WO2005093418A1 - Procede de detection specifique de substance de test a l'aide de courant photoelectrique et d'electrodes, cellule de mesure, dispositif de mesure et solution tampon destines a etre utilises a cet effet - Google Patents

Procede de detection specifique de substance de test a l'aide de courant photoelectrique et d'electrodes, cellule de mesure, dispositif de mesure et solution tampon destines a etre utilises a cet effet 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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WO
WIPO (PCT)
Prior art keywords
working electrode
substance
electrode
test substance
sensitizing dye
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PCT/JP2005/005715
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English (en)
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/ja
Application filed by Toto, Ltd. filed Critical Toto, Ltd.
Publication of WO2005093418A1 publication Critical patent/WO2005093418A1/fr

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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.

Abstract

La présente invention a trait à un procédé de détection et de quantification extrêmement sensible, appropriée et précise d'une substance de test présentant une capacité de liaison spécifique. Selon le procédé de l'invention, une solution d'échantillon contenant la substance de test est d'abord mise en contact, en présence d'un colorant sensibilisant, avec une électrode de travail comprenant un matériau de sonde apte à la liaison spécifique directe ou indirecte avec la substance de test à la surface, permettant ainsi la liaison spécifique directe ou indirecte de la substance de test au matériau de sonde, et la fixation du colorant sensibilisant à l'électrode de travail grâce à cette liaison. Ensuite, l'électrode de travail et une contre-électrode sont mises en contact avec un milieu électrolytique et l'électrode de travail est soumise à une photo-irradiation en vue d'induire la photoexcitation du colorant sensibilisant. Par la suite, un photocourant circulant entre l'électrode de travail et la contre-électrode provoqué par le transfert d'électrons depuis le colorant ainsi soumis à une photoexcitation vers l'électrode de travail est détecté.
PCT/JP2005/005715 2004-03-26 2005-03-28 Procede de detection specifique de substance de test a l'aide de courant photoelectrique et d'electrodes, cellule de mesure, dispositif de mesure et solution tampon destines a etre utilises a cet effet WO2005093418A1 (fr)

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CN107167443A (zh) * 2017-05-31 2017-09-15 上海市环境科学研究院 一种利用紫外光谱仪检测pcb77的方法
CN113311034A (zh) * 2021-05-14 2021-08-27 江苏大学 一种检测转基因作物中Cry1Ab蛋白的原位比率光电化学传感器的制备方法
CN117645416A (zh) * 2024-01-29 2024-03-05 潍坊市环境科学研究设计院有限公司 基于磁分离策略用环境雌激素的电极材料的制备方法及电极材料应用

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Publication number Priority date Publication date Assignee Title
JP5286786B2 (ja) * 2005-11-16 2013-09-11 三菱瓦斯化学株式会社 二重鎖ｄｎａ量の測定方法および測定用キット
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 (zh) * 2017-05-31 2017-09-15 上海市环境科学研究院 一种利用紫外光谱仪检测pcb77的方法
CN107167443B (zh) * 2017-05-31 2023-03-31 上海市环境科学研究院 一种利用紫外光谱仪检测pcb77的方法
CN113311034A (zh) * 2021-05-14 2021-08-27 江苏大学 一种检测转基因作物中Cry1Ab蛋白的原位比率光电化学传感器的制备方法
CN113311034B (zh) * 2021-05-14 2023-04-11 江苏大学 一种检测转基因作物中Cry1Ab蛋白的原位比率光电化学传感器的制备方法
CN117645416A (zh) * 2024-01-29 2024-03-05 潍坊市环境科学研究设计院有限公司 基于磁分离策略用环境雌激素的电极材料的制备方法及电极材料应用
CN117645416B (zh) * 2024-01-29 2024-04-02 潍坊市环境科学研究设计院有限公司 基于磁分离策略用环境雌激素的电极材料的制备方法及电极材料应用

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