JP2014059161A - Electrophoretic test tool and manufacturing method for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an electrophoretic test tool allowing a highly accurate and reliable electrophoresis.SOLUTION: An electrophoretic test tool includes a gel that has pH gradient. The gel includes a plurality of main regions, a connection region located between the two adjoining main regions, and a buffer zone located between the adjoining main regions and the connection region. The pH gradient of the main regions is gentler than pH gradient of the connection region, and pH gradient of the buffer zone is gentler than pH gradient of the main regions.

Description

  The present invention relates to an electrophoresis test device and a method for manufacturing the same.

  Electrophoresis is a separation analysis method that utilizes a phenomenon in which a charged substance in a medium moves in an electric field according to the electric charge when a voltage is applied to the medium such as a solution or a hydrophilic support immersed in the medium. It is. In particular, electrophoresis using gel as a medium (gel electrophoresis) is a technique for separating biopolymers such as proteins and nucleic acids, in the fields of life science such as biochemistry and molecular biology, and in the field of clinical testing. Widely used.

  There are mainly two methods for protein electrophoresis: separation based on protein size (molecular weight) and separation based on charge (isoelectric point). For separation by molecular weight, electrophoresis (SDS-PAGE method) using polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) is widely used. In the SDS-PAGE method, protein binds to SDS at a certain rate and its charge density becomes constant.In this state, the protein moves through the polyacrylamide having a network structure. It is separated according to its molecular weight.

  For separation by isoelectric point, electrophoresis (isoelectric focusing method) performed in the presence of a pH gradient is used. In isoelectric focusing, proteins are separated by gathering at a pH position equal to their isoelectric point in a pH gradient. In the past, amphoteric carriers have been used as the medium for isoelectric focusing, but in recent years, immobilized pH gradient (IPG) gels that do not collapse the pH gradient during energization are often used. ing.

  In recent years, a two-dimensional electrophoresis method combining the two electrophoresis methods has been used as part of proteome analysis for the purpose of comprehensively analyzing the structure and function of all proteins of an organism. In the two-dimensional electrophoresis method, isoelectric focusing is performed in the first dimension, and SDS-PAGE is performed in the subsequent second dimension. This has enabled thousands of proteins to be separated at once with high resolution.

  As described above, gel electrophoresis is an indispensable technique for separation and analysis of biopolymers such as proteins, but in any electrophoresis method, the accuracy and reproducibility of the analysis largely depend on the quality of the gel used. Therefore, in this field, it is desired to develop a technique capable of stably producing an electrophoretic test device equipped with a high-resolution gel.

  For example, Patent Document 1 discloses a gel sheet having a pH gradient by mixing two types of gel stock solutions having different concentrations in a stirring tank, and introducing the mixed solution into a gel container from the bottom to cause gelation (polymerization). A method of making is disclosed. In this case, an SDS-PAGE gel sheet having a predetermined pH gradient can be obtained by changing the ratio of each gel stock solution in the mixed solution to be introduced into the gel container.

  Further, Patent Document 2 discloses a gel plate manufacturing method (inkjet method) in which a monomer solution is applied on a plate. In this case, a liquid pool is formed on the plate, and two types of monomer solutions having different pH are mixed and applied to the liquid pool while changing the mixing ratio. Then, the gel plate for isoelectric focusing which has a predetermined pH gradient is obtained by apply | coating a polymerization initiator and gelatinizing a coating film.

  Moreover, the gel which raised the precision of a specific pH area is disclosed by patent document 3. FIG. In this case, three or more types of gels having a linear pH gradient are individually prepared, and three types of gels are joined and integrated with a gel not containing a pH buffer, thereby having three or more highly accurate linear pH gradients. It is thought that it is manufactured as a gel for electrophoresis.

Japanese Patent Laid-Open No. Sho 62-167659 JP 2012-2739 A US2008 / 0289963A1

In the electrophoresis using the gel of Patent Document 3, the interface between the gel having a pH gradient and the bonded gel not containing the pH buffer becomes a protein migration barrier, so that it is difficult to move the protein throughout the gel. Efficiency is expected to decline.
In the case of Patent Document 3, it is also conceivable to produce a gel using three types of gradient makers for forming three types of pH regions. In this case, two types of gel stock solutions having different pH values are introduced into a gel container from one type of gradient maker, and then a monomer that does not contain a pH buffer is introduced into the gel container, and then the remaining two types of gradients are introduced. It can operate similarly about a maker and can manufacture the gel for electrophoresis which consists of the gel part which has three types of pH gradient, and the joining gel part which does not contain the pH buff arrange | positioned among these. This gel seems to reduce the problem of protein migration barrier as described above, but the region length of the bonded gel must be adjusted only by the valve switching timing and the operation of the liquid feed pump. However, it is difficult to control the pH region and the pH position with high accuracy so as to obtain an ideal pH gradient.

  The present invention has been made in view of such problems, and provides an electrophoresis test device capable of performing electrophoresis with high accuracy and high reliability, and a manufacturing method capable of easily manufacturing the test device. The purpose is to do.

Thus, according to the present invention, comprising a gel having a pH gradient,
The gel includes a plurality of main regions, a connection region disposed between two adjacent main regions, and a buffer region disposed between the adjacent main regions and the connection region,
An electrophoretic test device is provided in which the pH gradient of the main region is gentler than the pH gradient of the connection region, and the pH gradient of the buffer region is gentler than the pH gradient of the main region.

According to another aspect of the present invention, a gel material application step for applying a gel material on a base material to form a liquid pool, and a pH buffer solution is applied to the liquid pool after the gel material application step. a pH buffer solution coating step, and a gelation step of gelling the coating film after the pH buffer solution coating step,
In the pH buffer solution application step, the application region on the substrate includes a plurality of main regions, a connection region located between two adjacent main regions, and between the adjacent main region and the connection region. Is divided into areas to be formed with a buffer area located at
In the region where the main region is to be formed, the pH buffer solution is applied while continuously and gently changing the coating amount per unit area of the pH buffer solution,
In the region where the connection region is to be formed, the pH buffer solution is applied so that the rate of change of the coating amount per unit area of the pH buffer solution is higher than the rate of change in the main region,
In the region where the buffer region is to be formed, manufacture of a test device for electrophoresis in which the pH buffer solution is applied so that the rate of change of the coating amount per unit area of the pH buffer solution is lower than the rate of change in the main region A method is provided.

The electrophoresis test device of the present invention simultaneously analyzes, for example, a first protein detected in a pH 5-6 region and a second protein detected in a pH 8-9 using a pH 4-10 gel. As in the case, it is suitable for electrophoresis of proteins that require analysis accuracy to be improved in a plurality of different regions, and an analysis result with high accuracy and high reliability can be obtained. In this case, the pH 5-6 portion and the pH 8-9 portion are formed as a main region with a gradual pH gradient that does not have a sharp pH gradient fluctuation point, and the connecting region does not require analysis accuracy. Can be formed.
By doing in this way, the length of the junction area | region unnecessary for a measurement in the electrophoresis direction of a gel can be shortened, and the main area | region required for a measurement can be lengthened. As a result, if the total length of the gel in the electrophoretic test device of the present invention is the same as the total length of the gel of the conventional product, the present invention can form a pH gradient more slowly than the conventional, and the analytical accuracy Will improve. In addition, since the buffer region having the smallest pH gradient is provided between the main region and the connection region, the pH gradient of the main region is hardly affected by the connection region, and the analysis accuracy is further improved.

The method for producing an electrophoresis test device according to the present invention comprises a pH buffer solution between a main region having a gradual pH gradient without a sharp pH gradient fluctuation point and a connection region having a rapidly changing pH gradient. The buffer region is formed by applying the pH buffer solution so that the change rate of the coating amount per unit area is lower than the change rate in the main region. As a result, the pH gradient of the main region is not easily affected by the pH gradient that changes rapidly in the connection region, and a gentle and ideal pH gradient can be easily formed in the main region.
Further, according to the production method of the present invention, in the same pH buffer solution application step, in order to form a gel by applying the pH buffer solution to the main region, the connection region, and the region where the buffer region should be formed in the puddle, In other words, since no boundary is formed between different gels, the protein moves smoothly during electrophoresis, and analysis efficiency does not decrease.

It is a perspective view which shows the state which can use the test device for isoelectric focusing of Embodiment 1 of this invention. It is a perspective view which shows the state which can be preserve | saved after drying the gel layer in the test device for isoelectric focusings of FIG. 1 (A). FIG. 2 is a conceptual diagram illustrating an electrophoresis test device and a manufacturing method thereof according to Embodiment 1. FIG. 3 is a partially enlarged view of FIG. 2. It is a block diagram which shows the apparatus which can manufacture the test device for electrophoresis of Embodiment 1. FIG. It is the schematic bottom view which looked at the inkjet head in the manufacturing apparatus of FIG. 4 from the downward direction. It is an enlarged view which shows the nozzle hole group of the inkjet head in the manufacturing apparatus of FIG. It is explanatory drawing which shows the state which apply | coats an acidic monomer solution to a base material using the manufacturing apparatus of FIG. It is explanatory drawing which shows the state which apply | coats a basic monomer solution to a base material continuously from FIG. 7 (A). It is explanatory drawing which shows the state which applies a polymerization initiator to a base material continuously from FIG. 7 (B). It is explanatory drawing which shows the state which the application | coating of the gel material liquid by the manufacturing apparatus of FIG. 4 was complete | finished.

<About electrophoresis test equipment>
The test device for electrophoresis of the present invention includes a gel having a pH gradient.
The gel includes a plurality of main regions, a connection region disposed between two adjacent main regions, and a buffer region disposed between the adjacent main regions and the connection regions.
The pH gradient of the main region is gentler than the pH gradient of the connection region, and the pH gradient of the buffer region is gentler than the pH gradient of the main region.

The test device for electrophoresis of the present invention may be configured as follows.
(1) The pH gradient in the buffer region may be a half or less of the pH gradient in the main region, and is preferably substantially zero (flat pH without a gradient). Here, the pH gradient can be obtained from the amount of change in pH value per unit distance in the X direction.
To stabilize the pH buffer amount after abrupt changes in pH buffer mixing conditions (cast method) and coating amount conditions (IJ method), wait for stabilization while maintaining a constant mixing state and coating amount state for a while. That is, it is desirable to provide a region where the pH gradient is substantially zero.
Another effect is that it is possible to prevent displacement in the pH region. That is, for example, when the pH of the main region is 5 to 6, if the pH of the buffer region at both ends of the main region is set to pH 5 and pH 6, respectively, both ends of the main region can be reliably set to pH 5 and pH 6, respectively. As a result, it is possible to prevent the positional shift of the pH gradient in the main region and improve the analysis accuracy.

(2) The length of the buffer region in the electrophoresis direction of the gel may be shorter than the length of the adjacent main region and the connection region. In this case, the length of the buffer region is not limited as long as the influence of the connection region on the main region can be reduced, and may be basically short.

(3) Since the main region is a substantial measurement region, it is desirable that the pH gradient of the main region is a linear gradient.

(4) Since the connection region is not a substantial measurement region, the pH gradient of the connection region may be a non-linear gradient.

  The form of the base material of the electrophoresis test device is not particularly limited, and examples thereof include an elongated plate and a chip molded into a predetermined shape. The material of the base material is not particularly limited as long as it can function as a base material for a test device for electrophoresis. For example, glass such as quartz glass and non-alkali glass, polyethylene terephthalate (PET), polymethacryl Examples thereof include resins such as acid methyl resin (PMMA), ceramics such as alumina, and low-temperature co-fired ceramic. Further, when the substrate is made of a hydrophobic material, the surface of the substrate on which the gel layer is formed may be subjected to a hydrophilic treatment, thereby improving the wettability of the monomer solution described below with respect to the substrate, and the monomer solution Adhesion between the gelled gel layer and the substrate is improved. Examples of the hydrophilic treatment include nitration using sulfuric acid, sulfonation using nitric acid, oxygen plasma treatment and the like.

  The material of the gel layer of the electrophoresis test device is not particularly limited as long as it can function as the gel layer of the electrophoresis test device. For example, as a general polyacrylamide gel material, acrylamide ( Monomer), bisacrylamide (crosslinking agent), pH adjusting material (pH buffer), tetramethylethylenediamine (TEMED: polymerization accelerator), polymerization initiator and pure water. As a polymerization initiator, when the monomer is thermally polymerized, a thermal polymerization initiator such as ammonium persulfate (APS) or benzoyl peroxide can be used. When the monomer is photopolymerized, riboflavins, 2,2-dimethoxy-2 -Photopolymerization initiators such as acetophenones such as phenylacetophenone, benzophenones, and azoisobutyronitrile can be used.

  In the present invention, the gel layer is formed by forming a puddle of a gel material solution on a base material, and applying a pH buffer solution on the puddle to cause gelation. At this time, as a liquid pool, a monomer solution (a gel material solution containing a polymerization initiator) to which a polymerization initiator is added in advance and a monomer solution (polymerization) in which a crosslinking agent other than the polymerization initiator, a polymerization accelerator, and the like are mixed are used. A case where a gel material liquid not containing an initiator) is used, and a case where a polymerization initiator is used.

  Therefore, in the present invention, “gel material liquid” means all of a gel material liquid to which a polymerization initiator has been added in advance, a gel material liquid not containing a polymerization initiator, and a polymerization initiator, unless otherwise specified. . Hereinafter, “a gel material solution to which a polymerization initiator has been added in advance” may be referred to as “a gel material solution containing a polymerization initiator”, and “a gel material solution that does not include a polymerization initiator” may be referred to as a “monomer solution”.

<About the manufacturing method of the electrophoresis test device>
The electrophoretic test device of the present invention includes a gel material application step in which a gel material is applied onto a substrate to form a coating film, and a pH buffer solution in which a pH buffer solution is applied onto the coating film after the gel material application step. A solution coating step and a gelation step of gelling the coating film after the pH buffer solution coating step.
In the pH buffer solution application step, the application region on the substrate includes a plurality of main regions, a connection region located between two adjacent main regions, and between the adjacent main region and the connection region. And a buffer region located in the region to be formed.
In the region where the main region is to be formed, the pH buffer solution is applied while continuously and gently changing the coating amount per unit area of the pH buffer solution.
In the region where the connection region is to be formed, the pH buffer solution is applied so that the change rate of the coating amount per unit area of the pH buffer solution is higher than the change rate in the main region.
In the region where the buffer region is to be formed, the pH buffer solution is applied so that the change rate of the coating amount per unit area of the pH buffer solution is lower than the change rate in the main region.

  In the present invention, the method for applying the gel material liquid on the substrate is not particularly limited, and any method can be used as long as the gel material liquid can be applied to a predetermined region on the upper surface of the substrate. Can be mentioned. Among these, it is preferable to use an ink jet apparatus provided with an ink jet head that discharges fine droplets with high accuracy and adheres them to a substrate. If an inkjet head is used, minute droplets can be applied to a predetermined area of an elongated base material with high accuracy and quantitatively. Therefore, the formation area of the gel layer to be obtained, the film thickness, the pH gradient, the concentration gradient, etc. Can be controlled easily and with high accuracy.

  Hereinafter, embodiments of an electrophoretic test device and a manufacturing method thereof according to the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to the embodiment.

(Embodiment 1)
FIG. 1 (A) is a perspective view showing a usable state of the isoelectric focusing test device of Embodiment 1 of the present invention, and FIG. 1 (B) is the isoelectric focusing of FIG. 1 (A). It is a perspective view which shows the state which can be preserve | saved after drying the gel layer in a test tool. FIG. 2 is a conceptual diagram illustrating the electrophoresis test device and the manufacturing method thereof according to Embodiment 1, and FIG. 3 is a partially enlarged view of FIG.

An electrophoretic test device GP ₁ shown in FIG. 1A is obtained by forming a bowl-shaped gel layer G ₁ on a substrate S. The gel layer G ₁ has a gently convex curved upper surface (surface opposite to the substrate) and a pH gradient in the longitudinal direction (hereinafter referred to as “X direction”) that is the electrophoresis direction. ing. The direction orthogonal to the X direction is the width direction of the substrate S (hereinafter referred to as “Y direction”). When the gel layer G ₁ is dried to a moisture content of 5% or less, the gel layer G ₁ is dried to form a dry film D _{1. The} isoelectric focusing test device GPD ₁ shown in FIG. can get.

In the present invention, the length and width of the gel layer G ₁ are the same as the length and width of the substrate S. The length and width of the substrate S are not particularly limited, but as an example, the length is about 50 to 250 mm, and the width is about 0.5 to 5 mm. The thickness of the gel layer G ₁ is not particularly limited, for example, about 195～1010Myuemu.
The thickness of the dry film D ₁ obtained by drying the gel layer G ₁ shrinks to 100 μm or less, but the length and width hardly change.

FIG. 2 includes an upper stage, a middle stage, and a lower stage. The upper part of FIG. 2 shows, in terms of area, changes in the coating amounts of an acidic monomer solution and a basic monomer solution, which are two types of pH buffer solutions used at the time of gel production described later. The middle part of FIG. 2 shows a change in the coating amount of the acidic monomer solution (thin line) and the basic monomer solution (thick line) in a line graph. The lower part of FIG. 2 shows the pH at the position in the X direction of the gel layer G ₁ with a line graph.

In FIG. 2, the application region is divided into regions A to K from one end S ₁ to the other end S ₂ of the substrate S, and the application amount of the acidic monomer solution and the basic monomer solution is controlled according to each region. It is shown that. Here, regions C, F and I correspond to regions where first to third main regions to be described later are to be formed, and regions A, B, D, E, G, H, J and K are buffer regions and connections which will be described later. This corresponds to a region where a region is to be formed. More specifically, the acidic side of region A and the basic side of region B are regions where a buffer region should be formed, and the remainder is a connection region, and the acidic side of region D and the basic side of region E form a buffer region The remaining area is the connection area, the acidic side of the area G and the basic side of the area H are the areas where the buffer area is to be formed and the remaining area is the connection area, the acidic side of the area J and the area K The basic side is a region where a buffer region is to be formed, and the remainder is a connection region.

As shown in FIG. 1 (A), FIG. 2 and FIG. 3, the gel layer G ₁ has a pH gradient in a predetermined pH range in the X direction. In the first embodiment, for example, a gel layer G ₁ is illustrates a case where a pH gradient with the range of the pH 3-10. Specifically, the gel layer G ₁ has a first main region having a pH of 3.35 to 4.75, a second main region having a pH of 5.45 to 6.15, and a third main region having a pH of 7.9 to 9.65. And a buffer region and a connection region disposed on both sides of each main region. Specifically, the gel layer G1 of this specific example can be formed by the method described later.
FIG. 3 shows only the second main region and the buffer regions and connection regions arranged on both sides of the second main region. Similarly, the buffer regions and connection regions are also provided on both sides of the first main region and both sides of the third main region. Is arranged.

In the gel layer G _{1 of the} first embodiment, the proteins are separated by electrophoresis into a first main region (pH 3.35 to 4.75), a second main region (pH 5.45 to 6.15), and a third main region (pH 7.9 to 9.65). The pH gradient of each main region provided in a relatively wide range is formed in a gentle linear shape (see the lower part of FIG. 2). Therefore, the gel layer G ₁ of the first embodiment, the acidic side of the first main region, between the first main region to the second main region, between the second main region and the third main region, and the third primary regions In a narrow region (buffer region and connection region) such as the basic side, high-precision separation of the protein is not expected, but the protein is also separated in these regions.

The characteristic structure of the gel layer G ₁ of Embodiment 1 is that the pH region (main region) to be analyzed in detail is divided into a plurality of points as described above, and the connection region where the pH changes rapidly between them. This is the point that buffer regions are arranged on the acidic side and the basic side. The pH of the buffer region is set to be equal to the pH at the end of the adjacent main region. Here, the significance of the buffer region will be described.

  When the buffer region is not provided on the acidic side of the second main region, the difference in pH between the acidic side end of the second main region and the basic side end of the first main region is large. The acidic side end of the region is more acidic than the set pH value. In addition, when the buffer region is not provided on the basic side of the second main region, the pH drop between the basic side end of the second main region and the acidic side end of the third main region is large, The basic side end of the second main region is more basic than the set pH value. As a result, the pH gradient between the acidic side end and the basic side end of the second main region becomes steep, and the length of the second main region in the X direction becomes short. As a result, the protein separation accuracy at the acidic side end and the basic side end of the second main region is lowered.

As described above, the cause that the acidic side end of the main region is more acidic than the set pH value and the basic side end is more basic than the set pH value is that the base S This is because the pH buffer diffuses before the gel material liquid applied to the gel is gelled. In particular, in the vicinity of the boundary where the pH value changes greatly, the coating amount of the acidic monomer solution and the basic monomer solution changes rapidly, so that the pH buffer is likely to diffuse.
Therefore, in the present invention, a buffer region having a pH equivalent to the pH of the acidic side end and the basic side end of the main region is provided on the acidic side and the basic side of the main region, so In this way, the influence of the pH change is prevented from directly affecting the main region. Thereby, a gentle pH gradient is formed in the main region over the set length range in the X direction. As a result, the protein separation accuracy at the acidic side end and the basic side end of the main region does not decrease.

Next, an apparatus capable of manufacturing the test tool GP ₁ shown in FIG. 1A will be described, and then a method of manufacturing the test tool GP ₁ using this apparatus will be described.

  FIG. 4 is a configuration diagram showing an apparatus capable of manufacturing the electrophoresis test device of the first embodiment. This test device manufacturing apparatus includes a stage 10 on which a base material S is set, an ink jet apparatus 30 as an application unit, a moving mechanism 40 that moves the stage 10 in a linear direction, and a sealable case 50 that houses these. And a control unit (not shown). The case 50 is provided with an opening / closing door (not shown).

The moving mechanism 40 includes a support base 40a that supports the stage 10, and the support base 40a can be reciprocated in a linear direction by a linear guide mechanism (not shown).
In FIG. 4, the support base 40a indicated by the solid line is in the standby position, and the support base 40a, the stage 10 and the substrate S travel straight to the position indicated by the two-dot chain line in the coating process. Thereby, the base material S set on the stage 10 passes just below the 1st-3rd inkjet heads 31b, 32b, and 33b mentioned later.

The ink jet device 30 includes an acidic solution discharge unit 31, a basic solution discharge unit 32, a polymerization initiator discharge unit 33, and a negative pressure adjustment unit 34.
The acidic solution discharge unit 31 includes a first tank 31a that stores the acidic monomer solution A, a first inkjet head 31b, and a first pipe 31c that sends the acidic monomer solution A from the first tank 31a to the first inkjet head 31b. And the acidic monomer solution A is supplied from the first tank 31a to the first inkjet head 31b using the water head difference. The acidic monomer solution A is set to a predetermined pH (for example, pH 2 to 7) by one or more kinds of pH buffers.

  The basic solution discharge unit 32 includes a second tank 32a that stores the basic solution B, a second inkjet head 32b, and a second pipe 32c that sends the basic solution B from the second tank 32a to the second inkjet head 32b. And the basic monomer solution B is supplied from the second tank 32a to the second inkjet head 32b using the water head difference. The basic monomer solution B is set to a predetermined pH (for example, pH 7 to 12) by one or more kinds of pH buffers.

  The polymerization initiator discharge unit 33 includes a third tank 33a that stores the polymerization initiator C, a third inkjet head 33b, and a third pipe 33c that sends the polymerization initiator C from the third tank 33a to the third inkjet head 33b. And the polymerization initiator C is supplied from the third tank 33a to the third inkjet head 33b using the water head difference.

  Examples of the first to third ink jet heads 31b to 33b include a thermal jet method, a piezo jet method, an electrostatic drive method, and the like, but each liquid in the ink jet device 30 (an acidic monomer solution A, a basic monomer solution B, a polymerization). When the initiator C) is cooled, it is desirable to use a piezo jet method or an electrostatic drive method without using a thermal jet method for applying heat to each liquid.

  The negative pressure adjusting unit 34 is connected to the first to third tanks 31a to 33a by pipes 35 to 37, manages the atmospheric pressure in the first to third tanks, and the first to third inkjet heads 31b. The insides of the first to third tanks 31a to 33a are adjusted to be constant at a predetermined pressure lower than the atmospheric pressure so that the liquid does not spill from the nozzle holes H (see FIG. 6) of .about.33b.

  The first to third ink jet heads 31b to 33b are integrated to form a set of ejection head units U, and the ejection head units U are fixed by a fixing member (not shown). And as shown in FIG. 5, although the 1st-3rd inkjet heads 31b-33b are arrange | positioned in a line on the movement locus | trajectory E of this base material S, the head arrangement | positioning order is not limited to this order. In the case of the first embodiment, the first to third inkjet heads 31b to 33b are arranged in this order from the upstream side in the moving direction of the substrate S.

  Further, as shown in FIGS. 5 and 6, a plurality of nozzles are arranged on the lower surface of the first to third inkjet heads 31 b to 33 b facing the movement locus E of the substrate S in a direction orthogonal to the direction of the movement locus E. The holes H are provided in one row. That is, the nozzle hole group HG in one row extends in a direction orthogonal to the direction of the movement locus E and with a length exceeding the width of the movement locus E. Although the nozzle hole diameter D and the nozzle hole interval P are not particularly limited, the diameter of the nozzle hole H is suitably about 10 to 100 μm, and the nozzle hole interval P is suitably about 100 to 200 μm. When the nozzle rows are arranged in a straight line, it is possible to use a method of apparently narrowing the nozzle hole interval by tilting the head direction. In the first to third ink jet heads 31b to 33b, the nozzle hole group HG may be provided in a plurality of rows of two or more.

Next, an example of a method for manufacturing the test tool GP ₁ using the test tool manufacturing apparatus having the above-described configuration will be described.
First, as shown in FIG. 4, an elongated rectangular base material S is set on the stage 10 in the standby position. On the base material S, a liquid pool L0 is formed in advance. As the liquid pool L0, a monomer solution obtained by diluting a monomer with pure water is used, and a crosslinking agent and a polymerization accelerator may be added to the monomer solution.

  Next, the coating process under normal temperature and atmospheric pressure based on a predetermined program is performed. That is, as shown in FIGS. 7A and 7B and FIGS. 8A and 8B, the support base 40a is intermittently moved in the direction of the arrow M by the moving mechanism 40, and the first to third directions. Minute droplets La, Lb, and Lc are intermittently ejected from the ink jet heads 31b to 33b, and the coating film L3 is formed on the liquid pool L0.

More specifically, as shown in FIG. 7A, when one end S _{1 of the} substrate S moves to a position directly below the nozzle hole group HG of the first inkjet head 31b of the inkjet apparatus 30, the first inkjet head 31b A small droplet La of the acidic monomer solution is discharged and applied onto the liquid pool L0. Thus, as shown in FIG. 7 (B), the coating film L1 of the mixed liquid of liquid pool L0 and an acidic monomer solution is formed at one end S ₁ side of the substrate S.

  In this case, the nozzle hole H that discharges the micro droplet La is selected from the nozzle hole group HG in the first inkjet head 31b so that the micro droplet La is not discharged onto the stage 10. The same applies to the second and third inkjet heads 32b and 33b. Further, the coating amount per unit area of the fine droplets La discharged from the first inkjet head 31b onto the substrate S is changed every time the substrate S moves intermittently by a predetermined distance (at predetermined discharge intervals). The amount is controlled to a predetermined amount, which will be described in detail later.

When one end S ₁ of the substrate S moves to a position just below the nozzle hole group HG of the second inkjet head 32b, a micro droplet Lb of the basic monomer solution is discharged from the second inkjet head 32b, and the coating film L1. It is applied on top. Thus, as shown in FIG. 8 (A), the coating film L2 of the liquid mixture of coating film L1 and the basic monomer solution is formed at one end S ₁ side of the substrate S. Also in this case, the coating amount per unit area of the micro droplets Lb ejected from the second inkjet head 32b is a predetermined amount every time the substrate S is intermittently moved by a predetermined distance (at a predetermined ejection interval). This will be described in detail later.

Then, when one end S ₁ of the substrate S moves to a position just below the nozzle hole group HG of the third inkjet head 33b, a minute droplet Lc of the polymerization initiator is discharged from the third inkjet head 33b, and on the coating film L2. To be applied. Thus, the coating film L2 and the polymerization initiator mixture coating film with L3 (FIG. 8 (B) refer) is formed at one end S ₁ side of the substrate S, one end S ₁ of the thus substrate S continuously coated film L3 toward the other end S ₂ side is gradually formed from the side. Then, when the other end S ₂ of the substrate S is sequentially passed through the first to third ink jet head 31B～33b, as shown in FIG. 8 (B), from the first to third ink jet head 31B～33b Droplet discharge stops sequentially.

Next, the application amount control of the acidic monomer solution and the basic monomer solution will be described with reference to FIG.
As shown in FIG. 2, the acidic monomer solution is controlled so that the coating amount decreases as it goes from one end S _{1 of the} substrate S to the other end S ₂ , and the basic monomer solution is the other end from the one end S _{1 of the} substrate S. the coating amount is controlled so as to increase toward the S _2.
More specifically, in the region A on the substrate S, the application amount of the acidic monomer solution is constant at 100%, and the application amount of the basic monomer solution is constant at 0%.
In region B, the application amount of the acidic monomer solution is constant at about 95%, and the application amount of the basic monomer solution is constant at about 5%.

In region C, the coating amount of the acidic monomer solution continuously decreases in the range of about 95 to 75%, and the coating amount of the basic monomer solution continuously increases in the range of about 5 to 25%.
In region D, the application amount of the acidic monomer solution is constant at about 75%, and the application amount of the basic monomer solution is constant at about 25%.
In the region E, the application amount of the acidic monomer solution is constant at about 65%, and the application amount of the basic monomer solution is constant at about 35%.

In the region F, the application amount of the acidic monomer solution continuously decreases in the range of about 65 to 55%, and the application amount of the basic monomer solution continuously increases in the range of about 35 to 45%.
In the region G, the application amount of the acidic monomer solution is constant at about 55%, and the application amount of the basic monomer solution is constant at about 45%.
In the region H, the application amount of the acidic monomer solution is constant at about 30%, and the application amount of the basic monomer solution is constant at about 70%.

In region I, the coating amount of the acidic monomer solution continuously decreases in the range of about 30 to 5%, and the coating amount of the basic monomer solution continuously increases in the range of about 70 to 95%.
In the region J, the application amount of the acidic monomer solution is constant at about 5%, and the application amount of the basic monomer solution is constant at about 95%.
In the region K, the application amount of the acidic monomer solution is constant at 0%, and the application amount of the basic monomer solution is constant at 100%.

That is, in the regions C, F, and I where the main regions are to be formed, the pH buffer solution is applied while continuously and gently changing the coating amount per unit area of the pH buffer solution.
Further, on the acidic side and the basic side of the regions A, B, D, E, G, H, J, and K where the buffer region is to be formed, the rate of change in the coating amount per unit area of the pH buffer solution is the main factor. The pH buffer solution is applied so as to be lower than the rate of change in the area.
Further, in the intermediate part of the regions A and B, the intermediate part of the regions D and E, the intermediate part of the regions G and H, and the intermediate part of the regions J and K to form the connection region, The pH buffer solution is applied so that the rate of change in the amount of coating is higher than the rate of change in the main region (see FIG. 3).
Here, “the rate of change of the coating amount per unit area of the pH buffer solution” means the degree of pH gradient.

By such coating amount control, the coating film L3 after the coating process has a pH gradient as shown in the lower part of FIG. In addition, a series of operation | movement of the test device manufacturing apparatus in such an application | coating process is performed when a control part controls each drive part based on a predetermined program.
Thereafter, the support base 40a returns to the standby position, and the coating process is completed.

After the coating process, the door of the case 50 is opened, the base material S is taken out and stored in the case for the gelation process, and the gelation process of the coating film L3 is performed at room temperature in the case. In addition, it takes about 3 to 5 hours to complete the gelation at room temperature. After completion of the gelation, the isoelectric focusing test device GP _{1 in} which a bowl-shaped gel layer G ₁ having rounded corners on the four ends is formed on the substrate S as shown in FIG. 1 (A). Is obtained.

Next, by drying the gel layer G ₁ of the obtained electrophoretic test device GP ₁ , the electrophoretic test device GPD ₁ of the present invention as a finished product shown in FIG. 1B is obtained. In this drying step, the method of drying the gel layer G ₁ is not particularly limited, and examples thereof include a method of heating the gel layer G ₁ with a heater or blowing hot air to the gel layer G ₁ for drying. Further, after the drying step, the dry film D ₁ may be carried out cooling step of cooling the -20 ° C. or less. Or you may perform a freeze-dry process instead of a drying process and a cooling process.

(Embodiment 2)
In Embodiment 1, the case where a monomer is thermally polymerized and gelled by using a thermal polymerization initiator such as ammonium persulfate (APS) or benzoyl peroxide as a polymerization initiator is illustrated. On the other hand, in Embodiment 2, photopolymerization initiators such as riboflavins, acetophenones such as 2,2-dimethoxy-2-phenylacetophenone, benzophenones, and azoisobutyronitrile are used as polymerization initiators. Polymerize to gel.

  In the case of the second embodiment, for example, in the apparatus for manufacturing an electrophoretic test device described with reference to FIG. 4, a light irradiator (not shown) is provided on the downstream side in the substrate transport direction (arrow M1 direction) of the ejection head unit U. The coating film on the material S is gelled by irradiating light. This light irradiator can irradiate light of a predetermined wavelength (for example, about 200 to 600 nm) with a predetermined light amount, and the irradiation light wavelength is appropriately set according to the type of the photopolymerization initiator to be used. Further, the light irradiator only needs to irradiate a part or the whole of the coating film on the substrate.

  In the manufacturing method of the electrophoresis test device of the second embodiment, a coating film is formed on a substrate in the same manner as in the first embodiment (explained in FIGS. 7A to 8B), and then the substrate is optically irradiated. Move to the irradiator side and irradiate the coating film with light to gel. At this time, when the light irradiator is of a type that irradiates a part of the coating film, the coating film is irradiated with light while moving the substrate from one end side to the other end side. When the light irradiator is of a type that irradiates light to the entire coating film, the substrate is moved directly below the light irradiator, and then the light is irradiated onto the coating film while the substrate is stationary.

(Other embodiments)
1. In Embodiment 1, although the case where the gel which has the 1st-3rd main region was manufactured was illustrated, the number of main regions may be two, or four or more.

2. In the first embodiment, the case where a coating film in a room temperature state is formed on the base material in the coating step is exemplified. However, the coating film is formed on the base material under cooling using an apparatus including a Peltier element and a tank cooling unit. It may be formed. In the first embodiment, the case where the coating film is formed in the air in the coating process is illustrated, but the coating film may be formed in a nitrogen atmosphere.

3. In the first embodiment, the case where the monomer solution and the polymerization initiator are individually applied onto the substrate is exemplified, but the gel material solution containing the polymerization initiator may be applied onto the substrate to form the liquid pool L0. . In this case, a gel material solution containing a polymerization initiator in a cooled state is used so that gelation of the gel material solution containing a polymerization initiator does not proceed during the coating process. In addition, the substrate may be cooled.

4). In the first embodiment, the case where the coating film L3 is formed by passing the substrate S once under the discharge head unit U is illustrated. However, the coating film L3 is formed by moving the substrate S one or more times. May be. In this case, the application timing of the polymerization initiator can be set, for example, at every movement, at a predetermined movement, or at the last movement.

D ₁ dry film GP ₁ isoelectric focusing test device (gel plate) ready for use
Test device for isoelectric focusing which can be stored in GPD ₁ G ₁ gel layer S base material A to K region

Claims (6)

comprising a gel having a pH gradient;
The gel includes a plurality of main regions, a connection region disposed between two adjacent main regions, and a buffer region disposed between the adjacent main regions and the connection region,
The electrophoresis test device according to claim 1, wherein the pH gradient of the main region is gentler than the pH gradient of the connection region, and the pH gradient of the buffer region is gentler than the pH gradient of the main region.   The electrophoresis test device according to claim 1, wherein the pH gradient in the buffer region is a half or less of the pH gradient in the main region.   The test device for electrophoresis according to claim 1 or 2, wherein a length of the buffer region in the electrophoresis direction of the gel is shorter than a length of the adjacent main region and the connection region.   The electrophoresis test device according to claim 1, wherein the pH gradient of the main region is a linear gradient.   The electrophoresis test device according to claim 1, wherein the pH gradient of the connection region is a non-linear gradient. A gel material application step of applying a gel material on a substrate to form a liquid pool, a pH buffer solution application step of applying a pH buffer solution onto the liquid pool after the gel material application step, and the pH buffer solution application Including a gelling step of gelling the coating film after the step,
In the pH buffer solution application step, the application region on the substrate includes a plurality of main regions, a connection region located between two adjacent main regions, and between the adjacent main region and the connection region. Is divided into areas to be formed with a buffer area located at
In the region where the main region is to be formed, the pH buffer solution is applied while continuously and gently changing the coating amount per unit area of the pH buffer solution,
In the region where the connection region is to be formed, the pH buffer solution is applied so that the rate of change of the coating amount per unit area of the pH buffer solution is higher than the rate of change in the main region,
In the region where the buffer region is to be formed, electrophoresis is characterized in that the pH buffer solution is applied so that the rate of change of the coating amount per unit area of the pH buffer solution is lower than the rate of change in the main region. Of manufacturing test equipment.