylindrical body, a metal structure disposed on the inner surface of the cylindrical body, the metal structure portion has metal particles arranged on the inner surface of the tube of the cylindrical body, the metal particles have an average diameter of 5 nm or more and 50 nm or less when viewed in a direction perpendicular to the inner surface of the cylindrical body, The average distance between the particle surfaces of adjacent metal particles is 3 nm or more and 90 nm or less.

[0016] In order to achieve the above object, a method for manufacturing a tubular device according to the present disclosure includes: A method for manufacturing the tubular device described above, a primer layer forming step of forming a primer layer containing methylolmelamine or a methylolmelamine derivative on the inner surface of the cylindrical body; a metal structure forming step of constructing the metal structure on the primer layer, The metal structure forming step includes a liquid passing step of maintaining a state in which a metal colloid solution is retained within the cylindrical body.

[0017] To achieve the above object, the protein crystallization method according to the present disclosure includes: A method for crystallizing a protein by counterdiffusion, comprising: The cylindrical device described above is used as a capillary. [Effects of the Invention]

[0018] According to the present disclosure, it is possible to provide a tubular device in which crystallization by the counterdiffusion method is more likely to occur, a method for producing this tubular device, and a method for crystallizing a protein using this tubular device. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a perspective view of a tubular device. [Figure 2] FIG. 2 is a schematic cross-sectional view of a tubular device. [Figure 3]FIG. 10 is a schematic diagram showing a state in which a part of the inner surface of the cylindrical device is developed into a flat surface. [Figure 4] 1 is an SEM image of point A in Example 1. [Figure 5] 1 is an SEM image of point B in Example 1. [Figure 6] 1 is an SEM image of point C in Example 1. [Figure 7] FIG. 10 is a Voronoi analysis diagram at point A in the first embodiment. [Figure 8] FIG. 1 is an explanatory diagram of a protein crystallization experiment using the counterdiffusion method. [Figure 9] 10 is an SEM image of point A in Example 2. [Figure 10] 10 is an SEM image of point B in Example 2. [Figure 11] 10 is an SEM image of point C in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0020] A tubular device, a method for manufacturing a tubular device, and a method for crystallizing a protein according to embodiments of the present disclosure will be described with reference to the drawings.

[0021] First, an outline of a tubular device, a method for manufacturing a tubular device, and a method for crystallizing a protein according to an embodiment of the present disclosure will be described.

[0022] FIG. 1 shows a tubular device 100 according to this embodiment.

[0023] The cylindrical device 100 comprises a long cylindrical body 1 and a metal structure 2 arranged on the inner surface of the cylindrical body 1, the metal structure 2 having metal particles 3 arranged on the inner surface of the cylindrical body 1, the metal particles 3 having an average diameter of 5 nm or more and 50 nm or less when viewed in a direction perpendicular to the inner surface of the cylindrical body 1, and the average particle surface distance between adjacent metal particles 3, 3 being 3 nm or more and 90 nm or less.

[0024] By using the tubular device 100 as a capillary, crystallization by the counterdiffusion method is facilitated. In other words, the tubular device 100 is suitable for use as a capillary in the counterdiffusion method.

[0025] The protein crystallization method according to this embodiment can be carried out by using the cylindrical device 100 as a capillary in the counterdiffusion method.

[0026] The manufacturing method for the tubular device according to this embodiment includes a primer layer forming process for forming a primer layer 21 containing methylolmelamine or a methylolmelamine derivative on the inner surface of the tubular body 1, and a metal structure forming process for constructing a metal structure part 2 on the primer layer 21, and the metal structure forming process includes a liquid passing process for maintaining a state in which a metal colloid solution is retained within the tubular body 1.

[0027] The tubular device 100 can be manufactured by the method for manufacturing a tubular device according to this embodiment.

[0028] The cylindrical device 100 will be described in detail below.

[0029] The cylindrical body 1 is a long, cylindrical or cylindrical member. A metal structure 2 is disposed on the inner surface of the cylindrical body 1.

[0030] The cylindrical body 1 may be formed of, for example, glass, polycarbonate, acrylic, acrylonitrile butadiene styrene polyimide, polyethylene terephthalate, polystyrene, and cycloolefin polymer. The cylindrical body 1 is preferably made of glass.

[0031] The inner diameter of the cylindrical body 1 is, for example, 0.2 mm or more and 2 mm or less.

[0032] The length of the cylindrical body 1 in the longitudinal direction of the cylinder (the same as the axial direction of the cylinder) is, for example, 50 mm or more and 300 mm or less.

[0033] As shown in Figures 2 and 3, the metal structure 2 is a structural part formed on the inner surface of the cylindrical body 1, and has an arrangement structure of metal particles 3 arranged on the inner surface of the cylindrical body 1. The metal structure 2 has a plurality (a large number) of metal particles 3 arranged on the inner surface of the cylinder of the cylindrical body 1. Note that Figure 2 is a schematic diagram of a cross section of the cylindrical device 100. Also, Figure 3 is a schematic diagram of a state in which part of the inner surface of the cylindrical device 100 (see Figure 1) is developed into a flat surface.

[0034] The metal structure 2 may have a primer layer 21 formed on the inner surface of the cylindrical body 1. The primer layer 21 is preferably a layer containing methylolmelamine or a methylolmelamine derivative. When the metal structure 2 has the primer layer 21, the metal particles 3 may be fixed onto the inner surface of the cylindrical body 1 via the primer layer 21.

[0035] The metal particles 3 are metal nanoparticles. The metal particles 3 are preferably nanoparticles of an element in Group IB or Group VIIIB of the periodic table. Specifically, the metal particles 3 are gold (Au), silver (Ag), copper (Cu), platinum (Pt), palladium (Pd), rhodium (Rh), iridium (Ir), ruthenium (Ru), nickel (Ni), cobalt (Co), and iron (Fe). In particular, the metal particles 3 are preferably colloidal nanoparticles of gold (Au), silver (Ag), or palladium (Pd).

[0036] The metal particles 3 preferably have a diameter of 5 nm or more and 50 nm or less when viewed in a direction perpendicular to the inner surface of the cylinder of the cylindrical body 1. In this case, the diameter refers to the equivalent diameter (the diameter of a circle having the same area as the projected area) calculated based on the projected area of ​​the metal particles 3 when viewed in a direction perpendicular to the inner surface of the cylinder of the cylindrical body 1. When the metal particles 3 have such a diameter, surface plasmon resonance occurs near the surface of the metal particles 3. As a result, when the cylindrical device 100 is used as a capillary (a narrow tube that holds a solution of a crystallization target such as a protein) in a counterdiffusion method, crystals of the crystallization target are more likely to form near the metal particles 3.

[0037] The average diameter (arithmetic mean value) of the metal particles 3 is preferably 5 nm or more and 50 nm or less. When the average diameter of the metal particles 3 is within this range, surface plasmon resonance is more likely to occur near the surface of the metal particles 3. Hereinafter, the average diameter of the metal particles 3 will be simply referred to as the average particle size.

[0038] The equivalent area circle diameter of the metal particles 3 may be determined by image analysis using an electron microscope image (SEM image) of the metal structure portion 2. When determining the equivalent area circle diameter of the metal particles 3, the image of the measurement object may be an SEM image taken with a field emission scanning electron microscope (manufactured by Hitachi High-Tech Corporation, model: SU8000). It is preferable to use an SEM image taken at a magnification of 100,000 times.

[0039] The metal particles 3 preferably have an average particle diameter of 5 nm to 50 nm in the state of the metal colloid solution used in the metal structure forming step. The average particle diameter of the metal particles 3 in this embodiment is a volume-based average particle diameter measured with a particle size measurement system (manufactured by Otsuka Electronics Co., Ltd., model: ELSZ1000) using dynamic light scattering (DLS). When the metal particles 3 have such a diameter, when the metal structure 2 is formed by the manufacturing method for a cylindrical device described below, the diameter of the metal particles 3 of the formed metal structure 2 when viewed in a direction perpendicular to the inner surface of the cylinder of the cylindrical body 1 can be 5 nm to 50 nm.

[0040] The average interparticle distance L between adjacent metal particles 3,3 is preferably 8 nm or more and 140 nm or less, which makes it easier for crystals of the material to be crystallized to form between the metal particles 3,3.

[0041] In this embodiment, the average interparticle distance L is a value obtained by dividing the area of ​​the image to be measured into Voronoi regions using the center of gravity of each metal particle 3 in the image as the generating point, calculating the average value (arithmetic mean value) of the area of ​​the Voronoi regions containing the center of gravity of each metal particle 3, and then calculating the square root of this average area value.

[0042] When determining the average interparticle distance L, an SEM image taken with a field emission scanning electron microscope (manufactured by Hitachi High-Technologies Corporation, model: SU8000) may be used as the image of the measurement target. The SEM image should be taken at a magnification of 100,000 times.

[0043] In this embodiment, the average value of the distance G between the particle surfaces of adjacent metal particles 3, 3 is a value obtained by subtracting the average particle diameter of the metal particles 3 from the average interparticle distance L. The distance G between the particle surfaces is the so-called interparticle gap.

[0044] The average value of the particle surface distance G is 3 nm or more and 90 nm or less. This makes it easier for surface plasmon resonance to occur near the surfaces of the metal particles 3, particularly between the metal particles 3, 3, and when the cylindrical device 100 is used as a capillary (a thin tube that holds a solution of a crystallization target such as a protein) for the counterdiffusion method, crystals of the crystallization target are more likely to form between the metal particles 3, 3.

[0045] When the metal structure 2 is divided into Voronoi regions using the center of gravity of each metal particle 3 as a kernel point, the average area (arithmetic mean value) of these Voronoi regions is preferably 1000 nm 2 More than 4000nm 2 or less, more preferably 1000 nm 2 More than 3000nm 2 As a result, surface plasmon resonance is more likely to occur near the surface of the metal particles 3, particularly between the metal particles 3, 3, and when the cylindrical device 100 is used as a capillary (a narrow tube for holding a solution of a crystallization target such as a protein) for a counterdiffusion method, crystals of the crystallization target are more likely to occur between the metal particles 3, 3. Specifically, when an area in an image of a measurement target is divided into Voronoi regions using the centers of gravity of the respective metal particles 3 in the image as kernel points, the metal structure section 2 is configured such that the average value of the area of ​​the Voronoi regions in the image is preferably 1000 nm 2 More than 4000nm 2 or less, more preferably 1000 nm 2 More than 3000nm2 The following is the result.

[0046] When the metal structure 2 is divided into Voronoi regions using the center of gravity of each metal particle 3 as a kernel point, the standard deviation of the area of ​​these Voronoi regions is preferably 200 nm 2 More than 2200nm 2 Less than 200 nm, more preferably 2 More than 2000nm 2 This makes it easier for surface plasmon resonance to occur uniformly near the surfaces of the metal particles 3, particularly between the metal particles 3, 3, and when the cylindrical device 100 is used as a capillary (a thin tube that holds a solution of a crystallization target such as a protein) for the counterdiffusion method, crystals of the crystallization target are more likely to occur between the metal particles 3, 3.

[0047] The method for manufacturing the cylindrical device will be described in detail below.

[0048] The primer layer forming step is a step of forming a primer layer containing methylolmelamine or a methylolmelamine derivative on the inner surface of the cylindrical body.

[0049] The methylol melamine derivative may be a compound obtained by condensing melamine with formaldehyde, a compound obtained by reacting methylol melamine with a lower alcohol to partially or completely etherify it, or a mixture thereof. Examples of the lower alcohol include aliphatic alcohols having 1 to 4 carbon atoms.

[0050] The methylol melamine derivative may be a condensate of a monomer or a polymer of dimer or higher of a methylol melamine derivative, or a mixture thereof. An example of a monomer of a methylol melamine derivative is an alkylated melamine such as hexamethoxymethylmelamine. Examples of condensates of polymers of a methylol melamine derivative include imino group-type methylated melamine resins, methylol group-type methylated melamine resins, methylol group-type methylated melamine resins, and fully alkylated methylated melamine resins.

[0051] An example of a method for forming a primer layer on the inner surface of the cylindrical body 1 is as follows.

[0052] One example of a method for forming a primer layer on the inner surface of the cylindrical body 1 is to prepare a primer solution containing methylol melamine or a methylol melamine derivative (hereinafter sometimes referred to as a primer) and pass this through the inside of the cylindrical body 1. In this method, the primer can be attached to the inner surface of the cylindrical body 1 by passing the primer solution through the inside of the cylindrical body 1.

[0053] After the primer is applied to the inner surface of the cylindrical body 1, excess primer solution is removed from inside the cylindrical body 1, and then the primer layer can be formed by heating and drying it in an oven or the like for a predetermined time (e.g., 5 to 20 minutes) at a predetermined temperature (e.g., 150°C to 200°C).

[0054] The primer solution may contain a base resin for fixing the primer to the inner surface of the cylindrical body 1 and an acid catalyst for crosslinking and curing (thermosetting) the primer and base resin. The primer solution may also contain an organic solvent such as cyclohexane as the solvent.

[0055] An example of a base resin is a thermoplastic saturated polyester.

[0056] The acid catalyst may be a sulfonic acid catalyst or a phosphoric acid catalyst, such as a dinonylnaphthalenedisulfonic acid catalyst, a dinonylnaphthalenesulfonic acid catalyst, p-toluenesulfonic acid, pyridine-p-toluenesulfonic acid, or a blocked acid catalyst thereof, or a blocked acid catalyst of phosphoric acid.

[0057] The metal structure forming step is a step of constructing the metal structure portion 2 on the primer layer 21. The metal structure forming step includes a liquid passing step of maintaining a state in which a metal colloid solution such as a gold colloid solution is retained inside the cylindrical body 1.

[0058] The metal colloid solution may be a colloidal solution of gold (Au), silver (Ag), copper (Cu), platinum (Pt), palladium (Pd), rhodium (Rh), iridium (Ir), ruthenium (Ru), nickel (Ni), cobalt (Co), and iron (Fe).

[0059] In the liquid passing step, the metal colloid solution is poured into the cylindrical body 1, and the cylindrical body 1 is filled with the metal colloid solution and maintained (left to stand) for 2 minutes to 20 minutes (for example, 10 minutes). In other words, the metal particles 3 are thereby captured by the methylol groups of the primer layer 21, and the metal structure portion 2 is formed on the primer layer 21.

[0060] In the liquid passing step, it is preferable to perform the operation of filling the interior of the cylindrical body 1 with the metal colloid solution and retaining it (maintaining the state where the metal colloid solution is retained in the cylindrical body 1) two or more times (for example, three times). In other words, in the liquid passing step, it is preferable to replace the metal colloid solution retained in the cylindrical body 1 one or more times (for example, twice). This allows the metal structure portion 2 to be formed uniformly in the axial direction of the cylindrical body 1. In the liquid passing step, the total time during which the interior of the cylindrical body 1 is filled with the metal colloid solution (i.e., retention time) is preferably 5 minutes or more and 60 minutes or less, and preferably 15 minutes or more and 60 minutes or less.

[0061] The liquid passing step is preferably carried out under temperature control, for example, at a temperature of 15° C. to 30° C., preferably 20° C. to 28° C. This allows the metal structure 2 to be formed uniformly over the entire inner surface of the cylindrical body 1. [Example]

[0062] The following describes, based on examples, the tubular device according to this embodiment, a method for manufacturing this tubular device, and a method for crystallizing a protein using this tubular device.

[0063] Example 1 In this example, a primer solution and a gold colloid solution were prepared as follows, and a cylindrical device was manufactured by forming a metal structure with gold nanoparticles arranged on the inner surface of a long cylindrical glass tube. This cylindrical device was then used as a capillary in a counterdiffusion method to conduct a protein crystallization experiment.

[0064] The primer solution for this example was prepared as follows: 0.8 wt % of a methylol melamine crosslinker (product name: CYMEL303LF, manufactured by Allnex, containing 99 wt % or more of alkylated melamine as the main component), 0.2 wt % of pyridine paratoluenesulfonic acid, and 2.5 wt % of a thermoplastic saturated polyester were dissolved in cyclohexane as a solvent.

[0065] The gold colloid solution was prepared as follows. A first solution was prepared by dissolving 0.3 g / L of tetrachloroauric(III) acid and 1.0 g / L of trisodium citrate dihydrate in ultrapure water as a solvent. An appropriate amount of sodium hydroxide was then added to this first solution to prepare a second solution with a pH adjusted to 11. This second solution was heated and stirred at 95°C for 30 minutes in a round-bottom flask to obtain a colloidal solution containing gold nanoparticles with an average particle size of 20 nm, which was used as the gold colloid solution of this example. The average particle size of the gold nanoparticles in the gold colloid solution was measured by volume using a particle size measurement system (manufactured by Otsuka Electronics Co., Ltd., model: ELSZ1000) using dynamic light scattering (DLS).

[0066] A clean glass tube having an inner diameter of 0.5 mm and a length of 15 cm was prepared.

[0067] A primer layer was formed on this glass tube as follows.

[0068] First, the primer solution was injected into the glass tube to fill the tube (cylinder), and after the primer solution had contacted the entire surface of the glass tube, the primer solution was allowed to flow out of the glass tube. Then, clean gas (air) was blown into the glass tube using an air gun to expel any excess primer solution remaining in the glass tube. The glass tube was then placed in a forced circulation oven under atmospheric pressure and heated and dried to form a primer layer. The temperature inside the oven was set to 180°C. The heating and drying time (the time the glass tube was placed in the oven) was 15 minutes. The glass tube was placed upright in the oven.

[0069] A metal structure portion with gold nanoparticles arranged thereon was formed on the glass tube on which the primer layer was formed (hereinafter simply referred to as the glass tube) in the following manner.

[0070] First, a gold colloid solution was injected into the glass tube, filling the glass tube (except for the portion where the metal structure was to be formed) with the gold colloid solution. Next, the glass tube was held for 10 minutes under temperature control while maintaining the gold colloid solution in the glass tube. After that, the gold colloid solution was drained from the glass tube. This series of operations (hereinafter referred to as the "replacement operation") was repeated three times to form the metal structure. While the glass tube was filled with the gold colloid solution, the temperature of the glass tube was controlled at 25±3°C. The inside of the glass tube was then rinsed with ultrapure water, and clean gas was blown into the glass tube to dry it, producing the cylindrical device of this example.

[0071] The observation points on this cylindrical device were 2 cm, 3 cm, and 4 cm from one end (three points spaced 1 cm apart in the axial direction of the cylindrical device), designated as points A, B, and C, respectively, and SEM images (magnification: 100,000 times) were obtained from points A to C. The SEM images were taken using a field-emission scanning electron microscope (Hitachi High-Tech Corporation, model: SU8000). Figures 4 to 6 show the SEM images from points A to C. Image analysis of these SEM images confirmed that gold nanoparticles with an average particle diameter of 20 nm were arranged on the inner surface of the glass tube.

[0072] These SEM images were further subjected to Voronoi analysis, in which the SEM images were divided into Voronoi regions using the centers of gravity of the gold nanoparticles as the generating points. As an example of the results of the Voronoi analysis, a diagram showing the division of the Voronoi regions at point A (hereinafter referred to as the Voronoi analysis diagram) is shown in Figure 7.

[0073] Based on the Voronoi analysis results, the average area and standard deviation of the Voronoi regions in each SEM image (average area and standard deviation of the Voronoi regions in three SEM images) were further calculated. The average interparticle distance was also calculated based on the Voronoi regions. Furthermore, the average interparticle distance was calculated based on the average interparticle distance and the average particle diameter of the gold nanoparticles. These results are shown in Table 1.

[0074] [Table 1]

[0075] The average interparticle distance of the gold nanoparticles determined for each SEM image was approximately 33 nm, and no variation in the adsorption density (arrangement density of the gold nanoparticles) was observed between observation points. In other words, no variation in the adsorption density was observed in the axial direction of the cylindrical device.

[0076] Next, a 50 mm length was cut out from one end of this cylindrical device, and the cylindrical device was used as a capillary for the counterdiffusion method and subjected to a protein crystallization experiment.

[0077] Figure 8 shows an explanatory diagram of a protein crystallization experiment using the counterdiffusion method.

[0078] In this example, a protein crystallization experiment using the counterdiffusion method was performed as follows. First, a protein solution 92 was filled into a cylindrical device 100 serving as a capillary, and the lower end of the capillary was closed with a gel layer 99. The lower end of the capillary was then immersed in a precipitant solution 93 (crystallization reagent). Specifically, the precipitant solution 93 was stored in a container 91, and the capillary was inserted into the container 91. The lower end of the capillary, together with the gel layer 99, was immersed in the precipitant solution 93 stored in the container 91. In this state, the protein solution 92 can diffuse into the precipitant solution 93 outside the capillary through the gel layer 99 (see diffusion flow F1), and the precipitant solution 93 can diffuse into the protein solution 92 inside the capillary through the gel layer 99 (see diffusion flow F2).

[0079] In this example, a lysozyme solution containing 20 mg / L of lysozyme and 600 mM (mmol / L) sodium chloride and 50 mM (mmol / L) acetic acid as buffer solutions, adjusted to a pH of about 4.5, was used as the protein solution.

[0080] As a precipitant solution (crystallization reagent), an acetate buffer solution containing 800 mM sodium chloride and 50 mM acetic acid and adjusted to a pH of about 4.5 was used.

[0081] The gel layer 99 was made of agarose gel, which is a porous material.

[0082] The crystallization experiment was carried out by leaving the sample to stand for 6 weeks under temperature control at 20±0.5° C. Four sets of crystallization experiments were carried out simultaneously under the same conditions (i.e., n=4).

[0083] Table 1 also shows the results of the crystallization experiment. The "average number of crystals generated" shown in Table 1 was calculated by counting the number of crystals obtained for each group and calculating the average value (arithmetic mean value) of the four groups. As shown in Table 1, in this example, an average of 2.5 crystals were obtained.

[0084] Example 2 Example 2 differs from Example 1 in that the amount of methylolmelamine crosslinking agent in the primer solution was reduced to 0.3 wt %. Otherwise, a cylindrical device was manufactured in the same manner as Example 1, and a crystallization experiment was conducted using this device.

[0085] 9 to 11 show SEM images of points A to C in this example. Table 1 also shows the average values ​​and standard deviations of the areas of the Voronoi regions in each SEM image in this example, as well as the results of the crystallization experiment.

[0086] As shown in Table 1, the standard deviation of the Voronoi area in Example 2 was particularly large compared to Example 1, indicating an increase in the variability in the adsorption density of gold nanoparticles. Furthermore, the average value of the Voronoi area was also large compared to Example 1, indicating a decrease in the adsorption density of gold nanoparticles. This result was also evident from the SEM images shown in Figures 9 to 11. The average number of crystals generated in the crystallization experiment was lower than in Example 1, which is thought to be due to an increase in the variability in the adsorption density of gold nanoparticles and a decrease in the adsorption density of gold nanoparticles.

[0087] Example 3 Example 3 differs from Example 1 in that the amount of tetrachloroauric(III) acid in the gold colloid solution was reduced to 0.03 g / L. Otherwise, a cylindrical device was produced in the same manner as Example 1, and a crystallization experiment was carried out using this device.

[0088] Table 1 shows the average value and standard deviation of the area of ​​the Voronoi region in each SEM image in this example, as well as the results of the crystallization experiment.

[0089] As shown in Table 1, it was found that the average value of the Voronoi area was particularly increased in Example 3 compared to Example 1, and the adsorption density of gold nanoparticles was significantly reduced. In this Example, the average number of crystals generated in the crystallization experiment was lower than in Examples 1 and 2. This is thought to be due to the significant decrease in the adsorption density of gold nanoparticles.

[0090] Example 4 Example 4 differs from Example 1 in that the holding time when forming the metal structure was extended to 30 minutes and the replacement operation was reduced to only one time.Otherwise, a cylindrical device was manufactured in the same manner as Example 1, and a crystallization experiment was conducted using this device.

[0091] Table 1 shows the average value and standard deviation of the area of ​​the Voronoi region in each SEM image in this example, as well as the results of the crystallization experiment.

[0092] As shown in Table 1, in Example 4, the average value of the Voronoi area is Increase The standard deviation of the Voronoi area also increased. In other words, the adsorption density of gold nanoparticles decreased and the variability in the adsorption density of gold nanoparticles increased. The average number of crystals generated in the crystallization experiment was lower than in Examples 1 and 2.

[0093] (Comparative Example 1) Comparative Example 1 differs from Example 1 in that the formation of the primer layer was omitted. Otherwise, a cylindrical device was manufactured in the same manner as Example 1, and a crystallization experiment was carried out using this device.

[0094] In Comparative Example 1, it was not possible to form a metal structure. This is thought to be because the formation of the primer layer was omitted. Therefore, it was not possible to calculate the average value and standard deviation of the area of ​​the Voronoi region in Comparative Example 1. Table 1 also shows the results of the crystallization experiment in this Comparative Example.

[0095] In this comparative example, no crystals were obtained in the crystallization experiment, which is thought to be because no metal structure was formed in the capillary.

[0096] As described above, when the cylindrical device according to this embodiment is used as a capillary for the counterdiffusion method, it has been found that crystallization is likely to occur in the counterdiffusion method when the average particle surface distance between adjacent metal particles in the metal structure is 3 nm to 90 nm, preferably 10 nm to 50 nm. 2 More than 2000nm 2 It has been found that crystallization is likely to occur in the counter diffusion method when the temperature is below 100°C.

[0097] In addition, the arithmetic mean value of the area of ​​the Voronoi region of the metal structure is 1000 nm 2 More than 4000nm 2 Below 1000 nm, preferably 2 More than 3300nm 2 It was found that crystallization is likely to occur in the counter-diffusion method when the standard deviation of the area of ​​the Voronoi region is 200 nm or less. 2 More than 2000nm 2 It has been found that crystallization is likely to occur in the counter diffusion method when the temperature is below 100°C.

[0098] Furthermore, it was found that forming a primer layer in the manufacturing of the cylindrical device according to this embodiment allows for the formation of a good metal structure, which in turn makes crystallization more likely to occur when the cylindrical device according to this embodiment is used as a capillary in the counterdiffusion method.

[0099] As described above, it is possible to provide a tubular device in which crystallization by the counterdiffusion method is more likely to occur, a method for producing this tubular device, and a method for crystallizing a protein using this tubular device.

[0100] It should be noted that the embodiments disclosed in this specification are merely examples, and the embodiments of the present disclosure are not limited to these, and can be modified as appropriate within the scope of the purpose of the present disclosure. [Industrial Applicability]

[0101] The present disclosure is applicable to a tubular device, a method for manufacturing a tubular device, and a method for crystallizing a protein. [Explanation of symbols]

[0102] 1: Cylindrical body 2:Metal structure 21: Primer layer 3: Metal particles 91: Container 92: Protein solution 93: Precipitant solution 99: Gel layer F1: Diffusion flow F2: Diffusion flow L: interparticle distance G: Distance between particle surfaces

Claims

1. A long cylindrical body, a metal structure disposed on the inner surface of the cylindrical body, the metal structure portion has metal particles arranged on the inner surface of the tube of the cylindrical body, When the metal structure portion is divided into Voronoi regions using the centers of gravity of the respective metal particles as generating points, the average area of ​​the Voronoi regions is 1000 nm 2 or more and 4000 nm 2 or less, the standard deviation of the area of ​​the Voronoi region is 2122 nm 2 or less; the average interparticle distance between adjacent metal particles is 33 nm or more and 63 nm or less; A cylindrical device, wherein the average particle surface distance between adjacent metal particles is 13 nm or more and 43 nm or less.

2. The tubular device according to claim 1 , wherein the tubular body is made of glass.

3. The cylindrical device according to claim 1 , wherein the inner diameter of the cylindrical body is 0.2 mm or more and 2 mm or less.

4. A cylindrical device described in any one of claims 1 to 3, wherein the metal particles have an average diameter of 50 nm or less when viewed in a direction perpendicular to the inner surface of the cylindrical body.

5. The metal structure has a standard deviation of the area of ​​the Voronoi region of 200 nm ２ 2000nm or more ２ 5. The tubular device according to claim 4, wherein:

6. The tubular device according to claim 1 , wherein the metal particles comprise gold.

7. the metal structure has a primer layer formed on the inner surface of the cylindrical body, The tubular device according to claim 1 , wherein the metal particles are fixed to the inner surface of the tubular body via the primer layer.

8. A method for manufacturing the tubular device according to any one of claims 1 to 3, comprising: a primer layer forming step of forming a primer layer containing methylolmelamine or a methylolmelamine derivative on the inner surface of the cylindrical body; a metal structure forming step of constructing the metal structure on the primer layer, The method for manufacturing a cylindrical device, wherein the metal structure forming step includes a liquid passing step of maintaining a metal colloid solution in a state where it is retained within the cylindrical body.

9. The method for producing a cylindrical device according to claim 8 , wherein the residence time in the liquid passing step is 5 minutes or more and 60 minutes or less.

10. A method for crystallizing a protein by counterdiffusion, comprising: A method for crystallizing a protein, using the tubular device according to claim 1 as a capillary.

Images