JP2006119111A - Specific detection method for test substance using photocurrent, electrode used therefor, measuring cell and measurement device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for sensitively, easily and accurately detecting and quantifying a test substance having a specific bonding property. <P>SOLUTION: A sample liquid containing the test substance is made to contact with a working electrode having a probe substance directly or indirectly, specifically bondable to the test substance on the surface under coexistence of a sensitizing dye, the test substance is made to directly or indirectly, specifically bond to the probe substance, and the sensitizing dye is made to be fixed to the working electrode by the bonding. The working electrode and a counter electrode are made to contact with a electrolyte medium, light is irradiated on the working electrode to photoexcite the sensitizing dye, and photocurrent flowing between the working electrode and the counter electrode by electron movement to the working electrode from the photoexcited sensitizing dye is detected. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

Background of the Invention

The present invention relates to a method for specifically detecting a test substance having specific binding properties such as a nucleic acid, an exogenous endocrine disrupting substance, an antigen, etc. by using a photocurrent, an electrode used therefor, a measurement cell, And a measuring device.

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)).

  On the other hand, in recent years, damage to the reproductive system and nervous system of exogenous endocrine disrupting substances (environmental hormones) such as dioxins has become a social problem. Currently, the detection of exogenous endocrine disrupting toxicity is carried out by various methods, but such substances are toxic at very low concentrations of the order of only 10 ppt. Therefore, a method for detecting an exogenous endocrine disrupting substance in such a low concentration range is desired.

  In particular, an exogenous endocrine disrupting substance binds to a target DNA via a protein such as a receptor, thereby affecting the expression of the DNA and causing toxicity. That is, the exogenous endocrine disrupting substance does not directly bind to DNA but binds indirectly to DNA via a protein such as a receptor. Therefore, in conventional methods such as prescreening using DNA binding properties, the evaluation of the binding is not easy.

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 recently fixed a sensitizing dye on a working electrode through direct or indirect specific binding between a test substance and a probe substance, and generated a photocurrent generated by photoexcitation of the sensitizing dye. By detecting it, the knowledge that a test substance can be detected and quantified with high sensitivity, simply and accurately was obtained. In addition, the inventors have also found that a plurality of samples can be individually measured on a single working electrode, or a plurality of types of analytes can be analyzed simultaneously.

  Accordingly, an object of the present invention is to detect and quantify a test substance having specific binding properties with high sensitivity, simply and accurately.

And the specific detection method of the test substance using the photocurrent of the present invention,
Preparing a sample solution containing a test substance, a working electrode having a probe substance capable of specifically binding directly or indirectly to the test substance, and a counter electrode;
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 the counter electrode to an electrolyte medium; 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. Is detected.

The electrode of the present invention is an electrode used as a working electrode in the above method,
A conductive substrate;
An electron-accepting layer comprising an electron-accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation, formed on the conductive substrate;
It is equipped with.

Furthermore, the measurement cell of the present invention is a measurement cell used in the above method,
The working electrode;
A counter electrode;
An electrolyte medium in contact with the working electrode and the counter electrode.

The measuring device of the present invention is a measuring device used in the above method,
The measurement cell;
A light source for irradiating light on the surface of the working electrode;
And an ammeter for measuring a current flowing between the working electrode and the counter electrode.

Detailed description of the invention

Specific Detection of Test Substance Using Photocurrent In the method of the present invention, first, a sample solution containing a test substance, a working electrode, and a counter electrode 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 medium, when the sensitizing dye is photoexcited by irradiating the working electrode with light, electron transfer occurs from the photoexcited sensitizing dye to the electron acceptor. 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 method of 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 method of the present invention, it is possible to select a test substance and a probe substance that can specifically bind to each other. 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. The steps of specific binding of the test substance to the working electrode in this embodiment are shown in FIGS. As shown in these figures, a single-stranded nucleic acid 1 as a test substance is hybridized with a single-stranded nucleic acid 4 having complementarity as a probe substance carried on the working electrode 3, A double-stranded nucleic acid 7 is formed.

  When a single-stranded nucleic acid is used as a test substance, it only needs to have a portion complementary to the nucleic acid that is the probe substance, and the length of the base pairs constituting the test substance is not limited. It preferably has a complementary portion of 15 bp or more. According to the method of the present invention, even when a nucleic acid having a base length of 200 bp, 500 bp, and 1000 bp is relatively long, a specific binding formation between the nucleic acid of the probe substance and the test substance can be performed with high sensitivity. Can be detected as

  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 method of 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 specific binding process of the test substance to the working electrode in this embodiment is shown in FIG. As shown in FIG. 2, a ligand 10 as a test substance first specifically binds to a receptor protein molecule 11 that is a mediator. Then, the receptor protein molecule 13 to which the ligand is bound specifically binds to a double-stranded nucleic acid 14 as a probe substance.

  According to the method of the present invention, a plurality of identical test substances derived from different acquisition routes are simultaneously reacted to one probe substance, and a difference in the amount of the test substance due to the origin of the sample is determined. It is also possible to quantify the test substance derived from the acquisition route. Specific application examples include expression profile analysis by competitive hybridization on a microarray. In this method, in order to analyze the difference in expression pattern of a specific gene among cells, a test substance labeled with different fluorescent dyes is competitively hybridized with the same probe substance. In the present invention, by using such a technique, an advantage that has not been obtained in the past can be obtained, in which an expression difference analysis between cells can be performed electrochemically.

Sensitizing dye In the method of the present invention, in order to detect the presence of a test substance by photocurrent, the test substance is directly or indirectly specifically bound to the probe substance in the presence of the sensitizing dye. Thus, the sensitizing dye is fixed to the working electrode by the binding. Therefore, in the method of the present invention, the test substance 1 or the mediating substance 11 can be labeled with the sensitizing dyes 2 and 12 in advance as shown in FIG. 1 (a) and FIG. In addition, when using a sensitizing dye 8 that can be intercalated with a conjugate 7 (for example, a double-stranded nucleic acid after hybridization) of a test substance and a probe substance as shown in FIG. By adding a sensitizing dye to the solution, the sensitizing dye can be fixed to the probe substance.

  According to a preferred embodiment of the present invention, when the test substance is a single-stranded nucleic acid, it is preferable to label one sensitizing dye per molecule of the test substance. The labeling position in the single-stranded nucleic acid should be either the 5 ′ end or the 3 ′ end of the single-stranded nucleic acid from the viewpoint of easily forming a specific bond between the test substance and the probe substance. From the viewpoint of further simplifying the labeling step, the 5 ′ end of the test substance is more preferable.

  According to another preferred embodiment of the present invention, in order to increase the amount of sensitizing dye supported per molecule of the test substance, two or more sensitizing dyes are labeled per molecule of the test substance. preferable. As a result, the amount of dye supported per unit specific surface area of the working electrode on which the electron accepting material is formed can be increased, and the photocurrent response can be observed with higher sensitivity.

  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.

  In the embodiment in which individual detection of a plurality of test substances is performed, the sensitizing dye that labels each test substance may be any one that can be excited by different wavelengths of light. It is only necessary that each test substance can be excited individually by selecting. For example, when a plurality of sensitizing dyes corresponding to a plurality of test substances are used and light having different excitation wavelengths is irradiated for each sensitizing dye, signals are detected individually even if the plurality of probes are on the same spot. It becomes possible. In the method of the present invention, the number of test substances is not limited, but considering the wavelength of light emitted from the light source and the absorption characteristics of the sensitizing dye, 1 to 5 types may be appropriate. The sensitizing dye that can be used in this embodiment only needs to be photoexcited in the wavelength region of the irradiation light, and the absorption maximum is not necessarily in the wavelength region. In addition, the presence or absence of the light absorption reaction of the sensitizing dye at a specific wavelength can be measured using an ultraviolet-visible spectrophotometer (for example, UV-3150 manufactured by Shimadzu Corporation).

  Specific examples of the sensitizing dye include metal complexes and organic dyes. Preferred examples of metal complexes 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 the organic dye include metal-free phthalocyanine, 9-phenylxanthene dye, cyanine dye, methocyanine dye, xanthene dye, triphenylmethane dye, acridine dye, oxazine dye, coumarin dye, Examples include merocyanine dyes, rhodacyanine dyes, polymethine dyes, and indigo dyes. As another preferred example of the sensitizing dye, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, Cy9 manufactured by Amersham Biosciences; AlexaFluor 355, AlexaFluor 405, AlexaFluor 430 manufactured by Molecular Probes, Inc. , AlexaFluor 488, AlexaFluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680, Alexa Fluor 680, Alexa Fluor 680, Alex Fluor 680, Alex Fluor 680 633, DY-635, DY-636, EVOblue10, EVOblue30, DY-647, DY-650, DY-651, DYQ-660, DYQ-661 and the like.

  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.

Working electrode and production thereof The working electrode used in the present invention is an electrode having the probe substance on its surface, and is an electrode that can accept electrons emitted by a sensitizing dye immobilized via the probe substance in response to photoexcitation. is there. 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 comprises a conductive substrate, and an electron accepting layer is formed on the conductive substrate. An electrode of this embodiment is shown in FIGS. The working electrode 4 shown in FIGS. 1 and 2 includes a conductive substrate 5 and an electron accepting layer 6 formed on the conductive substrate and containing an electron accepting substance. A probe substance is supported on the surface of the electron accepting layer 6.

  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 conductor (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, this A level 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, Ta 2 O 3, In 2 O 3, strontium titanate, CdS, ZnS, PbS, Bi 2 S 3, CdSe, CdTe, GaP, InP, GaAs, CuInS 2, CuInSe, a C60, more _{preferably, TiO 2, ZnO, SnO 2} , Fe 2 O 3, WO 3, Nb 2 O 5, Ta 2 O 3, in 2 O 3, strontium titanate, CdS, PbS, CdSe, InP GaAs, CuInS ₂ , CuInSe ₂ , most preferably TiO ₂ . Note that the semiconductors listed above may be either intrinsic semiconductors or impurity semiconductors.

  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 substrate, by using these materials, only the electron-accepting layer can function as a working electrode without using a conductive substrate. Can do.

  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 substrate 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 preferred that the working electrode further comprises a conductive substrate, and the electron accepting layer is formed on the conductive substrate. The conductive base material usable in the present invention is not only a material having conductivity on the support itself, such as a metal such as titanium, but also a material having a conductive material layer on the surface of a glass or plastic support. It's okay. When a conductive substrate having this conductive material layer is used, 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 substrate, the conductive substrate can be omitted. Further, in the present invention, the conductive substrate is not limited as long as it is a material that can ensure conductivity, and includes a thin film-like or spot-like conductive material layer that does not have strength as a support by itself. To do.

According to a preferred embodiment of the present invention, the conductive substrate is substantially transparent, specifically, the light transmittance is preferably 10% or more, more preferably 50% or more, and still more preferably. 70% or more. As a result, the cell is configured such that light is irradiated from the back side of the working electrode (that is, the conductive substrate), and the light transmitted through the working electrode (that is, the conductive substrate and the electron accepting layer) excites the sensitizing dye. can do. 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 the preferable aspect of this invention, it is preferable that the surface resistance of an electroconductive base material is 100 ohms / cm < ² > or less, More preferably, it is 40 ohms / cm < ² > or less. The lower limit of the surface resistance of the conductive substrate 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 on a conductive substrate include a method of coating a dispersion or colloidal solution of an electron-accepting material on a conductive support, and a precursor of semiconductor fine particles on the conductive support. And a method of obtaining a fine particle film by being hydrolyzed with moisture in the air (sol-gel method), a sputtering method, a CVD method, a PVD method, and a vapor deposition method. 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, 2761419, 2761791, 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. The coating amount of the semiconductor fine particles per 1 m ² of the conductive base material 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 apply heat treatment after coating the semiconductor fine particles on the conductive substrate. 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 base material having a low melting point or softening point, such as a polymer film, is used, the film is formed by a method that does not use high-temperature treatment in order to prevent deterioration due to heat. The film is preferably formed, and examples of such a film forming method include a method such as pressing, low-temperature heating, electron beam irradiation, microwave irradiation, electrophoresis, sputtering, CVD, PVD, and vapor deposition.

  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. According to a preferred embodiment of the present invention, a material that crosslinks between the working electrode and the nucleic acid probe can be used in order to firmly fix the nucleic acid probe to the working electrode. 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, it is preferable that the probe substance is divided and supported on each of the plurality of regions separated from each other on the working electrode, and the light irradiation by the light source is performed individually on 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 preferred aspect of the present invention, a plurality of types of probe substances can be carried on each of a plurality of regions separated from each other on the working electrode. 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, a different probe substance can be carried in each of a plurality of regions separated from each other on the working electrode. 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.

Counter electrode The counter electrode used in the present invention is not particularly limited as long as a current can flow between the electrode and the working electrode when it is brought into contact with the electrolyte medium. Insulating support such as glass, plastic, ceramics, etc. A body or a metal or conductive oxide vapor-deposited can be used. Moreover, the metal thin film as a 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 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.

Measurement method and apparatus In the method of the present invention, 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. To fix the sensitizing dye to the working electrode.

  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.

  In the method of the present invention, a working electrode in which a test substance is fixed together with a sensitizing dye is brought into contact with an electrolyte medium together with a counter electrode, and the working electrode is irradiated with light to photoexcite the sensitizing dye, thereby being photoexcited. The photocurrent flowing between the working electrode and the counter electrode due to the electron transfer from the sensitizing dye to the working electrode is detected. The relative positional relationship between the working electrode and the counter electrode is not limited as long as the working electrode and the counter electrode are not electrically short-circuited to each other and are in contact with the electrolyte medium. Alternatively, they may be spaced apart from each other on the same plane. When the working electrode and the counter electrode are arranged on the same plane and spaced apart from each other, both electrodes are provided on the insulating substrate to prevent an electrical short circuit between the working electrode and the counter electrode. Is desirable.

  An example of such a measurement cell is shown in FIG. The measurement cell 21 shown in FIG. 3 is formed by filling an electrolyte solution 24 into a gap formed by being sandwiched between a working electrode 22 and a counter electrode 23. The working electrode 22 includes a conductive base material 26 and an electron accepting layer 27, and is disposed so that the electron accepting layer 27 side is in contact with the electrolytic solution 24. By inserting an insulating spacer 25 between the working electrode 22 and the counter electrode 23, a space for accommodating the electrolytic solution 24 is secured. The distance between the electrodes is preferably short in order to efficiently perform the oxidation-reduction cycle, and is preferably several tens of μm in view of work accuracy. Further, if a so-called MEMS-like manufacturing method is used, it is possible to make the distance between electrodes closer to each other.

The electrolyte medium used in the present invention can comprise an electrolyte, a solvent, and optionally an additive. Preferable examples of the electrolyte include a combination of I ₂ and iodide (as iodide, metal iodide such as LiI, NaI, KI, CsI, CaI ₂ , tetraalkylammonium iodide, pyridinium iodide, imidazolium iodide). Quaternary ammonium compounds such as iodine salts), Br ₂ and bromide combinations (bromides include metal bromides such as LiBr, NaBr, KBr, CsBr, CaBr ₂ , or quaternary ammonium compounds such as tetraalkylammonium bromide and pyridinium bromide). Bromine salts), metal complexes such as ferrocyanate-ferricyanate and ferrocene-ferricinium ions, sulfur compounds such as sodium polysulfide, alkylthiol-alkyldisulfides, and viologens Examples thereof include dyes, hydroquinone-quinones, and the like. More preferable is an electrolyte in which I ₂ and iodine salt of a quaternary ammonium compound such as LiI, pyridinium iodide, imidazolium iodide and the like are combined. The electrolytes described above may be used in combination. Particularly preferred electrolyte media in the present invention are those comprising lithium ions.

  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.

According to a preferred embodiment of the present invention, an aqueous electrolyte solution can be used. Thereby, it becomes possible to measure appropriately without denaturing or deactivating biomolecules such as proteins. Further, there is an advantage that it is possible to prevent deterioration of the flow path of the electrolytic solution and the volatilization of the electrolytic solution and to easily perform waste liquid treatment. According to a preferred embodiment of the present invention, the aqueous electrolyte preferably comprises a supporting electrolyte, a reducing agent (electron donor), and water as a solvent. The supporting electrolyte is not limited as long as it dissociates into ions when dissolved in water to give conductivity, and does not inhibit the target electrode reaction. Preferred examples include NaCl, Na ₂ SO ₄ , KCl. , K ₂ SO _{4 and the} like. Preferable examples of the reducing agent (electron donor) include EDTA, triethanolamine, oxalic acid, hydroquinone and the like.

  According to a preferred embodiment of the present invention, the electrolyte medium can be used after gelation (solidification). Examples of the gelation method can be carried out by techniques such as addition of a polymer, addition of an oil gelling agent, polymerization including polyfunctional monomers, and a crosslinking reaction of the polymer. Examples of the polymer used in the matrix of the gel electrolyte include polyacrylonitrile and polyvinylidene fluoride.

  As shown in FIG. 3, a light source 28 is disposed above the working electrode 22 via a light source cover 29. That is, the cell is configured such that light is irradiated from the back side of the working electrode 22 (that is, the conductive substrate), and the light transmitted through the working electrode (that is, the conductive substrate and the electron accepting layer) excites the sensitizing dye. Has been. However, it goes without saying that light may be irradiated from the back side of the counter electrode by configuring the counter electrode with a light-transmitting material, or light may be irradiated in parallel to the working electrode and the counter electrode. 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 range using a spectroscope and a band pass filter as needed.

  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 the wavelength selection means include a spectroscope, a colored glass filter, an interference filter, a band pass 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.

  According to a preferred embodiment of the present invention, it is preferable that the light emitted from the light source is essentially free of ultraviolet light, or the light irradiation from the light source is performed through a means for removing the ultraviolet light. As a result, the background current due to photoexcitation of the electron-accepting substance itself, that is, noise, which can be generated when the irradiation light includes ultraviolet light having a wavelength of 400 nm or less can be effectively suppressed, and more accurate measurement can be performed. . In general, since sensitizing dyes can be excited by absorption of visible light, even if ultraviolet rays are removed, photocurrent can be detected with high sensitivity by irradiation with visible light.

  Preferable examples of the means for removing ultraviolet rays include an optical filter and a spectroscope. By using an optical filter or a spectroscope, the wavelength of irradiation light can be controlled, and only the sensitizing dye can be excited while preventing the photoexcitation of the working electrode itself. Examples of preferred optical filters include colored glass filters such as ultraviolet cut filters. As an example of a preferable spectroscope, there is a spectroscope having a built-in diffraction grating in that precise wavelength control is possible.

  Preferred examples of light sources that emit light that is essentially free of ultraviolet light include lasers, inorganic electroluminescence (EL) elements, organic electroluminescence (EL) elements, and light emitting diodes (LEDs), most preferably Light emitting diode (LED). According to the LED, it is possible to irradiate controlled light with a narrow wavelength distribution, and there are also advantages such as small size, light weight, low power consumption, and long life.

According to a more preferable aspect of the present invention, light having a wavelength shorter than the cutoff wavelength shown in Table 1 calculated by substituting the known band gap for the electron accepting material to be used in the following equation is removed. Is preferred. Thereby, generation | occurrence | production of a background current can be suppressed effectively according to the characteristic of an electron accepting substance.
Band gap (eV) = hν = hc / λ = 1239.8 / λ (mm)
(H: Planck constant, c: speed of light)

Table 1
Electron acceptor band gap Suitable cutoff wavelength
(EV) (nm)
Rutile type titanium oxide 3.2 387
Anatase type titanium oxide 3.0 413
Zinc oxide 3.1 400
Strontium titanate 3.2 387
Tin oxide 3.5 354
Tungsten oxide 2.8 443
Niobium oxide 3.1 400
Iron oxide 2.2 564

  Note that since the electron-accepting material may contain an impurity level, the cutoff wavelength may be set longer than the wavelength shown in Table 1 for the sake of completeness. When the working electrode is composed of a plurality of electron accepting substances, it is preferable to remove wavelengths shorter than the cutoff wavelength of the component having the narrowest band gap among the constituent components.

  As shown in FIG. 4, an ammeter 30 is connected between the working electrode 21 and the counter electrode 22, and the photocurrent flowing through the system by light irradiation is measured by the 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 concentration of the test substance 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. 5 shows the step of immobilizing the test substance and the second test substance on the probe substance in this embodiment. As shown in FIG. 5, the antigen 41 labeled with a sensitizing dye and the antigen 42 not labeled with the dye compete with each other and specifically bind to the antibody 43. Therefore, as the amount of the antigen 42 that is not dye-labeled increases, the amount of the dye-labeled antigen 43 that specifically binds to the antibody decreases. Therefore, the detection current value decreases as the second analyte concentration increases. You can get a line.

Measurement Method and Apparatus Using Flow Type Measurement Cell and Patterning Electrode As an example of a preferred embodiment of the method and apparatus of the present invention, a measurement method and apparatus using a flow type measurement cell and a patterning electrode will be described. FIG. 6 shows the overall structure of the apparatus. The apparatus 50 shown in FIG. 6 includes a flow type measurement cell 51, a light source 52, an electrolyte solution tank 53, a cleaning solution tank 54, a supply pump 55, an ammeter 56, and a discharge pump 57. . The flow-type measurement cell 51 includes a patterned working electrode 58 and a counter electrode 59 opposite to the working electrode. The electrolytic solution or the cleaning solution is accommodated between and flows between the working electrode 58 and the counter electrode 59. A flow path is formed. That is, the electrolytic solution or the cleaning solution supplied into the measurement cell 51 by the supply pump 55 passes through the flow path while being in contact with the working electrode 58 and the counter electrode 59, and is then discharged out of the measurement cell 51 by the discharge pump 57. It is configured to be. The control of the series of operations and the analysis of the photocurrent value can be performed by a control analysis device (not shown).

  The working electrode 58 has a plurality of regions separated from each other, on which a probe substance is supported on the electron-accepting layer, and is scanned with light emitted from a light source, and the sample of each substance in each region is scanned. It is configured so that detection or quantification can be performed continuously in one operation. Examples of the working electrode thus patterned are shown in FIGS. 7 (a) to (d) and FIGS. 8 (a) to (c).

  In the working electrode 58 shown in FIGS. 7A and 7B, a plurality of spots 60 carrying the probe substance 58c are vertically and horizontally arranged on the electron accepting layer 58b formed on the entire surface of the conductive substrate 58a. It has been patterned. A lead wire 61 is applied to the conductive base material of the working electrode 58, and the working electrode 58 as a whole is connected to the ammeter 56 via the lead wire 61. According to this working electrode 58, the photocurrent generated can be measured for each spot by sequentially irradiating each spot with light. In addition, since the structure of the electrode is relatively simple, it is easy to manufacture the electrode, and there is an advantage that a conventional DNA chip manufacturing technique can be used. Further, as a modification, the electron accepting layer 58b itself is formed in a spot shape as shown in FIG. 7C, and the probe material 58c is carried thereon, or as shown in FIG. 7D. The conductive base material may be omitted, and the spot-like working electrode 58 may be constituted by only the electron-accepting layer 58b, the probe material 58c may be supported thereon, and the lead wire 61 may be provided on the electron-accepting layer 58b. The latter has the advantage that the manufacturing process is simplified and the manufacturing cost can be reduced. The light source 52 used for this working electrode is a light source that moves vertically and horizontally on the working electrode 58 as shown in FIG. 9, or corresponds to each spot of the working electrode 58 as shown in FIG. Then, a plurality of light sources may be arranged, and each light source may be turned on and off in turn.

  The working electrode 58 ′ shown in FIGS. 8A and 8B is obtained by patterning a plurality of spots 60 ′ composed of a conductive base material 58a ′ and an electron accepting layer 58b ′ on the insulating substrate 58d ′ in the vertical and horizontal directions. The probe material 58c ′ is supported on the electron accepting layer 58b ′. A lead wire 61 ′ is individually applied to the conductive base material of each spot 60 ′, and each spot 60 ′ is connected to the ammeter 56 via the lead wire 61 ′. According to this working electrode 58 ', the photocurrent generated in each spot can be measured simultaneously and individually simply by irradiating the entire surface of the working electrode with light simultaneously. Further, since the photocurrent at each spot can be measured individually, there is an advantage that the photocurrent generated at other spots is not picked up as noise. Further, as a modification, as shown in FIG. 8C, the conductive base material is omitted, and the spot-like working electrode 58 ′ is formed only by the electron accepting layer 58b ′, and the probe substance 58c ′ is formed thereon. The lead wire 61 ′ may be provided on the electron-accepting layer 58b ′, which is advantageous in that the manufacturing process can be simplified and the manufacturing cost can be reduced. As in the case of the working electrode of FIG. 6, the light source 52 used for the working electrode 58 ′ is a light source that moves vertically and horizontally on the working electrode 58, or a plurality of light sources 52 corresponding to each spot of the working electrode 58. These light sources may be arranged, and each light source may be turned on and off in turn.

An example of a measurement method using this apparatus will be described below.
First, 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 sensitizing dye is fixed to the working electrode 58 by this binding. . At this time, spot pattern masking shown in FIG. 7 is applied to the electron-accepting layer of the working electrode to obtain a working electrode 58 in which a plurality of spots 60 carrying the probe substance are patterned in the vertical and horizontal directions. The working electrode 58 thus obtained is attached to the flow type measurement cell 51.

  Next, the supply pump 55 is operated to feed the electrolytic solution from the electrolytic solution tank 53 into the measuring cell 51. After the flow path in the measuring cell is filled with the electrolytic solution, the feeding is stopped. Light is applied to the working electrode 58 from the light source 52, and the photocurrent generated between the working electrode 58 and the counter electrode 59 is measured by an ammeter 56. In the measurement of the photocurrent value, it is preferable to adopt a value several tens of seconds after the start of irradiation, at which the photocurrent value becomes stable. Then, in a control analysis device (not shown), the test substance concentration is calculated by comparing the obtained current value with a calibration curve between the test substance concentration and the current value prepared in advance. After the measurement of the photocurrent, the supply pump 55 is operated to supply the cleaning liquid from the cleaning liquid tank 53 into the measuring cell 51, and at the same time, the electrolytic solution in the measuring cell 51 is discharged by operating the discharge pump 57. After the electrolyte solution in the flow channel in the cell is replaced with the cleaning solution, the liquid feeding and draining are stopped. Thereby, the next measurement can be performed in the same procedure as described above using the measurement cell 51 cleaned with the cleaning liquid.

  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, by dissolving NH ₂ modified DNA having the nucleotide sequence of _5'-NH 2 -AACGTCGTGACTGGG 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 a base sequence of 5'-Rho-CCCAGCTACGACGTT was dissolved in a buffer (2X SSC, 0.03% SDS) to obtain rhodamine having respective concentrations of 28.6 μM and 286 μM. A modified 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 2 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 2
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

  Furthermore, 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.

Using the working electrode thus obtained and a platinum electrode as a counter electrode, a measurement cell as shown in FIGS. 3 and 4 was assembled as follows.
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. In this way, a sandwich type measurement 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-Cho 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. 6 shows the change over time of the detected current when using the 28.6 μM rhodamine-modified DNA solution, and FIG. 7 shows the change over time of the detected 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. 8 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. 8, the current value varied 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 measurement cell was constructed using the working electrode, and measurement was performed in the same manner as in Example 2. FIG. 6 shows the change over time of the detected current when using the 28.6 μM rhodamine-modified DNA solution, and FIG. 7 shows the change over time of the detected 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. 6 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 5'-Rho-CCCAGTCACGACGTTT base sequence and PNA having 5'-CCCGTCACGACGGTTT base sequence as a competitor (2X SSC, 0.03% 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. 6 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: Preparation of calibration curve (1) Preparation of test substance and probe substance The following bases having a 3 'end labeled with rhodamine B as a dye-labeled test substance (hereinafter also referred to as test DNA) A 15-base nucleobase having a sequence (3 ′ rhodamine DNA) was prepared. Further, as a probe substance (hereinafter also referred to as probe DNA), a 15-base nucleobase having a complementary strand to the test DNA (DNA having a 5 ′ end modified with an amino group (hereinafter referred to as 5′-NH ₂ -DNA)) That is, this probe DNA and test DNA can form a double-stranded DNA by a hybridization reaction.
Test DNA (3 ′ rhodamine DNA):
3 'Rho-TTGCAGCACTGACCC 5'
Probe DNA (5′-NH ₂ -DNA):
5 ′ NH ₂ -AACGTCGGTACTGGGG 3 ′

(2) Production of working electrode and loading of probe substance First, 1 g of fine titania powder (Showa Titanium Co., F2, average particle size 60 nm, anatase: rutile = 4: 6), and 1 g of an organic vehicle having the following composition: Was gradually kneaded in an automatic mortar, and 1 g of a solvent (α terpineol: butyl carbitol = weight ratio 60:40) was gradually added 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

Mask the edge frame on the conductive surface of fluorine-doped tin oxide (F-SnO ₂ : FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 15Ω / □) with a metal metal screen mask, and A film having a thickness of 120 μm and a size of 5 mm × 5 mm was produced using the paste. The obtained film was dried at 60 ° C. for 3 hours and then baked at 500 ° C. for 30 minutes to obtain a working electrode having a titanium oxide porous film as an electron-accepting layer. The working electrode thus obtained was irradiated with UV light overnight with a BLB lamp to remove dirt and residual organic matter.

Subsequently, 5′-NH ₂ -DNA was dissolved in 50 mM HEPES (pH 7.0) as a probe DNA to prepare an aqueous solution. This solution was previously held at 95 ° C. for 3 minutes and then heat-denatured by cooling on ice (2 ° C.) for 3 minutes or more.

A spacer-perforated tape was affixed on the electron-accepting layer of the working electrode obtained earlier, and air remaining on the tape adhesive surface was removed using the tip of a tweezers. On this tape, a silicon sheet having an opening of 5 mm × 5 mm square was placed and brought into close contact. The opening was charged with 25 μl of the previously prepared 5′-NH ₂ -DNA solution (200 μM). 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 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 container was kept at 60 ° C. for 6 hours to incubate 5′-NH ₂ -DNA. Thereafter, the DNA solution was removed, and the electrode surface was gently washed with running water for 2 seconds, and then air was blown to scatter the remaining water. Thus, a working electrode carrying the probe DNA was obtained.

  A new silicon seal was placed on the working electrode carrying the probe substance, and 25 μl of 1 μM diethanolamine was loaded into the opening as a blocking agent. In order to prevent bubbles from entering the blocking agent as much as possible, the glass plate was covered with a lid from directly above 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 again lightly washed with running water for 2 seconds and then air was blown to scatter the remaining water. Thus, a working electrode subjected to blocking was obtained.

(3) Hybridization 3 ′ rhodamine DNA as a dye-labeled test DNA is dissolved in an HEPES aqueous solution, and each concentration is 0, 10, 40, 400, 4000, 15000, 25000, 30000, 40000, and 80000 nM. A 3 ′ rhodamine DNA solution was prepared.
Each concentration of 3 ′ rhodamine DNA solution was loaded onto a 25 μl working electrode. In order to prevent bubbles from entering the solution as much as possible, it was covered with a glass plate from directly above and placed in a plastic container whose vapor pressure was adjusted with wet paper or the like. Thus, hybridization was performed by incubating at 60 ° C. for 15 hours.

The working electrode thus hybridized was immersed in a washing solution and washed with gentle 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 working electrode thus obtained and a platinum electrode as a counter electrode, a measurement cell as shown in FIGS. 3 and 4 was assembled as follows.
First, a platinum electrode was prepared by sputtering a platinum thin film on a 1 mm thick glass substrate through a chromium layer for ensuring adhesion. 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 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. After 1 drop (5 μL) of this electrolyte solution was dropped onto the platinum electrode, the working electrode was placed so that its electron-accepting layer was opposed to the platinum electrode. In this way, a sandwich type measurement cell was obtained in which the spacer and the electrolyte were sandwiched between the working electrode and the counter electrode.

(5) Measurement of photocurrent The lead wire of the working electrode and the lead wire of the counter electrode were connected to a potentiostat (HSV-100, manufactured by Hokuto Denko Corporation). Light is guided from a 250W xenon lamp (LA-250Xe, manufactured by Hayashi Watch Industry Co., Ltd.) using a liquid light guide, and light with a wavelength of 430 nm or less can be removed in order to avoid light absorption and photocurrent generation by titanium oxide as a substrate. The surface of the working electrode was irradiated with light through an ultraviolet cut filter (Asahi Techno Glass Co., Ltd., Y-43). At this time, the current value flowing between the working electrode and the counter electrode was measured over time by an ammeter. The current value was measured for 60 seconds, but the light irradiation was performed only for 30 seconds after 10 seconds from the start of current measurement.

(6) Preparation of calibration curve From the concentration of the test DNA and the value of the detected photocurrent, the calibration curve shown in FIGS. 14 and 15 was obtained. As shown in FIG. 14, in the low concentration range where the test DNA concentration is 0 to 4000 nM, a proportional relationship was observed between the test DNA concentration and the current value. In addition, as shown in FIG. 15, in the high concentration range where the test DNA concentration is 1000 to 100,000 nM, a linear relationship was obtained between the logarithm of the test DNA concentration and the current value. Therefore, by using these calibration curves, the concentration of the unknown test DNA can be accurately known based on the measured photocurrent value. That is, according to the method of the present invention, DNA can be quantified.

Example 7: Example using an electrolyte medium containing lithium ions The following electrolytic solutions A and B containing two kinds of counter cations were used as the electrolytic solution, and the test DNA solution (5'-NH ₂ -DNA solution) ) Was carried out in the same manner as in Example 6 except that the concentration was 200 μM.
Electrolyte A: A solution in which 0.06 M I ₂ and 0.6 M TPA ⁺ I ⁻ are dissolved in a mixed solvent of 40% by volume ethylene carbonate and 60% by volume acetonitrile (tetrapropylaminemonium cation ((n-C ₃ H ₇ ) Electrolyte solution having 4N ⁺ ; TPA ⁺ ) as a counter cation)
Electrolytic solution B: a solution in which 0.05 M I ₂ and 0.5 M Li ⁺ I ⁻ are dissolved in acetonitrile (electrolytic solution using lithium ions as counter cations)

  In addition, as a blank, the photocurrent value was also measured for a working electrode as fabricated, in which neither the probe DNA nor the test DNA was immobilized.

The measured photocurrent values (stable current values) were as shown in Table 4.
Table 4
Electrolyte Photocurrent value (mA)
Example of the present invention Blank
A (TPA ⁺ ) 1.4 0.3
B (Li ⁺ ) 1.7 0.4

  As can be seen from Table 4, both electrolytic solutions A and B contained iodine and iodide, and even when any of them was used, the photocurrent could be detected with high sensitivity. In particular, in the electrolytic solution B containing lithium ions as a counter cation, the photocurrent tended to increase by 20 to 35% as compared with the electrolytic solution A not containing lithium ions. That is, it can be seen that by using lithium ions as the counter cation in the electrolyte medium, the detected photocurrent can be increased and the test DNA can be detected with high sensitivity.

Example 8: Example using titanium oxide or strontium titanate as an electron accepting substance As in Example 6, except that a working electrode preparation paste was prepared as follows and the concentration of the test DNA solution was 200 μM. The test was conducted.

That is, the working electrode preparation paste was prepared by gradually mixing a solvent (0.5 g) while kneading 0.5 g of an oxide semiconductor shown below and 0.5 g of an organic vehicle similar to that used in Example 6 in an automatic mortar. It was performed by adding 1 g of α-terpineol: butyl carbitol = weight ratio 60:40). This series of mixing was carried out for a total of 5 hours.
TiO ₂ : Showa Titanium Co., F-2, average particle size of about 60 nm, anatase: rutile = 2: 8 mixed phase ZnO: CI Kasei Co., Ltd. NanotekZnO, average particle size of about 30 nm
Nb ₂ O ₅ : manufactured by Taki Chemical Co., Ltd., powder obtained by evaporating and drying niobium oxide sol, average particle diameter of about 10 nm
WO ₃ : Wako Pure Chemical Industries, average particle size of about 100 nm
SrTiO ₃ : A toluene solution of strontium 2-ethylhexanoate and an isopropyl alcohol solution of titanium tetraisopropoxide were mixed at a molar ratio (Sr: Ti) of 1: 1 and evaporated to dryness at 100 ° C. Self-made powder by baking at 850 ° C., average particle size of about 200 nm
In ₂ O ₃ : Wako Pure Chemical Industries, average particle size of about 100 nm

  In addition, as a blank, the photocurrent value was also measured for a working electrode as fabricated, in which neither the probe DNA nor the test DNA was immobilized.

The measured photocurrent values (stable current values) were as shown in Table 5. Table 4 shows not only actual measured values but also corrected current values obtained by subtracting measured background current values for blanks.
Table 5
Electron acceptor average particle diameter Photocurrent (mA)
(Nm) Measured value Blank Current value after correction TiO ₂ 60 1.53 0.04 1.49
ZnO 30 0.59 0.03 0.56
WO ₃ 100 0.24 0.10 0.14
Nb ₂ O ₅ 10 0.025 0.010 0.015
SrTiO ₃ 200 0.045 0.0006 0.045
In ₂ O ₃ 100 0.30 0.07 0.23

  As can be seen from Table 5, the photocurrent could be detected when any of the above oxide semiconductors was used as the electron acceptor. In particular, titanium oxide shows the highest value together with the measured value of the photocurrent and the corrected current value, indicating that it is an excellent electron accepting material. In addition, strontium titanate has an excellent S / N ratio because the background current value measured for the blank is extremely low, and it can be seen that the response current caused by the test DNA can be detected with high accuracy.

Example 9: Wavelength dependence of background current in various oxide semiconductors For five types of oxide semiconductors below, using a spectrophotometer (manufactured by Shimadzu Corporation, UV-3150), diffuse reflection (DR) spectra were obtained. It was measured.
SrTiO ₃ : A toluene solution of strontium 2-ethylhexanoate and an isopropyl alcohol solution of titanium tetraisopropoxide were mixed at a molar ratio (Sr: Ti) of 1: 1 and evaporated to dryness at 100 ° C. Self-made powder by baking at 850 ° C., average particle size of about 200 nm
F3: titanium oxide manufactured by Showa Titanium Co., average particle size of about 50 nm, anatase: rutile = 4: 6 titanium oxide P-25: Nippon Aerosil Co., Ltd., average particle size of about 25 nm, anatase: rutile = 7: 3 titanium oxide ST- 01: manufactured by Ishihara Sangyo Co., Ltd., average particle size of about 7 nm, anatase type titanium oxide ZnO: manufactured by CI Kasei Co., Ltd., NanotekZnO, average particle size of 30 nm

  The result was as shown in FIG. As shown in FIG. 16, it can be seen that all the measured oxide powders absorb light having a wavelength in the ultraviolet region, but hardly absorb light having a wavelength of 410 nm or more. This means that the background current derived from the light absorption of the electron accepting substance itself can be suppressed as much as possible by controlling the wavelength of the irradiation light using a cut filter or a bandpass filter. In particular, as can be seen from FIG. 16, strontium titanate hardly absorbs light having a wavelength of 400 nm or more, which is consistent with the result of Example 8 in which the background current is extremely low.

Example 10: Influence on measurement by dye labeling position in test DNA Two types of 3 'rhodamine DNA and 5' rhodamine DNA shown below were used as dye-labeled test DNA, and the test DNA solution The test was performed in the same manner as in Example 6 except that the concentration was 200 μM.
Test DNA (3 ′ rhodamine DNA):
3 'Rho-TTGCAGCACTGACCC 5'
Test DNA (5 ′ rhodamine DNA):
3 'TTGCAGCACTGACCC-Rho5'

All of these test DNAs can form double-stranded DNA by hybridization reaction with 5′-NH ₂ -DNA which is a probe DNA.

For comparison, a 15-base nucleobase (T-rhodamine DNA) having the following base sequence, which has no complementary strand to the probe DNA and labeled with rhodamine B and has the following base sequence, is used instead of the test DNA. The same test as above was performed.
T-rhodamine DNA:
5 'TTTTTTTTTTTTTTT-Rho3'

  In addition, as a blank, the photocurrent value was also measured for a working electrode as fabricated, in which neither the probe DNA nor the test DNA was immobilized.

The measured photocurrent values (stable current values) were as shown in Table 6.
Table 6
Test DNA Photocurrent value (mA)
3 'Rhodamine DNA 1.53
5 ′ Rhodamine DNA 1.28
T-Rhodamine DNA (Comparative Example) 0.29
Blank (comparative example) 0.04

As shown in Table 6, almost the same photocurrent was observed both when the 3 ′ end of the test DNA was dye-labeled and when the 5 ′ end of the test DNA was dye-labeled. From this result, it can be seen that the test DNA can be detected with high sensitivity regardless of whether the 3 ′ end or the 5 ′ end of the test DNA is labeled with a dye.
On the other hand, as can be seen from Table 6, when T-rhodamine DNA having no complementary strand with the probe DNA was used, almost no photocurrent derived from the dye was detected. That is, it can be seen that no hybridization reaction occurred between T-rhodamine DNA and probe DNA.
In addition, it can be seen from the results of the blank test that almost no photocurrent is detected when an as-prepared working electrode in which neither the probe DNA nor the test DNA is immobilized is used.
Therefore, the generation of the photocurrent is basically caused by the photoexcitation reaction of the sensitizing dye labeled on the test DNA and the electron transfer reaction to the electron accepting substance, and the photocurrent value is the double-stranded DNA on the electrode. It can be seen that this depends on the rate of formation.

Example 11: Measurement of photocurrent action spectrum The procedure of Example 6 was repeated except that the concentration of the test DNA solution was 40 μM and the photocurrent action spectrum was measured in place of the photocurrent measurement. The test was conducted.
That is, the measurement of the action spectrum of the photocurrent was performed using a wavelength variable monochromatic light source (manufactured by Spectrometer). IPCE (Incident Photon to Current Efficiency) was calculated using the following formula based on a report by M. Gratzel et al. (J. Am. Chem. Soc. 115, 6382, 1993). The center wavelength was set to each wavelength, and the half width was 20 nm. Photon flux (Photon Flux) was measured using a spectrum radiometer (USR-40D manufactured by USHIO INC.).
IPCE = 1250 × photocurrent density (μA / cm ² ) / [wavelength (nm) × photon flux (W / m ² )]

  The measured action spectrum (IPCE wavelength dependence) was as shown in FIG. FIG. 17 also shows the absorption spectrum of rhodamine B, which is the sensitizing dye used. As can be seen from FIG. 17, the obtained action spectrum showed a profile similar to the absorption spectrum of the sensitizing dye (rhodamine B). This suggests that the photocurrent is due to excitation of the sensitizing dye. That is, since the absorption center wavelength of rhodamine B is seen to be 520 nm, it is considered that the photocurrent value can be increased by increasing the amount of light in this wavelength region. On the other hand, in the wavelength region of 420 nm or less, the photocurrent tended to increase, but this photocurrent is a photocurrent caused by photoexcitation of titanium oxide itself, which is an electron accepting substance. This current is generated regardless of the presence or absence of hybridization between the test DNA and the probe DNA, and becomes so-called noise. Therefore, it can be seen that it is effective to remove light of 420 nm or less as much as possible in order to realize high accuracy of the sensor.

Example 12: Relationship between light source filter and background current A test was conducted in the same manner as in Example 6 except that the following six types of optical filters were used and hybridization was not performed.
Y-43 filter: Asahi Techno Glass, Y-43, UV-cut filter
550nm-40hw filter: Optline, A06-8200843, center wavelength 550nm, half width 40nm
20nm-220hw filter: Asahi Spectroscope, PB0620-220, center wavelength 620nm, half-value width 220nm
600nm-260hw filter: Asahi Spectroscopic Co., PB060-260, center wavelength 600nm, half width 260nm
620nm-260hw filter: Asahi Spectroscopic Co., PB0620-260, center wavelength 620nm, half width 260nm
600nm-300hw filter: Asahi Spectroscopic Co., Ltd., PB0600-300, center wavelength 600nm, half width 300nm

Further, the light absorption amount of the dye was measured as follows. First, the DNA-labeled dye adsorbed on the titanium oxide electrode was measured using a spectrophotometer (manufactured by Shimadzu Corporation, UV-3100). Based on the diffuse reflection method using an integrating sphere, the reflectance (R%) of the dye was calculated. At this time, barium sulfate powder was used as a reference material having a reflectance of 100%. Then, the absorption rate α (%) was calculated based on the formula α (%) = 100−R (%).
Subsequently, the light absorption amount was calculated based on the following formula.

The measured background photocurrent value (stable current value) and light absorption were as shown in Table 7.
Table 7
Filter Background photocurrent light absorption
(ΜA) (mW / cm ² )
Y-43 filter 46.6 4.3
550nm-40hw filter 1.5 0.57
20nm-220hw filter 11.8 1.6
600nm-260hw filter 26.4 3.6
620nm-260hw filter 16.4 2.7
600nm-300hw filter 42.1 4.1

  In the present example in which the hybridization reaction is not performed, it is desirable that the current value is essentially zero because photoexcitation of the sensitizing dye does not occur. However, as shown in Table 7, a photocurrent due to photoexcitation of titanium oxide itself, which is an electron acceptor, is observed as a background current. Further, as can be seen from Table 7, when a 550 nm-40 hw filter with a small amount of ultraviolet light is used, the background current can be reduced, but the light absorption amount of the dye is smaller than other filters. Therefore, it can be seen that the use of a 550 nm-40 hw filter is preferable for reducing the background, while the use of a Y-43 filter is effective for obtaining a large photocurrent value.

  For reference, the spectral distribution of the light source when the above six types of filters are used in a 250 W xenon lamp light source (LA250Xe, manufactured by Hayashi Watch Industry Co., Ltd.) is measured using a spectral radiometer (USHIO, USR-40D). did. The result was as shown in FIG. FIG. 19 shows an enlarged view of the wavelength region of 350 to 550 nm in the spectrum distribution diagram shown in FIG.

Example 13: Relationship between light source filter and S / N ratio Except that the Y-43 filter and 550 nm-40 hw filter used in Example 11 were used as optical filters, and the concentration of the test DNA solution was 40 μM. The test was conducted in the same manner as in FIG.

  The time-dependent change of the measured background photocurrent value was as shown in FIG. As shown in FIG. 20, the maximum value of the photocurrent of the Y-43 filter was 3.4 mA, and the maximum value of the 550 nm-40 hw filter was 1.1 mA. That is, the value of the photocurrent when using the 550 nm-40 hw filter was as small as about 1/3 that when using the Y-43 filter. This is because the 550 nm-40 hw filter is a filter that can reduce the background current as can be seen from the result of Example 12, but also the intensity of visible light is weakened, so the light absorption amount of the sensitizing dye is reduced. it is conceivable that.

  Here, the ratio of the maximum value of the photocurrent / the background current is considered as an index of the signal / noise ratio (S / N ratio) of the sensor. When the 550 nm-40 hw filter is used, the background current is reduced to 1/30 as compared with the Y-43 filter, while the maximum value of the photocurrent is 1/3. Therefore, the S / N ratio of the 550 nm-40 hw filter is improved by (1/3) / (1/30) = 10 times compared to the Y-43 filter. The same evaluation was performed on other filters, and it was found that the S / N ratio of the 550 nm-40 hw filter was the best.

Example 14: Irradiation light intensity dependency The test was conducted in the same manner as in Example 6 except that the concentration of the test DNA solution was 40 μM and the intensity of the light source was changed. Further, the light intensity was measured using a spectrum radiometer (USR-40d, manufactured by USHIO INC.).

The relationship between the measured photocurrent value (stable current value) and the light intensity was as shown in FIG. As shown in FIG. 21, the linearity can be confirmed up to the region of light intensity of 800 mW / cm ² , and it has been found that the reaction rate of the photocurrent is light quantity rate control in which the reaction speed is proportional to the first power of the light intensity. In general, in order to function with high accuracy and stability as a sensor, it is desirable to perform evaluation under a light amount rate control, in which the reaction rate is not a diffusion rate rate proportional to the 1/2 power of the light intensity. ing.

Example 15: Examination of LED light source The test was carried out in the same manner as in Example 6 except that the probe DNA was supported as follows, hybridization was not performed, and the following LED was used as the light source. .
Red LED: Oasis RED, TOL-50aURsCEs
Yellow LED: Nichia, YELLOW NSPY-500S
White LED: Nichia, WHITE NSPW500BS
Blue LED: Toyoda Gosei, BLUE E1L51-3B
Green LED: Toyoda Gosei, GREEN E1L51-3G
In addition, when the spectrum distribution of these LED was measured using the spectrum radiometer (USHIO, USR-40D), the result as shown in FIG. 22 was obtained.

That is, loading of probe DNA was performed as follows. First, a working electrode before carrying the probe DNA was prepared in the same manner as in Example 6. The working electrode thus obtained was irradiated with UV light overnight with a BLB lamp to remove dirt and residual organic matter. Then, the aqueous solution is prepared of _5'-NH 2 -3 'rhodamine DNA shown below as probe DNA was dissolved in 50mM HEPES (pH7.0). This solution was previously held at 95 ° C. for 3 minutes and then heat-denatured by cooling on ice (2 ° C.) for 3 minutes or more.
Probe DNA (5′-NH ₂ -3 ′ rhodamine DNA):
5 ′ NH ₂ -AACGTCGGTACTGGGG 3 ′ Rho

In order to improve the binding force between the probe DNA and the electron accepting substance (titanium oxide) on the electron accepting layer of the working electrode obtained previously, surface treatment with a silane coupling agent was performed. That is, a solution obtained by dissolving 0.5 wt% of a silane coupling agent (manufactured by Shin-Etsu Chemical Co., Ltd., KBM-403) in isopropanol was reacted with the surface of titanium oxide at 75 ° C. for 5 minutes, and then washed with isopropanol. Dried. A spacer-perforated tape was affixed to the surface of the working electrode thus obtained, and air remaining on the tape adhesive surface was removed using the tip of a tweezers. On this tape, a silicon sheet having an opening of 5 mm × 5 mm square was placed and brought into close contact. This opening was charged with 25 μl of the previously prepared 5′-NH ₂ -3 ′ rhodamine DNA solution (40 μM). 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 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 container was incubated at 60 ° C. for 15 hours to incubate 5 ′ amine-3 ′ rhodamine DNA. Thereafter, the DNA solution was removed, and the electrode surface was gently washed with running water for 2 seconds, and then air was blown to scatter the remaining water. Thus, a working electrode carrying the probe DNA was obtained.

  In this way, a new silicon seal was placed on the working electrode carrying the probe substance, and 25 μl of 10 μM diethanolamine was injected into the opening as a blocking agent. In order to prevent bubbles from entering the blocking agent as much as possible, the glass plate was covered with a lid from directly above 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 again lightly washed with running water for 2 seconds and then air was blown to scatter the remaining water. Thus, a working electrode subjected to blocking was obtained.

  Moreover, the photocurrent was also measured for a blank electrode in which the probe DNA was not immobilized on the working electrode. The ratio of the photocurrent of the working electrode to which the probe DNA was immobilized and the photocurrent of the Planck electrode was evaluated as the S / N ratio.

  FIG. 23 shows the measured photocurrent, blank current, difference between photocurrent and blank current, and S / N ratio. As can be seen from FIG. 23, in the yellow and green LEDs, the blank current could be kept low, and good results were obtained. Since blue and white LEDs slightly include wavelengths of 400 nm or less, the background current value derived from titanium oxide detected as noise increased. On the other hand, although the red LED had the strongest light intensity, no photocurrent was detected because it was longer than the excitation wavelength of rhodamine used as the dye.

Example 16: Detection of test DNA using LED light source Test DNA solution concentrations were 10, 20, and 30 μM, probe DNA was carried as follows, and the following LEDs were used as light sources Except for the above, photocurrent was measured in the same manner as in Example 6.
Yellow LED: Nichia, YELLOW NSPY-500S
Green LED: Toyoda Gosei, GREEN E1L51-3G

  That is, loading of probe DNA was performed as follows. First, in the same manner as in Example 6, a working electrode before carrying the probe substance was produced. The working electrode thus obtained was irradiated with UV light overnight with a BLB lamp to remove dirt and residual organic matter.

Next, the same 5′-NH ₂ -DNA as that used in Example 6 was dissolved in 50 mM HEPES (pH 7.0) as a probe DNA to prepare an aqueous solution. This solution was previously held at 95 ° C. for 3 minutes and then heat-denatured by cooling on ice (2 ° C.) for 3 minutes or more.

In order to improve the binding force between the probe DNA and the electron accepting substance (titanium oxide) on the electron accepting layer of the working electrode obtained previously, surface treatment with a silane coupling agent was performed. That is, a solution obtained by dissolving 0.5 wt% of a silane coupling agent (Shin-Etsu Chemical, KBM-403) in isopropanol was reacted with the surface of the electron-accepting layer (titanium oxide) at 75 ° C. for 5 minutes, and then washed with isopropanol. And dried. Spacer perforated tape was affixed to the surface of the working electrode thus obtained, and air remaining on the tape adhesive surface was removed using the tip of a tweezers. On this tape, a silicon sheet having an opening of 5 mm × 5 mm square was placed and brought into close contact. 25 μl of the previously prepared 5′-NH ₂ -DNA solution (200 μM) 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 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 container was kept at 60 ° C. for 6 hours to incubate 5′-NH ₂ -DNA. Thereafter, the DNA solution was removed, and the electrode surface was gently washed with running water for 2 seconds, and then air was blown to scatter the remaining water. Thus, a working electrode carrying the probe DNA was obtained.

  In this way, a new silicon seal was placed on the working electrode carrying the probe substance, and 25 μl of 10 μM diethanolamine was injected into the opening as a blocking agent. In order to prevent bubbles from entering the blocking agent as much as possible, the glass plate was covered with a lid from directly above 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 again lightly washed with running water for 2 seconds and then air was blown to scatter the remaining water. Thus, a working electrode subjected to blocking was obtained.

  The relationship between the measured photocurrent and the test DNA concentration was as shown in FIG. When yellow and green LEDs were used as light sources, a photocurrent depending on the test DNA concentration was observed, and a photocurrent superior to that of a blank having a concentration of 0 μM without hybridization was observed. From these results, it can be seen that the yellow and green LED light sources are suitable for quantitative measurement of the test DNA.

Example 17: Dye labeling of test DNA using ruthenium complex succinimidyl ester As a sensitizing dye for labeling a test DNA, commercially available as a dye for dye-modifying a protein having an amino group, A ruthenium complex succinimidyl ester (Ru-ONSu) represented by the formula was prepared.

Modification to the DNA-terminal amine using this sensitizing dye was performed as follows. First, the sensitizing dye was reacted with 15-mer ssDNA having an amino group at the 3 ′ end under the following conditions.
Ru-ONSu: 200 nmol / 25 μl
ssDNA: 20 nmol / 25 μl
Buffer: 50 mM HEPES (pH 7.0)
TAPS (pH 8.0)
TAPS (pH 9.0)

Each solution was shaken at room temperature for 15 hours, followed by gel filtration followed by ethanol precipitation to purify the dye-modified DNA. The concentration of Ru-ONSu was measured with an absorption spectrum at 458 nm. Further, the concentration of ssDNA was measured using a quantitative kit manufactured by Molecular Probes. Based on the obtained measurement results, the modification ratio (molar ratio) of Ru-ONSu to ssDNA was calculated. The obtained results were as shown in Table 8.
Table 8
Buffer modification ratio (Ru / DNA)
HEPES (pH 7.0) 5.6
TAPS (pH 8.0) 3.1
TAPS (pH 9.0) 4.4

  From the results shown in Table 8, it can be seen that succinimidyl ester derivatives can also be used as labeling dyes for DNA. As can be seen from the results in Table 8, the succinimidyl ester derivative appears to bind not only to the terminal amine but also to the amine in the nucleobase. It can also be seen that the labeling dye can be introduced.

Example 18: Measurement of action spectrum of photocurrent Using 3 ′ AlexaFluor ^(R) 647 DNA shown below as a dye-labeled test DNA and 5′-NH ₂ -DNA-2 as a probe DNA The action spectrum of the photocurrent was measured in the same manner as in Example 11 except that the concentration of the test DNA solution was 40 μM.
Test DNA (3 ′ Alexa Fluora 647 DNA):
3 'AlexaFluora 647-CCATGATGTCAGTTT 5'
Probe DNA (5′-NH ₂ -DNA-2):
5 'NH2-GGTACTACAGTCAAA 3'
These test DNA and probe DNA can form double-stranded DNA by a hybridization reaction.

The measured action spectrum (IPCE wavelength dependence) was as shown in FIG. As can be seen from Figure 25, the action spectrum obtained showed the same profile and the absorption spectrum of the AlexaFluor ^(R) 647 which is a sensitizing dye. This suggests that the photocurrent is due to excitation of the sensitizing dye. Since the absorption center wavelength of AlexaFluor ^(R) 647 appears to be 640 nm, it can be separated from the aforementioned absorption center wavelength (520 nm) of the rhodamine B dye, and the photocurrent derived from each dye can be observed. Thus, by labeling each DNA with a plurality of dyes having different absorption wavelengths and irradiating light that can excite those dyes separately, it becomes possible to simultaneously observe the formation of double-stranded DNA as a photocurrent. .

Example 19: Evaluation of fine structure of working electrode provided with titanium oxide porous film as electron-accepting layer A working electrode comprising a titanium oxide porous film as an electron-accepting layer was produced in the same manner as in Example 6. The cross section and surface of the obtained working electrode were observed with a scanning electron microscope (S-4100, manufactured by Hitachi, Ltd.). FIG. 26 shows an SEM image obtained for the cross section of the working electrode, and FIG. 27 shows an SEM image obtained for the titanium oxide porous membrane surface of the working electrode. From the image shown in FIG. 26, it can be seen that the porous titanium oxide electrode has a film thickness of about 20 μm and a uniform film structure with a very smooth surface. In addition, from the image shown in FIG. 27, the obtained porous electrode has a relatively large meso fine particle in which particles corresponding to primary particles (particle size: about 40 to 50 nm) of the used titanium oxide powder are well dispersed. It was found to be a good porous membrane with pores. Therefore, in the obtained porous membrane, it is considered that even a DNA molecule having a relatively large molecular size easily diffuses into the pores.

In addition, the pore distribution of the obtained titanium oxide porous membrane was measured using a pore distribution measuring device (Belsorp28SA, manufactured by Bell Japan). At this time, a sample obtained by peeling the porous titanium oxide film from the fired working electrode was used as a measurement sample. The surface area of the sample was calculated from the BET equation, and the pore diameter was calculated from the DH equation. As a result, a BET specific surface area of 19.8 m ² / g was relatively high, and a curve with a peak in the pore distribution at 67.8 nm was obtained. From these results, it was found that the obtained titanium oxide porous membrane had a very porous structure in which relatively large pores were uniformly opened. In such a large pore, it is considered that diffusion of a relatively bulky DNA molecule (for example, a single-stranded DNA having a width of about 2 nm) easily occurs.

Example 20: Optimization of oxide semiconductor electrode for LED light source (1) Preparation of test substance and probe substance As a dye-labeled test substance (hereinafter also referred to as test DNA), the 5 'end is replaced with a fluorescent dye Cy5 A labeled 25-base nucleobase (5′Cy5 DNA) having the following base sequence was prepared. In addition, a 25-base nucleobase (5′-NH ₂ -DNA) labeled with an amine at the 5 ′ end having a complementary strand to the test DNA was prepared as a probe substance (hereinafter also referred to as probe DNA). That is, this probe DNA and test DNA can form a double-stranded DNA by a hybridization reaction.
Test DNA (5′Cy5 DNA):
3′-TGGAAGTAGTTTTTGTAGTAGTAG-Cy5-5 ′
Probe DNA (5′-NH ₂ -DNA):
5'NH ₂ -ACCTTCATCAAAAACATCATCATCC-3 '

(2) Production of working electrode and loading of probe substance Use of probe DNA shown in (1) above as probe substance, titania fine powder or oxide semiconductor fine powder shown below as an alternative, A working electrode carrying probe DNA was obtained in the same manner as in Example 6 except that the film thickness when applying the paste was 60 μm.
TiO ₂ : Showa Titanium Co., F-3, average particle size 50 nm
ZnO: NanotekZnO, manufactured by CI Kasei Co., Ltd.)
Nb ₂ O ₅ : manufactured by Taki Chemical Co., Ltd., powder obtained by evaporating and drying niobium oxide sol
WO ₃ : Wako Pure Chemical Industries, Ltd.
SrTiO ₃ : Powder obtained by hydrolysis and firing of Sr (NO ₃ ) ₂ + Ti (OiPr) ₄
In ₂ O ₃ : Wako Pure Chemical Industries
Ta ₂ O ₅ : Sigma-Aldrich Japan

Table 9 shows band gaps, average particle diameters, and background currents of the various oxide semiconductors used. The background current is a photocurrent of a bare electrode to which neither the probe DNA nor the test DNA is adsorbed.
Table 9
Oxide semiconductor Band gap (eV) Average grain size (nm) Background current (nA)
Titanium oxide (anatase) 3.2 50 3
Zinc oxide 3.1 30 25
Niobium oxide 3.1 10 20
Tungsten oxide 2.8 100 25
Strontium titanate 3.2 200 0
Indium oxide 3.8 100 15
Tantalum oxide 3.9 500 0

  The working electrode carrying the probe substance was placed in a plastic container whose vapor pressure was adjusted with wet paper or the like, and placed in saturated steam at 60 ° C. for 30 minutes. The electrode thus exposed to vapor was heat-treated at 90 ° C. for 3 minutes, and a new silicon seal was placed on the electrode.

(3) Hybridization Hybridization was carried out in the same manner as in Example 6 except that a 10 μM 5′Cy5 DNA solution was used as the test DNA solution and that the incubation temperature was 50 ° C.

(4) Assembly of measurement cell A sandwich type measurement cell was assembled in the same manner as in Example 6.

(5) Measurement of photocurrent Photocurrent was measured in the same manner as in Example 6 except that a red LED (manufactured by CCS, HLV-27-NR-R) was used as a light source, and an ultraviolet cut filter was not used. Measurements were made.
The result was as shown in FIG. As shown in FIG. 28, when Cy-5 dye-labeled DNA was used as a test DNA under irradiation with a red LED, it was found that high photocurrent values were obtained at indium oxide, zinc oxide, and titanium oxide electrodes. . In addition, it was found that other oxide semiconductor electrodes, tungsten oxide, niobium oxide, strontium titanate, and tantalum oxide were effective as DNA sensors because photocurrent values higher than the background current value were obtained. From the above results, it can be seen that not only the titanium oxide electrode is necessarily the optimum electrode but also the optimum electrode can be changed according to various conditions such as the light source, the labeling dye, and the DNA.

Example 21: Example using a working electrode composed only of an electron-accepting layer (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 25-base nucleobase (Cy5-labeled ssDNA) was prepared.
Dye-labeled probe DNA (Cy5-labeled ssDNA):
5 'NH2-ACCTTCATCAAAAACATCATCATCC-Cy5-3'

(2) Preparation of working electrode and support of dye-labeled DNA Fluorine-doped tin oxide (F-SnO ₂ : FTO) -coated glass (manufactured by AI Special Glass Co., Ltd., U membrane) as a working electrode composed of only an electron accepting layer Sheet resistance: 15Ω / □) and tin-doped indium oxide (Sn—InO 2: ITO) coated glass (manufactured by Toyo Seimitsu Kogyo Co., Ltd., 100Ω / □) were prepared. These glasses were washed with acetone and water, and irradiated with ultraviolet rays for 30 minutes in an oxygen atmosphere to remove dirt and residual organic matter. A spacer-perforated tape was applied to the cleaned glass, and air remaining on the tape adhesive surface was removed using the tip of a tweezers. On this tape, a silicon sheet having an opening with a size of 5 mm × 5 mm square was placed and adhered.

  Subsequently, the Cy5-labeled ssDNA solution prepared at each concentration of 0, 1, 10, 50 μM was held at 95 ° C. for 5 minutes, immediately transferred to ice and held for 10 minutes to denature the DNA, and the electrode prepared earlier The upper opening was loaded with 25 μl. 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. Then, the glass solution was covered with a glass plate so that air bubbles would not enter into the DNA solution, housed in a plastic container whose vapor pressure was adjusted with wet paper or the like, and incubated at 60 ° C. overnight. . After incubation, the DNA solution was removed, the electrode surface was gently washed with running water for about 2 seconds, and then air was blown to scatter the remaining water. Thereafter, the electrode on which DNA was adsorbed was washed under the conditions shown in Table 3 of Example 6, and the remaining water was scattered by finally blowing air.

(3) Assembly of measurement cell Using the working electrode thus obtained and a platinum electrode as a counter electrode, a flow type measurement cell as shown in FIG. 6 was assembled. At that time, the working electrode was placed opposite to the platinum counter electrode, and a 500 μm thick silicon sheet was inserted between them to prevent a short circuit between the two electrodes and form a space filled with the electrolyte. . The silicon sheet has a hole sufficiently larger than 5 mm × 5 mm square, and has a structure in which the electrolyte solution fed here accumulates and the DNA immobilized on the working electrode comes into contact. The working electrode was connected to a potentiostat (BAS Co., Ltd., ALS Modl832A) via a spring probe in electrical contact, and the platinum electrode was connected to a lead wire connected to the end.
As an electrolytic solution, 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. This electrolytic solution was filled between the working electrode and the platinum counter electrode incorporated in the flow type measurement cell described above.

(4) Photocurrent measurement Light generated from an LED (CLV, HLV-27-NR-R) fixed to a flow type measurement cell is irradiated on the surface of the working electrode, and between the working electrode and the platinum counter electrode. The flowing current was measured over time. The measurement was performed 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.
The photocurrent values measured for each concentration of Cy5-labeled ssDNA were as shown in FIG. As shown in FIG. 29, when FTO or ITO is used as the electron-accepting layer, it can function sufficiently as a working electrode without further providing a conductive base material thereunder. That is, it was confirmed that FTO and ITO function not only as an electron-accepting layer but also as a conductive substrate.

Example 22: Measurement of protein (1) Preparation of test substance and probe substance As a test substance labeled with dye, HSA (human serum albumin) labeled with rhodamine was prepared. In addition, an anti-HSA antibody (rabbit anti-HSA serum, Japan Biotest Laboratories) was prepared as a probe substance.

(2) Production of working electrode and loading of probe substance Production of the working electrode and removal of dirt and residual organic substances were carried out in the same manner as in Example 6.
A spacer-perforated tape was affixed on the obtained working electrode, and air remaining on the tape adhesive surface was removed using the tip of a tweezers. On this tape, a silicon sheet having an opening with a size of 5 mm × 5 mm square was placed and adhered.
Next, an aqueous solution was prepared by dissolving an anti-HSA antibody (rabbit anti-HSA serum, Japan Biotest Laboratories) as a probe substance in a 50 mM HEPES buffer (pH 7.0) to 7.68 mg / ml. This solution was loaded at 25 μl / electrode into the opening of the silicon seal on the working electrode, covered with a glass plate, and allowed to stand overnight at 4 ° C. to solidify. The working electrode thus obtained was washed 3 times with 50 mM HEPES buffer (pH 7.0). After washing, 50 mM HEPES buffer solution (pH 7.0) containing 0.2% casein was loaded at 25 μl / electrode into an opening where a silicon seal was placed on the surface of the working electrode, covered with a glass plate, and 90 ° C. at 90 ° C. Incubated for minutes and blocked.
Tetramethylrhodamine was labeled with HSA using FluoReporter Tetramethylrhodamine Protein Labeling kit (Molecular Probes) as a labeling kit. According to the manufacturer's protocol, dye labeling reaction, purification, and separation were performed to obtain 1.5 ml of a rhodamine-labeled HSA solution with a labeling ratio of 1.78 at a concentration of 3.78 mg / ml.

(3) Loading of test substance Next, the working electrode after blocking was washed three times with a 50 mM HEPES buffer solution (pH 7.0) containing 0.05% Tween 20 (hereinafter T-HEPES). Rhodamine-labeled HSA solutions at various concentrations of 0.1, 0.33, 1.0, and 3.3 mg / ml were prepared by serial dilution of rhodamine-labeled HSA with a blocking solution. Each concentration of rhodamine-labeled HSA solution was loaded at 25 μl / electrode into the opening of the silicon seal on the working electrode, covered with a glass plate, and incubated at 30 ° C. for 90 minutes. Then, it washed 3 times with T-HEPES and rinsed with ultrapure water.

(4) Assembly of measurement cell A flow type measurement cell was assembled in the same manner as in Example 21 except that an aqueous solution in which components consisting of 100 mM ethylenediaminetetraacetic acid, 100 mM NaCl, and 100 mM Na ₂ SO ₄ were used was used as the electrolyte. It was.

(5) Photocurrent measurement Using a green LED (CLV, HLV-24GR-NR-3W) and a condensing microfiber head (CCS, HFR-25-30) fixed to a flow-type measurement cell Light was irradiated from 3 cm above the electrode surface, and the current flowing between the working electrode and the platinum counter electrode was measured over time. The measurement was performed for 180 seconds, but the 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.
The photocurrent value measured for each concentration of HSA solution was as shown in FIG. It was found that a calibration curve having a high correlation coefficient can be drawn when the antigen concentration is in the range of 0.1 to 3.3 mg / ml, and the protein can be quantified.

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 measurement cell by which the light source is arrange | positioned, and the part 21 enclosed with the dotted line in a figure is a measurement cell. FIG. 4 is a plan view of the measurement cell shown in FIG. 3. 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 an example of the apparatus using the flow type measurement cell and the patterned working electrode. It is a figure which shows an example of the patterned working electrode, (a) is a top view of a working electrode, (b) is sectional drawing of a working electrode, (c) is sectional drawing of the working electrode of another aspect, (D) shows sectional drawing of the working electrode of another aspect, respectively. It is a figure which shows another example of the patterned working electrode, (a) is a top view of a working electrode, (b) is sectional drawing of a working electrode, (c) is sectional drawing of the working electrode of another aspect. Respectively. It is a figure which shows an example of the light source used for the patterned working electrode. It is a figure which shows another example of the light source used for the working electrode patterned. 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 nM, 28.6 micromol, and 286 micromol which were obtained in Example 2. FIG. 10 is a diagram showing a calibration curve in a low concentration range obtained for test substance DNA in Example 6. FIG. 6 is a diagram showing a calibration curve in a high concentration range obtained for test substance DNA in Example 6. It is a figure which shows the result of the light absorption measurement of the various oxide semiconductors obtained in Example 9 by the diffuse reflection spectrum. FIG. 10 is a diagram showing an action spectrum obtained in Example 11. It is a figure which shows the spectral distribution of the light irradiated through the various optical filters used in Example 12. FIG. It is the figure which expanded the wavelength range of 350-550 nm in the spectrum distribution map shown by FIG. It is a figure which shows the time-dependent change of the background photocurrent value obtained in Example 13 when various optical filters are used. It is a figure which shows the relationship between the photocurrent value obtained in Example 14, and light intensity. It is a figure which shows the spectrum distribution of various LED used in Example 15. FIG. It is a figure which shows the difference between a photocurrent, a blank current, a photocurrent and a blank current, and an S / N ratio when various LEDs obtained in Example 15 are used. It is a figure which shows the relationship between the photocurrent obtained in Example 16, and a test DNA density | concentration. 19 is a diagram showing an action spectrum obtained in Example 18. FIG. 20 is a SEM image obtained in Example 19 and obtained for a cross section of the working electrode. FIG. 20 is an SEM image obtained for the surface of the porous titanium oxide film of the working electrode obtained in Example 19. FIG. It is a figure which shows the photocurrent value obtained about various oxide semiconductor electrodes obtained in Example 20. FIG. 22 is a graph showing the relationship between photocurrent and Cy5-labeled ssDNA concentration obtained in Example 21. 22 is a graph showing the relationship between photocurrent and rhodamine-labeled HSA concentration obtained in Example 22. FIG.

Claims (64)

A specific detection method for a test substance using photocurrent,
Preparing a sample solution containing a test substance, a working electrode having a probe substance capable of specifically binding directly or indirectly to the test substance, and a counter electrode;
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 the counter electrode to an electrolyte medium; 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. Detecting the method. A specific detection method for a test substance using photocurrent,
A working electrode comprising a probe substance to which a test substance is directly or indirectly specifically bound, and a sensitizing dye fixed by the binding, and a counter electrode are brought into contact with the electrolyte medium;
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. Detecting the method.   The method according to claim 1 or 2, wherein the test substance is previously labeled with the sensitizing dye.   The method according to claim 1 or 2, wherein the sensitizing dye is capable of intercalating with a conjugate of the test substance and the probe substance.   The method according to any one of claims 1 to 4, wherein 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.   The method according to claim 5, wherein the complementary nucleic acid has a complementary portion of 15 bp or more with respect to the nucleic acid.   The method according to claim 5 or 6, wherein the test substance is a nucleic acid previously labeled with the sensitizing dye, and one sensitizing dye is labeled per molecule of the test substance.   The method according to claim 5 or 6, wherein the test substance is a nucleic acid previously labeled with the sensitizing dye, and two or more sensitizing dyes are labeled per molecule of the test substance.   The sample further comprises a mediator that is specifically labeled with the sensitizing dye and capable of specifically binding to the test substance, and a binding substance between the mediator and the test substance is the probe substance. The method according to claim 1 or 2, wherein the method specifically binds to.   The method according to claim 9, wherein the test substance is a ligand, the mediator is a receptor protein molecule, and the probe substance is a double-stranded nucleic acid.   The method according to claim 9 or 10, wherein the test substance is an exogenous endocrine disrupting substance.   The step of detecting the photocurrent further comprises measuring a current value or an electric quantity and calculating a test substance concentration in the sample solution from the obtained current value or electric quantity. 12. The method according to any one of 11 above.   The step of calculating the test substance concentration in the sample solution from the obtained current value or electric quantity includes a calibration curve between the test substance concentration and the current value or electric quantity prepared in advance, and the obtained current value or electric quantity. The method according to claim 12, wherein the method is performed by comparing the quantity of electricity. The test substance is previously labeled with the sensitizing dye,
The sample solution further comprises a second test substance that is not labeled with the sensitizing dye and can specifically bind to the probe substance, whereby the test substance and the second test substance are included. Allowing the substance to compete with and specifically bind to the probe substance; and
The step of detecting the photocurrent further comprises measuring a current value or an electric quantity, and calculating a second analyte concentration in the sample solution from the obtained current value or electric quantity. Item 3. The method according to Item 1 or 2.   The step of calculating the second test substance concentration in the sample solution from the obtained current value or electric quantity is a calibration curve of the second test substance concentration and the current value or electric quantity prepared in advance, The method according to claim 14, wherein the method is performed by comparing the obtained current value or electric quantity.   The method according to claim 14 or 15, wherein the test substance and the second test substance are antigens, and the probe substance is an antibody.   The method according to any one of claims 14 to 16, wherein the second test substance has a property that it is easier to specifically bind to the probe substance than the test substance.   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 the probe material is supported on the surface of the electron accepting layer. The method according to claim 1.   19. The method of claim 18, wherein the electron accepting material is a material having an energy level lower than the energy level of the lowest unoccupied molecular orbital (LUMO) of the sensitizing dye.   The electron acceptor material comprises at least one selected from the group consisting of titanium oxide, zinc oxide, tin oxide, niobium oxide, indium oxide, tungsten oxide, tantalum oxide, and strontium titanate. The method described in 1.   20. A method according to claim 18 or 19, wherein the electron acceptor is titanium oxide or strontium titanate.   20. The method according to claim 18 or 19, wherein the electron accepting material is indium-tin composite oxide (ITO) or fluorine doped tin oxide (FTO).   The method according to any one of claims 1 to 22, wherein the working electrode further comprises a conductive substrate, and the electron-accepting layer is formed on the conductive substrate.   The method according to any one of claims 1 to 23, wherein the sensitizing dye is a metal complex dye or an organic dye.   25. A method according to any one of claims 4 to 24, wherein the intercalable sensitizing dye is acridine orange or ethidium bromide. Two or more kinds of the test substances are present, and the test substances are labeled with different sensitizing dyes that can be excited by light of different wavelengths,
The method according to any one of claims 1 to 25, wherein each test substance is individually detected by irradiating light having a different wavelength for each sensitizing dye.   27. The probe material according to any one of claims 1 to 26, wherein the probe substance is divided and supported in a plurality of regions separated from each other on the working electrode, and the light irradiation is performed on each region individually. The method described in 1.   28. The method according to claim 27, wherein the electron-accepting layer is formed over the entire surface of the conductive substrate, and a photocurrent flowing through the entire conductive substrate is detected.   The working electrode further includes an insulating substrate, and the spot made of the conductive base material and the electron-accepting layer is formed on the insulating substrate by being divided into a plurality of regions separated from each other. The method according to any one of claims 1 to 27, wherein the photocurrent flowing for each conductive substrate in each region is individually detected.   The working electrode further comprises an insulating substrate, and a spot made of the electron accepting layer is formed on the insulating substrate by being divided into a plurality of regions separated from each other. 28. A method according to any one of claims 1 to 27, wherein the photocurrent flowing through each layer is individually detected.   31. The measurement of a plurality of sample liquids is simultaneously performed by supporting a plurality of types of probe substances on each of a plurality of regions separated from each other on the working electrode. Method.   31. The measurement of a plurality of types of test substances is simultaneously performed by loading different probe substances for each of a plurality of areas separated from each other on the working electrode. the method of.   The method according to any one of claims 1 to 32, wherein the electrolyte medium comprises lithium ions.   34. A method according to any one of claims 1 to 33, wherein the light is substantially free of ultraviolet light.   The method according to any one of claims 1 to 33, wherein the light irradiation is performed through a means for removing ultraviolet rays.   36. The method of claim 35, wherein the means for removing ultraviolet light is an optical filter or a spectroscope.   37. The light according to any one of claims 34 to 36, wherein the light is light emitted from at least one light source selected from the group consisting of a laser, an inorganic electroluminescence (EL) element, and an organic electroluminescence (EL) element. The method according to item.   38. A method according to any one of claims 34 to 37, wherein the light is light emitted from a light source that is a light emitting diode (LED). An electrode used as a working electrode in the method according to any one of claims 1-38,
A conductive substrate;
An electron-accepting layer comprising an electron-accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation, formed on the conductive substrate;
With an electrode.   40. The electrode according to claim 39, further comprising a probe substance supported on the electron-accepting layer and capable of specifically binding directly or indirectly to the test substance.   41. The electrode according to claim 39 or 40, wherein the electron-accepting material is a material having an energy level lower than the energy level of the lowest unoccupied molecular orbital (LUMO) of the sensitizing dye.   The electron-accepting material comprises at least one selected from the group consisting of titanium oxide, zinc oxide, tin oxide, niobium oxide, indium oxide, tungsten oxide, tantalum oxide, and strontium titanate. The electrode according to any one of the above.   The electrode according to any one of claims 39 to 41, wherein the electron-accepting substance is titanium oxide.   The electrode according to any one of claims 39 to 41, wherein the electron-accepting substance is strontium titanate.   The electrode according to any one of claims 39 to 44, wherein the electron-accepting layer has a thickness of 0.1 to 200 µm.   The electrode according to any one of claims 39 to 45, wherein the electron-accepting layer has porosity.   47. The electrode according to any one of claims 39 to 46, wherein the conductive substrate is transparent.   The electrode according to any one of claims 39 to 47, wherein the probe substance is supported by being divided into a plurality of regions separated from each other on the electron-accepting layer.   49. The electrode according to claim 48, wherein a lead wire is connected to the conductive substrate, wherein the electron-accepting layer is formed over the entire surface of the conductive substrate.   An insulating substrate, and a spot formed of the conductive base material and the electron-accepting layer is formed on the insulating substrate by being divided into a plurality of regions separated from each other. The electrode according to claim 48, wherein a lead wire is individually connected for each conductive substrate. A measurement cell used in the method according to any one of claims 1 to 38,
A working electrode according to any one of claims 39 to 50;
A counter electrode;
A measurement cell comprising: an electrolyte medium in contact with the working electrode and the counter electrode.   52. The measurement cell according to claim 51, further comprising a spacer inserted between the working electrode and the counter electrode.   53. The measuring cell according to claim 51 or 52, wherein the electrolyte medium is filled between the working electrode and the counter electrode.   The measurement cell according to any one of claims 51 to 54, wherein the electrolyte medium contains lithium ions. A measuring apparatus used in the method according to any one of claims 1 to 38,
A measurement cell according to any one of claims 51 to 54;
A light source for irradiating light on the surface of the working electrode;
A measuring apparatus comprising: an ammeter that measures a current flowing between the working electrode and the counter electrode.   56. The apparatus according to claim 55, wherein the light source comprises a plurality of light sources capable of emitting light of different wavelengths depending on the type of sensitizing dye.   56. The apparatus according to claim 55, wherein the light source further comprises wavelength selection means, and is capable of irradiating light of different wavelengths depending on the type of sensitizing dye.   58. Apparatus according to any one of claims 55 to 57, wherein the light source is a light source that emits light that is substantially free of ultraviolet light.   58. Apparatus according to any one of claims 55 to 57, further comprising means for removing ultraviolet light between the light source and the working electrode.   60. The apparatus of claim 59, wherein the means for removing ultraviolet light is an optical filter or a spectroscope.   61. The apparatus according to any one of claims 58 to 60, wherein the light source is at least one selected from the group consisting of a laser, an inorganic electroluminescence (EL) element, and an organic electroluminescence (EL) element.   61. The apparatus according to any one of claims 58 to 60, wherein the light source is a light emitting diode (LED).   63. Apparatus according to any one of claims 55 to 62, wherein the light source further comprises a scanning mechanism for scanning over the working electrode.   The apparatus according to any one of claims 55 to 63, wherein the ammeter further includes means for calculating a concentration of the test substance in the sample solution from the obtained amount of current or amount of electricity.