WO2008059848A1 - Structure of micro/nanoconstruction, bioinspection chip utilizing the same and process for producing them - Google Patents

Abstract

A simple and easy process for direct production molding of a structure of nanoconstruction or microconstruction from a synthesized resin material; and a biosensing disk utilizing the structure and a bioinspection chip utilizing the same. The process for producing a structure of nanoconstruction or microconstruction comprises the steps of placing a powdery resin on a surface of original plate; heating the original plate and the resin at a temperature of from the glass transition temperature of the resin to the melting temperature thereof; pressing the resin against the original plate; and cooling the resin to the glass transition temperaturethereof or below and removing the original plate to thereby form a reversal construction of nanoconstruction or microconstruction with respect to the original plate. Further, there is provided a biosensing chip obtained by causing the structure to adsorb an antibody and providing the backside thereof with a light shielding layer, and provided a bioinspection chip having the biosensing chip disposed inside a flow channel side substratum and inside a lid side substratum.

Description

 Specification

 Micro / nano-structures and bio-detection chips and methods for producing them

 Technical field

 The present invention relates to a structure having a nanostructure and a microstructure, a biosensing disk, and a method for manufacturing a biotest chip using the same.

 Background art

 [0002] One of the basic technologies that support nano technology is microfabrication technology. A typical example is a semiconductor microfabrication technique represented by lithography. Ultra-fine structures can be formed using deep ultraviolet rays or electron beams, and devices and processes for next-generation nano-lithography have been widely studied.

 Under such circumstances, a nanoimprint method has been proposed in which the press working technique used for mass production of compact discs or the like is applied to the formation of nanostructures (see, for example, Patent Document 1). This is a technique in which a mold having a fine structure (hereinafter also referred to as “original” or “mold”) is pressed onto a polymer resin, thereby transferring the fine structure of the mold onto the resin on the substrate. FIG. 20 is a diagram for explaining such a conventional nanoimprint method.

[0004] Referring to FIG. 20, a master 904 is prepared for a resin layer 902 formed on a substrate 900.

 (a). Next, the master 904, the substrate 900, and the resin layer 902 are heated to a predetermined temperature, and the master 904 is pressed onto the resin layer 902 at a temperature higher than the Tg of the resin layer 902 (b). The whole is cooled in the pressed state, and when the temperature becomes lower than Tg of the resin layer 902, the original 904 is removed. Through this process, the shape of the original is transferred to the resin layer.

[0005] This nanoimprint method does not require expensive equipment and ancillary processes, and can produce a transfer pattern with a resolution of 10 nm or less, so that integrated microstructures can be formed at once. Therefore, it is attracting attention as a next generation semiconductor ultrafine processing technology.

[0006] In the nanoimprint method, exposure and development of a photosensitive resin (resist), which was impossible with conventional fine processing, become unnecessary. In addition, when the resin itself is the object to be processed, there is an advantage that it is economical and harmful waste and discharge are reduced because steps such as etching are unnecessary. Furthermore, since the mold once created is repeatedly used, even if it has a complicated structure such as a curved surface structure, a large number of replicas can be created once the original plate is prepared. Also

It is also possible to mold with a high aspect ratio structure (aspect ratio of 3 or more), which was difficult to mold with conventional injection molding.

 [0007] Therefore, it has a feature that it can be easily transferred and produced even with a high aspect ratio structure of micro and nano structures that cannot be efficiently produced by conventional processing. In addition, the material to be processed can be selected from thermoplastics such as acrylic, PET, PC, and PB, depending on the application, enabling micro and nano processing of a wide variety of materials. On the other hand, it is also possible to create fine lattices on glass and metal surfaces, and its industrial application range is expanding!

 [0008] As one of the fields of use, there is a field of bioassays. This is a technique in which a biological product such as an antibody is adsorbed on a micro- or nano-structure on a resin to determine the presence or absence of a sample or quantification. For example, Patent Document 2 discloses a method for distributing a sample to a microscale device, bringing the sample into contact with a target material, and detecting an interaction between the sample and the target material.

 Patent Document 1: Japanese Unexamined Patent Publication No. 2006-219752

 Patent Document 2: Special Table 2007—527784

 Disclosure of the invention

 Problems to be solved by the invention

 [0009] However, in the conventional nanoimprint method, a polymer polymer dissolved in a solvent is applied on a substrate and used! /, Therefore, for a resin containing an active group such as a carboxyl group, the solvent is not used. In some cases, they bind to each other and deactivate, thereby impairing the functionality of the resin.

 [0010] For example, since an antibody is a protein, if there is a carboxyl group on the resin surface, the N-terminal can form an amide bond and be adsorbed on the resin surface. However, if the carboxyl group is bound in the resin as described above, the antibody cannot be bound.

 [0011] In addition, the conventional resin having a nano-structure has a problem in that it is difficult to determine the chromaticity of the reaction solution that has caused an enzyme reaction on the resin because it transmits light to some extent.

[0012] In addition, when embossing or thick-film resin molding that directly presses a resin plate is used, if the resin fluidity is insufficient, a master (mold) with a high aspect ratio structure is particularly perfect. However, high pressure is required.

[0013] On the other hand, since the resin melts when heated close to the melting temperature, the fluidity increases, and the deep groove portion of the large master plate (mold) of the fluid coiling is not filled with the resin and spreads to the periphery.

 [0014] The present invention has been made in view of the force and the circumstances, and provides a simple method for producing a structure having a nanostructure and a microstructure. In addition, a biosensing disk having an antibody adsorbed on the structure and a method for producing the same are provided. Furthermore, a bio-test chip using the bio-sensing disc and a method for manufacturing the same are provided.

 Means for solving the problem

[0015] In order to solve the above problems, a method for producing a structure having a nanostructure and a microstructure of the present invention includes:

 A material placement step of placing a powdery resin on the surface of the original,

 A material heating step of heating the original plate and the resin above the glass transition temperature and below the melting temperature of the resin;

 A pressing step of pressing the resin on the original plate;

 Cooling the original plate and the resin to below the glass transition temperature, removing the original plate, and forming a reversal structure of the micro / nano structure of the original plate. Provided is a method for manufacturing a structure having a

[0016] The resin in the material arranging step is preferably a thermoplastic resin! In the material arranging step, a molding frame may be installed around the resin to surround the fine structure of the original plate. Furthermore, the size of the powdery resin in the material arranging step is preferably a powder or particles smaller than the pattern size of the original plate. The material placement step is preferably performed under reduced pressure or in a vacuum atmosphere.

 [0017] Further, a method for producing a biosensing disk in which an antibody is adsorbed to a structure having a nanostructure and a microstructure of the present invention includes:

 A material placement step of placing a powdery resin on the surface of the original,

A material heating step of heating the original plate and the resin above the glass transition temperature and below the melting temperature of the resin; A pressing step of pressing the resin on the original plate;

 A cooling step for cooling the original plate and the resin to a glass transition temperature or lower;

 Removing the original plate to obtain a structure having a micro-nano structure which is an inverted structure of the original plate; and

 A light shielding layer forming step of forming a light shielding layer on the back surface of the structure,

 The present invention also provides a method for producing a nanosensing disk comprising an antibody adsorption step of adsorbing an antibody to the micro / nano structure of the structure.

[0018] Further, the present invention provides a method for producing a biotest chip using a structure in which an antibody is adsorbed to a structure having a micro-nano structure.

 A flow path forming step of forming a flow path on the substrate to obtain a flow path side substrate;

 An adhesive layer forming step of forming an adhesive layer on the substrate to obtain a lid side substrate;

 A disposing step of disposing a structure in which an antibody is fixed to a micro-nano structure at a predetermined position of the lid-side substrate;

 The present invention provides a method for manufacturing a biotest chip, which includes a bonding step of pressing the flow path side substrate and the lid side substrate.

[0019] Further, the biosensing disc of the present invention comprises:

 Has a micro 'nano structure on one surface,

 A disc having a light shielding layer formed on the other surface;

 A biosensing disk having an antibody adsorbed on the surface of the micro-nano structure is provided.

[0020] Further, the biotest chip of the present invention comprises:

 A flow path side substrate in which a flow path is formed;

 A lid side substrate having an adhesive layer formed on the surface;

 The present invention provides a biotest chip comprising a structure having a micro / nano structure adsorbed with an antibody on its surface.

 The invention's effect

[0021] In the method for producing a structure having a nanostructure and a microstructure according to the present invention, a polymer resin powder, an oligomer powder, or the like directly purified to an original plate (mold) is directly molded without being subjected to secondary processing. Therefore, efficient micro and nano structure can be manufactured.

[0022] Further, in the production method of the present invention, since the resin is molded without dissolving the resin in the solvent, the active group is not deactivated by the residual solvent.

[0023] In the production method of the present invention, by selecting a resin powder having a diameter smaller than the pattern size of the original (mold), molding can be performed at a low pressure. In addition, molding with a low pressure is possible even for molding with a high aspect ratio structure.

[0024] Since the light-shielding layer is formed on the back surface of the biosensing disk of the present invention, the color of the reaction solution can be easily identified when assembled. In addition, when used in a bio-test chip, the disk can be bonded with ultraviolet light while being placed inside the chip. BEST MODE FOR CARRYING OUT THE INVENTION

 [0025] A method for producing a structure having a nanostructure and a microstructure of the present invention will be described below with reference to FIG.

 (Embodiment 1)

 The original plate 3 used in the production method of the present invention is provided with a micro and nano structure consisting of fine protrusions or grooves (FIG. 1 (a)). The material of the original plate 3 is preferably silicon, quartz glass, sapphire, SiC, Ni, or super steel. This micro and nano structure corresponds to the nano structure on the structure to be molded using the original plate produced by this method.

[0027] Here, "micro, nanostructure" means that the minimum size (for example, one side, height, and diameter) is a chromano-noskenore (if it is f line, 1 _{μm to} 25 μ ηι ^ Ο Ο μm to 250 μm, or 100 μm to 2500 μm) or any one or more arbitrary shapes (eg, polygonal cylinder, cylinder, flat plate, nanometer scale (eg, 10 nm to 500 nm, 50 nm to 1000 nm, or 100 nm to 1000 nm), It is a structure having a polygonal cone and a cone. The nanostructure may be a structure in which one shape or two or more shapes (including those different in size) are repeatedly arranged.

[0028] In one embodiment, the microstructure is a cylindrical array arrangement IJ (eg, height 100 m to 500 m, repeat pitch 100 Hm to 300 11 m). In another embodiment, the microstructure is a plate array (eg, thickness 2 μm to 20 μm, height 10 ^ m to 500 μm, repeat pitch 3 μm to 40 μm). In this specification, the term "micro'nanostructure" includes both microstructure and nanostructure. Huh. Micro'nanostructures may include either microstructures or nanostructure forces, or both. Therefore, “micro, nanostructure” can also be called “micro'nanostructure”.

 [0029] Micro'nanostructures are methods commonly used for microfabrication, for example in the field of semiconductor manufacturing, such as photolithographic techniques, electron beam (EB) lithography techniques, and / or etching techniques, particularly reactive ions. It can be produced using dry etching techniques such as etching (RIE) or plasma etching, and / or electron beam direct writing.

 [0030] Around the original plate 3, a metal mold 1 is provided for adjusting the thickness of the resin after molding. Examples of the metal type 1 material include aluminum, tungsten, iron, and stainless steel. The metal mold 1 here may be referred to as “molding frame 1”.

 [0031] The material of the resin powder 2 is preferably a purified product of a thermoplastic resin such as acrylic, polycarbonate, or pet. It is desirable that the size of the powder be as small as possible or less than the pattern size of the original plate. Further, when a substance is actively adsorbed to the micro / nano structure, a resin that can be surface-modified so that the substance can be easily adsorbed may be used as the resin powder. For example, when trying to adsorb proteins, etc., if there are a lot of carboxyl groups on the surface of the micro-nano structure, the ability to create an amino group and amide bond with the protein and immediately adsorb strongly. Touch with S.

 [0032] The amount of the resin powder 2 is the sum of the volume of the hollow pattern portion of the original 3 and the volume of the metal mold 1 surrounding the upper surface of the original when the resin reaches the glass transition temperature or higher. Larger amounts are preferred. More preferably, an amount exceeding 200% of the volume, more preferably an amount exceeding 120% of the volume, is placed on the master 3.

 [0033] After the resin powder 2 is placed on the original 3, the atmosphere may be reduced in pressure or vacuum. Alternatively, an inert gas atmosphere may be used.

 [0034] Generation of bubbles during molding can be suppressed or eliminated by reducing the pressure or applying a vacuum. In addition, chemical reactions with atmospheric gas components can be suppressed and eliminated. Alternatively, the chemical reaction can be suppressed or eliminated by using an inert gas atmosphere.

[0035] The pressing surface of the original plate is pressed with a flat metal pressing plate 4 (aluminum, tungsten, iron, stainless steel, silicon, SiC, quartz, etc.) while the resin powder is softened by heating (Fig. L (b)). This process uses a technology called “thermal nanoimprinting” Use. Thus, the techniques, conditions, equipment, etc. used in this process (and related processes) will generally be described briefly below in the same way as those normally used in thermal nanoimprinting.

 [0036] The heating is preferably performed at a temperature from the glass transition temperature (Tg) or higher to the melting temperature of the thermoplastic resin. More preferably, the heating is performed up to a temperature 10 ° C to 100 ° C higher than the glass transition temperature, more preferably 10 ° C to 80 ° C higher, more preferably 30 ° C to 60 ° C higher.

 [0037] The pressure at the time of pressing varies depending on the thermoplastic resin to be used, but is generally lMPa to lOOMPa, preferably lMPa to 50 MPa, more preferably IMPa to 10 MPa, more preferably 2 MPa to 5 MPa. .

 [0038] After pressing, leave the pressure and temperature at the time of pressing (Fig. L (c)). In general, the time is 30 seconds to 30 minutes, preferably 1 minute to 20 minutes, more preferably 2 minutes to 5 minutes. At this time, the viscosity of the thermoplastic resin decreases, and the thermoplastic resin is filled along with the fine structure of the original plate (mold) with the passage of time, and the inverted structure of the micro and nano structures is temporarily fixed. However, in this state, when the resin is released from the original plate, the transferred inverted structure disappears due to the viscosity of the resin over time.

 [0039] After a certain period of time, the original and the resin are cooled while maintaining the pressure during pressing.

 When the temperature of the resin falls below the glass transition temperature, the pressing pressure is gradually released. At this time, the viscosity of the thermoplastic resin increases, and the inverted structure of the micro and nano structures is fixed. At the same time, an inert gas such as air or nitrogen may be introduced from the outside to return to atmospheric pressure.

Yes

 [0040] When the temperature of the resin is 10 ° C to 200 ° C, preferably 20 ° C to; 100 ° C, more preferably 40 ° C to 80 ° C below the glass transition temperature, (Fig. L (d)).

FIG. 2 shows a graph summarizing an example of temperature and pressure processes. The horizontal axis is time (minutes), the left vertical axis is temperature (° C), and the right vertical axis is pressure (MPa). First, raise the pressure to 140 ° C over about 5 minutes without applying pressure. Then preheat at 140 ° C for about 2 minutes. Then apply lMPa pressure and maintain the pressure for 3 minutes. Cooling is performed after heating and pressurization. In the example of FIG. 2, the pressure is reduced from the start of cooling, and the pressure is gradually reduced. When the temperature reaches about 45 ° C, the pressure is reduced to zero. [0042] The original plate may be subjected to a surface treatment such as application of a release agent composed of a fluororesin or the like in order to improve releasability.

 In a preferred embodiment, the micro / nano structure is transferred by pressing the original plate against the polymer resin film. When pressing, it is preferable to heat and soften the polymer resin film.

 In another embodiment, the polymer or polymer is applied to the original plate and then polymerized or cured to transfer the micro-nano structure to the polymer resin.

 As the polymer resin, a resin generally used for molding, for example, a thermoplastic resin, a thermosetting resin, or a photocurable resin can be used.

 Such a structure having a micro'nano structure is useful as a microreactor, biochip, optical element, or micromachine.

 [0045] For example, a binding partner (for example, an antigen and an antibody, an enzyme and a substrate, a receptor) that forms a microreactor or a microfluidic device or a microwell with a micronanostructure and specifically binds to the micronanostructure. And one of the ligand, the polynucleotide strand and its complementary strand) can be used to detect the other binding partner present in a small amount of sample, or the binding reaction can be accelerated to increase the time required for detection. It can be shortened. Such microdevices can be used as immunoassay chips, DNA chips, diagnostic detection kits, chemical analysis chips, and microsensors.

 [0046] Alternatively, the flow of fluid in the flow path is controlled by forming nanostructures in the flow path of a microreactor or microfluidic device formed of micro'nanostructures, or a plurality of fluids It can promote mixing or function as a size filter.

 [0047] In addition, an antireflection structure with a conical nanostructure and a photonic crystal structure with an array of rectangular parallelepiped nanostructures can be used for IJ.

[0048] Details of the technology necessary for the implementation of the present invention are described in textbooks, academic literature, patent application publications or patents (for example, “Nanoimprint Basics and Technology Development / Application Deployment—Nanoimprint Fundamental Technologies”). And the latest technological developments "edited by Yoshihiko Hirai, issued July 3, 2006 Frontier Publishing Co., Ltd., Stephen Y. Chou, Peter R. Krauss and Preston J. Renstrom," Nanoimprint Lithography, J. Vac. Sci. Technol. B14 (6), Nov / Dec 1996, pp. 4129-4133, see US Pat. No. 5,772,905.

 Examples 1 and 2 will be described later as examples of the present embodiment.

 [0049] (Embodiment 2)

 In this embodiment, a case where a resin structure having a micro'nano structure using the manufacturing method shown in Embodiment 1 is applied to biosensing will be described.

 [0050] Biosensing refers to a method for detecting a substance using a biological product. The substance to be detected is also called an antigen. In particular, an enzyme linked immunosorbent assay (hereinafter referred to as “ELISA”) is a method used for detecting or quantifying the concentration of an antibody or antigen. In recent years, ELISA has also been performed for environmental substance testing and is a popular detection method. There are two types of ELISA: direct adsorption and sandwich.

 [0051] In the direct adsorption method, a solution containing an antigen to be detected is directly brought into contact with a plastic tube or a microphone plate plate well, and the antigen is adsorbed on its wall surface. After that, the force that does not adsorb the antigen is covered, and the wall surface portion is covered with a protein unrelated to the antigen to be detected. Hereinafter, the solid surface on which the antigen or antibody is adsorbed is referred to as “solid surface”. This is called blocking. Next, an antibody that specifically adsorbs to the antigen to be detected is added. The antibody that did not bind to the antigen is washed away, and the antibody adsorbed to the antigen on the solid surface is quantified by an enzymatic reaction.

 [0052] On the other hand, in the sandwich method, an antibody that specifically adsorbs to an antigen to be detected is first bound to a solid surface in advance. Next, after blocking the solid surface, a solution containing the antigen to be detected is added, and the antigen in the solution is bound to the antibody on the solid surface. After washing away unwanted proteins and antigens that did not bind to the antibody, add the labeled antibody and quantify the target antigen bound to the solid phase.

[0053] Biosensing using ELISA can detect only a protein to be detected even if a protein other than the protein to be detected is present in the solution. In this method, if as much antibody as possible can be adsorbed on the solid surface, a large amount of antigen can be captured, and the detection sensitivity becomes high. Since the resin structure having a micro / nano structure of the present invention can have a surface area higher than that of a flat structure, it can be used as a solid surface on which antibodies are adsorbed in ELISA. [0054] There are no particular limitations on the micro-nanostructure that can be used in the present invention. Shapes such as strips, cylinders, and triangular pyramids may be used. These patterns can also be mixed and used. In addition, as a material that can be used, a material that can adsorb antibody preferentially as well as the materials described in Embodiment 1 can be used. For example, since an antibody is a protein, it has an amino group and a carboxyl group. It is possible to use materials having functional groups that actively adsorb these functional groups on the surface. In the examples according to this embodiment, a copolymer of polymethyl methacrylate and methacrylic acid is shown. In the present specification, a copolymer of methyl methacrylate and methacrylic acid is referred to as a “PMMA copolymer”.

 [0055] In the application of biosensing according to the present embodiment, the overall shape is not limited. The portion having a microphone opening 'nanostructure may also include a curved surface that is not only a flat surface. For example

, Film shape, square shape, cylindrical shape, and the like, but are not limited thereto. However, a film-like one can be suitably used because it is easy to produce. In this embodiment, a film-like example will be described. In the present specification, a film in which a micro-nano structure is formed on the surface of a resin is called a “resin disk”. Resin disks using PMMA copolymers are called “copolymer resin disks”. A resin disc in which an antibody is immobilized on the surface of a micro-nano structure is called an “antibody-immobilized resin disc”. The antibody-fixing resin disc is

 [0056] The antibody is obtained from an immune cell produced by introducing an antigen into the body of the organism and the organism produces against the antigen. Here, the antibody can be obtained even if the antigen is a non-protein substance. Therefore, a substance to be detected that can be detected by an antibody is not limited to a protein. Hereinafter, the substance to be detected is also called a specimen, and includes substances other than proteins.

 Examples 3, 4, 5, and 6 will be described later as examples according to the present embodiment.

(Embodiment 3)

The resin structure having a micro / nano structure of the present invention is very useful in biosensing because it has an excellent antibody adsorption ability as described in Embodiment 2. Therefore, an embodiment in which the resin disk having the micro'nano structure shown in the second embodiment is used for a biochip will be described. [0057] The bio-test chip of the present invention has a flow path formed on a substrate, and a micro-nanostructured resin disk or an antibody-immobilized resin disk (hereinafter referred to as "antibody fixing") in the middle of the flow path. This is called a “resin disk”. In other words, these are simply resin discs and antibody-fixed resin discs using the terms already defined. This channel is further covered with another substrate to prevent contamination in the channel. In addition, several injection holes are formed on the surface of the substrate to inject the substance into the biodetection chip. Hereinafter, the substrate on which the flow path is formed is referred to as a flow path side substrate, and the other substrate is referred to as a lid side substrate. Note that it is preferable to form the flow path and the injection hole on one of the substrates because it saves the manufacturing effort. However, the flow path can be made on two substrates.

 FIG. 3 shows the appearance of the biotest chip 20 of the present invention. The bio-test chip 20 covers the flow path side substrate 21 formed with the flow path with the lid side substrate 22. The biotest chip shown here is a square with a side of approximately 3cm. An injection hole 23 is formed in the middle or end of the flow path. Necessary substances are supplied from the injection hole into the biotest chip.

 [0059] The flow path is a passage formed between two closely contacting substrates, and an end of which is formed an induction hole or a liquid reservoir. It may be connected to another flow path via a passive valve. The noble valve is a portion that is narrowed to the extent that a film can be formed by the surface tension of the liquid. Liquid normally cannot pass through the passive valve due to its surface tension, but can pass through the passive valve by applying air pressure. At least one detection unit 24 is provided in the flow path. A resin disc or an antibody-fixed resin disc is disposed in the detection unit.

FIG. 4 shows an example of the flow path 30. The flow path 30 includes a sample liquid injection part 32, an antibody 'labeled antibody injection part 35, a washing liquid injection part 34, a substrate injection part 31, a detection part 36, a drainage suction part 37, and a reaction liquid storage part 38. The substrate injection part 31, the cleaning liquid injection part 34, and the antigen / labeled antibody injection part 35 have an air injection part 33. A passive valve 39 is located between the flow path and the injection part. An air hole for applying air pressure to pass through the passive valve is denoted by reference numeral 33. In the present specification, the passage leading to the detection unit 36 is also referred to as a flow path 30. [0061] Both the flow path side substrate and the lid side substrate are preferably made of resin. This is because if it is made of resin, it is easy to process and has high productivity. In addition, since the purpose of the biopsy chip of the present invention is to assemble a sample by an enzyme reaction, it is preferable that the biopsy chip is a material that transmits light used for the assay. In particular, when a reaction solution reservoir for observing the reaction solution is provided, it is preferable that the light used for both the flow path side substrate and the lid side substrate is a material through which light passes. Specifically, acryl, polycarbonate and the like are preferable.

 [0062] However, only the part of the reaction liquid storage part has optical transparency! /, May! / Further, one of the flow path side substrate and the lid side substrate may be light transmissive, and the other may be formed with a reflective surface that reflects light. That is, the flow path side substrate and the lid side substrate may not be entirely formed of the same material, and may be formed of a plurality of partially different materials. Further, the flow path side substrate and the lid side substrate may be made of different materials.

 [0063] When the flow path side substrate and the lid side substrate are formed by thermocompression bonding, it is desirable that the respective softening temperatures are lower than the softening temperature of the resin disk. This is because when the flow path side substrate and the lid side substrate are subjected to thermocompression bonding, heating at a temperature higher than the Tg of the resin disk may cause the micronano structure of the resin disk to melt.

 [0064] If the softening temperature of one of the flow path side substrate and the lid side substrate is lower than the softening temperature of the other, the substrate with the higher softening temperature is embedded in the substrate with the lower softening temperature, so that the bonding is easier.

 [0065] As the resin disk used in the bio-test chip of the present invention, those shown in Embodiments 1 and 2 and the examples thereof can be used. As for the fine structure of the surface, L / S type, pillar type and other appropriate patterns can be used as long as they are not particularly limited. The antibody may or may not be immobilized. If the antibody is not immobilized, inject the antibody and cross-linking agent into the chip before use, and adsorb the antibody to the micro-nanostructure part. In this case, it is necessary to thoroughly clean the inside of the flow path after the adsorption.

[0066] However, in the case where the flow path side substrate and the lid side substrate are bonded with a photo-curable adhesive and irradiated with ultraviolet rays (hereinafter also referred to as "UV light"), the antibody-immobilized resin disk is used. When used, it is desirable to prevent UV light from directly irradiating the area where the antibody is immobilized. The antibody is a protein, and when the structure is destroyed by UV light, the protein is detected. This is because the binding force to the case may be deactivated.

 Therefore, in addition to the resin disks shown in the first and second embodiments and the examples, a light shielding layer may be formed below the resin disk. The light-shielding layer may be formed by applying a reflective material after the resin disk is fabricated, and a reflective film may be formed by sputtering or other methods such as vapor deposition on the side. May be. In other words, the light shielding layer on the back side of the resin disk may be a reflective layer.

 More specifically, a metal thin film such as gold (Au), silver (Ag), aluminum, and rhodium (Rh) can be suitably used as the reflective layer. In the case of a metal thin film, the thickness should be 5 nm or more, preferably lOnm or more. Further, if it becomes too thick, warping occurs and peeling occurs, so that it is preferably 1/10 or less of the thickness of the resin disk. This is to prevent UV light from transmitting. Alternatively, a composition comprising a metal oxide such as titanium oxide or zinc oxide and a binding resin may be applied and formed as a reflective layer by a method such as screen printing. In the case of a paint-like composition, since a large stress is hardly generated by application, the thickness may be several meters.

 [0069] Since the resin disk having such a light-shielding layer looks good in the color of the reaction solution of the enzyme reaction with the antibody bound to the micro'nano structure part, it can be used simply as an antibody-fixing resin disk for IJ. I can do it. In particular, a transparent resin having a large number of functional groups on the resin surface, such as the PMMA copolymer of the present invention, can be obtained by simply protecting the antibody from UV light during curing as described above. The presence of the light shielding layer is effective at the point where the degree of coloring is easy to check! /, And! /.

 [0070] Further, UV light does not pass through the surface of the flow path side substrate or the lid side substrate where the resin disk is disposed, or a light shielding layer may be formed. In this case, it goes without saying that a light emitting layer is formed on the surface of the substrate on which the UV light is irradiated.

 [0071] The biotest chip of the present invention can be made as a whole with a size of about several centimeters square, and is used when assembling a small amount of specimen. In particular, the antibody can be adsorbed to a small area with high density by the resin disk shown in Embodiment 2 and the example, and the detection sensitivity becomes very high.

[0072] Next, how to use the bio-test chip will be described. Refer to Fig. 4. A resin disc on which an antibody that binds differently is adsorbed is provided. The blocking liquid is injected from the blocking liquid injection section. The blocking solution is injected for the purpose of binding a protein unrelated to the specimen to the portion where the protein binds on the wall surface from the flow path to the detection section.

 Next, a solution containing the sample is injected from the sample solution injection unit 32. The sample solution passes through the flow path, reaches the detection unit 36, and touches the antibody bound to the resin disk. The specimen in the specimen solution binds to the antibody on the resin disc. Then, the antigen 'labeled antibody is injected into the biotest chip from the antigen' labeled antibody injection unit 35 and further adsorbed to the specimen adsorbed to the antibody bound to the resin disk.

 [0074] PBS or the like is injected from the cleaning liquid injection section 34 and thoroughly washed. Then, a colored solution such as TMBZ is introduced from the substrate introduction part 31. The colored solution reacts with the label of the antigen 'labeled antibody and changes its color. After a predetermined time, the reaction solution passes through the colorimeter introduction path and is taken out to the reaction solution reservoir 38 or the outside of the inspection chip as it is. The reaction solution is measured for luminescence with a colorimeter. As described above, the biotest chip of the present invention can detect and quantify the specimen with a single chip.

 FIG. 5 is a conceptual diagram of the biotest chip as seen from the cross section. Between the flow path side substrate 21 and the lid side substrate 22 adhered by the adhesive layer 49, there is an introduction portion 35 for a specimen, a labeled antigen, a washing liquid, and the like. An injection hole is provided here. The introduction part passes through a narrower channel and reaches the detection part 36 where the antibody-immobilized resin disk is disposed. Although a part of the flow path is shown, it is indicated by reference numeral 30 here. The detection unit further leads to a portion 37 for storing the reaction solution and drainage.

 [Embodiment 4]

 Next, a method for manufacturing the biotest chip of the present invention will be described. In the biodetection chip of the present invention, a flow path and an injection hole are formed in a transparent resin plate serving as a substrate, and then an antibody-immobilized resin disk is disposed on the surface of the substrate on the lid side. Are made by bonding.

[0077] There are two basic manufacturing methods. The first production method is a method in which the lid side substrate on which the antibody-immobilized resin disk is disposed and the flow path side substrate are bonded to each other with a photocurable adhesive. is there. Next, the second production method is a method in which the lid side substrate on which the antibody-immobilized resin disk is disposed and the flow path side substrate are bonded together by thermocompression bonding. Hereinafter, each form and its Example are demonstrated.

 First, a method for bonding a lid side substrate on which a resin disc with an antibody is disposed and a flow path side substrate with a photocurable adhesive will be described.

 [0079] 1. Channel master

 The flow path original plate is for forming a flow path side substrate. A channel is formed by forming a dent on one surface of the flow path side substrate material whose both surfaces are flat. At this time, it is the original flow path that makes the substrate material dent. Since it is preferable to use a resin as the substrate material in terms of cost, mass productivity, and the like, it is preferable that the flow path original plate is made of a metal material that is harder than the resin and has a property that is difficult to deform. Among metal materials, materials that are easily processed and inexpensive are suitable. Since the flow path is not an ultra-fine structure of nanometer order formed on the resin disk, the flow path original plate need not have so high accuracy. Specifically, nickel, iron, copper, zinc, ano-remium, etc. are preferably used. There is no particular limitation on how to make the flow path original plate. Processing methods such as forging, cutting and etching can be used as appropriate.

 [0080] 2.Press machine

 The press machine is used to apply pressure when the flow path side substrate and the lid side substrate are bonded. Therefore, it is preferable to have a mechanism capable of adjusting the temperature of both the upper board and the lower board. It is preferable that the pressure has a pressurizing capacity of about 1 OMPa. Further, at the time of bonding, it is preferable to have a function of depressurizing the space including the upper and lower boards while being pressurized and heated for the purpose of defoaming. In addition, it is preferable to use a photocurable adhesive when bonding the two substrates. Therefore, a press machine having a function of irradiating the sample with UV light while heating and pressing is suitable.

 [0081] 3. Fabrication of flow path side substrate

The original plate is fixed to the lower plate of the press, and a height control material for height control is placed around it. The height regulating material is for preventing excessive deformation of the substrate material by pressing. When viewed from the side of the press machine, it pressurizes materials that are difficult to deform. Therefore, a sticky metal material is preferable so as not to increase the load on the press. Specifically, Aluminum Yu Muya copper.

 Next, the substrate material is placed on the original plate and pressed while being heated on the upper plate. If preheating is performed at this time, it is easy to process.

 [0082] Cooling should not be rapid! /. This is because if the flow path side substrate with processing strain is rapidly cooled, a large warp or cracking occurs due to the stress. It is preferable to combine natural cooling and air cooling. Even if such slow cooling is performed, stress remains in the substrate. A smoothing process may be performed to relieve the stress.

 [0083] In the smoothing process, the processed flow path side substrate is generally sandwiched between two smooth surfaces, and the annealing process is performed. However, other methods may be used depending on the characteristics of the resin used.

 [0084] After forming the flow path, an injection hole is formed. From here, the sample is injected into the test chip. The injection hole may be formed either before or after the smoothing process. More preferably, the injection hole is formed before the smoothing treatment.

 The production of the flow path side substrate is not limited to this method. For example, it may be produced by injection molding using a mold.

 [0085] 4.Preparation of lid side substrate

 As the material for the lid side substrate, a material whose surface is flat in advance is used. This is because even if either the flow path side substrate or the lid side substrate is warped, the respective substrates do not adhere to each other, and problems such as liquid leakage occur when performing the assembly.

 Then, an adhesive layer is formed on one surface of the lid side substrate. The adhesive layer is a layer for bonding the flow path side substrate and the foot side substrate. An antibody-fixing resin disk is disposed inside the two substrates bonded together. Since antibodies are proteins, the presence of a large amount of volatile solvent inside may lead to inactivation of antibodies. Therefore, it is preferable to bond the lid side substrate after sufficiently evaporating the solvent even if the adhesive contains a solvent. A thermosetting adhesive can also be used, but at the time of bonding, the antibody is present inside, so that the antibody may be deactivated at a high temperature.

[0087] The photocurable adhesive contains a solvent when forming the adhesive layer! / Even after the adhesive layer is formed, The solvent can be sufficiently volatilized. In addition, since the curing is performed by light, the temperature rise during bonding is small. Therefore, it is preferable to use a photo-curable adhesive when the antibody is fixed to a resin disk with a micro-nano structure in advance.

 [0088] After forming the adhesive layer, the antibody-immobilized resin disk is disposed at a predetermined position. The disk can be fixed to the adhesive layer by the tackiness of the adhesive layer itself. However, it can be fixed by other methods such as double-sided tape or instant adhesive.

 [0089] 5. Adhesion between substrates

 The flow path side substrate is placed on the lid side substrate, and the pressure is applied while heating. When the adhesive layer is formed of a photo-curable adhesive, predetermined light may be irradiated.

 As described above, the force S can be used to produce the biotest chip of the present invention using a photocurable adhesive.

 [0090] Although an example in which the antibody-fixing resin disk is disposed on the lid side substrate has been shown, a resin disk on which the antibody is not fixed may be used. If the antibody is not fixed, the antibody and the cross-linking agent are injected into the chip after bonding the flow path side substrate and the lid substrate. This is because the antibody is adsorbed to the micro-nano structure of the resin disk within the chip. This method has the freedom that the antibody can be selected later, but on the other hand, it also increases the time and effort required to absorb the antibody in each chip. Examples relating to the present embodiment will be described later as Examples 7, 8, and 9.

[0091] (Embodiment 5)

 Next, a manufacturing method by a method in which the lid side substrate on which the antibody-immobilized resin disk is disposed and the flow path side substrate are bonded by thermocompression bonding will be described.

 1. The original plate, 2. Press machine, 3. Production of the flow path side substrate may be exactly the same as in the third embodiment.

[0092] 4. Preparation of lid side substrate

 In this embodiment, it is not necessary to form an adhesive layer simply by preparing a material having a flat surface on the lid side substrate. If the lid side substrate is prepared, it is possible to move to bonding both substrates.

[0093] 5. Adhesion between substrates

UV irradiation is performed on the inside of the flow path side substrate and the lid side substrate. This is a biotest This is to disinfect the inner wall surface when it comes to the top, and to modify the bonding surface of the two substrates with UV light to facilitate bonding.

 Next, the antibody-immobilized resin disk is disposed on the flow path side substrate. The bonding method is not particularly limited. The antibody-immobilized resin disk may have a light shielding layer on the back surface.

 Next, these are pressurized while being heated by a press. In this case, the temperature of the press board on the lid side substrate is made higher than the temperature of the press board pushing the flow path side substrate. The reason why the temperature gradient is applied in this way is to prevent the antibody-immobilized resin disk disposed on the flow path side substrate from being exposed to a high temperature, and to ensure that the flow path is formed because the flow path side substrate does not deform so much.

Yes

 [0096] When both the two substrates are heated close to the resin melting temperature, the adhesion becomes easy. The entire substrate cannot retain its shape, and part of the flow path may be clogged. Therefore, it is preferable to increase the temperature on the lid side substrate in which no flow path is formed. Example 10 will be described later as an example according to the present embodiment.

 Example

 [0097] Hereinafter, an example of an embodiment of the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

<Example 1>

 This example is an example according to the first embodiment.

 A silicon crystal substrate was used as the original plate. The micro-nano structure on the pressing surface of the original plate is a lattice-like groove structure with a width of 2 m, a depth of 12 m, and a repetition pitch of 3.5 μm. This micro / nano structure was fabricated by UV exposure and plasma etching. The part with the pattern is within a circular shape with a diameter of 7mm.

 [0099] An aluminum forming frame having a hole with an inner diameter of 7.5 mm was installed so as to surround the pattern portion of the original plate. The thickness of the thin plate is 0.5mm.

 [0100] PMMA (polymethyl methacrylate) (a reagent sold by Aldrich) was used as the thermoplastic resin. The weight average molecular weight Mw was 996,000 or 350,000. 0.3 to 0.6 gram was placed on the pattern on the original.

[0101] Subsequently, after making a vacuum atmosphere, the original plate and the resin are heated to 140 to 145 ° C. here Installed the original plate on a hot plate. An aluminum pressing plate was placed on the resin powder, and pressed with a pressure of 2 to 20 MPa for 3 to 10 minutes. Air was introduced with the original plate pressed, and cooling started at the same time. Cooling was performed by cooling the hot plate with water. After about 10 minutes, the pressure was released after cooling to 90 ° C.

 [0102] Subsequently, the original plate was left as it was, and further cooling was continued to cool the resin temperature to 60-30 ° C. Thereafter, the resin structure having a micro-nano structure in which the pattern of the original plate was reversed was released from the original plate.

 FIG. 6 shows a scanning electron microscope (hereinafter referred to as “SEM”) photograph of the obtained resin structure having a micro-nano structure. Figure 6 (a) is an SEM photograph taken at a magnification of 850x and Figure 6 (b) at a magnification of 4000x. The arrows at the bottom left of the photo correspond to 11.7m and 2.5m respectively. It can be observed from Fig. 6 (b) that the plate-like structures are arranged in an orderly manner. The plate-like structure appears to be slightly thicker near the substrate. However, other than this, it can be seen that the edges and corners are formed without sagging. The thickness of this plate was approximately 2111, the height was 12 m, and the repeat pitch was 3.5 m. This coincided with the size of the lattice-like groove structure of the original silicon crystal substrate. In addition, as shown in FIG. 6 (a), there was no pattern loss or sagging over a large area.

 <Example 2>

 This example is an example according to the first embodiment.

 In this example, the possibility of using a polymethyl methacrylate-methacrylic acid copolymer as a functional resin film in the production method of the present invention was examined. This copolymer was synthesized by the applicant. This copolymer can have many carboxyl groups on the surface when formed into a film. Therefore, proteins such as biological products can be strongly adsorbed.

 [0105] As in Example 1, an original plate and a metal frame were prepared. A Muckel plate was used as the original plate. The micro-nano structure on the pressing surface of the original plate is a lattice-like groove structure with a width of 20 μm, a depth of 50 μm, and a repeating pitch of about 30 μm.

[0106] A polymethyl methacrylate-monomethacrylic acid copolymer was used as the thermoplastic resin. This was placed in the pattern on the nickel mold by 0.3-0.6 grams. [0107] Subsequently, after making a vacuum atmosphere, the original and the resin are heated to 150 ° C. Here, the original plate was placed on the hot plate. An aluminum pressing plate was placed on the resin powder, and pressed against it at a pressure of 2 to 20 MPa for 3 to 10 minutes. Air was introduced with the original plate pressed, and cooling was started at the same time. Cooling was performed by cooling the hot plate with water. After about 10 minutes, the pressure was released after cooling to 90 ° C.

 [0108] Subsequently, the original plate was left as it was, and further cooling was continued to cool the resin temperature to 60-30 ° C.

 Thereafter, a resin structure having a micro-nano structure in which the pattern of the original plate was reversed was released from the original plate.

[0109] FIG. 7 shows an SEM photograph of the obtained resin structure having a micro-nano structure. Figure 7 (a) is an SEM photograph taken at a magnification of 110, and Figure 7 (b) is taken at a magnification of 500. The arrows in the lower left of the photo correspond to 90 · 9 m and 20.0 m, respectively. It can be observed from Fig. 7 (b) that the plate-like structures are arranged in an orderly manner. It can be seen that the plate-like structure is formed without sagging at the edges or corners. The thickness of the plate was approximately 20 mm 111, the height was 50 mm, and the repeat pitch was 30 Hm. This coincided with the size of the grid-like groove structure of the original nickel plate. In addition, as shown in Fig. 7 (a), there was no pattern loss or sagging over a large area.

 [0110] <Example 3>

 This example is an example according to the second embodiment.

 A resin disk having a micro'nano structure having a diameter of 6 mm and a thickness of about 500 m was prepared by the method for manufacturing a structure having a micro'nano structure shown in the first embodiment. Resin discs with micro / nano structures were called “resin discs”. The resin used is PMMA. The formed pattern has a lattice-like groove structure (hereinafter referred to as “L / S structure”) having a width of 2 m, a depth of 12 m, and a repeating pitch of 3.5 μm. This is the same as that created in Example 1.

[0111] A 96-well plate for ELISA (diameter: about 7 mm) with this resin disc spread was injected with a solution of HRP (Horseradish Peroxidase) -labeled antibody in varying concentrations and a cross-linking agent at 37 degrees. Left for 2 hours. HRP-labeled antibody concentrations are 0.01, 0.1, 1.0, 1 It was changed to 0, 100, 1000, lOOOOng / mL. The HRP-labeled antibody is an antibody modified with an HPR label, and is adsorbed on the solid phase surface of the groove structure of the resin disk.

 [0112] Thereafter, the plate was washed 5 times with PBS (phosphate buffered saline), the resin disc was replaced with a new ELIS A plate well, and the color former (TMBZ: 3, 3, 5, 5, 5'-Tetramethylbe nzidine ). The HRP-labeled antibody is adsorbed on the solid phase surface of the resin disc, and TMBZ is oxidized by the HRP label and turns blue.

 [0113] After 30 minutes, a stopper (sulfuric acid: H 2 SO 4) was added to stop the reaction of TMBZ. TMBZ is anti

 twenty four

 When response stops, it turns yellow. Then, only the reaction solution was put into a well of a new ELISA plate, and the absorbance was measured with a plate reader. The measurement wavelength is 450 nm. Separately, a flat resin plate that does not form micro-nano structures and a comparative example using the wall of the well of the 96-well plate for ELISA as a solid phase surface without using a resin disc were also prepared. The ELISA 96-well plate was made of polystyrene.

 [0114] Figure 8 shows the results. Figure 8 shows the relationship between the antibody concentration (ng / mL) brought into contact with the solid surface and the absorbance of the reaction solution at a wavelength of 450 nm. In the graph, the circle 10 is a resin disk, the oblique square 11 is a flat resin plate, and the square 12 is a direct case.

 [0115] When the antibody concentration is low, there is almost no difference in absorbance, and even when the antibody concentration is high, there is no difference between the samples. This is because when the antibody concentration is low, only a small amount cannot be adsorbed. Also, when the antibody concentration is high, the TMBZ reaction is saturated!

 [0116] The most sensitive detection is from several (ng / mL) to several tens (ng / mU of antibody concentration)

 This was (region 13). In particular, when the antibody concentration was 10 (ng / mU), the absorbance was about 3 times that of the case where the antibody was directly adsorbed to the well.

 [0117] <Example 4>

 This example is an example according to the second embodiment.

Next, the antibody adsorption ability of the polymethyl methacrylate-methacrylic acid copolymer used in Example 2 was confirmed. Polymethyl methacrylate-methacrylic acid copolymer was called PMMA copolymer. PMMA-based copolymers have the ability to have many carboxyl groups on the surface when formed into a film. Therefore, it was considered that the antibody adsorption capacity was increased. [0118] Fig. 9 shows the production reaction and rough structure of this copolymer. This copolymer is a product formed by condensation reaction between methyl methacrylate and methacrylic acid at a ratio of M: N! The condensation reaction was 1.5 hours at 60 ° C. This copolymer has force carboxyl groups in the part that was methacrylic acid. This carboxyl group exists on the resin surface.

 FIG. 10 shows a conceptual diagram of the surface state of this resin. Figure 10 (a) shows the case of a general resin. Common resins such as PMMA and PS (polystyrene) have few on the surface even if carboxyl groups 17 are present in the structure 16. Therefore, there are few antibodies 15 adsorbed on the resin surface!

 On the other hand, FIG. 10 (b) is a conceptual diagram in the case of PMMA copolymer resin. This copolymer has many carboxyl groups on the surface of the resin, which is only 18 in the resin. The carboxyl group on the resin surface can be strongly bound by forming a peptide bond with the amino group of the amino acid forming the antibody 15. This embodiment will be described below.

 [0121] Polymethyl methacrylate-methacrylic acid copolymer using the mold production method of the present invention

 (: PMMA-based copolymer) with micro / nanostructure! /, Plate-shaped resin plate (hereinafter referred to as “copolymer resin plate”) to the well of ELISA 96-well plate, HRP labeled antibody (lOng / mL) and a crosslinking agent were injected and left at 37 ° C. for 1 hour.

 [0122] 1-Ethyl-3 (3-dimethylaminopropyl) carpositimide (hereinafter referred to as “EDC”) was used as the crosslinking agent. EDC promotes the amide bond between the carboxyl group on the surface of the copolymer resin plate and the N-terminus of the antibody.

 [0123] Thereafter, the plate was thoroughly washed with PBS, the resin disc was replaced with a new ELISA plate well, and reacted with TMBZ. After 15 minutes, a stopper (sulfuric acid) was added, and only the reaction solution was placed in a well of a new ELISA plate, and the absorbance was measured with a plate reader. The measurement wavelength is 450 nm. As a result, the absorbance was confirmed to be 5 times that of the case where the wall of the well was directly used as the solid surface.

 [0124] <Example 5>

 This example is an example according to the second embodiment.

Next, an example of antibody adsorption ability when a micro'nano structure is formed on a copolymer resin plate will be described. Resin disk with micro 'nano structure formed on copolymer resin plate Was called a copolymer resin disk.

 The prepared samples are (1) No micro / nanostructure! /, PMMA resin flat plate, (2) PMMA resin disk with L / S structure formed on one side, (3) No micro / nanostructure! /, Copolymer resin plate, (4) copolymer resin disk with pillar structure formed on one side. PMMA having a weight average molecular weight Mw of 996 k was used. The pillar structure is a columnar continuous pattern. In this example, a pillar structure in which a columnar structure having a diameter of 2111 and a height of 4 m was formed at a pitch of 4.O ^ m was used. For copolymer resin plates and copolymer resin disks using PMMA copolymer resins, the antibody adsorption ability was confirmed when a crosslinking agent was used during antibody adsorption and when it was not used.

 The procedure for measuring the antibody adsorption capacity is the same as in Example 4. The results are shown in Table 1.

[table 1]

Table 1 shows the values of the resin material, the antibody adsorption capacity in the case of a flat plate without micro structure, and the antibody adsorption capacity in the case of forming micro structure. The magnification in the table indicates the detection intensity (absorbance) obtained from each sample, and the wall of the well is directly fixed. This is the value divided by the detected intensity obtained by the existing method for the phase surface (relative intensity; also referred to as “sensitization effect”). A / R is the aspect ratio of the microstructure. The figures for the surface area increase effect in the right column are the net effects due to the theoretically predicted surface area increase. In the microstructure column, SEM photographs of the microstructure are also shown.

 [0126] Referring to the upper part of Table 1, when looking at PMMA, if a resin flat plate is used, a resin disc that has an L / S structure with a force aspect ratio of 6 that has the same sensitivity as the existing method is used. 3 times the sensitization effect was observed. The surface area increasing effect is 2.5 times.

 Next, refer to the middle row of Table 1. This is the result when a PMMA copolymer is used and no crosslinking agent is used during antibody adsorption. In the table, the PMMA copolymer is described as “PMMA base Co-polymer”. First, when using a copolymer resin plate with no micro-nano structure, the antibody adsorption capacity was comparable to that of the existing method. In addition, when a copolymer resin disk to which a pillar structure with an external ratio of 2 was transferred was used, the sensitization effect was doubled. In these verifications, since no cross-linking agent was used, only a simple surface area increasing effect (1.8 times) appeared, and the measured values agreed well with the theoretical expected values.

 [0127] Next, refer to the lower part of Table 1. This is the result of using a PMMA copolymer and using a crosslinking agent during antibody adsorption. By using a cross-linking agent, the surface of the PMMA copolymer and the antibody are strongly adsorbed, so it was thought that the ability to maximize the antibody fixing performance of this resin could be achieved. First, when the copolymer resin plate was used, the sensitizing effect was 5 times that of the existing method. Furthermore, when copolymer resin discs with transferred aspect ratio 2 pillar structures were used, a sensitizing effect of 8 times was confirmed. From this result, it was shown that the antibody was immobilized at a higher density than the existing method. The surface area increasing effect is 1.8 times.

 <Example 6>

 This example is an example according to the second embodiment.

Next, in order to further enhance the antibody adsorption capacity, an example in which UV ozone treatment was performed on a resin disk having a micro-nano structure will be described. UV treatment is known to have a surface modification effect on the substance, and antibody adsorption ability was expected by performing UV treatment before adsorbing antibodies. The prepared samples are (1) PMMA resin flat plate, (2) PMMA resin disc, (3) copolymer resin plate, and (4) copolymer resin disc. In this example, an L / S structure with an aspect ratio of 6 was formed on the resin disc for both PMMA and PMMA copolymer. Each sample was subjected to UV treatment, and then the crosslinker and antibody were adsorbed together on the sample surface. The antibody adsorption procedure is the same as in Example 4. For the UV treatment, a surface treatment device with an ultraviolet lamp was used, and a resin flat plate and a resin disc were placed on a sample table 62 mm below the lamp and irradiated for 6 minutes. As the ultraviolet lamp, a synthetic quartz lamp that generates light having a wavelength of 184.9 and a wavelength of 253.7 nm was used.

 Table 2 shows the results of antibody immobilization performance when using PMMA and PMMA-based resin plates and resin disks with micro-nanostructures.

[Table 2]

Look at PMMA, referring to the upper part of Table 2. Here, since the fine structure is the same pattern, the surface area increasing effect is all the same value (2.5 times). First, using a resin flat plate, the sensitization effect was three times that of the existing method. Furthermore, when using a resin disc that was copied from an L / S structure with an aspect ratio of 6, the sensitization effect was 7 times higher. On the other hand, referring to the lower part of Table 2, PMMA-based copolymers have an aspect ratio of 7 times when resin flat plates are used. In the case of using a disk to which the L / S structure of 6 was transferred, a 20-fold sensitization effect was obtained. These sensitizing effects are considered to be a surface area increasing effect, an effect of a combination of a PMMA copolymer and a cross-linking agent (hereinafter referred to as “high density antibody fixing effect”), and a synergistic effect of UV ozone treatment. The breakdown of each effect can be thought of as follows based on theoretical values and measured values.

 [0130] The surface area increasing effect is approximately 2.5 times under the condition of an aspect ratio of 6. The high-density antibody fixing effect is 4 to 5 times that of the PMMA copolymer used in the examples. The UV ozone treatment effect is 1.4 to 1.8 times.

 [0131] From the above, it was found that the resin structure having a micro-nano structure of the present invention is very effective when applied to biosensing.

 [0132] <Example 7>

 This example is an example according to the fourth embodiment.

 Referring to Fig. 11 (a), first, Ni Denki's original flow channel plate 40, which is the prototype of the flow channel structure, is placed on the lower press plate 44 so that the flow channel structure is on top, and the flow channel is placed on it. A substrate material acrylic plate 41 to be formed was placed. Note that the upper press plate 45 and the lower press plate 44 are omitted in FIG. 11 (a).

 [0133] Next, a stainless steel back plate 42 was placed on the acrylic plate 41 of the substrate material. The back plate can be suitably used because it does not easily generate fine powder due to corrosion. However, the present invention is not limited to this. Around the original plate, an aluminum height control material 43 was installed to adjust the thickness after molding.

 [0134] Referring to Fig. 11 (b), the temperature was then raised to 160 ° C for both the upper and lower presses. At this time, the pressure in the container (chamber one: not shown) surrounding the press board was reduced. This is to suppress and eliminate the generation of bubbles during molding. After both the press upper and lower plates reached 160 ° C, they were held for about 10 minutes for preheating. By this preheating, it was possible to soften the acrylic plate, improve the deformation and fluidity of the substrate material, and promote the filling of the original plate into the microstructure.

[0135] After preheating was completed, pressing was performed at a pressure of 3 to 4 MPa for about 10 minutes. After pressing, cooling was performed while maintaining the caloric pressure state. The cooling was not forced cooling, but the heating power of the press was turned off and cooling was performed by natural cooling. When the temperature dropped to about 100 degrees, air was introduced and cooling was performed by blowing air. After that, when it reaches about 40-50 ° C, it is depressurized. The sample was taken out. By performing slow cooling at the time of molding, it was possible to greatly suppress the deformation of the substrate material in the subsequent heat process.

 [0136] Finally, an injection hole for introducing the solution was drilled. In this way, a flow path side substrate 21 having a flow path and an injection hole was obtained.

 [0137] As described above, the flow path side substrate 21 formed the flow path by press working. For this reason, stress remains on the substrate immediately after the flow path is formed, which may warp the flow path forming surface side. It is also necessary to smooth the surface 25 where the flow path side substrate 21 adheres to the lid side substrate. Therefore, in this process, a process of relieving stress and eliminating warping was performed.

 [0138] Referring to Fig. 12, the smooth surface of silicon wafer 46 was placed on the lower press plate 44 with the smooth surface facing upward. On the silicon wafer 46, the flow path side substrate 21 was placed so that the surface to which the flow path was transferred faced down. Next, the upper and lower press plates 44 and 45 were heated to 90 ° C., and the inside of the container (chamber one: not shown) covering the upper and lower press plates 44 and 45 was decompressed.

 [0139] After reaching 90 ° C for both upper and lower presses, pressing was performed at a pressure of 0.8 MPa for 20-30 minutes. After the press, the heating device was turned off and cooled while maintaining the pressurized state. Cooling was forcibly performed by applying wind. When the temperature reached about 70 ° C, air was introduced, the pressure was released, and the sample was taken out. By performing this step, a flow path side substrate having a smooth adhesive surface 25 and no warpage was obtained.

 [0140] Next, an adhesive layer 49 was formed on the lid side substrate, and was bonded to the flow path side substrate.

 Refer to Figure 13 (a). First, the adhesive layer 49 was formed on the acrylic lid side substrate 22 with a photocurable resin (hereinafter also referred to as “UV curable adhesive”). Specifically, the lid side substrate 48 is vacuum-adsorbed and fixed to the spin coater sample stage, and a few drops of “Henkel LOCTITE Visible Cure Visible Light Curing Type 3105” are added, with a rotational speed of 1000 ( Rotation / minute) for 20 seconds, followed by spin coating by rotating for 2 seconds (rotation / minute) for 60 seconds

Yes

[0141] The lid side substrate coated with the UV curable adhesive was left on a hot plate set at 80 ° C and beta-treated for 20 minutes. The beta lid-side substrate was placed under an ultraviolet light source and irradiated with ultraviolet light for 10 minutes to cure the adhesive. In this way, the adhesive layer 49 having a thickness of 500 nm was formed on the surface of the acrylic lid side substrate 22. [0142] Next, referring to FIG. 13 (b), an antibody-fixing resin disk 26 made of a PMMA copolymer is placed on the adhesive layer 49 of the lid-side substrate 22 so as not to destroy the micro-nano structure. Even pressed lightly on. It was easily fixed by the tackiness of UV curing adhesive. The antibody-immobilized resin disk 26 was tested for adhesion using double-sided tape or instant adhesive.

 Next, with reference to FIG. 13 (c), the adhesive surface 25 of the flow path side substrate 21 and the adhesive layer 49 on the lid side substrate 22 were made to face each other and placed on the press lower platen 44. A carbon foil 50 having a thickness of 0.5 mm was placed thereon. In this process, when carbon oil was used, it was possible to press uniformly.

 [0144] Both the upper and lower press plates 44 and 45 were heated to 36 ° C, and the inside of the container (chamber one: not shown) covering the upper and lower press plates was depressurized. This is in order to eliminate the generation of bubbles during bonding, “suppression of contamination”.

 [0145] Pressing was performed at a pressure of 3 to 4 MPa for 10 minutes. After the press, the outside air was introduced and the pressure was released, and the sample was taken out. In this way, the biotest chip of the present invention could be obtained.

 <Example 8>

 This example is an example according to the fourth embodiment.

 FIG. 14 shows an outline of the manufacturing method of this example. Referring to FIG. 14 (a), the flow path side substrate 22 was prepared in the same manner as in Example 7. Thereafter, on the part directly above the antibody-fixing resin disc, a light-shielding film 52 was formed by screen-printing a paint in which titanium oxide and resin were dispersed in a solvent to a thickness of several meters.

 Referring to FIG. 14 (b), the lid-side substrate 22 was produced in the same manner as in Example 7. However, the UV treatment performed at the end of the formation process of the adhesive layer 49 is not performed in this embodiment! Only the UV curing adhesive is spin coated on the lid side substrate 22. An antibody-fixing resin disk 26 made of PMMA copolymer was disposed on the UV curable adhesive layer 51.

Referring to FIG. 14 (c), the flow path side substrate 21 and the lid side substrate 22 were overlapped and pressed at the same pressure of 3 to 4 MPa as in Example 7 for 10 minutes. During the pressing process, side-force ultraviolet rays (UV light) were applied to the flow path side substrate. After the press was completed, outside air was introduced and pressure was released, and the sample was taken out.

[0148] In this example, the adhesive layer 49 was not completely formed on the lid-side substrate 22, and is shown in Fig. 14 (c). The adhesive layer is cured while heating and pressing during the bonding process. The bio-test chip of the present invention thus obtained was able to adhere more firmly than in Example 7. In addition, since the light shielding film 52 is formed on the force flow path side substrate 21 that has been subjected to UV treatment since the antibody-fixing resin disc is mounted, the antibody may not be deactivated by UV light!

<Example 9>

 This example is an example according to the fourth embodiment.

 FIG. 15 shows an outline of this embodiment. The flow path side substrate 21 was produced in the same manner as in Example 7. The lid-side substrate 22 was produced in exactly the same way as in Example 8 (FIG. 15 (a)). That is, only the layer 51 of the UV curable adhesive was formed on the lid side substrate.

 Referring to FIG. 15 (b), an antibody-fixing resin disk 26 made of PMMA copolymer was placed on the lid-side substrate 22. At this time, a light shielding layer 56 for shielding ultraviolet light and visible light was previously formed on the resin disk bonding surface. The light shielding layer 56 is a gold vapor-deposited film and has a thickness of 0.5 m. Then, the adhesive surface 25 of the flow path side substrate 21 and the lid side substrate 22 were opposed to each other and placed on the press lower plate 44.

 [0150] Referring to Fig. 15 (c), both upper and lower press plates 44 and 45 were heated to 36 ° C, and the inside of the container (chamber one: not shown) covering the upper and lower press plates was depressurized. This is to suppress and eliminate the generation of bubbles during the crimping. Then, ultraviolet light was irradiated from the lid side substrate side, and pressing was performed at a pressure of 3 to 4 MPa for 10 minutes.

 [0151] By doing so, the UV curing adhesive layer 51 of the antibody-fixing resin disk can also be cured. At this time, since the light shielding layer 56 is formed on the back surface of the antibody-fixing resin disc, the antibody is not deactivated by UV light.

 [0152] <Example 10>

 This example is an example according to the fifth embodiment.

 Figure 16 shows an overview of this example. Referring to FIG. 16 (a), the flow path side substrate 21 was produced in the same procedure as in Example 7. Neither the adhesive layer nor the UV curable adhesive layer is formed on the lid side substrate 22. UV ozone treatment was applied to the inner surface of these two substrates including the bonding surface for 10 minutes.

[0153] An antibody-immobilized resin disk was attached to a predetermined position inside the flow path side substrate using an instantaneous adhesive. Adhesion was also possible with double-sided tape. And the adhesive surface of the flow path side substrate, The lid side substrate was placed on the press lower platen 44 so that the adhesive surfaces of the lid side substrate faced each other and the flow path side substrate faced up. A stainless steel back plate 42 was placed thereon, and a carbon oil 50 was placed thereon. By using carbon oil, it can be pressed uniformly.

[0154] The upper press plate 45 was set to 36 ° C and the lower press plate 44 was set to 120 ° C, and the inside of the container (chamber one: not shown) covering the upper and lower press plates was depressurized. This is to eliminate the generation of bubbles during the crimping process.

 [0155] Pressing was performed at a pressure of 0 · 8 MPa for 10 minutes. After pressing for 10 minutes, it was cooled in the pressurized state, and when it reached about 70 degrees, it was introduced into the atmosphere, depressurized, and the sample was taken out. As a comparative example, an inspection chip was manufactured in the same manner with the upper and lower press plates at 100 ° C.

 [0156] Figures 17 and 18 show the results. FIG. 17 is an SEM photograph of a cross section of the reaction part 36. The production conditions are the case where the press upper plate 45 that pressed the flow path side substrate 21 is crimped at 36 ° C, and the press lower plate that pressed the lid side substrate 22 is pressed at 120 ° C, which is the sample of this example. . The magnifications are 30 times in Fig. 17 (a), 200 times in (b), and 1000 times in (c). The arrows in the lower left of the photo correspond to 333 m and δθ μ ΐ ^ 10 m, respectively.

 [0157] Referring to FIG. 17 (a), the reaction part 36 is visible in the center of the photograph. The lid side substrate 22 is on the upper side, and the flow path side substrate 21 is on the lower side. Here, the adhesion portion 60 between the lid side substrate 22 and the flow path side substrate 21 was observed. As the magnification was increased to 200 times (b) and 1000 times (c), it was found that the flow path side substrate was slightly indented into the lid side substrate. At the joint 60, the wall surface was a straight line or a gentle arc. That is, the lid side substrate was deformed by the flow path side substrate.

 [0158] Fig. 18 is a cross-sectional SEM photograph of the reaction zone 36 of the comparative sample fabricated at 100 ° C for both the upper and lower presses. The magnifications of (a), (b), and (c) and the size of the arrow at the lower left of the photograph are the same as in FIG. From the observation in Fig. 18 (a), the penetration of the channel side substrate 21 into the lid side substrate 22 was slightly shallow, and the side wall 62 was greatly bent in an arc shape. As a result, it was found that deformation due to softening of the acrylic material, which is the substrate material, progressed during molding, and that it was not an appropriate temperature condition for bonding a flow path having a fine structure such as a noble valve.

FIG. 19 is a transmission microscope photograph showing the state of the passive valve 39 in the case of FIGS. 17 and 18. This passive valve 39 is connected between the antibody-labeled antibody injection part 35 and the flow path 30. It is. Figure 19 (&) shows the case where the flow path side substrate is molded at 36 ° and the lid side substrate is molded at 120 ° C. (B) shows the case where the flow path side substrate is molded at 100 ° C and the lid side substrate is molded at 100 ° C. The arrow in Fig. 19 (a) shows the trajectory when the solution flows in the biotest chip.

 [0160] Referring to Fig. 19 (a), when the flow path side substrate was 36 ° C and the lid side substrate was 120 ° C, the three passive valves 39 were firmly formed. In other words, three passages were created between the antibody-labeled antibody injection part 35 and the flow path 30. On the other hand, referring to FIG. 19 (b), when both the flow path side substrate and the lid side substrate were manufactured at 100 ° C., the passive valve 39 was crushed! /.

 [0161] That is, when an acrylic plate is used as a substrate material and a flow path side substrate and a lid side substrate are bonded only by thermocompression bonding without forming an adhesive layer, the temperature is at least between the flow path side substrate and the lid side substrate. A degree gradient must be set. In other words, a bio-test chip in which the flow path side substrate and the lid side substrate are bonded only by thermocompression bonding between the adhesive layers, and the passive valve can be molded without collapsing! It is possible to assume that there is a temperature gradient between the substrates. In both cases where the upper and lower sides of the press panel were 80 ° C and 90 ° C, unbonded portions remained.

 [0162] The above embodiments and examples are described as examples for easy understanding of the present invention, and the present invention is not limited to the specific configurations described in this specification or the accompanying drawings. It should be noted that the present invention is not limited only to the composition. The specific configurations, means, and methods described herein may be changed to equivalents without departing from the spirit and scope of the present invention.

 Industrial applicability

[0163] The present invention is useful as a microreactor, biochip, optical element, and micromachine.

 Brief Description of Drawings

 FIG. 1 is a diagram for explaining a method for producing a structure having a micro and nano structure according to the present invention.

 FIG. 2 is a diagram for explaining the time progression of temperature and pressing pressure in the method of the present invention.

FIG. 3 is a view showing a result of manufacturing a structure having a high aspect ratio micro'nano structure using an acrylic material. FIG. 4 is a view showing a result of producing a structure having a high aspect ratio micro ′ nanostructure using a methyl methacrylate-methacrylic acid copolymer.

 FIG. 5 is a graph showing the effect of the amount of antibody adsorbed on a structure having a micro'nano structure.

 FIG. 6 is a conceptual diagram showing the production of a copolymer of methyl methacrylate-methacrylic acid.

 FIG. 7 is a diagram for explaining how antibodies are adsorbed on the surface of a resin.

 FIG. 8 is a photograph showing an example of a biotest chip of the present invention.

 FIG. 9 is a diagram showing an example of a flow path of a biotest chip.

 FIG. 10 is a view showing a cross section of the biopsy chip of the present invention.

 FIG. 11 is a diagram illustrating a method for manufacturing a flow path side substrate.

 FIG. 12 is a diagram for explaining smoothing of the flow path side substrate.

 FIG. 13 is a diagram illustrating a method for manufacturing a biotest chip according to the present invention.

 FIG. 14 is a diagram illustrating a method for manufacturing a biotest chip according to the present invention.

 FIG. 15 is a diagram illustrating a method for manufacturing a biotest chip according to the present invention.

 FIG. 16 is a diagram for explaining a method for manufacturing the biotest chip of the present invention by thermocompression bonding.

 FIG. 17 is a photograph showing a cross section of a bio-test chip by thermocompression bonding with a temperature gradient.

 FIG. 18 is a photograph showing a cross section of a bio-test chip by thermocompression bonding without applying a temperature gradient

[Fig. 19] A photograph showing the effect of temperature gradient in the passive valve section.

FIG. 20 is a diagram for explaining a conventional nanoimprint method.

Explanation of symbols

 1 Metal mold

 2 Resin powder

 3 Original edition

 4 Pressing plate

 20 Bio test chip

 21 Channel substrate

22 Lid side substrate Injection hole detector

Flow path

Antibody · Labeled antibody injection part Detection part Flow path master

Lower press

Press top

Silicone adhesive layer

Light shielding film

Shading layer

Claims

The scope of the claims

 [1] A material placement step of placing powdered resin on the surface of the original plate,

 A material heating step of heating the original plate and the resin above the glass transition temperature and below the melting temperature of the resin;

 A pressing step of pressing the resin on the original plate;

 Cooling the original plate and the resin below the glass transition temperature, removing the original plate, and forming a reversal structure of the micro / nano structure of the original plate, A method for producing a structure having the same.

 [2] The method according to claim 1, wherein the resin in the material arranging step is a thermoplastic resin.

 [3] The manufacturing of the structure according to any one of claims 1 and 2, wherein in the material arranging step, a molding frame is installed so as to surround the fine structure of the original plate around the resin. Method.

 [4] The manufacturing of the structure according to any one of claims 1 to 3, wherein a size of the powdery resin in the material arranging step is a powder or particles smaller than a pattern size of the original plate. Method.

 [5] The method according to any one of claims 1 to 4, wherein the pressurizing step is performed under reduced pressure or in a vacuum atmosphere.

6. The method for producing a structure according to any one of claims 1 to 4, wherein the resin in the material arranging step is a copolymer of polymethyl methacrylate and methacrylic acid.

[7] A material placement step of placing powdered resin on the surface of the original plate,

 A material heating step of heating the original plate and the resin above the glass transition temperature and below the melting temperature of the resin;

 A pressing step of pressing the resin on the original plate;

 A cooling step for cooling the original plate and the resin to a glass transition temperature or lower;

Removing the original plate to obtain a structure having a micro-nano structure which is an inverted structure of the original plate; and A light shielding layer forming step of forming a light shielding layer on the back surface of the structure,

 A method for producing a nano-sensing disk, comprising: an antibody adsorption step of adsorbing an antibody to the micro / nano structure of the structure.

8. The method for manufacturing a biosensing disk according to claim 7, wherein the resin in the material arranging step is a thermoplastic resin.

[9] The manufacturing of the bio-sensing disk according to any one of claims 7 and 8, wherein in the material arranging step, a molding frame is installed so as to surround the fine structure of the original plate around the resin. Method.

[10] The biosensing disk according to any one of claims 7 to 9, wherein the size of the powdery resin in the material arranging step is powder or particles smaller than the pattern size of the original plate. Manufacturing method.

[11] The method of manufacturing a biosensing disk according to any one of [7] to [10], wherein the pressurizing step is performed in a reduced pressure or a vacuum atmosphere.

[12] The process for producing a biosensing disk according to any one of claims 7 to 11, wherein the resin in the material arranging step is a copolymer of polymethyl methacrylate and methacrylic acid.

 [13] The method for manufacturing a biosensing disk according to any one of [7] to [12], wherein the light shielding layer is a reflective layer that reflects light.

[14] The method for producing a biosensing disk according to any one of [7] to [13], wherein the antibody adsorption step is a step of bringing a solution containing a cross-linking agent and an antibody into contact with the structure.

 [15] The method for producing a biosensing disk according to claim 14, wherein the cross-linking agent is 1-ethyl-3- (3-dimethylaminopropyl) carpositimide.

[16] The request according to any one of claims 7 to 14, wherein the antibody adsorption step includes a step of irradiating the structure with UV light, and a step of bringing the structure into contact with a solution containing a crosslinking agent and an antibody. A method for producing a biosensing disk described in the claim.

[17] The antibody adsorption step includes irradiating the structure with UV light;

17. The step of bringing the structure into contact with a solution containing a crosslinking agent and an antibody. Manufacturing method of the manufactured biosensing disc.

[18] has a micro 'nanostructure on one surface,

 A disc having a light shielding layer formed on the other surface;

 A biosensing disc having an antibody adsorbed on the surface of the micro'nano structure.

19. The biosensing disc structure according to claim 18, wherein the biosensing disc is made of resin.

 20. The biosensing disk according to claim 18, wherein the resin has a carboxyl group on the surface.

21. The biosensing disk according to any one of claims 18 to 20, wherein the resin is a copolymer of polymethyl methacrylate and methacrylic acid.

[22] The micro 'nano structure is any one of L / S structure and pillar structure.

A biosensing disk according to claim 1.

23. The biosensing disk according to any one of claims 18 to 22, wherein the light shielding layer is a metal thin film having a thickness of 10 nm or more.

24. The light shielding layer is a film of a composition comprising metal oxide fine particles and a resin.

Any one of 23 forces, The biosensing disk described in claim 1.

[25] The disk according to any one of claims 18 to 24, wherein the disk is transparent.

[26] a flow path forming step of forming a flow path on the substrate to obtain a flow path side substrate;

 An adhesive layer forming step of forming an adhesive layer on the substrate to obtain a lid side substrate;

 A disposing step of disposing a structure having a micro-nano structure in which an antibody is fixed at a predetermined position of the lid-side substrate;

 A method for producing a biotest chip, comprising a bonding step of pressing the flow path side substrate and the lid side substrate.

[27] The method for producing a biotest chip according to [26], further comprising a step of irradiating the adhesive layer with UV light.

[28] a flow path forming step of forming a flow path on the substrate to obtain a flow path side substrate;

 An adhesive layer forming step of forming an adhesive layer on the substrate to obtain a lid side substrate;

 An arrangement step of arranging the biosensing disk according to any one of claims 18 to 25 at a predetermined position of the lid-side substrate,

 A method for producing a biotest chip, comprising a bonding step of pressing the flow path side substrate and the lid side substrate.

 29. The method for producing a biotest chip according to claim 28, wherein the bonding step is a step of irradiating UV light from the lid side substrate side while pressing the flow path side substrate and the lid side substrate.

 [30] including a step of forming a light shielding layer on the surface of the flow path side substrate at a position corresponding to the position where the structure is disposed on the lid side substrate;

 27. The method for manufacturing a biotest chip according to claim 26, wherein the bonding step is a step of irradiating UV light from the side of the flow path side substrate while pressurizing the flow path side substrate and the lid side substrate.

 [31] a flow path forming step of forming a flow path on the substrate to obtain a flow path side substrate;

 A disposing step of disposing a structure having a micro-nano structure in which an antibody is fixed at a predetermined position of the flow path side substrate;

 A method for manufacturing a biotest chip, comprising a step of bringing the flow path side substrate and the lid side substrate into contact with each other and pressurizing the lid side substrate at a temperature higher than the temperature of the flow path side substrate.

[32] The flow path side substrate and the lid side substrate are made of acrylic, and the temperature of the lid side substrate is set to 1

32. The method for producing a bio-test chip according to claim 31, wherein the temperature of the flow path side substrate is set to 20 °, and the temperature of the flow path side substrate is 38 ° or less.

[33] The flow path forming step includes:

 A process of obtaining a flow path side substrate by pressing the flow path original plate against the substrate and performing pressure molding;

 The method for producing a biotest chip according to any one of claims 26 to 32, comprising a step of smoothing the flow path side substrate.

[34] a channel-side substrate on which a channel is formed; A lid side substrate having an adhesive layer formed on the surface;

 A bio-test chip comprising a micro / nano structure having an antibody adsorbed on its surface

[35] The biotest according to claim 34, wherein the structure is disposed on the lid side substrate!

36. The biotest chip according to claim 34, wherein the adhesive layer is a photocurable adhesive.

[37] a channel substrate on which a channel is formed;

 A lid side substrate deformed by the flow path side substrate;

 A bio-test chip comprising a micro / nano structure having an antibody adsorbed on its surface

38. The biotest according to claim 37, wherein the structure is disposed on the flow path side substrate!

[39] The biotest chip according to any one of claims 34 to 38, wherein a passive valve is formed in the flow path.

[40] The biotest chip according to any one of claims 34 to 39, wherein an injection hole is formed in the flow path side substrate.

41. The flow path side substrate and the lid side substrate pass at least a specific wavelength of light.

The biotest chip according to any one of claims 34 to 40.

[42] The bio-test chip according to any one of claims 34 to 41, wherein the structure is the biosensing disk according to any one of claims 18 to 25.