Immunosensors, methods for regenerating antibodies

Abstract

To provide an immunosensor that can be repeatedly and continuously used and can be reduced in a size, and an antibody recycling method in the immunosensor.SOLUTION: An immunosensor 1 comprises: a recycling unit 2 that has at least one flow channel 21, at least one liquid storage unit 22, an electrolytic film 23 that is interposed between the flow channel 21 and liquid storage unit 22, a first electrode 24 that is arranged in the flow channel 21, and a second electrode 25 that is arranged in the liquid storage unit 22; voltage application means 3 that applies a voltage to the two electrodes; an antibody 4 that is arranged in the flow channel 21; and detection means 5 that detects an antigen 110 captured by the antibody 4. The flow channel 21 and the liquid storage unit 22 are adjacent and joined to each other with the electrolytic film 23 therebetween in an area 7. The flow channel 21 is a flow channel that causes a sample solution 120 including the antigen 110 to flow, and the liquid storage unit 22 is a container or a flow channel that stores liquid 130.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an immunosensor and a method for regenerating antibodies in an immunosensor. [Background technology]

[0002] To realize advanced preventive medicine, smartwatches and other devices incorporating immunosensors are being developed as wearable devices. An immunosensor is a biosensor that uses a protein (antibody) that specifically binds to a particular biomarker (antigen). Immunosensors detect specific proteins in blood, etc., using antigen-antibody reactions. In an immunosensor, a primary antibody is immobilized on a substrate beforehand. Then, a sample such as a liquid containing an antigen is passed through, and the antigen is captured by the primary antibody. Furthermore, a secondary antibody, which is a labeling substance, is bound to the antigen, sandwiching the antigen between the primary and secondary antibodies. In this state, the specific protein (antigen) in the sample is quantified by measuring the absorbance, fluorescence intensity, and mass change of the secondary antibody (label), as well as the absorbance and oxidation-reduction current of the reaction product between the labeling substance (enzyme) and the substrate.

[0003] Antigens detected by wearable devices with built-in immunosensors include, for example, lifestyle-related disease markers. Tumor markers that suggest the presence of cancer include, for example, CEA and AFP (alpha-fetoprotein). Biomarkers that suggest heart disease include, for example, oxidized LDL and C-reactive protein (CRP). Biomarkers that suggest dementia include, for example, amyloid-beta.

[0004] In Japan, which has become a super-aging society, the current medical system, which relies on symptomatic treatment, is reaching its limits. Therefore, there is a need to build a healthy and long-lived society, prevent lifestyle-related diseases, and move away from symptomatic treatment. As a means of solving these problems, wearable devices with built-in immunosensors that can monitor the health of the user (wearer) on a daily basis are considered to be effective.

[0005] Representative immunosensors include, for example, sensors using ELISA (a method for quantifying trace amounts of biological substances using antigen-antibody reactions), sensors using immunochromatography such as pregnancy tests, and sensors using surface plasmon resonance (SPR) (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mazher-Iqbal Mohammed, Marc PY Desmulliez, “Lab-on-a-chip based immunosensor principles and techniques for the detection of cardiac biomarkers: a review”, Lab on a chip 2011, 11, 569-595. [Overview of the project] [Problems that the invention aims to solve]

[0007] Conventional immunosensors are essentially disposable and cannot be used continuously. This is because, after detecting an antigen, it was necessary to dissociate the antigen from the antibody and regenerate the immunosensor by delivering a regeneration solution (such as acid or alkali) into the immunosensor using a liquid delivery pump or valve. This method of dissociating the antigen contributed to the complexity and size of the immunosensor, as well as the consumption of reagents (need for replacement). In other words, conventional regeneration methods hindered the miniaturization of immunosensors.

[0008] The present invention has been made in view of the above circumstances, and aims to provide an immunosensor that can be used repeatedly and continuously and can be further miniaturized, and a method for regenerating antibodies in the immunosensor. [Means for solving the problem]

[0009] The present invention has the following aspects. [1] A regeneration unit having at least one flow channel, at least one liquid containment section, an electrolytic film interposed between the flow channel and the liquid containment section, a first electrode disposed in the flow channel, and a second electrode disposed in the liquid containment section. Voltage applying means for applying voltage to the first electrode and the second electrode, The antibody placed in the aforementioned channel, The system comprises a detection means for detecting the antigen captured by the antibody, The flow channel and the liquid containment section are joined to each other in adjacent regions via the electrolytic film. The aforementioned channel is a channel for flowing a sample solution containing an antigen, and the aforementioned liquid containment section is a container or channel for containing a liquid, wherein the immunosensor is an immunosensor. [2] The immunosensor according to [1], further comprising two or more regeneration units along the longitudinal direction of the flow path. [3] The immunosensor according to [1], further comprising a pH detection unit for detecting the pH of the sample solution present in the region including the first electrode and its vicinity. A method for regenerating antibodies in an immunosensor as described in any of [4][1] to [3], A method for regenerating an antibody, comprising: applying a voltage to the first electrode and the second electrode using the voltage application means; moving hydrogen ions or hydroxide ions contained in the liquid in the liquid containment section to the sample solution in the channel via the electrolytic membrane; or moving hydrogen ions or hydroxide ions contained in the sample solution in the channel to the liquid in the liquid containment section; thereby controlling the pH of the sample solution present in the channel, including the first electrode and its surrounding area. [5] The method for regenerating an antibody according to [4], wherein the pH of the sample solution present in the region including the first electrode and its vicinity is controlled to be between 1 and 3.5. [6] The method for regenerating an antibody according to [4], wherein the pH of the sample solution present in the region including the first electrode and its vicinity is controlled to 10 or higher. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide an immunosensor that can be repeatedly and continuously used and can be further miniaturized, and a method for regenerating an antibody in the immunosensor.

Brief Description of the Drawings

[0011] [Figure 1] FIG. 1 shows a schematic configuration of an immunosensor according to an embodiment of the present invention, and is a schematic diagram showing a cross-section along the height direction of the immunosensor. [Figure 2] FIG. 2 shows a schematic configuration of a first modification of the immunosensor according to an embodiment of the present invention, and is a schematic diagram showing a cross-section of the immunosensor. [Figure 3] FIG. 3 shows a schematic configuration of a second modification of the immunosensor according to an embodiment of the present invention, and is a schematic diagram showing a cross-section of the immunosensor. [Figure 4] FIG. 4 shows a schematic configuration of a third modification of the immunosensor according to an embodiment of the present invention, and is a schematic diagram showing a cross-section of the immunosensor. [Figure 5] FIG. 5 shows a schematic configuration of a fourth modification of the immunosensor according to an embodiment of the present invention, and is a schematic diagram showing a cross-section of the immunosensor. [Figure 6] FIG. 6 shows a schematic configuration of a fifth modification of the immunosensor according to an embodiment of the present invention, and is a schematic diagram showing a cross-section of the immunosensor.

Embodiments for Carrying Out the Invention

[0012] Embodiments of the immunosensor of the present invention and a method for regenerating an antibody in the immunosensor will be described. Note that this embodiment is specifically described to better understand the gist of the invention, and does not limit the present invention unless otherwise specified.

[0013] [Immunosensor] FIG. 1 shows a schematic configuration of an immunosensor according to an embodiment of the present invention, and is a schematic diagram showing a cross-section along the height direction of the immunosensor. As shown in Figure 1, the immunosensor 1 of this embodiment comprises a regeneration unit 2, a voltage application means 3, an antibody 4, and a detection means 5. The regeneration unit 2 includes a flow path 21, a liquid containment unit 22, an electrolytic film 23, a first electrode 24, and a second electrode 25. Furthermore, the immunosensor 1 in this embodiment may include a substrate 6 that supports the regeneration unit 2.

[0014] In this embodiment, the flow path 21 and the liquid containment section 22 are arranged adjacent to each other. Flow channel 21 is a channel through which the sample solution 120 containing the antigen 110 flows. The liquid storage section 22 is a container or flow path that holds the liquid 130.

[0015] The electrolytic film 23 is interposed between the flow channel 21 and the liquid containment section 22. More specifically, as shown in Figure 1, the flow channel 21 and the liquid containment section 22 are joined via the electrolytic film 23 in adjacent regions 7. In other words, it is sufficient that parts of the flow channel 21 and the liquid containment section 22 are joined to each other via the electrolytic film 23 in region 7, but they do not need to be joined in other parts, and they do not need to be adjacent in other parts.

[0016] The first electrode 24 is positioned within the flow channel 21. Preferably, the first electrode 24 is positioned within the flow channel 21 facing the electrolytic film 23. The second electrode 25 is located within the liquid containment section 22. The position of the second electrode 25 within the liquid containment section 22 is not particularly limited, but it is preferable that the second electrode 25 be located opposite the electrolytic film 23, and more preferably opposite the first electrode 24. Whether the first electrode 24 and the second electrode 25 are positioned opposite each other or not, there is almost no difference in the antibody 4 regeneration ability of the immunosensor 1.

[0017] The voltage application means 3 is connected to the first electrode 24 and the second electrode 25, and applies a voltage to these two electrodes to generate an electric field E from the second electrode 25 (liquid containment section 22) toward the first electrode 24 (flow channel 21), or an electric field E from the first electrode 24 (flow channel 21) toward the second electrode 25 (liquid containment section 22).

[0018] In this embodiment, the antibody 4 is immobilized on the surface 24a of the first site of the first electrode 24.

[0019] The detection means 5 detects the antigen 110 captured by the antibody 4.

[0020] [Flow path] The channel 21 is not particularly limited as long as it can carry the sample solution 120 containing the antigen 110, but it is preferably a microchannel formed on the channel substrate 8 by, for example, hydrogel molding, photolithography, soft lithography, cutting, joining, or a combination of these microprocessing methods. In other words, the channel 21 is preferably a microchannel formed on the channel substrate 8 provided on one surface 6a of the substrate 6. The microchannel is a channel with an inner diameter (maximum diameter) on the order of nanometers to millimeters. Because the channel 21 is a microchannel, the antigen 110 can be detected with high sensitivity by the immunosensor 1. Furthermore, the antigen 110 bound to the antibody 4 can be easily dissociated by the regeneration method described later. Examples of the flow channel substrate 8 include silicone rubber, various resins other than silicone rubber, glass, and the like.

[0021] [Liquid storage section] The liquid containment section 22, like the flow path 21, is a container or flow path formed on the flow path substrate 8 by a microfabrication method such as hydrogel molding, photolithography, soft lithography, cutting, joining, or a combination of these microfabrication methods. In other words, the liquid containment section 22 is a container or flow path formed on the flow path substrate 8 provided on one surface 6a of the substrate 6.

[0022] [Electrolytic membrane] The electrolytic membrane 23 is a porous membrane having numerous pores with an inner diameter (maximum diameter) on the order of nanometers, and contains hydrogen ions (H) contained in the liquid 130 in the liquid containment section 22. + ) or hydroxide ion (OH - ) can pass through, and hydrogen ions (H) contained in the sample solution 120 + ) or hydroxide ion (OH - It is an ion exchange membrane that can permeate ions. The material of the electrolytic film 23 is hydrogen ions (H) contained in the liquid 130 in the liquid containment section 22. + ) or hydroxide ion (OH - ) can pass through, and hydrogen ions (H) contained in the sample solution 120 + ) or hydroxide ion (OH - The material is not particularly limited as long as it can permeate ), but examples include Nafion®, polyethersulfone, polyvinylidene fluoride, polyurethane, polyamide-imide, and polyimide.

[0023] [1st electrode] The first electrode 24 is not particularly limited, as long as it can immobilize the antibody 4 on its surface 24a and does not degrade due to the sample solution 120 and ions such as hydrogen ions and hydroxide ions. Examples of the first electrode 24 include metal electrodes made of metals such as gold and platinum, and carbon electrodes.

[0024] [Second electrode] The second electrode 25 is not particularly limited as long as it does not degrade due to the liquid 130 and ions such as hydrogen ions and hydroxide ions. Examples of the second electrode 25 include metal electrodes made of metals such as gold and platinum, and carbon electrodes.

[0025] [Voltage application means] The voltage application means 3 is not particularly limited as long as it can apply voltage to the first electrode 24 and the second electrode 25. Examples of the voltage application means 3 include a voltage generator and a voltage-current generator.

[0026] [antibody] Antibody 4 is not particularly limited as long as it can capture antigen 110 contained in sample solution 120, but examples include anti-CEA antibody, anti-AFP (α-fetoprotein) antibody, antioxidant LDL antibody, anti-C-reactive protein (CRP) antibody, anti-amyloid β antibody, etc.

[0027] [Detection means] The detection means 5 is selected according to the application of the immunosensor 1, that is, the type of antigen 110 to be detected by the immunosensor 1. To detect the antigen 110, methods are used in which a secondary antibody, which is a labeling substance, is bound to the antigen 110 captured by the antibody (primary antibody) 4, or in which a secondary antibody, which is a labeling substance, is not used. In methods using secondary antibodies, the detection means 5 is one that can perform a detection method appropriate to the secondary antibody. Examples of detection means 5 include a device that measures the fluorescence emitted by the secondary antibody (labeled), a device that measures the absorbance of the substance produced by the reaction of the secondary antibody (labeled) and the substrate, and a device that measures the oxidation-reduction current of the substance produced by the reaction of the secondary antibody (labeled) and the substrate. Methods that do not use secondary antibodies, i.e., methods that directly measure the amount of antigen 110 bound to antibody 4, include, for example, surface plasmon resonance (SPL) spectroscopy, quartz crystal microbalance spectroscopy, electrochemical impedance spectroscopy, and surface acoustic wave spectroscopy. Figure 1 illustrates a case in which the amount of antigen 110 bound to antibody 4 is directly measured by surface plasmon resonance spectroscopy. In this case, the detection means 5 includes a light source 51 that irradiates the first electrode 24 with white light, and a detector 52 that detects the amount by which the wavelength of light attenuated by surface plasmon resonance changes due to the binding of antigen 110 and antibody 4.

[0028] [substrate] The substrate 6 is not particularly limited as long as it can be provided with a flow channel substrate 8 on one of its surfaces 6a, but examples include glass substrates, resin substrates, etc.

[0029] [antigen] The antigen 110 is not particularly limited. For example, tumor markers suggesting the presence of cancer such as CEA and AFP (α-fetoprotein), biomarkers suggesting heart diseases such as oxidized LDL and C-reactive protein (CRP), and biomarkers suggesting dementia such as amyloid β can be mentioned.

[0030] [Sample solution] The sample solution 120 is a solution containing the antigen 110. Specifically, examples of the sample solution 120 include body fluids such as blood, interstitial fluid, sweat, and saliva, and physiological saline such as phosphate-buffered saline and Tris-buffered saline.

[0031] [Liquid] The liquid 130 is not particularly limited as long as hydrogen ions (H + ) or hydroxide ions (OH - ) are generated when a voltage is applied to the first electrode 24 and the second electrode 25 by the voltage application means 3. For example, water, an aqueous sodium chloride solution, an aqueous copper sulfate solution, ethanol, etc. can be mentioned.

[0032] [Method of using the immunosensor] The method of using the immunosensor 1 of the present embodiment will be described.

[0033] "The first method of use" Flow the sample solution 120 containing the antigen 110 through the flow path 21. When the sample solution 120 is flowed through the flow path 21, the antigen 110 contained in the sample solution 120 is captured by the antibody 4 immobilized on the first electrode 24. Next, the antigen 110 is qualitatively or quantitatively analyzed by the detection means 5. Specifically, white light is irradiated from the light source 51 to the first electrode 24, and the detector 52 detects the amount by which the wavelength of the light attenuated by surface plasmon resonance changes due to the binding of the antigen 110 and the antibody 4, thereby qualitatively or quantitatively analyzing the antigen 110. After the detection of the antigen 110 is completed, the liquid 130 is injected into the liquid storage part 22. Note that the liquid 130 may be injected into the liquid storage part 22 simultaneously with flowing the sample solution 120 containing the antigen 110 through the flow path 21. Next, the voltage application means 3 applies a voltage to the first electrode 24 and the second electrode 25, generating an electric field E from the second electrode 25 toward the first electrode 24. This then causes hydrogen ions (H) contained in the liquid 130 in the liquid containment section 22 to be transmitted through the electrolytic film 23. + Either the hydroxide ions (OH) in the sample solution 120 in the channel 21 move into the sample solution 120 in the channel 21, or the hydroxide ions (OH) contained in the sample solution 120 in the channel 21 move into the sample solution 120 in the channel 21. - The antigen 110 captured by the antibody 4 moves to the liquid 130 in the liquid containment section 22. As a result, the pH of the sample solution 120 in region α, including the first electrode 24 and its surroundings, in the flow path 21 becomes less than 7 (acidic). Consequently, the acidic sample solution 120 causes the antigen 110 captured by the antibody 4 to dissociate, and the antibody 4 is regenerated to a state where the antigen 110 is not bound. This makes it possible for the antibody 4 to capture the antigen 110 contained in the sample solution 120 again.

[0034] "Second method of use" Similar to the first method of use, when a sample solution 120 containing the antigen 110 is flowed through the channel 21, the antigen 110 contained in the sample solution 120 is captured by the antibody 4 immobilized on the first electrode 24. Next, the antigen 110 is qualitatively or quantitatively determined by the detection means 5, similar to the first method of use. After the detection of antigen 110 is complete, liquid 130 is injected into the liquid container 22, in the same manner as in the first method of use. Next, the voltage application means 3 applies a voltage to the first electrode 24 and the second electrode 25, generating an electric field E from the first electrode 24 to the second electrode 25. This generates hydroxide ions (OH) contained in the liquid 130 in the liquid containment section 22, which are transmitted through the electrolytic film 23. - ) moves into the sample solution 120 in the channel 21, or hydrogen ions (H) contained in the sample solution 120 in the channel 21 move into the sample solution 120 in the channel 21. +The antigen 110 moves into the liquid 130 in the liquid containment section 22. As a result, the pH of the sample solution 120 in region α, including the first electrode 24 and its surroundings, in the flow path 21 becomes greater than 7 (alkaline). Consequently, the acidic sample solution 120 dissociates the antigen 110 captured by the antibody 4, and the antibody 4 is regenerated to a state where the antigen 110 is not bound. This makes it possible for the antibody 4 to capture the antigen 110 contained in the sample solution 120 again.

[0035] According to the immunosensor 1 of this embodiment, as in the first method described above, the voltage application means 3 applies a voltage to the first electrode 24 and the second electrode 25, generating an electric field E directed from the second electrode 25 to the first electrode 24, thereby making the pH of the sample solution 120 present in the region α including the first electrode 24 and its surroundings within the channel 21 less than 7. As a result, the acidic sample solution 120 dissociates the antigen 110 captured by the antibody 4, allowing the antibody 4 to be regenerated into a state where the antigen 110 is not bound. Furthermore, according to the immunosensor 1 of this embodiment, as in the second method described above, the voltage application means 3 applies a voltage to the first electrode 24 and the second electrode 25, generating an electric field E directed from the first electrode 24 to the second electrode 25, thereby making the pH of the sample solution 120 present in the region α including the first electrode 24 and its surroundings within the channel 21 greater than 7. As a result, the alkaline sample solution 120 can dissociate the antigen 110 captured by the antibody 4, thereby regenerating the antibody 4 to a state where the antigen 110 is not bound. Furthermore, unlike conventional methods, there is no need to use a liquid delivery pump or valve to deliver a regeneration solution such as acid or alkali into the channel 21 where the antibody 4 is located, thus enabling easy regeneration of the antibody 4. Consequently, the immunosensor 1 can be used repeatedly and continuously without being disposable. In addition, since antibody 4 can be regenerated without the use of large-scale equipment, the immunosensor 1 can be miniaturized.

[0036] [Other embodiments] The present invention is not limited to the embodiments described above. The immunosensor of the present invention may employ, for example, the modified forms shown in Figures 2 to 5. In the figures showing each modified form, the same reference numerals are used for parts that are the same as those in the embodiments described above, and their descriptions are omitted; only the differences are described.

[0037] [First variation] As shown in Figure 2, in the first modified immunosensor 200, the antibody 4 is located in a region separate from the first electrode 24 within the channel 21. Furthermore, the antibody 4 is not immobilized on the surface 24a of the first electrode 24. In the immunosensor 300, since the antibody 4 is located in a region separate from the first electrode 24 within the channel 21, the waveguide mode detection method can be applied to detect the antigen 110.

[0038] [Second variation] As shown in Figure 3, the third modified immunosensor 300 may have a configuration in which the channel 21 (channel 21A), the liquid containment section 22, and the channel 21 (channel 21B) are arranged adjacent to each other in this order. In the second modified immunosensor 300, the liquid containment section 22 is sandwiched between the channels 21A and 21B. Furthermore, the channel 21A and the liquid containment section 22 are joined via an electrolytic membrane 23. In addition, the channel 21B and the liquid containment section 22 are joined via an electrolytic membrane 23. According to the second modified immunosensor 300, the antibodies in channel 21A and the antibodies in channel 21B can be regenerated using the liquid 130 in the liquid containment section 22. Therefore, antibody regeneration can be performed more efficiently. Note that the regeneration of antibodies in channel 21A and the regeneration of antibodies in channel 21B may be performed simultaneously or individually. Furthermore, in the second modified immunosensor 300, the first electrode 24 may also have a first site 24A for immobilizing the antibody 4 and a second site 24B that functions to control the pH of the sample solution 120. Also, in the second modified immunosensor 300, the antibody 4 may be provided in a region separate from the first electrode 24 within the flow channel 21, and the antibody 4 may not be immobilized on the surface 24a of the first electrode 24.

[0039] [Third variation] As shown in Figure 4, the fourth modified immunosensor 400 may have two sets of channel sets 410, each consisting of one channel 21 and one liquid storage section 22. The third modified immunosensor 400 includes a first set 410A consisting of a channel 21A and a liquid storage section 22A joined via an electrolytic membrane 23, and a second set 410B consisting of a channel 21B and a liquid storage section 22B joined via an electrolytic membrane 23. Furthermore, the liquid storage sections 22A of the first set 410A and the channel 21B of the second set 410B, which are adjacent to each other, are joined via the electrolytic membrane 23. That is, in the third modified immunosensor 400, the channel 21A, liquid storage section 22A, channel 21B, and liquid storage section 22B are arranged adjacent to each other in this order. According to this embodiment, the antibodies in channel 21A and channel 21B can be regenerated using the liquid 130 in the liquid storage section 22A. Therefore, antibody regeneration can be performed more efficiently. The regeneration of antibodies in channel 21A and in channel 21B may be performed simultaneously or individually. Furthermore, the antibodies in channel 21B can be regenerated using at least one of the liquid 130A in liquid container 22A and the liquid 130B in liquid container 22B. Therefore, by using different types of liquids for liquid 130A in liquid container 22A and liquid 130B in liquid container 22B, antibody regeneration in channel 21B can be performed in stages. In addition, in the third modified immunosensor 400, the first electrode 24 may have a first site 24A for immobilizing the antibody 4 and a second site 24B that functions to control the pH of the sample solution 120. Also, in the third modified immunosensor 400, the antibody 4 may be provided in a region separate from the first electrode 24 in channel 21, and the antibody 4 may not be immobilized on the surface 24a of the first electrode 24.

[0040] [Fourth variation] As shown in Figure 5, the immunosensor 500 of the fourth modified example may have two or more regeneration units 2 along the longitudinal direction of the flow path 21. The longitudinal direction of the flow path 21 is the direction in which the sample solution 120 flows within the flow path 21. In the immunosensor 500 of the fourth modified example, for example, the sample solution 120 is flowed in the direction of the arrow shown in Figure 5, perpendicular to the height direction of the immunosensor 500. In this case, the sample solution 120 first comes into contact with the antibody 4 immobilized on the first electrode 24, and then comes into contact with the antibody 4 immobilized on the first electrode 24. Here, if different antibodies 4 are used as the antibodies 4 immobilized on the first electrode 24, each antibody 4 can capture different antigens contained in the sample solution 120. Furthermore, the regeneration of the antibody 4 in the regeneration unit 2A having the first electrode 24 and the regeneration of the antibody 4 in the regeneration unit 2B having the first electrode 24 can be performed separately. Therefore, one immunosensor 500 can repeatedly and continuously detect two or more antigens. In addition, in the immunosensor 500 of the fourth modified example, the first electrode 24 may have a first site 24A for immobilizing the antibody 4 and a second site 24B that functions to control the pH of the sample solution 120. In addition, in the immunosensor 500 of the fourth modified example, the antibody 4 may be provided in a region separate from the first electrode 24 in the flow channel 21, and the antibody 4 may not be immobilized on the surface 24a of the first electrode 24.

[0041] [Fifth variation] As shown in Figure 6, the fifth modified immunosensor 600 may include a pH detection unit 610 for detecting the pH of the sample solution 120 present in the region α including the first electrode 24 and its surroundings. The pH detection unit 610 is connected to the voltage application means 3. Examples of the pH detection unit 610 include a pH meter. By including the pH detection unit 610, it is not necessary to use a pH indicator to detect the pH of the sample solution 120. Furthermore, by including the pH detection unit 610, the pH of the sample solution 120 can be detected immediately, and the pH of the sample solution 120 can be more easily controlled by adjusting the voltage applied to the first electrode 24 and the second electrode 25 by the voltage application means 3. Note that the first to fourth modified versions may also include a pH detection unit.

[0042] [Methods for regenerating antibodies] The antibody regeneration method according to one embodiment of the present invention is the antibody regeneration method in the immunosensor according to the above-mentioned embodiment of the present invention.

[0043] "The first method of regeneration" In this embodiment, the antibody regeneration method involves, for example, applying a voltage to the first electrode 24 and the second electrode 25 using the voltage application means 3 in the immunosensor 1, generating an electric field E from the second electrode 25 toward the first electrode 24, thereby moving hydrogen ions contained in the liquid 130 in the liquid containment section 22 to the sample solution 120 in the channel 21 via the electrolytic membrane 23, or moving hydroxide ions contained in the sample solution 120 in the channel 21 toward the liquid 130 in the liquid containment section 22, thereby controlling the pH of the sample solution 120 in the channel 21, including the first electrode 24 and its surroundings, to less than 7.

[0044] The voltage applied to the first electrode 24 and the second electrode 25 is not particularly limited, but is preferably between 1V and 5V, and more preferably between 2V and 4V. If the voltage is less than 1V, the amount of ion movement is small, making it difficult to adequately adjust the pH of the sample solution 120 in the region α including the first electrode 24 and its surroundings. If the voltage exceeds 5V, the amount of gas generated near the first electrode 24 increases, increasing the risk of damage to the antibody 4.

[0045] The time for which voltage is applied to the first electrode 24 and the second electrode 25 is not particularly limited, but is preferably 1 minute or more and 10 minutes or less, and more preferably 1 minute or more and 5 minutes or less.

[0046] Within the flow path 21, it is preferable to control the pH of the sample solution 120 in region α, including the first electrode 24 and its surroundings, to 1-3.5, and more preferably to 2-3. By controlling the pH of the sample solution 120 to 1-3.5, the dissociation of the antigen 110 from the antibody 4 can be further promoted.

[0047] The sample solution 120 may contain a pH indicator. The pH indicator is not particularly limited as long as it can indicate a range from alkaline (pH 14) to acidic (pH 1), but examples include thymol blue, methyl red, and phenolphthalein. Thymol blue is yellow in neutral solutions and changes to red in acidic solutions. Thymol blue is yellow at pH 3, and the closer it is to red, the lower the pH and the higher the acidity. If the sample solution 120 contains a pH indicator, the pH of the sample solution 120 present in region α including the first electrode 24 and its surroundings can be confirmed by applying voltage to the first electrode 24 and the second electrode 25. The pH indicator allows for a decision on whether to continue applying voltage to the first electrode 24 and the second electrode 25 or to stop applying voltage to the first electrode 24 and the second electrode 25.

[0048] Furthermore, the immunosensor 1 may include a pH detection unit, such as a pH meter, for detecting the pH of the sample solution 120 present in region α including the first electrode 24 and its surroundings. If a pH detection unit is included, the pH of the sample solution 120 present in region α including the first electrode 24 and its surroundings can be confirmed by applying voltage to the first electrode 24 and the second electrode 25. The pH detection unit can immediately determine whether to continue applying voltage to the first electrode 24 and the second electrode 25 or to stop applying voltage to the first electrode 24 and the second electrode 25.

[0049] According to the antibody regeneration method of this embodiment, by applying voltage to the first electrode 24 and the second electrode 25 and controlling the pH of the sample solution 120 present in the region α including the first electrode 24 and its surroundings to less than 7, the antigen 110 captured by the antibody 4 can be dissociated, and the antibody 4 can be regenerated to a state in which the antigen 110 is not bound. Furthermore, unlike conventional methods, there is no need to deliver a regeneration solution such as acid or alkali into the flow path 21 where the antibody 4 is located using a liquid delivery pump or valve, thus enabling easy regeneration of the antibody 4. Consequently, the immunosensor 1 can be used repeatedly and continuously. In addition, antibody 4 can be regenerated without using large-scale equipment.

[0050] "The second method of regeneration" In this embodiment, the antibody regeneration method involves applying a voltage to the first electrode 24 and the second electrode 25 using the voltage application means 3, generating an electric field E from the first electrode 24 to the second electrode 25, thereby moving hydroxide ions contained in the liquid 130 in the liquid containment section 22 to the sample solution 120 in the flow channel 21 via the electrolytic membrane 23, or moving hydrogen ions contained in the sample solution 120 in the flow channel 21 to the liquid 130 in the liquid containment section 22, thereby controlling the pH of the sample solution 120 in the region α including the first electrode 24 and its surroundings within the flow channel 21 to be greater than 7.

[0051] The voltage applied to the first electrode 24 and the second electrode 25 is not particularly limited, but is preferably between 1V and 5V, and more preferably between 2V and 4V. If the voltage is less than 1V, the amount of ion movement is small, making it difficult to adequately adjust the pH of the sample solution 120 in the region α including the first electrode 24 and its surroundings. If the voltage exceeds 5V, the amount of gas generated near the first electrode 24 increases, increasing the risk of damage to the antibody 4.

[0052] The time for which voltage is applied to the first electrode 24 and the second electrode 25 is not particularly limited, but is preferably 1 minute or more and 10 minutes or less, and more preferably 1 minute or more and 5 minutes or less.

[0053] Within the flow path 21, it is desirable to control the pH of the sample solution 120 in region α, including the first electrode 24 and its surroundings, to 10 or higher, more preferably to 10-12, and even more preferably to 11-12. By controlling the pH of the sample solution 120 to 10 or higher and 12, the dissociation of the antigen 110 from the antibody 4 can be further promoted.

[0054] The sample solution 120 may contain a pH indicator. The pH indicator is not particularly limited as long as it can indicate a range from alkaline (pH 14) to acidic (pH 1), but examples include alizarin yellow, thymol blue, methyl red, and phenolphthalein. Alizarin yellow is yellow in neutral solutions and changes to orange in alkaline solutions. Alizarin yellow is yellow at pH 10, and the closer it is to orange, the higher the pH and the higher the alkalinity. If the sample solution 120 contains a pH indicator, the pH of the sample solution 120 in region α, including the first electrode 24 and its surroundings, can be confirmed by applying voltage to the first electrode 24 and the second electrode 25. The pH indicator allows for a decision on whether to continue applying voltage to the first electrode 24 and the second electrode 25 or to stop applying voltage to the first electrode 24 and the second electrode 25.

[0055] In the second regeneration method, as in the second regeneration method, the immunosensor 1 may also include a pH detection unit, such as a pH meter, for detecting the pH of the sample solution 120 present in the region α including the first electrode 24 and its surroundings.

[0056] According to the antibody regeneration method of this embodiment, by applying voltage to the first electrode 24 and the second electrode 25 and controlling the pH of the sample solution 120 present in the region α including the first electrode 24 and its surroundings to more than 7, the antigen 110 captured by the antibody 4 can be dissociated, and the antibody 4 can be regenerated to a state in which the antigen 110 is not bound. Furthermore, as in the conventional method, it is not necessary to deliver a regeneration solution such as acid or alkali into the flow path 21 in which the antibody 4 is located using a liquid delivery pump or valve, so the regeneration of the antibody 4 can be performed simply. Therefore, the immunosensor 1 can be used repeatedly and continuously. In addition, the antibody 4 can be regenerated without using large-scale equipment. [Industrial applicability]

[0057] Because the immunosensor of the present invention can be used repeatedly and continuously, it is possible to miniaturize the immunosensor and realize a wearable device with a built-in immunosensor. [Explanation of Symbols]

[0058] 1,200,300,400,500,600 Immunosensor 2 Playback section 3. Voltage application means 4 Antibodies 5. Detection means 6 circuit boards 7 areas 8. Flow channel substrate 21 Flow channels 22 Liquid storage section 23 Electrolytic membrane 24 1st electrode 24A 1st part 24B 2nd part 25 2nd electrode 110 Antigen 120 Sample Solutions 130 liquid

Claims

1. The device comprises at least one channel, at least one liquid containment section, an electrolytic membrane interposed between the channel and the liquid containment section, a first electrode disposed in the channel, a second electrode disposed in the liquid containment section, a voltage applying means for applying a voltage to the first electrode and the second electrode, an antibody disposed in the channel, and a detection means for detecting a physical or chemical amount corresponding to the amount of antigen bound to the antibody. The flow channel and the liquid containment section are joined to each other in adjacent regions via the electrolytic film. The channel is a channel through which a sample solution containing an antigen flows, and the liquid containment section is a container or channel that contains an electrolyte capable of supplying hydrogen ions or hydroxide ions when a voltage is applied by the voltage application means. By applying a voltage between the first electrode and the second electrode using the voltage application means, hydrogen ions or hydroxide ions in the electrolyte in the liquid containment section move to the sample solution in the channel via the electrolytic film, or hydrogen ions or hydroxide ions in the sample solution in the channel move into the liquid containment section. An immunosensor in which, within the flow channel, a change in the pH of the sample solution present in the region including the first electrode and its surroundings causes the antigen captured by the antibody to dissociate, and the antibody is regenerated to a state where the antigen is not bound.

2. The immunosensor according to claim 1, further comprising a pH detection unit for detecting the pH of the sample solution present in a region including the first electrode and its surrounding area.

3. A method for regenerating an antibody in an immunosensor according to claim 1 or 2, A method for regenerating antibodies, comprising: applying a voltage to the first electrode and the second electrode using the voltage application means; moving hydrogen ions or hydroxide ions contained in the liquid in the liquid containment section to the sample solution in the channel via the electrolytic membrane; or moving hydrogen ions or hydroxide ions contained in the sample solution in the channel to the liquid in the liquid containment section; thereby controlling the pH of the sample solution present in the channel, including the first electrode and its surrounding area.

4. The antibody regeneration method according to claim 3, wherein the pH of the sample solution present in the region including the first electrode and its surrounding area is controlled to be between 1 and 3.

5.

5. The method for regenerating an antibody according to claim 3, wherein the pH of the sample solution present in the region including the first electrode and its surrounding area is controlled to 10 or higher.

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