JP5564800B2 - Device with reaction field - Google Patents

Description

  The present invention relates to a device having a field (reaction field) for causing some kind of reaction to a minute sample such as a biological sample.

  In an antigen-antibody reaction detection method by a conventional ELISA (Enzyme-Linked ImmunoSorbent Assay) method, antigen detection is performed by a dye reaction based on an enzyme reaction immobilized on an antibody during a secondary antibody reaction. At that time, it is a necessary condition that an efficient reaction proceeds by an antibody reaction and a sequential dye coloring reaction. As a result of the dye reaction, a method of measuring the absorbance of the dye by a change in light transmittance is generally used (for example, see Non-Patent Document 1).

  However, the amplification rate is often insufficient when the amount of antigen present is extremely small, which has a problem in detection sensitivity. In a general specimen test, for example, 3 mL (milliliter) of a reagent and 20 μL (microliter) of a specimen are mixed to induce a dye reaction based on an enzyme reaction, and the absorbance of the reagent solution that changes due to the reaction is monitored. In the same way, especially when dealing with a very small amount of an analyte sample (for example, a liquid containing glucose in the range of 2 nL (nanoliter) to 200 nL, or whole blood containing glucose, plasma, serum, etc.) Is used as a reaction liquid at the same ratio, the reaction liquid with 3 μL reacts quickly with the sample liquid because the volume of the reagent liquid on the droplets is small. Occur. As a result, the droplet disappears in a shorter time than the time required for completion of the reaction, and thus the absorbance of the reactive dye cannot be measured. Also, in a microdroplet reaction, it is required to set the temperature to about 37 ° C. in order to accelerate the reaction (especially in a reagent system mainly composed of an enzyme reaction). Etc. were major technical issues. For this reason, the technique which introduce | transduces a liquid into a reaction field efficiently and rapidly was calculated | required. Such a technical problem is not limited to measurement related to a biological sample, and is common in various scenes where similar measurement is performed.

Osamu Osayama et al., “Clinical Chemistry”, 2nd edition, Ishiyaku Publishing Co., Ltd., January 2006

  A specific aspect of the present invention has an object to provide a technique capable of efficiently and promptly introducing a liquid in the case of performing measurement using a very small amount of liquid.

  A device having a reaction field according to the present invention includes a substrate having a recess on one surface side and a covering body having a through hole, and the covering body is in the through hole and inside the through hole in a plan view. The device is a device having a reaction field that has a plurality of projecting portions projecting toward the surface, and the covering body is provided on one surface side of the substrate so that the through hole and the concave portion overlap each other.

  According to such a configuration, the dropped liquid easily collects in the protrusions having high surface energy, and the collected liquid enters the recesses from the gaps between the protrusions, so that a very small amount of liquid is efficiently introduced into the recesses. It can be introduced quickly and well. This effect becomes more prominent when the gap between the protrusions is narrowed to such an extent that a capillary force is generated.

  Preferably, each of the plurality of protrusions has a corner. More preferably, the corner is an acute angle.

  By making each protrusion have a corner, that is, a portion protruding sharply, the effect of drawing the liquid into the recess is more emphasized. This effect is further enhanced by making the corners acute.

  Preferably, the inner wall surface defining the recess is made higher than the lyophilicity of the surface of the covering.

  Since the surface energy difference is provided by surface modification of each of the inner wall surface of the recess and the surface of the film, the inlet of the sample liquid is narrowed by providing the film on the recess, It is not difficult to introduce the sample solution.

  Preferably, the inner wall surface of the recess is modified to be lyophilic, and the surface of the covering is modified to be liquid repellent.

  Thereby, it is easy to ensure a larger difference in surface energy between the inner wall surface of the recess and the surface of the film.

  Preferably, the through hole is arranged so as to be substantially in the center of the recess in plan view.

  Thereby, it is easy to maintain the mechanical strength of the film. In addition, the effect of suppressing the evaporation of the sample liquid by the film is expected to be uniform.

  Preferably, the concave portion has a substantially hemispherical shape.

  Thereby, it is expected that the reaction of the sample solution in the recess as a reaction field becomes more uniform.

  Preferably, the substrate is a transparent substrate.

  Accordingly, it becomes easy to perform various measurements by irradiating the sample liquid introduced into the recess with light.

  Moreover, it is also preferable that the device further includes a reflective film provided on a surface of the covering body facing the substrate. More preferably, the reflective film is provided in a range overlapping with the concave portion, for example.

  Thereby, when the substrate is transparent, light can be incident from the back side of the substrate, and the reflected light obtained by reflecting the light by the metal layer can be detected on the back side of the substrate. Therefore, optical measurement becomes easy.

It is a schematic cross section which shows the structure of the device which has the reaction field of one Embodiment. It is a figure which shows the schematic top view of a recessed part and a covering. It is the figure which showed the other aspect about the shape of the projection part. It is a schematic cross section for demonstrating the introduction process of the sample to the device shown in FIG. It is a typical perspective view which shows the structure of the device of another aspect. It is process sectional drawing for demonstrating an example of the manufacturing method of a device. It is process sectional drawing for demonstrating an example of the manufacturing method of a device. It is typical sectional drawing explaining the structure of the device which concerns on a modified example, and its usage condition.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a schematic cross-sectional view showing the structure of a device having a reaction field according to one embodiment (hereinafter simply referred to as “device”). The device 1 shown in FIG. 1 includes a substrate 10 having a recess 12 on one surface side, and a covering 14 provided on one surface of the substrate 10.

  The substrate 10 is a transparent substrate such as a plastic substrate or a quartz glass substrate. Use of a transparent substrate as the substrate 10 is convenient for optical measurement described in detail later. The thickness of the substrate 10 is, for example, about 0.05 mm to 2.5 mm. When optical measurement is not necessary, the substrate 10 may be opaque, and for example, a semiconductor substrate such as a silicon substrate can be used as the substrate 10.

  The concave portion 12 is provided on one surface side of the substrate 10 as described above. The recess 12 is formed using, for example, a photolithography technique and an etching technique. In the present embodiment, the recess 12 is formed in a substantially hemispherical shape. The diameter D1 of the recess 12 is, for example, about 0.1 mm to 5.0 mm. The recess 12 functions as a minute reaction field for introducing a sample (reagent solution).

  The covering 14 is provided on one surface of the substrate 10 so as to cover the recess 12. The covering 14 is made of a small piece such as a plastic substrate or a glass substrate. The covering 14 has a through-hole 16 and is provided so that the through-hole 16 and the recess 12 overlap each other. The through hole 16 in the present embodiment has a diameter D2 smaller than the diameter D1 of the recess 12. The diameter D2 of the through hole 16 is, for example, about 10 μm.

  FIG. 2 is a schematic plan view of the recess 12 and the covering 14. As shown in FIG. 2, the covering 14 has a larger area than the opening of the recess 12 and is provided so as to cover the recess 12. In the illustrated example, the covering body 14 has a rectangular shape, but the shape is not limited thereto. Further, in the present embodiment, as shown in FIG. 2, the through hole 16 is arranged so as to be substantially at the center in the plan view of the recess 12.

  Here, the shape of the covering 14 and the through hole 16 will be described in detail with reference to FIG. As shown in FIG. 2, the covering body 14 includes a plurality of protrusions 14 a that protrude toward the inside of the through hole 16 in plan view. An example covering 14 shown in FIG. 2 has eight protrusions 14a. In order to avoid complication of the drawing, only one protrusion 14a is denoted by a reference numeral in FIG. Each of the protrusions 14a in the example shown in FIG. 2 has a corner (a portion protruding sharply). Each protrusion 14a has a size of about 50 μm, for example. The pitch between the protrusions 14a is, for example, about 100 μm.

  FIG. 3 is a diagram showing another aspect of the shape of the protrusion. 3A is four protrusions, FIG. 3B is five protrusions, FIG. 3C is 16 protrusions, and FIGS. 3D and 3E are more numerous. The structural example of the coating | covering body 14 which has this protrusion part is shown. Each protrusion has a corner. Furthermore, each protrusion shown in FIGS. 3C to 3E is formed with an acute corner. In other words, the covering body 14 in the present embodiment is formed in a star bust shape in the plan view of the through hole 16. In addition, although it is a preferable aspect that each projection part has a corner | angular part and these corner | angular parts are acute angles, respectively, it is not an essential condition. Each protrusion may have a shape without a corner. The diameter D2 (see FIG. 1) of the through hole 16 is, for example, about 10 μm. Here, the diameter D2 of the through hole 16 in the present embodiment is a diameter with respect to the circumference of a circle passing through the tip of each protrusion 14a.

  Next, the characteristics of the surfaces of the recess 12 and the covering 14 will be described. A surface treatment corresponding to the polarity of the sample (reagent solution) to be introduced into the recess 12 is performed on the surface of the inner wall of the recess 12. Specifically, in the case of introducing a lyophilic (hydrophilic) reaction reagent and a sample, a lyophilic process (hydrophilic process) is performed on the surface of the inner wall of the recess 12. That is, the surface of the inner wall of the recess 12 is modified such that the surface of the inner wall that defines the recess 12 is relatively more lyophilic than the surface of the covering 14. In the present embodiment, the surface of the inner wall of the recess 12 is modified to be lyophilic, and the surface of the covering 14 is modified to be lyophobic. The lyophilic treatment can be realized by using, for example, a PEG (polyethylene glycol) molecular film agent. Specifically, the inner wall surface of the recess 12 is reacted with an amino-based silanization reagent, and then reacted with a PEG-based reagent terminal-functionalized with NHS ester. Thereby, the lyophilicity of the surface of the inner wall of the recess 12 is increased. In addition, the liquid repellency treatment can be realized by modifying the surface of the above-described covering 14 using a fluorine-based silanizing agent, for example.

  FIG. 4 is a schematic cross-sectional view for explaining the process of introducing the sample into the device shown in FIG. As illustrated, the sample solution 20 is introduced into the recess 12 through the through hole 16 provided in the covering 14 on the substrate 10. For example, a very small amount of the sample solution 20 is dropped into the through hole 16 in a plurality of times. For example, a droplet discharge method (inkjet method) is suitable for dropping a very small amount of the sample liquid 20. The minute amount means, for example, that the diameter of the droplet is smaller than the diameter of the through hole 16. At this time, it is more desirable to drop the sample solution 20 at a position shifted from the center m of the through hole 16 as shown in the figure. For example, the sample solution 20 may be dropped onto the edge portion of the through hole 16 as shown in the drawing. Due to the effect of the protrusions 14 a arranged along the outer edge of the through-hole 16, the sample liquid 20 is likely to gather on the protrusions 14 a having a high surface energy. Since the collected liquid enters the recess 12 through the gap between the protrusions 14a, a very small amount of liquid can be introduced into the recess 12 efficiently and promptly. This effect becomes more prominent when the gap between the protrusions 14a is narrowed to such an extent that a capillary force is generated. Furthermore, in this embodiment, since the surface of the covering body 14 is liquid repellent, the sample liquid 20 adhering to the edge portion of the through-hole 16 can be quickly removed from the surface of the covering body 14 and the recess 12 having an lyophilic inner wall surface. Get into. In this way, by utilizing the difference in surface energy between the recess 12 and the covering 14, a small amount of the sample solution 20 can be more reliably introduced into the recess 12. If the sample liquid introduced into the recess 12 contains air, the air can be removed by slightly vibrating the substrate 10.

  FIG. 5 is a schematic perspective view showing the configuration of a device according to another aspect. The device 1 described above has one recess 12, a covering 14, and a through hole 16, whereas the device 1 a shown in FIG. 5 is provided corresponding to the plurality of recesses 12 and each recess 12. A covering body 114 having a plurality of through holes 16. That is, in the device 1 a shown in FIG. 4, the covering body that covers each recess 12 is shared by one. In addition, you may provide a coating body corresponding to each recessed part 12 and each through-hole 16 separately. Further, in the device 1a of the example shown in FIG. 5, the concave portions 12 and the through holes 16 are arranged in a matrix, but the arrangement state is not limited to this.

  Next, a preferred example of the method for manufacturing the device 1 according to this embodiment will be described. The device 1a can be manufactured similarly.

  FIG. 6 is a process cross-sectional view for explaining an example of the manufacturing method of the device 1. Briefly described, as shown in FIG. 6A, the device 1 according to the present embodiment individually forms a substrate 10 having a recess 12 and a covering body 14 having a through hole 16, respectively. Thereafter, as shown in FIG. 6B, the substrate 10 and the covering body 14 can be bonded together. Hereinafter, some specific embodiments will be described.

  One specific embodiment is an embodiment using resin (plastic) injection molding. In this case, for example, polystyrene, which is one of general-purpose resins, is injection-molded, so that the substrate 10 having the recesses 12 and the covering 14 having the through holes 16 as shown in FIG. Each is formed, and each protrusion 14a is also formed. Next, as shown in FIG. 6B, the substrate 10 and the cover 14 are bonded together. For example, a solvent such as xylene is uniformly applied on one surface of the substrate 10 by a method such as spin coating (for example, a film thickness of about 1 μm). When one surface of the substrate 10 is dissolved, the covering 14 is placed on the substrate 10 and pressed. Thereby, the substrate 10 and the cover 14 are joined (fused). Since the solvent xylene or the like evaporates from the through hole 16, the solvent does not remain in the recess 12. The resin is not limited to polystyrene, and polycarbonate, polymethyl methacrylate, SNP, TPX, and the like can also be used. In addition to the above, various methods such as a method using an adhesive, a method using frictional heat, a method using ultrasonic welding, and a method using vibration welding are appropriately used as a method for bonding the substrate 10 and the cover 14 together. Can be adopted.

  Moreover, the aspect which uses a glass substrate as another specific aspect is mentioned. In this case, the substrate 10 having the recesses 12 is formed by performing wet etching on the glass substrate (FIG. 6A). Further, by performing wet etching on another glass substrate, a covering body 14 having a through hole 16 and provided with each projection 14a in the through hole 16 is formed (FIG. 6A). As an etching solution, for example, ammonium monohydrogen difluoride (for example, a concentration of about 5%) can be used. By performing isotropic etching using such an etching solution, a hemispherical recess 12 can be formed in the glass substrate, and a through-hole 16 having a protrusion 14a can be formed in another glass substrate. . The etching rate is, for example, about 50 nm / min. Next, the substrate 10 and the cover 14 are bonded together (FIG. 6B). The bonding of the substrate 10 made of a glass substrate and the covering body 14 can be performed, for example, by direct bonding by applying heat and pressure. Alternatively, the substrate 10 and the cover 14 may be bonded together using an adhesive such as so-called water glass (an aqueous solution having a high concentration of sodium silicate).

  Further, as another specific embodiment, there is an embodiment in which a glass substrate and a semiconductor substrate (silicon substrate) are used in combination. In this case, the substrate 10 having the recess 12 is formed by performing wet etching on the glass substrate in the same manner as described above (FIG. 6A). Further, the covering body 14 having the through holes 16 is formed by etching a semiconductor substrate such as a silicon substrate (FIG. 6A). After that, the substrate 10 made of a glass substrate and the covering body 14 made of a semiconductor substrate are bonded together (FIG. 6B). Here, for example, the substrate 10 and the covering body 14 can be bonded together by anodic bonding.

  FIG. 7 is a process cross-sectional view for explaining another example of the method for manufacturing the device 1. Hereinafter, this aspect will be described in detail.

  As shown in FIG. 7A, the glass substrate is etched to form the substrate 10 having the recesses 12. Specific conditions are the same as above. Next, as shown in FIG. 7B, a sacrificial layer (sacrificial material) 22 made of resin or the like is embedded in the recess 12 of the substrate 10. At this time, the sacrificial layer 22 is formed so that one surface of the substrate 10 is flat as illustrated.

  Next, as shown in FIG. 7C, a layer 24 made of resin or the like is formed on one surface of the substrate 10. The formation of the layer 24 can be performed by, for example, a spin coating method. Next, the layer 24 is etched to remove unnecessary portions. Thereby, the covering body 14 which has the through-hole 16 and in which each projection part 14a was provided in the said through-hole is formed on one surface of the board | substrate 10 (FIG.7 (D)).

  Next, the sacrificial layer 22 embedded in the recess 12 of the substrate 10 is removed by a method such as plasma etching. Thereby, as shown in FIG. 7E, the device 1 according to the present embodiment is completed.

  According to the present embodiment as described above, the liquid easily collects in the protrusions 14a having a high surface energy, and the collected liquid enters the recesses 12 through the gaps between the protrusions 14a. Can be introduced into the recess 12 efficiently and promptly. This effect becomes more prominent when the gap between the protrusions 14a is narrowed to such an extent that a capillary force is generated.

  In addition, the covering 14 provided so as to overlap the concave portion 12 is in a state where the opening portion of the concave portion 12 is blocked to some extent, so that the volume of the reaction field by the concave portion 12 is secured and introduced into the concave portion 12. The evaporation of the sample 20 can be suppressed.

  In addition, since the surface energy difference is provided by surface modification of the inner wall surface of the recess 12 and the surface of the covering 14, the provision of the covering 14 on the recess 12 makes the sample liquid introduction port Despite being narrowed, it is not difficult to introduce the sample solution.

  Next, an aspect of the modified implementation will be described. The modes described below are particularly suitable for performing measurement on a sample solution by an optical technique.

  FIG. 8 is a schematic cross-sectional view illustrating a configuration of a device according to a modified example and a usage mode thereof. The basic configuration of the device 1b shown in FIG. 8 is the same as that of the device 1 according to the above-described embodiment (see FIG. 1 and the like). The components common to both are given the same reference numerals, and detailed description of the components is omitted.

  In the device 1b shown in FIG. 8, a reflective film 15 is provided on the back surface (the surface facing the recess 12) of the covering 14. The reflective film 15 is disposed so as to overlap with at least a part of the recess 12. Further, the reflective film 15 is formed on the back surface of the covering 14 so as not to interfere with the bonding of the covering 14 and the substrate 10. The reflective film 15 is a film made of a metal such as gold. The manufacturing method of the device 1b having such a reflective film 15 is basically the same as that of the above-described embodiment (see FIG. 6). Specifically, the reflective film 15 may be formed in advance by forming a metal film at a predetermined position on one surface of the substrate or the like to be the covering 14 and shaping the metal film by appropriate etching or the like.

  Next, an example of how the device 1b is used will be described. As shown in FIG. 8, light is incident from the back side of the substrate 10 toward the sample solution 20 in the recess 12. As illustrated, incident light is incident on the back surface of the substrate 10 at an angle and is reflected by the reflective film 15 of the covering 14. The reflected light is collected in a measurement light collection 32 made up of a CCD or the like as shown in the figure. The light detection device 30 detects the intensity of the light (reflected light) collected on the measurement light collecting plate 32. Thereby, the change of the sample solution 20 in the recess 12 can be measured. For example, a dye reaction using an enzyme reaction can be monitored. At this time, detection information is amplified by preparing a plurality of light sources and allowing light to enter from each of them, so that highly sensitive detection is possible. As the reactive reagent, for example, a blood glucose level test reagent is used. Among these reagents, glucose oxidase, peroxydehydrogenase, and 4-aminoantipyrine (dye precursor) are present, and react with blood sugar (glucose) contained in serum to develop color. By introducing the reagent and serum into the recess 12 using, for example, the necessary amounts described above, highly sensitive detection becomes possible.

  In addition, this invention is not limited to the content of embodiment mentioned above, In the range of the summary of this invention, it can change and implement variously. For example, in the above-described embodiment, a sensor (so-called biosensor) for handling a biological sample is exemplified as a typical application example of a device having a reaction field according to the present invention. It is not limited to this.

  In the above description, the embodiment in which the surface modification is further combined with the covering body having a plurality of protrusions arranged around the through hole is exemplified. However, the surface modification is not necessarily combined. Not necessary.

  DESCRIPTION OF SYMBOLS 1 ... Device which has reaction field, 10 ... Board | substrate, 12 ... Recessed part, 14 ... Covering body, 14a ... Projection part, 15 ... Reflection film, 16 ... Through-hole, 20 ... Sample liquid

Claims (11)

A substrate having a recess on one side;
A covering having a through hole for introducing the sample liquid into the recess;
With
In plan view, the cover member is disposed along the outer edge of the through hole has a butt raised portion that protrudes toward the inside of the through hole,
The protrusion has an acute corner in plan view,
The said covering body is a device which has a reaction field provided so that the said through-hole and the said recessed part may overlap with the one surface side of the said board | substrate. The device having a reaction field according to claim 1, wherein the number of the protrusions is 16 or more. The device having a reaction field according to claim 1, wherein the number of the protrusions is 16 or more and 32 or less.   The device having a reaction field according to any one of claims 1 to 3, wherein the sample liquid is guided to the concave portion by a capillary force at the corner portion. The device having a reaction field according to any one of claims 1 to 4 , wherein the lyophilicity of the inner wall surface of the recess is higher than the lyophilicity of the surface of the covering. The device having a reaction field according to claim 5 , wherein the inner wall surface of the recess is modified to be lyophilic, and the surface of the covering is modified to be liquid repellent. The device having a reaction field according to any one of claims 1 to 6 , wherein the through-hole of the covering body is arranged in accordance with a substantially center in a plan view of the concave portion. The device having a reaction field according to any one of claims 1 to 7 , wherein the concave portion is substantially hemispherical. The device having a reaction field according to any one of claims 1 to 8 , wherein the substrate is a transparent substrate. The device having a reaction field according to any one of claims 1 to 9 , further comprising a reflective film provided on a surface of the covering body facing the substrate. The device having a reaction field according to claim 10 , wherein the reflective film is provided in a range overlapping with the concave portion.

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