WO2010024446A1 - 光電流による被検物質の特定的検出に用いられる電極部材 - Google Patents
光電流による被検物質の特定的検出に用いられる電極部材 Download PDFInfo
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- WO2010024446A1 WO2010024446A1 PCT/JP2009/065246 JP2009065246W WO2010024446A1 WO 2010024446 A1 WO2010024446 A1 WO 2010024446A1 JP 2009065246 W JP2009065246 W JP 2009065246W WO 2010024446 A1 WO2010024446 A1 WO 2010024446A1
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- Y02P70/50—Manufacturing or production processes characterised by the final manufactured product
Definitions
- the present invention relates to an electrode member used for specific detection of a test substance having specific binding properties such as a nucleic acid, an exogenous endocrine disrupting substance, and an antigen using a photocurrent, and a method for producing the same.
- the photocurrent generated by photoexcitation of the sensitizing dye can be used to detect test substances (biomolecules such as DNA and proteins).
- test substances biomolecules such as DNA and proteins.
- Proposals for use have been made (see, for example, Japanese Patent Application Laid-Open No. 2002-181777 (Patent Document 1), Japanese Patent Application Laid-Open No. 2005-251426 (Patent Document 2), and Japanese Patent Application Publication No. 2006-507491 (Patent Document 3).
- a current generated by irradiating excitation light to a dye immobilized on an electrode is measured, and the amount of dye bonded from the detected amount of current is measured. Therefore, compared with the conventional method of detecting fluorescence as an image, the apparatus can be miniaturized, and the test substance can be detected to some extent simply.
- Nucleic acids, exogenous endocrine disruptors, antigens and other substances are generally present in low concentrations in the sample.
- an electrode used for detection of a test substance by photocurrent is ideally high in light transmittance, biomolecule loading and photoelectron conversion efficiency.
- simply using a working electrode having such characteristics does not necessarily provide a sufficient measured value in the measurement of a test substance in a low concentration range. There wasn't.
- the present inventors have recently used a semiconductor and a test substance having specific binding properties with higher sensitivity by using a semiconductor and another kind of substance on the surface of the semiconductor as an electron accepting substance in the electrode member. It was detected that it was possible to quantitate with higher accuracy.
- the present invention is based on such knowledge.
- an object of the present invention is to provide an electrode member that can detect a test substance having specific binding properties with higher sensitivity and quantify it with higher accuracy.
- the electrode member according to the present invention is an electrode member used for specific detection of a test substance by photocurrent, and has at least a conductive base material and an electron accepting substance on the conductive base material.
- the electron-accepting material is a first material layer made of a semiconductor, and a second material made of a semiconductor or a metal or metal oxide of a different type from the semiconductor, supported on the surface of the first material layer. And at least.
- the detection sensitivity of a test substance at a low concentration can be improved, and the measurement accuracy can be improved.
- FIG. 4 is a diagram showing photocurrent values obtained in Example 1.
- 6 is a diagram showing photocurrent values obtained in Example 2.
- FIG. FIG. 6 is a diagram showing photocurrent values obtained in Example 4.
- FIG. 10 is a diagram showing photocurrent values obtained in Example 5.
- 4 is a TEM analysis photograph of the ZnO sputter electrode obtained in Example 6 at a magnification of 450,000 times.
- 7 is a TEM analysis photograph of the ZnO sputter electrode obtained in Example 6 at a magnification of 1.8 million.
- 7 is a TEM analysis photograph of the ZnO nitric acid-treated electrode obtained in Example 6 at a magnification of 450,000 times.
- 4 is a TEM analysis photograph of the ZnO nitric acid treated electrode obtained in Example 6 at a magnification of 1.8 million.
- a specific detection method of a test substance using a photocurrent includes a sample liquid containing a test substance and a probe substance that can bind directly or indirectly to the test substance.
- a working electrode provided on the surface and a counter electrode are prepared, and in the presence of a sensitizing dye, the sample liquid is brought into contact with the working electrode, and the test substance is directly or indirectly specific to the probe substance. Bonding, fixing the sensitizing dye to the working electrode, bringing the working electrode and the counter electrode into contact with an electrolyte medium, and irradiating the working electrode with light to irradiate the working electrode and the counter electrode Detecting photocurrent flowing between the electrodes.
- Electrode Member The electrode member according to the present invention is used as a working electrode by immobilizing a test substance on the electrode surface in the above-described specific detection method of the test substance by photocurrent.
- Conductive substrate As the conductive substrate constituting the electrode member according to the present invention, not only a conductive material but also a conductive material is supported on the surface of a non-conductive support such as glass or plastic. It is also possible to have a configuration that ensures conductivity.
- conductive materials include metals such as platinum, gold, silver, copper, aluminum, rhodium, and indium; conductive ceramics such as carbon, carbide, and nitride; fluorine on indium and indium-tin tin composite oxides and tin oxide Conductive metal oxides such as those doped, tin oxide doped antimony, zinc oxide doped gallium, or zinc oxide doped aluminum, more preferably indium-tin It is a composite oxide (I T O) and a metal oxide (F T O) in which tin oxide is doped with fluorine.
- metals such as platinum, gold, silver, copper, aluminum, rhodium, and indium
- conductive ceramics such as carbon, carbide, and nitride
- Conductive metal oxides such as those doped, tin oxide doped antimony, zinc oxide doped gallium, or zinc oxide doped aluminum, more preferably in
- the electron accepting substance itself also functions as a conductive base material
- the conductive base material includes a thin film-like or spot-like shape having no strength as a support itself.
- the conductive substrate is substantially transparent, specifically, the light transmittance is preferably 10% or more, more preferably 50% or more, and still more preferably. 70% or more.
- the cell is configured such that light is irradiated from the back side of the working electrode (ie, the conductive substrate), and the light transmitted through the working electrode (ie, the conductive substrate and the electron accepting layer) excites the sensitizing dye. can do.
- the thickness of the conductive equipment is preferably about 0.02 to 10 ⁇ m.
- the surface resistance of an electroconductive base material is 100 ohm / cm ⁇ 2 > or less, More preferably, it is 40 ohm / cm ⁇ 2 > or less.
- the lower limit of the surface resistance of the conductive substrate is not particularly limited, but will usually be about 0.1 ⁇ / cm 2 .
- Electron-accepting material comprises an electron-accepting material placed on the conductive substrate, the electron-accepting material comprising a layer of a first material made of a semiconductor and a layer of the first material. It is comprised at least from the semiconductor of the kind different from the said semiconductor supported on the surface of the layer, or the 2nd substance which consists of a metal or a metal oxide.
- the first substance is a semiconductor, more preferably an oxide semiconductor, still more preferably a metal oxide semiconductor, and most preferably an n-type metal oxide semiconductor.
- the semiconductor may have a structure such as a porous body or an uneven surface shape, whereby a working electrode having a large surface area can be produced, and the amount of immobilized probes is increased. The advantage of being able to be made is obtained.
- the electron acceptor in the present invention comprises an electron acceptor capable of accepting electrons emitted by a sensitizing dye fixed via a probe substance in response to photoexcitation. That is, the electron-accepting substance is a substance that can take an energy level at which electrons can be injected from the photoexcited labeling dye.
- the energy level (A) capable of injecting electrons from the photoexcited labeling dye means, for example, a conduction band (conduction band: CB) when a semiconductor is used as the electron-accepting material.
- CB conduction band
- a metal is used as the electron-accepting material, it means the Fermi level.
- the level of A is a level that is lower than the energy level of the lowest unoccupied molecular orbital (LUMO) of the sensitizing dye, in other words, Any material having an energy level lower than the LUMO energy level of the sensitizing dye may be used.
- LUMO lowest unoccupied molecular orbital
- Preferred examples of the electron accepting material include simple semiconductors such as silicon and germanium; oxides such as titanium, tin, zinc, iron, tungsten, zirconium, hafnium, strontium, indium, cerium, yttrium, lanthanum, vanadium, niobium, and tantalum.
- Perovskite type semiconductors such as strontium titanate, calcium titanate, sodium titanate, barium titanate, potassium niobate; sulfide semiconductors of cadmium, zinc, lead, silver, antimony, bismuth; cadmium, lead selenide semiconductors Cadmium telluride semiconductors; phosphide semiconductors such as zinc, gallium, indium and cadmium; gallium arsenide, copper-indium selenide, copper-indium-sulfide compound semiconductors, and more preferably silicon Emissions, TiO 2, SnO 2, Fe 2 O 3, WO 3, ZnO, Nb 2 O 5, strontium titanate, indium oxide, CdS, ZnS, PbS, Bi 2 S 3, CdSe, CdTe, GaP, InP, GaAs CuInS 2 , CuInSe, C60, more preferably TiO 2 , ZnO, SnO 2 , Fe 2 O 3 , WO
- the potential of the conduction band of the semiconductor is preferably lower than the LUMO potential of the sensitizing dye, more preferably the LUMO of the sensitizing dye> the conduction band of the semiconductor> the redox of the electrolyte.
- ITO indium-tin composite oxide
- FTO fluorine-doped tin oxide
- ITO and FTO have the property of functioning not only as an electron accepting substance but also as a conductive base material. Therefore, when these materials are used, it is possible to constitute a conductive electron accepting layer. Yes, it can be used without introducing a conductive substrate.
- the second substance constituting the electron-accepting material of the electrode member according to the present invention comprises a semiconductor of a different type from the semiconductor of the first substance, or a metal or metal oxide.
- Specific examples of the semiconductor constituting the second substance include those exemplified as the semiconductor of the first substance.
- Examples of the metal include gold, platinum, silver, copper, aluminum, rhodium, indium and nickel.
- the metal includes not only a so-called narrow metal having a metal bond but also a “metal element”.
- Non-semiconductor oxides such as titanium, tin, zinc, iron, tungsten, zirconium, hafnium, strontium, indium, cerium, yttrium, lanthanum, vanadium, niobium, and tantalum are used as the metal oxide constituting the second substance. Is mentioned.
- This second substance is supported on the surface of the first substance layer.
- “supported on the surface” is not particularly limited as long as the layer of the first substance and the second substance exist in electrical or physical contact with each other. It may be in a form that is formed, or may be placed on a part of the surface of the layer of the first substance so as to partially cover the surface. In the latter case, the second substance may be formed by applying it to the surface of the layer of the first substance and then removing it partially. Furthermore, the present invention includes a presence form in which the second substance is localized on the surface of the layer of the first substance in the form of particles.
- the surface element ratio (substance ratio) of the metal element constituting the second substance to the metal element constituting the first substance is more than 0 and less than 1, more preferably this The surface element ratio is more than 0 and less than 0.27, more preferably more than 0 and less than 0.15, and most preferably more than 0 and less than 0.10.
- the measurement of the surface existing element ratio can be obtained by analysis using X-ray photoelectron analysis.
- the coating thickness of the second substance is preferably more than 0 and less than 2 nm.
- a mode in which the first substance is detected in a region having a certain depth from the surface of the layer of one substance is also included.
- the second substance is preferably present in a region having a depth of 4 nm inward from the surface of the first substance, and more preferably in a region of 2 nm or less.
- the semiconductor or metal or metal oxide as the electron accepting substance may be either single crystal or polycrystalline.
- Manufacturing method of electrode member As a preferable method of forming an electron accepting material on a conductive substrate, a dispersion or colloidal solution of an electron accepting material is applied on a conductive support, and a precursor of semiconductor fine particles is made conductive. Examples of the method include a method of obtaining a fine particle film by applying on a 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 material includes a method of grinding with a mortar, a method of dispersing while grinding using a mill, or a method of synthesizing a semiconductor. Examples thereof include a method in which it is precipitated as fine particles in a solvent and used as it is.
- the dispersion medium include water or various organic solvents (for example, methanol, ethanol, isopropyl alcohol, dichloromethane, acetone, acetonitrile, ethyl acetate, etc.).
- a polymer, a surfactant, an acid, a chelating agent, or the like may be used as a dispersion aid as necessary.
- Preferred examples of the application method of the electron acceptor dispersion or colloidal solution include the roller method as the application system, the dip method, the air knife method as the metering system, the blade method, etc., and the application and metering in the same part.
- the thickness of the semiconductor of the first material is preferably 0.1 to 200 ⁇ m, more preferably 0.8. It is 1 to 100 ⁇ m, more preferably 1 to 30 ⁇ m, and most preferably 2 to 25 ⁇ m.
- the electron accepting material comprises indium-tin composite oxide (ITO) or metal oxide (FTO) in which tin oxide is doped with fluorine (FTO)
- the thickness of the electron accepting material layer Is preferably 1 nm or more, more preferably 10 nm to 1 ⁇ m.
- the method of supporting the second substance on the surface of the layer of the first substance may be appropriately determined according to the method for forming the first substance, and for example, a vapor deposition method can be used.
- the electrode member according to another preferred embodiment of the present invention is manufactured by removing a part of the second material deposited on the surface of the first material layer.
- Examples of the method for removing the second metal or metal oxide include physical removal and chemical removal.
- the physical removal method is a method using heat, ultrasonic waves, electrochemical removal, removal by a seal or the like.
- the chemical removal is removal using dissolution with an acid, an alkaline solution or a chemical.
- the second substance is zinc oxide
- an acidic solution for example, nitric acid, hydrochloric acid, acetic acid, hydrogen peroxide solution, sulfuric acid, organic sulfonic acid, citric acid solution, etc., and buffers adjusted to a pH of less than 2.9 such as succinic acid buffer and acetic acid buffer are also used. I can do it.
- it is possible to shorten the treatment time by preferably setting the pH of the acidic solution to less than 2.9, more preferably 2.5 or less. At this time, the immersion time of the electrode in the acidic solution may be 1 minute or longer.
- Test substance The test substance in the present invention is not particularly limited as long as it is a substance having specific binding properties with the probe substance.
- a probe substance capable of specifically binding directly or indirectly to a test substance is supported on the surface of the electrode member, preferably an electron accepting substance, so that the test substance is probed. It becomes possible to detect by binding specifically to a substance directly or indirectly.
- the electrode member used in the present invention is preferably an electrode having on its surface a probe material that can be directly or indirectly bonded to the test substance.
- Suitable probe substances include biomolecules. That is, the probe substance specifically binds not only to a substance that directly binds specifically to the test substance, but also to a conjugate obtained by specifically binding the test substance to a mediator such as a receptor protein molecule. It may be a possible substance.
- the sample solution is brought into contact with the working electrode in the presence of the sensitizing dye, and the test substance is directly or indirectly specifically bound to the probe substance, and the sensitizing dye is fixed to the working electrode by this binding.
- the sensitizing dye is a substance that can emit electrons to the working electrode in response to photoexcitation.
- the probe substance is the primary antibody and the sensitizing dye is the secondary antibody.
- the sensitizing dye is labeled with a second test substance that can specifically bind to the probe substance.
- the test substance and the probe substance that can specifically bind to each other can be selected. That is, according to a preferred embodiment of the present invention, it is preferable that a substance having specific binding properties be a test substance and a substance that specifically binds to the test substance be carried on the working electrode as a probe substance. As a result, the test substance can be directly bound and detected on the working electrode.
- preferred examples of the combination of the test substance and the probe substance include a single-stranded nucleic acid and a single-stranded nucleic acid combination complementary to the nucleic acid, an antigen and an antibody, and a receptor protein and a ligand. The combination of these is mentioned.
- 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 specific binding properties is used as a test substance, a substance that specifically binds to the test substance is used as a mediator, and It is preferable to carry a substance capable of binding to the working electrode as a probe substance. As a result, even a substance that cannot specifically bind to the probe substance can be detected by specifically binding indirectly to the working electrode via the mediator substance.
- preferred examples of the combination of the test substance, the mediator, and the probe substance include a ligand, a receptor protein molecule that can accept the ligand, and two molecules that can specifically bind to the receptor protein molecule.
- a combination of nucleic acids in a strand is mentioned.
- the ligand include exogenous endocrine disrupting substances (environmental hormones).
- An exogenous endocrine disrupting substance is a substance that binds to DNA or a peptide through a receptor protein molecule and produces a toxicity by affecting its gene expression. According to the method of the present invention, It is possible to easily monitor the binding of a protein such as a receptor to DNA or peptide.
- the loading of the probe substance on the electrode member can be performed according to a known method.
- a single-stranded nucleic acid when used as a probe substance, it can be carried out by binding a nucleic acid probe directly or indirectly to the working electrode surface.
- the nucleic acid probe into which the functional group has been introduced can be immobilized on the carrier as it is by an immobilization reaction.
- Introduction of a functional group at the end of a nucleic acid can be performed using an enzymatic reaction or a DNA synthesizer.
- an amino group, a carboxyl group, a thiol group, a hydroxyl group, a phosphate group, or a diol group can be suitably used as a functional group for immobilizing the probe substance to the working electrode.
- a material that crosslinks between the working electrode and the probe substance can also be used in order to firmly fix the probe substance to the working electrode.
- a crosslinking material include silane coupling agents, titanate coupling agents, and conductive polymers such as polythiophene, polyacetylene, polypyrrole, and polyaniline.
- the probe substance can be immobilized efficiently by a simple operation called physical adsorption.
- the physical adsorption of the probe substance on the electrode surface can be performed, for example, as follows. First, the electrode surface is cleaned with ultrapure water and acetone using an ultrasonic cleaner. Thereafter, a buffer solution containing the probe substance is dropped on the electrode, and after standing, the probe substance is adsorbed on the electrode surface by washing.
- the detection apparatus basically has the configuration shown in FIG. That is, the counter electrode 2 was fixed to the opposing member 1, and the electrode member according to the present invention was placed as the working electrode 4 through the electrolyte pad 3. Further, light was applied to the working electrode by the light source 5, and laser was applied to each spot on the electrode by the electrode unit 6 to obtain a photocurrent.
- Example 1 Specific detection of proteins by photocurrent using different working electrodes
- Fluorine-doped tin oxide (F-SnO 2 : FTO) coated glass manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12 ⁇ / cm 2 , shape: 50 mm ⁇ 26 mm
- ZnO / FTO electrode ZnO is 0.44 nm (sputtering time 20 seconds, 50 W, sputter rate 1.3 nm / min), 1.08 nm (sputtering time 50 seconds, 50 W, sputter rate 1) on the above FTO electrode by sputtering. 3 nm / min)
- ZnO / FTO * electrode An electrode was prepared by depositing ZnO to a thickness of 50 nm (200 W, sputtering time: 8 minutes, sputtering rate: 6.25 nm / min) on the above FTO electrode by sputtering. Approximate). This electrode was subjected to ultrasonic cleaning for 1 minute in order of acetone, ultrapure water, and acetone, immersed in a 1M nitric acid solution (pH 0.2), and shaken for 5 minutes. Thereafter, it was sufficiently rinsed with ultrapure water to make this electrode ZnO / FTO * .
- the working electrode prepared by immobilizing the probe protein was subjected to ultrasonic cleaning for 1 minute each in the order of acetone, ultrapure water, and acetone. Thereafter, an adhesive seal (thickness: 0.5 mm) in which an opening having a diameter of 3 mm was formed was placed and adhered.
- a goat-derived antibody (anti-luciferase polyclonal antibody: manufactured by Promega) was prepared at 10 ⁇ g / ml.
- the solvent used was a 10 mM phosphate buffer (pH 7) containing 250 mM NaCl and 0.05% Tween20. 5 ⁇ l each of this protein solution was dropped into the opening by the seal on the electrode and incubated at 37 ° C. for 30 minutes. Thereafter, the electrode was washed by shaking in ultrapure water for 10 minutes.
- Binding reaction dye-labeled antigen of test protein (Cy5-anti-goat antibody (rabbit): manufactured by Chemicon) was prepared at 100 ng / ml. At this time, as a solvent, a 10 mM phosphate buffer (pH 7) containing 250 mM NaCl and 0.05% Tween 20 was used. 5 ⁇ l of the prepared antigen solution was dropped into the seal opening on the working electrode on which the probe protein had previously been immobilized, and incubated at 37 ° C. for 1 hour. Thereafter, the seal on the electrode was peeled off, and the surface was washed with ultrapure water.
- test substance binding working electrode prepared by the above-described method and a counter electrode formed by depositing platinum on a glass plate were prepared.
- An electrolyte sheet containing an electrolytic solution (0.4M tetrapropylammonium iodide) was sandwiched between both electrodes and adhered.
- the surface of the working electrode on which the protein was immobilized and the platinum deposition surface of the counter electrode were arranged to face each other.
- the working electrode was irradiated with a laser light source (output 120 mW, diameter of the irradiated region 1 mm, wavelength 650 nm red laser), and the current value observed at that time was recorded. The result was as shown in FIG.
- Example 2 Partial removal by nitric acid treatment Production of electrode As a substrate for working electrode, fluorine-doped tin oxide (F-SnO2: FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12 ⁇ / cm 2 , shape: 50 mm ⁇ 26 mm) Prepared.
- An electrode (ZnO / FTO) in which ZnO was sputtered to a thickness of 50 nm was prepared by the method described in Example 1. The electrode was subjected to ultrasonic cleaning for 1 minute in order of acetone, ultrapure water, and acetone, and then immersed in a nitric acid solution adjusted to each concentration.
- Nitric acid was prepared to the following 8 pH (concentration). Table 1 shows the immersion time of the prepared nitric acid solution and the electrode. After dipping in each solution, the electrode was thoroughly rinsed and washed with ultrapure water.
- An adhesive seal (thickness: 0.5 mm) having an opening with a diameter of 3 mm was placed on and brought into close contact with the electrode produced by immunoassay .
- Cy5-labeled anti-goat antibody prepared in 10 ng / ml, 100 ng / ml, 1 ⁇ g / ml (10 mM phosphate buffer, 0.05% Tween 20, 250 mM NaCl) was added dropwise to the seal opening at 60 ° C. at 60 ° C. Incubation was performed for a minute.
- Example 3 Surface elemental analysis of electrode ZnO / FTO electrode (ZnO film thickness 0.44 nm, 1.03 nm, 50 nm) prepared in Example 1 and ZnO / FTO * electrode (nitric acid treatment concentration: 1 M, 5 mM, 3.2 mM) 2 mM, 0.1 mM) and a total of nine types of FTO electrodes were subjected to surface elemental analysis using X-ray photoelectron analysis. The analysis was performed using a PHI1800 type X-ray photoelectron spectrometer manufactured by ULVAC-PHI under the conditions of an X-ray source MgK ⁇ (100 w) analysis region of 0.8 ⁇ 2.0 mm. The results were as shown in Table 2.
- Example 4 Specific detection of protein by photocurrent using nitric acid-treated electrodes on various sputter surfaces Production of working electrode Fluorine-doped tin oxide (F-SnO 2 : FTO) coated glass as a substrate for FTO electrode working electrode (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12 ⁇ / cm 2 , shape: 50 mm ⁇ 26 mm) Prepared.
- F-SnO 2 Fluorine-doped tin oxide
- FTO Fluorine-doped tin oxide
- ITO / FTO electrode An electrode (thickness was estimated from the sputtering rate) was prepared by depositing ITO on the FTO electrode to a thickness of 50 nm (sputtering time 8 minutes, 100 W, sputtering rate 6.25 nm / min) by sputtering.
- WO 3 / FTO electrode Prepare an electrode (film thickness is estimated from the sputtering rate) by depositing WO 3 to 50 nm (sputtering time 8 minutes, 100 W, sputtering rate 6.25 nm / min) on the above FTO electrode by sputtering. did.
- SrTiO 3 / FTO electrode An electrode (the film thickness is estimated from the sputtering rate) on which the SrTiO 3 film is deposited to 50 nm (sputtering time 8 minutes, 100 W, sputtering rate 6.25 nm / min) on the above FTO electrode by sputtering. did.
- ITO / FTO, WO 3 / FTO, and SrTiO 3 / FTO electrodes were each ultrasonically cleaned in the order of acetone, ultrapure water, and acetone for 1 minute each to give a 1M nitric acid solution (pH 0.2). And was shaken for 15 minutes. Sufficiently rinsed with subsequent ultra-pure water, the electrode respectively ITO / FTO *, WO 3 / FTO *, and the SrTiO 3 / FTO *.
- the working electrode prepared by immobilizing the probe protein was subjected to ultrasonic cleaning for 1 minute each in the order of acetone, ultrapure water, and acetone. Thereafter, an adhesive seal (thickness: 0.5 mm) in which an opening having a diameter of 3 mm was formed was placed and adhered.
- a goat-derived antibody (anti-luciferase polyclonal antibody: manufactured by Promega) was prepared at 10 ⁇ g / ml.
- the solvent used was a 10 mM phosphate buffer (pH 7) containing 250 mM NaCl and 0.05% Tween20. 5 ⁇ l each of this protein solution was dropped into the opening by the seal on the electrode and incubated at 37 ° C. for 30 minutes. Thereafter, the electrode was washed by shaking in ultrapure water for 10 minutes.
- Binding reaction dye-labeled antigen of test protein (Cy5-anti-goat antibody (rabbit): manufactured by Chemicon) was prepared at 100 ng / ml. At this time, as a solvent, a 10 mM phosphate buffer (pH 7) containing 250 mM NaCl and 0.05% Tween 20 was used. 5 ⁇ l of the prepared antigen solution was dropped into the seal opening on the working electrode on which the probe protein had previously been immobilized, and incubated at 37 ° C. for 1 hour. Thereafter, the seal on the electrode was peeled off, and the surface was washed with ultrapure water.
- test substance binding working electrode prepared by the above-described method and a counter electrode formed by depositing platinum on a glass plate were prepared.
- An electrolyte sheet containing an electrolytic solution (0.4M tetrapropylammonium iodide) was sandwiched between both electrodes and adhered.
- the surface of the working electrode on which the protein was immobilized and the platinum deposition surface of the counter electrode were arranged to face each other.
- the working electrode was irradiated with a laser light source (output 120 mW, diameter of the irradiated region 1 mm, wavelength 650 nm red laser), and the current value observed at that time was recorded. The result was as shown in FIG. From these results, it was shown that even when ITO, WO 3 , or SrTiO 3 was used as the second substance, specific detection of the test protein by photocurrent was possible after the nitric acid treatment.
- Example 5 Specific detection of a test substance by sandwich immunoassay using a prepared electrode Production of working electrode Fluorine-doped tin oxide (F-SnO 2 : FTO) coated glass as a substrate for FTO electrode working electrode (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12 ⁇ / cm 2 , shape: 50 mm ⁇ 26 mm) Prepared.
- F-SnO 2 Fluorine-doped tin oxide
- FTO Fluorine-doped tin oxide
- ZnO / FTO electrode An electrode (thickness was estimated from the sputtering rate) was prepared by depositing ZnO at a thickness of 50 nm (sputtering time 8 minutes, 100 W, sputtering rate 6.25 nm / min) on the FTO electrode.
- the above ZnO / FTO electrode was subjected to ultrasonic cleaning for 1 minute each in the order of acetone, ultrapure water, and acetone, immersed in 1M nitric acid solution (pH 0.2) and shaken for 15 minutes. Thereafter, it was sufficiently rinsed with ultrapure water to make this electrode ZnO / FTO * .
- the working electrode prepared by immobilizing the probe protein was subjected to ultrasonic cleaning for 1 minute each in the order of acetone, ultrapure water, and acetone. Thereafter, an adhesive seal (thickness: 0.5 mm) in which an opening having a diameter of 3 mm was formed was placed and adhered.
- Anti-Prostate Specific antigen antibody (Fitzgerald) was prepared at 15 ⁇ g / ml. At this time, 10 mM phosphate buffer (pH 7.4) was used as a solvent. 5 ⁇ l each of the antibody solution was dropped into the opening by the electrode field seal and incubated at 37 ° C. for 10 minutes. Thereafter, the electrode was washed by shaking in 10 mM phosphate buffer for 10 minutes.
- Test Protein Binding Reaction Cy5-labeled anti-Prostate Specific Antigen antibody (manufactured by Biodesign) was prepared at 5 ⁇ g / ml. At this time, 10 mM phosphate buffer (pH 7), 150 mM NaCl, 0.05% Tween 20 was used as the solvent. Antigen (Prostate Specific Antigen) is added to the prepared antigen solution at each concentration, and after sufficient agitation by pipetting, 5 ⁇ l is added dropwise at 37 ° C. to the seal opening on the working electrode to which the probe protein has been immobilized previously. Incubation was for 10 minutes. Thereafter, the seal on the electrode was removed, and the surface was washed with a buffer solution and ultrapure water.
- test substance binding working electrode prepared by the above-described method and a counter electrode formed by depositing platinum on a glass plate were prepared.
- An electrolyte sheet containing an electrolytic solution (0.4M tetrapropylammonium iodide) was sandwiched between both electrodes and adhered.
- the surface of the working electrode on which the protein was immobilized and the platinum deposition surface of the counter electrode were arranged to face each other.
- the working electrode was irradiated with a laser light source (output 120 mW, diameter of the irradiated region 1 mm, wavelength 650 nm red laser), and the current value observed at that time was recorded. The result was as shown in FIG. From this result, it was shown that a test substance can be detected using a sandwich immunoassay by photocurrent.
- Example 6 Analysis of ZnO nitric acid-treated electrode by TEM
- the TEM and EDS analysis of the ZnO sputter electrode and the ZnO nitric acid-treated electrode prepared by the method shown in Example 1 were performed.
- TEM analysis a JEM-2010F field emission type transmission electron microscope manufactured by JEOL Ltd. was used, and measurement was performed at an acceleration voltage of 200 kV.
- EDS analysis a Nolan UTW type Si (Li) semiconductor detector was used, and an analysis region of 1 nm was analyzed.
- the TEM observation photograph of the ZnO sputter electrode was as shown in FIG. 6 and FIG.
- ZnO / FTO a ZnO layer of about 50 nm was observed on the FTO layer.
- Zn was observed on the FTO layer side (spots 3, 4, 7) of the ZnO layer / FTO layer interface.
- spot 5 is considered to be a Zn concentration near the detection limit of EDS, which is difficult to discuss, but a slight Zn-L peak is observed and Zn may exist.
- Zn was not observed within about 10 nm from the interface (spot 6).
- a TEM observation photograph of the ZnO nitric acid treated electrode was taken. It was as shown in FIG. 8 and FIG. No special layers such as the remainder of the ZnO layer were observed on the FTO layer surface. From the EDS spectrum, Zn may exist on the surface (spots 1 and 4) of the FTO layer. Zn was not observed inside the approximately 2 nm FTO layer (spot 2). Similarly, there is a possibility that Zn exists in the grain boundary (spot 5), and no Zn was observed inside the approximately 10 nm FTO layer (spot 6).
- the Zn concentration is below the detection limit (% order) of EDS. From the above analysis, it is considered that Zn is diffused by about 4 nm to the FTO layer side of the ZnO layer / FTO layer interface at the ZnO sputter electrode. It is done. On the other hand, in the nitric acid-treated electrode, there is a possibility that Zn exists in the portion of the surface of the FTO layer less than 2 nm. From this, it is presumed that the Zn diffusion portion on the surface of the FTO layer is removed together with the ZnO layer of about 50 nm by the nitric acid treatment.
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Abstract
Description
光電流を用いた被検物質の特異的検出方法は、被検物質を含む試料液と該被検物質と直接または間接的に結合可能なプローブ物質を表面に備えた作用電極と、対極とを用意し、増感色素の共存下、前記試料液を前記作用電極に接触させて、前記プローブ物質に前記被検物質を直接又は間接的に特異的に結合させ、該結合により前記増感色素を前記作用電極に固定させ、前記作用電極と前記対電極とを電解質媒体に接触させ、そして、前記作用電極に光を照射して前記作用電極と前記対電極との間に流れる光電流を検出することを含んでなるものである。
本発明による電極部材は上記光電流による被検物質の特異的検出法において、被検物質を電極表面に固定化することで、作用電極として用いられる。
本発明による電極部材を構成する導電性基材としては、導電性を有する材料のみならず、ガラス、プラスチックのような非導電性の支持体の表面に導電性を有する材料を担持させ導電性を確保した構成のものであってもよい。
本発明による電極部材は、上記導電性基材上に置かれた電子受容物質を有してなり、この電子受容物質は、半導体からなる第一物質の層と、この第一物質の層の表面に担持された、前記半導体とは異なる種類の半導体または金属もしくは金属酸化物からなる第二物質とから少なくとも構成される。
導電性基材上への電子受容物質の好ましい形成方法としては、電子受容物質の分散液またはコロイド溶液を導電性支持体上に塗布する方法、半導体微粒子の前駆体を導電性支持体上に塗布し空気中の水分によって加水分解して微粒子膜を得る方法(ゾル-ゲル法)、スパッタリング法、CVD法、PVD法、蒸着法などが挙げられる。電子受容物質としての半導体微粒子の分散液を作製する方法としては、前述のゾル-ゲル法の他、乳鉢ですり潰す方法、ミルを使って粉砕しながら分散する方法、あるいは半導体を合成する際に溶媒中で微粒子として析出させそのまま使用する方法等が挙げられる。このときの分散媒としては水または各種の有機溶媒(例えばメタノール、エタノール、イソプロピルアルコール、ジクロロメタン、アセトン、アセトニトリル、酢酸エチル等)が挙げられる。分散の際、必要に応じてポリマー、界面活性剤、酸、もしくはキレート剤などを分散助剤として使用してもよい。
本発明における被検物質としては、プローブ物質と特異的な結合性を有する物質であれば特に限定はない。本発明による電極部材にあっては、被検物質と直接または間接的に特異的に結合可能なプローブ物質を電極部材表面、好ましくは電子受容物質に担持させておくことにより、被検物質をプローブ物質に直接または間接的に特異的に結合させて検出することが可能となる。
本発明に用いる電極部材は、被検物質と直接または間接的に結合が可能なプローブ物質を表面に備えた電極とされることが好ましい。好適なプローブ物質としては生体分子が挙げられる。すなわち、プローブ物質は、被検物質と直接、特異的に結合する物質のみならず、被検物質を受容体蛋白質分子等の媒介物質に特異的に結合させて得られる結合体と特異的に結合可能な物質であってよい。ついで、増感色素の共存下、試料液を作用電極に接触させて、プローブ物質に被検物質を直接または間接的に特異的に結合させ、この結合により増感色素を作用電極に固定させる。増感色素は、光励起に応じて作用電極に電子を放出可能な物質であり、被検物質の特異的結合法がサンドイッチ法である場合、プローブ物質は1次抗体で、増感色素は2次抗体に標識されてなり、被検物質の特異的検出法が競合法である場合、増感色素はプローブ物質に特異的に結合可能な第二の被検物質に標識される。
作用電極の作製
FTO電極
作用電極用の基材として、フッ素をドープした酸化スズ(F-SnO2:FTO)コートガラス(エイアイ特殊硝子社製、U膜、シート抵抗:12Ω/cm2、形状:50mm×26mm)を用意した。
上記のFTO電極上にスパッタ法によってZnOを0.44nm(スパッタ時間20秒、50W、スパッタレート1.3nm/min)、1.08nm(スパッタ時間50秒、50W、スパッタレート1.3nm/min)50nm(スパッタ時間8分、100W、スパッタレート6.25nm/min)に成膜した電極(膜厚はスパッタレートから概算)を用意した。
上記のFTO電極上にスパッタ法によってZnOを厚さ50nm(200W、スパッタ時間8分、スパッタレート6.25nm/min)に成膜した電極を用意した(膜厚はスパッタレートから概算)。この電極をアセトン、超純水、アセトンの順に1分間ずつ超音波洗浄を行い、1M硝酸溶液(pH0.2)に浸漬し5分間振盪を行った。その後超純水で十分すすぎ、この電極をZnO/FTO*とした。
作製した作用電極をそれぞれアセトン、超純水、アセトンの順に1分ずつ超音波洗浄を行った。その後、直径3mmの大きさの開口部が形成された粘着性シール(厚さ:0.5mm)を載置して密着させた。ヤギ由来抗体(抗ルシフェラーゼ ポリクローナル抗体 : Promega社製)を10μg/mlになるように調製した。この時溶媒は250mM NaCl、0.05%Tween20含有 10mMリン酸緩衝液(pH7)を用いた。このタンパク質溶液を、電極上のシールによる開口部にそれぞれ5μlずつ滴下し、37℃で30分間インキュベートを行った。その後、電極を超純水中で10分間振盪し、洗浄を行った。
色素標識抗原(Cy5-抗ヤギ抗体(ウサギ): chemicon製)を100ng/mlに調製した。この時溶媒は、250mM NaCl、0.05%Tween20含有10mMリン酸緩衝液(pH7)を用いた。調製した抗原溶液を、先にプローブタンパク質を固定化した作用電極上のシール開口部に5μlずつ滴下し、37℃で1時間インキュベートを行った。その後電極上のシールを剥がし、超純水で表面を流し洗浄した。
上述の方法で作製した、被検物質結合作用電極と、ガラス板上に白金が蒸着されてなる対電極とを用意した。両電極間に電解液(0.4Mテトラプロピルアンモニウムヨージド)を含有した電解質シートを挟み、密着させた。この時、作用電極のタンパク質が固定化された面と対電極の白金蒸着面とが対向するように配置した。両電極を電気化学アナライザーに接続した状態で、作用電極にレーザー光源(出力120mW、照射領域の直径1mm 波長650nm赤色レーザー)を照射し、そのときに観察される電流値を記録した。結果は図2に示されるとおりであった。
電極の作製
作用電極用の基材として、フッ素をドープした酸化スズ(F-SnO2:FTO)コートガラス(エイアイ特殊硝子社製、U膜、シート抵抗:12Ω/cm2、形状:50mm×26mm)を用意した。この電極に膜厚50nmでZnOをスパッタした電極(ZnO/FTO)を例1に記載した方法で作製した。この電極をアセトン、超純水、アセトンの順に1分間ずつ超音波洗浄した後、各濃度に調製した硝酸溶液に浸漬した。硝酸を次の8つのpH(濃度)に調製した。調製した硝酸溶液と電極の浸漬時間を表1に示した。各溶液に浸漬後、電極を超純水十分濯ぎ洗浄を行った。
作製した電極に直径3mmの大きさの開口部が形成された粘着性シール(厚さ:0.5mm)を載置して密着させた。この開口部に10μg/ml(10mMリン酸緩衝液[pH7] 0.05% Tween20、250mM NaCl)に調製したヤギ由来抗体溶液を5μlずつ滴下し、37℃で30分間インキュベートした。その後、超純水中で10分間振盪し洗浄した。その後10ng/ml、100ng/ml、1μg/ml(10mMリン酸緩衝液、0.05%Tween20、250mM NaCl)に調製したCy5標識抗ヤギ抗体をシール開口部に5μlずつ滴下し、37℃で60分間インキュベートを行った。
例1に記載した条件を用い、光電流の測定を行った。その結果は、図3に示されるとおりであった。この結果より、硝酸のpHに依存し作製された電極の特性が変化することが判った。
例1で作製したZnO/FTO電極(ZnO膜厚0.44nm、1.03nm、50nm)と、ZnO/FTO*電極(硝酸処理濃度:1M、5mM、3.2mM、2mM、0.1mM)、さらにFTO電極の計9種類の電極をX線光電子分析法を用いて表面元素分析を行った。分析はアルバック・ファイ製PHI1800型 X線光電子分光装置を用い、X線源 MgKα(100w) 分析領域 0.8×2.0mmの条件で行った。その結果は表2に示されるとおりであった。
作用電極の作製
FTO電極
作用電極用の基材として、フッ素をドープした酸化スズ(F-SnO2:FTO)コートガラス(エイアイ特殊硝子社製、U膜、シート抵抗:12Ω/ cm2、形状:50mm×26mm)を用意した。
上記のFTO電極上にスパッタ法によってITOを50nm(スパッタ時間8分、100W、スパッタレート6.25nm/min)に成膜した電極(膜厚はスパッタレートから概算)を用意した。
上記のFTO電極上にスパッタ法によってWO3を50nm(スパッタ時間8分、100W、スパッタレート6.25nm/min)に成膜した電極(膜厚はスパッタレートから概算)を用意した。
上記のFTO電極上にスパッタ法によってSrTiO3を50nm(スパッタ時間8分、100W、スパッタレート6.25nm/min)に成膜した電極(膜厚はスパッタレートから概算)を用意した。
上記のITO/FTO、WO3/FTO、SrTiO3/FTO電極をそれぞれ、アセトン、超純水、アセトンの順に1分間ずつ超音波洗浄を行い、1M硝酸溶液(pH0.2)に浸漬し15分間振盪を行った。その後超純水で十分すすぎ、この電極をそれぞれITO/FTO*、WO3/FTO*、SrTiO3/FTO*とした。
作製した作用電極をそれぞれアセトン、超純水、アセトンの順に1分ずつ超音波洗浄を行った。その後、直径3mmの大きさの開口部が形成された粘着性シール(厚さ:0.5mm)を載置して密着させた。ヤギ由来抗体(抗ルシフェラーゼ ポリクローナル抗体 : Promega社製)を10μg/mlになるように調製した。この時溶媒は250mM NaCl、0.05%Tween20含有 10mMリン酸緩衝液(pH7)を用いた。このタンパク質溶液を、電極上のシールによる開口部にそれぞれ5μlずつ滴下し、37℃で30分間インキュベートを行った。その後、電極を超純水中で10分間振盪し、洗浄を行った。
色素標識抗原(Cy5-抗ヤギ抗体(ウサギ): chemicon製)を100ng/mlに調製した。この時溶媒は、250mM NaCl、0.05%Tween20含有 10mMリン酸緩衝液(pH7)を用いた。調製した抗原溶液を、先にプローブタンパク質を固定化した作用電極上のシール開口部に5μlずつ滴下し、37℃で1時間インキュベートを行った。その後電極上のシールを剥がし、超純水で表面を流し洗浄した。
上述の方法で作製した、被検物質結合作用電極と、ガラス板上に白金が蒸着されてなる対電極とを用意した。両電極間に電解液(0.4Mテトラプロピルアンモニウムヨージド)を含有した電解質シートを挟み、密着させた。この時、作用電極のタンパク質が固定化された面と対電極の白金蒸着面とが対向するように配置した。両電極を電気化学アナライザーに接続した状態で、作用電極にレーザー光源(出力120mW、照射領域の直径1mm 波長650nm赤色レーザー)を照射し、そのときに観察される電流値を記録した。結果は図4に示されるとおりであった。この結果より、第二物質として、ITO、WO3、SrTiO3を用いた場合においても、硝酸処理を行った後に、光電流による被検タンパク質の特異的検出が可能であることが示された。
作用電極の作製
FTO電極
作用電極用の基材として、フッ素をドープした酸化スズ(F-SnO2:FTO)コートガラス(エイアイ特殊硝子社製、U膜、シート抵抗:12Ω/ cm2、形状:50mm×26mm)を用意した。
上記のFTO電極上にスパッタ法によってZnOを50nm(スパッタ時間8分、100W、スパッタレート6.25nm/min)に成膜した電極(膜厚はスパッタレートから概算)を用意した。
上記のZnO/FTO電極を、アセトン、超純水、アセトンの順に1分間ずつ超音波洗浄を行い、1M硝酸溶液(pH0.2)に浸漬し15分間振盪を行った。その後超純水で十分すすぎ、この電極をZnO/FTO*とした。
作製した作用電極をそれぞれアセトン、超純水、アセトンの順に1分ずつ超音波洗浄を行った。その後、直径3mmの大きさの開口部が形成された粘着性シール(厚さ:0.5mm)を載置して密着させた。抗Prostate Specific antigen抗体(Fitzgerald社製)を15μg/mlになるように調製した。この時溶媒には、10mMリン酸緩衝液(pH7.4)を用いた。抗体溶液を電極場のシールによる開口部にそれぞれ5μlずつ滴下し、37℃で10分間インキュベートを行った。その後、電極を10mM リン酸緩衝液中で10分間振盪し、洗浄を行った。
Cy5標識抗Prostate Specific Antigen抗体(Biodesign社製)を5μg/mlに調製した。この時溶媒は、10mMリン酸緩衝液(pH7)、150mM NaCl、0.05%Tween20を用いた。調製した抗原溶液に抗原(Prostate Specific Antigen)を各濃度で添加し、ピペッテイングによって十分撹拌したのちに、先にプローブタンパク質を固定化した作用電極上のシール開口部に5μlずつ滴下し、37℃で10分間インキュベートを行った。その後電極上のシールを剥がし、緩衝液と超純水で表面を流し洗浄した。
上述の方法で作製した、被検物質結合作用電極と、ガラス板上に白金が蒸着されてなる対電極とを用意した。両電極間に電解液(0.4Mテトラプロピルアンモニウムヨージド)を含有した電解質シートを挟み、密着させた。この時、作用電極のタンパク質が固定化された面と対電極の白金蒸着面とが対向するように配置した。両電極を電気化学アナライザーに接続した状態で、作用電極にレーザー光源(出力120mW、照射領域の直径1mm 波長650nm赤色レーザー)を照射し、そのときに観察される電流値を記録した。結果は図5に示されるとおりであった。この結果より、光電流によるサンドイッチイムノアッセイを用いた被検物質の検出が可能であることが示された。
例1に示した方法で作製した、ZnOスパッタ電極と、ZnO硝酸処理電極のTEMおよび、EDS分析を実施した。TEM分析には日本電子製 JEM-2010F型 電界放射型透過電子顕微鏡を用い、加速電圧200kVで測定を行った。またEDS分析にはノーラン製 UTW型Si(Li)半導体検出器を用い分析領域1nmの分析を実施した。
Claims (13)
- 光電流による被検物質の特異的検出に用いられる電極部材であって、
導電性基材と、該導電性基材上の電子受容物質とを少なくとも有してなり、
前記電子受容物質が、半導体からなる第一物質の層と、該第一物質の層の表面に担持された、前記半導体とは異なる種類の半導体または金属もしくは金属酸化物からなる第二物質とから少なくともなる、電極部材。 - 前記第二物質が、前記第一物質の層の表面に被着された後、それを一部除去する工程に付されてなるものである、請求項1に記載の電極部材。
- 前記第一物質を構成する金属元素に対する、前記第二物質を構成する金属元素の表面存在元素比(物質量比)が0超過、1未満である、請求項1に記載の電極部材。
- 前記表面存在元素比が、0.27未満である、請求項3に記載の電極部材。
- 前記第二物質が、前記第一物質の表面を部分的に被覆してなり、かつ第二物質の被覆膜厚が0超過、2nm未満である、請求項1に記載の電極部材。
- 前記第二物質が、前記第一物質の表面から内部へ4nmの深さの領域に存在する、請求項1に記載の電極部材。
- 前記第一物質が、インジウム-スズ複合酸化物またはフッ素がドープされた酸化スズである、請求項1に記載の電極部材。
- 前記第二物質が、酸化亜鉛、インジウム-スズ複合酸化物、酸化タングステン、またはチタン酸ストロンチウムである、請求項1に記載の電極部材。
- 前記電子受容物質にプローブ物質がさらに担持されてなる、請求項1に記載の電極部材。
- 請求項1に記載の電極部材の製造方法であって、
導電性基材上に、半導体を含んでなる層を形成して、前記第一物質の層を形成する工程と、
前記層の表面に前記半導体とは異なる半導体または金属もしくは金属酸化物を被着させ、前記第二物質を被着させる工程とを少なくとも含んでなる、方法。 - 前記第二物質を被着させた後、それを一部除去する工程に付する、請求項10に記載の方法。
- 前記除去が、第二物質を酸性水溶液と接触させることにより行われる、請求項11に記載の方法。
- 前記酸性水溶液のpHが2.9未満のものである、請求項12に記載の方法。
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US (1) | US20110193187A1 (ja) |
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JP2012225885A (ja) * | 2011-04-22 | 2012-11-15 | Nara Institute Of Science & Technology | 被検物質の電気化学的検出方法 |
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EP2515112B1 (en) | 2011-04-22 | 2015-08-12 | Sysmex Corporation | Method for electrochemically detecting analyte |
TWI583947B (zh) * | 2013-12-16 | 2017-05-21 | 聖高拜塑膠製品公司 | 電極及製造電極的方法 |
US10468528B2 (en) * | 2014-04-16 | 2019-11-05 | Taiwan Semiconductor Manufacturing Company, Ltd. | FinFET device with high-k metal gate stack |
US9721955B2 (en) | 2014-04-25 | 2017-08-01 | Taiwan Semiconductor Manufacturing Company, Ltd. | Structure and method for SRAM FinFET device having an oxide feature |
US9224736B1 (en) | 2014-06-27 | 2015-12-29 | Taiwan Semicondcutor Manufacturing Company, Ltd. | Structure and method for SRAM FinFET device |
CN110088609A (zh) | 2016-11-30 | 2019-08-02 | 美国圣戈班性能塑料公司 | 电极及电极制造方法 |
CN113533473B (zh) * | 2021-06-22 | 2024-02-06 | 武汉纺织大学 | 含有金属-有机骨架的工作电极及其制备方法和应用 |
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JP2012225885A (ja) * | 2011-04-22 | 2012-11-15 | Nara Institute Of Science & Technology | 被検物質の電気化学的検出方法 |
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JP2013068624A (ja) | 2013-04-18 |
CN102203596A (zh) | 2011-09-28 |
JP5344079B2 (ja) | 2013-11-20 |
EP2327979A1 (en) | 2011-06-01 |
US20110193187A1 (en) | 2011-08-11 |
JPWO2010024446A1 (ja) | 2012-01-26 |
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