WO2012121229A1 - 微生物検出用センサーおよびその製造方法 - Google Patents
微生物検出用センサーおよびその製造方法 Download PDFInfo
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- WO2012121229A1 WO2012121229A1 PCT/JP2012/055611 JP2012055611W WO2012121229A1 WO 2012121229 A1 WO2012121229 A1 WO 2012121229A1 JP 2012055611 W JP2012055611 W JP 2012055611W WO 2012121229 A1 WO2012121229 A1 WO 2012121229A1
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- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
- C25D5/00—Electroplating characterised by the process; Pretreatment or after-treatment of workpieces
- C25D5/60—Electroplating characterised by the structure or texture of the layers
- C25D5/605—Surface topography of the layers, e.g. rough, dendritic or nodular layers
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T428/00—Stock material or miscellaneous articles
- Y10T428/24—Structurally defined web or sheet [e.g., overall dimension, etc.]
- Y10T428/24479—Structurally defined web or sheet [e.g., overall dimension, etc.] including variation in thickness
Definitions
- the present invention relates to a sensor for detecting microorganisms and a method for producing the same.
- a technique capable of detecting the pathogenic bacteria quickly and with high sensitivity is required.
- methods for detecting and identifying microorganisms include ELISA methods and Western blotting methods. These include, for example, an antigen-antibody reaction between an antibody (primary antibody) and a microorganism-specific protein, and then a labeled secondary antibody is reacted with the antibody (primary antibody) to cause chemiluminescence of the secondary antibody or ATP. This is a method of detecting by monitoring the hydrolysis reaction.
- Patent Document 1 describes a method for detecting anionic molecules (ATP, amino acids, etc.) derived from microorganisms using the electrochemical properties of a polymer having a molecular template.
- none of the above methods is a method for detecting microorganisms themselves.
- the ELISA method is not easy because it is necessary to prepare an antibody against a protein unique to a microorganism.
- An object of the present invention is to provide a novel microorganism detection sensor that can detect microorganisms quickly and easily and with high sensitivity, and a method for producing the same.
- the present invention includes a detection unit having a detection electrode and a polymer layer that is arranged on the detection electrode and includes a three-dimensional structure template complementary to the three-dimensional structure of the microorganism to be detected. It is a sensor that detects microorganisms based on the capture state of microorganisms.
- the polymer layer is a polymerization process that forms a polymer layer on the detection electrode by polymerizing monomers in the presence of microorganisms to be detected, and partially destroying the microorganisms incorporated in the polymer layer And a peroxidation process in which the polymer layer is peroxidized to release microorganisms from the polymer layer.
- a preferred form of the sensor further includes a counter electrode, and an AC voltage is applied between the detection electrode and the counter electrode of the detection unit in a state where the detection unit and the counter electrode are in contact with the sample solution, and the sample is subjected to dielectrophoresis. It is configured to guide the microorganisms in the solution toward the detection unit.
- the application time of the AC voltage is not particularly limited as long as microorganisms in the sample solution are guided in the direction of the detection unit.
- a preferred form of the sensor further includes a crystal resonator having the detection electrode of the detection unit as one electrode, and measures the change in the mass of the polymer layer from the change in the resonance frequency of the crystal resonator to determine the capture state of the microorganism. To detect.
- the monomer is preferably selected from the group consisting of pyrrole, aniline, thiophene and derivatives thereof.
- the surface on which the polymer layer of the detection electrode is formed is preferably a rough surface.
- the microorganism As the microorganism, a microorganism having a negative or excessive charge on the entire surface or the surface is preferable.
- the microorganism is a bacterium, and in this case, the destruction step is a step of performing a lysis treatment.
- the bacterium include Pseudomonas aeruginosa, Acinetobacter, and Escherichia coli.
- the present invention also relates to a sensor for detecting a microorganism having a detection portion having a detection electrode and a polymer layer provided on the detection electrode and having a three-dimensional template complementary to the three-dimensional structure of the microorganism.
- a polymerization process in which a monomer layer is polymerized in the presence of a microorganism to be detected to form a polymer layer in which the microorganism is incorporated on the detection electrode, and the microorganism incorporated in the polymer layer is partially And a step of peroxidizing the polymer layer to release microorganisms from the polymer layer.
- the senor further includes a counter electrode, and the polymerization step is performed by applying a voltage between the detection electrode and the counter electrode under contact with the monomer solution, This is a step of electrolytic polymerization of monomers.
- the releasing step is performed by applying a voltage between the detection electrode and the counter electrode under contact with a solution in a neutral to alkaline range to pass the polymer layer. This is a step of oxidizing treatment.
- the preferred form of the production method includes a roughening step of performing a roughening treatment on the surface of the detection electrode on which the polymer layer is formed.
- the senor of the present invention it is possible to detect microorganisms quickly and easily with high sensitivity.
- a sensor capable of detecting microorganisms quickly and easily with high sensitivity is provided.
- the preferable preparation process of the polymer layer of the sensor concerning this invention is shown typically, (a) before a polymerization process, (b) after a polymerization process, (c) after a destruction process, (d) after a peroxidation process.
- the sensor of this invention it is a schematic diagram which shows the outline of a mode that the target microorganism is capture
- FIG. 2 is a diagram showing an electron micrograph of the surface of a polypyrrole layer after the polymerization step of Example 1.
- FIG. 4 is a graph showing the relationship between time and current in the polymerization process of Example 1, and the relationship between time and the resonance frequency of the crystal resonator. 4 is a graph showing the relationship between time and mass change in Example 1.
- 2 is an electron micrograph of the surface of a peroxidized polypyrrole layer after the lysis step and the peroxidation step of Example 1.
- FIG. It is an electron micrograph of the peroxidation polypyrrole layer surface after a lysis process and a peroxidation process at the time of changing lysis conditions from Example 1.
- the sensor of the present invention includes a detection part having a detection electrode and a polymer layer that is arranged on the detection electrode and has a three-dimensional template complementary to the three-dimensional structure of the microorganism, Microorganisms are detected based on the captured state.
- the polymer layer of the sensor of the present invention is a polymer that forms a polymer layer on the detection electrode by polymerizing monomers in the presence of microorganisms to be detected (hereinafter also referred to as “target microorganisms”).
- the manufacturing method includes a step, a destruction step for partially destroying microorganisms incorporated in the polymer layer, and a peroxidation step for peroxidizing the polymer layer to release microorganisms from the polymer layer.
- FIG. 1 is a cross-sectional view schematically showing a preferred production process of a polymer layer of a sensor according to the present invention.
- FIG. 1 shows an embodiment in which pyrrole is used as a monomer.
- a solution 12 containing a microorganism 13 and pyrrole is prepared in an environment in contact with the detection electrode 11.
- electrolysis using the detection electrode 11 as an anode and a counter electrode (not shown) as a cathode is performed, and polypyrrole (FIG.
- pyrrole 1B is formed on the detection electrode 11 by an oxidative polymerization reaction of pyrrole.
- Py is an abbreviation for polypyrrole.
- Microorganisms 13 are taken into the formed polymer layer 14.
- the pyrrole itself has a positive charge in order to emit electrons to the detection electrode 11 in the polymerization process, and in order to compensate for this positive charge, the microorganism 13 whose whole or surface charge is in a state of excessive negative charge. Is believed to be incorporated into the polymer layer 14.
- a destruction step for destroying a part of the microorganisms 13 taken in the polymer layer 14 is performed.
- the destruction step can be performed by, for example, addition of a degrading enzyme, temperature adjustment, ultrasonic treatment, ozone treatment, presence of residual chlorine, or bacteriophage treatment.
- the destruction step can be performed by a lysis treatment using a degrading enzyme such as lysozyme (hereinafter, the destruction step by the lysis treatment is also referred to as “lysis step”).
- lysis step a degrading enzyme such as lysozyme
- the polymer layer 14 is peroxidized.
- the polypyrrole constituting the polymer layer 14 becomes peroxide polypyrrole (“Oppy” in FIG. 1D is an abbreviation for peroxide polypyrrole) and becomes electrically neutral. Released from layer 14.
- the region where the microorganisms 13 exist in the polymer layer 14 becomes a template 15 having a three-dimensional structure complementary to the three-dimensional structure of the microorganisms 13.
- This peroxidation step (St3) also causes hardening of the polymer layer 14 and stabilizes the template 15 of the microorganism 13.
- the peroxidation step (St3) is preferably performed by preparing the solution 12 in a neutral to alkaline range and applying a voltage between the detection electrode 11 and a counter electrode (not shown).
- the laminate of the polymer layer 14 having the template 15 formed in this way and the detection electrode 11 constitutes the detection unit 17 in the sensor of the present invention.
- the three-dimensional structure of the template formed may vary depending on the solution composition of the peroxidation reaction and the voltage for causing the peroxidation reaction.
- a template having a close three-dimensional structure is formed by the microorganism 13 to be detected under conditions where the peroxidation reaction proceeds gradually.
- the microorganism 13 to be detected is not particularly limited as long as the whole or surface of the microorganism has a negative charge excess, such as Escherichia genus of Escherichia coli, Pseudomonas genus such as Pseudomonas aeruginosa, Acinetobacter calocoaceticus, etc.
- viruses examples include hepatitis A virus, adenovirus, rotavirus, and norovirus, candidia as fungi, and cryptosporidium as protozoa.
- the total or surface charge of the microorganism varies depending on the water quality of the solution 12 such as pH.
- the solution 12 may be made alkaline in order to make the negative charge excessive when forming the template or measuring.
- the monomer used as the raw material for the polymer layer is not limited to pyrrole, and other examples include aniline, thiophene. And derivatives thereof.
- the material of the detection electrode 11 is not particularly limited, and is a gold electrode, a multilayer electrode of gold and chromium, a multilayer electrode of gold and titanium, a silver electrode, a multilayer electrode of silver and chromium, and a silver and titanium layer. Examples include multilayer electrodes, lead electrodes, platinum electrodes, carbon electrodes, and the like.
- the surface on which the polymer layer 14 of the detection electrode 11 is formed is preferably subjected to a roughening treatment. Since the surface on which the polymer layer 14 of the detection electrode 11 is formed is a rough surface, the adhesion to the polymer layer 14 is improved, and the surface area of the electrode is increased. For example, when a gold electrode is used as the detection electrode 11, it is possible to perform a roughening process in which the gold electrode surface is subjected to plasma etching and then gold nanoparticles are fixed to roughen the surface.
- FIG. 2 is a schematic diagram showing an outline of how a target microorganism is captured by a template.
- FIG. 2A shows a case where the microorganism 13a in the sample solution is a target microorganism
- FIG. 2B shows a case where the microorganism 13b in the sample solution is not a target microorganism.
- a sample solution is prepared in an environment in contact with the detection unit 17 including the polymer layer 14 and the detection electrode 11 and the counter electrode 16. Then, an AC voltage is applied between the detection electrode 11 and the counter electrode 16, and microorganisms in the sample solution are moved toward the detection unit 17 by dielectrophoresis.
- the configuration of the counter electrode 16, adjustment of the applied voltage, and preparation of the sample solution are performed so that the microorganisms move toward the detection electrode 11 by dielectrophoresis.
- the microorganism 13a having a three-dimensional structure complementary to the three-dimensional structure of the template 15 is captured in the template 15 (FIG. 2A), but is not complementary to the template 15.
- the microorganism 13b is not captured in the template 15 (FIG. 2B).
- turbidity other than microorganisms such as mud and iron rust is contained in water, they are not captured because they are different from the template 15 in three-dimensional shape, charged state, etc. and are not complementary.
- dielectrophoresis can be performed under conditions where microorganisms are collected on the electrode but other turbid substances are not collected.
- dielectrophoresis can be performed under conditions where microorganisms are collected on the electrode but other turbid substances are not collected.
- it is necessary to change the conditions of dielectrophoresis such as frequency in accordance with changes in water quality such as water conductivity.
- water quality such as water conductivity.
- Detection of target microorganisms When the microorganisms 13a are trapped in the template 15, for example, a change in mass, a change in conductive characteristics, a change in capacitance, a change in light reflectance, a change in temperature, and the like occur in the laminate composed of the polymer layer 14 and the detection electrode 11.
- a change in mass, a change in conductive characteristics, a change in capacitance, a change in light reflectance, a change in temperature, and the like occur in the laminate composed of the polymer layer 14 and the detection electrode 11.
- the capture state of the microorganism in the template 15 is detected.
- the target microorganism can be detected based on the captured state. With such detection, rapid and sensitive detection of the target microorganism can be achieved.
- the mass change detection method there is a detection method for detecting a change in the resonance frequency of the crystal resonator.
- a crystal resonator microbalance (QCM) sensor which is a preferred example of the sensor of
- FIG. 3 is a schematic diagram showing a schematic configuration of the QCM sensor.
- the QCM sensor 33 includes a cell 27 for holding a solution, a crystal resonator 32 disposed at the bottom of the cell 27, an oscillation circuit 22, and a controller 21 having a frequency counter.
- the crystal unit 32 is formed by sequentially stacking the detection unit 17, the crystal piece 24, and the counter electrode (second counter electrode) 23 manufactured by the process illustrated in FIG. 1.
- the QCM sensor 33 further includes a counter electrode (first counter electrode) 16 immersed in the sample solution 31 and a reference electrode 30, and is connected to the detection electrode 11 and the counter electrode 16 of the detection unit 17.
- An AC power supply 29 is provided.
- the sample solution 31 is added into the cell 27. Then, by applying an AC voltage between the detection electrode 11 and the counter electrode 16 by the AC power source 29, the microorganisms contained in the sample solution 31 are moved in the direction of the detection unit 17 by dielectrophoresis. At the same time, the oscillation circuit 22 applies an AC voltage between the detection electrode 11 and the counter electrode 23 to vibrate the crystal piece 24. When microorganisms are trapped in the mold 15 of the polymer layer 14, the mass of the detection unit 17 changes, and the resonance frequency of the crystal piece 24 changes. The frequency counter in the controller 21 receives the signal from the oscillation circuit 22 and measures the resonance frequency value. The capture state of the microorganism is detected from the change in the resonance frequency value.
- a polymer layer can be formed on the detection electrode 11 in accordance with the roughening treatment of the surface of the detection electrode 11 and the process shown in FIG.
- a crystal resonator in which the detection electrode 11, the crystal piece 24, and the counter electrode 23 are stacked in this order is arranged at the bottom of the cell 27, and a DC power supply is connected instead of the AC power supply 29.
- the progress of the formation of the polymer layer can be confirmed by monitoring the change in the resonance frequency of the crystal resonator together with the formation of the polymer layer.
- the templates according to the present invention are individually formed and combined, or a template corresponding to a plurality of microorganisms is contained in a single template. By forming simultaneously, it is also possible to detect a plurality of types of microorganisms simultaneously.
- bacteria can be detected in several minutes to several tens of minutes, and can be detected much more quickly than in the culture method.
- it is a device such as a water purifier, a water server or an automatic ice making device. Easy to incorporate and automate.
- it can be used in water purification plants and beverage / food factories as a bacteria inspection tool for water quality testing and food testing. More specifically, it is possible to automatically detect bacteria in the apparatus such as the water storage tank and the piping path and notify the user, or take measures such as sterilization and washing automatically.
- the polymer layer in the above-mentioned sensor has a three-dimensional structure template complementary to the three-dimensional structure of the microorganism, a microorganism capturing device, a microorganism shape recognition device, a microorganism tracking device, It can also be used for a catalyst carrier utilizing the porous material.
- the polymer layer is produced using an electrochemical measurement system (Model 842B, manufactured by ALS), the detection electrode is a gold electrode (corresponding to one electrode 11 of the crystal resonator), and the reference electrode is used.
- the detection electrode is a gold electrode (corresponding to one electrode 11 of the crystal resonator)
- the reference electrode is used.
- Ag / AgCl saturated KCl
- a counter electrode was a Pt rod (diameter 1 mm, length 4 cm, manufactured by Niraco Co., Ltd.).
- the potential is a value relative to the potential of the reference electrode.
- a quartz crystal resonator (electrode area 0.196 cm 2 , fundamental vibration frequency 9 MHz, AT cut, square type, manufactured by Seiko EG & G Co., Ltd.) provided with gold electrodes on both surfaces was used.
- Example 1 Pseudomonas aeruginosa (zeta potential: ⁇ 33.87 mV) was used as the microorganism to be detected.
- Example 2 Acinetobactor calcoaceticus (zeta potential: ⁇ 28.14 mV)
- Example 3 Escherichia coli was used.
- Example 5 Pseudomonas aeruginosa, Escherichia coli, Acinetobactor calcoaceticus, and Serratia marcescens were used.
- 4 and 5 show electron micrographs of Pseudomonas aeruginosa and Acinetobacter, respectively. From the micrographs shown in FIGS. 4 and 5, it can be seen that the shape of Pseudomonas aeruginosa is bowl-shaped, and the shape of Acinetobacter is more nearly spherical.
- Example 1 ⁇ Production of sensor> (Roughening process of gold electrode) The surface of the gold electrode was roughened on the surface of the gold electrode of the quartz crystal laminate according to the following procedure in order to improve the adhesion to the polypyrrole peroxide layer. 1. The gold electrode surface was etched for 30 seconds by a plasma etching apparatus (SEDE / meiwa fossis). 2. A crystal resonator was placed at the bottom of the cell 27 of the QCM sensor 33 as shown in FIG. Thereafter, 500 ⁇ L of a solution containing 30 nm citrate-protected gold nanoparticles (0.0574 wt%) was added to the cell 27 and left at room temperature for 24 hours. 3.
- SEDE plasma etching apparatus
- a peroxide polypyrrole layer was prepared on the gold electrode according to the following procedure. 1. A 0.1 M pyrrole aqueous solution containing Pseudomonas aeruginosa and phosphate buffer (0.2 M, pH 2.56) was prepared as a modification solution. 2. The modification solution was added into the cell 27 of the QCM sensor 33 where the gold electrode subjected to the roughening treatment was disposed, and the first counter electrode and the reference electrode were inserted into the modification solution. 3. Polypyrrole was deposited on the gold electrode by constant potential electrolysis (+0.975 V, 90 seconds) in the modification solution, and a polypyrrole layer was produced (polymerization step).
- the resonance frequency of the crystal resonator was also monitored. 4). Lysozyme (10 mg / mL) was added dropwise to the prepared polypyrrole layer and shaken for 120 minutes, and then a 10% solution of a nonionic surfactant (trade name: triton) was added and shaken for 80 minutes (lysis process). 5. After cleaning the polypyrrole layer several times with ultrapure water, a 0.1M NaOH solution is added into the cell 27, and a constant potential +975 mV is applied for 120 seconds to perform a peroxidation treatment to obtain a peroxidized polypyrrole layer. (Peroxidation process). In the peroxidation process, the resonance frequency of the crystal resonator was also monitored.
- FIG. 6 shows an electron micrograph of the surface of the polypyrrole layer after the polymerization step. It was observed that Pseudomonas aeruginosa was taken up on the surface of the polypyrrole layer.
- FIG. 7 is a graph showing the relationship between time and current in the polymerization process, and the relationship between time and the resonance frequency of the crystal resonator. The time at the start of constant potential electrolysis is set to 0 seconds.
- FIG. 8 is a graph showing the relationship between time and mass change by calculating the mass change amount of the crystal resonator from the change amount of the resonance frequency shown in FIG. From these graphs, it can be seen that the mass of the surface of the crystal resonator increases in proportion to the electrolysis time, and a sufficient mass change, that is, a sufficient polymerization of the polypyrrole layer is achieved in 90 seconds.
- FIG. 9 shows an electron micrograph of the surface of the peroxidized polypyrrole layer after the lysis step and the peroxidation step. It can be seen that Pseudomonas aeruginosa was not observed on the surface of the peroxide polypyrrole layer, and thus Pseudomonas aeruginosa was released from the surface of the peroxide polypyrrole layer.
- FIG. 10 shows the surface of the peroxidized polypyrrole layer produced by changing the conditions of the shaking time after dropping lysozyme in the lysis step and the shaking time after adding the nonionic surfactant. The electron micrograph of is shown.
- FIG. 10 shows the surface of the peroxidized polypyrrole layer produced by changing the conditions of the shaking time after dropping lysozyme in the lysis step and the shaking time after adding the nonionic surfactant. The electron micrograph of is shown.
- FIGS. 10 (a) shows a case where the shaking time after adding lysozyme is 30 minutes and a shaking time after addition of the nonionic surfactant is 20 minutes.
- FIG. 10 (b) shows a case where lysozyme is dropped.
- FIG. 10C shows the shaking time after adding lysozyme is 90 minutes.
- the electron micrograph when the shaking time after adding an activator is 60 minutes is shown. From FIGS. 10 (a) to 10 (c), it can be seen that release of Pseudomonas aeruginosa is not sufficient under these conditions, and therefore the conditions of the lysis step of Example 1 are suitable conditions.
- FIG. 11 is a graph showing the relationship between time and current in the peroxidation process and the relationship between time and the resonance frequency of the crystal unit.
- the time at the constant potential application time in the peroxidation step is set to 0 seconds. It can be seen that the current value decreases with time and the peroxidation process proceeds. It can also be seen that the resonance frequency increases and the mass of the electrode surface decreases. This is understood to be due to the release of Pseudomonas aeruginosa.
- Detection experiment Microorganisms were detected using a QCM sensor, which was prepared as described above and provided with a quartz crystal resonator having a Pseudomonas aeruginosa template and a peroxide polypyrrole layer formed on the surface of the cell. A sample solution containing microorganisms was added to the cell. Thereafter, an alternating voltage was applied between the gold electrode and the first counter electrode, and microorganisms were concentrated on the surface of the peroxide polypyrrole layer by dielectrophoresis.
- An alternating voltage (waveform: sine wave, voltage: 2 Vpp, frequency: 10 MHz) was generated by a waveform generator (7075, manufactured by Hioki Electric Co., Ltd.). Further, the voltage was amplified 10 times by an amplifier (HAS4101, manufactured by NF Circuit Design Block Co., Ltd.) and applied as 20 Vpp. In addition, the resonance frequency of the crystal resonator during voltage application was monitored.
- FIG. 12 is a graph showing the relationship between the AC voltage application time and the resonance frequency of the crystal resonator. From the results shown in FIG. 12, it was found that the resonance frequency was greatly reduced in the detection experiment in which the sample solution containing Pseudomonas aeruginosa was added. A decrease in the resonance frequency means an increase in the mass of the surface of the crystal unit, and the mass of the surface of the crystal unit is increased by the dielectrophoretic force acting on Pseudomonas aeruginosa and being incorporated into the peroxide polypyrrole layer mold. it is conceivable that. On the other hand, almost no change was observed for the Acinetobacter having different shapes as in the blank.
- Acinetobacter different from the shape of the template is not as easily incorporated into the peroxidized polypyrrole layer as Pseudomonas aeruginosa, and it can be determined that the sensor recognizes the type of bacteria with high accuracy.
- Example 2 ⁇ Production of sensor> The gold electrode was roughened in the same manner as in Example 1 except that Acinetobacter was used instead of Pseudomonas aeruginosa in Example 1, and the above polymerization process, lysis process, and peroxidation process were further performed. .
- FIG. 13 shows an electron micrograph of the surface of the polypyrrole layer after the polymerization step. It was observed that Acinetobacter was taken into the surface of the polypyrrole layer.
- FIG. 14 is a graph showing the relationship between time and current in the polymerization process, and the relationship between time and the resonance frequency of the crystal resonator. The time at the start of constant potential electrolysis is set to 0 seconds. From this graph, it can be seen that the mass of the surface of the crystal resonator increased in proportion to the electrolysis time.
- FIG. 15 shows an electron micrograph of the surface of the peroxidized polypyrrole layer after the lysis step and the peroxidation step. It can be seen that no Acinetobacter was observed on the surface of the peroxide polypyrrole layer, and thus Acinetobacter was released from the surface of the peroxide polypyrrole layer.
- FIG. 16 is a graph showing the relationship between time and current in the peroxidation process, and the relationship between time and the resonance frequency of the crystal resonator.
- the time at the constant potential application time in the peroxidation step is set to 0 seconds. It can be seen that the current value decreases with time and the peroxidation process proceeds. It can also be seen that the resonance frequency increases and the mass of the electrode surface decreases. This is understood to be due to the release of Acinetobacter.
- Detection experiment Microorganisms were detected using a QCM sensor, which was prepared as described above, and was provided with a quartz crystal resonator having a peroxide polypyrrole layer having an Acinetobacter template on the surface thereof, at the bottom of the cell.
- the experimental conditions were the same as in Example 1.
- FIG. 17 is a graph showing the relationship between the AC voltage application time and the resonance frequency of the crystal resonator. From the results shown in FIG. 17, it was found that in the detection experiment in which the sample solution containing Acinetobacter was added, the resonance frequency was greatly reduced. The decrease in resonance frequency means an increase in the mass of the quartz crystal surface, and it is thought that the mass of the quartz crystal surface increased by the dielectrophoretic force acting on the Acinetobacter and being taken into the mold of the peroxide polypyrrole layer. It is done. On the other hand, almost no change was observed for Pseudomonas aeruginosa having different shapes as in the blank.
- Pseudomonas aeruginosa which is different from the shape of the template, is not easily incorporated into the peroxidized polypyrrole layer as much as Acinetobacter, and it can be determined that the sensor recognizes the type of bacteria with high accuracy.
- Example 3 ⁇ Production of sensor> The gold electrode was roughened in the same manner as in Example 1 except that Escherichia coli was used in place of Pseudomonas aeruginosa in Example 1, and the above polymerization process, lysis process, and peroxidation process were further performed. .
- Detection experiment Microorganisms were detected using a QCM sensor provided on the bottom of the cell with a quartz crystal formed on the surface and having a polypyrrole peroxide layer having an Escherichia coli template formed on the surface. As measurement samples, solutions of Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter were used.
- FIG. 18 is a graph showing the relationship between the AC voltage application time and the resonance frequency of the crystal resonator. From the results shown in FIG. 18, it was found that in the detection experiment in which the sample solution containing E. coli was added, the resonance frequency was greatly reduced. A decrease in the resonance frequency means an increase in the mass of the quartz crystal surface, and it is thought that the mass of the quartz crystal surface has increased due to the dielectrophoretic force acting on E. coli and being incorporated into the peroxide polypyrrole layer mold. It is done. On the other hand, almost no change was observed for Pseudomonas aeruginosa and Acinetobacter as in the blank.
- Pseudomonas aeruginosa and Acinetobacter which are different from the shape of the template, are not as easily incorporated into the peroxidized polypyrrole layer as E. coli, and it can be determined that the sensor recognizes the type of bacteria with high accuracy.
- Example 4 ⁇ Production of sensor> Using Pseudomonas aeruginosa, the gold electrode roughening step was performed in the same manner as in Example 1, and the above-described polymerization step, lysis step, and peroxidation step were further performed.
- Detection experiment Microorganisms were detected using a QCM sensor, which was prepared as described above and provided with a quartz crystal resonator having a Pseudomonas aeruginosa template and a peroxide polypyrrole layer formed on the surface of the cell. Two kinds of measurement samples were used: a solution (a) in which solutions of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter and Serratia were mixed, and a solution (b) in which solutions of Escherichia coli, Acinetobacter and Serratia were mixed.
- FIG. 19 is a graph showing the relationship between the AC voltage application time and the resonance frequency of the crystal resonator. From the results shown in FIG. 19, it was found that in the detection experiment in which the sample solution containing Pseudomonas aeruginosa was added, the resonance frequency was greatly reduced. A decrease in the resonance frequency means an increase in the mass of the surface of the crystal unit, and the mass of the surface of the crystal unit is increased by the dielectrophoretic force acting on Pseudomonas aeruginosa and being incorporated into the peroxide polypyrrole layer mold. it is conceivable that.
- Example 5 ⁇ Production of sensor> Using a modification solution containing all of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter, and Serratia bacteria, a roughening process of the gold electrode is performed in the same manner as in Example 1, and the above polymerization process, lysis process, and peroxidation process are further performed. I did it.
- Detection experiment Microorganisms were detected using a QCM sensor provided on the bottom of the cell with a quartz crystal having a peroxide polypyrrole layer having a template containing four types of microorganisms formed on the surface, prepared as described above. As measurement samples, four types of solutions containing Pseudomonas aeruginosa, Escherichia coli, Acinetobacter, and Serratia were used.
- (result) 20 to 23 are graphs showing the relationship between the AC voltage application time and the resonance frequency of the crystal resonator. 20 to 23 show the results of detection experiments in which sample solutions each containing Pseudomonas aeruginosa, Escherichia coli, Acinetobacter, and Serratia bacteria were added, and the resonance frequency was greatly reduced regardless of which sample solution was added. I found out that Accordingly, it can be determined that a plurality of types of microorganisms are detected by a sensor having a plurality of types of microorganism templates.
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Abstract
Description
[センサーにおけるポリマー層の作製]
図1は、本発明にかかるセンサーのポリマー層の好ましい作製工程を模式的に示す断面図である。図1では、モノマーとしてピロールを用いる場合の実施形態を示す。まず、図1(a)に示すように、検出用電極11に接触する環境下に、微生物13およびピロールを含む溶液12を準備する。重合工程(St1)では、検出用電極11を陽極とし、対電極(不図示)を陰極とする電気分解を行い、ピロールの酸化的重合反応により、検出用電極11上にポリピロール(図1(b)中「PPy」は、ポリピロールの略である)からなるポリマー層14を形成する。形成されたポリマー層14には、微生物13が取り込まれる。ピロールは、重合工程で検出用電極11に電子を放出するためにそれ自体は陽電荷を有し、この陽電荷を補償するために、全体または表面の電荷が負電荷過剰の状態にある微生物13がポリマー層14中に取り込まれると考えられる。
図2は、鋳型へ標的微生物が捕捉される様子の概略を示す模式図である。図2(a)は試料溶液中の微生物13aが標的微生物である場合を示し、図2(b)は試料溶液中の微生物13bが標的微生物でない場合を示す。図2(a),(b)に示すように、まず、ポリマー層14と検出用電極11とからなる検出部17と、対電極16とに接触する環境下に、試料溶液を準備する。そして、検出用電極11と対電極16間との間に交流電圧を印加し、誘電泳動により試料溶液中の微生物を検出部17の方向に移動させる。なお、微生物が誘電泳動により検出用電極11の方に向かって移動するように、対電極16の構成、印加電圧の調整、試料溶液の調製を行なう。微生物が検出用電極11の方向に移動すると、鋳型15の三次元構造と相補的な立体構造の微生物13aは鋳型15内に捕捉されるが(図2(a))、鋳型15と相補的でない微生物13bは鋳型15内に捕捉されない(図2(b))。また、微生物以外の、たとえば、泥、鉄さびといった濁質が水に含まれていた場合であっても、それらも鋳型15と三次元的形状、荷電状態等が異なり相補的でないため、捕捉されない。そのため、標的微生物と他の濁質の識別が可能である。微生物と他の濁質との分離は、誘電泳動によっても可能である(微生物を電極に集めるが他の濁質は集めないような条件で誘電泳動を行うことができる)が、誘電泳動で微生物と他の濁質を分離するためには、水の導電率などの水質の変化に応じて、周波数などの誘電泳動の条件を変える必要がある。本発明のセンサの場合、対象物の形状で識別できるため、水質の影響を受けにくい。
微生物13aが鋳型15内に捕捉されると、ポリマー層14および検出用電極11からなる積層体に、たとえば、質量変化、導電特性変化、電気容量変化、光反射率変化、温度変化等が生じる。本発明のセンサーにおいては、このような変化を検出して、微生物の鋳型15への捕捉状態を検出する。そして、捕捉状態に基づいて標的微生物の検出が可能となる。このような検出により、標的微生物の迅速かつ高感度の検出が達成され得る。質量変化の検出方法の具体例として、水晶振動子の共振周波数の変化を検出する検出方法が挙げられる。以下、本発明のセンサーの好ましい一例である、水晶振動子マイクロバランス(QCM)センサーについ説明する。
図3は、QCMセンサーの概略構成を示す模式図である。QCMセンサー33は、溶液を保持するセル27と、セル27の底部に配置された水晶振動子32と、発振回路22と、周波数カウンタを有するコントローラ21とを備える。水晶振動子32は、図1に示した工程により作製された検出部17と、水晶片24と、対電極(第2対電極)23とが順に積層されてなる。QCMセンサー33は、さらに、試料溶液31内に浸漬される対電極(第1対電極)16と、参照電極30とを備え、検出部17の検出用電極11と対電極16とに接続された交流電源29を備える。
<センサーの作製>
(金電極の粗面化工程)
金電極の表面に、過酸化ポリピロール層との密着性向上のため以下の手順にしたがい水晶振動子積層体の金電極表面の粗面化処理を行なった。
1.プラズマエッチング装置(SEDE/meiwa fosis)により、金電極表面に30秒間エッチングを行なった。
2.水晶振動子を、図3に示すようなQCMセンサー33のセル27の底部に設置した。その後、30nmのクエン酸保護金ナノ粒子(0.0574wt%)を含んだ溶液500μLをセル27に添加し、室温で24時間放置した。
3.金電極を純水で洗浄後、臭化ヘキサデシルトリメチルアンモニウム溶液(0.1M)9mL、塩化金(III)酸四塩化物(0.01M)250μL、NaOH(0.1M)50μL、アスコルビン酸(0.1M)50μLを混合して出来た溶液(成長液)500μLをセル27に添加し、室温で24時間静置した。
4.セル27内の溶液を除去し、金電極を超純水で洗浄した。
以下の手順に従って金電極上に過酸化ポリピロール層を作製した。
1.緑膿菌、リン酸緩衝液(0.2M、pH2.56)を含む0.1Mのピロール水溶液を調製して修飾溶液とした。
2.上記で粗面化処理を施した金電極が配置されているQCMセンサー33のセル27内に、修飾溶液を添加し、修飾溶液内に第1対電極および参照電極を差し込んだ。
3.修飾溶液中において定電位電解(+0.975V、90秒間)することで金電極上にポリピロールを析出させ、ポリピロール層を作製した(重合工程)。重合工程においては、水晶振動子の共振周波数のモニターも行なった。
4.作製したポリピロール層にリゾチーム(10mg/mL)を滴下し、120分間振盪し、その後非イオン性界面活性剤(商品名:triton)の10%溶液を添加し、80分間振盪した(溶菌工程)。
5.超純水で数回のポリピロール層の洗浄を行った後、セル27内に0.1MのNaOH溶液を添加し、定電位+975mVを120秒間印加して過酸化処理を施し過酸化ポリピロール層を得た(過酸化工程)。過酸化工程においては、水晶振動子の共振周波数のモニターも行なった。
図6は、重合工程後のポリピロール層表面の電子顕微鏡写真を示す。ポリピロール層の表面に緑膿菌が取り込まれた様子が観察された。図7は、重合工程における時間と電流の関係、および時間と水晶振動子の共振周波数の関係を示すグラフである。定電位電解開始時点の時間を0秒とする。また、図8は、図7に示した共振周波数の変化量から水晶振動子の質量変化量を算出し、時間と質量変化の関係を示すグラフである。これらのグラフから、電解時間に比例して水晶振動子表面の質量が増加し、90秒間で十分な質量変化、すなわち十分なポリピロール層の重合が達成されることがわかる。
(検出実験)
上述のようにして作製した、緑膿菌鋳型を有する過酸化ポリピロール層が表面に形成された水晶振動子をセルの底部に備えたQCMセンサーを用いて微生物の検出を行なった。セル内に微生物を含む試料溶液を添加した。その後、金電極と第1対電極間に交流電圧を印加し、誘電泳動により微生物を過酸化ポリピロール層の表面へ濃縮させた。波形発生装置(7075、日置電機(株)製)により、交流電圧(波形:正弦波、電圧:2Vpp、周波数:10MHz)を発生させた。さらに増幅器(HAS4101,(株)エヌエフ回路設計ブッロク製)で電圧を10倍に増幅し、20Vppとして印加した。また、電圧印加時の水晶振動子の共振周波数をモニタリングした。
図12は、交流電圧印加時間と水晶振動子の共振周波数の関係を示すグラフである。図12に示す結果から、緑膿菌を含む試料溶液を添加した検出実験では、共振周波数が大きく減少することが分かった。共振周波数の減少は水晶振動子表面の質量の増加を意味しており、緑膿菌に誘電泳動力が作用し過酸化ポリピロール層の鋳型に取り込まれることで水晶振動子表面の質量が増加したものと考えられる。一方、形状の異なるアシネトバクターに対してはブランクと同様にほとんど変化が見られなかった。よって、鋳型の形状とは異なるアシネトバクターは緑膿菌ほど容易に過酸化ポリピロール層に取り込まれていないと考えられ、センサーは細菌の種類を高精度に認識していると判断できる。
<センサーの作製>
実施例1の緑膿菌に代えて、アシネトバクターを用いた点以外は、実施例1と同様に金電極の粗面化工程を行ない、さらに上述の重合工程、溶菌工程、過酸化工程を行なった。
図13は、重合工程後のポリピロール層表面の電子顕微鏡写真を示す。ポリピロール層の表面にアシネトバクターが取り込まれた様子が観察された。図14は、重合工程における時間と電流の関係、および時間と水晶振動子の共振周波数の関係を示すグラフである。定電位電解開始時点の時間を0秒とする。このグラフから、電解時間に比例して水晶振動子表面の質量が増加したことがわかる。
(検出実験)
上述のようにして作製した、アシネトバクター鋳型を有する過酸化ポリピロール層が表面に形成された水晶振動子をセルの底部に備えたQCMセンサーを用いて微生物の検出を行なった。実験条件は、実施例1と同様とした。
図17は、交流電圧印加時間と水晶振動子の共振周波数の関係を示すグラフである。図17に示す結果から、アシネトバクターを含む試料溶液を添加した検出実験では、共振周波数が大きく減少することが分かった。共振周波数の減少は水晶振動子表面の質量の増加を意味しており、アシネトバクターに誘電泳動力が作用し過酸化ポリピロール層の鋳型に取り込まれることで水晶振動子表面の質量が増加したものと考えられる。一方、形状の異なる緑膿菌に対してはブランクと同様にほとんど変化が見られなかった。よって、鋳型の形状とは異なる緑膿菌はアシネトバクターほど容易に過酸化ポリピロール層に取り込まれていないと考えられ、センサーは細菌の種類を高精度に認識していると判断できる。
<センサーの作製>
実施例1の緑膿菌に代えて、大腸菌を用いた点以外は、実施例1と同様に金電極の粗面化工程を行ない、さらに上述の重合工程、溶菌工程、過酸化工程を行なった。
(検出実験)
上述のようにして作製した、大腸菌鋳型を有する過酸化ポリピロール層が表面に形成された水晶振動子をセルの底部に備えたQCMセンサーを用いて微生物の検出を行なった。測定サンプルとしては、緑膿菌・大腸菌・アシネトバクターのそれぞれの溶液を用いた。
図18は、交流電圧印加時間と水晶振動子の共振周波数の関係を示すグラフである。図18に示す結果から、大腸菌を含む試料溶液を添加した検出実験では、共振周波数が大きく減少することが分かった。共振周波数の減少は水晶振動子表面の質量の増加を意味しており、大腸菌に誘電泳動力が作用し過酸化ポリピロール層の鋳型に取り込まれることで水晶振動子表面の質量が増加したものと考えられる。一方、形状の異なる緑膿菌やアシネトバクターに対してはブランクと同様にほとんど変化が見られなかった。よって、鋳型の形状とは異なる緑膿菌やアシネトバクターは大腸菌ほど容易に過酸化ポリピロール層に取り込まれていないと考えられ、センサーは細菌の種類を高精度に認識していると判断できる。
<センサーの作製>
緑膿菌を用いて、実施例1と同様に金電極の粗面化工程を行ない、さらに上述の重合工程、溶菌工程、過酸化工程を行なった。
(検出実験)
上述のようにして作製した、緑膿菌鋳型を有する過酸化ポリピロール層が表面に形成された水晶振動子をセルの底部に備えたQCMセンサーを用いて微生物の検出を行なった。測定サンプルとしては、緑膿菌・大腸菌・アシネトバクター・セラチア菌の各溶液を混合した溶液(a)と、大腸菌・アシネトバクター・セラチア菌の各溶液を混合した溶液(b)の2種類を用いた。
図19は、交流電圧印加時間と水晶振動子の共振周波数の関係を示すグラフである。図19に示す結果から、緑膿菌を含む試料溶液を添加した検出実験では、共振周波数が大きく減少することが分かった。共振周波数の減少は水晶振動子表面の質量の増加を意味しており、緑膿菌に誘電泳動力が作用し過酸化ポリピロール層の鋳型に取り込まれることで水晶振動子表面の質量が増加したものと考えられる。一方、形状の異なる大腸菌やアシネトバクターやセラチア菌に対してはブランク(c)と同様にほとんど変化が見られなかった。よって、鋳型の形状とは異なる大腸菌やアシネトバクターやセラチア菌は緑膿菌ほど容易に過酸化ポリピロール層に取り込まれていないと考えられ、センサーは細菌の種類を高精度に認識していると判断できる。
<センサーの作製>
緑膿菌、大腸菌、アシネトバクター、及びセラチア菌の全てを含む修飾溶液を用いて、実施例1と同様に金電極の粗面化工程を行ない、さらに上述の重合工程、溶菌工程、過酸化工程を行なった。
(検出実験)
上述のようにして作製した、4種類の微生物を含む鋳型を有する過酸化ポリピロール層が表面に形成された水晶振動子をセルの底部に備えたQCMセンサーを用いて微生物の検出を行なった。測定サンプルとしては、緑膿菌、大腸菌、アシネトバクター、セラチア菌をそれぞれを含む4種類の溶液を用いた。
図20~図23は、交流電圧印加時間と水晶振動子の共振周波数の関係を示すグラフである。図20~図23は、緑膿菌、大腸菌、アシネトバクター、セラチア菌をそれぞれを含む試料溶液を添加した検出実験における結果を示し、いずれの試料溶液を添加した場合であっても共振周波数が大きく減少することが分かった。これより、複数種の微生物の鋳型を有するセンサーによって、複数種の微生物が検出されていると判断できる。
Claims (14)
- 検出用電極と、前記検出用電極上に配置され、検出対象の微生物の立体構造に相補的な三次元構造の鋳型を備えたポリマー層とを有する検出部を備え、
前記鋳型への前記微生物の捕捉状態に基づいて前記微生物を検出するセンサーであって、
前記ポリマー層は、検出対象とする微生物の存在下でモノマーを重合して前記微生物を取り込んだ状態の前記ポリマー層を前記検出用電極上に形成する重合工程、前記ポリマー層に取り込まれた微生物を部分的に破壊する破壊工程、および前記ポリマー層を過酸化処理して前記ポリマー層から前記微生物を放出する過酸化工程を有する製造方法により形成される、センサー。 - 対電極をさらに備え、
前記検出部と前記対電極とを試料溶液に接触させた状態で、前記検出部の検出用電極と前記対電極間に交流電圧を印加し、誘電泳動により前記試料溶液中の微生物を前記検出部の方向に導く、請求項1に記載のセンサー。 - 前記検出部の前記検出用電極を一方の電極とする水晶振動子をさらに備え、
前記水晶振動子の共振周波数の変化から前記ポリマー層の質量の変化を測定して前記微生物の捕捉状態を検出する、請求項1または2に記載のセンサー。 - 前記モノマーが、ピロール、アニリン、チオフェンおよびそれらの誘導体からなる群から選択される、請求項1~3のいずれかに記載のセンサー。
- 前記モノマーが、ピロールまたはその誘導体からなる、請求項4に記載のセンサー。
- 前記検出用電極の前記ポリマー層の形成面が粗面である、請求項1~5のいずれかに記載のセンサー。
- 前記微生物は、全体または表面の電荷が負電荷過剰の状態にある、請求項1~6のいずれかに記載のセンサー。
- 前記微生物は細菌であり、前記破壊工程において溶菌処理を行なう請求項1~7のいずれかに記載のセンサー。
- 前記細菌は、緑膿菌、アシネトバクターまたは大腸菌である、請求項8に記載のセンサー。
- 検出用電極と、前記検出用電極上に配置され、微生物の立体構造に相補的な三次元構造の鋳型を備えたポリマー層とを有する検出部を備えた微生物を検出するセンサーの製造方法であって、
検出対象とする微生物の存在下でモノマーを重合して前記微生物を取り込んだ状態の前記ポリマー層を前記検出用電極上に形成する重合工程、
前記ポリマー層に取り込まれた微生物を部分的に破壊する破壊工程、および
前記ポリマー層を過酸化処理して前記ポリマー層から前記微生物を放出する工程、を有する製造方法。 - 前記センサーは対電極をさらに備え、
前記重合工程は、前記モノマーの溶液の接触下にある前記検出用電極と前記対電極との間に電圧を印加して、前記モノマーを電解重合する、請求項10に記載の製造方法。 - 前記過酸化工程は、中性からアルカリ性の範囲内の溶液の接触下にある前記検出用電極と前記対電極との間に電圧を印加して、前記ポリマー層を過酸化処理する、請求項10または11に記載の製造方法。
- 前記検出用電極の前記ポリマー層の形成面に粗面化処理を行なう粗面化工程を有する、請求項10~12のいずれかに記載の製造方法。
- 微生物の立体構造に相補的な三次元構造の鋳型を備えたポリマー層であって、
前記ポリマー層は、前記微生物の存在下でモノマーを重合して前記ポリマー層を形成する重合工程、前記ポリマー層に取り込まれた微生物を部分的に破壊する破壊工程、および前記ポリマー層から前記微生物を放出する過酸化工程を有する製造方法により製造される、ポリマー層。
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103454331A (zh) * | 2013-09-06 | 2013-12-18 | 南京理工大学 | 过氧化pedot/go修饰电极及其对农药吡虫啉的电化学检测方法 |
WO2014156584A1 (ja) * | 2013-03-28 | 2014-10-02 | シャープ株式会社 | 微生物検出用センサー、その製造方法、およびポリマー層 |
JP2015042958A (ja) * | 2013-08-26 | 2015-03-05 | 公立大学法人大阪府立大学 | 被検出微生物を検出する検出方法 |
WO2015166977A1 (ja) * | 2014-05-02 | 2015-11-05 | 公立大学法人大阪府立大学 | がん細胞検出用高分子膜及びその製造方法、並びにそれを用いたがん細胞検出装置 |
JP2017187463A (ja) * | 2016-04-05 | 2017-10-12 | シャープ株式会社 | センサ装置、検出方法、及びセンサユニット |
WO2017175879A1 (ja) * | 2016-04-05 | 2017-10-12 | シャープ株式会社 | センサ装置、検出方法、及びセンサユニット |
US9890991B2 (en) | 2013-03-14 | 2018-02-13 | Whirlpool Corporation | Domestic appliance including piezoelectric components |
JP2020091218A (ja) * | 2018-12-06 | 2020-06-11 | 東ソー株式会社 | 機能性物質固定化粒子の保持方法 |
KR20200074785A (ko) * | 2018-12-17 | 2020-06-25 | 한국과학기술연구원 | 수직 나노갭 전극을 이용한 유전영동 방법에 의한 고효율 바이오연료 생산 균주의 선별방법 |
Families Citing this family (5)
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---|---|---|---|---|
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CN108241056A (zh) * | 2016-12-23 | 2018-07-03 | 财团法人金属工业研究发展中心 | 生物检测装置 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003524417A (ja) * | 2000-02-11 | 2003-08-19 | バイオメリュー・インコーポレイテッド | バルク液から微生物を表面培養するための装置および方法 |
JP2009058232A (ja) * | 2007-08-29 | 2009-03-19 | Atect Corp | 分子鋳型を有するポリマーを備えたセンサー |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2161653C2 (ru) * | 1998-08-24 | 2001-01-10 | ФАРМАКОВСКИЙ Дмитрий Александрович | Способ количественного электрохимического анализа биомолекул |
US20040126814A1 (en) * | 2000-08-21 | 2004-07-01 | Singh Waheguru Pal | Sensor having molecularly imprinted polymers |
US6582971B1 (en) * | 2000-08-21 | 2003-06-24 | Lynntech, Inc. | Imprinting large molecular weight compounds in polymer composites |
US20090012446A1 (en) * | 2007-07-03 | 2009-01-08 | Xinyan Cui | Devices, systems and methods for release of chemical agents |
US20120258444A1 (en) * | 2010-11-18 | 2012-10-11 | Therrien Joel M | Acoustic wave (aw) sensing devices using live cells |
-
2012
- 2012-03-06 CN CN201280011897.7A patent/CN103459583B/zh not_active Expired - Fee Related
- 2012-03-06 US US14/003,613 patent/US9206461B2/en active Active
- 2012-03-06 EP EP12754214.0A patent/EP2684946B1/en active Active
- 2012-03-06 WO PCT/JP2012/055611 patent/WO2012121229A1/ja active Application Filing
- 2012-03-06 JP JP2013503547A patent/JP6014582B2/ja active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003524417A (ja) * | 2000-02-11 | 2003-08-19 | バイオメリュー・インコーポレイテッド | バルク液から微生物を表面培養するための装置および方法 |
JP2009058232A (ja) * | 2007-08-29 | 2009-03-19 | Atect Corp | 分子鋳型を有するポリマーを備えたセンサー |
Non-Patent Citations (5)
Title |
---|
HIROMI MIZOBATA ET AL.: "Bunshi Igata Kasanka Polypyrrole-maku o Mochiita QCM Kenshutsuki no Kaihatsu", FLOW INJECTION BUNSEKI KOENKAI KOEN YOSHISHU, vol. 48TH, 2009, pages 45 - 46, XP008170729 * |
HIROMI MIZOBATA ET AL.: "Bunshi Igata Kasanka Polypyrrole-maku o Riyo shita ATP no Kenshutsu", ABSTRACTS OF THE SYMPOSIUM OF THE JAPAN SOCIETY FOR ANALYTICAL CHEMISTRY, vol. 71ST, May 2010 (2010-05-01), pages 57, XP008170731 * |
HIROMI MIZOBATA ET AL.: "Bunshi Igata Kasanka Polypyrrole-maku o Riyo shita Rinsanki Gan'yu Busshitsu no Kenshutsu", THE JAPAN SOCIETY FOR ANALYTICAL CHEMISTRY NENKAI KOEN YOSHISHU, vol. 59TH, September 2010 (2010-09-01), pages 358, XP008170730 * |
TAKEDA S. ET AL.: "A Highly Sensitive Amperometric Adenosine Triphosphate Sensor Based on Molecularly Imprinted Overoxidized Polypyrrole.", J. FLOW INJECTION ANAL., vol. 25, no. 1, 2008, pages 77 - 79, XP055124178 * |
YU NAKADOI ET AL.: "Bunshi Igata Kasaka Polypyrrole-maku o Mochiita Virus Sensor no Kaihatsu", EXTENDED ABSTRACTS, JAPAN SOCIETY OF APPLIED PHYSICS AND RELATED SOCIETIES, vol. 58, 9 March 2011 (2011-03-09), pages 12 - 407, XP008170719 * |
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