WO2005093418A1 - Method of specifically detecting test substance by using photocurrent and electrodes, measurement cell, measurement device and buffer solution to be used therefor - Google Patents

Method of specifically detecting test substance by using photocurrent and electrodes, measurement cell, measurement device and buffer solution to be used therefor Download PDF

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Publication number
WO2005093418A1
WO2005093418A1 PCT/JP2005/005715 JP2005005715W WO2005093418A1 WO 2005093418 A1 WO2005093418 A1 WO 2005093418A1 JP 2005005715 W JP2005005715 W JP 2005005715W WO 2005093418 A1 WO2005093418 A1 WO 2005093418A1
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WIPO (PCT)
Prior art keywords
working electrode
substance
electrode
test substance
sensitizing dye
Prior art date
Application number
PCT/JP2005/005715
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French (fr)
Japanese (ja)
Inventor
Masahiro Miyauchi
Hiromasa Tokudome
Shuji Sonezaki
Hiroshi Ishikawa
Makoto Bekki
Koki Kanehira
Hitoshi Oohara
Yoko Yamada
Yumi Ogami
Original Assignee
Toto, Ltd.
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Priority claimed from JP2004277869A external-priority patent/JP2006090893A/en
Application filed by Toto, Ltd. filed Critical Toto, Ltd.
Publication of WO2005093418A1 publication Critical patent/WO2005093418A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • the present invention provides a method for specifically detecting a test substance having a specific binding property, such as a nucleic acid, an exogenous endocrine disrupting substance, or an antigen, using a photocurrent, an electrode used for the method, a measuring cell, a measuring device, And a buffer solution.
  • a test substance having a specific binding property such as a nucleic acid, an exogenous endocrine disrupting substance, or an antigen
  • Genetic diagnosis which analyzes DNA in biological samples, holds promise as a new prevention and diagnosis method for various diseases.
  • the following techniques have been proposed as techniques for performing such DNA analysis easily and accurately.
  • a double-stranded recognizer such as an intercalator is added.
  • exogenous endocrine disrupting substances bind to target DNA via proteins such as receptors. This affects the expression of the DNA, etc., and causes toxicity. That is, exogenous endocrine disruptors do not directly bind to DNA, but bind to indirect DNA via proteins such as receptors. Therefore, the evaluation of the binding is not easy in conventional methods such as pre-staring using DNA binding.
  • the present inventors have recently immobilized a sensitizing dye on a working electrode through direct or indirect specific binding between a test substance and a probe substance, and generated the photosensitizing dye by photoexcitation. It has been found that by detecting photocurrent, the analyte can be detected and quantified easily and accurately with high sensitivity. In addition, it has been found that a plurality of samples can be individually measured on a single working electrode, or that a plurality of types of test substances can be simultaneously analyzed.
  • an object of the present invention is to detect and quantify a test substance having a specific binding property with high sensitivity in a simple and accurate manner.
  • a sample liquid containing a test substance, a working electrode having a probe substance capable of directly or indirectly specifically binding to the test substance on its surface, and a counter electrode are prepared.
  • the sample solution is brought into contact with the working electrode to specifically or directly bind the test substance to the probe substance, and the binding causes the sensitizing dye to act on the probe substance.
  • the working electrode is irradiated with light to excite the sensitizing dye, and a photocurrent flowing between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode.
  • the electrode of the present invention is an electrode used as a working electrode in the above method, comprising: a conductive base material;
  • the measurement cell of the present invention is a measurement cell used in the above method, wherein the working electrode;
  • the measuring device of the present invention is a measuring device used in the above method
  • the measurement cell The measurement cell,
  • the buffer solution of the present invention is a buffer solution used in contact with a working electrode, which is used in the above method,
  • FIG. 1 Fixation of a test substance to 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 labeled with a sensitizing dye in advance, and
  • b shows the addition of a sensitizing dye capable of intercalating to a double-stranded nucleic acid. Each case is shown.
  • FIG. 4 is a diagram showing a step of immobilizing a test substance on a probe substance when the quality is a double-stranded nucleic acid.
  • FIG. 3 is a view showing a measurement cell in which a light source is arranged, and a portion 21 surrounded by a dotted line in the figure is a measurement cell.
  • FIG. 4 is a plan view of the measuring cell shown in FIG. 3.
  • FIG. 5 A step of immobilizing a test substance on a probe substance when a test substance and a second test substance having specific binding properties competing with each other are antigens and the probe substance is an antibody.
  • FIG. 6 is a diagram showing an example of an apparatus using a flow-type measurement cell and a patterned working electrode.
  • FIG. 7 is a diagram showing an example of a patterned working electrode, wherein (a) is a plan view of the working electrode, (b) is a cross-sectional view of the working electrode, and (c) is a working electrode of another embodiment. A cross-sectional view is shown, and (d) is a cross-sectional view of a working electrode of still another embodiment.
  • FIG. 8 is a view showing another example of a patterned working electrode, wherein (a) is a plan view of the working electrode, (b) is a cross-sectional view of the working electrode, and (c) is another embodiment. Sectional views of the working electrode are shown.
  • FIG. 9 is a diagram showing an example of a light source used for a patterned working electrode.
  • FIG. 10 is a view showing another example of the light source used for the patterned working electrode.
  • FIG. 11 is a diagram showing the change over time in the detection current when a 28.6 M rhodamine-modified DNA solution obtained in Examples 2 to 5 is used as a sample solution by hand.
  • FIG. 12 shows a change with time of a detection current when a 286 M rhodamine-modified DNA solution obtained in Examples 2 to 5 was used as a sample solution.
  • Fig. 13 is a diagram showing the detection current in a steady state when the rhodamine-modified DNA solutions at OnM, 28.6M, and 286 ⁇ M obtained in Example 2 were used as sample solutions. is there.
  • FIG. 14 is a diagram showing a calibration curve in a low concentration range obtained for a test substance DNA in Example 6.
  • FIG. 15 A calibration curve in the high concentration range obtained for the test substance DNA in Example 6 FIG.
  • FIG. 16 is a diagram showing the results of light absorption measurement of various oxide semiconductors by diffuse reflection spectrum, obtained in Example 9.
  • FIG. 17 is a view showing an action spectrum obtained in Example 11!
  • FIG. 18 is a view showing a spectral distribution of light irradiated through various optical filters used in Example 12.
  • FIG. 19 is an enlarged view of a wavelength range of 350 to 550 nm in the spectrum distribution diagram shown in FIG.
  • FIG. 20 is a diagram showing a temporal change of a background photocurrent value when using various optical filters obtained in Example 13.
  • FIG. 21 is a view showing the relationship between the photocurrent value and the light intensity obtained in Example 14.
  • FIG. 22 is a view showing the spectrum distribution of various LEDs used in Example 15.
  • FIG. 23 is a diagram showing a photocurrent, a blank current, a difference between a photocurrent and a blank current, and an SZN ratio obtained in Example 15 when various LEDs are used.
  • FIG. 24 is a view showing the relationship between the photocurrent and the test DNA concentration obtained in Example 16.
  • FIG. 25 is a view showing an action spectrum obtained in Example 18.
  • FIG. 26 is an SEM image obtained for a cross section of a working electrode obtained in Example 19.
  • FIG. 27 is an SEM image obtained of a surface of a titanium oxide porous membrane of a working electrode obtained in Example 19.
  • FIG. 28 is a diagram showing photocurrent values obtained for various oxide semiconductor electrodes obtained in Example 20.
  • FIG. 29 is a view showing the relationship between the photocurrent and the concentration of Cy5-labeled ssDNA obtained in Example 21.
  • FIG. 30 shows the relationship between the photocurrent and the concentration of rhodamine-labeled HSA obtained in Example 22.
  • FIG. 31 is a view showing a photocurrent value obtained in Example 25 when each buffer was used as a cleaning solution for a working electrode. Detailed description of the invention
  • 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 having on its surface a probe substance that can specifically or directly bind to a test substance. That is, the probe substance specifically binds not only to a substance that directly and specifically binds 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 bondable substance.
  • the sample solution is brought into contact with the working electrode to specifically or directly bind the test substance to the probe substance, and this binding fixes the sensitizing dye to the working electrode. Let it.
  • a sensitizing dye is a substance capable of emitting electrons to a working electrode in response to photoexcitation.
  • the sensitizing dye may be labeled with a test substance or a mediator in advance, or may be intercalated with a conjugate of a test substance and a probe substance. When using a sensitizing dye that can be curated, it should simply be added to the sample solution.
  • the working electrode and the counter electrode are brought into contact with the electrolyte medium, the working electrode is irradiated with light to excite the sensitizing dye, whereby the electron transfer from the photoexcited sensitizing dye to the electron acceptor substance occurs.
  • the analyte can be detected with high sensitivity.
  • the detected current has a high correlation with the concentration of the test sample in the sample solution, quantitative measurement of the test sample can be performed based on the measured current amount or electric amount.
  • 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.
  • the probe substance capable of specifically binding directly or indirectly to the test substance is supported on the surface of the working electrode, so that the test substance is directly or indirectly attached to the probe substance. It becomes possible to detect by binding specifically.
  • a test substance and a probe substance that can specifically bind to each other can be selected. That is, according to a preferred embodiment of the present invention, a substance having specific binding property is used as a test substance, and specifically binds to the test substance. It is preferable that the substance to be carried is carried on the working electrode as a probe substance. Thus, the test substance can be directly and specifically bound to the working electrode for detection.
  • preferred examples of the combination of the test substance and the probe substance include a single-stranded nucleic acid, a combination of single-stranded nucleic acids having complementarity to the nucleic acid, and a combination of an antigen and an antibody. .
  • the test substance is preferably a single-stranded nucleic acid
  • the probe substance is preferably a single-stranded nucleic acid having complementarity to the nucleic acid.
  • FIGS. 1 (a) and (b) The steps of specific binding of the test substance to the working electrode in this embodiment are shown in FIGS. 1 (a) and (b).
  • a single-stranded nucleic acid 1 as a test substance is hybridized with a complementary single-stranded nucleic acid 4 as a probe substance carried on a working electrode 3, and Form double-stranded nucleic acid 7.
  • the length of base pairs constituting the test substance is not limited as long as it has a portion complementary to the nucleic acid as a probe substance. It is preferable that the substance has a complementary portion of 15 bp or more to the nucleic acid. According to the method of the present invention, even for a nucleic acid having a relatively long chain length having a base pair of 200 bp, 500 bp, and 100 bp, the specific bond formation between the nucleic acid of the probe substance and the nucleic acid of the test substance can be detected with high sensitivity. It can be detected as a current.
  • a sample solution containing a single-stranded nucleic acid as a test substance includes blood such as peripheral venous blood, leukocytes, serum, urine, feces, semen, saliva, cultured cells, and tissues such as various organ cells. It can be prepared by extracting a nucleic acid from various sample samples containing a nucleic acid, such as a cell, by a known method. At this time, the cells in the sample can be destroyed by applying a physical action such as shaking or ultrasonic waves to the carrier by applying an external force. In addition, by using a nucleic acid extraction solution, cellular force and nucleic acid can be released.
  • blood such as peripheral venous blood, leukocytes, serum, urine, feces, semen, saliva, cultured cells, and tissues such as various organ cells. It can be prepared by extracting a nucleic acid from various sample samples containing a nucleic acid, such as a cell, by a known method. At this time, the cells in the sample can be destroyed by
  • nucleic acid elution solution examples include a solution containing a surfactant such as SDS, Triton-X, Tween-20, saponin, EDTA, protease, and the like.
  • a surfactant such as SDS, Triton-X, Tween-20, saponin, EDTA, protease, and the like.
  • a typical method for amplifying the offspring is a method using an enzyme such as polymerase chain reaction (PCR).
  • enzymes used in the gene amplification method include DNA-dependent DNA polymerases such as DNA polymerase and Taq polymerase, DNA-dependent RNA polymerases such as RNA polymerase I, and Q-polymerases.
  • An example is an RNA-dependent RNA polymerase such as ⁇ -replicase, which is preferably a PCR method using Taq polymerase in that amplification can be continuously repeated only by adjusting the temperature.
  • a nucleic acid can be specifically labeled with a sensitizing dye during the amplification.
  • a sensitizing dye can be carried out by incorporating aminoallyl-modified dUTP into DNA. This molecule is incorporated with the same efficiency as unmodified dUTP.
  • the fluorescent dye activated by N-hydroxysuccinimide (N-hydroxysuccinimide) reacts specifically with the modified dUTP, yielding a test substance uniformly labeled with the sensitizing dye. .
  • nucleic acid obtained as described above! / which is obtained by first purifying a purified nucleic acid solution at 90 to 98 ° C, preferably at 95 ° C or higher.
  • a single-stranded nucleic acid can be prepared by heat denaturation at a temperature.
  • the test substance and the probe substance may indirectly and specifically bind. That is, according to another preferred embodiment of the present invention, a substance having a specific binding property is used as a test substance, and a substance that specifically binds to the test substance is allowed to coexist as a mediator, and the substance having a specific binding property is used. It is preferable that a substance that can be bound to the target be supported on the working electrode as a probe substance. Thus, even a substance that cannot specifically bind to the probe substance can be specifically and indirectly bound to the working electrode via the mediator to be detected.
  • Preferred examples of the combination of the test substance, the mediator, and the probe substance in this embodiment include a ligand, a receptor protein molecule capable of accepting the ligand, and a ligand capable of specifically binding to the receptor protein molecule. Combinations of single-stranded nucleic acids are included.
  • Preferred examples of ligands include exogenous endocrine disruptors (environmental hormones).
  • An exogenous endocrine disrupting substance is a substance that binds to DNA via a receptor protein molecule and affects its gene expression to cause toxicity. According to the method of the present invention, the substance is produced by a test substance. Simple binding of proteins such as receptors to DNA It can be monitored on flights.
  • the ligand 10 as a test substance first specifically binds to a receptor protein molecule 11 which is a mediator. Then, the receptor protein molecule 13 to which the ligand is bound specifically binds to the double-stranded nucleic acid 14 as a probe substance.
  • a single probe substance is reacted with a plurality of the same test substances derived from different acquisition routes simultaneously, and the difference in the test substance amount due to the sample origin is determined. It is also possible to quantify the test substance derived from the intended access route.
  • a specific application example is the expression profile analysis by competitive hybridization on a microarray. In this method, test substances labeled with different fluorescent dyes are hybridized competitively with the same probe substance in order to analyze differences in the expression pattern of specific genes between cells. Things. In the present invention, the use of such a technique provides an unprecedented advantage that the analysis of the expression difference between cells can be performed electrochemically.
  • the test substance in order to detect the presence of a test substance by photocurrent, the test substance is directly or indirectly specifically bound to a probe substance in the presence of a sensitizing dye.
  • the binding immobilizes the sensitizing dye on the working electrode.
  • FIG. 1 (a) and FIG. 2 there is a test substance 1! /, And a mediator substance 11 is labeled in advance with sensitizing dyes 2 and 12. be able to.
  • FIG. 1 (a) and FIG. 2 there is a test substance 1! /, And a mediator substance 11 is labeled in advance with sensitizing dyes 2 and 12. be able to.
  • a conjugate of a test substance and a probe substance 7 for example, double-stranded nucleic acid after hybridization
  • a sensitizing dye capable of intercalation
  • a sensitizing dye can be immobilized on the probe substance by adding a sensitizing dye to the sample solution.
  • the test substance 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 of the single-stranded nucleic acid is set at 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. It is more preferable to use a 5′-terminal of the test substance from the viewpoint of further simplifying the labeling step.
  • two or more sensitizing dyes are labeled with 2 or more sensitizing dyes per molecule of the test substance. It is preferred that As a result, the amount of dye carried per unit specific surface area in the working electrode on which the electron accepting substance 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 capable of emitting electrons to the working electrode in response to photoexcitation, capable of transitioning to a photoexcited state by irradiation with a light source, and capable of transitioning from the excited state to the working electrode. Any material can be used as long as it can take an electron state that allows electron injection. Therefore, the sensitizing dye to be used is not particularly limited as long as it can take the above-mentioned electronic state between the working electrode, and particularly the electron accepting layer. Therefore, various kinds of sensitizing dyes can be used. No need to use.
  • a sensitizing dye that labels each test substance may be one that can be excited by light of different wavelengths. It suffices if each of the test substances can be individually excited by selecting the above wavelength. For example, when using multiple sensitizing dyes corresponding to multiple analytes and irradiating light with different excitation wavelengths for each sensitizing dye, signals can be detected individually even if multiple probes are on the same spot. This is possible.
  • the number of test substances is not limited. However, in consideration of the wavelength of light emitted from the light source and the absorption characteristics of the sensitizing dye, 1 to 5 kinds may be appropriate.
  • the sensitizing dye which can be used in this embodiment does not necessarily have to have its absorption maximum in the wavelength region of the irradiation light as long as it is photo-excited within the wavelength region of the irradiation light.
  • the presence or absence of a light absorption reaction of a sensitizing dye at a specific wavelength can be measured using an ultraviolet-visible spectrophotometer (for example, UV-3150, manufactured by Shimadzu Corporation).
  • the sensitizing dye include a metal complex and an organic dye.
  • Preferred examples of the metal complex include metal phthalocyanines such as copper phthalocyanine and tital phthalocyanine; chlorophyll or a derivative thereof; hemin; Complexes of osmium, iron and zinc (eg, cis-succinate-bis (2,2, -biviridyl-4,4, -dicarboxylate) ruthenium ()) can be mentioned.
  • organic dyes include metal-free phthalocyanine and 9-phen -Luxanthene dyes, cyanine dyes, metalocyanine dyes, xanthene dyes, triphenylenediolemethane dyes, ataridine dyes, oxazine dyes, coumarin dyes, melocyanin dyes, mouth dashyanine dyes, polymethine dyes, And indigo dyes.
  • the sensitizing dye manufactured by Amersham Biosciences
  • Preferred examples of the sensitizing dye capable of intercalating a double-stranded nucleic acid include ataridin orange and ethidium bromide.
  • double-stranded nucleic acid labeled with the sensitizing dye is formed simply by adding the nucleic acid to the sample solution after hybridization, so that the single-stranded nucleic acid is labeled in advance. No need to do.
  • the working electrode used in the present invention is an electrode provided with the above-mentioned probe substance on its surface, and is an electrode capable of accepting electrons emitted by the sensitizing dye fixed via the probe substance in response to photoexcitation. Therefore, the configuration and material of the working electrode are not limited as long as the electron transfer occurs between the working electrode and the sensitizing dye used, and various configurations and materials may be used.
  • the working electrode has an electron accepting layer containing an electron accepting substance capable of accepting an electron emitted by the sensitizing dye in response to photoexcitation, and the surface of the electron accepting layer
  • a probe substance is provided.
  • the working electrode further includes a conductive base material, and an electron receiving layer is formed on the conductive base material.
  • the electrodes of this embodiment are 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. And the electron accepting layer 6
  • the probe substance is carried on the surface of the substrate.
  • the electron-accepting layer in the present invention comprises an electron-accepting substance capable of accepting electrons emitted by a sensitizing dye fixed via a probe substance in response to photoexcitation.
  • the electron accepting substance can be a substance capable of taking an energy level at which an electron can be injected from a photoexcited labeling dye.
  • the energy level (A) at which electron injection from the photoexcited labeling dye is possible means, for example, a conductor (conduction band: CB) when a semiconductor is used as the electron accepting material.
  • CB conductor
  • a metal is used as the electron accepting material, it means the Fermi level.
  • the electron accepting substance used in the present invention has a level lower than the LUMO energy level of the sensitizing dye of A, in other words, lower than the LUMO energy level of the sensitizing dye! What is necessary is just to have an energy level.
  • the electron accepting substance include simple semiconductors such as silicon and germanium; titanium, tin, zinc, iron, tungsten, zirconium, hafnium, strontium, indium, cerium, yttrium, lanthanum, vanadium, and niobium.
  • Oxide semiconductors such as titanium and tantalum; perovskite semiconductors such as strontium titanate, calcium titanate, sodium titanate, barium titanate and potassium niobate; cadmium, zinc, lead, silver, antimony and bismuth Sulfide semiconductors; cadmium and lead selenide semiconductors; cadmium telluride semiconductors; zinc, gallium, indium, cadmium and other phosphorus nitride semiconductors; gallium arsenide, copper indium selenium nitride, copper indium sulfide compound semiconductors; gold , Platinum, silver, copper, aluminum Metals such as rhodium, indium and nickel; organic polymers such as polythiophene, polyphosphorus, polyacetylene and polypyrrole; and molecular inorganic substances such as C60 and C70, more preferably silicon, TiO, SnO, Fe0, WO , ZnO, Nb O
  • CuInS and CuInSe most preferably TiO.
  • the above listed semiconductors are: , May be misaligned,.
  • ITO indium-tin composite oxide
  • FTO fluorine-doped tin oxide
  • the semiconductor or metal may be either single crystal or polycrystal, but the polycrystal is more porous than the more dense one. Are preferred. This increases the specific surface area, adsorbs a large amount of the test substance and the sensitizing dye, and allows the test substance to be detected with higher sensitivity. Therefore, according to a preferred embodiment of the present invention, the electron accepting layer has porosity, and the diameter of each hole is preferably 3 to 1000 nm, more preferably 10 to 100 nm.
  • the surface area in a state where the electron-accepting layer is formed on the conductive substrate is preferably at least 10 times the projected area, more preferably at least 100 times the projected area. It is preferred that The upper limit of this surface area is not particularly limited, but will usually be about 1000 times.
  • the particle size of the fine particles of the electron-accepting substance constituting the electron-accepting layer is preferably 5 to 200 nm, more preferably 8 to 200 nm as primary particles as an average particle size using the diameter when the projected area is converted into a circle. 100100 nm, more preferably 20-60 nm.
  • the average particle diameter of the fine particles (secondary particles) of the electron accepting substance in the dispersion is preferably 0.01 to: LOO ⁇ m.
  • an electron accepting layer may be formed by using fine particles of an electron accepting substance having a large particle size, for example, about 300 nm.
  • the working electrode further includes a conductive substrate, and that the electron-accepting layer is formed on the conductive substrate.
  • the conductive substrate usable in the present invention include not only those having conductivity on the support itself, such as metals such as titanium, but also those having a conductive material layer on the surface of a glass or plastic support. You may be there.
  • the electron accepting layer is formed on the conductive material layer.
  • the conductive material constituting the conductive material layer include platinum, gold, silver, copper, and gold.
  • Metals such as lumidium, rhodium and indium; conductive ceramics such as carbon, carbide, and nitride; and indium oxide composite oxides, tin oxide doped with fluorine, tin oxide doped with antimony, and acids
  • Conductive metal oxides such as gallium-doped aluminum or zinc oxide-doped aluminum; more preferably, indium-tin composite oxide (ITO); ), A metal oxide (FTO) in which tin oxide is doped with fluorine.
  • ITO indium-tin composite oxide
  • FTO metal oxide
  • the conductive substrate can be omitted.
  • the conductive substrate is not limited as long as it is a material that can secure conductivity, and includes a thin-film or spot-shaped conductive material layer that does not itself have strength as a support.
  • the conductive substrate is not limited as long as it is a material that can secure conductivity, and includes a thin-film or spot-shaped conductive material layer that does not itself have strength as a support
  • the conductive substrate is substantially transparent, specifically, the light transmittance is preferably 10% or more, more preferably 50% or more. And even more preferably more than 70%.
  • the thickness of the conductive material layer is preferably about 0.02 to LO / zm.
  • the conductive substrate has a surface resistance of 100 QZcm 2 or less, more preferably 40 QZcm 2 or less. Lower limit of the surface resistance of the conductive substrate is not particularly limited, will usually 0. l Q Zcm 2 about.
  • Examples of preferable methods for forming the electron-accepting layer on the conductive substrate include a method in which a dispersion or colloid solution of an electron-accepting substance is applied on a conductive support, and a method in which a precursor of semiconductor fine particles is electrically conductive.
  • the method includes a method in which a fine particle film is obtained by coating on a porous support and hydrolyzing with moisture in the air (sol-gel method), a sputtering method, a CVD method, a PVD method, and a vapor deposition method.
  • a method of preparing a dispersion of semiconductor fine particles as an electron accepting substance a method of grinding in a mortar, a method of dispersing while grinding using a mill, or a method of synthesizing a semiconductor in a solvent when synthesizing a semiconductor. And then use as it is as fine particles.
  • the dispersion medium include water and various organic solvents (eg, methanol, ethanol, isopropyl alcohol, dichloromethane, acetone, acetonitrile, ethyl acetate, etc.).
  • organic solvents eg, methanol, ethanol, isopropyl alcohol, dichloromethane, acetone, acetonitrile, ethyl acetate, etc.
  • polymer, surfactant, acid, Alternatively, a chelating agent or the like may be used as a dispersion aid.
  • Preferable examples of the method of applying the dispersion liquid or colloid solution of the electron acceptor include a roller method and a dip method as an ablation method, an air knife method and a blade method as a metering system, and applications and metering.
  • the slide hopper method described in, for example, Japanese Patent Publication No. 58-4589 [disclosed in Japanese Patent Publication No. 58-4589, US Patent Nos. 2,681,294, 27,61419, 2761791, etc.] Structural methods, curtain methods, spin methods, and spray methods are mentioned.
  • the thickness of the electron-accepting layer is preferably 0.1 to 200 111, more preferably 0.1. ⁇ : LOO / zm, more preferably 1 to 30 / ⁇ , most preferably 2 to 25 / ⁇ .
  • the coating amount of the semiconductor fine particles per conductive substrate lm 2 is more preferably preferably in the range of 0. 5 ⁇ 400G instrument 5: is LOOG.
  • the electron-accepting substance comprises indium-tin composite oxide (ITO) or metal oxide (FTO) obtained by doping tin oxide with fluorine
  • the thickness of the receiving layer is preferably lnm or more, more preferably ⁇ ⁇ ⁇ ! 11 ⁇ m.
  • a preferred heat treatment temperature is 40 to 700 ° C, more preferably 100 to 600 ° C.
  • the preferable heating time is about 10 minutes to 10 hours.
  • a conductive substrate such as a polymer film having a low melting point and a low softening point
  • high-temperature treatment is performed to prevent deterioration due to heat.
  • film forming methods that are preferable to perform film formation without using a method include pressing, low-temperature heating, electron beam irradiation, microwave irradiation, electrophoresis, sputtering, CVD, PVD, vapor deposition, and the like. Is mentioned.
  • the probe substance is carried on the surface of the electron accepting layer of the working electrode thus obtained.
  • the loading of the probe substance on the working electrode can be performed according to a known method.
  • an oxidized layer is formed on the surface of the working electrode, and the nucleic acid probe and the working electrode are bonded via the oxidized layer.
  • the nucleic acid probe can be fixed to the working electrode by introducing a functional group into the terminal of the nucleic acid.
  • the nucleic acid probe into which the functional group has been introduced can be directly immobilized on the carrier by the immobilization reaction.
  • Introduction of a functional group to the end of a nucleic acid can be performed using an enzyme reaction or a DNA synthesizer.
  • the enzyme used in the enzymatic reaction include terminal dexoxynucleotidyl transferase, poly A polymerase, polynucleotide polymerase, DNA polymerase, polynucleotide adenyl transferase, and RNA ligase.
  • functional groups can be introduced by polymerase chain reaction (PCR), nick translation, or random primer method. The functional group can be introduced at the 3 'end, 5' end, or a random position, which can be introduced into any part of the nucleic acid.
  • amines, carboxylic acids, sulfonic acids, thiols, hydroxyl groups, phosphoric acids, and the like can be suitably used as functional groups for immobilizing nucleic acid probes on working electrodes.
  • a material that bridges between the working electrode and the nucleic acid probe in order to firmly fix the nucleic acid probe to the working electrode, it is also possible to use a material that bridges between the working electrode and the nucleic acid probe.
  • Preferred examples of such a cross-linking material include silane coupling agents, titanate coupling agents, and conductive polymers such as polythiophene, polyacetylene, polypyrrole, and polyaline.
  • immobilization of a nucleic acid probe can be efficiently performed by a simpler operation called physical adsorption.
  • 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 solution containing a nucleic acid probe, and the nucleic acid probe is adsorbed on the surface of the carrier.
  • the buffer solution of the present invention described later as this buffer solution the detection sensitivity of the test substance by the working electrode can be improved.
  • a blocking agent As a usable blocking agent, any substance can be used as long as it is capable of filling a site on the surface of the electron-accepting layer after the nucleic acid probe has been adsorbed and can be adsorbed to the electron-accepting substance by chemical adsorption or physical adsorption. It is preferably, but not limited to, a substance having a functional group that can be adsorbed via a chemical bond.
  • preferable blocking agents include those capable of adsorbing on titanium oxide such as carboxylic acid group, phosphoric acid group, sulfonic acid group, hydroxyl group, amino group, pyridyl group and amide. Substances having various functional groups.
  • a probe substance is supported on the working electrode in a plurality of sections separated from each other, and light irradiation by a light source is individually performed on each area.
  • a plurality of samples can be measured on one working electrode, it is possible to perform DNA chip integration and the like.
  • a plurality of areas separated from each other, on which a probe substance is supported on a working electrode are patterned, and each of the areas is scanned while being irradiated with light. It is preferable that the detection or quantification of the test substance is continuously performed in a single operation in the sample in the region.
  • 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. Accordingly, it is possible to simultaneously measure a large number of samples in a number obtained by multiplying the number of regions by the number of types of probe substances in each region.
  • a different probe substance can be carried in each of a plurality of regions separated from each other on the working electrode.
  • a number of types of probe substances corresponding to the number of the divided areas can be carried, so that a large number of types of test substances can be measured at the same time. Since this embodiment can analyze a different test substance for each region, it can be preferably used for multi-item analysis of single nucleotide polymorphism analysis (SNPs).
  • SNPs single nucleotide polymorphism analysis
  • the counter electrode used in the present invention has an electrode between it and the working electrode when it comes into contact with the electrolyte medium.
  • the material is not particularly limited as long as it can flow, and a material obtained by depositing a metal or a conductive oxide on an insulating support such as glass, plastic, and ceramics can be used. Further, it can be formed by forming a metal thin film as a counter electrode by a method such as vapor deposition or sputtering so as to have a thickness of 5 ⁇ m or less, preferably in a range of 3 nm to 3 ⁇ m.
  • Preferred examples of the material usable for the counter electrode include conductive polymers such as platinum, gold, noradium, nickel, carbon, and polythiophene, and conductive ceramics such as oxides, carbides, and nitrides, and are more preferable. Is platinum and carbon, most preferably platinum. These materials can be formed into a thin film by the same method as the method for forming the electron accepting layer.
  • a sample solution is brought into contact with a working electrode in the coexistence of a sensitizing dye, and a test substance is directly or indirectly specifically bound to a probe substance. Is fixed to the working electrode. At this time, the detection sensitivity of the test substance by the working electrode can be improved by using the buffer solution of the present invention described later as the solvent of the sample solution.
  • hybridization with a single-stranded nucleic acid as a probe substance is performed.
  • a reaction can be performed.
  • the temperature of the hybridization reaction is preferably in the range of 37 to 72 ° C, but the optimum temperature varies depending on the base sequence and length of the probe used.
  • a conjugate of a test substance and a probe substance for example, double-stranded nucleic acid after hybridization, sensitizing color that can be intercalated.
  • a conjugate can be specifically labeled with the sensitizer by adding a sensitizer to the sample solution.
  • a test substance which is not bound to the working electrode can be used.
  • the substance is removed.
  • the cleaning liquid may further include a surfactant.
  • the working electrode on which the test substance is fixed together with the sensitizing dye is brought into contact with the electrolyte medium together with the counter electrode, and the working electrode is irradiated with light to excite the sensitizing dye. Then, a photocurrent flowing between the working electrode and the counter electrode due to the electron transfer from the photoexcited sensitizing dye to the working electrode is detected.
  • the relative positions of the working electrode and the counter electrode are not limited as long as they are not electrically short-circuited with each other and are not limited as long as they are in contact with the electrolyte medium. They may be arranged separately on the same plane. When the working electrode and the counter electrode are arranged separately on the same plane, both electrodes are provided on an insulating substrate to prevent an electrical short circuit between the working electrode and the counter electrode. Desired,.
  • FIG. 3 shows an example of such a measuring cell.
  • the measuring cell 21 shown in FIG. 3 has an electrolyte 24 filled in a gap formed between the working electrode 22 and the counter electrode 23.
  • the working electrode 22 includes a conductive base material 26 and an electron accepting layer 27, and is arranged so that the electron accepting layer 27 side is in contact with the electrolyte 24.
  • a space for accommodating the electrolytic solution 24 is secured by inserting the insulator 25 between the working electrode 22 and the counter electrode 23.
  • the distance between the electrodes is preferably short in order to efficiently carry out the cycle of oxidation reduction, and is preferably several tens / zm in view of the workability. Further, if a so-called MEMS-like manufacturing method is used, it is possible to make the distance between the electrodes closer.
  • the electrolyte medium used in the present invention can include an electrolyte, a solvent, and optionally an additive.
  • Preferred examples of the electrolyte include a combination of I and iodide.
  • Br and bromide such as quaternary ammonium compound iodine salts such as laalkylammonium iodide, pyridinium iodide, imidazolym iodide
  • Metal bromide such as LiBr, NaBr, KBr, CsBr, CaBr, or tetraalkyl
  • Bromide salts of quaternary ammonium compounds such as ammonium-bromobromide and pyridi-bromobromide
  • metal complexes such as ferroscrite-ferricyanate and fuecopene-phenylene-dimion, sodium polysulfide, and alkyl.
  • Thiol alkyl disulphide Examples of such compounds include a diol compound, a porogen dye, and a hydroquinone-quinone, and more preferably, quaternary ammonium such as I and Lil, pyridinodimoxide, and imidazolymoxide.
  • electrolyte that combines a compound iodine salt.
  • the above-mentioned electrolytes may be used in combination.
  • the electrolyte medium contains lithium ions.
  • the electrolyte concentration of the electrolytic solution is preferably 0.1 to 15 M, more preferably 0.2 to: LOM.
  • the preferable concentration of iodine is 0.01-0.5M.
  • the solvent include water, alcohols (methanol, ethanol, etc.), aprotic polar solvents (for example, -tolyls such as acetonitrile, carbonates such as propylene carbonate and ethylene carbonate, Dimethylformamide, dimethylsulfoxide, sulfolane, 1,3-dimethylimidazolinone, 3-methyloxazolidinone, and heterocyclic compounds such as dialkylimidazolidium salts). It is also possible to use the buffer solution of the present invention described later as a solvent for the electrolyte medium, thereby improving the detection sensitivity of the test substance by the working electrode.
  • aprotic polar solvents for example, -tolyls such as acetonitrile, carbonates such as propylene carbonate and ethylene carbonate, Dimethylformamide, dimethylsulfoxide, sulfolane, 1,3-dimethylimidazolinone, 3-methyloxazolidinone, and heterocyclic compounds such as dial
  • an aqueous electrolyte solution can be used. This makes it possible to measure appropriately without denaturing or inactivating biomolecules such as proteins. In addition, there is an advantage that the deterioration of the flow path of the electrolytic solution and the like and the volatilization of the electrolytic solution can be prevented, and the waste liquid can be easily treated.
  • 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 to give conductivity when dissolved in water and does not inhibit the intended electrode reaction. SO and the like.
  • Examples include EDTA, triethanolamine, oxalic acid, hydroquinone and the like.
  • the electrolyte medium may be used after being gelled (solidified).
  • the gelling method include polymer addition, oil gelling agent addition, polymerization including polyfunctional monomers, and cross-linking reaction of the polymer.
  • polymers used for the gel electrolyte matrix include polyacrylonitrile and polyvinylidene. And fluoride.
  • a light source 28 is disposed above a working electrode 22 via a light source cover 29. That is, by irradiating light from the back side of the working electrode 22 (that is, the conductive substrate), the cell is formed such that the light transmitted through the working electrode (that is, the conductive substrate and the electron accepting layer) excites the sensitizing dye. It is configured. However, it goes without saying that light can be irradiated from the back side of the counter electrode by forming the counter electrode with a translucent material, or that the working electrode and the counter electrode can be irradiated in parallel. .
  • 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, and is preferably a fluorescent light, a black light, a germicidal lamp, an incandescent lamp, a low-pressure mercury lamp.
  • Light bulbs, xenon lamps, halogen lamps, metal halide lamps, LEDs (white, blue, green, red), sunlight and the like can be mentioned. Further, if necessary, only light in a specific wavelength region may be irradiated using a spectroscope or a bandpass filter.
  • a wavelength selection means is used from a light source. It is possible to excite a plurality of dyes individually by irradiating light of a specific wavelength through the light source.
  • the wavelength selecting unit include a spectroscope, a color glass filter, an interference filter, a bandpass filter, and the like.
  • a plurality of light sources capable of irradiating light of different wavelengths depending on the type of the sensitizing dye may be used.
  • the light source include a laser beam irradiated with light of a specific wavelength and an LED. May be used.
  • the light may be guided using quartz, glass, or a liquid light guide.
  • the light emitted from the light source is essentially free of ultraviolet light, or the irradiation of light from the light source is performed through means for removing ultraviolet light.
  • sensitizing dyes can generally be excited by absorption of visible light, so even if ultraviolet light is removed, It is possible to detect photocurrent with high sensitivity by irradiation.
  • Preferable examples of the means for removing ultraviolet light include an optical filter and a spectroscope.
  • an optical filter or a spectroscope By using an optical filter or a spectroscope, the wavelength of irradiation light can be controlled, and it becomes possible to excite only the sensitizing dye while preventing photo-excitation of the working electrode itself.
  • Preferred examples of the optical filter include a color glass filter such as an ultraviolet cut filter.
  • An example of a preferable spectroscope is a spectrometer having a built-in diffraction grating because strict wavelength control is possible.
  • EL inorganic electroluminescent
  • EL organic electroluminescent
  • LED light emitting diode
  • LED light emitting diode
  • the cutoff wavelength shown in Table 1 is calculated by substituting the known band gap for the electron acceptor used in the following equation. It is preferable to remove light having a shorter wavelength. Thereby, generation of a knock ground current can be effectively suppressed according to the characteristics of the electron accepting substance.
  • the cutoff wavelength may be set to a longer wavelength than the wavelength shown in Table 1 for completeness.
  • the working electrode is composed of a plurality of electron-accepting substances, it is preferable that the band gap is narrowest among the constituent components, the wavelength is shorter than the cutoff wavelength of the component, and the wavelength is removed.
  • an ammeter 30 is connected between the working electrode 21 and the counter electrode 22, and a photocurrent flowing through the system due to light irradiation is measured by the ammeter.
  • the current value at that time reflects the amount of the sensitizing dye trapped on the working electrode.
  • the ammeter further includes a means for calculating the concentration of the test substance in the sample liquid from the obtained current amount or electric quantity.
  • a current value in the step of detecting a photocurrent, can be measured, and a concentration of a test substance in a sample solution can be calculated from the obtained current value or electric quantity. .
  • This calculation of the test substance concentration can be performed by comparing the calibration curve of the test substance concentration and the current value or the amount of electricity prepared in advance with the obtained current value or the amount of electricity.
  • the current value is different from the amount of the sensitizing dye trapped on the working electrode. Because it is reflected, an accurate current value corresponding to the concentration of the test substance can be obtained, making it suitable for quantitative measurement.
  • a test substance previously labeled with a sensitizing dye can be used as a competitor to specifically bind to a probe substance not labeled with a sensitizing dye.
  • the second analyte can be quantitatively determined.
  • the second test substance preferably has a property of more easily binding to the probe substance than the labeled test substance. Competition of these two analytes for specific binding to the probe substance provides a correlation between the detected current value and the concentration of the second analyte. That is, as the number of non-dye-labeled second test substances increases, the number of competitors that specifically bind to the probe substance decreases. A calibration curve with decreasing values can be obtained. Therefore, detection and quantification of the second test substance not labeled with the sensitizing dye can be performed.
  • the test substance and the second test substance are preferably antigens, and the probe substance is preferably an antibody.
  • FIG. 5 shows a step of fixing the test substance and the second test substance to the probe substance in this embodiment.
  • antigen 41 labeled with a sensitizing dye and antigen 42 not dye-labeled compete with each other to specifically bind to antibody 43. Therefore, as the amount of the non-dye-labeled antigen 42 increases, the amount of the dye-labeled antigen 43 that specifically binds to the antibody decreases, so that the detection current value decreases as the concentration of the second analyte increases.
  • a calibration curve can be obtained.
  • FIG. 6 shows the overall structure of the device.
  • the apparatus 50 shown in FIG. 6 includes a flow type measurement cell 51, a light source 52, an electrolyte 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 facing the working electrode, and contains an electrolytic solution or a cleaning solution between the working electrode 58 and the counter electrode 59; A flow path that can flow is formed.
  • the electrolyte or cleaning solution supplied into the measurement cell 51 by the supply pump 55 After passing through the flow path while contacting the measurement electrode 58 and the counter electrode 59, the gas is discharged to the outside of the measurement cell 51 by the discharge pump 57.
  • the control of these series of operations and the analysis of the photocurrent value can be performed by a control analyzer (not shown).
  • the working electrode 58 is formed by patterning a plurality of regions separated from each other on which a probe substance is supported on an electron-accepting layer. It is configured so that detection or quantification of a test substance can be continuously performed by a single operation on a sample.
  • FIGS. 7 (a) to 7 (d) and FIGS. 8 (a) to 8 (c) show examples of the working electrode patterned in this manner.
  • the working electrode 58 shown in FIGS. 7 (a) and 7 (b) has a plurality of spots 60 carrying a probe substance 58c on an electron accepting layer 58b formed on the entire surface of a conductive substrate 58a. It is patterned vertically and horizontally. Then, a lead wire 61 is provided on the conductive base material of the working electrode 58, and the entire working electrode 58 is connected to the ammeter 56 via the lead wire 61. According to the working electrode 58, the generated photocurrent can be measured for each spot by sequentially irradiating each spot with light. In addition, since the configuration of the electrodes is relatively simple, it is easy to manufacture the electrodes, and there is an advantage that a conventional DNA chip manufacturing technology can be used.
  • the electron accepting layer 58b itself is formed in a spot shape and the probe substance 58c is carried thereon, or as shown in FIG. 7 (d).
  • the conductive base material may be omitted, and the spot-like working electrode 58 may be constituted only by the electron accepting layer 58b, the probe substance 58c may be carried thereon, and the lead wire 61 may be provided on the electron accepting layer 58b.
  • the latter has the advantage that the manufacturing process can be simplified and the manufacturing cost can be reduced.
  • the light source 52 used for the working electrode corresponds to a force that 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 are arranged, and each light source is turned on and off in order.
  • the working electrode 58 'shown in Figs. 8 (a) and 8 (b) has a conductive base material 58a, an electron-accepting layer 58b, and a plurality of spots 60 also having a force on an insulating substrate 58d' in the vertical and horizontal directions.
  • the probe substance 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 of the spots 60 is connected through the lead wire 61.
  • Pot 60 ' is connected to ammeter 56. According to the working electrode 58 ', the photocurrent generated in each spot can be measured simultaneously and individually simply by simultaneously irradiating the entire surface of the working electrode with light.
  • the photocurrent at each spot can be measured individually, there is an advantage that the photocurrent generated at another spot is not picked up as noise.
  • the conductive base material is omitted, and a spot-like working electrode 58 'is constituted only by the electron accepting layer 58b, and the probe substance 58c' is placed thereon.
  • the manufacturing process for supporting and supporting the electron receiving layer 58b 'with the lead wire 61' can be simplified and the manufacturing cost can be reduced.
  • the light source 52 used for the working electrode 58 ′ corresponds to a force that is a light source that moves vertically and horizontally on the working electrode 58 or each spot of the working electrode 58, as in the case of the working electrode in FIG.
  • a plurality of light sources may be arranged in order to turn on and off each light source in turn.
  • the sample solution is brought into contact with the working electrode to specifically or indirectly specifically bind the test substance to the probe substance.
  • the spot pattern shown in FIG. 7 is masked on 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 mounted on the flow type measurement cell 51.
  • the supply pump 55 is operated to feed the electrolytic solution from the electrolytic solution tank 53 into the measuring cell 51, and after the flow path in the measuring cell is filled with the electrolytic solution, the liquid supply is stopped.
  • Light is emitted from the light source 52 to the working electrode 58, and a photocurrent generated between the working electrode 58 and the counter electrode 59 is measured by the ammeter 56.
  • the photocurrent value it is preferable to adopt a value several tens of seconds after the start of irradiation, at which the photocurrent value is stabilized.
  • the analyte concentration is calculated by comparing the obtained current value with a previously prepared calibration curve of the analyte concentration and the current value.
  • the supply pump 55 is operated to feed the cleaning liquid from the cleaning liquid tank 53 into the measurement cell 51, and at the same time, the electrolyte in the measurement cell 51 is discharged by operating the discharge pump 57, and the measurement is performed. After replacing the washing solution with the electrolyte in the flow path in the cell, stop feeding and draining. to this Thus, the next measurement can be performed in the same procedure as above using the measurement cell 51 cleaned with the cleaning liquid.
  • a buffer solution containing a carboxyl group, a phosphate group, and an amino group-free buffer and a solvent are used as the buffer solution used in contact with the working electrode.
  • it is used.
  • Examples of use in contact with the working electrode include a process of immobilizing the probe substance on the working electrode, a process of immobilizing the test substance on the working electrode via the probe substance, and an immobilization of the test substance. Examples include a subsequent process of cleaning the working electrode, a step of detecting a photocurrent using the working electrode, and the like.
  • the buffer is not limited as long as it has a chemical structure not containing a carboxyl group, a phosphate group, and an amino group and has a buffering action.
  • R 1 is an alkylene group having 1 to 4 carbon atoms, which may be substituted with a hydroxyl group
  • X is a sulfonic acid group or a salt thereof
  • A is O or YR 2 — N (here Wherein R 2 has the same meaning as R 1
  • Y is a sulfonic acid group or a salt thereof, or a hydroxyl group).
  • the alkylene group is preferably an ethylene group! /.
  • buffers include 2- [4- (2-hydroxyethyl) 1-piperazyl] ethanesulfonic acid (HEPES), piperazine-1,4-bis (2ethanesulfonic acid) ( PIPES), piperazine-1,4-bis (2 ethanesulfonic acid), sesquisodium salt (PIP ES sesquisodium), and 2-morpholinoethanesulfonic acid (MES)
  • HEPES 2- [4- (2-hydroxyethyl) 1-piperazyl] ethanesulfonic acid
  • PIPES piperazine-1,4-bis (2 ethanesulfonic acid)
  • PIPES piperazine-1,4-bis (2 ethanesulfonic acid
  • sesquisodium salt PIP ES sesquisodium
  • MES 2-morpholinoethanesulfonic acid
  • the alkylene group is preferably a propylene group.
  • Such a buffer include 3- [4- (2-hydroxyethyl) 1-piradizyl] propanesulfonic acid (EPPS), 2-hydroxy-1- [4- (2-hydroxyethyl). Tyl) 1-piperazyl] propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-13 morpholinopropanesulfonic acid (MOPSO), and piperazine-1,4 Bis (2-hydroxyl-3-propanesulfonic acid) (POPSO).
  • EPPS 3- [4- (2-hydroxyethyl) 1-piradizyl] propanesulfonic acid
  • HEPPSO 2-hydroxy-1- [4- (2-hydroxyethyl).
  • Tyl) 1-piperazyl] propanesulfonic acid HEPPSO
  • MOPS 3-morpholinopropanesulfonic acid
  • MOPSO 2-hydroxy-13 morpholinopropanesulfonic acid
  • POPSO piperazine-1,4 Bis (2-hydroxyl-3-prop
  • the concentration of the buffer is preferably 1 to 200 mM, more preferably 1 to 100 mM, and still more preferably 10 to 50 mM.
  • the solvent used for the buffer solution is not limited as long as it does not inhibit the properties of the test substance and the working electrode.
  • Preferred examples include water and alcohol, and more preferred is water. is there.
  • a buffer solution in order to stably retain a test substance having a specific binding property such as a nucleic acid, an exogenous endocrine disrupting substance, or an antigen to improve the measurement accuracy, a buffer solution
  • the pH is preferably adjusted to 5.0 to 9.0, more preferably 6.0 to 8.0, and even more preferably 6.5 to 7.5.
  • the use of the buffer solution is not limited as long as it is a solution used in contact with the working electrode used in the method of the present invention. Preferred uses include a test substance as described above.
  • the solvent examples include a solvent for a sample solution, a solvent for a solution containing a probe substance capable of directly or indirectly specifically binding to a test substance, a solvent for an electrolyte medium, and a washing solution for a working electrode or a measurement cell.
  • Example 1 Fabrication of working lightning pole
  • raw materials having the following composition were thoroughly mixed using an automatic mortar, and then dried at 150 ° C. for 6 hours to obtain a mixture.
  • An edge frame of a glass substrate (manufactured by Asahi Glass) on which a fluorine-doped SnO film is formed has a width of about 63
  • the paste was squeegee printed, and dried at 60 ° C for 2 hours.
  • the obtained glass substrate was placed in a firing furnace, the temperature of the furnace was raised to 500 ° C. over about 17 minutes, and the temperature was maintained 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 on which an electron receiving layer containing titanium oxide was formed was obtained.
  • NA was dissolved in a buffer (3X SSC) to prepare a 286 ⁇ NH-modified DNA solution.
  • the DNA solution was sufficiently distributed to the four corners of the opening of the seal. Subsequently, the DNA solution was directly covered with a glass plate so as to prevent bubbles from entering the DNA solution as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like.
  • the NH-modified DNA was incubated in the container at 60 ° C. for 2 hours. Then remove the DNA solution and gently run the electrode with running water.
  • a silicone seal similar to that used in Example 1 was placed on the surface of the working electrode, and an ordamine-modified DNA solution of each concentration was injected into the opening at a rate of 35 / zl.
  • the solution was directly covered with a glass plate so as to prevent bubbles from entering the solution as much as possible, and the solution was placed in a plastic container whose vapor pressure was adjusted with moistened paper or the like.
  • hybridization was performed by incubating overnight (12 hours) at 60 ° C.
  • the working electrode thus subjected to hybridization was immersed in a washing solution, and washed while being slowly shaken.
  • the cleaning liquids shown in Table 2 below were used, and each cleaning liquid was cleaned at the cleaning time, the number of cleaning times, and the temperature shown in the following table.
  • the washing container was replaced every time the washing solution was changed.
  • 2X SSC Aqueous solution containing 0.3 M sodium salt and 0.03 M sodium citrate (PH 7.0)
  • washing was performed twice by slightly raising and lowering the working electrode with ethanol in the liquid. After the second washing, air was blown quickly to disperse residual water without wiping with paper etc.
  • a measurement cell as shown in FIGS. 3 and 4 was assembled as follows.
  • a platinum electrode was prepared by forming a platinum thin film on a glass substrate by sputtering. A 500 m thick silicon sheet 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 platinum-coated end of the platinum electrode so that current could be taken out. The working electrode was also connected to the ammeter via a lead wire.
  • the electrolytic solution a mixed solution prepared by dissolving 0.05M of iodine and 0.5M of tetrapropylammonium-moxide in a mixed solvent of ethylene carbonate and acetonitrile having a volume ratio of 8: 2 was prepared. After 5 L of this electrolytic solution was dropped on the platinum electrode, the working electrode was placed such that its electron-accepting layer faced the platinum electrode. In this manner, a sandwich-type measurement cell in which the spacer and the electrolyte were sandwiched between the working electrode and the counter electrode was obtained.
  • FIG. 6 shows the change over time in the detection current when using a 28.6 ⁇ rhodamine-modified DNA solution
  • FIG. 7 shows the change over time using the 286 ⁇ rhodamine-modified DNA solution.
  • Row 3 Probe unmatched work ffl Lightning used.
  • Example 1 For comparison, the titania oxide before supporting the NH-modified DNA prepared in Example 1 was also used.
  • a measurement cell was constructed using a working electrode on which only an electron-accepting layer containing an ion was formed, and the measurement was carried out in the same manner as in Example 2.
  • Figure 6 shows the change over time in the detection current when a 28.6 M rhodamine-modified DNA solution was used
  • Figure 7 shows the change over time in the detection current when a 286 M rhodamine-modified DNA solution was used.
  • the titanium oxide itself was excited by a small amount of powerful UV light that could not be removed by the UV cut filter, and a photocurrent was observed.However, in Example 2 using the rhodamine-modified DNA solution, The photocurrent was significantly lower than in the case.
  • Example 4 Measurement when working electrode is blocked
  • a working electrode carrying a probe substance was obtained in the same manner as in Example 1.
  • the same silicon seal as that used in Example 1 was placed on the electrode surface again, and 35 ⁇ l of 10 ⁇ l of diethanolamine was injected into the opening as a blocking agent. Bubbles as much as possible in the blocking agent It was placed in a plastic container whose top was covered with a glass plate and whose vapor pressure was adjusted with moistened paper. Then, the blocking agent was incubated at 60 ° C for 30 minutes. After the electrode surface was lightly washed again with running water, air was blown to disperse residual water.
  • Rhodamine-modified DNA having the nucleotide sequence of CCCAGTCACGACGTT and PNA having the nucleotide sequence of 5'-CCCAGTCACGACGTTT as a competitor are combined with a sofa (2X SSC, 0.03% SDS).
  • a solution containing 6 M rhodamine modification and 200 M PNA and a solution containing 286 ⁇ ⁇ rhodamine modification and 200 ⁇ M PNA were prepared.
  • test DNA As a dye-labeled test substance (hereinafter also referred to as test DNA), 15 nucleobases (3 'rhodamine DNA) having the following base sequence and labeled at the 3' end with rhodamine B were prepared.
  • probe DNA As a probe substance (hereinafter, also referred to as probe DNA), a 15-nucleotide base having a complementary strand to the above-described test DNA (a DNA whose 5 ′ end is modified with an amino group (hereinafter, 5′-NH-DNA)) That is, the probe DNA and the test DNA were
  • Double-stranded DNA can be formed by a bridging reaction.
  • Test DNA (3, Rhodamine DNA): 3 'Rho TTGCAGCACTGACCC 5'
  • Fluorine-doped tin oxide (F—SnO: FTO) coated glass manufactured by AI Special Glass Co., Ltd.
  • An aqueous solution was prepared by dissolving. This solution was previously kept at 95 ° C for 3 minutes, and then heat-denatured by cooling on ice (2 ° C) for 3 minutes or more.
  • a perforated tape for a spacer was stuck on the electron-accepting layer of the working electrode obtained earlier, and air remaining on the tape-adhering surface was removed using a tip of a piset.
  • a silicon sheet having an opening having a size of 5 mm ⁇ 5 mm square was placed and brought into close contact with each other. 251 of the previously prepared 5'-NH-DNA solution (200 / zM) was loaded into the opening.
  • the tip of the pipette tip was used to sufficiently spread the DNA solution to the four corners of the opening of the silicon seal. Then, the DNA solution was covered with a glass plate from above so as to prevent air bubbles from entering the DNA solution as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. Incubate 5'-NH-DNA in this container at 60 ° C for 6 hours
  • 3 'rhodamine DNA as test DNA labeled with dye is dissolved in HEPES aqueous solution, and 3' rhodamine DNA solution having each concentration of 0, 10, 40, 400, 4000, 15000, 25000, 30000, 40000, and 80,000 nM Was prepared.
  • a 25 ⁇ l working electrode was loaded with each concentration of 3 ′ rhodamine DNA solution.
  • the solution was placed directly above the glass plate with a lid so as to prevent air bubbles from entering the solution as much as possible, and the solution was placed in a plastic container whose vapor pressure was adjusted with wet paper or the like.
  • hybridization was performed by incubating at 60 ° C. for 15 hours.
  • the working electrode thus subjected to hybridization was immersed in a cleaning solution, and washed while being slowly shaken.
  • the cleaning liquids shown in Table 3 below were used, and each cleaning liquid was cleaned at the cleaning time, the number of cleaning times, and the temperature shown in the following table.
  • the washing container was replaced every time the washing solution was changed. Further, the working electrode was rinsed with water for 5 seconds, and air was quickly blown to disperse residual water.
  • HEPES 50 mM, pH 7.0 solution of 2- [4- (2-hydroxyethyl) -11-biperazinyl] ethanesulfonic acid (manufactured by Dojindo Laboratories)
  • a measuring cell as shown in FIGS. 3 and 4 was assembled as follows.
  • a platinum electrode was prepared by sputtering a platinum thin film on a lmm-thick glass substrate via a chromium layer for ensuring adhesion.
  • a 500 / zm-thick silicon sheet 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.
  • a lead wire was connected to the platinum-coated end of the platinum electrode so that current could be taken out.
  • the working electrode was also connected to the ammeter via a lead wire.
  • a mixed solution was prepared by dissolving iodine (0.06M) and tetrapropylammonium-dymoxide (0.6M) in a mixed solvent of acetonitrile and ethylene carbonate having a volume ratio of 4: 6. After one drop (5 ⁇ L) of this electrolyte was dropped on the platinum electrode, the working electrode was placed so that its electron-accepting layer faced the platinum electrode. Thus, a sandwich-type measurement cell in which the spacer and the electrolytic solution were sandwiched between the working electrode and the counter electrode was obtained.
  • the lead wire of the working electrode and the lead wire of the counter electrode were connected to a potentiostat (Hokuto Denko Corporation, HS V-100).
  • Light is guided from a 250W xenon lamp (manufactured by Hayashi Watch Industry Co., Ltd., LA-250Xe) using a liquid light guide, and the light absorption and photocurrent by the substrate Sidani Titanium
  • the working electrode surface was irradiated with light through an ultraviolet cut filter (Y-43, manufactured by Asahi Techno Glass Co., Ltd.) capable of removing light having a wavelength of 430 nm or less.
  • an ultraviolet cut filter Y-43, manufactured by Asahi Techno Glass Co., Ltd.
  • the calibration curves shown in FIGS. 14 and 15 were obtained. As shown in FIG. 14, in the low concentration range where the test DNA concentration was 0 to 4000 nM, a proportional relationship was observed between the test DNA concentration and the current value. Further, as shown in FIG. 15, a linear relationship was obtained between the logarithm of the test DNA concentration and the current value in the high concentration range of the test DNA concentration of 1000 to: LOOOOOnM. 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 Thunder solution containing lithium ion ⁇ : body
  • electrolytes A and B containing the following two types of counter cations were used as the electrolyte and that the concentration of the test DNA solution (5,1-NH-DNA solution) was 200 / zM
  • Electrolyte A 0.06M I and 0.6M in a mixed solvent of 40% by volume ethylene carbonate and 60% by volume acetonitrile
  • Electrolyte B a solution of 0.05M I and 0.5M Li + I— dissolved in acetonitrile
  • the measured photocurrent value (stable current value) was as shown in Table 4.
  • both electrolytic solutions A and B contained iodine and iodide, and in each case, photocurrent could be detected with high sensitivity.
  • the photocurrent tended to increase by 20 to 35% compared to the electrolyte solution A containing no lithium ions.
  • the detected photocurrent can be increased and the test DNA can be detected with high sensitivity.
  • Example 6 The test was carried out in the same manner as in Example 6, except that the working electrode preparation paste was prepared as follows, and the concentration of the test DNA solution was set to 200 ⁇ M.
  • Nb O Taki Chemical Co., Ltd., powder obtained by evaporating and drying niobium oxide sol, average particle size of about 10 ⁇
  • Diffuse reflection (DR) spectra of the following five types of semiconductors were measured using a spectrophotometer (UV-3150, manufactured by Shimadzu Corporation).
  • Powders prepared by mixing isopropyl alcohol solution of poxide in a molar ratio (Sr: Ti) of 1: 1, evaporating to dryness at 100 ° C, and baking at 850 ° C, average particle size About 2 OOnm F3: Showa Titanium Co., Ltd., average particle size about 50 nm, anatase: rutile 4: 6 titanium oxide
  • Example 10 Influence of ⁇ depending on the position of elementary label in inspected DNA
  • Example 6 Same as Example 6, except that the following two types of dye-labeled test DNA, 3 'rhodamine DNA and 5' rhodamine DNA, were used, and the test DNA solution concentration was 200 M The test was performed.
  • a double-stranded DNA can be formed by the hybridization reaction.
  • the measured photocurrent value (stable current value) was as shown in Table 6.
  • 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.
  • results of the blank test show that almost no photocurrent is detected when the working electrode is used as is, in which neither the probe DNA nor the test DNA is immobilized.
  • the generation of 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 acceptor, and the photocurrent value is the double-stranded DNA on the electrode. It turns out that it depends on the rate of formation of.
  • the concentration of the test DNA solution was set to 40 M. Instead of the photocurrent measurement, The test was performed in the same manner as in Example 6 except that the action spectrum of the photocurrent was measured.
  • IPCE 1250 X photocurrent density AZcm 2 ) Z [wavelength (nm) X photon flux (W / m 2 )]
  • FIG. 17 shows the absorption spectrum of the sensitizing dye rhodamine B used.
  • the resulting action spectrum showed a profile similar to that of the sensitizing dye (rhodamine B). This suggests that the photocurrent is due to the excitation of the sensitizing dye. That is, since the absorption center wavelength of rhodamine B is 520 nm, it is considered that the photocurrent value can be increased by increasing the amount of light in this wavelength range. On the other hand, the photocurrent tended to increase in the wavelength region of 420 nm or less.
  • 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, resulting in so-called noise. Therefore, it can be seen that it is effective to remove light having a wavelength of 420 nm or less as much as possible in order to increase the accuracy of the sensor.
  • Example 12 Relationship between light source filter and background current
  • the test was performed in the same manner as in Example 6, except that the following six types of optical filters were used, and that no hybridization was performed.
  • Y-43 filter Asahi Techno Glass Co., Ltd., Y-43, UV cut filter
  • 550nm-40hw filter Optoline, A06-8200843, center wavelength 550nm, half width 40nm
  • 20nm-220hw filter manufactured by Asahi Spectroscopy, PB0620-220, center wavelength 620nm, half width 220nm
  • 600nm-260hw filter Asahi Spectroscopy, PB060_260, center wavelength 600nm, half width 260nm
  • 620nm-260hw filter manufactured by Asahi Spectroscopy, PB0620_260, center wavelength 620nm, half width 260nm
  • 600nm-300hw filter Asahi Spectroscopy, PB0600-300, center wavelength 600nm, half width 300nm
  • the measured background photocurrent value (stable current value) and the amount of light absorption were as shown in Table 7.
  • the spectral distribution of the light source when the above six types of filters are used for a 250W xenon lamp light source is shown by a spectral radiometer (USIO-40D).
  • the results were as shown in FIG. Fig. 19 shows an enlarged view of the wavelength range from 350 to 550nm in the spectrum distribution diagram shown in Fig. 18.
  • Example 13 Cabinet of light source filter S / N ratio
  • Example 6 The test was performed in the same manner as in Example 6, except that the Y-43 filter and the 550 nm-40hw filter used in Example 11 were used as the optical filters, and the concentration of the test DNA solution was set to 40 M.
  • the time-dependent change of the measured background photocurrent value was as shown in FIG. As shown in Figure 20, the maximum photocurrent of the Y-43 filter was 3.4 mA, and the maximum value of the 550 nm-40hw filter was 1.1 mA. That is, the value of the photocurrent when using the 550nm-40hw filter was as small as about 1Z3 when using the Y-43 filter. This is because the 550nm-40hw filter is a filter that can reduce the background current as well as the component of the result of Example 12, but also weakens the intensity of visible light. It is thought that it became small.
  • the ratio of the maximum value Z of the photocurrent to the background current is considered.
  • the same evaluation was performed for other filters, and it was found that the S / N ratio of the 550nm-40hw filter was the best! /.
  • the test was performed in the same manner as in Example 6, except that the concentration of the test DNA solution was set to 40 M and the intensity of the light source was changed.
  • the light intensity was measured using a spectral radiometer (USR-40d, manufactured by Shio Denki).
  • the relationship between the measured photocurrent value (stable current value) and the light intensity was as shown in Fig. 21. As shown in FIG. 21, the linearity was confirmed up to the region of light intensity of 800 mWZcm 2 , and it was found that the reaction speed of the photocurrent was light rate-determined in proportion to the first power of the light intensity. In general, in order for a sensor to function with high accuracy and stability, it is considered desirable to evaluate the reaction under a light-rate control, in which the reaction rate does not become diffusion-controlled in proportion to the 1Z2 power of light intensity. I have.
  • Example 6 The test was carried out in the same manner as in Example 6, except that the probe DNA was carried as follows, hybridization was not carried out, and the following LED was used as a light source.
  • Red LED Oasis RED, TOL-50aURsCEs
  • aqueous solution was prepared by dissolving in ES (pH 7.0). Keep this solution in advance at 95 ° C for 3 minutes Then, it was heat denatured by cooling on ice (2 ° C) for 3 minutes or more.
  • Probe DNA (5, -NH3, rhodamine DNA):
  • a surface treatment with a silane coupling agent was performed on the electron-accepting layer of the working electrode obtained above in order to improve the bonding force between the probe DNA and the electron-accepting substance (titanium oxide). That is, a solution in which 0.5 wt% of a silane coupling agent (manufactured by Shin-Etsu-Danigaku Co., Ltd., KBM-403) was dissolved in isopropanol was allowed to react at 75 ° C. for 5 minutes on the surface of the titanium oxide, It was washed with isopropanol and dried.
  • a silane coupling agent manufactured by Shin-Etsu-Danigaku Co., Ltd., KBM-403
  • a perforated tape for spacer was stuck on the surface of the working electrode thus obtained, and air remaining on the tape-adhered surface was removed using a tip of tweezers.
  • a silicon sheet having an opening having a size of 5 mm ⁇ 5 mm square was placed and brought into close contact with each other.
  • the 5,1-NH-3, rhodamine DNA solution (40) 40
  • a new silicone seal was placed on the working electrode carrying the probe substance in this manner, and 25 ⁇ l of 10 ⁇ l of diethanolamine was injected into the opening as a blocking agent.
  • the container 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. Then, it was kept at 60 ° C for 30 minutes to incubate the blocking agent. After the electrode surface was again lightly washed with running water for 2 seconds, air was blown to disperse residual water. Thus, a blocked working electrode was obtained.
  • the photocurrent was measured for a blank electrode in which the probe DNA was not immobilized on the working electrode.
  • the ratio between the photocurrent of the working electrode on which the probe DNA was immobilized and the photocurrent of the plank electrode was evaluated as the SZN ratio.
  • the loading of the probe DNA was performed as follows, and the following LED was used as the light source, the same as in Example 6 Photocurrent measurements were made.
  • An aqueous solution was prepared by dissolving in HEPES (pH 7.0). This solution was kept at 95 ° C for 3 minutes in advance, and then heat-denatured by cooling on ice (2 ° C) for 3 minutes or more.
  • a surface treatment with a silane coupling agent was performed on the electron-accepting layer of the working electrode obtained above in order to improve the bonding strength between the probe DNA and the electron-accepting substance (titanium oxide). That is, a solution of 0.5 wt% of a silane coupling agent (Shin-Etsu Chemical, KBM-403) dissolved in isopropanol is allowed to react at 75 ° C for 5 minutes on the surface of the electron-accepting layer (titanium oxide), and then the solution is mixed with isopropanol. Washed and dried.
  • a silane coupling agent Shin-Etsu Chemical, KBM-403
  • a perforated tape for a spacer was stuck on the surface of the working electrode thus obtained, and air remaining on the tape-adhering surface was removed with the tip of tweezers.
  • a silicon sheet having an opening having a size of 5 mm ⁇ 5 mm square was placed on the tape and brought into close contact therewith.
  • a new silicone seal was placed on the working electrode carrying the probe substance in this manner, and 25 ⁇ l of 10 ⁇ l of diethanolamine was injected into the opening as a blocking agent.
  • the container 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. Then, it was kept at 60 ° C for 30 minutes to incubate the blocking agent. After the electrode surface was again lightly washed with running water for 2 seconds, air was blown to disperse residual water. Thus, a blocked working electrode was obtained.
  • Example 17 Dye labeling of target DNA using ruthenium complex succinimidyl ester
  • a protein having an amino group commercially available as dyes order to dye modified Ru, ruthenium complex Sukushin'imiji glycol ester represented by the following formula - the (R U ONSu) Prepared.
  • succinimidyl ester derivatives were used as DNA labeling dyes. It turns out that it is also available. As can be seen from Table 8, the succinimidyl ester derivative appears to bind not only to the terminal amine but also to the amine in the nucleobase.
  • this dye can introduce a plurality of labeling dyes into one molecule of DNA.
  • the action spectrum of the photocurrent was measured in the same manner as in Example 11, except that the concentration of the A solution was set to 40 / zM.
  • Probe DNA (5, -NH -DNA-2):
  • test DNA and the probe DNA can form a double-stranded DNA by a hybridization reaction.
  • the measured action spectrum (wavelength dependence of IPCE) was as shown in Fig. 25.
  • the obtained action spectrum showed a profile similar to that of the sensitizing dye AlexaFluor (R) 647. This suggests that the photocurrent is due to the excitation of the sensitizing dye.
  • the absorption center wavelength of AlexaFluor (R) 647 is considered to be 640 nm, the photocurrent derived from each dye can be observed separately from the absorption center wavelength (520 nm) of the rhodamine B dye described above. Therefore, it is possible to simultaneously observe the formation of double-stranded DNA as a photocurrent by labeling each DNA with multiple dyes having different absorption wavelengths and irradiating light that can excite these dyes separately. Become.
  • Example 19 Evaluation of the fine structure of a working lightning electrode with a titanium oxide porous film as a thunderbolt layer
  • Example 6 In the same manner as in Example 6, a working electrode provided with a titanium oxide porous film as an electron-accepting layer was produced. The cross section and surface of the obtained working electrode were observed with a scanning electron microscope (S-4100, manufactured by Hitachi, Ltd.).
  • Figure 26 shows the SEM image obtained for the cross section of the working electrode.
  • FIG. 27 shows SEM images obtained on the surface of the titanium oxide porous membrane of the working electrode, respectively. From the image shown in FIG. 26, it can be seen that the film thickness of the porous titanium oxide electrode is about 20 / zm, and the surface has a very smooth and uniform film structure. In addition, from the image shown in FIG.
  • the obtained porous electrode shows that the particles corresponding to the primary particles (particle diameter: about 40 to 50 nm) of the titanium oxide powder used are relatively well dispersed. A good porous membrane with pores helped. Therefore, in the obtained porous membrane, even if it is a DNA molecule having a relatively large molecular size, it is considered that diffusion into pores easily occurs.
  • the pore distribution of the obtained titanium oxide porous membrane was measured using a pore distribution measuring device (Belsorp28SA, manufactured by Nippon Bell). At this time, as the measurement sample, the working electrode strength after firing was obtained by peeling the porous titanium oxide film. The surface area of the sample was calculated by the BET equation, and the pore diameter was calculated by the DH equation. As a result, the BET specific surface area was 19.8 m 2 Zg, and a relatively high pore distribution was obtained with a curve having a peak at 67.8 nm. From these results, it was a component that the obtained titanium oxide porous membrane had a very porous structure in which relatively large pores were uniformly opened. In such large pores, diffusion of relatively bulky V ⁇ DNA molecules (for example, single-stranded DNA having a width of about 2 nm) is considered to occur easily.
  • V ⁇ DNA molecules for example, single-stranded DNA having a width of about 2 nm
  • Example 20 Commercialization of an oxide semiconductor lightning pole used for an LED light source
  • test DNA As a dye-labeled test substance (hereinafter also referred to as test DNA), a 25-nucleobase (5 'Cy5 DNA) labeled at the 5' end with a fluorescent dye Cy5 and having the following base sequence is available. did.
  • probe DNA As a probe substance (hereinafter, also referred to as probe DNA), a 25-nucleotide base (5'-NH-DNA) having a complementary chain to the above-mentioned test DNA and labeled with an amine at the 5 'end is used.
  • the probe DNA and the test DNA can form a double-stranded DNA by a hybridization reaction.
  • TiO Showa Titanium, F-3, average particle size 50nm
  • Nb O Taki Chemical Co., Ltd., powder obtained by evaporating and drying niobium oxide sol
  • Table 9 shows the band gap, average particle size, and background current of the various oxide semiconductors used. Note that the background current is a photocurrent of a bare electrode which is not adsorbed to both the probe DNA and the test DNA.
  • the hybridization was carried out in the same manner as in Example 6, except that the M5 and Cy5 DNA solutions were used as the test DNA solution, and the temperature of the incubation was 50 ° C.
  • a sandwich-type measuring cell was assembled in the same manner as in Example 6.
  • the photocurrent was measured in the same manner as in Example 6, except that a red LED (CCS, HLV-27-NR-R) was used as the light source, and that no power was applied using an ultraviolet light filter.
  • a red LED CCS, HLV-27-NR-R
  • FIG. 28 when Cy-5 dye-labeled DNA was used as the test DNA under irradiation with red LED, high photocurrent values were obtained at the indium oxide, oxidized zinc oxide, and oxidized titanium electrodes. It turned out to be.
  • other oxide semiconductor electrodes, tungsten oxide, niobium oxide, strontium titanate, and tantalum oxydioxide also have a photocurrent value higher than the background current value, so that they are effective as DNA sensors. It turned out to be. From the above results, it can be seen that the titanium oxide electrode is not always the optimal electrode, and the optimal electrode can also be changed according to various conditions such as the light source, the labeling dye, and the DNA.
  • Example 2 ⁇ Work composed of only ⁇ layer ⁇ fl Lightning example
  • Cy5-labeled ssDNA As a dye-labeled probe DNA, a 25 base nucleobase (Cy5-labeled ssDNA) having the following base sequence and having a 3′-end labeled with Cy5 was prepared.
  • Fluorine-doped tin oxide (F—S ⁇ : FTO) coated glass (AI film, U film, sheet resistance: 15 ⁇ / port)
  • tin-doped indium oxide (Sn—In02: ITO) coated glass Toyo Seimitsu Kogyo KK Made by Shikisha Co., Ltd., 100 ⁇ . These glasses were washed with acetone and water, and irradiated with ultraviolet rays for 30 minutes in an oxygen atmosphere to remove stains and residual organic substances. Perforated tape for spacer was applied to the washed glass, and the air remaining on the tape-bonded surface was removed using the tip of tweezers. A silicon sheet having an opening having a size of 5 mm ⁇ 5 mm square was placed on and adhered to the tape.
  • the Cy5-labeled ssDNA solution prepared at each concentration of 0, 1, 10, and 50 ⁇ was kept at 95 ° C for 5 minutes, immediately transferred to ice and kept for 10 minutes to denature the DNA. Then, 251 was loaded into the opening on the electrode prepared above. At this time, the tip of the pipette tip was used to sufficiently spread the DNA solution to the four corners of the opening of the silicon seal. Then, cover directly with a glass plate to prevent bubbles from entering the DNA solution, and place in a plastic container whose vapor pressure has been adjusted with moistened paper, etc., at 60 ° C. Incubate overnight.
  • the DNA solution was removed, the electrode surface was gently washed with running water for about 2 seconds, and then air was blown to disperse residual water. Thereafter, the electrode on which the DNA was adsorbed was washed under the conditions shown in Table 3 of Example 6, and finally, air was blown to disperse the residual water.
  • a flow type measurement cell as shown in FIG. 6 was assembled.
  • the working electrode is placed so as to face the platinum counter electrode, and a silicon sheet with a thickness of 500 m is formed between the working electrode to prevent a short circuit between the electrodes and to form a space for filling the electrolyte. Inserted.
  • the silicon sheet has a hole that is sufficiently larger than 5 mm x 5 mm square, where the fed electrolyte is collected and the DNA immobilized on the working electrode comes into contact.
  • the working electrode was connected via a spring probe in electrical contact, and the platinum electrode was connected via a lead wire connected to the end to a potentiostat (B1S1S, ALS Modl832A).
  • a potentiostat B1S1S, ALS Modl832A
  • As an electrolytic solution a mixed solution prepared by dissolving iodine (0.6M) and tetrapropylammonium-dymoxide (0.6M) 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 incorporated in the flow-type measuring cell described above and the platinum counter electrode.
  • 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.
  • HSA human serum albumin labeled with rhodamine
  • an anti-HSA antibody (Egret anti-HSA serum, Japan Biotest Laboratory) was prepared as a probe substance.
  • a working electrode was prepared, and dirt and remaining organic substances were removed in the same manner as in Example 6.
  • a perforated tape for spacer was applied on the obtained working electrode, and a tape-adhering surface was applied using a tip of tweezers. The remaining air was removed.
  • a silicon sheet having an opening having a size of 5 mm ⁇ 5 mm square was placed and closely attached.
  • an aqueous solution was prepared by dissolving an anti-HSA antibody (Peacock anti-HSA serum, Japan Biotest Laboratories) as a probe substance in 50 mM HEPES buffer (pH 7.0) to 7.68 mg / ml.
  • This solution was loaded into the opening of the silicon seal on the working electrode in the form of a 25 1Z electrode, covered with a glass plate, and left at 4 ° C overnight to solidify.
  • the working electrode thus obtained was washed three times with a 50 mM HEPES buffer (pH 7.0).
  • HSA was labeled with tetramethylrhodamine using a FluoReporter Tetramethylrhodamine Protein Labeling kit (Molecular Probes) as a labeling kit. According to the manufacturer's protocol, the labeling reaction, purification, and separation of the dye were performed to obtain a rhodamine-labeled HSA solution having a labeling ratio of 1.
  • Rhodamine-labeled HSA was serially diluted with a blocking solution to prepare rhodamine-labeled HSA solutions at concentrations of 0.1, 0.33, 1.0, and 3.3 mg / ml. Each concentration of rhodamine-labeled HSA solution was loaded into the opening of the silicon seal on the working electrode by 25 ⁇ l electrode, covered with a glass plate, and incubated at 30 ° C for 90 minutes. Then, it was washed three times with T-HEPES and rinsed with ultrapure water.
  • Example 21 The procedure was the same as in Example 21 except that an aqueous solution in which lOOmM ethylenediaminetetraacetic acid, lOOmM NaCl, and lOOmM NaSO were also dissolved was used as the electrolyte.
  • a row type measuring cell was assembled.
  • the photocurrent measured for each concentration of the HSA solution was as shown in FIG.
  • a calibration curve with a high correlation coefficient could be drawn when the antigen concentration was in the range of 0.1 to 3.3 mgZml, which proved that protein quantification was possible.
  • Example 23 Examination of the solution used in the process of immobilizing the probe material First, in the same manner as in Example 1, a working electrode on which an electron-accepting layer containing titanium oxide was formed was obtained. 5, NH— AACGTCGTGACTGGG 3, with Rho base sequence 3, mouth
  • the rhodamine-modified DNA was dissolved in buffer 1 or 2 shown below to prepare a 200 M rhodamine-modified DNA solution. This solution was previously kept at 95 ° C for 3 minutes, and then denatured by cooling on ice.
  • Buffer l 50mM HEPES aqueous solution, pH 7.0
  • Buffer 2 2X SSC: Aqueous solution containing 0.3M sodium chloride and 0.03M sodium citrate, pH 7.0, having a carboxyl group in the chemical structure
  • a silicon seal having a thickness of 700 ⁇ m and having an opening of 5 mm x 5 mm square was formed on the electron-accepting layer of the working electrode obtained above. 351 of this solution with a mouth-modified DN ⁇ solution of 200 ⁇ at the opening was injected. At this time, the DNA solution was sufficiently distributed to the four corners of the opening of the silicon seal using the tip of the pipette tip. Subsequently, the DNA solution was covered directly above with a glass plate so as to prevent bubbles from entering the DNA solution as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. The container was kept at 60 ° C.
  • a new silicone seal was placed on the working electrode carrying the probe substance, and 25 ⁇ l of 10 ⁇ l of diethanolamine was loaded as a blocking agent into the opening.
  • the container was placed in a plastic container whose top pressure was covered with a glass plate and whose vapor pressure was adjusted with moistened paper or the like. Then, it was kept at 60 ° C for 30 minutes to incubate the blocking agent. After the electrode surface was again lightly washed with running water for 2 seconds, air was blown to disperse residual water. Thus, a blocked working electrode was obtained.
  • a cell was prepared and the photocurrent was measured in the same manner as in Example 2, except that the steps of No., Ibridization and the subsequent washing step were not performed.
  • the photocurrent value was measured by measuring a stabilized current value when irradiated with light and a stabilized current value when not irradiated with light, and calculating the difference between these current values.
  • Row 24 Examination of the sample liquid used in the process of causing the probe to make an inspection or
  • a working electrode carrying a probe substance and being subjected to blocking was obtained in the same manner as in Example 6, except that 'amino-modified DNA was used.
  • Buffer 2 2X SSC: Aqueous solution containing 0.3M sodium chloride and 0.03M sodium citrate, pH 7.0, having a carboxyl group in the chemical structure
  • Example 25 Consideration of a solution used as a cleaning solution for a working lightning electrode
  • Buffer 1, 2 or 3 shown below was used as a washing solution in a formulation based on the following table, and each washing solution was washed at the washing time, washing frequency and temperature shown in the following table. The washing container was replaced every time the washing solution was changed.
  • Buffer l 50 mM HEPES aqueous solution, pH 7.0
  • Buffer 2 2X SSC: aqueous solution containing 0.3 M sodium chloride and 0.03 M sodium citrate, pH 7.0), having a carboxyl group in the chemical structure
  • Knoffer 3 150 mM phosphate buffer (PBS), pH 7.0, which is not a buffer solution of the present invention because it has a phosphate group in its chemical structure.
  • the obtained photocurrent value was as shown in FIG. From the results shown in Fig. 31, when the HEPES aqueous solution (buffer 1) was used as the washing solution for the working electrode, the aqueous solution of the carboxyl group-containing SSC aqueous solution (buffer 2) and the phosphate group-containing PBS (buffer 3) was used. In comparison, the photocurrent value is significantly higher.

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Abstract

A method of highly sensitively, conveniently and accurately detecting and quantifying a test substance having a specific binding ability is disclosed. According to this method, a sample solution containing the test substance is first brought into contact, in the coexistence of a sensitizing dye, with a working electrode having a probe material capable of directly or indirectly binding specifically to the test substance on the surface, thus allowing the test substance to directly or indirectly bind specifically to the probe material, and fixing the sensitizing dye to the working electrode owing to this binding. Next, the working electrode and a counter electrode are brought into contact with an electrolytic medium and the working electrode is photo irradiated to thereby induce the photoexcitation of the sensitizing dye. Then a photocurrent flowing between the working electrode and the counter electrode caused by the electron transfer from the thus photoexcited sensitizing dye to the working electrode is detected.

Description

明 細 書  Specification
光電流を用いた被検物質の特異的検出方法、それに用いられる電極、測 定用セル、測定装置、および緩衝溶液  Specific detection method of analyte using photocurrent, electrodes, measuring cell, measuring device, and buffer solution used in the method
発明の背景  Background of the Invention
[0001] 発明の分野  [0001] Field of the Invention
本発明は、光電流を用いて、核酸、外因性内分泌攪乱物質、抗原等の特異的結合 性を有する被検物質を特異的に検出する方法、それに用いられる電極、測定用セル 、測定装置、および緩衝溶液に関する。  The present invention provides a method for specifically detecting a test substance having a specific binding property, such as a nucleic acid, an exogenous endocrine disrupting substance, or an antigen, using a photocurrent, an electrode used for the method, a measuring cell, a measuring device, And a buffer solution.
[0002] 普晋 術  [0002] Jujin Ju
生体試料中の DNAを解析する遺伝子診断法が、各種病気の新たな予防および診 断法として、有望視されている。このような DNA解析を簡便かつ正確に行う技術とし て、以下のものが提案されている。  Genetic diagnosis, which analyzes DNA in biological samples, holds promise as a new prevention and diagnosis method for various diseases. The following techniques have been proposed as techniques for performing such DNA analysis easily and accurately.
[0003] 被検体 DNAを、これと相補的な塩基配列を有し、かつ蛍光物質を標識された DN Aプローブとハイブリダィズさせ、その際の蛍光シグナルを検出する、 DNAの分析方 法が知られている（例えば、特開平 7— 107999号公報および特開平 11— 315095 号公報参照）。この方法にあっては、ハイブリダィゼーシヨンによる二本鎖 DNAの形 成を色素の蛍光により検出する。  [0003] There is known a DNA analysis method in which a test DNA is hybridized with a labeled DNA probe having a nucleotide sequence complementary to this and a fluorescent substance, and a fluorescent signal at that time is detected. (See, for example, JP-A-7-107999 and JP-A-11-315095). In this method, the formation of double-stranded DNA by hybridization is detected by the fluorescence of the dye.
[0004] また、一本鎖に変性された遺伝子サンプルを、これに相補性を有する一本鎖の核 酸プローブとハイブリダィゼーシヨンさせた後、インターカレータ等の二本鎖認識体を 添加して電気化学的に検出する方法が知られている（例えば、特許公報第 257344 3号および表面科学 Vol. 24, No. 11. Pp. 671-676, 2003参照）。  [0004] Further, after a single-stranded denatured gene sample is hybridized with a single-stranded nucleic acid probe having complementarity thereto, a double-stranded recognizer such as an intercalator is added. (See, for example, Patent Publication No. 2573443 and Surface Science Vol. 24, No. 11. Pp. 671-676, 2003).
[0005] 一方、近年、ダイォキシンを始めとする外因性内分泌撹乱物質 (環境ホルモン)の 生殖系および神経系等への障害が社会問題化している。現在、外因性内分泌撹乱 毒性の検出は様々な方法によって行われている力 そのような物質はわずか lOppt レベル程度の極めて低い濃度で毒性を示す。このため、そのような低濃度範囲にお ける外因性内分泌撹乱物質の検出方法が望まれている。  [0005] On the other hand, in recent years, disorders of the reproductive system and nervous system of exogenous endocrine disrupting substances (environmental hormones) such as dioxin have become a social problem. Currently, detection of exogenous endocrine disrupting toxicity is done by a variety of methods. Such substances are toxic at very low concentrations, on the order of only lOppt levels. Therefore, a method for detecting an exogenous endocrine disrupting substance in such a low concentration range is desired.
[0006] 特に、外因性内分泌撹乱物質は、受容体等のタンパク質を介して標的 DNAに結 合し、それにより当該 DNAの発現等に影響を与え、毒性を生じる。すなわち、外因 性内分泌撹乱物質は、 DNAに直接的に結合するのではなぐ受容体等のタンパク 質を介して間接的 DNAに結合する。そのため、 DNA結合性を用いたプレスタリー- ング等の従来の方法にあっては、その結合の評価は容易ではな 、。 [0006] In particular, exogenous endocrine disrupting substances bind to target DNA via proteins such as receptors. This affects the expression of the DNA, etc., and causes toxicity. That is, exogenous endocrine disruptors do not directly bind to DNA, but bind to indirect DNA via proteins such as receptors. Therefore, the evaluation of the binding is not easy in conventional methods such as pre-staring using DNA binding.
[0007] ところで、増感色素を用いて光力 電気エネルギーを発生させる太陽電池が知られ ている（例えば、特開平 1 220380号公報参照)。この太陽電池は、多結晶の金属 酸化物半導体を有し、かつその表面積に広範囲にわたり増感色素の層が形成され てなるものである。し力しながら、このような電極を生物化学的な分析に応用しょうとす る試みは未だなされて ヽな 、。 [0007] By the way, a solar cell that generates photo-electric energy by using a sensitizing dye is known (for example, see Japanese Patent Application Laid-Open No. 122380/1990). This solar cell has a polycrystalline metal oxide semiconductor and has a sensitizing dye layer formed over a wide area over its surface area. However, no attempt has been made to apply such an electrode to biochemical analysis.
発明の概要  Summary of the Invention
[0008] 本発明者らは、今般、被検物質とプローブ物質との直接または間接的な特異的結 合を介して作用電極に増感色素を固定させ、この増感色素を光励起させて発生する 光電流を検出することにより、被検物質を高感度で簡便かつ正確に検出および定量 出来るとの知見を得た。また、一枚の作用電極上で、複数の試料を個別に測定したり 、あるいは複数種類の被検物質の分析を同時に行えるとの知見も得た。  [0008] The present inventors have recently immobilized a sensitizing dye on a working electrode through direct or indirect specific binding between a test substance and a probe substance, and generated the photosensitizing dye by photoexcitation. It has been found that by detecting photocurrent, the analyte can be detected and quantified easily and accurately with high sensitivity. In addition, it has been found that a plurality of samples can be individually measured on a single working electrode, or that a plurality of types of test substances can be simultaneously analyzed.
[0009] したがって、本発明は、特異的結合性を有する被検物質を高感度で簡便かつ正確 に検出および定量することを目的としている。  [0009] Therefore, an object of the present invention is to detect and quantify a test substance having a specific binding property with high sensitivity in a simple and accurate manner.
[0010] そして、本発明の光電流を用いた被検物質の特異的検出方法は、  [0010] Then, the method of the present invention for specific detection of a test substance using photocurrent,
被検物質を含む試料液と、該被検物質と直接または間接的に特異的に結合可能 なプローブ物質を表面に備えた作用電極と、対電極とを用意し、  A sample liquid containing a test substance, a working electrode having a probe substance capable of directly or indirectly specifically binding to the test substance on its surface, and a counter electrode are prepared.
増感色素の共存下、前記試料液を前記作用電極に接触させて、前記プローブ物 質に前記被検物質を直接または間接的に特異的に結合させ、該結合により前記増 感色素を前記作用電極に固定させ、  In the presence of a sensitizing dye, the sample solution is brought into contact with the working electrode to specifically or directly bind the test substance to the probe substance, and the binding causes the sensitizing dye to act on the probe substance. Fixed to the electrode,
前記作用電極と前記対電極とを電解質媒体に接触させ、そして、  Contacting the working electrode and the counter electrode with an electrolyte medium; and
前記作用電極に光を照射して前記増感色素を光励起させ、該光励起された増感 色素から前記作用電極への電子移動に起因して前記作用電極と前記対電極との間 に流れる光電流を検出すること  The working electrode is irradiated with light to excite the sensitizing dye, and a photocurrent flowing between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. Detecting
を含んでなるものである。 [0011] また、本発明の電極は、上記方法において作用電極として用いられる電極であって 導電性基材と、 . [0011] Further, the electrode of the present invention is an electrode used as a working electrode in the above method, comprising: a conductive base material;
該導電性基材上に形成される、前記増感色素が光励起に応じて放出する電子を 受容可能な電子受容物質を含んでなる電子受容層と、  An electron-accepting layer formed on the conductive substrate, the electron-accepting layer including an electron-accepting substance capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation;
を備えたものである。  It is provided with.
[0012] さらに、本発明の測定用セルは、上記方法に用いられる測定用セルであって、 上記作用電極と、  [0012] Further, the measurement cell of the present invention is a measurement cell used in the above method, wherein the working electrode;
対電極と、  A counter electrode,
前記作用電極および前記対電極が接触される電解質媒体と  An electrolyte medium with which the working electrode and the counter electrode are contacted;
を備えたものである。  It is provided with.
[0013] また、本発明の測定装置は、上記方法に用いられる測定装置であって、  [0013] The measuring device of the present invention is a measuring device used in the above method,
上記測定用セルと、  The measurement cell,
前記作用電極の表面に光を照射する光源と、  A light source for irradiating light to the surface of the working electrode,
前記作用電極と前記対電極との間を流れる電流を測定する電流計と  An ammeter for measuring a current flowing between the working electrode and the counter electrode;
を備えたものである。  It is provided with.
[0014] さらに、本発明の緩衝溶液は、上記方法において用いられる、作用電極との接触 下で使用される緩衝溶液であって、  Further, the buffer solution of the present invention is a buffer solution used in contact with a working electrode, which is used in the above method,
カルボキシル基、リン酸基、およびアミノ基を含まない緩衝剤と、  A buffer not containing a carboxyl group, a phosphate group, and an amino group;
溶媒と  Solvent and
を含んでなるものである。  .
図面の簡単な説明  Brief Description of Drawings
[0015] [図 1]被検物質が一本鎖の核酸であり、プローブ物質が前記核酸に対して相補性を 有する一本鎖の核酸である場合における、被検物質のプローブ物質への固定ィ匕ェ 程を示す図であり、（a)は被検物質が予め増感色素で標識されてなる場合を、（b)は 二本鎖の核酸にインターカレーシヨン可能な増感色素を添加した場合をそれぞれ示 す。  [FIG. 1] Fixation of a test substance to 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 labeled with a sensitizing dye in advance, and (b) shows the addition of a sensitizing dye capable of intercalating to a double-stranded nucleic acid. Each case is shown.
[図 2]被検物質がリガンドであり、媒介物質が受容体蛋白質分子であり、プローブ物 質が二本鎖の核酸である場合における、被検物質のプローブ物質への固定ィ匕工程 を示す図である。 [Figure 2] The test substance is a ligand, the mediator is a receptor protein molecule, and the probe substance FIG. 4 is a diagram showing a step of immobilizing a test substance on a probe substance when the quality is a double-stranded nucleic acid.
[図 3]光源が配置された測定用セルを示す図であり、図中の点線で囲まれる部分 21 が測定用セルである。  FIG. 3 is a view showing a measurement cell in which a light source is arranged, and a portion 21 surrounded by a dotted line in the figure is a measurement cell.
[図 4]図 3に示される測定用セルの平面図である。  FIG. 4 is a plan view of the measuring cell shown in FIG. 3.
[図 5]互いに競合する特異的結合性を有する被検物質および第二の被検物質が抗 原であり、プローブ物質が抗体である場合の、被検物質のプローブ物質への固定ィ匕 工程を示す図である。  [FIG. 5] A step of immobilizing a test substance on a probe substance when a test substance and a second test substance having specific binding properties competing with each other are antigens and the probe substance is an antibody. FIG.
[図 6]フロー型測定用セルおよびパターユングされた作用電極を用いた装置の一例 を示す図である。  FIG. 6 is a diagram showing an example of an apparatus using a flow-type measurement cell and a patterned working electrode.
[図 7]パターニングされた作用電極の一例を示す図であり、 (a)は作用電極の平面図 を、（b)は作用電極の断面図を、（c)は別の態様の作用電極の断面図を、（d)はさら に別の態様の作用電極の断面図をそれぞれ示す。  FIG. 7 is a diagram showing an example of a patterned working electrode, wherein (a) is a plan view of the working electrode, (b) is a cross-sectional view of the working electrode, and (c) is a working electrode of another embodiment. A cross-sectional view is shown, and (d) is a cross-sectional view of a working electrode of still another embodiment.
[図 8]パターニングされた作用電極の他の一例を示す図であり、 (a)は作用電極の平 面図を、（b)は作用電極の断面図を、（c)は別の態様の作用電極の断面図をそれぞ れ示す。  FIG. 8 is a view showing another example of a patterned working electrode, wherein (a) is a plan view of the working electrode, (b) is a cross-sectional view of the working electrode, and (c) is another embodiment. Sectional views of the working electrode are shown.
[図 9]パターユングされた作用電極に用いられる光源の一例を示す図である。  FIG. 9 is a diagram showing an example of a light source used for a patterned working electrode.
[図 10]パターユングされた作用電極に用いられる光源の他の一例を示す図である。 FIG. 10 is a view showing another example of the light source used for the patterned working electrode.
[図 11]例 2〜5において得られた、 28. 6 Mローダミン修飾 DNA溶液を試料液とし 手用いた場合の検出電流の経時変化を示す図である。 FIG. 11 is a diagram showing the change over time in the detection current when a 28.6 M rhodamine-modified DNA solution obtained in Examples 2 to 5 is used as a sample solution by hand.
[図 12]例 2〜5において得られた、 286 Mローダミン修飾 DNA溶液を試料液として 用 、た場合の検出電流の経時変化を示す。  FIG. 12 shows a change with time of a detection current when a 286 M rhodamine-modified DNA solution obtained in Examples 2 to 5 was used as a sample solution.
[図 13]例 2において得られた、 OnM、 28. 6 M、および 286 μ Μの各濃度のローダ ミン修飾 DNA溶液を試料液として用いた場合の、定常状態にある検出電流を示す 図である。  [Fig. 13] Fig. 13 is a diagram showing the detection current in a steady state when the rhodamine-modified DNA solutions at OnM, 28.6M, and 286µM obtained in Example 2 were used as sample solutions. is there.
[図 14]例 6において被検物質 DNAについて得られた、低濃度域における検量線を 示す図である。  FIG. 14 is a diagram showing a calibration curve in a low concentration range obtained for a test substance DNA in Example 6.
[図 15]例 6において被検物質 DNAについて得られた、高濃度域における検量線を 示す図である。 [FIG. 15] A calibration curve in the high concentration range obtained for the test substance DNA in Example 6 FIG.
[図 16]例 9において得られた、拡散反射スペクトルによる各種酸ィ匕物半導体の光吸 収測定の結果を示す図である。  FIG. 16 is a diagram showing the results of light absorption measurement of various oxide semiconductors by diffuse reflection spectrum, obtained in Example 9.
[図 17]例 11にお!/、て得られたアクションスペクトルを示す図である。  FIG. 17 is a view showing an action spectrum obtained in Example 11!
[図 18]例 12にお 、て使用した各種光学フィルタを介して照射した光のスペクトル分 布を示す図である。  FIG. 18 is a view showing a spectral distribution of light irradiated through various optical filters used in Example 12.
[図 19]図 18に示されるスペクトル分布図における 350〜550nmの波長域を拡大した 図である。  FIG. 19 is an enlarged view of a wavelength range of 350 to 550 nm in the spectrum distribution diagram shown in FIG.
[図 20]例 13において得られた、各種光学フィルタを用いた場合におけるバックグラン ド光電流値の経時変化を示す図である。  FIG. 20 is a diagram showing a temporal change of a background photocurrent value when using various optical filters obtained in Example 13.
[図 21]例 14において得られた、光電流値と、光強度との関係を示す図である。  FIG. 21 is a view showing the relationship between the photocurrent value and the light intensity obtained in Example 14.
[図 22]例 15にお 、て使用した各種 LEDのスペクトル分布を示す図である。  FIG. 22 is a view showing the spectrum distribution of various LEDs used in Example 15.
[図 23]例 15において得られた、各種 LEDを使用した場合における、光電流、ブラン ク電流、光電流とブランク電流の差、および SZN比を示す図である。  FIG. 23 is a diagram showing a photocurrent, a blank current, a difference between a photocurrent and a blank current, and an SZN ratio obtained in Example 15 when various LEDs are used.
[図 24]例 16において得られた、光電流と、被験 DNA濃度との関係を示す図である。  FIG. 24 is a view showing the relationship between the photocurrent and the test DNA concentration obtained in Example 16.
[図 25]例 18において得られた、アクションスペクトルを示す図である。  FIG. 25 is a view showing an action spectrum obtained in Example 18.
[図 26]例 19において得られた、作用電極の断面について得られた SEM画像である  FIG. 26 is an SEM image obtained for a cross section of a working electrode obtained in Example 19.
[図 27]例 19において得られた、作用電極の酸ィ匕チタン多孔質膜表面について得ら れた SEM画像である。 FIG. 27 is an SEM image obtained of a surface of a titanium oxide porous membrane of a working electrode obtained in Example 19.
[図 28]例 20にお 、て得られた、各種酸化物半導体電極につ!、て得られた光電流値 を示す図である。  FIG. 28 is a diagram showing photocurrent values obtained for various oxide semiconductor electrodes obtained in Example 20.
[図 29]例 21において得られた、光電流と、 Cy5標識 ssDNA濃度との関係を示す図 である。  FIG. 29 is a view showing the relationship between the photocurrent and the concentration of Cy5-labeled ssDNA obtained in Example 21.
[図 30]例 22において得られた、光電流と、ローダミン標識 HSA濃度との関係を示す 図である。  FIG. 30 shows the relationship between the photocurrent and the concentration of rhodamine-labeled HSA obtained in Example 22.
[図 31]例 25において得られた、作用電極の洗浄液として各バッファを使用した場合 の光電流値を示す図である。 発明の具体的説明 FIG. 31 is a view showing a photocurrent value obtained in Example 25 when each buffer was used as a cleaning solution for a working electrode. Detailed description of the invention
[0016] 光 流を用いた被檢物皙の特異的枪出  [0016] Specific detection of inspected material using light flow
本発明の方法にあっては、まず、被検物質を含む試料液と、作用電極と、対電極と を用意する。本発明に用いる作用電極は、被検物質と直接または間接的に特異的に 結合可能なプローブ物質を表面に備えた電極である。すなわち、プローブ物質は、 被検物質と直接、特異的に結合する物質のみならず、被検物質を受容体蛋白質分 子等の媒介物質に特異的に結合させて得られる結合体と特異的に結合可能な物質 であってよい。次いで、増感色素の共存下、試料液を作用電極に接触させて、プロ ーブ物質に被検物質を直接または間接的に特異的に結合させ、この結合により増感 色素を作用電極に固定させる。増感色素は、光励起に応じて作用電極に電子を放 出可能な物質であり、被検物質あるいは媒介物質に予め標識させておくか、あるい は被検物質およびプローブ物質の結合体にインターカレーシヨン可能な増感色素を 用いる場合には試料液に単に添加すればょ 、。  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 having on its surface a probe substance that can specifically or directly bind to a test substance. That is, the probe substance specifically binds not only to a substance that directly and specifically binds 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 bondable substance. Next, in the co-presence of a sensitizing dye, the sample solution is brought into contact with the working electrode to specifically or directly bind the test substance to the probe substance, and this binding fixes the sensitizing dye to the working electrode. Let it. A sensitizing dye is a substance capable of emitting electrons to a working electrode in response to photoexcitation. The sensitizing dye may be labeled with a test substance or a mediator in advance, or may be intercalated with a conjugate of a test substance and a probe substance. When using a sensitizing dye that can be curated, it should simply be added to the sample solution.
[0017] そして、作用電極と対電極とを電解質媒体に接触させた後、作用電極に光を照射 して増感色素を光励起させると、光励起された増感色素から電子受容物質へ電子移 動が起こる。この電子移動に起因して作用電極と対電極との間に流れる光電流を検 出することにより、被検物質を高い感度で検出することができる。また、この検出電流 は試料液中の被検試料濃度との高 、相関関係を有して 、るので、測定された電流 量または電気量に基づき被検試料の定量測定を行うことができる。  [0017] Then, after the working electrode and the counter electrode are brought into contact with the electrolyte medium, the working electrode is irradiated with light to excite the sensitizing dye, whereby the electron transfer from the photoexcited sensitizing dye to the electron acceptor substance occurs. Happens. By detecting the photocurrent flowing between the working electrode and the counter electrode due to the electron transfer, the analyte can be detected with high sensitivity. In addition, since the detected current has a high correlation with the concentration of the test sample in the sample solution, quantitative measurement of the test sample can be performed based on the measured current amount or electric amount.
[0018] 被檢物晳およびプローブ物質  [0018] Inspection material and probe substance
本発明の方法における被検物質としては、特異的な結合性を有する物質であれば 限定されず、種々の物質であってよい。このような被検物質であれば、被検物質と直 接または間接的に特異的に結合可能なプローブ物質を作用電極表面に担持させて おくことにより、被検物質をプローブ物質に直接または間接的に特異的に結合させて 検出することが可能となる。  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. With such a test substance, the probe substance capable of specifically binding directly or indirectly to the test substance is supported on the surface of the working electrode, so that the test substance is directly or indirectly attached to the probe substance. It becomes possible to detect by binding specifically.
[0019] すなわち、本発明の方法にあっては、被検物質およびプローブ物質として互いに 特異的に結合可能なものを選択することができる。すなわち、本発明の好ましい態様 によれば、特異的な結合性を有する物質を被検物質とし、被検物質と特異的に結合 する物質をプローブ物質として作用電極に担持させるのが好ましい。これにより、作 用電極上に被検物質を直接、特異的に結合させて検出することができる。この態様 における、被検物質およびプローブ物質の組合せの好ましい例としては、一本鎖の 核酸および核酸に対して相補性を有する一本鎖の核酸の組合せ、ならびに抗原お よび抗体の組合せが挙げられる。 That is, in the method of the present invention, a test substance and a probe substance that can specifically bind to each other can be selected. That is, according to a preferred embodiment of the present invention, a substance having specific binding property is used as a test substance, and specifically binds to the test substance. It is preferable that the substance to be carried is carried on the working electrode as a probe substance. Thus, the test substance can be directly and specifically bound to the working electrode for detection. In this embodiment, preferred examples of the combination of the test substance and the probe substance include a single-stranded nucleic acid, a combination of single-stranded nucleic acids having complementarity to the nucleic acid, and a combination of an antigen and an antibody. .
[0020] 本発明のより好ましい態様によれば、被検物質を一本鎖の核酸とし、プローブ物質 を核酸に対して相補性を有する一本鎖の核酸とするのが好まし 、。この態様におけ る被検物質の作用電極への特異的結合の工程を図 1 (a)および (b)に示す。これら の図に示されるように、被検物質としての一本鎖の核酸 1は、作用電極 3上に担持さ れたプローブ物質としての相補性を有する一本鎖の核酸 4とハイブリダィズされて、 二本鎖の核酸 7を形成する。  According to a more preferred embodiment of the present invention, the test substance is preferably a single-stranded nucleic acid, and the probe substance is preferably 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. 1 (a) and (b). As shown in these figures, a single-stranded nucleic acid 1 as a test substance is hybridized with a complementary single-stranded nucleic acid 4 as a probe substance carried on a working electrode 3, and Form double-stranded nucleic acid 7.
[0021] 一本鎖の核酸を被検物質とする場合、プローブ物質である核酸と相補性部分を有 していればよぐ被検物質を構成する塩基対の長さは限定されないが、プローブ物質 が核酸に対して 15bp以上の相補性部分を有するのが好ましい。本発明の方法によ れば、 200bp、 500bp、 lOOObpの塩基対を有する比較的鎖長の長い核酸であって も、高感度にプローブ物質と被検物質の核酸同士の特異的結合形成を光電流として 検出することができる。  [0021] When a single-stranded nucleic acid is used as a test substance, the length of base pairs constituting the test substance is not limited as long as it has a portion complementary to the nucleic acid as a probe substance. It is preferable that the substance has a complementary portion of 15 bp or more to the nucleic acid. According to the method of the present invention, even for a nucleic acid having a relatively long chain length having a base pair of 200 bp, 500 bp, and 100 bp, the specific bond formation between the nucleic acid of the probe substance and the nucleic acid of the test substance can be detected with high sensitivity. It can be detected as a current.
[0022] 被検物質としての一本鎖の核酸を含む試料液は、末梢静脈血のような血液、白血 球、血清、尿、糞便、***、唾液、培養細胞、各種臓器細胞のような組織細胞等の、 核酸を含有する各種検体試料から、公知の方法により核酸を抽出して作製すること ができる。このとき、検体試料中の細胞の破壊は、例えば、振とう、超音波等の物理 的作用を外部力 加えて担体を振動させることにより行なうことができる。また、核酸 抽出溶液を用いて、細胞力も核酸を遊離させることもできる。核酸溶出溶液の例とし ては、 SDS、 Triton-X、 Tween-20のような界面活性剤、サポニン、 EDTA、プロテア ゼ等を含む溶液が挙げられる。これらの溶液を用いて核酸を溶出する場合、 37°C 以上の温度でインキュベートすることにより反応を促進することができる。  [0022] A sample solution containing a single-stranded nucleic acid as a test substance includes blood such as peripheral venous blood, leukocytes, serum, urine, feces, semen, saliva, cultured cells, and tissues such as various organ cells. It can be prepared by extracting a nucleic acid from various sample samples containing a nucleic acid, such as a cell, by a known method. At this time, the cells in the sample can be destroyed by applying a physical action such as shaking or ultrasonic waves to the carrier by applying an external force. In addition, by using a nucleic acid extraction solution, cellular force and nucleic acid can be released. 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 promoted by incubating at a temperature of 37 ° C or higher.
[0023] 本発明のより好ましい態様によれば、被検物質とする遺伝子の含有量が微量であ る場合には、公知の方法により遺伝子を増幅した後検出を行なうのが好ましい。遺伝 子を増幅する方法としては、ポリメラーゼチェインリアクション (PCR)等の酵素を用い る方法が代表的であろう。ここで、遺伝子増幅法に用いられる酵素の例としては、 DN Aポリメラ一ゼ、 Taqポリメラ一ゼのような DNA依存型 DNAポリメラ一ゼ、 RNAポリメ ラーゼ Iのような DNA依存型 RNAポリメラーゼ、 Q βレプリカーゼのような RNA依存 型 RNAポリメラ ゼが挙げられ、好ましくは温度を調節するだけで連続して増幅を繰 り返すことができる点で、 Taqポリメラーゼを用いる PCR法である。 According to a more preferred embodiment of the present invention, when the content of the gene as the test substance is very small, it is preferable to perform detection after amplifying the gene by a known method. Heredity A typical method for amplifying the offspring is a method using an enzyme such as polymerase chain reaction (PCR). Here, examples of enzymes used in the gene amplification method include DNA-dependent DNA polymerases such as DNA polymerase and Taq polymerase, DNA-dependent RNA polymerases such as RNA polymerase I, and Q-polymerases. An example is an RNA-dependent RNA polymerase such as β-replicase, which is preferably a PCR method using Taq polymerase in that amplification can be continuously repeated only by adjusting the temperature.
[0024] 本発明の好ましい態様によれば、上記増幅時に特異的に核酸を増感色素で標識 することが出来る。一般的には、 DNAにアミノアリル修飾 dUTPを取り込ませることに より行うことができる。この分子は未修飾の dUTPと同じ効率で取り込まれる。次の力 ップリング段階において、 N—ヒドロキシサクシンアミド（N- hydroxysuccinimide)により 活性化された蛍光色素が修飾 dUTPと特異的に反応し、均一に増感色素で標識さ れた被検物質が得られる。  According to a preferred embodiment of the present invention, a 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, the fluorescent dye activated by N-hydroxysuccinimide (N-hydroxysuccinimide) reacts specifically with the modified dUTP, yielding a test substance uniformly labeled with the sensitizing dye. .
[0025] 本発明の好ま 、態様によれば、上記のようにして得られた核酸の粗抽出液ある!/、 は精製した核酸溶液をまず 90〜98°C、好ましくは 95°C以上の温度で熱変性を施し、 一本鎖核酸を調製することができる。  [0025] According to a preferred embodiment of the present invention, there is a crude extract of nucleic acid obtained as described above! /, Which is obtained by first purifying a purified nucleic acid solution at 90 to 98 ° C, preferably at 95 ° C or higher. A single-stranded nucleic acid can be prepared by heat denaturation at a temperature.
[0026] 本発明の方法にあっては、被検物質とプローブ物質が間接的に特異的に結合する ものであってもよい。すなわち、本発明の別の好ましい態様によれば、特異的な結合 性を有する物質を被検物質とし、この被検物質と特異的に結合する物質を媒介物質 として共存させ、この媒介物質と特異的に結合可能な物質をプローブ物質として作 用電極に担持させるのが好ましい。これにより、プローブ物質に特異的に結合できな い物質であっても、媒介物質を介して作用電極上に間接的に特異的に結合させて 検出することができる。この態様における、被検物質、媒介物質、およびプローブ物 質の組合せの好ましい例としては、リガンド、このリガンドを受容可能な受容体蛋白質 分子、およびこの受容体蛋白質分子と特異的に結合可能な二本鎖の核酸の組合せ が挙げられる。リガンドの好ましい例としては、外因性内分泌攪乱物質 (環境ホルモン )が挙げられる。外因性内分泌撹乱物質とは、受容体蛋白質分子を介して DNAに結 合し、その遺伝子発現に影響して毒性を生じる物質であるが、本発明の方法によれ ば、被検物質によりもたらされる受容体等のタンパク質の DNAに対する結合性を簡 便にモニタリングすることができる。この態様における被検物質の作用電極への特異 的結合の工程を図 2に示す。図 2に示されるように、被検物質としてのリガンド 10は、 まず、媒介物質である受容体蛋白質分子 11に特異的に結合する。そして、リガンド が結合された受容体蛋白質分子 13が、プローブ物質としての二本鎖の核酸 14に特 異的に結合する。 [0026] In the method of the present invention, the test substance and the probe substance may indirectly and specifically bind. That is, according to another preferred embodiment of the present invention, a substance having a specific binding property is used as a test substance, and a substance that specifically binds to the test substance is allowed to coexist as a mediator, and the substance having a specific binding property is used. It is preferable that a substance that can be bound to the target be supported on the working electrode as a probe substance. Thus, even a substance that cannot specifically bind to the probe substance can be specifically and indirectly bound to the working electrode via the mediator to be detected. Preferred examples of the combination of the test substance, the mediator, and the probe substance in this embodiment include a ligand, a receptor protein molecule capable of accepting the ligand, and a ligand capable of specifically binding to the receptor protein molecule. Combinations of single-stranded nucleic acids are included. Preferred examples of ligands include exogenous endocrine disruptors (environmental hormones). An exogenous endocrine disrupting substance is a substance that binds to DNA via a receptor protein molecule and affects its gene expression to cause toxicity. According to the method of the present invention, the substance is produced by a test substance. Simple binding of proteins such as receptors to DNA It can be monitored on flights. FIG. 2 shows the steps of specific binding of the test substance to the working electrode in this embodiment. As shown in FIG. 2, the ligand 10 as a test substance first specifically binds to a receptor protein molecule 11 which is a mediator. Then, the receptor protein molecule 13 to which the ligand is bound specifically binds to the double-stranded nucleic acid 14 as a probe substance.
[0027] 本発明の方法によれば、 1つのプローブ物質に対し、異なる入手経路に由来する 複数の同一被検物質を同時に反応させ、サンプルの由来による被検物質量の差異 を判断することにより、目的とする入手経路に由来する被検物質を定量することも可 能である。具体的な適用例としては、マイクロアレイ上での競合的ハイブリダィゼーシ ヨンによる発現プロフィール解析が挙げられる。これは、細胞間での特定遺伝子の発 現パターンの差異を解析するため、別々の蛍光色素で標識された被検物質を、同一 プローブ物質に対して競合的にハイブリダィゼーシヨンを行わせるものである。本発 明においては、このような手法を用いることにより、細胞間での発現差異解析が電気 化学的に行えるという、従来に無い利点が得られる。  According to the method of the present invention, a single probe substance is reacted with a plurality of the same test substances derived from different acquisition routes simultaneously, and the difference in the test substance amount due to the sample origin is determined. It is also possible to quantify the test substance derived from the intended access route. A specific application example is the expression profile analysis by competitive hybridization on a microarray. In this method, test substances labeled with different fluorescent dyes are hybridized competitively with the same probe substance in order to analyze differences in the expression pattern of specific genes between cells. Things. In the present invention, the use of such a technique provides an unprecedented advantage that the analysis of the expression difference between cells can be performed electrochemically.
[0028] 增感色素 [0028] 增 Sensitive dye
本発明の方法にあっては、被検物質の存在を光電流で検出するために、増感色素 の共存下、プローブ物質に被検物質を直接または間接的に特異的に結合させて、 該結合により増感色素を作用電極に固定させる。そのために、本発明の方法にあつ ては、図 1 (a)および図 2に示されるように被検物質 1ある!/、は媒介物質 11に予め増 感色素 2, 12で標識しておくことができる。また、図 1 (b)に示されるように被検物質お よびプローブ物質の結合体 7 (例えばノ、イブリダィゼーシヨン後の二本鎖核酸）〖こイン ターカレーシヨン可能な増感色素 8を用いる場合には、試料液に増感色素を添加す ることにより、プローブ物質に増感色素を固定させることができる。  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 a probe substance in the presence of a sensitizing dye. The binding immobilizes the sensitizing dye on the working electrode. For this purpose, in the method of the present invention, as shown in FIG. 1 (a) and FIG. 2, there is a test substance 1! /, And a mediator substance 11 is labeled in advance with sensitizing dyes 2 and 12. be able to. In addition, as shown in FIG. 1 (b), a conjugate of a test substance and a probe substance 7 (for example, double-stranded nucleic acid after hybridization) sensitizing dye capable of intercalation When using 8, a sensitizing dye can be immobilized on the probe substance by adding a sensitizing dye to the sample solution.
[0029] 本発明の好ましい態様によれば、被検物質が一本鎖の核酸の場合、被検物質 1分 子につき増感色素を一つ標識するのが好ましい。一本鎖の核酸における標識位置 は、容易に被検物質とプローブ物質の特異的な結合を形成させる観点から、一本鎖 の核酸の 5 '末端または 3，末端の 、ずれかの位置とするのが好ましく、標識工程をさ らに簡便にする観点から被検物質の 5'末端とするのがさらに好ましい。 [0030] また、本発明の別の好ましい態様によれば、被検物質 1分子あたりの増感色素担持 量を高める為、被検物質 1分子につき 2つ以上の増感色素を 2つ以上標識するのが 好ましい。これにより、電子受容物質の形成された作用電極における単位比表面積 あたりの色素担持量をより多くすることができ、より高感度に光電流応答を観測するこ とがでさる。 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 of the single-stranded nucleic acid is set at 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. It is more preferable to use a 5′-terminal of the test substance from the viewpoint of further simplifying the labeling step. According to another preferred embodiment of the present invention, in order to increase the amount of sensitizing dye carried per molecule of the test substance, two or more sensitizing dyes are labeled with 2 or more sensitizing dyes per molecule of the test substance. It is preferred that As a result, the amount of dye carried per unit specific surface area in the working electrode on which the electron accepting substance is formed can be increased, and the photocurrent response can be observed with higher sensitivity.
[0031] 本発明に用いる増感色素は、光励起に応じて作用電極に電子を放出可能な物質 であり、光源の照射による光励起状態への遷移が可能であり、かつ励起状態から作 用電極に電子注入できる電子状態を採りうるものであればよい。したがって、用いる 増感色素は、作用電極、特に電子受容層との間において上記電子状態をとることが できるものであればよいことから、多種の増感色素が使用可能であり、高価な色素を 使用する必要がない。  The sensitizing dye used in the present invention is a substance capable of emitting electrons to the working electrode in response to photoexcitation, capable of transitioning to a photoexcited state by irradiation with a light source, and capable of transitioning from the excited state to the working electrode. Any material can be used as long as it can take an electron state that allows electron injection. Therefore, the sensitizing dye to be used is not particularly limited as long as it can take the above-mentioned electronic state between the working electrode, and particularly the electron accepting layer. Therefore, various kinds of sensitizing dyes can be used. No need to use.
[0032] 複数の被検物質の個別検出を行う態様にあっては、各々の被検物質に標識する増 感色素は、それぞれ異なる波長の光で励起できるものであればよぐ例えば、照射光 の波長を選択することにより各被検物質を個別に励起できればよい。例えば、複数の 被検物質に対応する複数の増感色素を用い、各増感色素毎に異なる励起波長の光 を照射すると、複数のプローブが同一スポット上であっても個別に信号を検出するこ とが可能となる。本発明の方法において、被検物質の数は限定されないが、光源か ら照射される光の波長と増感色素の吸収特性を考慮すると、 1〜5種類が適当であろ う。この態様において使用可能な増感色素は、照射光の波長領域内において光励 起しさえすればよぐ必ずしもその吸収極大が該波長領域にある必要はない。なお、 特定波長における増感色素の光吸収反応の有無は、紫外可視スぺクトロフォトメータ 一 (例えば、島津製作所社製、 UV— 3150)を用いて測定することができる。  [0032] In an embodiment in which a plurality of test substances are individually detected, a sensitizing dye that labels each test substance may be one that can be excited by light of different wavelengths. It suffices if each of the test substances can be individually excited by selecting the above wavelength. For example, when using multiple sensitizing dyes corresponding to multiple analytes and irradiating light with different excitation wavelengths for each sensitizing dye, signals can be detected individually even if multiple probes are on the same spot. This is possible. In the method of the present invention, the number of test substances is not limited. However, in consideration of the wavelength of light emitted from the light source and the absorption characteristics of the sensitizing dye, 1 to 5 kinds may be appropriate. The sensitizing dye which can be used in this embodiment does not necessarily have to have its absorption maximum in the wavelength region of the irradiation light as long as it is photo-excited within the wavelength region of the irradiation light. The presence or absence of a light absorption reaction of a sensitizing dye at a specific wavelength can be measured using an ultraviolet-visible spectrophotometer (for example, UV-3150, manufactured by Shimadzu Corporation).
[0033] 増感色素の具体例としては、金属錯体ゃ有機色素が挙げられる。金属錯体の好ま しい例としては、銅フタロシアニン、チタ-ルフタロシアニン等の金属フタロシアニン； クロロフィルまたはその誘導体;へミン、特開平 1 220380号公報や特表平 5— 50 4023号公報に記載のルテニウム、オスミウム、鉄及び亜鉛の錯体 (例えばシスージ シァネート—ビス（2、 2，—ビビリジル— 4、 4，—ジカルボキシレート）ルテニウム（Π) )があげられる。有機色素の好ましい例としては、メタルフリーフタロシアニン、 9—フエ -ルキサンテン系色素、シァニン系色素、メタロシアニン系色素、キサンテン系色素、 トリフエ二ノレメタン系色素、アタリジン系色素、ォキサジン系色素、クマリン系色素、メロ シァニン系色素、口ダシァニン系色素、ポリメチン系色素、インジゴ系色素等が挙げら れる。また、増感色素の別の好ましい例としては、アマシャムバイオサイエンス社製の[0033] Specific examples of the sensitizing dye include a metal complex and an organic dye. Preferred examples of the metal complex include metal phthalocyanines such as copper phthalocyanine and tital phthalocyanine; chlorophyll or a derivative thereof; hemin; Complexes of osmium, iron and zinc (eg, cis-succinate-bis (2,2, -biviridyl-4,4, -dicarboxylate) ruthenium ()) can be mentioned. Preferred examples of organic dyes include metal-free phthalocyanine and 9-phen -Luxanthene dyes, cyanine dyes, metalocyanine dyes, xanthene dyes, triphenylenediolemethane dyes, ataridine dyes, oxazine dyes, coumarin dyes, melocyanin dyes, mouth dashyanine dyes, polymethine dyes, And indigo dyes. Further, as another preferred example of the sensitizing dye, manufactured by Amersham Biosciences
Cy3、 Cy3. 5、 Cy5、 Cy5. 5、 Cy7、 Cy7. 5、 Cy9 ;モルキュラープローブ社製の A lexaFluor355、 AlexaFluor405、 AlexaFluor430、 AlexaFluor488、 AlexaFlu or532、 AlexaFluor546、 AlexaFluor555、 AlexaFluor568、 AlexaFluor594、 AlexaFluor633、 AlexaFluor647、 AlexaFluor660、 AlexaFluor680、 AlexaF luor700、 AlexaFluor750 ;Dyomics社製の DY— 610、 DY— 615、 DY— 630、 DY— 631、 DY— 633、 DY— 635、 DY— 636、 EVObluelO、 EVOblue30、 DY — 647、 DY— 650、 DY— 651、 DYQ— 660、 DYQ— 661力挙げられる。 .. Cy3, Cy3 5, Cy5 , Cy5 5, C y 7, Cy7 5, C y 9;. Mol Molecular Probe Co. A lexaFluor355, AlexaFluor405, AlexaFluor430, AlexaFluor488 , AlexaFlu or532, AlexaFluor546, AlexaFluor555, AlexaFluor568, AlexaFluor594, AlexaFluor633, AlexaFluor647, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750; Dyomics DY—610, DY—615, DY—630, DY—631, DY—633, DY—635, DY—636, EVObluelO, EVOblue30, DY — 647, DY—650, DY—651, DYQ—660, DYQ—661.
[0034] 二本鎖核酸にインターカレーシヨン可能な増感色素の好ましい例としては、アタリジ ンオレンジ、ェチジゥムブロマイドが挙げられる。このような増感色素を用いる場合、 核酸のハイブリダィゼーシヨン後に試料液に添加するだけで増感色素で標識された 二本鎖核酸が形成されるので、予め一本鎖の核酸を標識する必要が無い。  Preferred examples of the sensitizing dye capable of intercalating a double-stranded nucleic acid include ataridin orange and ethidium bromide. When such a sensitizing dye is used, double-stranded nucleic acid labeled with the sensitizing dye is formed simply by adding the nucleic acid to the sample solution after hybridization, so that the single-stranded nucleic acid is labeled in advance. No need to do.
[0035] 作用雷極およびその製诰  [0035] Action lightning pole and its production
本発明に用いる作用電極は、上記プローブ物質を表面に備えた電極であり、プロ ーブ物質を介して固定された増感色素が光励起に応じて放出する電子を受容可能 な電極である。したがって、作用電極の構成および材料は、使用される増感色素との 間で上記電子移動が生じるものであれば限定されず、種々の構成および材料であつ てよい。  The working electrode used in the present invention is an electrode provided with the above-mentioned probe substance on its surface, and is an electrode capable of accepting electrons emitted by the sensitizing dye fixed via the probe substance in response to photoexcitation. Therefore, the configuration and material of the working electrode are not limited as long as the electron transfer occurs between the working electrode and the sensitizing dye used, and various configurations and materials may be used.
[0036] 本発明の好ましい態様によれば、作用電極が増感色素が光励起に応じて放出する 電子を受容可能な電子受容物質を含んでなる電子受容層を有し、この電子受容層 の表面にプローブ物質が備えられてなるのが好ましい。また、本発明のより好ましい 態様によれば、作用電極が導電性基材をさらに含んでなり、この導電性基材上に電 子受容層が形成されてなるのが好ましい。この態様の電極は図 1および 2に示される 。図 1および 2に示される作用電極 4は、導電性基材 5と、この導電性基材上に形成さ れ、電子受容物質を含んで成る電子受容層 6とを備えてなる。そして、電子受容層 6 の表面にプローブ物質が担持される。 According to a preferred embodiment of the present invention, the working electrode has an electron accepting layer containing an electron accepting substance capable of accepting an electron emitted by the sensitizing dye in response to photoexcitation, and the surface of the electron accepting layer Preferably, a probe substance is provided. Further, according to a more preferred aspect of the present invention, it is preferable that the working electrode further includes a conductive base material, and an electron receiving layer is formed on the conductive base material. The electrodes of this embodiment are 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. And the electron accepting layer 6 The probe substance is carried on the surface of the substrate.
[0037] 本発明における電子受容層は、プローブ物質を介して固定された増感色素が光励 起に応じて放出する電子を受容可能な電子受容物質を含んでなる。すなわち、電子 受容物質は、光励起された標識色素からの電子注入が可能なエネルギー準位を取 り得る物質であることができる。ここで、光励起された標識色素からの電子注入が可 能なエネルギー準位 (A)とは、例えば、電子受容性材料として半導体を用いる場合 には、伝導体 (コンダクシヨンバンド: CB)を意味し、電子受容性材料として金属を用 いる場合には、フェルミ準位を意味し、電子受容性材料として有機物もしくは C60等 の分子状無機物を用いる場合には、最低非占有分子軌道 (Lowest Unoccupied Molecular OrbitakLUMO)を意味する。すなわち、本発明に用いる電子受容物質は 、この Aの準位力 増感色素の LUMOのエネルギー準位よりも卑な準位、換言すれば 、増感色素の LUMOのエネルギー準位よりも低!、エネルギー準位を有するものであ ればよい。  [0037] The electron-accepting layer in the present invention comprises an electron-accepting substance capable of accepting electrons emitted by a sensitizing dye fixed via a probe substance in response to photoexcitation. That is, the electron accepting substance can be a substance capable of taking an energy level at which an electron can be injected from a photoexcited labeling dye. Here, the energy level (A) at which electron injection from the photoexcited labeling dye is possible 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 substance or a molecular inorganic substance such as C60 is used as the electron accepting material, the lowest unoccupied molecular orbital (Lowest Unoccupied Molecular Orbital) is used. OrbitakLUMO). That is, the electron accepting substance used in the present invention has a level lower than the LUMO energy level of the sensitizing dye of A, in other words, lower than the LUMO energy level of the sensitizing dye! What is necessary is just to have an energy level.
[0038] 電子受容物質の好ましい例としては、シリコン、ゲルマニウムなどの単体半導体;チ タン、スズ、亜鉛、鉄、タングステン、ジルコニウム、ハフニウム、ストロンチウム、インジ ゥム、セリウム、イットリウム、ランタン、バナジウム、ニオブ、タンタル等の酸化物半導 体；チタン酸ストロンチウム、チタン酸カルシウム、チタン酸ナトリウム、チタン酸バリウ ム、ニオブ酸カリウム等のぺロブスカイト型半導体;カドミウム、亜鉛、鉛、銀、アンチモ ン、ビスマスの硫化物半導体;カドミウム、鉛のセレン化物半導体;カドミウムのテルル 化物半導体;亜鉛、ガリウム、インジウム、カドミウム等のリンィ匕物半導体;ガリウムヒ素 、銅 インジウム セレンィ匕物、銅 インジウム 硫化物の化合物半導体;金、白金 、銀、銅、アルミニウム、ロジウム、インジウム、ニッケル等の金属；ポリチォフェン、ポリ ァ-リン、ポリアセチレン、ポリピロール等の有機物ポリマー； C60、 C70等の分子状 無機物が挙げられ、より好ましくは、シリコン、 TiO、 SnO、 Fe 0、 WO、 ZnO、 Nb O  [0038] Preferable examples of the electron accepting substance include simple semiconductors such as silicon and germanium; titanium, tin, zinc, iron, tungsten, zirconium, hafnium, strontium, indium, cerium, yttrium, lanthanum, vanadium, and niobium. Oxide semiconductors such as titanium and tantalum; perovskite semiconductors such as strontium titanate, calcium titanate, sodium titanate, barium titanate and potassium niobate; cadmium, zinc, lead, silver, antimony and bismuth Sulfide semiconductors; cadmium and lead selenide semiconductors; cadmium telluride semiconductors; zinc, gallium, indium, cadmium and other phosphorus nitride semiconductors; gallium arsenide, copper indium selenium nitride, copper indium sulfide compound semiconductors; gold , Platinum, silver, copper, aluminum Metals such as rhodium, indium and nickel; organic polymers such as polythiophene, polyphosphorus, polyacetylene and polypyrrole; and molecular inorganic substances such as C60 and C70, more preferably silicon, TiO, SnO, Fe0, WO , ZnO, Nb O
2 2 2 3 3 2 5 2 2 2 3 3 2 5
、 Ta O、 In O、チタン酸ストロンチウム、 CdSゝ ZnSゝ PbSゝ Bi S、 CdSeゝ CdTe、 GaP、, Ta O, In O, strontium titanate, CdS ゝ ZnS ゝ PbS ゝ Bi S, CdSe ゝ CdTe, GaP,
2 3 2 3 2 3 2 3 2 3 2 3
InPゝ GaAsゝ CuInS、 CuInSeゝ C60であり、さらに好ましくは、 TiO、 ZnO、 SnO、 Fe O  InP ゝ GaAs ゝ CuInS, CuInSe ゝ C60, more preferably TiO, ZnO, SnO, FeO
2 2 2 2 3 2 2 2 2 3
、 WO、 Nb O、 Ta O、 In O、チタン酸ストロンチウム、 CdS、 PbS、 CdSe、 InP、 GaAs, , WO, NbO, TaO, InO, strontium titanate, CdS, PbS, CdSe, InP, GaAs,
3 2 5 2 3 2 3  3 2 5 2 3 2 3
CuInS、 CuInSeであり、最も好ましくは TiOである。なお、上記の列挙した半導体は、
Figure imgf000015_0001
、ずれであってもよ 、。 CuInS and CuInSe, most preferably TiO. The above listed semiconductors are:
Figure imgf000015_0001
, May be misaligned,.
[0039] また、本発明の別の好ま 、態様によれば、電子受容物質として、インジウム-スズ 複合酸ィ匕物 (ITO)またはフッ素がドープされた酸化スズ (FTO)を用いることができる 。ITOおよび FTOは電子受容層のみならず導電性基材としても機能する性質を有 するため、これらの材料を使用することにより導電性基材を用いることなく電子受容層 のみで作用電極として機能させることができる。  Further, 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 acceptor. Since ITO and FTO have the property of functioning not only as an electron-accepting layer but also as a conductive substrate, using these materials allows the electron-accepting layer to function as a working electrode without using a conductive substrate. be able to.
[0040] 電子受容物質として半導体または金属を用いる場合、その半導体または金属は単 結晶および多結晶のいずれであってもよいが、多結晶体が好ましぐさらに緻密なも のよりも多孔性を有するものが好ましい。これにより、比表面積が大きくなり、被検物 質および増感色素を多く吸着させて、より高い感度で被検物質を検出することができ る。したがって、本発明の好ましい態様によれば、電子受容層が多孔性を有しており 、各孔の径が 3〜1000nmであるのが好ましぐより好ましくは、 10〜100nmである。  [0040] When a semiconductor or metal is used as the electron accepting substance, the semiconductor or metal may be either single crystal or polycrystal, but the polycrystal is more porous than the more dense one. Are preferred. This increases the specific surface area, adsorbs a large amount of the test substance and the sensitizing dye, and allows the test substance to be detected with higher sensitivity. Therefore, according to a preferred embodiment of the present invention, the electron accepting layer has porosity, and the diameter of each hole is preferably 3 to 1000 nm, more preferably 10 to 100 nm.
[0041] 本発明の好ま 、態様によれば、電子受容層を導電性基材上に形成した状態での 表面積は、投影面積に対して 10倍以上であることが好ましぐさらに 100倍以上であ ることが好ましい。この表面積の上限には特に限定されないが、通常 1000倍程度で あろう。電子受容層を構成する電子受容物質の微粒子の粒径は、投影面積を円に 換算したときの直径を用いた平均粒径で一次粒子として 5〜200nmであることが好 ましぐより好ましくは 8〜100nmであり、さらに好ましくは 20〜60nmである。また、 分散物中の電子受容性物質の微粒子（二次粒子）の平均粒径としては 0. 01〜： LOO μ mであることが好ましい。また、入射光を散乱させて光捕獲率を向上させる目的で、 粒子サイズの大きな、例えば 300nm程度の電子受容物質の微粒子を併用して、電 子受容層を形成してもよい。  [0041] According to a preferred embodiment of the present invention, the surface area in a state where the electron-accepting layer is formed on the conductive substrate is preferably at least 10 times the projected area, more preferably at least 100 times the projected area. It is preferred that The upper limit of this surface area is not particularly limited, but will usually be about 1000 times. The particle size of the fine particles of the electron-accepting substance constituting the electron-accepting layer is preferably 5 to 200 nm, more preferably 8 to 200 nm as primary particles as an average particle size using the diameter when the projected area is converted into a circle. 100100 nm, more preferably 20-60 nm. The average particle diameter of the fine particles (secondary particles) of the electron accepting substance in the dispersion is preferably 0.01 to: LOO μm. Further, 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 substance having a large particle size, for example, about 300 nm.
[0042] 本発明の好ま 、態様によれば、作用電極が導電性基材をさらに含んでなり、電子 受容層が導電性基材上に形成されてなるのが好ましい。本発明に使用可能な導電 性基材としては、チタン等の金属のように支持体そのものに導電性があるもののみな らず、ガラスもしくはプラスチックの支持体の表面に導電材層を有するものであってよ い。この導電材層を有する導電性基材を使用する場合、電子受容層はその導電材 層上に形成される。導電材層を構成する導電材の例としては、白金、金、銀、銅、ァ ルミ-ゥム、ロジウム、インジウム等の金属;炭素、炭化物、窒化物等の導電性セラミツ タス；およびインジウムースズ複合酸化物、酸化スズにフッ素をドープしたもの、酸化 スズにアンチモンをドープしたもの、酸ィ匕亜鈴にガリウムをドープしたもの、または酸 化亜鉛にアルミニウムをドープしたもの等の導電性の金属酸ィ匕物が挙げられ、より好 ましくは、インジウム-スズ複合酸ィ匕物 (ITO)、酸化スズにフッ素をドープした金属酸 化物 (FTO)である。ただし、前述した通り、電子受容層自体が導電性基材としても機 能する場合にあっては導電性基材は省略可能である。また、本発明において、導電 性基材は、導電性を確保できる材料であれば限定されず、それ自体では支持体とし ての強度を有しない薄膜状またはスポット状の導電材層も包含するものとする。 According to a preferred embodiment of the present invention, it is preferable that the working electrode further includes a conductive substrate, and that the electron-accepting layer is formed on the conductive substrate. Examples of the conductive substrate usable in the present invention include not only those having conductivity on the support itself, such as metals such as titanium, but also those having a conductive material layer on the surface of a glass or plastic support. You may be there. 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 platinum, gold, silver, copper, and gold. Metals such as lumidium, rhodium and indium; conductive ceramics such as carbon, carbide, and nitride; and indium oxide composite oxides, tin oxide doped with fluorine, tin oxide doped with antimony, and acids Conductive metal oxides such as gallium-doped aluminum or zinc oxide-doped aluminum; more preferably, indium-tin composite oxide (ITO); ), A 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 secure conductivity, and includes a thin-film or spot-shaped conductive material layer that does not itself have strength as a support. And
[0043] 本発明の好ましい態様によれば、導電性基材が実質的に透明、具体的には、光の 透過率が 10%以上であるのが好ましぐより好ましくは 50%以上であり、さらに好まし くは 70%以上である。これにより、作用電極の裏側 (すなわち導電性基材)から光を 照射させて、作用電極 (すなわち導電性基材および電子受容層）を透過した光が増 感色素を励起するようにセルを構成することができる。また、本発明の好ましい態様 によれば、導電材層の厚みは、 0. 02〜： LO /z m程度であるのが好ましい。さらに、本 発明の好ましい態様によれば、導電性基材の表面抵抗が 100 Q Zcm2以下であり、 さらに好ましくは 40 Q Zcm2以下であるのが好ましい。導電性基材の表面抵抗の下 限は特に限定されないが、通常 0. l Q Zcm2程度であろう。 [0043] 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 even more preferably more than 70%. This configures the cell so that light is emitted from the back side of the working electrode (ie, the conductive substrate), and the light transmitted through the working electrode (ie, the conductive substrate and the electron-accepting layer) excites the sensitizing dye. can do. According to a preferred embodiment of the present invention, the thickness of the conductive material layer is preferably about 0.02 to LO / zm. Further, according to a preferred embodiment of the present invention, the conductive substrate has a surface resistance of 100 QZcm 2 or less, more preferably 40 QZcm 2 or less. Lower limit of the surface resistance of the conductive substrate is not particularly limited, will usually 0. l Q Zcm 2 about.
[0044] 導電性基材上への電子受容層の好ましい形成方法の例としては、電子受容物質 の分散液またはコロイド溶液を導電性支持体上に塗布する方法、半導体微粒子の前 駆体を導電性支持体上に塗布し空気中の水分によって加水分解して微粒子膜を得 る方法 (ゾルーゲル法）、スパッタリング法、 CVD法、 PVD法、蒸着法などが挙げられ る。電子受容物質としての半導体微粒子の分散液を作成する方法としては、前述の ゾルーゲル法の他、乳鉢ですり潰す方法、ミルを使って粉砕しながら分散する方法、 あるいは半導体を合成する際に溶媒中で微粒子として析出させそのまま使用する方 法等が挙げられる。このときの分散媒としては水または各種の有機溶媒 (例えばメタノ ール、エタノール、イソプロピルアルコール、ジクロロメタン、アセトン、ァセトニトリル、 酢酸ェチル等）が挙げられる。分散の際、必要に応じてポリマー、界面活性剤、酸、 もしくはキレート剤などを分散助剤として使用してもよい。 [0044] Examples of preferable methods for forming the electron-accepting layer on the conductive substrate include a method in which a dispersion or colloid solution of an electron-accepting substance is applied on a conductive support, and a method in which a precursor of semiconductor fine particles is electrically conductive. The method includes a method in which a fine particle film is obtained by coating on a porous support and hydrolyzing with moisture in the air (sol-gel method), a sputtering method, a CVD method, a PVD method, and a vapor deposition method. In addition to the above-mentioned sol-gel method, a method of preparing a dispersion of semiconductor fine particles as an electron accepting substance, a method of grinding in a mortar, a method of dispersing while grinding using a mill, or a method of synthesizing a semiconductor in a solvent when synthesizing a semiconductor. And then use as it is as fine particles. Examples of the dispersion medium include water and various organic solvents (eg, methanol, ethanol, isopropyl alcohol, dichloromethane, acetone, acetonitrile, ethyl acetate, etc.). During dispersion, polymer, surfactant, acid, Alternatively, a chelating agent or the like may be used as a dispersion aid.
[0045] 電子受容物質の分散液またはコロイド溶液の塗布方法の好ましい例としては、アブ リケーシヨン系としてローラ法、ディップ法、メータリング系としてエアーナイフ法、ブレ ード法等、またアプリケーションとメータリングを同一部分でできるものとして、特公昭 58— 4589号公報【こ開示されて!ヽるワイヤーノ一法、米国特許 2681294号、同 27 61419号、同 2761791号等に記載のスライドホッパ法、エタストルージョン法、カー テン法、スピン法、スプレー法が挙げられる。  [0045] Preferable examples of the method of applying the dispersion liquid or colloid solution of the electron acceptor include a roller method and a dip method as an ablation method, an air knife method and a blade method as a metering system, and applications and metering. And the slide hopper method described in, for example, Japanese Patent Publication No. 58-4589 [disclosed in Japanese Patent Publication No. 58-4589, US Patent Nos. 2,681,294, 27,61419, 2761791, etc.] Structural methods, curtain methods, spin methods, and spray methods are mentioned.
[0046] 本発明の好ましい態様によれば、電子受容層が半導体微粒子力 なる場合、電子 受容層の膜厚が 0. 1〜200 111でぁるのが好ましぐょり好ましくは0. 1〜： LOO /z m であり、さらに好ましくは 1〜30 /ζ πι、最も好ましくは 2〜25 /ζ πιである。これにより、 単位投影面積当たりのプローブ物質および固定される増感色素量を増加して光電 流量を多くするとともに、電荷再結合による生成した電子の損失をも低減することが できる。また、導電性基材 lm2当たりの半導体微粒子の塗布量は 0. 5〜400gである のが好ましぐより好ましくは 5〜： LOOgである。 According to a preferred embodiment of the present invention, when the electron-accepting layer is a semiconductor fine particle, the thickness of the electron-accepting layer is preferably 0.1 to 200 111, more preferably 0.1. ~: LOO / zm, more preferably 1 to 30 / ζπι, most preferably 2 to 25 / ζπι. Thus, the amount of the probe substance and the amount of the sensitizing dye fixed per unit projected area can be increased to increase the photoelectric flow rate, and also reduce the loss of electrons generated by charge recombination. The coating amount of the semiconductor fine particles per conductive substrate lm 2 is more preferably preferably in the range of 0. 5～400G instrument 5: is LOOG.
[0047] 本発明の好ま 、態様によれば、電子受容物質がインジウム-スズ複合酸化物 (IT O)または酸化スズにフッ素をドープした金属酸ィ匕物 (FTO)を含んでなる場合、電子 受容層の膜厚が lnm以上であるのが好ましぐより好ましくは ΙΟηπ！〜 1 μ mである。  According to a preferred embodiment of the present invention, when the electron-accepting substance comprises indium-tin composite oxide (ITO) or metal oxide (FTO) obtained by doping tin oxide with fluorine, The thickness of the receiving layer is preferably lnm or more, more preferably よ り ηπ! 11 μm.
[0048] 本発明の好ましい態様によれば、半導体微粒子を導電性基材上に塗布した後に 加熱処理を施すのが好ましい。これにより、粒子同士を電気的に接触させ、また、塗 膜強度の向上や支持体との密着性を向上させることができる。好ましい加熱処理温 度は、 40〜700°Cであり、より好ましくは 100〜600°Cである。また、好ましい加熱処 理時間は 10分〜 10時間程度である。  [0048] According to a preferred embodiment of the present invention, it is preferable to perform a heat treatment after applying the semiconductor fine particles on the conductive base material. As a result, the particles can be brought into electrical contact with each other, the coating film strength can be improved, and the adhesion to the support can be improved. A preferred heat treatment temperature is 40 to 700 ° C, more preferably 100 to 600 ° C. The preferable heating time is about 10 minutes to 10 hours.
[0049] また、本発明の別の好ま 、態様によれば、ポリマーフィルムなど融点や軟ィ匕点の 低い導電性基材を用いる場合にあっては、熱による劣化を防止するため、高温処理 を用いない方法により膜形成を行うのが好ましぐそのような膜形成方法の例として、 プレス、低温加熱、電子線照射、マイクロ波照射、電気泳動、スパッタリング、 CVD、 PVD、蒸着等の方法が挙げられる。  According to another preferred embodiment of the present invention, when a conductive substrate such as a polymer film having a low melting point and a low softening point is used, high-temperature treatment is performed to prevent deterioration due to heat. Examples of such film forming methods that are preferable to perform film formation without using a method include pressing, low-temperature heating, electron beam irradiation, microwave irradiation, electrophoresis, sputtering, CVD, PVD, vapor deposition, and the like. Is mentioned.
[0050] こうして得られた作用電極の電子受容層の表面にはプローブ物質が担持される。 作用電極へのプローブ物質の担持は公知の方法に従い行うことができる。本発明の 好ましい態様によれば、プローブ物質として一本鎖の核酸を用いる場合には、作用 電極表面に酸化層を形成させておき、この酸化層を介して核酸プロ ブと作用電極 とを結合させることにより行うことができる。このとき、核酸プローブの作用電極への固 定化は、核酸の末端に官能基を導入することにより行うことができる。これにより、官 能基が導入された核酸プローブはそのまま固定ィ匕反応により担体上に固定化される ことができる。核酸末端への官能基の導入は、酵素反応もしくは DNA合成機を用い て行なうことができる。酵素反応において用いられる酵素としては、例えば、タ—ミナ ルデォキシヌクレオチジルトランスフェラーゼ、ポリ Aポリメラーゼ、ポリヌクレオチド力 イネース、 DNAポリメラーゼ、ポリヌクレオチドアデ-ルトランスフェラーゼ、 RNAリガ —ゼを挙げることができる。また、ポリメラ一ゼチェインリアクション (PCR法）、ニックト ランスレーシヨン、ランダムプライマー法により官能基を導入することもできる。官能基 は、核酸のどの部分に導入されてもよぐ 3'末端、 5'末端もしくはランダムな位置に導 人することができる。 The probe substance is carried on the surface of the electron accepting layer of the working electrode thus obtained. The 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 the probe substance, an oxidized layer is formed on the surface of the working electrode, and the nucleic acid probe and the working electrode are bonded via the oxidized layer. Can be performed. At this time, the nucleic acid probe can be fixed to the working electrode by introducing a functional group into the terminal of the nucleic acid. Thus, the nucleic acid probe into which the functional group has been introduced can be directly immobilized on the carrier by the immobilization reaction. Introduction of a functional group to the end of a nucleic acid can be performed using an enzyme reaction or a DNA synthesizer. Examples of the enzyme used in the enzymatic reaction include terminal dexoxynucleotidyl transferase, poly A polymerase, polynucleotide polymerase, DNA polymerase, polynucleotide adenyl transferase, and RNA ligase. In addition, functional groups can be introduced by polymerase chain reaction (PCR), nick translation, or random primer method. The functional group can be introduced at the 3 'end, 5' end, or a random position, which can be introduced into any part of the nucleic acid.
[0051] 本発明の好ましい態様によれば、核酸プローブの作用電極への固定ィ匕のため官能 基として、ァミン、カルボン酸、スルホン酸、チオール、水酸基、リン酸等が好適に使 用できる。また、本発明の好ましい態様によれば、核酸プローブを作用電極に強固に 固定ィ匕するためには、作用電極と核酸プローブの間を架橋する材料を使用すること も可能である。そのような架橋材料の好ましい例としては、シランカップリング剤、チタ ネートカップリング剤や、ポリチォフェン、ポリアセチレン、ポリピロール、ポリア-リン 等の導電性ポリマーが挙げられる。  [0051] According to a preferred embodiment of the present invention, amines, carboxylic acids, sulfonic acids, thiols, hydroxyl groups, phosphoric acids, and the like can be suitably used as functional groups for immobilizing nucleic acid probes on working electrodes. Further, according to a preferred embodiment of the present invention, in order to firmly fix the nucleic acid probe to the working electrode, it is also possible to use a material that bridges between the working electrode and the nucleic acid probe. Preferred examples of such a cross-linking material include silane coupling agents, titanate coupling agents, and conductive polymers such as polythiophene, polyacetylene, polypyrrole, and polyaline.
[0052] 本発明の好ましい態様によれば、核酸プローブの固定化を物理吸着という、より簡 単な操作で効率よく行うことも可能である。電極表面への核酸プローブの物理吸着は 、例えば、以下のように行なうことができる。まず、電極表面を、超音波洗浄器を用い て蒸留水およびアルコ―ルで洗浄する。その後、電極を核酸プロ—ブを含有する緩 衝液に挿入して核酸プロ—ブを担体表面に吸着させる。この緩衝液として後述する 本発明の緩衝溶液を用いることにより、作用電極による被検物質の検出感度を向上 させることがでさる。 [0053] また、核酸プローブの吸着後、ブロッキング剤を添加することにより、非特異的な吸 着を抑制することができる。使用可能なブロッキング剤としては、核酸プローブが吸着 して ヽな 、電子受容層表面のサイトを埋めることができ、かつ電子受容物質に対して 化学吸着あるいは物理吸着等により吸着可能な物質であれば限定されないが、好ま しくは化学結合を介して吸着可能な官能基を有する物質である。例えば、酸化チタン を電子受容層として用いる場合における好ましいブロッキング剤の例としては、カル ボン酸基、リン酸基、スルホン酸基、水酸基、アミノ基、ピリジル基、アミド等の酸化チ タンに吸着可能な官能基を有する物質が挙げられる。 According to a preferred embodiment of the present invention, immobilization of a nucleic acid probe can be efficiently performed by a simpler operation called physical adsorption. 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 solution containing a nucleic acid probe, and the nucleic acid probe is adsorbed on the surface of the carrier. By using the buffer solution of the present invention described later as this buffer solution, the detection sensitivity of the test substance by the working electrode can be improved. [0053] Further, after the nucleic acid probe is adsorbed, non-specific adsorption can be suppressed by adding a blocking agent. As a usable blocking agent, any substance can be used as long as it is capable of filling a site on the surface of the electron-accepting layer after the nucleic acid probe has been adsorbed and can be adsorbed to the electron-accepting substance by chemical adsorption or physical adsorption. It is preferably, but not limited to, a substance having a functional group that can be adsorbed via a chemical bond. For example, when titanium oxide is used as the electron-accepting layer, preferable blocking agents include those capable of adsorbing on titanium oxide such as carboxylic acid group, phosphoric acid group, sulfonic acid group, hydroxyl group, amino group, pyridyl group and amide. Substances having various functional groups.
[0054] 本発明の好ましい態様によれば、作用電極上にプローブ物質が互いに分離された 複数の領域毎に区分されて担持されてなり、光源による光照射が各領域に対して個 別に行われるのが好ましい。これにより、複数の試料を一枚の作用電極上で測定す ることができるので、 DNAチップの集積ィ匕等が可能となる。本発明のより好ましい態 様によれば、作用電極上にプローブ物質が担持された、互いに分離された複数の領 域がパター-ングされており、光源力 照射される光でスキャニングしながら、各領域 の試料について被検物質の検出または定量を一度の操作で連続的に行うことが好ま しい。  According to a preferred embodiment of the present invention, a probe substance is supported on the working electrode in a plurality of sections separated from each other, and light irradiation by a light source is individually performed on each area. Is preferred. Thus, since a plurality of samples can be measured on one working electrode, it is possible to perform DNA chip integration and the like. According to a more preferred aspect of the present invention, a plurality of areas separated from each other, on which a probe substance is supported on a working electrode, are patterned, and each of the areas is scanned while being irradiated with light. It is preferable that the detection or quantification of the test substance is continuously performed in a single operation in the sample in the region.
[0055] 本発明のより好ましい態様によれば、作用電極上の互いに分離された複数の領域 の各領域に複数種類のプローブ物質を担持させることができる。これにより、領域の 個数に、各領域毎のプローブ物質の種類数を乗じた数の、多数のサンプルの測定を 同時に行うことができる。  According to a more preferred embodiment 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. Accordingly, it is possible to simultaneously measure a large number of samples in a number obtained by multiplying the number of regions by the number of types of probe substances in each region.
[0056] 本発明のより好ましい態様によれば、作用電極上の互いに分離された複数の領域 の各領域毎に異なるプローブ物質を担持させることができる。これにより、区分された 領域の数に相当する種類数のプローブ物質を担持させることができるので、多種類 の被検物質の測定を同時に行うことができる。この態様は、各領域毎に異なる被検物 質の分析が可能なため、一塩基多型の解析 (SNPs)の多項目解析に好ましく利用 することができる。  According to a more preferred 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, a number of types of probe substances corresponding to the number of the divided areas can be carried, so that a large number of types of test substances can be measured at the same time. Since this embodiment can analyze a different test substance for each region, it can be preferably used for multi-item analysis of single nucleotide polymorphism analysis (SNPs).
[0057] 対電極  [0057] Counter electrode
本発明に用いる対電極は、電解質媒体に接触させた場合に作用電極との間に電 流が流れることができるものであれば特に限定されず、ガラス、プラスチック、セラミツ タス等の絶縁性の支持体に、金属もしくは導電性の酸化物を蒸着したものが使用可 能である。また、対電極としての金属薄膜を 5 μ m以下、好ましくは 3nm〜3 μ mの範 囲の膜厚になるように、蒸着やスパッタリングなどの方法により形成して作成すること もできる。対電極に使用可能な材料の好ましい例としては、白金、金、ノラジウム、二 ッケル、カーボン、ポリチォフェン等の導電性ポリマー、酸化物、炭化物、窒化物等の 導電性セラミックス等が挙げられ、より好ましくは、白金、カーボンであり、最も好ましく は白金である。これらの材料は電子受容層の形成方法と同様の方法により薄膜形成 が可能である。 The counter electrode used in the present invention has an electrode between it and the working electrode when it comes into contact with the electrolyte medium. The material is not particularly limited as long as it can flow, and a material obtained by depositing a metal or a conductive oxide on an insulating support such as glass, plastic, and ceramics can be used. Further, it can be formed by forming a metal thin film as a counter electrode by a method such as vapor deposition or sputtering so as to have a thickness of 5 μm or less, preferably in a range of 3 nm to 3 μm. Preferred examples of the material usable for the counter electrode include conductive polymers such as platinum, gold, noradium, nickel, carbon, and polythiophene, and conductive ceramics such as oxides, carbides, and nitrides, and are more preferable. Is platinum and carbon, most preferably platinum. These materials can be formed into a thin film by the same method as the method for forming the electron accepting layer.
[0058] 沏 I よび 置 [0058] 沏 I and placement
本発明の方法にあっては、増感色素の共存下、試料液を作用電極に接触させて、 プローブ物質に被検物質を直接または間接的に特異的に結合させ、この結合により 増感色素を前記作用電極に固定させる。このとき、試料液の溶媒として後述する本 発明の緩衝溶液を用いることにより、作用電極による被検物質の検出感度を向上さ せることができる。  In the method of the present invention, a sample solution is brought into contact with a working electrode in the coexistence of a sensitizing dye, and a test substance is directly or indirectly specifically bound to a probe substance. Is fixed to the working electrode. At this time, the detection sensitivity of the test substance by the working electrode can be improved by using the buffer solution of the present invention described later as the solvent of the sample solution.
[0059] 本発明の好ましい態様によれば、増感色素で予め標識された一本鎖の核酸を被検 物質とする場合、プローブ物質である一本鎖核酸との間でハイブリダィゼーシヨン反 応を行なうことができる。ハイブリダィゼーシヨン反応の好まし 、温度は 37〜72°Cの 範囲であるが、その最適温度は使用するプローブの塩基配列や長さ等により異なる  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, hybridization with a single-stranded nucleic acid as a probe substance is performed. A reaction can be performed. The temperature of the hybridization reaction is preferably in the range of 37 to 72 ° C, but the optimum temperature varies depending on the base sequence and length of the probe used.
[0060] 本発明の別の好ま 、態様によれば、被検物質およびプローブ物質の結合体 (例 えばノ、イブリダィゼーシヨン後の二本鎖核酸）にインターカレーシヨン可能な増感色 素を用いる場合には、試料液に増感色素を添加することにより結合体を特異的に増 感色素で標識することができる。 [0060] According to another preferred embodiment of the present invention, a conjugate of a test substance and a probe substance (for example, double-stranded nucleic acid after hybridization, sensitizing color that can be intercalated). When a sensitizer is used, the conjugate can be specifically labeled with the sensitizer by adding a sensitizer to the sample solution.
[0061] 本発明の好ましい態様によれば、プローブ物質に被検物質を直接または間接的に 特異的に結合させた作用電極を洗浄液で洗浄することにより、作用電極に結合しな 力つた被検物質を除去するのが好ましい。このときの洗浄液として、本発明の緩衝溶 液を用いることにより、作用電極による被検物質の検出感度を向上させることができる 。また、この洗浄液は、界面活性剤をさらに含むものであってよい。 According to a preferred embodiment of the present invention, by washing a working electrode in which a test substance is directly or indirectly specifically bound to a probe substance with a washing solution, a test substance which is not bound to the working electrode can be used. Preferably, the substance is removed. By using the buffer solution of the present invention as the washing solution at this time, the detection sensitivity of the test substance by the working electrode can be improved. . Further, the cleaning liquid may further include a surfactant.
[0062] 本発明の方法にあっては、被検物質が増感色素と共に固定された作用電極を、対 電極と共に電解質媒体に接触させ、作用電極に光を照射して増感色素を光励起さ せ、光励起された増感色素から作用電極への電子移動に起因して作用電極と対電 極との間に流れる光電流を検出する。作用電極および対電極の相対的な位置関係 は、互いに電気的に短絡することなぐなおかつ電解質媒体に接触しさえしていれば 限定されるものではなぐ互いに対向させて配置してもよいし、あるいは同一平面上 に互いに離間させて配置してもよい。なお、作用電極および対電極が同一平面上に 互いに離間させて配置される場合には、作用電極と対電極との間の電気的な短絡を 防止するために絶縁基板上に両電極が設けられるのが望ま 、。  In the method of the present invention, the working electrode on which the test substance is fixed together with the sensitizing dye is brought into contact with the electrolyte medium together with the counter electrode, and the working electrode is irradiated with light to excite the sensitizing dye. Then, a photocurrent flowing between the working electrode and the counter electrode due to the electron transfer from the photoexcited sensitizing dye to the working electrode is detected. The relative positions of the working electrode and the counter electrode are not limited as long as they are not electrically short-circuited with each other and are not limited as long as they are in contact with the electrolyte medium. They may be arranged separately on the same plane. When the working electrode and the counter electrode are arranged separately on the same plane, both electrodes are provided on an insulating substrate to prevent an electrical short circuit between the working electrode and the counter electrode. Desired,.
[0063] このような測定用セルの一例を図 3に示す。図 3に示される測定用セル 21は、作用 電極 22と対電極 23とにより挟まれて形成された空隙内に電解液 24が充填されてな る。作用電極 22は、導電性基材 26と電子受容層 27とを備えてなり、電子受容層 27 側を電解液 24に接触させるように配置される。作用電極 22と対電極 23との間には絶 ぺーサ 25が挿入されることにより、電解液 24を収容する空間が確保されている。 電極間の距離は酸ィ匕還元のサイクルを効率良く行わせるためには短!、方が好ましく 、工作的な精度との兼ね合いから数十/ z mであることが望ましい。また、いわゆる MEMS的な製造方法を利用するのであれば、より近接した電極間距離とすることも可 能である。  FIG. 3 shows an example of such a measuring cell. The measuring cell 21 shown in FIG. 3 has an electrolyte 24 filled in a gap formed between the working electrode 22 and the counter electrode 23. The working electrode 22 includes a conductive base material 26 and an electron accepting layer 27, and is arranged so that the electron accepting layer 27 side is in contact with the electrolyte 24. A space for accommodating the electrolytic solution 24 is secured by inserting the insulator 25 between the working electrode 22 and the counter electrode 23. The distance between the electrodes is preferably short in order to efficiently carry out the cycle of oxidation reduction, and is preferably several tens / zm in view of the workability. Further, if a so-called MEMS-like manufacturing method is used, it is possible to make the distance between the electrodes closer.
[0064] 本発明において用いる電解質媒体は、電解質、溶媒、および所望により添加物を 含んでなるものであることができる。電解質の好ましい例としては、 Iとヨウ化物の組み  [0064] The electrolyte medium used in the present invention can include an electrolyte, a solvent, and optionally an additive. Preferred examples of the electrolyte include a combination of I and iodide.
2  2
合わせ（ヨウ化物としては Lil、 Nal、 KI、 Csl、 Calなどの金属ヨウ化物、あるいはテト  Combination (as metal iodide such as Lil, Nal, KI, Csl, Cal, etc.
2  2
ラアルキルアンモニゥムョーダイド、ピリジニゥムョーダイド、イミダゾリゥムョーダイドな ど 4級アンモ-ゥム化合物のヨウ素塩など）、 Brと臭化物の組み合わせ (臭化物とし  Combination of Br and bromide (as bromide), such as quaternary ammonium compound iodine salts such as laalkylammonium iodide, pyridinium iodide, imidazolym iodide
2  2
ては LiBr、 NaBr、 KBr、 CsBr、 CaBrなどの金属臭化物、あるいはテトラアルキル  Metal bromide such as LiBr, NaBr, KBr, CsBr, CaBr, or tetraalkyl
2  2
アンモ-ゥムブロマイド、ピリジ-ゥムブロマイドなど 4級アンモ-ゥム化合物の臭素塩 など）のほか、フエロシアン酸塩—フェリシアン酸塩やフエ口セン—フエリシ-ゥムィォ ンなどの金属錯体、ポリ硫化ナトリウム、アルキルチオール アルキルジスルフイドな どのィォゥ化合物、ピオロゲン色素、ヒドロキノンーキノン等が挙げられ、より好ましく は、 Iと Lilやピリジニゥムョーダイド、イミダゾリゥムョーダイドなど 4級アンモニゥム化Bromide salts of quaternary ammonium compounds, such as ammonium-bromobromide and pyridi-bromobromide), metal complexes such as ferrosyanate-ferricyanate and fuecopene-phenylene-dimion, sodium polysulfide, and alkyl. Thiol alkyl disulphide Examples of such compounds include a diol compound, a porogen dye, and a hydroquinone-quinone, and more preferably, quaternary ammonium such as I and Lil, pyridinodimoxide, and imidazolymoxide.
2 2
合物のヨウ素塩を組み合わせた電解質である。上述した電解質は混合して用いても ょ 、。本発明にお 、て特に好まし 、電解質媒体はリチウムイオンを含んでなるもので ある。  It is an electrolyte that combines a compound iodine salt. The above-mentioned electrolytes may be used in combination. In the present invention, particularly preferably, the electrolyte medium contains lithium ions.
[0065] 本発明の好ましい態様によれば、電解液の電解質濃度は 0. 1〜15Mであるのが 好ましぐより好ましくは 0. 2〜： LOMである。また、電解質にヨウ素を添加する場合に おける、好ましいヨウ素の添加濃度は 0. 01-0. 5Mである。  According to a preferred embodiment of the present invention, the electrolyte concentration of the electrolytic solution is preferably 0.1 to 15 M, more preferably 0.2 to: LOM. When iodine is added to the electrolyte, the preferable concentration of iodine is 0.01-0.5M.
[0066] 好まし!/、溶媒の例としては、水、アルコール (メタノール、エタノール等）、非プロトン 性の極性溶媒 (例えばァセトニトリルなどの-トリル類、炭酸プロピレンや炭酸ェチレ ンなどのカーボネート類、ジメチルホルムアミド、ジメチルスルホキシド、スルホラン、 1 , 3—ジメチルイミダゾリノンや 3—メチルォキサゾリジノン、ジアルキルイミダゾリゥム塩 などの複素環化合物、等)が挙げられる。また、電解質媒体の溶媒として後述する本 発明の緩衝溶液を用いることも可能であり、それにより作用電極による被検物質の検 出感度を向上させることができる。  [0066] Preferred! / Examples of the solvent include water, alcohols (methanol, ethanol, etc.), aprotic polar solvents (for example, -tolyls such as acetonitrile, carbonates such as propylene carbonate and ethylene carbonate, Dimethylformamide, dimethylsulfoxide, sulfolane, 1,3-dimethylimidazolinone, 3-methyloxazolidinone, and heterocyclic compounds such as dialkylimidazolidium salts). It is also possible to use the buffer solution of the present invention described later as a solvent for the electrolyte medium, thereby improving the detection sensitivity of the test substance by the working electrode.
[0067] 本発明の好ましい態様によれば、水系の電解液が用いることができる。これにより、 蛋白質等の生体分子を変性または失活させることなく適切に測定することが可能とな る。また、電解液の流路等の劣化および電解液の揮発を防止できるとともに、廃液処 理も容易に行えるとの利点もある。本発明の好ましい態様によれば、水系電解質は、 支持電解質と、還元剤 (電子供与体)と、溶媒としての水とを含んでなるのが好ましい 。支持電解質としては、水に溶解した時にイオンに解離して伝導性を与え、かつ目的 とする電極反応を阻害しないものであれば限定されず、好ましい例としては、 NaCl、 Na SO、 KC1、 K SOなどが挙げられる。還元剤（電子供与体）の好ましい例として According to a preferred embodiment of the present invention, an aqueous electrolyte solution can be used. This makes it possible to measure appropriately without denaturing or inactivating biomolecules such as proteins. In addition, there is an advantage that the deterioration of the flow path of the electrolytic solution and the like and the volatilization of the electrolytic solution can be prevented, and the waste liquid can be easily treated. 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 to give conductivity when dissolved in water and does not inhibit the intended electrode reaction. SO and the like. Preferred examples of the reducing agent (electron donor)
2 4 2 4 2 4 2 4
は、 EDTA、トリエタノールァミン、シユウ酸、ヒドロキノンなどが挙げられる。  Examples include EDTA, triethanolamine, oxalic acid, hydroquinone and the like.
[0068] 本発明の好ましい態様によれば、電解質媒体はゲル化（固体化)させて使用するこ ともできる。ゲルイ匕の方法の例としては、ポリマー添加、オイルゲル化剤添加、多官能 モノマー類を含む重合、ポリマーの架橋反応等の手法により行うことができる。ゲル電 解質のマトリクスに使用されるポリマーの例としては、ポリアクリロニトリル、ポリビ-リデ ンフルオリド等が挙げられる。 [0068] According to a preferred embodiment of the present invention, the electrolyte medium may be used after being gelled (solidified). Examples of the gelling method include polymer addition, oil gelling agent addition, polymerization including polyfunctional monomers, and cross-linking reaction of the polymer. Examples of polymers used for the gel electrolyte matrix include polyacrylonitrile and polyvinylidene. And fluoride.
[0069] 図 3に示されるように作用電極 22の上方には光源 28が光源カバー 29を介して配 置される。すなわち、作用電極 22の裏側 (すなわち導電性基材)から光を照射させて 、作用電極 (すなわち導電性基材および電子受容層）を透過した光が増感色素を励 起するようにセルが構成されている。もっとも、対電極を透光性の材料で構成すること により光を対電極の裏側から照射してもよぐあるいは、作用電極および対電極に平 行に光を照射してもよいのは言うまでもない。本発明に用いる光源としては、標識色 素を光励起できる波長の光を照射できるものであれば限定されず、好まし 、例として は、蛍光灯、ブラックライト、殺菌ランプ、白熱電球、低圧水銀ランプ、高圧水銀ラン プ、キセノンランプ、水銀 キセノンランプ、ハロゲンランプ、メタルハライドランプ、 LE D (白色、青、緑、赤）、レーザー光、太陽光を用いることができ、より好ましくは、蛍光 灯、白熱電球、キセノンランプ、ハロゲンランプ、メタルハライドランプ、 LED (白色、 青、緑、赤）、太陽光等を挙げることができる。また、必要に応じて、分光器やバンドパ スフィルタを用いて特定波長領域の光のみを照射してもよ 、。  As shown in FIG. 3, a light source 28 is disposed above a working electrode 22 via a light source cover 29. That is, by irradiating light from the back side of the working electrode 22 (that is, the conductive substrate), the cell is formed such that the light transmitted through the working electrode (that is, the conductive substrate and the electron accepting layer) excites the sensitizing dye. It is configured. However, it goes without saying that light can be irradiated from the back side of the counter electrode by forming the counter electrode with a translucent material, or that the working electrode and the counter electrode can be irradiated in parallel. . 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, and is preferably a fluorescent light, a black light, a germicidal lamp, an incandescent lamp, a low-pressure mercury lamp. , High-pressure mercury lamp, xenon lamp, mercury xenon lamp, halogen lamp, metal halide lamp, LED (white, blue, green, red), laser light, sunlight, and more preferably fluorescent light, incandescent light Light bulbs, xenon lamps, halogen lamps, metal halide lamps, LEDs (white, blue, green, red), sunlight and the like can be mentioned. Further, if necessary, only light in a specific wavelength region may be irradiated using a spectroscope or a bandpass filter.
[0070] 本発明の好ましい態様によれば、互いに異なる光波長で励起可能な二種以上の増 感色素を用いて複数種類の被検物質を個別に検出する場合、光源から波長選択手 段を介して特定波長の光を照射することにより、複数の色素を個別に励起することが 可能である。波長選択手段の例としては、分光器、色ガラスフィルタ、干渉フィルタ、 バンドパスフィルタ等が挙げられる。また、増感色素の種類に応じて異なる波長の光 を照射可能な複数の光源を用いてもよぐこの場合の好ましい光源の例としては、特 定波長の光が照射されるレーザー光や LEDを用いてもよい。また、作用極に光を効 率よく照射するため、石英、ガラス、液体ライトガイドを用いて導光してもよい。  [0070] According to a preferred embodiment of the present invention, when a plurality of types of test substances are individually detected using two or more types of sensitizing dyes excitable at mutually different light wavelengths, a wavelength selection means is used from a light source. It is possible to excite a plurality of dyes individually by irradiating light of a specific wavelength through the light source. Examples of the wavelength selecting unit include a spectroscope, a color glass filter, an interference filter, a bandpass filter, and the like. In addition, a plurality of light sources capable of irradiating light of different wavelengths depending on the type of the sensitizing dye may be used. In this case, preferable examples of the light source include a laser beam irradiated with light of a specific wavelength and an LED. May be used. In order to efficiently irradiate the working electrode with light, the light may be guided using quartz, glass, or a liquid light guide.
[0071] 本発明の好ましい態様によれば、光源力 放射される光がもともと紫外線を実質的 に含まな!/、か、または光源からの光の照射が紫外線を除去する手段を介して行われ るのが好ましい。これにより、照射光に 400nm以下の波長の紫外線が含まれる場合 に発生しうる電子受容物質自体の光励起によるバックグランド電流、すなわちノイズを 効果的に抑制して、より精度の高い測定が可能となる。なお、増感色素は一般的に 可視光の吸収により励起されることができるため、紫外線を除去したとしても可視光の 照射により高い感度で光電流を検出することが可能である。 [0071] According to a preferred embodiment of the present invention, the light emitted from the light source is essentially free of ultraviolet light, or the irradiation of light from the light source is performed through means for removing ultraviolet light. Preferably. This effectively suppresses the background current due to photoexcitation of the electron acceptor itself, which can occur when the irradiation light contains ultraviolet light with a wavelength of 400 nm or less, that is, noise, and enables more accurate measurement. . It should be noted that sensitizing dyes can generally be excited by absorption of visible light, so even if ultraviolet light is removed, It is possible to detect photocurrent with high sensitivity by irradiation.
[0072] 紫外線を除去する手段の好ま 、例としては、光学フィルタ、および分光器が挙げ られる。光学フィルタまたは分光器を用いることにより、照射光の波長を制御すること ができ、作用電極自体の光励起を防止しつつ、増感色素のみを励起することが可能 となる。好ましい光学フィルタの例としては、紫外線カットフィルタ等の色ガラスフィル タが挙げられる。好ましい分光器の例としては、厳密な波長制御が可能な点で、回折 格子が内蔵された分光器が挙げられる。  [0072] Preferable examples of the means for removing ultraviolet light include an optical filter and a spectroscope. By using an optical filter or a spectroscope, the wavelength of irradiation light can be controlled, and it becomes possible to excite only the sensitizing dye while preventing photo-excitation of the working electrode itself. Preferred examples of the optical filter include a color glass filter such as an ultraviolet cut filter. An example of a preferable spectroscope is a spectrometer having a built-in diffraction grating because strict wavelength control is possible.
[0073] もともと紫外線を実質的に含まない光を放出する光源の好ましい例としては、レー ザ、無機エレクト口ルミネッセンス（EL)素子、有機エレクト口ルミネッセンス（EL)素子 、発光ダイオード (LED)が挙げられるが、最も好ましくは発光ダイオード (LED)であ る。 LEDによれば、波長分布の狭い制御された光を照射することができ、小型、軽量 、低消費電力、および長寿命といった利点も得られる。  [0073] Preferable examples of the light source that emits light substantially free from ultraviolet light include a laser, an inorganic electroluminescent (EL) element, an organic electroluminescent (EL) element, and a light emitting diode (LED). And most preferably a light emitting diode (LED). According to the LED, controlled light having a narrow wavelength distribution can be emitted, and advantages such as small size, light weight, low power consumption, and long life can be obtained.
[0074] 本発明のより好ま U、態様によれば、使用する電子受容物質につ!、ての既知のバ ンドギャップを下記式に代入して算出される、表 1に示されるカットオフ波長よりも短い 波長の光を除去することが好ましい。これにより、電子受容物質の特性に応じて、ノ ッ クグランド電流の発生を効果的に抑制できる。  [0074] According to a more preferred embodiment of the present invention, the cutoff wavelength shown in Table 1 is calculated by substituting the known band gap for the electron acceptor used in the following equation. It is preferable to remove light having a shorter wavelength. Thereby, generation of a knock ground current can be effectively suppressed according to the characteristics of the electron accepting substance.
バンドギャップ（eV) = h v = c/ λ = 1239. 8/ λ (mm) (h:プランク定数、 c :光速）  Band gap (eV) = h v = c / λ = 1239.8 / λ (mm) (h: Planck constant, c: speed of light)
[0075] [表 1] [Table 1]
表 1 table 1
電子受容物質 バンドギャップ 好適なカットオフ波長  Electron acceptor Band gap Suitable cut-off wavelength
Le V)_ ( n m) ルチル型酸化チタン 3 . 2 3 8 7 アナ夕一ゼ型酸化チタン 3 . 0 4 1 3 酸化亜鉛 3 , 1 4 0 0 チタン酸ストロンチウム 3 . 2 3 8 7 酸化スズ 3 . 5 3 5 4 酸化タングステン 2 . 8 4 4 3 酸化ニオブ 3 . 1 4 0 0 酸化鉄 2 . 2 5 6 4  Le V) _ (nm) Rutile-type titanium oxide 3.2 3 8 7 Ana-type titanium oxide 3.0 4 1 3 Zinc oxide 3, 1400 Strontium titanate 3.2 3 8 7 Tin oxide 3 5 3 5 4 Tungsten oxide 2.8 4 4 3 Niobium oxide 3.1 4 0 0 Iron oxide 2.2 5 6 4
[0076] なお、電子受容物質には不純物準位を含む場合があるため、万全を期して、カット オフ波長を表 1に示される波長よりも長波長側に設定しても構わない。また、作用電 極が複数の電子受容物質で構成されている場合には、構成成分のうち最もバンドギ ヤップが狭 、成分のカットオフ波長よりも短 、波長を除去するのが好まし 、。 Note that since the electron accepting substance may include an impurity level, the cutoff wavelength may be set to a longer wavelength than the wavelength shown in Table 1 for completeness. When the working electrode is composed of a plurality of electron-accepting substances, it is preferable that the band gap is narrowest among the constituent components, the wavelength is shorter than the cutoff wavelength of the component, and the wavelength is removed.
[0077] 図 4に示されるように、作用電極 21および対電極 22間には電流計 30が接続され、 光照射により系内を流れる光電流が電流計により測定される。これにより、被検物質 を検出することができる。その際の電流値は作用電極上にトラップされた増感色素の 量を反映する。例えば、被検物質が核酸の場合、相補性のある核酸間で形成された 二本鎖の量が、電流値となり反映される。したがって、得られた電流値から被検物質 を定量することができる。したがって、本発明の好ましい態様によれば、電流計が、得 られた電流量または電気量カゝら試料液中の被検物質濃度を算出する手段をさら〖こ 備えてなるのが好ましい。  [0077] As shown in Fig. 4, an ammeter 30 is connected between the working electrode 21 and the counter electrode 22, and a photocurrent flowing through the system due to light irradiation is measured by the ammeter. As a result, the test substance can be detected. The current value at that time reflects the amount of the sensitizing dye trapped on the working electrode. For example, when the test substance is a nucleic acid, the amount of double strand formed between complementary nucleic acids is reflected as a current value. Therefore, the analyte can be quantified from the obtained current value. Therefore, according to a preferred embodiment of the present invention, it is preferable that the ammeter further includes a means for calculating the concentration of the test substance in the sample liquid from the obtained current amount or electric quantity.
[0078] 本発明の好ましい態様によれば、光電流を検出する工程が、電流値を測定し、得ら れた電流値または電気量から試料液中の被検物質濃度を算出することができる。こ の被検物質濃度の算出は、予め作成された被検物質濃度と電流値または電気量と の検量線と、得られた電流値または電気量とを対比することにより行うことができる。 本発明の方法にあっては、電流値は作用電極上にトラップされた増感色素の量が反 映されるので、被検物質濃度に対応した正確な電流値が得られるため、定量測定に 適する。 According to a preferred embodiment of the present invention, in the step of detecting a photocurrent, a current value can be measured, and a concentration of a test substance in a sample solution can be calculated from the obtained current value or electric quantity. . This calculation of the test substance concentration can be performed by comparing the calibration curve of the test substance concentration and the current value or the amount of electricity prepared in advance with the obtained current value or the amount of electricity. In the method of the present invention, the current value is different from the amount of the sensitizing dye trapped on the working electrode. Because it is reflected, an accurate current value corresponding to the concentration of the test substance can be obtained, making it suitable for quantitative measurement.
[0079] 本発明の別の好ましい態様によれば、予め増感色素で標識された被検物質を競合 物質として用いて、増感色素で標識されていない、プローブ物質に特異的に結合可 能な第二の被検物質を定量することができる。第二の被検物質はプローブ物質に標 識済被検物質よりも特異的に結合しやすい性質を有するのが好ましい。これら二種 類の被検物質を競合させてプローブ物質に特異的に結合させると、検出される電流 値と第二の被検物質の濃度との間に相関関係が得られる。つまり、色素標識されて いない第二の被検物質の数が増加するにつれ、プローブ物質に特異的に結合する 競合物質の数が減少するため、第二の被検物質濃度の増加につれて、検出電流値 が減少する検量線を得ることができる。したがって、増感色素で標識されていない第 二の被検物質の検出および定量が可能となる。  According to another preferred embodiment of the present invention, a test substance previously labeled with a sensitizing dye can be used as a competitor to specifically bind to a probe substance not labeled with a sensitizing dye. The second analyte can be quantitatively determined. The second test substance preferably has a property of more easily binding to the probe substance than the labeled test substance. Competition of these two analytes for specific binding to the probe substance provides a correlation between the detected current value and the concentration of the second analyte. That is, as the number of non-dye-labeled second test substances increases, the number of competitors that specifically bind to the probe substance decreases. A calibration curve with decreasing values can be obtained. Therefore, detection and quantification of the second test substance not labeled with the sensitizing dye can be performed.
[0080] 本発明のより好ましい態様によれば、被検物質および第二の被検物質が抗原であ り、プローブ物質が抗体であるのが好ましい。この態様における被検物質および第二 の被検物質のプローブ物質への固定ィ匕工程を図 5に示す。図 5に示されるように、増 感色素で標識された抗原 41と、色素標識されていない抗原 42とが競合して抗体 43 に特異的に結合する。したがって、色素標識されていない抗原 42が増加するにつれ 、抗体に特異的に結合する色素標識された抗原 43が減少するため、第二の被検物 質濃度の増加につれて、検出電流値が減少する検量線を得ることができる。  [0080] 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 a step of fixing the test substance and the second test substance to the probe substance in this embodiment. As shown in FIG. 5, antigen 41 labeled with a sensitizing dye and antigen 42 not dye-labeled compete with each other to specifically bind to antibody 43. Therefore, as the amount of the non-dye-labeled antigen 42 increases, the amount of the dye-labeled antigen 43 that specifically binds to the antibody decreases, so that the detection current value decreases as the concentration of the second analyte increases. A calibration curve can be obtained.
[0081] フロー型測定用セルおよびパターユング電極を用いた測定方法および装置 Measurement Method and Apparatus Using Flow Type Measurement Cell and Putter-Jung Electrode
本発明の方法および装置の好まし 、実施態様の一例として、フロー型測定用セル およびパターユング電極を用いた測定方法および装置について説明する。図 6に、 装置の全体構造を示す。図 6に示される装置 50は、フロー型測定用セル 51と、光源 52と、電解液タンク 53と、洗浄液タンク 54と、供給ポンプ 55と、電流計 56と、排出ポ ンプ 57とを備えてなる。フロー型測定用セル 51は、パターユングされた作用電極 58 と、作用電極に対向する対電極 59とを備えてなり、作用電極 58および対電極 59の 間に、電解液または洗浄液を収容しかつ流すことができる流路が形成される。すなわ ち、供給ポンプ 55により測定用セル 51内に供給された電解液または洗浄液は、作 用電極 58および対電極 59に接触しながら流路を通過した後、排出ポンプ 57により 測定用セル 51外に排出されるように構成されている。これら一連の動作の制御およ び光電流値の解析は図示しない制御解析装置により行われることができる。 As 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 pattern Jung electrode will be described. Figure 6 shows the overall structure of the device. The apparatus 50 shown in FIG. 6 includes a flow type measurement cell 51, a light source 52, an electrolyte tank 53, a cleaning solution tank 54, a supply pump 55, an ammeter 56, and a discharge pump 57. Become. The flow-type measurement cell 51 includes a patterned working electrode 58 and a counter electrode 59 facing the working electrode, and contains an electrolytic solution or a cleaning solution between the working electrode 58 and the counter electrode 59; A flow path that can flow is formed. In other words, the electrolyte or cleaning solution supplied into the measurement cell 51 by the supply pump 55 After passing through the flow path while contacting the measurement electrode 58 and the counter electrode 59, the gas is discharged to the outside of the measurement cell 51 by the discharge pump 57. The control of these series of operations and the analysis of the photocurrent value can be performed by a control analyzer (not shown).
[0082] 作用電極 58は、電子受容層上にプローブ物質が担持された、互いに分離された 複数の領域がパターユングされており、光源力も照射される光でスキャニングしなが ら、各領域の試料にっ ヽて被検物質の検出または定量を一度の操作で連続的に行 えるように構成されて 、る。このようにパターユングされた作用電極の例を図 7 (a)〜（ d)および図 8 (a)〜（c)に示す。  The working electrode 58 is formed by patterning a plurality of regions separated from each other on which a probe substance is supported on an electron-accepting layer. It is configured so that detection or quantification of a test substance can be continuously performed by a single operation on a sample. FIGS. 7 (a) to 7 (d) and FIGS. 8 (a) to 8 (c) show examples of the working electrode patterned in this manner.
[0083] 図 7 (a)および (b)に示される作用電極 58は、導電性基材 58aの全面に形成された 電子受容層 58b上に、プローブ物質 58cが担持された複数のスポット 60が縦横方向 にパター-ングされたものである。そして、この作用電極 58の導電性基材にリード線 61が施され、このリード線 61を介して作用電極 58全体が電流計 56に接続されてい る。この作用電極 58によれば、各スポットに順次光照射を行うことにより、発生する光 電流を各スポット毎に測定することができる。また、電極の構成が比較的簡単なため 電極の作製が容易であり、従来の DNAチップの製造技術を利用できるとの利点もあ る。また、変形例として、図 7 (c)に示されるように電子受容層 58b自体をスポット状に 形成してその上にプローブ物質 58cを担持させる、あるいは、図 7 (d)に示されるよう に導電性基材を省略して電子受容層 58bのみでスポット状の作用電極 58を構成し てその上にプローブ物質 58cを担持させ、かつ電子受容層 58bにリード線 61を施し てもよく、特に後者にあっては、製造工程が簡略化されるとともに製造コストも低減で きるとの利点がある。この作用電極に使用される光源 52としては、図 9に示されるよう に作用電極 58上を縦横方向に移動する光源である力、または図 10に示されるように 作用電極 58の各スポットに対応して複数の光源が配列させておき、各光源を順に点 灯および消灯させるものであってよ 、。  The working electrode 58 shown in FIGS. 7 (a) and 7 (b) has a plurality of spots 60 carrying a probe substance 58c on an electron accepting layer 58b formed on the entire surface of a conductive substrate 58a. It is patterned vertically and horizontally. Then, a lead wire 61 is provided on the conductive base material of the working electrode 58, and the entire working electrode 58 is connected to the ammeter 56 via the lead wire 61. According to the working electrode 58, the generated photocurrent can be measured for each spot by sequentially irradiating each spot with light. In addition, since the configuration of the electrodes is relatively simple, it is easy to manufacture the electrodes, and there is an advantage that a conventional DNA chip manufacturing technology can be used. As a modification, as shown in FIG. 7 (c), the electron accepting layer 58b itself is formed in a spot shape and the probe substance 58c is carried thereon, or as shown in FIG. 7 (d). The conductive base material may be omitted, and the spot-like working electrode 58 may be constituted only by the electron accepting layer 58b, the probe substance 58c may be carried thereon, and the lead wire 61 may be provided on the electron accepting layer 58b. The latter has the advantage that the manufacturing process can be simplified and the manufacturing cost can be reduced. The light source 52 used for the working electrode corresponds to a force that 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 are arranged, and each light source is turned on and off in order.
[0084] 図 8 (a)および (b)に示される作用電極 58 'は、絶縁基板 58d'上に導電性基材 58 a，および電子受容層 58b，力もなる複数のスポット 60，が縦横方向にパターユングさ れており、電子受容層 58b '上にプローブ物質 58c 'が担持される。そして、各スポット 60，の導電性基材には個別にリード線 61，が施され、このリード線 61，を介して各ス ポット 60'が電流計 56に接続されている。この作用電極 58 'によれば、作用電極の全 面に光を同時に照射するだけで、各スポットに発生する光電流を同時にかつ個別に 測定することができる。また、各スポットにおける光電流を個別に測定することができ るので、他のスポットで発生した光電流をノイズとして拾うことが無 、との利点もある。 また、変形例として、図 8 (c)に示されるように導電性基材を省略して電子受容層 58b ，のみでスポット状の作用電極 58 'を構成してその上にプローブ物質 58c'を担持さ せ、かつ電子受容層 58b 'にリード線 61 'を施してもよぐ製造工程が簡略ィ匕されると ともに製造コストも低減できるとの利点がある。この作用電極 58 'に使用される光源 5 2は、図 6の作用電極の場合と同様、作用電極 58上を縦横方向に移動する光源であ る力、または作用電極 58の各スポットに対応して複数の光源が配列させておき、各 光源を順に点灯および消灯させるものであってよい。 [0084] The working electrode 58 'shown in Figs. 8 (a) and 8 (b) has a conductive base material 58a, an electron-accepting layer 58b, and a plurality of spots 60 also having a force on an insulating substrate 58d' in the vertical and horizontal directions. The probe substance 58c 'is supported on the electron accepting layer 58b'. Then, a lead wire 61 is individually applied to the conductive base material of each spot 60, and each of the spots 60 is connected through the lead wire 61. Pot 60 'is connected to ammeter 56. According to the working electrode 58 ', the photocurrent generated in each spot can be measured simultaneously and individually simply by simultaneously irradiating the entire surface of the working electrode with light. Further, since the photocurrent at each spot can be measured individually, there is an advantage that the photocurrent generated at another spot is not picked up as noise. As a modification, as shown in FIG. 8 (c), the conductive base material is omitted, and a spot-like working electrode 58 'is constituted only by the electron accepting layer 58b, and the probe substance 58c' is placed thereon. There is an advantage that the manufacturing process for supporting and supporting the electron receiving layer 58b 'with the lead wire 61' can be simplified and the manufacturing cost can be reduced. The light source 52 used for the working electrode 58 ′ corresponds to a force that is a light source that moves vertically and horizontally on the working electrode 58 or each spot of the working electrode 58, as in the case of the working electrode in FIG. A plurality of light sources may be arranged in order to turn on and off each light source in turn.
[0085] この装置を使用した測定方法の一例について以下に説明する。 [0085] An example of a measuring method using this device will be described below.
まず、増感色素の共存下、試料液を作用電極に接触させて、プローブ物質に被検 物質を直接または間接的に特異的に結合させ、この結合により増感色素を作用電極 58に固定させる。このとき、作用電極の電子受容層に図 7に示されるスポットパターン のマスキングを施して、プローブ物質が担持された複数のスポット 60が縦横方向にパ ターニングされた作用電極 58を得る。こうして得られた作用電極 58をフロー型測定 用セル 51に装着する。  First, in the coexistence of a sensitizing dye, the sample solution is brought into contact with the working electrode to specifically or indirectly specifically bind the test substance to the probe substance. . At this time, the spot pattern shown in FIG. 7 is masked on 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 mounted on the flow type measurement cell 51.
[0086] 次いで、供給ポンプ 55を作動させて電解液タンク 53から電解液を測定用セル 51 内に送り込み、測定用セル内の流路を電解液で満たした後、送液を停止する。光源 52から作用電極 58に光を照射し、電流計 56により作用電極 58および対電極 59間 に発生する光電流を測定する。この光電流値の測定にあっては、光電流値が安定す る、照射開始後数十秒後の値を採用するのが好ましい。そして、図示しない制御解 析装置において、得られた電流値と、予め作成された被検物質濃度と電流値との検 量線とを対比することにより被検物質濃度が算出される。光電流の測定終了後、供給 ポンプ 55を作動させて洗浄液タンク 53から洗浄液を測定用セル 51内に送り込むと 同時に、測定用セル 51内の電解液を排出ポンプ 57を作動させて排出し、測定用セ ル内の流路内の電解液で洗浄液で置換した後、送液および排液を停止する。これに より、洗浄液で洗浄ィ匕された測定用セル 51を用いて、次の測定を上記同様の手順で 行うことができる。 [0086] Next, the supply pump 55 is operated to feed the electrolytic solution from the electrolytic solution tank 53 into the measuring cell 51, and after the flow path in the measuring cell is filled with the electrolytic solution, the liquid supply is stopped. Light is emitted from the light source 52 to the working electrode 58, and a photocurrent generated between the working electrode 58 and the counter electrode 59 is measured by the 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 is stabilized. Then, in a control analyzer (not shown), the analyte concentration is calculated by comparing the obtained current value with a previously prepared calibration curve of the analyte concentration and the current value. After the photocurrent measurement is completed, the supply pump 55 is operated to feed the cleaning liquid from the cleaning liquid tank 53 into the measurement cell 51, and at the same time, the electrolyte in the measurement cell 51 is discharged by operating the discharge pump 57, and the measurement is performed. After replacing the washing solution with the electrolyte in the flow path in the cell, stop feeding and draining. to this Thus, the next measurement can be performed in the same procedure as above using the measurement cell 51 cleaned with the cleaning liquid.
[0087] 作用雷極 の接触下で使用される緩衝溶液 [0087] Buffer solution used under contact with working lightning pole
本発明の好ましい態様によれば、作用電極との接触下で使用される緩衝溶液とし て、カルボキシル基、リン酸基、およびアミノ基を含まない緩衝剤と、溶媒とを含んで なる緩衝溶液を用いるのが好ましい。作用電極との接触下での使用の例としては、プ ローブ物質を作用電極に固定ィ匕させる処理、被検物質をプローブ物質を介して作用 電極に固定化させる処理、被検物質の固定化後の作用電極を洗浄する処理、作用 電極を用いて光電流を検出する工程等が挙げられる。そして、上記緩衝剤を含む緩 衝溶液を用いることにより、測定対象物および作用電極の特性を阻害することなぐ 光電流による被検物質の検出感度を飛躍的に向上させることができる。この傾向は、 作用電極の表面が酸ィ匕チタンやチタン酸ストロンチウム力 なる場合にぉ 、て、特に 顕著である。  According to a preferred embodiment of the present invention, a buffer solution containing a carboxyl group, a phosphate group, and an amino group-free buffer and a solvent are used as the buffer solution used in contact with the working electrode. Preferably, it is used. Examples of use in contact with the working electrode include a process of immobilizing the probe substance on the working electrode, a process of immobilizing the test substance on the working electrode via the probe substance, and an immobilization of the test substance. Examples include a subsequent process of cleaning the working electrode, a step of detecting a photocurrent using the working electrode, and the like. By using a buffer solution containing the above buffer, it is possible to dramatically improve the detection sensitivity of a test substance by a photocurrent without impairing the characteristics of the measurement object and the working electrode. This tendency is particularly remarkable when the surface of the working electrode is made of titanium oxide or strontium titanate.
[0088] 上記緩衝溶液を使用すると検出感度が飛躍的に向上する理由は定かではな！/ヽが 、一般的な生化学分野で使用される、リン酸緩衝液、およびァミンもしくはカルボン酸 を主成分とする緩衝溶液と異なり、作用電極との相互作用が起こりにくいためではな いか、と考えられる。すなわち、緩衝剤がカルボキシル基、リン酸基、およびアミノ基を 含むと、これらの基が作用電極に対して何らかの相互作用をして、作用電極上のプロ ーブ物質の剥離や色素から作用電極への電子注入の阻害を引き起こし、その結果 検出電流値の値を低下を招くのではないかと考えられるが、本発明はこれに限定さ れるものではない。  [0088] It is not clear why the detection sensitivity is dramatically improved by using the above buffer solution! / ヽ, unlike phosphate buffers and buffer solutions containing amine or carboxylic acid as the main components, which are commonly used in the field of biochemistry. Conceivable. That is, if the buffer contains a carboxyl group, a phosphate group, and an amino group, these groups will interact with the working electrode in some way, exfoliating the probe substance on the working electrode and removing the dye from the working electrode. It is thought that this may cause the inhibition of electron injection into the semiconductor device, resulting in a decrease in the value of the detected current value, but the present invention is not limited to this.
[0089] 上記緩衝剤は、カルボキシル基、リン酸基、およびアミノ基を含まな ヽ化学構造を 有し、かつ緩衝作用を有するものである限り限定されないが、好ましい緩衝剤として は、下記式 (I) :  [0089] The buffer is not limited as long as it has a chemical structure not containing a carboxyl group, a phosphate group, and an amino group and has a buffering action. I):
[化 1]  [Chemical 1]
(I)(I)
Figure imgf000029_0001
(式中、 R1はヒドロキシル基で置換されていてもよい、炭素数が 1〜4のアルキレン基 であり、 Xはスルホン酸基またはその塩であり、 Aは Oまたは YR2— N (ここで、 R2は R1 と同義であり、 Yはスルホン酸基もしくはその塩またはヒドロキシル基である） ) で表される化合物が挙げられる。これらの緩衝剤は、核酸、外因性内分泌攪乱物質 、抗原等の特異的結合性を有する被検物質を安定に保持させて測定精度を向上さ せるといった利点を有する。
Figure imgf000029_0001
(Wherein, R 1 is an alkylene group having 1 to 4 carbon atoms, which may be substituted with a hydroxyl group, X is a sulfonic acid group or a salt thereof, and A is O or YR 2 — N (here Wherein R 2 has the same meaning as R 1 , and Y is a sulfonic acid group or a salt thereof, or a hydroxyl group). These buffers have the advantage of stably retaining test substances having specific binding properties, such as nucleic acids, exogenous endocrine disruptors, antigens, etc., and improving measurement accuracy.
[0090] 本発明の好まし 、態様によれば、アルキレン基がエチレン基であるのが好まし!/、。 [0090] According to a preferred embodiment of the present invention, the alkylene group is preferably an ethylene group! /.
このような緩衝剤の具体例としては、 2— [4— (2 ヒドロキシェチル） 1—ピぺラジ -ル]エタンスルホン酸（HEPES)、ピペラジン一 1, 4 ビス（2 エタンスルホン酸） ( PIPES)、ピペラジン一 1, 4 ビス（2 エタンスルホン酸）、セスキナトリウム塩（PIP ES sesquisodium)、および 2—モルフオリノエタンスルホン酸 (MES)が挙げられる  Specific examples of such a buffer include 2- [4- (2-hydroxyethyl) 1-piperazyl] ethanesulfonic acid (HEPES), piperazine-1,4-bis (2ethanesulfonic acid) ( PIPES), piperazine-1,4-bis (2 ethanesulfonic acid), sesquisodium salt (PIP ES sesquisodium), and 2-morpholinoethanesulfonic acid (MES)
[0091] 本発明の好ましい態様によれば、アルキレン基がプロピレン基であるのが好ましい。 [0091] According to a preferred embodiment of the present invention, the alkylene group is preferably a propylene group.
このような緩衝剤の具体例としては、 3— [4— (2—ヒドロキシェチル） 1—ピぺラジ -ル]プロパンスルホン酸（EPPS)、 2 ヒドロキシ一 3— [4— (2 ヒドロキシェチル)一 1 -ピペラジ -ル]プロパンスルホン酸（HEPPSO)、 3 -モルフォリノプロパンスルホ ン酸（MOPS)、 2 ヒドロキシ一 3 モルフォリノプロパンスルホン酸（MOPSO)、お よびピぺラジン一 1, 4 ビス（2 ヒドロキシル 3 プロパンスルホン酸）（POPSO) が挙げられる。  Specific examples of such a buffer include 3- [4- (2-hydroxyethyl) 1-piradizyl] propanesulfonic acid (EPPS), 2-hydroxy-1- [4- (2-hydroxyethyl). Tyl) 1-piperazyl] propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-13 morpholinopropanesulfonic acid (MOPSO), and piperazine-1,4 Bis (2-hydroxyl-3-propanesulfonic acid) (POPSO).
[0092] 本発明の好ましい態様によれば、緩衝剤の濃度を l〜200mMとするのが好ましく 、より好ましくは l〜100mM、さらに好ましくは 10〜50mMである。  [0092] According to a preferred embodiment of the present invention, the concentration of the buffer is preferably 1 to 200 mM, more preferably 1 to 100 mM, and still more preferably 10 to 50 mM.
[0093] 上記緩衝溶液に用いる溶媒は、被検物質および作用電極の特性を阻害しな!ヽもの であれば限定されないが、好ましい例としては、水およびアルコールが挙げられ、より 好ましくは水である。  [0093] The solvent used for the buffer solution is not limited as long as it does not inhibit the properties of the test substance and the working electrode. Preferred examples include water and alcohol, and more preferred is water. is there.
[0094] 本発明の好ま ヽ態様によれば、核酸、外因性内分泌攪乱物質、抗原等の特異的 結合性を有する被検物質を安定に保持させて測定精度を向上させるためには、緩衝 溶液の pHを 5, 0〜9. 0とするの力好ましく、より好ましくは 6. 0〜8. 0、さらに好まし くは 6. 5〜7. 5とする。 [0095] 上記緩衝溶液の用途は、本発明の方法に用いられる作用電極との接触下で使用 される溶液であれば限定されないが、好ましい用途としては、上述の通り、被検物質 を含有する試料液の溶媒、被検物質と直接または間接的に特異的に結合可能なプ ローブ物質を含む溶液の溶媒、電解質媒体の溶媒、および作用電極ないし測定用 セルの洗浄液等が挙げられる。 [0094] According to a preferred embodiment of the present invention, in order to stably retain a test substance having a specific binding property such as a nucleic acid, an exogenous endocrine disrupting substance, or an antigen to improve the measurement accuracy, a buffer solution The pH is preferably adjusted to 5.0 to 9.0, more preferably 6.0 to 8.0, and even more preferably 6.5 to 7.5. [0095] The use of the buffer solution is not limited as long as it is a solution used in contact with the working electrode used in the method of the present invention. Preferred uses include a test substance as described above. Examples of the solvent include a solvent for a sample solution, a solvent for a solution containing a probe substance capable of directly or indirectly specifically binding to a test substance, a solvent for an electrolyte medium, and a washing solution for a working electrode or a measurement cell.
実施例  Example
[0096] 以下の実施例によって本発明をさらに詳細に説明する。なお、本発明はこれら実施 例に限定されるものではない。  [0096] The present invention will be described in more detail with reference to the following examples. Note that the present invention is not limited to these examples.
[0097] 例 1 :作用雷極の作製 [0097] Example 1: Fabrication of working lightning pole
まず、以下の配合を有する原料を自動乳鉢を用いて充分に混合した後、 150°Cで 6時間乾燥させて混合物を得た。  First, raw materials having the following composition were thoroughly mixed using an automatic mortar, and then dried at 150 ° C. for 6 hours to obtain a mixture.
a テルビネオール 60重量0 /0 a Terubineoru 60 weight 0/0
2—（2 ブトキシエトキシ）エタノール 15重量0 /0 2- (2-butoxyethoxy) ethanol 15 wt 0/0
ェチルセルロース 25重量％  Ethyl cellulose 25% by weight
[0098] 得られた混合物 0. 5gに粒状酸化チタン（日本エアロゾル社製 P25、平均粒径 20 〜25nm) lgを添加して混合した後、先に得られた混合物 0. 5gをさらに添加して混 合した。その後、 OC—テルビネオールを加えて再度混合して、ペーストを得た。これら 一連の混合は自動乳鉢を用いて合計 3時間行った。  [0098] To 0.5 g of the obtained mixture was added and mixed with granular titanium oxide (P25, manufactured by Nippon Aerosol Co., Ltd., average particle size: 20 to 25 nm), and then 0.5 g of the previously obtained mixture was further added. Mixed. Thereafter, OC-terbineol was added and mixed again to obtain a paste. This series of mixing was performed using an automatic mortar for a total of 3 hours.
[0099] フッ素ドープされた SnO膜が形成されたガラス基板 (旭硝子製)の縁枠を幅約 63 [0099] An edge frame of a glass substrate (manufactured by Asahi Glass) on which a fluorine-doped SnO film is formed has a width of about 63
2  2
mのテープでマスキングし、上記ペーストをスキージ印刷した後、 60°Cで 2時間乾 燥させた。得られたガラス基板を焼成炉内に入れ、約 17分間かけて 500°Cまで炉内 温度を上昇させ、この温度で 30分間保持した後、放冷した。炉内温度が 100°Cに達 した時点でガラス基板をエタノールに浸漬した。こうして、酸化チタンを含んでなる電 子受容層が形成された作用電極を得た。  After masking with a m tape, the paste was squeegee printed, and dried at 60 ° C for 2 hours. The obtained glass substrate was placed in a firing furnace, the temperature of the furnace was raised to 500 ° C. over about 17 minutes, and the temperature was maintained 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 on which an electron receiving layer containing titanium oxide was formed was obtained.
[0100] 次いで、 5， -NH — AACGTCGTGACTGGGの塩基配列を有する NH修飾 D [0100] Next, NH-modified D having the nucleotide sequence of 5, -NH—AACGTCGTGACTGGG
2 2 twenty two
NAをバッファ（3X SSC)に溶解して、 286 μ Μの NH修飾 DNA溶液を調製した。 NA was dissolved in a buffer (3X SSC) to prepare a 286 μΜ NH-modified DNA solution.
2  2
この溶液を、予め 95°Cで 3分間保持した後、氷上で冷却させることにより、変性させて ヽ 7こ o [0101] 先に得られた作用電極の電子受容層上に、 5mm X 5mm角の大きさの開口部が 形成された、厚さ 700 μ mのシリコンシールを載置した。この開口部〖こ 286 μ Μの Ν Η修飾 DN Α溶液 35 1注入した。このとき、ピペットチップの先端を用いて、シリコンAfter pre-holding this solution at 95 ° C for 3 minutes, it is denatured by cooling on ice. [0101] On the electron-accepting layer of the working electrode obtained above, a silicon seal having a thickness of 700 µm and having an opening of 5 mm x 5 mm square was formed. 351 μl of this Η-modified DNΑ solution at 286 μΜ of the opening was injected. At this time, use the tip of the pipette tip to
2 2
シールの開口部の四隅まで充分に DNA溶液が行き渡るようにした。続いて、 DNA 溶液中に気泡が極力入らな 、ようにガラス板で真上力 覆 、、湿らせた紙等で蒸気 圧が調整されたプラスチック容器に収容した。この容器中に 60°Cで 2時間保持して、 NH修飾 DN Aをインキュベートした。その後、 DN A溶液を除去し、流水で軽く電極 The DNA solution was sufficiently distributed to the four corners of the opening of the seal. Subsequently, the DNA solution was directly covered with a glass plate so as to prevent bubbles from entering the DNA solution as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. The NH-modified DNA was incubated in the container at 60 ° C. for 2 hours. Then remove the DNA solution and gently run the electrode with running water.
2 2
表面を洗浄した後、空気を吹き付けて残水を飛散させた。こうして、プローブ物質が 担持された作用電極を得た。  After cleaning the surface, air was blown to disperse residual water. Thus, a working electrode carrying the probe substance was obtained.
[0102] 例 2：ローダミン修飾 DNAの枪出  [0102] Example 2: Extraction of rhodamine-modified DNA
5， -Rho CCCAGTCACGACGTTの塩基配列を有するローダミン修飾 DNA をバッファ（2X SSC、 0. 03% SDS)に溶解して、 28. 6 μ Μおよび 286 μ Μの各 濃度を有するローダミン修飾 DNA溶液を作製した。  5. Dissolve rhodamine-modified DNA having the base sequence of -Rho CCCAGTCACGACGTT in buffer (2X SSC, 0.03% SDS) to prepare rhodamine-modified DNA solutions with concentrations of 28.6 μΜ and 286 μΜ did.
例 1で使用したものと同様のシリコンシールを作用電極表面に載置し、各濃度の口 ーダミン修飾 DNA溶液を 35 /z lずつ、開口部に注入した。溶液中に気泡が極力入ら な 、ようにガラス板で真上力 覆 、、湿らせた紙等で蒸気圧を調整したプラスチック 容器に入れた。こうして、 60°Cで一晩（12時間)インキュベートさせて、ハイブリダィゼ ーシヨンを行った。  A silicone seal similar to that used in Example 1 was placed on the surface of the working electrode, and an ordamine-modified DNA solution of each concentration was injected into the opening at a rate of 35 / zl. The solution was directly covered with a glass plate so as to prevent bubbles from entering the solution as much as possible, and the solution was placed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. Thus, hybridization was performed by incubating overnight (12 hours) at 60 ° C.
[0103] こうしてハイブリダィゼーシヨンが施された作用電極を洗浄液に浸し、ゆっくりと揺ら しながら洗浄した。洗浄液としては下記表 2に示されるものを使用し、各洗浄液につ いて下記表に示される洗浄時間、洗浄回数、および温度で洗浄を行った。なお、洗 浄液を変更する毎に洗浄容器を交換した。  [0103] The working electrode thus subjected to hybridization was immersed in a washing solution, and washed while being slowly shaken. The cleaning liquids shown in Table 2 below were used, and each cleaning liquid was cleaned at the cleaning time, the number of cleaning times, and the temperature shown in the following table. The washing container was replaced every time the washing solution was changed.
[表 2] 表 2 [Table 2] Table 2
洗浄液 1回当たりの 洗浄回数 温度  Number of washings per washing solution Temperature
洗浄時間  Cleaning time
2X SSC、 0. 2% SD S 6分間 3回 至 ¾m 2X SSC, 0.2% SD S 6 minutes 3 times ¾m
0. 2 X SSC、 0. 2 % S D S 6分間 3回 0.2 X SSC, 0.2% SDS 3 times for 6 minutes
0. 2 X SSC、 0. 2 % S D S 13分間 1回 60°C 0.2 X SSC, 0.2% SDS 13 minutes once 60 ° C
0. 2 X SSC、 0. 2% SDS 6分間 2回 室温0.2 X SSC, 0.2% SDS twice for 6 minutes Room temperature
0. 2 X s s c 6分間 1回 室 ½a 注） 2X S S C： 0. 3 M塩ィ匕ナトリウムおよび 0. 03 Mクェン酸ナトリウム 含有水溶液 （ P H 7. 0 ) 0.2 X s sc once for 6 minutes Room a Note) 2X SSC: Aqueous solution containing 0.3 M sodium salt and 0.03 M sodium citrate (PH 7.0)
SDS：硫酸ドデシルナトリウム  SDS: Sodium dodecyl sulfate
[0104] さらに、作用電極をエタノールで軽く液中で上下させることにより、 2回洗浄を行った 。 2回目の洗浄後は紙等で拭き取らずに、素早く空気を吹き付けて残水を飛散させた [0104] Further, washing was performed twice by slightly raising and lowering the working electrode with ethanol in the liquid. After the second washing, air was blown quickly to disperse residual water without wiping with paper etc.
[0105] こうして得られた作用電極と、対電極としての白金電極とを用いて、図 3および図 4 に示されるような測定用セルを以下のようにして組み立てた。 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.
まず、白金電極として、ガラス基板上に白金薄膜をスパッタリングにより形成したもの を用意した。白金電極の白金膜上に、厚さ 500 mのシリコンシートを載置した。この シリコンシートは作用電極と対電極との接触による短絡を防ぐためのスぺーサ一であ る。このとき、白金電極の白金で被膜された端部にリード線を接続して、電流を取り出 し可能に構成した。作用電極もリード線を介して電流計と接続した。  First, a platinum electrode was prepared by forming a platinum thin film on a glass substrate by sputtering. A 500 m thick silicon sheet 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 platinum-coated end of the platinum electrode so that current could be taken out. The working electrode was also connected to the ammeter via a lead wire.
電解液として、体積比が 8： 2のエチレンカーボネートとァセトニトリルの混合溶媒に ヨウ素 0.05Mとテトラプロピルアンモ-ゥムョーダイド 0.5Mを溶解した混合液を用 意した。この電解液を白金電極に 5 L滴下した後、作用電極をその電子受容層が 白金電極と対向するように、載置した。こうして、スぺーサ一および電解液を作用電 極および対電極とで挟持されてなる、サンドイッチ型の測定用セルを得た。  As the electrolytic solution, a mixed solution prepared by dissolving 0.05M of iodine and 0.5M of tetrapropylammonium-moxide in a mixed solvent of ethylene carbonate and acetonitrile having a volume ratio of 8: 2 was prepared. After 5 L of this electrolytic solution was dropped on the platinum electrode, the working electrode was placed such that its electron-accepting layer faced the platinum electrode. In this manner, a sandwich-type measurement cell in which the spacer and the electrolyte were sandwiched between the working electrode and the counter electrode was obtained.
[0106] 作用電極のリード線と対電極のリード線とを電流計 (ALSモデル 832A、ディアル電 気化学アナライザー）に接続した。光源 (林時計社製、 LA— 250XE)から液体ライト ガイドを用いて導光し、紫外線カットフィルタ (Y-43、旭テクノグラス）を介して作用電 極表面の 1. 5cm上方から白色光を 30秒間作用電極表面に照射した。このとき、作 用電極と対電極との間に流れる電流値を電流計により経時的に測定した。 [0106] Connect the lead wire of the working electrode and the lead wire of the counter electrode with an ammeter (ALS model 832A, (Chemical analyzer). Light was guided from a light source (LA-250XE, manufactured by Hayashi Watch Co., Ltd.) using a liquid light guide, and white light was emitted 1.5 cm above the working electrode surface through an ultraviolet cut filter (Y-43, Asahi Techno Glass). The working electrode surface was irradiated for 30 seconds. At this time, the value of the current flowing between the working electrode and the counter electrode was measured over time with an ammeter.
[0107] 図 6に 28. 6 μ Μローダミン修飾 DNA溶液を用いた場合の検出電流の経時変化を 、図 7に 286 Μローダミン修飾 DNA溶液を用いた場合の検出電流の経時変化を 示す。これらの図に示されるように、増感色素で標識づけられた DNAと、これと相補 性を有する DNAとをノ、イブリダィゼーシヨンさせると、光の照射により大きな電流が流 れることが分力ゝる。 FIG. 6 shows the change over time in the detection current when using a 28.6 μΜ rhodamine-modified DNA solution, and FIG. 7 shows the change over time using the 286 場合 rhodamine-modified DNA solution. As shown in these figures, when DNA labeled with a sensitizing dye and DNA complementary to the DNA are subjected to hybridization, a large current may flow due to light irradiation. Power.
[0108] また、 28. 6 μ Μおよび 286 μ Μの各ローダミン修飾 DNA溶液を用いた場合につ いて、電流値が定常状態になった時点における電流値を別途測定したところ、図 8に 示される通りの結果が得られた。また、参照のため、ローダミン修飾 DNAの固定ィ匕を 行わなカゝつた場合についても同様にして測定を行った。その結果も図 8に併せて示 す。図 8に示されるように、ローダミン修飾 DNAの濃度に依存して電流値が変化した 。したがって、本発明の方法によれば定量分析が可能なことが分かる。  [0108] In addition, when the current values reached a steady state when the respective rhodamine-modified DNA solutions of 28.6 µm and 286 µm were used, the current values were separately measured. The results were as expected. In addition, for reference, the same measurement was performed for a case where the immobilization of rhodamine-modified DNA was not performed. The results are also shown in FIG. As shown in FIG. 8, the current value changed depending on the concentration of the rhodamine-modified DNA. Therefore, it is understood that quantitative analysis is possible according to the method of the present invention.
[0109] ί列 3 :プローブ 相 させない作 ffl雷 使用した沏 I定  [0109] Row 3: Probe unmatched work ffl Lightning used.
また、比較のため、例 1で作製された、 NH修飾 DNAを担持させる前の、酸化チタ  For comparison, the titania oxide before supporting the NH-modified DNA prepared in Example 1 was also used.
2  2
ンを含んでなる電子受容層のみが形成された作用電極を用 ヽて測定用セルを構成 して、例 2と同様に測定を行った。図 6に 28. 6 Mローダミン修飾 DN A溶液を用い た場合の検出電流の経時変化を、図 7に 286 Mローダミン修飾 DNA溶液を用い た場合の検出電流の経時変化を示す。これらの図に示されるように、紫外線カツトフ ィルタで除去できな力つた若干量の紫外線によって酸ィ匕チタン自身が励起され、光 電流が観測されるものの、ローダミン修飾 DNA溶液を用いた例 2の場合と比べて、 光電流は著しく低力つた。  A measurement cell was constructed using a working electrode on which only an electron-accepting layer containing an ion was formed, and the measurement was carried out in the same manner as in Example 2. Figure 6 shows the change over time in the detection current when a 28.6 M rhodamine-modified DNA solution was used, and Figure 7 shows the change over time in the detection current when a 286 M rhodamine-modified DNA solution was used. As shown in these figures, the titanium oxide itself was excited by a small amount of powerful UV light that could not be removed by the UV cut filter, and a photocurrent was observed.However, in Example 2 using the rhodamine-modified DNA solution, The photocurrent was significantly lower than in the case.
[0110] 例 4：作用電極にブロッキングを施した場合の測定 [0110] Example 4: Measurement when working electrode is blocked
例 1と同様にして、プローブ物質が担持された作用電極を得た。この電極表面に例 1で使用したものと同様のシリコンシールを再度載置して、開口部にブロッキング剤と して 10 μ Μのジエタノールァミン 35 μ 1を注入した。ブロッキング剤中に気泡が極力 入らな 、ようにガラス板で真上力 覆 、、湿らせた紙等で蒸気圧を調整したプラスチ ック容器に入れた。そして、 60°Cで 30分間保持して、ブロッキング剤をインキュベート した。電極表面を再度流水で軽く洗浄した後、空気を吹き付けて残水を飛散させた。 A working electrode carrying a probe substance was obtained in the same manner as in Example 1. The same silicon seal as that used in Example 1 was placed on the electrode surface again, and 35 μl of 10 μl of diethanolamine was injected into the opening as a blocking agent. Bubbles as much as possible in the blocking agent It was placed in a plastic container whose top was covered with a glass plate and whose vapor pressure was adjusted with moistened paper. Then, the blocking agent was incubated at 60 ° C for 30 minutes. After the electrode surface was lightly washed again with running water, air was blown to disperse residual water.
[0111] こうしてプローブ物質がブロッキングされた作用電極を用いて、例 2と同様にして、口 ーダミン修飾 DNAのインキュベーションによるハイブリダィゼーシヨン、および光電流 測定を行った。その結果は図 6および図 7に示される通りであった。これらの図に示さ れるように、ブロッキングが施された作用電極を用いた場合には、それが施されない 例 2の場合と比べて、著しく電流値が低下した。  Using the working electrode on which the probe substance was blocked in this manner, hybridization by incubation of mouth-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 the blocking was used, the current value was significantly reduced as compared with the case of Example 2 where the working electrode was not subjected to the blocking.
[0112] 例 5：競合物質 PNAとの共存下におけるローダミン修飾 DNAの検出  [0112] Example 5: Detection of rhodamine-modified DNA in the presence of competitor PNA
5， -Rho CCCAGTCACGACGTTの塩基配列を有するローダミン修飾 DNA と、競合物質として 5'— CCCAGTCACGACGTTTの塩基配列を有する PNAとを ノ ソファ（2X SSC, 0. 03% SDS)【こ溶解して、 28. 6 Mローダミン修飾および 2 00 M PNA含有溶液と、 286 μ Μローダミン修飾および 200 μ M PNA含有溶 液とを作製した。  5, Rhodamine-modified DNA having the nucleotide sequence of CCCAGTCACGACGTT and PNA having the nucleotide sequence of 5'-CCCAGTCACGACGTTT as a competitor are combined with a sofa (2X SSC, 0.03% SDS). A solution containing 6 M rhodamine modification and 200 M PNA and a solution containing 286 μ μ rhodamine modification and 200 μM PNA were prepared.
この試料溶液を用いたこと以外は、例 2と同様〖こして、ローダミン修飾 DNAのインキ ュベーシヨンによるノ、イブリダィゼーシヨン、および光電流測定を行った。その結果は 図 6および図 7に示される通りであった。これらの図に示されるように、ハイブリダィゼ ーシヨン時に競合すると考えられる塩基配列を有する PNAを共存させた場合、 PNA が共存しな 、例 2の場合と比べて、電流値が低下した。  Except that this sample solution was used, measurement of rhodamine-modified DNA by incubation was performed in the same manner as in Example 2, and the hybridization and photocurrent were measured. The results were as shown in FIGS. As shown in these figures, when PNA having a base sequence considered to compete during hybridization was allowed to coexist, the current value was lower than in Example 2 where PNA did not coexist.
[0113] 例 6 :枪量線の作成 [0113] Example 6: Creating a radiation curve
( 1) 被検物質およびプローブ物質の準備  (1) Preparation of test substance and probe substance
色素標識された被検物質 (以下、被検 DNAともいう）として、 3'末端をローダミン B で標識された、以下の塩基配列を有する 15塩基の核酸塩基（3'ローダミン DNA)を 用意した。また、プローブ物質（以下、プローブ DNAともいう）として上記被検 DNAと 相補鎖を有する 15塩基の核酸塩基 (5'末端をァミノ基で修飾した DNA (以下、 5' - NH—DNAという）を用意した。すなわち、このプローブ DNAと被検 DNAは、ハイ As a dye-labeled test substance (hereinafter also referred to as test DNA), 15 nucleobases (3 'rhodamine DNA) having the following base sequence and labeled at the 3' end with rhodamine B were prepared. As a probe substance (hereinafter, also referred to as probe DNA), a 15-nucleotide base having a complementary strand to the above-described test DNA (a DNA whose 5 ′ end is modified with an amino group (hereinafter, 5′-NH-DNA)) That is, the probe DNA and the test DNA were
2 2
ブリダィゼーシヨン反応により二本鎖 DNAを形成することができる。  Double-stranded DNA can be formed by a bridging reaction.
被検 DNA(3，ローダミン DNA)： 3 ' Rho TTGCAGCACTGACCC 5 ' Test DNA (3, Rhodamine DNA): 3 'Rho TTGCAGCACTGACCC 5'
プローブ DNA (5 ' -NH -DNA)：  Probe DNA (5'-NH-DNA):
2  2
5 ' NH - AACGTCGTG ACTGGG 3 '  5 'NH-AACGTCGTG ACTGGG 3'
2  2
[0114] (2) 作用電極の作製およびプローブ物質の担持  (2) Preparation of working electrode and loading of probe substance
まず、チタ-ァ微粉末（昭和タイタ-ゥム社製、 F2、平均粒径 60nm、アナターゼ： ルチル =4 : 6) lgと、以下の配合を有する有機ビヒクル lgとを自動乳鉢で混練しなが ら、徐々に溶媒（ αテルビネオール：ブチルカルビトール =重量比 60 : 40) lgを添加 して、酸ィ匕チタンペーストを得た。これら一連の混合は合計 5時間行われた。  First, lg of titanium fine powder (manufactured by Showa Tita-Pharm, F2, average particle size 60 nm, anatase: rutile = 4: 6) lg and an organic vehicle lg having the following composition are not kneaded in an automatic mortar. Meanwhile, a solvent (α terbineol: butyl carbitol = weight ratio: 60:40) lg was gradually added to obtain a titanium oxide paste. This series of mixing was performed for a total of 5 hours.
a テルビネオール 65重量％  a Terbineol 65% by weight
ブチノレカノレビトーノレ 15重量％  Butinorekanorebitonore 15% by weight
ポリビュルブチラール 20重量％  20% by weight of polybutyral
[0115] フッ素ドープされた酸化スズ (F— SnO ： FTO)コートガラス (エイアイ特殊硝子社製  [0115] Fluorine-doped tin oxide (F—SnO: FTO) coated glass (manufactured by AI Special Glass Co., Ltd.)
2  2
、 U膜、シート抵抗： 15 ΩΖ口）の導電面上の縁枠を金属メタルスクリーンマスクでマ スキングし、上記ペーストを用いて厚さ 120 m、大きさ 5mm X 5mmの膜を作製した 。得られた膜を 60°Cで 3時間乾燥させた後、 500°Cで 30分間焼成を行い、酸化チタ ン多孔質膜を電子受容層として備えた作用電極を得た。こうして得られた作用電極 に BLBランプで一晩紫外線照射を施し、汚れおよび残存有機物の除去を行った。  , U film, sheet resistance: 15 ΩΖ) The edge frame on the conductive surface was masked with a metal metal screen mask, and a film having a thickness of 120 m and a size of 5 mm × 5 mm was prepared using the above paste. After the obtained film was dried at 60 ° C. for 3 hours, it was baked at 500 ° C. for 30 minutes to obtain a working electrode provided with a titanium oxide porous film as an electron accepting layer. The working electrode thus obtained was irradiated with ultraviolet light overnight using a BLB lamp to remove dirt and residual organic matter.
[0116] 次いで、プローブ DNAとして 5，一 NH — DNAを 50mM HEPES (pH7. 0)に溶 [0116] Next, 5,1-NH—DNA as probe DNA was dissolved in 50 mM HEPES (pH 7.0).
2  2
解させて水溶液を調製した。この溶液を、予め 95°Cで 3分間保持した後、氷上（2°C) で 3分間以上冷却させることにより、熱変性させておいた。  An aqueous solution was prepared by dissolving. This solution was previously kept at 95 ° C for 3 minutes, and then heat-denatured by cooling on ice (2 ° C) for 3 minutes or more.
[0117] 先に得られた作用電極の電子受容層上に、スぺーサー用穴あきテープを貼り、ピ ンセットの先を用いてテープ接着面に残存する空気を除去した。このテープ上に、 5 mm X 5mm角の大きさの開口部が形成されたシリコンシートを載置して密着させた。 この開口部に先に調製した 5 '— NH — DNA溶液（200 /z M)を 25 1装填した。こ [0117] A perforated tape for a spacer was stuck on the electron-accepting layer of the working electrode obtained earlier, and air remaining on the tape-adhering surface was removed using a tip of a piset. On this tape, a silicon sheet having an opening having a size of 5 mm × 5 mm square was placed and brought into close contact with each other. 251 of the previously prepared 5'-NH-DNA solution (200 / zM) was loaded into the opening. This
2  2
のとき、ピペットチップの先端を用いて、シリコンシールの開口部の四隅まで充分に D NA溶液が行き渡るようにした。続いて、 DNA溶液中に気泡が極力入らないようにガ ラス板で真上から蓋をして、湿らせた紙等で蒸気圧が調整されたプラスチック容器に 収容した。この容器中に 60°Cで 6時間保持して、 5 '—NH —DNAをインキュベート  At this time, the tip of the pipette tip was used to sufficiently spread the DNA solution to the four corners of the opening of the silicon seal. Then, the DNA solution was covered with a glass plate from above so as to prevent air bubbles from entering the DNA solution as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. Incubate 5'-NH-DNA in this container at 60 ° C for 6 hours
2 した。その後、 DNA溶液を除去し、流水で軽く電極表面を 2秒間洗浄した後、空気を 吹き付けて残水を飛散させた。こうして、プローブ DNAが担持された作用電極を得 た。 2 did. Thereafter, the DNA solution was removed, the electrode surface was washed lightly with running water for 2 seconds, and air was blown to disperse the remaining water. Thus, a working electrode carrying the probe DNA was obtained.
[0118] 上記プローブ物質が担持された作用電極に新たなシリコンシールを載置し、開口 部にブロッキング剤として 1 μ Μのジエタノールァミン 25 μ 1を装填した。ブロッキング 剤中に気泡が極力入らな 、ようにガラス板で真上力 蓋をして、湿らせた紙等で蒸気 圧を調整したプラスチック容器に入れた。そして、 60°Cで 30分間保持して、ブロッキ ング剤をインキュベートした。電極表面を再度流水で軽く 2秒間洗浄した後、空気を 吹き付けて残水を飛散させた。こうしてブロッキングが施された作用電極を得た。 [0118] placing a new silicon seal to the working electrode to the probe substance is supported, was charged with diethanol § Min 25 mu 1 of 1 mu Micromax as a blocking agent in the openings. In order to prevent air bubbles from entering the blocking agent as much as possible, the lid was covered directly with a glass plate and placed in a plastic container whose vapor pressure was adjusted with wet paper or the like. Then, it was kept at 60 ° C for 30 minutes to incubate the blocking agent. After the electrode surface was again lightly washed with running water for 2 seconds, air was blown to disperse residual water. Thus, a blocked working electrode was obtained.
[0119] (3)ハイブリダィゼーシヨン  [0119] (3) Hybridization
色素標識された被検 DNAとしての 3 'ローダミン DNAを HEPES水溶液に溶解し て、 0、 10、 40、 400、 4000、 15000、 25000、 30000、 40000、 80000nMの各 濃度を有する 3'ローダミン DNA溶液を作製した。  3 'rhodamine DNA as test DNA labeled with dye is dissolved in HEPES aqueous solution, and 3' rhodamine DNA solution having each concentration of 0, 10, 40, 400, 4000, 15000, 25000, 30000, 40000, and 80,000 nM Was prepared.
各濃度の 3'ローダミン DNA溶液を 25 μ 1作用電極に装填した。溶液中に気泡が極 力入らな 、ようにガラス板で真上力 蓋をして、湿らせた紙等で蒸気圧を調整したプ ラスチック容器に入れた。こうして、 60°Cで 15時間インキュベートさせて、ハイブリダィ ゼーシヨンを行った。  A 25 μl working electrode was loaded with each concentration of 3 ′ rhodamine DNA solution. The solution was placed directly above the glass plate with a lid so as to prevent air bubbles from entering the solution as much as possible, and the solution was 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.
[0120] こうしてハイブリダィゼーシヨンが施された作用電極を洗浄液に浸し、ゆっくりと揺ら しながら洗浄した。洗浄液としては下記表 3に示されるものを使用し、各洗浄液につ いて下記表に示される洗浄時間、洗浄回数、および温度で洗浄を行った。なお、洗 浄液を変更する毎に洗浄容器を交換した。さらに、作用電極を水で 5秒間洗い流し、 素早く空気を吹き付けて残水を飛散させた。  [0120] The working electrode thus subjected to hybridization was immersed in a cleaning solution, and washed while being slowly shaken. The cleaning liquids shown in Table 3 below were used, and each cleaning liquid was cleaned at the cleaning time, the number of cleaning times, and the temperature shown in the following table. The washing container was replaced every time the washing solution was changed. Further, the working electrode was rinsed with water for 5 seconds, and air was quickly blown to disperse residual water.
[表 3] 表 [Table 3] table
洗浄液 1回当たりの 洗浄回数 温度  Number of washings per washing solution Temperature
洗浄時間  Cleaning time
HEPE S、 0. l%Twe e n20 6分間 6回  HEPE S, 0.1% Tween 20 6 min 6 times
HEPE S、 0. l%Twe e n20 13分間 1回 60°C  HEPE S, 0.1% Tween 20 once for 13 minutes 60 ° C
HEPE S 6分間 2回  HEPE S twice for 6 minutes
超純水 15分間 1回  Ultrapure water once for 15 minutes
注） HEPES： 2— [4一（2—ヒドロキシェチル） 一 1—ビペラジニル]ェタン スルホン酸 （同仁化学研究所製） の 50mM、 pH7. 0の溶液  Note) HEPES: 50 mM, pH 7.0 solution of 2- [4- (2-hydroxyethyl) -11-biperazinyl] ethanesulfonic acid (manufactured by Dojindo Laboratories)
0. l%Twe en20： SERVA社製の界面活性剤を超純水に 0. lvol% で希釈したもの  0. l% Tween20: SERVA surfactant diluted with ultrapure water at 0.1 lvol%
[0121] (4)測定用セルの組み立て [0121] (4) Assembly of measurement cell
こうして得られた作用電極と、対電極としての白金電極とを用いて、図 3および図 4 に示されるような測定用セルを以下のようにして組み立てた。  Using the working electrode thus obtained and a platinum electrode as a counter electrode, a measuring cell as shown in FIGS. 3 and 4 was assembled as follows.
まず、白金電極として、厚さ lmmのガラス基板上に、密着性確保のためのクロム層 を介して、白金薄膜をスパッタリングにより形成したものを用意した。白金電極の白金 膜上に、厚さ 500 /zmのシリコンシートを載置した。このシリコンシートは作用電極と対 電極との接触による短絡を防ぐためのスぺーサ一である。このとき、白金電極の白金 で被膜された端部にリード線を接続して、電流を取り出し可能に構成した。作用電極 もリード線を介して電流計と接続した。  First, a platinum electrode was prepared by sputtering a platinum thin film on a lmm-thick glass substrate via a chromium layer for ensuring adhesion. A 500 / zm-thick silicon sheet 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 platinum-coated end of the platinum electrode so that current could be taken out. The working electrode was also connected to the ammeter via a lead wire.
電解液として、体積比が 4： 6のァセトニトリルと炭酸エチレンの混合溶媒にヨウ素 0. 06Mとテトラプロピルアンモ-ゥムョーダイド 0.6Mを溶解した混合液を用意した。こ の電解液を白金電極に 1滴（5 μ L)滴下した後、作用電極をその電子受容層が白金 電極と対向するように、載置した。こうして、スぺーサ一および電解液を作用電極およ び対電極とで挟持されてなる、サンドイッチ型の測定用セルを得た。  As an electrolytic solution, a mixed solution was prepared by dissolving iodine (0.06M) and tetrapropylammonium-dymoxide (0.6M) in a mixed solvent of acetonitrile and ethylene carbonate having a volume ratio of 4: 6. After one drop (5 μL) of this electrolyte was dropped on the platinum electrode, the working electrode was placed so that its electron-accepting layer faced the platinum electrode. Thus, a sandwich-type measurement cell in which the spacer and the electrolytic solution were sandwiched between the working electrode and the counter electrode was obtained.
[0122] (5)光電流の測定 [0122] (5) Photocurrent measurement
作用電極のリード線と対電極のリード線とをポテンシォスタツト（北斗電工社製、 HS V— 100)に接続した。 250Wキセノンランプ (林時計工業社製、 LA— 250Xe)から 液体ライトガイドを用いて導光し、基材である酸ィ匕チタンによる光吸収および光電流 発生を避けるため 430nm以下の波長の光を除去できる紫外線カットフィルタ (旭テク ノグラス社製、 Y— 43)を介して作用電極表面に光を作用電極表面に照射した。この とき、作用電極と対電極との間に流れる電流値を電流計により経時的に測定した。電 流値の測定は 60秒間行った力 光の照射は電流の測定開始 10秒後から 30秒間の み行った。 The lead wire of the working electrode and the lead wire of the counter electrode were connected to a potentiostat (Hokuto Denko Corporation, HS V-100). Light is guided from a 250W xenon lamp (manufactured by Hayashi Watch Industry Co., Ltd., LA-250Xe) using a liquid light guide, and the light absorption and photocurrent by the substrate Sidani Titanium In order to avoid generation, the working electrode surface was irradiated with light through an ultraviolet cut filter (Y-43, manufactured by Asahi Techno Glass Co., Ltd.) capable of removing light having a wavelength of 430 nm or less. At this time, the value of the current flowing between the working electrode and the counter electrode was measured over time with an ammeter. The current value was measured for 60 seconds, and the irradiation of light was performed only for 30 seconds from 10 seconds after the start of the current measurement.
[0123] (6)検量線の作成  [0123] (6) Preparation of calibration curve
被検 DNAの濃度と、検出された光電流の値とから、図 14および 15に示される検量 線が得られた。図 14に示されるように、被検 DNA濃度が 0〜4000nMの低濃度域 では、被検 DNA濃度と電流値との間に比例関係が見られた。また、図 15に示される ように、被検 DNA濃度が 1000〜： LOOOOOnMの高濃度域では、被検 DNA濃度の 対数と電流値との間にリニアな関係が得られた。したがって、これらの検量線を用い ることにより、未知の被検 DNAの濃度を、測定された光電流値に基づいて正確に知 ることができる。すなわち、本発明の方法によれば、 DNAの定量が可能である。  From the concentration of the test DNA and the value of the detected photocurrent, the calibration curves shown in FIGS. 14 and 15 were obtained. As shown in FIG. 14, in the low concentration range where the test DNA concentration was 0 to 4000 nM, a proportional relationship was observed between the test DNA concentration and the current value. Further, as shown in FIG. 15, a linear relationship was obtained between the logarithm of the test DNA concentration and the current value in the high concentration range of the test DNA concentration of 1000 to: LOOOOOnM. 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.
[0124] 例 7：リチウムイオン 含む雷解皙^:体 用いた例  [0124] Example 7: Thunder solution containing lithium ion ^: body Example
電解液として以下の二種類の対カチオンを含有する電解液 Aおよび Bを使用したこ と、および被検 DNA溶液（5，一 NH—DNA溶液）の濃度を 200 /z Mとしたこと以外  Except that electrolytes A and B containing the following two types of counter cations were used as the electrolyte and that the concentration of the test DNA solution (5,1-NH-DNA solution) was 200 / zM
2  2
は例 6と同様にして試験を行った。  Was tested in the same manner as in Example 6.
電解液 A： 40容量％炭酸エチレンおよび 60容量％ァセトニトリルの混合溶媒中、 0 . 06M Iおよび 0. 6M ΤΡΑ  Electrolyte A: 0.06M I and 0.6M in a mixed solvent of 40% by volume ethylene carbonate and 60% by volume acetonitrile
2 +Γが溶解された液（テトラプロピルアミンモ-ゥムカ チオン（(n— C H ) 4N+ ;TPA+)を対カチオンとして有する電解液）  2 + Γ-dissolved solution (electrolyte solution with tetrapropylamine mo-dumcatione ((n-C H) 4N +; TPA +) as counter cation)
3 7  3 7
電解液 B:ァセトニトリル中、 0. 05M Iおよび 0. 5M Li+I—が溶解した液（リチウ  Electrolyte B: a solution of 0.05M I and 0.5M Li + I— dissolved in acetonitrile
2  2
ムイオンを対カチオンとする電解液）  Electrolyte with cation as counter cation)
[0125] また、ブランクとして、プローブ DNAおよび被検 DNAのいずれも固定化されていな V、、作製されたままの作用電極につ!、ても光電流値を測定した。  [0125] In addition, as a blank, the photocurrent value was measured for V, in which neither the probe DNA nor the test DNA was immobilized, or for the working electrode as produced.
[0126] 測定された光電流値 (安定電流値）は表 4に示される通りであった。  The measured photocurrent value (stable current value) was as shown in Table 4.
[表 4] 電解液 光電流値 （mA) [Table 4] Electrolyte Photocurrent value (mA)
本発明例 ブランク  Invention Example Blank
A ( T P A + ) 1 . 4 0 . 3 A (TPA + ) 1.4.0.3
B ( L i +) 1 . 7 0 . 4 B (L i + ) 1.7.0.4
[0127] 表 4から分力るように、電解液 Aおよび Bのいずれもヨウ素とヨウ化物を含んでおり、 そのいずれを用いた場合も、高感度に光電流を検出することができた。特に、リチウ ムイオンを対カチオンとして含有する電解液 Bでは、リチウムイオンを含まな 、電解液 Aと比べて、 20〜35%も光電流が増加する傾向が見られた。つまり、リチウムイオン を対カチオンとして電解質媒体に使用することで、検出される光電流を増加させて、 被検 DNAを高感度に検出できることが分かる。  [0127] As can be seen from Table 4, both electrolytic solutions A and B contained iodine and iodide, and in each case, photocurrent could be detected with high sensitivity. In particular, in the case of the electrolyte solution B containing lithium ions as a counter cation, the photocurrent tended to increase by 20 to 35% compared to the electrolyte solution A containing no lithium ions. In other words, it can be seen that by using lithium ions as counter cations in the electrolyte medium, the detected photocurrent can be increased and the test DNA can be detected with high sensitivity.
[0128] ί列 8 : 早^^ して酸化チタンまたはチタン酴ストロンチウム fflいた ί列  [0128] Row 8: Titanium oxide or titanium 酴 strontium ffl
作用電極作製用ペーストを以下のように調製したこと、および被検 DNA溶液の濃 度を 200 μ Μとしたこと以外は例 6と同様にして試験を行った。  The test was carried out in the same manner as in Example 6, except that the working electrode preparation paste was prepared as follows, and the concentration of the test DNA solution was set to 200 µM.
[0129] すなわち、作用電極作製用ペーストの作製は、以下に示される酸化物半導体 0. 5 gと、例 6で使用したものと同様の有機ビヒクル 0. 5gとを自動乳鉢で混練しながら、徐 々に溶媒（ αテルビネオール：ブチルカルビトール =重量比 60 :40) lgを添カ卩するこ とにより行った。これら一連の混合は合計 5時間行われた。  [0129] That is, the production of the working electrode paste was performed by kneading 0.5 g of the oxide semiconductor shown below and 0.5 g of the same organic vehicle as that used in Example 6 in an automatic mortar. It was carried out by gradually adding a solvent (α terbineol: butyl carbitol = weight ratio 60:40) lg. This series of mixing was performed for a total of 5 hours.
TiO：昭和タイタ-ゥム社製、 F— 2、平均粒子径約 60nm、アナターゼ:ルチル = 2 TiO: Showa Tita-Pum, F-2, average particle size about 60 nm, anatase: rutile = 2
2 2
: 8の混合相  : 8 mixed phases
ZnO :シーアィ化成社製、 NanotekZnO、平均粒子径約 30nm  ZnO: Shika Chemical Co., Ltd., NanotekZnO, average particle size about 30 nm
Nb O：多木化学社製、酸化ニオブゾルを蒸発乾固した粉末、平均粒子径約 10η Nb O: Taki Chemical Co., Ltd., powder obtained by evaporating and drying niobium oxide sol, average particle size of about 10η
2 5 twenty five
m  m
WO：和光純薬社製、平均粒子径約 lOOnm  WO: Wako Pure Chemical Industries, average particle size about lOOnm
3  Three
SrTiO： 2—ェチルへキサン酸ストロンチウムのトルエン溶液とチタンテトライソプロ SrTiO: toluene solution of strontium 2-ethylhexanoate and titanium tetraisopro
3 Three
ポキシドのイソプロピルアルコール溶液をモル比（Sr :Ti)で 1： 1の割合で混合し、 10 0°Cで蒸発乾固させた後、 850°Cで焼成する方法で自製した粉末、平均粒子径約 2 OOnm In O：和光純薬社製、平均粒子径約 lOOnm Powders prepared by mixing isopropyl alcohol solution of poxide in a molar ratio (Sr: Ti) of 1: 1, evaporating to dryness at 100 ° C, and baking at 850 ° C, average particle size About 2 OOnm In O: Wako Pure Chemical Industries, average particle size about 100 nm
2 3  twenty three
[0130] また、ブランクとして、プローブ DNAおよび被検 DNAのいずれも固定化されていな [0130] As a blank, neither the probe DNA nor the test DNA was immobilized.
V、、作製されたままの作用電極につ!、ても光電流値を測定した。 V. With respect to the working electrode as manufactured, the photocurrent value was also measured.
[0131] 測定された光電流値 (安定電流値）は表 5に示される通りであった。なお、表 4には[0131] The measured photocurrent value (stable current value) was as shown in Table 5. Table 4 shows
、実測値のみならず、ブランクについて計測されたバックグランド電流値を差し引いた 補正後の電流値も示す。 In addition to the measured values, the corrected current values obtained by subtracting the background current values measured for the blanks are also shown.
[表 5] 表 — 5  [Table 5] Table — 5
電子受容物質 平均粒子径 光電流値 （mA)  Electron acceptor Average particle size Photocurrent (mA)
(腹） 実測値 ブランク 補正後の電流値 (Belly) Measured value Blank Current value after correction
T i 02 6 0 1. 5 3 0. 04 1. 49 T i 0 2 6 0 1.5.3 0.04 1.49
ZnO 3 0 0. 5 9 0. 03 0. 56  ZnO 3 0 0.5 9 0.03 0.56
W03 1 0 0 0. 2 4 0. 10 0. 14 W0 3 1 0 0 0.2 4 0.10 0.14
Nb205 1 0 0. 0 2 5 0. 01 0 0. 01 5Nb 2 0 5 1 0 0. 0 2 5 0.01 0 0.01 5
S r T i Oa 2 0 0 0. 0 4 5 0. 00 06 0. 04 5S r T i Oa 2 0 0 0. 0 4 5 0.00 06 0.04 5
I risC 1 0 0 0. 3 0 0. 07 0. 23 I risC 1 0 0 0.3 0 0.07 0.23
[0132] 表 5から分かるように、上記いずれの酸化物半導体を電子受容物質として使用した 場合にも、光電流を検出することができた。特に、光電流の実測値および補正後の 電流値と共に酸ィ匕チタンが最も高 ヽ値を示し、優れた電子受容物質であることが分 かる。また、チタン酸ストロンチウムは、ブランクについて計測されたバックグランド電 流値が極めて低いことから SZN比に優れており、高い精度で被検 DNAに起因する 応答電流を検出できることが分かる。 [0132] As can be seen from Table 5, a photocurrent could be detected when any of the above oxide semiconductors was used as an electron acceptor. In particular, titanium oxide shows the highest value together with the actually measured photocurrent value and the corrected current value, indicating that it is an excellent electron acceptor. Also, strontium titanate has an excellent SZN ratio because the background current value measured for the blank is extremely low, indicating that the response current due to the test DNA can be detected with high accuracy.
[0133] 例 9：各糠酸化物半導体におけるバックグランド雷流の波長依存件  Example 9: Wavelength dependence of background lightning current in each bran oxide semiconductor
以下の 5種類の酸ィ匕物半導体について、分光光度計 (島津製作所社製、 UV-31 50)を使用して、拡散反射 (DR)スペクトルを測定した。  Diffuse reflection (DR) spectra of the following five types of semiconductors were measured using a spectrophotometer (UV-3150, manufactured by Shimadzu Corporation).
SrTiO ： 2—ェチルへキサン酸ストロンチウムのトルエン溶液とチタンテトライソプロ SrTiO: Toluene solution of strontium 2-ethylhexanoate and titanium tetraisopro
3 Three
ポキシドのイソプロピルアルコール溶液をモル比（Sr :Ti)で 1： 1の割合で混合し、 10 0°Cで蒸発乾固させた後、 850°Cで焼成する方法で自製した粉末、平均粒子径約 2 OOnm F3 :昭和タイタ-ゥム社製、平均粒子径約 50nm、アナターゼ:ルチル =4 : 6の酸 化チタン Powders prepared by mixing isopropyl alcohol solution of poxide in a molar ratio (Sr: Ti) of 1: 1, evaporating to dryness at 100 ° C, and baking at 850 ° C, average particle size About 2 OOnm F3: Showa Titanium Co., Ltd., average particle size about 50 nm, anatase: rutile = 4: 6 titanium oxide
P- 25 :日本ァエロジル社製、平均粒子径約 25nm、アナターゼ：ルチル = 7 : 3の 酸化チタン  P- 25: Nippon Aerosil Co., Ltd., average particle size about 25 nm, anatase: rutile = 7: 3 titanium oxide
ST— 01 :石原産業社製、平均粒子径約 7nm、アナターゼ型酸化チタン ZnO :シーアィ化成社製、 NanotekZnO、平均粒子径 30nm  ST-01: manufactured by Ishihara Sangyo Co., Ltd., average particle size about 7 nm, anatase type titanium oxide ZnO: manufactured by C Kasei, NanotekZnO, average particle size: 30 nm
[0134] その結果は図 16に示される通りであった。図 16に示されるように、測定した全ての 酸ィ匕物粉末は全て紫外領域の波長の光を吸収する力 410nm以上の波長の光を ほとんど吸収しないことが分かる。このことは、照射光の波長をカットフィルタやバンド パスフィルタを用 V、て制御することにより、電子受容物質自体の光吸収に由来するバ ックグランド電流を極力抑制できることを意味する。特に、図 16から分力るように、チタ ン酸ストロンチウムは 400nm以上の波長の光をほとんど吸収しておらず、ノ ックグラ ンド電流が極めて低 、と!、う例 8の結果と一致する。 [0134] The result was as shown in FIG. As shown in FIG. 16, it can be seen that all of the measured acid oxide powders hardly absorb light having a wavelength of 410 nm or more, which absorbs light having a wavelength in the ultraviolet region. This means that by controlling the wavelength of the irradiation light using a cut filter or a bandpass filter, the background current due to the light absorption of the electron acceptor itself can be suppressed as much as possible. In particular, as can be seen from FIG. 16, strontium titanate hardly absorbs light having a wavelength of 400 nm or more, and the knock ground current is extremely low. This is consistent with the result of Example 8.
[0135] 例 10 :被檢 DNAにおける 素標識位置による沏 への影響 [0135] Example 10: Influence of 標識 depending on the position of elementary label in inspected DNA
色素標識された被検 DNAとして以下に示される 3'ローダミン DNAおよび 5'ローダ ミン DNAの二種類を使用したこと、および被検 DNA溶液の濃度を 200 Mとしたこ と以外は例 6と同様にして試験を行った。  Same as Example 6, except that the following two types of dye-labeled test DNA, 3 'rhodamine DNA and 5' rhodamine DNA, were used, and the test DNA solution concentration was 200 M The test was performed.
被検 DNA(3，ローダミン DNA)：  Test DNA (3, Rhodamine DNA):
3 ' Rho TTGCAGCACTGACCC 5 '  3 'Rho TTGCAGCACTGACCC 5'
被検 DNA(5，ローダミン DNA)：  Test DNA (5, Rhodamine DNA):
3' TTGCAGCACTGACCC -Rho5'  3 'TTGCAGCACTGACCC -Rho5'
[0136] なお、これらの被検 DNAはいずれも、プローブ DNAである 5，一 NH— DNAとノヽ [0136] Note that all of these test DNAs were probe DNAs such as 5,1-NH-DNA and
2  2
イブリダィゼーシヨン反応により二本鎖 DNAを形成することができるものである。  A double-stranded DNA can be formed by the hybridization reaction.
[0137] 比較のため、被検 DNAの代わりに、プローブ DNAとの相補鎖を持たない、 3 '末端 をローダミン Bで標識された、以下の塩基配列を有する 15塩基の核酸塩基 (T一口一 ダミン DNA)を用いて、上記同様の試験を行った。 [0137] For comparison, instead of the test DNA, a 15-nucleotide base having the following base sequence (T bit A test similar to the above was performed using Damin DNA).
T—ローダミン DNA:  T—rhodamine DNA:
5' T T T T T TTTTT-Rho3' [0138] また、ブランクとして、プローブ DNAおよび被検 DNAのいずれも固定化されていな V、、作製されたままの作用電極につ!、ても光電流値を測定した。 5 'TTTTT TTTTT-Rho3' [0138] Further, as a blank, the photocurrent value was measured for V, in which neither the probe DNA nor the test DNA was immobilized, and for the working electrode as produced.
[0139] 測定された光電流値 (安定電流値）は表 6に示される通りであった。  The measured photocurrent value (stable current value) was as shown in Table 6.
[表 6] 表 6  [Table 6] Table 6
被検 DNA 光電流値 （mA)  Test DNA photocurrent (mA)
3' ローダミン DNA 1. 53  3 'rhodamine DNA 1.53
5 ' ローダミン DN A 1. 28  5 'Rhodamine DN A 1.28
T—ローダミン DNA (比較例） 0. 29  T-rhodamine DNA (comparative example) 0.29
ブランク （比較例） 0. 04  Blank (Comparative example) 0.04
[0140] 表 6に示される通り、被検 DNAの 3 '末端を色素標識した場合と、被検 DNAの 5' 末端を色素標識した場合の両方において、ほぼ同等の光電流が観測された。この結 果から、被検 DNAの 3'末端および 5'末端のどちらに色素標識しても、高感度に被 検 DNAを検出できることが分かる。 [0140] 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.
一方、表 6から分力るように、プローブ DNAと相補鎖を持たない T—ローダミン DN Aを使用した場合には、色素に由来する光電流がほとんど検出されな力つた。すなわ ち、 T—ローダミン DNAとプローブ DNAとの間にハイブリダィゼーシヨン反応が起こ らな力つたことが分かる。  On the other hand, as shown in 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 was found that the hybridization reaction between the T-rhodamine DNA and the probe DNA was strong.
また、ブランク試験の結果から、プローブ DNAおよび被検 DNAのいずれも固定化 されていない、作製されたままの作用電極を使用した場合には、ほとんど光電流が検 出されないことが分かる。  In addition, the results of the blank test show that almost no photocurrent is detected when the working electrode is used as is, in which neither the probe DNA nor the test DNA is immobilized.
したがって、光電流の発生は基本的に被検 DNAに標識された増感色素の光励起 反応および電子受容物質への電子移動反応によって起こるものであり、その光電流 値は電極上の二本鎖 DNAの形成の割合に依存することが分かる。  Therefore, the generation of 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 acceptor, and the photocurrent value is the double-stranded DNA on the electrode. It turns out that it depends on the rate of formation of.
[0141] 例 11：光雷流のアクションスペクトルの測定 [0141] Example 11: Measurement of action spectrum of lightning current
被検 DNA溶液の濃度を 40 Mとしたこと、光電流の測定の代わりに以下に述べる 光電流のアクションスペクトルの測定を行ったこと以外は例 6と同様にして試験を行つ た。 The concentration of the test DNA solution was set to 40 M. Instead of the photocurrent measurement, The test was performed in the same manner as in Example 6 except that the action spectrum of the photocurrent was measured.
すなわち、光電流のアクションスペクトルの測定は、波長可変単色光源 (分光計器 製）を用いて行った。 IPCE (Incident Photon to Current Efficiency：光電変換効率）を M. Gratzelらの報告（J. Am. Chem. Soc. 115, 6382, 1993)に基づき、下記式を用い て算出した。中心波長を各波長に設定し、半値幅を 20nmとした。フオトンフラックス（ Photon Flux)はスペクトルラディオメ一ター（ゥシォ電機社製、 USR-40D)を用いて 測定した。  That is, the measurement of the action spectrum of the photocurrent was performed using a wavelength-tunable monochromatic light source (manufactured by Spectrometer). Based on the report of M. Gratzel et al. (J. Am. Chem. Soc. 115, 6382, 1993), IPCE (Incident Photon to Current Efficiency) was calculated using the following equation. The center wavelength was set for each wavelength, and the half width was set to 20 nm. The photon flux was measured using a spectral radiometer (USR-40D, manufactured by Dashio Electric Co., Ltd.).
IPCE = 1250 X光電流密度 AZcm2)Z [波長（nm) Xフオトンフラックス（ W/m2) ] IPCE = 1250 X photocurrent density AZcm 2 ) Z [wavelength (nm) X photon flux (W / m 2 )]
[0142] 測定されたアクションスペクトル (IPCEの波長依存性）は図 17に示される通りであつ た。図 17には、使用した増感色素であるローダミン Bの吸収スペクトルの併せて示し てある。図 17から分力るように、得られたアクションスペクトルは、増感色素（ローダミ ン B)の吸収スペクトルと同様のプロファイルを示した。これは、光電流が増感色素の 励起に起因することを示唆する。すなわち、ローダミン Bの吸収中心波長は 520nmと 見られるので、この波長域の光量を増カロさせることで、光電流値を増大できるものと 考えられる。一方、 420nm以下の波長領域では光電流が上昇する傾向が見られた 力 この光電流は電子受容物質である酸化チタン自身の光励起に起因する光電流 である。この電流は、被検 DNAおよびプローブ DNAのハイブリダィゼーシヨンの有 無にかかわらず発生し、いわゆるノイズとなる。したがって、センサの高精度化を実現 させるためには、 420nm以下の光を極力除去することが有効であることが分かる。  [0142] The measured action spectrum (wavelength dependence of IPCE) was as shown in Fig. 17. FIG. 17 also shows the absorption spectrum of the sensitizing dye rhodamine B used. As can be seen from FIG. 17, the resulting action spectrum showed a profile similar to that of the sensitizing dye (rhodamine B). This suggests that the photocurrent is due to the excitation of the sensitizing dye. That is, since the absorption center wavelength of rhodamine B is 520 nm, it is considered that the photocurrent value can be increased by increasing the amount of light in this wavelength range. On the other hand, the photocurrent tended to increase in the wavelength region of 420 nm or less. Force 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, resulting in so-called noise. Therefore, it can be seen that it is effective to remove light having a wavelength of 420 nm or less as much as possible in order to increase the accuracy of the sensor.
[0143] 例 12：光源フィルタとバックグランド電流との関係 [0143] Example 12: Relationship between light source filter and background current
光学フィルタとして以下の 6種類を使用したこと、およびノヽイブリダィゼーシヨンを行 わな力つたこと以外は例 6と同様にして試験を行った。  The test was performed in the same manner as in Example 6, except that the following six types of optical filters were used, and that no hybridization was performed.
Y- 43フィルタ：朝日テクノグラス社製、 Y- 43、紫外線カットフィルタ  Y-43 filter: Asahi Techno Glass Co., Ltd., Y-43, UV cut filter
550nm- 40hwフィルタ：ォプトライン社製、 A06-8200843、中心波長 550nm、半値幅 40nm  550nm-40hw filter: Optoline, A06-8200843, center wavelength 550nm, half width 40nm
20nm-220hwフィルタ：朝日分光社製、 PB0620-220,中心波長 620nm、半値幅 220nm 20nm-220hw filter: manufactured by Asahi Spectroscopy, PB0620-220, center wavelength 620nm, half width 220nm
600nm-260hwフィルタ：朝日分光社製、 PB060_260、中心波長 600nm、半値幅 260nm  600nm-260hw filter: Asahi Spectroscopy, PB060_260, center wavelength 600nm, half width 260nm
620nm-260hwフィルタ：朝日分光社製、 PB0620_260、中心波長 620nm、半値幅 260nm  620nm-260hw filter: manufactured by Asahi Spectroscopy, PB0620_260, center wavelength 620nm, half width 260nm
600nm-300hwフィルタ：朝日分光社製、 PB0600-300、中心波長 600nm、半値幅 300nm  600nm-300hw filter: Asahi Spectroscopy, PB0600-300, center wavelength 600nm, half width 300nm
[0144] また、色素の光吸収量を以下のようにして測定した。まず、酸化チタン電極上に吸 着した DNAにラベルした色素を分光光度計 (島津製作所社製、 UV- 3100)を用い て測定した。積分球を使用した拡散反射法に基づき、色素の反射率 (R%)を算出し た。このとき、反射率 100%の参照物質として、硫酸バリウムの粉末を用いた。そして 、式 a (%) = 100—R(%)に基づき、吸収率 α (%)を算出した。  [0144] The light absorption of the dye was measured as follows. First, the dye labeled on the DNA adsorbed on the titanium oxide electrode was measured using a spectrophotometer (UV-3100, manufactured by Shimadzu Corporation). The reflectance (R%) of the dye was calculated based on the diffuse reflection method using an integrating sphere. At this time, barium sulfate powder was used as a reference material having a reflectance of 100%. Then, based on the equation a (%) = 100-R (%), the absorption rate α (%) was calculated.
次いで、光吸収量を下記式に基づき、算出した。  Next, the amount of light absorption was calculated based on the following equation.
[数 1] 光吸収量 = j" |l及収率 （Q%)x光源の照度 （mW/cm2} [Equation 1] Light absorption = j "| l and yield (Q%) x Illuminance of light source (mW / cm 2 }
[0145] 測定されたバックグランド光電流値 (安定電流値)および光吸収量は表 7に示される 通りであった。 The measured background photocurrent value (stable current value) and the amount of light absorption were as shown in Table 7.
[表 7] 表 7  [Table 7] Table 7
フィル夕 バックグランド光電流値 光吸収量  Fill evening background photocurrent light absorption
( z A)_ (mW c m2 )(z A) _ (mW cm 2 )
Y-43フィル夕 4 6 . 6 4 . 3Y-43 Phil Yu 4 6. 6 4. 3
550nm- 40hwフィル夕 1 . 5 0 . 5 7 550nm-40hw fill 1.5.0.5.7
20nm-220hwフィル夕 1 1 . 8 1 . 6 20nm-220hw fill evening 1 1.8 1.6
600nm- 260hwフィルタ 2 6 . 4 3 . 6600nm-260hw filter 26.44.3
620nm- 260hwフィル夕 1 6 . 4 2 . 7620nm-260hw fill 16.4.2.7
600nm- 300hwフィルタ 4 2 . 1 4 . 1 [0146] ノ、イブリダィゼーシヨン反応を行わない本例にあっては、増感色素の光励起が起こ らないことから、電流値は本来ゼロとなることが望ましい。しかし、表 7に示されるように 、電子受容物質である酸化チタン自身の光励起による光電流力 Sバックグランド電流と して観測されてしまう。また、表 7から分力るように、紫外線量の少ない 550nm- 40hwフ ィルタを使用した場合にはバックグランド電流を低減できるが、色素の光吸収量が他 のフィルタよりも少なくなつてしまう。したがって、バックグランドの低減には 600nm-300hw filter 4 2 .1 4.1 No. In the present example in which no hybridization reaction is performed, it is desirable that the current value be essentially zero since photoexcitation of the sensitizing dye does not occur. However, as shown in Table 7, it is observed as a photocurrent force S background current due to photoexcitation of titanium oxide itself, which is an electron accepting substance. Also, as can be seen from Table 7, when a 550 nm-40hw filter with a small amount of ultraviolet light is used, the background current can be reduced, but the amount of light absorbed by the dye is smaller than that of other filters. Therefore, to reduce the background
550nm-40hwフィルタの使用が好まし!/、一方、大きな光電流の値を得るためには Y-43 フィルタの使用が有効であることが分かる。  The use of a 550nm-40hw filter is preferred! / On the other hand, it can be seen that the use of a Y-43 filter is effective for obtaining a large photocurrent value.
[0147] 参考のため、上記 6種類のフィルタを 250Wのキセノンランプ光源（林時計工業社 製、 LA250Xe)に使用した場合の光源のスペクトル分布を、スペクトルラディォメータ 一（ゥシォ電機、 USR— 40D)を用いて測定した。その結果は図 18に示される通りで あった。また、図 18に示されるスペクトル分布図における 350〜550nmの波長域を 拡大した図を図 19に示す。  [0147] For reference, the spectral distribution of the light source when the above six types of filters are used for a 250W xenon lamp light source (manufactured by Hayashi Tokei Kogyo Co., Ltd., LA250Xe) is shown by a spectral radiometer (USIO-40D). ). The results were as shown in FIG. Fig. 19 shows an enlarged view of the wavelength range from 350 to 550nm in the spectrum distribution diagram shown in Fig. 18.
[0148] 例 13：光源フィルタ S /N比 の閣係  [0148] Example 13: Cabinet of light source filter S / N ratio
光学フィルタとして例 11で用いた Y-43フィルタおよび 550nm- 40hwフィルタを使用し たこと、および被検 DNA溶液の濃度を 40 Mとしたこと以外は例 6と同様にして試 験を行った。  The test was performed in the same manner as in Example 6, except that the Y-43 filter and the 550 nm-40hw filter used in Example 11 were used as the optical filters, and the concentration of the test DNA solution was set to 40 M.
[0149] 測定されたバックグランド光電流値の経時変化は図 20に示される通りであった。図 20〖こ示されるよう〖こ、 Y- 43フィルタの光電流の最大値は 3. 4mA、 550nm-40hwフィ ルタの最大値は 1. 1mAとなった。すなわち、 550nm-40hwフィルタを使用した場合の 光電流の値は Y-43フィルタを使用した場合の 1Z3程度と小さ力つた。これは、 550nm- 40hwフィルタは、例 12の結果力 分力るようにバックグランド電流を低減でき るフィルタである一方、可視光の強度も弱くしてしまうため、増感色素の光吸収量が 小さくなつたためと考えられる。  [0149] The time-dependent change of the measured background photocurrent value was as shown in FIG. As shown in Figure 20, the maximum photocurrent of the Y-43 filter was 3.4 mA, and the maximum value of the 550 nm-40hw filter was 1.1 mA. That is, the value of the photocurrent when using the 550nm-40hw filter was as small as about 1Z3 when using the Y-43 filter. This is because the 550nm-40hw filter is a filter that can reduce the background current as well as the component of the result of Example 12, but also weakens the intensity of visible light. It is thought that it became small.
[0150] ここで、センサのシグナル Zノイズ比（SZN比）の指標として、光電流の最大値 Z バックグランド電流の比を考える。 550nm- 40hwフィルタを用いると、 Y-43フィルタよりも ノ ックグランド電流が 1Z30に低減される一方、光電流の最大値が 1Z3となる。した がって、 550nm-40hwフィルタの SZN比は Y-43フィルタに比較して、（lZ3) Z (lZ 30) = 10倍向上することになる。他のフィルタに関しても同様な評価をおこなったとこ ろ、 550nm- 40hwフィルタの S/N比が最も優れて!/、ることが分かった。 Here, as an index of the signal Z noise ratio (SZN ratio) of the sensor, the ratio of the maximum value Z of the photocurrent to the background current is considered. When the 550nm-40hw filter is used, the knock ground current is reduced to 1Z30 compared to the Y-43 filter, while the maximum value of the photocurrent is 1Z3. Therefore, the SZN ratio of the 550nm-40hw filter is (lZ3) Z (lZ 30) = 10 times better. The same evaluation was performed for other filters, and it was found that the S / N ratio of the 550nm-40hw filter was the best! /.
[0151] 例 14 :照射光の強度依存件  [0151] Example 14: Irradiation intensity dependence
被検 DNA溶液の濃度を 40 Mとしたこと、および光源の強度を変化させたこと以 外は例 6と同様にして試験を行った。また、光強度をスペクトルラディオメ一ター（ゥシ ォ電機社製、 USR—40d)を用いて測定した。  The test was performed in the same manner as in Example 6, except that the concentration of the test DNA solution was set to 40 M and the intensity of the light source was changed. The light intensity was measured using a spectral radiometer (USR-40d, manufactured by Shio Denki).
[0152] 測定された光電流値 (安定電流値）と、光強度との関係は図 21に示される通りであ つた。図 21に示されるように、光強度 800mWZcm2の領域まで線形性が確認でき、 光電流が反応速度は光強度の 1乗に比例する光量律速であることが判明した。なお 、一般的に、センサとして高精度でかつ安定に機能させるためには、反応速度が光 強度の 1Z2乗に比例する拡散律速とはならない、光量律速下で評価することが望ま しいとされている。 [0152] The relationship between the measured photocurrent value (stable current value) and the light intensity was as shown in Fig. 21. As shown in FIG. 21, the linearity was confirmed up to the region of light intensity of 800 mWZcm 2 , and it was found that the reaction speed of the photocurrent was light rate-determined in proportion to the first power of the light intensity. In general, in order for a sensor to function with high accuracy and stability, it is considered desirable to evaluate the reaction under a light-rate control, in which the reaction rate does not become diffusion-controlled in proportion to the 1Z2 power of light intensity. I have.
[0153] 例 15 : LED光源の枪討  [0153] Example 15: Discussion of LED light source
プローブ DNAの担持を以下の通り行ったこと、ハイブリダィゼーシヨンを行わなかつ たこと、および光源として以下の LEDを使用したこと以外は、例 6と同様にして試験を 行った。  The test was carried out in the same manner as in Example 6, except that the probe DNA was carried as follows, hybridization was not carried out, and the following LED was used as a light source.
赤色 LED :オアシス RED、 TOL-50aURsCEs  Red LED: Oasis RED, TOL-50aURsCEs
黄色 LED :日亜化学、 YELLOW NSPY- 500S  Yellow LED: Nichia, YELLOW NSPY-500S
白色 LED :日亜化学、 WHITE NSPW500BS  White LED: Nichia Chemical, WHITE NSPW500BS
青色 LED :豊田合成、 BLUE E1L51-3B  Blue LED: Toyoda Gosei, BLUE E1L51-3B
緑色 LED :豊田合成、 GREEN E1L51- 3G  Green LED: Toyoda Gosei, GREEN E1L51-3G
なお、これらの LEDのスペクトル分布をスペクトルラディオメ一ター（ゥシォ電機、 U SR— 40D)を用いて測定したところ、図 22に示される通りの結果が得られた。  When the spectral distribution of these LEDs was measured using a spectral radiometer (Disho Electric, USR-40D), the results shown in FIG. 22 were obtained.
[0154] すなわち、プローブ DNAの担持は以下の通り行った。まず、例 6と同様にしてプロ ーブ DNAが担持される前の作用電極を作製した。こうして得られた作用電極に BLB ランプで一晩紫外線照射を施し、汚れおよび残存有機物の除去を行った。次いで、 プローブ DNAとして以下に示される 5'—NH—3'ローダミンDNAを50mM HEP [0154] That is, loading of the probe DNA was performed as follows. First, a working electrode before carrying probe DNA was prepared in the same manner as in Example 6. The working electrode thus obtained was irradiated with ultraviolet light using a BLB lamp overnight to remove dirt and residual organic matter. Then, 5'-NH-3 'rhodamine DNA shown below was used as probe DNA in 50 mM HEP.
2  2
ES (pH7. 0)に溶解させて水溶液を調製した。この溶液を、予め 95°Cで 3分間保持 した後、氷上（2°C)で 3分間以上冷却させることにより、熱変性させておいた。 An aqueous solution was prepared by dissolving in ES (pH 7.0). Keep this solution in advance at 95 ° C for 3 minutes Then, it was heat denatured by cooling on ice (2 ° C) for 3 minutes or more.
プローブ DNA(5， -NH 3，ローダミンDNA)：  Probe DNA (5, -NH3, rhodamine DNA):
2  2
5' NH -AACGTCGTGACTGGG 3'Rho  5 'NH -AACGTCGTGACTGGG 3'Rho
2  2
[0155] 先に得られた作用電極の電子受容層上に、プローブ DNAと電子受容物質 (酸ィ匕 チタン)の結合力を向上させるために、シランカップリング剤による表面処理を行った 。すなわち、イソプロパノール中にシランカップリング剤（信越ィ匕学社製、 KBM-403 )を 0. 5wt%溶解させた液を、 75°Cで 5分間酸ィ匕チタンの表面に反応させた後、イソ プロパノールで洗净して乾燥した。こうして得られた作用電極表面にスぺーサ用穴あ きテープを貼り、ピンセットの先を用いてテープ接着面に残存する空気を除去した。こ のテープ上に、 5mm X 5mm角の大きさの開口部が形成されたシリコンシートを載置 して密着させた。この開口部に先に調製した 5，一 NH— 3，ローダミン DNA溶液 (40  [0155] A surface treatment with a silane coupling agent was performed on the electron-accepting layer of the working electrode obtained above in order to improve the bonding force between the probe DNA and the electron-accepting substance (titanium oxide). That is, a solution in which 0.5 wt% of a silane coupling agent (manufactured by Shin-Etsu-Danigaku Co., Ltd., KBM-403) was dissolved in isopropanol was allowed to react at 75 ° C. for 5 minutes on the surface of the titanium oxide, It was washed with isopropanol and dried. A perforated tape for spacer was stuck on the surface of the working electrode thus obtained, and air remaining on the tape-adhered surface was removed using a tip of tweezers. On this tape, a silicon sheet having an opening having a size of 5 mm × 5 mm square was placed and brought into close contact with each other. The 5,1-NH-3, rhodamine DNA solution (40
2  2
μ Μ)を 25 μ 1装填した。このとき、ピペットチップの先端を用いて、シリコンシールの 開口部の四隅まで充分に DNA溶液が行き渡るようにした。続いて、 DNA溶液中に 気泡が極力入らな 、ようにガラス板で真上力 蓋をして、湿らせた紙等で蒸気圧が調 整されたプラスチック容器に収容した。この容器中に 60°Cで 15時間保持して、 5'アミ ン— 3'ローダミン DNAをインキュベートした。その後、 DNA溶液を除去し、流水で軽 く電極表面を 2秒間洗浄した後、空気を吹き付けて残水を飛散させた。こうして、プロ ーブ DNAが担持された作用電極を得た。  μΜ) was loaded in an amount of 25 μl. At this time, the tip of the pipette tip was used to sufficiently spread the DNA solution to the four corners of the opening of the silicon seal. Subsequently, the DNA solution was covered with a glass plate so as to prevent bubbles from entering as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. The container was kept at 60 ° C. for 15 hours, and 5′-amine-3′-rhodamine DNA was incubated. Thereafter, the DNA solution was removed, and the electrode surface was lightly washed with running water for 2 seconds, and then air was blown to disperse residual water. Thus, a working electrode carrying probe DNA was obtained.
[0156] こうしてプローブ物質が担持された作用電極に新たなシリコンシールを載置し、開 口部にブロッキング剤として 10 μ Μのジエタノールァミン 25 μ 1を注入した。ブロッキ ング剤中に気泡が極力入らな 、ようにガラス板で真上から蓋をして、湿らせた紙等で 蒸気圧を調整したプラスチック容器に入れた。そして、 60°Cで 30分間保持して、プロ ッキング剤をインキュベートした。電極表面を再度流水で軽く 2秒間洗浄した後、空気 を吹き付けて残水を飛散させた。こうしてブロッキングが施された作用電極を得た。  [0156] A new silicone seal was placed on the working electrode carrying the probe substance in this manner, and 25 µl of 10 µl of diethanolamine was injected into the opening as a blocking agent. In order to prevent air bubbles from entering the blocking agent as much as possible, the container 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. Then, it was kept at 60 ° C for 30 minutes to incubate the blocking agent. After the electrode surface was again lightly washed with running water for 2 seconds, air was blown to disperse residual water. Thus, a blocked working electrode was obtained.
[0157] また、ブランクとして、作用電極に上記プローブ DNAを固定ィ匕しないブランク電極 についても光電流を測定した。プローブ DNAを固定ィ匕した作用電極の光電流と、プ ランク電極の光電流との比を SZN比として評価した。  [0157] Further, as a blank, the photocurrent was measured for a blank electrode in which the probe DNA was not immobilized on the working electrode. The ratio between the photocurrent of the working electrode on which the probe DNA was immobilized and the photocurrent of the plank electrode was evaluated as the SZN ratio.
[0158] 測定された光電流、ブランク電流、光電流とブランク電流の差、および SZN比を図 23に示す。図 23から分かるように、黄色および緑色の LEDにおいて、ブランク電流 を低く抑えることができ、良好な結果が得られた。青色および白色の LEDは 400nm 以下の波長を若干含むため、ノイズとして検出される酸ィ匕チタン由来のノックグランド 電流値が大きくなつた。一方、赤色の LEDは最も光強度が強いにもかかわらず、色 素として用いたローダミンの励起波長よりも長波長のため、光電流が検出されなかつ た。 [0158] Measured photocurrent, blank current, difference between photocurrent and blank current, and SZN ratio See Figure 23. As can be seen from FIG. 23, in the yellow and green LEDs, the blank current was able to be kept low, and good results were obtained. Since blue and white LEDs contain a small wavelength of 400 nm or less, the knock ground current value derived from titanium oxide, which is detected as noise, has increased. On the other hand, although the red LED has the highest light intensity, no photocurrent was detected because the wavelength was longer than the excitation wavelength of rhodamine used as a colorant.
[0159] 例 16： LED光源を用!ヽた被檢 DNAの檢出  [0159] Example 16: Use of LED light source!
被検 DNA溶液の濃度を 10、 20、および 30 μ Μとしたこと、プローブ DNAの担持 を以下の通り行ったこと、および光源として以下の LEDを使用したこと以外は、例 6と 同様にして光電流の測定を行った。  Except that the concentration of the test DNA solution was 10, 20, and 30 μΜ, the loading of the probe DNA was performed as follows, and the following LED was used as the light source, the same as in Example 6 Photocurrent measurements were made.
黄色 LED :日亜化学、 YELLOW NSPY- 500S  Yellow LED: Nichia, YELLOW NSPY-500S
緑色 LED :豊田合成、 GREEN E1L51- 3G  Green LED: Toyoda Gosei, GREEN E1L51-3G
[0160] すなわち、プローブ DNAの担持は以下の通り行った。まず、例 6と同様にしてプロ ーブ物質が担持される前の作用電極を作製した。こうして得られた作用電極に BLB ランプで一晩紫外線照射を施し、汚れおよび残存有機物の除去を行った。 [0160] That is, loading of the probe DNA was performed as follows. First, in the same manner as in Example 6, a working electrode before the probe substance was carried was produced. The working electrode thus obtained was irradiated with ultraviolet light using a BLB lamp overnight to remove dirt and residual organic matter.
[0161] 次いで、プローブ DNAとして例 6で使用したものと同じ 5'—NH—DNAを 50mM [0161] Next, the same 5'-NH-DNA as that used in Example 6 was used as the probe DNA at 50 mM.
2  2
HEPES (pH7. 0)に溶解させて水溶液を調製した。この溶液を、予め 95°Cで 3分 間保持した後、氷上（2°C)で 3分間以上冷却させることにより、熱変性させておいた。  An aqueous solution was prepared by dissolving in HEPES (pH 7.0). This solution was kept at 95 ° C for 3 minutes in advance, and then heat-denatured by cooling on ice (2 ° C) for 3 minutes or more.
[0162] 先に得られた作用電極の電子受容層上に、プローブ DNAと電子受容物質 (酸ィ匕 チタン)の結合力を向上させるために、シランカップリング剤による表面処理を行った 。すなわち、イソプロパノール中にシランカップリング剤（信越化学、 KBM— 403)を 0 . 5wt%溶解させた液を 75°Cで 5分間電子受容層（酸化チタン）の表面に反応させ た後、イソプロパノールで洗浄して乾燥した。こうして得られた作用電極表面にスぺー サ用穴あきテープを貼り、ピンセットの先を用いてテープ接着面に残存する空気を除 去した。このテープ上に、 5mm X 5mm角の大きさの開口部が形成されたシリコンシ 一トを載置して密着させた。この開口部に先に調製した 5'—NH—DNA溶液（200 [0162] A surface treatment with a silane coupling agent was performed on the electron-accepting layer of the working electrode obtained above in order to improve the bonding strength between the probe DNA and the electron-accepting substance (titanium oxide). That is, a solution of 0.5 wt% of a silane coupling agent (Shin-Etsu Chemical, KBM-403) dissolved in isopropanol is allowed to react at 75 ° C for 5 minutes on the surface of the electron-accepting layer (titanium oxide), and then the solution is mixed with isopropanol. Washed and dried. A perforated tape for a spacer was stuck on the surface of the working electrode thus obtained, and air remaining on the tape-adhering surface was removed with the tip of tweezers. A silicon sheet having an opening having a size of 5 mm × 5 mm square was placed on the tape and brought into close contact therewith. The previously prepared 5'-NH-DNA solution (200
2  2
μ Μ)を 25 μ 1注入した。このとき、ピペットチップの先端を用いて、シリコンシールの 開口部の四隅まで充分に DNA溶液が行き渡るようにした。続いて、 DNA溶液中に 気泡が極力入らな 、ようにガラス板で真上力 蓋をして、湿らせた紙等で蒸気圧が調 整されたプラスチック容器に収容した。この容器中に 60°Cで 6時間保持して、 5'— N H—DN Aをインキュベートした。その後、 DN A溶液を除去し、流水で軽く電極表面μΜ) was injected at 25 μl. At this time, the tip of the pipette tip was used to sufficiently spread the DNA solution to the four corners of the opening of the silicon seal. Then, in the DNA solution The container was covered with a glass plate so as to prevent bubbles from entering as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. The container was kept at 60 ° C for 6 hours, and 5′-NH-DNA was incubated. Then remove the DNA solution and gently run the electrode surface with running water.
2 2
を 2秒間洗浄した後、空気を吹き付けて残水を飛散させた。こうして、プローブ DNA が担持された作用電極を得た。  After washing for 2 seconds, air was blown to disperse residual water. Thus, a working electrode carrying the probe DNA was obtained.
[0163] こうしてプローブ物質が担持された作用電極に新たなシリコンシールを載置し、開 口部にブロッキング剤として 10 μ Μのジエタノールァミン 25 μ 1を注入した。ブロッキ ング剤中に気泡が極力入らな 、ようにガラス板で真上から蓋をして、湿らせた紙等で 蒸気圧を調整したプラスチック容器に入れた。そして、 60°Cで 30分間保持して、プロ ッキング剤をインキュベートした。電極表面を再度流水で軽く 2秒間洗浄した後、空気 を吹き付けて残水を飛散させた。こうしてブロッキングが施された作用電極を得た。  [0163] A new silicone seal was placed on the working electrode carrying the probe substance in this manner, and 25 µl of 10 µl of diethanolamine was injected into the opening as a blocking agent. In order to prevent air bubbles from entering the blocking agent as much as possible, the container 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. Then, it was kept at 60 ° C for 30 minutes to incubate the blocking agent. After the electrode surface was again lightly washed with running water for 2 seconds, air was blown to disperse residual water. Thus, a blocked working electrode was obtained.
[0164] 測定された光電流と被検 DNA濃度との関係は図 24に示される通りであった。黄色 および緑色 LEDを光源として用いた場合、被検 DNA濃度に依存した光電流が観測 され、ノ、イブリダィゼーシヨンを行わな力つた濃度 0 Mのブランクよりも優位な光電 流が観測された。これらの結果から、黄色および緑色 LED光源は被検 DNAの定量 測定に適して 、ることが分かる。  [0164] The relationship between the measured photocurrent and the test DNA concentration was as shown in FIG. When yellow and green LEDs were used as the light source, a photocurrent depending on the DNA concentration of the test sample was observed, and a photoelectric current superior to that of the 0 M blank, which did not perform the hybridization and was not observed, was observed. Was. These results indicate that the yellow and green LED light sources are suitable for quantitative measurement of the test DNA.
[0165] 例 17：ルテニウム錯体スクシンィミジルエステルを用いた被枪 DNAの色素標識  Example 17: Dye labeling of target DNA using ruthenium complex succinimidyl ester
被検 DNAを標識するための増感色素として、アミノ基を持つ蛋白質を色素修飾す るための色素として市販されて 、る、下記式で表されるルテニウム錯体スクシンイミジ ルエステル (RU— ONSu)を用意した。 As a sensitizing dye for labeling the test DNA, a protein having an amino group commercially available as dyes order to dye modified, Ru, ruthenium complex Sukushin'imiji glycol ester represented by the following formula - the (R U ONSu) Prepared.
[化 2] [Chemical 2]
Figure imgf000051_0001
Figure imgf000051_0001
Ru-ONSu Ru-ONSu
[0166] この増感色素を用いた DNA末端ァミンへの修飾を以下の通り行った。まず、上記 増感色素を以下の条件で 3'末端にアミノ基を持つ 15merの ssDNAと反応させた。 [0166] Modification to DNA terminal amine using this sensitizing dye was performed as follows. First, the sensitizing dye was reacted with a 15-mer ssDNA having an amino group at the 3 'end under the following conditions.
Ru-ONSu: 200nmol/25 μ 1  Ru-ONSu: 200 nmol / 25 μ 1
ssDNA: 20nmol/25 μ 1  ssDNA: 20 nmol / 25 μ 1
緩衝液： 50mM HEPES(pH7. 0)  Buffer: 50 mM HEPES (pH 7.0)
TAPS(pH8. 0)  TAPS (pH 8.0)
TAPS(pH9. 0)  TAPS (pH 9.0)
[0167] 各溶液を室温で 15時間振盪させた後、ゲルろ過、続、てエタノール沈殿を行 、、 色素修飾 DNAを精製した。 Ru— ONSuの濃度を 458nmの吸収スペクトルで測定し た。また、 ssDNAの濃度を MolecularProbes社の定量キットを用いて測定した。得 られた測定結果を基に、 ssDNAに対する Ru— ONSuの修飾比（モル比）を算出し た。得られた結果は表 8に示される通りであった。  [0167] 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. In addition, the concentration of ssDNA was measured using a quantitative kit from Molecular Probes. Based on the measurement results obtained, the modification ratio (molar ratio) of Ru-ONSu to ssDNA was calculated. The results obtained are as shown in Table 8.
[表 8] 表 8  [Table 8] Table 8
緩衝液 修飾比 （Ru/DNA)  Buffer modification ratio (Ru / DNA)
HEPE S ( H 7. 0) 5. 6  HEPE S (H 7.0) 5.6
TAPS (pH 8. 0) 3. 1  TAPS (pH 8.0) 3.1
TAPS lpH9. 0) 4. 4 表 8に示される結果から、スクシンィミジルエステル誘導体を DNAの標識色素とし ても利用可能なことが分かる。表 8の結果力も分力るように、スクシンィミジルエステル 誘導体は末端ァミンのみならず核酸塩基中のァミンにも結合していると見られるためTAPS lpH 9.0) 4.4 Based on the results shown in Table 8, succinimidyl ester derivatives were used as DNA labeling dyes. It turns out that it is also available. As can be seen from Table 8, the succinimidyl ester derivative appears to bind not only to the terminal amine but also to the amine in the nucleobase.
、この色素によれば 1分子の DNAに対して複数個の標識色素を導入できることも分 かる。 It can also be seen that this dye can introduce a plurality of labeling dyes into one molecule of DNA.
[0169] 例 18：光電流のアクションスペクトルの測定  [0169] Example 18: Measuring action spectrum of photocurrent
色素標識された被検 DNAとして以下に示される 3 ' AlexaFluor(R)647DNAを使用 し、かつプローブ DNAとして 5' -NH—DNA— 2を使用したこと、および被検 DN 3 'AlexaFluor (R) 647 DNA shown below was used as the dye-labeled test DNA, and 5'-NH-DNA-2 was used as the probe DNA.
2  2
A溶液の濃度を 40 /z Mとしたこと以外は例 11と同様にして光電流のアクションスぺク トルの測定を行った。  The action spectrum of the photocurrent was measured in the same manner as in Example 11, except that the concentration of the A solution was set to 40 / zM.
被検 DN A ( 3 ' AlexaFluora647DNA)：  Tested DN A (3 'AlexaFluora647DNA):
3' AlexaFluora647 - CCATGATGTCAGTTT 5'  3 'AlexaFluora647-CCATGATGTCAGTTT 5'
プローブ DNA(5， -NH -DNA- 2)：  Probe DNA (5, -NH -DNA-2):
2  2
5' NH2 - GGTACTACAGTCAAA 3'  5 'NH2-GGTACTACAGTCAAA 3'
なお、これらの被検 DNAとプローブ DNAは、ハイブリダィゼーシヨン反応により二 本鎖 DNAを形成することができるものである。  The test DNA and the probe DNA can form a double-stranded DNA by a hybridization reaction.
[0170] 測定されたアクションスペクトル (IPCEの波長依存性）は図 25に示される通りであつ た。図 25から分かるように、得られたアクションスペクトルは、増感色素である AlexaF luor(R)647の吸収スペクトルと同様のプロファイルを示した。これは、光電流が増感色 素の励起に起因することを示唆する。 AlexaFluor(R)647の吸収中心波長は 640nm と見られるので、前述したローダミン B色素の吸収中心波長（520nm)と分離して、そ れぞれの色素由来の光電流を観測できる。よって、複数の吸収波長の異なる色素を 各々の DNAに標識し、それらの色素を別々に励起できる光を照射することで、同時 に二本鎖 DNAの形成を光電流として観測することが可能となる。 [0170] The measured action spectrum (wavelength dependence of IPCE) was as shown in Fig. 25. As can be seen from FIG. 25, the obtained action spectrum showed a profile similar to that of the sensitizing dye AlexaFluor (R) 647. This suggests that the photocurrent is due to the excitation of the sensitizing dye. Since the absorption center wavelength of AlexaFluor (R) 647 is considered to be 640 nm, the photocurrent derived from each dye can be observed separately from the absorption center wavelength (520 nm) of the rhodamine B dye described above. Therefore, it is possible to simultaneously observe the formation of double-stranded DNA as a photocurrent by labeling each DNA with multiple dyes having different absorption wavelengths and irradiating light that can excite these dyes separately. Become.
[0171] 例 19：酸化チタン多孔皙膜を雷子^容層 して備えた作用雷極の微細構诰の評  [0171] Example 19: Evaluation of the fine structure of a working lightning electrode with a titanium oxide porous film as a thunderbolt layer
M  M
例 6と同様にして、酸化チタン多孔質膜を電子受容層として備えた作用電極の作製 を行った。得られた作用電極の断面および表面を走査型電子顕微鏡 (S-4100型、 日 立製作所製）により観察した。作用電極の断面について得られた SEM画像を図 26 に、作用電極の酸ィ匕チタン多孔質膜表面について得られた SEM画像を図 27に、そ れぞれ示す。図 26に示される画像から、多孔質酸化チタン電極の膜厚は約 20 /z m であり、表面が非常に平滑な均一な膜構造を有することが分かる。また、図 27に示さ れる画像から、得られた多孔質電極は、使用した酸化チタン粉末の一次粒子 (粒径： 約 40〜50nm)に相当する粒子が良好に分散した、比較的大きなメソ細孔を有する 良好な多孔質膜であることが分力つた。従って、得られた多孔質膜にあっては、比較 的分子サイズの大き 、DNA分子であっても細孔内への拡散が容易に起こるものと考 えられる。 In the same manner as in Example 6, a working electrode provided with a titanium oxide porous film as an electron-accepting layer was produced. The cross section and surface of the obtained working electrode were observed with a scanning electron microscope (S-4100, manufactured by Hitachi, Ltd.). Figure 26 shows the SEM image obtained for the cross section of the working electrode. FIG. 27 shows SEM images obtained on the surface of the titanium oxide porous membrane of the working electrode, respectively. From the image shown in FIG. 26, it can be seen that the film thickness of the porous titanium oxide electrode is about 20 / zm, and the surface has a very smooth and uniform film structure. In addition, from the image shown in FIG. 27, the obtained porous electrode shows that the particles corresponding to the primary particles (particle diameter: about 40 to 50 nm) of the titanium oxide powder used are relatively well dispersed. A good porous membrane with pores helped. Therefore, in the obtained porous membrane, even if it is a DNA molecule having a relatively large molecular size, it is considered that diffusion into pores easily occurs.
[0172] また、得られた酸化チタン多孔質膜の細孔分布を、細孔分布測定装置（日本ベル 製、 Belsorp28SA)を用いて測定した。このとき、測定サンプルとして、焼成後の作用 電極力も酸ィ匕チタン多孔質膜を剥離させたものを用いた。サンプルの表面積は BET 式より、細孔径は DH式により算出した。その結果、 BET比表面積は 19. 8m2Zgと 比較的高ぐ細孔分布は 67. 8nmにピークを持つ曲線が得られた。これらの結果か ら、得られた酸化チタン多孔質膜は、比較的大きな細孔が均一に開いた非常にポー ラスな構造を有することが分力つた。このような大きな細孔内にあっては、比較的嵩高 Vヽ DNA分子（例えば一本鎖 DN Aの幅が約 2nmのもの）の拡散が容易に起こるもの と考えられる。 [0172] Further, the pore distribution of the obtained titanium oxide porous membrane was measured using a pore distribution measuring device (Belsorp28SA, manufactured by Nippon Bell). At this time, as the measurement sample, the working electrode strength after firing was obtained by peeling the porous titanium oxide film. The surface area of the sample was calculated by the BET equation, and the pore diameter was calculated by the DH equation. As a result, the BET specific surface area was 19.8 m 2 Zg, and a relatively high pore distribution was obtained with a curve having a peak at 67.8 nm. From these results, it was a component that the obtained titanium oxide porous membrane had a very porous structure in which relatively large pores were uniformly opened. In such large pores, diffusion of relatively bulky V ヽ DNA molecules (for example, single-stranded DNA having a width of about 2 nm) is considered to occur easily.
[0173] 例 20 :LED光源に針する酸化物半 体雷極の最商化  [0173] Example 20: Commercialization of an oxide semiconductor lightning pole used for an LED light source
(1)被検物質およびプローブ物質の準備  (1) Preparation of test substance and probe substance
色素標識された被検物質 (以下、被検 DNAともいう）として、 5'末端を蛍光色素 Cy 5で標識された、以下の塩基配列を有する 25塩基の核酸塩基（5 ' Cy5DNA)を用 意した。また、プローブ物質（以下、プローブ DNAともいう）として上記被検 DNAと相 補鎖を有する 5'末端にアミンを標識した 25塩基の核酸塩基 (5' -NH— DNA)を  As a dye-labeled test substance (hereinafter also referred to as test DNA), a 25-nucleobase (5 'Cy5 DNA) labeled at the 5' end with a fluorescent dye Cy5 and having the following base sequence is available. did. As a probe substance (hereinafter, also referred to as probe DNA), a 25-nucleotide base (5'-NH-DNA) having a complementary chain to the above-mentioned test DNA and labeled with an amine at the 5 'end is used.
2  2
用意した。すなわち、このプローブ DNAと被検 DNAは、ハイブリダィゼーシヨン反応 により二本鎖 DNAを形成することができる。  Prepared. That is, the probe DNA and the test DNA can form a double-stranded DNA by a hybridization reaction.
被検 DNA (5， Cy5DNA)：  Test DNA (5, Cy5DNA):
3， 一 TGGAAGTAGTTTTTGTAGTAGTAGG一 Cy 5— 5 ' プローブ DNA(5' -NH -DNA)： 5' NH - ACCTTCATCAAAAACATCATCATCC 3 ' 3, one TGGAAGTAGTTTTTGTAGTAGTAGG one Cy5—5 'probe DNA (5'-NH-DNA): 5 'NH-ACCTTCATCAAAAACATCATCATCC 3'
2  2
[0174] (2)作用電極の作製およびプローブ物質の担持  (2) Preparation of working electrode and support of probe substance
プローブ物質として上記（1)に示したプローブ DNAを使用したこと、チタ-ァ微粉 末またはその代わりとして以下に示される酸ィ匕物半導体微粉末を使用したこと、およ びペーストを塗布する際の膜厚を 60 μ mとしたこと以外は例 6と同様にしてプローブ DNAが担持された作用電極を得た。  The use of the probe DNA shown in (1) above as the probe substance, the use of titer fine powder or the following oxide semiconductor powder as an alternative, and the application of paste A working electrode carrying a probe DNA was obtained in the same manner as in Example 6, except that the film thickness of was set to 60 μm.
TiO：昭和タイタ-ゥム社製、 F-3、平均粒径 50nm  TiO: Showa Titanium, F-3, average particle size 50nm
2  2
ZnO :シーアィ化成社製、 NanotekZnO)  (ZnO: Shika Chemical Co., Ltd., NanotekZnO)
Nb O：多木化学社製、酸化ニオブゾルを蒸発乾固した粉末  Nb O: Taki Chemical Co., Ltd., powder obtained by evaporating and drying niobium oxide sol
2 5  twenty five
WO：和光純薬社製  WO: Wako Pure Chemical Industries
3  Three
SrTiO ： Sr(NO ) +Ti(OiPr)を加水分解後焼成して得た粉末  SrTiO: Powder obtained by sintering Sr (NO 2) + Ti (OiPr) after hydrolysis
3 3 2 4  3 3 2 4
In O：和光純薬社製  In O: Wako Pure Chemical Industries
2 3  twenty three
Ta〇：シグマアルドリッチジャパン社製  Ta〇: Sigma-Aldrich Japan
2 5  twenty five
[0175] 用いた各種酸ィ匕物半導体のバンドギャップ、平均粒径、およびバックグランド電流 を表 9に示す。なお、バックグランド電流とは、プローブ DNAおよび被検 DNAのどち らも吸着して ヽな 、剥き出し（Bare)の電極の光電流である。  Table 9 shows the band gap, average particle size, and background current of the various oxide semiconductors used. Note that the background current is a photocurrent of a bare electrode which is not adsorbed to both the probe DNA and the test DNA.
[表 9]  [Table 9]
¾ 9  ¾ 9
酸化物半導体 バンドギャップ (eV) 平均粒径 (nm) バックグランド電流 (nA ) 酸化チタン （アナ夕ーゼ） 3.2 50 3 酸化亜鉛 3. 1 30 25 酸化ニオブ 3. 1 10 20 酸化タングステン 2.8 100 25 チタン酸ストロンチウム 3.2 200 0 酸化インジウム 3.8 100 15 酸化タンタル 3. 9 500 0 上記プローブ物質が担持された作用電極を、湿らせた紙等で蒸気圧が調整された プラスチック容器に入れて、 60°Cで 30分間、飽和水蒸気中に置いた。こうして蒸気 に触れさせた電極に 90°Cで 3分間加熱処理を行 、、新たなシリコンシールを電極に 載せた。 Oxide semiconductor Band gap (eV) Average particle size (nm) Background current (nA) Titanium oxide (anaze) 3.2 50 3 Zinc oxide 3.1 30 25 Niobium oxide 3.1 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 Place the working electrode carrying the above probe substance in a plastic container whose vapor pressure has been adjusted with moistened paper, etc. For 30 minutes in saturated steam. The electrode exposed to the vapor was heated at 90 ° C for 3 minutes, and a new silicone seal was applied to the electrode. I put it.
[0177] (3)ハイブリダィゼーシヨン  [0177] (3) Hybridization
被検 DNA溶液として Mの 5，Cy5DNA溶液を使用したこと、およびインキュ ペートの温度を 50°Cとしたこと以外は例 6と同様にしてノ、イブリダィゼーシヨンを行つ た。  The hybridization was carried out in the same manner as in Example 6, except that the M5 and Cy5 DNA solutions were used as the test DNA solution, and the temperature of the incubation was 50 ° C.
[0178] (4)測定用セルの組み立て  (4) Assembly of Measurement Cell
例 6と同様にしてサンドイッチ型の測定用セルを組み立てた。  A sandwich-type measuring cell was assembled in the same manner as in Example 6.
[0179] (5)光電流の測定 (5) Photocurrent measurement
光源として赤色 LED (CCS社製、 HLV- 27- NR- R)を用いたこと、および紫外線力 ットフィルタを使用しな力つたこと以外は例 6と同様にして光電流の測定を行った。 その結果は、図 28に示される通りであった。図 28に示されるように、赤色 LED照射 下で Cy— 5色素標識 DNAを被検 DNAとして用いた場合、酸化インジウム、酸ィ匕亜 鉛、および酸ィ匕チタン電極において高い光電流値が得られることが判明した。また、 その他の酸化物半導体電極、酸化タングステン、酸化ニオブ、チタン酸ストロンチウ ム、および酸ィ匕タンタルにお 、てもバックグランド電流値以上の光電流値が得られて いるので DNAセンサーとして有効であることが判明した。上記結果から、必ずしも酸 化チタン電極だけが最適な電極であるとは限らず、光源、標識色素、および DNA等 の諸条件に応じて最適な電極も変化し得ることが分かる。  The photocurrent was measured in the same manner as in Example 6, except that a red LED (CCS, HLV-27-NR-R) was used as the light source, and that no power was applied using an ultraviolet light filter. The result was as shown in FIG. As shown in FIG. 28, when Cy-5 dye-labeled DNA was used as the test DNA under irradiation with red LED, high photocurrent values were obtained at the indium oxide, oxidized zinc oxide, and oxidized titanium electrodes. It turned out to be. In addition, other oxide semiconductor electrodes, tungsten oxide, niobium oxide, strontium titanate, and tantalum oxydioxide also have a photocurrent value higher than the background current value, so that they are effective as DNA sensors. It turned out to be. From the above results, it can be seen that the titanium oxide electrode is not always the optimal electrode, and the optimal electrode can also be changed according to various conditions such as the light source, the labeling dye, and the DNA.
[0180] 例 2ί :雷早 ^^層のみで構成される作 ffl雷 用いた例 [0180] Example 2ί: Work composed of only ^^ layer ^ fl Lightning example
(1) 色素標識プローブ DNAの準備  (1) Preparation of dye-labeled probe DNA
色素標識されたプローブ DNAとして、 3'末端を Cy5で標識された、以下の塩基配 列を有する 25塩基の核酸塩基 (Cy5標識 ssDNA)を用意した。  As a dye-labeled probe DNA, a 25 base nucleobase (Cy5-labeled ssDNA) having the following base sequence and having a 3′-end labeled with Cy5 was prepared.
色素標識プローブ DNA(Cy5標識 ssDNA)：  Dye-labeled probe DNA (Cy5-labeled ssDNA):
5， NH2 - ACCTTCATCAAAAACATCATCATCC Cy5 3 '  5, NH2-ACCTTCATCAAAAACATCATCATCC Cy5 3 '
[0181] (2)作用電極の準備および色素標識 DNAの担持 (2) Preparation of working electrode and loading of dye-labeled DNA
電子受容層のみで構成される作用電極として、フッ素をドープした酸化スズ (F— S ηθ： FTO)コートガラス (エイアイ特殊硝子社製、 U膜、シート抵抗： 15 Ω /口）およ Fluorine-doped tin oxide (F—S ηθ: FTO) coated glass (AI film, U film, sheet resistance: 15 Ω / port)
2 2
びスズをドープした酸化インジウム（Sn— In02 :ITO)コートガラス (東洋精密工業株 式会社製、 100 ΩΖ口）を用意した。これらのガラスをアセトンと水で洗浄し、酸素雰 囲気下で紫外線照射を 30分間施して、汚れおよび残存有機物の除去を行った。洗 浄されたガラスにスぺーサ用穴あきテープを貼り、ピンセットの先を用いてテープ接 着面に残存する空気を除去した。このテープ上に 5mm X 5mm角の大きさの開口部 が形成されたシリコンシートを戴置して密着させた。 And tin-doped indium oxide (Sn—In02: ITO) coated glass (Toyo Seimitsu Kogyo KK Made by Shikisha Co., Ltd., 100 Ω). These glasses were washed with acetone and water, and irradiated with ultraviolet rays for 30 minutes in an oxygen atmosphere to remove stains and residual organic substances. Perforated tape for spacer was applied to the washed glass, and the air remaining on the tape-bonded surface was removed using the tip of tweezers. A silicon sheet having an opening having a size of 5 mm × 5 mm square was placed on and adhered to the tape.
[0182] 続いて、 0、 1、 10、 50 μ Μの各濃度に調製した Cy5標識 ssDNA溶液を 95°Cで 5 分間保持した後、直ちに氷上に移して 10分間保持して DNAを変性させ、先に用意 した電極上の開口部に 25 1を装填した。この時ピペットチップの先端を用いて、シリ コンシールの開口部の四隅まで充分に DNA溶液が行き渡るようにした。そして、 DN A溶液中に気泡が入らな ヽようにガラス板で真上力ゝら蓋をして、湿らせた紙等で蒸気 圧が調製されたプラスチック容器に収容して、 60°Cで一晩インキュベートさせた。イン キュベート後、 DNA溶液を除去し、流水で軽く電極表面を約 2秒間洗浄した後、空 気を吹き付けて残水を飛散させた。その後、例 6の表 3に示した条件で DNAを吸着 させた電極を洗浄し、最後に空気を吹き付けて残水を飛散させた。  [0182] Subsequently, the Cy5-labeled ssDNA solution prepared at each concentration of 0, 1, 10, and 50 μΜ was kept at 95 ° C for 5 minutes, immediately transferred to ice and kept for 10 minutes to denature the DNA. Then, 251 was loaded into the opening on the electrode prepared above. At this time, the tip of the pipette tip was used to sufficiently spread the DNA solution to the four corners of the opening of the silicon seal. Then, cover directly with a glass plate to prevent bubbles from entering the DNA solution, and place in a plastic container whose vapor pressure has been adjusted with moistened paper, etc., at 60 ° C. Incubate overnight. After the 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 disperse residual water. Thereafter, the electrode on which the DNA was adsorbed was washed under the conditions shown in Table 3 of Example 6, and finally, air was blown to disperse the residual water.
[0183] (3)測定用セルの組み立て  [0183] (3) Assembly of measurement cell
こうして得られた作用電極と、対電極としての白金電極とを用いて、図 6に示される ようなフロー型測定セルを組み立てた。その際、作用電極を白金対電極と対向させて 配置するとともに、その間には、両電極間の短絡を防止しかつ電解液を充填する空 間を形成するための膜厚 500 mのシリコンシートを挿入した。シリコンシートには 5 mm X 5mm角よりも十分大きい穴が空いており、ここに送液された電解液が溜まり、 作用電極上に固定化された DNAが接触する構造になっている。作用電極は電気的 に接しているスプリングプローブを介して、白金電極は端部に接続されたリード線を 介してポテンシォスタツト（ビ一'ェ一'エス株式会社、 ALS Modl832A)に接続した。 電解液として、体積比が 4： 6のァセトニトリルと炭酸エチレンの混合溶媒にヨウ素 0. 06Mとテトラプロピルアンモ-ゥムョーダイド 0. 6Mを溶解した混合液を用意した。こ の電解液を先に述べたフロー型測定セルに組み込まれた作用電極と白金対電極の 間に充填させた。  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 this time, the working electrode is placed so as to face the platinum counter electrode, and a silicon sheet with a thickness of 500 m is formed between the working electrode to prevent a short circuit between the electrodes and to form a space for filling the electrolyte. Inserted. The silicon sheet has a hole that is sufficiently larger than 5 mm x 5 mm square, where the fed electrolyte is collected and the DNA immobilized on the working electrode comes into contact. The working electrode was connected via a spring probe in electrical contact, and the platinum electrode was connected via a lead wire connected to the end to a potentiostat (B1S1S, ALS Modl832A). . As an electrolytic solution, a mixed solution prepared by dissolving iodine (0.6M) and tetrapropylammonium-dymoxide (0.6M) 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 incorporated in the flow-type measuring cell described above and the platinum counter electrode.
[0184] (4)光電流の測定 フロー型測定セルに固定した LED (CCS社製、 HLV-27-NR-R)力 生じた光を作 用電極表面に照射し、作用電極と白金対電極との間に流れる電流を経時的に測定 した。測定は 180秒間行ったが、光の照射は電流の測定開始 60秒後から 60秒間の み行った。観測した光電流について、 120秒後の光電流値から 180秒後の光電流値 を差し引くことにより補正を行った。 [0184] (4) Photocurrent measurement LED (CCS, HLV-27-NR-R) fixed to a flow-type measuring cell Force Generates light on the working electrode surface and changes the current flowing between the working electrode and the platinum counter electrode over time. It was measured. The measurement was performed for 180 seconds, but the light irradiation was performed only for 60 seconds after 60 seconds from the start of the current measurement. The observed photocurrent was corrected by subtracting the photocurrent value after 180 seconds from the photocurrent value after 120 seconds.
各濃度の Cy5標識 ssDNAについて測定されたた光電流値は、図 29に示される通 りであった。図 29に示されるように、 FTOまたは ITOを電子受容層として用いた場合 、その下層に導電性基材を更に設けること無ぐ作用電極として十分に機能させるこ とが出来る。すなわち、 FTOおよび ITOは電子受容層のみならず導電性基材として も機能することが確認された。  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.
[0185] 例 22 :蛋白皙の測定 [0185] Example 22: Measurement of protein
(1)被検物質およびプローブ物質の準備  (1) Preparation of test substance and probe substance
色素標識された被検物質として、ローダミンで標識した HSA (ヒト血清アルブミン)を 用意した。また、プローブ物質として、抗 HSA抗体 (ゥサギ抗 HSA血清、日本バイオ テスト研究所)を用意した。  HSA (human serum albumin) labeled with rhodamine was prepared as a dye-labeled test substance. In addition, an anti-HSA antibody (Egret anti-HSA serum, Japan Biotest Laboratory) was prepared as a probe substance.
[0186] (2)作用電極の作製およびプローブ物質の担持 [0186] (2) Preparation of working electrode and support of probe substance
例 6と同様にして作用電極の作製、および汚れおよび残存有機物の除去を行った 得られた作用電極上に、スぺーサ用穴あきテープを貼り、ピンセットの先を用いてテ ープ接着面に残存する空気を除去した。このテープ上に、 5mm X 5mm角の大きさ の開口部が形成されたシリコンシートを戴置して密着させた。  A working electrode was prepared, and dirt and remaining organic substances were removed in the same manner as in Example 6. A perforated tape for spacer was applied on the obtained working electrode, and a tape-adhering surface was applied using a tip of tweezers. The remaining air was removed. On this tape, a silicon sheet having an opening having a size of 5 mm × 5 mm square was placed and closely attached.
次いで、プローブ物質として抗 HSA抗体（ゥサギ抗 HSA血清、 日本バイオテスト研 究所）を 50mM HEPES緩衝液 (pH7. 0)に 7. 68mg/mlになるように溶解させて 水溶液を調製した。この溶液を、作用電極上のシリコンシールの開口部に 25 1Z電 極ずつ装填して、ガラス板で覆い、 4°Cで一晩放置して固相化した。こうして得られた 作用電極を 50mM HEPES緩衝液 (pH 7. 0)で 3回洗浄した。洗浄後、 0. 2%カゼ インを含む 50mM HEPES緩衝液 (pH 7. 0)をシリコンシールを作用電極表面に載 置した開口部に 25 μ 1Ζ電極ずつ装填して、ガラス板で覆い 30°Cで 90分インキュべ ートし、ブロッキングを行った。 Next, an aqueous solution was prepared by dissolving an anti-HSA antibody (Peacock anti-HSA serum, Japan Biotest Laboratories) as a probe substance in 50 mM HEPES buffer (pH 7.0) to 7.68 mg / ml. This solution was loaded into the opening of the silicon seal on the working electrode in the form of a 25 1Z electrode, covered with a glass plate, and left at 4 ° C overnight to solidify. The working electrode thus obtained was washed three times with a 50 mM HEPES buffer (pH 7.0). After washing, load a 50 μM HEPES buffer (pH 7.0) containing 0.2% casein into the opening where the silicone seal was placed on the working electrode surface in 25 μΖ 1Ζ electrodes, cover with a glass plate, and cover 30 °. Incubate at C for 90 minutes And blocked.
標識キットとして FluoReporter Tetramethylrhodamine Protein Labeling kit ( Molecular Probes社）を用いて、テトラメチルローダミンを HSAに標識した。製造元の プロトコルに従い、色素の標識反応、精製、および分離を行い、標識比 1のローダミン 標識 HSA溶液を 3. 78mg,ml濃度で 1. 5ml得た。  HSA was labeled with tetramethylrhodamine using a FluoReporter Tetramethylrhodamine Protein Labeling kit (Molecular Probes) as a labeling kit. According to the manufacturer's protocol, the labeling reaction, purification, and separation of the dye were performed to obtain a rhodamine-labeled HSA solution having a labeling ratio of 1.
[0187] (3)被検物質の装填 [0187] (3) Loading of test substance
次いで、ブロッキング後の作用電極を 0. 05% Tween20を含む 50mM HEPES 緩衝液 (pH7. 0) (以下 T— HEPES)で 3回洗浄した。ローダミン標識 HSAをブロッ キング液で段階希釈することにより、 0. 1, 0. 33、 1. 0、および 3. 3mg/mlの各濃 度のローダミン標識 HSA溶液を作製した。各濃度のローダミン標識 HSA溶液を作 用電極上のシリコンシールの開口部に 25 μ 1Ζ電極ずつ装填して、ガラス板で覆い、 30°Cで 90分間インキュベートした。その後、 T— HEPESで 3回洗浄し、超純水でリ ンスした。  Next, the working electrode after blocking was washed three times with 50 mM HEPES buffer solution (pH 7.0) containing 0.05% Tween 20 (hereinafter referred to as T-HEPES). Rhodamine-labeled HSA was serially diluted with a blocking solution to prepare rhodamine-labeled HSA solutions at concentrations of 0.1, 0.33, 1.0, and 3.3 mg / ml. Each concentration of rhodamine-labeled HSA solution was loaded into the opening of the silicon seal on the working electrode by 25 µl electrode, covered with a glass plate, and incubated at 30 ° C for 90 minutes. Then, it was washed three times with T-HEPES and rinsed with ultrapure water.
[0188] (4)測定用セルの組み立て  (4) Assembly of Measurement Cell
電解液として lOOmMエチレンジァミン四酢酸、 lOOmM NaCl、および lOOmM Na SO力もなる成分を溶解した水溶液を使用したこと以外は例 21と同様にしてフ The procedure was the same as in Example 21 except that an aqueous solution in which lOOmM ethylenediaminetetraacetic acid, lOOmM NaCl, and lOOmM NaSO were also dissolved was used as the electrolyte.
2 4 twenty four
ロー型測定セルを組み立てた。  A row type measuring cell was assembled.
[0189] (5)光電流の測定 [0189] (5) Photocurrent measurement
フロー型測定セルに固定した緑色 LED (CCS社製、 HLV-24GR-NR-3W)と集光 マイクロファイバヘッド（CCS社製、 HFR-25-30)を用いて作用電極表面の 3cm上方 力ゝら光を照射し、作用電極と白金対電極の間に流れる電流を経時的に測定した。測 定は 180秒間行った力 光の照射は電流の測定開始 60秒後から 60秒間のみ行つ た。観測した光電流は 120秒後の光電流値から 180秒後の光電流値を差し引くこと で補正を行った。  Using a green LED (CCS, HLV-24GR-NR-3W) fixed to a flow-type measuring cell and a condensing microfiber head (CCS, HFR-25-30), force 3 cm above the surface of the working electrode. Then, the current flowing between the working electrode and the platinum counter electrode was measured over time. The measurement was performed for 180 seconds, and the light irradiation was performed only for 60 seconds after 60 seconds from the start of the current measurement. The observed photocurrent was corrected by subtracting the photocurrent value after 180 seconds from the photocurrent value after 120 seconds.
各濃度の HSA溶液について測定された光電流値は、図 30に示される通りであつ た。抗原濃度が 0. 1〜3. 3mgZmlの範囲で相関係数の高い検量線を引くことがで き、蛋白質の定量が可能であることが分力つた。  The photocurrent measured for each concentration of the HSA solution was as shown in FIG. A calibration curve with a high correlation coefficient could be drawn when the antigen concentration was in the range of 0.1 to 3.3 mgZml, which proved that protein quantification was possible.
[0190] 例 23 :プローブ物皙を作用雷極表 こ固定化する工程に用いる溶液の檢討 まず、例 1と同様にして酸化チタンを含んでなる電子受容層が形成された作用電極 を得た。 5， NH— AACGTCGTGACTGGG 3，Rhoの塩基配列を有する 3，口 [0190] Example 23: Examination of the solution used in the process of immobilizing the probe material First, in the same manner as in Example 1, a working electrode on which an electron-accepting layer containing titanium oxide was formed was obtained. 5, NH— AACGTCGTGACTGGG 3, with Rho base sequence 3, mouth
2  2
ーダミン修飾 DNAを以下に示されるバッファ 1または 2に溶解して、 200 Mのロー ダミン修飾 DNA溶液を調製した。この溶液を、予め 95°Cで 3分間保持した後、氷上 で冷却させることにより、変性させておいた。  The rhodamine-modified DNA was dissolved in buffer 1 or 2 shown below to prepare a 200 M rhodamine-modified DNA solution. This solution was previously kept at 95 ° C for 3 minutes, and then denatured by cooling on ice.
バッファ l : 50mM HEPES水溶液、 pH7. 0  Buffer l: 50mM HEPES aqueous solution, pH 7.0
バッファ 2 : 2X SSC : 0. 3M塩化ナトリウムおよび 0. 03Mクェン酸ナトリウム含有 水溶液、 pH7. 0、化学構造中にカルボキシル基を有する  Buffer 2: 2X SSC: Aqueous solution containing 0.3M sodium chloride and 0.03M sodium citrate, pH 7.0, having a carboxyl group in the chemical structure
[0191] 先に得られた作用電極の電子受容層上に、 5mm X 5mm角の大きさの開口部が 形成された、厚さ 700 μ mのシリコンシールを載置した。この開口部〖こ 200 μ Μの口 ーダミン修飾 DN Α溶液を 35 1注入した。このとき、ピペットチップの先端を用いて、 シリコンシールの開口部の四隅まで充分に DNA溶液が行き渡るようにした。続いて、 DNA溶液中に気泡が極力入らな ヽようにガラス板で真上カゝら覆 ヽ、湿らせた紙等で 蒸気圧が調整されたプラスチック容器に収容した。この容器中に 60°Cで 15時間保持 して、ローダミン修飾 DNAをインキュベートした。その後、 DNA溶液を除去し、流水 で軽く電極表面を洗浄した後、空気を吹き付けて残水を飛散させた。こうして、プロ一 ブ物質が担持された作用電極を得た。  [0191] On the electron-accepting layer of the working electrode obtained above, a silicon seal having a thickness of 700 µm and having an opening of 5 mm x 5 mm square was formed. 351 of this solution with a mouth-modified DNΑ solution of 200 μΜ at the opening was injected. At this time, the DNA solution was sufficiently distributed to the four corners of the opening of the silicon seal using the tip of the pipette tip. Subsequently, the DNA solution was covered directly above with a glass plate so as to prevent bubbles from entering the DNA solution as much as possible, and housed in a plastic container whose vapor pressure was adjusted with moistened paper or the like. The container was kept at 60 ° C. for 15 hours to incubate the rhodamine-modified DNA. Thereafter, the DNA solution was removed, the electrode surface was lightly washed with running water, and air was blown to disperse residual water. Thus, a working electrode carrying the probe substance was obtained.
[0192] 上記プローブ物質が担持された作用電極に新たなシリコンシールを載置し、開口 部にブロッキング剤として 10 μ Μのジエタノールァミン 25 μ 1を装填した。ブロッキン グ剤中に気泡が極力入らな 、ようにガラス板で真上力も蓋をして、湿らせた紙等で蒸 気圧を調整したプラスチック容器に入れた。そして、 60°Cで 30分間保持して、ブロッ キング剤をインキュベートした。電極表面を再度流水で軽く 2秒間洗浄した後、空気 を吹き付けて残水を飛散させた。こうしてブロッキングが施された作用電極を得た。  [0192] A new silicone seal was placed on the working electrode carrying the probe substance, and 25 µl of 10 µl of diethanolamine was loaded as a blocking agent into the opening. In order to prevent air bubbles from entering the blocking agent as much as possible, the container was placed in a plastic container whose top pressure was covered with a glass plate and whose vapor pressure was adjusted with moistened paper or the like. Then, it was kept at 60 ° C for 30 minutes to incubate the blocking agent. After the electrode surface was again lightly washed with running water for 2 seconds, air was blown to disperse residual water. Thus, a blocked working electrode was obtained.
[0193] ノ、イブリダィゼーシヨンおよびその後の洗浄工程を行わな力つたこと以外は、例 2と 同様にしてセルの作製および光電流の測定を行った。なお、光電流値の測定は、光 照射時の安定化電流値と未照射時の安定化電流値とを測定して、これらの電流値の 差を算出することにより行った。  [0193] A cell was prepared and the photocurrent was measured in the same manner as in Example 2, except that the steps of No., Ibridization and the subsequent washing step were not performed. The photocurrent value was measured by measuring a stabilized current value when irradiated with light and a stabilized current value when not irradiated with light, and calculating the difference between these current values.
[0194] また、ブランク試験として、プローブ物質（ローダミン修飾 DNA)が固定化されて！/、 な ヽ作用電極にっ ヽても光電流を測定した。 [0194] As a blank test, a probe substance (rhodamine-modified DNA) was immobilized! /, The photocurrent was also measured with the working electrode.
[0195] 得られた光電流値と、ブランク試験の電流値との比力 SZN比を算出した。その結 果は表 10に示される通りであった。  [0195] The specific force SZN ratio between the obtained photocurrent value and the current value in the blank test was calculated. The results are shown in Table 10.
[表 10] 表 1 0  [Table 10] Table 10
緩衝溶液 緩衝剤 S /N比 バッファ 1 H E P E S 6 . 7 6  Buffer solution Buffer S / N ratio Buffer 1 H E P E S 6. 7 6
ノ、"ヅファ 2 S S C 1^ 0 6  No, "ヅ FA 2 S S C 1 ^ 0 6
[0196] 表 10に示される結果から、プローブ物質を作用電極表面に固定ィ匕する工程に用い る溶液として、 HEPES (バッファ 1)を使用した場合、カルボキシル基を有する SSC ( ノ ッファ 2)と比べて、 SZN比が格段に高 、ことが分かる。 [0196] From the results shown in Table 10, when HEPES (buffer 1) was used as the solution used in the step of immobilizing the probe substance on the surface of the working electrode, it was compared with SSC having a carboxyl group (Noffer 2). In comparison, it can be seen that the SZN ratio is much higher.
[0197] ί列 24 :プローブ に被檢 を または にキ寺¾ に させる工程 に使用される試料液の枪討  [0197] Row 24: Examination of the sample liquid used in the process of causing the probe to make an inspection or
プローブ物質として 5， -ΝΗ AACGTCGTGACTGGGの塩基酉己歹 IJを有する 5  As a probe substance, 5-has AACGTCGTGACTGGG base IJ 5
2  2
'ァミノ修飾 DNAを用いたこと以外は例 6と同様にして、プローブ物質が担持され、か つ、ブロッキングが施された作用電極を得た。  A working electrode carrying a probe substance and being subjected to blocking was obtained in the same manner as in Example 6, except that 'amino-modified DNA was used.
[0198] 次いで、試料液として 5， AACGTCGTGACTGGG 3 'Rhoの塩基配列を有す る 3'ローダミン修飾 DNAを以下に示されるバッファ 1または 2に溶解した 200 μ Μの 溶液を用いたこと、および 60°Cで 15時間インキュベーションを行ったこと以外は例 2 と同様にして、ハイブリダィゼーシヨン、作用電極の洗浄、セルの作製、および光電流 の測定を行った。なお、光電流値の測定は、光照射時の安定化電流値と未照射時 の安定化電流値とを測定して、これらの電流値の差を算出することにより行った。 バッファ l : 50mM HEPES水溶液、 pH7. 0 [0198] Next, as a sample solution, a 200 μΜ solution of 3 ′ rhodamine-modified DNA having the base sequence of AACGTCGTGACTGGG 3 ′ Rho dissolved in buffer 1 or 2 shown below was used; Hybridization, washing of the working electrode, fabrication of the cell, and measurement of the photocurrent were performed in the same manner as in Example 2 except that incubation was performed at 15 ° C for 15 hours. The photocurrent value was measured by measuring a stabilized current value when irradiated with light and a stabilized current value when not irradiated, and calculating the difference between these current values. Buffer l: 50mM HEPES aqueous solution, pH 7.0
バッファ 2 : 2X SSC : 0. 3M塩化ナトリウムおよび 0. 03Mクェン酸ナトリウム含有 水溶液、 pH7. 0、化学構造中にカルボキシル基を有する  Buffer 2: 2X SSC: Aqueous solution containing 0.3M sodium chloride and 0.03M sodium citrate, pH 7.0, having a carboxyl group in the chemical structure
[0199] また、ブランク試験として、プローブ物質（5'ァミノ修飾 DNA)および被検物質（3' ローダミン修飾 DNA)が固定ィ匕されて ヽな ヽ作用電極につ!ヽても光電流を測定した 得られた光電流値と、ブランク試験の電流値との比力 SZN比を算出した。その結 果は表 11に示される通りであった。 [0199] As a blank test, the photocurrent was measured even when the probe substance (5'-amino-modified DNA) and the test substance (3'-rhodamine-modified DNA) were immobilized on a working electrode. did The specific force SZN ratio between the obtained photocurrent value and the current value of the blank test was calculated. The results are shown in Table 11.
[表 11]  [Table 11]
緩衝溶液 緩衝剤 S N比 Buffer solution Buffer S N ratio
バッファ 1 HEPE S 5. 76  Buffer 1 HEPE S 5.76
バッファ 2 SSC 2. 07  Buffer 2 SSC 2.07
[0201] 表 11に示される結果から、プローブ物質に被検物質を直接または間接的に特異的 に結合させる工程に使用される溶液として、 HEPES (バッファ 1)を使用した場合、力 ルポキシル基を有する SSC (バッファ 2)と比べて、 SZN比が格段に高いことが分か る。 [0201] From the results shown in Table 11, when HEPES (buffer 1) was used as the solution used in the step of directly or indirectly specifically binding the test substance to the probe substance, It can be seen that the SZN ratio is much higher than the SSC (buffer 2).
[0202] 例 25:作用雷極の洗浄液 して使用される溶液の枪討  [0202] Example 25: Consideration of a solution used as a cleaning solution for a working lightning electrode
ノ、イブリダィゼーシヨン後の作用電極の洗浄を以下の通り行ったこと以外は、例 7と 同様にして実験を行った。  No. 7 An experiment was performed in the same manner as in Example 7, except that the working electrode was washed after the hybridization.
すなわち、ハイブリダィゼーシヨンが施された作用電極を洗浄液に浸し、ゆっくりと 揺らしながら洗浄した。洗浄液としては以下に示されるバッファ 1、 2または 3を下記表 に基づく配合で使用し、各洗浄液について下記表に示される洗浄時間、洗浄回数、 および温度で洗浄を行った。なお、洗浄液を変更する毎に洗浄容器を交換した。 バッファ l:50mM HEPES水溶液、 pH7.0  That is, the working electrode to which the hybridization was applied was immersed in a cleaning solution, and washed while being slowly shaken. Buffer 1, 2 or 3 shown below was used as a washing solution in a formulation based on the following table, and each washing solution was washed at the washing time, washing frequency and temperature shown in the following table. The washing container was replaced every time the washing solution was changed. Buffer l: 50 mM HEPES aqueous solution, pH 7.0
バッファ 2:2X SSC:0.3M塩化ナトリウムおよび 0.03Mクェン酸ナトリウム含有 水溶液、 pH7.0)、化学構造中にカルボキシル基を有する  Buffer 2: 2X SSC: aqueous solution containing 0.3 M sodium chloride and 0.03 M sodium citrate, pH 7.0), having a carboxyl group in the chemical structure
ノッファ 3:150mM リン酸緩衝液 (PBS)、pH7.0、化学構造中にリン酸基を有 するため本発明の緩衝溶液ではな ヽ  Knoffer 3: 150 mM phosphate buffer (PBS), pH 7.0, which is not a buffer solution of the present invention because it has a phosphate group in its chemical structure.
[0203] [表 12] 表 1 2 [0203] [Table 12] Table 1 2
洗浄液 1回当たりの 洗浄回数 温度 洗浄時間  Number of washings per washing solution Temperature Washing time
界面活性剤 0 . 1 %を含むバッファ 1〜3 6分間 6回 室温 界面活性剤 0 . 1 %を含むバッファ 1〜3 1 3分間 1回 6 0 °C ノ、"ヅファ 1〜 3のみ 6分間 2回  Buffer containing 0.1% surfactant 1-3 times 6 minutes 6 times Room temperature Surfactant containing 0.1% buffer 1-3 1 3 minutes 1 time 60 ° C No, only 1 ヅ 3 for 6 minutes Twice
[0204] また、比較のため、ノ ッファ 1〜3の代わりに水を使用して、上記同様の洗浄を行つ た電極にっ ヽても光電流を測定した。 [0204] For comparison, the photocurrent was measured also on the electrode that had been cleaned in the same manner as described above, using water instead of the knockers 1-3.
[0205] 得られた光電流値は、図 31に示される通りであった。図 31に示される結果から、作 用電極の洗浄液として、 HEPES水溶液 (バッファ 1)を使用した場合、カルボキシル 基を有する SSC水溶液 (バッファ 2)やリン酸基を有する PBS (バッファ 3)の水溶液と 比べて、光電流値が格段に高いことが分かる。 [0205] The obtained photocurrent value was as shown in FIG. From the results shown in Fig. 31, when the HEPES aqueous solution (buffer 1) was used as the washing solution for the working electrode, the aqueous solution of the carboxyl group-containing SSC aqueous solution (buffer 2) and the phosphate group-containing PBS (buffer 3) was used. In comparison, the photocurrent value is significantly higher.

Claims

請求の範囲 The scope of the claims
[1] 光電流を用いた被検物質の特異的検出方法であって、  [1] A method for specific detection of an analyte using photocurrent,
被検物質を含む試料液と、該被検物質と直接または間接的に特異的に結合可能 なプローブ物質を表面に備えた作用電極と、対電極とを用意し、  A sample liquid containing a test substance, a working electrode having a probe substance capable of directly or indirectly specifically binding to the test substance on its surface, and a counter electrode are prepared.
増感色素の共存下、前記試料液を前記作用電極に接触させて、前記プローブ物 質に前記被検物質を直接または間接的に特異的に結合させ、該結合により前記増 感色素を前記作用電極に固定させ、  In the presence of a sensitizing dye, the sample solution is brought into contact with the working electrode to specifically or directly bind the test substance to the probe substance, and the binding causes the sensitizing dye to act on the probe substance. Fixed to the electrode,
前記作用電極と前記対電極とを電解質媒体に接触させ、そして、  Contacting the working electrode and the counter electrode with an electrolyte medium; and
前記作用電極に光を照射して前記増感色素を光励起させ、該光励起された増感 色素から前記作用電極への電子移動に起因して前記作用電極と前記対電極との間 に流れる光電流を検出すること  The working electrode is irradiated with light to excite the sensitizing dye, and a photocurrent flowing between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. Detecting
を含んでなる、方法。  A method comprising:
[2] 光電流を用いた被検物質の特異的検出方法であって、  [2] A method for specific detection of a test substance using a photocurrent,
被検物質が直接または間接的に特異的に結合したプローブ物質を表面に備え、か っ該結合により増感色素が固定されてなる作用電極と、対電極とを電解質媒体に接 触させ、  A probe electrode to which a test substance is directly or indirectly specifically bound is provided on the surface, and a working electrode having a sensitizing dye immobilized by the binding and a counter electrode are brought into contact with an electrolyte medium,
前記作用電極に光を照射して前記増感色素を光励起させ、該光励起された増感 色素から前記作用電極への電子移動に起因して前記作用電極と前記対電極との間 に流れる光電流を検出すること  The working electrode is irradiated with light to excite the sensitizing dye, and a photocurrent flowing between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. Detecting
を含んでなる、方法。  A method comprising:
[3] 前記プローブ物質を表面に備えた作用電極が、前記プローブ物質を含む溶液を前 記作用電極に接触させることにより得られる、請求項 1または 2に記載の方法。  3. The method according to claim 1, wherein the working electrode provided with the probe substance on its surface is obtained by bringing a solution containing the probe substance into contact with the working electrode.
[4] 前記作用電極と対電極とを電解質媒体に接触させる前に、前記作用電極を洗浄液 で洗浄する工程をさらに含んでなる、請求項 1〜3のいずれか一項に記載の緩衝溶 液。  4. The buffer solution according to any one of claims 1 to 3, further comprising a step of washing the working electrode with a washing solution before bringing the working electrode and the counter electrode into contact with an electrolyte medium. .
[5] 前記被検物質が予め前記増感色素で標識されてなる、請求項 1〜4のいずれか一 項に記載の方法。  [5] The method according to any one of claims 1 to 4, wherein the test substance is previously labeled with the sensitizing dye.
[6] 前記増感色素が前記被検物質とプローブ物質との結合体にインターカレーシヨン 可能なものである、請求項 1〜5のいずれか一項に記載の方法。 [6] The sensitizing dye is intercalated to a conjugate of the test substance and the probe substance. 6. The method according to any one of the preceding claims, wherein the method is possible.
[7] 前記被検物質が一本鎖の核酸であり、前記プローブ物質が前記核酸に対して相補 性を有する一本鎖の核酸である、請求項 1〜6のいずれか一項に記載の方法。 [7] The method according to any one of [1] to [6], 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. Method.
[8] 前記相補性を有する核酸が、前記核酸に対して 15bp以上の相補性部分を有する[8] The nucleic acid having complementarity has a complementary portion of 15 bp or more with respect to the nucleic acid.
、請求項 7に記載の方法。 The method of claim 7.
[9] 前記被検物質が予め前記増感色素で標識されてなる核酸であり、前記被検物質 1 分子につき前記増感色素力 S1つ標識されている、請求項 7または 8に記載の方法。 [9] The method according to claim 7, wherein the test substance is a nucleic acid previously labeled with the sensitizing dye, and one molecule of the test substance is labeled with one sensitizing dye S. .
[10] 前記被検物質が予め前記増感色素で標識されてなる核酸であり、前記被検物質 1 分子につき前記増感色素が 2つ以上標識されている、請求項 7または 8に記載の方 法。 10. The method according to claim 7, wherein the test substance is a nucleic acid previously labeled with the sensitizing dye, and two or more sensitizing dyes are labeled for one molecule of the test substance. Method.
[11] 前記試料が、予め前記増感色素で標識されてなる、前記被検物質と特異的に結合 可能な媒介物質をさらに含んでなり、該媒介物質と前記被検物質との結合物が前記 プローブ物質に特異的に結合する、請求項 1〜4のいずれか一項に記載の方法。  [11] The sample further comprises an intermediary substance which can be specifically bound to the test substance, which is previously labeled with the sensitizing dye. The method according to any one of claims 1 to 4, wherein the method specifically binds to the probe substance.
[12] 前記被検物質がリガンドであり、前記媒介物質が受容体蛋白質分子であり、前記プ ローブ物質が二本鎖の核酸である、請求項 11に記載の方法。  12. The method according to claim 11, wherein the test substance is a ligand, the mediator is a receptor protein molecule, and the probe substance is a double-stranded nucleic acid.
[13] 前記被検物質が外因性内分泌攪乱物質である、請求項 11または 12に記載の方 法。  13. The method according to claim 11, wherein the test substance is an exogenous endocrine disrupting substance.
[14] 前記光電流を検出する工程が、電流値または電気量を測定し、得られた電流値ま たは電気量力 前記試料液中の被検物質濃度を算出することをさらに含んでなる、 請求項 1〜13のいずれか一項に記載の方法。  [14] The step of detecting the photocurrent further comprises measuring a current value or an electric quantity, and calculating an obtained current value or an electric quantity force, a concentration of the test substance in the sample liquid. A method according to any one of claims 1 to 13.
[15] 得られた電流値または電気量から前記試料液中の被検物質濃度を算出する工程 力 予め作成された被検物質濃度と電流値または電気量との検量線と、得られた電 流値または電気量とを対比することにより行われる、請求項 14に記載の方法。  [15] Step of calculating the concentration of the test substance in the sample solution from the obtained current value or electric quantity The power of the calibration curve between the test substance concentration and the current value or the electric quantity prepared in advance, and the obtained electric power 15. The method according to claim 14, which is performed by comparing with a flow value or an electric quantity.
[16] 前記被検物質が予め前記増感色素で標識されてなり、  [16] The test substance is previously labeled with the sensitizing dye,
前記試料液が、前記増感色素で標識されていない、前記プローブ物質に特異的に 結合可能な第二の被検物質をさらに含んでなり、それにより、前記被検物質および 第二の被検物質を前記プローブ物質に競合させて特異的に結合させ、そして、 前記光電流を検出する工程が、電流値または電気量を測定し、得られた電流値ま たは電気量カゝら前記試料液中の第二の被検物質濃度を算出することをさらに含んで なる、請求項 1〜4のいずれか一項に記載の方法。 The sample solution further comprises a second test substance that is not labeled with the sensitizing dye and that can specifically bind to the probe substance, whereby the test substance and the second test substance The step of allowing a substance to compete with and specifically bind to the probe substance, and detecting the photocurrent measures a current value or an electric quantity, and obtains the obtained current value. The method according to any one of claims 1 to 4, further comprising calculating a concentration of a second test substance in the sample liquid by measuring an amount of electricity.
[17] 得られた電流値または電気量から前記試料液中の第二の被検物質濃度を算出す る工程が、予め作成された第二の被検物質濃度と電流値または電気量との検量線と 、得られた電流値または電気量とを対比することにより行われる、請求項 16に記載の 方法。 [17] The step of calculating the second test substance concentration in the sample liquid from the obtained current value or electric quantity includes the step of calculating the second test substance concentration prepared in advance and the current value or electric quantity. 17. The method according to claim 16, which is performed by comparing a calibration curve with an obtained current value or electric quantity.
[18] 前記被検物質および前記第二の被検物質が抗原であり、前記プローブ物質が抗 体である、請求項 16または 17に記載の方法。  [18] The method according to claim 16 or 17, wherein the test substance and the second test substance are antigens, and the probe substance is an antibody.
[19] 前記第二の被検物質が、前記被検物質よりも前記プローブ物質に特異的に結合し やすい性質を有する、請求項 16〜 18のいずれか一項に記載の方法。 [19] The method according to any one of claims 16 to 18, wherein the second test substance has a property of more specifically binding to the probe substance than the test substance.
[20] 前記作用電極が、前記増感色素が光励起に応じて放出する電子を受容可能な電 子受容物質を含んでなる電子受容層を有し、該電子受容層の表面に前記プローブ 物質が担持されてなる、請求項 1〜19のいずれか一項に記載の方法。 [20] The working electrode has an electron accepting layer containing an electron accepting substance capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation, and the probe substance is provided on the surface of the electron accepting layer. The method according to any one of claims 1 to 19, wherein the method is carried.
[21] 前記電子受容物質が、前記増感色素の最低非占有分子軌道 (LUMO)のヱネル ギー準位よりも低 、エネルギー準位を有する物質である、請求項 20に記載の方法。 [21] The method according to claim 20, wherein the electron accepting substance is a substance having an energy level lower than the energy level of the lowest unoccupied molecular orbital (LUMO) of the sensitizing dye.
[22] 前記電子受容物質が、酸化チタン、酸化亜鉛、酸化スズ、酸化ニオブ、酸化インジ ゥム、酸化タングステン、酸ィ匕タンタル、およびチタン酸ストロンチウム力もなる群から 選択される少なくとも一種を含んでなる、請求項 20または 21に記載の方法。 [22] The electron accepting substance contains 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. 22. A method according to claim 20 or claim 21.
[23] 前記電子受容物質が酸ィ匕チタンまたはチタン酸ストロンチウムである、請求項 20ま たは 21に記載の方法。 23. The method according to claim 20, wherein the electron accepting substance is titanium oxide or strontium titanate.
[24] 前記電子受容物質力 Sインジウムースズ複合酸ィ匕物 (ITO)またはフッ素がドープさ れた酸化スズ (FTO)である、請求項 20または 21に記載の方法。  [24] The method according to claim 20 or 21, wherein the electron accepting substance is S-indium oxide complex oxide (ITO) or fluorine-doped tin oxide (FTO).
[25] 前記作用電極が導電性基材をさらに含んでなり、前記電子受容層が該導電性基 材上に形成されてなる、請求項 1〜24のいずれか一項に記載の方法。  [25] The method according to any one of claims 1 to 24, wherein the working electrode further comprises a conductive substrate, and the electron accepting layer is formed on the conductive substrate.
[26] 前記増感色素が金属錯体色素または有機色素である、請求項 1〜25のいずれか 一項に記載の方法。  26. The method according to claim 1, wherein the sensitizing dye is a metal complex dye or an organic dye.
[27] 前記インターカレーシヨン可能な増感色素力 アタリジンオレンジまたはェチジゥム ブロマイドである、請求項 6〜26の 、ずれか一項に記載の方法。 [27] The method according to any one of claims 6 to 26, wherein the intercalable sensitizing dye is atalidine orange or ethidium bromide.
[28] 前記被検物質が二種以上存在し、前記各被検物質が互いに異なる波長の光で励 起可能な異なる増感色素で標識され、 [28] There are two or more kinds of the test substances, and each of the test substances is labeled with a different sensitizing dye capable of being excited by light having a different wavelength.
各増感色素毎に異なる波長の光を照射することにより、前記各被検物質を個別に 検出する、請求項 1〜27のいずれか一項に記載の方法。  The method according to any one of claims 1 to 27, wherein each of the test substances is individually detected by irradiating light having a different wavelength to each sensitizing dye.
[29] 前記作用電極上に前記プローブ物質が互いに分離された複数の領域毎に区分さ れて担持されてなり、前記光照射が各領域に対して個別に行われる、請求項 1〜28 の!、ずれか一項に記載の方法。 29. The method according to claim 1, wherein the probe substance is supported on the working electrode by being divided into a plurality of regions separated from each other, and the light irradiation is individually performed on each region. !, The method according to any one of the above.
[30] 前記電子受容層が前記導電性基材の全面にわたって形成されてなり、前記導電 性基材全体を流れる光電流を検出する、請求項 29に記載の方法。 30. The method according to claim 29, wherein the electron accepting layer is formed over the entire surface of the conductive substrate, and detects a photocurrent flowing through the entire conductive substrate.
[31] 前記作用電極が絶縁基板をさらに備えてなり、該絶縁基板上に、前記導電性基材 および前記電子受容層からなるスポットが、互いに分離された複数の領域毎に区分 されて形成されてなり、該各領域の導電性基材毎に流れる光電流を個別に検出する[31] The working electrode further includes an insulating substrate, and spots made of the conductive base material and the electron accepting layer are formed on the insulating substrate so as to be divided into a plurality of regions separated from each other. And individually detects a photocurrent flowing for each conductive substrate in each region.
、請求項 1〜27のいずれか一項に記載の方法。 28. The method according to any one of claims 1 to 27.
[32] 前記作用電極が絶縁基板をさらに備えてなり、該絶縁基板上に、前記電子受容層 力 なるスポットが、互いに分離された複数の領域毎に区分されて形成されてなり、 該各領域の電子受容層毎に流れる光電流を個別に検出する、請求項 1〜27のいず れか一項に記載の方法。 [32] The working electrode further comprises an insulating substrate, and the electron accepting layer is formed on the insulating substrate by dividing the spot into a plurality of regions separated from each other. The method according to any one of claims 1 to 27, wherein a photocurrent flowing in each of the electron receiving layers is individually detected.
[33] 前記作用電極上の互いに分離された複数の領域の各領域に複数種類のプローブ 物質が担持させることにより、複数の試料液の測定を同時に行う、請求項 29〜32の33. The method according to claim 29, wherein a plurality of types of probe substances are carried on each of a plurality of regions separated from each other on the working electrode, whereby a plurality of sample liquids are simultaneously measured.
V、ずれか一項に記載の方法。 V, the method according to any one of the preceding claims.
[34] 前記作用電極上の互いに分離された複数の領域の各領域毎に異なるプローブ物 質が担持させることにより、複数種類の被検物質の測定を同時に行う、請求項 29〜334. The method according to claim 29, wherein a plurality of types of test substances are simultaneously measured by carrying a different probe substance in each of the plurality of regions separated from each other on the working electrode.
2の 、ずれか一項に記載の方法。 2. The method according to any one of the preceding items.
[35] 前記電解質媒体がリチウムイオンを含む、請求項 1〜34のいずれか一項に記載の 方法。 [35] The method according to any one of claims 1 to 34, wherein the electrolyte medium includes lithium ions.
[36] 前記光が紫外線を実質的に含まない、請求項 1〜35のいずれか一項に記載の方 法。  [36] The method according to any one of claims 1 to 35, wherein the light does not substantially include ultraviolet light.
[37] 前記光の照射が、紫外線を除去する手段を介して行われる、請求項 1〜35のいず れか一項に記載の方法。 [37] The method according to any one of claims 1 to 35, wherein the irradiation of the light is performed through means for removing ultraviolet rays. The method according to any one of the preceding claims.
[38] 前記紫外線を除去する手段が、光学フィルタまたは分光器である、請求項 37に記 載の方法。  [38] The method according to claim 37, wherein the means for removing ultraviolet rays is an optical filter or a spectroscope.
[39] 前記光が、レーザ、無機エレクト口ルミネッセンス (EL)素子、および有機エレクト口 ルミネッセンス (EL)素子力 なる群力 選択される少なくとも一つの光源力 放出さ れた光である、請求項 36〜38のいずれか一項に記載の方法。  39. The light emitted from a laser, an inorganic electroluminescent (EL) element, and an organic electroluminescent (EL) element at least one light source selected from the group consisting of: 39. The method according to any one of -38.
[40] 前記光が、発光ダイオード (LED)である光源力も放出された光である、請求項 36 〜39の!、ずれか一項に記載の方法。  [40] The method according to any one of claims 36 to 39, wherein the light is light emitted by a light source which is a light emitting diode (LED).
[41] 請求項 1〜40のいずれか一項に記載の方法において作用電極として用いられる電 極であって、  [41] An electrode used as a working electrode in the method according to any one of claims 1 to 40,
導電性基材と、  A conductive substrate,
該導電性基材上に形成される、前記増感色素が光励起に応じて放出する電子を 受容可能な電子受容物質を含んでなる電子受容層と、  An electron-accepting layer formed on the conductive substrate, the electron-accepting layer including an electron-accepting substance capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation;
を備えた、電極。  An electrode comprising:
[42] 前記電子受容層上に担持される、前記被検物質と直接または間接的に特異的に 結合可能なプローブ物質をさらに備えた、請求項 41に記載の電極。  42. The electrode according to claim 41, further comprising a probe substance carried on the electron accepting layer and capable of directly or indirectly specifically binding to the test substance.
[43] 前記電子受容物質が、前記増感色素の最低非占有分子軌道 (LUMO)のヱネル ギー準位よりも低 、エネルギー準位を有する物質である、請求項 41または 42に記載 の電極。  43. The electrode according to claim 41, wherein the electron accepting substance is a substance having an energy level lower than the energy level of the lowest unoccupied molecular orbital (LUMO) of the sensitizing dye.
[44] 前記電子受容物質が、酸化チタン、酸化亜鉛、酸化スズ、酸化ニオブ、酸化インジ ゥム、酸化タングステン、酸ィ匕タンタル、およびチタン酸ストロンチウム力もなる群から 選択される少なくとも一種を含んでなる、請求項 41〜43のいずれか一項に記載の電 極。  [44] The electron accepting substance contains 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. An electrode according to any one of claims 41 to 43.
[45] 前記電子受容物質が酸ィ匕チタンである、請求項 41〜43のいずれか一項に記載の 電極。  [45] The electrode according to any one of claims 41 to 43, wherein the electron accepting substance is titanium oxide.
[46] 前記電子受容物質がチタン酸ストロンチウムである、請求項 41〜43のいずれか一 項に記載の電極。  46. The electrode according to claim 41, wherein the electron accepting substance is strontium titanate.
[47] 前記電子受容層の膜厚が 0. 1〜200 μ mである、請求項 41〜46のいずれか一項 に記載の電極。 47. The electron-accepting layer according to claim 41, wherein the thickness of the electron-accepting layer is 0.1 to 200 μm. An electrode according to claim 1.
[48] 前記電子受容層が多孔性を有する、請求項 41〜47のいずれか一項に記載の電 極。  [48] The electrode according to any one of claims 41 to 47, wherein the electron accepting layer has porosity.
[49] 前記導電性基材が透明である、請求項 41〜48のいずれか一項に記載の電極。  [49] The electrode according to any one of claims 41 to 48, wherein the conductive substrate is transparent.
[50] 前記プローブ物質が、前記電子受容層上の、互いに分離された複数の領域毎に 区分されて担持されてなる、請求項 41〜49のいずれか一項に記載の電極。 50. The electrode according to any one of claims 41 to 49, wherein the probe substance is carried on the electron-accepting layer by being divided into a plurality of regions separated from each other.
[51] 前記電子受容層が前記導電性基材の全面にわたって形成されてなる、前記導電 性基材にリード線が接続されてなる、請求項 50に記載の電極。 51. The electrode according to claim 50, wherein the electron accepting layer is formed over the entire surface of the conductive substrate, and a lead wire is connected to the conductive substrate.
[52] 絶縁基板をさらに備えてなり、該絶縁基板上に、前記導電性基材および前記電子 受容層からなるスポットが、互いに分離された複数の領域毎に区分されて形成されて なり、該各領域の導電性基材毎に個別にリード線が接続されてなる、請求項 50に記 載の電極。 [52] The apparatus further comprises an insulating substrate, wherein spots comprising the conductive base material and the electron accepting layer are formed on the insulating substrate so as to be divided into a plurality of regions separated from each other. 51. The electrode according to claim 50, wherein a lead wire is individually connected to each conductive base material in each region.
[53] 請求項 1〜40のいずれか一項に記載の方法に用いられる測定用セルであって、 請求項 41〜52のいずれか一項に記載の作用電極と、  [53] A measurement cell used in the method according to any one of claims 1 to 40, wherein the working electrode according to any one of claims 41 to 52,
対電極と、  A counter electrode,
前記作用電極および前記対電極が接触される電解質媒体と  An electrolyte medium with which the working electrode and the counter electrode are contacted;
を備えた、測定用セル。  A measurement cell comprising:
[54] 前記作用電極と前記対電極との間に挿入されるスぺーサをさらに備えた、請求項 5 [54] The device according to claim 5, further comprising a spacer inserted between the working electrode and the counter electrode.
3に記載の測定用セル。 3. The measuring cell according to 3.
[55] 前記電解質媒体が前記作用電極と前記対電極との間に充填されてなる、請求項 555. The method according to claim 5, wherein the electrolyte medium is filled between the working electrode and the counter electrode.
3または 54に記載の測定用セル。 54. The measuring cell according to 3 or 54.
[56] 前記電解質媒体がリチウムイオンを含む、請求項 53〜56のいずれか一項に記載 の測定用セル。 [56] The measuring cell according to any one of [53] to [56], wherein the electrolyte medium contains lithium ions.
[57] 請求項 1〜40のいずれか一項に記載の方法に用いられる測定装置であって、 請求項 53〜56のいずれか一項に記載される測定用セルと、  [57] A measuring device used in the method according to any one of claims 1 to 40, wherein the measuring cell according to any one of claims 53 to 56,
前記作用電極の表面に光を照射する光源と、  A light source for irradiating light to the surface of the working electrode,
前記作用電極と前記対電極との間を流れる電流を測定する電流計と  An ammeter for measuring a current flowing between the working electrode and the counter electrode;
を備えた、測定装置。 A measuring device comprising:
[58] 前記光源が、増感色素の種類に応じて異なる波長の光を照射可能な複数の光源 を備えた、請求項 57に記載の装置。 58. The apparatus according to claim 57, wherein the light source includes a plurality of light sources capable of irradiating light having different wavelengths according to the type of the sensitizing dye.
[59] 前記光源が、波長選択手段をさらに備えてなり、増感色素の種類に応じて異なる波 長の光を照射可能とされてなる、請求項 57に記載の装置。 59. The apparatus according to claim 57, wherein the light source further comprises a wavelength selection means, and is capable of irradiating light having a different wavelength depending on the type of the sensitizing dye.
[60] 前記光源が、紫外線を実質的に含まない光を放出する光源である、請求項 57〜5 [60] The light source according to claim 57, wherein the light source emits light substantially free of ultraviolet rays.
9の 、ずれか一項に記載の装置。  Device according to any one of the preceding claims.
[61] 前記光源と前記作用電極との間に、紫外線を除去する手段をさらに備えた、請求 項 57〜59の!、ずれか一項に記載の装置。 [61] The apparatus according to any one of [57] to [57], further comprising a means for removing ultraviolet light between the light source and the working electrode.
[62] 前記紫外線を除去する手段が、光学フィルタまたは分光器である、請求項 61に記 載の装置。 62. The apparatus according to claim 61, wherein said means for removing ultraviolet light is an optical filter or a spectroscope.
[63] 前記光源が、レーザ、無機エレクト口ルミネッセンス (EL)素子、および有機エレクト 口ルミネッセンス (EL)素子力もなる群力も選択される少なくとも一つである、請求項 6 0〜62の!ヽずれか一項に記載の装置。  63. The misalignment according to claim 60, wherein the light source is at least one selected from the group consisting of a laser, an inorganic electroluminescent (EL) element, and an organic electroluminescent (EL) element. An apparatus according to claim 1.
[64] 前記光源が、発光ダイオード (LED)である、請求項 60〜62の!ヽずれか一項に記 載の装置。  [64] The apparatus according to any one of claims 60 to 62, wherein the light source is a light emitting diode (LED).
[65] 前記光源が、前記作用電極上を走査するための走査機構をさらに備えてなる、請 求項 57〜64の!、ずれか一項に記載の装置。  [65] The apparatus according to any one of claims 57 to 64, wherein the light source further comprises a scanning mechanism for scanning the working electrode.
[66] 前記電流計が、得られた電流量または電気量から試料液中の被検物質濃度を算 出する手段をさらに備えてなる、請求項 57〜65のいずれか一項に記載の装置。 [66] The apparatus according to any one of [57] to [65], wherein the ammeter further comprises means for calculating the concentration of the test substance in the sample solution from the obtained current amount or electric amount. .
[67] 請求項 1〜40のいずれか一項に記載の方法において用いられる、作用電極との接 触下で使用される緩衝溶液であって、 [67] A buffer solution used in contact with a working electrode, which is used in the method according to any one of claims 1 to 40,
カルボキシル基、リン酸基、およびアミノ基を含まない緩衝剤と、  A buffer not containing a carboxyl group, a phosphate group, and an amino group;
溶媒と  Solvent and
を含んでなる緩衝溶液。  A buffer solution comprising:
[68] 前記緩衝剤が、下記式 (I)： [68] The buffer according to the following formula (I):
[化 1]
Figure imgf000070_0001
[Chemical 1]
Figure imgf000070_0001
(式中、 R1はヒドロキシル基で置換されていてもよい、炭素数が 1〜4のアルキレン基 であり、 Xはスルホン酸基またはその塩であり、 Aは Oまたは YR2— N (ここで、 R2は R1 と同義であり、 Yはスルホン酸基もしくはその塩またはヒドロキシル基である） ) で表される化合物である、請求項 67に記載の緩衝溶液。 (Wherein, R 1 is an alkylene group having 1 to 4 carbon atoms, which may be substituted with a hydroxyl group, X is a sulfonic acid group or a salt thereof, and A is O or YR 2 — N (here The buffer solution according to claim 67, wherein R 2 has the same meaning as R 1 , and Y is a sulfonic acid group or a salt thereof or a hydroxyl group.
[69] 請求項 1および 3〜39のいずれか一項に記載される試料液の溶媒として使用され る、請求項 67または 68に記載の緩衝溶液。 [69] The buffer solution according to claim 67 or 68, which is used as a solvent for the sample solution according to any one of claims 1 and 3 to 39.
[70] 請求項 3〜40の 、ずれか一項に記載されるプローブ物質を含む溶液の溶媒として 使用される、請求項 67または 68に記載の緩衝溶液。 [70] The buffer solution according to claim 67 or 68, which is used as a solvent for a solution containing the probe substance according to any one of claims 3 to 40.
[71] 電解質をさらに含んでなり、請求項 1〜40のいずれか一項に記載される電解質媒 体として使用される、請求項 67または 68に記載の緩衝溶液。 [71] The buffer solution according to claim 67 or 68, further comprising an electrolyte, which is used as the electrolyte medium according to any one of claims 1 to 40.
[72] 請求項 4〜40のいずれか一項に記載される洗浄液として使用される、請求項 67ま たは 68に記載の緩衝溶液。 [72] The buffer solution according to claim 67 or 68, which is used as the washing solution according to any one of claims 4 to 40.
PCT/JP2005/005715 2004-03-26 2005-03-28 Method of specifically detecting test substance by using photocurrent and electrodes, measurement cell, measurement device and buffer solution to be used therefor WO2005093418A1 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501492B2 (en) 2009-03-18 2013-08-06 Toto Ltd. Measurement device used for specifically detecting substance to be examined using photocurrent, sensor unit used for same, and method for specifically detecting substance to be examined using photocurrent
JP5286786B2 (en) * 2005-11-16 2013-09-11 三菱瓦斯化学株式会社 Method for measuring the amount of double-stranded DNA and kit for measurement
CN107167443A (en) * 2017-05-31 2017-09-15 上海市环境科学研究院 A kind of method that utilization ultraviolet spectrometer detects PCB77
CN113311034A (en) * 2021-05-14 2021-08-27 江苏大学 Preparation method of in-situ ratio photoelectrochemical sensor for detecting Cry1Ab protein in transgenic crops
CN117645416A (en) * 2024-01-29 2024-03-05 潍坊市环境科学研究设计院有限公司 Preparation method of electrode material based on environmental estrogen for magnetic separation strategy and application of electrode material

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06186164A (en) * 1987-11-04 1994-07-08 Igen Inc Electrochemical luminescence rhenium moiety and using method thereof
JP2000510233A (en) * 1996-04-25 2000-08-08 ペンス，インコーポレイテッド Biosensor device and method
WO2002004935A1 (en) * 2000-07-06 2002-01-17 Asahi Kasei Kabushiki Kaisha Molecule detecting sensor
JP2003516165A (en) * 1999-12-09 2003-05-13 モトローラ・インコーポレイテッド Methods and compositions for electrical detection of nucleic acid reactions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06186164A (en) * 1987-11-04 1994-07-08 Igen Inc Electrochemical luminescence rhenium moiety and using method thereof
JP2000510233A (en) * 1996-04-25 2000-08-08 ペンス，インコーポレイテッド Biosensor device and method
JP2003516165A (en) * 1999-12-09 2003-05-13 モトローラ・インコーポレイテッド Methods and compositions for electrical detection of nucleic acid reactions
WO2002004935A1 (en) * 2000-07-06 2002-01-17 Asahi Kasei Kabushiki Kaisha Molecule detecting sensor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KON ET AL: "Shikiso no Intercalation o Riyo shita DNA no Hikari Denki Kagakuteki Kenshutsu.", THE CHEMICAL SOCIETY OF JAPAN KOEN YOKOSHU., vol. 79, no. 1, 2001, pages 8 *
NAKAMURA ET AL: "Koden Henkan ni yoru Atarashii DNA Nihonsa Kenshutsuho.", THE CHEMICAL SOCIETY OF JAPAN KOEN YOKOSHU., vol. 81, no. 2, 2002, pages 947 *
TAGAKI ET AL: "DEVELOPMENT OF A GENE DETECTION ASSAY BASED ON PHOTO-INDUCED ELECTION TRANSFER.", PEPTIDE SCIENCE., vol. 2003, no. 10, 10 March 2004 (2004-03-10), pages 453 - 454, XP009116342 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5286786B2 (en) * 2005-11-16 2013-09-11 三菱瓦斯化学株式会社 Method for measuring the amount of double-stranded DNA and kit for measurement
US8501492B2 (en) 2009-03-18 2013-08-06 Toto Ltd. Measurement device used for specifically detecting substance to be examined using photocurrent, sensor unit used for same, and method for specifically detecting substance to be examined using photocurrent
CN107167443A (en) * 2017-05-31 2017-09-15 上海市环境科学研究院 A kind of method that utilization ultraviolet spectrometer detects PCB77
CN107167443B (en) * 2017-05-31 2023-03-31 上海市环境科学研究院 Method for detecting PCB77 by using ultraviolet spectrometer
CN113311034A (en) * 2021-05-14 2021-08-27 江苏大学 Preparation method of in-situ ratio photoelectrochemical sensor for detecting Cry1Ab protein in transgenic crops
CN113311034B (en) * 2021-05-14 2023-04-11 江苏大学 Preparation method of photoelectrochemical sensor for detecting in-situ ratio of Cry1Ab protein in transgenic crops
CN117645416A (en) * 2024-01-29 2024-03-05 潍坊市环境科学研究设计院有限公司 Preparation method of electrode material based on environmental estrogen for magnetic separation strategy and application of electrode material
CN117645416B (en) * 2024-01-29 2024-04-02 潍坊市环境科学研究设计院有限公司 Preparation method of electrode material based on environmental estrogen for magnetic separation strategy and application of electrode material

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