JP4873219B2 - Electrode unit used for specific detection of test substance using photocurrent generated by photoexcitation of sensitizing dye, and sensor cell and measurement apparatus using the same - Google Patents

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to an electrode unit used in a method for specifically detecting a test substance having specific binding properties such as a nucleic acid, an exogenous endocrine disrupting substance, and an antigen using a photocurrent, and uses the same. The present invention relates to a sensor cell and a measuring apparatus.

BACKGROUND ART Gene diagnostic methods for analyzing DNA in biological samples are promising as new preventive and diagnostic methods for various diseases. The following are proposed as techniques for performing such DNA analysis simply and accurately.

  There is known a DNA analysis method in which a sample DNA is hybridized with a DNA probe having a base sequence complementary thereto and a fluorescent substance labeled, and a fluorescent signal at that time is detected (for example, Patent Document 1 (Japanese Patent Laid-Open No. 7-107999) and Patent Document 2 (Japanese Patent Laid-Open No. 11-315095). In this method, the formation of double-stranded DNA by hybridization is detected by fluorescence of the dye.

  In addition, a gene sample denatured into a single strand is hybridized with a complementary single-stranded nucleic acid probe, and then a double-stranded recognizer such as an intercalator is added for electrochemical detection. (See, for example, Patent Document 3 (Patent Publication No. 2573443) and Non-Patent Document 1 (Surface Science Vol. 24, No. 11. Pp. 671-676, 2003)). The cell used in this method has a so-called flow cell structure in which an electrolytic solution flows in the constructed sensor cell because a sensor cell is constructed such that the electrode is immersed after injecting the sample solution into the cell container. Absent.

By the way, a solar cell that generates electrical energy from light using a sensitizing dye is known (see, for example, Patent Document 4 (Japanese Patent Laid-Open No. 1-220380)). This solar cell has a polycrystalline metal oxide semiconductor and has a sensitizing dye layer formed over its surface area over a wide range. However, no attempt has been made to apply such an electrode to biochemical analysis.
JP-A-7-107999 Japanese Patent Laid-Open No. 11-315095 Japanese Patent No. 2573443 Japanese Patent Laid-Open No. 1-220380 Surface Science Vol. 24, No. 11. Pp. 671-676, 2003

Summary of the Invention

  The present inventors detect a photocurrent generated by immobilizing a sensitizing dye on a working electrode through direct or indirect specific binding between a test substance and a probe substance and photoexciting the sensitizing dye. Thus, it has been found that a test substance can be detected and quantified with high sensitivity, simply and accurately. The inventors of the present invention are now an electrode unit having both a working electrode and a counter electrode on the same surface as an electrode material used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. As a result, it was found that the degree of freedom in sensor cell design and material selection was greatly expanded, and the productivity, performance, and ease of use of the sensor cell could be greatly improved.

  Therefore, the present invention greatly expands the freedom of sensor cell design and material selection used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye, and the sensor cell productivity, performance, It is an object of the present invention to provide an electrode unit that can greatly improve ease of use, and a sensor cell and a measuring apparatus using the electrode unit.

And according to the present invention, an electrode unit used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
An insulating substrate;
A working electrode provided with an electron-accepting layer that is provided locally on the insulating substrate and includes an electron-accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation;
A counter electrode provided on the same surface of the insulating substrate as the working electrode and spaced apart from the working electrode.

Further, according to the present invention, a sensor cell used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
The electrode unit;
A facing member provided facing the electrode unit;
A spacer having an opening that forms a flow path by being sandwiched between the electrode unit and the facing member;
A supply port that is formed in at least one of the electrode unit, the opposing member, and the spacer, and communicates with the flow path;
And at least one of the electrode unit, the facing member, and the spacer, and a discharge port that is formed at a position different from the supply port and communicates with the flow path, the electron-accepting layer and the pair An electrode is exposed in the flow path.

In addition, the measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye according to the present invention is as follows:
The sensor cell;
An ammeter for measuring a current flowing between the working electrode and the counter electrode, and a liquid feeding means for controlling supply and stop of the liquid into the flow path are provided.

Detailed description of the invention

Electrode Unit The electrode unit according to the present invention is used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. FIG. 1 shows an example of the electrode unit of the present invention. As shown in FIG. 1, the electrode unit 1 of the present invention includes an insulating substrate 2, a working electrode 3, and a counter electrode 4. The insulating substrate 2 used in the present invention is an insulating substrate so as not to short-circuit the working electrode 2 and the counter electrode 3. The working electrode 3 is provided locally on the insulating substrate 2 and includes an electron accepting layer containing an electron accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation. The counter electrode 4 is provided on the same surface as the working electrode 3 of the insulating substrate 2 and is separated from the working electrode 3. Lead wires 2 ′ and 4 ′ are provided so as to extend from each of the working electrode 2 and the counter electrode 4.

  As described above, the electrode unit according to the present invention is an integrated electrode member having a working electrode and a counter electrode on the same plane, and by using this, the degree of freedom of sensor cell design and material selection is remarkably increased. Spreading can greatly improve the productivity, performance and ease of use of sensor cells. That is, when a general electrode arrangement in which the two electrodes of the working electrode and the counter electrode are opposed to each other is to be used for specific detection of a test substance using photocurrent, the presence of the two electrodes Arrangement is not easy, and for example, the substrate of the working electrode is made of a transparent material and light must be radiated from the backside thereof, and there are considerable restrictions on the design and material selection of the sensor cell. On the other hand, since the electrode unit according to the present invention is an integral electrode member and does not require two electrodes to face each other, a configuration in which the light source faces the surface of the electrode unit can be easily adopted. As a result, the working electrode is not limited to a transparent material, and can be made of an opaque material such as ceramics or plastic, so that the degree of freedom in selecting an electrode material is expanded. In addition, the direct irradiation of the working electrode surface from the light source can eliminate the loss of light caused by the transparency of the transparent electrode material that occurs when irradiating from the back surface of the electrode, so that more accurate measurement can be expected. Furthermore, since the electrode unit according to the present invention is an integrated electrode member, the working electrode, the counter electrode, and the lead wire can be formed by one-step conductive patterning, which improves the productivity of the electrode. . In addition, since the material facing the electrode unit does not need to have conductivity, a commonly used material such as transparent plastic or glass can be used, and the productivity of the cell is improved.

  According to a preferred embodiment of the present invention, as shown in FIG. 1, the working electrode 3 and the counter electrode 4 are preferably formed in a spot shape divided into a plurality of regions separated from each other. In particular, by providing a plurality of working electrodes, it is possible to simultaneously measure a plurality of samples. In this embodiment, it is preferable that the lead wire 3 ′ is individually provided from each of the plurality of working electrodes 3 in that individual detection can be performed for each spot.

  The insulating substrate used in the present invention is an insulating substrate for preventing the working electrode and the counter electrode from being short-circuited, and examples of the insulating substrate include glass, ceramics, and plastics. The insulating substrate used in the present invention may be either transparent or opaque. This is because when the cell is constructed using the electrode unit according to the present invention, the light source can be arranged directly facing the surface of the electrode unit, so that the working electrode can be irradiated with light even if the insulating substrate is opaque. This is because.

  The working electrode used in the present invention has a probe substance that can specifically or directly bind to a test substance on its surface, so that a sensitizing dye immobilized via the probe substance is released in response to photoexcitation. It is an electrode that can accept electrons. Accordingly, the configuration and material of the working electrode are not limited as long as the above-described electron transfer occurs with the sensitizing dye to be used, and may be various configurations and materials.

  According to a preferred embodiment of the present invention, the working electrode has an electron accepting layer containing an electron accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation, and a probe material is provided on the surface of the electron accepting layer. Is preferably provided. According to a more preferred aspect of the present invention, it is preferable that the working electrode further includes a conductive material layer, and an electron accepting layer is formed on the conductive material layer.

  The electron-accepting layer in the present invention comprises an electron-accepting material that can accept electrons emitted by a sensitizing dye immobilized via a probe material in response to photoexcitation. That is, the electron-accepting substance can be a substance that can take an energy level in which electrons can be injected from the photoexcited labeling dye. Here, the energy level (A) capable of injecting electrons from the photoexcited labeling dye means, for example, a conduction band (conduction band: CB) when a semiconductor is used as the electron-accepting material. When a metal is used as the electron-accepting material, it means the Fermi level. When an organic material or a molecular inorganic material such as C60 is used as the electron-accepting material, the lowest unoccupied molecular orbital (Lowest Unoccupied Molecular Orbital: LUMO). That is, in the electron acceptor used in the present invention, the level of A is lower than the LUMO energy level of the sensitizing dye, in other words, lower than the LUMO energy level of the sensitizing dye. Any material having an energy level may be used.

Preferred examples of the electron accepting material include simple semiconductors such as silicon and germanium; oxides such as titanium, tin, zinc, iron, tungsten, zirconium, hafnium, strontium, indium, cerium, yttrium, lanthanum, vanadium, niobium, and tantalum. Semiconductors: Perovskite type semiconductors such as strontium titanate, calcium titanate, sodium titanate, barium titanate, potassium niobate; cadmium, zinc, lead, silver, antimony, bismuth sulfide semiconductors; cadmium, lead selenide semiconductors Cadmium telluride semiconductors; phosphide semiconductors such as zinc, gallium, indium and cadmium; gallium arsenide, copper-indium selenide, copper-indium-sulfide compound semiconductors; gold, platinum, silver, copper, aluminum Rhodium, indium, a metal such as nickel, polythiophene, polyaniline, polyacetylene, organic polymer polypyrrole; is C60, C70, etc. molecular inorganic, and more preferably include _{silicon, TiO 2, SnO 2, Fe} 2 O 3, WO ₃ , ZnO, Nb ₂ O ₅ , strontium titanate, indium oxide, CdS, ZnS, PbS, Bi ₂ S ₃ , CdSe, CdTe, GaP, InP, GaAs, CuInS ₂ , CuInSe, C60, more preferably _{, TiO 2, ZnO, SnO 2} , Fe 2 O 3, WO 3, Nb 2 O 5, strontium titanate, CdS, PbS, CdSe, InP , GaAs, a CuInS _2, CuInSe _2, and most preferably in TiO ₂ is there. Note that the semiconductors listed above may be either intrinsic semiconductors or impurity semiconductors.

  According to a preferred embodiment of the present invention, the electron acceptor is preferably a semiconductor, more preferably an oxide semiconductor, still more preferably a metal oxide semiconductor, and most preferably an n-type metal oxide semiconductor. . According to this aspect, electrons can be efficiently extracted from the dye by utilizing the band gap of the semiconductor. In addition, by using a semiconductor having a structure such as a porous body or an uneven surface shape, a working electrode having a large surface area can be produced, and the amount of immobilized probes can be increased.

  According to a preferred embodiment of the present invention, the potential of the conduction band of the semiconductor is preferably lower than the LUMO potential of the sensitizing dye, more preferably the LUMO of the sensitizing dye> the conduction band of the semiconductor> the redox of the electrolyte. Potential> potential satisfying the relationship of sensitizing dye HOMO. By having such a relationship, it becomes possible to efficiently extract electrons.

  According to a preferred embodiment of the present invention, when the electron accepting layer is made of a semiconductor, the layer surface may be cationized. By cationization, it becomes possible to adsorb the probe substance (DNA, protein, etc.) to the electron accepting layer with high efficiency. The cationization can be performed by, for example, allowing a silane coupling agent such as aminosilane, a cationic polymer, a quaternary ammonium compound, or the like to act on the surface of the electron accepting layer.

  According to another preferred embodiment of the present invention, indium-tin composite oxide (ITO) or fluorine-doped tin oxide (FTO) can be used as the electron accepting material. Since ITO and FTO have the property of functioning not only as an electron-accepting layer but also as a conductive material layer, by using these materials, it is possible to function as a working electrode using only the electron-accepting layer without using a conductive material layer. .

  When a semiconductor or metal is used as the electron-accepting substance, the semiconductor or metal may be single crystal or polycrystalline, but a polycrystalline body is preferable, and a porous material is more preferable than a dense material. As a result, the specific surface area is increased, and a large amount of the test substance and sensitizing dye can be adsorbed to detect the test substance with higher sensitivity. Therefore, according to the preferable aspect of this invention, it is preferable that the electron-accepting layer has porosity, and the diameter of each hole is 3-1000 nm, More preferably, it is 10-100 nm.

  According to a preferred aspect of the present invention, the surface area in a state where the electron accepting layer is formed on the conductive material layer is preferably 10 times or more, more preferably 100 times or more with respect to the projected area. The upper limit of the surface area is not particularly limited, but will usually be about 1000 times. The particle diameter of the fine particles of the electron accepting material constituting the electron accepting layer is preferably 5 to 200 nm as a primary particle in terms of an average particle diameter using a diameter when the projected area is converted into a circle, and more preferably 8 to It is 100 nm, More preferably, it is 20-60 nm. The average particle diameter of the electron-accepting substance fine particles (secondary particles) in the dispersion is preferably 0.01 to 100 μm. In addition, for the purpose of improving the light capture rate by scattering incident light, an electron accepting layer may be formed by using fine particles of an electron accepting material having a large particle size, for example, about 300 nm.

  According to a preferred embodiment of the present invention, it is preferable that the working electrode further includes a conductive material layer, and the electron accepting layer is formed on the conductive material layer. Examples of the conductive material constituting the conductive material layer include metals such as platinum, gold, silver, copper, aluminum, rhodium, and indium; conductive ceramics such as carbon, carbide, and nitride; and indium-tin composite oxides, Examples include conductive metal oxides such as tin oxide doped with fluorine, tin oxide doped with antimony, zinc oxide doped with gallium, or zinc oxide doped with aluminum. Are indium-tin composite oxide (ITO) and metal oxide (FTO) in which tin oxide is doped with fluorine. However, as described above, when the electron-accepting layer itself also functions as a conductive material layer, the conductive material layer can be omitted.

According to a preferred embodiment of the present invention, the conductive material layer is substantially transparent, specifically, the light transmittance is preferably 10% or more, more preferably 50% or more, and further preferably 70. % Or more. Moreover, according to the preferable aspect of this invention, it is preferable that the thickness of a electrically-conductive material layer is about 0.02-10 micrometers. Furthermore, according to a preferred embodiment of the present invention, the surface resistance of the conductive material layer is 100 Ω / cm ² or less, more preferably 40 Ω / cm ² or less. The lower limit of the surface resistance of the conductive material layer is not particularly limited, but will usually be about 0.1 Ω / cm ² .

  Examples of a preferable method for forming an electron-accepting layer include a method of applying a dispersion or colloidal solution of an electron-accepting substance, a method of applying a precursor of semiconductor fine particles, and hydrolyzing with moisture in the air to obtain a fine particle film (sol -Gel method), sputtering method, CVD method, PVD method, vapor deposition method and the like. As a method for preparing a dispersion of semiconductor fine particles as an electron acceptor, in addition to the sol-gel method described above, a method of grinding with a mortar, a method of dispersing while grinding using a mill, or a semiconductor synthesis Examples thereof include a method in which it is precipitated as fine particles in a solvent and used as it is. Examples of the dispersion medium include water or various organic solvents (for example, methanol, ethanol, isopropyl alcohol, dichloromethane, acetone, acetonitrile, ethyl acetate, etc.). At the time of dispersion, a polymer, a surfactant, an acid, a chelating agent, or the like may be used as a dispersion aid as necessary.

  Preferred examples of the application method of the electron acceptor dispersion or colloidal solution include the roller method as the application system, the dip method, the air knife method as the metering system, the blade method, etc., and the application and metering in the same part. The wire bar method disclosed in Japanese Patent Publication No. 58-4589, the slide hopper method, the extrusion method, the curtain method, the spin method, and the spray method described in US Pat. Nos. 2,681,294, 2,761,419, 2,761791, etc. Is mentioned.

According to a preferred aspect of the present invention, when the electron accepting layer is composed of semiconductor fine particles, the thickness of the electron accepting layer is preferably 0.1 to 200 μm, more preferably 0.1 to 100 μm, and still more preferably. Is 1-30 μm, most preferably 2-25 μm. As a result, the probe substance per unit projected area and the amount of sensitizing dye to be fixed can be increased to increase the photoelectric flow rate, and the loss of generated electrons due to charge recombination can also be reduced. Further, the coating amount of the semiconductor fine particles per 1 m ² is preferably 0.5 to 400 g, more preferably 5 to 100 g.

  According to a preferred embodiment of the present invention, when the electron-accepting material comprises indium-tin composite oxide (ITO) or metal oxide (FTO) doped with fluorine in tin oxide, the thickness of the electron-accepting layer is 1 nm. It is preferable that it is above, More preferably, it is 10 nm-1 micrometer.

  According to a preferred embodiment of the present invention, it is preferable to perform heat treatment after applying the semiconductor fine particles. Thereby, particle | grains can be made to contact electrically and the improvement of coating-film intensity | strength and adhesiveness with a support body can be improved. A preferable heat treatment temperature is 40 to 700 ° C, more preferably 100 to 600 ° C. Moreover, preferable heat processing time is about 10 minutes-10 hours.

  According to another preferred embodiment of the present invention, when a conductive material layer having a low melting point or softening point is used, such as a polymer film, the film is formed by a method that does not use high-temperature treatment in order to prevent deterioration due to heat. Examples of such film formation methods include methods such as pressing, low-temperature heating, electron beam irradiation, microwave irradiation, electrophoresis, sputtering, CVD, PVD, and vapor deposition.

  According to a preferred aspect of the present invention, when the working electrode does not have a conductive material layer, the lead wire is preferably made of the same material as the electron-accepting layer, and when the working electrode has a conductive material layer, The lead wire is preferably made of the same material as the conductive material layer. According to this aspect, since the lead wire can be made of the same material as the conductive material layer or the electron-accepting layer, the conductive pattern of the electrode and the lead wire can be formed in one step when the electrode unit is manufactured, and the production of the electrode Improves. For example, when the electron accepting material includes indium-tin composite oxide (ITO) or metal oxide (FTO) in which tin oxide is doped with fluorine, prepare a glass substrate on which an ITO or FTO film is formed. The conductive pattern can be formed with extremely high productivity by processing the surface in one step so as to leave the electrode and lead wire portions by etching by sandblasting or the like.

  A probe substance is supported on the surface of the electron accepting layer of the working electrode thus obtained. Loading of the probe substance on the working electrode can be performed according to a known method. According to a preferred embodiment of the present invention, when a single-stranded nucleic acid is used as a probe substance, an oxidized layer is formed on the surface of the working electrode, and the nucleic acid probe and the working electrode are connected via the oxidized layer. This can be done by bonding. At this time, the nucleic acid probe can be immobilized on the working electrode by introducing a functional group at the end of the nucleic acid. As a result, the nucleic acid probe into which the functional group has been introduced can be immobilized on the carrier as it is by an immobilization reaction. Introduction of a functional group at the end of a nucleic acid can be performed using an enzymatic reaction or a DNA synthesizer. Examples of the enzyme used in the enzymatic reaction include terminal deoxynucleotidyl transferase, poly A polymerase, polynucleotide kinase, DNA polymerase, polynucleotide adenyl transferase, RNA ligase. Can be mentioned. Moreover, a functional group can also be introduce | transduced by a polymerase chain reaction (PCR method), a nick translation, and a random primer method. The functional group may be introduced at any part of the nucleic acid, and can be introduced at the 3 ′ end, 5 ′ end or at a random position.

  According to a preferred embodiment of the present invention, amine, carboxylic acid, sulfonic acid, thiol, hydroxyl group, phosphoric acid and the like can be suitably used as the functional group for immobilizing the nucleic acid probe to the working electrode. Moreover, according to the preferable aspect of this invention, in order to fix | immobilize a diffusion probe firmly to a working electrode, it is also possible to use the material which bridge | crosslinks between a working electrode and a diffusion probe. Preferable examples of such a crosslinking material include silane coupling agents, titanate coupling agents, and conductive polymers such as polythiophene, polyacetylene, polypyrrole, and polyaniline.

  According to a preferred embodiment of the present invention, the nucleic acid probe can be immobilized efficiently by a simple operation called physical adsorption. The physical adsorption of the nucleic acid probe to the electrode surface can be performed, for example, as follows. First, the electrode surface is cleaned with distilled water and alcohol using an ultrasonic cleaner. Thereafter, the electrode is inserted into a buffer containing the nucleic acid probe to adsorb the nucleic acid probe onto the surface of the carrier.

  Moreover, nonspecific adsorption | suction can be suppressed by adding a blocking agent after adsorption | suction of a nucleic acid probe. The blocking agent that can be used is not limited as long as it can fill a site on the surface of the electron accepting layer on which the nucleic acid probe is not adsorbed and can adsorb the electron accepting material by chemical adsorption or physical adsorption. However, it is preferably a substance having a functional group that can be adsorbed via a chemical bond. For example, preferable examples of the blocking agent in the case of using titanium oxide as an electron-accepting layer include functional groups that can be adsorbed to titanium oxide such as carboxylic acid group, phosphoric acid group, sulfonic acid group, hydroxyl group, amino group, pyridyl group, and amide. Examples thereof include substances having a group.

  According to a preferred aspect of the present invention, the working electrode is formed in a spot shape divided into a plurality of regions separated from each other, or a plurality of regions in which the probe substance is separated from each other on one working electrode. It is preferable that the light is irradiated by the light source individually for each region. Thereby, since a plurality of samples can be measured on one working electrode, integration of DNA chips and the like are possible. According to a more preferable aspect of the present invention, a plurality of regions separated from each other, on which a probe substance is supported on the working electrode, is patterned, and a sample in each region is scanned while scanning with light emitted from a light source. It is preferable that the detection or quantification of the test substance is continuously performed in one operation.

  According to a more preferable aspect of the present invention, a plurality of spot-like working electrodes divided into a plurality of regions separated from each other, or a plurality of regions in a plurality of regions separated from each other on one working electrode. Various types of probe materials can be supported. As a result, a large number of samples can be simultaneously measured by multiplying the number of regions by the number of types of probe substances for each region.

  According to a more preferable aspect of the present invention, each spot-like working electrode divided into a plurality of areas separated from each other, or each area of a plurality of areas separated from each other on one working electrode. Different probe materials can be carried. As a result, the number of types of probe substances corresponding to the number of the divided areas can be carried, so that multiple types of analytes can be measured simultaneously. This aspect can be preferably used for multi-item analysis of single nucleotide polymorphism analysis (SNPs) because different test substances can be analyzed for each region.

  The counter electrode used in the present invention is not particularly limited as long as an electric current can flow between the counter electrode and the working electrode when it is brought into contact with the electrolytic solution, and may be a metal or a conductive oxide layer. it can. According to a preferred aspect of the present invention, the metal thin film as the counter electrode can be formed by a method such as vapor deposition or sputtering so as to have a film thickness of 5 μm or less, preferably in the range of 3 nm to 3 μm. Preferable examples of materials that can be used for the counter electrode include conductive polymers such as platinum, gold, palladium, nickel, carbon, and polythiophene, and conductive ceramics such as oxide, carbide, and nitride, and more preferably. Platinum and carbon, and most preferably platinum. These materials can be formed into a thin film by the same method as the electron accepting layer.

Sensor Cell The sensor cell according to the present invention is used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. 2 and 3 show an example of the sensor cell of the present invention. As shown in FIGS. 2 and 3, the sensor cell 10 of the present invention includes an electrode unit 1, a facing member 11, and a spacer 12. In the present invention, the spacer 12 is a member having an opening 12 a that constitutes a flow path by being sandwiched between the electrode unit 1 and the opposing member 11. That is, in the present invention, the facing member is a member that is provided to face the electrode unit and constitutes the bottom surface or the top surface of the flow path together with the spacer. Here, the flow path is a space for flowing and storing a liquid such as an electrolytic solution. The electrode unit according to the present invention comprises a working electrode having an electron-accepting layer and a counter electrode on the same plane, and is arranged so that the electron-accepting layer and the counter electrode of the working electrode are exposed to the flow path. The The working electrode and the facing member are provided so as to face each other and form a flow path by a combination with a spacer. That is, as shown in FIG. 2, the flow path 12 a is defined by the spacer 12, the electrode unit 1, and the pressing member as the opposing member 11. According to the above configuration, since the electron-accepting layer and the counter electrode of the working electrode are exposed to each other in the flow path, the assembly functions as a sensor cell for photocurrent measurement when the flow path is filled with the electrolytic solution. Can do. In addition, since the sensor cell can be easily disassembled and reassembled, operations such as sample replacement can be performed very simply.

  Then, at least one of the electrode unit, the facing member, and the spacer is formed with a supply port and a discharge port communicating with the flow channel, respectively, so that various treatment liquids such as an electrolytic solution can be placed in the flow channel. It is made to be able to flow. This means that the supply port and the discharge port communicating with the flow path may be provided on any of the upper surface, the side surface, and the bottom surface of the sensor cell. For example, as shown in FIG. 2, a liquid such as an electrolytic solution can be supplied into the sensor cell from the supply port 11a of the opposing member 11, and after flowing through the flow path 12a, can be discharged from the discharge port 11b. Therefore, according to the sensor cell of the present invention, after the sensor cell is constructed, an electrolytic solution for electrochemical measurement can be poured into the flow path. Accordingly, it is possible to effectively prevent leakage of the electrolyte during cell construction, which is likely to occur in a conventional sensor cell in which an electrode or the like is arranged after the electrolyte is injected into the cell container. In particular, in the conventional sensor cell, the electrode must be immersed in a cell container filled with a predetermined amount of electrolyte solution or the electrolyte solution must be sandwiched between the electrodes, so that the electrolyte solution spills or leaks. there is a possibility. If there is such a loss of the electrolytic solution, the amount of the electrolytic solution in the sensor cell varies with each measurement, which may reduce the measurement accuracy. On the other hand, according to the sensor cell of the present invention, the electrolyte can be filled in the cell without any gaps, and the leakage of the electrolyte filled in the cell can be effectively prevented. It can be kept constant with high accuracy. As a result, it is considered that the measurement accuracy can be improved.

Furthermore, according to the sensor cell of the present invention, not only the electrolytic solution but also various processing solutions that can be used for the sensor cell can be passed through the flow path. For example, a sample solution containing a test substance or a reagent solution containing a reagent can be passed through the flow path. Further, after the measurement, the cleaning liquid can be passed through the flow path. Therefore, according to the sensor cell of the present invention, various processes such as the exchange of the electrolyte accompanying the exchange of the sample, the supply of the sample liquid containing the test substance, the supply of the reagent solution, and the cleaning liquid in the cell (particularly the working electrode). Can be performed by a very simple method of simply flowing the treatment liquid into the system. In other words, the sample solution or reagent solution used for the measurement can be automatically sent to the electrode, so that the contact time between the solution and the electrode can be easily controlled, and variation between each measurement is reduced. Measurement accuracy can be improved.
According to a preferred embodiment of the present invention, the sensor cell is preferably a flow cell. By adopting the flow cell, the smaller the volume, the easier the liquid exchange and the unnecessary mixing between the solutions can be reduced. According to a more preferred embodiment of the present invention, it is preferable to insert an air segment to prevent mixing between different solutions when changing to a different solution. The introduction of the air segment can be performed by appropriately devising a valve mechanism such as providing a branch pipe for taking in air in the liquid feeding means.

  The opposing member used in the present invention is a member provided to face the electrode unit, and is not an electrode, so conductivity is not required. Therefore, as the facing member, materials generally used for the sensor cell such as plastic and glass can be used, so that the productivity of the sensor cell can be improved. According to a preferred aspect of the present invention, the facing member can be transparent. Thereby, light can be irradiated from the working electrode surface side by providing a light source inside or outside the opposing member.

  The spacer used in the present invention is a member having an opening that constitutes a flow path by being sandwiched between the electrode unit and the opposing member, and defines the interval between the electrode unit and the opposing member. Therefore, it is preferable to use a member that can effectively prevent liquid leakage as the spacer, and examples thereof include packing. Preferred materials for the packing include silicone rubber sheet, fluoro rubber, natural rubber, styrene / butadiene rubber, butadiene rubber, ethylene / propylene rubber, butyl rubber, nitrile rubber, chloroprene rubber, chlorosulfonated polyethylene rubber, urethane rubber, acrylic Examples include rubber, hydrin rubber, hydrogenated nitrile rubber, polysulfide rubber, and polydimethylsiloxane (PDMS). In the sensor cell of the present invention, the opposing member and the spacer may be easily removable from each other, but the opposing member and the spacer may be integrated using the same or different materials.

  According to a preferred embodiment of the present invention, as shown in FIGS. 2 and 3, it is preferable that the spacer 12 is disposed on the electrode unit 1 and the opposing member 11 is disposed on the spacer 12. In this embodiment, as shown in FIGS. 2 and 3, it is preferable to reinforce the strength of the electrode unit by further providing a support member 13 that supports the electrode unit 1 from the back surface.

  According to a preferred aspect of the present invention, as shown in FIGS. 2 and 3, it is preferable that the facing member 11 includes a supply port 11 a and a discharge port 11 b that communicate with a flow channel for forming a flow channel. That is, a space formed by the opening 12a of the spacer 12 being sandwiched between the electrode unit 1 and the opposing member 11 serves as a flow path, and a liquid such as an electrolyte is supplied into the flow path 12a through the supply port 11a and the discharge port 11b. Can be efficiently supplied and discharged.

  According to a preferred aspect of the present invention, the facing member can be transparent. Thereby, a light source can be provided in the inside or the outside of the opposing member, and the surface of the working electrode can be irradiated with light.

  According to a preferred aspect of the present invention, it is preferable that the facing member 11 is a pressing member that presses down so that the electrode unit and the spacer are in close contact with each other. By using this pressing member, the constituent members of the cell can be pressed and brought into close contact with each other, so that leakage of liquid such as electrolyte can be effectively prevented. In addition, since the working electrode can be easily attached and detached by providing the pressing member, an apparatus excellent in operability and maintainability can be provided. In order for the counter member to function as a pressing member, the counter member is given sufficient weight by itself or by other components such as a light source to hold down the electrode unit and the spacer so that the counter member is in close contact with each other. Alternatively, a fixing means for pressing down the electrode unit and the spacer so as to closely contact each other may be provided on the opposing member.

  According to a preferred aspect of the present invention, the pressing member can further include an opening or a light transmitting portion for allowing light for light excitation to pass through. According to a preferred aspect of the present invention, a plurality of openings or light-transmitting portions may be provided so that a plurality of spots of the test substance can be individually irradiated with light for each spot. In order to prevent light leakage, the pressing member may have light blocking properties except for the opening and the light transmitting portion.

  According to a preferred embodiment of the present invention, it is preferable that the pressing member 11 further includes a light source 14 as shown in FIGS. The light source used in the present invention is not limited as long as it can irradiate light having a wavelength capable of photoexciting the labeling dye. Preferred examples include fluorescent lamps, black lights, sterilizing lamps, incandescent lamps, low pressure mercury lamps, high pressure mercury. Lamps, xenon lamps, mercury-xenon lamps, halogen lamps, metal halide lamps, LEDs (white, blue, green, red), laser light and sunlight can be used, more preferably fluorescent lamps, incandescent lamps, xenon lamps. , Halogen lamp, metal halide lamp, LED (white, blue, green, red), sunlight and the like. Moreover, you may irradiate only the light of a specific wavelength area | region using a spectroscope and a band pass filter as needed. The light source used in the present invention includes not only a light source position that is fixed, but also a light source position that can move parallel to the surface of the working electrode.

  The light source 14 shown in FIGS. 2 and 3 is a moving light source provided with a driving means (not shown) such as a motor for moving in a direction parallel to the electrode unit 1. The light can be individually irradiated while sequentially moving on the working electrode. Thus, accurate individual measurement of a plurality of spots of test substances can be automatically and efficiently performed. Further, as another preferred embodiment, instead of making the light source movable, a driving means for moving the electrode unit in a direction parallel to the working electrode is provided in the sensor cell so that the working electrode moves relative to the light source. May be further provided. As another preferred embodiment, instead of making the light source movable, the pressing member is controlled so as to be opened only when light irradiation is necessary for each of the plurality of openings or light transmitting portions. A plurality of light shielding shutters may be further provided. Or as another suitable aspect, you may provide a some light source separately with respect to each of a some opening part or a translucent part instead of making a light source a movable type. In any of the above preferred embodiments, as in the embodiments shown in FIGS. 2 and 3, accurate individual measurement of a plurality of spots of test substances can be performed automatically and efficiently.

  According to a preferred aspect of the present invention, as shown in FIG. 3, the pressing member 11 can further include liquid feeding means 16 and 16 at positions corresponding to the supply port 11a and the discharge port 11b.

  According to a preferred aspect of the present invention, the counter substrate 11 is exposed or protruded so as to be electrically connectable to each of the lead wire 3 ′ extending from the working electrode 3 and the lead wire 4 ′ extending from the counter electrode 4. The contact probe 15 provided outside and extending to the outside of the counter substrate 11 is further provided, so that an electrical connection can be easily ensured. According to this aspect, since it is possible to ensure electrical connection to each of the working electrode and the counter electrode simply by assembling the sensor cell, when replacing the electrode unit, the work of wiring the working electrode and the counter electrode to the lead wire is reduced. Since it becomes unnecessary, the electrode can be replaced very easily. The contact probe used in this embodiment is not limited as long as it can be electrically connected, and a commercially available contact probe or spring probe can be preferably used.

  4 and 5 show another example of a sensor cell in which a spacer is arranged on the electrode unit and a counter member is arranged on the spacer. The sensor cell 20 shown in FIGS. 4 and 5 is the same as the sensor cell shown in FIGS. 1 and 2 in that the spacer 22 is arranged on the working electrode 1, but the facing member 21 arranged on the spacer 22 is transparent. The pressing member 27 is separately provided on the opposing member 21. Further, the supply ports 1a and 23a and the discharge ports 1b and 23b communicating with the flow path are formed in the electrode unit 21 and the support member 23, not in the facing member. Accordingly, liquid feeding is performed from the liquid feeding means 26 below the sensor cell via the supply ports 1a and 23a and the discharge ports 1b and 23b. According to this aspect, since the opposing member has translucency, by providing the light source 24 on the outside of the opposing member, it is possible to irradiate light from the surface side of the working electrode while ensuring excellent airtightness in the cell. it can.

  According to a preferred embodiment of the present invention, as shown in FIGS. 4 and 5, the uppermost part of the sensor cell 20 is further provided with a pressing member 27 that presses down the working electrode, the opposing member, and the spacer so as to closely contact each other. Preferably. The structure of the pressing member 27 is basically the same as that of the pressing member 11 as the opposing member shown in FIGS. 2 and 3, and includes a light source 24 and a contact probe 25. However, in the illustrated example, since the liquid is fed from below the sensor cell, the supply port and the discharge port are not provided. By using this pressing member, the constituent members of the cell can be pressed and brought into close contact with each other, so that leakage of liquid such as electrolyte can be effectively prevented.

  According to a preferred aspect of the present invention, as shown in FIG. 4, the pressing member 27 further includes at least one fixing means 28 selected from the group consisting of screws, springs, and magnets. It is preferable that the pressing member, the electrode unit, the opposing member, the spacer, and / or the supporting base material are detachably fixed to each other. By using this coupling means, the constituent members of the cell can be more closely adhered, and thus leakage of the electrolyte can be more effectively prevented. Moreover, since the coupling means is easily removable, sample exchange and maintenance can be easily performed.

  According to a preferred aspect of the present invention, the pressing member may have a light shielding property, and can be manufactured using a known material having a light shielding property. For example, when the pressing member has a plurality of openings and is a light-shielding member excluding the openings, when light is irradiated to one opening by a light source, It is possible to effectively prevent generation of a photocurrent caused by a test substance other than the irradiation target due to light leakage. In other words, by forming a plurality of electron-accepting layers and / or analytes in spots at positions on the working electrode corresponding to a plurality of openings, only one spot affects other spots. The light can be irradiated individually and accurately.

  According to another preferable aspect of the present invention, a spacer can be disposed on the opposing member, and an electrode unit can be disposed on the spacer. An example of the sensor cell of this embodiment is shown in FIGS. The sensor cell shown in FIGS. 6 and 7 differs from the embodiment shown in FIGS. 2 to 5 in that the working electrode 1 is disposed on the spacer 32, but the flow path 32a is the spacer 32, the electrode unit 1, and the opposing member. It is the same in that it is partitioned by 31. In this embodiment, since the opposing member 33 is not an electrode, the strength can be sufficiently secured by itself without requiring a support member.

  In the embodiment shown in FIGS. 6 and 7, the supply port 31 a and the discharge port 31 b communicating with the flow path are respectively formed in the opposing member 31, thereby allowing various kinds of electrolytes and the like in the flow path 32 a. The treatment liquid can be made to flow. That is, as shown in FIG. 7, a liquid such as an electrolytic solution is supplied from the supply port 31a into the sensor cell via the liquid feeding means 36, flows through the flow path 32a, and then is discharged from the discharge port 31b. it can. In addition, a pressing member 37 is further provided on the uppermost portion of the sensor cell 30 to press down the working electrode, the opposing member, and the spacer so as to closely contact each other. The structure of the pressing member 37 is basically the same as that of the pressing member 11 as the opposing member shown in FIGS. 2 and 3, and includes a movable light source 34. In the illustrated example, liquid is fed from below the sensor cell. Therefore, it does not have a supply port and a discharge port, and does not have a contact probe. The facing member 31 is exposed or protruded so as to be electrically connectable to each of the lead wire 3 ′ extending from the working electrode 3 and the lead wire 4 ′ extending from the counter electrode 4. By further including a contact probe 35 extending to the outside, an electrical connection can be easily secured.

  FIGS. 8 and 9 show another example of the sensor cell in which the spacer is arranged on the opposing member and the electrode unit is arranged on the spacer. The sensor cell shown in FIGS. 8 and 9 is basically the same as the sensor cell shown in FIGS. 6 and 7 except that the liquid is supplied from above the sensor cell. That is, in the embodiment shown in FIGS. 8 and 9, the supply ports 1 a and 47 a and the discharge ports 1 b and 47 b communicating with the flow path are formed in the electrode unit 1 and the pressing member 47, respectively. Various treatment liquids such as an electrolytic solution can be allowed to flow in the passage. That is, as shown in FIG. 9, a liquid such as an electrolytic solution is supplied from the supply ports 47a and 1a into the sensor cell via the liquid supply means 46, flows through the flow path 42a, and then is discharged from the discharge ports 1b and 47b. Can be done.

  FIGS. 10 and 11 show another example of the sensor cell in which the spacer is arranged on the facing member and the electrode unit is arranged on the spacer. The sensor cell shown in FIGS. 10 and 11 is provided with a spacer 52 and a contact probe 55 having a height sufficient to form a supply port 52a and a discharge port 52b on the side surface of the opposing member 51. The cartridge is configured as a body type cartridge. The electrode unit 1 is disposed on the cartridge, and the pressing member 57 is disposed on the electrode unit 1. The facing member 51 is provided with a light transmitting portion 58 for transmitting light from a light source provided therebelow, and a liquid leakage preventing packing 59 is provided on the light transmitting portion 58. According to this aspect, since the spacer and the counter member can be handled in an integrated cartridge unit, when exchanging the sample, the next measurement can be performed simply by replacing the electrode unit, and the operation is simple. Become.

  According to the preferred embodiment of the present invention, as shown in FIGS. 10 and 11, the light source 54 can be provided immediately below the light transmitting portion 58 of the facing member 51. That is, in this aspect, the light source is disposed below the translucent sensor cell. By arranging the light source below as described above, restrictions on the weight and size of the light source and the mechanism for controlling the light source are eased, and the degree of freedom in designing the light source is increased. Therefore, this aspect is suitable for use of a light source that requires complicated control, such as a movable light source that moves in the vertical and horizontal directions.

Specific detection of test substance using photocurrent As described above, the electrode unit and sensor cell of the present invention are used for specific detection of a test substance using photocurrent generated by photoexcitation of a sensitizing dye. A specific detection method for a test substance using a photocurrent generated by photoexcitation of the sensitizing dye will be specifically described below.

  In this method, first, a sample solution containing a test substance, and an electrode unit including a working electrode and a counter electrode according to the present invention are prepared. The working electrode used in the present invention is an electrode provided on the surface with a probe substance capable of specifically binding directly or indirectly to a test substance. That is, the probe substance specifically binds not only to a substance that directly binds specifically to the test substance, but also to a conjugate obtained by specifically binding the test substance to a mediator such as a receptor protein molecule. It may be a possible substance. Next, in the presence of the sensitizing dye, the sample solution is brought into contact with the working electrode to specifically bind the test substance directly or indirectly to the probe substance, and the sensitizing dye is fixed to the working electrode by this binding. Sensitizing dyes are substances that can emit electrons to the working electrode in response to photoexcitation, and can be pre-labeled to the test substance or mediator, or intercalated to the conjugate of the test substance and the probe substance. When a sensitizing dye is used, it may be simply added to the sample solution.

  Then, after bringing the working electrode and the counter electrode into contact with the electrolyte solution in the sensor cell, when the sensitizing dye is photoexcited by irradiating the working electrode with light, electron transfer from the photoexcited sensitizing dye to the electron accepting substance occurs. Occur. By detecting the photocurrent flowing between the working electrode and the counter electrode due to this electron movement, the test substance can be detected with high sensitivity. Further, since the detected current has a high correlation with the test sample concentration in the sample solution, the test sample can be quantitatively measured based on the measured current amount or electric quantity.

Test substance and probe substance The test substance in the present invention is not limited as long as it has a specific binding property, and may be various substances. In such a test substance, the test substance is directly or indirectly attached to the probe substance by supporting on the surface of the working electrode a probe substance that can specifically or directly bind to the test substance. It becomes possible to detect it by specifically binding to.

  That is, in the present invention, substances that can specifically bind to each other can be selected as the test substance and the probe substance. That is, according to a preferred embodiment of the present invention, it is preferable that a substance having specific binding properties be a test substance and a substance that specifically binds to the test substance be carried on the working electrode as a probe substance. As a result, the test substance can be directly bound and detected on the working electrode. Preferable examples of the combination of the test substance and the probe substance in this embodiment include a single-stranded nucleic acid and a combination of single-stranded nucleic acids having complementarity to the nucleic acid, and a combination of antigen and antibody.

  According to a more preferred embodiment of the present invention, it is preferable that the test substance is a single-stranded nucleic acid and the probe substance is a single-stranded nucleic acid having complementarity to the nucleic acid. More preferably, the probe substance has a complementary portion of 15 bp or more to the nucleic acid. The specific binding process of the test substance to the working electrode in this embodiment is shown in FIGS. As shown in these figures, a single-stranded nucleic acid 121 as a test substance is hybridized with a single-stranded nucleic acid 124 having complementarity as a probe substance carried on the working electrode 123, and A double-stranded nucleic acid 127 is formed.

  Sample liquids containing single-stranded nucleic acids as test substances are blood such as peripheral venous blood, white blood cells, serum, urine, feces, semen, saliva, cultured cells, tissue cells such as various organ cells, etc. Nucleic acids can be extracted from various specimen samples containing nucleic acids by a known method. At this time, destruction of the cells in the specimen sample can be performed, for example, by applying a physical action such as shaking or ultrasonic waves from the outside and vibrating the carrier. In addition, nucleic acid can be released from cells using a nucleic acid extraction solution. Examples of the nucleic acid elution solution include a solution containing a surfactant such as SDS, Triton-X, Tween-20, saponin, EDTA, protease, and the like. When nucleic acids are eluted using these solutions, the reaction can be accelerated by incubating at a temperature of 37 ° C. or higher.

  According to a more preferred embodiment of the present invention, when the content of a gene as a test substance is very small, detection is preferably performed after the gene is amplified by a known method. A typical method for amplifying a gene would be a method using an enzyme such as polymerase chain reaction (PCR). Examples of enzymes used in the gene amplification method include DNA polymerases, DNA-dependent DNA polymerases such as Taq polymerase, and DNA-dependent RNA polymerases such as RNA polymerase I. And RNA-dependent RNA polymerases such as Qβ replicase, and the PCR method using Taq polymerase is preferred in that amplification can be repeated continuously only by adjusting the temperature.

  According to a preferred embodiment of the present invention, the nucleic acid can be specifically labeled with a sensitizing dye during the amplification. Generally, it can be carried out by incorporating aminoallyl-modified dUTP into DNA. This molecule is incorporated with the same efficiency as unmodified dUTP. In the next coupling step, a fluorescent dye activated by N-hydroxysuccinimide specifically reacts with the modified dUTP, and a test substance uniformly labeled with a sensitizing dye is obtained.

  According to a preferred embodiment of the present invention, the nucleic acid crude extract solution or purified nucleic acid solution obtained as described above is first subjected to heat denaturation at a temperature of 90 to 98 ° C., preferably 95 ° C. or higher, to obtain a single strand. Nucleic acids can be prepared.

  In the present invention, the test substance and the probe substance may indirectly bind specifically. That is, according to another preferred embodiment of the present invention, a substance having specific binding properties is used as a test substance, a substance that specifically binds to the test substance is used as a mediator, and It is preferable to carry a substance capable of binding to the working electrode as a probe substance. Thereby, even a substance that cannot specifically bind to the probe substance can be detected by specifically binding indirectly to the working electrode via the mediator substance. In this embodiment, preferred examples of the combination of the test substance, the mediator, and the probe substance include a ligand, a receptor protein molecule that can accept the ligand, and two molecules that can specifically bind to the receptor protein molecule. A combination of nucleic acids in a strand is mentioned. Preferable examples of the ligand include exogenous endocrine disrupting substances (environmental hormones). An exogenous endocrine disrupting substance is a substance that binds to DNA via a receptor protein molecule and produces toxicity by affecting its gene expression. According to the method of the present invention, the receptor produced by a test substance is used. It is possible to easily monitor the binding of a protein such as a body to DNA. The process of specific binding of the test substance to the working electrode in this embodiment is shown in FIG. As shown in FIG. 13, a ligand 130 as a test substance first specifically binds to a receptor protein molecule 131 that is a mediator. The receptor protein molecule 133 to which the ligand is bound specifically binds to the double-stranded nucleic acid 134 as the probe substance.

  According to a preferred embodiment of the present invention, the test substance can be two or more kinds. According to the method of the present invention, by using a plurality of sensitizing dyes and irradiating light having different excitation wavelengths for each sensitizing dye, it is possible to individually detect a plurality of types of test substances. .

Sensitizing dye In the present invention, in order to detect the presence of the test substance by photocurrent, in the presence of the sensitizing dye, the test substance is directly or indirectly specifically bound to the probe substance, The sensitizing dye is fixed to the working electrode by the binding. Therefore, in the present invention, the test substance 121 or the mediator 131 can be labeled with the sensitizing dyes 122 and 132 in advance as shown in FIGS. In addition, as shown in FIG. 12B, when using a sensitizing dye 128 that can be intercalated with a conjugate 127 of a test substance and a probe substance (for example, a double-stranded nucleic acid after hybridization), a sample is used. By adding a sensitizing dye to the solution, the sensitizing dye can be fixed to the probe substance.

  The sensitizing dye used in the present invention is a substance that can emit electrons to the working electrode in response to photoexcitation, can be changed to the photoexcited state by irradiation with a light source, and can be injected into the working electrode from the excited state. Anything that can take a state may be used. Therefore, any sensitizing dye can be used as long as it can take the above-described electronic state with the working electrode, particularly the electron accepting layer. Therefore, various sensitizing dyes can be used, and expensive dyes can be used. There is no need to use it.

  Specific examples of the sensitizing dye include metal complexes and organic dyes. Preferred examples of the metal complex include metal phthalocyanines such as copper phthalocyanine and titanyl phthalocyanine; chlorophyll or derivatives thereof; hemin, ruthenium, osmium, iron described in JP-A-1-220380 and JP-A-5-504023, and Zinc complexes such as cis-dicyanate-bis (2,2′-bipyridyl-4,4′-dicarboxylate) ruthenium (II)) are mentioned. Preferred examples of organic dyes include metal-free phthalocyanine, 9-phenylxanthene dyes, cyanine dyes, methocyanine dyes, xanthene dyes, triphenylmethane dyes, acridine dyes, oxazine dyes, coumarin dyes, Examples include merocyanine dyes, rhodacyanine dyes, polymethine dyes, and indigo dyes.

  Preferable examples of the sensitizing dye capable of intercalating with a double-stranded nucleic acid include acridine orange and ethidium bromide. When such a sensitizing dye is used, a double-stranded nucleic acid labeled with a sensitizing dye is formed simply by adding to the sample solution after hybridization of the nucleic acid. Therefore, it is necessary to label the single-stranded nucleic acid in advance. No.

Measurement method and apparatus In the measurement method using the electrode unit or sensor cell of the present invention, first, in the presence of a sensitizing dye, the sample solution is brought into contact with the working electrode, and the test substance is directly or indirectly applied to the probe substance. The sensitizing dye is fixed to the working electrode by this binding.

  According to a preferred embodiment of the present invention, when a single-stranded nucleic acid previously labeled with a sensitizing dye is used as a test substance, a hybridization reaction can be performed with a single-stranded nucleic acid that is a probe substance. . The preferred temperature for the hybridization reaction is in the range of 37-72 ° C., but the optimum temperature varies depending on the base sequence and length of the probe used.

  According to another preferred embodiment of the present invention, when a sensitizing dye that can be intercalated is used for a conjugate of a test substance and a probe substance (for example, a double-stranded nucleic acid after hybridization), it is added to the sample solution. By adding a sensitizing dye, the conjugate can be specifically labeled with a sensitizing dye.

  The working electrode thus fixed with the sensitizing dye is brought into contact with the electrolyte together with the counter electrode, and the sensitizing dye is photoexcited by irradiating the working electrode with light, from the photoexcited sensitizing dye to the working electrode. The photocurrent flowing between the working electrode and the counter electrode due to the electron movement is detected. The sensor cell of the present invention is used as the sensor cell at that time.

  An example of a measurement method using the sensor cell shown in FIGS. 2 and 3 will be described. First, the test substance is assembled so as to have the cell structure shown in FIG. 2 using the electrode unit 1 including the working electrode 3 and the counter electrode 4 fixed together with the sensitizing dye, the counter member 11, and the spacer 12. . At this time, the electrode unit 1 is disposed so that the electron accepting layer side of the working electrode 3 is in contact with the electrolytic solution. Thus, the space formed by the opening 12a of the spacer 12 being sandwiched between the electrode unit 1 and the opposing member 11 serves as a flow path, and a liquid such as an electrolytic solution is supplied through the supply port 11a and the exhaust port 11b of the opposing member 11. It can be efficiently supplied and discharged into the flow path 12a. That is, liquid supply means 16 such as a liquid supply pump and a check valve are provided at the supply port 12b and the discharge port 12c, and the liquid supply is stopped after the electrolyte is filled in the flow path prior to measurement. As a result, at the time of measurement, a certain amount of electrolyte is reliably filled in the flow path. That is, according to the sensor cell of the present invention, the electrolyte can be filled in the cell without any gap, and leakage of the electrolyte filled in the cell can be effectively prevented. Can be kept constant, and the measurement accuracy can be improved.

The electrolytic solution used in the present invention can comprise an electrolyte, a solvent, and optionally an additive. Preferred examples of the electrolyte include iodide alone, or a combination of I ₂ and iodide (as iodide, metal iodide such as LiI, NaI, KI, CsI, CaI ₂ , tetraalkylammonium iodide, pyridinium iodide) Iodine salt of quaternary ammonium compound such as imidazolium iodide, etc .; bromide alone, or a combination of Br ₂ and bromide (as bromide, metal bromide such as LiBr, NaBr, KBr, CsBr, CaBr ₂ or tetraalkylammonium bromide) And bromine salts of quaternary ammonium compounds such as pyridinium bromide); metal complexes such as ferrocyanate-ferricyanate and ferrocene-ferricinium ion; sodium polysulfide, alkylthiol-alkyldisulfur Sulfur compounds such as fido; viologen dyes; hydroquinone-quinones, and the like, and more preferably electrolytes in which I ₂ and iodine salts of quaternary ammonium compounds such as LiI, pyridinium iodide, and imidazolium iodide are combined. The electrolytes described above may be used in combination. The electrolyte preferably has a higher redox potential than HOMO of the sensitizing dye.

  According to the preferable aspect of this invention, it is preferable that the electrolyte concentration of electrolyte solution is 0.1-15M, More preferably, it is 0.2-10M. Further, when iodine is added to the electrolyte, a preferable iodine concentration is 0.01 to 0.5M.

  Examples of preferred solvents include water, alcohol (methanol, ethanol, etc.), aprotic polar solvents (eg, nitriles such as acetonitrile, carbonates such as propylene carbonate and ethylene carbonate, dimethylformamide, dimethyl sulfoxide, sulfolane, 1 , 3-dimethylimidazolinone, 3-methyloxazolidinone, heterocyclic compounds such as dialkylimidazolium salts, and the like. In a system using an oxide semiconductor for the electron-accepting layer, when an iodide is used for the electrolyte and an aprotic polar solvent is used for the solvent, the detection accuracy increases, and DNA single nucleotide polymorphisms (SNPs) are analyzed. It becomes possible.

  As shown in FIG. 2, a light source 14 is disposed above the electrode unit 1 via a pressing member 11 as a counter member. The light source used in the present invention is as described above.

  According to a preferred aspect of the present invention, when two or more kinds of test substances are individually detected using two or more sensitizing dyes that can be excited at different light wavelengths, a specific wavelength is detected from the light source via the wavelength selection means. It is possible to excite a plurality of dyes individually by irradiating the light. Examples of wavelength selection means include a spectroscope, a colored glass filter, an interference filter, a bandpass filter, and the like. In addition, a plurality of light sources capable of irradiating light of different wavelengths depending on the type of sensitizing dye may be used. Examples of preferable light sources in this case include laser light and LED irradiated with light of a specific wavelength. It may be used. Further, in order to efficiently irradiate the working electrode with light, light may be guided using quartz, glass, or a liquid light guide.

  As shown in FIGS. 1 and 2, a contact probe 45 is connected to lead wires 3a and 4a extending from the working electrode 3 and the counter electrode 4, respectively, and the contact probe 15 is connected to an ammeter so that light irradiation is performed. The photocurrent flowing through the system is measured by an ammeter. Thereby, the test substance can be detected. The current value at that time reflects the amount of sensitizing dye trapped on the working electrode. For example, when the test substance is a nucleic acid, the amount of double strands formed between complementary nucleic acids is reflected as the current value. Therefore, the test substance can be quantified from the obtained current value. Therefore, according to a preferred aspect of the present invention, it is preferable that the ammeter further comprises means for calculating the concentration of the test substance in the sample solution from the obtained amount of current or electricity.

  According to a preferred aspect of the present invention, the step of detecting the photocurrent can measure the current value, and calculate the concentration of the test substance in the sample liquid from the obtained current value or electric quantity. The calculation of the test substance concentration can be performed by comparing a calibration curve between the test substance concentration and the current value or the electric quantity prepared in advance with the obtained current value or the electric quantity. In the method of the present invention, since the current value reflects the amount of the sensitizing dye trapped on the working electrode, an accurate current value corresponding to the test substance concentration can be obtained, which is suitable for quantitative measurement. .

  According to another preferred embodiment of the present invention, a test substance previously labeled with a sensitizing dye is used as a competitor, and a second substance capable of specifically binding to a probe substance that is not labeled with a sensitizing dye. The test substance can be quantified. It is preferable that the second test substance has a property of easily binding to the probe substance more specifically than the labeled test substance. When these two kinds of test substances are made to compete and specifically bind to the probe substance, a correlation is obtained between the detected current value and the concentration of the second test substance. That is, as the number of second analytes that are not dye-labeled increases, the number of competitors that specifically bind to the probe substance decreases, so that the detection current increases as the second analyte concentration increases. A calibration curve with decreasing values can be obtained. Therefore, it is possible to detect and quantify the second test substance that is not labeled with a sensitizing dye.

  According to a more preferred embodiment of the present invention, the test substance and the second test substance are preferably antigens, and the probe substance is preferably an antibody. FIG. 14 shows the step of immobilizing the test substance and the second test substance on the probe substance in this embodiment. As shown in FIG. 14, the antigen 141 labeled with a sensitizing dye and the antigen 142 not labeled with the dye compete with each other and specifically bind to the antibody 143. Therefore, as the amount of the antigen 142 that is not labeled with the dye increases, the amount of the dye-labeled antigen 143 that specifically binds to the antibody decreases. Therefore, the detection current value decreases as the second analyte concentration increases. You can get a line.

  The following examples further illustrate the present invention. The present invention is not limited to these examples.

Example 1 Production of Working Electrode First, raw materials having the following composition were mixed thoroughly using an automatic mortar and then dried at 150 ° C. for 6 hours to obtain a mixture.
α-Terpineol 60% by weight
2- (2-butoxyethoxy) ethanol 15% by weight
Ethylcellulose 25% by weight

  After adding and mixing 1 g of granular titanium oxide (P25 manufactured by Nippon Aerosol Co., Ltd., average particle size 20 to 25 nm) to 0.5 g of the obtained mixture, 0.5 g of the previously obtained mixture was further added and mixed. . Thereafter, α-terpineol was added and mixed again to obtain a paste. The series of mixing was performed for 3 hours in total using an automatic mortar.

The edge frame of a glass substrate (manufactured by Asahi Glass) on which a fluorine-doped SnO ₂ film was formed was masked with a tape having a width of about 63 μm, and the paste was squeegee-printed and then dried at 60 ° C. for 2 hours. The obtained glass substrate was put in a baking furnace, the furnace temperature was raised to 500 ° C. over about 17 minutes, held at this temperature for 30 minutes, and then allowed to cool. When the furnace temperature reached 100 ° C., the glass substrate was immersed in ethanol. Thus, a working electrode having an electron accepting layer containing titanium oxide was obtained.

_Then, the NH ₂ modified DNA having the nucleotide sequence of the 105-NH 2 -AACGTCGTGACTGGG dissolved in buffer (3X SSC), was prepared NH ₂ modified DNA solution 286MyuM. This solution was previously denatured by being kept at 95 ° C. for 3 minutes and then cooled on ice.

On the electron-accepting layer of the working electrode obtained previously, a silicon seal with a thickness of 700 μm in which an opening having a size of 5 mm × 5 mm square was formed was placed. 35 μl of 286 μM NH ₂ modified DNA solution was injected into this opening. At this time, the tip of the pipette tip was used so that the DNA solution was sufficiently distributed to the four corners of the opening of the silicon seal. Subsequently, the DNA solution was covered from above with a glass plate so as to prevent bubbles from entering as much as possible, and stored in a plastic container whose vapor pressure was adjusted with wet paper or the like. The NH ₂ -modified DNA was incubated in this container at 60 ° C. for 2 hours. Thereafter, the DNA solution was removed, the surface of the electrode was washed lightly with running water, and then the remaining water was scattered by blowing air. Thus, a working electrode carrying the probe substance was obtained.

Example 2: Detection of rhodamine-modified DNA Rhodamine-modified DNA having the nucleotide sequence of 105-Rho-CCCAGCTCACGACGTT was dissolved in a buffer (2X SSC, 0.03% SDS) to modify rhodamine having respective concentrations of 28.6 μM and 286 μM. A DNA solution was prepared.
A silicon seal similar to that used in Example 1 was placed on the surface of the working electrode, and 35 μl of each concentration of rhodamine-modified DNA solution was injected into the opening. The solution was covered with a glass plate from directly above so that air bubbles would not enter the solution as much as possible, and placed in a plastic container whose vapor pressure was adjusted with wet paper or the like. In this way, hybridization was performed by incubating overnight (12 hours) at 60 ° C.

The working electrode thus hybridized was immersed in a washing solution and washed with gentle shaking. The cleaning liquid shown in the following table was used, and each cleaning liquid was cleaned at the cleaning time, the number of cleanings, and the temperature shown in the following table. The cleaning container was replaced every time the cleaning liquid was changed.
Table 1
Number of cleanings per cleaning liquid Temperature
Cleaning time
2X SSC, 0.2% SDS, 6 minutes, 3 times, room temperature 0.2X SSC, 0.2% SDS, 6 minutes, 3 times, room temperature, 0.2X SSC, 0.2% SDS, 13 minutes, 1 time, 60 ° C
0.2X SSC, 0.2% SDS, 6 minutes, 2 times, room temperature
0.2X SSC 6 minutes once Room temperature
Note) 2X SSC: aqueous solution containing 0.3 M sodium chloride and 0.03 M sodium citrate (pH 7.0)
SDS: sodium dodecyl sulfate

  Further, the working electrode was washed twice by gently moving it up and down with ethanol. After the second washing, the remaining water was scattered by quickly blowing air without wiping with paper or the like.

A sensor cell was assembled as follows using the working electrode thus obtained and a platinum electrode as a counter electrode.
First, a platinum electrode prepared by sputtering a platinum thin film on a glass substrate was prepared. A silicon sheet having a thickness of 500 μm was placed on the platinum film of the platinum electrode. This silicon sheet is a spacer for preventing a short circuit due to contact between the working electrode and the counter electrode. At this time, a lead wire was connected to the end of the platinum electrode coated with platinum so that current could be taken out. The working electrode was also connected to an ammeter via a lead wire.
As an electrolytic solution, a mixed solution was prepared by dissolving 0.05M iodine and 0.5M tetrapropylammonium iodide in a mixed solvent of ethylene carbonate and acetonitrile having a volume ratio of 8: 2. After 5 μL of this electrolytic solution was dropped on the platinum electrode, the working electrode was placed so that its electron-accepting layer was opposed to the platinum electrode. Thus, a sandwich type sensor cell was obtained in which the spacer and the electrolyte were sandwiched between the working electrode and the counter electrode.

  The working electrode lead wire and the counter electrode lead wire were connected to an ammeter (ALS model 832A, Dial Electrochemical Analyzer). Light is guided from a light source (manufactured by Hayashi Watch Co., Ltd., LA-250XE) using a liquid light guide, and white light is emitted from 1.5 cm above the working electrode surface through an ultraviolet cut filter (Y-43, Asahi Techno Glass). The surface of the working electrode was irradiated for 2 seconds. At this time, the current value flowing between the working electrode and the counter electrode was measured over time by an ammeter.

  FIG. 15 shows the change over time in the detection current when using the 28.6 μM rhodamine-modified DNA solution, and FIG. 16 shows the change over time in the detection current when using the 286 μM rhodamine-modified DNA solution. As shown in these figures, it can be seen that when a DNA labeled with a sensitizing dye is hybridized with a DNA having complementarity with the DNA, a large current flows due to light irradiation.

  In addition, when the 28.6 μM and 286 μM rhodamine-modified DNA solutions were used, the current value at the time when the current value reached a steady state was measured separately, and the results shown in FIG. 13 were obtained. For reference, the same measurement was performed when rhodamine-modified DNA was not immobilized. The results are also shown in FIG. As shown in FIG. 17, the current value changed depending on the concentration of rhodamine-modified DNA. Therefore, it can be seen that quantitative analysis is possible according to the method of the present invention.

Example 3: Measurement using a working electrode not loaded with a probe substance Also, for comparison, only the electron-accepting layer made of titanium oxide was formed in Example 1 before loading NH ₂ -modified DNA. A sensor cell was constructed using the working electrode, and measurement was performed in the same manner as in Example 2. FIG. 15 shows the change over time in the detection current when using the 28.6 μM rhodamine-modified DNA solution, and FIG. 16 shows the change over time in the detection current when using the 286 μM rhodamine-modified DNA solution. As shown in these figures, titanium oxide itself is excited by a certain amount of ultraviolet rays that could not be removed by the ultraviolet cut filter, and a photocurrent is observed, but compared with the case of Example 2 using a rhodamine-modified DNA solution. The photocurrent was extremely low.

Example 4: A working electrode carrying a probe substance was obtained in the same manner as in Measurement Example 1 where the working electrode was blocked . A silicon seal similar to that used in Example 1 was again placed on the electrode surface, and 35 μl of 10 μM diethanolamine was injected into the opening as a blocking agent. It was covered with a glass plate from directly above so that air bubbles would not enter the blocking agent as much as possible, and placed in a plastic container whose vapor pressure was adjusted with wet paper or the like. And it hold | maintained at 60 degreeC for 30 minute (s), and incubated the blocking agent. The electrode surface was lightly washed again with running water, and then air was blown to scatter the remaining water.

  Using the working electrode thus blocked with the probe substance, hybridization by incubation of rhodamine-modified DNA and photocurrent measurement were performed in the same manner as in Example 2. The results were as shown in FIG. 15 and FIG. As shown in these figures, when the working electrode subjected to blocking was used, the current value was remarkably reduced as compared with the case of Example 2 where it was not applied.

Example 5: Detection of rhodamine-modified DNA in the presence of competitor PNA Rhodamine-modified DNA having the base sequence of 105-Rho-CCCAGCTACGACGTT and PNA having the base sequence of 105-CCCGTCACGACGTT as a competitor are buffered (2X SSC, 0.08.6% SDS) to prepare 28.6 μM rhodamine-modified and 200 μM PNA-containing solution and 286 μM rhodamine-modified and 200 μM PNA-containing solution.
Except for using this sample solution, hybridization by incubation of rhodamine-modified DNA and photocurrent measurement were performed in the same manner as in Example 2. The results were as shown in FIG. 15 and FIG. As shown in these figures, when PNA having a base sequence that is considered to compete during hybridization coexists, the current value decreased compared to Example 2 where PNA does not coexist.

Example 6: Individual measurement of test substance in multiple spots using electrode unit (1) Preparation of dye-labeled probe DNA The dye-labeled probe DNA has the following base sequence labeled with Cy5 at the 3 'end A 15-base nucleobase (Cy5-labeled ssDNA, manufactured by Proligo) was prepared.
Dye-labeled probe DNA (Cy5-labeled ssDNA):
5′NH ₂ -AACGTCGTGACTGGGG-Cy5-3 ′

(2) Preparation of electrode unit and support of dye-labeled probe DNA First, titania fine powder (Showa Titanium, F2, average particle size 60 nm, anatase: rutile = 4: 6) 1 g and an organic vehicle having the following composition 1 g of a solvent (α terpineol: butyl carbitol = weight ratio 60:40) was gradually added while kneading 1 g with an automatic mortar to obtain a titanium oxide paste. This series of mixing was carried out for a total of 5 hours.
α-Terpineol 65% by weight
Butyl carbitol 15% by weight
Polyvinyl butyral 20% by weight

Fluorine-doped tin oxide (F-SnO ₂ : FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 15Ω / □) is a circular spot having a diameter of 3 mm as shown in FIG. Etching was performed by sandblasting so as to leave 6 points at intervals of 2 mm and to leave lead wires extending from each spot. Five of the circular spots thus formed were used as working electrodes having an FTO film as an electron accepting layer. On the other hand, a platinum film was formed by vapor deposition on the remaining one circular spot to form a counter electrode. Thus, an electrode unit provided with a working electrode and a counter electrode was obtained on a glass substrate of FTO-coated glass which is an insulating substrate.

  Next, Cy5-labeled ssDNA as dye-labeled probe DNA was dissolved in 50 mM HEPES (pH 7.0) to prepare aqueous solutions with Cy5-labeled ssDNA concentrations of 0 μM, 1 μM, and 10 μM. This solution was preliminarily kept at 95 ° C. for 5 minutes, and then immediately denatured by cooling on ice for 10 minutes or more.

A spacer-perforated tape (hole diameter: 3 mm, number of spots: 6, thickness: 50 μm) is pasted on the electron accepting layer of the working electrode obtained earlier, and remains on the tape adhesive surface using the tip of the tweezers. To remove the air. On this tape, a silicon sheet (thickness: 1 mm) in which six openings with a diameter of 3 mm were formed was placed and adhered. 3 μl of each concentration of Cy5-labeled ssDNA solution prepared previously was loaded into the opening. As shown in FIG. 1, numbers are assigned to the six spots, and the concentration of the Cy5-labeled ssDNA solution loaded in each spot is as shown in Table 2 below.
Table 2
Spot number I II III IV V
DNA concentration 0M 1nM 10nM 100nM 1μM

  At this time, the tip of the pipette tip was used so that the DNA solution was sufficiently distributed to every corner of the opening of the silicon seal. Subsequently, the glass solution was covered with a glass plate so that air bubbles would not enter the DNA solution as much as possible, and then stored in a plastic container whose vapor pressure was adjusted with wet paper or the like. The Cy5-labeled ssDNA was incubated in this container at 50 ° C. overnight. Thereafter, the DNA solution was removed, the surface of the electrode was washed lightly with running water, and then the remaining water was scattered by blowing air. Thus, a working electrode carrying the dye-labeled probe DNA was obtained.

Thus, the working electrode carrying the dye-labeled probe DNA was immersed in a washing solution and washed while gently shaking. The cleaning liquid shown in Table 3 below was used, and each cleaning liquid was cleaned at the cleaning time, the number of times of cleaning, and the temperature shown in the following table. The cleaning container was replaced every time the cleaning liquid was changed. Further, the working electrode was rinsed with water for 5 seconds, and air was quickly blown to scatter the remaining water.
Table 3
Number of cleanings per cleaning liquid Temperature
Cleaning time
HEPES, 0.1% Tween20 6 minutes 6 times, room temperature HEPES, 0.1% Tween20 13 minutes, 1 time 60 ° C
HEPES 6 minutes 2 times room temperature
Ultrapure water 15 minutes once Room temperature
Note) HEPES: 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (manufactured by Dojindo Laboratories), 50 mM, pH 7.0 0.1% Tween 20: Surface activity manufactured by SERVA Agent diluted to 0.1 vol% in ultrapure water

(4) Assembly of measurement cell Using the electrode unit thus obtained, a measurement cell as shown in FIGS. 2 and 3 was assembled as follows.
First, a silicon sheet having a film thickness of 500 μm was inserted between the electrode unit and the pressing member. The silicon sheet is for creating a space for filling the electrolytic solution, and has an opening sufficiently large to encompass all spots, and the electrolytic solution into which the opening is fed Constitutes a flow path in which is accumulated. Lead wires extending from each working electrode and the counter electrode were respectively connected to a potentiostat (ALS Modl832A) via a spring probe in electrical contact. A pressing member is placed on the electrode unit, and a red laser light source (Audio Technica, SU-31: wavelength 635 nm) is passed through this holding member through an ant stage for moving the light source (manufactured by Suruga Seiki Co., Ltd., B05-41M). ) Was attached. In this way, a sandwich type measurement cell was obtained in which the spacer and the electrolyte were sandwiched between the electrode unit and the pressing member.
Next, a mixed solution in which 0.06M iodine and 0.6M tetrapropylammonium iodide were dissolved in a mixed solvent of acetonitrile and ethylene carbonate having a volume ratio of 4: 6 was prepared as an electrolytic solution. This electrolytic solution was filled between the working electrode and the pressing member.

(5) Measurement of photocurrent Using a moving light source consisting of a red laser fixed to an ant stage for moving the light source, light is irradiated individually for each spot from 3 cm above the surface of the working electrode, and the working electrode and platinum The current flowing between the counter electrodes was measured over time. The current value was measured for 180 seconds, but light irradiation was performed only for 60 seconds after 60 seconds from the start of current measurement. The observed photocurrent was corrected by subtracting the photocurrent value after 180 seconds from the photocurrent value after 120 seconds.

  As a result, the photocurrent detected at the time of light irradiation for each spot was as shown in FIG. As shown in FIG. 18, it can be seen that the value of the photocurrent detected when each spot was irradiated with light was highly correlated with the concentration of the Cy5-labeled ssDNA aqueous solution loaded in the spot irradiated with light. . That is, it can be seen that the photocurrent depending on the concentration of the test sample can be measured using an electrode unit having a working electrode and a counter electrode on the same surface.

It is a figure which shows an example of the working electrode of this invention. It is a figure which shows an example of the sensor cell of this invention, and is a figure which shows the state before an assembly. It is a figure which shows the state after the assembly of the sensor cell shown by FIG. It is a figure which shows another example of the sensor cell of this invention, and is a figure which shows the state before an assembly. It is a figure which shows the state after the assembly of the sensor cell shown by FIG. It is a figure which shows another example of the sensor cell of this invention, and is a figure which shows the state before an assembly. It is a figure which shows the state after the assembly of the sensor cell shown by FIG. It is a figure which shows another example of the sensor cell of this invention, and is a figure which shows the state before an assembly. It is a figure which shows the state after the assembly of the sensor cell shown by FIG. It is a figure which shows another example of the sensor cell of this invention, and is a figure which shows the state before an assembly. It is a figure which shows the state after the assembly of the sensor cell shown by FIG. FIG. 4 is a diagram showing a process of immobilizing a test substance on a probe substance when the test substance is a single-stranded nucleic acid and the probe substance is a single-stranded nucleic acid complementary to the nucleic acid. (A) shows the case where the test substance is previously labeled with a sensitizing dye, and (b) shows the case where a sensitizing dye capable of intercalation is added to a double-stranded nucleic acid. It is a figure which shows the immobilization process to the probe substance of a test substance in case a test substance is a ligand, a mediator is a receptor protein molecule, and a probe substance is a double-stranded nucleic acid. It is a figure which shows the fixation process to the probe substance of a test substance in case the test substance and the 2nd test substance which have the specific binding property which mutually compete are antigens, and a probe substance is an antibody. It is a figure which shows a time-dependent change of the detection electric current at the time of using the 28.6 micromol rhodamine modification DNA solution obtained in Examples 2-5 as a sample liquid. The time-dependent change of the detection electric current at the time of using 286 micromol rhodamine modification DNA solution obtained in Examples 2-5 as a sample liquid is shown. It is a figure which shows the detection electric current in a steady state at the time of using the rhodamine modification DNA solution of each density | concentration of 0 micromol, 28.6 micromol, and 286 micromol which were obtained in Example 2. FIG. FIG. 10 is a diagram showing detected current values in each spot working electrode obtained in Example 6.

Claims (13)

An electrode unit used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
An insulating substrate;
A working electrode provided with an electron-accepting layer that is provided locally on the insulating substrate and includes an electron-accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation;
An electrode unit comprising: a counter electrode provided on the same plane as the working electrode of the insulating substrate and spaced apart from the working electrode.   The electrode unit according to claim 1, wherein the working electrode and the counter electrode are formed in a spot shape divided into a plurality of regions separated from each other.   3. The electrode unit according to claim 1, further comprising a probe substance that is supported on the electron-accepting layer and can be directly or indirectly specifically bound to the test substance.   The electron acceptor is selected from the group consisting of titanium oxide, zinc oxide, tin oxide, niobium oxide, strontium titanate, indium oxide, indium-tin composite oxide (ITO), and fluorine-doped tin oxide (FTO). The electrode unit according to claim 1, wherein the electrode unit is at least one kind. A sensor cell used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
The electrode unit according to any one of claims 1 to 4,
A facing member provided facing the electrode unit;
A spacer having an opening that forms a flow path by being sandwiched between the electrode unit and the facing member;
A supply port that is formed in at least one of the electrode unit, the opposing member, and the spacer, and communicates with the flow path;
And at least one of the electrode unit, the facing member, and the spacer, and a discharge port that is formed at a position different from the supply port and communicates with the flow path, the electron-accepting layer and the pair A sensor cell in which an electrode is exposed to the flow path.   The sensor cell according to claim 5, wherein the spacer is disposed on the electrode unit, and the facing member is disposed on the spacer.   The sensor cell according to claim 6, wherein the facing member is a pressing member that holds down the electrode unit and the spacer so as to closely contact each other.   The sensor cell according to claim 6 or 7, wherein the facing member is transparent.   The sensor cell according to claim 5, wherein the spacer is disposed on the facing member, and the electrode unit is disposed on the spacer.   The pressing electrode according to any one of claims 5, 6, 8, and 9, further comprising a pressing member that presses the working electrode, the opposing member, and the spacer downward so that the working electrode, the facing member, and the spacer are in close contact with each other. Sensor cell.   The sensor cell according to any one of claims 7 to 10, wherein the pressing member further includes an opening or a light-transmitting part for allowing light for light excitation to pass through. A specific detection method of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
The sensor cell according to any one of claims 5 to 11,
Prepare a sample solution containing the test substance,
A probe substance capable of specifically binding directly or indirectly with the test substance is immobilized on the surface of a working electrode provided in the electrode unit of the sensor cell ;
In the presence of a sensitizing dye, the sample solution is brought into contact with the working electrode to specifically bind the test substance directly or indirectly to the probe substance, and the binding causes the sensitizing dye to bind to the working electrode. Fixed to
Contacting the working electrode and a counter electrode in an electrode unit of the sensor cell with an electrolyte; and
Light is applied to the working electrode to excite the sensitizing dye, and a photocurrent flows between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. A method for specific detection of a test substance, comprising detecting
A measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A sensor cell according to any one of claims 5 to 11,
A measuring apparatus comprising: an ammeter that measures a current flowing between the working electrode and the counter electrode; and a liquid feeding unit that controls supply and stop of the liquid into the flow path.

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