JP2005345243A - Nucleic acid detecting substrate and nucleic acid detecting method using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid detector. <P>SOLUTION: A nucleic acid chain detecting substrate is equipped with a substrate, a plurality of the electrode regions formed on the substrate, a plurality of the probe fixing electrodes and signal extraction electrodes formed on each of the electrode regions and a frame surrounding the probe fixing electrodes and/or the signal extraction electrodes to be formed into a container shape and characterized in that the probe fixing electrodes and the signal extraction electrodes are connected by signal wiring, and the substrate having the electrode regions and the reaction container are closely bonded. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nucleic acid detection substrate.

In recent years, genetic testing techniques using nucleic acid chain-immobilized arrays (DNA arrays) have attracted attention (Beattie et al. 1993, Fodor et al. 1991, Khrapko et al. 1989, Southern et al. 1994). The DNA array is composed of several cm square glass or silicon arrays on which 10 ¹ to 10 ⁵ different types of DNA probes are immobilized, and is labeled with fluorescent dyes or radioisotopes (RI) on the array. The sample gene is reacted, or a mixture of unlabeled sample gene and labeled oligonucleotide is reacted by sandwich hybridization. When a sequence complementary to the DNA probe on the array is present in the sample, a signal (fluorescence intensity, RI intensity) derived from the label is obtained at a specific site on the array. If the sequence and position of the immobilized DNA probe are known in advance, the base sequence present in the sample gene can be easily examined. Since a large amount of information on a base sequence can be obtained with a small amount of sample, a DNA array is expected not only as a gene detection technique but also as a sequencing technique (Pease et al., 1994, Parinov et al., 1996, FIG. 1). Although the above DNA array is excellent in detecting many kinds of genes for one sample, it is not necessarily suitable for processing many samples. Currently, adaptation to multi-sample gene detection has been reported by combining it with a micro-tarter plate-like container, but this requires a large and expensive optical detector (FIG. 2).

  On the other hand, a nucleic acid chain detection method based on an electrochemical technique has also been reported (Patent Document 1). These methods do not require sample gene labeling, and the detection also measures an electric signal, so that a complicated system such as fluorescence detection is not required, and a reduction in size of the system can be expected. However, the handling of the electrodes has become complicated for the detection of a large number of samples, and it has not been fully supported. Recently, detection of multiple genes x multiple samples using a combination of a stick-like electrode with a large number of probes immobilized and a microtiter plate-like container has been reported, but the mechanism of robotics is the handling of stick-like electrodes. There is a drawback that the apparatus becomes necessary and the apparatus becomes large (FIG. 3).

As described above, high-throughput detection of heterogeneous genes x multisamples and multigene genes x multisamples is desired in future tailor-made medicine, but none of the current technologies meet all specifications, and new means Was desired.
Japanese Patent No. 2573443

  As described above, the current gene detection apparatus has a problem that it is difficult to detect heterogeneous genes × multiple samples or various genes × multiple samples. In order to solve the above problems, an object of the present invention is to provide a nucleic acid detection device.

  The present invention is a nucleic acid chain detection substrate, a substrate, a plurality of electrode regions formed on the substrate, a plurality of probe-immobilized electrodes and signal extraction electrodes formed on the electrode region, and the probe-immobilization A frame formed in a container shape surrounding the electrode and / or signal extraction electrode, the probe-immobilized electrode and the signal extraction electrode are connected by a signal wiring, and the electrode region The substrate having the reaction container and the reaction container are in close contact with each other to provide a nucleic acid chain detection substrate.

  The present invention also provides the nucleic acid chain detection substrate, wherein the electrode region is further formed with a counter electrode.

  Furthermore, the present invention provides the nucleic acid chain detection substrate, wherein the electrode region is further formed with a reference electrode.

  Furthermore, the present invention provides a nucleic acid chain detection substrate, which is the above-described nucleic acid detection substrate and has a microtiter plate shape.

  The present invention also relates to a method for detecting a nucleic acid chain having a specific base sequence present in a sample, the probe immobilized on the probe-immobilized electrode of the nucleic acid detection substrate using the nucleic acid detection substrate. And a step of performing a hybridization reaction with a nucleic acid chain present in the sample, and a step of electrochemically detecting the presence or absence of hybridization between the probe and the nucleic acid chain. provide.

  Furthermore, the present invention provides the above detection method, further comprising a step of causing a double-stranded DNA recognition substance to act in order to perform the electrochemical detection.

  Furthermore, the present invention provides the above detection method, wherein the double-stranded DNA recognition substance is an intercalating agent.

  The present invention also provides a nucleic acid detection system for carrying out the above method, wherein the nucleic acid detection device detects nucleic acid using at least a nucleic acid extraction unit, a nucleic acid amplification unit, and the nucleic acid detection substrate. An apparatus comprising a nucleic acid detection unit is provided.

  Furthermore, the present invention is an upper nucleic acid detection apparatus, which is a tip rack, a reagent rack, a waste liquid rack, a movable dispenser capable of dispensing a reagent, and a movable capable of performing current measurement. A nucleic acid detection unit, wherein the nucleic acid amplification unit and the detection unit include a thermal cycler, and the nucleic acid detection device further includes a vacuum pump, a syringe pump, and an electric control unit. Providing equipment.

  Hereinafter, the nucleic acid detection substrate of the present invention will be described in detail with reference to the drawings.

  The nucleic acid chain detection device of the present invention includes a substrate 2, a plurality of electrode regions 12 formed on the substrate 2, a plurality of probe-immobilized electrodes 8 and signal extraction electrodes 9 formed on the electrode region 12, and a probe-immobilized electrode. A nucleic acid chain detection device comprising a frame 13 for partitioning 8.

  The material of the substrate 2 used in the present invention is not particularly limited, and any material known to those skilled in the art can be used. For example, inorganic insulating materials such as glass, quartz glass, silicon, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. Polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin Organic materials such as polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. Further, the substrate may be flat, uneven, or porous. Further, the substrate surface is preferably covered with an insulating film 10.

  A plurality of electrode regions 12 are formed on the substrate 2.

  The shape of the electrode region 12 is not particularly specified, but as shown in FIG. 4 (B), it is 36 mm (3 × 2) so that it can correspond to the format of a commercially available 24, 48, 96, 384, 1536 hole microtiter plate. 18 mm (6 × 4), 9 mm (12 × 8), 4.5 mm (24 × 16), and 2.25 (48 × 32) mm. The electrode region 12 may be patterned directly on the substrate 2 or may be used by previously preparing an electrode region on another substrate and attaching it to the substrate on which it is used. The electrode region 12 includes a probe-immobilized electrode 8 in which a nucleic acid probe is immobilized on a working electrode, and a signal extraction electrode 9 for extracting a signal from the probe-immobilized electrode 8 to the outside via a signal wiring 11 It is configured. Further, the probe-immobilized electrode 8 is an electrode in which a nucleic acid probe is immobilized on a working electrode, as in the case of a conventional product.

  The shape of the probe-immobilized electrode 8 on which the nucleic acid probe is immobilized and the signal extraction electrode 9 are not particularly limited, but 36 mm (3 × 2), 18 mm (6 × 4), 9 mm (12 × 8), 4.5 mm (24 × 16), and 2.25 (48 × 32) mm are desirable. The signal extraction electrode 9 is preferably located in the vicinity of the probe-immobilized electrode 8, but if it is formed on the same substrate, it can be used without any problem even if it is the edge of the substrate, for example. For example, as shown in FIG. 4 (A), 4 × 4 = 16 probe-immobilized electrodes 8 and signal extraction electrodes 9 can be formed as the probe-immobilized electrode 8 and the signal extraction electrode 9. . Further, as shown in FIG. 4 (A), not only is the probe-immobilized electrode 8 and the signal extraction electrode 9 formed in parallel, but also the probe-immobilized electrode 8 is surrounded as shown in FIG. A signal extraction electrode 9 may be disposed.

  In addition, since a plurality of probe-immobilized electrodes 8 are created in the electrode region 12, by immobilizing nucleic acid probes having different base sequences for each probe-immobilized electrode 8, it is possible to target multiple types of targets at once. An inspection can be performed. The number of probe-immobilized electrodes 8 and signal extraction electrodes 9 may be the same, or the number of signal extraction electrodes 9 may be small. In the case of the same number, it is desirable that the probe-immobilized electrode 8 and the signal extraction electrode 9 are directly connected one-to-one through the signal wiring 11 (see, for example, FIG. 4). When the number of signal extraction electrodes 9 is small, a switching element is introduced so that a plurality of working electrodes can be switched (see, for example, FIG. 12). The switching method may be a matrix method used for liquid crystal display, or may be an active matrix method using a MOSFET. Also, a MOS image sensor type scanning circuit can be used. The probe immobilization electrode 8 and the signal extraction electrode 9 are desirably close to each other, but are not particularly limited.

  Further, if a reference electrode and / or a counter electrode is provided for each electrode region 12 to form a three-electrode system, detection accuracy can be improved (see, for example, FIG. 8).

  Here, the material of each electrode is not particularly limited, and any material known to those skilled in the art can be used. For example, gold alloys, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium, tungsten and other simple metals and alloys thereof, carbon such as graphite and glassy carbon, and oxides or compounds thereof Can be used. In particular, it is desirable to use gold as the material of the probe nucleic acid-immobilized electrode 8.

  These electrodes can also be produced by plating, printing, sputtering, vapor deposition or the like. When vapor deposition is performed, the electrode film can be formed by a resistance heating method, a high frequency heating method, or an electron beam heating method. When sputtering is performed, the electrode film can be formed by DC bipolar sputtering, bias sputtering, asymmetrical AC sputtering, getter sputtering, or high frequency sputtering. Here, when gold is used for the electrode, the orientation index of the (111) plane of the gold crystal structure is important. The orientation index is determined from the following equation by the method of Willson.

Orientation index (hkl) = IF _(hkl) / IFR _(hkl)
hkl: Face index
IF _(hkl) ; relative strength of (hkl) plane
IFR _(hkl) ; IF _(hkl) as standard gold on the ASTM card
Here, in the case of the nucleic acid chain detection electrode, the orientation index is preferably 1 or more. Furthermore, the orientation index is desirably 2 or more. In order to improve the orientation, it is also effective to heat the substrate during vapor deposition or sputtering. The heating temperature is not particularly limited, but is preferably in the range of 50 ° C to 500 ° C. By controlling the orientation, it is possible to uniformly control the amount of immobilized probes. In addition, when the electrode material such as gold is deposited or sputtered on a substrate such as glass, titanium, chromium, copper, nickel, or an alloy thereof is used alone or in combination as an adhesive layer between the substrate and gold. Accordingly, a stable electrode layer can be formed.

  When patterning the electrode region 12 on the substrate 2, it is necessary to make each electrode area uniform in order to increase detection accuracy. As a means for controlling the electrode area to be constant, a photolithography technique can be used. The exposed area is patterned by photolithography to control the area constant. The insulating material used in the present invention is not particularly limited, but is preferably a photopolymer or a photoresist material. As the resist material, a light exposure photoresist, a far ultraviolet photoresist, an X-ray photoresist, and an electron beam photoresist are used. Photoresist for photoexposure includes cyclized rubber, polycinnamic acid, and novolac resin as main raw materials. For the deep ultraviolet photoresist, cyclized rubber, phenol resin, polymethylisopropenyl ketone (PMIPK), polymethyl methacrylate (PMMA), or the like is used. In addition, COP, metal acrylate, and other substances described in the Thin Film Handbook (Ohm Co.) can be used for the X-ray resist, and the substances described in the above documents such as PMMA can be used for the electron beam resist. Can do. The resist used here is desirably 100 mm or more and 1 mm or less. By covering the electrode with a photoresist and performing lithography, the area can be made constant. As a result, the amount of DNA probe immobilized becomes uniform between the respective electrodes, enabling measurement with excellent reproducibility. Conventionally, the resist material is generally removed finally, but the electrode for the probe-immobilized electrode 8 can be used as a part of the electrode without removing the resist material. In this case, it is necessary to use a substance having high water resistance for the resist material to be used. An insulating layer formed on the electrode can be used other than a photoresist material. For example, oxides such as Si, Ti, Al, Zn, Pb, Cd, W, Mo, Cr, Ta, and Ni, nitrides, carbides, and other alloys can be used. After these materials are formed into a thin film by sputtering, vapor deposition, CVD, or the like, the electrode exposed portion is patterned by photolithography, and the area is controlled to be constant.

  When lithography is used, a highly accurate electrode can be manufactured, but there are cases where manufacturing costs are slightly increased. A printed circuit board can also be used to produce electrodes at low cost. In this case, it is possible to use polyimide or glass epoxy resin as a substrate material. The electrode formed on the printed circuit board can be selected from the electrode materials described above.

  The material of the probe fixed to the probe-immobilized electrode 8 is not particularly limited, but DNA, RNA, PNA, methylphosphonate skeleton nucleic acid, and other nucleic acid analogs can be used. These chimeric nucleic acids may also be used. The length of the probe to be used is not particularly limited, but is 8 to 200 bases, preferably 10 to 100 bases, and more preferably 15 to 50 bases.

  The method for immobilizing the probe to the working electrode in order to produce the probe-immobilized electrode 8 is not particularly limited, but can be immobilized using methods known to those skilled in the art. For example, it can be easily performed by utilizing a bond between a thiol group introduced into a probe and gold. In addition, it can be immobilized by physical adsorption, chemical adsorption, hydrophobic bond, embedding, and covalent bond. For example, it can also be covalently bonded onto the substrate using a condensing agent such as carbodiimide via an amino group introduced at the end of the probe. Moreover, it can also fix by an ionic bond by coat | covering an anionic organic substance on a board | substrate. Furthermore, when biotin is introduced at the end of the probe, it can be immobilized by a biotin-avidin bond. Alternatively, a thiol group can be introduced at the end of the probe, SS can be formed between the thiol-containing substance coated on the substrate, and the probe can be firmly fixed. In these cases, immobilization can be facilitated by modifying the electrode surface with molecules having functional groups in advance. It is desirable to introduce a spacer between the probe and the terminal modification group in order to suppress steric hindrance between the substrate surface and the probe. The spacer molecule is not particularly limited, but may be an alkane skeleton, an alkyne skeleton, an alkene skeleton, an ethylene glycol skeleton, or a nucleic acid chain. The molecular structure may be linear or branched. The length of the spacer portion is not particularly limited, but is preferably 10 to 500, preferably 20 to 200, and more preferably 50 to 100 in terms of a carbon-carbon bond. Furthermore, in order to suppress non-specific adsorption of nucleic acids and intercalating agents on the electrode surface, the electrode surface should be mercaptoethanol, mercaptohexanol, mercaptoheptanol, mercaptoethylene glycol, mercapto oligoethylene glycol, mercapto polyethylene glycol, carbon It is desirable to coat with a mercaptan such as an alkanethiol having a chain of 30 to 50, a lipid such as stearylamine, a surfactant, albumin, a nucleic acid or the like.

  When the nucleic acid probe is immobilized on the electrode, the probe can be immobilized relatively easily by using an immobilization apparatus called a DNA spotter or DNA arrayer. At this time, in order not to damage the surface of the electrode, it is preferable to use an ink jet type or electrostatic type spotter. Alternatively, the nucleic acid chain can be directly synthesized on the electrode surface.

  Further, the step of forming the probe-immobilized electrode 8 by immobilizing the probe to the electrode is preferably performed before the substrate 2 and the frame 13 are brought into close contact with each other, but can be immobilized even after the contact. Further, the nucleic acid detection substrate of the present invention is not necessarily provided in a state where the probe is immobilized on the probe-immobilized electrode 8, and may be provided as a substrate on which the probe is not immobilized. In this case, when the nucleic acid detection substrate is used, a desired probe is immobilized on the substrate as described above.

  The nucleic acid detection substrate of the present invention surrounds the probe-immobilized electrode 8 and / or the signal extraction electrode 9 in addition to the electrode region 12 (probe-immobilized electrode 8 and signal extraction electrode 9) formed on the substrate 2 in plural. And a frame 13 formed in a container shape. In order to perform the gene detection reaction with the probe-immobilized electrode 8, it is necessary to react the sample solution with the electrode. At this time, the frame 13 is used so that the sample solution is held on the electrode. Form wells / containers. That is, in the nucleic acid detection substrate of the present invention, the reaction well 15 and / or the signal extraction well 16 are formed by the frame 13 for each probe-immobilized electrode 8 and / or signal extraction electrode 9. (See, for example, FIGS. 5, 6, and 7).

  The material of the frame 13 is not particularly limited. For example, an inorganic insulating material such as glass, quartz glass, silicon, alumina, sapphire, forsterite, silicon carbide, silicon oxide, silicon nitride, or the like can be used. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, Teflon (registered trademark) resin, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, Organic materials such as polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone, etc. Can be used.

  In addition, the shape of the well / container formed by the frame 13 is not particularly limited, but 36 mm (3 × 2), 18 mm so as to be compatible with a commercially available 24, 48, 96, 354, 1536 hole microtiter plate format. It is desirable to form with a pitch of (6 × 4), 9 mm (12 × 8), 4.5 mm (24 × 16), and 2.25 (48 × 32) mm. Therefore, the nucleic acid detection substrate of the present invention is provided as an electrode-integrated microtiter plate in which the electrode region 12 is formed in the microtiter plate format and the frame 3 is formed in the microtiter plate format. You can also.

  Also, the shape of each well / container is not particularly limited, and may be circular or polygonal. In addition, by preparing the frame 13 without the bottom of the well / container and preparing the substrate 2 on which the electrode region 12 is patterned, the substrate 2 is used in close contact with each other, so that the nucleic acid detection substrate of the present invention can be easily produced. I will. At this time, it is desirable that the well / container formed by the frame 13 and the electrode region 12 have the same pitch. In order to bring the frame 13 and the substrate 2 into close contact with each other, a strong contact method such as adhesion, pressure bonding, or the like that prevents liquid leakage is desirable, but it is also possible to make close contact using silicon packing or the like.

  In addition, the nucleic acid detection substrate of the present invention does not necessarily have to be provided integrally with the substrate 2 on which the electrode region 12 is formed and the frame 13, and may be provided in a state where they are separated. In this case, when the nucleic acid detection substrate is used, the substrate 2 and the frame 13 are used in close contact as described above.

  The well / container formed by the frame 13 is subjected to a detection reaction by dispensing a hybridization solution, a washing solution, an electrochemical measurement solution, etc., but if the liquid touches the signal extraction electrode 9 during the reaction, an accurate signal is obtained. Measurement becomes impossible. Therefore, it is desirable to cover the well of the signal extraction electrode 9 until just before the current measurement. The shape of the cover is not particularly limited, but it may be a lid with a shape in which the reaction well portion including the probe-immobilized electrode 8 is left open and the signal extraction well portion including the signal extraction electrode 9 is closed. The detection is performed by removing the lid immediately before the signal measurement. It is also possible to apply a thin film to the signal extraction well and first detect the signal from the signal extraction electrode 9 after breaking the film with a prober during signal measurement.

  By using the nucleic acid chain detection apparatus of the present invention, the presence or absence of a nucleic acid chain having a specific base sequence can be detected electrochemically. The electrochemical signal can be indexed by oxidation-reduction current change, oxidation-reduction potential change, capacitance change, resistance change, and electrochemiluminescence change. The effect of these signal changes is promoted by the combined use of a substance that specifically binds to a double-stranded nucleic acid chain such as an intercalating agent, the addition of metal particles such as gold colloid, and the like by metal ions such as silver ions. The effect can also be promoted by combining an antigen-antibody reaction or an enzyme label such as alkaline phosphatase or peroxidase. For example, an RNA / DNA recognition antibody can be used when an RNA molecule is used as a probe or when the target is an RNA molecule. Alternatively, the target can be hapten-labeled and recognized with an anti-hapten antibody. Finally, detection is performed by reacting with an enzyme-labeled secondary antibody. The substrate is preferably a substance that becomes electrochemically active after the catalytic reaction of an enzyme such as paraaminophenyl phosphate. Further, the efficiency can be further improved by combining with a mediator such as ferricyan ion. The double-stranded nucleic acid-specific binding substance used here is not particularly limited, but is a metal complex called a metallointercalator such as ruthenium, cobalt, and iron, Hoechst 33258, Hoechst 33342, acridine orange, ethidium bromide, bisintercalator, and Organic compounds such as tris intercalators and biopolymers such as antibodies or enzymes can also be used. It can also be detected by sandwich hybridization using a second nucleic acid probe labeled with a metal complex such as ferrocene.

  A series of operations and reaction conditions for nucleic acid chain detection using this nucleic acid chain detection electrode will be described below. First, a probe-fixed electrode 8 is formed by immobilizing a desired probe on the electrode on the substrate manufactured as described above. Here, a method for immobilizing DNA on a gold electrode is described. After the electrode is washed with deionized water, an activation treatment is performed. For activation, a 0.1 to 10 mmol / L sulfuric acid solution can be used. In this solution, the potential is scanned in the range of -0.5 to 2 V (vs Ag / AgCl) and in the range of 1 v / s to 100,000 v / s. Thereby, the electrode surface is activated to the state which can fix | immobilize a probe. Further, the activation process can be performed using an etching gas. In this case, radicals such as ozone, CF3, and oxygen can be used as the etching gas. In the probe used for immobilization, a thiol group is introduced at the 5 ′ or 3 ′ end. The thiolated probe is dissolved in a solution containing a reducing agent such as DTT until immediately before immobilization, and DTT is removed by gel filtration or extraction with ethyl acetate immediately before use. Immobilization was very simple, and the probe DNA was dissolved in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 in a range of 1 ng / ml to 1 mg / mL and activated. Immediately after immersion of the electrode. The immobilization reaction is performed in the range of 4 to 100 ° C. for about 10 minutes to overnight.

  After probe immobilization, it is desirable to store under conditions where no nucleolytic enzyme (nuclease) is present and, if possible, light shielding. However, in the short term, it can be stored in a wet state. The composition of the preservation solution is preferably the composition of the solution for performing the hybridization reaction, Tris-EDTA buffer solution or deionized water. Furthermore, the storage temperature is 4 ° C. or less, preferably −20 ° C. In addition, when the electrode after immobilization of the nucleic acid probe is stored for a long time, it is desirable to store it in a dry state. Although the method to make it dry is not specifically limited, It can carry out by freeze-drying or air drying. The gas phase is not particularly limited, but is desirably an inert gas such as argon, nitrogen, dry air, or a vacuum state. The operability of inspection can be improved by attaching a mark or barcode to each electrode.

  The nucleic acid chain detected by the nucleic acid detection substrate of the present invention is not particularly limited. For example, hepatitis virus (A, B, C, D, E, F, G type), HIV, influenza virus (A, B, C, D, E, F), herpes group virus, adenovirus, human polyoma virus , Human papillomavirus, human parvovirus, mumps virus, human rotavirus, enterovirus, Japanese encephalitis virus, smallpox virus, coronavirus, SARS, dengue virus, rubella virus, HTLV, virus infection, Staphylococcus aureus, hemolytic Streptococcus, pathogenic E. coli, Vibrio parahaemolyticus, Helicobacter pylori, Campylobacter, Vibrio cholerae, Shigella, Salmonella, Bacillus anthracis, Yersinia, Neisseria, Listeria, Leptospira, Legionella, Spirocheta, Mycoplasma pneumonia, Rickettsia, Chlamydia, Malaria Dysentery candy It can be used for detection of bacterial infections such as liver, pathogenic fungi, parasites, and fungi.

  Furthermore, it can also be used for type determination (genotyping) of the infectious disease-causing microorganism. For example, hepatitis C virus 1a, 1b, 2a, 2b, 3a, 3b, and 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, which are associated with human papillomavirus oncogenesis 54, 56, 58, 59, 66, 68, 69, and 6, 11, 34, 40, 42, 43, 44, 70, etc. not associated with carcinogenesis. Furthermore, drug resistance genes can also be detected, for example, drug resistance genes of Mycobacterium tuberculosis, AIDS virus, and respiratory infection-causing bacteria. In addition, hereditary diseases, retinoblastoma, Wilms tumor, familial colon polyposis, hereditary non-polyposis colon cancer, neurofibromatosis, familial breast cancer, xeroderma pigmentosum, brain tumor, oral cancer, esophageal cancer, Tumors such as stomach cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, thyroid tumor, breast tumor, urological tumor, male organ tumor, female organ tumor, skin tumor, bone / soft tissue tumor, leukemia, lymphoma, and solid tumor Sexual illness can be examined. In addition to medical treatment, it can be applied to all food testing, quarantine, pharmaceutical testing, forensic medicine, agriculture, livestock, fishery, forestry, etc. that require genetic testing. Furthermore, restriction enzyme fragment multiple systems (RFLP), single nucleotide multiple systems (SNPs), microsatellite sequences, and the like can also be detected. It can also be used for analysis of unknown base sequences.

  Therefore, the sample to be reacted with the probe-immobilized electrode is not particularly limited. For example, blood, serum, leukocytes, urine, feces, semen, saliva, tissue, cultured cells, sputum, food, soil, drainage, wastewater, air, etc. Can be used.

  In order to detect nucleic acids contained in these samples, nucleic acid components are first extracted from the samples. The extraction method is not particularly limited. For example, a liquid-liquid extraction method such as a phenol-chloroform method or a solid-liquid extraction method using a carrier can be used. In addition, commercially available nucleic acid extraction methods such as QIAamp (manufactured by QIAGEN) and Smaitest (manufactured by Sumitomo Metals) can also be used. These samples are dispensed to a microtiter plate or the like and used for gene detection. When a gene is extracted, if a microtiter plate or the like holding a hydrophobic membrane is used, the detection operation can be more easily performed. The extracted nucleic acid is preferably dissolved in an appropriate solution.

  Next, a hybridization reaction is performed between the nucleic acid extracted from the sample and the probe immobilized on the probe-immobilized electrode. The reaction is carried out in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In the reaction solution, hybridization accelerators such as dextran sulfate, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant can be added. Next, the extracted nucleic acid is added to the reaction solution and heat denatured at 90 ° C. or higher. Contact between the reaction solution containing the nucleic acid and the probe-immobilized electrode can be performed immediately after denaturation or after rapid cooling to 0 ° C. During the reaction, the reaction rate can be increased by an operation such as stirring or shaking. The reaction temperature may be in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. The hybridization reaction can be controlled electrochemically, and by applying a positive potential to the electrode, what was conventionally required from several hours to several days can be shortened to several minutes. At this time, the potential can be swept at a constant speed, applied in pulses, or a constant potential can be applied. The electric potential is applied between the signal extraction electrode and the probe fixed electrode formed in the electrode region. The potential is applied by controlling the current and voltage using a potentiostat, a digital multimeter, a function generator or the like. After the hybridization reaction, the probe-immobilized electrode is washed. For washing, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.

  After washing, the presence or absence of hybridization is detected electrochemically. In order to electrochemically detect a double-stranded nucleic acid, first, a double-stranded nucleic acid recognition substance is allowed to act on the formed double-stranded nucleic acid. For example, an intercalating agent that selectively binds to the formed double-stranded part is allowed to act. Here, the intercalating agent used as the double-stranded nucleic acid recognizing substance is not particularly limited. For example, bisintercalators such as Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisacridine, trisintercalator, Alternatively, a polyintercalator or the like can be used. The concentration of the intercalating agent varies depending on the type, but is generally used in the range of 1 ng / ml to 1 mg / ml. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 is used. Specifically, an intercalating agent is added to the reaction well formed on the nucleic acid detection substrate to react with the double-stranded nucleic acid, and then the well is washed and subjected to electrochemical measurement. In the measurement, a potential higher than the potential at which the intercalating agent electrochemically reacts is applied, and the reaction current value derived from the intercalating agent is measured. At this time, the potential can be swept at a constant speed, applied in pulses, or a constant potential can be applied. The potential is applied between the signal extraction electrode and the probe-immobilized electrode. The potential is applied by controlling the current and voltage using a potentiostat, a digital multimeter, a function generator or the like. After the hybridization reaction, the concentration of the target gene is calculated from the calibration curve based on the current value obtained from the intercalating agent. In order to take out an electrical signal from such a connection terminal to the outside, it can be performed using a prober as in the conventional case. Further, as shown in FIG. 10 or 11, by arranging the plurality of probers in the same arrangement as the connection terminals formed on the nucleic acid detection substrate, electrical signals from the plurality of connection terminals are simultaneously taken out. be able to.

  According to the nucleic acid detection substrate, it is possible to simultaneously detect the presence of nucleic acid chains in a plurality of samples by using samples derived from different specimens for each reaction well formed on the substrate.

  In addition, as described above, a nucleic acid chain detection device can be used to automate and detect the presence or absence of nucleic acids using the nucleic acid detection substrate of the present invention. For example, as shown in FIG. 13, an apparatus in which a chip rack 29, a reagent rack 30, a waste liquid rack 31, a nucleic acid extraction unit 32, a nucleic acid amplification unit 33, and a nucleic acid detection unit 35 are installed on a stage 36, A movable dispenser 24 for dispensing reagents and a movable prober set 22 for current measurement are attached to the rail 28, and a vacuum pump 25 used for nucleic acid extraction, The syringe pump 26 used for the injection and an electric control unit 27 are attached to the apparatus for controlling and measuring the electric system. The nucleic acid amplification unit 34 and the detection unit 35 include a thermal cycler 33 capable of changing the temperature. This is a device that incorporates this mechanism. Nucleic acid extraction can also be performed using magnetic fine particles, and in that case, a magnetic separation unit using a magnet is incorporated.

  For example, in the apparatus shown in FIG. 13, the dispenser 24 has an arm, and each of the tip rack 29, the reagent rack 30, the waste liquid rack 31, the nucleic acid extraction unit 32, the nucleic acid amplification unit 33, and the nucleic acid detection unit 35 is provided. The prober set 22 can move through the rail 28 and can contact the signal readout electrode 9 of the nucleic acid detection substrate disposed in the detection unit 35 to perform current measurement. The nucleic acid extraction unit 32, the nucleic acid amplification unit 33, and the nucleic acid detection unit 35 can perform a nucleic acid extraction step from the sample, an amplification step of the extracted nucleic acid, and a nucleic acid detection step using the nucleic acid detection substrate of the present invention, respectively. These units may be integrated. In each step, necessary chips and reagents are used from the chip rack 29 and the reagent rack 30, and the waste liquid is discarded in the waste liquid rack 31. In each operation, the vacuum pump 25 and the syringe pump 26 are used to suck and discharge the solution. Further, the electric control unit 27 controls the electric system to read the electric signal from the signal reading electrode and calculate the concentration of the nucleic acid based on the current value.

  The overall flow of the nucleic acid detection process by the nucleic acid detection apparatus is shown in FIG. FIG. 15 shows the operation of the extraction process performed in the nucleic acid extraction unit 32. FIG. 16 shows the operation of the amplification process performed in the nucleic acid amplification unit 33. FIG. 17 shows the operation of preparing a sample for detection. FIG. 18 shows the operation of the detection process performed in the nucleic acid detection unit 35.

  Those skilled in the art can easily understand the configuration and use of each unit of the nucleic acid detection apparatus having the above arrangement. A person skilled in the art will be able to easily create an apparatus having such a configuration.

Electrode Patterning Substrate FIG. 4 is a diagram showing an example of a substrate on which electrodes are patterned. In FIG. 4B, a thin film of titanium (500 mm) and gold (2000 mm) is laminated on a glass substrate 2 (d = 0.8 mm) by sputtering, and the probe-fixed electrode portion 8 (0.5 mm) and the signal extraction electrode 9 are resisted. It has an insulated structure. Forty-eight 48 × 6 × 8 electrode regions 12 each including the probe-fixed electrode 8 and the signal extraction electrode 9 are formed. FIG. 4A is an enlarged view of the electrode region 12, and 16 probe-immobilized electrodes 8 are formed in 4 × 4. The signal extraction electrodes 9 are directly coupled to each probe-fixed electrode 8 by signal wirings 11, and the number of signal extraction electrodes 9 is 16 × 4 × 4. The thiolated nucleic acid probe was immobilized on the opening of the insulating film 10 with a thiol / gold bond. This substrate can immobilize 16 different types of probes. By using such a substrate, it is possible to simultaneously detect the presence or absence of a plurality of nucleic acids for a plurality of samples by electrochemical means.

Nucleic acid probe-immobilized electrode 8 holder and electrode-integrated microtiter plate FIGS. 5 and 6 are diagrams showing an example of a nucleic acid probe-immobilized electrode holder and electrode-integrated microtiter plate. A plastic frame 13 molded from polypropylene was attached to the substrate 2 produced by the method shown in FIG. 4 with an epoxy resin adhesive to produce an electrode-integrated microtiter plate (FIG. 5A). The plastic frame 13 is divided into 96 reaction cells of 12 × 8, and is designed so that 48 probe-immobilized electrodes and 48 terminal portions are opened (FIG. 6). FIG. 5B is a cross-sectional view after the plastic frame 13 is bonded. A reaction well 15 and a signal readout well 16 are respectively formed. The probe-fixed electrode region and the signal extraction electrode region are separated. A sample can be added to the reaction well 16, and the probe-immobilized electrode 8 and the nucleic acid contained in the sample can be hybridized to detect the hybridized nucleic acid. By using such a substrate, it is possible to simultaneously detect the presence or absence of a plurality of nucleic acids in a plurality of samples by electrochemical means and the same operation as that in a conventional microtiter plate.

Electrode Patterning Substrate FIG. 7 is a diagram showing another example of a substrate on which the electrode region 12 is patterned. FIG. 7 (B) shows a thin film of titanium (500 mm) and gold (2000 mm) deposited on glass substrate 2 (d = 0.8 mm) by sputtering, probe-immobilized electrode portion 8 (0.5 mm) and signal extraction electrode. 9 is insulated with resist. 20 electrode regions of 4 × 5 are formed of the probe-immobilized electrode 8 and the signal extraction electrode 9 (FIG. 8). FIG. 7A is an enlarged view of the electrode region. 8 × 8 = 64 probe-immobilized electrodes 8 are formed. In each electrode region, four counter electrodes 18 and three reference electrodes 19 are patterned. The signal extraction electrodes 9 are directly coupled to each probe-fixed electrode 8 by signal wirings 11, and 80 signal extraction electrodes 9 are formed. The thiolated nucleic acid probe was immobilized on the opening of the insulating film 10 with a thiol / gold bond. In this substrate, 64 different types of probes can be immobilized on one reaction well 15.

Electrode integrated microtiter plate FIG. 9 shows an example of a gene detection reaction using an electrode integrated microtiter plate. A substrate 2 on which electrodes are patterned is attached to the bottom surface of the microtiter plate. In the reaction well 15, the probe-immobilized electrode 8 and the reaction solution 20 are in direct contact, and the reaction solution 20 is held in the reaction well 15. In the signal extraction well 16, the signal extraction electrode 9 is exposed, and an electrochemical signal is detected from the outside via the prober 22, and transferred to the detector.

FIG. 10 is a diagram showing an example of a schematic diagram of a prober for taking out an electrical signal from the signal extraction electrode 9 to the outside. A spring is built in the prober 22, so that the signal extraction electrode 9 and the prober 22 can be reliably contacted. FIG. 10A is a prober for use in the examples described in FIGS. 4 to 6, where 4 × 4 = 16 is one unit. FIG. 10B is a view of the prober as viewed from above, and electric wiring is individually led out from the end of the prober and connected to an electrochemical measuring instrument for signal detection. By using such a prober set, signals can be simultaneously read from a plurality of signal extraction wells of the nucleic acid detection substrate of the present invention.

FIG. 11 is a diagram showing an example of a schematic diagram of a prober for taking out an electrical signal from the signal extraction electrode 9 to the outside. FIG. 11A is a prober used in the embodiment shown in FIG. 7, where 80 units form one unit. FIG. 11B is a view of the prober as viewed from above. Electrical wiring is individually led out from the end of the prober and connected to an electrochemical measuring instrument for signal detection.

Prober FIG. 12 is a view showing an example of a substrate on which the electrode region 12 is patterned. FIG. 12B shows a structure in which a CMOS type transistor circuit, a probe fixing electrode 8 made of a gold thin film and a signal extraction electrode 9 are formed on a silicon substrate 2 (d = 0.2 mm) and insulated with a resist. . 6 × 8 = 48 electrode regions 12 each including the probe fixing electrode 8 and the signal extraction electrode 9 are formed. FIG. 12A is an enlarged view of the electrode region 12, and 4 × 4 = 16 probe immobilization regions 8 are formed. Each probe-immobilized electrode 8 is connected to a switching element and has a mechanism that can be controlled by active matrix control.

Detection System FIG. 13 is a diagram showing an example of a nucleic acid detection apparatus using the nucleic acid detection substrate of the present invention. FIG. 13 is a top view of the apparatus, and the stage 36 is provided with a chip rack 29, a reagent rack 30, a waste rack 31, a nucleic acid extraction unit 32, a nucleic acid amplification unit 33, and a nucleic acid detection unit 35. ing. A movable dispenser 24 for dispensing reagents and a movable prober set 22 for measuring current are attached to the rail 28. A vacuum pump 25 used for nucleic acid extraction, a syringe pump 26 used for dispensing, and an electric system control / measurement unit 27 are attached to the apparatus. The nucleic acid amplification unit 34 and the detection unit 35 incorporate a mechanism of a thermal cycler 33 that can change the temperature. Nucleic acid extraction can also be performed using magnetic fine particles, and in that case, a magnetic separation unit using a magnet is incorporated.

  Fig. 14 shows the overall flow. FIG. 15 shows the operation of the extraction process, FIG. 16 shows the operation of the amplification process, FIG. 17 shows the operation of sample preparation for detection, and FIG. 18 shows the operation of the detection process.

The figure which showed the probe fixed chip | tip of the prior art. The figure which showed the probe fixed microtiter plate of the prior art. The figure which showed the nucleic acid detection by the nucleic acid detection board | substrate of a prior art. The figure which showed an example of patterning of an electrode area | region in one aspect of the nucleic acid detection board | substrate of this invention. The figure which showed an example of the plastic frame-electrode integrated microtiter plate in one aspect of the nucleic acid detection board | substrate of this invention. The figure which showed an example of the electrode integrated microtiter plate in one aspect of the nucleic acid detection board | substrate of this invention. The figure which showed an example of the board | substrate which patterned the electrode area | region in one aspect of the nucleic acid detection board | substrate of this invention. The figure which showed an example of the board | substrate which patterned the electrode area | region in one aspect of the nucleic acid detection board | substrate of this invention. The figure which showed an example of the detection using the electrode integrated microtiter plate in one aspect of the nucleic acid detection board | substrate of this invention. The figure which showed an example of the probe for reading an electric current from the nucleic acid detection board | substrate of this invention. The figure which showed an example of the probe for reading an electric current from the nucleic acid detection board | substrate of this invention. The figure which showed an example of the board | substrate which patterned the electrode area | region in one aspect of the nucleic acid detection board | substrate of this invention The figure which showed an example of the apparatus for detecting a nucleic acid using the nucleic acid detection board | substrate of this invention. Overall flow diagram. The flowchart of an extraction process. The flowchart of an amplification process. Flow chart of sample preparation. The flowchart of a detection process.

Explanation of symbols

1 ... probe, 2 ... substrate, 4 ... fluorescent label, 5 ... microtiter plate, 6 ... electrode, 7 ... electrode-integrated container, 8 ... probe-immobilized electrode 9 ... Signal extraction electrode, 10 ... Insulating film, 11 ... Signal wiring, 12 ... Electrode region, 13 ... Frame, 14 ... Glass chip with Au pattern, 15. ..Reaction well, 16 ... Signal extraction well, 17 ... Signal extraction electrode, 18 ... Counter electrode, 19 ... Reference electrode, 20 ... Sample solution (sample, cleaning solution, intercalating agent) Solution), 21 ... nozzle, 22 ... prober, 23 ... switching circuit, 24 ... dispenser, 25 ... vacuum pump, 26 ... syringe pump, 27 ... electrical control Unit, 28 ... Rail, 29 ... Chip rack, 30 ... Reagent rack, 31 ... Waste liquid rack, 32 ... Extraction unit, 33 ... Thermal sensor Ecler, 34 ... Amplification unit, 35 ... Detection unit

Claims (9)

A nucleic acid chain detection substrate comprising:
A substrate,
A plurality of electrode regions formed on the substrate;
A plurality of probe-immobilized electrodes and signal extraction electrodes formed in the electrode region;
A frame formed in a container shape surrounding the probe-immobilized electrode and / or the signal extraction electrode,
The probe-immobilized electrode and the signal extraction electrode are connected by a signal wiring, and the substrate having the electrode region and the reaction container are in close contact with each other.   2. The nucleic acid chain detection substrate according to claim 1, wherein a counter electrode is further formed in the electrode region.   The nucleic acid chain detection substrate according to claim 1, wherein a reference electrode is further formed in the electrode region.   4. The nucleic acid chain detection substrate according to claim 1, wherein the nucleic acid chain detection substrate is in the form of a microtiter plate. A method for detecting a nucleic acid chain having a specific base sequence present in a sample,
Hybridization of a probe immobilized on a probe-immobilized electrode of the nucleic acid detection substrate using the nucleic acid detection substrate according to any one of claims 1 to 4 and a nucleic acid chain present in the sample Carrying out a reaction;
Electrochemically detecting the presence or absence of hybridization between the probe and the nucleic acid chain;
A method comprising the steps of: The detection method according to claim 4,
A detection method further comprising a step of allowing a double-stranded DNA recognition substance to act in order to perform the electrochemical detection. The detection method according to claim 5, comprising:
The double-stranded DNA recognition substance is an intercalating agent. A nucleic acid detection apparatus for performing the method according to any one of claims 4 to 6,
The nucleic acid detection system includes at least a nucleic acid extraction unit, a nucleic acid amplification unit, and a nucleic acid detection unit for detecting a nucleic acid using the nucleic acid detection substrate according to any one of claims 1 to 4. Equipment. 9. The nucleic acid detection device according to claim 8, wherein the nucleic acid detection device performs a chip rack, a reagent rack, a waste liquid rack, a movable dispenser capable of dispensing a reagent, and current measurement. It has a movable prober set that can
The nucleic acid amplification unit and the detection unit each include a thermal cycler, and the nucleic acid detection device further includes a vacuum pump, a syringe pump, and an electric control unit.

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