WO2023157711A1 - Inspection chip, reaction detection device, and reaction detection method - Google Patents

Inspection chip, reaction detection device, and reaction detection method Download PDF

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Publication number
WO2023157711A1
WO2023157711A1 PCT/JP2023/003927 JP2023003927W WO2023157711A1 WO 2023157711 A1 WO2023157711 A1 WO 2023157711A1 JP 2023003927 W JP2023003927 W JP 2023003927W WO 2023157711 A1 WO2023157711 A1 WO 2023157711A1
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chamber
group
chambers
reaction
reaction detection
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PCT/JP2023/003927
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French (fr)
Japanese (ja)
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清一郎 東
宏明 花房
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国立大学法人広島大学
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Publication of WO2023157711A1 publication Critical patent/WO2023157711A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems

Definitions

  • the present invention relates to a test chip, a reaction detection device, and a reaction detection method, and more particularly to a test chip, a reaction detection device, and a reaction detection method for detecting reactions related to nucleic acid amplification.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • a detection method using a fluorescent substance is used to detect nucleic acid amplification products.
  • detection methods using fluorescent substances have problems such as the need to hybridize a fluorescent probe and the detection process is complicated, and the fluorescence detector is expensive.
  • Other detection methods have been developed (eg, US Pat.
  • Patent Document 1 proton release is detected by an ion selective field effect transistor (ISFET) during the primer elongation step of nucleic acid amplification such as the PCR method.
  • ISFET ion selective field effect transistor
  • in order to detect changes in the pH value due to released protons it is necessary to inject a solution containing a nucleic acid as a specimen into a microvolume chamber and measure the pH value for each chamber. .
  • the present invention has been made in view of the circumstances described above, and aims to provide a test chip, a reaction detection device, and a reaction detection method that can easily and quickly detect a nucleic acid amplification reaction.
  • the test chip includes: a plurality of chambers having ion-sensitive field effect transistors and reference electrodes for detecting the pH value of the analyte;
  • the chamber is The sources and drains of the ion-sensitive field effect transistors are grouped by a first group consisting of a plurality of first groups connected to each other, and the reference electrodes are grouped by a first group consisting of a plurality of second groups connected to each other. grouped by groups, The chambers of each of the first groups included in each of the second groups are wired so that there is no more than one.
  • the reference electrode is arranged on a lid covering the opening of the chamber, You can do it.
  • the inspection chip comprising a channel that communicates the adjacent chambers with each other; You can do it.
  • the volume of the chamber is 10 pL or less, You can do it.
  • the reaction detection device includes: A test chip according to the first aspect; a control unit that is connected to the test chip and detects a reaction of the specimen in the chamber; The control unit selecting one said chamber by selecting one said first group from said first group and selecting one said second group from said second group; By measuring the pH value with the ion sensitive field effect transistor, the reaction of the analyte in the selected chamber is detected.
  • reaction detection method Injecting a specimen into the chamber of the test chip according to the first aspect, selecting one said chamber by selecting one said first group from said first group and selecting one said second group from said second group; By measuring the pH value with the ion sensitive field effect transistor, the reaction of the analyte in the selected chamber is detected.
  • the selection of chambers and the presence or absence of reactions accompanied by changes in pH value occurring in each chamber can be electrically performed using ion-sensitive field effect transistors. Therefore, it is possible to detect the nucleic acid amplification reaction easily and quickly.
  • FIG. 1 is a perspective view showing the configuration of a reaction detection device according to an embodiment of the present invention
  • FIG. It is a side view of a test chip concerning an embodiment. It is a figure which shows the structure of the chamber which concerns on embodiment, (A) is a top view, (B) is side sectional drawing. (A) shows an array of chambers in a chamber layer, and (B) is an enlarged view of (A) showing one chamber.
  • FIG. 10 is a side cross-sectional view of a chamber with a lid having a reference electrode; It is a conceptual diagram which shows the wiring example of ISFET which concerns on embodiment, and a reference electrode. It is a functional block diagram showing the configuration of a control unit according to the embodiment.
  • FIG. 6 is a flowchart showing the flow of reaction detection processing according to the embodiment; 4 is a graph showing an example of the relationship between the amount of specimen and the pH value; It is a conceptual diagram which shows the relationship between selection of a chamber and the output of ISFET.
  • FIG. 10 is a diagram showing an example of annularly arranged chambers;
  • FIG. 4 is a schematic diagram showing chamber layers in a flow-down configuration;
  • FIG. 4 is a schematic diagram showing the chamber layers of a multi-layer flow-down structure;
  • test chip the reaction detection device, and the reaction detection method according to the embodiments of the present invention will be described as a reaction detection device that detects an amplification product contained in a sample by detecting a proton release reaction in nucleic acid amplification by the RCA method.
  • a reaction detection device that detects an amplification product contained in a sample by detecting a proton release reaction in nucleic acid amplification by the RCA method.
  • the reaction detection apparatus 1 includes a test chip 10 having a plurality of chambers 12 into which specimens are injected, and a detection unit 50 connected to the test chip 10 to detect a target substance. Prepare.
  • the inspection chip 10 includes a substrate 15 having a sensor and a chamber layer 11 in which a microcapacity chamber 12 is formed.
  • ISFETs 16 which are a plurality of ion-sensitive field effect transistors (Ion Selective Field Effect Transistors) as sensors.
  • a wiring 17 for operating the ISFET 16 is formed.
  • the substrate 15 includes connection terminals 15a for connecting the wiring 17 and the circuits in the detection unit 50.
  • the material of the substrate 15 is not particularly limited, and a semiconductor such as silicon, a glass substrate, a plastic substrate, or the like can be used.
  • the substrate 15 according to this embodiment is a plastic substrate.
  • the ISFET 16 is formed by a known method.
  • An insulating layer 18 is formed on the ISFET 16 .
  • a slit 18a is formed in the insulating layer 18, and the gate portion of the ISFET 16 is opened.
  • the size of the slit 18 a is not particularly limited, and may be set to a size corresponding to the size of the gate portion of the ISFET 16 .
  • the slit 18a has a rectangular shape with a side of 0.5 ⁇ m to 1000 ⁇ m (micrometers).
  • the chambers 12 are containers into which specimens are injected, and are formed on the substrate 15 so as to contain one ISFET 16 each.
  • a reference electrode 19 is formed on the substrate 15 , and the reference electrode 19 is opened inside each chamber 12 so that a voltage can be applied.
  • the chamber layer 11 including the chamber 12 is made of epoxy resin on the substrate 15 using photolithography.
  • the method of making the chamber layer 11 is not limited to photolithography.
  • chamber layer 11 may be molded onto substrate 15 .
  • the chamber layer 11 may be created by a three-dimensional printer such as stereolithography using an ultraviolet curable resin.
  • the chamber layer 11 including the chamber 12 is formed on the substrate 15, the present invention is not limited to this. It is also possible to
  • the chamber 12 is formed as a microcapacity container in order to detect a minute amount of protons released during nucleic acid amplification by the RCA method as a change in pH value.
  • the volume of chamber 12 is preferably 10 pL or less, more preferably 1 pL or less.
  • the shape of the chamber 12 is not particularly limited. Chamber 12 according to the present embodiment is formed in a cylindrical shape.
  • the inner diameter of chamber 12 is preferably between 1 ⁇ m and 1000 ⁇ m in diameter.
  • the height of the chamber 12 is preferably 1 ⁇ m to 1000 ⁇ m.
  • the chamber 12 according to this embodiment has a diameter of 20 ⁇ m, a height of 16 ⁇ m, and a capacity of about 5 pL (picoliters).
  • FIG. 4(A) is an enlarged view of the surface of the inspection chip 10
  • FIG. 4(B) is an enlarged view of one chamber 12 in FIG. 4(A).
  • the chambers 12 are arranged in a matrix, more specifically, in an alternating matrix.
  • the sample is distributed in a fixed amount to each chamber 12 .
  • the chamber layer 11 according to the present embodiment has a channel 13 , and the adjacent chambers 12 communicate with each other through the channel 13 . Thereby, the specimen introduced into the chamber 12 is diffused to other chambers 12 through the channel 13 .
  • a coating agent may be applied to the inner walls of the chamber 12 and the channel 13 to improve the fluidity of the specimen.
  • the coating agent is not particularly limited, and for example, a vitreous coating agent such as polysilazane, a hydrophobic coating agent, a water-repellent coating agent, and the like can be used.
  • the distance between the adjacent chambers 12 is not particularly limited, it is preferable that the channels 13 are arranged so that the length of the channels 13 is 1000 ⁇ m or less from the viewpoint of the fluidity of the sample and the rapidity of the detection process. In the present embodiment, since the chambers 12 are arranged in an alternate matrix, the distance between the chambers 12 can be shortened and the fluidity of the sample can be improved.
  • the reference electrode 19 is formed on the substrate 15 in the present embodiment, it is not limited to this, and may be formed so that a voltage can be applied to each chamber 12 .
  • a reference electrode 19 arranged on a lid 20 that covers the openings of the chambers 12 may be configured to be inserted into each chamber 12 .
  • the wiring of the reference electrode 19 and the wiring of the ISFET 16 can be divided between the lid portion 20 and the substrate 15, so that the wiring of the substrate 15 is facilitated and the structure can be simplified.
  • Each chamber is grouped into a first group group A and a second group group B, as shown in the conceptual diagram of FIG. Specifically, the sources and drains of the ISFETs 16 in each chamber 12 are connected for each first group Ai (i is a natural number from 1 to n) to form a first group A.
  • the sources and drains of the ISFETs 16 of the first group Ai are connected to the power supply in the detection unit 50, and voltages can be selectively applied to the ISFETs 16 of the first group Ai by the controller 51, which will be described later.
  • the chambers 12 arranged in a matrix form a first group Ai for each column in one of the directions (horizontal direction in FIG. 6) in which the chambers 12 are arranged in series. Thereby, a voltage can be simultaneously applied to the ISFETs 16 in the same first group Ai.
  • each chamber 12 is connected to each second group Bj (j is a natural number from 1 to m) to form a second group B.
  • the reference electrodes 19 of the second group Bj are connected to the power supply in the detection unit 50, and a voltage can be selectively applied to the reference electrodes 19 of the second group Bj by the control section 51, which will be described later.
  • the chambers 12 arranged in a matrix form a second group Bj for each row in a direction orthogonal to the first group Ai (vertical direction in FIG. 6). Thereby, the voltage can be applied simultaneously to the reference electrodes 19 in the same second group Bj.
  • each second group Bj is wired so as to include one or less chambers 12 of each first group Ai.
  • each first group Ai is wired so as to include one or less chambers 12 of each second group Bj. Accordingly, by selecting either one of the first group Ai and one of the second group Bj, it is possible to select one or less chambers 12 and measure the pH value without selecting a plurality of chambers 12 at the same time. can be done. That is, in the present embodiment, the ISFET 16 is wired in an active matrix system, the chambers 12 are sequentially selected, and the pH value is measured to determine whether or not the amplified product of the nucleic acid to be detected is present in the chambers 12. can judge.
  • the detection unit 50 is connected to the test chip 10 and detects the nucleic acid amplification reaction by measuring the pH value inside each chamber 12 .
  • the detection unit 50 includes a control section 51, a storage section 52, a display section 53, an input section 54, and a reader 55, as shown in the functional block diagram of FIG.
  • the control unit 51 is composed of a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), etc., and controls the operation of the reaction detection device 1 as a whole. In addition, the control unit 51 sequentially selects the first group Ai and the second group Bj for the chambers 12 formed in the test chip 10, thereby measuring the pH value of the sample in each chamber 12 and amplifying the nucleic acid. Detects the presence or absence of reaction.
  • a CPU Central Processing Unit
  • ROM Read Only Memory
  • RAM Random Access Memory
  • the control unit 51 reads various operation programs and data stored in the ROM of the control unit 51, the storage unit 52, etc. into the RAM and operates the CPU, thereby realizing each function of the control unit 51 shown in FIG. Let Thereby, the control unit 51 operates as a selection unit 511 that selects the chamber 12 to be measured and a measurement unit 512 that measures the pH value of the selected chamber 12 .
  • the selection unit 511 sequentially selects the first group Ai and the second group Bj according to a predetermined order. Accordingly, the chamber 12 to be measured is selected from among the chambers 12 formed in the chamber layer 11 .
  • the measurement unit 512 applies voltage to the ISFET 16 and the reference electrode 19 of the chamber 12 selected by the selection unit 511 to measure the pH value of the sample in the chamber 12 .
  • the storage unit 52 is a non-volatile memory such as a hard disk or flash memory, and stores programs for selecting the chambers 12, pH values measured for each chamber 12, and the like.
  • the display unit 53 is a display device provided in the detection unit 50, such as a liquid crystal monitor.
  • the display unit 53 displays information such as the measured pH value and the number of chambers 12 in which the amplified product of the nucleic acid to be detected has been confirmed.
  • the input unit 54 is an input device for inputting the start and end of reaction detection processing, various set values of the reaction detection device 1, and the like.
  • the input unit 54 is a keyboard, touch panel, mouse, etc. provided in the detection unit 50 .
  • the reader 55 connects the connection terminal 15a provided on the inspection chip 10 to the power supply circuit, switch circuit, etc. in the detection unit 50 via contacts. Thereby, the controller 51 applies a voltage to the ISFET 16 and the reference electrode 19 associated with the selected chamber 12 and measures the pH value of the sample in the chamber 12 .
  • a specimen to be detected by the reaction detection device 1 is prepared. Specifically, a nucleic acid amplification process such as the RCA method or the PCR method is performed to generate a sample that causes a primer extension reaction. In the present embodiment, a solution containing an amplification product obtained by amplifying DNA of a given virus by the RCA method is used as a specimen. Then, the sample is injected into the chamber 12 of the inspection chip 10 to prepare for inspection (step S11). At this time, the specimen solution injected into some of the chambers 12 of the chamber layer 11 is diffused to all the chambers 12 of the chamber layer 11 via the channel 13 .
  • a nucleic acid amplification process such as the RCA method or the PCR method is performed to generate a sample that causes a primer extension reaction.
  • a solution containing an amplification product obtained by amplifying DNA of a given virus by the RCA method is used as a specimen.
  • the sample is injected into the chamber 12 of the inspection chip 10 to prepare for inspection (step
  • the test chip 10 into which the sample is injected is connected to the detection unit 50 via the reader 55, and reaction detection is started.
  • reaction detection is started, the selector 511 of the controller 51 scans the chamber 12 to detect the nucleic acid amplification reaction. Specifically, the selector 511 selects one second group Bj from the second group B, and applies voltage to the reference electrodes 19 of the chambers 12 belonging to the second group Bj (step S12).
  • the selection unit 511 selects one first group Ai from the first group group A (step S13).
  • the measurement unit 512 measures the drain current of the first group Ai. Thereby, the measuring unit 512 measures the pH value of one chamber 12 belonging to the first group Ai and the second group Bj (step S14).
  • the release amount and release rate of protons that change the pH value differ depending on the method used for nucleic acid amplification. For example, when viruses are detected using the Rha-RCA (RNase H-assisted RCA) method, about 1 ⁇ 10 4 protons are generated in 5 minutes by performing nucleic acid amplification for one virus.
  • the volume of the chamber 12 is preferably 10 pL or less. , is more preferably 1 pL or less. As described above, the volume of chamber 12 according to this embodiment is approximately 5 pL.
  • the selection unit 511 sequentially switches the first group Ai until all the first groups Ai from the first group A have been selected (NO in step S15), and the measurement unit 512 detects the drain current from the first group Ai. Measure the pH value. Thereby, the measuring unit 512 can sequentially measure the pH values of the chambers 12 belonging to the selected second group Bj.
  • the selection unit 511 selects another second group Bj from the second group B, and selects the newly selected second group Bj. A voltage is applied to the reference electrodes 19 of the chambers 12 belonging to group Bj.
  • the selection unit 511 and the measurement unit 512 select all the second groups Bj of the second group B, and repeat the selection and measurement of the second groups Bj until the measurement is completed (NO in step S16).
  • FIG. 10 is a conceptual diagram showing the flow of nucleic acid amplification reaction detection within the chamber 12.
  • the voltage is applied to the reference electrodes 19 of the second group B2 selected by the selector 511.
  • drain current values measured by the measuring unit 512 when the first group Ai of the first group group A is sequentially selected.
  • the chambers 12 belonging to the first group A3 and the second group B2 contain amplification products of nucleic acids to be detected.
  • the pH value in the chamber 12 changes due to protons released during the primer elongation step in the RCA method. Therefore, as shown in FIG.
  • FIG. 10 indicates that the chamber 12 contains the amplification product of the nucleic acid to be detected. In this way, by sequentially scanning a plurality of chambers 12 arranged in an active matrix system, the nucleic acid amplification reaction can be rapidly detected.
  • the first group A consisting of a plurality of first groups Ai that share the wiring of the source and drain of the ISFET 16, and the reference
  • the first group Ai and the second group Bj are sequentially selected for the small-capacity chambers 12 specified by the second group B consisting of a plurality of second groups Bj sharing the wiring of the electrodes 19, and the selection is performed.
  • the pH value of the chamber 12 is measured.
  • the selection of the chamber 12 can be electrically performed by the switch circuit, so that the reaction can be detected at high speed.
  • the detection since the presence or absence of reaction can be electrically detected by measuring the pH value using the ISFET 16, the detection can be performed inexpensively and easily compared to the detection of amplification products using a fluorescence detector.
  • each chamber since the volume of each chamber is as small as 10 pL or less, it is possible to accurately change the pH value based on proton release by the primer extension reaction.
  • the chambers 12 according to the present embodiment are communicated with each other by the flow paths 13, it is possible to rapidly diffuse the specimen to each chamber 12, thereby improving the detection efficiency.
  • the chambers 12 are arranged in a matrix, and the ISFETs 16 and the reference electrodes 19 are wired in an active matrix system, but this is not the only option.
  • the chambers 12 may be arranged in an annular shape consisting of a plurality of concentric circles.
  • a group of chambers 12 arranged on circles of the same diameter is defined as a first group Ai, and the sources and drains are connected to form a first group A.
  • a second group B may be formed by connecting the reference electrodes 19 to the group of the chambers 12 arranged in the radial direction of the annular arrangement as the second group Bj.
  • the sample injected into the chamber 12 is homogenized through the channel 13 in the present embodiment, it is not limited to this.
  • the sample solution may be flowed down and injected into each chamber 12 .
  • a sloped portion 12a that slopes downward from the upper edge of the chamber 12 may be formed.
  • the chamber layer 11 is one layer in the present embodiment, the present invention is not limited to this, and a plurality of chamber layers 11 may be laminated.
  • the chambers 12 of each layer are provided with the above-described inclined portions 12a so that the specimen overflowing from the chambers 12 of the upper chamber layer 11 sequentially flows into the chambers 12 of the lower chamber layer 11. can be configured to This makes it possible to easily equalize the amount of specimen injected into the chamber 12 of each layer.
  • the detection unit 50 including the reader 55 is connected to the inspection chip 10, but this is not the only option.
  • a program for operating as a control unit 51, a storage unit 52, a display unit 53, and an input unit 54 is installed in a mobile communication terminal such as a smartphone, and the detection unit 50 is detected by connecting the mobile communication terminal and the reader 55. It may be operated as As a result, reaction detection can be easily performed using a general smartphone or the like.
  • detection data can be transmitted to the server via a network, so that a large amount of detection data can be quickly collected and analyzed. Become.
  • the present invention is suitable for reaction detection that detects reactions related to nucleic acid amplification.
  • it is suitable for reaction detection when rapid and inexpensive detection of amplification products is required, such as detection of viruses associated with infectious diseases.
  • reaction detection device 10 inspection chip, 11 chamber layer, 12 chamber, 12a inclined portion, 13 flow path, 15 substrate, 15a connection terminal, 16 ISFET, 17 wiring, 18 insulating layer, 18a slit, 19 reference electrode, 20 lid section, 50 detection unit, 51 control section, 511 selection section, 512 measurement section, 52 storage section, 53 display section, 54 input section, 55 reader

Abstract

An inspection chip (10) comprises a plurality of chambers (12) each having a reference electrode (19) and an ISFET (16) for detecting the pH value of a specimen, the chambers (12) being grouped into a first group collection A comprising a plurality of first groups Ai in which the source and drain of the ISFET (16) are connected to each other, and a second group collection B comprising a plurality of second groups Bj in which the reference electrodes (19) are connected to each other. The inspection chip is also wired so that there is no more than one chamber (12) in each first group Ai included in each second group Bj. Through this configuration, selection of a chamber (12) and the presence/absence of a reaction accompanying a change in pH value occurring in each chamber (12) can be easily and rapidly detected using the ISFET (16).

Description

検査チップ、反応検出装置及び反応検出方法Inspection chip, reaction detection device and reaction detection method
　本発明は、検査チップ、反応検出装置及び反応検出方法に関し、より詳細には核酸の増幅に係る反応を検出する検査チップ、反応検出装置及び反応検出方法に関する。 The present invention relates to a test chip, a reaction detection device, and a reaction detection method, and more particularly to a test chip, a reaction detection device, and a reaction detection method for detecting reactions related to nucleic acid amplification.
　従来、病原性細菌、ウイルス等を検出する方法として、ポリメラーゼ連鎖反応（ＰＣＲ：Polymerase Chain Reaction）法、ローリングサークル増幅（ＲＣＡ：Rolling Circle Amplification）法等による核酸増幅を用いる方法が開発されている。一般的に、核酸の増幅産物の検出には、蛍光物質を用いた検出方法が用いられている。しかしながら、蛍光物質を用いた検出方法には、蛍光プローブのハイブリダイズ等が必要で検出に係る工程が複雑であること、蛍光検出器が高価であること等の課題があるため、蛍光物質を用いない他の検出方法が開発されている（例えば、特許文献１）。 Conventionally, as methods for detecting pathogenic bacteria, viruses, etc., methods using nucleic acid amplification such as the polymerase chain reaction (PCR) method and the rolling circle amplification (RCA) method have been developed. In general, a detection method using a fluorescent substance is used to detect nucleic acid amplification products. However, detection methods using fluorescent substances have problems such as the need to hybridize a fluorescent probe and the detection process is complicated, and the fluorescence detector is expensive. Other detection methods have been developed (eg, US Pat.
特表２０１０－５１９９１４号公報Japanese Patent Publication No. 2010-519914
　特許文献１では、ＰＣＲ法等の核酸増幅のプライマー伸長段階におけるプロトン放出をイオン感応性電界効果トランジスタ（ＩＳＦＥＴ：Ion Selective Field Effect Transistor）によって検出する。特許文献１のように、放出されるプロトンによるｐＨ値の変化を検出するためには、検体となる核酸を含む溶液を微少容量のチャンバに注入し、チャンバごとにｐＨ値を計測する必要がある。 In Patent Document 1, proton release is detected by an ion selective field effect transistor (ISFET) during the primer elongation step of nucleic acid amplification such as the PCR method. As in Patent Document 1, in order to detect changes in the pH value due to released protons, it is necessary to inject a solution containing a nucleic acid as a specimen into a microvolume chamber and measure the pH value for each chamber. .
　しかしながら、核酸増幅に係るプロトン放出を精度よく検出するためには、例えば１０００ｐＬ（ピコリットル）容量のチャンバ内のｐＨ値を数万個単位で測定することが求められる。このように多数のチャンバについて、個々にｐＨ値を測定するためには、スイッチを含む複雑な回路が必要となり、また測定に要する時間は長くなる。 However, in order to accurately detect the proton emission associated with nucleic acid amplification, it is required to measure the pH value in a chamber with a capacity of 1000 pL (picoliters) in units of tens of thousands. In order to individually measure the pH values of such a large number of chambers, complicated circuits including switches are required, and the time required for measurement is long.
　本発明は、上述の事情に鑑みてなされたものであり、容易かつ迅速に核酸増幅反応を検出することができる検査チップ、反応検出装置及び反応検出方法を提供することを目的とする。 The present invention has been made in view of the circumstances described above, and aims to provide a test chip, a reaction detection device, and a reaction detection method that can easily and quickly detect a nucleic acid amplification reaction.
　上記目的を達成するために、この発明の第１の観点に係る検査チップは、
　検体のｐＨ値を検出するイオン感応性電界効果トランジスタ及び参照電極を有する複数のチャンバを備え、
　前記チャンバは、
　前記イオン感応性電界効果トランジスタのソース及びドレインが互いに接続された複数の第１グループからなる第１グループ群によってグルーピングされるとともに、前記参照電極が互いに接続された複数の第２グループからなる第２グループ群によってグルーピングされ、
　それぞれの前記第２グループに含まれる、それぞれの前記第１グループの前記チャンバは１つ以下となるように配線されている。 In order to achieve the above object, the test chip according to the first aspect of the present invention includes:
a plurality of chambers having ion-sensitive field effect transistors and reference electrodes for detecting the pH value of the analyte;
The chamber is
The sources and drains of the ion-sensitive field effect transistors are grouped by a first group consisting of a plurality of first groups connected to each other, and the reference electrodes are grouped by a first group consisting of a plurality of second groups connected to each other. grouped by groups,
The chambers of each of the first groups included in each of the second groups are wired so that there is no more than one.
　また、前記参照電極は、前記チャンバの開口部を覆う蓋部に配置されている、
　こととしてもよい。 Further, the reference electrode is arranged on a lid covering the opening of the chamber,
You can do it.
　また、検査チップは、
　隣接する前記チャンバを互いに連通する流路を備える、
　こととしてもよい。 Also, the inspection chip
comprising a channel that communicates the adjacent chambers with each other;
You can do it.
　また、前記チャンバの容量は１０ｐＬ以下である、
　こととしてもよい。 Further, the volume of the chamber is 10 pL or less,
You can do it.
　また、本発明の第２の観点に係る反応検出装置は、
　第１の観点に係る検査チップと、
　前記検査チップと接続され、前記チャンバ内の検体の反応を検出する制御部と、を備え、
　前記制御部は、
　前記第１グループ群から１つの前記第１グループを選択するとともに、前記第２グループ群から１つの前記第２グループを選択することにより、１つの前記チャンバを選択し、
　前記イオン感応性電界効果トランジスタでｐＨ値を測定することにより、選択された前記チャンバ内の検体の反応を検出する。 Further, the reaction detection device according to the second aspect of the present invention includes:
A test chip according to the first aspect;
a control unit that is connected to the test chip and detects a reaction of the specimen in the chamber;
The control unit
selecting one said chamber by selecting one said first group from said first group and selecting one said second group from said second group;
By measuring the pH value with the ion sensitive field effect transistor, the reaction of the analyte in the selected chamber is detected.
　また、本発明の第３の観点に係る反応検出方法では、
　第１の観点に係る検査チップのチャンバに検体を注入し、
　前記第１グループ群から１つの前記第１グループを選択するとともに、前記第２グループ群から１つの前記第２グループを選択することにより、１つの前記チャンバを選択し、
　前記イオン感応性電界効果トランジスタでｐＨ値を測定することにより、選択された前記チャンバ内の検体の反応を検出する。 Further, in the reaction detection method according to the third aspect of the present invention,
Injecting a specimen into the chamber of the test chip according to the first aspect,
selecting one said chamber by selecting one said first group from said first group and selecting one said second group from said second group;
By measuring the pH value with the ion sensitive field effect transistor, the reaction of the analyte in the selected chamber is detected.
　本発明の検査チップ、反応検出装置及び反応検出方法によれば、チャンバの選択及び各チャンバで生じるｐＨ値の変化を伴う反応の有無を、イオン感応性電界効果トランジスタを用いて電気的に行うことができるので、容易かつ迅速に核酸増幅反応を検出することが可能である。 According to the test chip, the reaction detection device, and the reaction detection method of the present invention, the selection of chambers and the presence or absence of reactions accompanied by changes in pH value occurring in each chamber can be electrically performed using ion-sensitive field effect transistors. Therefore, it is possible to detect the nucleic acid amplification reaction easily and quickly.
本発明の実施の形態に係る反応検出装置の構成を示す斜視図である。1 is a perspective view showing the configuration of a reaction detection device according to an embodiment of the present invention; FIG. 実施の形態に係る検査チップの側面図である。It is a side view of a test chip concerning an embodiment. 実施の形態に係るチャンバの構造を示す図であり、（Ａ）は平面図、（Ｂ）は側面断面図である。It is a figure which shows the structure of the chamber which concerns on embodiment, (A) is a top view, (B) is side sectional drawing. （Ａ）はチャンバ層におけるチャンバの配列を示す図であり、（Ｂ）は１つのチャンバを示す（Ａ）の拡大図である。(A) shows an array of chambers in a chamber layer, and (B) is an enlarged view of (A) showing one chamber. 参照電極を有する蓋部を備えるチャンバの側面断面図である。FIG. 10 is a side cross-sectional view of a chamber with a lid having a reference electrode; 実施の形態に係るＩＳＦＥＴ及び参照電極の配線例を示す概念図である。It is a conceptual diagram which shows the wiring example of ISFET which concerns on embodiment, and a reference electrode. 実施の形態に係る制御ユニットの構成を示す機能ブロック図である。It is a functional block diagram showing the configuration of a control unit according to the embodiment. 実施の形態に係る反応検出処理の流れを示すフローチャートである。6 is a flowchart showing the flow of reaction detection processing according to the embodiment; 検体の量とｐＨ値との関係の例を示すグラフである。4 is a graph showing an example of the relationship between the amount of specimen and the pH value; チャンバの選択とＩＳＦＥＴの出力との関係を示す概念図である。It is a conceptual diagram which shows the relationship between selection of a chamber and the output of ISFET. 環状配置されたチャンバの例を示す図である。FIG. 10 is a diagram showing an example of annularly arranged chambers; 流下式構造のチャンバ層を示す概略図である。FIG. 4 is a schematic diagram showing chamber layers in a flow-down configuration; 多層流下式構造のチャンバ層を示す概略図である。FIG. 4 is a schematic diagram showing the chamber layers of a multi-layer flow-down structure;
　以下、本発明の実施の形態に係る検査チップ、反応検出装置及び反応検出方法について、ＲＣＡ法による核酸増幅におけるプロトン放出反応を検出することにより、検体に含まれる増幅産物を検出する反応検出装置を例に、図面を参照して詳細に説明する。 Hereinafter, the test chip, the reaction detection device, and the reaction detection method according to the embodiments of the present invention will be described as a reaction detection device that detects an amplification product contained in a sample by detecting a proton release reaction in nucleic acid amplification by the RCA method. An example will be described in detail with reference to the drawings.
　本実施の形態に係る反応検出装置１は、図１に示すように、検体が注入される複数のチャンバ１２を有する検査チップ１０、検査チップ１０と接続されて対象物質を検出する検出ユニット５０を備える。 As shown in FIG. 1, the reaction detection apparatus 1 according to the present embodiment includes a test chip 10 having a plurality of chambers 12 into which specimens are injected, and a detection unit 50 connected to the test chip 10 to detect a target substance. Prepare.
　図２に示すように、検査チップ１０は、センサを有する基板１５、微小容量のチャンバ１２が形成されたチャンバ層１１を備える。 As shown in FIG. 2, the inspection chip 10 includes a substrate 15 having a sensor and a chamber layer 11 in which a microcapacity chamber 12 is formed.
　図３（Ａ）、（Ｂ）に示すように、基板１５のチャンバ層１１と接する側の面には、センサとしての複数のイオン感応性電界効果トランジスタ（Ion Selective Field Effect Transistor）であるＩＳＦＥＴ１６と、ＩＳＦＥＴ１６を動作させるための配線１７が形成されている。また、図２に示すように、基板１５は、配線１７と検出ユニット５０内の回路とを接続するための接続端子１５ａを備える。基板１５の材料は、特に限定されず、シリコン等の半導体、ガラス基板、プラスチック基板等を用いることができる。本実施の形態に係る基板１５はプラスチック基板である。 As shown in FIGS. 3A and 3B, on the surface of the substrate 15 in contact with the chamber layer 11, ISFETs 16, which are a plurality of ion-sensitive field effect transistors (Ion Selective Field Effect Transistors) as sensors. , a wiring 17 for operating the ISFET 16 is formed. In addition, as shown in FIG. 2, the substrate 15 includes connection terminals 15a for connecting the wiring 17 and the circuits in the detection unit 50. As shown in FIG. The material of the substrate 15 is not particularly limited, and a semiconductor such as silicon, a glass substrate, a plastic substrate, or the like can be used. The substrate 15 according to this embodiment is a plastic substrate.
　ＩＳＦＥＴ１６は、公知の方法で形成される。ＩＳＦＥＴ１６上には、絶縁層１８が形成されている。また、絶縁層１８には、スリット１８ａが形成されており、ＩＳＦＥＴ１６のゲート部が開口されている。スリット１８ａの大きさは特に限定されず、ＩＳＦＥＴ１６のゲート部の大きさに対応する寸法とすればよい。例えば、スリット１８ａは、一辺を０．５μｍ～１０００μｍ（マイクロメートル）とする長方形である。 The ISFET 16 is formed by a known method. An insulating layer 18 is formed on the ISFET 16 . A slit 18a is formed in the insulating layer 18, and the gate portion of the ISFET 16 is opened. The size of the slit 18 a is not particularly limited, and may be set to a size corresponding to the size of the gate portion of the ISFET 16 . For example, the slit 18a has a rectangular shape with a side of 0.5 μm to 1000 μm (micrometers).
　チャンバ１２は、検体が注入される容器であり、それぞれ１つのＩＳＦＥＴ１６が含まれるように、基板１５上に形成される。また、基板１５には、参照電極１９が形成されており、参照電極１９は各チャンバ１２の内部で電圧印加できるように開口されている。 The chambers 12 are containers into which specimens are injected, and are formed on the substrate 15 so as to contain one ISFET 16 each. A reference electrode 19 is formed on the substrate 15 , and the reference electrode 19 is opened inside each chamber 12 so that a voltage can be applied.
　チャンバ１２を含むチャンバ層１１は、基板１５上にフォトリソグラフィを用いてエポキシ樹脂で作成される。チャンバ層１１の作成方法は、フォトリソグラフィに限定されない。例えば、チャンバ層１１は、基板１５上にモールド成形されることとしてもよい。また、チャンバ層１１は、紫外線硬化樹脂を用いた光造形等の３次元プリンタによって作成されることとしてもよい。また、チャンバ１２を含むチャンバ層１１は、基板１５上に形成されることとしたが、これに限られず、フォトリソグラフィ、モールド成形等によって作成されたチャンバ層１１を基板１５上に載置、接着することとしてもよい。 The chamber layer 11 including the chamber 12 is made of epoxy resin on the substrate 15 using photolithography. The method of making the chamber layer 11 is not limited to photolithography. For example, chamber layer 11 may be molded onto substrate 15 . Also, the chamber layer 11 may be created by a three-dimensional printer such as stereolithography using an ultraviolet curable resin. In addition, although the chamber layer 11 including the chamber 12 is formed on the substrate 15, the present invention is not limited to this. It is also possible to
　本実施の形態に係るチャンバ１２は、ＲＣＡ法による核酸増幅において放出される微量のプロトンをｐＨ値の変化として検出するため、微小容量の容器として形成される。チャンバ１２の容量は、好ましくは１０ｐＬ以下、より好ましくは１ｐＬ以下である。 The chamber 12 according to the present embodiment is formed as a microcapacity container in order to detect a minute amount of protons released during nucleic acid amplification by the RCA method as a change in pH value. The volume of chamber 12 is preferably 10 pL or less, more preferably 1 pL or less.
　チャンバ１２の形状は特に限定されない。本実施の形態に係るチャンバ１２は、円筒形状に形成されている。チャンバ１２の内径は、直径１μｍ～１０００μｍであることが好ましい。また、チャンバ１２の高さは、１μｍ～１０００μｍであることが好ましい。本実施の形態に係るチャンバ１２は、直径２０μｍ、高さ１６μｍであり、容量は約５ｐＬ（ピコリットル）である。 The shape of the chamber 12 is not particularly limited. Chamber 12 according to the present embodiment is formed in a cylindrical shape. The inner diameter of chamber 12 is preferably between 1 μm and 1000 μm in diameter. Also, the height of the chamber 12 is preferably 1 μm to 1000 μm. The chamber 12 according to this embodiment has a diameter of 20 μm, a height of 16 μm, and a capacity of about 5 pL (picoliters).
　図４（Ａ）は、検査チップ１０の表面の拡大図であり、図４（Ｂ）は、図４（Ａ）のうち１つのチャンバ１２を拡大した図である。図４（Ａ）に示すように、チャンバ１２は、マトリックス状、より詳細には交互配列のマトリックス状に配置されている。 FIG. 4(A) is an enlarged view of the surface of the inspection chip 10, and FIG. 4(B) is an enlarged view of one chamber 12 in FIG. 4(A). As shown in FIG. 4A, the chambers 12 are arranged in a matrix, more specifically, in an alternating matrix.
　ｐＨ値の変化を精度よく検出するため、各チャンバ１２には、検体が定量で配分されることが好ましい。本実施の形態に係るチャンバ層１１は流路１３を備え、隣接するチャンバ１２は流路１３によって相互に連通されている。これにより、チャンバ１２に導入された検体は、流路１３を通じて他のチャンバ１２へと拡散される。 In order to accurately detect changes in the pH value, it is preferable that the sample is distributed in a fixed amount to each chamber 12 . The chamber layer 11 according to the present embodiment has a channel 13 , and the adjacent chambers 12 communicate with each other through the channel 13 . Thereby, the specimen introduced into the chamber 12 is diffused to other chambers 12 through the channel 13 .
　チャンバ１２及び流路１３の内壁には、検体の流動性を向上させるため、コーティング剤が塗布されることとしてもよい。コーティング剤は特に限定されず、例えばポリシラザン等のガラス質コーティング剤、疎水性コーティング剤、撥水性コーティング剤等を用いることができる。隣接するチャンバ１２間の距離は特に限定されないが、検体の流動性及び検出処理の迅速性の観点から、流路１３の長さが１０００μｍ以下となるように配置されることが好ましい。本実施の形態では、チャンバ１２を交互配列のマトリックス状に配置しているので、チャンバ１２間の距離を短くし、検体の流動性を向上させることができる。 A coating agent may be applied to the inner walls of the chamber 12 and the channel 13 to improve the fluidity of the specimen. The coating agent is not particularly limited, and for example, a vitreous coating agent such as polysilazane, a hydrophobic coating agent, a water-repellent coating agent, and the like can be used. Although the distance between the adjacent chambers 12 is not particularly limited, it is preferable that the channels 13 are arranged so that the length of the channels 13 is 1000 μm or less from the viewpoint of the fluidity of the sample and the rapidity of the detection process. In the present embodiment, since the chambers 12 are arranged in an alternate matrix, the distance between the chambers 12 can be shortened and the fluidity of the sample can be improved.
　本実施の形態では、参照電極１９は基板１５上に形成されることとしたが、これに限られず、各チャンバ１２内に電圧を印加可能なように形成されていればよい。例えば、図５の断面図に示すように、チャンバ１２の開口部を覆う蓋部２０に配置された参照電極１９が、各チャンバ１２に挿入されるように構成されていてもよい。これにより、参照電極１９の配線とＩＳＦＥＴ１６の配線とを、蓋部２０と基板１５とに分けることができるので、基板１５の配線が容易となり、構造を簡素化することが可能となる。 Although the reference electrode 19 is formed on the substrate 15 in the present embodiment, it is not limited to this, and may be formed so that a voltage can be applied to each chamber 12 . For example, as shown in the cross-sectional view of FIG. 5, a reference electrode 19 arranged on a lid 20 that covers the openings of the chambers 12 may be configured to be inserted into each chamber 12 . As a result, the wiring of the reference electrode 19 and the wiring of the ISFET 16 can be divided between the lid portion 20 and the substrate 15, so that the wiring of the substrate 15 is facilitated and the structure can be simplified.
　各チャンバは図６の概念図に示すように、第１グループ群Ａ及び第２グループ群Ｂにグルーピングされている。具体的には、各チャンバ１２のＩＳＦＥＴ１６のソース及びドレインは、第１グループＡｉ（ｉは１からｎの自然数）ごとに接続され、第１グループ群Ａを構成している。第１グループＡｉのＩＳＦＥＴ１６のソース及びドレインは、検出ユニット５０内の電源に接続され、後述する制御部５１によってそれぞれの第１グループＡｉのＩＳＦＥＴ１６に選択的に電圧を印加することができる。本実施の形態では、マトリックス状に配置されたチャンバ１２について、チャンバ１２が直列配置されたいずれかの方向（図６の水平方向）の列ごとに第１グループＡｉが構成される。これにより、同一の第１グループＡｉ内のＩＳＦＥＴ１６に同時に電圧印加することができる。 Each chamber is grouped into a first group group A and a second group group B, as shown in the conceptual diagram of FIG. Specifically, the sources and drains of the ISFETs 16 in each chamber 12 are connected for each first group Ai (i is a natural number from 1 to n) to form a first group A. The sources and drains of the ISFETs 16 of the first group Ai are connected to the power supply in the detection unit 50, and voltages can be selectively applied to the ISFETs 16 of the first group Ai by the controller 51, which will be described later. In the present embodiment, the chambers 12 arranged in a matrix form a first group Ai for each column in one of the directions (horizontal direction in FIG. 6) in which the chambers 12 are arranged in series. Thereby, a voltage can be simultaneously applied to the ISFETs 16 in the same first group Ai.
　また、各チャンバ１２の参照電極１９は、第２グループＢｊ（ｊは１からｍの自然数）ごとに接続され、第２グループ群Ｂを構成している。第２グループＢｊの参照電極１９は、検出ユニット５０内の電源に接続されており、後述する制御部５１によってそれぞれの第２グループＢｊの参照電極１９に選択的に電圧を印加することができる。本実施の形態では、マトリックス状に配置されたチャンバ１２について、第１グループＡｉと直交する方向（図６の垂直方向）の列ごとに第２グループＢｊが構成される。これにより、同一の第２グループＢｊ内の参照電極１９に同時に電圧印加することができる。 In addition, the reference electrodes 19 of each chamber 12 are connected to each second group Bj (j is a natural number from 1 to m) to form a second group B. The reference electrodes 19 of the second group Bj are connected to the power supply in the detection unit 50, and a voltage can be selectively applied to the reference electrodes 19 of the second group Bj by the control section 51, which will be described later. In this embodiment, the chambers 12 arranged in a matrix form a second group Bj for each row in a direction orthogonal to the first group Ai (vertical direction in FIG. 6). Thereby, the voltage can be applied simultaneously to the reference electrodes 19 in the same second group Bj.
　言い換えると、それぞれの第２グループＢｊには、各第１グループＡｉのチャンバ１２が１つ以下含まれるように配線されている。また、それぞれの第１グループＡｉには、各第２グループＢｊのチャンバ１２が１つ以下含まれるように配線されている。これにより、第１グループＡｉのいずれかと第２グループＢｊのいずれかとを選択することで、同時に複数のチャンバ１２を選択することなく、１つ以下のチャンバ１２を選択してｐＨ値を計測することができる。すなわち、本実施の形態では、アクティブマトリックス方式でＩＳＦＥＴ１６を配線し、チャンバ１２を順次選択してｐＨ値を測定することにより、チャンバ１２内に検出対象の核酸の増幅産物が存在するか否かを判定することができる。 In other words, each second group Bj is wired so as to include one or less chambers 12 of each first group Ai. Also, each first group Ai is wired so as to include one or less chambers 12 of each second group Bj. Accordingly, by selecting either one of the first group Ai and one of the second group Bj, it is possible to select one or less chambers 12 and measure the pH value without selecting a plurality of chambers 12 at the same time. can be done. That is, in the present embodiment, the ISFET 16 is wired in an active matrix system, the chambers 12 are sequentially selected, and the pH value is measured to determine whether or not the amplified product of the nucleic acid to be detected is present in the chambers 12. can judge.
　検出ユニット５０は、検査チップ１０と接続され、各チャンバ１２内のｐＨ値を測定することによって、核酸増幅反応の検出を行う。検出ユニット５０は、図７の機能ブロック図に示すように、制御部５１、記憶部５２、表示部５３、入力部５４、リーダー５５を備える。 The detection unit 50 is connected to the test chip 10 and detects the nucleic acid amplification reaction by measuring the pH value inside each chamber 12 . The detection unit 50 includes a control section 51, a storage section 52, a display section 53, an input section 54, and a reader 55, as shown in the functional block diagram of FIG.
　制御部５１は、ＣＰＵ(Central Processing Unit)、ＲＯＭ(Read Only Memory)、ＲＡＭ(Random Access Memory)等から構成されており、反応検出装置１全体の動作を制御する。また、制御部５１は、検査チップ１０に形成されたチャンバ１２について、第１グループＡｉと第２グループＢｊとを順次選択することにより、各チャンバ１２内の検体のｐＨ値を測定し、核酸増幅反応の有無を検出する。 The control unit 51 is composed of a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), etc., and controls the operation of the reaction detection device 1 as a whole. In addition, the control unit 51 sequentially selects the first group Ai and the second group Bj for the chambers 12 formed in the test chip 10, thereby measuring the pH value of the sample in each chamber 12 and amplifying the nucleic acid. Detects the presence or absence of reaction.
　制御部５１は、制御部５１のＲＯＭ、記憶部５２等に記憶されている各種動作プログラム及びデータをＲＡＭに読み込んでＣＰＵを動作させることにより、図７に示される制御部５１の各機能を実現させる。これにより、制御部５１は、測定対象のチャンバ１２を選択する選択部５１１、選択されたチャンバ１２のｐＨ値を測定する測定部５１２として動作する。 The control unit 51 reads various operation programs and data stored in the ROM of the control unit 51, the storage unit 52, etc. into the RAM and operates the CPU, thereby realizing each function of the control unit 51 shown in FIG. Let Thereby, the control unit 51 operates as a selection unit 511 that selects the chamber 12 to be measured and a measurement unit 512 that measures the pH value of the selected chamber 12 .
　選択部５１１は、予め定められた順序にしたがって、第１グループＡｉと第２グループＢｊとを順次選択する。これにより、チャンバ層１１に形成されたチャンバ１２のうち、測定対象となるチャンバ１２を選択する。 The selection unit 511 sequentially selects the first group Ai and the second group Bj according to a predetermined order. Accordingly, the chamber 12 to be measured is selected from among the chambers 12 formed in the chamber layer 11 .
　測定部５１２は、選択部５１１で選択されたチャンバ１２について、ＩＳＦＥＴ１６及び参照電極１９に電圧を印加してチャンバ１２内の検体のｐＨ値を測定する。 The measurement unit 512 applies voltage to the ISFET 16 and the reference electrode 19 of the chamber 12 selected by the selection unit 511 to measure the pH value of the sample in the chamber 12 .
　記憶部５２は、ハードディスク、フラッシュメモリ等の不揮発性メモリであり、チャンバ１２を選択するためのプログラム、チャンバ１２ごとに測定されたｐＨ値等を記憶する。 The storage unit 52 is a non-volatile memory such as a hard disk or flash memory, and stores programs for selecting the chambers 12, pH values measured for each chamber 12, and the like.
　表示部５３は、検出ユニット５０に備えられた表示用デバイスであり、例えば液晶モニタである。表示部５３は、測定されたｐＨ値、検出対象の核酸の増幅産物が確認されたチャンバ１２の数等の情報を表示する。 The display unit 53 is a display device provided in the detection unit 50, such as a liquid crystal monitor. The display unit 53 displays information such as the measured pH value and the number of chambers 12 in which the amplified product of the nucleic acid to be detected has been confirmed.
　入力部５４は、反応検出処理の開始、終了、反応検出装置１の各種設定値等を入力するための入力デバイスである。入力部５４は検出ユニット５０に備えられたキーボード、タッチパネル、マウス等である。 The input unit 54 is an input device for inputting the start and end of reaction detection processing, various set values of the reaction detection device 1, and the like. The input unit 54 is a keyboard, touch panel, mouse, etc. provided in the detection unit 50 .
　リーダー５５は、検査チップ１０に設けられた接続端子１５ａと、検出ユニット５０内の電源回路、スイッチ回路等とを接点を介して接続する。これにより、制御部５１は、選択したチャンバ１２に係るＩＳＦＥＴ１６、参照電極１９に電圧を印加するとともに、チャンバ１２内の検体のｐＨ値を測定する。 The reader 55 connects the connection terminal 15a provided on the inspection chip 10 to the power supply circuit, switch circuit, etc. in the detection unit 50 via contacts. Thereby, the controller 51 applies a voltage to the ISFET 16 and the reference electrode 19 associated with the selected chamber 12 and measures the pH value of the sample in the chamber 12 .
　続いて、本実施の形態に係る反応検出装置１による核酸増幅反応の検出方法について、図８のフローチャートを参照しつつ説明する。 Next, a method for detecting a nucleic acid amplification reaction by the reaction detection device 1 according to this embodiment will be described with reference to the flowchart of FIG.
　まず、準備工程として、反応検出装置１で検出を行う検体を準備する。具体的には、ＲＣＡ法、ＰＣＲ法等の核酸増幅処理を実施してプライマー伸長反応を生じる検体を生成する。本実施の形態では、検体として、所定のウイルスに係るＤＮＡをＲＣＡ法によって増幅させた増幅産物を含む溶液を用いる。そして、検査チップ１０のチャンバ１２に検体を注入し、検査の準備を行う（ステップＳ１１）。この際、チャンバ層１１の一部のチャンバ１２へ注入された検体の溶液は、流路１３を介して、チャンバ層１１の全てのチャンバ１２へと拡散される。 First, as a preparation step, a specimen to be detected by the reaction detection device 1 is prepared. Specifically, a nucleic acid amplification process such as the RCA method or the PCR method is performed to generate a sample that causes a primer extension reaction. In the present embodiment, a solution containing an amplification product obtained by amplifying DNA of a given virus by the RCA method is used as a specimen. Then, the sample is injected into the chamber 12 of the inspection chip 10 to prepare for inspection (step S11). At this time, the specimen solution injected into some of the chambers 12 of the chamber layer 11 is diffused to all the chambers 12 of the chamber layer 11 via the channel 13 .
　検体が注入された検査チップ１０は、リーダー５５を介して検出ユニット５０に接続され、反応検出が開始される。反応検出が開始されると、制御部５１の選択部５１１は、チャンバ１２を走査して核酸増幅反応の検出を行う。具体的には、選択部５１１は、第２グループ群Ｂから１つの第２グループＢｊを選択し、第２グループＢｊに属するチャンバ１２の参照電極１９に電圧を印加する（ステップＳ１２）。 The test chip 10 into which the sample is injected is connected to the detection unit 50 via the reader 55, and reaction detection is started. When reaction detection is started, the selector 511 of the controller 51 scans the chamber 12 to detect the nucleic acid amplification reaction. Specifically, the selector 511 selects one second group Bj from the second group B, and applies voltage to the reference electrodes 19 of the chambers 12 belonging to the second group Bj (step S12).
　また、選択部５１１は、第１グループ群Ａから１つの第１グループＡｉを選択する（ステップＳ１３）。測定部５１２は、第１グループＡｉのドレイン電流を測定する。これにより、測定部５１２は、第１グループＡｉ及び第２グループＢｊに属する１つのチャンバ１２のｐＨ値を測定する（ステップＳ１４）。 Also, the selection unit 511 selects one first group Ai from the first group group A (step S13). The measurement unit 512 measures the drain current of the first group Ai. Thereby, the measuring unit 512 measures the pH value of one chamber 12 belonging to the first group Ai and the second group Bj (step S14).
　ｐＨ値を変化させるプロトンの放出量、放出速度は、核酸増幅に用いる方法によって異なる。例えば、Ｒｈａ－ＲＣＡ（RNase H-assisted RCA）法を用いてウイルスを検出する場合、ウイルス１個について核酸増幅を行うことによって５分間で生成されるプロトンは１×１０４個程度である。図９に示すように、ＩＳＦＥＴ１６の感度の観点から、放出されるプロトンによってｐＨ値が０．２５程度変化するようにチャンバ１２の容量を設定すると、チャンバ１２の容量は１０ｐＬ以下であることが好ましく、１ｐＬ以下であることがより好ましい。上述したように、本実施の形態に係るチャンバ１２の容量は大凡５ｐＬである。 The release amount and release rate of protons that change the pH value differ depending on the method used for nucleic acid amplification. For example, when viruses are detected using the Rha-RCA (RNase H-assisted RCA) method, about 1×10 4 protons are generated in 5 minutes by performing nucleic acid amplification for one virus. As shown in FIG. 9, from the viewpoint of the sensitivity of the ISFET 16, when the volume of the chamber 12 is set so that the pH value changes by about 0.25 due to the emitted protons, the volume of the chamber 12 is preferably 10 pL or less. , is more preferably 1 pL or less. As described above, the volume of chamber 12 according to this embodiment is approximately 5 pL.
　選択部５１１は、第１グループ群Ａから全ての第１グループＡｉを選択し終えるまで（ステップＳ１５のＮＯ）、第１グループＡｉを順次切り替え、測定部５１２は、第１グループＡｉのドレイン電流からｐＨ値を測定する。これにより、測定部５１２は、選択されている第２グループＢｊに属するチャンバ１２のｐＨ値を順次測定することができる。 The selection unit 511 sequentially switches the first group Ai until all the first groups Ai from the first group A have been selected (NO in step S15), and the measurement unit 512 detects the drain current from the first group Ai. Measure the pH value. Thereby, the measuring unit 512 can sequentially measure the pH values of the chambers 12 belonging to the selected second group Bj.
　選択された第２グループＢｊのチャンバ１２について測定が終了すると（ステップＳ１５のＹＥＳ）、選択部５１１は、第２グループ群Ｂから別の第２グループＢｊを選択し、新たに選択された第２グループＢｊに属するチャンバ１２の参照電極１９に電圧を印加する。選択部５１１及び測定部５１２は、第２グループ群Ｂの全ての第２グループＢｊを選択し、測定が終了するまで、第２グループＢｊの選択と測定を繰り返す（ステップＳ１６のＮＯ）。 When the measurement of the chambers 12 of the selected second group Bj is completed (YES in step S15), the selection unit 511 selects another second group Bj from the second group B, and selects the newly selected second group Bj. A voltage is applied to the reference electrodes 19 of the chambers 12 belonging to group Bj. The selection unit 511 and the measurement unit 512 select all the second groups Bj of the second group B, and repeat the selection and measurement of the second groups Bj until the measurement is completed (NO in step S16).
　全ての第２グループＢｊを選択し、ｐＨ値の測定が終了すると（ステップＳ１６のＹＥＳ）、反応検出処理は終了する。 When all the second groups Bj are selected and the pH value measurement is finished (YES in step S16), the reaction detection process is finished.
　図１０は、チャンバ１２内の核酸増幅反応検出の流れを示す概念図である。図１０の例では、選択部５１１で選択された第２グループＢ２の参照電極１９に電圧が印加されている。そして、第１グループ群Ａの第１グループＡｉを順次選択した場合に測定部５１２で測定されるドレイン電流値が示されている。本例では、第１グループＡ３及び第２グループＢ２に属するチャンバ１２に検出対象となる核酸の増幅産物が含まれている。そして、ＲＣＡ法におけるプライマー伸長段階で放出されるプロトンによって、当該チャンバ１２内のｐＨ値が変化している。したがって、図１０に示すように、第２グループＢ２のチャンバ１２を順次スキャンした場合の検出電流は第１グループＡ３が選択された時点で大きくなり、他のチャンバ１２と異なる。これにより、このチャンバ１２に検出対象の核酸の増幅産物が含まれていることがわかる。このように、アクティブマトリックス方式で配置された複数のチャンバ１２を順次走査することにより、迅速に核酸増幅反応の検出を行うことができる。 FIG. 10 is a conceptual diagram showing the flow of nucleic acid amplification reaction detection within the chamber 12. FIG. In the example of FIG. 10, the voltage is applied to the reference electrodes 19 of the second group B2 selected by the selector 511. In the example of FIG. Also shown are drain current values measured by the measuring unit 512 when the first group Ai of the first group group A is sequentially selected. In this example, the chambers 12 belonging to the first group A3 and the second group B2 contain amplification products of nucleic acids to be detected. The pH value in the chamber 12 changes due to protons released during the primer elongation step in the RCA method. Therefore, as shown in FIG. 10, when the chambers 12 of the second group B2 are sequentially scanned, the detection current increases when the first group A3 is selected, and differs from the other chambers 12. FIG. This indicates that the chamber 12 contains the amplification product of the nucleic acid to be detected. In this way, by sequentially scanning a plurality of chambers 12 arranged in an active matrix system, the nucleic acid amplification reaction can be rapidly detected.
　以上、詳細に説明したように、本実施の形態に係る反応検出装置１によれば、ＩＳＦＥＴ１６のソース、ドレインの配線を共通にする複数の第１グループＡｉからなる第１グループ群Ａと、参照電極１９の配線を共通にする複数の第２グループＢｊからなる第２グループ群Ｂとによって特定される微小容量のチャンバ１２について、第１グループＡｉと第２グループＢｊとを順次選択して、選択されたチャンバ１２のｐＨ値を測定する。これにより、チャンバ１２の選択をスイッチ回路によって電気的に行うことができるので、高速に反応検出を行うことができる。また、ＩＳＦＥＴ１６を用いたｐＨ値の測定によって、反応の有無を電気的に検出できるので、蛍光検出器を用いた増幅産物の検出と比較して、安価で容易に検出を行うことができる。 As described in detail above, according to the reaction detection device 1 according to the present embodiment, the first group A consisting of a plurality of first groups Ai that share the wiring of the source and drain of the ISFET 16, and the reference The first group Ai and the second group Bj are sequentially selected for the small-capacity chambers 12 specified by the second group B consisting of a plurality of second groups Bj sharing the wiring of the electrodes 19, and the selection is performed. The pH value of the chamber 12 is measured. As a result, the selection of the chamber 12 can be electrically performed by the switch circuit, so that the reaction can be detected at high speed. In addition, since the presence or absence of reaction can be electrically detected by measuring the pH value using the ISFET 16, the detection can be performed inexpensively and easily compared to the detection of amplification products using a fluorescence detector.
　また、本実施の形態では、各チャンバの容量を１０ｐＬ以下と微小な容量としているので、プライマー伸長反応によるプロトン放出に基づくｐＨ値の変化を精度よく行うことが可能となる。 In addition, in the present embodiment, since the volume of each chamber is as small as 10 pL or less, it is possible to accurately change the pH value based on proton release by the primer extension reaction.
　また、本実施の形態に係るチャンバ１２は流路１３で連通されているので、検体を各チャンバ１２に迅速に拡散させることが可能となり、検出効率を向上させることができる。 In addition, since the chambers 12 according to the present embodiment are communicated with each other by the flow paths 13, it is possible to rapidly diffuse the specimen to each chamber 12, thereby improving the detection efficiency.
　本実施の形態では、チャンバ１２はマトリックス状に配置されており、ＩＳＦＥＴ１６及び参照電極１９がアクティブマトリックス方式で配線されていることとしたが，これに限られない。例えば、図１１に示すように、チャンバ１２は、複数の同心円からなる環状に配置されることとしてもよい。この場合、図１１に示すように、同径の円上に配置されたチャンバ１２のグループを第１グループＡｉとしてソース、ドレインを接続し、第１グループ群Ａを構成すればよい。また、環状配置の半径方向に配置されているチャンバ１２のグループを第２グループＢｊとして参照電極１９を接続し、第２グループ群Ｂを構成すればよい。 In this embodiment, the chambers 12 are arranged in a matrix, and the ISFETs 16 and the reference electrodes 19 are wired in an active matrix system, but this is not the only option. For example, as shown in FIG. 11, the chambers 12 may be arranged in an annular shape consisting of a plurality of concentric circles. In this case, as shown in FIG. 11, a group of chambers 12 arranged on circles of the same diameter is defined as a first group Ai, and the sources and drains are connected to form a first group A. As shown in FIG. A second group B may be formed by connecting the reference electrodes 19 to the group of the chambers 12 arranged in the radial direction of the annular arrangement as the second group Bj.
　本実施の形態では、チャンバ１２に注入された検体は、流路１３を通じて均一化されることとしたが、これに限られない。例えば、図１２に示すように、検体の溶液を流下させて各チャンバ１２に注入することとしてもよい。この場合、チャンバ１２の上縁部から外側下方に傾斜する傾斜部１２ａを形成してもよい。チャンバ１２の容量以上に注入された検体がチャンバ１２からあふれることにより、各チャンバ１２の検体量を所定の量に均一化することができる。これにより、ｐＨ値の変化をより精度よく検出することができる。さらに、流下式構造により各チャンバ１２に検体を均等に配分できる場合、チャンバ１２同士を連通する流路１３は形成されていなくてもよいので、チャンバ層１１の構造をより簡素化することができる。また、あふれた検体がチャンバ層１１の底部に集められることにより、余分な検体を容易に回収することができる。 Although the sample injected into the chamber 12 is homogenized through the channel 13 in the present embodiment, it is not limited to this. For example, as shown in FIG. 12, the sample solution may be flowed down and injected into each chamber 12 . In this case, a sloped portion 12a that slopes downward from the upper edge of the chamber 12 may be formed. By overflowing the chamber 12 with the sample injected in excess of the volume of the chamber 12, the amount of sample in each chamber 12 can be uniformed to a predetermined amount. Thereby, the change in pH value can be detected more accurately. Furthermore, when the sample can be evenly distributed to each chamber 12 by the flow-down structure, the flow path 13 communicating between the chambers 12 does not need to be formed, so the structure of the chamber layer 11 can be further simplified. . In addition, since the overflowing specimen is collected at the bottom of the chamber layer 11, excess specimen can be easily recovered.
　また、本実施の形態では、チャンバ層１１は１層であることとしたが、これに限られず、複数のチャンバ層１１が積層されることとしてもよい。この場合、図１３に示すように、各層のチャンバ１２が上述した傾斜部１２ａを備え、上側のチャンバ層１１のチャンバ１２からあふれた検体が下側のチャンバ層１１のチャンバ１２へ順次流入するように構成してもよい。これにより、容易に各層のチャンバ１２に注入される検体の量を均一化することができる。 In addition, although the chamber layer 11 is one layer in the present embodiment, the present invention is not limited to this, and a plurality of chamber layers 11 may be laminated. In this case, as shown in FIG. 13, the chambers 12 of each layer are provided with the above-described inclined portions 12a so that the specimen overflowing from the chambers 12 of the upper chamber layer 11 sequentially flows into the chambers 12 of the lower chamber layer 11. can be configured to This makes it possible to easily equalize the amount of specimen injected into the chamber 12 of each layer.
　また、本実施の形態では、リーダー５５を備える検出ユニット５０が検査チップ１０と接続されることとしたが、これに限られない。例えば、スマートフォン等の携帯通信端末に、制御部５１、記憶部５２、表示部５３、入力部５４として動作するためのプログラムをインストールし、携帯通信端末とリーダー５５とを接続することによって検出ユニット５０として動作させることとしてもよい。これにより、一般的なスマートフォン等を用いて容易に反応検出を行うことができる。また、検出ユニット５０に、通信機能を有する携帯通信端末を用いることにより、ネットワークを介して検出データをサーバに送信することができるので、迅速に大量の検出データを収集、分析することが可能となる。 Also, in the present embodiment, the detection unit 50 including the reader 55 is connected to the inspection chip 10, but this is not the only option. For example, a program for operating as a control unit 51, a storage unit 52, a display unit 53, and an input unit 54 is installed in a mobile communication terminal such as a smartphone, and the detection unit 50 is detected by connecting the mobile communication terminal and the reader 55. It may be operated as As a result, reaction detection can be easily performed using a general smartphone or the like. Moreover, by using a mobile communication terminal having a communication function as the detection unit 50, detection data can be transmitted to the server via a network, so that a large amount of detection data can be quickly collected and analyzed. Become.
　この発明は、この発明の広義の精神と範囲を逸脱することなく、様々な実施の形態及び変形が可能とされるものである。また、上述した実施の形態は、この発明を説明するためのものであり、この発明の範囲を限定するものではない。すなわち、この発明の範囲は、実施の形態ではなく、特許請求の範囲によって示される。そして、特許請求の範囲内及びそれと同等の発明の意義の範囲内で施される様々な変形が、この発明の範囲内とみなされる。 Various embodiments and modifications of the present invention are possible without departing from the broad spirit and scope of the present invention. Moreover, the embodiment described above is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.
　本発明は、核酸増幅に係る反応を検出する反応検出に好適である。特に、感染症に係るウイルスの検出等、迅速で安価に増幅産物を検出することが求められる場合の反応検出に好適である。 The present invention is suitable for reaction detection that detects reactions related to nucleic acid amplification. In particular, it is suitable for reaction detection when rapid and inexpensive detection of amplification products is required, such as detection of viruses associated with infectious diseases.
１　反応検出装置、１０　検査チップ、１１　チャンバ層、１２　チャンバ、１２ａ　傾斜部、１３　流路、１５　基板、１５ａ　接続端子、１６　ＩＳＦＥＴ、１７　配線、１８　絶縁層、１８ａ　スリット、１９　参照電極、２０　蓋部、５０　検出ユニット、５１　制御部、５１１　選択部、５１２　測定部、５２　記憶部、５３　表示部、５４　入力部、５５　リーダー 1 reaction detection device, 10 inspection chip, 11 chamber layer, 12 chamber, 12a inclined portion, 13 flow path, 15 substrate, 15a connection terminal, 16 ISFET, 17 wiring, 18 insulating layer, 18a slit, 19 reference electrode, 20 lid section, 50 detection unit, 51 control section, 511 selection section, 512 measurement section, 52 storage section, 53 display section, 54 input section, 55 reader

Claims (6)

  1. 　検体のｐＨ値を検出するイオン感応性電界効果トランジスタ及び参照電極を有する複数のチャンバを備え、
    　前記チャンバは、
    　前記イオン感応性電界効果トランジスタのソース及びドレインが互いに接続された複数の第１グループからなる第１グループ群によってグルーピングされるとともに、前記参照電極が互いに接続された複数の第２グループからなる第２グループ群によってグルーピングされ、
    　それぞれの前記第２グループに含まれる、それぞれの前記第１グループの前記チャンバは１つ以下となるように配線されている、
    　ことを特徴とする検査チップ。     a plurality of chambers having ion-sensitive field effect transistors and reference electrodes for detecting the pH value of the analyte;
    The chamber is
    The sources and drains of the ion-sensitive field effect transistors are grouped by a first group consisting of a plurality of first groups connected to each other, and the reference electrodes are grouped by a first group consisting of a plurality of second groups connected to each other. grouped by groups,
    each said first group included in each said second group is wired so that there is no more than one said chamber;
    An inspection chip characterized by:
  2. 　前記参照電極は、前記チャンバの開口部を覆う蓋部に配置されている、
    　ことを特徴とする請求項１に記載の検査チップ。     The reference electrode is arranged on a lid covering the opening of the chamber,
    The inspection chip according to claim 1, characterized in that:
  3. 　隣接する前記チャンバを互いに連通する流路を備える、
    　ことを特徴とする請求項１又は２に記載の検査チップ。     comprising a channel that communicates the adjacent chambers with each other;
    The test chip according to claim 1 or 2, characterized in that:
  4. 　前記チャンバの容量は１０ｐＬ以下である、
    　ことを特徴とする請求項１から３のいずれか一項に記載の検査チップ。     the chamber has a volume of 10 pL or less;
    The inspection chip according to any one of claims 1 to 3, characterized in that:
  5. 　請求項１から４のいずれか一項に記載の検査チップと、
    　前記検査チップと接続され、前記チャンバ内の検体の反応を検出する制御部と、を備え、
    　前記制御部は、
    　前記第１グループ群から１つの前記第１グループを選択するとともに、前記第２グループ群から１つの前記第２グループを選択することにより、１つの前記チャンバを選択し、
    　前記イオン感応性電界効果トランジスタでｐＨ値を測定することにより、選択された前記チャンバ内の検体の反応を検出する、
    　ことを特徴とする反応検出装置。     The inspection chip according to any one of claims 1 to 4;
    a control unit that is connected to the test chip and detects a reaction of the specimen in the chamber;
    The control unit
    selecting one said chamber by selecting one said first group from said first group and selecting one said second group from said second group;
    detecting the reaction of an analyte in the selected chamber by measuring the pH value with the ion-sensitive field effect transistor;
    A reaction detection device characterized by:
  6. 　請求項１から４のいずれか一項に記載の検査チップの前記チャンバに検体を注入し、
    　前記第１グループ群から１つの前記第１グループを選択するとともに、前記第２グループ群から１つの前記第２グループを選択することにより、１つの前記チャンバを選択し、
    　前記イオン感応性電界効果トランジスタでｐＨ値を測定することにより、選択された前記チャンバ内の検体の反応を検出する、
    　ことを特徴とする反応検出方法。     Injecting a sample into the chamber of the test chip according to any one of claims 1 to 4,
    selecting one said chamber by selecting one said first group from said first group and selecting one said second group from said second group;
    detecting the reaction of an analyte in the selected chamber by measuring the pH value with the ion-sensitive field effect transistor;
    A reaction detection method characterized by:
PCT/JP2023/003927 2022-02-15 2023-02-07 Inspection chip, reaction detection device, and reaction detection method WO2023157711A1 (en)

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Publication number Priority date Publication date Assignee Title
JP2006322943A (en) * 2000-09-29 2006-11-30 Toshiba Corp Nucleic acid detecting sensor
JP2003004697A (en) * 2000-12-22 2003-01-08 Seiko Epson Corp Sensor cell
US20080012007A1 (en) * 2004-04-01 2008-01-17 Nanyang Technological University Addressable Transistor Chip For Conducting Assays
US20110315559A1 (en) * 2006-12-28 2011-12-29 Gordon Holt Quality control methods for the manufacture of polymer arrays
JP2020165979A (en) * 2008-10-22 2020-10-08 ライフ テクノロジーズ コーポレーション Integrated sensor arrays for biological and chemical analysis
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