JP5686954B2 - device - Google Patents

Description

  The present invention relates to a device having a field (micro 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 side and a cover having a through hole, and the cover has the through hole and the recess on one side of the substrate. The covering body is a device having a reaction field having a contact angle control region in which the contact angle of the liquid becomes smaller as it is closer to the through hole on the surface not facing the substrate. is there.

  According to such a configuration, the liquid dropped on the cover is directed toward the through hole by changing the contact angle (in other words, wettability) in the vicinity of the through hole on the surface of the cover continuously or stepwise. Therefore, a very small amount of liquid can be efficiently and promptly introduced into the recess.

  Preferably, the contact angle control region has a plurality of sub-regions, and the contact angle of the liquid in each of the plurality of sub-regions is different.

  Thereby, a contact angle can be changed in steps. By forming such a sub-region, it is easier to form the contact angle control region.

  The plurality of sub-regions described above are provided in an annular shape, for example, around the through hole.

  By setting it as such a sub-region, it becomes possible to move a liquid more reliably toward a through-hole.

  Preferably, the through hole is disposed at a substantially center in the plan view of the recess.

  Thereby, it is easy to maintain the mechanical strength of the covering. In addition, the effect of suppressing the evaporation of the sample liquid by the covering 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 expanded the vicinity of the through-hole of a covering, and was shown typically. 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 a figure explaining an example of the optical measuring method using the device which has a reaction field.

  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 having a diameter smaller than the diameter of the recess 12, and is provided so that the through-hole 16 and the recess 12 overlap each other. The diameter D2 of the through hole 16 is, for example, about 10 μm. The cover 14 has a contact angle on the surface that is not in contact with one surface of the substrate 10 such that the closer to the through hole 16, the smaller the liquid contact angle, and the farther from the through hole 16, the relatively larger contact angle. It has a control area 22. Details of the contact angle control region 22 will be described later.

  FIG. 2 shows 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.

  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 covering 14 using, for example, a fluorine-based silanizing agent. The liquid repellency treatment on the surface of the covering 14 will be described in detail below.

  FIG. 3 is an enlarged view schematically showing the vicinity of the through hole 16 of the covering 14. Specifically, FIG. 3A is a schematic plan view of the covering 14, and FIG. 3B is a schematic cross-sectional view corresponding to the line bb shown in FIG. 3A of the covering 14. As shown in FIGS. 3A and 3B, the covering body 14 has a liquid contact angle relative to the surface (the surface on the side not in contact with the substrate 10) closer to the through hole 16 in plan view. The contact angle control region 22 has a contact angle that is relatively larger as it is smaller and farther from the through hole 16. The contact angle control region 22 in the present embodiment includes annular sub-regions 22a, 22b, and 22c provided concentrically with the through hole 16 as a center. The plurality of sub-regions 22a, 22b, and 22c have different contact angles. Specifically, the innermost sub-region 22a has a contact angle of 80 ° (this is a numerical value when the liquid is water, the same applies hereinafter). A sub-region 22b provided outside the sub-region 22a has a contact angle of 100 °. The sub-region 22c provided on the outermost side has a contact angle of 130 °. The width of each sub-region 22a-22c is about 5 micrometers-10 micrometers, for example. An example of a method for forming such a contact angle control region 22 will be briefly described. For example, a thiol molecule containing a saturated alkyl fluorine chain or a thiol molecule containing only an alkyl chain is used alone or in combination. The formation of each sub-region can be realized by repeating the surface decomposition reaction by UV irradiation (for example, ultraviolet irradiation with a wavelength of 157 nm) through a mask and the creation of a new molecular film.

  In addition, the numerical value of the contact angle mentioned above is an example, and is not limited to these. Further, the number of sub-regions is not limited to three. If the number of sub-regions is larger, a smoother droplet flow effect is expected. Furthermore, the shape of each sub-region is not necessarily concentric.

  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. In the present embodiment, since the surface of the covering 14 is liquid repellent, the sample liquid 20 adhering to the edge portion of the through-hole 16 is quickly transferred from the surface of the covering 14 to the recess 12 whose inner wall surface is lyophilic. Get in. 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. As described above with reference to FIG. 3, the contact angle of the liquid on the surface of the covering body 14 is made smaller as being closer to the through hole (that is, the wettability becomes higher). The dropped sample solution 20 is likely to gather toward the through hole 16. Thereby, a very small amount of liquid can be efficiently and promptly introduced into the recess. 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 has a plurality of recesses 12 and a plurality of corresponding through holes 16. ing. Moreover, in the device 1a shown in FIG. 5, the covering body which covers each recessed part 12 is made common. That is, the covering body 114 of the present embodiment has a plurality of through holes 16 and is provided across a plurality of recesses. A plurality of coverings may be provided corresponding to each recess 12 and each through hole 16. 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 are bonded together, and the covering body 14 is further subjected to a liquid repellency treatment. 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. Form each one. 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). Moreover, the cover 14 which has the through-hole 16 is formed by performing wet etching with respect to another glass substrate (FIG. 6 (A)). 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, the hemispherical recess 12 can be formed in the glass substrate, and the through-hole 16 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 14 having the through-hole 16 is formed on one surface of the substrate 10 (FIG. 7D).

  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 dropped on the covering 14 is caused to change by changing the contact angle (in other words, wettability) in the vicinity of the through hole 16 on the surface of the covering 14. Since it becomes easy to gather toward 16, it becomes possible to introduce a small amount of liquid into the recess efficiently and promptly.

  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.

  DESCRIPTION OF SYMBOLS 1 ... Device which has reaction field, 10 ... Board | substrate, 12 ... Recessed part, 14 ... Covering body, 15 ... Reflecting film, 16 ... Through-hole, 20 ... Sample liquid, 22 ... Contact angle control area, 22a, 22b, 22c ... Sub region

Claims (6)

A substrate having a plurality of recesses on one surface side;
A covering having a through hole;
With
Wherein the covering body, the and the through hole is provided with a plurality such that the through holes are superposed on the concave portion, the diameter of the through hole in plan view, is formed smaller than the diameter of the corresponding recess ,
The covering body is in the plane of the side not facing the substrate, it has a contact angle control region comprising a plurality of annular sub-region disposed concentrically around the through hole, the sub-region before Symbol annular The device in which the contact angle control region is formed so that the contact angle of the liquid when the liquid is arranged becomes smaller as the contact angle is closer to the through hole.   The device according to claim 1, wherein the through hole is disposed at a substantially center in a plan view of the recess.   The device according to claim 1, wherein the recess is substantially hemispherical.   The device according to claim 1, wherein the substrate is a transparent substrate.   The device according to any one of claims 1 to 4, further comprising a reflective film provided on a surface of the covering body facing the substrate.   The device according to claim 5, wherein the reflective film is provided in a range overlapping with the concave portion.

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