Patent Application
20250137999
The present invention relates to a solution supply device for supplying a solution to a detection device for detecting a substance to be detected in a liquid sample, a detection set, and a detection method.
In recent years, Point of Care Test (POCT) reagents that use antigen-antibody reactions or the like to test for infectious diseases and pregnancy and to measure blood glucose levels and the like have attracted attention. It is possible for tests and measurements using POCT reagents to determine results in a short period of time. In addition, methods using POCT reagents are simple and POCT reagents are inexpensive. Since POCT reagents have these characteristics, POCT reagents are widely used in medical check-ups at the stage when symptoms are mild, regular check-ups, or the like. In addition, POCT reagents are also an important diagnostic tool in home health care, which is expected to increase in the future.
In examinations or diagnosis using an examination kit, which has a type of POCT reagent, a liquid sample such as blood is introduced into the examination kit and a specific substance to be detected included in the liquid sample is detected. The immunochromatography method is a commonly used method for detecting specific substances to be detected in liquid samples. In the immunochromatography method, a liquid sample is dropped onto a membrane carrier provided in the examination kit such that, in the process of the liquid sample moving over the membrane carrier, the substance to be detected in the liquid sample binds to a labeling substance. Furthermore, the substance to be detected binds specifically and selectively to the substance fixed in the examination kit (referred to below as the “detection substance”). The resulting change in color, weight, or the like occurring in the examination kit is detected. The detection substance may be referred to as a reagent.
Nitrocellulose membranes are often used as membrane carriers for moving liquid samples (refer to Patent Document 1 below). Nitrocellulose membranes have a large number of micropores with a diameter of approximately several μm and the liquid sample moves through these pores due to capillary force.
However, nitrocellulose membranes are derived from natural products and the flow velocity of the liquid sample through the membrane varies according to the membrane because the pore size and the manner in which the pores are connected to each other in the membrane are not uniform. When variations occur in the flow velocity, the time required to detect the substance to be detected also varies. As a result, there is a possibility that an incorrect judgment may be made in which the substance to be detected is not detected before the substance to be detected binds to the labeling substance or reagent.
To solve the problem described above, a method was devised for artificially creating micro-flow channels for a liquid sample (refer to Patent Documents 2 and 3 described below). By using this method, it is possible to produce a membrane carrier which has a uniform structure. As a result, it is possible to reduce the possibility of an incorrect judgment being made in which the substance to be detected is not detected before the substance to be detected binds to the labeling substance or reagent.
In the immunochromatography method, a liquid sample is dropped onto a membrane carrier provided in an examination kit such that, in the process of the liquid sample moving over the membrane carrier, the substance to be detected in the liquid sample binds to the labeling substance. Furthermore, the substance to be detected binds specifically and selectively to a detection substance fixed in the examination kit. The resulting change in color, weight, or the like occurring in the examination kit is detected. There is a well-known method (color change detection method) for detecting a substance to be detected, in which, for a substance to be detected which is bound to a labeling substance such as colored latex particles, fluorescent particles, or metal colloidal particles, an optical measuring instrument such as an absorbance meter is used to detect color changes in a detection zone caused by binding to a reagent fixed in the detection zone. In addition, there is also a method (electrochemical immunochromatography method) in which the concentration of a biomarker is detected by being converted into an electrochemically active substance concentration.
In the electrochemical immunochromatography method, there are issues in that the deployment of a plurality of types of solutions, such as reaction liquids, cleaning solutions, and secondary reaction liquids is necessary, the time and effort required for the use thereof is increased, and the detection time is increased and these have been obstacles to the widespread use of examination kits using the electrochemical immunochromatography method. That is, for examination kits using the electrochemical immunochromatography method, there was a demand for a POCT reagent (examination kit) that is capable of determining results in a short period of time, that has an easy use method, and that is inexpensive. Even in a case of using the color change detection method, there were similar issues with methods for which the deployment of a plurality of types of solutions was necessary.
The present invention was created in view of the circumstances described above and has an object of providing a technique that makes it possible to save time and effort during use and shorten detection time in a case where a plurality of types of solutions such as a reaction liquid, a cleaning solution, and a secondary reaction liquid are deployed in an examination technique using the immunochromatography method.
According to the present invention, the following techniques are provided.
[1]
A solution supply device supplying a plurality of solutions including a reaction liquid to a detection device having a flow channel that transports the reaction liquid and is provided over a substrate formed of a resin, a solid-phase part provided in the flow channel and on which antibodies or antigens are solid-phased, a detection part for detecting reaction of the reaction liquid with the antibodies or the antigens, and a fine uneven structure that has a plurality of protrusions and is formed integrally with the substrate in a region where the flow channel is provided, the solution supply device including a solution unit having a plurality of solution accommodating parts accommodating the respective plurality of solutions, a communicating unit having a plurality of communicating parts provided respectively for the solution accommodating parts and communicating with an inside and an outside of the solution accommodating parts to supply the solutions to the detection device, and a positioning part for arranging the communicating parts in predetermined positions for communicating with the solution accommodating parts.
[2]
The solution supply device according to [1], in which the communicating part is a tube formed to have one end with a needle shape, and the one end is made to pierce the solution accommodating parts to supply the solution accommodated in the solution accommodating parts to the detection device through the tube.
[3]
The solution supply device according to [1] or [2], in which the communicating unit has a plurality of arrangement parts functioning as the positioning part and accommodating the respective plurality of solution accommodating parts,
The solution supply device according to any one of [1] to [3], in which the communicating parts each have an adjustment part that adjusts a position at which the solution is supplied to the detection device.
[5]
The solution supply device according to any one of [1] to [4], in which at least one solution accommodating part of the plurality of solution accommodating parts is fillable with a solution, and remaining solution accommodating parts are filled with a predetermined solution in advance.
[6]
The solution supply device according to any one of [1] to [5], in which, by performing one operation of arranging the communicating parts at the predetermined positions for communicating with the solution accommodating parts, the plurality of solution accommodating parts provided in the solution unit communicate simultaneously through the corresponding communicating parts.
[7]
A solution supply device supplying a plurality of solutions including a reaction liquid to a detection device having a flow channel that transports the reaction liquid and is provided over a substrate formed of a resin, a solid-phase part provided in the flow channel and on which antibodies or antigens are solid-phased, a detection part for detecting reaction of the reaction liquid with the antibodies or the antigens, and a fine uneven structure that has a plurality of protrusions and is formed integrally with the substrate in a region where the flow channel is provided, the solution supply device including a solution unit having a plurality of solution accommodating parts accommodating the respective plurality of solutions, and a communicating unit having a plurality of communicating parts provided respectively for the solution accommodating parts and communicating with an inside and an outside of the solution accommodating parts to supply the solutions to the detection device, in which the solution unit and the communicating unit are configured such that, by performing one operation that allows the communicating parts to communicate with the solution accommodating parts, all the solution accommodating parts communicate simultaneously to start supply of the solution.
[8]
The solution supply device according to [7], in which the communicating part is a tube formed to have one end with a needle shape, and the one end is made to pierce the solution accommodating parts to supply the solution accommodated in the solution accommodating parts to the detection device through the tube.
[9]
The solution supply device according to [7] or [8], in which the communicating unit has a plurality of arrangement parts accommodating the respective plurality of solution accommodating parts, the communicating parts are provided in the respective arrangement parts, and, when the solution accommodating parts are accommodated in the arrangement parts, the communicating parts communicate between the inside and the outside of the solution accommodating parts to supply the solution to the detection device.
The solution supply device according to any one of [7] to [9], in which the communicating parts each have an adjustment part that adjusts a position at which the solution is supplied to the detection device.
The solution supply device according to any one of [7] to [10], in which at least one solution accommodating part of the plurality of solution accommodating parts is fillable with a solution, and remaining solution accommodating parts are filled with a predetermined solution in advance.
A detection set including a detection device having a flow channel that transports a reaction liquid and is provided over a substrate formed of a resin, a solid-phase part provided in the flow channel and on which antibodies or antigens are solid-phased, a detection part for detecting reaction of the reaction liquid with the antibodies or the antigens, and a fine uneven structure that has a plurality of protrusions and is formed integrally with the substrate in a region where the flow channel is provided, and the solution supply device according to any one of [1] to [11], which supplies a plurality of solutions including the reaction liquid to the detection device.
[13]
The detection set according to [12], in which the detection part is provided closer to another end side of the flow channel than the solid-phase part.
[14]
The detection set according to [12] or [13], in which the fine uneven structure has a first uneven portion in which the plurality of protrusions are provided relatively loosely, and a second uneven portion in which the plurality of protrusions are provided relatively densely, and
The detection set according to [14], in which the first uneven portion is provided closer to the one end side of the flow channel than the second uneven portion.
[16]
The detection set according to [14] or [15], further including a buffer region in which the protrusions are not provided at a boundary between the first uneven portion and the second uneven portion.
[17]
The detection set according to any one of [14] to [16], in which a step or a slope is provided at the boundary between the first uneven portion and the second uneven portion, and a region of the step or the slope on a first uneven portion side is higher than a region on a second uneven portion side.
[18]
The detection set according to any one of [14] to [17], further including a recessed region provided with a recess at a boundary between the first uneven portion and the second uneven portion.
[19]
The detection set according to any one of [14] to [18], in which, when the first uneven portion and the second uneven portion are adjacent to each other, a ratio (P1/P2) of a pitch (P1) between the protrusions in the first uneven portion and a pitch (P2) between the protrusions in the second uneven portion is 1.1 or more and 5 or less.
[20]
The detection set according to any one of [12] to [19], further including a region in which the protrusions are provided in a diamond-shaped lattice shape.
[21]
The detection set according to any one of [12] to [20], further including a region in which the protrusions are provided in a regular lattice shape.
[22]
The detection set according to any one of [12] to [21], in which the protrusions are provided as cones.
[23]
The detection set according to any one of [12] to [22], further including an introduction part for introducing the solution into the flow channel, in which the solution introduced into the flow channel is formed of a plurality of types of solutions, and the introduction part is provided at a plurality of locations according to the plurality of types of solutions.
[24]
The detection set according to any one of [12] to [23], in which an electrode portion is provided in the detection part, and the detection part detects the reaction of the reaction liquid with the antibodies or the antigens based on a current flowing through the electrode portion.
[25]
The detection set according to [24], in which the electrode portion is formed on the protrusion of the fine uneven structure, a maximum peak height Rp of a roughness curve of the electrode portion is 0.005 μm or more and 10 μm or less, and an average length RSm of a roughness curve element is 0.01 μm or more and 15 μm or less.
[26]
The detection set according to [24] or [25], in which the electrode portion has a conductive film layer in which a conductive substance is formed on the protrusions of the fine uneven structure by at least one of sputtering, vacuum evaporation, laser ablation, and CVD.
[27]
The detection set according to any one of [24] to [26], in which the electrode portion has a printed layer of paste including conductor particles on the protrusions of the fine uneven structure.
[28]
The detection set according to any one of [24] to [27], in which the electrode portion has a working electrode and a counter electrode separated from the working electrode, and the working electrode is provided at the same position as the counter electrode or upstream of the counter electrode with respect to a flow channel direction.
[29]
The detection set according to [28], in which the counter electrode is provided over an entire flow channel in a width direction.
[30]
The detection set according to [28] or [29], in which the working electrode is provided over an entire flow channel in a width direction.
[31]
The detection set according to any one of [28] to [30], in which the working electrode is formed as a comb-shaped electrode.
[32]
The detection set according to any one of [28] to [31], in which the electrode portion further has a reference electrode.
[33]
A detection method for detecting a reaction of a liquid sample with an antibody using the detection set according to any one of [12] to [32].
According to the present invention, it is possible to provide a technique that makes it possible to save time and effort during use and shorten detection time in a case where a plurality of types of solutions such as a reaction liquid, a cleaning solution, and a secondary reaction liquid are deployed in an examination technique using the immunochromatography method.
A description will be given below of an embodiment of the present invention.
The examination kit 18 has the function of detecting the substance to be detected in the liquid sample.
As described in detail below, the examination kit 18 is a type of POCT reagent. A liquid sample, such as blood, is introduced into the examination kit 18 and a specific substance to be detected included in the liquid sample is detected. The immunochromatography method is applied as the method for detecting a specific substance to be detected from the liquid sample.
In the present embodiment, a description will be given of the examination kit 18 which is configured using the electrochemical immunochromatography method, in which the concentration of a biomarker is converted to an electrochemically active substance concentration for detection.
As described above, in the electrochemical immunochromatography method, the deployment of a plurality of types of solutions (liquid samples) such as a reaction liquid, a cleaning solution, and a secondary reaction liquid, is necessary, and, generally, the time and effort required for use is increased and the detection time is increased. In the present embodiment, a flow channel 2 of the membrane carrier 3 is divided into a plurality of areas (here, three areas of first to third fine structure regions 31 to 33) and the flow velocity of the solution is controlled to be different in each area. As the structure for making the flow velocity different, the fine structures formed in the membrane carrier 3, that is, structures that cause a capillary action that determines the speed at which the solution is transported, were set for each area.
The details are described below.
As shown in
The surface of the membrane carrier 3 has, in order from the left side in the figure, a cleaning solution zone 3x, where a cleaning solution is dropped, a droplet zone 3z, where the liquid sample is dropped, and a detection zone 3y for detecting the substance to be detected in the liquid sample. Although not shown here, an absorbent pad for absorbing excess solution is provided on the downstream side from the membrane carrier 3 (on the right side in the figure).
The cleaning solution zone 3x is exposed at a first opening 18b of the housing 18a. The droplet zone 3z is exposed at a third opening 18d of the housing 18a. The detection zone 3y is exposed at a second opening 18c of the housing 18a. The cleaning solution may be dropped in the droplet zone 3z, in which case the first opening 18b may be omitted. In a case of a plurality of types of solutions, introduction ports (openings) are provided according to those solutions. That is, the introduction ports are provided according to what kind of solution is to be moved, at what timing, and at what speed. A plurality of solutions may be dropped at a particular introduction port and the timing thereof may be the same or different.
In the detection zone 3y, an electrode portion 20 is provided for detection using the electrochemical detection method. The electrode portion 20 is, for example, two electrodes (a two-electrode system) formed of a working electrode 25 on the upstream side in the travel direction d and a counter electrode 26 on the downstream side thereof. The electrode portion 20 may be a three-electrode system having a reference electrode 27, as described below. A measuring device 21 is connected to the electrode portion 20. The measuring device 21 may be a general measuring device or may be configured as a device in which a predetermined application is introduced in a mobile terminal such as a smart phone.
As shown in
Due to capillary action, the space between the plurality of protrusions 8 functions as the flow channel 2 that transports the liquid sample along the surface of the membrane carrier 3. In other words, due to the capillary action, gaps in the fine structure 7 function as the flow channel 2 for transporting the liquid sample along the surface of the membrane carrier 3. The plurality of protrusions 8 are formed in rows in regular alignment on the surface of the membrane carrier 3 in a regular or translationally symmetric manner, such as in a lattice arrangement (for example, a diamond-shaped lattice arrangement or a regular lattice arrangement).
The protrusions 8 are, for example, cones and, here, have a circular cone shape, as shown in
The fine structure 7 generates capillary action. Due to the capillary action of the fine structure 7, the liquid sample is transported through the fine structure 7 from the cleaning solution zone 3x or the droplet zone 3z on the left side of the figure to the detection zone 3y (along the travel direction d in
In the present embodiment, as shown in
The speed at which the solution is transported is understood from Poiseuille's equation, which describes the flow between parallel plates. For example, in the fine structure 7 that generates the capillary action phenomenon, for example, in a structure in which a plurality of the protrusions 8 are arranged, the narrower a distance 5 between the protrusions 8, the greater the transport speed of the solution. That is, by appropriately setting the looseness and density of the fine structure (the arrangement of the protrusions 8 shown in
When the substance to be detected in the liquid sample reaches the detection zone 3y, it is detected as a current value by the measuring device 21 through the electrode portion 20 (the working electrode 25 and the counter electrode 26) provided in the detection zone 3y. That is, a potential difference is applied between the working electrode 25 and the counter electrode 26 of the electrode portion 20 and the oxidation current is measured by the measuring device 21. In a case where the color change detection method is used, the substance to be detected is detected according to the color change of the detection zone 3y.
The membrane carrier 3 including the fine structure 7 (a plurality of the protrusions 8) is formed of, for example, thermoplastic plastic. That is, it is possible to produce the membrane carrier 3 having the fine structure 7 by processing a membrane base material formed of thermoplastic plastic by thermal imprinting. The thermoplastic plastic forming the membrane carrier 3 may be, for example, at least one selected from the group consisting of polyester-based resins, polyolefin-based resins, polystyrene-based resins, polycarbonate-based resins, fluorinated resins, and acrylic-based resins. Specific thermoplastic plastics may be, for example, at least one formed of polyethylene terephthalate (PET), cyclo-olefin polymers (COP), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyvinylidene fluoride (PVDF), and polymethyl methacrylate (PMMA).
The glass transition point Tg or melting point Tm of the thermoplastic plastics described above may be 80° C. to 180° C. The storage modulus of the thermoplastic plastic at a temperature 20° C. higher than the glass transition point Tg may be 1.0 Pa or more and 1.0×107 Pa or less. The storage modulus of the thermoplastic plastic at a temperature 20° C. higher than the melting point Tm may be 1.0 Pa or more and 1.0×107 Pa or less.
In a case where the glass transition or melting of the thermoplastic plastic occurs at a temperature of less than 80° C. and the storage modulus of the thermoplastic plastic at a temperature 20° C. higher than the glass transition point or melting point is 1.0×107 Pa or less, it is difficult in practice to use the thermoplastic plastic as a solid at room temperature and difficult to produce a membrane carrier by thermal imprinting.
In a case where the glass transition or melting of the thermoplastic plastic occurs at a temperature higher than 180° C., the molding temperature during thermal imprinting becomes high and the productivity of the membrane carrier decreases. In other words, in a case where the temperature required to soften the thermoplastic plastic during thermal imprinting is higher than 180° C., the productivity of the membrane carrier is reduced.
In a case where the storage modulus of the thermoplastic plastic at a temperature 20° C. higher than the glass transition point or melting point is 1.0×107 Pa or less, it is possible to keep the molding pressure required to produce the fine structure small and the production efficiency is improved because it is possible to carry out production under relatively mild conditions.
It is possible to form the protrusions 8 of the cones (here, circular cones) by thermal imprinting using a mold. In a case of forming a cone using a mold, in comparison with forming a grooved flow channel (line-and-space structure) using a mold, the volume of metal to be scraped from the surface of the metal member during mold production is greatly reduced and the processing cost of the mold is reduced. In contrast, production of a mold for forming a line-and-space structure requires a large volume of metal to be scraped from the metal member.
In addition, the upper part of the cone is thinner than the bottom surface of the cone. Accordingly, in a case where a mold is used to form a cone, in comparison with a case where a column having the same bottom surface as the cone is formed using a mold, the volume of metal that is scraped from the surface of the metal member during the production of the mold is greatly reduced and the processing cost of the mold is reduced.
Furthermore, the porosity of the fine structure with regularly aligned cones is greater than the porosity of a line-and-space structure. In addition, the porosity of the fine structure with regularly aligned cones is greater than the porosity of a structure with a plurality of regularly aligned columns having the same bottom surface as the cone. Therefore, according to the fine structure with regularly aligned cones, it is possible to increase the flow velocity of the liquid sample, which is advantageous for the detection of the substance to be detected.
As described above, it is possible to freely select the shape of a bottom surface 10 of the cone (protrusion 8), which may be a circular cone as shown in
A diameter 4 of the bottom surface 10 of the protrusion 8 is, for example, 10 μm to 1000 μm. In a case where the diameter 4 of the bottom surface 10 of the protrusion 8 is smaller than 10 μm, the fine processing cost of the mold is increased and it is also difficult to uniformly produce numerous fine structures 7 on the surface of the membrane carrier 3, which has a large area. Accordingly, the fine structure 7 that is excessively small is not suitable for practical use. In a case where the diameter 4 of the bottom surface 10 of the fine structure 7 is smaller than 10 μm, the capillary force, which is necessary to move the liquid sample, tends to weaken. In a case where the diameter 4 of the bottom surface 10 of the fine structure 7 is larger than 1000 μm, the volume of metal to be scraped from the metal member during production of the mold becomes larger and the production cost of the mold and membrane carrier 3 is increased. In addition, in a case where the diameter 4 of the bottom surface 10 of the fine structure 7 is larger than 1000 μm, the area of the flow channel 2 in the membrane carrier 3 is also increased, which greatly increases the size of the examination kit 18, which is disadvantageous for the transportation of the examination kit 18 itself. In a case where the protrusion 8 (the fine structure 7) is a circular cone, the diameter 4 of the bottom surface 10 of the protrusion 8 may be the diameter 4 of the bottom surface 10 (circle) of the circular cone.
A height 6 of the protrusion 8 is, for example, 10 μm to 500 μm. In a case where the height 6 of the protrusion 8 is lower than 10 μm, the capillary force, which is necessary to move the liquid sample, tends to weaken. In a case where the height 6 of the protrusion 8 is higher than 500 μm, it is difficult to completely fill the thermoplastic plastic into the recesses of the mold (the depressions corresponding to the shapes of the protrusions 8 of the fine structure 7) during thermal imprinting.
The overall shape of the membrane carrier 3 is not particularly limited, but may be, for example, polygonal, such as a square, circular, or oval. In a case where the membrane carrier 3 is a rectangle, a length L1 of the membrane carrier 3 may be, for example, 2 mm to 100 mm, and a width L2 of the membrane carrier 3 may be, for example, 3 mm to 100 mm. In addition, widths L21 to L23 of the first to third fine structure regions 31 to 33 may each be, for example, 1 mm to 50 mm. The thickness of the membrane carrier 3 excluding the height 6 of the fine structure 7 (that is, the protrusions 8) may be, for example, 0.1 mm to 10 mm.
An aspect ratio Lv/Lh of the protrusion 8 may be 1/10 or more and 2/1 or less. In a case where the aspect ratio Lv/Lh is smaller than 1/10, the contact area between the liquid sample and the flow channel 2 is small and the capillary force decreases, which tends to make it difficult to move the liquid sample. In a case where the aspect ratio Lv/Lh is larger than 2/1, the productivity of the membrane carrier 3 by thermal imprinting decreases. In a case where the protrusion 8 is a cone (more specifically, a circular cone), as in the present embodiment, a length Lh of the protrusion 8 in the horizontal direction may be the diameter 4 of the bottom surface 10 of the protrusion 8. In addition, a length Lv of the protrusion 8 in the orthogonal direction may be the height 6 of the protrusion 8 from the flat portion 9 of the membrane carrier 3.
A ratio D2/D1 of the diameter 4 (D1) of the bottom surface of the protrusion 8 and a distance (D2) between the nearest centers of the protrusions 8 may be greater than 1 and 5 or less. The ratio D2/D1 may not be 1 or less. In a case where the ratio D2/D1 is greater than 5, the contact area between the liquid sample and the flow channel 2 decreases, the capillary force decreases, and the liquid sample tends to be difficult to move. In a case where the protrusion 8 is a circular cone, as in the present embodiment, the diameter 4 (D1) of the bottom surface 10 of the protrusion 8 may be the diameter of the bottom surface of the circular cone and a distance D2 between the nearest centers may be the distance between the vertices of a pair of adjacent protrusions 8 (circular cones). The diameter 4 (D1) of the bottom surface 10 of the protrusions 8 may match the length Lh of the protrusion 8 in the horizontal direction described above. Accordingly, the aspect ratio Lv/Lh may be expressed as Lv/D1.
In addition, in a case where the pitches (distance between vertices) between the protrusions 8 of the fine uneven structures are compared between adjacent zones, the ratio (P1/P2) of a pitch P1 of the zone of the fine uneven structure configured relatively loosely to a pitch P2 of the zone of the fine uneven structure configured relatively densely is 1.1 or more and 5 or less. Here, in a case where the pitch between the protrusions 8 in the first fine structure region 31 is P11, the pitch between the protrusions 8 in the second fine structure region 32 is P21, and the pitch between the protrusions 8 in the third fine structure region 33 is P23, the ratio (P11/P21) is 1.1 or more and 5 or less and the ratio (P21/P23) is 1.1 or more and 5 or less. The ratio (P1/P2) is set according to the speed at which the solution is to be moved. For the lower limit of the ratio (P1/P2), in a case of being small, there will be no difference in speed between zones and the significance of providing a difference in the degree of looseness and density of the fine uneven structure will decrease. From this viewpoint, the ratio (P1/P2) is preferably 1.2 or higher and more preferably 1.3 or higher. For the upper limit, in a case of being excessively large, the difference in the solution movement speed between zones becomes excessively large, making it difficult to adjust the speed throughout the examination kit 18. From this viewpoint, an upper limit of 4 or less is preferable and 3 or less is more preferable.
Referring to
In the arrangement example shown in
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The electrode portion 20 (the working electrode 25 and the counter electrode 26 in the case of a two-electrode system and the reference electrode 27 in the case of three electrodes) may be formed by a conductive substance being provided on the protrusions 8 of the fine structure 7. The conductive substance is not particularly limited and examples thereof include gold, silver, platinum, palladium, carbon, graphene, carbon nanotubes (CNTs), composite materials thereof, and the like. The reference electrode 27 is not particularly limited and examples thereof include an Ag/AgCl electrode.
The conductive substance of the protrusion 8 is, for example, a conductor film formed using at least one of sputtering, vacuum evaporation, laser ablation, and chemical vapor deposition (CVD), or a printed layer formed of a paste (ink) including conductor particles by a method such as ink jet printing or screen printing. The working electrode 25 may be surface modified with thiols or the like for antibody fixation.
In such a case, a maximum peak height Rp of the roughness curve of the electrode portion 20 is 0.005 μm or more and 10 μm or less and an average length RSm of the roughness curve elements is 0.01 μm or more and 15 μm or less. Setting such a surface roughness makes it possible to generate favorable capillary force and to increase the amount of signal obtained due to the increased surface area of the electrode portion 20. It is possible to calculate the surface roughness by analyzing SEM images as shown in
The method for manufacturing the examination kit 18 obtains the examination kit 18 by the following steps.
There is provided a step of producing the membrane carrier 3 having a fine structure (plurality of protrusions 8) corresponding to the shape of the recesses by applying the surface of a mold in which a plurality of recesses are formed to a membrane base material made of thermoplastic plastic and heating the base material.
The method for manufacturing the examination kit 18 is further provided with a step of fixing a reagent or a labeling substance to the detection zone 3y of the surface of the membrane carrier 3 with the fine structure 7, more specifically, to a solid-phase part 50 of the third fine structure region 33.
The fine production method of the mold used in the thermal imprinting step may be, for example, etching, photolithography, machine cutting, laser machining, or the like. It is possible to select a fine production method suitable for the processing size and processing range.
Before performing the thermal imprinting, it is desirable to perform a mold release treatment. In the mold release treatment, for example, a monolayer may be produced on the mold surface to reduce the surface energy. As a result, the membrane carrier 3 formed of thermoplastic plastic is easily detached from the surface of the mold after the thermal imprinting.
The thermal imprinting method may be either a flat press method or a roll method. In the flat press method, the mold is overlaid with base materials formed of thermoplastic plastic between parallel upper and lower stages which face each other, and these are placed between the stages. The mold and base materials are then heated and pressed through the stages. The flat press method is superior in the point of providing favorable molding accuracy. The roll method uses a heated roll mold and molding is performed by the squeezing pressure between the rolls. The roll method has excellent productivity.
Conditions such as the molding temperature, molding pressure, and transfer time when performing thermal imprinting may be selected according to the size of the fine processing, the shape of the fine structure (the protrusions 8), the size of the processing range, and the like. For example, in the case of the flat press method, the molding temperature may be 20° C. to 50° C. higher than the glass transition point Tg or 20° C. to 50° C. higher than the melting point Tm. The molding pressure may be 1 MPa to 10 MPa. The transfer time (time to hold the mold and base materials while applying pressure) may be 3 to 10 minutes. Thermal imprinting under the above conditions makes accurate transfer of the fine structure of the mold to the base material surface easy.
Depending on the type of thermoplastic plastic forming the membrane carrier 3 and the type of reagent (detection substance), it may be difficult to fix the reagent (detection substance) to the solid-phase part 50 of the membrane carrier 3. In such a case, by applying an appropriate surface treatment to the detection zone 3y only in advance, the reagent (detection substance) is easily fixed to the detection zone 3y (that is, the solid-phase part 50) of the membrane carrier 3.
The surface treatment method of the detection zone 3y is not limited at all and may be, for example, various plasma treatments, UV treatments, UV/ozone treatments, or various methods such as surface Modified Example using 3-aminopropyltriethoxysilane or glutaraldehyde.
The reagent (detection substance) fixed in the detection zone 3y may be, for example, an antibody. For example, in
The antibody is a substance that causes an antigen-antibody reaction with the substance to be detected. The antibody may be a polyclonal antibody or a monoclonal antibody. The substance to be detected is not limited at all and may be any substance capable of causing an antigen-antibody reaction with the antibody, such as various pathogens and various clinical markers. Specifically, the substance to be detected may be, for example, virus antibodies for the influenza virus, norovirus, adenovirus, RS virus, HAV, HBs, HIV, and the like. The substance to be detected may be a bacterial antigen for MRSA, group A streptococci, group B streptococci, Legionella spp., or the like, or toxins produced by bacteria or the like. The substance to be detected may be mycoplasma, Chlamydia trachomatis, or a hormone such as human chorionic gonadotropin. The substance to be detected may be a C-reactive protein, myoglobin, cardiac troponin, various tumor markers, pesticides, environmental hormones, and the like. In particular, in a case where there is an urgent need to detect substances to be detected such as the influenza virus, norovirus, C-reactive protein, myoglobin, and cardiac troponin and to take therapeutic measures for diseases caused by these substances, the usefulness of the examination kit 18 according to the present embodiment is particularly great. The substance to be detected may be an antigen able to induce an immune reaction by itself. The substance to be detected may be a hapten that is not able to induce an immune reaction by itself, but is able to bind to an antibody by an antigen-antibody reaction with the antibody.
Referring to the chart shown in
First, the examination kit 18 and the solutions to be used (a reaction liquid, a cleaning solution, and a secondary reaction liquid) are prepared. As described above, an antibody 51 is fixed in the solid-phase part 50 of the third fine structure region 33.
When the reaction liquid is dropped from the droplet zone 3z into the second fine structure region 32, the reaction liquid moves to the third fine structure region 33 due to the capillary action of the fine structure 7.
A detection target 91 and a detection target (labeling body) 92 in the reaction liquid are fixed by reacting with the antibody 51. Excess reaction liquid is absorbed by a water-absorbing pad; however, some of a detection target 91a and a detection target (labeling body) 92a are not fixed and remain on the third fine structure region 33.
Subsequently, a cleaning solution 93 is dropped from the cleaning solution zone 3x to clean the detection target 91a and the detection target (labeling body) 92a that are not fixed to the solid-phase part 50 and remain on the third fine structure region 33. The detection target (labeling body) 92a has an alkaline phosphatase (ALP) labeling body.
After cleaning, a secondary reaction liquid (for example, p-aminophenyl phosphate 94) dropped into the second fine structure region 32 moves to the third fine structure region 33 due to the capillary action of the fine structure 7. The p-aminophenyl phosphate 94 reacts with the detection target (labeling body) 92 fixed on the antibody 51 to produce an electrically active substance (here, p-aminophenol 95). This substance correlates (is proportional) to the amount (concentration) of the detection target (labeling body) 92 fixed on the antibody 51. Accordingly, it is possible to accurately and stably measure the concentration of the target to be measured by the value of the oxidation current measured at the electrode portion 20.
According to the present embodiment, in the examination kit 18 applying the immunochromatography method, it is possible to set the speed at which the solution is moved (speed due to the capillary action) in the flow channel 2 of the fine structure 7 of the membrane carrier 3 to be different in a plurality of regions. As a result, even in a case where it is necessary to deploy a plurality of types of solutions such as a reaction liquid, a cleaning solution, and a secondary reaction liquid, it is possible to adjust the timing of the deployment of these solutions according to the mode of use. Accordingly, it is possible to eliminate the time and effort for timing adjustments and the like, which would normally be necessary, and it is possible to carry out stable and appropriate examination. Specifically, using a specific jig (solution supply device) or the like, it is possible to apply drops simultaneously to a plurality of different locations, in consideration of the timing at which each solution is deployed. That is, it is possible to deploy each solution in only one operation. A description will be given of a configuration using a jig (solution supply device) in the fourth embodiment described below.
Referring to
As shown in the figures, the membrane carrier 103 has a rectangular shape with a predetermined length L10 and a width L20. The membrane carrier 103 is provided with, from the left side, a first fine structure region 131 (width L201), a second fine structure region 132 (width L202), a third fine structure region 133 (width L203), and a fourth fine structure region 134 (width L204). As in the first embodiment, in these regions, the looseness and density of the protrusions in the fine structure are different, resulting in different speeds due to capillary action.
Specifically, the first fine structure region 131 is set to be the loosest (region A11), next, the third fine structure region 133 is the second loosest (region A13), the second fine structure region 132 is the third loosest (region A12), and the fourth fine structure region 134 is the densest (region A14). In addition, the fourth fine structure region 134 is provided with a solid-phase part 150.
In addition, a buffer region with a predetermined width L31 is provided at the second boundary 142 between the second fine structure region 132 and the third fine structure region 133. A fine structure (that is, the protrusions) is not provided in the buffer region. Similarly, a buffer region of a predetermined width L32 is provided at the third boundary 143 between the third fine structure region 133 and the fourth fine structure region 134. By providing such a buffer region, it is possible to absorb differences in the amount of solution transported in each region and prevent backflow or the like from being generated. For example, in the second fine structure region 132 and the third fine structure region 133, the fine structure of the third fine structure region 133 on the downstream side is loose. Accordingly, the solution has a higher movement speed in the second fine structure region 132. As a result, when there is no buffer region at the second boundary 142, backflow may occur depending on the amount of solution being deployed. However, as in the present embodiment, by providing a buffer region in which no capillary force is generated, it is possible to prevent backflow from being generated due to the movement speed of the solution and the amount of solution deployed.
In the present embodiment, referring to
In a membrane carrier 203 shown in
In a membrane carrier 303 shown in
In a membrane carrier 403 shown in
In a membrane carrier 503 shown in
In a membrane carrier 603 shown in
In a membrane carrier 703 shown in
It is possible to combine the configurations in
Referring to
The examination set 1 is provided with the examination kit 18 described above and the solution supply device 60 that supplies each solution to the examination kit 18. The solution supply device 60 accommodates a plurality of solutions (here, first to third solutions 98a to 98c) used for examination. As the solution, it is possible to use the solution shown in the examination method of the first embodiment (refer to
The solution supply device 60 is provided with the solution unit 80 and the communicating unit 70, is arranged so as to cover the top surface of the examination kit 18, and is operated by a user (here, the person in charge of the examination) to supply the solutions (the first to third solutions 98a to 98c) described above to predetermined regions of the membrane carrier 3 of the examination kit 18.
A detailed description will be given below.
The solution unit 80 has a plate-shaped plate 82 that is rectangular in the top surface view and a plurality of solution accommodating parts 85 provided in a concave shape in the plate 82.
The plate 82 and the solution accommodating parts 85 are integrally provided with a resin material. As the resin material, for example, it is possible to use a thermoplastic plastic such as polypropylene or polyethylene as exemplified as the material of the membrane carrier 3. The thickness of the plate 82 and the solution accommodating parts 85 is set such that the solution unit 80 has a certain rigidity, while the thickness of a bottom surface 86 is set such that it is possible for the communicating unit 70 having the injection needle structure to perforate therethrough.
As shown in
The solution accommodating parts 85 (first to third solution accommodating parts 85a to 85c) have a bottomed cylindrical shape. The size of the solution accommodating parts 85 is appropriately set according to the amount of solution to be accommodated. In addition, the shape of the solution accommodating parts 85 is not limited to a cylindrical shape, and may be a rectangular tube shape. The inner diameter of the solution accommodating parts 85 is large enough to be able to accommodate the arrangement part 75 and is set to be substantially the same as the outer diameter of the arrangement part 75. The depth of the solution accommodating parts 85 is set such that the bottom surface 86 of the solution accommodating parts 85 comes into contact with a bottom surface 76 of the arrangement parts 75 when the solution accommodating parts 85 (the first to third solution accommodating parts 85a to 85c) are fitted into the arrangement parts 75 (the first to third arrangement parts 75a to 75c) described below.
The communicating unit 70 has a plate-shaped plate 72 that is rectangular in the top surface view, a frame-like frame 71 that extends downward from the periphery of the plate 72, a plurality of arrangement parts 75 provided in a concave shape in the plate 72, and a communicating part 77 provided in the arrangement part 75. The frame 71, the plate 72, and the arrangement part 75 are integrally provided with a resin material. As the resin material, for example, it is possible to use a thermoplastic plastic such as polypropylene or polyethylene similarly to the solution unit 80. The thicknesses of the frame 71, the plate 72, and the arrangement part 75 are set such that the communicating unit 70 has a certain rigidity.
The frame 71 functions to separate the plate 72 and the examination kit 18 by a predetermined distance and has the function of appropriately fixing the communicating unit 70 to the examination kit 18. As the fixing function, it is possible to adopt a configuration in which the frame lower end portion 73 of the frame 71 is fitted to a step portion 19 provided on the periphery of the examination kit 18.
As shown in
The arrangement parts 75 (the first to third arrangement parts 75a to 75c) have a bottomed cylindrical shape. The size of the cylindrical shape is set such that it is possible to fit and accommodate the solution accommodating parts 85 (the first to third solution accommodating parts 85a to 85c) therein. The shape of the arrangement parts 75 is not limited to a cylindrical shape, but may be a rectangular tube shape as long as it is possible to fit and accommodate the solution accommodating parts 85 therein. The inner diameter of the arrangement parts 75 is set to be substantially the same as the outer diameter of the solution accommodating parts 85. The depth of the arrangement parts 75 is set such that the bottom surface 86 of the solution accommodating parts 85 comes into contact with the bottom surface 76 of the arrangement parts 75 when fitted into the solution accommodating parts 85 (the first to third solution accommodating parts 85a to 85c).
The communicating parts 77 (first to third communicating parts 77a to 77c) have the structure of a tube (straight tube), that is, an injection needle in which the upper end (a needle tip 79) in the figure is formed into a needle shape. Here, the bottom surface 76 of the arrangement parts 75 communicates in the orthogonal direction such that the needle tip 79 of the injection needle is on the inner side of the arrangement parts 75. When the arrangement parts 75 are fitted into the solution accommodating parts 85, the communicating part 77 perforates the bottom surface 86 of the solution accommodating parts 85. In other words, the arrangement parts 75 (the first to third arrangement parts 75a to 75c) and the solution accommodating parts 85 (the first to third solution accommodating parts 85a to 85c) function as positioning parts for arranging the communicating parts 77 (the first to third communicating parts 77a to 77c) at predetermined positions communicating the solution accommodating parts 85 (the first to third solution accommodating parts 85a to 85c). Due to this, the solutions accommodated in the solution accommodating parts 85 pass through the communicating parts 77 and drip onto the examination kit 18.
The communicating parts 77 are formed of a resin material or metal, similarly to a typical injection needle. It is possible to use a thermoplastic plastic such as polypropylene or polyethylene as the resin material. It is possible to use stainless steel as the metal. The size of the communicating parts 77 is not particularly limited as long as the bottom surfaces 86 of the solution accommodating parts 85 are perforated such that it is possible for the solution to drip smoothly, but it is possible to set the inner diameter of the communicating part 77 to, for example, 1 mm to 2 mm.
The position on the examination kit 18 at which the solution is dropped, more specifically, the position on the membrane carrier 3, is determined by the position of the solution accommodating parts 85, that is, the position of the communicating parts 77. Accordingly, the introduction ports (the first opening 18b to the third opening 18d) described in the first embodiment (refer to
In addition, the number of communicating parts 77 is not limited to one for each arrangement part 75, but may be a plurality, or may be different for each arrangement part 75.
Referring to
First, as shown in
Subsequently, as shown in
Next, as shown in
As above, according to the present embodiment, it is possible to start supplying a plurality of solutions (the first to third solutions 98a to 98c) to the examination kit 18 simultaneously with one operation. That is, in a case of deploying a plurality of types of solutions such as a reaction liquid, a cleaning solution, and a secondary reaction liquid, it is possible to save the time and effort of the user during use and to shorten the detection time. In addition, it is possible to maintain a constant timing for developing a plurality of types of solutions.
It is possible to adopt various configurations for the solution supply device 60 without being limited to the configuration described above. Referring to
In the solution supply device 60 of Modified Example 1 shown in
For the solution supply device 60 of Modified Example 2 shown in
Although the embodiments (first to fourth embodiments) of the present invention were described with reference to the drawings, these are examples of the present invention and it is also possible to adopt various configurations (Modified Examples) other than those described above. For example, although the flow channel 2 was provided as a fine structure (fine uneven structure) of the membrane carrier 3 over a substrate formed of resin, it is possible to adopt various configurations, materials, and the like as long as it is possible to provide fine structures (fine uneven structures) with different looseness and density in regions of the flow channel 2.
The characteristics of the present invention will be briefly summarized as follows.
(1) The solution supply device of the present embodiment supplies a plurality of solutions including a reaction liquid to a detection device having a flow channel that transports the reaction liquid and is provided over a substrate formed of a resin, a solid-phase part provided in the flow channel and on which antibodies or antigens are solid-phased, a detection part for detecting reaction of the reaction liquid with the antibodies or the antigens, and a fine uneven structure that has a plurality of protrusions and is formed integrally with the substrate in a region where the flow channel is provided, the solution supply device including a solution unit having a plurality of solution accommodating parts accommodating the respective plurality of solutions, a communicating unit having a plurality of communicating parts provided respectively for the solution accommodating parts and communicating with an inside and an outside of the solution accommodating parts to supply the solutions to the detection device, and a positioning part for arranging the communicating parts in predetermined positions for communicating with the solution accommodating parts.
(2) The communicating part is a tube formed to have one end with a needle shape, and the one end is made to pierce the solution accommodating parts to supply the solution accommodated in the solution accommodating parts to the detection device through the tube.
(3) The communicating unit has a plurality of arrangement parts functioning as the positioning part and accommodating the respective plurality of solution accommodating parts, the communicating parts are provided in the respective arrangement parts, and, when the solution accommodating parts are accommodated in the arrangement parts, the communicating parts communicate between the inside and the outside of the solution accommodating parts to supply the solution to the detection device.
(4) The communicating parts each have an adjustment part that adjusts a position at which the solution is supplied to the detection device.
(5) At least one solution accommodating part of the plurality of solution accommodating parts is fillable with a solution, and remaining solution accommodating parts are filled with a predetermined solution in advance.
(6) By performing one operation of arranging the communicating parts at the predetermined positions for communicating with the solution accommodating parts, the plurality of solution accommodating parts provided in the solution unit communicate simultaneously through the corresponding communicating parts.
(7) The solution supply device of the present embodiment supplies a plurality of solutions including a reaction liquid to a detection device having a flow channel that transports the reaction liquid and is provided over a substrate formed of a resin, a solid-phase part provided in the flow channel and on which antibodies or antigens are solid-phased, a detection part for detecting reaction of the reaction liquid with the antibodies or the antigens, and a fine uneven structure that has a plurality of protrusions and is formed integrally with the substrate in a region where the flow channel is provided, the solution supply device including a solution unit having a plurality of solution accommodating parts accommodating the respective plurality of solutions, and a communicating unit having a plurality of communicating parts provided respectively for the solution accommodating parts and communicating with an inside and an outside of the solution accommodating parts to supply the solutions to the detection device, in which the solution unit and the communicating unit are configured such that, by performing one operation that allows the communicating parts to communicate with the solution accommodating parts, all the solution accommodating parts communicate simultaneously to start supply of the solutions.
(8) The communicating part is a tube formed to have one end with a needle shape and the one end is made to pierce the solution accommodating parts to supply the solution accommodated in the solution accommodating parts to the detection device through the tube.
(9) The solution supply device according to claim 7 or 8, in which the communicating unit has a plurality of arrangement parts accommodating the respective plurality of solution accommodating parts, the communicating parts are provided in the respective arrangement parts, and, when the solution accommodating parts are accommodated in the arrangement parts, the communicating parts communicate between the inside and the outside of the solution accommodating parts to supply the solutions to the detection device.
(10) The communicating parts each have an adjustment part that adjusts a position at which the solution is supplied to the detection device.
(11) At least one solution accommodating part of the plurality of solution accommodating parts is fillable with a solution, and remaining solution accommodating parts are filled with a predetermined solution in advance.
(12) The detection set of the present invention includes a detection device having a flow channel that transports a reaction liquid and is provided over a substrate formed of a resin, a solid-phase part provided in the flow channel and on which antibodies or antigens are solid-phased, a detection part for detecting reaction of the reaction liquid with the antibodies or the antigens, and a fine uneven structure that has a plurality of protrusions and is formed integrally with the substrate in a region where the flow channel is provided, and the solution supply device described above, which supplies a plurality of solutions including the reaction liquid to the detection device.
(13) The detection part is provided closer to another end side (that is, the downstream side) of the flow channel than the solid-phase part.
(14) The fine uneven structure has a first uneven portion in which the plurality of protrusions are provided relatively loosely, and a second uneven portion in which the plurality of protrusions are provided relatively densely, and the first uneven portion and the second uneven portion are provided closer to one end side (that is, the upstream side) of the flow channel than the solid-phase part.
(15) The first uneven portion is provided closer to one end side (that is, the upstream side) of the flow channel than the second uneven portion.
(16) There is a buffer region in which the protrusions are not provided at a boundary between the first uneven portion and the second uneven portion.
(17) A step or a slope is provided at the boundary between the first uneven portion and the second uneven portion, and a region of the step or the slope on a first uneven portion side is higher than a region on a second uneven portion side.
(18) There is a recessed region provided with a recess at a boundary between the first uneven portion and the second uneven portion.
(19) When the first uneven portion and the second uneven portion are adjacent to each other, a ratio (P1/P2) of a pitch (P1) between the protrusions in the first uneven portion and a pitch (P2) between the protrusions in the second uneven portion is 1.1 or more and 5 or less.
(20) A region in which the protrusions are provided in a diamond-shaped lattice shape is provided.
(21) A region in which the protrusions are provided in a regular lattice shape is provided.
(22) The protrusions are provided as cones.
(23) An introduction part for introducing the solution into the flow channel is further provided, in which the solution introduced into the flow channel is formed of a plurality of types of solutions, and the introduction part is provided at a plurality of locations according to the plurality of types of solutions.
(24) An electrode portion is provided in the detection part, and the detection part detects the reaction of the reaction liquid with the antibodies or the antigens based on a current flowing through the electrode portion.
(25) The electrode portion is formed on the protrusion of the fine uneven structure, a maximum peak height Rp of a roughness curve of the electrode portion is 0.005 μm or more and 10 μm or less, and an average length RSm of a roughness curve element is 0.01 μm or more and 15 μm or less.
(26) The electrode portion has a conductive film layer in which a conductive substance is formed on the protrusions of the fine uneven structure by at least one of sputtering, vacuum evaporation, laser ablation, and CVD.
(27) The electrode portion has a printed layer of paste including conductor particles on the protrusions of the fine uneven structure.
(28) The electrode portion has a working electrode and a counter electrode separated from the working electrode, and the working electrode is provided at the same position as the counter electrode or upstream of the counter electrode with respect to a flow channel direction.
(29) The counter electrode is provided over an entire flow channel in a width direction.
(30) The working electrode is provided over an entire flow channel in a width direction.
(31) The working electrode is formed as a comb-shaped electrode.
(32) The electrode portion further has a reference electrode.
(33) The detection method of the present embodiment detects a reaction of a liquid sample with an antibody using the detection set described above.
A specific description will be given of the present invention using Examples (Examples 1 and 2), but the present invention is not limited to these Examples. In the following Examples, experiments were conducted to evaluate the control of solution flow velocity when a fine structure was formed with a plurality of regions of different looseness and density in the membrane carrier.
In the present Example, a description will be given of an experiment for quantitative evaluation of the form of the solution swapping when a tricolor aqueous solution is deployed on the membrane carrier.
(1) In order to confirm the technique of controlling solution deployment by changing the fine structure, in the membrane carrier 3 with the configuration shown in
The fine structure of the mold surface was transferred to the surface of a membrane base material formed of thermoplastic plastic by the following thermal imprinting step. In the thermal imprinting step, X-300 manufactured by SCIVAX was used. In the thermal imprinting step, the surface of the mold with the fine structure (a plurality of recesses) was applied to a membrane base material made of thermoplastic plastic and the mold and base material were pressed while being heated. The molding temperature was 180° C. The applied pressure was 5.5 MPa. The transfer time was 5 minutes. After the fine structure transfer, the mold and base material were cooled to 140° C. while pressure was applied to the mold and base material. The pressure was released after cooling. The membrane carrier of Example 1 was obtained by the above thermal imprinting step. The membrane carrier had a surface that included a plurality of circular cones (fine structures) and flat portions. The shape and size of the protrusions (circular cones) on the surface of the membrane carrier matched the shape and size of the recesses (inverted circular cones) formed on the mold.
The protrusion 8 is a circular cone structure with a diameter 4 and a height 6 which are both 30 μm.
The length L1 of the membrane carrier 3 is 5 mm and respective widths L21 to L23 of the first to third fine structure regions 31 to 33 are each 20 mm.
(2) A water-absorbing pad used in Navi-Flu was attached to the edge of the region of the third fine structure region 33, so as to overlap therewith by 5 mm. Furthermore, the conjugate pads used in Navi-Flu were fixed at the points where the distance between the fine structures changed (positions corresponding to the first boundary 41 and the second boundary 42 in
(3) The conjugate pads were numbered “1”, “2”, and “3” in order of proximity to a water-absorbing pad and aqueous solutions of the compositions shown in Table 1 were dropped on each pad. The amount of dropped solution was determined in consideration of the distance to the water-absorbing pad and the amount trapped in the conjugate pads during deployment. In order to simplify the operation and result verification of this experiment, drops were added to the pads in the order of “1”, “2”, and “3” every 10 seconds.
(4) Video was taken of the manner in which the solution of each color was deployed and the color change at the midpoint between drop point 1 and the water-absorbing pad was analyzed as an image.
The imaged video was converted into images every 10 seconds and imported into image analysis software (software name “Image J”). Images at 30, 140, and 310 seconds after the start of the test are shown in
The measurement point was set at the midpoint between the drop point 1 and the water-absorbing pad and the RGB display data for that point was recorded. Next, the component ratio of each RGB color was calculated according to Equation 1. As an example, Table 2 shows the conversion data for each red (R), green (G), and blue (B) aqueous solution.
Furthermore, in order to quantitatively evaluate the examination results and the flow manner, the following equation 2 was investigated as a method to quantitatively evaluate the mixing ratio. This was calculated by determining how the RGB component ratio of each measurement point was able to be realized according to the extent of mixing the component ratios of the monochromatic colors in Table 2. The solver function of the spreadsheet software Excel was used to minimize the error E between the measured values and calculated values.
x, y, and z are determined such that ε expressed in Equation 2 is minimized.
Here, R_r, R_g, and R_b are the R, G, and B component ratios of the red solution, respectively. G and B mean the same for the green solution and blue solution, respectively. r, g, and b are the measured values for the R, G, and B component ratios at the measurement point, respectively. x, y, and z are the mixing ratios of the red, green, and blue solutions at the measurement point, respectively, and were restricted such that x+y+z=1.
As a result of analyzing the data in
Based on these results, the temporal changes in the mixing ratios of the solutions were quantitatively evaluated by image analysis. That is, it was confirmed that, by appropriately setting the loose and dense state of the fine structure (protrusion spacing) in each region of the membrane carrier, it is possible to adjust the movement speed of the solution in the flow channels of the membrane carrier and to deploy a plurality of solutions.
The results of a similar test and evaluation carried out using nitrocellulose instead of imprinted sheets (Comparative Example) are shown in
Compared to
Although it is potentially possible to produce a device with a structure designed to have a short determination time and no mixing of solutions, it is not possible to perform adjustment of the flow velocity or the like, and the design freedom is low. On the other hand, in a case where the membrane carrier is formed of imprinted sheets as shown in Example 1, there is a high degree of freedom in adjusting the flow velocity and the like.
According to Example 1, in the case of the imprinted sheet membrane carrier, it is possible to control the flow velocity by adjusting the fine structure and material and it is possible to respond flexibly to market needs.
(1) To confirm the technique of controlling solution deployment by changing the fine structure, for the membrane carrier 103 of the second embodiment with the configuration shown in
The protrusion 8 is a circular cone structure with the diameter 4 and the height 6 which are both 32 μm.
The length L1 of the membrane carrier 103 is 5 mm, the width L201 of the first fine structure region 131=15 mm, the width L202 of the second fine structure region 132=30 mm, the width L203 of the third fine structure region 133=45 mm, and the width L204 of the fourth fine structure region 134=40 mm.
In a side surface view, the second fine structure region 132, the third fine structure region 133, and the fourth fine structure region 134 are inclined at an inclination angle of 2.2°.
The first boundary 141 is a continuous boundary between the first fine structure region 131 and the second fine structure region 132 without a buffer region.
The second boundary 142 is the width L31=0.15 mm of the buffer region (unprocessed region).
The third boundary 143 is the width L32=0.20 mm of the buffer region (unprocessed region).
In Experiment 1, using the same experiment and analysis method as in Example 1, the color change at a predetermined point from the RGB component was analyzed to quantitatively confirm the manner in which the liquid was replaced.
Specifically, the color change at a point 5 mm from the most downstream part (the right-side edge of the fourth fine structure region 134 shown in the figure) was analyzed from the RGB components. That is, the solution mixing ratio during the deployment was evaluated based on the RGB component ratio of each solution.
In this experiment, as shown in
The solutions used were as follows.
In Example 3, for the purpose of making the test system closer to actual use conditions, the test conditions of Experiment 2 in Example 2 were partially changed and a CRP detection test was carried out in which human serum was mixed with the sample solution.
In the structure of the test specimen (membrane carrier 103) corresponding to
The length L1 of the membrane carrier 103 is 5 mm, the width L201 of the first fine structure region 131=36.95 mm, the width L202 of the second fine structure region 132=5 mm, the width L203 of the third fine structure region 133=40 mm, and the width L204 of the fourth fine structure region 134=40 mm.
In a side surface view, the second fine structure region 132, the third fine structure region 133, and the fourth fine structure region 134 are inclined at an inclination angle of 2.1°.
The first boundary 141 is a continuous boundary between the first fine structure region 131 and the second fine structure region 132 without a buffer region.
At the second boundary 142, the width L31 of the buffer region (unprocessed region)=0.15 mm.
At the third boundary 143, the width L32 of the buffer region (unprocessed region)=0.15 mm.
1 μL of an anti-CRP antibody solid-phase solution was dropped at a position of 17.5 mm from the most downstream end of the test specimen and dried at 45° C. in the atmosphere for 1 hour to set 25 ng of the anti-CRP antibody as a solid-phase.
As shown in
Ten minutes after all solutions were deployed, the water-absorbing pad and the imprinted sheet (the membrane carrier 103) were separated to prevent backflow of the solution and then the fluorescence intensity of the antibody solid-phase part was measured.
The fluorescence intensity measurement results for each test are shown in
It was possible to achieve an equivalent minimum detection sensitivity and a measurement range of three digits or more (detection range) regardless of the presence or absence of serum.
The fluorescence intensity tended to decrease with the presence of serum. It is assumed that the protein in the serum (up to 80 mg/mL) inhibited the reaction between the antibody and CRP.
In Example 3, for simplicity, a flow channel that functions in a case where one worker continuously drops three types of solutions was designed; however, it is easy to modify the design of the present example to a flow channel design that functions in a case where three types of solutions are dropped simultaneously using a jig or the like.
Specifically, in order to adjust the 6-second time difference from dropping the CRP solution to dropping the fluorescent labeled anti-CRP antibody solution, when the flow velocity in the third fine structure region 133 is set to V (mm/s), the flow channel length of the third fine structure region 133 may be increased by 6×Vmm. The time difference from dropping the CRP solution to dropping the developing solution is also able to be adjusted by increasing the flow channel length of the first fine structure 131 under the same considerations.
As described above, it may be said that it is clear that, when the flow channel design of Example 3 is slightly modified, it is possible to obtain an equivalent detection performance even in a case where three types of solutions are dropped simultaneously using a jig or the like.
In addition, in Examples 2 and 3, preferable examples include examples having a plurality of types of fine structure regions having different flow velocities. On the other hand, it is also possible to obtain the same effect by applying the solution backflow prevention structure (refer to
In Example 4, electrochemical detection tests were carried out on the imprinted sheet based on the results of Examples 1 to 3. In this Example, from the viewpoint of confirming whether it was possible to perform the electrochemical detection appropriately or not, all solutions were dropped at the same position in the fourth fine structure region 134 with different drop timings.
The membrane carrier 103 with the same structure as the test specimen produced in Example 3 was prepared. The length L1 of the membrane carrier 103 is 5 mm, the width L201 of the first fine structure region 131=36.95 mm, the width L202 of the second fine structure region 132=5 mm, the width L203 of the third fine structure region 133=40 mm, and the width L204 of the fourth fine structure region 134=40 mm.
(2) Electrode Portion (Working electrode and Counter Electrode)
An imprinted sheet was attached to a substrate formed by attaching polyimide tape on a SUS plate and gold was vacuum evaporated through a mask processed into an electrode shape as the electrode portion 20.
The electrode shape is as follows.
In the same manner as in Example 3, 1 μL of an anti-CRP antibody solid-phase solution was dropped on a position 5 mm upstream position from the working electrode and dried at 45° C. in the atmosphere for 1 hour to set 25 ng of the anti-CRP antibody as a solid-phase.
Between the electrode portion 20 (working electrode and counter electrode) and the substrate was made to be conductive with silver paste (Dotite D-550) and the substrate was clipped between the alligator clips of an electrochemical measuring device (1252A manufactured by Solartron). The solution was deployed by applying a potential of +50 mV between the working electrode and the counter electrode and the current values were plotted against time (refer to
The dropped solutions were as follows.
This application claims priority based on Japanese Application No. 2021-139004, filed Aug. 27, 2021, the entire disclosure of which is hereby incorporated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-139004 | Aug 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/030094 | 8/5/2022 | WO |
