Gene detection tools and gene detection kits

Abstract

Provided is a gene detection tool with which it is possible to quickly collect and add a test sample through a simple operation and also to reduce the amount of waste.　This gene detection tool 2 comprises a container body 21 for accommodating a gene synthesis reaction solution 10, a container lid 22 for sealing the container body 21, and an electrode part 23 and a sample collection part 24 that are provided to the container lid 22 and inserted into the container body 21. The sample collection part 24 is configured to be capable of suctioning a test sample that may include a gene to be detected.

Description

[Technical Field]

[0001] The present invention relates to a gene detection tool and a gene detection kit. [Background technology]

[0002] Hybridization methods have been widely used as a technique for detecting genes having specific base sequences, and one known method is an electrochemical detection method that uses an electrode-type sensor (see, for example, Patent Document 1).

[0003] Patent Document 1 proposes that amplification reaction and electrochemical measurement of a test sample that may contain a target gene be carried out in the same amplification reaction tube equipped with electrodes. The amplification reaction tube has electrodes embedded in its wall, allowing electrochemical measurement of the solution in the tube.

[0004] In the measurement procedure disclosed in Patent Document 1, a nucleic acid amplification buffer is first poured into an amplification reaction tube, and a test sample is then added. After the lid of the amplification reaction tube containing the nucleic acid amplification buffer and the test sample is closed and sealed, the tube is placed in an amplification device. Next, the solution in the tube is heated by the amplification device to amplify the target gene, and the reduction current of the solution in the tube is measured.

[0005] However, Patent Document 1 has the problem that it requires the complicated steps of collecting a test sample in a sample collection tool separate from the amplification reaction tube and adding the collected test sample to the amplification reaction tube. Furthermore, when handling patient-derived specimens (e.g., saliva samples and blood samples) at medical institutions, clinical trial institutions, etc., amplification reaction tubes and sample collection tools are basically disposable, so there are also the problems of using sample collection tools increasing costs and generating a lot of waste from testing. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-098963 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a gene detection tool and a gene detection kit that enable quick collection and addition of a test sample with simple operations and that also enable a reduction in the amount of waste. [Means for solving the problem]

[0008] One embodiment of the gene detection tool of the present invention comprises a container body for containing a gene synthesis reaction solution, a container lid for sealing the container body, and an electrode section and a sample collection section provided on the container lid and inserted into the container body, wherein the sample collection section is configured to be able to suck up a test sample that may contain a gene to be detected.

[0009] According to the gene detection tool of the above embodiment, by providing a sample collection section on the container lid, there is no need to use a sample collection tool, so that the test sample can be collected and added quickly with simple operations and the amount of waste can be reduced.

[0010] In the gene detection tool of the above embodiment, the sample collection part may be provided at the tip of the electrode part.

[0011] According to this embodiment, the operation of contacting the sample collection part with the test sample to collect the sample is facilitated, and when the container lid is attached to the container body, the sample collection part (test sample) can be reliably immersed in the gene synthesis reaction solution contained in the container body. For example, if the test sample is a patient's saliva, the saliva sample can be easily and simply sucked and collected by the sample collection part by contacting the sample collection part provided at the tip of the electrode part with the saliva in the patient's oral cavity. Furthermore, by providing the sample collection part at the tip of the electrode part, the genetic detection tool can have a simple structure.

[0012] In the gene detection tool of the above embodiment, the electrode unit includes a plurality of electrodes provided on an insulating substrate, an insulating layer covering the electrodes, and a sensor unit in which a portion of the plurality of electrodes is exposed, and the sample collection unit is provided on the sensor unit. do.

[0013] The gene detection tool of the above embodiment According to the method, by covering multiple electrodes with an insulating layer to prevent erroneous detection, and by providing a sample collection section on top of a sensor section that exposes a portion of the electrodes, the electrode section and sample collection section can be made compact and simple in structure.

[0014] In this embodiment, the sample collection section may be formed by a recess in the insulating layer that opens to the outer surface of the electrode section, and at least a portion of the inner wall surface of the recess may be hydrophilic. Here, "at least a portion of the inner wall surface is hydrophilic" means that the contact angle of the inner wall surface with the test sample (aqueous liquid) is less than 90 degrees, making it easily wettable. The hydrophilicity of the inner wall surface may be determined by either the chemical composition or the surface roughness of the inner wall surface.

[0015] According to this aspect, the sample collection section can be realized with a simple structure by forming it from a part of the insulating layer, and since the inner wall surface of the sample collection section is hydrophilic, the test sample can be reliably sucked into the sample collection section.

[0016] In the gene detection tool of the above embodiment, the electrode section may include a working electrode and a reference electrode, or a working electrode, a reference electrode and a counter electrode, and the electrodes may include a metal layer formed on the insulating substrate, a carbon layer formed on the metal layer, and an upper adhesive layer formed between the upper surface of the metal layer and the carbon layer, and the upper adhesive layer may be made of silicon.

[0017] According to this aspect, each electrode has a metal layer, which reduces electrical resistance and improves measurement sensitivity. Furthermore, by covering the metal layer with a carbon layer, oxidation-reduction of the metal layer can be prevented, improving measurement sensitivity and reproducibility. Furthermore, by providing an upper adhesive layer made of silicon between the upper surface of the metal layer and the carbon layer, adhesion between the metal layer and the carbon layer is improved. Furthermore, silicon has a higher electrical resistivity than metal, which suppresses hydrogen generation on the upper surface of the metal layer during measurement and prevents peeling between the metal layer and the carbon layer, improving measurement sensitivity and reproducibility.

[0018] A gene detection kit according to one embodiment of the present invention includes the gene detection tool according to the above embodiment and the gene synthesis reaction solution, wherein the gene synthesis reaction solution includes at least a primer capable of synthesizing a gene using the target gene as a template.

[0019] According to the gene detection kit of the above embodiment, the gene detection tool of the above embodiment allows for quick and simple collection and addition of a test sample, facilitating gene detection operations in medical institutions, testing institutions, etc. Furthermore, patients themselves can easily and reliably collect and add their own specimen samples (e.g., saliva samples or blood samples) at home, etc., thereby improving versatility and convenience. [Effects of the Invention]

[0020] The present invention can provide a gene detection tool and a gene detection kit that enable quick collection and addition of test samples with simple operations and that can reduce the amount of waste. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a schematic longitudinal sectional view showing one embodiment of a gene detection tool. [Figure 2] FIG. [Figure 3] 1A and 1B are schematic diagrams showing an enlarged view of the tip of the electrode part of the same embodiment, in which (A) is a plan view showing the tip without the insulating layer, (B) is a cross-sectional view corresponding to the BB position in (A), and (C) is a cross-sectional view corresponding to the CC position in (A). [Figure 4] 10A and 10B are enlarged schematic views showing the tip of an electrode part of another embodiment, in which (A) is a plan view showing the tip without the insulating layer, and (B) is a cross-sectional view corresponding to the DD position of (A). [Figure 5] 10(A) and 10(B) are schematic cross-sectional views showing the tip of an electrode part of still another embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0022] An embodiment of the gene detection tool of the present invention will be described with reference to the drawings. Fig. 1 is a schematic longitudinal sectional view showing the embodiment. Fig. 2 is an exploded side view showing the embodiment. Fig. 3 is a schematic enlarged view of the tip of the electrode part of the embodiment, where (A) is a plan view, (B) is a sectional view corresponding to the position BB in (A), and (C) is a sectional view corresponding to the position CC in (A).

[0023] The gene detection tool 2 comprises a container body 21 for containing a gene synthesis reaction solution 10, a container lid 22 for sealing the container body 21, and an electrode part 23 and a sample collection part 24 that are provided on the container lid 22 and inserted into the container body 21. The sample collection part 24 is configured to be able to suck up a test sample that may contain a target gene to be detected.

[0024] The container body 21 has the form of a bottomed cylindrical member with one closed end and the other open end, and is what is commonly called a tube. The container lid 22 is configured to be attachable to the container body 21 so as to seal the opening 211 of the container body 21. In this embodiment, the container lid 22 has a cylindrical lid portion 221 that is fitted into the opening 211 of the container body 21, a lid top surface portion 222 that closes one end side of the lid cylindrical portion 221, and a substantially circular lid flange portion 223 that protrudes outward from the upper outer peripheral surface of the lid cylindrical portion 221.

[0025] The electrode unit 23 is fixed to the container lid 22 so as to penetrate the lid top surface 222 of the container lid 22. As also shown in Fig. 3, the electrode unit 23 includes a plurality of electrodes 232, 233, and 234 provided on an insulating base material 231, an insulating layer 235 covering the electrodes 232, 233, and 234, and a sensor unit 236 in which portions of the electrodes 232, 233, and 234 are exposed.

[0026] In this embodiment, the insulating base material 231 has a substantially rectangular flat plate shape in a plan view. A working electrode 232, a counter electrode 233, and a reference electrode 234 are provided and insulated from one another on the insulating base material 231. The electrodes 232, 233, and 234 are formed of, for example, thin gold films, and are provided from near one end of the insulating base material 231 to near the other end in the longitudinal direction.

[0027] An insulating layer 235 that insulates the working electrode 232, the counter electrode 233, and the reference electrode 234 from one another is formed on the insulating base material 231. The insulating layer 235 has an adhesive layer (not shown) on the surface facing the insulating base material 231. The insulating layer 235 is attached to the insulating base material 231 so as to cover the electrodes 232, 233, and 234, and is embedded between the electrodes 232, 233, and 234, while also covering the side surfaces of the electrodes 232, 233, and 234.

[0028] For example, the thickness of insulating base material 231 is about 0.35 mm, the thickness of electrodes 232, 233, and 234 is about 100 nm, and the thickness of insulating layer 235 is about 0.1 mm. Note that the thicknesses of insulating base material 231, electrodes 232, 233, and 234, and insulating layer 235 are shown schematically in Figures 3(B) and 3(C).

[0029] A sampling section 24 formed by a recess 235a in an insulating layer 235 that opens to the tip surface (outer surface) of the electrode section 23 is provided on one end side of the electrode section 23. One ends of the working electrode 232, counter electrode 233, and reference electrode 234 are exposed within the sampling section 24. That is, one ends of the working electrode 232, counter electrode 233, and reference electrode 234 are not covered with the insulating layer 235 and form a sensor section 236. In other words, the sampling section 24 is provided at the tip end of the electrode section 23 and on the sensor section 236. In the sensor section 236, a silver-silver chloride layer 237 is formed on the upper surface of one end side of the reference electrode 234.

[0030] At least a portion of the inner wall surface of the recess 235a of the insulating layer 235 that forms the sample collection section 24 is hydrophilic. In this embodiment, the inner wall surface of the top surface 241 of the sample collection section 24 is hydrophilic, making it easier to suck and collect a test sample that may contain a target gene. Note that the surface of the insulating base material 231 and the surfaces of the electrodes 232, 233, and 234 exposed within the sample collection section 24 may also be hydrophilic.

[0031] 1, the container body 21 can be sealed by fitting the tubular lid portion 221 of the container lid 22 into the opening 211 of the container body 21. When the container lid 22 is attached to the container body 21, the tip of the electrode portion 23 (the end portion where the sensor portion 236 and the sampling portion 24 are provided) is arranged near the bottom of the container body 21. This ensures that the sensor portion 236 and the sampling portion 24 are immersed in the gene synthesis reaction solution 10 contained in the container body 21.

[0032] The other end of electrode section 23 is provided to protrude upward from container lid 22. At the other end of electrode section 23, the other ends of electrodes 232, 233, 234 (ends opposite sensor section 236) are exposed, and a terminal section 238 is formed. A connector 8 connected to an electrochemical measurement device is detachably attached to terminal section 238.

[0033] The operation for carrying out gene detection using the gene detection tool 2 of the above embodiment will be described below. First, the gene synthesis reaction solution 10 is placed in the container body 21. The gene synthesis reaction solution 10 may be sealed in advance in the container body 21 using a cap or film (not shown) separate from the container lid 22.

[0034] The person conducting the test (for example, the patient himself / herself) picks up the container lid 22, brings the tip of the electrode section 23 into contact with a test sample that may contain the target gene, and aspirates and collects the test sample into the sample collection section 24. For example, the patient himself / herself brings the tip of the electrode section 23 into contact with saliva in his / her own oral cavity, and collects a saliva sample into the sample collection section 24 as the test sample.

[0035] Next, the test implementer attaches the container lid 22 held in his / her hand to the container body 21 to seal the container body 21, and immerses the tip of the electrode part 23 in the gene synthesis reaction liquid 10. The test sample in the sample collection part 24 diffuses into the gene synthesis reaction liquid 10. The gene synthesis reaction liquid 10 also enters the sample collection part 24, and the gene synthesis reaction liquid 10 comes into contact with the sensor part 236. In this way, the test implementer can quickly and easily collect and add the test sample using the container lid 22 having the sample collection part 24.

[0036] Next, the entire gene detection tool 2, or at least the container body 21, is placed in an appropriate incubator (for example, a PCR device), and the terminal 238 of the electrode part 23 is connected to a connector 8 that is connected to an electrochemical measurement device.

[0037] The incubator is operated to amplify the target gene in the test sample, and the amplification of the target gene is detected intermittently or continuously by the electrochemical measurement device. Generally, when a gene amplification process is required before detecting the target gene, the detection process of the target gene is performed after operating the incubator until the time expected to be required for amplifying the target gene (preset amplification time) has elapsed.

[0038] In contrast, when the gene detection tool 2 is used, the target gene can be electrochemically detected while being amplified. This allows the test to be terminated when the target gene is electrochemically detected, even before the predetermined amplification time has elapsed, thereby shortening the test time. Note that if the target gene is not detected even after the predetermined amplification time has elapsed since the start of the gene amplification process, it can be determined that the test sample does not contain the target gene.

[0039] In the genetic detection tool of the present invention, the shape of the container body is not limited to the cylindrical shape shown in Figures 1 and 2, and may be any other shape, such as a prismatic shape. Furthermore, the genetic detection tool of the present invention is not limited to a monochannel type having only one combination of a container body and a container lid, but may be a multichannel type having two or more combinations of a container body and a container lid. In the case of a multichannel type, testing can be performed under the same conditions.

[0040] Furthermore, the dimensions of the gene detection tool of the present invention are not particularly limited, but for example, in the case of a substantially cylindrical gene detection tool 2, the total length of the gene detection tool 2 (the total length from the bottom end of the container body 21 to the end on the terminal portion 238 side of the electrode portion 23) can be approximately 20 to 60 mm, and the total length of the container body 21 can be approximately 10 to 50 mm. The inner diameter of the container body 21 can be, for example, approximately 3 to 10 mm. However, the gene detection tool of the present invention can also be smaller or larger than the above dimensions.

[0041] The material of the gene detection tool of the present invention is not particularly limited, and it can be molded from, for example, a plastic material (e.g., polyolefin, particularly polyethylene, polyester, particularly polyethylene terephthalate, or polyacrylate, particularly polymethyl methacrylate). It is preferable to manufacture the gene detection tool from a transparent material so that the reaction state in the reaction chamber and the test state in the detection chamber can be observed from the outside.

[0042] Furthermore, the material of insulating substrate 231 of electrode portion 23 is not particularly limited, and examples thereof include polyimide (PI), glass, polyethylene terephthalate (PET), methacrylic resin (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), polyoxymethylene (POM), ABS resin (ABS), etc. However, the material of insulating substrate 231 is not limited to these, and may be ceramics, quartz, etc. Furthermore, the shape, thickness, and size of insulating substrate 231 are not particularly limited.

[0043] The target gene to be detected to which the present invention can be applied is not particularly limited, as long as it is a gene for which a primer for a gene synthesis reaction can be designed and for which a gene synthesis reaction can be carried out using the primer. For example, it can be a naturally occurring gene (e.g., a gene derived from an animal, plant, microorganism, or virus), or an artificially produced gene (e.g., a chemically synthesized gene or a gene produced by genetic engineering). In this specification, "gene" includes both DNA and RNA.

[0044] Furthermore, the test sample to which the present invention can be applied is not particularly limited as long as it has the potential to contain the target gene, and examples thereof include biological samples (e.g., animal (including human) body fluids (e.g., saliva, blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, or sputum) or excrement (e.g., feces), organs, tissues, or animals and plants themselves or dried forms thereof) or environmental samples (e.g., river water, lake water, seawater, or soil). Because the present invention allows testing operations to be performed in a sealed state, it is particularly suitable for application to dangerous test samples or test samples that may be hazardous (e.g., test samples from virally infected patients).

[0045] The gene synthesis reaction solution 10 may also contain an electrochemically active substance capable of specifically binding to genes. The electrochemically active substance specifically binds to genes and generates a detectable electrochemical signal. That is, the electrochemically active substance specifically binds to genes but not to substances other than genes, and exhibits oxidation or reduction activity in electrochemical measurement. When a specific voltage is applied to a sample solution containing genes bound to an electrochemically active substance, a signal proportional to the amount of gene-binding substance is obtained.

[0046] The electrochemically active substance is not particularly limited as long as it is a gene-binding substance that can specifically bind to genes and is electrochemically active, but is preferably, for example, an intercalating agent that can specifically bind to double-stranded DNA and is electrochemically active. Note that "specifically binds to double-stranded DNA" means that it does not bind to single-stranded DNA but binds to genes.

[0047] Examples of the electrochemically active substance include bisbenzimide derivatives, ferrocene derivatives, quinone derivatives, indophenol derivatives, acridine derivatives, flavin derivatives, viologen derivatives, ruthenium complexes, osmium complexes, cobalt complexes, platinum complexes, copper complexes, actinomycin D, dounomycin, or derivatives thereof.

[0048] In a test using the gene detection tool 2, a gene synthesis reaction is carried out using a test sample that may contain a target gene and a gene synthesis reaction solution that contains at least a primer capable of synthesizing a gene using the target gene as a template. This gene synthesis reaction is not particularly limited as long as it is a reaction that can synthesize a gene that binds to a gene-binding substance using the target gene in the test sample as a template, but examples include a gene amplification reaction, a replication reaction, a transcription reaction, and a reverse transcription reaction, and the gene amplification reaction is preferred.

[0049] Any known method can be used for the gene amplification reaction, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN), transcription-mediated amplification (TMA), strand displacement amplification (SDA), ligase chain reaction (LCR), or nucleic acid sequence-based amplification (NASBA).

[0050] The gene synthesis reaction itself can be carried out in exactly the same way as a normal gene synthesis reaction. For example, when PCR is carried out as the gene synthesis reaction, it can be carried out in the same way as a normal PCR.

[0051] When the gene to be detected is DNA, PCR can be performed, for example, by using a heat-stable DNA polymerase (e.g., Taq polymerase) to perform an initial denaturation reaction (e.g., at 97°C for 2 to 3 minutes), followed by repeating (e.g., 15 to 45 times) an amplification cycle consisting of (1) a DNA denaturation step (90 to 94°C for 30 seconds), (2) an annealing step between single-stranded DNA and a primer (50 to 55°C for 30 seconds), and (3) a DNA synthesis step using the heat-stable DNA polymerase (70 to 75°C for 1 to 2 minutes).

[0052] When the gene to be detected is RNA, reverse transcription PCR (RT-PCR) can be used, for example. That is, after reverse transcription using a reverse transcriptase and an oligo(dT) primer, an initial denaturation reaction and subsequent amplification cycles can be repeated using a thermostable DNA polymerase (e.g., Taq polymerase) in the same manner as in the case of DNA.

[0053] In a test using the gene detection tool 2, for example, by electrochemically detecting the target gene after the completion of the synthesis step (3) and before the denaturation step (1), the test can be terminated with a positive result when the target gene is detected even before the completion of a specified number of amplification cycles. An electrochemical measurement device connected to the terminal portion 238 of the electrode portion 23 of the gene detection tool 2 can measure the electrochemical response by measuring the current value generated when a potential is applied to the solution in the container body 21 (for example, the gene synthesis reaction solution 10 to which the test sample has been added).

[0054] Here, examples of methods for measuring electrochemical responses using an electrochemical measurement device include various electrochemical measurement methods, such as linear sweep voltammetry (LSV), couloamperometry (CA), coulochronometry (CC), and cyclic voltammetry (CV).

[0055] In detection utilizing the electrochemical response to gene synthesis reaction solution 10 containing a test sample and an electrochemically active substance capable of specifically binding to a gene, the presence or absence of a target gene in the test sample can be determined based on the following principle. That is, the electrochemically active substance can specifically bind to a gene and is electrochemically active. Therefore, when comparing the electrochemical response when the test sample is added to gene synthesis reaction solution 10 containing no gene or only a small amount of gene with the electrochemical response when the test sample is added to gene synthesis reaction solution 10 containing a large amount of gene, the gene-binding substance binds to the gene in the latter case, resulting in a decrease in the electrochemical response compared to the former, resulting in an electrochemically detectable difference.

[0056] Therefore, when a large amount of gene is synthesized by a gene synthesis reaction in the container body 21, the electrochemical response is reduced compared to the electrochemical response in a comparative test (for example, a test in which the gene synthesis reaction is not performed). In this case, it can be determined that the target gene is present in the test sample.

[0057] On the other hand, if the electrochemical response is not different from that in the comparative test when a large amount of gene is not present even after the gene synthesis reaction, it can be determined that the target gene is not present in the test sample.

[0058] For example, when PCR is performed as the gene synthesis reaction, an intercalating agent that can specifically bind to double-stranded DNA by inserting (i.e., intercalating) between adjacent base pairs in double-stranded DNA can be used as the gene-binding substance.

[0059] When DNA is amplified by PCR and a large amount of double-stranded DNA is present in the gene synthesis reaction solution 10 to which the test sample has been added, the electrochemical response is reduced compared to the electrochemical response in the comparative test. In this case, it can be determined that the target gene is present in the test sample. On the other hand, when DNA is not amplified by PCR and a large amount of double-stranded DNA is not present, there is no difference between the electrochemical response in the comparative test and the electrochemical response in the comparative test. In this case, it can be determined that the target gene is not present in the test sample.

[0060] In a test using the gene detection tool of the present invention, the presence or absence of the target gene may be determined by presetting a threshold value for the current value generated when a potential is applied. In this case, a comparative test is not required, which simplifies the test procedure and reduces the test cost.

[0061] The gene detection kit of the present invention includes a gene synthesis reaction solution in addition to the gene detection tool of the present invention described above. Here, the gene synthesis reaction solution may include an electrochemically active substance capable of specifically binding to a gene. In the gene detection kit of the present invention, the gene synthesis reaction solution may be stored in a container separate from the gene detection tool, or may be sealed in the container itself.

[0062] Next, a modified example of the electrode unit 23 will be described with reference to Fig. 4. In the embodiment shown in Fig. 4, each of the working electrode 232, the counter electrode 233, and the reference electrode 234 includes a metal layer 41 formed on an insulating substrate 231, a carbon layer 42 formed to cover the metal layer 41, a lower adhesive layer 43 formed between the insulating substrate 231 and the metal layer 41, and an upper adhesive layer 44 formed between the upper surface of the metal layer 41 and the carbon layer 42.

[0063] The lower adhesive layer 43 is a thin film made of, for example, silicon, that prevents peeling between the insulating base material 231 and the metal layer 41. The material for the lower adhesive layer 43 may be any material that has good adhesion to the insulating base material 231 and the metal layer 41, and in addition to silicon, for example, chromium, titanium, or tungsten can be used.

[0064] The lower adhesive layer 43 may be formed of a surface treatment layer obtained by performing a surface treatment on the surface of the insulating substrate 231 to improve adhesion to the metal layer 41. Examples of such surface treatments include plasma treatment, corona treatment, flame treatment, etching treatment, steam treatment, and ion beam treatment.

[0065] The metal layer 41 is made of a material having a lower electrical resistivity than the carbon layer 42, and is formed on the lower adhesive layer 43. The metal layer 41 is intended to reduce the electrical resistance between one end and the other end of each of the working electrode 232, the counter electrode 233, and the reference electrode 234 (between the sensor portion 236 and the terminal portion 238). Examples of materials that can be used for the metal layer 41 include silver, ruthenium, tantalum, titanium, copper, aluminum, platinum, niobium, zirconium, alloys of these elements, and alloys of these elements with carbon.

[0066] The upper adhesive layer 44 is formed on the upper surface of the metal layer 41, and is a thin film that prevents the upper surface of the metal layer 41 from peeling off from the carbon layer 42, and is made of silicon.

[0067] The carbon layer 42 is formed on the metal layer 41 via an upper adhesive layer 44. The carbon layer 42 is made of, for example, amorphous carbon or diamond-like carbon (DLC).

[0068] Carbon has the following properties, making it suitable for use in carbon layer 42 to protect metal layer 41: (1) excellent stability even in a vacuum at 3000°C (in air at 500°C), (2) resistance to chemical attack, (3) impermeability to gases and solutions, (4) excellent hardness and strength, (5) excellent electrical conductivity, (6) resistance to moisture from metal salts, etc., (7) good compatibility with blood and tissues, and (8) isotropy of physical and chemical properties.

[0069] The lower adhesive layer 43, the metal layer 41, the upper adhesive layer 44, and the carbon layer 42 are preferably manufactured by vapor deposition, since this allows for highly accurate control of the shape and film thickness of each layer. Vapor deposition methods that can be used here include physical vapor deposition (PVD) methods such as vacuum deposition, ion plating, and sputtering, as well as chemical vapor deposition (CVD). However, the manufacturing method for each layer is not limited to vapor deposition, and may also be a printing method such as screen printing or inkjet printing.

[0070] A second insulating layer 45 is formed on the insulating base material 231 so as to surround the contours of the lower adhesive layer 43, the metal layer 41, and the upper adhesive layer 44 in a plan view. The side surfaces of the metal layer 41 are covered with the second insulating layer 45. In this embodiment, the side surfaces of the lower adhesive layer 43, the side surfaces of the upper adhesive layer 44, and the side surfaces of the carbon layer 42 are also covered with the second insulating layer 45. The lower surface of the second insulating layer 45 is in contact with the insulating base material 231. The lower adhesive layer 43, the metal layer 41, and the upper adhesive layer 44 are surrounded by the insulating base material 231 and the second insulating layer 45, and are therefore isolated from the ambient atmosphere.

[0071] The material of second insulating layer 45 is not particularly limited, and examples thereof include silicon oxide film (SiO2), silicon nitride film (Si3N4), aluminum oxide (Al2O3), etc. However, second insulating layer 45 is not limited to being made of these materials, and may be any insulating material that can insulate the side surfaces of electrodes 232, 233, and 234 (at least the side surfaces of metal layer 41) from the ambient atmosphere and is impermeable to moisture.

[0072] The height position (thickness) of the upper surface of the second insulating layer 45 may be such that the second insulating layer 45 can cover at least the side surface of the metal layer 41. However, in order to increase the contact area between the second insulating layer 45 and the carbon layer 42, it is preferable that the height position of the upper surface of the second insulating layer 45 is approximately the same as the height position of the upper surface of the carbon layer 42. This can reliably prevent moisture from penetrating into the metal layer 41 from between the second insulating layer 45 and the carbon layer 42.

[0073] In this embodiment, the electrodes 232, 233, and 234 each include a metal layer 41 formed on an insulating substrate 231, a carbon layer 42 formed on the insulating substrate 231 to cover the metal layer 41, and a lower adhesive layer 43 formed between the insulating substrate 231 and the metal layer 41. The electrodes 232, 233, and 234 have a low electrical resistance due to the metal layer 41, thereby improving measurement sensitivity. Covering the upper surface of the metal layer 41 with the carbon layer 42 and the side surfaces of the metal layer 41 with the second insulating layer 45 prevents oxidation-reduction of the metal layer 41, thereby improving measurement sensitivity and reproducibility. Furthermore, providing an upper adhesive layer 44 made of silicon between the upper surface of the metal layer 41 and the carbon layer 42 improves adhesion between the metal layer 41 and the carbon layer 42. Furthermore, silicon has a higher electrical resistivity than metals, which suppresses hydrogen generation on the upper surface of the metal layer 41 during measurement. Furthermore, since the side surfaces of the metal layer 41 are covered with the second insulating layer 45, moisture does not reach the side surfaces, preventing hydrogen generation on the side surfaces of the metal layer 41. This prevents peeling between the insulating base material 231 and the metal layer 41, improving measurement sensitivity and reproducibility.

[0074] Furthermore, since the electrodes 232, 233, and 234 are provided with a lower adhesive layer 43 formed between the insulating substrate 231 and the metal layer 41, it is possible to prevent a decrease in adhesion between the insulating substrate 231 and the metal layer 41 during measurement, thereby improving measurement sensitivity and reproducibility.

[0075] The metal layer 41, the carbon layer 42, and the adhesive layers 43 and 44 are formed by vapor deposition, and are formed to have the same shape in a plan view. By forming the layers 41, 42, 43, and 44 by vapor deposition, the shape and film thickness of the layers 41, 42, 43, and 44 can be controlled with high precision, and the stability of the overall electrical resistance of each of the electrodes 232, 233, and 234 can be improved.

[0076] The lower adhesive layer 43 is made of silicon. Silicon has good adhesion to glass and to metals, so it can strengthen the adhesion between the metal layer 41 and the insulating base material 231. The upper adhesive layer 44 is also made of silicon. Silicon has good adhesion to metals and to carbon, so it can strengthen the adhesion between the metal layer 41 and the carbon layer 42.

[0077] The electrode unit 23 is equipped with the working electrode 232, the reference electrode 234, and the counter electrode 233, and is therefore applicable to three-electrode electrochemical measurements. The working electrode 232, the reference electrode 234, and the counter electrode 233 can have low electrical resistance, can prevent oxidation-reduction of the metal layer 41, and can prevent peeling of the metal layer 41, thereby improving measurement sensitivity and reproducibility.

[0078] The electrode unit 23 may be applicable to two-electrode electrochemical measurements using a working electrode and a reference electrode. If both the working electrode and the reference electrode are configured with electrodes having a metal layer, a carbon layer, and an adhesive layer, the electrical resistance of both the working electrode and the reference electrode can be reduced, oxidation-reduction of the metal layer can be prevented, and peeling of the metal layer can be prevented, thereby improving measurement sensitivity and reproducibility.

[0079] Next, a description will be given of a manufacturing example of the electrode part 23 shown in Fig. 5. On a glass substrate having a thickness of about 2500 nm (2.5 µm) as the insulating base material 231, a silicon layer having a thickness of about 20 nm was formed as the lower adhesive layer 43 by a sputtering method using a metal mask having an opening pattern corresponding to the lower adhesive layer formation region. Note that the film thickness of the lower adhesive layer 43 made of silicon is not particularly limited.

[0080] Using a metal mask having the same opening pattern as the opening pattern corresponding to the region of the metal mask where the lower adhesive layer is to be formed, a silver layer having a thickness of about 150 nm was formed as metal layer 41 on lower adhesive layer 43 by sputtering.

[0081] Thereafter, using a metal mask having the same opening pattern as the opening pattern corresponding to the lower adhesive layer formation region, a silicon layer having a thickness of about 20 nm was formed as upper adhesive layer 44 by sputtering on metal layer 41. The thickness of upper adhesive layer 44 made of silicon is not particularly limited.

[0082] Next, using a metal mask having the same opening pattern as the opening pattern corresponding to the lower adhesive layer formation region, a carbon layer 42 having a thickness of approximately 1000 nm was formed by sputtering on the upper adhesive layer 44. In this way, a working electrode 232, a counter electrode 233, and a reference electrode 234 each having a lower adhesive layer 43, a metal layer 41, an upper adhesive layer 44, and a carbon layer 42, respectively, were formed.

[0083] Here, after the insulating substrate 231 was carried into the chamber of a sputtering device, the lower adhesive layer 43, the metal layer 41, the upper adhesive layer 44, and the carbon layer 42 were formed on the insulating substrate 231 using the same metal mask without carrying it out of the chamber. This shortens the time required to form the lower adhesive layer 43, the metal layer 41, the upper adhesive layer 44, and the carbon layer 42, and also prevents foreign matter from adhering between the layers. Furthermore, the metal layer 41, the carbon layer 42, and the adhesive layers 43 and 44 are formed to have the same shape in a plan view.

[0084] The line width (the dimension in the width direction perpendicular to the longitudinal direction) of the electrodes 232, 233, and 234 is about 1.0 mm, and the interval between the electrodes 232, 233, and 234 is about 0.5 mm.

[0085] Using a sputtering method, a metal mask having an opening pattern that opens around the area where the lower adhesive layer is to be formed was used to form a second insulating layer 45 having a thickness of approximately 1200 nm on the insulating base material 231 so as to cover the side surfaces (side surfaces of the electrodes 232, 233, and 234) of the lower adhesive layer 43, the metal layer 41, the upper adhesive layer 44, and the carbon layer 42. The second insulating layer 45 is formed so as to surround the peripheries of the electrodes 232, 233, and 234 and to be embedded between the electrodes 232, 233, and 234.

[0086] In this way, by forming the lower adhesive layer 43, metal layer 41, upper adhesive layer 44, carbon layer 42, and second insulating layer 45 by vapor deposition (here, sputtering) using a metal mask with an opening pattern, patterning by etching or lift-off is not required after deposition of each layer, thereby reducing manufacturing costs.

[0087] A silver layer having a thickness of approximately 100 nm was formed by a film formation method on the upper surface of the carbon layer 42 on one end side of the reference electrode 234, and then chlorination treatment was performed to form a silver-silver chloride layer 237. In this manner, the electrode part 23 was produced. The silver-silver chloride layer 237 may be formed after the second insulating layer 45 is formed, or may be formed before the second insulating layer 45 is formed.

[0088] Finally, the insulating layer 235 is attached onto the insulating base material 231 so as to cover the electrodes 232, 233, 234 and the second insulating layer 45, thereby forming the sensor portion 236 and the sampling portion 24.

[0089] The thickness of the metal layer 41 is not particularly limited, but is preferably 50 nm or more and 1000 nm or less. If the thickness of the metal layer 41 is thinner than 50 nm, the electrodes 232, 233, and 234 will have high resistance, resulting in reduced measurement sensitivity. If the thickness of the metal layer 41 is thicker than 1000 nm, when the metal layer 41 is formed by a vapor deposition method (e.g., a sputtering method), it will take a long time to form the metal layer 41, resulting in reduced production efficiency.

[0090] It should be noted that the manufacturing cost can be reduced by providing regions for a plurality of electrode portions 23 on one insulating base material 231, forming the plurality of electrode portions 23 simultaneously, and then separating each electrode portion 23 into individual pieces.

[0091] 5(A), in the electrode unit 23, a surface treatment layer 46 formed by performing a surface treatment to improve adhesion on the surface of the insulating base material 231 may be formed as a lower adhesive layer, and the metal layer 41 and the second insulating layer 45 may be formed on the surface treatment layer 46. This improves adhesion between the insulating base material 231 and the metal layer 41 and second insulating layer 45, reliably prevents moisture from penetrating between the insulating base material 231 and the second insulating layer 45, and more reliably prevents hydrogen generation on the side surface of the metal layer 41 and peeling of the second insulating layer 45 during measurement.

[0092] 5(B), the insulating layer 235 may be formed of a lower insulating layer 235b cut out at a portion where the sample collecting section 24 will be formed, and an upper insulating layer 235c formed on the lower insulating layer 235b so as to cover the cutout portion of the lower insulating layer 235b. The lower surface of the upper insulating layer 235c that forms the top surface 241 of the sample collecting section 24 is hydrophilic, which ensures that the test sample can be sucked and collected into the sample collecting section 24. In this way, the insulating layer 235 may be formed of multiple layers.

[0093] The configuration of the insulating layer 235 shown in FIG. 5(B) is also applicable to the insulating layer 235 of the electrode section 23 shown in FIGS. 4 and 5(A).

[0094] The present invention is not limited to the above-described embodiment and can be embodied in various forms. For example, the attachment structure of the container lid 22 to the container body 21 may be a structure in which the container lid 22 fits onto the outer periphery of the container body 21, or a screw-type connection structure.

[0095] Also, As a reference example of the present invention, The sample collection section 24 may be provided at a position on the electrode section 23 that is different from the sensor section 236, or may be provided on the container lid 22 at a position different from the electrode section 23.

[0096] Furthermore, the electrode section 23 may have a configuration that includes the working electrode 232 and the reference electrode 234 as electrodes, but does not include the counter electrode 233, and is applicable to two-electrode electrochemical measurements. [Explanation of symbols]

[0097] 2 Gene detection tool, 10 Gene synthesis reaction solution, 21 Container body, 22 Container lid, 23 Electrode part, 24 Sample collection part, 41 Metal layer, 42 Carbon layer, 44 Upper adhesive layer, 231 Insulating base material, 232 Working electrode, 233 Counter electrode, 234 Reference electrode, 235 Insulating layer, 235a Recess, 236 Sensor part

Claims

1. a container body for containing a gene synthesis reaction solution containing at least a primer capable of synthesizing a gene using the target gene as a template; a container lid that seals the container body; an electrode portion and a sample collecting portion provided on the container lid and inserted into the container body, the sample collection unit is configured to be able to aspirate and collect a test sample that may contain the target gene, and add the sample to the gene synthesis reaction solution in the container body; the electrode unit includes a plurality of electrodes provided on an insulating substrate, an insulating layer covering the electrodes, and a sensor unit in which portions of the plurality of electrodes are exposed; The sample collection unit is provided on the sensor unit. Gene detection tools.

2. The sample collection unit is provided at the tip of the electrode unit. The gene detection tool according to claim 1 .

3. the sample collection section is formed by a recess in the insulating layer that opens to the outer surface of the electrode section, and at least a part of the inner wall surface of the recess is hydrophilic. The gene detection tool according to claim 1 or 2.

4. the electrode unit includes, as the electrodes, a working electrode and a reference electrode, or a working electrode, a reference electrode, and a counter electrode; the electrode comprises a metal layer formed on the insulating substrate, a carbon layer formed on the metal layer, and an upper adhesion layer formed between an upper surface of the metal layer and the carbon layer; The upper adhesive layer is formed of silicon. The gene detection tool according to any one of claims 1 to 3.

5. A gene detection kit comprising the gene detection tool according to any one of claims 1 to 4 and the gene synthesis reaction solution.

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