Method and apparatus for treating a target substance by electrophoresis

Abstract

An embodiment according to the present disclosure provides a method for treating a target substance by electrophoresis, said method comprising: providing a separator that permits pass through of the target substance; introducing, to a first side of the separator, a first solution containing the target substance; introducing a second solution to a second side of the separator; and causing, by electrophoresis, a selective transfer of the target substance from the first solution through the separator to the second solution.

Description

Method and apparatus for processing target materials by electrophoresis

[0001] The present invention relates to a method and apparatus for processing a target material by electrophoresis.

[0002] Many techniques are known for separating substances in solution.

[0003] Electrophoresis is one such technique. Various electrophoretic methods have been developed. For example, two-dimensional electrophoresis is a technique in which target substances are separated by electrophoresis in two sequential directions, and is widely used in protein analysis. Generally, isoelectric focusing is performed in a linear direction using a polyacrylamide gel (first dimension), and then SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate the substances based on molecular weight in a direction perpendicular to the first dimension (second dimension). This technique requires two electrophoresis runs and also requires 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 involves concentrating a sample by centrifugal ultrafiltration and then diluting it with a buffer to perform buffer exchange. As such, dialysis generally requires large equipment, a large amount of solvent (buffer), and a long processing time (several hours to a full day). Centrifugal ultrafiltration is prone to clogging and is not suitable for crude samples containing many impurities.

[0005] Because dialysis is a phenomenon based on osmotic pressure, it is not possible to concentrate a target substance by itself. To achieve concentration, 50% to 60% glycerol is mixed with the external solution. This causes the water in the internal solution to be expelled into the external solution. However, glycerol enters and mixes with the internal solution containing the target substance. 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, because glycerol is a nonionic substance and has no charge, it cannot be separated using electrophoresis.

[0006] As can be seen from the above examples, there is a demand for simpler and more efficient methods for separating substances.

[0007] According to one embodiment of the present disclosure, there is provided a method for processing a target substance by electrophoresis, comprising: providing a separator that allows passage of the target substance; introducing a first solution containing the target substance to a first side of the separator; introducing a second solution to a second side of the separator; and selectively transferring the target substance from the first solution through the separator to the second solution by electrophoresis.

[0008] According to one embodiment of the present disclosure, there is provided an apparatus (device) for processing a target substance by electrophoresis. According to one embodiment, the apparatus (device) includes: a first solution chamber (input chamber); a second solution chamber (elution chamber) adjacent to the first solution chamber (input chamber); and a separator that separates the first solution chamber from the second solution chamber, allows passage of the target substance from the first solution chamber to the second solution chamber, and is electrically conductive; wherein the first solution chamber is filled with a first solution containing the target substance; the second solution chamber is filled with a second solution; and an electric field is applied from outside the first solution chamber and the second solution chamber to selectively migrate the target substance from the first solution chamber through the separator to the second solution chamber by electrophoresis.

[0009] This allows, for example, the target substance to be separated simply and efficiently by electrophoresis, and the device can be made compact.

[0010]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0011] Schematic diagram of an electrophoresis device according to an embodiment. Schematic diagram of an electrophoresis device according to an embodiment. Schematic diagram of an electrophoresis device according to an embodiment. Schematic diagram of an electrophoresis device according to an embodiment. Schematic diagram of an electrophoresis device according to an embodiment. Graph showing the results of concentration ratio according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. Graph showing the abundance ratio of albumin and amylase in the above example. Graph showing the relationship between the concentration of a counter substance in an initial eluate and the concentration of albumin in the eluate after electrophoresis processing according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. Electrophoresis gel photograph showing the results of electrophoresis processing according to an example. 1A and 1B are schematic diagrams of an electrophoresis unit according to an embodiment, and a pipette cartridge including the electrophoresis unit according to an embodiment.

[0012] In some embodiments, the target substance may be a substance contained in a subject's subject solution. According to some embodiments of the present disclosure, the target substance may be processed and subjected to testing in the subject.

[0013] In some embodiments, the subject may include or be a human. In some embodiments, the subject may include or be a non-human animal. The subject may include or be a mammal. The subject may be, for example, without limitation, a working animal, a livestock animal, a pet animal, or a wild animal. The subject may be a plant or a microorganism.

[0014] The sample containing the target substance may be a solution. The "solution" may be a solution derived from a subject. The "solution" may be a body fluid, a solution derived from a body fluid, or a diluted solution of a body fluid. The solution may be a non-body fluid (non-body fluid-derived) solution, or a mixture of a body fluid or a body fluid-derived solution and a non-body fluid-derived solution. The solution may be a lysate of microparticles, cells, extracellular vesicles, etc., a culture medium, etc. The solution may be a solution used for sample measurement, or a solution used for calibration measurement. For example, the solution may be a standard solution or a calibration solution. For example, the solution may be a liquid that intentionally or deliberately does not contain the target substance to be measured, in order to be used for calibration, etc. The sample to be measured may be a specimen. The solution may be a solution containing a chemical substance.

[0015] The "body fluid" may be lymph, tissue fluid such as interstitial fluid, intercellular fluid, or interstitial fluid, or may be body cavity fluid, serous cavity fluid, pleural effusion, peritoneal fluid, pericardial fluid, cerebrospinal fluid (spinal fluid), synovial fluid, or aqueous humor (aqueous humor). The body fluid may be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, or intestinal fluid, or may be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, or milk. The body fluid may be animal or human body fluid. The "body fluid" may also be a solution. The solution may contain a physiological buffer solution such as phosphate-buffered saline (PBS) or 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.

[0016] The solution may contain a target substance. The solution may have the potential to contain a target substance. In some embodiments, the target substance may be a molecule, ion, macromolecule, biomolecule, etc. The target substance may comprise a biomolecule. The target substance may be a biological substance. The target substance may be a protein, a glycated protein, etc. For example, the solution may be tears, and the target substance may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin contained in 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. The albumin may be oxidized albumin (HNA) or reduced albumin (HMA). In some embodiments, the substance of interest may be Advanced Glycation End Products (AGEs). In some embodiments, the substance of interest may be glycated lipids.

[0017] As used herein, the term "albumin" includes both glycated (glycoalbumin) and non-glycated albumins, and includes both oxidized and reduced albumins, unless otherwise specified.

[0018] The solution containing the target substance may be a crude sample. As used herein, the term "crude sample" generally refers to a biological sample that is believed to contain the target substance and has not undergone a procedure for isolating or purifying the target substance. The term "crude sample" does not exclude lysates of microorganisms, cells, extracellular vesicles, etc., or culture fluids.

[0019] As used herein, the term "contaminant" generally refers to a substance that is not the target substance to be recovered, regardless of whether it is an impurity or a contaminant, unless otherwise indicated.

[0020] <Separator> As used herein, the term "separator" generally refers to a membrane, member, or region that separates two solutions. A separator allows a substance of interest contained in one solution to pass through to the other solution. Alternatively, a separator allows impurities contained in one solution to pass through to the other solution, while preventing the passage of the substance of interest.

[0021] As used herein, the phrase "separator partitions the solution compartment" or similar expressions means that the amount of permeation (the passage of the solvent through the separator) of the solvent or buffer contained in the solution compartment is sufficiently small relative to the dialysis (the passage of the solute through the separator) of the target substance during a desired separation or other operation. This does not require that no trace of permeation occurs.

[0022] The separator of the present disclosure preferably has electrical conductivity suitable for electrophoresis. However, the components or materials constituting the separator do not necessarily have electrical conductivity. As used herein, the expression "the separator divides a solution chamber" or similar expressions should be interpreted to mean that the target substance can pass through the separator during electrophoresis.

[0023] In some embodiments, the separator may comprise a gel. Examples of gels include, but are not limited to, agarose gels, polyacrylamide gels (PAGE), and the like.

[0024] In some embodiments, the separator may comprise filter paper or cellulose. In some embodiments, the separator may comprise a resin (e.g., polytetrafluoroethylene (PTFE)).

[0025] In some embodiments, the separator may include multiple different materials. In some embodiments, the separator may include a filter paper (such as a cellulose membrane or other membrane) and a gel. For example, the gel may be positioned on the inlet chamber side of the filter paper. The filter paper provides physical support for the gel.

[0026] Buffer Exchange: In some embodiments, the solvents of the solutions on either side of the separator may be different. In some embodiments, the first buffer in the first solution compartment may be different from the second buffer in the second solution compartment. In this case, a buffer exchange is performed on the target substance.

[0027] The solvent of the solution may be a buffer solution. Examples of buffer solutions include, but are not limited to, Tris-glycine buffer, phosphate buffer, barbital Na buffer, etc.

[0028] Concentration In some embodiments, the volumes of the solution chambers or the volumes of the solutions on either side of the separator 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 may be smaller than the volume of the first solution chamber or the amount of the first solution (first buffer) in the first solution chamber. This allows for concentration of the target substance. Not all of the target substance in the first solution chamber (input chamber) will necessarily be transferred to the second solution chamber (elution chamber) by electrophoresis. The volume of the second solution or the second solution chamber, or the properties of the second solution or the second solution chamber, may be configured so that the concentration of the target substance in the second solution after electrophoresis is higher than the concentration of the target substance in the initial first solution.

[0029] The term "concentration ratio" as used herein is defined as the ratio C1 / C0 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.

[0030] During processing, the concentration of the target substance in the second solution increases. Accordingly, the osmotic pressure increases in the reverse direction, from the second solution chamber to the first solution chamber. This suggests the existence of a concentration limit. As a solution to this problem, in some embodiments, a substance (counter substance) that migrates to the first solution chamber by electrophoresis may be added to the second solution. The counter substance preferably has an opposite charge to that of the target substance in the solution. Examples of counter substances include, but are not limited to, biological macromolecules such as proteins and artificial macromolecules.

[0031] Selectivity In some embodiments, the separator may be selective. That is, the separator may have the ability to pass a molecule of interest while not allowing, inhibiting, or blocking the passage of other molecules of interest or contaminants. For example, the separator may be or include a semipermeable membrane or a dialysis membrane. The semipermeable membrane or dialysis membrane may be selected from cellulose esters, regenerated cellulose, and the like.

[0032] For example, the separator may be chemically selective. In some embodiments, the separator may allow contaminants to pass through.

[0033] In some embodiments, the separator may contain a substance that provides selective electrophoresis (sometimes referred to herein as a "selectivity substance"). The separator may contain a substance capable of purifying 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 may be either Type I or Type II. Examples of hydrophobic resins include compounds having functional groups such as butyl groups, phenyl groups, and hexyl groups. The separator may contain a compound or mixture of compounds having functional groups that exhibit multiple different interactions (e.g., ion exchange, hydrophobic binding, affinity binding, etc.). The separator may also contain a compound used in a mixed mode. These substances inhibit, do not allow, or block the passage of impurities. This can increase the degree of purification of the target substance, for example, compared to when no resin is used.

[0034] For example, the selectivity substance may be contained in or supported by a gel. For example, the selectivity substance may be supported on or near the surface of a semipermeable membrane. In some embodiments, the selectivity substance may be disposed in a portion other than the separator. For example, the selectivity substance may be provided in the first solution or the first solution chamber. In some embodiments, the selectivity substance may be mixed into the first solution before electrophoresis.

[0035] <Multiple Electrophoresis> In some embodiments, a first solution chamber, a second solution chamber, a third solution chamber, a first separator disposed between the first and second solution chambers, and a second separator disposed between the second and third solution chambers may be provided. An electrode pair may be disposed outside the first and third solution chambers. In some embodiments, the first separator allows the target substance to pass through, while the second separator does not. In some embodiments, the second separator may not allow the target substance to pass through, but may allow some of the impurities (e.g., substances smaller than the target substance) to pass through. As a result, the target substance moves from the first solution chamber through the first separator to the second solution chamber and is retained there. Meanwhile, some of the impurities are retained in the first solution chamber. Another portion of the impurities passes through the second solution chamber and the second separator and enters the third solution chamber. This allows, for example, further purification of the target substance. In some embodiments, two or more separators may be used. In some embodiments, three or more solution chambers or solutions may be used.

[0036] In some embodiments, the target substance that has undergone electrophoretic processing (such as separation, buffer exchange, or concentration) may be transported by further electrophoresis to another location (such as a collection chamber, a sensor, or a chamber for performing another process).

[0037] In some embodiments, the processed target substance may be transported by electrophoresis to a sensor that detects the target substance. For example, the device may further include a sensor. For example, the device may be configured to be coupled to the sensor. For example, the third solution chamber may be a space in which a sensor that detects the target substance is disposed. The present disclosure provides an electrophoresis apparatus, device, or system.

[0038] In some embodiments, a solution containing a target substance can be introduced into such a sensing device, subjected to electrophoretic processing (separation, purification, buffer exchange, concentration, etc.), and the processed target substance can be detected by the sensing device. This allows, for example, detection or measurement of low concentrations of a target substance with high efficiency and / or high sensitivity. Using such an electrophoresis apparatus, device, or system, for example, low-concentration proteins such as albumin in saliva can be concentrated and purified, and further detected or measured with high sensitivity or accuracy.

[0039] <Selectivity by Isoelectric Point> In some embodiments, selectivity for the target substance and impurities may be achieved depending on the pH of the input solution. The pH of the input solution may be set to be between the isoelectric points of the target substance and the impurities. For example, if a negatively charged target substance in the input solution is to be attracted to a positive electrode, the pH of the input solution may be set to be higher than the isoelectric point of the target substance. The surface of the target substance becomes negatively charged and is attracted to the positive electrode. The pH of the input solution may be set so that the isoelectric point of the target molecule in the input solution is lower than that of the target molecule and higher than that of the impurities. For example, the impurities may migrate in the opposite direction to the target substance. For example, even if the impurities migrate to the same electrode, the amount of migration may be sufficiently smaller than that of the target substance. For example, the impurities may not migrate at all. This allows purification or separation of the target substance.

[0040] <Performing Multiple Processes> In some embodiments, multiple processes such as buffer exchange, concentration, purification, and separation may be performed simultaneously. The multiple processes may be selected from the group consisting of buffer exchange, concentration, purification, and separation. The times of the multiple processes may or may not overlap with each other. As used herein, the expression "performing multiple processes simultaneously" means that multiple processes are performed during a single one-dimensional electrophoresis process, unless otherwise specified.

[0041] Some embodiments of the present disclosure do not preclude two-dimensional or multiple-dimensional electrophoresis. Any single-dimensional electrophoresis may be used for the processing electrophoresis of any embodiment of the present disclosure. In some embodiments, multiple-dimensional processing electrophoresis may be used.

[0042] In some embodiments, a treatment electrophoresis may be performed intermittently. For example, the positive and negative electrodes may be switched periodically or irregularly. This may, for example, reduce problems such as separator clogging and / or allow for efficient or effective treatment electrophoresis. Such switching of the electric field direction should not be considered multi-dimensional electrophoresis. In this disclosure, unless otherwise specified, a series of electrophoresis treatments performed with the same electrode pair is considered to be one-dimensional electrophoresis.

[0043] <Embodiments> Several embodiments will be described below with reference to the drawings.

[0044] <Embodiment 1> An apparatus 100 according to an embodiment of the present disclosure and its operation will be described using Fig. 1 . As 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 separator 111 that separates the first solution chamber 101 from the second solution chamber 102. A first buffer 131 and a target molecule 141 are input into the first solution chamber 101. The second solution chamber 102 is filled with a second buffer 132. The electrophoresis apparatus 100 shown in Fig. 1 further includes an electrode pair 121, 122. The electrode pair 121, 122 are arranged on either side of the first solution chamber 101 and the second solution chamber 102. In other words, the electrode pair 121, 122 is arranged so that under the electric field generated by the electrode pair 121, 122, the target molecule 141 in the first solution chamber 101 moves from the first solution chamber 101 through the separator 111 to the second solution chamber 102.

[0045] The electrode pair 121, 122 is connected to a DC power supply 123. In Figure 1A, the switch 125 is open.

[0046] 1B, switch 125 is closed, charging electrode 121 negatively and electrode 122 positively, generating an electric field between them. Under this electric field, electrophoresis occurs. That is, negatively charged target molecules 141 begin to move within first buffer 131 toward electrode 122. The target molecules 141 pass through separator 111 and enter second buffer 132 in second solution chamber 102.

[0047] 1C, electrophoresis is carried out for a predetermined time until a sufficient amount of target molecules 141 enters the second buffer 132 in the second solution chamber 102. The switch 125 is opened to remove the electric field and terminate the process.

[0048] As a result, the target molecule 141 that was originally contained in the first buffer 131 is now contained in the second buffer 132. A buffer exchange has been performed. In some embodiments, the concentration of the target molecule 141 can be increased.

[0049] <Embodiment 2> An apparatus 200 according to an embodiment of the present disclosure and its operation will be described using Fig. 2. As shown in Fig. 2A, the electrophoresis apparatus 200 includes a first solution chamber (input chamber) 201, an adjacent second solution chamber (elution chamber) 202, and a separator 211 that separates the first solution chamber 201 from the second solution chamber 202. A first buffer 231, a target molecule 241, and impurities 242 are input into the first solution chamber 201. The second solution chamber 202 is filled with a second buffer 232.

[0050] The electrophoresis device 200 shown in Figure 2 further includes electrode pairs 221 and 222. In Figure 2A, the switch 225 is open.

[0051] In this embodiment, the target molecule 241 and the impurities 242 have different isoelectric points. The pH of the first buffer 231 is between the isoelectric points. That is, the isoelectric point of the target molecule 241 is lower than the pH of the first buffer 231, and the isoelectric point of the impurities 242 is higher than the pH of the first buffer 231.

[0052] 2B , switch 225 is closed to charge electrode 221 negatively and electrode 222 positively, generating an electric field between them. Under this electric field, electrophoresis occurs. That is, negatively charged target molecules 241 begin to move within first buffer 231 toward electrode 222. The target molecules 241 pass through separator 211 and enter second buffer 232 in second solution chamber 202. Meanwhile, positively charged impurities 242 begin to move within first buffer 231 toward electrode 221. In other words, impurities 242 do not move toward electrode 222 or separator 211.

[0053] 2C, electrophoresis is carried out for a predetermined time until a sufficient amount of target molecules 241 enters the second buffer 232 in the second solution chamber 202. Impurities 242 remain in the first solution chamber (input chamber) 201 or the first buffer 231. The switch 225 is opened to remove the electric field and terminate the process.

[0054] As a result, the target molecule 241 originally contained in the first buffer 231 is contained in the second buffer 232. The second buffer 232 does not contain the impurities 242. In other words, the target molecule 241 can be purified or separated. Furthermore, the buffer has been exchanged. In some embodiments, the concentration of the target molecule 241 can be further increased.

[0055] Embodiment 3 An apparatus 300 according to an embodiment of the present disclosure and its operation will be described with reference to FIG. 3. As shown in FIG. 3A, the electrophoresis apparatus 300 includes a first solution chamber (input chamber) 301, an adjacent second solution chamber (elution chamber) 302, and a separator 311 that separates the first solution chamber 301 from the second solution chamber 302. A first buffer 331, a target molecule 341, and impurities 342 are input into the first solution chamber 301. The second solution chamber 302 is filled with a second buffer 332. The electrophoresis apparatus 300 shown in FIG. 3 further includes electrode pairs 321 and 322. In FIG. 3A, a switch 325 is open.

[0056] In this embodiment, the separator 311 has selectivity. That is, the separator 311 has the ability to pass the target molecule 341 and not the impurities 342. For example, it is assumed that the typical impurities 342 have a larger size than the target molecule 341. The separator 311 is a semipermeable membrane, and its pore size is larger than the target molecule 341 and smaller than the impurities 342.

[0057] As shown in FIG. 3B , switch 325 is closed, charging electrode 321 negatively and electrode 322 positively, generating an electric field between them. Under this electric field, electrophoresis occurs. That is, target molecule 341 begins to move within first buffer 331 toward electrode 322. Target molecule 341 passes through separator 311 and enters second buffer 332 in second solution chamber 302. In FIG. 3 , it is assumed that impurities 342 have the same charge as target molecule 341. Impurities 342 begin to move within first buffer 331 toward electrode 322. However, they cannot pass through separator 311 and enter second buffer 332 in second solution chamber 302, and therefore remain in first buffer 331.

[0058] 3C, electrophoresis is carried out for a predetermined time until a sufficient amount of target molecules 341 enters the second buffer 332 in the second solution chamber 302. Impurities 342 remain in the first solution chamber (input chamber) 301 or the first buffer 331. The switch 325 is opened to remove the electric field and terminate the process.

[0059] As a result, the target molecule 341 that was originally contained in the first buffer 331 is now contained in the second buffer 332. The second buffer 332 does not contain the impurities 342. In other words, the target molecule 341 can be purified or separated. Furthermore, buffer exchange has been performed. In some embodiments, the concentration of the target molecule 341 can be further increased.

[0060] <Embodiment 4> An apparatus 400 according to an embodiment of the present disclosure and its operation will be described using Fig. 4. As shown in Fig. 4A, the electrophoresis apparatus 400 includes a first solution chamber (input chamber) 401, an adjacent second solution chamber (elution chamber) 402, and a first separator 411 that separates the first solution chamber 401 from the second solution chamber 402. A first buffer 431, a target molecule 441, and impurities 442 and 443 are input into the first solution chamber 401. The second solution chamber 402 is filled with a second buffer 432.

[0061] 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 separator 412 separating the second solution chamber 402 from the third solution chamber 403 .

[0062] Electrophoresis device 400 of Figure 4 further includes electrode pairs 421 and 422. In Figure 4A, switch 425 is open.

[0063] In this embodiment, the first separator 411 and the second separator 412 are selective. That is, the first separator 411 has the ability to pass the target molecule 441 and not the impurities 442. For example, it is assumed that the impurities 442 have a larger size than the target molecule 441. For example, the first separator 411 is a semipermeable membrane, and its pore size is larger than the target molecule 441 and smaller than the impurities 442. In this example, the impurities 443 can pass through the first separator 411.

[0064] The second separator 412 has the ability to pass the impurities 443 but not the target molecules 441. For example, it is assumed that typical impurities 443 have a size smaller than the target molecules 441. For example, the second separator 412 is a semipermeable membrane, and the pore size thereof is smaller than the target molecules 441 and larger than the impurities 443. In this embodiment, the impurities 443 can pass through the first separator 411.

[0065] 4B , switch 425 is closed, charging electrode 421 negatively and electrode 422 positively, generating an electric field between them. Under this electric field, electrophoresis occurs. That is, target molecule 441 and like-charged impurities 442 and 443 begin to move within first buffer 431 toward electrode 422. Target molecule 441 and impurities 443 pass through first separator 411 and enter second buffer 432 in second solution chamber 402. Impurities 442 cannot pass through first separator 411 and cannot enter second buffer 432 in second solution chamber 402, and therefore remain in first buffer 431.

[0066] The target molecule 441 and the impurities 443 move within the second buffer 432 toward the electrode 422. The impurities 443 pass through the second separator 412 and enter the third buffer 433 of the third solution chamber 403. The target molecule 441 cannot pass through the second separator 412 and cannot enter the third buffer 433 of the third solution chamber 403, and therefore remains in the second buffer 432.

[0067] 4C , electrophoresis is carried out for a predetermined time until a sufficient amount of target molecules 441 enters the second buffer 432 in the second solution chamber 402. Impurities 442 remain in the first solution chamber (input chamber) 401 or the first buffer 431, and impurities 443 enter the third solution chamber (output chamber) 403 or the third buffer 433. Switch 425 is opened, discontinuing the electric field and completing the process.

[0068] As a result, the target molecule 441, which was originally contained in the first buffer 431, is now contained in the second buffer 432. The second buffer 432 does not contain the impurities 442 and 443. In other words, the target molecule 441 can be purified or separated. Furthermore, buffer exchange has been performed. In some embodiments, the concentration of the target molecule 441 can be further increased.

[0069] <Embodiment 5> An apparatus 500 according to an embodiment of the present disclosure and its operation will be described using Fig. 5. As shown in Fig. 5A, the electrophoresis apparatus 500 includes a first solution chamber (input chamber) 501, an adjacent second solution chamber (first elution chamber) 502, and a first separator 511 that separates the first solution chamber 501 from the second solution chamber 502. A first buffer 531 and a target molecule 541 are input into the first solution chamber 501. The second solution chamber 502 is filled with a second buffer 532.

[0070] 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 separator 512 separating the second solution chamber 502 from the third solution chamber 503. The third solution chamber 503 is filled with a second buffer 533.

[0071] The electrophoresis device 500 of Figure 5 further includes a first electrode pair 521a, 522a, which are arranged to perform electrophoresis with respect to the first solution chamber 501, the first separator 511, and the second solution chamber 502. The second separator and the third solution chamber are arranged perpendicular or at an angle to the electric field generated by the first electrode pair 521a, 522a. The electrophoresis device 500 of Figure 5 further includes a second electrode pair 521b, 522b, which are arranged to perform electrophoresis with respect to the second solution chamber 502, the second separator 512, and the third solution chamber 503. In some embodiments, the first solution chamber 501, the first separator 511, the second solution chamber 502, and the first electrode pair 521a, 522a may be configured to perform primary electrophoresis, and the second solution chamber 502, the second separator 512, the third solution chamber 503, and the second electrode pair 521b, 522b may be configured to perform secondary electrophoresis. In FIG. 5, the switches are omitted.

[0072] 5B, when a voltage is applied to the first electrode pair 521a, 522a, an electric field is generated between them. Under this electric field, electrophoresis occurs. That is, the target molecule 541 begins to move within the first buffer 531 toward the electrode 522a. The target molecule 541 passes through the first separator 511 and enters the second buffer 532 in the second solution chamber 502.

[0073] Electrophoresis is carried out for a predetermined time until a sufficient amount of target molecules 541 enters the second buffer 532 in the second solution chamber 502. The electric field is then removed to terminate the primary electrophoresis.

[0074] As a result, the molecule of interest 541 that was originally contained in the first buffer 531 is now contained in the second buffer 532. A buffer exchange has been performed. In some embodiments, the concentration of the molecule of interest 541 can be increased.

[0075] Next, as shown in FIG. 5C , the voltage across the first electrode pair 521 a, 522 a is released, and a voltage is applied to the second electrode pair 521 b, 522 b to generate an electric field between them. Under this electric field, electrophoresis occurs. That is, the target molecule 541 begins to move within the second buffer 532 toward the electrode 522 b. The target molecule 541 passes through the second separator 512 and enters the third buffer 533 in the third solution chamber 503.

[0076] Electrophoresis is carried out for a predetermined time until a sufficient amount of target molecules 541 enters the third buffer 533 in the third solution chamber 503. The electric field is then removed to terminate the secondary electrophoresis.

[0077] As a result, the target molecule 541, which was initially contained in the first buffer 531, is now contained in the third buffer 533 via the second buffer 532. Two buffer exchanges have been performed. In some embodiments, the concentration of the target molecule 541 can be further increased.

[0078] In some embodiments, separation or purification may be achieved by primary electrophoresis and secondary electrophoresis. In some embodiments, separation or purification may be achieved primarily by primary electrophoresis and not by secondary electrophoresis. In some embodiments, concentration may be achieved by primary electrophoresis and secondary electrophoresis. In some embodiments, concentration may be achieved primarily by primary electrophoresis and not by secondary electrophoresis. In some embodiments, buffer exchange may be achieved by primary electrophoresis and secondary electrophoresis. In some embodiments, buffer exchange may be achieved primarily by primary electrophoresis and not by secondary electrophoresis.

[0079] In some embodiments, the primary electrophoresis may perform one or more of the following processes on the target molecules contained in the input solution: separation, purification, concentration, and buffer exchange; and the secondary electrophoresis may then move the processed target molecules to another location. For example, the secondary electrophoresis may move the processed target molecules to a system, sensor, flow path, space, or other location where the target molecules are measured. The third solution chamber in this embodiment may be a solution chamber for a system, sensor, flow path, space, or other location where the target molecules are measured, or may be a solution chamber fluidically connected to such a system, sensor, flow path, space, or other location.

[0080] <Various Embodiments> In addition to the above-described embodiment, various other embodiments are possible.

[0081] In some embodiments, contaminants may be allowed to pass through the separator while preventing the target material from passing through the separator.

[0082] In some embodiments, other substances (also referred to as "binding substances") may be bound to the substance of interest or the impurities. In some embodiments, the binding substances may be designed or selected so that complexes of the binding substance with the substance of interest or the impurities cannot pass through the size-selective separator. In some embodiments, the binding substances may be designed or selected so that the isoelectric point of the complex between the binding substance and the substance of interest is different from the isoelectric point of the substance of interest. For example, the substance of interest may be designed so that it does not migrate alone, but its complex with the binding substance migrates, or vice versa.

[0083] In some embodiments, the device may include an input chamber and two adjacent elution chambers. A first elution chamber, a first separator, an input chamber, a second separator, and a second elution chamber may be arranged in this order between the electrode pair in the direction of the electric field. For example, a first target substance may pass from the input chamber through the first separator to the first elution chamber, and a second target substance may pass from the input chamber through the second separator to the second elution chamber. The pH of the input solution may be set to be between the isoelectric points of the first target substance and the second target substance. Two target substances can be separated, purified, and concentrated, and their buffers can be exchanged by applying an electric field in a single operation.

[0084] In some embodiments, the direction of the electric field may be reversed. For example, application of a unidirectional electric field may cause the target substance to migrate, while some contaminants (e.g., small molecules) may also migrate to the elution chamber. For example, if the contaminants are small molecules, it may be effective to temporarily apply a reverse electric field (a backward electric field). Application of a reverse electric field causes the target substance and small molecules that have migrated to the elution chamber to migrate again toward the input chamber. Here, small molecules with smaller molecular weights than the target substance migrate faster than the target substance and can return to the input chamber. However, the target substance does not migrate as quickly as small molecules and cannot return to the input chamber. In other words, application of an appropriate reverse electric field in addition to application of a positive electric field can promote the reverse migration of contaminants relative to the target substance, thereby increasing the concentration and / or purity of the target substance in the elution chamber.

[0085] <Examples> Several examples will be described below. Preparation and measurement steps and conditions common to the examples are as follows. Any differences will be explained in the section for each example.

[0086] Sample Preparation: Human serum albumin (HSA, hereafter sometimes referred to as "albumin") (Cosmo Bio), 10x Tris-glycine buffer (Wako), and purified water (DW) were mixed to prepare a diluted HSA solution. The volume ratio of HSA, Tris-glycine buffer, and DW was 1:2:17 for a 20-fold dilution and 2:1:7 for a 5-fold dilution.

[0087] <Preparation of Tubes for Process Electrophoresis> For process electrophoresis, a plastic tube with an inner diameter of approximately 6-8 mm and a length of several centimeters was used. When two solution chambers were provided, two interlocking tubes (a loading chamber tube and an elution chamber tube) were used. These were tightly connected, and a separator or a membrane supporting the separator was tightly sandwiched between them. When a semipermeable membrane was used as the separator, it was tightly sandwiched between the two tubes. When a gel was used as the separator, a 0.45 μm polytetrafluoroethylene resin-coated membrane (PTFE membrane) or a 0.2 μm cellulose acetate membrane was used as a gel support, tightly sandwiched between the two tubes. Liquid gel was dropped onto the surface of the loading chamber and allowed to stand until solidified. Alternatively, when no support was used, one end of the elution chamber tube was closed with Saran Wrap (registered trademark, Asahi Kasei Corporation), and the gel was introduced through the opening at the other end. After the gel was allowed to solidify, the Saran Wrap was removed and the elution chamber tubing was connected to the input chamber tubing.

[0088] A solution containing the target substance was placed in the input chamber, and a buffer solution (Tris-glycine, approximately pH 8.3-9.0) was placed in the elution chamber. After the solution was placed in each end of the tube, they were sealed with a dialysis membrane with a molecular weight cutoff of 12 kDa to 14 kDa to prevent air bubbles from entering. The volume of elution chamber V1 was adjusted by the length of the elution chamber and was approximately 35 μL to 260 μL. The buffer solution in the elution chamber was Tris-glycine buffer solution (Wako, #201-18601, pH 8.3-9.0).

[0089] <Process Electrophoresis> The electrophoresis tube filled with the solution was immersed in an electrophoresis tank filled with 1x Tris-glycine buffer. This was placed in a chromatography chamber maintained at 4°C and kept sufficiently cool. In the electrophoresis tank, the positive electrode was placed at the end of the elution chamber of the electrophoresis tube, and the negative electrode was placed at the end of the input chamber. A voltage of 150 V was applied to these electrodes for 120 minutes.

[0090] <Measurement of albumin absorbance> Absorbance was measured using DM-JACK (registered trademark, Minaris Medical Co., Ltd.). Prior to measurement, a calibration curve was prepared using a standard substance, if necessary. The pre-electrophoresis sample and eluted sample were diluted with 1x Tris-glycine buffer so that the estimated absorbance was approximately within the range of the calibration curve.

[0091] <Measurement Electrophoresis> Measurement electrophoresis was performed using SDS-PAGE. After process electrophoresis, the eluate was removed from the elution chamber. For saliva samples, 30 μL of eluate was mixed with 10 μL of 4x Loading Buffer (Wako, #196-16142). For serum samples, 5 μL of 25- to 50-fold diluted eluate was added with 5 μL of 4x Loading Buffer (Wako, #196-16142) and 10 μL of DW. This sample was heated at 95°C for 10 minutes. Next, 8 μL was dropped onto a 10% polyacrylamide gel (DRC, #NTH-525P). Electrophoresis was performed at 180 V for 60 minutes, followed by staining, destaining, and photographing.

[0092] Example 1: Concentration Ideally, if all molecules of the target substance initially loaded into the loading chamber were to migrate to the elution chamber, the ratio (V1 / V0) of the volume of the loading chamber (V0) to the volume of the elution chamber (V1) would be the concentration factor (C1 / C0). However, in reality, not all of the target substance migrates to the elution chamber. For example, some target substances may adhere to the separator or the inner wall of the loading chamber. For example, some target substances may not pass through the separator within a predetermined time. For example, some target substances may leak out of the electrophoresis tube. However, in general, the concentration factor can be considered to be a function of the ratio of the volumes of the loading chamber and the elution chamber. To confirm this, in this example, the ratio of the volumes of the loading chamber and the elution chamber was varied (V0 / V1 = 6 to 56), and the concentration factor of albumin (HSA) as the target substance was determined by absorbance measurement.

[0093] The HSA input was prepared at two concentrations: 5x diluted and 20x diluted. In this example, agarose gel was used as the separator. The concentration of each HSA was determined by absorbance using a DM-JACK. The concentration rate was calculated as the ratio of the concentration (C0) before input (before process electrophoresis) to the concentration (C1) after process electrophoresis (obtained from the elution chamber).

[0094] As shown in Figure 6, in both the 5x and 20x dilutions, the concentration ratio was observed to be approximately linear with respect to the volume ratio. Not all of the albumin in the input solution was transferred to the elution compartment. However, this linear relationship indicates that the concentration ratio can be easily determined or controlled by the structure of the electrophoresis device.

[0095] Example 2 Purification Using Agarose Gel In this example, albumin, a target substance, was purified from saliva and serum using agarose gel as a separator.

[0096] Saliva samples were prepared as follows: 3.6 mL of saliva sample was obtained from the inventor and filtered through cotton. This was mixed with 0.4 mL of 1x Tris-glycine buffer (Wako, #201-18601, pH 8.3-8.9). Serum samples were prepared in the same manner as in Example 1.

[0097] The separator was made by dissolving agarose gel in 1x Tris-glycine buffer (both manufactured by Wako) at a volume ratio of 1%. This was supported by a filter unit made of Ultrafree MC-HV PVDF Φ0.45 μm (manufactured by Merck).

[0098] Process electrophoresis was carried out at an applied voltage of 100 V for 240 minutes.

[0099] Figure 7 shows the electrophoresis gel photograph. 1 and 2 correspond to the original saliva sample and the eluate after process electrophoresis, respectively. 3 and 4 correspond to the original serum sample and the eluate after process electrophoresis, respectively. Albumin is indicated by a black arrow, and major contaminants are indicated by a white arrow.

[0100] In the original saliva sample (1), bands of albumin (black arrow) and impurities (white arrow, corresponding to amylase) were observed. On the other hand, in the saliva sample (2) after process electrophoresis, the bands of the impurities became extremely faint, while the band of albumin became dark. Based on the band intensity, the concentration rate of this process electrophoresis was approximately 35 times. In other words, the impurities were removed, and the target substance, albumin, was concentrated.

[0101] In the original serum sample (3), bands of albumin (black arrow) and impurities (white arrow, corresponding to IgG light-chain) were observed. On the other hand, in the serum sample (4) after process electrophoresis, the bands of the impurities became fainter and the band of albumin became darker. Based on the band intensities, the concentration rate of this process electrophoresis was approximately 2.1 times.

[0102] In both saliva and serum samples, the target substance, albumin, was concentrated and contaminants were removed.

[0103] Example 3 Purification Using Polyacrylamide Gel In this example, albumin, a target substance, was purified from serum using polyacrylamide gel as a separator.

[0104] Serum samples were 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).

[0105] 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 placed in an elution chamber tube, one end of which was sealed with Saran Wrap. After solidification, the Saran Wrap was removed, and the elution chamber tube was connected to the input chamber tube.

[0106] Process electrophoresis was carried out at an applied voltage of 150 V for 150 minutes.

[0107] Measurement electrophoresis was performed as follows. First, after process electrophoresis, the eluate was removed from the elution chamber. 1 μL of this eluate, diluted 25-fold with 1× Tris-glycine buffer (pH 8.5), was mixed with 5 μL of 4× Loading Buffer (Wako, #196-16142) and 14 μL of ultrapure water. 5 μL of the original serum sample, diluted 25-fold, was mixed with 5 μL of 4× Loading Buffer (Wako, #196-16142) and 10 μL of ultrapure water. The mixture was heated at 95°C for 10 minutes. 8 μL of this mixture was dropped onto a 10% polyacrylamide gel (DRC, #NTH-525P). Electrophoresis was performed at 180 V for 60 minutes.

[0108] The electrophoresis gel photograph is shown in Figure 8. 1 and 2 correspond to the original serum sample and the eluate after process electrophoresis, respectively.

[0109] Compared to the original serum sample (1), the albumin band (black arrow) in the serum sample (2) after process electrophoresis was darker, and the bands of impurities (white arrow) were significantly fainter. Based on the band intensities, the concentration rate of this process electrophoresis was approximately 6.5 times higher. This means that the impurities were removed, and the target substance, albumin, was concentrated.

[0110] Example 4 Purification Using a Dialysis Membrane In this example, albumin, a target substance, was purified from serum using a dialysis membrane (semipermeable membrane) as a separator.

[0111] 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.

[0112] Process electrophoresis was carried out at an applied voltage of 150 V for 120 minutes.

[0113] Measurement electrophoresis was performed as follows. First, after process electrophoresis, the eluate was removed from the elution chamber and diluted 25-fold with 1x Tris-glycine buffer (pH 8.6). 5 μL of the 10x diluted eluate, 5 μL of 4x 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 dropped onto a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0114] A photograph of the electrophoresis gel is shown in Figure 9. 1 and 2 correspond to the original serum sample and the eluate after process electrophoresis, respectively.

[0115] Compared to the original serum sample (1), the albumin band (black arrow) in the serum sample (2) after process electrophoresis was darker, and the bands of impurities (white arrow) were almost completely eliminated. The molecular weight of HSA is approximately 66 kDa. Process electrophoresis confirmed that HSA smaller than 100 kDa passed through the separator, while molecules larger than this did not. Note that molecules larger than 100 kDa are contained in serum but not in saliva. Amylase is contained in saliva but not in serum.

[0116] Example 5: Purification based on isoelectric point In this example, albumin was separated by setting the pH of the input solution between the isoelectric point of the target substance (albumin) (pI = 4.9) and the isoelectric point of the impurity (hemoglobin) (pI = approximately 7). Agarose gel was used as the separator.

[0117] Samples were prepared by mixing 20 μL of serum (Cosmo Bio Co., Ltd., #12181450), 800 μL of HbA1c (Hitachi Chemical Diagnostics Systems Co., Ltd., MetaboLead Control 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.

[0118] The separator was prepared by introducing 1% agarose gel into 10 mM sodium phosphate buffer, 1x Tris-glycine buffer (pH 6.5), or 1x Tris-glycine buffer (pH 8.5). 250 μL of this gel was supported on a Gel Ultrafree MC-HV PVDF Φ0.45 μm filter unit (Merck).

[0119] Process electrophoresis was carried out at 130 mA for 140 minutes.

[0120] Measurement electrophoresis was performed as follows. First, after process electrophoresis, the eluate was removed from the elution chamber. 0.8 μL of the eluate was mixed with 5 μL of 4x Loading Buffer (Wako, #196-16142), and 14.2 μL of DW. 4 μL of the original mixed sample solution was mixed with 5 μL of 4x Loading Buffer (Wako, #196-16142), and 11 μL of DW. The mixture was heated at 95°C for 10 minutes. 5 μL of the mixture was dropped onto a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0121] Figure 10 shows an electrophoresis gel photograph. 1 and 2 correspond to the original sample (1) and the eluate (2) after process electrophoresis when the input solution pH was 8.5. Both albumin (pI = 4.9) and hemoglobin (pI = approximately 7) migrated to the elution chamber by process electrophoresis. Because both are negatively charged at pH 8.5, they migrated toward the positive electrode and entered the elution chamber. 3, 4, and 5 correspond to the original sample (3), the eluate (4) after process electrophoresis, and the eluate (5) after reverse process electrophoresis when the input solution pH was 6.5. As can be seen from the eluate (4) after process electrophoresis, hemoglobin did not migrate to the elution chamber by process electrophoresis. As can be seen from the case where a reverse voltage was applied (5), the hemoglobin migrated in the reverse direction (toward the negative electrode). That is, it was confirmed that albumin migrated toward the positive electrode and hemoglobin migrated toward the negative electrode. Thus, it was demonstrated that the target substance can be separated from the impurities by setting the pH of the input solution between the isoelectric points of the target substance and the impurities.

[0122] Example 6 Addition of Resin In this example, a resin was added to a separator, and albumin, a target substance, was purified from saliva.

[0123] The saliva samples were prepared as follows: 12.6 mL of saliva was obtained from the inventors and filtered through cotton, which was then mixed with 1.4 mL of 1x Tris-glycine buffer (pH 7.5).

[0124] Each resin was added to this. The addition was carried out as follows: First, the additives were equilibrated with 1x Tris-glycine buffer, and as much of the supernatant as possible was removed. 2% agarose gel dissolved in each resin, which had been kept at 65°C, was added and mixed. The resin and agarose gel were thoroughly suspended, and approximately 250 μL of this was introduced into the separator section and allowed to cool sufficiently to solidify. A cellulose acetate membrane filter Φ0.2 μm (Advantec Toyo) was used to support the agarose mixed gel.

[0125] The additives used in this example are as follows: Cation exchange carrier "SP": TOYOPEARL SP-650M, TOSOH, #0007997 Anion exchange carrier "SQ": TOYOPEARL SuperQ-650M, TOSOH, #0017227 Hydroxyapatite "HA": CHT Ceramic Hydroxyapatite, Bio-Rad, #1582200

[0126] Process electrophoresis was carried out at 130 mA for 120 minutes.

[0127] Measurement electrophoresis was performed as follows. First, after process electrophoresis, the eluate was removed from the elution chamber. 10 μL of the eluate was mixed with 5 μL of 4x Loading Buffer (Wako, #196-16142), and 5 μL of DW. The mixture was heated at 95°C for 10 minutes. 3 μL of the mixture was dropped onto a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0128] Figure 11 shows the electrophoresis gel photographs of the additives SP and HA. 1, 2, 3, and 4 correspond to the original saliva sample (1), the eluate without resin additive (2), the eluate with SP (3), and the eluate with HA (4), respectively.

[0129] In the original saliva sample (1), bands of albumin (black arrow) and contaminants (white arrow, corresponding to amylase) were observed. The bands in the eluate (2) without resin additive were darker than those in the original saliva sample (1) because of the concentration. Without the additive, amylase removal was ineffective. On the other hand, in the eluate (3) with the addition of SP, the albumin band remained nearly the same, while the amylase band became weaker. SP was found to effectively block the passage of amylase into the elution chamber. Furthermore, in the eluate (4) with the addition of HA, the albumin band remained nearly the same, while the amylase band became significantly weaker. SP was found to even more effectively block the passage of amylase into the elution chamber.

[0130] Figure 12 shows an electrophoresis gel photograph of the additive SQ. 1, 2, and 3 correspond to the original saliva sample (1), the eluate without resin additive (2), and the eluate with SQ (3), respectively. In the eluate with SQ (4), the albumin band was almost the same as or slightly fainter than in the eluate without resin additive (2), and the amylase band had almost disappeared. It was found that SQ very effectively blocked the passage of amylase into the elution chamber.

[0131] The abundance ratio of HSA and the two amylases contained in each solution was determined from the band densities in the electrophoresis gel photographs shown in Figures 11 and 12 (Figure 13). "Amylase_up" and "Amylase_down" correspond to the two bands that appear below the albumin peak, with "Amylase_up" referring to the band closest to albumin and "Amylase_down" referring to the band farther from albumin. The solutions shown here are the original saliva, the eluate after process electrophoresis without a resin additive, and the eluate after process electrophoresis with the additive SP, HA, or SQ added.

[0132] Figure 13 shows a graph summarizing the results of Figures 11 and 12. The original saliva contains a very high amount of amylase relative to HSA. Here, HSA and two amylases are compared. In particular, Amylase_down is high (A). Without resin, albumin is concentrated, and amylase remains unchanged, becoming relatively low relative to albumin (B). This shows the concentration rate and impurity removal rate without a resin additive. It can be seen that adding an additive further removes impurities (C-E). In particular, it was found that using SQ as an additive can remove most of the amylase (E).

[0133] Example 7: Improvement of concentration rate In this example, agarose gel was used as a separator, and a counter substance was added to the eluate in advance, to concentrate albumin as a target substance from a serum sample.

[0134] A 20-fold diluted solution of HSA was prepared as a serum sample. 100 μL of 1% agarose was used as a separator. A cellulose acetate membrane filter Φ0.2 μm (Advantec Toyo) was used to support the agarose mixed gel.

[0135] The counter substances used in this example are as follows: RNase A (MACHEREY-NAGEL). A protein with a molecular weight of 13.7 kDa or less, with an isoelectric point of 8.6. Lysozyme (derived from egg white) (Nacalai Tesque). An enzyme with a molecular weight of 14 kDa and an isoelectric point of 11. Both have an opposite charge to albumin and move in the opposite direction to albumin during electrophoresis. These substances were added at multiple concentrations to the Tris-glycine buffer solution, which was the elution solution.

[0136] Process electrophoresis was carried out at 150V for 240 minutes.

[0137] The eluate was then removed from the elution chamber and subjected to electrophoresis. The concentration of each albumin was determined from the density of the albumin band in the electrophoresis image.

[0138] FIG. 14 shows the relationship between the concentration of the counter substance in the initial eluate and the concentration of albumin in the eluate after process electrophoresis when the counter substance is lysozyme (A) and RNase A (B). In both cases, the albumin concentration in the eluate increased almost in proportion to the concentration of the counter substance. The migration of albumin increased the albumin concentration in the eluate and decreased the albumin concentration in the input solution, resulting in a decrease in osmotic pressure. This indicates that there is a limit to the albumin concentration in the eluate. However, it was revealed that the migration of the counter substance in the opposite direction to albumin reduces the problem of decreased osmotic pressure and allows a higher final albumin concentration to be achieved.

[0139] Example 8 Separation and Concentration of Albumin in Saliva In this example, albumin, a target substance, was purified and concentrated from saliva using polyacrylamide gel as a separator.

[0140] As the electrophoresis buffer, a tris-glycine buffer (pH 6.0, hereinafter referred to as 1x "TMG buffer") whose pH had been adjusted with maleic acid was introduced into the elution chamber.

[0141] The saliva samples were prepared as follows: Saliva was obtained from the inventor and filtered through 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 of each was introduced into the input chamber.

[0142] The separator was prepared as follows: First, 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 were mixed to prepare a 10% polyacrylamide gel. 160 μL of this gel was supported on a CELLLOSE ACETATE Φ0.2 μm filter unit (ADVANTEC).

[0143] Process electrophoresis was carried out at room temperature at 25 V (current maximum approximately 24 mA) for 180 minutes.

[0144] Measurement electrophoresis was performed as follows. First, after process 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 dropped onto a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0145] Figure 15 shows a photograph of the electrophoresis gel. 1, 2, and 3 correspond to the original saliva sample (1), and the input solution (2) and eluate (3) after process electrophoresis, respectively. Although some amylase (1) present in the saliva was detected in the eluate (3), most of it remained in the input chamber (2). In contrast, most of the albumin was able to be transferred to the eluate (3). This confirmed that the albumin in the saliva was at least separated from the amylase.

[0146] The intensity of the albumin band in the eluate after process electrophoresis was approximately 12.7 times that in the saliva sample before process electrophoresis. In other words, this process electrophoresis enabled albumin to be concentrated 12.7 times. The volume of the input chamber was 2.5 mL, and the volume of the elution chamber was 150 μL, and the ratio was 17 times without taking osmotic pressure into consideration. Therefore, this result indicates that this method can concentrate albumin to a high concentration.

[0147] Example 9 Separation of Salivary Albumin and Macromolecules 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 the saliva.

[0148] The electrophoresis buffer and saliva sample were the same as those used in Example 8. The pH of the eluent and separator gel was adjusted to 8.5.

[0149] The separator was prepared by adding 50 μL of 1.5% agarose gel and solidifying it to form a support, followed by adding 110 μL of a mixture of 10% polyacrylamide gel and 1× TMG buffer (pH 8.5) and leaving it to stand at 30°C for 20 minutes.

[0150] Process electrophoresis was carried out at room temperature for 30 minutes at 100 V (maximum current of approximately 24 mA). 1x TMG buffer (pH 7.5) was used as the electrophoresis buffer.

[0151] Measurement electrophoresis was performed as follows. First, after process 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 dropped onto a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0152] Figure 16 shows a photograph of the electrophoresis gel. 1, 2, and 3 correspond to the original saliva sample (1), and the input solution (2) and eluate (3) after process electrophoresis, respectively. Although some amylase and lactoferrin (1) present in the saliva were detected in the eluate (3), most of them remained in the input chamber (2). In contrast, most of the albumin was able to be transferred to the eluate (3). This confirmed that the albumin in the saliva was at least separated from the amylase and lactoferrin.

[0153] The saliva sample contained macromolecules or macrocomplexes (1). These macromolecules are presumed to contain mucin. Thus, the migration of the macromolecules to the elution chamber was prevented. For example, adjusting the pH of the input chamber can prevent the migration of the macromolecules to the elution chamber. For example, using a separator with a relatively narrow mesh structure, such as polyacrylamide, can prevent the migration of the macromolecules to the elution chamber. These methods may also be combined.

[0154] Lactoferrin has an isoelectric point of 8.8, and is slightly positively charged or uncharged at pH 8.5 of the solution in this example. Therefore, it is thought that lactoferrin either migrated in the opposite direction to albumin or did not migrate at all, or at least did not migrate into the elution chamber.

[0155] Although removal of small molecules was not intended in this example, it is possible. For example, the separator may contain a resin that captures small molecules or slows the migration speed of small molecules (such as a resin used in gel filtration chromatography or size exclusion chromatography, such as Sephacryl (registered trademark)). For example, a small molecule capture resin may be mixed into the separator gel. For example, a polyacrylamide gel layer may be laminated with a layer containing a small molecule capture resin. With this configuration, the separator captures not only relatively large molecules such as mucin and lactoferrin, but also small molecule impurities, allowing the target substance to pass through. This allows the target substance (e.g., albumin) to be purified.

[0156] Example 10: Separation of albumin and hemoglobin in whole blood In this example, polyacrylamide gel was used as a separator to separate both albumin and hemoglobin, which were target substances, from a whole blood sample.

[0157] As the electrophoresis buffer, a tris-glycine buffer (pH 5.2, hereinafter referred to as 1x "TMG buffer") whose pH had been adjusted with maleic acid was used. This 1x TMG buffer was introduced into the elution chamber.

[0158] The whole blood sample was prepared as follows: 30 μL of fingertip blood (whole blood) was collected from the inventor in a blood collection tube, mixed with 270 μL of 1×TMG buffer (containing 0.1% Triton X-100), and left to stand at room temperature for 1 hour. This resulted in hemolysis of the whole blood sample. 300 μL of this hemolyzed sample was diluted with 11,700 μL of 1×TMG buffer.

[0159] The separator was prepared as follows: First, 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 were mixed to prepare a 10% polyacrylamide gel. 160 μL of this gel was supported on a CELLLOSE ACETATE Φ0.2 μm filter unit (ADVANTEC).

[0160] Process electrophoresis was carried out under cooling at 90V for 20 minutes, then at 80V for 60 minutes, then at 90V for 20 minutes.

[0161] Measurement electrophoresis was performed as follows. First, after process 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 dropped onto a 5-20% polyacrylamide gel (DRC, #NTH-576HP10). Electrophoresis was performed at 180 V for 60 minutes.

[0162] Albumin and hemoglobin migrate in opposite directions at this pH. Ideally, two elution chambers should be arranged in the direction of the applied voltage relative to the input chamber. However, in the setup of this example, one elution chamber was provided relative to the input chamber. Therefore, the voltage application method was changed for albumin and hemoglobin. Hereinafter, in the setup of this example, the voltage application direction for eluting albumin is referred to as the "forward direction," and that for eluting hemoglobin is referred to as the "backward direction."

[0163] A photograph of the electrophoresis gel is shown in Figure 17. 1 to 5 correspond to the original whole blood sample (1), the input solution (2) and eluate (3) after forward process electrophoresis, and the input solution (4) and eluate (5) after backward process electrophoresis, respectively.

[0164] As is clear from bands (3) and (5) in the eluate, albumin and hemoglobin were separated in the eluate. Furthermore, at least albumin was simultaneously concentrated. As can be seen from these results, this method makes it possible to simultaneously separate and purify albumin and hemoglobin.

[0165] 18 shows a longitudinal cross-sectional view of an electrophoresis unit 600 according to one embodiment. Electrophoresis unit 600 includes, in the longitudinal direction, an electrophoresis chamber 651, a top plate 652 covering it from above, and a separator 610 disposed within electrophoresis chamber 651. An input chamber 601 containing a sample solution and an elution chamber 601 containing an eluate are defined by electrophoresis chamber 651, top plate 652, and separator 610 via separator 610. The volumes of input chamber 601 and eluate, or the ratio thereof, may be set based on a desired concentration rate.

[0166] The electrophoresis tank 651 may have a circular or U-shaped cross section perpendicular to its length. This allows for a uniform application of an electric field, reducing waste in the movement of substances. It also allows for a large contact area when contacting a heat sink or fluid (air) for temperature control from the outside. The electrophoresis tank 651 may be configured to receive contact with a heat sink, irradiation with electromagnetic waves such as light, or blown air from the outside. This may allow for adjustment of the temperature of the solution inside. The electrophoresis tank 651 may be formed from a highly thermally conductive material.

[0167] The electrophoresis tank 651 has electrodes 621, 622 on both end surfaces in the longitudinal direction. The electrodes 621, 622 may be fixed to the electrophoresis tank 651. For example, the electrodes 621, 622 may be formed from flat metal plates. The flat metal plates 621, 622 may be bonded to both end surfaces of the electrophoresis tank 651. For example, the electrodes 621, 622 may be formed by depositing a material such as platinum by plating. The electrodes 621, 622 may be configured as external members so as to be in contact with or in close proximity to both end surfaces of the electrophoresis tank 651 during the electrophoresis process.

[0168] Example 12: Cartridge Figure 19 shows an example of a pipette cartridge 700 including the electrophoresis unit 600 of the above-described example. Figure 19(A) shows a top view of the pipette cartridge 700, and Figure 19(B) shows a cross-sectional view taken along the line A-A in (A). The pipette cartridge 700 is provided with an electrophoresis unit fixing portion 710 for fitting and fixing the electrophoresis unit 600 to a plate (main body) 701. The electrophoresis unit fixing portion 710 is opened in the plate 701, and the electrophoresis unit 600 is inserted into the opening. The electrophoresis unit 600 is fixed by locking pins 711 and 712 fixed to the plate 701 at the four corners of the opening.

[0169] Electrode contact leaf springs 713 and 714 are disposed at both longitudinal ends of the electrophoresis unit fixing portion 710. The electrodes 621 and 622 at both ends of the electrophoresis unit 600 inserted and fixed in the opening of the electrophoresis unit fixing portion 710 contact the electrode contact leaf springs 713 and 714, respectively, forming an electrical connection therebetween. The leaf springs 713 and 714 are connected via electric wires 733 and 734 to connectors 731 and 732 fixed to the leading end of the plate 701 in the longitudinal direction. The connectors 731 and 732 abut against stoppers that stop the tip of the pipette cartridge 700 when the pipette cartridge 700 is inserted tip-first into an automatic pipette device body (not shown). Contacts (not shown) for the connectors 731 and 732 are provided on their end faces. Establishing an electrical connection between the connectors 731 and 732 and their contacts allows power to be supplied to the electrophoresis unit 600 .

[0170] 19, the electrical connection between the electrophoresis unit 600 and the outside is established by the leaf springs 713 and 714 and the connectors 731 and 732, but the form of the electrical connection is not limited to this. For example, a power supply unit having a leaf spring may be disposed within the main body of the automatic pipette device, and this may move relative to the inserted cartridge 700, so that the leaf spring contacts the electrodes 621 and 622 at both ends of the electrophoresis unit 600.

[0171] In some embodiments, the pipette cartridge may be provided with a measurement well, a well for containing a solution required for the measurement, holes for holding pipette tips, tubing, etc. In some embodiments, the pipette cartridge may be equipped with a sensor.

[0172] The pipette cartridge 700 shown in FIG. 19 includes a sample container holder 721 for holding a sample container to be subjected to the electrophoresis process, a sample tip holder 722 for holding a sample tip for removing a sample from a sample container and introducing it into the input chamber of the electrophoresis unit 600, and an eluate tip holder 723 for arranging an eluate tip for removing a solution from the elution chamber after the electrophoresis process. The pipette cartridge 700 also includes a buffer well 731 for containing a buffer used in the electrophoresis process. The buffer well 731 is sealed with a sealing membrane 732 until use, sealing off the buffer (not shown) inside. The sealing membrane is, for example, an aluminum membrane, which can be easily penetrated by a pipette tip to obtain the buffer inside. The pipette cartridge 700 also includes an eluate tube holder 714 for holding a tube for collecting the eluate after the process. The treated solution after the electrophoresis process can be transferred into the eluate tube and then moved together with the tube from the pipette cartridge 700 to another location.

[0173] For example, the pipette cartridge 700 and / or the automatic pipette device may be configured to control the temperature of the electrophoresis unit 600. For example, a temperature-controlled heat sink may be disposed within the automatic pipette device, which may move relative to the inserted cartridge 700 and be in close contact with the electrophoresis chamber 651 of the electrophoresis unit 600. Alternatively, for example, the automatic pipette device may apply a temperature-controlled fluid (e.g., cooled air) to the surface of the electrophoresis chamber 651. This allows for efficient and appropriate temperature control of the solution during the electrophoresis process. In some embodiments, the temperature of the electrophoresis chamber 651 and the temperature of the solution during the electrophoresis process may be measured and automatically controlled.

[0174] In some embodiments, a pipette cartridge (unmounted) for mounting an electrophoresis unit may be provided, while in some embodiments, a pipette cartridge (including an electrophoresis unit) may be provided with an electrophoresis unit mounted thereon.

[0175] The present disclosure also provides the following embodiments: A001 A method for processing a target substance by electrophoresis, comprising: providing a separator that allows the passage of a target substance; introducing a first solution containing the target substance to a first side of the separator; introducing a second solution to a second side of the separator; and selectively transferring the target substance from the first solution through the separator to the second solution by electrophoresis. A001b A method for processing a target substance by electrophoresis, comprising: providing a separator that allows the passage of one of a target substance and impurities; introducing a first solution containing the target substance and the impurities to the first side of the separator; introducing a second solution to the second side of the separator; and selectively transferring one of the target substance and the impurities from the first solution through the separator to the second solution by electrophoresis. A002: The method according to A001, A001b, or any of the embodiments, wherein the method of processing the target substance comprises at least one of concentrating the target substance; exchanging the buffer of the target substance; and purifying the target substance. A003: The method according to A001, or any of the embodiments, wherein the method of processing the target substance comprises simultaneously performing two or more of concentrating the target substance; exchanging the buffer of the target substance; and purifying the target substance. A004: The method according to A001, or any of the embodiments, wherein the method of processing the target substance comprises simultaneously concentrating the target substance and purifying the target substance. A005: The method according to A003 or A004, or any of the embodiments, wherein the simultaneously performing comprises performing by one-dimensional electrophoresis. A007 The method of any one of A001 to A003 or any embodiment, wherein the separator comprises a material selected from the group consisting of a gel, a semipermeable membrane, and a resin.A008: The method of any one of A001 to A007, or any embodiment, wherein the separator comprises an agarose gel. A011: The method of A001, or any embodiment, wherein the second volume of the second solution is smaller than the first volume of the first solution. A012: The method of A001 or A011, or any embodiment, wherein the method concentrates the biomolecule. A015: The method of any one of A001 to A012, or any embodiment, wherein the method further comprises introducing into the second solution a substance (counter substance) that migrates in the opposite direction to the target substance by electrophoresis, and wherein selectively moving the target substance by electrophoresis simultaneously comprises electrophoretically moving the counter substance from the second solution through the separator to the first solution. A016: The method of A015, or any embodiment, wherein the counter substance has an opposite charge to the target substance. A017 The method according to A015 or A016, or any of the embodiments, wherein the counter substance is a polymer. A018 The method according to A017, or any of the embodiments, wherein the counter substance is a biopolymer. A019 The method according to A018, or any of the embodiments, wherein the counter substance is a protein or an enzyme. A021 The method according to A001, or any of the embodiments, wherein the first solvent (first buffer) of the first solution and the second solvent (second buffer) of the second solution are different. A031 The method according to A001, or any of the embodiments, wherein the separator selectively passes the target substance or allows the target substance to selectively pass through. A031b The method according to A001b, or any of the embodiments, wherein the separator selectively passes the impurities or allows the target substance to selectively pass through.A032 The method according to A031, or any embodiment, wherein the separator has the ability to suppress, avoid, or block the passage of at least one contaminant. A041 The method according to A031 or A032, or any embodiment, wherein the separator comprises a gel that selectively allows the target substance to pass through. A042 The method according to A041, or any embodiment, wherein the gel is a polyacrylamide gel. A051 The method according to A031 or A032, or any embodiment, wherein the separator comprises a semi-permeable membrane that selectively allows the target substance to pass through. A061 The method according to any one of A001 to A051 or any embodiment, wherein the separator, the first solution, or the first solution chamber (input chamber) comprises a selectivity substance capable of allowing the target substance to selectively pass through the separator and / or suppressing, avoiding, or blocking at least one contaminant from passing through the separator. A061b The method according to any one of A001b to A051 or any embodiment, wherein the separator, the first solution, or the first solution chamber (input chamber) comprises a selectivity substance capable of allowing at least one contaminant to selectively pass through the separator and / or suppressing, avoiding, or blocking the target substance from passing through the separator. A062 The method according to any one of A001 to A047 or any embodiment, further comprising mixing a selectivity substance into the first solution prior to the electrophoresis. A063: The method according to A061 or A062, or any of the embodiments, wherein the selectivity material has the ability to purify proteins. A064: The method according to any one of A061 to A063, or any of the embodiments, wherein the selectivity material 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.A071 A001 or the method according to any of the embodiments, wherein the pH of the first solution is greater (higher) / less (lower) than the isoelectric point of the target substance, the target substance having a negative / positive charge, and the selective migration by electrophoresis comprises applying an electric field so that the first solution side is negative / positive and the second solution side is positive / negative. A072 A071 or the method according to any of the embodiments, wherein the pH of the first solution is the same as or higher than the isoelectric point of at least one of the impurities contained in the first solution. A081 A method for processing a target substance by electrophoresis, comprising: providing a first separator that allows the passage of the target substance; introducing a first solution containing the target substance to a first side of the first separator; introducing a second solution to a second side of the first separator and a first side of a second separator; introducing a third solution to a second side of the second separator; selectively transferring the target substance from the first solution through the first separator to the second solution by electrophoresis; and transferring the target substance from the second solution through the second separator to the third solution by electrophoresis. A081b A method for processing a target substance by electrophoresis, comprising: 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; introducing a first solution containing the first target substance and the second target substance between the first and second separators; selectively transferring the first target substance from the first solution through the first separator to an opposite side of the first separator by electrophoresis; and transferring the second target substance from the first solution through the second separator to an opposite side of the second separator by electrophoresis. A082 The method of A081b, or any embodiment, wherein the pH of the solution is between the isoelectric points of the first target substance and the second target substance.A091: The method according to any one of A001 to A082 or any embodiment, wherein the first solution contains the target substance and small molecular weight impurities having a smaller molecular weight than the target substance, and the electrophoresis comprises repeatedly applying a forward electric field in a direction in which the target substance and the small molecular weight impurities pass through the separator and move into the second solution, and repeatedly applying a reverse electric field. A092: The method according to A091 or any embodiment, wherein repeatedly applying the forward electric field and the reverse electric field comprises promoting the return of the small molecular weight impurities from the first solution. A101: The method according to any one of A001 to A081b or any embodiment, wherein the first solution is a solution derived from a living organism. A102: The method according to any one of A001 to A081b or any embodiment, wherein the first solution is a crude sample. A103: The method according to A101 or 102, or any of the embodiments, wherein the first solution is a biologically derived solution selected from the group consisting of blood, saliva, tears, urine, and interstitial fluid. A111: The method according to any of A001 to A103, or any of the embodiments, wherein the target substance is a biomolecule contained in the first solution. A112: The method according to any of A001 to A103, or any of the embodiments, wherein the target substance is albumin contained in the first solution.

[0176] B001 An apparatus (device) for processing a target substance by electrophoresis, comprising: a first solution chamber (input chamber); a second solution chamber (elution chamber) adjacent to the first solution chamber (input chamber); and a separator that separates the first solution chamber from the second solution chamber, allows the target substance to pass from the first solution chamber to the second solution chamber, and is electrically conductive; wherein the first solution chamber is filled with a first solution containing the target substance; the second solution chamber is filled with a second solution; and an electric field is applied from outside the first solution chamber and the second solution chamber, selectively moving the target substance from the first solution chamber through the separator to the second solution chamber by electrophoresis. B001b An apparatus (device) for processing a target substance by electrophoresis, comprising: a first solution chamber (input chamber); a second solution chamber (elution chamber) adjacent to the first solution chamber (input chamber); and a separator that separates the first solution chamber from the second solution chamber, allows either the target substance or impurities to pass from the first solution chamber to the second solution chamber, and is electrically conductive; wherein the first solution chamber is filled with a first solution containing the target substance and the impurities; the second solution chamber is filled with a second solution; and an electric field is applied from outside the first solution chamber and the second solution chamber to selectively migrate either the target substance or the impurities from the first solution chamber through the separator to the second solution chamber by electrophoresis. B002 The apparatus (device) according to B001, further comprising an electrode pair configured to apply an electric field from outside the first solution chamber and the second solution chamber. B011 The device according to B001 or B002, or any of the embodiments, wherein the second volume of the second solution chamber is smaller than the first volume of the first solution chamber. B021 The device according to B001 or B002, or any of the embodiments, wherein the first solvent (first buffer) of the first solution and the second solvent (second buffer) of the second solution are different from each other.B031 The device according to B001 or B002, or any of the embodiments, wherein the separator selectively passes the target substance or allows the target substance to selectively pass through. B032 The device according to B031, or any of the embodiments, wherein the separator has the ability to suppress, avoid, or block the passage of at least one impurity. B041 The device according to B031 or A032, or any of the embodiments, wherein the separator comprises a gel that selectively passes the target substance. B042 The device according to B041, or any of the embodiments, wherein the gel is a polyacrylamide gel. B051 The device according to B031 or B032, or any of the embodiments, wherein the separator comprises a semipermeable membrane that selectively passes the target substance. B061 The device of any one of B001 to B051 or any embodiment, wherein the separator, the first solution, or the first solution chamber (input chamber) comprises a selectivity substance capable of allowing the target substance to selectively pass through the separator and / or inhibiting, avoiding, or blocking at least one contaminant from passing through the separator. B062 The device of any one of B031 to B047 or any embodiment, wherein the selectivity substance is mixed with the first solution prior to the electrophoresis. B063 The device of B051 or B052 or any embodiment, wherein the selectivity substance is a resin for protein purification. B064 A method of the device according to any one of B051 to B053 or any embodiment, wherein the selectivity substance or the protein purification resin is selected from the group consisting of hydroxyapatite, a cation exchange carrier, and an anion exchange carrier.B081 An apparatus (device) for processing a target substance by electrophoresis, comprising: a first solution chamber (input chamber); a second solution chamber (first elution chamber) adjacent to the first solution chamber (input chamber); a third solution chamber (second elution chamber) adjacent to the second solution chamber (first elution chamber); a first separator that separates the first solution chamber from the second solution chamber and allows the target substance to pass from the first solution chamber to the second solution chamber (and has electrical conductivity); and a second separator that separates the second solution chamber from the third solution chamber and allows the target substance to pass from the second solution chamber to the third solution chamber (and has electrical conductivity).

[0177] C001 A method for processing a target substance by electrophoresis, comprising: providing an electrophoresis device according to any one of embodiments B001 to B064, or any embodiment; filling the first solution chamber with a first solution containing the target substance; filling the second solution chamber with a second solution; and applying an electric field from outside the first solution chamber and the second solution chamber, thereby selectively moving the target substance from the first solution chamber through the separator to the second solution chamber by electrophoresis. C081 A method for processing a target substance by electrophoresis, comprising: providing an electrophoresis device as described in B081 or any of the embodiments; filling the first solution chamber with a first solution containing the target substance; filling the second solution chamber with a second solution; filling the third solution chamber with a third solution; 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 through the first separator to the second solution chamber by electrophoresis; and applying an electric field from outside the second solution chamber and the third solution chamber to move the target substance from the second solution chamber through the second separator to the third solution chamber by electrophoresis.

[0178] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the above specification, the descriptions and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is therefore contemplated that the present invention shall encompass all such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for processing a target substance by electrophoresis, comprising: providing a separator that allows passage of the target substance, the separator comprising a gel; introducing a first solution containing the substance of interest onto a first side of the separator; introducing a second solution onto a second side of the separator; and selectively transferring the target substance from the first solution through the separator to the second solution by electrophoresis; A method for providing the above.

2. 10. The method of claim 1, The method for treating the target substance comprises: concentrating said substance of interest; exchanging the buffer of the subject material; and Purifying the target substance. Doing multiple things at the same time, method.

3. 3. The method of claim 2, said simultaneously performing comprises performing in one dimension electrophoresis; method.

4. 10. The method of claim 1, the second volume of the second solution is less than the first volume of the first solution; method.

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

6. 6. The method of claim 5, The counter substance has an opposite charge to the target substance. method.

7. 10. The method of claim 1, the first solvent (first buffer) of the first solution is different from the second solvent (second buffer) of the second solution; method.

8. 10. The method of claim 1, The separator selectively passes the target substance or allows the target substance to selectively pass through. method.

9. (delete)

10. 10. The method of claim 1, The gel is a polyacrylamide gel. method.

11. 9. The method of claim 8, The separator includes a semipermeable membrane that selectively allows the target substance to pass through. method.

12. 10. The method of claim 1, The separator or the first solution chamber (input chamber) is provided with a selective substance capable of selectively allowing the target substance to pass through the separator and / or inhibiting, avoiding, or blocking the passage of at least one impurity through the separator. method.

13. 10. The method of claim 1, further comprising mixing a selectivity substance into the first solution prior to the electrophoresis. method.

14. 14. The method of claim 12 or 13, The selectivity agent has the ability to purify a protein. method.

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

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

17. 17. The method of claim 16, The pH of the first solution is equal to or higher than the isoelectric point of at least one of the contaminants contained in the first solution; method.

18. 10. The method of claim 1, The first solution is a biologically derived solution. method.

19. 20. The method of claim 18, The first solution is a crude sample. method.

20. 20. The method of claim 18 or 19, The first solution is a biologically derived solution selected from the group consisting of blood, saliva, tears, urine, and interstitial fluid. method.

21. 21. The method of claim 20, the target substance is a biomolecule contained in the first solution; method.

22. 22. The method of claim 21, the target substance is albumin contained in the first solution; method.

23. A device for processing a target substance by electrophoresis, comprising: First solution chamber; a second solution chamber adjacent to the first solution chamber; and a separator that separates the first solution chamber from the second solution chamber, allows a target substance to pass from the first solution chamber to the second solution chamber, and is electrically conductive, the separator comprising a gel; Equipped with 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 migrate the target substance from the first solution chamber through the separator to the second solution chamber by electrophoresis; Devices configured to:

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

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

26. 25. A device according to claim 23 or 24, The separator selectively passes the target substance or allows the target substance to selectively pass through. device.

27. (delete)

28. 27. The device of claim 26, The separator includes a semipermeable membrane that selectively allows the target substance to pass through. device.

29. 25. A device according to claim 23 or 24, the separator or the first solution chamber comprises a selective material capable of selectively allowing the target substance to pass through the separator and / or inhibiting, avoiding, or blocking the passage of at least one contaminant through the separator; device.

30. 25. A device according to claim 23 or 24, configured to mix the selectivity substance with a first solution prior to the electrophoresis; device.

31. A device for processing a target substance by electrophoresis, comprising: 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 separates the first solution chamber from the second solution chamber, allows a target substance to pass from the first solution chamber to the second solution chamber, and is electrically conductive, the separator comprising a gel; and a second separator that separates the second solution chamber from the third solution chamber, allows the target substance to pass from the second solution chamber to the third solution chamber, and is electrically conductive, the second separator comprising a gel; 1. A device comprising:

32. 1. A method for processing 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 substance of interest; filling the second solution chamber with a second solution; and applying an electric field from outside the first solution chamber and the second solution chamber; selectively transferring the target substance from the first solution chamber through the separator to the second solution chamber by electrophoresis; A method for providing the above.