WO2014041908A1

WO2014041908A1 - Electrophoresis test tool and method for producing same

Info

Publication number: WO2014041908A1
Application number: PCT/JP2013/070460
Authority: WO
Inventors: 政俊中川
Original assignee: シャープ株式会社
Priority date: 2012-09-14
Filing date: 2013-07-29
Publication date: 2014-03-20
Also published as: JP2014059160A

Abstract

Provided is an electrophoresis test tool which enables high-precision and high-reliability electrophoresis. The electrophoresis test tool is characterized in comprising a gel having a pH gradient from the acidic side to the basic side, and in that the gel comprises a main region and buffer regions adjacent to the main region, and the pH values of the buffer regions are substantially constant.

Description

Electrophoresis test device and method for manufacturing the same

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. As an isoelectric focusing medium, an amphoteric carrier has been used in the past, but in recent years, an immobilized pH gradient (Immobilized pH Gradient: IPG) gel that does not collapse during energization is often used. ing.

In recent years, as a part of proteome analysis aiming at comprehensive analysis of the structure and function of all proteins of living organisms, a two-dimensional electrophoresis method combining the two electrophoresis methods has been used. 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 separating and analyzing biopolymers such as proteins. However, in any electrophoresis method, the accuracy and reproducibility of 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 concentration 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 concentration gradient can be obtained by changing the ratio of each gel stock solution in the mixed solution to be introduced into the gel container. Moreover, using the gradient maker described in Patent Document 1, two types of gel stock solutions having different pHs are mixed in a stirring tank, and the mixture is introduced into the gel container from the bottom to cause gelation, thereby adjusting the pH gradient. The gel sheet which has can be produced. In this case, a gel sheet having a predetermined pH gradient is obtained by changing the ratio of each gel stock solution in the mixed solution to be introduced into the gel container, and the gel sheet is elongated by cutting the gel sheet with a predetermined width in the pH gradient direction. By pasting on the plate, a gel plate for isoelectric focusing is obtained.

In the gel plate manufacturing method described in Patent Document 1, it was difficult to control the pH gradient in the gel container, and it was difficult to obtain a stable quality gel plate. Therefore, Patent Document 2 discloses a gel plate manufacturing method (inkjet method) in which a monomer solution is applied onto a plate as a technique capable of accurately managing a pH gradient. That is, after forming a liquid pool on the substrate and discharging the monomer solution into the liquid pool, a gel layer is formed on the substrate by applying a polymerization initiator to gel the coating film. In this case, a gel plate for isoelectric focusing having a predetermined pH gradient can be obtained by applying two types of monomer solutions having different pHs to the pool while changing the coating amount.

Japanese Patent Laid-Open No. 62-167659 JP 2012-2739 A

FIG. 9 (A) is a conceptual diagram illustrating the electrophoresis test device and the manufacturing method of Patent Document 2, and FIG. 9 (B) is a state immediately after applying the pH buffer solution by the method shown in FIG. 9 (A). It is a conceptual diagram explaining a state. FIGS. 9A and 9B are composed of an upper stage, a middle stage, and a lower stage, respectively. The upper part of FIGS. 9A and 9B shows the change in application amount of the acidic monomer solution and the basic monomer solution, which are two types of pH buffer solutions used at the time of gel production described later, in terms of area. The middle part of FIGS. 9A and 9B is a line graph showing the change in the coating amount of the acidic monomer solution (thin line) and the basic monomer solution (thick line). In the lower part of FIGS. 9A and 9B, the pH at the position of the gel layer in the electrophoresis direction is shown by a line graph.

In the case of Patent Document 2, the pH value increases linearly from one end S ₁ to the other end S ₂ of the substrate S so as to have a pH gradient as shown in the lower line graph of FIG. 9A. As described above, the acidic monomer solution and the basic monomer solution are applied to the liquid pool while changing the coating amount with the inkjet head (see the upper and middle stages of FIG. 9A). However, as shown in the upper part of FIG. 9B, in such ink-jet coating, the coating start end region I on the one end S ₁ side and the coating end region on the other end S ₂ side of the base material S that particularly increases the amount of solution. In II, the liquid film shape cannot be maintained at a right angle, and the amount of the solution decreases. Therefore, as shown in the middle part of FIG. 9B, the balance of the solution amount is lost from the beginning, and as shown in the lower part of FIG. 9B, the pH positions in the application start region I and the application end region II. Deviation (pH value deviation) may occur, and the pH gradient may be reversed. Thus, when the reverse gradient of the pH gradient is formed at both ends in the electrophoresis direction of the gel, the protein separation accuracy by electrophoresis using this gel is lowered. Such a reverse gradient of the pH gradient does not occur only by ink jet coating, but may also occur by coating using a pipetter, a dispenser, or the like.

In general, during electrophoresis in which a predetermined voltage is applied by contacting electrodes at both ends of the gel in the electrophoresis direction, impurities in the gel are attracted to one electrode side (positive electrode side or negative electrode side) and collected. The gel end where the impurities accumulate is a portion that is inherently unsuitable for protein separation. Here, the “impurities” include residual compounds at the time of gel chip production (unreacted monomer compounds, polymerization agents, etc.), impurities contained in the evaluation protein sample, impurities in electrophoresis reagents (electrophoresis buffer), and the like.

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 from the acidic side to the basic side,
The gel includes a main region and a buffer region adjacent to the main region;
There is provided an electrophoretic test device in which the pH value of the buffer region is substantially constant (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 is divided into a main region and a region where a buffer region adjacent to the main region is 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 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 gel in the electrophoretic test device of the present invention includes a main region having a linear pH gradient, and a buffer region adjacent to (preferably both) at least one of the acidic side and basic side ends of the main region. The pH gradient of the buffer region is gentler than the pH gradient of the main region, and the pH value of the buffer region is substantially constant. For example, if the main region has a linear pH gradient of pH 3 to 10, a buffer region constant at pH 3 is provided on the acidic side of the main region, and a buffer region constant at pH 10 is provided on the basic side of the main region. be able to.

As described with reference to FIG. 9B, in the gel formed by the conventional coating method, the pH gradient in the coating start region I and the coating end region II (that is, the pH gradient in the main region) is reversed. However, in the present invention, at least one (preferably both) of the application start region I and the application end region II is intentionally used as a buffer region, so that the pH gradient is reversed at the end of the main region in the electrophoresis direction. A linear pH gradient can be formed without forming a gradient.
In addition, the electrophoresis test device of the present invention is advantageous in that the gel end is unsuitable for protein separation because impurities in the gel accumulate during electrophoresis, and the length of the main region in the electrophoresis direction is convenient. Is not substantially shortened.
Therefore, the electrophoresis test device of the present invention can perform highly accurate separation and measurement of proteins in the main region of the gel during electrophoresis, and can obtain highly reliable analysis results.

Further, according to the method for manufacturing an electrophoretic test device of the present invention, the pH gradient in the main region is not affected even if the solution application amount changes in the application start region I and the application end region II. As a result, it is possible to manufacture an electrophoretic test device including a gel capable of performing highly accurate protein separation measurement.

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. It is a conceptual diagram explaining the state immediately after apply | coating pH buffer solution by the method shown to FIG. 2 (A). 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. 3 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. 6 (A). It is explanatory drawing which shows the state which apply | coats a polymerization initiator to a base material continuously from FIG. 6 (B). It is explanatory drawing which shows the state which the application | coating of the gel material liquid by the manufacturing apparatus of FIG. 3 was complete | finished. 6 is a conceptual diagram illustrating an electrophoresis test device and a manufacturing method thereof according to Embodiment 2. FIG. It is a conceptual diagram explaining the state immediately after apply | coating pH buffer solution by the method shown to FIG. 8 (A). It is a conceptual diagram explaining the test device for electrophoresis of the past (patent document 2), and its manufacturing method. It is a conceptual diagram explaining the state immediately after apply | coating pH buffer solution by the method shown to FIG. 9 (A).

<About electrophoresis test equipment>
The electrophoresis test device of the present invention comprises a gel having a pH gradient from the acidic side to the basic side,
The gel includes a main region and a buffer region adjacent to the main region.
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 number of the main regions is not particularly limited, and may be one or plural.

(2) The buffer region may be disposed adjacent to the acidic side end and the basic side end in the main region. When there is one main region, buffer regions are disposed at both ends in the electrophoresis direction of the gel, and a main region having a single continuous pH gradient is disposed between the buffer regions at both ends. When there are a plurality of main regions, buffer regions are arranged at both ends in the electrophoresis direction of the gel, a plurality of main regions having a pH gradient are arranged between the buffer regions at both ends, and also between two adjacent main regions. A buffer area is arranged.

(3) The pH gradient in the buffer region may be a half or less of the pH gradient in the main region.

(4) The pH gradient of the main region may be a 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), ammonium persulfate (APS: polymerization initiator) and pure water.

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 comprises a gel material application step for applying a gel material on a substrate to form a liquid pool, and a pH buffer for applying a pH buffer solution onto the liquid pool 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 is divided into a main region and a region where a buffer region adjacent to the main region is 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 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 onto 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. For example, a pipetter, a dispenser, an inkjet device, etc. 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. 2A is a conceptual diagram illustrating the electrophoresis test device and the manufacturing method thereof according to Embodiment 1, and FIG. 2B is a pH buffer solution applied by the method shown in FIG. It is a conceptual diagram explaining the state immediately after.

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, but is, for example, about 195 to 1010 μm.
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.

図 Figures 2 (A) and 2 (B) are composed of an upper stage, a middle stage and a lower stage, respectively. The upper part of FIGS. 2 (A) and 2 (B) shows the change in application amount of the acidic monomer solution and the basic monomer solution, which are two types of pH buffer solutions used at the time of gel production described later, in terms of area. The middle part of FIGS. 2 (A) and 2 (B) 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. In the lower part of FIGS. 2A and 2B, the pH at the position of the gel layer in the electrophoresis direction is shown by a line graph.

Further, in FIG. 2, the application region is divided into regions A to C 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, the region B corresponds to a region where a main region described later is to be formed, and the regions A and C correspond to regions where a buffer region described later is to be formed. In other words, the acidic side and the basic side of the region B where the main region is to be formed are the regions A and C where the buffer region is to be formed.

As shown in FIGS. 1A, 2A and 2B, the gel layer G ₁ has a pH gradient in a predetermined pH range in the X direction (electrophoresis direction). In Embodiment 1, the gel layer G ₁ has a main region having a pH gradient of pH 3 to 10, a pH 3 buffer region adjacent to the acidic side end of the main region, and a pH 10 adjacent to the basic side end of the main region. The case of having a buffer region is illustrated.

The characteristic structure of the gel layer G ₁ of Embodiment 1 is that buffer regions are arranged on the acidic side and basic side of the pH region (main region) to be analyzed in detail as described above. The pH of the buffer region is set to be equal to the pH at the end of the adjacent main region. By providing a buffer region having a pH equivalent to the pH of the acidic side and basic side end of the main region on the acidic side and basic side of the main region, in FIGS. 9 (A) and (B) The reverse gradient of the pH gradient that occurs at both ends of the described main region can be prevented. As a result, a gel in which the separation accuracy of the protein at the acidic side end and the basic side end of the main region does not deteriorate is obtained.

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. 3 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. 3, 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. As a result, the substrate S set on the stage 10 passes directly below first to third inkjet heads 31b, 32b, and 33b, which will be described 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 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 (acidic monomer solution A, basic monomer solution B, polymerization in the ink jet device 30). 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 controls 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 drip from the nozzle holes H (see FIG. 5).

The first to third ink jet heads 31b to 33b are integrated to form a set of discharge head units U, which are fixed by a fixing member (not shown). As shown in FIG. 4, the first to third ink jet heads 31b to 33b are arranged in a line on the movement locus E of the base material S, but the head arrangement order is not limited to this order. In 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. 4 and 5, a plurality of nozzles are formed on the lower surfaces of the first to third inkjet heads 31b to 33b 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. The nozzle hole diameter D and the nozzle hole interval P are not particularly limited, but 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 inkjet 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. 3, 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, a coating process under normal temperature and atmospheric pressure based on a predetermined program is performed. That is, as shown in FIGS. 6A and 6B and FIGS. 7A and 7B, the support 40a is intermittently moved in the direction of the arrow M by the moving mechanism 40, and the first to third Minute droplets La, Lb, and Lc are intermittently ejected from the inkjet heads 31b to 33b, and a coating film L3 is formed on the liquid pool L0.

More specifically, as shown in FIG. 6 (A), 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. 6 (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. 7 (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. 7 (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 sequentially passes through the first to third inkjet heads 31b to 33b, as shown in FIG. 7B, the first to third inkjet heads 31b to 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 FIGS. 2 (A) and 2 (B).
As shown in FIG. 2A, 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 one end S _{1 of the} substrate S. The coating amount is controlled to increase from the other end S ₂ toward the other end 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 the region B, the application amount of the acidic monomer solution continuously decreases in the range of 100 to 0%, and the application amount of the basic monomer solution continuously increases in the range of 0 to 100%.
In region C, 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 region B 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.
Further, in the regions A and C 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.
Here, “the rate of change of the coating amount per unit area of the pH buffer solution” means the degree of pH gradient.

The coating film immediately after the acidic and basic monomer solution is applied by such coating amount control is applied to the regions A and C in which the buffer regions are to be formed, as shown in the upper and middle parts of FIG. Although the shape collapses from the right-angle shape, the pH values of A and C in these regions are not changed. 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. Note that 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. In the present invention, the isoelectric focusing test device GP ₁ is an incomplete product.

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, a cooling step for cooling the dry film D ₁ to −20 ° C. or less may be performed. Or you may perform a freeze-dry process instead of a drying process and a cooling process.

(Embodiment 2)
FIG. 8A is a conceptual diagram illustrating the electrophoresis test device and the manufacturing method thereof according to Embodiment 2, and FIG. 8B is a diagram immediately after applying a pH buffer solution by the method shown in FIG. It is a conceptual diagram explaining a state.
FIGS. 8A and 8B are composed of an upper stage, a middle stage, and a lower stage, respectively. The upper part of FIGS. 8A and 8B 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 during gel production described later. The middle part of FIGS. 8A and 8B 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 FIGS. 8A and 8B shows the pH at the position of the gel layer in the electrophoresis direction as a line graph.

The gel in the electrophoresis test device of the second embodiment is the same as that of the first embodiment except that the gel has a plurality of main regions and a buffer region disposed between two adjacent main regions. Specifically, the gel has an acidic buffer region (pH 3), a basic buffer region (pH 10), and a first main region (pH 3 to 5.5) disposed between the buffer regions on both ends. ), The second main region (pH 5.5 to 8.5) and the third main region (pH 8.5 to 10), and the buffer region (pH 5.5) disposed between the first main region and the second main region. ) And a buffer region (pH 8.5) disposed between the second main region and the third main region.

In the case of a gel having a long pH range such as pH 3 to 10, there is a possibility that the intermediate pH value may be shifted even if there is no pH value shift at the start point (pH 3) and the end point (pH 10). On the other hand, if the gel structure as in Embodiment 2 is used, positions where pH values at two points (pH 5.5, pH 8.5) are determined can be included between

pH

3 and 10, as described above. It is possible to suppress an intermediate pH value shift.

(Embodiment 3)
In Embodiments 1 and 2, 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 exemplified. On the other hand, in Embodiment 3, 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 Embodiment 3, for example, in the apparatus for manufacturing an electrophoretic test device described with reference to FIG. 3, 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 having 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 method for manufacturing the electrophoresis test device of the third embodiment, a coating film is formed on a substrate in the same manner as in the first embodiment (description in FIGS. 6A to 7B), 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 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.

2. 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.

3. 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
GPD ₁ storage device for isoelectric focusing that can be stored G ₁ gel layer S base material AC region

Claims

Comprising a gel with a pH gradient from the acidic side to the basic side,
The gel includes a main region and a buffer region adjacent to the main region;
A test device for electrophoresis, wherein the pH value of the buffer region is substantially constant.
The electrophoretic test device according to claim 1, wherein the number of the main regions is one.
3. The electrophoresis test device according to claim 1, wherein the buffer region is disposed adjacent to an acidic side end and a basic side end in the main region.
The electrophoresis test device according to any one of claims 1 to 3, wherein the pH gradient in the buffer region is a gradient equal to or less than half of the pH gradient in the main region.
The electrophoresis test device according to any one of claims 1 to 4, wherein the pH gradient of the main region is a 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 is divided into a main region and a region where a buffer region adjacent to the main region is 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 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.
The pH buffer solution application step includes a first step of applying a first pH buffer solution having a first pH as the pH buffer solution on the liquid pool, and a first pH buffer solution different from the first pH as the pH buffer solution. Applying a second pH buffer solution having a pH of 2 onto the pool,
In the first step, the buffer region adjacent to the acidic side end of the main region should be formed in one direction without changing the coating amount per unit area of the first pH buffer solution, Applying the first pH buffer solution to the main region in one direction while changing the coating amount per unit area,
In the second step, the buffer region that is to be formed adjacent to the basic side end of the main region is applied in one direction without changing the coating amount per unit area of the second pH buffer solution. The method for producing an electrophoretic test device according to claim 6, wherein the second pH buffer solution is applied to the main region in one direction while changing an application amount per unit area.