Electrochemical lateral flow immunoassay method, sensor therefor, and method of manufacturing the same

Abstract

The present application aims to provide an electrochemical lateral flow immunoassay method and a sensor used therefor, which can achieve a short reaction time, excellent sensitivity, high-precision quantitative measurement and electrical detection by controlling the flow of a sample solution. The technical solution is that an electrode portion 5, a conductive portion 7 for conducting current from the electrode portion 5, and a connecting portion 12 connected to an electrical detector 4 for detecting the current value are arranged on a support body 2 made of a resin sheet; and a plurality of pad types 8, 10, 11, 13, 15, 16 are arranged in a partially laminated manner on the support body 2, so that the sample solution flows through the plurality of pad types, and the flow is controlled at the position of the electrode portion 5 to perform electrochemical detection. Then, the flow is controlled by a flow rate control pad 15, a flow path portion fiber pad 10, or flow rate control protrusions 27a, 27b, 27c.

Description

Technical Field

[0001] This invention relates to an electrochemical lateral flow immunosensor detection method and its manufacturing method for detecting antigens or antibodies in body fluids, proteins in food raw materials / processed products, etc. Background Technology

[0002] In recent years, "Point of Care Testing" has been implemented in clinics or small hospitals, which involves physical examinations during patient diagnosis. Visual identification (qualitative) products based on blood biochemistry testing systems consisting of dry chemical reagents and small colorimeters for organ classification abnormality diagnosis, and for the diagnosis of infections using immunochromatographic test strips with mucosal fluid or blood samples collected from the pharynx or nose as samples, have also been introduced into circulation.

[0003] Immunochromatographic test strips are used to detect a test substance in a sample through an antigen-antibody reaction. An immunochromatographic test strip is an article in which an antibody or antigen, which is the test substance, is immobilized on an insoluble in vivo membrane carrier serving as the chromatography medium. A detection zone, serving as the stationary phase, is formed. A mobile phase containing a conjugate (detection reagent), i.e., a marker that has bound an antibody or antigen that can bind to the test substance, is used. The test substance reacts specifically with the conjugate in the mobile phase. Furthermore, in the detection zone of the stationary phase, the test substance bound to the conjugate reacts specifically with the antibody or antigen immobilized in the detection zone. Colloidal metal particles such as gold colloid or colored latex particles are typically used as markers, so the presence of the test substance in the sample is detected by the color at the detection zone.

[0004] Regarding the structure of immunochromatographic test strips, as described in Patent Document 1, they are generally configured with: a sample pad for supplying the sample, a coupling pad for the mobile phase (i.e., the coupling agent), an insoluble in vivo membrane carrier that facilitates the development of the composite of the sample and the coupling agent and has a detection section for detection, and an absorption pad for absorbing the sample developed by the insoluble in vivo membrane carrier.

[0005] The inventors of this application disclosed in Patent Document 2 the following method (method for determining a test substance): An active electrode with a first antibody specifically binding to the test substance immobilized is prepared, and a metal particle serving as a marker is formed by binding the second antibody, which specifically binds to the test substance, to the surface of the active electrode. The test substance, i.e., antigen or antibody, and the antibody or antigen already bound to the marker are supplied to the surface of the active electrode to induce an antigen-antibody reaction. Accordingly, an amount of the metal particle corresponding to the test substance in the test solution is accumulated near the surface of the active electrode. The potential of the active electrode is controlled to allow the upper... The metal particles undergo electrochemical oxidation, and the unreacted metal particles bound to the second antibody are removed by dropping an aqueous chloride solution such as NaCl, KCl, or HCl (which also serves as an electrolyte and a washing solution for removing unreacted markers bound to antibodies or antigens) onto the active electrode. This process suppresses noise from the antibody or antigen being measured, or even from impurities in the measurement solution. Under this noise-suppressed state, the current value generated when the oxidized metal microparticles are electrochemically reduced is measured, and the presence or concentration of the test substance is determined based on the current value.

[0006] It should also be noted that Patent Document 2 describes "...the test paper and the printed electrode are aligned such that the working electrode of the printed electrode contacts the determination part", and also describes "a thin sheet 26 with an absorbent pad 25 and an inner membrane 22 disposed on the inner side". Figure 4 As shown in (a), primary antibody 2 is immobilized in a given region on the surface of in vivo membrane 22 to form a determination section (immobilization region) 23. Antibody specifically binding to secondary antibody 4, which is labeled with metal microparticles 5, is immobilized on the surface of in vivo membrane 22 downstream of determination section 23 to form control section 24. Furthermore, it is also described that "at least determination section 23 in in vivo membrane 22 is aligned with the active electrode 1," and "after aligning at least determination section 23 in in vivo membrane 22 with the active electrode 1," "as shown in (a)..." Figure 6 The printed electrode shown is positioned so that the test strip and the printed electrode are aligned in a manner that the electrode contacts the determination section. However, according to the verification results of Patent Document 2, at the aforementioned electrode position, the proportion of the test substance in the test solution that does not show antigen-antibody activity is high, and it cannot be said that the test substance in the test solution can be accurately quantified.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: International Patent Publication 2002037099A1

[0010] Patent Document 2: Japanese Invention Patent No. 5187759 Summary of the Invention

[0011] Technical issues

[0012] However, existing immunochromatographic test strips rely on visually judging the color generated by the reaction between the capture antibody, the marker antibody, and the antigen in the test solution, all coated linearly on the surface of an insoluble in vivo membrane carrier, for qualitative analysis, or using optical instruments for semi-quantitative analysis. This approach suffers from the difficulty in accurately and precisely quantifying the amount of the tested substance. Therefore, there is a desire for a smaller, portable testing device that can perform immediate and accurate quantitative analysis.

[0013] Furthermore, based on the above verification, Patent Document 2 cannot accurately measure speed, flow pattern, flow rate, etc., without controlling the electrode location, and it is also impossible to achieve a structure that allows for simple connection of the measuring device and immediate detection. Consequently, there is no consideration regarding the manufacturing method of such a measuring device (sensor).

[0014] Furthermore, using adhesives to fix the pads is not advisable. In particular, if adhesives are used on the electrode sections (active electrode, counter electrode, and active electrode), the reaction product of the antigen (or antibody) in the sample solution with the antibody (or antigen) bound to the gold colloid cannot react with the antibody (or antigen) immobilized on the electrode (active electrode). Therefore, in principle, adhesives should not be used. Thus, when placing pads on the aforementioned electrode sections, a very difficult problem to solve is ensuring, without using adhesives, the minimum necessary flow rate and time for the antigen-antibody reaction, so that the sample solution can reliably flow to the electrode surface.

[0015] Therefore, the present invention aims to provide an electrochemical lateral-flow immunoassay method and its sensor, which can achieve quantitative determination and electrodetection with short reaction time, excellent sensitivity, and high accuracy by controlling the flow of the sample solution. Furthermore, the present invention also aims to provide a low-cost manufacturing method that allows for the efficient and practical assembly of existing immunochromatographic test strip pads onto a support with printed electrode portions using existing manufacturing equipment for mass production. Further, the present invention aims to provide a lateral-flow immunosensor and its manufacturing method, according to which the pad can be fixed without adhesive when placed onto the support with printed electrode portions, and the sample solution can reliably flow onto the electrode portions.

[0016] Technical solution

[0017] The present invention preferably includes a flow rate control pad on each electrode section, which is used to allow the test substance in the test solution to approach the electrode section as the test solution moves toward the electrode section through the fiber pad of the flow path section, and to release the test solution at an appropriate rate to obtain the necessary minimum reaction time, so as to complete the antigen-antibody reaction (sandwich analysis) at the active electrode.

[0018] According to the present invention, by providing the aforementioned flow control pad corresponding to the position of the electrode portion of the support body made of resin sheet, the flow of the sample solution at the electrode portion position can be controlled to ensure the necessary minimum reaction time, thereby improving the sensitivity of the electrode portion and enabling rapid, simple, and highly accurate quantitative determination. In other words, by providing the aforementioned flow control pad, the flow of the sample solution at the electrode portion position can be controlled. The flow of the sample solution flowing through the pad can be controlled within the minimum reaction time and speed necessary for the antigen-antibody reaction near the electrode surface. Therefore, microflow paths or pumps for this flow control are unnecessary, and a portable, small current detection instrument can be used for highly sensitive, rapid, simple, and highly accurate quantitative determination. Thus, this flow control, maintaining a constant amount and speed of sample solution near the electrode surface while ensuring close contact with the electrode portion, is achieved.

[0019] The sample pad, coupling pad, absorbent pad, and support in this side-flow immunosensor can be manufactured using the same materials as those used in commonly used immunochromatographic test strips, as shown in Patent Document 1.

[0020] However, this invention differs from existing immunochromatographic test strips in that a sample pad, coupling pad, flow path fiber pad, flow rate control pad, and absorbent pad are locally stacked on the electrode-printed support (described later) for manufacturing. Regarding the pads, a portion of the coupling pad is stacked on the upper flow side of the flow path fiber pad, and a portion of the flow rate control pad is stacked on the lower flow side of the flow path fiber pad. Alternatively, the flow path fiber pad can be disposed on top of the electrode portion and stacked on top of that electrode portion. The flow path fiber pad and flow rate control pad are made of a material different from that used in conventional immunochromatography.

[0021] The present invention is characterized in that, unlike the general immunochromatography described above, the flow path fiber pad is configured to contact a portion below the downstream side of the coupling pad, and the flow rate control pad, which ensures the completion of the antigen-antibody reaction (sandwich assay) at the active electrode, is configured on top of the electrode portion, contacting a portion below the downstream side of the flow path fiber pad. Preferably, the flow path fiber pad functions to move the sample solution toward the electrode portion while an immunological reaction occurs in the coupling pad. The coupling pad contains and dries an antibody or antigen that has undergone an immunological reaction with the antigen or antibody in the sample solution, bound to colloidal metal particles such as gold.

[0022] According to the present invention, by means of the above-mentioned flow control pad, the flow rate of the sample solution is controlled from the shortest necessary reaction time required for the antigen-antibody reaction, thereby enabling rapid and accurate electrical detection at the active electrode.

[0023] In addition, an electrolysis / cleaning fluid hole can be provided in the upper box 22 to supply electrolysis / cleaning fluid to the fiber pad of the flow path. Supplying electrolysis / cleaning fluid from the fiber pad of the flow path can more effectively clean unreacted substances.

[0024] It should also be noted that in this electrochemical side-flow immunosensor, even if the electrolyte / cleaning solution is supplied from the sample well, unreacted substances can be cleaned, and measurement results with little difference can be obtained.

[0025] The present invention further preferably uses different antibodies or antigens in this electrochemical side-flow immunosensor to induce an immunological reaction in the test substance bound to the marker. Specifically, the antibody or antigen inducing an immunological reaction in the test substance bound to the marker and the antibody or antigen inducing an immunological reaction in the test substance immobilized on the surface of the active electrode are respectively antibodies or antigens that recognize different sites of the test substance. By using different antibodies or antigens for the marker and the active electrode, specificity and sensitivity are improved. That is, the antibodies used as research or product raw materials are polyclonal antibodies and monoclonal antibodies, which have different manufacturing methods or properties, and are therefore used differently according to their intended use. Monoclonal antibodies can only bind to one recognized site (Epitope) of the target protein, while polyclonal antibodies can bind to multiple Epitopes of the same protein, thus allowing one polyclonal antibody to recognize multiple Epitopes of a protein.

[0026] The present invention also preferably includes a cartridge for housing the aforementioned electrochemical side-flow immunosensor, wherein the cartridge is provided with a flow rate control protrusion for controlling the flow by pressing the flow rate control pad from above to perform a gating action. It should be noted that the above-mentioned objective can be achieved by using one or more flow rate control protrusions, or by pressing partially or completely onto the flow rate control pad.

[0027] According to the present invention, the flow rate control protrusion provided on the tray can control the flow rate control pad provided on the electrode from both above and below, ensuring the minimum flow rate and time necessary for the antigen-antibody reaction, and reliably distributing the sample solution on the surface of the electrode.

[0028] The present invention also preferably includes a tray for housing the electrochemical side-flow immunosensor, with the connecting part protruding outward from the tray, the connecting part being inserted into an electrical measuring device for detecting the current of the electrode, and connected to its circuit to measure the current generated at the electrode.

[0029] According to the present invention, while keeping the lateral flow immunosensor inside the tray, an electrical measuring device for detecting the current of the electrode portion can be inserted into the connecting portion protruding outward, thereby forming a circuit to immediately measure the current generated by the electrode.

[0030] This invention is also an electrochemical side-flow immunosensor, characterized in that an electrode portion, a conductive portion for conducting current from the electrode portion, and a connection portion for connecting to an electrical measuring device for measuring the current value are disposed on a resin sheet support. Furthermore, multiple pads are disposed on the support, allowing the sample solution to flow through these pads, with flow controlled at the electrode portion to ensure the minimum reaction time necessary for electrochemical detection. Preferably, the flow control pad is disposed along the surface of the electrode portion on the support or on the electrode portion of a fiber pad disposed on the electrode portion. The flow rate control pad is used to control the flow mode of the sample solution, including velocity and / or flow rate, within a given range by pressing the upper and lower flow sides of the flow control pad from above with appropriate pressure using a flow control protrusion.

[0031] The present invention preferably includes the following steps: forming an active electrode, a reference electrode, a counter electrode, and a connecting portion for connecting the conductive portion for conducting current to an electrical measuring device by printing conductive carbon on a resin sheet support; further, printing silver / silver chloride on the conductive carbon on the counter electrode; immobilizing a capture antibody or antigen on the surface of the active electrode; and placing a flow path fiber pad on the support that moves toward the active electrode while reacting with the antibody or antigen bound to the marker in the sample solution, and a flow rate control pad that controls the flow of the sample solution to promote an antigen / antibody reaction between the reactants in the sample solution and the capture antibody or antigen immobilized on the active electrode on the electrode portion.

[0032] According to the present invention, the manufacturing process can be performed more efficiently by using existing printing techniques such as screen printing to print electrical wiring structures on a resin sheet support and to print silver / silver chloride on conductive carbon, to immobilize antibodies or antigens on the surface of the active electrode, and to stack multiple pads on the support using existing devices.

[0033] Furthermore, according to the present invention, by providing the aforementioned flow control pad corresponding to the position of the electrode portion of the resin sheet support, it is possible to control the flow of the sample solution at the electrode portion to ensure the necessary minimum reaction time, thereby improving the sensitivity at the electrode portion and enabling rapid, convenient, and highly accurate quantitative determination. In other words, by providing the aforementioned flow control pad, the flow of the sample solution at the electrode portion can be controlled. The flow of the sample solution can be controlled from the minimum reaction time and speed necessary for the antigen-antibody reaction near the electrode surface, thus eliminating the need for a microfluidic path or pump for flow control, and enabling highly sensitive, rapid, convenient, and accurate quantitative determination using a portable, small current detection instrument.

[0034] This invention is also an electrochemical side-flow immunosensor, characterized in that a resin sheet support is provided with an active electrode made of printed conductive carbon, a counter electrode made of printed conductive carbon and a reference electrode made of printed silver / silver chloride arranged opposite each other, a conductive part made of printed conductive carbon for conducting the current generated by the electrodes, and a connecting part for conducting the current generated by the electrodes to an electrical measuring device. Furthermore, multiple pads are provided on the electrodes of the support, so that the sample solution flows through the multiple pads, and the flow is controlled at the position of the electrodes to perform electrochemical detection.

[0035] According to the present invention, the flow of the sample solution can be controlled at the positions of the above three electrode sections to ensure sufficient antigen-antibody reaction time, thus enabling accurate electrochemical measurement.

[0036] The present invention also preferably includes a step of arranging pads on the aforementioned support; the pads include: a sample pad for absorbing the dripped sample solution; a conjugate pad for dissolving and linking antibodies or antigens bound to impregnated gold colloids while absorbing the sample from the sample pad; a flow path fiber pad for moving towards the active electrode side while reacting the antigens or antibodies in the sample with the antibodies or antigens bound to the gold colloids; a sandwich-type flow control pad for forming an antigen / antibody reaction between the moving reactants and the capture antibodies immobilized on the active electrode; and an absorbent pad for absorbing the reaction residue liquid of the sample solution.

[0037] The invention is further characterized in that the sample pad, coupling pad, flow path fiber pad, flow rate control pad, and absorbent pad are fixed from the upper flow side; specifically, the sample pad, with the remaining portion below the distance between the stacked coupling pads, is fixed with adhesive on the support; the coupling pad is stacked with a portion of the upper flow side of the flow path fiber pad, and the remaining portion below is fixed with adhesive on the support; the flow path fiber pad, with a portion remaining stacked with a portion of the upper flow side of the flow rate control pad and the electrode stack, the remaining portion below is fixed with adhesive on the support; the flow rate control pad is in contact with the active electrode, the counter electrode, and the reference electrode (but without the support). (Fixed with adhesive on the surface), the position reaches the uppermost part of the conductive part from which the electrical insulation has been printed, and is fixed below it with adhesive on the conductive part from which the electrical insulation has been printed; or, a flow path fiber pad is disposed up to the surface of the electrode part, and is fixed below it with adhesive outside the electrode part, and a flow rate limiting pad is stacked on the upper surface of the electrode part (without adhesive fixation), and the lower part beyond this is fixed below with adhesive on the support; an absorbent pad is stacked on the upper part of the lower flow side of the flow rate control pad, the position reaches the end of its lower flow part, and is fixed below it with adhesive on the support above which the insulation has been printed on the conductive part.

[0038] This invention is also a lateral flow immunosensor used in a tray assembly for detecting antigens or antibodies in body fluids, proteins in food raw materials / processed products, etc. Its features include: a flow rate control pad for controlling the flow of the sample solution, including speed and / or flow rate, provided on a resin sheet support; an absorbent pad for absorbing the reaction residue liquid of the sample solution; and a flow path fiber pad for moving the sample solution while reacting with the antibodies or antigens bound to the markers; and a flow rate control protrusion provided in the tray for controlling the flow by pressing the flow rate control pad, flow path fiber pad, and / or absorbent pad from above to act as a gate; thereby fixing the pads.

[0039] The present invention can also be exemplified as a method for manufacturing a side-flow immunosensor, characterized in that a flow rate control pad for controlling the flow mode of the test solution, including speed and / or flow rate, an absorbent pad for absorbing the reaction residue liquid of the test solution, and a flow path fiber pad for moving the test solution while reacting with the antibody or antigen bound to the marker of the test solution; a flow rate control protrusion for controlling the flow by pressing the flow rate control pad, the flow path fiber pad, and / or the absorbent pad from above to act as a gate; thereby fixing the above-mentioned pads.

[0040] According to the present invention, by using a flow rate control protrusion to control the flow by pressing the pad from above to act as a gate, the pad can be fixed without the use of adhesives or the like.

[0041] Furthermore, as a manufacturing method, a method is adopted to first manufacture multiple pads (connecting the pads to each other) and then attach them together to the electrode layer, thereby enabling more efficient manufacturing.

[0042] It is also preferable to provide a protrusion on the above-mentioned tray box for pressing the flow rate control pad, flow path fiber pad, sample pad, coupling pad and / or the above-mentioned absorbent pad from above, so as to position or flip the pads and fix the stacked parts.

[0043] The effects of the invention

[0044] According to the present invention, by using a flow rate control protrusion to control the flow by pressing the pad from above, the pad can be fixed without the use of adhesives or the like. Furthermore, even when the pad is placed on a support with printed electrode portions, the sample solution can be reliably delivered to the electrode portions. The flow rate control protrusion can also be formed according to the shape of the electrode portions to control the flow pattern of the sample solution, including its velocity and / or flow rate.

[0045] Furthermore, the manufacturing method of the electrochemical lateral flow immunosensor of the present invention can be used to automatically manufacture the sensor using existing manufacturing equipment, and products with uniform performance can be obtained. Moreover, as a manufacturing method, methods such as first connecting multiple pads and then attaching them together to the electrode layer can be employed, thereby enabling more efficient manufacturing. Attached Figure Description

[0046] Figure 1 This is a top view showing the electrochemical side-flow immunosensor according to an embodiment of the present invention, in which conductive carbon is printed on the electrodes, conductive parts and connecting parts, and an electrical insulator is also printed on the conductive parts.

[0047] Figure 2 This is a cross-sectional view illustrating the pad-like laminated structure of the electrochemical side-flow immunosensor according to the above embodiment, showing a portion of each of the following components stacked together: the flowmeter sample pad, the coupling pad, the flow path fiber pad, the flow rate control pad, and the absorbent pad. Figure 2 (a) is an example of placing a flow rate limiting pad on the electrode section. Figure 2 (b) is an example where a flow rate limiting pad is placed on top of the electrode section where the fiber pad in the flow path section is placed.

[0048] Figure 3 These are a top view and a perspective view of the upper box of the storage tray for storing the side-flow immunosensor according to the above embodiment; wherein Figure 3 (a) is a top view of its upper box. Figure 3(b) is the inside view of the lower box; 28 is the sample hole, and 29 is the cleaning / electrolyte hole; in addition, in the inside view of the upper box, 27 is the flow rate control protrusion, showing its front and rear sides. Figure 3 (c) is a three-dimensional view of the top and inside of the upper box and the top of the lower box.

[0049] Figure 4 The electrochemical side-flow immunosensor of the above embodiment is shown in two states ((a) and (b)) housed in a tray. It is a side view showing the structure in which a certain pressure is applied from inside the upper tray to the flow control pad 15 to contribute to flow control. In addition, the above-mentioned protrusion 27 is located on the flow control pad 15. The number of protrusions can be one, which is used to press the entire surface; or there can be two or more, which are used to press the two ends. The number is not limited, as long as the above-mentioned flow control can be achieved.

[0050] Figure 5 This is a diagram showing the positional relationship between the flow rate control protrusion and the electrode portion inside the upper casing of the side-flow immunosensor according to the above embodiment.

[0051] Figure 6 This is an explanatory diagram illustrating an example of using the lateral flow immunosensor according to the above-described embodiment, wherein... Figure 6 (a) illustrates an example of the immunosensor of the present invention and its connection portion being inserted into a portable small instrument for measuring reduction current. Figure 6 (b) shows the circuit diagram.

[0052] Figure 7 This is a diagram illustrating one example of the test solution inspection principle of the electrochemical side-flow immunosensor described in the above embodiment. The diagram shows that when the sample is dripped onto the sample pad, the antibody bound to the dried gold colloid contained in the dissolution coupling pad flows while reacting with the antigen in the sample, and reacts with the capture antibody that has been immobilized on the electrode (active electrode) to form a sandwich-like structure, in which the gold colloid corresponding to the amount of antigen in the sample is captured.

[0053] Figure 8 This is a diagram illustrating an example of multiple pads of the electrochemical side-flow immunosensor described above, pressed from above.

[0054] Figure 9 This is a diagram illustrating an example of multiple pads of the electrochemical side-flow immunosensor described above, pressed from above.

[0055] Figure Labels

[0056] 1: Electrochemical side-flow immunosensor; 2: Support (a support on which electrodes are printed); 2-1, 2-2: Electrochemical side-flow immunosensor; 3: Carrier (a carrier with electrodes, wiring, and other electrical internal structures); 4: Electrical measuring device (instrument); 5: Electrode section; 5a: Actual electrode; 5b: Counter electrode; 5c: Reference electrode; 6: Circuit; 7: Conductive part; 8: Sample pad; 9: Electrical insulation; 10: Flow path fiber pad (inner membrane of the flow path); 11: Coupling pad; 12: Connector; 13: Sample well (sample well) 14: Supply hole for the material solution; 15: Control section; 16: Flow control pad; 17: Absorption pad; 28: Measurement of reduction current of metal nanoparticles on the electrode; 29: Upper box; 20: Upper box top; 21: Upper box inside; 22: Lower box; 23: Lower box inside; 27a, 27b, 27c: Flow control protrusions; 27c: Pad pressing body; 28: Sample hole; 29: Cleaning solution / electrolyte hole; 30: Disk box; 31-1, 31-2: Disk box for storing the side-flow immunosensor; P1~P3: Flow control protrusions. Detailed Implementation

[0057] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0058] The electrochemical side-flow immunosensors 1, 2-1, and 2-2 of this invention are sensors 1, 2-1, and 2-2 such as: Figure 1 As shown, electrodes are printed on a support 2 on a resin sheet, i.e., a carrier 3, such as... Figure 2 As shown in (a) and (b), the sample that may contain the substance being tested is spread laterally (lateral flow) on the support, and the substance being tested is quantified accordingly.

[0059] Sensors 1, 2-1, and 2-2 are sensors in which an electrode portion 5, a conductive portion 7 for conducting current from the electrode portion 5, and a connection portion 12 connected to an electrical measuring instrument 4 for measuring the current value are disposed on a resin sheet carrier 3. Furthermore, pads 8, 10, 11, 15, and 16 are partially stacked on the support, allowing the sample solution to flow through multiple pads. The flow is controlled at the position of the electrode portion 5 for electrochemical detection. Figure 2 The two configurations shown in reference numerals 2-1 and 2-2. Figure 2 (a) is an example in which a flow rate limiting pad 15 is disposed on the electrode portion 5. Figure 2 (b) is an example in which a flow rate limiting pad 10 is disposed on the electrode section 5 between the flow path fiber pad 11 and the electrode section 5.

[0060] The assay method for the test substance using the electrochemical immunosensor of this invention, such as... Figure 1 , Figure 5 as well as Figure 6As shown, an electrode 5a with a first capture antibody or antigen specifically binding to the test substance and a second antibody or antigen specifically binding to the test substance bound to metal particles (labels) are prepared. A test solution and the label are supplied to the surface of the electrode 5a to induce an antigen-antibody reaction. Accordingly, the amount of metal particles corresponding to the test substance in the test solution is gathered near the surface of the electrode 5a. The electrode 5a is potential-controlled to oxidize the metal particles electrochemically. At the same time, unreacted metal particles bound to the second antibody are removed by flushing the electrode 5a with an aqueous chloride solution such as NaCl, KCl, or HCl (which also serves as an electrolyte and cleaning solution). The noise caused by the antibody or antigen used for testing and even impurities in the test solution is suppressed. Under the state of suppressed noise, the reduction current value generated when the oxidized metal particles are reduced electrochemically is measured. The presence or concentration of the test substance is quantified based on the current value.

[0061] This side-flow immunosensor 2, including the sample pad 8, conjugate pad 11, absorbent pad 16, and support 2, can be manufactured using the same materials as those used in the sample pad, conjugate pad, and absorbent pad of commonly used immunochromatographic test strips as shown in Patent Document 2. In an immunochromatographic immunoassay, the sample travels through the device by capillary force, binding to a conjugate pre-impregnated with primary antibody in the conjugate pad 11, and to secondary antibody immobilized on the in vivo membrane as a determination baseline, and is captured on the spot. Primary antibody that has not yet bound to the sample passes through the determination baseline unchanged and flows to the next control baseline, where it is captured. As for the binding principle, in addition to the sandwich assay, inhibition assay can also be used. In the lateral flow immunoassay of the present invention, due to the need for multifunctionality, it is necessary to combine multiple different components. Generally, the sample pad 8, the conjugate release pad 11, the reaction in vivo membrane, and the absorbent pad 16 are necessary. However, in addition, the flow path fiber pad 11 and the flow rate control pad 15 are also necessary. Depending on the specific application, a blood cell separator filter may also be required. In this embodiment, the following structure can be adopted: an absorbent pad 16 and a dense liner sheet disposed on the inner side of the reaction in vivo membrane; a primary antibody is immobilized in a given area on the surface of the in vivo membrane to form an immobilized area (judgment section); an antibody specifically binding to a secondary antibody labeled with metal microparticles is immobilized on the surface of the in vivo membrane flowing down to the judgment section, serving as a control section (flow rate limiting pad 15). That is, at least the judgment section and the active electrode 5a in the reaction in vivo membrane overlap. After this overlap, the test strip and the printed electrode 5 are overlapped in such a way that the active electrode 5a of the printed electrode 5 contacts the judgment section.

[0062] Furthermore, the electrochemical side-flow immunosensor 2-1 or 2-2 of this embodiment can be manufactured as follows: electrodes and wiring are formed between an electrically insulating portion of a carrier that is a synthetic resin sheet, forming a support with an electrical structure. On this basis, it is manufactured using the same material as the sample pad 8, coupling pad 11, and absorbent pad 16 used in commonly used immunochromatographic test strips. Figure 2 ).

[0063] However, the electrochemical side-flow immunosensor 2-1 or 2-2 of the present invention differs from existing immunochromatographic test strips in that it is manufactured by partially stacking a sample pad 8, a coupling pad 11, a flow path fiber pad 10, a flow rate control pad 15, and an absorption pad 16 on a support 2 formed by printing or the like on a carrier 3 covering the electrode portion 5, as described later. It should be noted that the sample pad 8 and the coupling pad 11 are disposed on the aforementioned support 2, which is not printed or covered with carbon, silver / silver chloride.

[0064] Next, the process includes printing silver / silver chloride on conductive carbon on the reference electrode 5c and immobilizing antibodies or antigens on the surface of the active electrode 5. The active electrode 5a of the electrode portion 5 is formed along the full width of the resin sheet support 2. The reference electrode 5c and the counter electrode 5b are respectively disposed on the left and right ends of the support 2 on the rear side (downstream side) of the active electrode 5a. Each electrode portion 5a, 5b, and 5c is led out by a conductive portion 7 and connected to the connecting portion 12 on the downstream side. The connecting portion 12 is inserted into the electrical measuring device 4 (the connecting portion 12 is also exposed from the trays 31-1 and 31-2), and electrical measurements are performed through the electrical measuring device 4. Figure 5 ).

[0065] (Electrode portion printed on the support)

[0066] Manufactured according to the following procedures: Figure 1 and Figure 2 As shown, conductive carbon is printed onto the support 2 of the synthetic resin sheet in the shape indicated by the blackening, forming the substrate of the active electrode 5a, the reference electrode 5c, and the counter electrode 5b, the conductive part 7 for conducting current, and the connecting part 12 for connecting with the electrical measuring instrument 4. Furthermore, silver / silver chloride is coated onto the conductive carbon on the reference electrode 5c by printing or the like. The active electrode 5a is formed along the full width of the carrier 3 of the resin sheet. The reference electrode 5c and the counter electrode 5b are respectively disposed on the left and right ends of the carrier 3 of the electrical internal structure on the rear side (downstream side) of the active electrode 5a. Each electrode part 5a to 5c is led out by the conductive part 7 and connected to the connecting part 12 on the downstream side. Next, the support 2 is manufactured according to the following steps: forming the conductive part 7 for conducting current and the connecting part 12 for connecting with the electrical measuring instrument 4; further, silver / silver chloride is coated onto the conductive carbon on the reference electrode 5b by printing or the like. Figure 1In this process, conductive carbon is printed onto a carrier 3 made of synthetic resin or similar material, forming an electrode section 5 and a connection section 12 to the electrical measuring device 4. Electrode (active electrode 5a and counter electrode 5b) 5, a conductive section 7, and a connection section 12 to the instrument are then formed. A reference electrode 5c is coated with silver / silver chloride through printing.

[0067] Materials used for the conductive carbon particle paste printed on the support 2 include, for example, Ketjen black, graphite sheets, carbon nanotubes, and fullerenes; however, Ketjen black is preferred due to cost considerations. The silver / silver chloride printed on the reference electrode 5c, using a paste composed of silver chloride crystal microparticles, exhibits good adhesion to the coating surface. The carrier 3 for the resin sheet can be a sheet made of various resins, such as 0.1–0.5 mm thick polypropylene, or a substrate-like material, as long as it allows for the stacking of circuits and pads.

[0068] (Carrier: The carrier of the circuit)

[0069] The printed circuit support 2 used in this invention is formed by the carrier 3 constituting the electrode portion 5, wiring, etc. The carrier 3 constituting the electrodes, wiring, etc. can be manufactured as follows: conductive carbon is printed on the three electrode portions 5 (5a, 5b, 5c), the conductive portions 7 that conduct current from these electrodes, and the connection portion 12 that connects to the electrical measuring device 4 that measures their current values. Then, silver / silver chloride is printed on the reference electrode. Furthermore, an electrical insulating material 9 (polyurethane resin, polyamide resin, polyester resin, phenolic resin, etc.) is covered on the three conductive portions 7 led out from the electrode portions 5 (5a, 5b, 5c) by printing or the like.

[0070] (Identification font)

[0071] The marker used in this invention can be any electrolytic metal particle (microparticles, colloidal particles, quantum dots, etc. of gold, platinum, silver, copper, rhodium, palladium, etc.), with a particle size preferably between 20 nm and 100 nm, and gold colloidal particles between 40 nm and 60 nm being particularly preferred. These gold colloidal particles can be manufactured using conventional methods, such as by adding a trisodium citrate aqueous solution dropwise to a heated tetrachloroalloy(III) hydrochloric acid aqueous solution and stirring.

[0072] (Fiber pad in the flow path section)

[0073] The flow path fiber pad 10 used in this invention is a nonwoven fabric formed from fibrous fine fibers. As the material, any thin nonwoven fabric with strong capillary force formed from glass fiber, resin fiber, carbon fiber, natural fiber, etc., is acceptable, with glass fiber being particularly preferred at a thickness of 0.3–1.0 mm. The flow path fiber pad 10 facilitates the flow of antigens or antibodies in the sample solution, guiding them towards the reaction region (above the active electrode) 5a while they react with the antibody or antigen bound to the marker in the conjugate pad.

[0074] (Flow control pad)

[0075] The flow control pad 15 used in this invention is a nonwoven fabric formed from fibrous fine fibers. The material used is a thin nonwoven fabric with weak capillary force, formed from glass fibers, resin fibers, carbon fibers, natural fibers, etc. Compared to the flow path fiber pad 10 with the same thickness of 0.3–1.0 mm glass fibers, the flow control pad 15 is preferably made of a nonwoven fabric of dense natural fibers with a thickness of 0.1–0.7 mm.

[0076] In this embodiment, the flow control pad 15 is positioned above the electrode section (active electrode, counter electrode, and reference electrode formed by printing silver / silver chloride) 5. Then, the flow of the substance being tested is controlled by pressing the flow control protrusion 27 located inside the upper compartment 22b of the trays 31-1 and 31-2 at two or more locations, or at one location covering the entire surface, from above the flow control pad 15.

[0077] In one embodiment, the flow rate control protrusion 27a on the upstream side is disposed corresponding to the position of the active electrode 5a, while the flow rate control protrusion 27b on the downstream side is disposed facing the positions of the reference electrode 5c and the counter electrode 5b. That is, at the position of the electrode portion 5, two points are pressed on the front side (upstream side) and the rear side (downstream side) of the flow rate control pad 15, thereby, between these two points (H1-H2), the active electrode side 5a, the reference electrode 5c, and the counter electrode 5b are pressed by the flow rate control protrusion 27a between the flow rate control pad 15. Figure 5 It should be noted that as long as they are not housed in trays 31-1 and 31-2, the flow control pads 15 of the electrochemical side-flow immunosensors 2-1 and 2-2 of this application will not be pressed by the aforementioned flow control protrusions 27.

[0078] (Manufacturing method of electrochemical side-flow immunosensor)

[0079] The manufacturing method of the immune sensor 2-1 of the present invention is as follows: an adhesive is applied to the support body 2, except for the three types of electrode parts 5; first, the lowermost flow part, excluding the three types of electrode parts 5 corresponding to the position of the flow control pad 15, is fixed to the support body 2; then, the lower flow side of the flow path fiber pad 10 is stacked on top of the flow control pad, and the remaining lower part is fixed with the adhesive on the support body; then, a portion of the lower flow side of the coupling pad 11 is stacked on top of the upper flow side of the flow path fiber pad 10; the lower flow side of the sample pad 8 is stacked on top of the upper flow side of the coupling pad 11, and the remaining lower part is fixed with the adhesive on the support body.

[0080] The manufacturing method of the immune sensor 2-2 of the present invention is as follows: an adhesive is applied to the support 2, except for the three electrode parts; first, the bottom part of the flow path fiber pad 10, excluding the electrode part, is fixed with the adhesive on the support; then, the bottom part of the flow rate limiting pad 15 is stacked on top of the electrode part of the flow path fiber pad 10, and its lowest flow part is fixed with the adhesive on the support 2; a portion of the bottom part of the coupling pad 11 on the lower flow side is stacked on top of the upper flow side of the flow path fiber pad 10; the bottom part of the sample pad 8 on the lower flow side is stacked on top of the upper flow side of the coupling pad 11, and the remaining bottom part is fixed with the adhesive on the support.

[0081] It should be noted that the electrode portions 5 on the support can be printed using various methods. For example, a printing plate can be made by screen printing, which can print multiple (e.g., more than 50) single measurement portions of electrode portions 5 in a continuous shape at one time using conductive carbon or silver / silver chloride. By making a support with more than 50 electrodes printed on it, a continuous body of materials locally stacked together, which is the sensor, can be manufactured.

[0082] As an example of a mass production method, elongated materials can be pasted using an automated manufacturing device for immunochromatographic test strips sold by BioDot on the market. Then, a marker is coated onto the coupling pad 11 of one of these materials and dried. The material is then cut to the width of each immunosensor. In this way, the electrochemical side-flow immunosensor 1 of this patent can be mass-produced and supplied to the product at a lower cost.

[0083] Therefore, as a manufacturing method, manufacturing can be performed more efficiently by first connecting multiple pads 8, 10, 11, 13, 15, and 16 together and then attaching them to the electrode layer. In addition, flow rate control protrusions 27a and 27b are formed corresponding to the shapes of the electrode sections 5a, 5b, and 5c, which can not only press down the pads 8, 10, and 11, but also control the flow mode of the sample solution, including speed and / or flow rate.

[0084] (Measurement Principle)

[0085] The following describes a measurement method (an example of an antigen assay) using sensors 2-1 and 2-2 of the present invention.

[0086] like Figure 7 As shown, the sample (containing the antigen of the test subject) is dripped onto the sample pad 13, which contains antibodies bound to gold colloid. Figure 7 In section 1), the gold colloid binds antibodies and antigens (the analyte) while they flow upwards along the flow path, undergoing an antigen + antibody reaction. Figure 7In the middle 2), it binds to the capture antibody immobilized on the electrode part (support surface) 5 at its front, forming a sandwich type, and the unreacted material (the excess gold colloid bound to the antibody) is captured by the absorbent pad 16. Figure 7 (3) Subsequently, electrolyte (such as saline solution) is dripped from electrolyte hole 29 onto the fiber pad 10 of the flow path section until it reaches the desired consistency. Figure 7 In section 3, the “gold particle binding antibody + antigen + capture antibody (immobilized in electrode section 5)” part, after washing away excess unreacted gold colloid bound antibody relative to the amount of antigen in the sample, connects the electrode section 12 printed on the support 2 to the electrical measuring instrument 4, applies a certain voltage, measures the reduction current of the generated gold particles, and quantifies the concentration of the test subject in the blood.

[0087] According to the first embodiment, the antigen and the antibody bound to the gold colloid in the sample flow while undergoing an antigen-antibody reaction, approaching the capture antibody that has been immobilized on the electrode (active electrode). Thus, as it flows along the electrode, almost all of the antigen in the sample binds to the capture antibody.

[0088] Subsequently, the flow control pad 15, which is arranged above the electrode section 5, concentrates the amount of metal microparticles in the test solution corresponding to the amount of the test substance (antigen or antibody) on the active electrode 5a. After the metal microparticles are electrochemically oxidized, the current value generated when the oxidized metal is electrochemically reduced is detected in the electrode section 5. The presence or concentration of the test substance is determined based on the current value.

[0089] That is, for example, based on antigen-antibody reactions, the antibody that binds to the antigen or antibody in the sample and the amount of metal microparticles specifically reacts with the antigen, causing metal microparticles corresponding to the mass of the analyte to accumulate near the surface of the electrode. After the metal constituting the microparticles is electrochemically oxidized, the reduction current value at which the oxidized metal is reduced is measured. The intensity of this reduction current represents the amount of metal accumulated near the electrode, thus enabling the quantification or detection of antigens or antibodies in the analyte. It is important that the electrochemical oxidation of the metal microparticles is performed while metal microparticles are accumulated near the surface of the electrode. Therefore, the electrode portion is directly and closely attached to the support, using a flow-limiting pad or flow path fiber pad provided without adhesive. This ensures that all metal microparticles related to the reaction with the analyte are involved in electron transfer and acceptance at the electrode surface, resulting in highly sensitive and precise measurement of the analyte.

[0090] [Fabrication of an electrochemical side-flow immunosensor for CRP quantification]

[0091] (Preparation of anti-CRP monoclonal antibody solution)

[0092] The CRP antibodies used in the following experiments were two anti-human CRP monoclonal antibodies (No. 8 and No. 5) from Immuno Probe. Solutions were prepared by diluting each antibody to a concentration of 5 mg / ml using 10 mM Tris buffer.

[0093] (Example of CRP immune sensor fabrication)

[0094] 1) Regarding the fabrication of carrier 3, conductive carbon (Ketjen black manufactured by Lion Specialty Chemicals) was screen-printed onto the support (transparent polypropylene from Lohmann Precision) 2. Figure 1 and Figure 2 After the active electrode 5a, counter electrode 5b, reference electrode 5c, conductive part 7, and connecting part 12 are shown, silver / silver chloride (manufactured by BAS) is printed on the reference electrode 5c, and an electrical insulating part (polyurethane resin) is screen printed on the conductive part 7.

[0095] 2) The sample pad 8 is made of cellulose fiber (AHLSTROM). The sample pad 8 has many functions, including: adsorbing the sample and transporting it to the coupling release area or the internal membrane for analysis at a uniform and constant speed; reliably preventing the sample from spilling out of the equipment; filtering particles or cells from the sample; and impregnating the sample with chemicals, etc.

[0096] 3) Regarding the preparation of CRP monoclonal antibody-labeled gold colloidal particles: Add 1.0 ml of 1.1% tetrachloroalkanolite (I II) tetrahydrate (manufactured by Tanaka Precious Metals Co., Ltd.) aqueous solution and 2.44 ml of K2CO3 diluted to 10 mM (manufactured by Fujifilm and Koko Pure Chemical Industries Co., Ltd., product number: 162-03495) aqueous solution to 99 ml of distilled water. Mix and heat at 50°C for 3 minutes. Then add 1.0 ml of sodium citrate diluted to 1.1% (manufactured by Fujifilm and Koko Pure Chemical Industries Co., Ltd., product number: 191-01785) aqueous solution. Boil for 11 minutes and cool in distilled water ice to prepare gold colloidal particles with an average particle size of 40 nm.

[0097] Add 1 ml of CRP antibody solution diluted with 10 mM Tris buffer to prepare a 0.5 mg / ml solution of anti-human monoclonal antibody (ImmunoProbe, No. 8). After mixing and stirring, let stand at room temperature for 60 minutes. Then add 10% bovine serum albumin solution and disperse by sonication. Let stand at room temperature for 5 minutes, then centrifuge using a cooled centrifuge to remove the supernatant and obtain the precipitate. Add another 10 ml of 10% bovine serum albumin solution and disperse by sonication. Measure the absorbance (OD) of this solution at a wavelength of 520 nm. Adjust the concentration until the absorbance (OD) reaches approximately 9 to prepare the CRP monoclonal antibody-labeled gold colloidal solution.

[0098] 4) Regarding the fabrication of the conjugate pad 11, the CRP monoclonal antibody-labeled gold colloidal particle (conjugate) solution prepared in step 3) above is impregnated in an EMDMILIPORE GLASSFIBER DIAGNOSTICS PAD until saturated. The pad is then freeze-dried overnight to produce the conjugate pad 11. The conjugate pad 11 retains the conjugate during storage to maintain detection capabilities, and efficiently releases the conjugate as the sample moves along the pad.

[0099] 5) Immobilization of anti-human CRP monoclonal antibody on the active electrode. Anti-human CRP monoclonal antibody (Immuno Probe, No. 5) was diluted with 10 mM Tris buffer, and 4 μl of the solution was placed on the active electrode 5a. After standing overnight in the refrigerator, the residual antibody solution was blown away with a spray gun, and the anti-human CRP monoclonal antibody was immobilized on the active electrode.

[0100] 6) Sealing on the electrode sections. In order to prevent proteins and other substances other than the analyte in the sample solution (sample) from being adsorbed onto the three electrode sections 5, 10 μl of 2.5% casein boric acid aqueous solution (pH 8.5) was placed on each of the three electrode sections (active electrode 5a, counter electrode 5b, and reference electrode 5c). After standing for 1 hour at room temperature with normal humidity, the residual solution was blown away with a spray gun.

[0101] 7) Fabrication of electrochemical side-flow immunosensors

[0102] A support 2 with an adhesive attached to plastic is first fitted with a flow path fiber pad (glass fiber pad manufactured by AHLSTROM: product number 8964) 10, followed by a coupling pad 11 fabricated in step 4), and then a sample pad (glass fiber pad manufactured by AHLSTROM: product number 0238) 8. On the other hand, a flow rate control pad (cellulose long fiber nonwoven fabric manufactured by Asahi Kasei Corporation: product number SA28G) 15 is fitted at the upper end of the three electrode sections 5 on the downstream side of the flow path fiber pad 10, and then an absorption pad (cellulose fiber pad manufactured by AHLSTROM: product number 0270) 16 is fitted. This structure, formed by overlapping the constituent elements, is cut to the width of the printed electrode section units to produce an electrochemical side-flow immunosensor 1. This side-flow immunosensor 1 of the present invention is used as a testing device with a dedicated plastic casing during measurement. That is, as... Figure 3 As shown in (a) and 3(b), it is configured as an electrochemical side-flow immunosensor 1 used for inserting into a tray 30 having a supply hole 29 for supplying sample solution and cleaning solution to the upstream side.

[0103] Figure 4 In (a) and 4(b), it is configured as an electrochemical side-flow immunosensor 1 housed in a disk cassette 30. Figure 4 (a) is an example of making the flow rate control pad 15 closely connected to the electrode section 5. On the upstream side, the lower layer of the downstream end of the flow path fiber pad 10 is stacked on the upper layer of the upstream end of the flow rate control pad 15. On the downstream side, the lower layer of the upstream end of the absorbent pad 16 is stacked to guide the absorbent pad 16. Therefore, it is possible to make the flow rate control pad 15 closely connected to the electrode section 15, press it down with the front and rear (upstream and downstream) pads 11 and 16, and slow down the velocity of the sample solution on the flow rate control pad 15.

[0104] Figure 4 (b) is an example where a flow control pad 15 is stacked on top of the flow path fiber pad 10 and the electrode portion is laid on the surface of the electrode portion 5, so that they are in close contact. In this example, the upper part of the flow control pad 15 on the downstream side, which extends beyond the electrode portion, is stacked on the lower layer of the upper end of the absorbent pad 16, guiding the absorbent pad 16. Therefore, by stacking the flow control pad 15 on the upper layer of the flow path fiber pad 10 located on the electrode portion, the velocity of the sample solution can be slowed down, and by stacking a portion of the downstream side of the flow control pad 15 on the upper end of the absorbent pad 16, the sample solution can be moved.

[0105] In this way, by stacking a portion of the upper and lower sides of the flow rate control pad 15 with other pads, the following flow pattern can be controlled: the sample solution moves toward the surface of the electrode 5, while maintaining a constant amount and speed of the sample solution near the surface of the electrode 5 and maintaining close contact with the electrode.

[0106] 8) Cartridge Unit for Sideflow Immunosensors

[0107] The clip unit, i.e., the tray 30, for housing the side-flow immunosensor 1 of the present invention consists of an upper tray 22 and a lower tray 23, and is made of resins such as polypropylene, polyester, polystyrene, and propylene. The immunosensor 1 of the present invention is placed in a designated position in the lower tray 23, covering the upper tray 22, and the two trays are pressed together to fit. Inside the upper tray 22b, there is also a flow rate control protrusion 27 formed on the inner wall. The flow rate control protrusion 27 controls the flow rate by pressing the flow rate control pad 15 from above. Although two protrusions are provided in this embodiment, it is not limited to this; one or more can be provided. In addition, sample holes 28 and cleaning / electrolyte holes 29 are formed in the trays 31-1 and 31-2. Figure 3 (a)) Inside the upper box 22b, a flow rate control protrusion 27 is arranged, and the flow rate control protrusion 27, which controls the function of the flow rate control pad 15, is located on the flow rate control pad 15. Figure 3 (b)) Although cleaning can be performed by supplying cleaning solution / electrolyte to the sample well 28, the cleaning efficiency is excellent when the cleaning solution / electrolyte is supplied from the well / electrolyte well 29 near the electrode section 5, and the measurement efficiency is also improved when the electrolyte is supplied near the electrode section 5.

[0108] Figure 5 (a) and (b) are diagrams showing the positional relationship between the flow rate control protrusion 27 inside the upper box 22b and the electrode parts 5 of the electrochemical side-flow immunosensors 2-1 and 2-2. Figure 5 (a) An example is given in which the upstream flow rate control protrusion 27a is disposed above the active electrode 5a of the electrode section 5 and the downstream flow rate control protrusion 27b is disposed above the counter electrode 5b (or reference electrode 5c) of the electrode section 5; the region between the upstream flow rate control protrusion 27a and the downstream flow rate control protrusion 27b of the flow rate control pad 15 is pressed to form reaction regions H1 to H2, thereby controlling the flow mode of the sample solution, including velocity and / or flow rate.

[0109] Figure 5 In (b), the region between the upstream flow rate control protrusion 27a and the downstream flow rate control protrusion 27b of the electrode section 5 is designated as reaction zones H1 to H2 to control the flow pattern of the sample solution, including velocity and / or flow rate. It should be noted that, furthermore, the flow rate control protrusion 27b may be disposed on the counter electrode 5b (or reference electrode 5c) of the electrode section 5.

[0110] In this way, by pressing the upper or lower flow side of the flow control pad 15 with the flow control protrusions 27a and 27b, the flow control pad 15 can be controlled within a given range H1 to H2, so that the amount and speed of the sample solution flowing on the surface of the electrode part 5 are constant. Furthermore, using existing printing and lamination techniques, manufacturing can be performed with good efficiency through processes such as printing an electrical wiring structure made of conductive carbon on a resin sheet support, printing silver / silver chloride on the conductive carbon, immobilizing antibodies or antigens on the active electrode surface, and laminating multiple pads 8, 11, 10, 15, and 16 on the aforementioned support. The aforementioned flow control protrusions 27a and 27b can also be arranged in a sloping shape above the electrode part 5 (see...). Figure 9 (b)).

[0111] Figure 8 In another example of this embodiment, a flow rate control protrusion (P1 to P3) 27c is provided at a position other than the above-mentioned flow rate control protrusions 27a and 27b. By pressing the pads 8, 11, 10, 15, 16 below it or the electrode part 5, conductive part 7, etc. below it, the flow mode of the sample solution, including speed and / or flow rate, is controlled.

[0112] In addition, such as Figure 9 As shown in (a) and 9(b), it is best to install the pad pressing bodies P1 to P3 (27c) inside 22b of the upper box of the tray 30. The pad pressing body P1 is used to press the absorbent pad 16. When the absorbent pad 16 contains liquid and becomes soft, it is easy to deviate or have a slight backflow. The absorbent pad pressing body P1 is designed to prevent these from happening. In addition, the flow rate control protrusions P2 and P3 are designed to prevent the sample pad 8, coupling pad 11, flow path fiber pad 10, and flow rate control pad 15 from flipping or deviating. These flow rate control protrusions P1 to P3 can also be configured as a ramp shape. Figure 9 (b) The liquid flow is specified as a slope (inclined surface) to stabilize the flow rate. These can also be designed to press the electrode part 5 from above, similar to the flow rate control protrusions 27a and 27b mentioned above.

[0113] [Example 1]

[0114] The electrochemical lateral flow immunosensor 1, fabricated using the above method, was subjected to a CRP quantification test.

[0115] (Experimental method)

[0116] (1) Samples

[0117] CRP standard serum (manufactured by Kanto Chemical) was diluted with physiological saline (containing 1% bovine serum albumin) to prepare test samples with CRP concentrations of 15 ng / ml, 90 ng / ml, and 210 ng / ml. The diluted solution was also prepared to a CRP concentration of 0 mg / ml for use as a test sample.

[0118] (2) Steps

[0119] After adding 55 μl of each sample prepared as described in (1) to the sample supply well 29 of the electrochemical side-flow immunosensor 1 for 3 minutes, the sample was then added to the well 29 (which also serves as the electrolyte supply well) for 3 minutes. The electrolytic current (μA) corresponding to the CRP concentration was measured. The measurements were repeated 12 times for each of the four concentrations of sample solutions shown in Table 1. However, the 0 concentration sample solution was measured 10 times.

[0120] (Experimental Results)

[0121] The experimental results are shown in Table 1. The quantitative results are as follows: the average value corresponding to a CRP concentration of 15 ng / ml is 1.154 μA, the CRP concentration of 1 μA equivalent is 13.00 ng / ml, and the concentration corresponding to its standard deviation of 0.282 μA is 3.666 ng / ml; additionally, the average value corresponding to a CRP concentration of 90 ng / ml is 3.230 μA, the CRP concentration of 1 μA equivalent is 27.86 ng / ml, and the concentration corresponding to its standard deviation of 0.420 μA is 11.70 ng / ml. A more precise quantitative result is: the average value corresponding to a CRP concentration of 210 ng / ml is 3.993 μA, the CRP concentration of 1 μA equivalent is 52.59 ng / ml, and the concentration corresponding to its standard deviation of 0.707 μA is 37.18 ng / ml.

[0122] [Table 1]

[0123] *Unit of current: μA

[0124] 0ng / ml 15ng / ml 90ng / ml 210ng / ml 1st time 0.142 1.039 3.283 3.891 2nd time 0.038 0.910 3.495 3.040 3rd 0.100 1.286 3.260 3.632 4th 0.083 1.488 3.994 4.170 5th 0.021 1.206 2.862 4.839 6th 0.041 0.790 2.709 5.172 7th 0.125 1.299 3.704 4.555 8th 0.108 1.073 3.503 2.704 9th 0.054 0.766 2.870 3.751 10th 0.100 1.678 2.623 4.458 11th 0.997 3.512 3.727 12th 1.402 3.203 3.982 average value 0.081 1.154 3.230 3.993 Standard deviation 0.041 0.282 0.420 0.707

[0125] The above description primarily uses monoclonal antibodies as an example to illustrate this embodiment, but the present invention can also be applied to polyclonal antibodies. Furthermore, regarding the manufacturing method, it can also be done as follows: Separate the areas where adhesive is used from the areas where adhesive is not used (electrode sections); after applying adhesive to pads 10, 11, 15, and 16, place the pads onto the support 2, which has electrical internal structures such as electrodes and wiring. Alternatively, using a tray 30 design allows for pressing, eliminating the need for adhesive during manufacturing.

Claims

1. An electrochemical lateral flow immunosensor, characterized in that, In a resin-made sheet, an electrode portion, a conductive portion in which a printed conductive carbon that conducts current from the electrode portion is provided, and a connection portion that is connected to an electrical measurement device that measures the current value are provided on a support body. The electrode portion is composed of an active electrode made of a printed conductive carbon, a counter electrode made of a printed conductive carbon that is provided opposite to the active electrode, and a reference electrode made of a printed silver / silver chloride. A flow path portion fiber pad is provided on the support body. The flow path portion fiber pad moves toward the electrode side while reacting an antigen or an antibody in a sample solution with an antibody or an antigen that has been combined with a metal fine particle as an indicator. A flow rate control pad is disposed on the electrode portion in a manner connected to the flow path portion fiber pad. The flow rate control pad controls the flow of the sample solution including the speed and / or the flow rate by causing a reactant in the sample solution to perform an antigen / antibody reaction with a capture antibody or an antigen that has been immobilized on the active electrode. In the flow rate control pad, the flow of the sample solution including the speed and / or the flow rate is controlled by the position of the electrode portion. Electrochemical detection is performed on the amount of the metal fine particle corresponding to the amount of the antigen or the antibody in the detected substance. A cartridge case that houses an electrochemical lateral flow immunosensor is provided with a flow rate control protrusion that presses from above and controls the flow rate control pad like a water flow barrier. The flow rate control pad is disposed on the surface of the electrode portion of the support body, and the upstream side and / or the downstream side of the flow rate control pad are pressed from above by the flow rate control protrusion with a moderate pressure. The flow of the sample solution including the speed and / or the flow rate is controlled by pressing the active electrode side of the flow rate control pad on the upstream side and the reference electrode and the counter electrode on the downstream side of the flow rate control pad at the position of the electrode portion.

2. An electrochemical lateral flow immunosensor, characterized in that, An electrode portion formed by printing conductive carbon, a conductive portion for conducting current of the electrode portion by printing conductive carbon, and a connection portion for connecting an electrical measurement device for measuring the current value are provided on a support body of a resin-made sheet; and a flow path portion fiber pad that allows an antigen or an antibody in a sample solution to react with an antibody or an antigen that binds to a metal microparticle as a marker and move toward an electrode side is provided on the support body; a flow rate control pad for controlling a flow mode of the sample solution including speed and / or flow rate so that a reactant of the sample solution reacts with a capture antibody or an antigen fixed to the electrode in an antigen / antibody reaction is provided on the electrode portion in a manner connected to the flow path portion fiber pad; and the flow path portion fiber pad is provided on the flow rate control pad or the flow rate control pad is provided on the electrode portion via the flow path portion fiber pad, and when a sample solution containing an antigen or an antibody as a target substance reacts with a metal microparticle to which an antibody or an antigen against the substances is bound in an antigen / antibody reaction, a flow mode of the sample solution including speed and / or flow rate is controlled by the flow rate control pad provided on a surface of the electrode portion, and thus an amount of the metal microparticle corresponding to an amount of the antigen or the antibody in the target substance is electrochemically detected. A cassette housing for housing the electrochemical lateral flow immunosensor is provided, and a flow rate control protrusion for pressing from above and controlling the flow rate control pad like a water flow barrier is provided on the cassette housing; the flow rate control pad is provided on a surface of the electrode portion of the support body, and the upstream side and / or the downstream side of the flow rate control pad are pressed from above by the flow rate control protrusion with a moderate pressure; and a flow mode of the sample solution including speed and / or flow rate is controlled by pressing both a working electrode side on which the flow rate control pad is located on the upstream side and a reference electrode and a counter electrode on which the flow rate control pad is located on the downstream side.

3. The electrochemical lateral flow immunosensor according to claim 1 or 2, wherein the antibody is immobilized on the surface of the electrode. A sample pad, a conjugate pad, and an absorbent pad are further provided as pads, and the flow path portion fiber pad is provided in a manner continuous with the conjugate pad; the flow rate control pad for completing the antigen / antibody reaction on the working electrode is provided on the electrode portion in a manner laminated with the flow path portion fiber pad.

4. The electrochemical lateral flow immunosensor according to claim 1 or 2, wherein A sample pad, a conjugate pad, and an absorbent pad are further provided as pads, and the flow path portion fiber pad is provided in a manner continuous with the conjugate pad; the flow rate control pad for completing the antigen / antibody reaction on the working electrode is provided on the electrode portion; and a part of the downstream side of the flow path portion fiber pad is laminated with a part of the upstream side of the absorbent pad.

5. The electrochemical lateral flow immunosensor according to claim 1 or 2, wherein The flow path portion fiber pad has a function of moving the sample solution toward the electrode portion while allowing immunological reaction in a conjugate pad in which an antibody or an antigen that immunologically reacts with an antigen or an antibody in the sample solution is combined with a colloidal metal microparticle.

6. The electrochemical lateral flow immunosensor according to claim 1 or 2, wherein The antibody or antigen that has reacted immunologically with the substance to be tested in combination with the metal microparticles as the marker and the antibody or antigen that has reacted immunologically with the substance to be tested and has been immobilized on the above-mentioned working electrode are respectively antibodies or antigens that recognize different sites of the substance to be tested.

7. The electrochemical lateral flow immunosensor of claim 3, wherein the antibody is immobilized on the surface of the electrode by a self-assembled monolayer. A portion of the upstream side of the flow rate control pad is partially laminated with a portion of the lower surface of the flow path portion fiber pad, and the lower surface is in close contact with the three electrodes as the working electrode, the counter electrode and the reference electrode, but is not fixed with an adhesive; a portion of the lower surface of the upstream side of the absorbent pad is partially laminated on the upper surface of the downstream side of the flow rate control pad.

8. The electrochemical lateral flow immunosensor of claim 3, wherein the antibody is immobilized on the surface of the electrode by a self-assembled monolayer. A cartridge that houses the electrochemical lateral flow immunosensor is provided, and the connection portion protrudes from the cartridge to the outside, and the connection portion is inserted into the current sensing portion of the electrical measuring device that detects the current of the electrode portion, and is connected thereto, thereby forming a circuit to measure the current generated at the electrode.

9. The method for manufacturing an electrochemical lateral flow immunosensor according to any one of claims 1 to 8, wherein The process includes: The electrode portion including the working electrode, the counter electrode and the reference electrode formed by printing conductive carbon on the support of a resin sheet, and the connection portion for connecting the conductive portion that conducts the current to the electrical measuring device, further printing silver / silver chloride on the conductive carbon on the counter electrode; Immobilizing the antibody or antigen on the surface of the working electrode; A flow path portion fiber pad that moves the antigen or antibody in the sample solution in reaction with the antibody or antigen combined with the metal microparticles as the marker toward the working electrode side is disposed on the support, and a flow rate control pad that controls the flow mode of the sample solution including the speed and / or flow rate to promote the antigen / antibody reaction between the reactant of the sample solution and the capture antibody or antigen immobilized on the working electrode is disposed on the electrode portion.

10. The method of claim 9, wherein the method further comprises the step of: The process further includes the step of disposing the following pads on the support: ​ A sample pad that absorbs the sample solution dropped; A conjugate pad that absorbs the sample from the sample pad and dissolves the antibody or antigen combined with the marker composed of the gold colloid dried by immersion; The flow path portion fiber pad that moves the antigen or antibody in the sample in reaction with the antibody or antigen combined with the gold colloid toward the working electrode side; The flow rate control pad that allows the moved reactant to perform the antigen / antibody reaction with the capture antibody or antigen immobilized on the working electrode to complete the formation of a sandwich type; An absorbent pad that absorbs the reaction residue liquid of the sample solution.

11. The method for manufacturing an electrochemical lateral flow immunosensor according to claim 9 or 10, wherein As the pad, a sample pad, a conjugate pad, and an absorbent pad are further provided. A part of the lower flow side of the sample pad is laminated on a part of the upper flow side of the conjugate pad, and the lower surface thereof is fixed by an adhesive on the support. A part of the lower flow side of the conjugate pad is laminated on a part of the upper surface of the flow path section fiber pad, and the lower surface thereof is fixed by an adhesive on the support. A part of the lower flow side of the flow path section fiber pad, i.e., a part up to the working electrode / counter electrode / reference electrode, is laminated on a part of the upper flow side of the flow rate control pad, and the lower surface thereof is fixed by an adhesive on the support. A part of the upper flow side of the flow rate control pad is laminated on a part of the upper surface of the lower flow side of the flow path section fiber pad, and the lower surface thereof is in close contact with the three electrodes of the working electrode, counter electrode, and reference electrode, but is not fixed by an adhesive. The absorbent pad is laminated on a part of the lower surface of the upper flow side thereof, and the lower surface thereof is fixed by an adhesive on the support.

12. The method of claim 11, wherein the method further comprises the step of: The flow rate control pad is partially laminated on a part of the lower surface of the flow path section fiber pad on the upper flow side thereof, and the lower surface thereof is in close contact with the three electrodes of the working electrode, counter electrode, and reference electrode, but is not fixed by an adhesive. The absorbent pad is partially laminated on a part of the upper surface of the lower flow side of the flow rate control pad. ​

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