JP4613099B2 - Electrochemical detector - Google Patents

Description

  The present invention generally relates to an electrochemical detection apparatus that performs electrochemical measurement or analysis of the amount of a substance contained in a small amount of liquid, and more specifically, an electrochemical detection apparatus improved to achieve high sensitivity. About.

  In recent years, many people have been suffering from allergic symptoms caused by airborne pollutants, mites, fungi, volatile organic compounds, and other airborne microscopic substances and chemical substances. Therefore, development of microchannel devices for measuring the amount of proteins such as allergens that cause allergies is extensive. As such a device, a microchannel device as shown in FIG. 25 has been proposed (see, for example, Patent Document 1).

  As shown in FIG. 25, on a substrate 151 such as glass or silicon, a microchannel reaction vessel having a solid fine particle 152 having a diameter of 1 mm or less as a reaction solid phase and a longitudinal sectional area larger than the diameter of the solid fine particle 152. 153, a microchannel separation unit 154 having a vertical cross-sectional area smaller than the diameter of the solid microparticles 152, an introduction unit or a microchannel inflow unit 155, 156 for introducing the antigen and the labeled antibody separately to the reaction tank unit 153, the first A microchannel inflow portion 157 for introducing an antibody and a microchannel inflow portion 158 for introducing a buffer solution or a washing solution are provided. At the end of each microchannel inflow portion 155, 156, 157, 158, there are provided an injection hole portion 155A, 156A, 157A, 158A for the antigen, labeled antibody (second antibody), first antibody, and washing solution, A waste liquid section 154A is provided at the end of the microchannel separation section 154. The solid microparticles 152 serve as a reaction solid phase for immune antigen-antibody reaction. The solid microparticles 152 as the reaction solid phase do not flow into the microchannel separation unit 154 but are blocked so that only unreacted materials flow into the microchannel separation unit 154 and are separated.

  According to this conventional example, immunoanalysis can be easily performed with a short reaction time by using a small amount of sample. As an immunoassay method, solid microparticles 152 as a reaction solid phase are introduced into a microchannel reaction tank unit 153, and antigens and labeled antibodies introduced from the introduction units or microchannel inflow units 155 and 156 are transferred onto the solid microparticles 152. React with. Next, unreacted substances are separated by the microchannel separation unit 154 and analyzed by photothermal conversion analysis. Photothermal conversion analysis is performed by a thermal lens microscope.

  However, as described above, the proposed device uses the thermal lens method as a detection method, which is problematic from the viewpoint of downsizing the apparatus.

  On the other hand, a device combined with an electrochemical detection method has been studied (for example, see Patent Document 2). By combining this with the microchannel device of Patent Document 1, proteins such as allergens are electrically isolated. An electrochemical detection type microchannel device capable of detection can be realized.

  FIG. 26A is a top view of such an electrochemical detection type microchannel device, and FIG. 26B is a cross-sectional view taken along the line BB in FIG. Referring to these drawings, a microchannel injection hole 504A, a discharge hole 505A, a microreaction vessel 503, and microchannels 504 and 505 are formed on the surface of the substrate 501, and their depth is 100 μm, The width of the flow path is 200 μm. A damming portion 508 having a flow channel having a width smaller than the diameter of the fine particles is formed between the microreaction vessel 503 and the flow channel 505 so as to dam the fine particles 502 on which the antibody is immobilized. . In addition, on the surface of the lid 510, the electrodes 506, the connection pads 507, and the wirings 509 that electrically connect them are formed in a desired pattern on the microchannel 505.

  For example, a test solution containing allergen is injected from the injection hole 504A using an external pump or the like, and reacted with the immobilized microparticle 502 with an antibody that specifically reacts with the allergen to capture the allergen on the surface of the microparticle 502. . After washing with a buffer solution, a solution containing an antibody labeled with an enzyme is injected from the injection hole 504A to form a complex composed of an immobilized antibody-allergen-enzyme-attached antibody on the surface of the microparticles 502. After washing with a buffer solution, a substrate material that is converted into an electrode active substance by an enzyme is flowed and converted into an electrode active substance by an enzyme of the complex. By detecting the electrode active substance in the region where the electrode 506 is formed from the connection pad 507 as a change in current or potential, the concentration of the detection target substance in a very small amount of sample liquid can be known.

  Referring to FIGS. 26 and 27, the electrode 506 includes a comb-shaped working electrode 520, a reference electrode 521, and a counter electrode 522 as proposed in Patent Document 3, the surface of the lid 510. The electrode 506 has a thickness of 1 μm or less. The depth (space thickness) of the flow path 505 in the region where the electrode 506 is formed is set to about 15 μm to 1000 μm.

  In such a device structure, the redox reaction at the working electrode 520 occurs only in the electrode active material near the working electrode 520, and the electrode active material located away from the working electrode 520 flows without contributing to the reaction. End up. As a result, the obtained current value or voltage value is small, and there is a problem that the detection sensitivity of the device is low. In order to solve this, it is conceivable to stop the flow of the liquid and carry out the oxidation-reduction reaction. However, since the working electrode 520 has no action of attracting the electrode active substance, the electrode activity at a location away from the working electrode 520 In order for a substance to come to the vicinity of the working electrode 520, there is no choice but to rely on the diffusion action, and there is a problem that the measurement time becomes very long in order to obtain a large current value or voltage value.

  Patent Document 4 proposes an electrode structure in which the working electrode is three-dimensionally formed. In order to increase the current value, it is necessary to increase the number of comb-type working electrodes, but in the electrode structure disclosed in this document, many electrode walls are formed in a predetermined region. There is a concern that the ratio of the height and width of a single wall becomes large, and the yield in the microfabrication process is remarkably deteriorated, the strength of the formed wall is reduced, and defects are generated during use. There were problems such as.

JP 2001-4628 A

JP 2003-285298 A

JP-A-1-272958

JP 2004-93406 A

  The present invention has been made to solve the above-described problems, and an object thereof is to provide an electrochemical detection apparatus improved so as to increase the detection sensitivity of the device.

  Another object of the present invention is to provide an electrochemical detection apparatus improved so as to shorten the measurement time.

  Another object of the present invention is to provide an electrochemical detection device excellent in mass productivity.

The electrochemical detection apparatus according to the present invention includes a reaction unit that causes a reaction in the space, a detection unit having an electrode for performing electrochemical detection in the space, the reaction unit, and the detection unit. And the depth of the space of the detection unit is shallower than the depth of the flow path. In the detection unit, a convex portion that protrudes toward the other surface is provided on one of the upper surface and the lower surface. A concave depression is formed on the other surface of the detection unit so as to face the projection. The electrode for performing electrochemical detection is formed in the concave depression.

  According to a further preferred embodiment of the present invention, the side surface of the convex portion is tapered.

  According to a further preferred embodiment of the present invention, the side surface of the concave recess is tapered.

  According to a further preferred embodiment of the present invention, the upper surface of the convex portion and the surface on which the concave depression is formed are on the same plane.

In the detection unit, a film or a resin layer having an opening is provided on the other of the upper surface or the lower surface so that the concave depression is formed .

An electrochemical detection apparatus according to another aspect of the present invention includes a reaction unit that causes a reaction in the space, a detection unit that includes an electrode for performing electrochemical detection in the space, and the reaction unit. A flow path connecting the detection unit, and the depth of the detection unit space is shallower than the depth of the flow channel, and in the detection unit, one of the upper surface or the lower surface is provided in the space. In the electrochemical detection device provided with a convex part that rises toward the surface, in the detection part, one of the upper surface or the lower surface is provided with a connection pad connection part electrically connected to the connection pad, The said convex part is comprised with the base material piece which has thickness, the said electrode is provided in the surface of the said base material piece, and the 1st connection connected to the said connection pad connection part on the back surface of the said base material piece A thickness portion of the base material piece A through hole is formed so as to penetrate through the first through hole, and the first connection portion and the connection pad connection portion are electrically connected through the through hole. The first connecting portion provided on the connecting pad is attached to one of the upper surface or the lower surface of the detecting portion with an adhesive so as to contact the connecting pad connecting portion, thereby forming the convex portion. Yes.

In the present invention, there is provided a detection device including at least a reaction part that causes an immune reaction, an enzyme reaction, etc., a detection part on which an electrode for performing electrochemical detection is formed, and a flow path connecting them. since the depth of the space is shallower than the depth of the upper Kiryuro, the smaller the volume of the near detector, the density reduction due to diffusion can be reduced compared to the conventional, it is possible to obtain a large amount of current as a result .

  Also, in this configuration, compared to the conventional configuration, while the liquid flows through the detection unit, the amount reaching the electrode surface due to the diffusion force and the collision by the wall and the collision diffusion in the liquid due to it increases. The advantage that a larger amount of current can be obtained is obtained. Further, since a large amount of current can be obtained as described above, there is an effect that high sensitivity detection can be realized as an apparatus.

  In addition, in this configuration, the depth of the flow path can be set regardless of the distance between the electrode surface and the substrate surface opposite to the electrode surface. There is also an effect that an immune reaction and an enzyme reaction can be realized simultaneously. In addition, since the enzyme reaction efficiency is improved by the effect, the amount of the electrode active substance is increased, and as a result, a large current amount can be obtained as compared with the conventional detection device, and a synergistic effect that high sensitivity can be realized is achieved. Arise.

  In the present invention, in the detection unit, the first connection part electrically connected to the connection pad is provided on one of the upper surface or the lower surface, and the convex part is provided on the back surface of the detection unit. A second connecting portion connected to the connecting portion; a through hole penetrating the convex portion in the thickness direction; and the electrode provided on the surface for performing electrochemical detection, the through hole Since the electrode and the second connection portion are electrically connected to each other, there is a problem that the wiring connecting the electrode and the connection pad provided in the detection portion is cut by a step as in the prior art. The effect of disappearing occurs. Moreover, in this structure, when producing the said convex part with a film etc., after producing the structure which provided the several electrode and the 2nd connection part in the big film, cut and paste each structure one by one This makes it possible to produce an advantage in terms of mass productivity with respect to the conventional method of producing electrodes for each batch.

  Further, in the present invention, in the detection unit, a layer made of a film or a resin layer, in which an opening that exposes at least a part of the electrode is provided on a surface on which the electrode unit, the wiring, the connection pad, and the like are formed in advance. Since the structure is characterized in that it is formed, there is an effect that the problem that the wiring connecting the electrode and the connection pad provided in the detection portion and the connection pad is cut by a step is eliminated as in the prior art.

Embodiments of the present invention will be described below with reference to the drawings.
[Embodiment 1]

  FIG. 1 is a top view of the electrochemical detection type microchannel device according to the first exemplary embodiment, and FIG. 2 is a cross-sectional view taken along line II-II in FIG.

  As shown in FIG. 1, in the electrochemical detection type microchannel device according to the present invention, a lid 20 is provided on the substrate 1 so that the surface of the substrate 1 and the surface of the lid 20 face each other. A test solution containing at least a detection target substance to be measured, an introduction hole 2 for introducing a buffer solution, a reaction unit 3 for causing an immune reaction or an enzyme reaction, a detection unit 4, a test solution, a buffer solution A flow path 6, a flow path 7 and a flow path 8 are formed through which the discharge holes 5 flowing out of the flow path, the test solution connecting them, and the buffer solution flow.

  In the detection unit 4, a convex portion 22 that is a step is provided on the surface of the substrate 1. The structure of the substrate 1 in which the flow paths 6, 7, and 8 are formed may be manufactured as an integral structure by pouring light or a thermosetting resin into a mold in which a flow path pattern is formed as in the past, and solidifying it. Moreover, the thing of the integral structure formed by the hot embossing method using the type | mold which formed the flow-path pattern in the board | substrate which consists of a polymethacrylic acid resin and a polycarbonate resin may be used.

  Further, for example, as shown in FIG. 3, a support substrate 10 may be bonded with a film 11 having a pattern of holes corresponding to the flow path. In this case, a plurality of holes having a predetermined flow path pattern may be formed at a time on a large-area film, and then cut into a predetermined size and bonded to the support base. Compared with the method, there is an advantage from the viewpoint of mass productivity.

  Referring to FIG. 1 again, the thickness of the substrate 1 is about 0.1 mm to 5 mm. The introduction part 2 and the outflow part 5 may be holes having a diameter of 10 μm or more and a depth of 0.1 μm to 1 mm, and the shape is not particularly limited. The reaction part 3 is provided with an antibody that reacts specifically with the allergen. The antibody may be immobilized on an insoluble carrier such as a bead 12, or may be provided on a substrate. The reaction section 3 may have a cylindrical hole, a hemispherical shape, a rectangular parallelepiped hole, or a part of a flow path, and may have a size of 10 μm or more and a depth of 0.1 μm or more. It may be 1 mm. Further, when the antibody is immobilized on an insoluble carrier such as a bead 12 and the carrier is not immobilized on a substrate, a damming portion 13 is provided in which the cross-sectional area of the connection port to the channel 7 is smaller than the size of the insoluble carrier. What is necessary is just to make it the structure.

  By using this configuration, even when the test solution or the buffer solution flows into the reaction unit 3 through the flow channel 6 and flows out to the flow channel 7, the carrier is prevented from flowing out of the reaction unit 3 together with the liquid. Can do. As another method, magnetic particles may be used as the insoluble carrier and fixed to the reaction unit 3 by a magnet (not shown) separately provided outside the substrate 1 or the lid 20. In this description, although it is described as an antibody material for simplicity, the material to be used may be any material that specifically reacts with and captures the detection target substance, such as a monoclonal antibody, polychlore, and the like. An antibody material such as an antibody, an imprinting polymer, or an artificial antibody material such as an aptamer material may be used.

  The flow paths 6, 7, and 8 have a width of 0.1 μm to 1 mm, and a depth of 0.1 μm to 1 mm, preferably 1 μm to 500 μm. The cross-sectional shape may be rectangular or trapezoidal, and the bottom of the flow path may be round like a part of a circle. The lid 20 can be made of a photocurable resin, glass, plastic material, silicon substrate, metal substrate, film, or a combination thereof. On the surface of the lid 20, the detection electrode 14 is formed at a position corresponding to the convex portion 22.

  The detection unit, which is a feature of the present invention, will be described in detail with reference to FIG. In the present invention, the electrode 14 is formed on the surface of the lid 20 so as to face the convex portion 22. The distance between the surface of the electrode 14 in contact with the liquid and the surface 21 of the convex portion 22 (the depth of the space of the detection unit) is at least the depth of the reaction unit 3 (the thickness of the space) or the flow path connected to the detection unit 4 It is characterized by being smaller than the smallest of 7 or 8 depth (space depth).

  The width of the flat portion of the convex portion 22 formed on the substrate 1 may be equal to or larger than the size of the region in contact with the liquid of the working electrode, and the length in the direction along the flow path is not particularly limited. When the convex part 22 is provided between the flow path 7 and the flow path 8, the shape of the convex part 22 may have a wall surface perpendicular | vertical with respect to the direction through which a liquid flows like FIG. Further, as shown in FIG. 5, the convex portion 22 may be tapered so that the liquid flows smoothly. By providing the taper, there is no problem that the flow stops near the side surface of the convex portion 22 or the flow velocity distribution occurs, and when bubbles are generated, the bubbles stop near the side surface of the convex portion 22 and the liquid flows. There is an effect that the problem that the device is lost or the pressure at the time of liquid feeding becomes high and the device is damaged does not occur. The width and length of the convex portion 22 are determined from the viewpoint of minimizing the degree of dilution of the electrode active substance in the flow path and maximizing the signal amount due to the oxidation-reduction reaction at the electrode, the strength of the entire apparatus, and the electrode size. It should be optimally designed based on the required size of the detector. Further, the distance between the surface of the electrode 14 and the opposing substrate surface 21 should be optimized as well, but in practice, it is designed to be 0.05 μm or more from the viewpoint of processing accuracy.

  For example, a method of detecting an antigen such as an allergen using the detection apparatus of the present invention will be briefly described. With reference to FIG. 1 and FIG. 2, the flow path 6, 7, 8, the reaction part 3, and the detection part 4 of a detection apparatus are previously filled with buffer solution. Next, a buffer solution is injected from the introduction hole 2 and the inside of the apparatus is washed with the buffer solution. Next, a plurality of beads 12 on which an antibody material has been immobilized in advance are injected from the introduction hole 2 together with a buffer solution, and then protein non-specificity on the surfaces of the flow paths 6, 7, 8, the reaction part 3, and the detection part 4 In order to prevent mechanical adsorption, an albumin aqueous solution was flowed to form an albumin film on the surface.

  Next, a test solution containing an antigen is introduced from the introduction hole 2 and moved to the reaction unit 3, and the antigen in the test solution is captured by the antibody material provided in the reaction unit 3 and then replaced with the test solution. Then, a buffer solution is introduced from the introduction hole 2 to wash the flow paths 6, 7, 8, the reaction unit 3, and the detection unit 4.

  Next, a buffer solution containing an antibody material labeled with an enzyme is injected from the introduction hole 2 and reacted with the antigen captured by the antibody immobilized on the reaction part 3 to be immobilized on the surface of the beads 12. A complex composed of an antibody-antigen-antibody-attached antibody material is formed. Since the antibody-attached antibody material flows in an excessive amount compared to the amount of antigen captured, in order to remove the unreacted antibody-attached antibody material, a buffer solution is introduced through the introduction hole 2 and the flow paths 6, 7, 8, The reaction unit 3 and the detection unit 4 are washed.

  Next, a buffer solution containing a substrate material that is converted into an electrode active substance by an enzyme used as a label is injected from the introduction hole 2, and a complex composed of an immobilized antibody-antigen-enzyme-attached antibody material formed in the reaction part and By making it react, the electrode active substance corresponding to the quantity of a composite_body | complex is produced. The electrode active substance enters the detection unit 4 from the reaction unit 3 through the flow path 7, and when a predetermined voltage is applied to the working electrode and the counter electrode in the detection unit 4, an oxidation-reduction reaction occurs and a current is generated. By measuring this current, the amount of allergen can be measured. As the enzyme material and the substrate material used as the label, known materials (for example, those disclosed in Patent Document 3 or Patent Document 5 (Japanese Patent Laid-Open No. 9-243590)) can be used.

  In the detection unit 4, an electric current is generated when the electrode active substance causes a redox reaction at the working electrode. That is, the magnitude of the current depends on the concentration of the electrode active material in the vicinity of the electrode 14. Usually, the electrode active substance generated in the reaction unit 3 is diluted before reaching the detection unit 4. In the conventional configuration, the depth of the flow path and the depth of the detection part are the same. For example, in order to increase the efficiency of the immune reaction or enzyme reaction in the reaction part, an antibody material or the like is immobilized on the surface of the beads. When the system was applied, the depth of the detection unit 4 was at least greater than the diameter of the beads in the reaction unit. For this reason, the electrode active substance is diluted in proportion to its volume in the detection unit, the concentration in the vicinity of the electrode is lowered, and the amount of current is also reduced. In the present invention, since the distance between the electrode surface and the substrate surface opposite to the electrode surface is set to be small regardless of the depth of the flow path, the volume in the vicinity of the detection unit 4 is small, and the concentration reduction due to diffusion is small compared to the conventional case. As a result, a large amount of current can be obtained.

  Further, the amount of the electrode active substance reaching the surface of the electrode 14 is determined by the diffusion force and the collision by the wall and the collision diffusion in the liquid by the collision while the liquid flows through the detection unit 4. And the distance between the opposite surface and the surface becomes smaller. In the present invention, since the distance is reduced compared to the conventional configuration, the amount of the electrode active substance that reaches and reacts with the electrode 14 is larger than the conventional configuration, and a large amount of current can be obtained compared to the conventional detection device. The advantage that you can do it. Further, since a large amount of current can be obtained as described above, there is an effect that high sensitivity detection can be realized as an apparatus.

  In the present invention, the depth of the flow paths 3, 7, 8 can be set regardless of the distance between the electrode surface and the substrate surface opposite to the electrode surface (depth of the detection unit space). These beads with antibodies can be used in combination, and there is an effect that a highly efficient immune reaction and enzyme reaction can be realized simultaneously. In addition, since the enzyme reaction efficiency is improved by the effect, the amount of the electrode active substance is increased, and as a result, a large current amount can be obtained as compared with the conventional detection device, and a synergistic effect that high sensitivity can be realized is achieved. Arise.

  As shown in FIG. 6, the detection unit of the present invention may have a width larger than the widths of both flow paths 7 and 8 connected to the detection unit 4. When the width of the detection part is the same as the width of the flow path, the pressure applied to the injection hole when the flow velocity is the same is inversely proportional to the cross-sectional area of the inlet of the detection part. That is, if the distance between the electrode surface and the substrate surface facing the electrode surface (depth of the detection unit space) is reduced by using the configuration of the present invention, the amount of signal to be detected increases, but it is applied to the flow path. There is concern that the pressure will increase and cause chip destruction. The configuration of the present invention is characterized in that the width of the detection unit 4 is increased, thereby making it possible to reduce the distance between the electrode surface and the substrate surface facing the electrode, while taking advantage of the feature of the present invention. There is an advantage that such an increase in pressure can be prevented. The width of the detection unit 4 to be actually set is designed based on the shape of the flow path of the entire apparatus and the pressure resistance of the apparatus. For example, if the cross-sectional area of the flow path is constant, the same cross-sectional area is used. Thus, the width of the detection unit 4 and the distance between the electrode surface and the substrate surface facing the electrode surface may be determined. It goes without saying that this value is determined so as to maximize the reactivity at the electrode 14 of the detection unit 4. In addition, in this configuration, the width of the electrode 14 to be formed can be increased and the detection current can be increased.

  An electrochemical detection type micro-channel device having such a detection part can be manufactured by a conventional photolithography method for forming a mold, a method in which the mold is combined with heat or a photocurable resin, a hot embossing method, or the like. Hereinafter, an example of a process of forming a detection part by combining a heat or a photocurable resin with a mold by a conventional photolithography method and using the mold will be briefly described with reference to FIGS. .

  As shown in FIGS. 7A and 7B, as a first step, a negative thick resist 602a is applied to the glass substrate 601. Next, as shown in FIG. 7C, the resist 602a is exposed to ultraviolet rays through a mask 604 that shields the desired region 603 from light. As shown in FIG. 7D, a resist 602a is developed and baked to form a convex portion 602 having a thickness corresponding to the detection portion (corresponding to the distance between the electrode surface and the substrate surface).

  As shown in FIG. 8A, as a second step, a thick film resist 605 is applied on the glass substrate 601 so as to cover the convex portion 602. Next, as shown in FIG. 8B, the resist 605 is exposed to ultraviolet rays through a mask 609 that shields a predetermined area 606 corresponding to the detection portion other than the flow path pattern. As shown in FIG. 8C, the resist 605 is developed and baked, whereby a convex portion 605 having a thickness corresponding to the distance between the electrode surface and the substrate surface on the surface of the glass 601 and the depth of the flow path. Thus, a mold 607 in which a convex portion 602 having a thickness corresponding to is formed can be obtained.

As Step 3, as shown in FIG. 9 (a), a viscous material 603a containing polydimethylsiloxane (PDMS) is poured into a mold 607, and then thermally cured as shown in FIG. 9 (b). The substrate 608 can be obtained by peeling the mold 607 and opening the injection hole and the discharge hole. Next, a lid in which a comb-shaped working electrode 520, a reference electrode 521, an electrode 14 including a counter electrode 522, a wiring 16, and a connection pad 15 having a predetermined pattern as shown in FIG. By attaching 20 to the substrate 608, a detection device as shown in FIGS. 1 and 2 can be manufactured.
[Embodiment 2]

  The present embodiment has the same configuration as that of the first embodiment except for the detection unit described below.

  The detection unit, which is a feature of the present embodiment, will be described in detail with reference to FIGS. As shown in these drawings, the first feature of the present embodiment is that the detection unit is provided with a convex part 122 that is a step raised toward the space on the surface of the substrate 101, and A concave depression 123 having a predetermined depth is formed on the surface so as to face the convex portion 122. The electrode 114 is formed in a concave region 123 formed on the surface of the lid 120 so as to face the convex portion 122, and the distance between the surface of the electrode 114 in contact with the liquid and the convex portion 122 facing the electrode surface. It is characterized in that (the thickness of the space of the detection unit) is smaller than at least the depth of the reaction unit 3 or the minimum depth of the flow path 107 or 108 connected to the detection unit 104.

  The second feature is that the upper surface 121 of the convex portion 122 formed on the substrate 101 is flush with the portion 120a which is the surface of the lid 120 and where the recess 123 is not formed. The width of the flat part of the convex part 122 should just be more than the magnitude | size of the area | region which contacts the liquid of a working electrode, and the length of the direction along the flow path is not specifically limited. The shape of the side wall of the convex portion 122 may be perpendicular to the direction of liquid flow as shown in FIGS. 10 and 13, or may be tapered so that the liquid flows smoothly as shown in FIGS. Further, the shape of the side wall of the recessed area of the lid 120 may be perpendicular to the liquid flow direction as shown in FIGS. 10 and 11, and a taper is provided so that the liquid flows smoothly as shown in FIGS. May be.

  The width and length of the convex portion 122 are determined from the viewpoint of minimizing the degree of dilution of the electrode active substance in the flow path and maximizing the signal amount due to the oxidation-reduction reaction at the electrode, the strength of the entire apparatus, and the electrode size. It should be optimally designed based on the required size of the detector. In addition, the distance between the surface of the electrode 114 and the surface of the convex portion 122 facing the electrode 114 should be optimized as well, but in practice, it is designed to be 0.05 μm or more from the viewpoint of processing accuracy. The

  For example, an antigen detection method using the detection apparatus having the configuration of the embodiment of the present invention is the same as the method described in the first embodiment. The effect is the same as that of the first embodiment, and the distance between the electrode surface and the substrate surface facing the electrode surface can be set small regardless of the depth of the flow path. Since the active substance concentration can be suppressed and the probability of reaching the electrode surface increases due to increased diffusion force and collision probability while flowing through the detector, the amount of electrode active substance that reacts with the electrode increases. The advantage that a large amount of current can be obtained as compared with this detection device is produced, and as a result, an effect that high-sensitivity detection can be realized occurs.

  Further, even in the configuration of the present embodiment, since the depth of the flow path can be set regardless of the distance between the electrode surface and the substrate surface facing the electrode surface, it can be used in combination with a bead with an antibody having a diameter larger than the distance, There is also an effect that a highly efficient immune reaction and enzyme reaction can be realized at the same time, and this effect improves the enzyme reaction efficiency, resulting in an increase in the amount of electrode active substance, resulting in a larger amount of current compared to conventional detection devices. This is the same as that of the first embodiment, in which a synergistic effect that high sensitivity can be realized can be realized.

  Further, the width of the detection unit 104 may be larger than the widths of both flow paths connected to the detection unit 104 as in the first embodiment. By increasing the width of the detection unit 104, as in the effect described in the first embodiment, while taking advantage of the feature of the present invention that the distance between the electrode surface and the substrate surface facing the electrode surface is reduced, it is applied to the flow path. The advantage is that an increase in pressure can also be prevented.

  Another advantage of the structure of this embodiment is the simplification of the manufacturing method. For example, the processing of the detection unit on the substrate side can be performed by a conventional photolithography method for producing a mold and a method for filling the mold with heat or a photocurable resin, as in the first embodiment. The method shown in FIG. That is, as shown in FIGS. 14A and 14B, a thick film resist 605 is applied to the glass substrate 601 as the first step. Next, as shown in FIG. 14C, the resist 605 is exposed to ultraviolet rays through a mask 609 that shields the desired region 606 from light. As shown in FIG. 14D, the resist 605 is developed and baked to form a mold. After that, the substrate 101 can be manufactured only by the same steps as in FIGS. 9A and 9B of Embodiment 1 using heat or a photocurable resin. Therefore, since the process corresponding to FIG. 8 of Embodiment 1 is not necessary, there is no fear of misalignment due to the alignment of the second photomask (the process of FIG. 8B), which is advantageous from the viewpoint of yield. Occurs.

  For processing on the lid side, a conventional photolithographic method is combined with an etching method, and a recess 123 is formed in a predetermined region of the lid 120 with reference to FIG. The electrode 114 including the comb-type working electrode 520, the reference electrode 521, and the counter electrode 522 may be formed in such a predetermined pattern. At the same time, wiring and connection pads may be formed. Thereafter, the region other than the region in contact with the liquid is covered with an insulating film, and the substrate is attached to complete. However, in this method, there is a concern that the wiring connecting the electrode 114 formed in the recess 123 and the connection pad is cut by the step of the recess 123.

  As a method for eliminating such a concern, as shown in FIG. 15, the surface of a silicon wafer, a glass substrate, a plastic substrate, or the like used as a lid 120 is disposed at a position corresponding to the convex portion 122 of the substrate 101. Through a process in which an etching method is combined with a photolithography method, an electrode portion 114 including a comb-type working electrode 520, a reference electrode 521, and a counter electrode 522 having a predetermined pattern as shown in FIG. 27, wiring (not shown), and connection pads (Not shown) is formed, and the detection unit 104 is configured. In this case, since it is formed on a plane, there is no concern that the wiring is cut. Next, a film 125 having a predetermined thickness with a hole having a predetermined size including an electrode portion is attached, and the convex portion 122, the flow paths 106, 107, 108, the injection hole 102, and the discharge hole 105 are further formed. A detection device having the structure of the present invention can be manufactured by bonding to the provided substrate 101.

Further, as shown in FIGS. 16 to 19, instead of pasting the film, for example, a photo-curing resin is applied to the lid 120 on which the electrode portion, the wiring, and the connection pad are formed by a conventional photolithography process to a predetermined thickness. 10 to FIG. 13 by coating only, exposing the region including at least a part of the electrode portion with ultraviolet light through a light-shielded mask, developing and baking to form a photocurable resin film 125. A detection device having the same configuration as that shown in FIG. 15 can be manufactured by bonding the substrate 101 provided with the projections, flow paths, injection, and discharge holes. 16 corresponds to FIG. 10, FIG. 17 corresponds to FIG. 11, FIG. 18 corresponds to FIG. 12, and FIG. 19 corresponds to FIG.
[Embodiment 3]

  This embodiment has the same configuration as that of the first embodiment except for the detection unit.

  Next, the detection unit, which is a feature of the present embodiment, will be described in detail with reference to FIGS. The first feature of the present embodiment is that the electrode 214 is formed on a convex region 222 having a predetermined height provided on the surface of the lid 220 so as to face the substrate 201 and is in contact with the liquid. The distance between the electrode surface 221 and the substrate surface 201a facing the electrode surface 221 (the thickness of the space of the detection unit) is at least the depth of the reaction unit or the flow path connected to the detection unit or the minimum depth of the channel It is small.

  The shape of the side wall of the convex region 222 may be perpendicular to the liquid flow direction as shown in FIG. 20, or may be tapered so that the liquid flows smoothly as shown in FIG. In addition, a recess in the recess as described in the second embodiment may be formed on the surface facing the protrusion region 222, or a taper may be formed on the side surface of the recess.

  The width and length of the convex region 222 are designed to minimize the degree of dilution of the electrode active substance in the flow path, maximize the signal amount due to the oxidation-reduction reaction at the electrode, the strength of the entire apparatus, and the electrode size. Should be optimally designed based on the size of the detection unit required by the manufacturer. Further, the distance between the electrode surface 221 and the substrate surface 201a facing the electrode surface 221 should be optimized as well, but in practice, it is designed to be 0.05 μm or more from the viewpoint of processing accuracy. The

  For example, an antigen detection method using the detection apparatus having the configuration of the present embodiment is the same as the method described in the first embodiment. The effect is the same as that of the first embodiment, and the distance between the electrode surface and the substrate surface facing the electrode surface can be set small regardless of the depth of the flow path. Since the active substance concentration can be suppressed and the probability of reaching the electrode surface increases due to increased diffusion force and collision probability while flowing through the detector, the amount of electrode active substance that reacts with the electrode increases. The advantage that a large amount of current can be obtained as compared with this detection device is produced, and as a result, an effect that high-sensitivity detection can be realized occurs.

  Further, even in the configuration of the present embodiment, since the depth of the flow path can be set regardless of the distance between the electrode surface and the substrate surface facing the electrode surface, it can be used in combination with a bead with an antibody having a diameter larger than the distance, There is also an effect that a highly efficient immune reaction and enzyme reaction can be realized at the same time, and this effect improves the enzyme reaction efficiency, resulting in an increase in the amount of electrode active substance, resulting in a larger amount of current compared to conventional detection devices. This is the same as in the first embodiment, in which a synergistic effect that high sensitivity can be realized is produced.

  Further, as in the first embodiment, the width of the detection unit may be larger than the widths of both flow paths connected to the detection unit. By increasing the width of the detection unit, the pressure applied to the flow path while taking advantage of the feature of the present invention that the distance between the electrode surface and the substrate surface facing the electrode surface is reduced, similar to the effect described in the first embodiment. The advantage that the increase of can also be prevented arises.

  In the structure of this embodiment, the convex region 222 is formed on the lid 220 by combining the conventional photolithography method with the etching method, and then the electrode 214 composed of a comb-type working electrode, a reference electrode, and a counter electrode in a predetermined pattern. It can be obtained by forming a wiring, a connection pad (not shown), and a substrate 201 on which a flow path, a reaction part, and a detection part (not shown) are formed. However, with this method, there is a concern that the wiring (not shown) connecting the electrode 214 formed in the convex region and the connection pad (not shown) is cut by the step of the convex region 222.

As a method for eliminating such a concern, for example, as shown in FIG. 22, a base having a predetermined pattern of electrode portions 214 and a plurality of through holes 251 formed of a comb-type working electrode, a reference electrode, and a counter electrode is formed on the surface. There is a configuration in which the material piece 250 is bonded to the lid 220 with an adhesive 252. Each electrode of the electrode part 214 is electrically connected to the first connection part 253 formed on the back surface of the base piece 250 through the through hole 251, and the first connection part 253 is formed on the surface of the lid 220. 2 It is electrically connected to a connection pad (not shown) through a connection part 254 and wiring (not shown). The advantage of this method is that many electrode-forming film pieces 250 can be produced at one time, and therefore, it is only necessary to cut it out and attach it to the lid, which is effective from the viewpoint of mass productivity.
(Example)

  With reference to FIG. 23, the manufacturing method of the apparatus concerning the Example of this invention is demonstrated concretely. 7 and 8, a mold is formed on the glass substrate using the thick film resist SU-8, and the thermosetting resin PDMS is formed in the same manner as in FIG. 9 using the mold. Thus, a substrate 401 having a convex portion 422 in which a flow path, liquid injection, and discharge holes were formed was manufactured. The size of the substrate 401 was 25 mm × 50 mm, the widths of the flow paths 406, 407, and 408 were set to 0.2 mm, and the depth was set to 0.05 mm. The height of the convex portion 422 formed on the detection unit 404 was set to 0.04 mm, that is, the depth, width, and length of 0.01 mm from the surface of the PDMS substrate. In the position corresponding to the detection portion on the surface of the lid 420, the electrode 414 including the comb-type working electrode 520, the counter electrode 521, and the reference electrode 522 as shown in FIG. Formed. The PDMS substrate 401 and the lid 420 were bonded together to obtain a study apparatus. A known technique (Patent Document 4) was used for electrode formation and materials. Each electrode is electrically connected to an extraction terminal 417 formed on the lid surface and a wiring 418. A diffusion unit 409 as shown in FIG. 24 is provided between the flow path 407 and the detection unit 404 so that the liquid contacts the entire electrode 414.

  Next, the effect of the present invention will be described. A phosphate buffer adjusted to pH 7.4 with 0.1 μM concentration of paraaminophenol as an electrode active substance is introduced from the injection hole 402 using the pump 460, and the flow paths 406, 407, 408 and the detection unit 404. After 3 minutes, the current value from the electrode 414 was measured 3 minutes later. Meanwhile, on one side of the comb electrode of the working electrode, paraaminophenol passes electrons to one side of the comb electrode and changes to paraquinoneimine. Paraquinoneimine receives electrons from the other comb electrode and returns to paraaminophenol, and this reaction is repeated to detect a large current value.

  As a comparison, an experiment similar to the above was performed with a conventional detection device having the same configuration as described above, except that the detection unit 404 was not provided with the step 422 and the depth was set to 0.05 mm, which was the same as that of the flow path. The obtained current value became 1/100 or less of the configuration of the present invention.

  From this, it was found that the configuration of the present invention is very effective for obtaining a large detection current.

  It should be understood that the embodiments disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

  As described above, according to the present invention, a large detection signal can be obtained, and a high-sensitivity electrochemical detection type micro-channel detection device can be provided. Because it can be applied to anything that can be detected automatically, its industrial significance is great.

1 is a top view of an electrochemical detection type microchannel device according to a first exemplary embodiment; It is sectional drawing which follows the II-II line in FIG. 6 is a cross-sectional view of an electrochemical detection type microchannel device according to a modification of the first embodiment. FIG. FIG. 3 is an enlarged cross-sectional view of a detection unit of the electrochemical detection type microchannel device according to the first exemplary embodiment. FIG. 6 is an enlarged cross-sectional view of a detection unit according to a modification of the electrochemical detection type microchannel device according to the first exemplary embodiment. FIG. 10 is a top view of a detection unit according to a further modification of the electrochemical detection type microchannel device according to the first exemplary embodiment. FIG. 3 is a cross-sectional view showing a first step of a method for manufacturing a detection unit of the electrochemical detection type microchannel device according to the first exemplary embodiment; FIG. 6 is a cross-sectional view showing a second step of the method of manufacturing the detection unit of the electrochemical detection type microchannel device according to the first embodiment. FIG. 6 is a cross-sectional view showing a third step of the method of manufacturing the detection unit of the electrochemical detection type microchannel device according to the first embodiment. FIG. 6 is an enlarged cross-sectional view of a detection unit of the electrochemical detection type microchannel device according to the second exemplary embodiment. FIG. 10 is an enlarged cross-sectional view of a detection unit according to a modification of the electrochemical detection type microchannel device according to the second exemplary embodiment. It is an expanded sectional view of the detection part concerning the further modification of the electrochemical detection type microchannel device concerning Embodiment 2. FIG. It is an expanded sectional view of the detection part concerning the further modification of the electrochemical detection type microchannel device concerning Embodiment 2. FIG. FIG. 10 is a cross-sectional view illustrating a method for manufacturing the detection unit of the electrochemical detection type microchannel device according to the second exemplary embodiment. FIG. 6 is a cross-sectional view according to a modification of the electrochemical detection type microchannel device according to the second exemplary embodiment. FIG. 6 is an enlarged cross-sectional view of another detection unit of the electrochemical detection type microchannel device according to the second exemplary embodiment. It is an expanded sectional view of the detection part concerning the further another modification of the electrochemical detection type microchannel device concerning Embodiment 2. FIG. It is an expanded sectional view of the detection part concerning the further another modification of the electrochemical detection type microchannel device concerning Embodiment 2. FIG. It is an expanded sectional view of the detection part concerning the further another modification of the electrochemical detection type microchannel device concerning Embodiment 2. FIG. FIG. 5 is an enlarged cross-sectional view of a detection unit of an electrochemical detection type microchannel device according to a third exemplary embodiment. FIG. 10 is an enlarged cross-sectional view of another detection unit of the electrochemical detection type micro-channel device according to the third exemplary embodiment. FIG. 10 is an enlarged cross-sectional view of still another detection unit of the electrochemical detection type microchannel device according to the third exemplary embodiment. (A) It is a top view of the electrochemical detection type | mold microchannel device concerning an Example. (B) It is sectional drawing which follows the BB line in Fig.23 (a). It is a top view of the diffusion part used for an example. It is a perspective view of the conventional electrochemical detection apparatus. (A) It is the upper surface schematic of another conventional electrochemical detection apparatus. (B) It is sectional drawing which follows the BB line in Fig.26 (a). It is the schematic which shows the structure of the electrode for the conventional electrochemical detection.

Explanation of symbols

1, 101, 201, 401, 501, 608 Substrate 2, 102, 402, 504A Inlet hole 3, 503 Reactor 4, 104, 404 Detector 5, 105, 405, 505A Discharge hole 6, 7, 8, 106, 107, 108, 406, 407, 408, 504, 505 Flow path 12, 112, 502 Bead 13, 508 Damping part 14, 114, 214, 414, 506 Electrode 15, 417, 507 Connection pad 16, 418, 509 Wiring 20, 120, 220, 420, 510 Lid 22, 122, 222 Protruding part 251 Through hole 252 Adhesive layer 253 First connecting part 254 Second connecting part 409 Diffusion part 520 Working electrode 521 Reference electrode 522 Counter electrode 601 Glass substrate 602, 605 Resist 603, 606 Shading area 604, 609 Mask 607

Claims (6)

A reaction part for causing a reaction in the space;
A detector having an electrode for electrochemical detection in the space;
A flow path connecting the reaction unit and the detection unit,
The depth of the detection unit space is shallower than the depth of the flow path,
In the detection unit, one of the upper surface or the lower surface is provided with a convex portion that rises toward the other surface ,
A concave recess is formed on the other surface of the detection unit so as to face the convex portion,
The electrochemical detection apparatus , wherein the electrode for performing electrochemical detection is formed in the concave depression . The electrochemical detection device according to claim 1 , wherein a side surface of the convex portion is tapered. The electrochemical detection device according to claim 2 , wherein a side surface of the concave depression is tapered. And the upper surface of the convex portion, the electrochemical detection apparatus according to any of claims 1 to 3, characterized in that on the same plane and the concave depression is formed faces. In the detecting unit, the other of the upper or lower surface, wherein as concave depression is formed, claim 1, characterized in that the film or resin layer having an opening is provided 4 2. The electrochemical detection device according to item 1 . A reaction part for causing a reaction in the space;
A detector having an electrode for electrochemical detection in the space;
A flow path connecting the reaction unit and the detection unit,
The depth of the detection unit space is shallower than the depth of the flow path,
In the detection unit, in one of the upper surface or the lower surface, an electrochemical detection device provided with a convex portion that rises toward the space,
In the detection unit, a connection pad connection part electrically connected to the connection pad is provided on one of the upper surface or the lower surface,
The convex portion is composed of a base material piece having a thickness,
The electrode is provided on the surface of the base piece,
A first connection portion connected to the connection pad connection portion is provided on the back surface of the base piece;
A through hole is formed so as to penetrate the thickness portion of the base piece,
The first connection portion and the connection pad connection portion are electrically connected through the through hole,
The base material piece is bonded to one of the upper surface and the lower surface of the detection unit so that the first connection portion provided on the back surface of the base material piece is in contact with the connection pad connection portion. An electrochemical detection device , wherein the convex portion is formed by being attached .

Images