WO2022176897A1

WO2022176897A1 - Device, chip, and substrate

Info

Publication number: WO2022176897A1
Application number: PCT/JP2022/006125
Authority: WO
Inventors: 周平青山; 雄斗秋山
Original assignee: デンカ株式会社
Priority date: 2021-02-19
Filing date: 2022-02-16
Publication date: 2022-08-25
Also published as: JPWO2022176897A1

Abstract

A device (100) according to the present invention captures and senses a detected substance in a liquid specimen, the device comprising: a base material (101); a flow path (103) which is provided to one surface of the base material (101) and along which the liquid specimen is fed; and a sensing zone (110) provided to one section of the flow path (103). A gel-like pillar (105) constituted by a gel-like substance is provided to the sensing zone (110), a captured substance (107) which specifically binds to the detected substance is held in the gel-like pillar (105), and the flow path (103) is formed by a plurality of flow path forming pillars (109) or microgrooves upstream or downstream of the sensing zone (110).

Description

Devices, Chips and Substrates

The present invention relates to devices, chips and substrates.

Patent Documents 1 to 3 disclose techniques related to devices for detecting components in specimens.
Patent Document 1 discloses an immunoassay system having a channel in which at least a part of a fine structure in which fine beads having a primary antibody immobilized on the surface are uniformly dispersed and held in a photocured hydrophilic resin is arranged. A microchip is described.

Patent Document 2 discloses a microstructure in which one type of specific binding reagent or one type of specimen mixed in a photo-cured hydrophilic resin is held by cross-linking in individual channels provided on a substrate. A microfluidic device is described in which a

U.S. Pat. No. 6,200,000 also discloses a diagnostic element comprising an inlet passageway; a retention port that embeds a diagnostic gel that includes a hole; and an outlet passageway, the inlet passageway and the outlet passageway being on either side of the retention port. Have been described.

WO2007/074756 WO2016/152702 Japanese translation of PCT publication No. 2013-515955

The present invention provides a device that has excellent detection sensitivity and can be used simply without using an additional device for liquid transfer.

According to the present invention, the device, chip and substrate shown below are provided.

[1] A device for capturing and detecting a substance to be detected in a liquid sample,
a base material;
a channel for transporting the liquid sample provided on one surface of the substrate;
a sensing zone provided in a portion of the flow path;
with
The detection zone is provided with gel-like pillars made of a gel-like substance,
a capture substance that specifically binds to the substance to be detected is held in the gel pillar;
The device, wherein the channel is formed by a plurality of channel-forming pillars or microgrooves upstream or downstream of the sensing zone.
[2] an induction zone is provided upstream of the detection zone to guide the liquid sample to the detection zone by capillary action;
The device according to [1], wherein in the guiding zone, the channel is formed by a plurality of the channel-forming pillars or the fine grooves.
[3] the shape of the flow path forming pillar is a cylinder, a cone, a truncated cone, a polygonal prism, a polygonal pyramid, or a truncated polygonal pyramid;
The device according to [1] or [2], wherein the shape of the gel-like pillars is cylindrical or polygonal.
[4] The device according to any one of [1] to [3], wherein the substance to be detected is an antigen, and the capturing substance is an antibody against the antigen.
[5] The device according to any one of [1] to [3], wherein the substance to be detected is a first antibody and the capturing substance is a second antibody specific to the first antibody. .
[6] The device according to any one of [1] to [5], wherein the trapping substance is covalently bonded to the gel-like substance.
[7] The device according to any one of [1] to [6], wherein the detection zone is provided with a plurality of the gel-like pillars in a direction perpendicular to the transport direction of the liquid sample.
[8] The device according to any one of [1] to [7], wherein the detection zone is provided with a plurality of the gel-like pillars in the liquid sample transfer direction of the channel.
[9] The device according to any one of [1] to [8], wherein the gel-like pillars are arranged in a lattice when the detection zone is viewed from above.
[10] The device according to any one of [1] to [9], wherein the gel-like pillars are cylindrical with a diameter of 10 μm or more and 1000 μm or less.
[11] The device according to any one of [1] to [10], wherein the gel-like pillars are cylindrical with a height of 10 μm or more and 1000 μm or less.
[12] The device according to any one of [1] to [11], further comprising a lid covering the channel.
[13] The material of the lid is selected from the group consisting of quartz glass, soda lime glass, borosilicate glass, poly(meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide and polyimide. The device of [12], comprising one or more of
[14] The device according to any one of [1] to [13], wherein a reservoir or a dam for the liquid sample is provided downstream of the detection zone.
[15] an induction zone is provided upstream of the detection zone to guide the liquid sample to the detection zone by capillary action;
The device of any one of [1] to [14], wherein the bottom surface a of the sensing zone is at a lower level than the bottom surface b of the induction zone.
[16] A device for capturing and detecting a substance to be detected in a liquid sample,
comprising a channel for transferring the liquid sample,
the channel has a detection zone in a part of the direction in which the liquid sample is transferred;
The detection zone is provided with a gel-like first pillar that is made of a gel-like substance and holds a capture substance that specifically binds to the substance to be detected. Or the device, wherein the downstream zone is provided with a plurality of second pillars different from said first pillars, or said zone is composed of micro-grooves.
[17] the flow path has an induction zone upstream of the detection zone that guides the liquid sample to the detection zone by capillary action;
The device of [16], wherein the induction zone is provided with the plurality of second pillars or the induction zone is composed of the microgrooves.
[18] A chip having the device according to any one of [1] to [17].
[19] A substrate used in a device that captures and detects a substance to be detected in a liquid sample,
a channel for transferring the liquid sample provided on one surface of the substrate;
a detection zone provided in a portion of the flow path;
with
The detection zone is provided with gel-like pillars made of a gel-like substance,
a capture substance that specifically binds to the substance to be detected is held in the gel pillar;
The substrate, wherein the channel is formed by a plurality of channel-forming pillars or fine grooves on the upstream side or downstream side of the detection zone.
[20] A long substrate,
a first zone provided in a part of the substrate in the longitudinal direction, made of a gel-like substance, and capable of arranging a gel-like first pillar holding a capture substance that specifically binds to a substance to be detected;
a second zone located on at least one side of the first zone in the longitudinal direction of the substrate and provided with a plurality of second pillars different from the first pillars or composed of fine grooves;
A substrate.

According to the present invention, it is possible to provide a device that has excellent detection sensitivity and can be used easily even without using an additional device for liquid transfer.

It is a perspective view showing an example of the configuration of the device in the embodiment. 1 is a cross-sectional view showing an example of the configuration of a device according to an embodiment; FIG. FIG. 4 is a top view showing an example of arrangement of adjacent gel-like pillars in the embodiment; FIG. 4 is a top view showing an example of arrangement of adjacent gel-like pillars in the embodiment; FIG. 4 is a top view showing an arrangement example of adjacent channel forming pillars in the embodiment. 1 is a cross-sectional view showing a configuration example of a device according to an embodiment; FIG. 1 is a top view showing an example of the configuration of a device according to an embodiment; FIG. It is a perspective view showing an example of the configuration of the device in the embodiment. It is a perspective view showing an example of the configuration of the device in the embodiment. It is a figure which shows the structure of the flow-path formation pillar in an Example. FIG. 10 is a diagram showing evaluation results of immunoassay. FIG. 10 is a diagram showing evaluation results of immunoassay. It is a perspective view showing an example of the configuration of the device in the embodiment.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The figures are schematic and do not correspond to actual dimensional proportions. In the present specification, "-" indicating a numerical range represents above and below, and includes both numerical values at both ends.

(device)
FIG. 1 is a perspective view showing an example of the configuration of the device according to this embodiment. A device 100 shown in FIG. 1 is a device that captures and detects a substance to be detected in a liquid sample. The device 100 includes a substrate 101, a channel 103 provided on one surface of the substrate 101 for transferring a liquid sample, a detection zone 110 (first zone) provided in a part of the channel 103, Prepare.
The detection zone 110 is provided with a gel pillar (first pillar) 105 made of a gel substance, and the gel pillar 105 holds a capture substance 107 that specifically binds to the substance to be detected. It is
On the upstream side or downstream side of the detection zone 110, the channel 103 is formed by a plurality of channel-forming pillars (a second pillar different from the first pillar) or fine grooves. An example is shown in which 103 is formed by a plurality of channel-forming pillars. In other words, the zone of the channel 103 upstream or downstream of the sensing zone 110 is provided with a plurality of channel-forming pillars, or such zone is composed of microgrooves.

The device 100 is provided with a guidance zone 120 upstream of the detection zone 110 to guide the liquid sample to the detection zone 110 by capillary action. formed by In other words, the guiding zone 120 of the channel 103 is provided with a plurality of channel-defining pillars 109 .
Further, the device 100 is provided downstream of the detection zone 110 with a discharge zone 130 for discharging the liquid sample from the detection zone 110 by capillary action. It is formed by forming pillars 111 . In other words, the discharge zone 130 of the channel 103 is provided with a plurality of channel-defining pillars 111 .

(Base material)
The base material 101 is specifically a substrate used as the base material of the device 100 . Examples of the shape of the base material 101 include a sheet shape and a plate shape. FIG. 1 shows an example of a device 100 having a sheet-like base material 101 .

Specific examples of the planar shape of the base material 101 include polygons such as squares, circles, and ellipses. When the base material 101 is rectangular, the vertical width (length in the lateral direction) of the base material 101 may be, for example, about 1 to 100 mm, and the width (length in the longitudinal direction) of the base material 101 may be, for example, It may be about 2 to 100 mm.

From the viewpoint of improving the strength of the device 100, the thickness of the base material 101 is, for example, 0.05 mm or more, preferably 0.1 mm or more.
From the viewpoint of thinning the device 100, the thickness of the substrate 101 is, for example, 5 mm or less, preferably 3 mm or less.

Specific examples of materials for the base material 101 include glass such as quartz glass, soda lime glass, borosilicate glass; and poly(meth)acrylate such as polymethyl(meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluorine One or two or more selected from the group consisting of resin materials such as resin, polyvinyl chloride, polyamide, and polyimide can be used.
Moreover, the material of the base material 101 is preferably a thermoplastic resin in that the base material 101 and the flow path forming pillars 109 can be integrally molded. The thermoplastic resin specifically includes one or more selected from the group consisting of polyester, polyolefin, polystyrene, polycarbonate, fluororesin and (meth)acrylic resin, more specifically polyethylene terephthalate. (PET), cycloolefin polymer (COP), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyethylene (PE) 1 or 2 or more.

In the device 100 shown in FIG. 1, the substrate 101 is a flat plate, and the gel-like pillars 105, the channel-forming pillars 109, and the channel-forming pillars 111 are arranged on the surface thereof. The shape is not limited to this.
FIG. 13 is a perspective view showing a configuration example of a device using a substrate having recesses. The basic configuration of the device 180 shown in FIG. 13 is similar to that of the device 100 shown in FIG. is formed.
In the example shown in FIG. 13, the concave portion 129 constitutes a channel for moving the liquid sample. The detection zone 110 may be provided in a part of the recess 129 in the longitudinal direction (transfer direction of the liquid sample). In the example shown in FIG. A detection zone 110 and an ejection zone 130 are provided in that order.
At this time, the bottom surfaces of gel-like pillar 105, channel-forming pillar 109, and channel-forming pillar 111 are positioned on the bottom surface of recess 129 as shown in FIG. 13, for example. In addition, the upper ends of the gel-like pillars 105, the channel-forming pillars 109, and the channel-forming pillars 111 may be positioned at the same level as the upper end of the side wall defining the recess 129, that is, the upper surface of the substrate 127, or at a different level. For example, it may be located below the upper surface of the base material 127 .
Also, in the induction zone 120, the detection zone 110 and the ejection zone 130, the width of the recesses 129 may be the same as shown in FIG. 13 or may be different. For example, the width of recesses 129 in sensing zone 110 may be greater than the width of recesses 129 in induction zone 120 and ejection zone 130 .

(detection zone)
Gel-like pillars 105 are provided in the detection zone (first zone) 110 . On the other hand, as shown in FIG. 1, it is preferable that the detection zone 110 is not provided with flow path forming pillars.
The gel pillar 105 is made of a gel substance. A gel-like substance is specifically a substance comprising a polymeric material having a three-dimensional network structure, which becomes gel-like when a liquid is applied. Therefore, the gel-like pillars 105 may be in a liquid-swollen state or in a non-liquid-swollen state, i.e., in a gel state when the liquid is applied to the detection zone 110 at a desired timing. may be

Specific examples of the material of the gel substance in the gel pillar 105 include polymer materials that form hydrogel. Examples of polymeric materials include (meth)acrylates such as polyethylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, polyurethane (meth)acrylate, gelatin (meth)acrylate, collagen (meth)acrylate; poly(meth)acrylate; ) One or more selected from the group consisting of acrylamide and collagen.
Here, (meth)acrylate is at least one of acrylate and methacrylate.

Examples of the shape of the gel-like pillar 105 include columnar bodies such as cylinders and polygonal columns; cones such as cones and polygonal pyramids; and truncated cones such as truncated cones and polygonal pyramids. These shapes do not need to be geometrically accurate shapes, and may be shapes with rounded corners, shapes with fine unevenness on the surface, and the like.
From the viewpoint of improving moldability, the shape of the gel-like pillars 105 is preferably cylindrical or polygonal.

When the gel-like pillars 105 are cylindrical, the diameter of the gel-like pillars 105 is preferably 10 μm or more, more preferably 50 μm or more, from the viewpoint of improving moldability.
Also, from the viewpoint of improving formability, the diameter of the gel-like pillars 105 is preferably 1000 μm or less, more preferably 500 μm or less.
For the diameter of the gel-like pillars 105, for example, arbitrary five gel-like pillars 105 are selected from the detection zone 110, and the average value of the diameters of the bottom surface or top surface of the selected five gel-like pillars 105 can be adopted. .

When the gel pillars 105 are columnar, the height of the gel pillars 105 is preferably 10 μm or more, more preferably 30 μm or more, from the viewpoint of improving moldability.
Also, from the viewpoint of improving formability, the height of the gel pillars 105 is preferably 1000 μm or less, more preferably 500 μm or less.
For the height of the gel-like pillars 105, for example, arbitrary five gel-like pillars 105 are selected from the detection zone 110, and the average value of the heights of the selected five gel-like pillars 105 can be adopted.

In the detection zone 110, the number of gel-like pillars 105 may be one or two or more.
In the detection zone 110, one gel-like pillar 105 may be provided in a direction perpendicular to the transfer direction X of the liquid sample (w in FIG. 1: width direction of the channel 103), or two or more gel-like pillars 105 may be provided. Shaped pillars 105 may be provided. From the viewpoint of improving the detection sensitivity and measurement accuracy of the substance to be detected, as shown in FIG. is provided. Also, in the detection zone 110, it is preferable that a plurality of gel-like pillars 105 are provided in the liquid sample transfer direction X (extending direction of the channel 103).

The planar arrangement of the plurality of gel-like pillars 105 may be regular or irregular.
From the viewpoint of improving the detection sensitivity and measurement accuracy of the substance to be detected, the gel pillars 105 are preferably arranged in a lattice when the detection zone 110 is viewed from above. More specifically, the lattice-like arrangement includes a square lattice and an oblique lattice such as a hexagonal lattice.

When a plurality of gel-like pillars 105 are provided, the distance between adjacent gel-like pillars 105 in the detection zone 110, that is, the closest distance between the gel-like pillars 105 is 0 μm or more. It is set accordingly. For example, when the gel-like pillars 105 are pillars, the closest distance is preferably greater than 0 μm, and when the gel-like pillars 105 are pyramidal or frustum-shaped, the closest distance is 0 μm or more.
From the viewpoint of moving the liquid such as the liquid sample more efficiently in the detection zone 110, the closest distance is, for example, 5 μm or more, preferably 10 μm or more.
In addition, the contact area between the liquid sample and the gel-like pillars 105 is increased, which increases the capillary force, making it easier to move the liquid sample. The distance is preferably 1000 μm or less, more preferably 500 μm or less.

Here, the "distance between adjacent gel-like pillars 105" is defined by the peripheral surfaces of the two closest gel-like pillars 105 on a line segment connecting the central points of the top view. Distance. Specifically, as the distance between adjacent gel-like pillars 105, five distances between arbitrary adjacent gel-like pillars 105 from the detection zone 110 are selected, and the average value of the selected five distances is adopted. can be done.
3(a), 3(b) and 4 are top views showing an arrangement example of the gel-like pillars 105. FIG. The planar shape of the gel-like pillar 105 is circular in FIGS. 3(a) and 4, and is rectangular in FIG. 3(b).
For example, in the arrangement of the cylinders arranged in a square lattice, the nearest distance is the distance indicated by the arrow in FIG. be. For example, as shown in FIG. 3A, the smaller the distance between the adjacent gel pillars 105, the smaller the gap between the adjacent gel pillars 105, and the greater the number of gel pillars 105 per unit area in top view. As a result, the contact area between the gel pillars 105 and the liquid sample can be increased, and the binding amount of the substance to be detected by the capture substance 107 can be increased. As a result, the detection sensitivity of the substance to be detected can be enhanced.
Regarding the detection sensitivity, more specifically, when the area of the detection zone 110 used for detecting the substance to be detected captured by the gel pillars 105 is larger than the size of the gel pillars 105 in plan view, By reducing the closest distance, the number of gel-like pillars 105 per unit area increases, and the intensity of the detection signal per unit area can be increased.
Further, in a plan view, the greater the number of gel pillars 105 per unit area, the higher the probability of contact between the gel pillars 105 and the substance to be detected. Capturing becomes possible, and as a result, the strength of the detection signal per unit area can be increased.
On the other hand, the greater the distance between adjacent gel-like pillars 105, the easier the molding.

Gel pillars 105 hold capture substances 107 . The trapping substance 107 may be chemically or physically immobilized on the gel pillars 105 . Also, the trapping substance 107 may be included in the gel pillars 105 or may be carried on the surface of the gel pillars 105 .
From the viewpoint of more stably holding trapping substance 107 in gel pillar 105 , trapping substance 107 is preferably covalently bonded to the gel substance in gel pillar 105 . At this time, the trapping substance 107 may be directly bound to the gel pillars 105 or may be bound to intervening molecules such as spacer molecules that are bound to the gel pillars 105 . Intervening molecules preferably include glycols such as polyethylene glycol, ethers, amines, esters, amides, alcohols, carboxylic acids, and the like.

Capture substance 107 is selected from substances that specifically bind to the substance to be detected. For example, when the substance to be detected is an antigen, the capture substance 107 may be an antibody against the antigen or an antigen-binding fragment thereof, preferably an antibody. Here, the antigen may be a substance having immunogenicity by itself, or may be a hapten.
Alternatively, the substance to be detected may be a first antibody, and the capture substance may be a second antibody specific to the first antibody.
When capture substance 107 is an antibody or antigen-binding fragment thereof, the antibody may be a polyclonal antibody or a monoclonal antibody.
When the capture substance 107 is an antibody or an antigen-binding fragment thereof, the substance to be detected may be a substance capable of antigen-antibody reaction with the antibody, such as various pathogens and various clinical markers. More specifically, the substances to be detected include virus antigens such as influenza virus, norovirus, adenovirus, respiratory syncytial virus, HAV, HBs, and HIV, and bacterial antigens such as MRSA, group A streptococcus, group B streptococcus, and Legionella spp. , toxins produced by bacteria, mycoplasma, chlamydia trachomatis, hormones such as human chorionic gonadotropin, C-reactive protein, myoglobin, cardiac troponin, various tumor markers, pesticides and environmental hormones. Its usefulness is even greater when the substance to be detected is an item requiring urgent detection and therapeutic measures, such as influenza virus, norovirus, C-reactive protein, myoglobin, and cardiac troponin. A substance to be detected is usually in a suspended or dissolved state in a liquid sample. A liquid sample may be, for example, a sample in which a substance to be detected is suspended or dissolved in a buffer.

Substances to be detected are not limited to antigens, and may be selected from the group consisting of, for example, proteins and peptides such as enzymes and antibodies; nucleic acids; polysaccharides; and glycoproteins. The capture substance 107 may be a substance having specificity for these substances to be detected. For example, capture substance 107 may be selected from the group consisting of proteins, nucleic acids, polysaccharides, glycoproteins.

The device 100 shows a configuration in which the bottom surface a of the detection zone 110 is at the same level as the bottom surface b of the induction zone 120. From the viewpoint of further increasing the detection sensitivity of the substance to be detected, the bottom surface a of the detection zone 110 may be at a level lower than the bottom surface b of the induction zone 120 .
Also, the bottom surface a of the sensing zone 110 may be at a lower level than the bottom surface c of the ejection zone 130 .

(induction zone)
The guidance zone (second zone) 120 is preferably provided with a plurality of channel-forming pillars 109 from the viewpoint of reliably guiding the liquid sample to the detection zone 110 by capillary action. The planar arrangement of the plurality of flow path forming pillars 109 may be regular or irregular.
From a similar point of view, the channel forming pillars 109 are preferably arranged in a lattice when the guide zone 120 is viewed from above. More specifically, the lattice-like arrangement includes a square lattice and an oblique lattice such as a hexagonal lattice.

Examples of the shape of the flow path forming pillar 109 include columnar bodies such as cylinders and polygonal columns; cones such as cones and polygonal pyramids; and truncated cones such as truncated cones and polygonal pyramids. These shapes do not need to be geometrically accurate shapes, and may be shapes with rounded corners, shapes with fine unevenness on the surface, and the like.
From the viewpoint of improving moldability, the shape of the flow path forming pillar 109 is preferably a cylinder, a cone, a truncated cone, a polygonal cylinder, a polygonal pyramid, or a truncated polygonal pyramid, more preferably a cylinder, a cone, or a truncated cone. A truncated cone is preferred.
The shape of the plurality of channel forming pillars 109 may be the same or different. From the viewpoint of producing a desired fine uneven structure with good reproducibility, it is preferable that the plurality of flow path forming pillars 109 have the same shape.

When the channel-forming pillar 109 is a truncated cone, the diameter of the bottom surface of the channel-forming pillar 109 is preferably 5 μm or more, more preferably 10 μm or more, from the viewpoint of improving formability.
In addition, from the viewpoint of improving formability, the diameter of the bottom surface of the flow path forming pillar 109 is preferably 1000 μm or less, more preferably 500 μm or less.
For the diameter of the bottom surface of the channel-forming pillar 109, for example, arbitrary five channel-forming pillars 109 are selected from the guiding zone 120, and the average value of the diameters of the bottom surfaces of the selected five channel-forming pillars 109 is adopted. be able to.

When the channel-forming pillar 109 is a truncated cone, the height of the channel-forming pillar 109 is preferably 10 μm or more, more preferably 25 μm or more, from the viewpoint of improving moldability.
Moreover, from the viewpoint of improving formability, the height of the flow path forming pillar 109 is preferably 1000 μm or less, more preferably 300 μm or less.
For the height of the channel-forming pillars 109, for example, arbitrary five channel-forming pillars 109 are selected from the induction zone 120, and the average value of the heights of the selected five channel-forming pillars 109 can be adopted. can.

The distance between the adjacent flow path forming pillars 109 in the guiding zone 120, that is, the closest distance between the flow path forming pillars 109 is appropriately set according to the shape of the flow path forming pillars 109, and is, for example, approximately 0 to 500 μm.
For example, when the channel-forming pillars 109 are cylindrical, the closest distance is preferably greater than 0 μm, and when the channel-forming pillars 109 are conical or frustoconical, the closest distance is 0 μm or more. Also, the closest distance may be, for example, 0.1 μm or more, or may be, for example, 2 μm or more.
The upper limit of the closest distance is preferably 500 μm or less, more preferably 100 μm or less, and even more preferably 50 μm or less. As a result, the contact area between the liquid sample and the substrate 101 and the channel-forming pillars 109 increases, increasing the capillary force and facilitating the movement of the liquid sample.

Here, the "distance between the adjacent flow path forming pillars 109" is defined by the peripheral surfaces of the two closest flow path forming pillars 109 on the line segment connecting the center points of the top view. distance As the distance between adjacent flow path forming pillars 109, specifically, five distances between any adjacent flow path forming pillars 109 from the induction zone 120 are selected, and the average value of the selected five distances is adopted. can do.
For example, when the closest distance between conical or truncated conical channel-forming pillars arranged in a hexagonal lattice is 0 μm, the adjacent channel-forming pillars 109 are arranged without gaps, for example, as shown in FIG. . FIG. 5 is a top view showing an arrangement example of the channel forming pillars 109. As shown in FIG. By arranging the channel-forming pillars 109 without gaps, the number of channel-forming pillars 109 per unit area is increased, and the capillary force is further increased, making it easier to transfer the liquid sample.

Specific examples of the material of the flow path forming pillar 109 include those described above as the material of the base material 101 . The material of the channel forming pillars 109 may be the same as or different from the material of the substrate 101 .
Further, for example, the channel forming pillar 109 and the base material 101 may be configured as a single member. That is, the device 100 does not have to have an interface between the base material 101 and the channel-forming pillars 109 .

(emission zone)
In the discharge zone (second zone) 130 , the channel 103 is formed by a plurality of channel-forming pillars 111 . In other words, the discharge zone 130 is provided with a plurality of channel forming pillars 111 . The configuration of the discharge zone 130 and the channel-forming pillars 111 can be, for example, according to the induction zone 120 and the channel-forming pillars 109, respectively.

Although not shown in FIG. 1, from the viewpoint of promoting discharge in the discharge zone 130, the discharge zone 130 of the device 100 may be discharged by overlapping an absorbent pad. Materials for the absorbent pad include paper and porous bodies. As for the arrangement of the absorbent pad, for example, it may be arranged so as to overlap the base material 101 in at least a part of the discharge zone 130 or may be arranged adjacent to the base material 101 .

Although the device 100 shown in FIG. 1 is not provided with a lid, the device may be provided with a lid that covers the channel.
FIG. 2 is a cross-sectional view showing another configuration example of the device according to this embodiment. The basic configuration of the device 150 shown in FIG. 2 is the same as the device 100 described above with reference to FIG. is different.
By providing the lid portion 113, drying of the gel-like pillars 105 in the detection zone 110 can be suppressed. Also. In the induction zone 120 provided with the channel-forming pillars 109 and the discharge zone 130 provided with the channel-forming pillars 111, the flow of the liquid sample by capillary action can be made more stable.
The lid portion 113 may cover the entire substrate 101 or may cover a portion of the substrate 101 .

From the viewpoint of improving the strength of the device 150, the thickness of the lid portion 113 is, for example, 5 μm or more, preferably 10 μm or more.
From the viewpoint of thinning the device 150, the thickness of the lid portion 113 is, for example, 1000 μm or less, preferably 200 μm or less.

From the viewpoint of improving visibility from above the device 150, the lid 113 is preferably made of a transparent material. More specifically, the material of the lid part 113 is glass such as quartz glass, soda lime glass, borosilicate glass; and poly(meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide , polyimide and other resins.

In the above example, the configuration in which the induction zone 120, the detection zone 110, and the discharge zone 130 are provided in this order from the upstream side of the device, that is, the liquid sample introduction side to the downstream side, was described as an example. At least one of the induction zone 120 and the ejection zone 130 need only be provided.
Also, the device may be provided with regions other than the regions described above.
For example, the device may be provided with a sample introduction upstream of the sensing zone 110 and more specifically upstream of the induction zone 120 .
6(a) to 6(d) are cross-sectional views showing examples of arrangement of devices.
As shown in FIG. 6( a ), the device may be provided with a planar portion 115 between the sensing zone 110 and the ejection zone 130 without flow channel forming pillars.
6(c), 6(d) or 6(b), the device may also include, downstream from the sensing zone 110, and more specifically upstream from the exhaust zone 130, a liquid A sample reservoir 119 or reservoir 121 or a damming portion 117 may be provided. None of the reservoir 119 or the reservoir 121 and the damming portion 117 is provided with flow path forming pillars.

In FIG. 6B, in the damming portion 117, the height (thickness of the substrate 101) gradually increases from the detection zone 110 toward the discharge zone 130, and there is a step at the boundary with the discharge zone 130. formed. Due to the presence of this step, the effect of preventing the liquid sample from flowing back from the discharge zone 130 to the detection zone 110, that is, the damming effect is exhibited.
6(c) and the reservoir 121 of FIG. 6(d) are provided with areas where the bottom surface of the substrate 101 is located at a level lower than the bottom surface of the substrate 101 in the detection zone 110. ing. Thereby, the moving speed of the liquid in the detection zone 110 can be adjusted.
6(a) to 6(d) can be appropriately combined to form a desired device. The moving speed of the liquid can be made more suitable.

Further, in the above example, in the induction zone 120 and the discharge zone 130, the channel 103 is configured by a plurality of channel-forming pillars 109 and a plurality of channel-forming pillars 111 (a plurality of flow channels in the channel 103). Although an example in which the passage-forming pillar 109 and a plurality of passage-forming pillars 111 are provided has been shown, the passage 103 may be configured by fine grooves.
FIG. 7 is a top view showing a configuration example of a device having fine grooves. Device 140 shown in FIG. 7 is an example in which the flow paths in induction zone 120 and discharge zone 130 shown in FIG.
The width of the

fine grooves

123 and 125 in the direction perpendicular to the direction of transfer of the liquid sample increases the contact area between the liquid sample and the channel 103, which increases the capillary force, so that the liquid sample can be moved. From the viewpoint of facilitating it, the thickness is preferably 2 μm or more, preferably 2000 μm or less, and more preferably 1000 μm or less. The widths of

microgrooves

123 and 125 may be the same or different.
Further, as a device having fine grooves, for example, the volume of

fine grooves

123 and 125 is 1000 μm width×20 mm length×1000 μm depth, and the volume of detection zone 110 is 2000 μm width×length. A shape having a size of 10 mm×1000 μm in depth and having gel-like pillars 105 arranged in the detection zone 110 can be exemplified.
In the example of FIG. 7, the detection zone 110 is a region wider than the

fine grooves

123 and 125 in the direction perpendicular to the transfer direction of the liquid sample. gel-like pillars 105 are regularly arranged.

Further, in the above example, an example in which a plurality of gel-like pillars 105 are provided in the detection zone 110 has been mainly described, but one gel-like pillar 105 may be provided in the detection zone 110 . In addition, it is preferable to provide one gel-like pillar 105 in the detection zone 110 from the viewpoints of easier fabrication, simpler detection system, and improved liquid fluidity. 8 and 9 are perspective views showing configuration examples of a device in which one gel-like pillar 105 is provided in the detection zone 110. FIG.
The basic configuration of device 160 shown in FIG. 8 and device 170 shown in FIG. 9 are both similar to the configuration of device 100 described above with reference to FIG. The difference is that a columnar gel pillar 105 and one rectangular parallelepiped gel pillar 105 are provided.
When one gel-like pillar 105 is provided in the detection zone 110, for example, the area of the gel-like pillar 105 may be larger than the areas of the channel-forming

pillars

109 and 111 in top view. .

Next, a method of manufacturing a device will be described by taking a device having the channel-forming pillar 109 and the channel-forming pillar 111 as an example.
The device can be obtained, for example, by forming gel-like pillars 105 and at least one of channel-forming pillars 109 and channel-forming pillars 111 on one surface of substrate 101 in a predetermined order.

Examples of the method for forming the channel-forming pillars 109 and the channel-forming pillars 111 include imprinting such as thermal imprinting and UV imprinting;
injection molding;
Pattern formation for UV curable resin by photolithography, soft lithography using UV curable resin patterned by photolithography as a template, pattern formation by etching using UV curable resin patterned by photolithography;
machine cutting; and laser processing. Among these methods, thermal imprinting and injection molding for thermoplastic resins are suitable as techniques for performing precision processing at low cost. Specific examples of the thermoplastic resin include those mentioned above as the material of the base material 101 .
When the shape of the flow path forming pillar 109 and the flow path forming pillar 111 is conical, in the case of a processing method using a mold such as imprinting or injection molding, the conical has a narrower top than the bottom. , the volume to be machined out at the time of manufacturing a mold is smaller than when manufacturing a columnar body with the same bottom surface, and the mold can be manufactured at a low cost. In this case, it becomes possible to detect the substance to be detected in the liquid sample at a lower cost.

As a method for forming the gel-like pillars 105, a liquid containing a UV curable resin and a polymerization initiator is applied between a pair of transparent materials (for example, glass) facing each other with a spacer interposed therebetween, and then a gel is formed. A photomask is provided in the region where the pillars 105 are formed, and UV light is applied to selectively cure the resin in the region provided with the photomask, thereby forming a pattern of the gel pillars 105 . A device is obtained by arranging the obtained gel-like pillars 105 at predetermined positions of the substrate 101 provided with the channel-forming pillars 109 and the channel-forming pillars 111 .
Also, at this time, a liquid containing a gelling agent is applied to a predetermined region of the surface of the base material 101 provided with at least one of the channel-forming pillars 109 and the channel-forming pillars 111, and then according to the procedure described above. By forming the gel pillars 105 , the gel pillars 105 may be formed directly on the substrate 101 .

(Detection method)
In this embodiment, the device can be suitably used for immunoassay. The device in this embodiment is provided with a sensing zone 110 with gel pillars 105 and at least one of an induction zone 120 with channel-forming pillars 109 or an ejection zone 130 with channel-forming pillars 111. Therefore, the substance to be detected in the liquid sample can be stably detected with excellent sensitivity. In addition, the device according to the present embodiment can be used without using an additional device such as a liquid-sending pump, so that the substance to be detected can be easily detected.

In this embodiment, the detection method using the device can be performed, for example, by the sandwich method. At this time, the detection method is, specifically,
A liquid sample containing a substance to be detected is introduced into the upstream side of the detection zone 110, specifically, the induction zone 120 or the upstream side of the induction zone 120, and guided to the detection zone 110 by capillary action. a step of capturing the substance to be detected in the gel-like pillars 105 by specific interaction with the capture substance 107 (step 11);
After the capturing step, a step of introducing a liquid containing a labeled antibody that specifically binds to the substance to be detected into the detection zone 110 and capturing the labeled antibody on the gel pillars 105 in which the substance to be detected is captured (step 12 ); and detecting or quantifying the substance to be detected in the liquid sample by detecting the labeled antibody captured by the gel pillars 105 (step 13).
including.
Also, between steps 11 and 12 and/or between steps 12 and 13, a buffer solution or the like is introduced upstream of the detection zone 110, specifically, upstream of the induction zone 120. and a further step of flushing the sensing zone 110 (step 14) may be performed.

Labeled antibodies used in immunoassays such as enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay can be used as the labeled antibody in step 12 . Labeled antibodies include, for example, fluorescence-labeled antibodies and enzyme-labeled antibodies.
Moreover, in step 13, a detection method can be used according to the type of labeled antibody used in step 12.
For example, when a fluorescence-labeled antibody is used in step 12 , the substance to be detected can be detected or quantified by measuring the presence or absence of fluorescence or fluorescence intensity in gel pillars 105 in step 13 .
Further, when an enzyme-labeled antibody is used in step 12, in step 13, a substrate for the enzyme immobilized on the labeled antibody is introduced into the detection zone 110, and the presence or absence of color development based on the substrate, the absorbance, the presence or absence of fluorescence, and the presence or absence of fluorescence The substance to be detected can be detected or quantified by measuring the intensity, the presence or absence of chemiluminescence, the chemiluminescence intensity, and the like.

(chip)
In this embodiment, the chip comprises the device in this embodiment described above. A chip may consist of a device, and may further have other members. A specific example of the other member is a housing that houses or holds the device.

(substrate)
In this embodiment, the substrate is used in the device in this embodiment described above. The structure described above for the base material 101 can be appropriately used for the substrate.
The substrate is elongated and has a first zone provided in a part of its longitudinal direction, and a second zone located on at least one side of the first zone (both sides in the substrate 101 shown in FIG. 1). 2 zones.
By arranging the gel-like pillars 105 in the first zone, it can function as the detection zone 110 of the device 100 . On the other hand, the second zone is provided with a plurality of channel-forming pillars (pillars different from the gel-like pillars 105) 109 and 111, or because the second zone is composed of fine grooves, the guiding zone of the device 100 120 and discharge zone 130 .

Although the embodiments of the present invention have been described above with reference to the drawings, these are examples of the present invention, and various configurations other than those described above can be adopted.

The present embodiment will be specifically described below with reference to examples and comparative examples, but the present embodiment is not limited to these examples.

(Examples 1 and 2)
In this example, a device in which an anti-CRP antibody was immobilized on gel-like pillars was produced, and CRP (C-reactive protein) in a liquid sample was detected.

(Fabrication of device)
A device having the schematic structure shown in FIG. 2 was fabricated.
1. Preparation of Induction Zone 120 and Ejection Zone 130 The base material 101 having the channel-forming pillars 109 and the channel-forming pillars 111 was produced by the following procedure. The shape and arrangement of the channel-defining pillars in both the induction zone 120 and the discharge zone 130 were the same.
A mold is pressed against a polymethyl methacrylate sheet (manufactured by Sumika Acrylic Co., Ltd., film thickness: about 200 μm), and thermal imprinting is performed under the conditions of a heating temperature of 160 ° C., a pressure of 5.5 MPa, and a pressing time of 3 minutes. The diameter of the bottom surface (hereinafter also referred to as "diameter") of 60 μm, the height of the pillar (hereinafter also referred to as "height") of 90 μm. 10(a) and 10(b) with an average distance of 62 μm, and the gap between the induction zone 120 and the discharge zone 130 is 4.5 mm. made.
Here, in the mold for thermal imprinting, truncated cone-shaped holes with an entrance diameter of 60 μm and a depth of 90 μm are arranged in a hexagonal grid arrangement with an average distance between the centers of the holes of 62 μm. , a nickel mold with a corresponding gap between the induction zone 120 and the ejection zone 130 of the substrate 101 of 4.5 mm. The holes were made by machining. The planar shape of the substrate 101 was 5 mm wide×30 mm long, the planar shape of the induction zone 120 was 5 mm wide×4.5 mm long, and the planar shape of the discharge zone 130 was 5 mm wide×21 mm long. 10(a) and 10(b) are a top view showing an optical microscope and a perspective view showing an SEM image of the channel forming pillar 109 and the channel forming pillar 111 in the obtained base material 101, respectively. In FIGS. 10(a) and 10(b), the laterally adjacent channel-forming pillars are arranged without gaps, and in FIGS. 10(a) and 10(b), the diagonally adjacent channel-forming pillars are They are arranged almost seamlessly.

2. Preparation of Gel Pillar 105 1 . Gel-like pillars 105 were formed on the substrate 101 obtained in 1. by the following procedure. The planar shape of the detection zone 110 was 5 mm wide×4.5 mm long.
2.1 Preparation of Polymer Solution For each of Examples 1 and 2, the following polymer solutions were prepared.
(a) Example 1
A solution obtained by diluting polyethylene glycol diacrylate (PEGDA, number average molecular weight 575, manufactured by Aldrich) with phosphate buffer solution (PBS) was treated with 2-hydroxy-2-methylpropiophenone as a photocuring initiator and as an antibody. A polymer solution (antibody concentration approximately 100 μg/mL, 20% PEGDA, 0.55% initiator) was prepared by adding anti-CRP antibody.
(b) Example 2
A solution obtained by diluting polyethylene glycol diacrylate (PEGDA, number average molecular weight 575, manufactured by Aldrich) with phosphate buffer solution (PBS) was treated with 2-hydroxy-2-methylpropiophenone as a photocuring initiator and as an antibody. By adding an anti-CRP antibody (PEG acrylated CRP antibody, Ac-PEG-Ab) modified with a PEG-Ac linker (PEG: molecular weight 2000), a polymer solution (antibody concentration of about 300 μg/mL, 20% PEGDA, 0.54% initiator) was prepared.
Ac: acrylic group PEG: polyethylene glycol group Ab: antibody molecule

2.2 Gelling 1. The polymer solution of each example was supplied to the gap between the induction zone 120 and the discharge zone 130 in the substrate 101 obtained in 1., and a cover glass (thickness of about 0.15 mm) was placed thereon. A mask (circular holes with a diameter of 400 μm arranged in a square lattice with a center-to-center distance of 800 μm) was covered, and an exposure apparatus (UIV270, manufactured by Ushio Inc.) was used. When the polymer solution of Example 2 was used, the gel-like pillars 105 were produced by light irradiation for 60 seconds. 2% Triton x-100/PBS was then used to wash away the uncured polymer solution.

3. Arrangement of Absorbent Pad Cut into a size of 5 mm in width and 40 mm in length at a position 10 mm from the end of the formation area (discharge zone 130) of the flow path forming pillar 111 in the substrate 101 on which the gel pillars 105 are formed. Absorbent pads (PVA sponge, A-3150HP, manufactured by Aion Co., Ltd., thickness of about 0.6 mm) were overlapped and fixed with tape.

(immunoassay)
Using the device obtained in each example, immunoassay was performed according to the following procedure.
1. 10 μL of an aqueous solution (CRP, concentration 10 μg/mL) obtained by diluting the antigen with 2% TritonX100/PBS was dropped onto the induction zone 120 on the substrate 101 and waited for 3 minutes.
2. 10 μL of an aqueous solution (FITC (fluorescein isothiocyanate)-labeled anti-CRP antibody, 20 μg/mL) obtained by diluting a fluorescence-labeled antibody with 2% Triton X100/PBS was dropped onto the induction zone 120 of the base material 101 and waited for 1 minute. This operation was repeated two more times (three times in total).
3. 10 μL of 2% Triton X100/PBS was dropped onto the induction zone 120 on the substrate 101 and waited for 1 minute. This operation was repeated once more (2 times in total).
4. It was confirmed that the aqueous solution had run off on the substrate 101 and was absorbed by the absorbent pad. (manufactured by Olympus Corporation).
The evaluation results of Examples 1 and 2 are shown in FIGS. 11(a) and 11(b), respectively. FIGS. 11(a) and 11(b), and FIGS. 12(a) and 12(b), which will be described later, show fluorescence microscope images of the gel-like pillars 105. FIG.

(Comparative example 1)
In Example 1, immunoassay 1. 1, except that 2% Triton X100/PBS was used instead of the aqueous solution diluted with the antigen. The results are shown in FIG. 12(a).

(Comparative example 2)
In Example 2, immunoassay 1. 2, except that 2% Triton X100/PBS was used instead of the aqueous solution diluted with the antigen. The results are shown in FIG. 12(b).

From FIGS. 11(a), 11(b), 12(a) and 12(b), in each example, CRP is captured by the gel pillars 105 by specific binding with the anti-CRP antibody. , can be stably detected.

This application claims priority based on Japanese Patent Application No. 2021-025325 filed on February 19, 2021, and the entire disclosure thereof is incorporated herein.

100 Device 101 Base material 103 Channel 105 Gel-like pillar (first pillar)
107 capture substance 109 channel-forming pillar (second pillar)
110 detection zone (first zone)
111 flow path forming pillar 113 lid portion 115 flat portion 117 damming portion 119 reservoir portion 120 induction zone (second zone)
121 reservoir 123 microgroove 125 microgroove 127 substrate 129 recess 130 discharge zone 140 device 150 device 160 device 170 device 180 device

Claims

A device for capturing and detecting a substance to be detected in a liquid sample,
a substrate;
a channel for transporting the liquid sample provided on one surface of the substrate;
a sensing zone provided in a portion of the flow path;
with
The detection zone is provided with gel-like pillars made of a gel-like substance,
a capture substance that specifically binds to the substance to be detected is held in the gel pillar;
The device, wherein the channel is formed by a plurality of channel-forming pillars or microgrooves upstream or downstream of the sensing zone.
an induction zone is provided upstream of the detection zone to guide the liquid sample to the detection zone by capillary action;
2. The device according to claim 1, wherein in said guiding zone, said channel is formed by a plurality of said channel-forming pillars or said microgrooves.
The shape of the flow path forming pillar is a cylinder, a cone, a truncated cone, a polygonal prism, a polygonal pyramid, or a truncated polygonal pyramid,
3. The device according to claim 1 or 2, wherein the shape of said gel-like pillars is cylindrical or polygonal.
The device according to any one of claims 1 to 3, wherein the substance to be detected is an antigen, and the capture substance is an antibody against the antigen.
The device according to any one of claims 1 to 3, wherein the substance to be detected is a first antibody and the capture substance is a second antibody specific to the first antibody.
The device according to any one of claims 1 to 5, wherein said capture substance is covalently bound to said gel-like substance.
The device according to any one of claims 1 to 6, wherein the detection zone is provided with a plurality of the gel-like pillars in a direction orthogonal to the transfer direction of the liquid sample.
The device according to any one of claims 1 to 7, wherein the detection zone is provided with a plurality of the gel-like pillars in the liquid sample transfer direction of the channel.
The device according to any one of claims 1 to 8, wherein the gel-like pillars are arranged in a lattice when the detection zone is viewed from above.
The device according to any one of claims 1 to 9, wherein the gel-like pillar has a cylindrical shape with a diameter of 10 µm or more and 1000 µm or less.
The device according to any one of claims 1 to 10, wherein the gel-like pillars are cylindrical with a height of 10 µm or more and 1000 µm or less.
The device according to any one of claims 1 to 11, further comprising a lid covering the channel.
1, wherein the material of the lid is selected from the group consisting of quartz glass, soda lime glass, borosilicate glass, poly(meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide and polyimide; 13. The device of claim 12, comprising a species or two or more species.
The device according to any one of claims 1 to 13, wherein a reservoir or a dam for the liquid sample is provided downstream of the detection zone.
an induction zone is provided upstream of the detection zone to guide the liquid sample to the detection zone by capillary action;
15. A device according to any one of the preceding claims, wherein the bottom surface a of the sensing zone is at a lower level than the bottom surface b of the induction zone.
A device for capturing and detecting a substance to be detected in a liquid sample,
comprising a channel for transferring the liquid sample,
the channel has a detection zone in a part of the direction in which the liquid sample is transferred;
The detection zone is provided with a gel-like first pillar that is made of a gel-like substance and holds a capture substance that specifically binds to the substance to be detected. Or the device, wherein the downstream zone is provided with a plurality of second pillars different from said first pillars, or said zone is composed of micro-grooves.
the flow path has an induction zone upstream of the detection zone that guides the liquid sample to the detection zone by capillary action;
17. The device of claim 16, wherein the induction zone is provided with the plurality of second pillars or is composed of the microgrooves.
A chip comprising the device according to any one of claims 1 to 17.
A substrate used in a device that captures and detects a substance to be detected in a liquid sample,
a channel for transferring the liquid sample provided on one surface of the substrate;
a detection zone provided in a portion of the flow path;
with
The detection zone is provided with gel-like pillars made of a gel-like substance,
a capture substance that specifically binds to the substance to be detected is held in the gel pillar;
The substrate, wherein the channel is formed by a plurality of channel-forming pillars or fine grooves on the upstream side or downstream side of the detection zone.
A long board,
a first zone provided in a part of the substrate in the longitudinal direction, made of a gel-like substance, and capable of arranging a gel-like first pillar holding a capture substance that specifically binds to a substance to be detected;
a second zone located on at least one side of the first zone in the longitudinal direction of the substrate and provided with a plurality of second pillars different from the first pillars or composed of fine grooves;
A substrate.