JP2013205088A - Solution application method and method of manufacturing test tool for isoelectric focusing - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a solution application method with which an application film having an ideal functional gradient is formed easily.SOLUTION: A solution application method includes a first step for unidirectionally applying a first solution to an application range on an applied material while changing an application amount per unit area, and a second step for unidirectionally applying a second solution to the application range on the applied material while changing an application amount per unit area. The first step and the second step are repeated multiple times, such that a total of application amounts per unit area of the first solution and the second solution is uniform over the entire application range.

Description

  The present invention relates to a solution coating method and a method for producing an isoelectric focusing test device.

  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 above two electrophoresis methods has been used as part of proteome analysis for the purpose of exhaustively 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 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. In addition, using the gradient maker described in Patent Document 1, two types of gel stock solutions having different pH 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 it at 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 is difficult to control the concentration gradient or pH gradient in the gel container, and it is difficult to obtain a stable quality gel plate. Therefore, Patent Document 2 discloses a gel plate manufacturing method in which a monomer solution is applied onto a plate as a technique capable of accurately managing a concentration gradient or pH gradient. That is, after forming a liquid pool on the plate 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, an SDS-PAGE gel plate having a predetermined concentration gradient or a predetermined pH gradient is prepared by mixing two types of monomer solutions having different concentrations or pHs and applying them to the pool while changing the mixing ratio. A gel plate for isoelectric focusing is obtained.

  In order to enable the gel layer produced as described in Patent Documents 1 and 2 to be stored for a long period of time, the gel layer is usually dried. And a gel layer is decompress | restored by making a dry film absorb and swell a sample solution at the time of use.

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

  When the acidic monomer solution and the basic monomer solution are individually applied onto the substrate using the discharge device as described in Patent Document 2, the total amount of application per unit area of the acidic and basic monomer solution is constant. When the coating film having a pH gradient in one direction is formed by applying once on the base material while changing the ratio of the coating amount of each solution, the following problem arises.

  That is, as shown in FIG. 9, when one solution is applied once in the X direction, it is applied while increasing the application amount stepwise, and the height of the coating film applied on the steps is When it becomes higher, the shape of the stair-like coating film collapses due to gravity and deforms to a uniform height. Alternatively, when the other solution is applied once in the X direction, it is applied while reducing the application amount stepwise, and the application amount becomes the largest in the initial stage of application. Shape collapses. FIG. 9 is a conceptual diagram for explaining the coating amount distribution in the conventional solution coating method, and the symbol S in FIG. 9 represents a substrate.

  As a result, each solution is applied to a specific region on the substrate S by an amount deviated from the assumed application amount, and the pH gradient of the application film greatly deviates from the ideal state. Therefore, since the gel layer obtained through the subsequent gelation step becomes a gel layer having a pH gradient greatly deviating from the ideal state, it is difficult to perform highly accurate and reliable isoelectric focusing. Become. As described above, when two different types of solutions are applied to a material to be coated to form a functional gradient such as pH and concentration, it has been difficult to obtain an ideal functional gradient by the conventional coating method.

  The present invention has been made in view of such problems, and provides a solution coating method that facilitates the formation of a coating film having an ideal functional gradient, and further provides high accuracy and high reliability. It is an object of the present invention to provide a method for producing a test device capable of performing electric point electrophoresis based on the solution application method.

Thus, according to the present invention, the first step of applying the first solution to the application area on the application material in one direction while changing the application amount per unit area, and the application process on the application material on the application area. And a second step of applying the two solutions in one direction while changing the application amount per unit area, and the total application amount per unit area of the first solution and the second solution over the entire application region. In order to be uniform, there is provided a solution coating method in which the first step and the second step are repeated a plurality of times.
In the solution coating method of the present invention, the first solution may be a first pH buffer solution having a first pH, and the second solution has a second pH having a second pH different from the first pH. It may be a buffer solution.
In the solution coating method of the present invention, the application of the first solution and the application of the second solution may be performed using an inkjet apparatus.

According to another aspect of the present invention, the method includes the step (A) and the step (B) based on the first step and the second step in the solution coating method,
The total coating amount per unit area of the acidic monomer solution as the first solution used in the step (A) and the basic monomer solution as the second solution used in the step (B) is uniform over the entire coating region. As described above, there is provided a method for producing an isoelectric focusing test device in which the steps (A) and (B) are repeated a plurality of times.
In the method for producing an isoelectric focusing test device of the present invention, by repeating the step (A) and the step (B) a plurality of times, the mixed solution of the acidic monomer solution and the basic monomer solution is mixed. A step (C) of applying a polymerization initiator on the coating film may be further included after forming the coating film on a substrate as a material to be coated.

According to the solution coating method of the present invention, since the first step and the second step are repeated a plurality of times, the height of the stepwise coating film formed per first and second step can be reduced. . As a result, the degree of liquid movement of the coating film formed per time of the first and second steps is reduced, and functional gradients such as pH and concentration are easily formed in the coating film.
Therefore, according to the method for manufacturing an electrophoretic test device based on the solution coating method of the present invention, it is easy to form a gel layer having an ideal pH gradient, and the isoelectric focusing is highly accurate and reliable. It becomes possible to manufacture a test device capable of performing electrophoresis.

It is a perspective view which shows Embodiment 1 of the test device manufactured with the manufacturing method of the test device for isoelectric focusing 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). It is a block diagram which shows the apparatus which can manufacture the test device for electrophoresis of Embodiment 1. FIG. FIG. 3 is a schematic bottom view of the ink jet head in the manufacturing apparatus of FIG. 2 as viewed from below. It is an enlarged view which shows the nozzle hole group of the inkjet head in the manufacturing apparatus of FIG. It is a conceptual diagram explaining the coating amount distribution in the solution coating method of the present invention. 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 1st application | coating process following FIG. 6 (B). It is explanatory drawing which shows the state which switches to the coating process of the 2nd time from the 1st time following FIG.6 (C). It is explanatory drawing which shows the 2nd application | coating process of the acidic monomer solution and basic monomer solution which continue from FIG. 7 (A). It is explanatory drawing which shows the state which switches to the coating process of the 2nd time from the 2nd time following FIG. 7 (B). It is explanatory drawing which shows the state which apply | coats a polymerization initiator after the nth application | coating process of an acidic monomer solution and a basic monomer solution. It is explanatory drawing which shows the state which complete | finished application | coating of the gel material liquid by the manufacturing apparatus of FIG. It is a conceptual diagram explaining the coating amount distribution by the conventional solution coating method.

(Embodiment 1)
FIG. 1 (A) is a perspective view showing Embodiment 1 of a test device GP ₁ manufactured by the method for manufacturing an isoelectric focusing test device of the present invention, and FIG. 1 (B) is FIG. 1 (A). it is a perspective view showing a storable state after the dry gel layer G ₁ in isoelectric test device GP ₁ of.

An isoelectric focusing 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 an upper surface which becomes gentle convex curved surface (a surface of the substrate opposite the side). Drying the gel layer G ₁ to a water content of 5%, dried film D ₁ and became 1 isoelectric point shown in (B) electrophoresis test device GPD ₁ gel layer G ₁ is dried is obtained It is done.

In the present invention, the length and width of the gel layer G ₁ is the same as the length and width of the substrate. The length and width of the substrate 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 film thickness of the gel layer G ₁ is not particularly limited, for example, about 195～1010Myuemu.
The thickness of the dried film obtained by drying the gel layer shrinks to 100 μm or less, but the length and width hardly change.

  The form of the base material S of the electrophoretic 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 substrate S is not particularly limited as long as it can function as a substrate for an electrophoresis test device. For example, glass such as quartz glass and alkali-free glass, polyethylene terephthalate (PET), poly Examples thereof include resins such as methyl methacrylate resin (PMMA), ceramics such as alumina, and low-temperature co-fired ceramic. When the substrate S is made of a hydrophobic material, the surface of the substrate S on which the gel layer is formed may be subjected to a hydrophilic treatment, thereby improving the wettability of the monomer solution with respect to the substrate S, and the monomer solution The adhesion between the gel layer in which the gel is gelled and the substrate S 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 G ₁ of the electrophoresis test device GP ₁ is not particularly limited as long as it can function as a gel layer of the electrophoresis test device. For example, as a material of a general polyacrylamide gel Examples include 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 G ₁ is formed by applying a gel material solution on the substrate S and causing it to gel. At this time, when a gel material solution (polymer material containing a polymerization initiator) to which a polymerization initiator has been added in advance is applied on the substrate S, a monomer solution (including a polymerization initiator) in which materials other than the polymerization initiator are mixed A non-gel material liquid) is applied onto the substrate S, and then a polymerization initiator may be applied onto the applied film. In the present invention, both cases are included.

  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”.

In the present invention, the method for applying the gel material liquid onto the substrate S 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 S. For example, a pipetter, dispenser, inkjet device Etc. 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, it is possible to apply fine droplets to a predetermined region of the elongated substrate S with high accuracy and quantitatively. Therefore, the formation region of the gel layer G ₁ to be obtained, the film thickness, the pH gradient, and The concentration gradient and the like can be controlled easily and with high accuracy.

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. 2 is a configuration diagram illustrating 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 has a support base 40a that supports the stage 10, and the support base 40a can be reciprocated in a linear direction (arrow M1, M2 direction) by a linear guide mechanism (not shown).
In FIG. 2, 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 can go 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 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 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 drip from the nozzle holes H (see FIG. 4) 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. 3, 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 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. 3 and 4, 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 device GP ₁ using the test device manufacturing apparatus having the above-described configuration will be described. The isoelectric focusing test device GP ₁ is manufactured based on the solution coating method of the present invention. Since it can be manufactured by the method, first, the solution coating method will be described.

FIG. 5 is a conceptual diagram illustrating the coating amount distribution in the solution coating method of the present invention.
The solution application method of the present invention includes, for example, a first step of applying the first solution in the X direction while changing the application amount per unit area in the application regions A to H on the application material Sx, and the application material Sx. A second step of applying the second solution in the X direction while changing the application amount per unit area in the application areas A to H, and the total application amount per unit area of the first solution and the second solution is In this method, the first step and the second step are repeated a plurality of times so as to be uniform over the entire coating region. In FIG. 5, “first” means the application amount of the first step and the first solution, and “second” means the application amount of the second step and the second solution.

  In FIG. 5, the application areas A to H are set by dividing the upper surface of the material Sx to be equally divided into eight in the X direction, one area corresponds to a unit area, and the first step and the second step are repeated five times. Illustrated. If the application direction is the X direction, it may be left to right or right to left. When applying from left to right, the application amount of the first solution increases stepwise from the application area A toward the application area H. In addition, the application amount of the second solution decreases stepwise from the application area A toward the application area H. The reverse is true when applying from right to left. Moreover, the sum total of the coating amount of the first solution and the coating amount of the second solution in the coating areas A to H is uniform. Moreover, you may perform a 1st process and a 2nd process alternately. Alternatively, the odd-numbered times may be performed after the first step is performed, the second step is performed, and the even-numbered times after the second step is performed, and the reverse may be performed.

  In this solution coating method, the number of coating areas, the number of times to repeat the first step and the second step, the composition of the first and second solutions, etc. are not particularly limited, but completely the same as the first and second steps performed at the first time. It is necessary to repeat the second and subsequent times under the same conditions. Thus, a coating film having an ideal functional gradient is formed on the material to be coated Sx by the first and second steps of the first time, and the coating film is stepwise while maintaining the ideal functional gradient after the second time. It gets thicker. Here, examples of the “functional gradient” include a pH gradient, a concentration gradient, and a particle size gradient.

  According to this solution coating method, the thickness of the coating film to be finally obtained is not formed by one application, but is gradually increased by a plurality of applications. The application amount of can be reduced. As a result, it is possible to suppress movement due to the deformation of the shape of the coating film in each application, and it is easy to form a coating film having an ideal functional gradient.

The isoelectric focusing test device GP ₁ of the manufacturing method includes a step based on the first step and the second step in the solution coating method (A) and step (B), the first solution used in step (A) Step (A) and Step (B) so that the total coating amount per unit area of the acidic monomer solution and the basic monomer solution as the second solution used in Step (B) is uniform over the entire coating region. ) Is repeated several times. Thereby, a coating film having an ideal pH gradient can be formed.

  In addition, the method for manufacturing the isoelectric focusing test device can be obtained by repeating steps (A) and (B) a plurality of times to form a coating film of a mixed solution in which an acidic monomer solution and a basic monomer solution are mixed. Further, it may further include a step (C) of applying a polymerization initiator on the coating film after being formed on a substrate as a material to be coated.

Hereinafter, 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. 2, an elongated rectangular base material S is set on the stage 10 in the standby position.

  Next, the coating process under normal temperature and atmospheric pressure based on a predetermined program is performed. That is, as shown in FIGS. 6 (A) to (C), FIGS. 7 (A) to (C) and FIGS. 8 (A) and (b), the support 40a is moved in one direction (in the direction of the arrow M1) by the moving mechanism 40. ) At a constant speed, and micro droplets La and Lb are intermittently ejected from the first and second inkjet heads 31b and 32b at regular time intervals to form a coating film on the substrate S. .

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 substrate S. Thus, as shown in FIG. 6 (B), the coating film L1 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.

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. 6 (C), the coating film L2 of the acidic monomer solution and the basic monomer mixture monomer solution are mixed and the solution is formed at one end S ₁ side of the substrate S.

Then, when the other end S ₂ of the substrate S moves to a position directly below the nozzle hole group HG of the first inkjet head 31b, the discharge of the fine droplet La of the acidic monomer solution is stopped, and the other end S _{2 of the} substrate S is stopped. 7 moves to a position directly below the nozzle hole group HG of the second inkjet head 32b, the support base 40a of the moving mechanism 40 moves in the reverse direction (arrow M2 direction) as shown in FIG. substrate S is moved in the opposite direction, the other end S ₂ of the substrate S is ejected microdroplets La acidic monomer solution resumes after moving to a position directly below the nozzle hole group HG of the first inkjet head 31b with (See FIG. 7B).

In this way, as shown in FIG. 7 (B), it is applied on the coating film L2 on the other end S ₂ side of the basic monomer solution and an acidic monomer solution and the substrate S and then mixed in the coating film L2 The coating film L3 is formed.

Then, when one end S ₁ of the substrate S moves to a position directly below the nozzle hole group HG of the second inkjet head 32b, the discharge of the fine droplet Lb of the basic monomer solution is stopped, and the one end S _{1 of the} substrate S is When the first inkjet head 31b has moved to a position just below the nozzle hole group HG, as shown in FIG. 7C, the support base 40a of the moving mechanism 40 moves in the reverse direction (arrow M1 direction) and moves together with the stage 10. The substrate S moves in the reverse direction.

  Thereafter, the operations described in FIGS. 6A to 7C are repeated a plurality of times. Thus, the acidic monomer solution and the basic monomer solution are applied on the substrate S a plurality of times while the substrate S repeats reciprocating movements a plurality of times in the directions of the arrow M1 and the arrow M2. Thereby, the coating film of the mixed solution of the acidic monomer solution and the basic monomer solution is formed on the substrate S while gradually increasing the thickness while maintaining an ideal pH gradient. Then, after the acidic monomer solution and the basic monomer solution were applied on the substrate S a predetermined number of times (n times), the discharge of the acidic monomer solution and the basic monomer solution was stopped and the thickness was increased on the substrate S. A coating film Ln is formed (see FIG. 8A).

Thereafter, microdroplets Lc of the polymerization initiator are uniformly applied from the third inkjet head 33b onto the coating film Ln. At this time, as shown in FIG. 8 (A), where the substrate S is one end S ₁ moves in the direction of arrow M has moved to a position directly below the nozzle hole group HG third inkjet head 33b, a third inkjet head A small droplet Lc of the polymerization initiator is discharged from 33b and applied onto the coating film Ln. Alternatively, the base material S may move in the direction opposite to the arrow M direction, and the polymerization initiator may be applied onto the coating film Ln from the other end S ₂ side. As a result, a coating film Lx of a gel material solution containing a polymerization initiator in which the coating film Ln (mixed monomer solution) and the polymerization initiator are mixed is formed on the substrate S as shown in FIG.

  Thereafter, when the base material S passes through the third inkjet head 33b, the ejection of the minute droplets Lc from the third inkjet head 33b is stopped, the support base 40a returns to the standby position, and the coating process ends. 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.

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 gelation, as shown in FIG. 1 (A), an isoelectric focusing test instrument GP _{1 in} which a bowl-shaped gel layer G having roundness at four ends is formed on a substrate S is obtained. can get.

Then, by drying the gel layer G ₁ of the isoelectric focusing test device GP ₁ , an isoelectric focusing test device GPD ₁ 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.

(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 base material is illustrated, but a gel material solution containing a polymerization initiator may be applied onto the base material. 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. For example, in the coating unit, the cooled monomer solution and the cooled polymerization initiator are mixed in the vicinity of the nozzle, and further, the vicinity of the nozzle is also cooled to cool the gel material solution containing the polymerization initiator before discharge. In addition, the substrate may be cooled.

3. A water film is formed in advance on the substrate, and the monomer solution and the polymerization initiator may be individually applied onto the water film as in the first embodiment, or the gel material solution containing the polymerization initiator may be applied. .

  The solution coating method of the present invention is suitable for forming a coating film and a gel layer having a pH gradient as described in Embodiment 1. In addition to this, for example, two types of solutions having different concentrations are used. The present invention can also be applied to a case where a coating film having a predetermined concentration gradient is formed by coating on a material to be coated. Furthermore, two kinds of fine particle dispersions in which fine particles having different particle diameters are dispersed in a solvent are applied onto a material to be coated to form a coating film having a particle diameter gradient in which the ratio of large particle diameters gradually increases. In this case, the solution coating method of the present invention can be applied.

A acidic monomer solution B basic monomer solution C polymerization initiator D ₁ dry film GP ₁ isoelectric focusing test device ready for use (gel plate)
GPD ₁ Storage device for isoelectric focusing that can be stored G ₁ Gel layer S Base material Sx Material to be coated

Claims (5)

  A first step of applying the first solution to the application region on the material to be applied in one direction while changing the application amount per unit area, and applying the second solution to the application region on the material to be applied per unit area A second step of applying in one direction while changing the amount, and the total amount of application per unit area of the first solution and the second solution is uniform over the entire application region. A solution coating method comprising repeating the first step and the second step a plurality of times.   2. The solution according to claim 1, wherein the first solution is a first pH buffer solution having a first pH, and the second solution is a second pH buffer solution having a second pH different from the first pH. Application method.   The solution application method according to claim 1, wherein the application of the first solution and the application of the second solution are performed using an ink jet apparatus. Including a step (A) and a step (B) based on the first step and the second step in the solution coating method according to claim 1,
The total coating amount per unit area of the acidic monomer solution as the first solution used in the step (A) and the basic monomer solution as the second solution used in the step (B) is uniform over the entire coating region. As described above, a method for producing an isoelectric focusing test device in which the step (A) and the step (B) are repeated a plurality of times.   After forming the coating film of the mixed solution in which the acidic monomer solution and the basic monomer solution are mixed on the base material as the coating material by repeating the step (A) and the step (B) a plurality of times. The method for producing an isoelectric focusing test device according to claim 4, further comprising a step (C) of applying a polymerization initiator on the coating film.