Method and apparatus for treating object substance by electrophoresis

Abstract

According to one embodiment of the present invention, provided is a method for treating a substance to be treated by electrophoresis, the method comprising: providing a separator through which the substance to be treated is allowed to pass; introducing a first solution containing a target substance into a first side of the separator; introducing a second solution into a second side of the separator; and selectively moving the object substance from the first solution to the second solution through the separator by electrophoresis.

Description

Technical Field

[0001] The present invention relates to a method and apparatus for treating a target substance by electrophoresis. Background Art

[0002] Various methods are known for separating substances in solution.

[0003] Electrophoresis is one such method. Various electrophoresis methods have been developed. For example, two-dimensional electrophoresis is a method for separating target substances by performing electrophoresis in two directions sequentially, and is widely used for protein analysis. Typically, polyacrylamide gels are used for linear separation (one-dimensional) by isoelectric electrophoresis, followed by separation based on molecular weight using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a direction perpendicular to this (second-dimensional). This method requires both two electrophoresis runs and the area of ​​the second-dimensional gel.

[0004] Dialysis is also a method for separating substances. Various dialysis methods have been developed. For example, discontinuous diafiltration is concentrated by centrifugal ultrafiltration and buffer exchange is performed by dilution with a buffer. As a result, dialysis usually requires large equipment, large amounts of solvent (buffer) and long processing times (several hours to a full day). Centrifugal ultrafiltration is prone to filter mesh clogging and is not suitable for crude samples containing a large amount of inclusions.

[0005] Dialysis is based on osmotic pressure and therefore cannot directly concentrate the target substance. To achieve this, 50% to 60% glycerol is mixed into the external solution. This causes water from the internal solution to be expelled into the external solution. However, glycerol can enter the internal solution containing the target substance and mix with it. Concentrated glycerol increases the viscosity of the solution. Therefore, the mixed glycerol may affect the analysis or measurement of the target substance contained in the internal solution. Furthermore, glycerol is a nonionic substance with no charge, making it unsuitable for separation using electrophoresis. Summary of the Invention

[0006] Problems to be solved by the invention

[0007] As can be understood from the above examples, there is a need for simpler and more efficient methods for separating substances.

[0008] Methods used to solve problems

[0009] According to one embodiment of the present invention, a method for treating a target substance by electrophoresis is provided. According to one embodiment, the method comprises:

[0010] Provide a partition that allows the target substance to pass through;

[0011] introducing a first solution containing the target substance into a first side of the separator;

[0012] introducing a second solution into the second side of the separator; and

[0013] The target substance is selectively moved from the first solution to the second solution through the separator by electrophoresis.

[0014] According to one embodiment of the present invention, there is provided an apparatus (device) for treating a target substance by electrophoresis. According to one embodiment, the apparatus (device) comprises:

[0015] First solution chamber (input chamber);

[0016] a second solution chamber (elution chamber) adjacent to the first solution chamber (input chamber); and

[0017] a separator that divides the first solution chamber and the second solution chamber, allows the target substance to pass from the first solution chamber to the second solution chamber, and has conductivity;

[0018] The device (device) is constructed in the following manner:

[0019] The first solution chamber is filled with a first solution containing the target substance;

[0020] The second solution chamber is filled with a second solution;

[0021] An electric field is applied from outside the first solution chamber and the second solution chamber to selectively move the target substance from the first solution chamber to the second solution chamber through the partition by electrophoresis.

[0022] As a result, for example, a target substance can be processed simply or efficiently by electrophoresis, and the device can also be miniaturized.

[0023] Further aspects and advantages of the present invention will be readily apparent to those skilled in the art from the following detailed description, which merely illustrates and describes exemplary embodiments of the present invention. It should be understood that the present invention is susceptible of other and different embodiments, and that some of its details may be modified in various obvious respects without departing from the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of an electrophoretic device according to one embodiment.

[0025] Figure 2 Schematic diagram of an electrophoretic device according to one embodiment.

[0026] Figure 3 Schematic diagram of an electrophoretic device according to one embodiment.

[0027] Figure 4Schematic diagram of an electrophoretic device according to one embodiment.

[0028] Figure 5 Schematic diagram of an electrophoretic device according to one embodiment.

[0029] Figure 6 This is a graph showing the results of the concentration rate of one example.

[0030] Figure 7 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0031] Figure 8 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0032] Figure 9 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0033] Figure 10 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0034] Figure 11 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0035] Figure 12 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0036] Figure 13 This is a graph showing the abundance ratio of albumin to amylase in the above examples.

[0037] Figure 14 Graph showing the relationship between the concentration of the counter substance in the initial eluate and the concentration of albumin in the eluate after electrophoresis in one example.

[0038] Figure 15 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0039] Figure 16 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0040] Figure 17 This is a photograph of an electrophoresis gel showing the results of electrophoresis treatment according to one embodiment.

[0041] Figure 18 Schematic diagram of an electrophoresis unit according to an embodiment.

[0042] Figure 19 FIG. 1 is a schematic diagram of a pipette cartridge containing an electrophoresis unit according to an embodiment. DETAILED DESCRIPTION

[0043] <Target substances>

[0044] In some embodiments, the target substance may be a substance contained in a target solution of a subject. According to some embodiments of the present invention, the treatment of the target substance may be performed for examination of the subject.

[0045] In some embodiments, the subject (subject) may include a human, or may be a human. In some embodiments, the subject may include an animal other than a human, or may be an animal other than a human. The subject may include a mammal, or may be a mammal. The subject may include, for example, but not limited to, working animals, livestock animals, pet animals, or wild animals. The subject may be a plant, or may be a microorganism.

[0046] The sample containing the target substance may be a solution. The "solution" may be a solution from the subject. The "solution" may be a body fluid, a solution derived from a body fluid, or a dilution of a body fluid. The solution may be a non-body fluid (non-body fluid derived) solution, or a body fluid or a mixture of a solution derived from a body fluid and a solution derived from a non-body fluid. The solution may be a disrupted solution, a culture medium, or the like of microparticles, cells, extracellular vesicles, etc. The solution may be a solution used in sample measurement, or a solution used in measurement for calibration. For example, the solution may be a standard solution or a calibration solution. For example, the solution may be a liquid that intentionally or intentionally does not contain the target substance for measurement for the purpose of calibration, etc. The sample to be measured may be a specimen. The solution may be a solution containing a chemical substance.

[0047] "Body fluid" can be lymph, can be tissue fluid such as interstitial fluid, intercellular fluid, interstitial fluid, can be body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (cerebrospinal fluid), joint fluid (synovial fluid), aqueous humor (aqueous humor). Body fluid can be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, intestinal fluid, can be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, breast milk. Body fluid can be animal body fluid or human body fluid. "Body fluid" can be a solution. The solution can include physiological buffers such as phosphate buffered saline (PBS) and N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer (TES) containing the target substance. The solution is not particularly limited as long as it contains the target substance.

[0048] The solution may contain the target substance. The solution may have the possibility of containing the target substance. In some embodiments, the target substance may be a molecule, an ion, a polymer, a biomolecule, etc. The target substance may have a biomolecule. The target substance may be a biomolecule. The target substance may be a protein, a glycated protein, etc. For example, the solution is tears, and the target substance may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin contained in the tears. Alternatively, the target substance may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin in blood, serum, or plasma, or may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin in interstitial fluid, urine, or saliva. Albumin may be oxidized albumin (HNA) or reduced albumin (HMA). In some embodiments, the target substance may be AGE (Advanced Glycation End Products, glycation end products, advanced glycation end products). In some embodiments, the target substance may be glycated lipid.

[0049] The term "albumin" used in this specification includes both glycated (glycated albumin) and non-glycated forms, unless otherwise specified, and includes both oxidized and reduced forms.

[0050] The solution containing the target substance may be a crude sample. As used herein, the term "crude sample" generally refers to a sample of biological origin believed to contain the target substance, which has not undergone isolation or purification steps for the target substance. "Crude sample" does not exclude disrupted solutions or culture solutions of microorganisms, cells, extracellular vesicles, etc.

[0051] The term "inclusions" used in this specification generally refers to substances that are not the target substance for recovery. Unless otherwise specified, they can be used regardless of whether they are impurities or inclusions.

[0052] <Separator>

[0053] The term "separator" as used herein generally refers to a membrane, member, or region that separates two solutions. A separator allows a target substance contained in one solution to pass into the other solution. Alternatively, a separator allows inclusions contained in one solution to pass into the other solution while preventing the target substance from passing.

[0054] The expression "a partition divides the solution chamber" or similar expressions used in this specification means that during the intended separation operation, the amount of permeation (solvent passing through the partition) of the solvent or buffer contained in the solution chamber is sufficiently small relative to the permeation (solute passing through the partition) of the target substance. This does not necessarily mean that even a small amount of permeation will not occur.

[0055] The separator of the present invention preferably has conductivity suitable for electrophoresis. The separator is not limited to components or materials that have conductivity. The phrase "a separator divides the solution chamber" or similar expressions used in this specification should be understood to refer to the degree to which the target substance can pass through the separator during electrophoresis.

[0056] In some embodiments, the separator may comprise a gel, and examples of gels include, but are not limited to, agarose gel, polyacrylamide gel (PAGE), and the like.

[0057] In some embodiments, the separator may comprise filter paper or cellulose. In some embodiments, the separator may comprise a resin (eg, polytetrafluoroethylene (PTFE)).

[0058] In some embodiments, the separator may comprise two or more different substances. In some embodiments, the separator may comprise filter paper (cellulose membrane, etc., or other membrane) and a gel. For example, the gel may be positioned on the inlet chamber side relative to the filter paper. The filter paper physically supports the gel.

[0059] Buffer exchange

[0060] In some embodiments, the solvents of the solutions on both sides of the separator can be different. In some embodiments, the first buffer of the first solution chamber and the second buffer of the second solution chamber can be different. In this case, the target substance can be buffer exchanged.

[0061] The solvent of the solution can be a buffer. Examples of buffers include, but are not limited to, Tris-glycine buffer, phosphate buffer, sodium barbital buffer, and the like.

[0062] <Concentration>

[0063] In some embodiments, the volume of the solution chambers on both sides of the separator or the volume of the solution may be different. For example, the volume of the second solution chamber or the amount of the second solution (second buffer) in the second solution chamber is less than the volume of the first solution chamber or the amount of the first solution (first buffer) in the first solution chamber. Thus, the target substance can be concentrated. It is not limited to that all the target substances in the first solution chamber (input chamber) are moved to the second solution chamber (elution chamber) due to electrophoresis. The volume of the second solution or the volume of the second solution chamber, or the properties of the second solution or the second solution chamber can be constructed in a way that the concentration of the target substance in the second solution after electrophoresis becomes higher than the concentration of the target substance in the initial first solution.

[0064] The term "concentration rate" used in this specification is defined as the ratio of the concentration C0 of the target substance in the first solution chamber (input chamber) before electrophoresis to the concentration C1 of the target substance in the second solution chamber (elution chamber) after electrophoresis, C1 / C0.

[0065] The concentration of the target substance in the second solution increases during the treatment. Along with this, the osmotic pressure from the second solution chamber to the first solution chamber, i.e. in the reverse direction, increases. This suggests the existence of a concentration limit. As one of the solutions to this problem, in some embodiments, a substance (countering substance) that moves to the first solution chamber due to electrophoresis can be added to the second solution. Preferably, the countering substance has an opposite charge to the target substance in the solution. Examples of countering substances include, but are not limited to, biological macromolecules such as proteins and artificial macromolecules.

[0066] <Selective>

[0067] In some embodiments, the separator can be selective. That is, the separator can have the ability to allow the passage of target molecules while not allowing, inhibiting, or blocking the passage of other target molecules or inclusions. For example, the separator can be a semipermeable membrane or a dialysis membrane, and can include a semipermeable membrane or a dialysis membrane. The semipermeable membrane or dialysis membrane can be selected from cellulose esters, regenerated cellulose, and the like.

[0068] For example, the separator can be chemically selective. In some embodiments, the separator can allow inclusions to pass through.

[0069] In some embodiments, the separator may contain a substance that can achieve selective electrophoresis (sometimes referred to as a "selective substance" in this specification). The separator may contain a substance that has the ability to purify proteins. The separator may contain a resin for protein purification. For example, the separator may be selected from hydroxyapatite, a cation exchange carrier, an anion exchange carrier, etc. Hydroxyapatite can be any of type I and type II. Examples of hydrophobic resins include compounds having butyl, phenyl, hexyl, etc. as functional groups. The separator may contain a compound or mixture having functional groups that can exert a variety of different interactions (ion exchange, hydrophobic binding, affinity binding, etc.). The separator may contain compounds used in a mixed mode. These substances inhibit, do not allow or block the passage of inclusions. As a result, the purity of the target substance can be improved, for example, compared to a case where no resin is present.

[0070] For example, the selective substance can be contained in or loaded on the gel. For example, the selective substance can be loaded on or near the surface of the semipermeable membrane. In some embodiments, the selective substance can be disposed in a portion other than the separator. For example, the selective substance can be disposed in the first solution or the first solution chamber. In some embodiments, the selective substance can be mixed into the first solution prior to electrophoresis.

[0071] <Multiple electrophoresis>

[0072] In some embodiments, a first solution chamber, a second solution chamber, a third solution chamber, a first separator disposed between the first solution chamber and the second solution chamber, and a second separator disposed between the second solution chamber and the third solution chamber may be provided. The electrode pair may be arranged on the outside of the first solution chamber and the third solution chamber. In some embodiments, the first separator allows the object substance to pass through, and the second separator does not allow the object substance to pass through. In some embodiments, the second separator may not allow the object substance to pass through, but allow a portion of inclusions (for example, a substance smaller than the object substance) to pass through. Thus, the object substance moves from the first solution chamber via the first separator to the second solution chamber and is retained there. On the other hand, a portion of the inclusions is retained in the first solution chamber. In addition, another portion of the inclusions enters the third solution chamber through the second solution chamber and the second separator. Thus, for example, further purification of the object substance can be performed. In some embodiments, more than two separators may be used. In some embodiments, more than three solution chambers or solutions may be used.

[0073] In some embodiments, further electrophoresis can be used to move the target substance that has been subjected to electrophoretic processing (separation, buffer exchange, concentration, etc.) to another location (a recovery chamber, a sensor, a chamber for performing other processes, etc.).

[0074] In some embodiments, electrophoresis can be used to move a processed target substance to a sensor for detection. For example, the device can also include a sensor. For example, the device can be configured to be connected to a sensor. For example, the third solution chamber can be a space where a sensor for detecting the target substance is located. The present invention provides an electrophoresis apparatus, device, or system.

[0075] In some embodiments, a solution containing a target substance can be introduced into such a sensing device for electrophoretic processing (separation, purification, buffer exchange, concentration, etc.), and the treated target substance can be detected by the sensing device. This allows, for example, efficient and / or highly sensitive detection or measurement of low concentrations of the target substance. By using such an electrophoretic apparatus, device, or system, for example, low-concentration proteins such as albumin in saliva can be concentrated and purified, and furthermore, can be detected or measured with high sensitivity or precision.

[0076] <Selectivity based on isoelectric point>

[0077] In some embodiments, selectivity for the target substance and inclusions can be generated according to the pH of the input liquid. The pH of the input liquid can be set between the isoelectric points of the target substance and the inclusions. For example, in the case of attracting the negatively charged target substance in the input liquid to the positive electrode, the pH of the input liquid is set to be higher than the isoelectric point of the target substance. The surface of the target substance is negatively charged and is attracted to the positive electrode. The pH in the input liquid can also be set as follows: the isoelectric point of the target molecule in the input liquid is smaller than the pH and larger than the isoelectric point of the inclusions. For example, the inclusions can move in the opposite direction to the target substance. For example, even if the inclusions move toward the same electrode, the amount of movement is sufficiently small compared to the target substance. For example, the inclusions can not move. In this way, the target substance can be purified or separated.

[0078] <Performing various treatments>

[0079] In some embodiments, multiple treatments such as buffer exchange, concentration, purification, and separation can be performed simultaneously. The multiple treatments can be selected from the group consisting of buffer exchange, concentration, purification, and separation. The timing of the multiple treatments may or may not overlap. As used herein, the phrase "concurrently performing multiple treatments" unless otherwise specified refers to performing multiple treatments during a single one-dimensional electrophoresis process.

[0080] In some embodiments of the present invention, two-dimensional or multi-dimensional electrophoresis is not excluded. In any one-dimensional electrophoresis, the treatment electrophoresis of any embodiment of the present invention is performed. In some embodiments, multi-dimensional treatment electrophoresis can be performed.

[0081] In some embodiments, a treatment electrophoresis can be performed intermittently. For example, the positive and negative electrodes can be switched periodically or irregularly. This can, for example, prevent problems such as separator mesh clogging and / or allow for efficient or effective treatment electrophoresis. This switching of the electric field direction should not be considered multidimensional electrophoresis. Unless otherwise specified in the present invention, a series of electrophoretic treatments performed using the same electrode pair is considered to be one-dimensional electrophoresis.

[0082] <Implementation Method>

[0083] Several embodiments are described below with reference to the accompanying drawings.

[0084] <Implementation Method 1>

[0085] use Figure 1 The device 100 and its operation according to one embodiment of the present invention will be described.

[0086] like Figure 1As shown in FIG. 1A , the electrophoresis apparatus 100 includes a first solution chamber (input chamber) 101, an adjacent second solution chamber (elution chamber) 102, and a partition 111 that separates the first solution chamber 101 from the second solution chamber 102. A first buffer solution 131 and target molecules 141 are loaded into the first solution chamber 101. The second solution chamber 102 is filled with a second buffer solution 132. Figure 1 The electrophoresis device 100 shown further includes an electrode pair 121, 122. The electrode pair 121, 122 is arranged to sandwich the first solution chamber 101 and the second solution chamber 102. In other words, the electrode pair 121, 122 is arranged such that, under the electric field formed by the electrode pair 121, 122, the target molecule 141 in the first solution chamber 101 moves from the first solution chamber 101 to the second solution chamber 102 through the partition 111.

[0087] The electrode pair 121 and 122 is connected to a DC power supply 123 . Figure 1 In A, switch 125 is open.

[0088] like Figure 1 As shown in FIG. 1B , switch 125 is closed, negatively charging electrode 121 and positively charging electrode 122, generating an electric field between them. Electrophoresis occurs under this electric field. Specifically, negatively charged target molecules 141 begin to migrate within first buffer solution 131 toward electrode 122. Target molecules 141 pass through partition 111 and enter second buffer solution 132 in second solution chamber 102.

[0089] like Figure 1 As shown in FIG. C, electrophoresis is performed for a predetermined time, and a sufficient amount of target molecules 141 enters the second buffer solution 132 of the second solution chamber 102. The switch 125 is opened to remove the electric field and terminate the process.

[0090] Thus, the target molecule 141 initially contained in the first buffer solution 131 is contained in the second buffer solution 132. Buffer exchange is performed. In some embodiments, the concentration of the target molecule 141 can be increased.

[0091] <Implementation Method 2>

[0092] use Figure 2 The device 200 and its operation according to one embodiment of the present invention will be described.

[0093] like Figure 2 As shown in FIG. 1A , the electrophoresis apparatus 200 includes a first solution chamber (input chamber) 201, an adjacent second solution chamber (elution chamber) 202, and a partition 211 that separates the first solution chamber 201 from the second solution chamber 202. A first buffer solution 231, target molecules 241, and inclusions 242 are introduced into the first solution chamber 201. The second solution chamber 202 is filled with the second buffer solution 232.

[0094] Figure 2 The electrophoresis device 200 shown further includes an electrode pair 221 , 222 . Figure 2 In A, switch 225 is open.

[0095] In this embodiment, the isoelectric points of the target molecule 241 and the inclusion 242 are different. The pH of the first buffer solution 231 is between these isoelectric points. That is, the isoelectric point of the target molecule 241 is lower than the pH of the first buffer solution 231, while the isoelectric point of the inclusion 242 is higher than the pH of the first buffer solution 231.

[0096] like Figure 2 As shown in Figure B, switch 225 is closed, negatively charging electrode 221 and positively charging electrode 222, generating an electric field between them. Electrophoresis occurs under this electric field. Specifically, negatively charged target molecules 241 begin to migrate within first buffer solution 231 toward electrode 222. Target molecules 241 pass through separator 211 and enter second buffer solution 232 in second solution chamber 202. Meanwhile, positively charged inclusions 242 begin to migrate within first buffer solution 231 toward electrode 221. In other words, inclusions 242 do not migrate toward electrode 222 or separator 211.

[0097] like Figure 2 As shown in FIG. C, electrophoresis is performed for a predetermined time, and a sufficient amount of target molecules 241 enter the second buffer solution 232 in the second solution chamber 202. Impurities 242 remain in the first solution chamber (input chamber) 201 or the first buffer solution 231. Switch 225 is opened to remove the electric field and terminate the process.

[0098] Thus, the target molecule 241 initially contained in the first buffer 231 is contained in the second buffer 232. The second buffer 232 is free of impurities 242. This allows the target molecule 241 to be purified or isolated. Furthermore, buffer exchange has been performed. In some embodiments, the concentration of the target molecule 241 can be further increased.

[0099] <Implementation Method 3>

[0100] use Figure 3 The device 300 and its operation according to one embodiment of the present invention will be described.

[0101] like Figure 3 As shown in FIG. 1A , the electrophoresis apparatus 300 includes a first solution chamber (input chamber) 301, an adjacent second solution chamber (elution chamber) 302, and a partition 311 that separates the first solution chamber 301 from the second solution chamber 302. A first buffer solution 331, target molecules 341, and inclusions 342 are introduced into the first solution chamber 301. The second solution chamber 302 is filled with a second buffer solution 332. Figure 3The electrophoresis device 300 shown further includes an electrode pair 321 , 322 . Figure 3 In A, switch 325 is open.

[0102] In this embodiment, separator 311 is selective. That is, separator 311 allows target molecules 341 to pass through, but prevents inclusions 342 from passing through. For example, assume that a typical inclusion 342 is larger than target molecules 341. Separator 311 is a semipermeable membrane with a pore size larger than target molecules 341 and smaller than inclusions 342.

[0103] like Figure 3 As shown in FIG. 3B , switch 325 is closed, negatively charging electrode 321 and positively charging electrode 322, generating an electric field between them. Electrophoresis occurs under this electric field. Specifically, target molecule 341 begins to migrate within first buffer solution 331 toward electrode 322. Target molecule 341 passes through partition 311 and enters second buffer solution 332 in second solution chamber 302. Figure 3 In the example, it is assumed that the impurity 342 has the same charge as the target molecule 341. The impurity 342 initially moves within the first buffer solution 331 toward the electrode 322. However, it cannot pass through the partition 311 and cannot enter the second buffer solution 332 of the second solution chamber 302, so it remains in the first buffer solution 331.

[0104] like Figure 3 As shown in FIG. C, electrophoresis is performed for a predetermined time, and a sufficient amount of target molecules 341 enter the second buffer solution 332 in the second solution chamber 302. Impurities 342 remain in the first solution chamber (input chamber) 301 or the first buffer solution 331. Switch 325 is opened to remove the electric field and terminate the process.

[0105] Thus, the target molecule 341 initially contained in the first buffer solution 331 is contained in the second buffer solution 332. The second buffer solution 332 is free of impurities 342. This allows the target molecule 341 to be purified or isolated. Furthermore, buffer exchange has been performed. In some embodiments, the concentration of the target molecule 341 can be further increased.

[0106] <Implementation Method 4>

[0107] use Figure 4 The device 400 and its operation according to one embodiment of the present invention will be described.

[0108] like Figure 4As shown in FIG. 1A , the electrophoresis apparatus 400 includes a first solution chamber (input chamber) 401, an adjacent second solution chamber (elution chamber) 402, and a first partition 411 that separates the first solution chamber 401 from the second solution chamber 402. A first buffer solution 431, target molecules 441, and inclusions 442 and 443 are introduced into the first solution chamber 401. The second solution chamber 402 is filled with a second buffer solution 432.

[0109] The electrophoresis apparatus 400 further includes a third solution chamber 403 (discharge chamber) adjacent to the second solution chamber (elution chamber) 402 , and a second partition 412 that partitions the second solution chamber 402 and the third solution chamber 403 .

[0110] Figure 4 The electrophoresis device 400 further includes an electrode pair 421 and 422 . Figure 4 In A, switch 425 is open.

[0111] In this embodiment, first separator 411 and second separator 412 are selective. That is, first separator 411 allows target molecules 441 to pass through, but excludes inclusions 442. For example, assume that inclusions 442 are typically larger than target molecules 441. For example, first separator 411 is a semipermeable membrane with a pore size larger than target molecules 441 and smaller than inclusions 442. In this embodiment, inclusions 443 are allowed to pass through first separator 411.

[0112] Second separator 412 has the ability to allow inclusions 443 to pass through but not target molecules 441. For example, assume that a typical inclusion 443 is smaller than target molecules 441. For example, second separator 412 is a semipermeable membrane with a pore size smaller than target molecules 441 and larger than inclusions 443. In this embodiment, inclusions 443 can pass through first separator 411.

[0113] like Figure 4 As shown in Figure B, switch 425 is closed, causing electrode 421 to become negatively charged and electrode 422 to become positively charged, generating an electric field between them. Electrophoresis occurs under this electric field. Specifically, target molecule 441 and like-charged inclusions 442 and 443 begin to migrate within first buffer solution 431 toward electrode 422. Target molecule 441 and inclusion 443 pass through first partition 411 and enter second buffer solution 432 in second solution chamber 402. Isolation 442 cannot pass through first partition 411 and cannot enter second buffer solution 432 in second solution chamber 402, thus remaining in first buffer solution 431.

[0114] Target molecule 441 and foreign substance 443 migrate within second buffer solution 432 toward electrode 422. Foreign substance 443 passes through second partition 412 and enters third buffer solution 433 within third solution chamber 403. Target molecule 441 cannot pass through second partition 412 and enter third buffer solution 433 within third solution chamber 403, and therefore remains within second buffer solution 432.

[0115] like Figure 4 As shown in FIG. 4C , electrophoresis is performed for a predetermined time, and a sufficient amount of target molecules 441 enter the second buffer solution 432 of the second solution chamber 402. Impurities 442 remain in the first solution chamber (input chamber) 401 or the first buffer solution 431, while impurities 443 enter the third solution chamber (discharge chamber) 403 or the third buffer solution 433. Switch 425 is opened to remove the electric field, thereby terminating the process.

[0116] Thus, the target molecule 441 initially contained in the first buffer solution 431 is contained in the second buffer solution 432. The second buffer solution 432 is free of impurities 442 and 443. This allows the target molecule 441 to be purified or isolated. Furthermore, buffer exchange has been performed. In some embodiments, the concentration of the target molecule 441 can be further increased.

[0117] <Implementation Method 5>

[0118] use Figure 5 The device 500 and its operation according to one embodiment of the present invention will be described.

[0119] like Figure 5 As shown in FIG. 1A , the electrophoresis apparatus 500 includes a first solution chamber (input chamber) 501, an adjacent second solution chamber (first elution chamber) 502, and a first partition 511 that separates the first solution chamber 501 from the second solution chamber 502. A first buffer solution 531 and target molecules 541 are loaded into the first solution chamber 501. The second solution chamber 502 is filled with a second buffer solution 532.

[0120] The electrophoresis apparatus 500 further includes a third solution chamber 503 (second elution chamber) adjacent to the second solution chamber (elution chamber) 502 and a second partition 512 that partitions the second solution chamber 502 from the third solution chamber 503. The third solution chamber 503 is filled with a second buffer solution 533.

[0121] Figure 5 The electrophoresis device 500 further includes a first electrode pair 521a and 522a arranged to perform electrophoresis on the first solution chamber 501, the first partition 511, and the second solution chamber 502. The second partition and the third solution chamber are arranged perpendicular to or at an angle to the electric field formed by the first electrode pair 521a and 522a. Figure 5The electrophoresis device 500 further includes a second electrode pair 521b, 522b configured to perform electrophoresis on the second solution chamber 502, the second partition 512, and the third solution chamber 503. In some embodiments, the first solution chamber 501, the first partition 511, the second solution chamber 502, and the first electrode pair 521a, 522a may be configured to perform electrophoresis once, and the second solution chamber 502, the second partition 512, the third solution chamber 503, and the second electrode pair 521b, 522b may be configured to perform electrophoresis twice. Figure 5 The switch is omitted.

[0122] like Figure 5 As shown in FIG. 2B , a voltage is applied to the first electrode pair 521a and 522a, generating an electric field between them. Electrophoresis occurs under this electric field. Specifically, the target molecule 541 begins to migrate within the first buffer solution 531 toward the electrode 522a. The target molecule 541 passes through the first partition 511 and enters the second buffer solution 532 in the second solution chamber 502.

[0123] Electrophoresis is performed for a predetermined time, and a sufficient amount of target molecules 541 enters the second buffer solution 532 in the second solution chamber 502. The electric field is removed, and one electrophoresis session ends.

[0124] Thus, the target molecule 541 initially contained in the first buffer solution 531 is contained in the second buffer solution 532. Buffer exchange is performed. In some embodiments, the concentration of the target molecule 541 can be increased.

[0125] Then, if Figure 5 As shown in Figure C, the voltage across first electrode pair 521a, 522a is removed, and a voltage is applied across second electrode pair 521b, 522b, generating an electric field therebetween. Electrophoresis occurs under this electric field. Specifically, target molecule 541 begins to migrate within second buffer solution 532 toward electrode 522b. Target molecule 541 passes through second partition 512 and enters third buffer solution 533 within third solution chamber 503.

[0126] Electrophoresis is performed for a predetermined time, and a sufficient amount of target molecules 541 enters the third buffer solution 533 in the third solution chamber 503. The electric field is removed, and the secondary electrophoresis ends.

[0127] Thus, the target molecule 541 initially contained in the first buffer solution 531 is contained in the third buffer solution 533 via the second buffer solution 532. Buffer exchange is performed twice. In some embodiments, the concentration of the target molecule 541 can be further increased.

[0128] In some embodiments, separation or purification can be performed by primary electrophoresis and secondary electrophoresis. In some embodiments, separation or purification can be performed primarily by primary electrophoresis, without secondary electrophoresis. In some embodiments, concentration can be performed by primary electrophoresis and secondary electrophoresis. In some embodiments, concentration can be performed primarily by primary electrophoresis, without secondary electrophoresis. In some embodiments, buffer exchange can be performed by primary electrophoresis and secondary electrophoresis. In some embodiments, buffer exchange can be performed primarily by primary electrophoresis, without secondary electrophoresis.

[0129] In some embodiments, the target molecules contained in the input solution can be subjected to one or more of the following treatments: separation, purification, concentration, and buffer exchange, by primary electrophoresis, and the treated target molecules can be moved to another location by secondary electrophoresis. For example, secondary electrophoresis can be used to move the treated target molecules to a system, sensor, flow path, or space, etc., where the target molecules are measured. The third solution chamber in this embodiment can be a solution chamber of the system, sensor, flow path, or space, etc., where the target molecules are measured, and can be a solution chamber fluidically connected to such a system.

[0130] <Various Implementation Methods>

[0131] In addition to the above-described embodiment, various embodiments can be adopted.

[0132] In some embodiments, inclusions can be allowed to pass through the separator without preventing the target substance from passing through the separator.

[0133] In some embodiments, the target substance or inclusion may be bound to another substance (also referred to as a "binding substance"). In some embodiments, the binding substance may be designed or selected so that the complex of the target substance or inclusion and the binding substance cannot pass through a size-selective separator. In some embodiments, the binding substance may be designed or selected so that the isoelectric point of the complex of the binding substance and the target substance is different from that of the target substance. For example, the design may be such that only the target substance does not migrate, but the complex with the binding substance does, or vice versa.

[0134] In some embodiments, there may be an input chamber and two elution chambers adjacent thereto. A first elution chamber, a first partition, an input chamber, a second partition, and a second elution chamber may be sequentially arranged between the electrode pairs along the direction of their electric field. For example, the first target substance may be moved from the input chamber to the first elution chamber through the first partition, and the second target substance may be moved from the input chamber to the second elution chamber through the second partition. The pH of the input liquid may be set between the isoelectric point of the first target substance and the isoelectric point of the second target substance. The two target substances may be separated, purified, and concentrated by a single electric field application operation, and their buffer solutions may be exchanged.

[0135] In some embodiments, the direction of the electric field can be switched. For example, sometimes by applying an electric field in one direction, while the target substance moves, a part of the inclusions (such as low molecules) moves to the elution chamber. For example, if the inclusion is a low molecule, it is effective to temporarily apply a reverse electric field (reverse electric field). Applying a reverse electric field causes the target substance and low molecules moved to the elution chamber to move again in the direction of the input chamber. Here, the low molecules with a molecular weight smaller than the target substance move faster than the target substance and can return to the input chamber. However, the target substance does not move like the low molecules and cannot return to the input chamber. That is, by applying a reverse electric field that is suitable relative to the application of a positive electric field, the reverse movement of the inclusions relative to the target substance can be promoted, and the concentration and / or purity of the target substance in the elution chamber can be increased.

[0136] <Example>

[0137] Several examples are described below. The common preparation steps, measurement steps, and conditions in the examples are as follows. Any differences from these are described in the individual examples.

[0138] Sample Preparation

[0139] Human serum albumin (HAS, hereinafter sometimes referred to as "albumin") (Cosmobio), 10x Tris-glycine buffer (Wako), and purified water (DW) were mixed to prepare an HSA dilution. For a 20-fold dilution, the volume ratio of HSA, Tris-glycine buffer, and DW was 1:2:17. For a 5-fold dilution, the volume ratio of HSA, Tris-glycine buffer, and DW was 2:1:7.

[0140] <Preparation of electrophoresis tubes>

[0141] During electrophoresis, a plastic tube with an inner diameter of about 6 to 8 mm and a length of several centimeters is used. When two solution chambers are set up, two interlocking tubes (input chamber tube and elution chamber tube) are used. These are tightly connected, and a separator or a membrane supporting the separator is tightly sandwiched between them. When a semipermeable membrane is used as a separator, a semipermeable membrane is tightly sandwiched between the two tubes. When a gel is used as a separator, a 0.45 μm tetrafluoroethylene resin coated membrane (PTEF membrane) or a 0.2 μm cellulose acetate membrane is tightly sandwiched between the two tubes as a support for the gel, and liquid gel is dripped onto the surface of the input chamber and allowed to stand until solidified. Alternatively, if no support is used, one end of the elution chamber tube is sealed with a saranlapp (registered trademark, Asahi Kasei Corporation) and the gel is introduced from the opening at the other end. After the gel is solidified, the saranlapp is removed and the elution chamber tube is connected to the input chamber tube.

[0142] A solution containing the target substance is added to the input chamber, and a buffer solution (Tris-glycine, pH approximately 8.3-9.0) is added to the elution chamber. After the solution is added, both ends of the tube are sealed with a dialysis membrane with a molecular weight cutoff of 12kDa to 14kDa to prevent the introduction of air bubbles. The volume of elution chamber V1 can be adjusted by the length of the elution chamber, ranging from approximately 35μL to 260μL. The buffer in the elution chamber is Tris-glycine buffer (Wako, #201-18601, pH 8.3-9.0).

[0143] <Processing Electrophoresis>

[0144] Immerse the electrophoresis tube filled with the solution in an electrophoresis tank filled with 1x Tris-glycine buffer. Place it in a chromatography chamber maintained at 4°C and keep it well cooled. In the electrophoresis tank, place the positive electrode at the end of the elution chamber of the electrophoresis tube, and the negative electrode at the end of the input chamber. Apply 150V to these electrodes for 120 minutes.

[0145] <Albumin absorbance measurement>

[0146] Absorbance was measured using DM-JACK (registered trademark, Minalis Medical Co., Ltd.). A calibration curve was prepared using standard substances as needed before measurement. The pre-run sample and eluted sample were diluted with 1× Tris-glycine buffer so that their absorbance roughly reached the range of the calibration curve.

[0147] <Electrophoresis measurement>

[0148] Electrophoresis for measurement was performed using SDS-PAGE. After electrophoresis, the eluate was removed from the elution chamber. In the case of saliva samples, 10 μL of 4× loading buffer (Wako, #196-16142) was mixed with 30 μL of the eluate. In the case of serum samples, 5 μL of 4× loading buffer (Wako, #196-16142) and 10 μL of DW were added to 5 μL of the eluate diluted 25-50 times. The sample was heated at 95°C for 10 minutes. Then, 8 μL was added dropwise to a 10% polyacrylamide gel (DRC, #NTH-525P). Electrophoresis was performed at 180V for 60 minutes, followed by staining, destaining, and photography.

[0149] <Example 1: Concentration>

[0150] Ideally, if all molecules of the target substance initially injected into the injection chamber are moved to the elution chamber, the ratio (V1 / V0) of the volume of the injection chamber (V0) to the volume of the elution chamber (V1) is the concentration rate (C1 / C0). However, in reality, not all target substances are moved to the elution chamber. For example, there are cases where a portion of the target substance adheres to the partition or the inner wall of the injection chamber. For example, there are cases where a portion of the target substance fails to pass through the partition within a predetermined time. For example, there are cases where a portion of the target substance leaks from the electrophoresis tube to the outside. However, it is generally believed that the concentration rate is a function of the volume ratio of the injection chamber to the elution chamber. In order to confirm this point, in this embodiment, the volume ratio of the injection chamber to the elution chamber is changed (V0 / V1=6~56) and the concentration rate of albumin (HSA) as the target substance is determined by absorbance measurement.

[0151] HSA was prepared at two concentrations: a 5x dilution and a 20x dilution. In this example, agarose gel was used as a separator. The concentration of each HSA was determined by absorbance using DM-JACK. The concentration rate was calculated as the ratio of the concentration before injection (before electrophoresis) (C0) to the concentration after electrophoresis (obtained from the elution chamber) (C1).

[0152] like Figure 6 As shown, a roughly linear relationship between the concentration rate and the volume ratio was observed for both the 5x and 20x dilutions. While not all albumin in the input solution migrates to the elution chamber, this linear relationship demonstrates that the concentration rate can be easily determined or controlled based on the electrophoresis device structure.

[0153] <Example 2: Purification using agarose gel>

[0154] In this example, albumin as a target substance was purified from saliva and serum using agarose gel as a separator.

[0155] Saliva samples were prepared as follows: 3.6 mL of saliva sample was obtained from the inventors and filtered through cotton wool. This was mixed with 0.4 mL of 1× Tris-glycine buffer (Wako, #201-18601, pH 8.3-8.9). Serum samples were prepared in the same manner as in Example 1.

[0156] The separator was prepared by dissolving agarose gel at a volume ratio of 1% in 1× Tris-glycine buffer (both from Wako) and supporting it using an Ultrafree MC-HV PVDF 0.45 μm (Merck) filter unit.

[0157] The electrophoresis was performed at an applied voltage of 100 V for 240 minutes.

[0158] Figure 7 Shown are photographs of electrophoresis gels. 1 and 2 correspond to the original saliva sample and the eluate after electrophoresis, respectively. 3 and 4 correspond to the original serum sample and the eluate after electrophoresis, respectively. Albumin is indicated by a black arrow, and major inclusions are indicated by white arrows.

[0159] In the case of the original saliva sample (1), bands for albumin (black arrow) and inclusions (white arrow, corresponding to amylase) were observed. On the other hand, in the case of the saliva sample (2) after electrophoresis, the bands for inclusions became extremely light, while the bands for albumin became darker. Based on the depth of the bands, the concentration rate of this electrophoresis treatment was approximately 35 times. In other words, inclusions were removed and the target substance, albumin, was concentrated.

[0160] In the case of the original serum sample (3), bands for albumin (black arrow) and inclusions (white arrow, corresponding to IgG light chains) were observed. On the other hand, in the case of the serum sample (4) after electrophoresis, the inclusion band became lighter, while the albumin band became darker. Based on the depth of the bands, the concentration rate of this electrophoresis treatment was approximately 2.1 times.

[0161] In the case of both saliva samples and serum samples, albumin, which is the target substance, is concentrated and impurities are removed.

[0162] <Example 3: Purification using polyacrylamide gel>

[0163] In this example, albumin as a target substance was purified from serum using polyacrylamide gel as a separator.

[0164] A serum sample was prepared by mixing 0.6 mL of serum (Cosmo Bio, #12181450), 0.6 mL of 1× Tris-glycine buffer (pH 8.5), and 4.8 mL of ultrapure water (DW).

[0165] The separator was prepared as follows. First, a 9% polyacrylamide gel was prepared by mixing 300 μL of ultrapure water, 50 μL of 1× Tris-glycine pH 8.5, 150 μL of a 30 w / v% acrylamide / Bis mixture, 2.5 μL of 10% APS, and 0.25 μL of TMED. This gel was added to an elution chamber tube sealed at one end with a saran latch. After solidification, the saran latch was removed and the elution chamber tube was connected to the input chamber tube.

[0166] The electrophoresis was performed at an applied voltage of 150 V for 150 minutes.

[0167] The electrophoresis measurement is performed as follows. First, after the electrophoresis is processed, the eluate is removed from the elution chamber. 1 μL of the eluate diluted 25 times with 1× Tris-glycine buffer (pH 8.5), 5 μL of 4× loading buffer (Wako, #196-16142) and 14 μL of ultrapure water are mixed. 5 μL of the original serum sample diluted 25 times, 5 μL of 4× loading buffer (Wako, #196-16142) and 10 μL of ultrapure water are mixed. The mixture is heated at 95°C for 10 minutes. 8 μL of it is added dropwise to a 10% polyacrylamide gel (DRC, #NTH-525P). Electrophoresis is performed at 180V for 60 minutes.

[0168] Figure 8 The electrophoresis gel is shown. 1 and 2 correspond to the original serum sample and the eluate after electrophoresis, respectively.

[0169] Compared to the original serum sample (1), the albumin band (black arrow) in the serum sample (2) after electrophoresis became darker, while the band of the inclusions (white arrow) became extremely lighter. Based on the depth of the bands, the concentration rate of this electrophoresis treatment was approximately 6.5 times. In other words, inclusions were removed and the target substance, albumin, was concentrated.

[0170] <Example 4: Purification using dialysis membrane>

[0171] In this example, albumin as a target substance was purified from serum using a dialysis membrane (semipermeable membrane) as a separator.

[0172] Serum samples were prepared in the same manner as in Example 3. A 100 kDa dialysis membrane (Biotech CE, TRIAL kit, 100 KD, 24 mm, 1 m (131417T)) was used as a separator.

[0173] The electrophoresis was performed at an applied voltage of 150 V for 120 minutes.

[0174] The electrophoresis was measured as follows. First, the eluate was taken out from the elution chamber after the electrophoresis and diluted 25 times with 1× Tris-glycine buffer (pH 8.6). 5 μL of the 10× diluted eluate, 5 μL of 4× loading buffer (Wako, #196-16142) and 10 μL of DW were mixed. The mixture was heated at 95°C for 10 minutes. 1 μL, 4 μL or 8 μL of the mixture was added dropwise to a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180V for 60 minutes.

[0175] Figure 9 The electrophoresis gel is shown. 1 and 2 correspond to the original serum sample and the eluate after electrophoresis, respectively.

[0176] Compared with the original serum sample (1), the band of albumin (black arrow) in the serum sample (2) after electrophoresis becomes darker, and the band of inclusions (white arrow) almost disappears. The molecular weight of HSA is about 66kDa. It can be confirmed that: by electrophoresis, HSA less than 100kDa passes through the separator, and molecules larger than it do not pass through the separator. It should be noted that large molecules of 100kDa are included in serum, but not in saliva. Amylase is included in saliva, but not in serum.

[0177] <Example 5: Purification based on isoelectric point>

[0178] In this example, the pH of the input solution was set between the isoelectric point of the target substance (albumin) (pI = 4.9) and the isoelectric point of the impurity (hemoglobin) (pI = approximately 7), and albumin was separated using agarose gel as a separator.

[0179] The sample was prepared by mixing 20 μL of serum (Cosmo Bio, #12181450), 800 μL of HbA1c (Hitachi Chemical Diagnostics System Co., Ltd., Metallocontroluol HbA1c, #058619), 1600 μL of 10 mM sodium phosphate buffer (pH = 6.5) or 1× Tris-glycine buffer (pH 8.5), and 5600 μL of DW.

[0180] Separators were prepared by introducing 1% agarose gel into 10 mM sodium phosphate buffer, 1× Tris-glycine buffer (pH 6.5) or 1× Tris-glycine buffer (pH 8.5). 250 μL of this gel was supported on a Gel Ultrafree MC-HVPVDF Φ 0.45 μm filter unit (Merck).

[0181] Treatment electrophoresis was performed at 130 mA for 140 minutes.

[0182] The electrophoresis measurement is performed as follows. First, the eluate is taken out from the elution chamber after the electrophoresis is processed. Mix 0.8 μL of the eluate, 5 μL of 4× loading buffer (Wako, #196-16142) and 14.2 μL of DW. Mix 4 μL of the original mixed sample solution, 5 μL of 4× loading buffer (Wako, #196-16142) and 11 μL of DW. Heat the mixture at 95°C for 10 minutes. Add 5 μL of it dropwise to a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Perform electrophoresis at 180V for 60 minutes.

[0183] Figure 10 The electrophoresis gel photographs are shown. 1 and 2 correspond to the original sample (1) when the input liquid pH is 8.5 and the eluate (2) after the treatment electrophoresis. Both albumin (pI = 4.9) and hemoglobin (pI = about 7) move to the elution chamber due to the treatment electrophoresis. At pH 8.5, both are negatively charged, so they move toward the positive electrode and enter the elution chamber. 3, 4, and 5 correspond to the original sample (3) when the input liquid pH is 6.5, the eluate (4) after the treatment electrophoresis, and the eluate (5) after the treatment electrophoresis in the opposite direction. It can be seen from the eluate (4) after the treatment electrophoresis that hemoglobin does not move to the elution chamber due to the treatment electrophoresis. It can be seen from the application of reverse voltage (5) that the hemoglobin moves in the opposite direction (towards the negative electrode). That is, it was confirmed that albumin moves toward the positive electrode and hemoglobin moves toward the negative electrode. This indicates that the target substance and the impurities can be separated by setting the pH of the input liquid between the isoelectric point of the target substance and the isoelectric point of the impurities.

[0184] <Example 6: Adding resin>

[0185] In this example, a resin was added to the separator to purify albumin as the target substance from saliva.

[0186] Saliva samples were prepared as follows: 12.6 mL of saliva was obtained from the inventors, filtered through cotton wool, and mixed with 1.4 mL of 1× Tris-glycine buffer (pH 7.5).

[0187] Each resin was added thereto. The addition was performed as follows. First, the additive was equilibrated with 1× Tris-glycine buffer, and the supernatant was removed as much as possible. The dissolved 2% agarose gel was added to each resin kept warm at 65°C and mixed. The resin and agarose gel were fully suspended, and about 250 μL of them were introduced into the separator part, and fully cooled to solidify. As a support for the agarose mixed gel, a cellulose acetate filter membrane Φ0.2 μm (Adbandek Toyo) was used.

[0188] The additives used in this example are as follows:

[0189] Cation exchange carrier "SP": TOYOPEARL SP-650M, TOSOH, #0007997

[0190] Anion exchange carrier "SQ": TOYOPEARL SuperQ-650M, TOSOH, #0017227

[0191] Hydroxyapatite "HA": CHT Ceramic Hydroxyapatite, Bio-Rad, #1582200

[0192] Treatment electrophoresis was performed at 130 mA for 120 minutes.

[0193] The electrophoresis was measured as follows. First, the eluate was removed from the elution chamber after the electrophoresis. 10 μL of the eluate, 5 μL of 4× loading buffer (Wako, #196-16142) and 5 μL of DW were mixed. The mixture was heated at 95°C for 10 minutes. 3 μL of the mixture was added dropwise to a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180V for 60 minutes.

[0194] Figure 11 Shown are electrophoresis gel images with SP and HA additives. 1, 2, 3, and 4 correspond to the original saliva sample (1), the eluate without resin additives (2), the eluate with SP added (3), and the eluate with HA added (4), respectively.

[0195] In the case of the original saliva sample (1), bands of albumin (black arrow) and inclusions (white arrow, equivalent to amylase) were observed. The bands of the eluate (2) without resin additives became darker than those of the original saliva sample (1) due to concentration. Amylase was not effectively removed without additives. On the other hand, in the case of the eluate (3) with the addition of SP, the bands of albumin were almost the same, and the bands of amylase became lighter. It can be seen that SP effectively blocked the entry of amylase into the elution chamber. Furthermore, in the case of the eluate (4) with the addition of HA, the bands of albumin were almost the same, and the bands of amylase became significantly lighter. It can be seen that SP further effectively blocked the entry of amylase into the elution chamber.

[0196] Figure 12 Shown are photographs of electrophoresis gels with the addition of SQ. 1, 2, and 3 correspond to the original saliva sample (1), the eluate without resin additives (2), and the eluate with SQ added (3), respectively. In the eluate with SQ added (4), the albumin band is almost the same as or slightly lighter than that in the eluate without resin additives (2), and the amylase band has almost disappeared. This indicates that SQ effectively blocks amylase from entering the elution chamber.

[0197] Depend on Figure 11 and 12 The ratio of HAS to the two amylases in each solution was calculated by looking at the band depths of the electrophoresis gel photograph shown in the figure. Figure 13 "Amylase_up" and "Amylase_down" correspond to the two bands appearing below the albumin peak, respectively. "Amylase_up" refers to the band close to albumin, and "Amylase_down" refers to the band farther from albumin. The solutions shown here are original saliva, the eluate after electrophoresis without resin additives, and the eluate after electrophoresis with additives SP, HA, or SQ.

[0198] Figure 13 Show summary Figure 11 and Figure 12 The graph is a result of the analysis. Original saliva contains a lot of amylase relative to HSA. Here, HSA and two amylases are compared. Amylase_down is particularly high (A). In the absence of resin, albumin is concentrated, amylase remains unchanged, and is relatively less than albumin (B). It shows the concentration rate and the removal rate of inclusions when there is no resin additive. It can be seen that by adding additives, inclusions are further removed (C to E). In particular, it can be seen that by using SQ as an additive, most of the amylase can be removed (E).

[0199] <Example 7: Improvement of Concentration Rate>

[0200] In this example, agarose gel was used as a separator, and a counter substance was added to the eluent in advance to concentrate albumin as the target substance from a serum sample.

[0201] A 20-fold dilution of HSA was prepared as a serum sample. 100 μL of 1% agarose was used as a separator. A 0.2 μm cellulose acetate filter (Adbandec Toyo) was used as a support for the agarose hybrid gel.

[0202] The counteracting substances used in this example are as follows:

[0203] RNase A (MACHEREY-NAGEL). Protein with a molecular weight of less than 13.7 kDa and an isoelectric point of 8.6.

[0204] Lysozyme (derived from egg white) (Nakarai Tesque Co., Ltd.) 14 kDa enzyme, isoelectric point 11.

[0205] All of them have an opposite charge to albumin and migrate in the opposite direction to albumin during electrophoresis. These were added to a Tris-glycine buffer as an eluent at various concentrations.

[0206] Electrophoresis was performed at 150 V for 240 minutes.

[0207] The eluate is then removed from the elution chamber and subjected to electrophoresis. The concentration of each albumin is determined based on the depth of the albumin bands in the electrophoresis gel photograph.

[0208] Figure 14 The relationship between the concentration of the antagonist in the initial eluent and the concentration of albumin in the eluent after the swimming is shown when the antagonist is lysozyme (A) and RNaseA (B). In either case, the concentration of albumin in the eluent increases substantially in proportion to the concentration of the antagonist. Due to the movement of albumin, the concentration of albumin in the eluent increases, and the concentration of albumin in the input liquid decreases, resulting in a decrease in osmotic pressure. Thus, there is a limit to the albumin concentration in the eluent. However, it is clear that the antagonist moves in the opposite direction to the albumin, thereby alleviating the problem of decreased osmotic pressure and achieving a higher final albumin concentration.

[0209] <Example 8: Separation and Concentration of Saliva Albumin>

[0210] In this example, albumin as a target substance was purified and concentrated from saliva using polyacrylamide gel as a separator.

[0211] The electrophoresis buffer used was a Tris-glycine buffer (pH 6.0, hereinafter referred to as 1× TMG buffer) whose pH was adjusted with maleic acid. This 1× TMG buffer was introduced into the elution chamber.

[0212] Saliva samples were prepared as follows: Saliva was obtained from the inventors and filtered through absorbent cotton. After filtration, 6 mL of the filtered saliva was mixed with 600 μL of 10-fold diluted TMG buffer (10×TMG buffer), and 2.5 mL was introduced into each injection chamber.

[0213] The separator was prepared as follows. First, 10% polyacrylamide gel was prepared by mixing 114 μL of ultrapure water, 20 μL of 1× TMG buffer (pH 6.0), 66 μL of a 30 w / v% acrylamide / Bis mixture, 2 μL of 10% APS, and 0.8 μL of TMED. 160 μL of this gel was supported on a CELLLOSE ACETATE Φ0.2 μm filter unit (ADVANTEC).

[0214] Treatment Electrophoresis was performed at room temperature at 25 V (current maximum of about 24 mA) for 180 minutes.

[0215] Electrophoresis was performed as follows. First, after electrophoresis, the eluate was removed from the elution chamber and 15 μL of the eluate was mixed with 5 μL of 4× loading buffer (Wako, #196-16142). The mixture was heated at 95°C for 10 minutes. 6 μL of the mixture was then added dropwise to a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0216] Figure 15 The following is a photograph of an electrophoresis gel. 1, 2, and 3 correspond to the original saliva sample (1), the input solution (2), and the eluate (3) after electrophoresis, respectively. Although some amylase (1) in the saliva can be detected in the eluate (3), almost all of it remains in the input chamber (2). In contrast, most of the albumin can move into the eluate (3). This confirms that at least the albumin in the saliva is separated from the amylase.

[0217] The depth of the albumin band in the eluate after electrophoresis was approximately 12.7 times greater than that in the saliva sample before electrophoresis. This indicates that albumin can be concentrated 12.7-fold using this electrophoresis method. Given an input chamber volume of 2.5 mL and an elution chamber volume of 150 μL, and ignoring osmotic pressure, this ratio is 17-fold. Therefore, this result demonstrates that this method can concentrate albumin to high concentrations.

[0218] <Example 9: Separation of Albumin and Macromolecules in Saliva>

[0219] In this example, polyacrylamide gel was used as a separator to purify albumin as a target substance from saliva and separate it from macromolecules in saliva.

[0220] The running buffer and saliva sample used were the same as those in Example 8. The pH of the eluent and the separator gel was adjusted to 8.5.

[0221] The separator was prepared by adding 50 μL of 1.5% agarose gel and solidifying it to form a support, into which 110 μL of a mixture of 10% polyacrylamide gel and 1× TMG buffer (pH 8.5) was introduced and allowed to stand at 30° C. for 20 minutes.

[0222] Treatment electrophoresis was performed at room temperature for 30 minutes at 100 V (maximum current: about 24 mA). 1× TMG buffer (pH 7.5) was used as the electrophoresis buffer.

[0223] Electrophoresis was performed as follows. First, after electrophoresis, the eluate was removed from the elution chamber and 15 μL of the eluate was mixed with 5 μL of 4× loading buffer (Wako, #196-16142). The mixture was heated at 95°C for 10 minutes. 3 μL of the mixture was then added dropwise to a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0224] Figure 16 The following is a photograph of an electrophoresis gel. 1, 2, and 3 correspond to the original saliva sample (1), the input solution (2), and the eluate (3) after electrophoresis treatment, respectively. Although amylase and lactoferrin (1) in saliva can be detected to some extent in the eluate (3), they essentially remain in the input chamber (2). In contrast, most albumin can migrate to the eluate (3). This confirms that at least the albumin in saliva is separated from amylase and lactoferrin.

[0225] There are giant molecules or giant complexes (1) in the saliva sample. It is speculated that the giant molecules contain mucin. Thus, it is possible to prevent the giant molecules from moving to the elution chamber. For example, by adjusting the pH of the input chamber, it is possible to prevent the giant molecules from moving to the elution chamber. For example, by using a partition having a small mesh structure such as polyacrylamide, it is possible to prevent the giant molecules from moving to the elution chamber. These can also be combined.

[0226] Lactoferrin has an isoelectric point of 8.8, and at the pH of 8.5 in the solution of this example, it carries a slight positive charge or is uncharged. Therefore, it is believed that lactoferrin either migrates in the opposite direction to albumin or does not migrate, at least not into the elution chamber.

[0227] In this embodiment, there is no intention to remove low molecules, but this is possible. For example, the separator may contain a resin that can capture low molecules or reduce the migration speed of low molecules (such as Sephak (registered trademark) resins used for gel filtration chromatography or size exclusion chromatography). For example, a low-molecular capture resin may be mixed in the gel of the separator. For example, a gel layer of polyacrylamide gel can be stacked with a layer containing a low-molecular capture resin. With such a structure, the separator not only captures larger molecules such as mucin and lactoferrin, but also captures low-molecular inclusions and allows the target substance to pass through. In this way, the target object substance (such as albumin) can be purified.

[0228] <Example 10: Separation of Albumin and Hemoglobin in Whole Blood>

[0229] In this example, polyacrylamide gel was used as a separator to separate both albumin and hemoglobin as target substances from a whole blood sample.

[0230] The electrophoresis buffer used was a Tris-glycine buffer (pH 5.2, hereinafter referred to as 1× TMG buffer) whose pH was adjusted with maleic acid. This 1× TMG buffer was introduced into the elution chamber.

[0231] Whole blood samples were prepared as follows. 30 μL of fingerstick blood (whole blood) was obtained from the inventors using a blood collection tube. This was mixed with 270 μL of 1× TMG buffer (containing 0.1% Triton X-100) and allowed to stand at room temperature for 1 hour. This allowed the whole blood sample to hemolyze. 300 μL of this hemolyzed sample was diluted with 11,700 μL of 1× TMG buffer.

[0232] The separator was prepared as follows. First, a 10% polyacrylamide gel was prepared by mixing 114 μL of ultrapure water, 20 μL of 1× TMG buffer (pH 5.2), 66 μL of a 30 w / v% acrylamide / Bis mixture, 2 μL of 10% APS, and 0.8 μL of TMED. 160 μL of this gel was supported on a CELLLOSE ACETATE Φ0.2 μm filter unit (ADVANTEC).

[0233] Processing Electrophoresis was performed at 90 V for 20 minutes, followed by 80 V for 60 minutes, and then at 90 V for 20 minutes under cooling.

[0234] Electrophoresis was performed as follows. First, after electrophoresis, the eluate was removed from the elution chamber and 15 μL of the eluate was mixed with 5 μL of 4× loading buffer (Wako, #196-16142). The mixture was heated at 95°C for 10 minutes. 3 μL of the mixture was then added dropwise to a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0235] Albumin and hemoglobin migrate in opposite directions at this pH. Originally, two elution chambers should be arranged relative to the input chamber along the direction of voltage application. However, in the configuration of this embodiment, a single elution chamber is provided relative to the input chamber. Therefore, the voltage application method for each of albumin and hemoglobin has been changed. Hereinafter, the voltage application direction for eluting albumin in the configuration of this embodiment will be referred to as the "forward direction," and the voltage application direction for eluting hemoglobin will be referred to as the "reverse direction."

[0236] Figure 17 The electrophoresis gel photographs are shown. 1 to 5 correspond to the original whole blood sample (1), the input solution (2) and the eluate (3) after the forward electrophoresis, and the input solution (4) and the eluate (5) after the reverse electrophoresis.

[0237] As can be seen from bands (3) and (5) of the eluate, albumin and hemoglobin were separated in the eluate. Furthermore, at least albumin was concentrated simultaneously. This result demonstrates that the present method is capable of simultaneously separating and purifying albumin and hemoglobin.

[0238] <Example 11: Electrophoresis Unit>

[0239] Figure 18 A cross-sectional view of an electrophoresis unit 600 along the longitudinal direction of one embodiment is shown. The electrophoresis unit 600 includes an electrophoresis tank 651, a top plate 652 covering the top plate 652, and a partition 610 disposed within the electrophoresis tank 651. An input chamber 601 for storing a sample solution and an elution chamber 601 for storing an eluent are defined by the electrophoresis tank 651, the top plate 652, and the partition 610, with the partition 610 interposed therebetween. The volumes of the input chamber 601 and the eluent, or their ratio, can be set based on the desired concentration ratio.

[0240] The cross-section of the electrophoretic tank 651 perpendicular to its length can be circular or U-shaped. In this way, the electric field can be applied uniformly, suppressing the loss during the movement of the substance. In addition, when contacting the radiator or fluid (air) for temperature control from the outside, a larger contact area can be adopted. The electrophoretic tank 651 can be constructed in a manner such that it can contact the radiator from the outside, irradiate electromagnetic waves such as light, receive air supply, etc. In this way, the temperature of the solution inside it can be adjusted. The electrophoretic tank 651 can be formed of a high thermal conductivity material.

[0241] The electrophoresis tank 651 has electrodes 621 and 622 at both end faces in the longitudinal direction. The electrodes 621 and 622 can be fixed to the electrophoresis tank 651. For example, the electrodes 621 and 622 can be formed using metal plates. The metal plates 621 and 622 can be bonded to both end faces of the electrophoresis tank 651. For example, the electrodes 621 and 622 can be formed by plating a material such as platinum. The electrodes 621 and 622 can be constructed as external components so as to contact or be close to both end faces of the electrophoresis tank 651 during electrophoresis processing.

[0242] <Example 12: Cartridge>

[0243] Figure 19 An example of a pipette cartridge 700 including the electrophoresis unit 600 of the above-described embodiment is included. Figure 19 (A) shows a top view of the pipette cartridge 700, Figure 19 (B) shows the AA cross-sectional view shown in (A). The pipette cartridge 700 is provided with an electrophoresis unit fixing portion 710, which is used to embed and fix the electrophoresis unit 600 on the plate (main body) 701. The electrophoresis unit fixing portion 710 has an opening in the plate 701, and the electrophoresis unit 600 is inserted into the opening. The electrophoresis unit 600 is fixed by fixing pins 711 and 712 fixed to the plate 701 at the four corners of the opening.

[0244] The electrophoresis unit fixing portion 710 is provided with leaf springs 713 and 714 for electrode contact at both ends thereof in the longitudinal direction. The electrodes 621 and 622 at both ends of the electrophoresis unit 600, which are inserted into the opening of the electrophoresis unit fixing portion 710 and fixed thereto, respectively contact the leaf springs 713 and 714 for electrode contact, thereby electrically connecting them. The leaf springs 713 and 714 are connected to the connectors 731 and 732 fixed to the front end of the length direction of the plate 701 via wires 733 and 734, respectively. With respect to the connectors 731 and 732, the pipette cartridge 700 is inserted from the front end into the main body of the automatic pipetting device (not shown) and abuts against a stopper that stops the front end of the pipette cartridge 700. The end face thereof is provided with a contact piece (not shown) with the connectors 731 and 732. By establishing an electrical connection between the connectors 731 and 732 and their contact pieces, power can be supplied to the electrophoresis unit 600.

[0245] Figure 19 The electrophoresis unit 600 is electrically connected to the outside world via leaf springs 713, 714 and connectors 731, 732, but the electrical connection method is not limited thereto. For example, a power supply unit having a leaf spring may be disposed within the main body of the automatic pipetting device, which moves relative to the inserted cartridge 700 and contacts the electrodes 621, 622 at both ends of the electrophoresis unit 600.

[0246] In some embodiments, the pipette cartridge may be provided with holes for measurement, holes for containing solutions required for measurement, holes for holding pipette tips, tubes, etc. In some embodiments, the pipette cartridge may be equipped with a sensor.

[0247] Figure 19 The illustrated pipette cartridge 700 includes a sample container carrier 721 for holding a container of a sample to be subjected to electrophoresis processing, a sample tip carrier 722 for holding a sample tip for removing the sample from the sample container and inserting it into the input chamber of the electrophoresis unit 600, and an eluent tip carrier 723 for disposing an eluent tip for removing the solution from the elution chamber after the electrophoresis processing. Furthermore, the pipette cartridge 700 may include a buffer well 731 for storing the buffer used in the electrophoresis processing. The buffer well 731 is sealed with a sealing film 732 before use, thereby sealing the buffer solution (not shown) inside. The sealing film may be, for example, an aluminum film, allowing the pipette tip to easily penetrate the aluminum film and access the buffer solution inside. The pipette cartridge 700 also includes an eluent tube carrier 714 for holding a tube for recovering the eluent after the processing. The processed solution after the electrophoresis processing can be transferred to the interior of the eluent tube and then moved from the pipette cartridge 700 to another location along with the tube.

[0248] For example, the pipette cartridge 700 and / or the automatic pipetting device can be configured in a manner that controls the temperature of the electrophoresis unit 600. For example, a temperature-controlled radiator can be configured in the automatic pipetting device, which moves relative to the inserted cartridge 700 and is in close contact with the electrophoresis tank 651 of the electrophoresis unit 600. Alternatively, for example, the automatic pipetting device can place a temperature-controlled fluid (e.g., cooling air) on the surface of the electrophoresis tank 651. In these ways, the temperature of the solution in the electrophoresis process can be efficiently controlled at an appropriate temperature. In some embodiments, the temperature of the electrophoresis tank 651 and the temperature of the solution in the electrophoresis process can be measured and automatically controlled.

[0249] In some embodiments, a (non-mounted) pipette cartridge for mounting an electrophoresis unit may be provided. In some embodiments, a pipette cartridge with (containing) an electrophoresis unit mounted thereon may be provided.

[0250] The present invention also provides the following embodiments:

[0251] A001

[0252] A method for treating a target substance by electrophoresis, comprising:

[0253] Provide a partition that allows the target substance to pass through;

[0254] introducing a first solution containing the target substance into a first side of the separator;

[0255] introducing a second solution into the second side of the separator; and

[0256] The target substance is selectively moved from the first solution to the second solution through the separator by electrophoresis.

[0257] A001b

[0258] A method for treating a target substance by electrophoresis, comprising:

[0259] providing a separator that allows one of the target substance and the inclusion to pass through;

[0260] introducing a first solution containing the target substance and the impurities into a first side of the separator;

[0261] introducing a second solution into the second side of the separator; and

[0262] One of the target substance and the impurities is selectively moved from the first solution to the second solution through the separator by electrophoresis.

[0263] A002

[0264] The method according to A001, A001b or any embodiment, wherein

[0265] The method for treating the target substance is a method of performing at least one of the following operations:

[0266] Concentrating the above-mentioned target substance;

[0267] exchanging the buffer solution of the target substance; and

[0268] The above-mentioned target substance is purified.

[0269] A003

[0270] The method according to A001 or any embodiment, wherein

[0271] The above method for treating the target substance is a method of simultaneously performing two or more of the following operations:

[0272] Concentrating the above-mentioned target substance;

[0273] exchanging the buffer solution of the target substance; and

[0274] The above-mentioned target substance is purified.

[0275] A004

[0276] The method according to A001 or any embodiment, wherein

[0277] The above method for treating the target substance is a method of simultaneously performing the following operations:

[0278] Concentrating the above-mentioned target substance; and

[0279] The above-mentioned target substance is purified.

[0280] A005

[0281] The method according to A003 or A004 or any embodiment, wherein

[0282] The aforementioned simultaneous performance includes performance by one-dimensional electrophoresis.

[0283] A007

[0284] The method according to any one of A001 to A003 or any one of the embodiments, wherein

[0285] The separator includes a material selected from the group consisting of a gel, a semipermeable membrane, and a resin.

[0286] A008

[0287] The method according to any one of A001 to A007 or any one of the embodiments, wherein

[0288] The separator comprises agarose gel.

[0289] A011

[0290] The method according to A001 or any embodiment, wherein

[0291] The second volume of the second solution is smaller than the first volume of the first solution.

[0292] A012

[0293] The method according to A001 or A011 or any embodiment, wherein

[0294] The above method concentrates the above biomolecules.

[0295] A015

[0296] The method according to any one of A001 to A012 or any one of the embodiments, wherein

[0297] The method further comprises introducing into the second solution a substance (counter substance) that migrates in a direction opposite to that of the target substance by electrophoresis,

[0298] The selectively moving the target substance by electrophoresis includes simultaneously moving the counter substance from the second solution to the first solution through the separator by electrophoresis.

[0299] A016

[0300] The method according to A015 or any embodiment, wherein

[0301] The counter substance has a charge opposite to that of the target substance.

[0302] A017

[0303] The method according to A015 or A016 or any embodiment, wherein

[0304] The counter substance is a macromolecule.

[0305] A018

[0306] The method according to A017 or any embodiment, wherein

[0307] The above-mentioned counteracting substance is a biopolymer.

[0308] A019

[0309] The method according to A018 or any embodiment, wherein

[0310] The above-mentioned counteracting substance is a protein or an enzyme.

[0311] A021

[0312] The method according to A001 or any embodiment, wherein

[0313] The first solvent (first buffer) of the first solution is different from the second solvent (second buffer) of the second solution.

[0314] A031

[0315] The method according to A001 or any embodiment, wherein

[0316] The separator selectively allows the target substance to pass therethrough or permits the selective passage of the target substance.

[0317] A031b

[0318] The method according to A001b or any embodiment, wherein

[0319] The separator selectively allows the foreign matter to pass therethrough or allows the selective passage of a target substance.

[0320] A032

[0321] The method according to A031 or any embodiment, wherein

[0322] The separator has the ability to inhibit, prevent or block the passage of at least one kind of inclusion.

[0323] A041

[0324] The method according to A031 or A032 or any embodiment, wherein

[0325] The separator includes a gel that selectively allows the target substance to pass therethrough.

[0326] A042

[0327] The method according to A041 or any embodiment, wherein

[0328] The above gel is polyacrylamide gel.

[0329] A051

[0330] The method according to A031 or A032 or any embodiment, wherein

[0331] The separator includes a semipermeable membrane that selectively allows the target substance to pass therethrough.

[0332] A061

[0333] The method according to any one of A001 to A051 or any one of the embodiments, wherein

[0334] The separator, the first solution, or the first solution chamber (input chamber) includes a selective substance capable of allowing the target substance to selectively pass through the separator and / or inhibiting, preventing, or blocking at least one foreign substance from passing through the separator.

[0335] A061b

[0336] The method according to any one of A001b to A051 or any one of the embodiments, wherein

[0337] The separator, the first solution, or the first solution chamber (input chamber) includes a selective substance capable of allowing at least one impurity to selectively pass through the separator and / or inhibiting, preventing, or blocking the target substance from passing through the separator.

[0338] A062

[0339] The method according to any one of items A001 to A047 or any one of embodiments, further comprising mixing a selective substance into the first solution before the electrophoresis.

[0340] A063

[0341] The method according to A061 or A062 or any embodiment, wherein

[0342] The aforementioned selective substances have the ability to purify proteins.

[0343] A064

[0344] A method according to any one of A061 to A063 or any embodiment, wherein

[0345] The selective substance or the protein purification resin is selected from the group consisting of hydroxyapatite, a cation exchange carrier, an anion exchange carrier, and a hydrophobic resin.

[0346] A071

[0347] The method according to A001 or any embodiment, wherein

[0348] The pH of the first solution is greater than (higher) or less than (lower) the isoelectric point of the target substance.

[0349] The above-mentioned target substance has negative / positive charge,

[0350] The selective movement by electrophoresis includes applying an electric field so that the first solution side is negative / positive and the second solution side is positive / negative.

[0351] A072

[0352] The method according to A071 or any embodiment, wherein

[0353] The pH of the first solution is equal to or higher than the isoelectric point of at least one of the inclusions contained in the first solution.

[0354] A081

[0355] A method for treating a target substance by electrophoresis, comprising:

[0356] providing a first separator allowing the target substance to pass through;

[0357] introducing a first solution containing the target substance into the first side of the first separator;

[0358] introducing a second solution into the second side of the first separator and the first side of the second separator;

[0359] introducing a third solution into the second side of the second separator;

[0360] selectively moving the target substance from the first solution to the second solution through the first separator by electrophoresis; and

[0361] The target substance is moved from the second solution to the third solution through the second separator by electrophoresis.

[0362] A081b

[0363] A method for treating a target substance by electrophoresis, comprising:

[0364] providing a first separator that allows a first target substance to pass through and a second separator that allows a second target substance to pass through;

[0365] introducing a first solution containing the first target substance and the second target substance between the first separator and the second separator;

[0366] selectively moving the first target substance from the first solution through the first separator to the opposite side of the first separator by electrophoresis; and

[0367] The second target substance is moved from the first solution to the opposite side of the second separator through the second separator by electrophoresis.

[0368] A082

[0369] The method according to A081b or any embodiment, wherein

[0370] The pH of the solution is between the isoelectric point of the first target substance and the isoelectric point of the second target substance.

[0371] A091

[0372] The method according to any one of A001 to A082 or any one of the embodiments, wherein

[0373] The first solution contains the target substance and low-molecular-weight impurities having a molecular weight smaller than that of the target substance.

[0374] The electrophoresis includes repeatedly applying a forward electric field and a reverse electric field in a direction in which the target substance and the low-molecular-weight impurities move toward the second solution through the separator.

[0375] A092

[0376] The method according to A091 or any embodiment, wherein

[0377] The step of repeatedly applying the forward electric field and the reverse electric field includes promoting the low molecular weight inclusions to return to the first solution.

[0378] A101

[0379] The method according to any one of A001 to A081b or any one of the embodiments, wherein

[0380] The first solution is a solution derived from a living organism.

[0381] A102

[0382] The method according to any one of A001 to A081b or any one of the embodiments, wherein

[0383] The first solution mentioned above is a crude sample.

[0384] A103

[0385] The method according to A101 or 102 or any embodiment, wherein

[0386] The first solution is a solution of biological origin selected from the group consisting of blood, saliva, tears, urine, and interstitial fluid.

[0387] A111

[0388] The method according to any one of A001 to A103 or any one of the embodiments, wherein

[0389] The target substance is the biomolecule contained in the first solution.

[0390] A112

[0391] The method according to any one of A001 to A103 or any one of the embodiments, wherein

[0392] The target substance is albumin contained in the first solution.

[0393] B001

[0394] An apparatus (device) for treating a target substance by electrophoresis, comprising:

[0395] First solution chamber (input chamber);

[0396] a second solution chamber (elution chamber) adjacent to the first solution chamber (input chamber); and

[0397] a separator that divides the first solution chamber and the second solution chamber, allows the target substance to pass from the first solution chamber to the second solution chamber, and has conductivity;

[0398] The device (device) is constructed in the following manner:

[0399] The first solution chamber is filled with a first solution containing the target substance;

[0400] The second solution chamber is filled with a second solution;

[0401] An electric field is applied from outside the first solution chamber and the second solution chamber to selectively move the target substance from the first solution chamber to the second solution chamber through the partition by electrophoresis.

[0402] B001b

[0403] An apparatus (device) for treating a target substance by electrophoresis, comprising:

[0404] First solution chamber (input chamber);

[0405] a second solution chamber (elution chamber) adjacent to the first solution chamber (input chamber); and

[0406] a separator that divides the first solution chamber and the second solution chamber, allows any of the target substance and inclusions to pass from the first solution chamber to the second solution chamber, and has conductivity;

[0407] The device (device) is constructed in the following manner:

[0408] The first solution chamber is filled with a first solution containing the target substance and the impurities;

[0409] The second solution chamber is filled with a second solution;

[0410] An electric field is applied from outside the first solution chamber and the second solution chamber to selectively move the target substance or the inclusions from the first solution chamber to the second solution chamber through the separator by electrophoresis.

[0411] B002

[0412] The apparatus (device) according to B001, wherein:

[0413] The invention further includes an electrode pair configured to apply an electric field from outside the first solution chamber and the second solution chamber.

[0414] B011

[0415] The apparatus (device) according to B001 or B002 or any embodiment, wherein

[0416] The second volume of the second solution chamber is smaller than the first volume of the first solution chamber.

[0417] B021

[0418] The apparatus (device) according to B001 or B002 or any embodiment, wherein

[0419] The first solvent (first buffer) of the first solution and the second solvent (second buffer) of the second solution are different from each other.

[0420] B031

[0421] The apparatus (device) according to B001 or B002 or any embodiment, wherein

[0422] The separator selectively allows the target substance to pass therethrough or permits the selective passage of the target substance.

[0423] B032

[0424] The apparatus (device) according to B031 or any embodiment, wherein

[0425] The separator has the ability to inhibit, prevent or block the passage of at least one kind of inclusion.

[0426] B041

[0427] The apparatus (device) according to B031 or A032 or any embodiment, wherein

[0428] The separator includes a gel that selectively allows the target substance to pass therethrough.

[0429] B042

[0430] The apparatus according to B041 or any embodiment, wherein

[0431] The above gel is polyacrylamide gel.

[0432] B051

[0433] The apparatus (device) according to B031 or B032 or any embodiment, wherein

[0434] The separator includes a semipermeable membrane that selectively allows the target substance to pass therethrough.

[0435] B061

[0436] The apparatus (device) according to any one or any embodiment of B001 to B051, wherein

[0437] The separator, the first solution, or the first solution chamber (input chamber) includes a selective substance capable of allowing the target substance to selectively pass through the separator and / or inhibiting, preventing, or blocking at least one foreign substance from passing through the separator.

[0438] B062

[0439] The apparatus (device) according to any one of items B031 to B047 or any one of embodiments B031 to B047 is configured such that the selective substance is mixed into the first solution before the electrophoresis.

[0440] B063

[0441] The apparatus (device) according to B051 or B052 or any embodiment, wherein

[0442] The above selective substance is a resin for protein purification.

[0443] B064

[0444] The apparatus (device) according to any one or any embodiment of B051 to B053, wherein

[0445] The selective substance or the protein purification resin is selected from the group consisting of hydroxyapatite, a cation exchange carrier, and an anion exchange carrier.

[0446] B081

[0447] An apparatus (device) for treating a target substance by electrophoresis, comprising:

[0448] First solution chamber (input chamber);

[0449] a second solution chamber (first elution chamber) adjacent to the first solution chamber (input chamber);

[0450] a third solution chamber (second elution chamber) adjacent to the second solution chamber (first elution chamber);

[0451] a first separator that divides the first solution chamber and the second solution chamber and allows the target substance to pass from the first solution chamber to the second solution chamber (and has conductivity); and

[0452] The second separator partitions the second solution chamber and the third solution chamber, and allows the target substance to pass from the second solution chamber to the third solution chamber (and has conductivity).

[0453] C001

[0454] A method for treating a target substance by electrophoresis, comprising:

[0455] Providing an electrophoresis device according to any one of embodiments B001 to B064 or any embodiment;

[0456] Filling the first solution chamber with a first solution containing a target substance;

[0457] Filling the second solution chamber with a second solution; and

[0458] An electric field is applied from outside the first solution chamber and the second solution chamber to selectively move the target substance from the first solution chamber to the second solution chamber through the partition by electrophoresis.

[0459] C081

[0460] A method for treating a target substance by electrophoresis, comprising:

[0461] Providing the electrophoresis device of B081 or any embodiment;

[0462] Filling the first solution chamber with a first solution containing a target substance;

[0463] Filling the second solution chamber with a second solution;

[0464] Filling the third solution chamber with a third solution;

[0465] applying an electric field from outside the first solution chamber and the second solution chamber to selectively move the target substance from the first solution chamber to the second solution chamber through the first partition by electrophoresis; and

[0466] An electric field is applied from outside the second solution chamber and the third solution chamber to cause the target substance to move from the second solution chamber to the third solution chamber through the second separator by electrophoresis.

[0467] Although preferred embodiments of the present invention are shown and described in this specification, such embodiments are provided as examples only, which will be apparent to those skilled in the art. The present invention is not intended to be limited by the specific examples provided in this specification. Although the present invention has been described with reference to the above description, the description of the embodiments of this specification and the accompanying drawings are not intended to be interpreted in a limiting sense. Without departing from the present invention, a number of variations, changes and substitutions may be conceived by those skilled in the art. Moreover, it should be understood that all aspects of the present invention are not limited to the specific descriptions, structures or relative proportions described in this specification that depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described in this specification may be used when implementing the present invention. It is therefore believed that the present invention also encompasses such alternatives, corrections, variations or equivalents. The above claims define the scope of the present invention and are intended to encompass the methods and structures in these claims and their equivalents.

Claims

1. A method for treating a target substance by electrophoresis, comprising: Provide a partition that allows the target substance to pass through; introducing a first solution containing the target substance into a first side of the separator; introducing a second solution into the second side of the separator; as well as The target substance is selectively moved from the first solution to the second solution through the separator by electrophoresis.

2. The method according to claim 1, wherein The method for treating the target substance is a method of simultaneously performing two or more of the following operations: concentrating the target substance; exchanging the buffer solution of the target substance; and The target substance is purified.

3. The method according to claim 2, wherein: The simultaneous performing includes performing by one-dimensional electrophoresis.

4. The method according to claim 1, wherein The second volume of the second solution is smaller than the first volume of the first solution.

5. The method according to any one or any embodiment of claims 1 to 4, wherein: The method further includes introducing into the second solution a counter substance that migrates in a direction opposite to the target substance by electrophoresis, Selectively moving the target substance by electrophoresis includes simultaneously moving the counter substance from the second solution to the first solution through the separator by electrophoresis.

6. The method according to claim 5, wherein: The counter substance has a charge opposite to that of the target substance.

7. The method according to claim 1, wherein The first solvent (first buffer) of the first solution is different from the second solvent (second buffer) of the second solution.

8. The method according to claim 1, wherein The separator selectively allows the target substance to pass therethrough or permits the selective passage of the target substance.

9. The method according to claim 8, wherein The separator includes a gel that selectively allows the target substance to pass therethrough.

10. The method according to claim 9, wherein: The gel is a polyacrylamide gel.

11. The method according to claim 8, wherein The separator includes a semipermeable membrane that selectively allows the target substance to pass therethrough.

12. The method according to claim 1, wherein The partition or the first solution chamber (input chamber) includes a selective substance having the ability to allow the target substance to selectively pass through the partition and / or to inhibit, prevent or block at least one foreign substance from passing through the partition.

13. The method according to claim 1, wherein The method further includes mixing a selective substance into the first solution before the electrophoresis.

14. The method according to claim 12 or 13, wherein: The selective substance has the ability to purify proteins.

15. The method according to claim 12 or 13, wherein: The selective substance is selected from the group consisting of hydroxyapatite, a cation exchange carrier, an anion exchange carrier and a hydrophobic resin.

16. The method according to claim 1, wherein The pH of the first solution is greater than (higher) / less than (lower) the isoelectric point of the target substance, The target substance has a negative / positive charge, The selective movement by electrophoresis includes applying an electric field such that the first solution side is negative / positive and the second solution side is positive / negative.

17. The method according to claim 16, wherein The pH of the first solution is the same as or higher than the isoelectric point of at least one of the inclusions contained in the first solution.

18. The method according to claim 1, wherein The first solution is a solution derived from an organism.

19. The method according to claim 18, wherein The first solution is a crude sample.

20. The method according to claim 18 or 19, wherein The first solution is a solution of biological origin selected from the group consisting of blood, saliva, tears, urine and interstitial fluid.

21. The method according to claim 20, wherein The target substance is a biomolecule contained in the first solution.

22. The method according to claim 21, wherein The target substance is albumin contained in the first solution.

23. A device for treating a target substance by electrophoresis, comprising: a first solution chamber; a second solution chamber adjacent to the first solution chamber; and a separator that divides the first solution chamber and the second solution chamber, allows the target substance to pass from the first solution chamber to the second solution chamber, and has conductivity; The device is constructed in the following manner: The first solution chamber is filled with a first solution containing the target substance; The second solution chamber is filled with a second solution; An electric field is applied from outside the first solution chamber and the second solution chamber to selectively move the target substance from the first solution chamber to the second solution chamber through the partition by electrophoresis.

24. The device according to claim 23, wherein The device further includes an electrode pair configured to apply an electric field from outside the first solution chamber and the second solution chamber.

25. The device according to claim 23 or 24, wherein The second volume of the second solution chamber is smaller than the first volume of the first solution chamber.

26. The device according to claim 23 or 24, wherein The separator selectively allows the target substance to pass therethrough or permits the selective passage of the target substance.

27. The device according to claim 26, wherein The separator includes a gel that selectively allows the target substance to pass therethrough.

28. The device according to claim 26, wherein The separator includes a semipermeable membrane that selectively allows the target substance to pass therethrough.

29. The device according to claim 23 or 24, wherein The partition or the first solution chamber includes a selective substance having the ability to allow the target substance to selectively pass through the partition and / or to inhibit, prevent or block at least one foreign substance from passing through the partition.

30. The device according to claim 23 or 24, which is constructed in such a manner that the selective substance is mixed into the first solution before the electrophoresis.

31. A device for treating a target substance by electrophoresis, comprising: a first solution chamber; a second solution chamber adjacent to the first solution chamber; a third solution chamber adjacent to the second solution chamber; a first separator that divides the first solution chamber and the second solution chamber, allows the target substance to pass from the first solution chamber to the second solution chamber, and has conductivity; as well as The second separator partitions the second solution chamber and the third solution chamber, allows the target substance to pass from the second solution chamber to the third solution chamber, and has conductivity.

32. A method for treating a target substance by electrophoresis, comprising: Providing an electrophoresis device according to any one of claims 23 to 31; filling the first solution chamber with a first solution containing a target substance; filling the second solution chamber with a second solution; as well as An electric field is applied from outside the first solution chamber and the second solution chamber to selectively move the target substance from the first solution chamber to the second solution chamber through the partition by electrophoresis.

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