JP4398953B2 - Nucleic acid detection sensor - Google Patents

Description

The present invention relates to a nucleic acid detection sensor that electrochemically detects whether or not a target nucleic acid chain in a test solution has a specific base sequence.

2. Description of the Related Art In recent years, gene testing techniques using nucleic acid chain fixed arrays (DNA arrays) have attracted attention as nucleic acid detection sensors. (See Non-Patent Document 1)
The DNA array refers to a glass or silicon array of several centimeters square on which 10 ¹ to 10 ⁵ kinds of different DNAs are immobilized. A test liquid gene labeled with a fluorescent dye or a radioisotope (RI) is reacted on the array, or a mixture of an unlabeled test liquid gene and a labeled oligonucleotide is reacted by sandwich hybridization. When a sequence complementary to the DNA on the array is present in the test solution, 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 are known in advance, the base sequence present in the test solution gene can be easily examined. DNA array
Since a lot of information on the base sequence can be obtained with a small amount of sample, it is highly expected as a sequence technique as well as a gene detection technique (see Non-Patent Document 2).

Methods for detecting nucleic acids bound to the array include a fluorescence detection method, an RI intensity detection method, an electrochemical detection method, and the like. Among them, the electrochemical method does not require sample gene labeling or a complicated system. Therefore, a reduction in the size of the system can be expected. In addition, since an electrode is used, there is an advantage that electrical reaction control can be easily performed.

In particular, among nucleic acid detection sensors using an electrochemical method, a sensor that constitutes a DNA array in which a plurality of electrodes to which different types of probe nucleic acid chains are immobilized is arranged in an XY matrix is a variety of nucleic acids. It is expected as a very useful technique that can detect a short time. However, this sensor must apply an equal voltage to a large number of nucleic acid chain fixed electrodes. Therefore, this sensor has problems such as a complicated circuit configuration and insufficient response speed and accuracy.
"Beattie et al. 1993, Fodor et al. 1991, Khrapko et al. 1989, Southern et al. 1994" "Pease et al. 1994, Parinov et al. 1996"

An object of this invention is to provide the nucleic acid detection sensor which can detect many types of nucleic acid at high speed and with high precision.

The nucleic acid detection sensor according to the present invention comprises:
Power supply,
A plurality of nucleic acid chain-immobilized electrodes on which probe nucleic acid chains are immobilized;
A counter electrode for passing an electric current between the nucleic acid chain-immobilized electrode;
A reference electrode provided for each of the nucleic acid chain fixed electrodes;
A first amplifier provided for each reference electrode and connected to the input electrode on the input side;
A reference resistor provided for each reference electrode and connected to the output of the first amplifier;
A second amplifier for connecting the reference resistor and the power source to the input side, connecting the counter electrode to the output side, and outputting power of a predetermined voltage to the counter electrode;
A third amplifier provided for each of the nucleic acid chain fixed electrodes, wherein the nucleic acid chain fixed electrode is connected to the input side, and the power source is connected to the output side;
It is a nucleic acid detection sensor, characterized in that it comprises a.

  Since the reference electrode is disposed on each nucleic acid chain fixed electrode, the measurement sensitivity is improved.

As described above, according to the present invention, it is possible to provide a nucleic acid detection sensor capable of detecting many kinds of nucleic acids at high speed and with high accuracy.

The nucleic acid detection sensor according to the embodiment of the present invention preferably includes the following components.
(1) The nucleic acid chain fixed electrode and the counter electrode are arranged to face each other.
(2) A reference electrode is arranged for each nucleic acid chain fixed electrode.
(3) The signal output line is shared by the switching element.

In the present specification, the “nucleic acid detection cell” is a unit compartment (unit) comprising a pair of nucleic acid chain fixed electrodes and a counter electrode in the nucleic acid detection sensor of the present invention in which a plurality of nucleic acid chain fixed electrodes are arranged. Cell).

A probe nucleic acid chain is immobilized on the nucleic acid chain fixed electrode so as to hybridize the target nucleic acid chain in the test solution. The nucleic acid chain fixed electrode functions as a working electrode in the nucleic acid detection sensor of the present invention. The “probe nucleic acid chain” refers to a nucleic acid chain immobilized (coupled) to a nucleic acid chain-immobilized electrode. The “target nucleic acid chain” means a nucleic acid chain that has a base sequence complementary to the probe nucleic acid chain and that undergoes a hybridization reaction with the probe nucleic acid chain, and is included in the test solution. To do.

  The counter electrode functions as an auxiliary electrode for flowing current between the nucleic acid chain fixed electrode.

Furthermore, the measurement method of the nucleic acid detection cell of the nucleic acid detection sensor of the present invention uses the nucleic acid chain fixed electrode and the counter electrode, and is arbitrarily arranged between the nucleic acid chain fixed electrode and the counter electrode. This is a two-electrode type electrochemical measurement in which a reference electrode is further connected to a two-electrode type nucleic acid chain-immobilized electrode or a counter electrode to detect an electrochemical change occurring between both electrodes. In the two-electrode system, since a current flows through the counter electrode, the carrier concentration at the electrode / interface that determines the potential of the counter electrode changes, so that the reference potential itself changes. On the other hand, in the three-electrode system, the current flows between the nucleic acid chain fixed electrode and the counter electrode, almost no current flows between the nucleic acid chain fixed electrode and the reference electrode, and between the counter electrode and the reference electrode. On the other hand, since a voltage is applied between the nucleic acid chain fixed electrode and the counter electrode so that a desired potential is applied, the reference potential (reference electrode potential) does not vary.

The knowledge about the target nucleic acid chain or the probe nucleic acid chain is obtained as follows by the nucleic acid detection sensor of the present invention. In the presence of a test solution containing a nucleic acid chain, a voltage is applied between the nucleic acid chain fixed electrode and the counter electrode in the nucleic acid detection cell. After hybridization is generated between the target nucleic acid strand and the probe nucleic acid strand, an electrochemical change occurring between the electrodes is detected. When the target nucleic acid strand hybridizes with the probe nucleic acid, an electrochemical change occurs between the electrodes. Therefore, if the change is detected, the probe nucleic acid strand or the target nucleic acid strand is
Whether or not it has a specific base sequence can be detected. The electrochemical change generated between the electrodes by the hybridization described above is preferably a current change resulting from the chemical change of the double-stranded recognizer when a double-stranded recognizer is added to the test solution. Thereby, the measurement can be performed easily and accurately.

A nucleic acid chain having a known base sequence is used as a probe nucleic acid chain to be immobilized on the nucleic acid chain-immobilized electrode. It may be detected whether or not a target nucleic acid chain that undergoes a hybridization reaction with the probe nucleic acid chain is present in the test solution. In addition, a nucleic acid chain having an unknown base sequence is used as a probe nucleic acid chain to be immobilized on the nucleic acid chain-immobilized electrode. Then, a nucleic acid chain having a known base sequence is contained in the test solution, and it is detected whether or not a target nucleic acid chain that undergoes a hybridization reaction with the probe nucleic acid chain exists in the test solution. In this way, knowledge on the sequence of the probe nucleic acid chain having the unknown base sequence may be obtained.

Typically, different types of probe nucleic acid strands are immobilized on each of the plurality of nucleic acid strand-immobilized electrodes. In order to supply different samples for each cell and test several samples at a time, the same type of probe nucleic acid chain may be immobilized.

Since each nucleic acid detection cell has one nucleic acid chain-immobilized electrode, the target nucleic acid chain or probe nucleic acid chain can be determined by examining which nucleic acid detection cell the target nucleic acid chain has hybridized to. The knowledge about the sequence of is obtained.

Therefore, a switching circuit, a decoder circuit, or a timing circuit for applying an electrical signal to each nucleic acid chain fixed electrode of each cell so that each nucleic acid detection cell operates independently;
It is desirable to arrange a circuit for outputting an electric signal from each nucleic acid chain fixed electrode and a switching circuit for outputting the electric signal from each nucleic acid chain fixed electrode to the outside.

A plurality of scanning lines are connected to a circuit such as the switching circuit for applying an electric signal to each nucleic acid chain fixed electrode of each cell. The scanning line is supplied with a signal for closing a switching element such as a transistor, preferably a thin film transistor, disposed between the nucleic acid chain fixed electrode and the signal line. In the present specification, the “signal line” means a conducting wire that transmits a signal indicating an electrical change from the nucleic acid chain fixed electrode that is a working electrode. When the switching element is closed by a signal from the scanning line, a voltage is applied to the nucleic acid chain fixed electrode to cause an electrochemical change. A change in voltage (or current) due to the change is transmitted through the signal line. In order to control such a plurality of electrodes, it is desirable to use a matrix system used for liquid crystal display. Further, an active matrix system using a MOSFET is desirable. A MOS image sensor type scanning circuit can also be used.

FIG. 01 shows a structure of a typical nucleic acid detection sensor provided with a circuit for applying a voltage to each nucleic acid chain fixed electrode. FIG. 01 shows the case of a two-electrode measurement method. In the nucleic acid detection sensor of FIG. 01, the switching element 10 connected to each nucleic acid chain fixed electrode 102.
3 is a scanning line driving circuit 107 for driving the scanning line 104 from the timing circuit 106.
Are opened and closed by sequentially receiving signals. The counter electrode 101 is a potentiostat circuit 11.
It is connected to a power source (not shown) through 0. When the switching element 103 is sequentially opened and closed, a voltage is applied to the nucleic acid chain fixed electrode 102 and the counter electrode 101). Thereby, the nucleic acid (not shown) hybridized with the nucleic acid chain fixed electrode 102 can be detected electrochemically. The electrochemical change is transmitted to the signal detection circuit 109 via the signal line 105 and detected.

As shown in FIG. 02, the signal line is preferably covered with an insulating material except for the contact with the switching element. FIG. 02 is a side view when the nucleic acid detection cell (dotted rectangle) in the nucleic acid detection sensor of FIG. 01 is cut parallel to the scanning line so as to cross the nucleic acid chain fixed electrode and the counter electrode. . In FIG. 02, a signal line 202 covered with an insulating film 203, a nucleic acid chain fixed electrode 204, and a counter electrode 205 are disposed on an insulating substrate 201.
Since 02 is immersed in the test liquid, the insulating film 203 is covered except for the intersection of the signal line 202 and the contact point of the switching element.

The arrangement of the signal lines, switching elements, and electrodes covered with the insulating material is not limited to the arrangement shown in FIG. FIG. 03 is an example of such an arrangement, and the nucleic acid chain fixed electrode 302 and the counter electrode (or reference electrode) 30 disposed on the insulating substrate 301.
3 are switching elements 304 and 305, respectively. The switching elements 304 and 305 are covered with insulating films 306 and 307 existing on both sides, respectively. If the switching element is placed under each electrode as shown in FIG. 03, the contact portion between the signal line (not shown) and the switching element is tested even when the test solution 308 is added to the upper surface of the nucleic acid chain fixed electrode and the counter electrode. Since it does not come into contact with the liquid 308, it has excellent insulating properties. In the arrangement of FIG. 04, the switching element is placed under each electrode as in FIG. 03, so that the insulation is excellent. However, in the arrangement shown in FIG. 03, the switching element is exposed on the back surface of the substrate. It is different from the arrangement.

Each electrode constituting the nucleic acid detection cell is preferably formed on an insulating substrate. As a material of the insulating substrate, for example, an inorganic insulating material such as glass, quartz glass, silicon, alumina, sapphire, forsterite, silicon carbide, silicon oxide, silicon nitride, or polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate , Unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate,
Polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile,
Organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicon resin, polyphenylene oxide, polysulfone can be used. Although there is, it is not limited to these.

It is desirable that each electrode, circuit, etc. are separated via an insulating material. 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 PMMA and other substances described in the above documents can be used for the electron beam resist. Is possible. 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. Thereby, the immobilized amount of the probe nucleic acid chain becomes uniform between the respective electrodes, and measurement with excellent reproducibility is possible. Conventionally, the resist material is generally finally removed. However, in the nucleic acid chain fixed electrode, the resist material can be used as a part of the electrode without being removed. In this case, it is necessary to use a substance having high water resistance for the resist material to be used. It is possible to use materials other than the photoresist material for the insulating layer formed on the electrode. For example, Si, Ti, Al, Zn, Pb, Cd, W,
It is also possible to use oxides such as Mo, Cr, Ta, and Ni, nitrides, carbides, and other alloys. 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.

On the insulating substrate, preferably 10 1 to 10 5 nucleic acid chain fixed electrodes are arranged. The preferred nucleic acid chain-immobilized electrode material is gold, but other materials can be used, such as gold alloys, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium,
A simple metal such as tungsten or an alloy thereof, carbon such as graphite or glassy carbon, or an oxide or a compound thereof can be used. 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 direct current bipolar sputtering, bias sputtering, asymmetrical alternating current 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 Willson method.

Orientation index (hkl) = IF (hkl) / IFR (hkl)
hkl; face index IF (hkl); (hkl) relative strength of face IFR (hkl); IF as standard gold described on the ASTM card (hkl)
Here, in the case of a nucleic acid chain fixed electrode for detecting a nucleic acid chain, the orientation index is required to be 1 or more, and the orientation index is preferably 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, the amount of nucleic acid chain immobilized can be controlled uniformly. In addition, when the above electrode material such as gold is vapor-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. Therefore, a stable electrode layer can be formed.

The shape of the nucleic acid chain fixed electrode is not particularly limited, and the shape as shown in FIGS. Use of the shapes of FIGS. 06 and 07 is advantageous because the contact area between the nucleic acid-immobilized electrode and the counter electrode (or reference electrode) is large. FIGS. 05 to 07 are enlarged views of the nucleic acid detection cell (dotted rectangle in FIG. 01). As in FIG. 01, the nucleic acid chain fixed electrodes 501, 601 and 701 are respectively switched. The signal lines 505, 605, and 705 are connected to each other through the elements 503, 603, and 703. The counter electrodes (or reference electrodes) 502, 602, and 702 are disposed in the vicinity of the nucleic acid chain fixed electrodes 501, 601, and 701.

In order to immobilize the probe nucleic acid chain on the nucleic acid chain-immobilized electrode, it is desirable to activate the electrode surface. Activation can be performed by a potential sweep in a sulfuric acid solution.

Activation can also be performed with mixed acid, aqua regia, and the like. The material constituting the probe nucleic acid chain is not particularly limited, but DNA, RNA, PNA, and other nucleic acid analogs can be used.

The method for immobilizing the probe nucleic acid chain is not particularly limited. For example, immobilization can be easily performed by utilizing a bond between a thiol group introduced into a probe nucleic acid chain and gold. In addition, immobilization is possible by physical adsorption, chemical adsorption, hydrophobic bond, embedding, covalent bond, and the like. Further, a condensing agent such as a biotin-avidin bond or carbodiimide can also be used. In these cases, immobilization can be facilitated by modifying the electrode surface with molecules having functional groups in advance. Furthermore, in order to suppress non-specific adsorption of nucleic acids and intercalating agents on the electrode surface, it is desirable to coat the electrode surface with a mercaptan such as mercaptoethanol or a lipid such as stearylamine.

Hereinafter, as an example, a method for immobilizing a probe nucleic acid chain to a nucleic acid chain immobilized electrode made of gold will be described. The electrode is washed with deionized water and then activated. 0.1-10mm for activation
Use an ol / L sulfuric acid solution. In this solution, -0.5 to 2 V (vs Ag / AgCl)
The potential is scanned in the range of 1 v / s to 100,000 v / s. Thereby, the electrode surface is activated to a state where the probe nucleic acid chain can be immobilized. A thiol group is introduced into the 5 ′ or 3 ′ end of the probe nucleic acid strand used for immobilization. The thiolated probe nucleic acid strand is dissolved in a solution of 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 is very simple and the ionic strength is 0
. Probe nucleic acid strands in a buffer solution in the range of 01 to 5 and pH 5 to 10
The electrode immediately after being dissolved and activated so as to be in the range of 1 mg / mL is immersed. The immobilization reaction is performed in the range of 4 to 100 ° C. for about 10 minutes to overnight.

It is desirable to store the electrode after immobilizing the probe nucleic acid chain in a condition where no nucleolytic enzyme (nuclease) is present, and to be shielded from light if possible. However, in the short-term case, 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 or deionized water. Furthermore, the storage temperature is 4 ° C. or lower, preferably −20 ° C. Moreover, when storing the nucleic acid chain | strand fixed electrode which fix | immobilized the probe nucleic acid chain | strand for a long period of time, it is desirable to preserve | save in a dry state. Although the method to make it dry is not specifically limited, It can carry out by freeze-drying, air drying, etc. The dry 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.

When the probe nucleic acid chain is immobilized on the electrode, the probe nucleic acid chain can be immobilized relatively easily by using an immobilization device 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. It is also possible to synthesize nucleic acid chains directly on the electrode surface.

One or more counter electrodes are arranged in the nucleic acid detection sensor of the present invention. In the case where a single counter electrode is disposed, a plurality of nucleic acid chain-immobilized electrodes commonly use a single counter electrode.

The distance between the nucleic acid chain fixed electrode and the counter electrode is not particularly limited as long as a desired voltage can be applied to the nucleic acid chain fixed electrode. In order to increase the response speed, for example, it is preferable to arrange at a distance within 1 cm.

In order to apply an equal voltage to all the nucleic acid chain fixed electrodes, it is preferable to arrange the counter electrode so as to be an equal distance from all the nucleic acid chain fixed electrodes.

The material used for the counter electrode is not particularly limited, and the material used for the nucleic acid chain fixed electrode can be used.

When measuring the nucleic acid detection sensor of the present invention by the three-electrode method, a reference electrode is arranged. Silver/
A silver chloride electrode, a mercury / mercury chloride electrode, or the like can be used as a reference electrode, but any other electrode can be used.

The reference electrode is generally disposed on the same substrate as the nucleic acid chain fixed electrode, but may be disposed at a site other than this.

The shape of the counter electrode or the reference electrode is not particularly limited, but a shape that increases the surface area and does not hinder the flow of the test solution is preferable in order to increase measurement accuracy. For example, such a condition is satisfied if the counter electrode or the reference electrode and the nucleic acid chain-immobilized electrode are in a comb shape.

The nucleic acid detection sensor according to the present invention preferably constitutes a nucleic acid detection system. The system includes one or a plurality of substrates on which a plurality of nucleic acid detection cells are formed, a sealed container for holding the substrates, and at least one opening for feeding a liquid, and a liquid. It is based on the structure provided with the container which has the space for storing A, and the terminal for connecting to an external apparatus.

On the system, there are a circuit for applying an electric signal to the counter electrode and the nucleic acid chain fixed electrode, a switching circuit for applying an electric signal to each counter electrode and each nucleic acid chain fixed electrode, and each nucleic acid chain fixed. It is desirable to include a circuit for outputting an electric signal from the electrode, a switching circuit for outputting the electric signal from each nucleic acid chain fixed electrode to the outside, a power source, a potentiostat, and a waveform generator. In addition, the system includes a MOSFET switching element at a specific position arranged on a matrix, a decoder circuit for outputting an electrical signal to the nucleic acid chain fixed electrode, a switching circuit, a timing circuit, a memory, an A / D converter, a waveform It is desirable to integrate circuits such as a generator, a power source, a potentiostat, and an electric signal detection circuit on one sensor.

In the nucleic acid detection system, a nucleic acid extraction mechanism, a nucleic acid purification mechanism, a nucleic acid amplification mechanism, and the like can be integrated. If a nucleic acid detection system provided with these mechanisms is used, a series of operations such as extraction, amplification, and detection of nucleic acids can be performed automatically.

FIG. 08A, FIG. 08B, and FIG. 08C show an example of a nucleic acid detection sensor that can constitute a nucleic acid detection system.

The nucleic acid detection sensors in FIGS. 08A, 08B, and 08C are arranged at a plurality of scanning lines 801, signal lines 802 arranged so as to be orthogonal to the scanning lines 801, and intersections of the scanning lines 801 and 802. A switching element 803 such as a thin film transistor, and a switching element 8
The first nucleic acid chain fixed electrode 804 connected to 03, a scanning line driving circuit 805 for driving each scanning line 801, and a signal line driving circuit 806 for driving each signal line 802 are installed. A substrate 807 (FIG. 08A) and a second substrate 809 (FIG. 08C) on which each counter electrode 808 is installed.
). Each counter electrode 808 is connected to a potentiostat (not shown). Note that in FIG. 08A and FIG. 08B, only one nucleic acid chain-immobilized electrode is written, but actually each rectangle surrounded by two adjacent scanning lines and two adjacent signal lines. One nucleic acid chain fixed electrode 804 is installed therein.

In order to detect the target nucleic acid in the test solution, the test solution is injected into a space interposed between the first substrate 807 and the second substrate 809, and then a driving signal is sent from the scanning line driving circuit 805 to the switching element 803. give. The switching element 803 is turned on by the driving signal output from the scanning line driving circuit 805, and the nucleic acid chain fixed electrode 804 and the signal line 802 are electrically connected. When the nucleic acid chain fixed electrode 804 and the signal line 802 are electrically connected, a voltage is applied between the nucleic acid chain fixed electrode 804 and the counter electrode 808. Thereby, for example, a substance such as an insertion agent inserted into the target nucleic acid hybridized with the nucleic acid chain fixed electrode 804 is oxidized. The current generated by the oxidation reaches the pad 810 provided at one end of the signal line 802 through the signal line 802, and is detected and quantified by an external device for current detection connected to the pad 810. The nucleic acid detection sensors shown in FIGS. 08A, 08B, and 08C are the same as the nucleic acid detection unit 8.
11. A scanning line driving circuit 805 and a signal line driving circuit 806 are integrally formed on a first substrate 807, and are used by being mounted on a nucleic acid detection system including a signal detection unit.

In addition, the counter electrode 905 may have a shape as shown in FIGS. 09A and 09B, and may be installed in the vicinity of the nucleic acid chain fixed electrode, being electrically connected to the scanning line 901. 09A and 09B
Then, the counter electrode 905 has a comb-shaped shape with each of the three branches, and is arranged so as to mesh with the nucleic acid chain fixed electrode 905 having the same shape.

Although FIG. 8A, FIG. 8B, FIG. 9A, and FIG. 9B show a nucleic acid detection sensor in which no reference electrode is provided, it is preferable to provide a reference electrode. As shown in FIGS. 9A and 9B, the reference electrode may be a comb electrode that meshes with the nucleic acid chain fixed electrode.

As for the circuit example of the embodiment in which the reference electrode is provided for each nucleic acid chain fixed electrode,
It will be described later.

FIG. 10 shows an outline of a nucleic acid detection system in which the nucleic acid detection sensor of the present invention is arranged.

A nucleic acid detection system 1007 shown in FIG. 10 includes a nucleic acid detection sensor 1001, a nucleic acid detection sensor fixing device 1002, an electric signal measurement device 1003, a CPU 1004, a power source 1005,
And a display device 1006.

In the above system, the nucleic acid detection sensor is normally installed on the substrate 1102 so as to be detachable by the connection terminal 1101 and stored in the container 1108 as shown in FIGS. 11A and 11B. As shown in FIG. 12, the board 1102 has, for example, a connection terminal insertion portion 1201 around it.
have. 11A and 11B, the test solution 1103 is the nucleic acid detection sensor 1.
The test liquid discharge port 1106 is closed so that 104 can be immersed, and the liquid is injected from a test liquid injection port 1105 provided at the bottom. Nucleic acid detection sensor 11 using test liquid 1103
After dipping 04, the nucleic acid contained in the test solution 1103 is hybridized to a nucleic acid chain fixed electrode (not shown) on the nucleic acid detection sensor 1104. When the nucleic acid detection sensor 1104 is heated during hybridization, the vaporized test liquid is discharged through the air hole 1107. If the target nucleic acid is contained in the test solution, the target nucleic acid hybridizes to a nucleic acid chain fixed electrode (not shown) on the nucleic acid detection sensor 1104. Therefore, test solution 11
Even after 03 is discharged from the test liquid discharge port 1106, it continues to bind to the nucleic acid chain fixed electrode. As shown in FIGS. 13A and 13B, the test liquid inlet 1305 and the test liquid outlet 1306 may be provided at a vertical position of the substrate 1302.

Hereinafter, an operation for obtaining knowledge about a target nucleic acid chain or a probe nucleic acid chain in a test solution using the nucleic acid detection sensor of the present invention will be described in detail.

First, a test solution containing a target nucleic acid chain is injected into a space interposed between the nucleic acid chain fixed electrode and the counter electrode.

The target nucleic acid chain to be detected is not particularly limited, and may be a nucleic acid chain such as a virus, a bacterium, a fungus, or a parasite, a gene causing a genetic disease, a marker gene for various diseases, or the like. For example, hepatitis virus (A, B, C, D, E, F, G type), HIV, influenza virus, herpes virus, adenovirus, human polyoma virus, human papilloma virus, human parvovirus, mumps virus, human Viral infections such as rotavirus, enterovirus, Japanese encephalitis virus, dengue virus, rubella virus, HTLV, Staphylococcus aureus, hemolytic streptococci, pathogenic E. coli, Vibrio parahaemolyticus, Helicobacter pylori, Campylobacter, Vibrio cholerae, Shigella , Salmonella, Yersinia, Neisseria gonorrhoeae, Listeria monocytogenes, Leptospira, Legionella, Spirochete, Mycoplasma pneumonia, Rickettsia, Chlamydia, Malaria, Shigella amoeba, pathogenic fungi, etc., can be used for detection of parasites and fungi. 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, Stomach cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, thyroid tumor, breast tumor,
It can also be used for examination of neoplastic diseases such as urological tumors, male organ tumors, female organ tumors, skin tumors, bone / soft tissue tumors, leukemias, lymphomas, solid tumors and the like. 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),
Detection of microsatellite arrays and the like is also possible. It can also be used for analysis of unknown base sequences.

The test fluid containing these target nucleic acids is not particularly limited, and for example, blood, serum, leukocytes, urine, stool, semen, saliva, tissue, cultured cells, sputum and the like can be used. From these test solutions, nucleic acid components are usually extracted. The extraction method is not particularly limited, and a liquid-liquid extraction method such as a phenol-chloroform method or a solid-liquid extraction method using a carrier can be used. Further, a commercially available nucleic acid extraction method QIAamp (manufactured by QIAGEN), Sumitest (manufactured by Sumitomo Metals), or the like can also be used.

After injecting the test solution into the space, a hybridization reaction is performed between the extracted nucleic acid component and the nucleic acid chain detection electrode. The reaction solution has an ionic strength in the range of 0.01-5, pH 5-10.
In a range of buffers. In this solution, it is possible to add dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant, and the like. The nucleic acid component extracted here is added and heat-denatured at 90 ° C. or higher. The insertion of the nucleic acid chain detection 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 is 10 ° C to 9 ° C.
The reaction is performed in the range of 0 ° C. and the reaction time is about 1 minute to overnight. The hybridization reaction can be controlled electrochemically, and by applying a positive potential to the nucleic acid chain-immobilized electrode, it has been possible to shorten what was conventionally required from several hours to several days to several minutes. On the other hand, when a negative potential is applied to the electrode surface, nonspecific binding can be removed.

  When the hybridization reaction is completed, the nucleic acid chain fixed 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, a double-stranded recognizer that selectively binds to the double-stranded part formed on the electrode surface (hybrid of probe nucleic acid strand and target nucleic acid strand), that is, an intercalator is allowed to act, and electrochemical measurement is performed. Do. The intercalating agent used here is not particularly limited. For example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator, polyintercalator and the like are used. It is possible. It is also possible to use metal complexes such as ruthenium, cobalt and iron called metallointercalators, organic compounds such as ethidium bromide, biopolymers such as antibodies and enzymes.

The concentration of the intercalating agent varies depending on the type, but is generally 1 ng / mL to 1 mg / mL
Use within the range. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 is used.

After the nucleic acid chain fixed electrode is reacted with the intercalating agent, it is washed and subjected to electrochemical measurement. Electrochemical measurement is performed by a three-electrode system, that is, a reference electrode, a counter electrode, and a working electrode. 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. For the measurement, current and voltage are controlled by using a potentiostat, a digital multimeter, a function generator or the like. Based on the obtained current value, the concentration of the target gene is calculated from the calibration curve.

The electrochemical signal can use a change in redox current, a change in redox potential, a change in capacitance, a change in resistance, and a change in electrochemiluminescence as an index. These signal changes are promoted by the combined use of substances that specifically bind to double-stranded nucleic acids such as intercalating agents.

The first nucleic acid detection sensor of the present invention is characterized in that the nucleic acid chain fixed electrode and the counter electrode are arranged to face each other so that the test solution flows between the nucleic acid chain fixed electrode and the counter electrode. .

A nucleic acid detection sensor constituting a conventional DNA array is shown in FIGS. 14C and 14D.
A plurality of nucleic acid chain-immobilized electrodes 140 on which probe nucleic acid chains 1402 having a known sequence are immobilized
1 and the counter electrode 1404 are disposed on the same substrate 1403, and the test liquid 1406 flows on the substrate 1403. In this arrangement, the distance between the counter electrode 1404 and each nucleic acid chain fixed electrode 1402 is different for each nucleic acid fixed electrode 1401. In such a configuration, the distance between the nucleic acid chain-immobilized electrode 1401 and the counter electrode 1404 may become far as shown at the left end in the figure, and the response speed becomes slow. Further, the distance between the counter electrode 1404 and each nucleic acid chain fixed electrode 1401 is equal to each nucleic acid chain fixed electrode 14.
Since it differs every 01, sufficient measurement accuracy cannot be achieved.

In contrast, in the first nucleic acid detection sensor of the present invention shown in FIGS. 14A and 14B,
The nucleic acid chain fixed electrode 1401 on which the probe nucleic acid chain 1402 is fixed and the counter electrode 1404 are plate-like electrodes and are arranged to face each other so that the test solution 1406 can be held between them. According to this arrangement, all the nucleic acid chain fixed electrodes 1401 on the first substrate 1403 are all on the second substrate 1405.
It can be arranged at an equal distance from the upper counter electrode 1404 and in the vicinity of the counter electrode 1404.
For this reason, if a nucleic acid detection sensor having electrodes arranged in such an arrangement is used, the target nucleic acid chain to be detected in the test solution 1406 hybridized with the probe nucleic acid chain 1402 on each nucleic acid chain fixed electrode 1401. It is possible to apply a voltage equal to all. Therefore, measurement accuracy and response speed are improved. In addition, since the test solution 1306 is injected between the first substrate 1303 and the second substrate 1305, the amount of necessary test solution can be reduced. Note that FIG.
A shows a nucleic acid detection sensor in which the reference electrode 1407 is not disposed on the first substrate 1403. FIG. 14B shows a nucleic acid detection sensor in which a reference electrode 1407 is arranged on a first substrate 1403.

In the first nucleic acid detection sensor of the present invention, when the nucleic acid chain fixed electrode is formed on the insulating substrate, the counter electrode is fixed with the nucleic acid chain so as to sandwich the flow path of the test solution together with the nucleic acid chain fixed electrode. It is arranged on a substrate different from the substrate on which the activating electrode is arranged.

The counter electrode and the nucleic acid chain fixed electrode may be arranged on different substrates, but the counter electrode is arranged at an equal distance from all the nucleic acid chain fixed electrodes in order to apply the same electrode to all the nucleic acid chain fixed electrodes. It is preferable to do. For example, when the nucleic acid chain fixed electrode is disposed on a plane, the counter electrode may be disposed on a plane parallel to the plane. When the nucleic acid chain fixed electrode is disposed on a spherical surface, the counter electrode may be disposed on a spherical surface concentric with the spherical surface.

In the first nucleic acid detection sensor of the present invention, the nucleic acid chain-immobilized electrode and the counter electrode both have flat surfaces, and the flat surfaces are arranged so that the flat surfaces face each other. Is desirable.

The first nucleic acid detection sensor of the present invention includes a plurality of nucleic acid detection cells, and each cell is provided with one or more nucleic acid chain fixed electrodes. One counter electrode may be provided for each nucleic acid chain fixed electrode. Common among a plurality of nucleic acid detection cells, that is, for example, one counter electrode may be provided for a plurality of nucleic acid chain fixed electrodes.

The distance from the counter electrode material and the nucleic acid chain fixed electrode to be arranged in the first nucleic acid detection sensor of the present invention is as described above.

When a reference electrode is further arranged on the first nucleic acid detection sensor of the present invention, it is generally arranged on the same substrate as the nucleic acid-immobilized electrode. You may arrange | position a reference electrode in parts other than this.

  The second nucleic acid detection sensor of the present invention is characterized in that a reference electrode is provided in each cell.

In the second nucleic acid detection sensor of the present invention, the counter electrode may be common to the plurality of nucleic acid chain-immobilized electrodes, or may be disposed for each nucleic acid detection cell. When a plurality of counter electrodes are arranged, the counter electrodes may be connected to either the signal line or the scanning line.

The materials of the nucleic acid chain fixed electrode, the counter electrode, and the reference electrode, the operation for measuring the nucleic acid, and the like are as described in the general configuration and usage of the nucleic acid detection sensor of the present invention described above. That is, by using an electrochemical reaction by a hybrid nucleic acid chain formed between a probe nucleic acid chain and a target nucleic acid chain, it is detected whether the probe nucleic acid chain or the target nucleic acid chain has a specific base sequence. .

Thus, if a reference electrode is provided for each nucleic acid chain fixed electrode, the uncompensated resistance between the nucleic acid chain fixed electrode and the reference electrode is reduced, and the measurement accuracy is improved. It is desirable to provide a reference electrode for each nucleic acid chain fixed electrode so that the potential can be controlled for each nucleic acid chain fixed electrode.

The second nucleic acid detection sensor of the present invention includes, for example, an operational amplifier 1607 functioning as a control amplifier, a voltage floor amplifier, and a current floor amplifier as shown in FIG.
, An operational amplifier 1608 and a potentiostat circuit for measuring a minute current provided with an operational amplifier 1609 is used. For simplicity, the potentiostat circuit of FIG. 15 shows only one nucleic acid chain-immobilized electrode, but actually, the second nucleic acid detection sensor of the present invention has a plurality of nucleic acid chain-immobilized electrodes. Electrodes are disposed.

This circuit includes three operational amplifiers each having the functions of a control amplifier, a voltage floor amplifier, and a current floor amplifier. These circuits differ from conventional circuits in that they are for measuring minute currents. Therefore, the potentiostat circuit that can be used in the nucleic acid detection sensor of the present invention may be used for measuring a minute current.

  The function of each operational amplifier in the circuit of FIG. 15 is as follows.

The operational amplifier 1607 forms a part of an inverting amplifier, and the counter electrode 1602 has ef (here, ef means the potential at the point f when the common potential is used as a reference, and so on).
By applying a voltage (1 + Zf / Rf) times that of ef to ea (ie Vcc)
(Where Zf represents the impedance of the electrochemical system from the counter electrode 1602 to the reference electrode 1603). Since the operational amplifier has negative feedback, ea is eb (
Common potential). In the figure, the common is grounded, but it is not always necessary to ground.

The operational amplifier 1608 has a function of amplifying the input power by Zin / Zout times (
Zin and Zout are input impedance and output impedance, respectively).
Since Zin is very high compared to Zout, the output power is significantly higher than the input power. Due to the function of the operational amplifier 1608, the internal resistance of the reference electrode 1603 can be ignored.

Since the operational amplifier 1609 also has a negative feedback, eg is equal to eh. Therefore, when the nucleic acid chain fixed electrode 1601 is connected to a signal by the switching element 1604, the potential of the nucleic acid chain fixed electrode 1601 is the common potential. Equal to the potential. Therefore, the operational amplifier 1609 is
It plays the role of maintaining the potential of the nucleic acid chain fixed electrode 1601 as the working electrode at a common potential. Assuming that the input voltage is V, if the resistance ratio between the point 0 and the point a (not shown) and the ratio of the resistance between the point a and the point f are set to 1, the potential of the reference electrode 1603 is -V. What is necessary is just to select suitably the resistance value of the resistance in a circuit, and the presence or absence of use of a resistance according to a desired gain. Since the potential of the nucleic acid chain fixed electrode 1601 is equal to the common potential, a voltage exactly equal to the input voltage is applied between the nucleic acid chain fixed electrode 1601 (working electrode) and the reference electrode 1603. Since the point g is virtually grounded, a current generated by applying a voltage to the nucleic acid chain fixed electrode 1601 by the switching element 1604 connected to the scanning line 1606 passes through the resistor 1610 from the point g on the signal line 1605. Point i is reached. Resistance 161
By measuring the voltage drop due to zero, the magnitude of the current can be measured.

When the resistor 1610 is placed between the point g and the point i, an error occurs in the potential of the nucleic acid chain fixed electrode 1601 due to the potential difference between both ends of the resistor. However, even if the resistor 1610 is placed between the points g and i,
Since eg is kept at a common potential, no error occurs in the potential of the nucleic acid chain fixed electrode 1601. Therefore, highly accurate electrochemical measurement is possible.

The circuit of FIG. 16 is another potentiostat circuit used for the second nucleic acid detection sensor, and has a function of keeping the voltage constant as in the circuit of FIG. Therefore, operational amplifier 170
7, 1708 and 1709 have the same functions as the corresponding operational amplifiers in the circuit of FIG.

In the nucleic acid detection sensor of the present embodiment, the reference electrode is arranged for each nucleic acid chain fixed electrode, as in FIG. 15, and therefore has a measurement accuracy compared to the conventional circuit.

In FIG. 16, for the sake of simplicity, only one reference electrode is drawn.
One or more nucleic acid chain fixed electrodes are arranged.

In the circuit of FIG. 16, since the potential applied to the scanning line also serves as the reference potential, the wiring that is output from the non-inverting input terminal of the operational amplifier 1708 is commonly used for a plurality of electrodes. It is not included in the number of wirings per detection cell.

As described above, the nucleic acid detection sensor of the present embodiment can achieve very high measurement sensitivity with simple wiring.

The circuit of FIG. 17 is still another potentiostat circuit used in the nucleic acid detection sensor of the second embodiment, and the circuit of FIG. 17 has a function of keeping the voltage constant as in the circuits of FIGS. 15 and 16. Have Therefore, the details of the functions of the potentiostats 1807, 1808, and 1809 are as described in FIG. 15 or FIG.

The circuit in FIG. 17 is different from the circuit in FIG. 16 in that the reference electrode 1803 is connected to the signal line 1804 instead of the scanning line 1806. For this reason, the circuit of FIG.
Is not connected to the scanning line 1806. Therefore, the reference potential of the reference electrode is equal to the scanning line 180.
The potential to be applied can be freely set. For this reason, in the circuit of FIG. 17, many kinds of intercalating agents can be used compared with the circuit of FIG.

In FIG. 17, the reference electrode 1803 is connected to the switching element 1804, but the switching element may be omitted.

In FIG. 17, the nucleic acid chain fixed electrode 1801 and the reference electrode 1803 are connected to the operational amplifier 180.
It arrange | positions so that the conducting wire connected to 8 non-inverting input terminals may be pinched | interposed. The reference electrode 1803 may be arranged on the same side as the nucleic acid chain fixed electrode 1801 so that both electrodes face each other.

As described above, the circuit of FIG. 17 can achieve high measurement sensitivity and can use many kinds of intercalating agents.

The third nucleic acid detection sensor of the present invention will be described with reference to FIGS.
The third nucleic acid detection sensor of the present invention is characterized in that a signal line is shared by a switching element. FIG. 18 is a top view of a normally used nucleic acid detection sensor. 4x
A nucleic acid chain-immobilized electrode 1901 on which a probe nucleic acid chain (not shown) is immobilized is arranged in the shape of 3 XY matrix. In the actual nucleic acid detection sensor, the counter electrode is located vertically above the plane on which the nucleic acid chain fixed electrode 1901 is disposed, but is omitted in FIG.

  Each nucleic acid chain fixed electrode 1901 forms a nucleic acid detection cell together with a counter electrode.

Each nucleic acid chain fixed electrode 1901 is connected to a signal line 1903 via a switching element 1902 such as a transistor. The signal line 1903 further includes an amplifier 1904 and A for amplifying the current from the nucleic acid chain fixed electrode 1901. / D converter 1905 is connected.

A timing pulse generator 19 is connected to the switching element 1902 via a scanning line 1906.
Since the clock signal is given from 09, the nucleic acid chain fixed electrode 1901 is scanned so as to be sequentially activated one column at a time from the left end in the direction of the arrow in the figure.

A counter 1908 and an X decoder 1907 in FIG. 18 control ON / OFF of signal lines. When the nucleic acid chain fixed electrode 1901 becomes active, the nucleic acid chain fixed electrode 1901 and the counter electrode (
A voltage is applied to the target nucleic acid hybridized on the nucleic acid chain fixed electrode 1901 to oxidize the intercalating agent inserted into the target nucleic acid. An electrical change generated during oxidation is amplified by the amplifier 1904 via the signal line 1903 and then A / D converted by the A / D converter 1905.

FIG. 19 is a diagram showing a circuit example of the third nucleic acid detection sensor of the present invention. The nucleic acid detection sensor of FIG. 19 is different from the nucleic acid detection sensor of FIG. 18 in that switching elements are arranged in the row direction and scanned from the top to the bottom in FIG.

In FIG. 19, the nucleic acid chain fixed electrode 2001 is arranged in a 4 × 3 XY matrix, and each nucleic acid chain fixed electrode 2001 and a counter electrode (not shown) constitute a nucleic acid detection cell. Yes.

Each nucleic acid chain fixed electrode 2001 is connected to a signal line 2004 via an amplifier 2002 and an electrode switching element 2003. A signal line switching element 2005 is connected to one end of each signal line 2004, and then the signal line 2004 becomes one and is connected to the A / D converter 2006.

The electrode switching element 2003 is sequentially supplied with an electrical signal via a signal line 2012 from a column direction scanning circuit constituted by an X decoder 2007 and a counter 2008. On the other hand, the signal line switching element 2005 is sequentially supplied with an electrical signal from a row direction scanning circuit constituted by a Y decoder 2009 and a counter 2010.

As shown in FIG. 20, if the clock signals generated from the timing pulse generator 2011 are supplied to the column direction scanning circuit and the row direction scanning circuit as an X direction clock signal and a Y direction clock signal, respectively, A voltage is applied from the end electrode) to the electrode in the first row, the second row, and the electrode in the first row, the third row, and the second row, the first row. The electrical change caused by the application of voltage is measured as a serial signal, and the output signal is A / D converted by an AD converter.

In the nucleic acid detection sensor of FIG. 19, a nucleic acid detection sensor using a scanning circuit composed of a decoder and a counter in order to sequentially detect an electric signal from the row direction is shown. As shown in FIG. 21, the decoder and counter of FIG. 19 can be replaced with a shift register circuit 2210. The configuration of the nucleic acid detection sensor of FIG. 21 is the same as that of FIG. 19 except that the decoder and counter are replaced with shift register circuits. Thus, the use of the shift register circuit simplifies the external circuit configuration.

The third nucleic acid detection sensor shown in FIGS. 19 and 21 also has an effect that the measurement can be speeded up as compared with the nucleic acid detection sensor shown in FIG.

The first to third nucleic acid detection sensors according to the present invention can be used alone or in appropriate combination.

The schematic diagram which shows the chip | tip for nucleic acid detection of the Example of this invention by which the some electrode is arrange | positioned. The figure which showed arrangement | positioning of the electrode and signal line in the chip | tip for nucleic acid detection of the Example of this invention. The figure which showed other arrangement | positioning of the electrode and signal wire | line in the chip | tip for nucleic acid detection of the Example of this invention. The figure which showed other arrangement | positioning of the electrode and signal wire | line in the chip | tip for nucleic acid detection of the Example of this invention. The enlarged view of the unit division of the chip | tip for nucleic acid detection of the Example of this invention by which the several electrode is arrange | positioned. The enlarged view of the unit division of the chip | tip for nucleic acid detection of the Example of this invention by which the several electrode is arrange | positioned. The enlarged view of the unit division of the chip | tip for nucleic acid detection of the Example of this invention by which the several electrode is arrange | positioned. FIG. 8A and FIG. 08B are views showing a nucleic acid detection chip that can be mounted on a nucleic acid detection system. FIG. 09A, FIG. 09B, and FIG. 09C are diagrams showing a nucleic acid detection chip that can be attached to a nucleic acid detection system. The figure which showed the nucleic acid detection system by which the chip | tip for nucleic acid detection is arrange | positioned. 11A and 11B are diagrams showing a nucleic acid detection chip according to an embodiment of the present invention housed in a container. The figure which shows the board | substrate which should mount | wear with the chip | tip for nucleic acid detection of the Example of this invention. 13A and 13B are diagrams showing a nucleic acid detection chip according to an embodiment of the present invention housed in a container. 14A to 14D are diagrams comparing the nucleic acid detection chip according to the example of the present invention in which electrodes are arranged at positions facing each other and a conventional nucleic acid detection chip in which electrodes are not arranged at positions facing each other. The figure which shows an example of the circuit applied to the Example of the 2nd chip | tip for nucleic acid detection of this invention. The figure which shows another example of the circuit applied to the Example of the 2nd chip | tip for nucleic acid detection of this invention. The figure which shows another example of the circuit applied to the Example of the 2nd chip | tip for nucleic acid detection of this invention. The figure which showed the structure of the chip | tip for nucleic acid detection of the Example of this invention arrange | positioned in the position which the electrode opposed. The figure which showed the wiring in the chip | tip for nucleic acid detection of the Example of this invention arrange | positioned in the position where the electrode opposed. The figure which showed the signal and output signal for applying a voltage to each unit division. The figure which showed the wiring in the chip | tip for nucleic acid detection of the Example of this invention arrange | positioned in the position where the electrode opposed.

Explanation of symbols

1401 ... Nucleic acid chain immobilized electrode 1402 ... Probe nucleic acid chain 1403 ... First substrate 1404 ... Counter electrode 1405 ... Second substrate 1406 ... Test solution 1407 ... Reference electrode

Claims (3)

Power supply,
A plurality of nucleic acid chain-immobilized electrodes on which probe nucleic acid chains are immobilized;
A counter electrode for passing an electric current between the nucleic acid chain-immobilized electrode;
A reference electrode provided for each of the nucleic acid chain fixed electrodes;
A first amplifier provided for each reference electrode and connected to the input electrode on the input side;
A reference resistor provided for each reference electrode and connected to the output of the first amplifier;
A second amplifier for connecting the reference resistor and the power source to the input side, connecting the counter electrode to the output side, and outputting power of a predetermined voltage to the counter electrode;
A third amplifier provided for each of the nucleic acid chain fixed electrodes, wherein the nucleic acid chain fixed electrode is connected to the input side, and the power source is connected to the output side;
Nucleic acid detection sensor, characterized in that it comprises a. The nucleic acid chain-immobilized electrode and the counter electrode are exposed to a test solution containing a nucleic acid chain, and a current change between the nucleic acid chain-immobilized electrode and the counter electrode is caused by hybridization of the probe nucleic acid chain and a nucleic acid chain in a test solution. The nucleic acid detection sensor according to claim 1, wherein 3. The nucleic acid detection sensor according to claim 2 , wherein a change in current between the nucleic acid chain-immobilized electrode and the counter electrode derived from a double-stranded recognizer added after the hybridization is detected.

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