Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
  • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
  • C12N11/087—Acrylic polymers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
  • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
  • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/795—Porphyrin- or corrin-ring-containing peptides
  • C07K14/80—Cytochromes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • 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
  • C12Q1/001—Enzyme electrodes
  • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
  • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
  • G01N2333/80—Cytochromes

Definitions

• Protein immobilization membrane Protein immobilization membrane, protein immobilization method, enzyme immobilization electrode, and biosensor
• the present invention relates to a technique for immobilizing a protein containing cytochrome on a material to be immobilized.
• a sensor configured to analyze a sample by an electrochemical method or an optical method is widely used as a nanosensor.
• An example of a biosensor for analyzing a sample by an electrochemical method is the biosensor 9 shown in FIG. 12 of the present application (see, for example, Patent Document 1).
• a cover 94 is bonded via a spacer 93 to a substrate 92 on which a working electrode 90 and a counter electrode 91 are formed.
• the biosensor 9 further has a channel 95 defined by a substrate 92, a spacer 93 and a cover 94.
• the flow path 95 is for moving the sample by capillary force, and a reagent part 96 is formed in the flow path 95.
• the reagent part 96 between the ends of the working electrode 90 and the counter electrode 91 It is formed so that it contains acid reductase.
• the oxidoreductase catalyzes a reaction in which, for example, the glucose force takes out electrons, and the electrons taken out from glucose are supplied to the working electrode 90.
• the amount of electrons supplied to the working electrode 90 can be measured as a response current using the working electrode 90 and the counter electrode 91.
• the material liquid containing an oxidoreductase is spotted on a target site of an object, the material liquid is dried to fix the acid reductase to the target site of the object. Use a trick.
• the second method is to use a cross-linking agent such as dartal aldehyde to target the target site with an acid.
• a cross-linking agent such as dartal aldehyde
• the third method is a method in which an acid reductase is immobilized together with a polymer in a state where a oxidoreductase is included in a polymer such as carbomethylcellulose (CMC).
• CMC carbomethylcellulose
• the fourth method uses a paste in which an acid reductase is dispersed in a conductive component such as a carbon paste, and this paste is applied to a target site of an object to immobilize the oxidoreductase. Is the method.
• Patent Document 1 Japanese Patent Publication No. 8-10208
• Non-Patent Document 1 Fumio Mizutani, “Application of enzyme modified electrode to sensor”, Analytical Science, Japan Society for Analytical Chemistry, September 1999, No. 48, No. 9, p809 ⁇ 821
• An object of the present invention is to fix a protein such as an acid reductase with good orientation and to express a desired activity appropriately and advantageously with a small amount of enzyme.
• Another object of the present invention is to make it possible to appropriately measure the concentration of a substrate such as glucose without using an electron transfer substance in a biosensor.
• a protein immobilization layer comprising a cell membrane-like structural layer and a protein that is immobilized on the cell membrane-like structural layer and contains cytochrome or a cytochrome complex.
• a capsule is provided.
• a first step of forming a cell membrane-like structural layer at a target site in the fixed member, and a cytochrome or cytochrome complex with respect to the cell membrane-like structural layer A second step of self-organizing the protein comprising: a protein immobilization method comprising:
• the protein immobilization method of the present invention further includes a third step that is performed prior to the first step and that performs a hydrophilic treatment on the target site.
• a base material and an enzyme-containing layer immobilized on the base material are provided, and the enzyme-containing layer includes a cell membrane-like structure layer, There is provided an enzyme-immobilized electrode comprising an enzyme having cytochrome as a subunit immobilized on a cell membrane-like structural layer in a self-organized state.
• a substrate and an enzyme-containing layer immobilized on the substrate are provided, and the enzyme-containing layer includes a cell membrane-like structural layer and the cell membrane-like layer. Fermentation using cytochromes immobilized as a subunit in the self-organized state of the structural layer And a biosensor is provided.
• the biosensor of the present invention further includes, for example, a flow path for moving a sample and a reagent part provided inside the flow path.
• the biosensor of the present invention is further provided with a working electrode and a counter electrode that are partly exposed in the flow path and are used to apply a voltage to the sample.
• a working electrode and a counter electrode that are partly exposed in the flow path and are used to apply a voltage to the sample.
• at least a part of the cell membrane-like structure layer is formed on the working electrode.
• the reagent part may also be formed as containing a color former!
• the reagent part includes, for example, a coloring layer containing a coloring agent, and a cell membrane-like structure layer and an enzyme-containing layer containing an enzyme.
• the cell membrane-like structural layer in the present invention includes, for example, a phospholipid polymer.
• a phospholipid polymer it is preferable to use a 2-methacryloyloxychetyl phosphorylcholine polymer.
• the cell membrane-like structural layer in the present invention is also preferably one into which a silane coupling agent is introduced.
• Tetraethoxysilane is preferably used as the silane coupling agent.
• a protein such as an enzyme in the present invention is, for example, CyGDH containing a subunit having glucose dehydrogenation activity and cytochrome C having an electron transfer function.
• FIG. 1 is an overall perspective view showing a biosensor according to a first embodiment of the present invention.
• FIG. 2 is an exploded perspective view of the biosensor shown in FIG.
• FIG. 3 is a cross-sectional view taken along the line ⁇ - ⁇ in FIG.
• FIG. 4 is an overall perspective view of a biosensor according to a second embodiment of the present invention.
• FIG. 5 is a cross-sectional view taken along line V—V in FIG.
• FIG. 6 is an AFM image showing the result of observation of the surface state of the carbon electrode by AFM in Example 1.
• FIG. 7 is an AFM image showing the result of observation by AFM of a state in which a single layer of phospholipid poly is formed on the surface of a carbon electrode in Example 1.
• FIG. 9 is a schematic diagram showing a schematic configuration of a current value measuring device used in Example 2.
• FIG. 10 is a graph showing measurement results of response current values in Example 2 as a time course.
• FIG. 11 is a graph showing the measurement result of the current value in Example 2 as a relationship with the glucose concentration.
• FIG. 12 is a cross-sectional view showing a main part of an example of a conventional biosensor.
• the biosensor XI shown in FIGS. 1 to 3 is configured to be disposable, and is used to measure a blood glucose level by being attached to a concentration measuring device (not shown).
• This biosensor XI is suitable for measuring blood glucose levels by an electrochemical method, and has a form in which a cover 3 is laminated on a long rectangular substrate 1 via a spacer 2. ing.
• each element 1 to 3 defines a cavity 4 extending in the longitudinal direction of the substrate 1 (Nl and N2 directions in the figure).
• the blood introduced from the introduction port 40 is converted into the longitudinal direction of the substrate 1 (Nl and N2 directions in the figure) using capillary action. It is for holding the introduced blood.
• the spacer 2 is for defining the distance from the upper surface 10 of the substrate 1 to the lower surface 30 of the cover 3, that is, the height dimension of the capillary 4, and is constituted by, for example, a double-sided tape.
• the spacer 2 is formed with a slit 20 having an open end.
• the slit 20 is for defining the width dimension of the barrier 4, and the open portion at the tip of the slit 20 constitutes an inlet 40 for introducing blood into the cavity 4.
• the cover 3 has an exhaust port 30 for exhausting the gas inside the pillar 4 to the outside.
• a cover 3 is made of a thermoplastic resin having high wettability such as vinylon or highly crystallized PVA.
• the substrate 1 is formed of an insulating grease material such as PET, and has a working electrode 11, a counter electrode 12, and an insulating film 13 on the upper surface 10 thereof. And the reagent part 14 is formed.
• the working electrode 11 and the counter electrode 12 are formed in an L-shape as a whole. More specifically, most of the working electrode 11 and the counter electrode 12 extend in the longitudinal direction of the substrate 1 (Nl and N2 directions in the figure), and the end portions 11a and 12a extend in the short direction of the substrate 1 ( (N3, N4 direction in the figure). On the other hand, the ends l ib and 12b of the working electrode 11 and the counter electrode 12 constitute a terminal part for contacting a terminal provided in a concentration measuring device (not shown).
• the working electrode 11 and the counter electrode 22 can be formed, for example, by screen printing using a carbon paste.
• the working electrode 11 and the counter electrode 12 can be formed using a conductive material other than carbon, and can also be formed by spin coating, thermal transfer, carbon rod slicing, vapor deposition, sputtering, or CVD.
• the insulating film 13 covers most of the working electrode 11 and the counter electrode 12 so as to expose the end portions 11a, 12a, l ib and 12b of the working electrode 11 and the counter electrode 12.
• the insulating film 13 has an opening 13a for exposing the end portions 11a and 12a of the working electrode 11 and the counter electrode 12.
• the opening 13a defines a region for forming the reagent part 14, and is formed in a long rectangular shape extending in the longitudinal direction of the substrate 1 (Nl and N2 directions in the figure).
• the insulating film 13 can be formed by screen printing using an ink containing a material having high water repellency or photolithography using a light-sensitive resin material.
• the reagent part 14 is provided so as to bridge the end parts 1 la and 12 a of the working electrode 11 and the counter electrode 12 in the opening 13 a of the insulating film 13.
• This reagent part 14 has a cell membrane-like structural layer 14A and a CyGDH layer 14B.
• the cell membrane-like structural layer 14A is for immobilizing CyGDH in an oriented state.
• This cell membrane-like structural layer 14A is a portion 14 ′ (hereinafter referred to as “exposed portion 14”) in which a solution containing a phospholipid polymer is exposed through the opening 13a of the insulating film 13 in the substrate 1, the working electrode 11, and the counter electrode 12. It can be formed by spotting ( ⁇ ) and drying.
• Examples of the phospholipid polymer include 2-methacryloyloxychetyl phosphorylcholine (
• MPC metal-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene-based polystyrene (for example, butyl methacrylate).
• a methacrylic acid ester for example, butyl methacrylate
• the phospholipid polymer for forming the cell membrane-like structural layer 14A may be a polymer containing monomer units having a structure similar to the phospholipid constituting the cell membrane in the molecule.
• Polymers other than MPC polymers can also be used.
• the phospholipid polymer it is preferable to use a polymer into which a silane coupling agent is introduced. This can increase the binding of the phospholipid polymer to the exposed portion 14 ⁇ .
• the exposed portion 14 ' is preferably subjected to a hydrophilic treatment.
• a hydrophilic group such as a hydroxyl group or a carboxyl group is introduced into the exposed portion 14 ', and the hydrophilic group is bonded to the silane coupling agent, thereby fixing the phospholipid polymer to the exposed portion 14' more firmly. be able to.
• the amount of the silane coupling agent in the polymer is, for example, 10 to 500 parts by weight with respect to 100 parts by weight of the polymer component.
• silane coupling agents include tetraethoxysilane, vinyltrichlorosilane, vinyltris (2-methoxyethoxy) silane, ⁇ —methacryloxypropyltrimethoxysilane, ⁇ —methacryloxypropyltriethoxysilane, j8 — (3, 4-Epoxycyclohexyl) etyltrimethoxysilane, ⁇ -glycidyl chloride, pill triethoxysilane, ⁇ - aminopropyltriethoxysilane, ⁇ - phenol ⁇ - Examples thereof include aminopropyltrimethoxysilane, ⁇ -chloropropyltrimethoxysilane, and ⁇ -mercaptopropyltrimethoxysilane.
• These silane coupling agents can be used alone or
• hydrophilic treatment of the exposed portion 14 ′ can be performed by various known methods.
• hydrophilic treatment examples include VUV treatment, UV treatment, corona discharge treatment, and plasma treatment.
• the CyGDH layer 14B is obtained by self-organizing and fixing CyGDH to the cell membrane-like structural layer 14A. Note that, in FIG. 3, the force in which Cy GDH is fixed on the surface of the cell membrane-like structural layer 14A is depicted. This is a schematic diagram shown for convenience in explaining the present plan. That is, the present inventors have confirmed that CyGDH is fixed in a self-organized manner with respect to the cell membrane-like structural layer 14A, but in what state CyGDH is with respect to the cell membrane-like structural layer 14A. It has not been confirmed at this stage, and since CyGDH derived from a microorganism belonging to Burkholderia's Sephacia described later is a transmembrane protein, it is not necessarily confirmed.
• CyGDH is not fixed only on the surface of the cell membrane-like structural layer 14A, but CyGDH penetrates the cell membrane-like structural layer 14A and can be immobilized on the cell membrane-like structural layer 14A. Is also included.
• the self-organized fixation of CyGDH to the cell membrane-like structural layer 14A is performed by, for example, immersing the substrate 1 having the cell membrane-like structural layer 14A formed on the exposed portion 14 'in an enzyme solution containing CyGDH.
• a certain process can be performed by spraying the enzyme solution on the cell membrane-like structural layer 14A and then drying it.
• CyGDH When CyGDH is fixed to the cell membrane-like structural layer 14A in a self-organizing manner, CyGDH is fixed in an oriented state, as can be inferred from the AFM image described later (see Fig. 8). I will be deceived. In other words, CyGDH is in a state where the active site of the ⁇ subunit is located on the surface layer of the reagent part 14, while cytochrome C is located near or in contact with the exposed part 14 ′ (working electrode 11). Fixed to the similar structure layer 14A.
• CyGDH used in the present invention refers to one containing at least an ⁇ subunit having glucose dehydrogenation activity and cytochrome C having an electron transfer function, Those having a subunit other than ⁇ subunit and cytochrome c are also included.
• An example of such CyGDH is disclosed in International Publication No. WO02 / 36779. CyGDH described in the earlier international application is derived from a microorganism belonging to Burkholderia 'sepasia, has a molecular weight of approximately 60 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and supplemented with FAD.
• the CyGDH of the present invention includes those obtained by using a transformant into which a gene encoding CyGDH collected from a microorganism belonging to Burkholderia's Sephacia has been transferred.
• CyGDH derived from a microorganism belonging to Burkholderia 'Sepacia is a transmembrane protein.
• CyGDH derived from the above microorganism originally exists in the cell membrane when such CyGDH is used, it exists in the cell membrane in a self-organized manner with respect to the cell membrane-like structural layer 14A.
• CyG DH can be immobilized in a state of orientation as in the case.
• Such self-organized immobilization of CyGDH can be achieved not only with CyGDH derived from microorganisms belonging to Burkholderia cepacia but also with CyGDH originally present in the cell membrane.
• Such a biosensor XI is attached to a concentration measuring device (not shown), and then supplied to blood 4 via the introduction port 40 of the biosensor XI.
• the blood glucose level can be automatically measured.
• the blood supply to the biosensor XI may be performed either before or after the biosensor XI is attached to the concentration measuring device (not shown). Usually, after incising the subject's skin to drain blood, the blood is attached to the inlet 40 of the biosensor XI.
• the concentration measuring device calculates the blood glucose level based on the response current value by measuring, for example, the amount of electron supply to the working electrode 11 as the response current value when the voltage is applied to the working electrode 11 and the counter electrode 12. Can do.
• CyGDH is immobilized with orientation, it is possible to suppress variation in the amount of CyGDH contained in the reagent part 14 and the orientation (position) of the active site for each biosensor XI. Can do. As a result, it is possible to suppress a variation in sensitivity for each of the nanosensors XI and perform an appropriate blood glucose level measurement.
• the biosensor XI since CyGDH is immobilized with orientation, cytochrome C exists at a position close to or in contact with the exposed portion 14 ′ (working electrode 11). Therefore, the reagent part 14 can efficiently supply electrons extracted from glucose to the working electrode 11. As a result, the biosensor XI can obtain an appropriate response current without using an electron transfer material such as a metal complex.
• the nanosensor X2 shown in FIG. 4 and FIG. 5 is configured to be suitable for measuring blood glucose level by an optical method, and thus the biosensor XI described above (see FIG. 1!). (See Figure 3).
• the nanosensor X2 has a configuration in which a cover 7 is laminated on a long rectangular substrate 5 via a pair of spacers 6.
• each element 5 to 7 defines a cavity 8 extending in the longitudinal direction of the substrate 5 (Nl and N2 directions in the figure).
• Kyapi Rally 8 is used to move the blood introduced from the introduction port 80 in the longitudinal direction of the substrate 5 (Nl and N2 directions in the figure) using capillary action and to hold the introduced blood. is there.
• a reagent part 51 is formed in the interior of the pillar 8. This reagent part 51 is obtained by forming a cell membrane-like structure layer 51B and a CyGHD layer 51C on the coloring layer 51A.
• the coloring layer 51A contains a coloring agent, and can be formed, for example, by applying a solution containing the coloring agent to a target site in the substrate 5 and then drying it.
• the color former that can be used in the present invention, for example, MTT (3- (4,5-Dimethy ⁇ 2-thiazolyl) -2,5-dipheny ⁇ 2H-tetrazolium bromide) ⁇ INT ( 2— (4— loaophen yl)-3- (4-nitrophenyl) -5-phenyto 2H- tetrazolium chloride), WST-4 (2- (4- lodopheny 1)-3- (2,4- dinitrophenyl)- 5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt), and 4AA (4-Aminoantipyrine).
• the cell membrane-like structural layer 51 and the 0011 layer 51 can be formed in the same manner as the biosensor XI described above (see FIGS. 1 to 3).
• the reagent part 51 includes the cell membrane-like structural layer 51B and the CyGDH layer 51C, the same as in the case of the biosensor XI described above (see FIGS. 1 to 3).
• the cell membrane similar structure layer 51B is in contact with the coloring layer 51A. Therefore, the reagent part 51 is in a state where CyGDH is oriented, that is, while the active site of the ⁇ subunit in CyGDH is present on the surface layer, cytochrome C in CyGDH is in contact with the coloring layer 51A, or on the coloring layer 51A. Immobilized in the cell membrane-like structure layer 51B in the state of being present nearby. Therefore, the biosensor X2 can enjoy the same effects as the previously described nanosensor XI (see FIGS. 1 to 3).
• the present invention is not limited to the above-described embodiment, and various modifications can be made.
• the present invention is not limited to a biosensor configured as a disposable, for example, a nanosensor that is used to continuously measure a blood glucose level by embedding at least an electrode part in a human body, and a concentration of a substrate other than glucose This can also be applied to biosensors of this type or enzyme electrodes for measuring the concentration of substrates such as glucose.
• a biosensor configured as a disposable, for example, a nanosensor that is used to continuously measure a blood glucose level by embedding at least an electrode part in a human body, and a concentration of a substrate other than glucose
• biosensors of this type or enzyme electrodes for measuring the concentration of substrates such as glucose.
• a carbon electrode, a phospholipid polymer layer, and a Cy GDH layer were formed on the surface of a PET substrate, and the surface properties before and after forming these layers were measured using an atomic force microscope (AFM). ) (Trade name “D-3100”; manufactured by Digital Instruments Inc.).
• the carbon electrode was formed by screen printing using carbon ink manufactured by Nippon Anchinson.
• Figure 6 shows the AFM image of the carbon electrode. As shown in Fig. 6, the surface of the carbon electrode layer was a relatively large uneven surface with carbon particles (average particle diameter of about lOOnm) appeared.
• the phospholipid polymer layer was formed by applying a VUV treatment (hydrophilic treatment) to the surface of the carbon electrode, and then applying the MPC polymer solution to the surface of the single bond electrode and drying it.
• the VUV treatment uses "MECL-M3-750" (EM 'D' Excimer Co., Ltd.), an excimer laser beam with a wavelength of 172 nm in the atmosphere, and an irradiation distance of lmm. This was done by irradiating the surface of the layer for 180 seconds.
• MPC polymer solution an MPC polymer (trade name “Rivisure”; manufactured by NOF Corporation) into which tetraethoxysilane as a silane coupling agent was introduced was used.
• Fig. 7 shows the AFM image after the formation of the phospholipid polymer layer.
• the diameter of the phospholipid part is 2 to 3 nm, which is smaller than that of the carbon particles, so the phospholipid polymer layer The surface of the layer was smoother than the carbon electrode layer! (See Figure 6).
• the CyGDH layer was formed by impregnating a carbon electrode on which a phospholipid polymer layer was formed with a CyGDH solution for 10 minutes.
• the concentration of CyGDH in the CyGDH solution was 100 UZ ⁇ based on the activity standard.
• the AFM image after the formation of the CyGDH layer is shown in FIG.
• CyGDH was immobilized on the phospholipid polymer so that at least a part of CyGDH appeared on the surface layer.
• CyGDH is immobilized on the phospholipid polymer layer with orientation.
• Example 1 As the proposed electrode, as in Example 1, a carbon electrode formed with a phospholipid polymer layer and CyGDH immobilized thereon was used.
• the comparative electrode was formed in the same manner as the present electrode except that it did not form a phospholipid polymer layer.
• the current value measuring device Y is provided with a working electrode Yl, a reference electrode 2 and a counter electrode 2, and these electrodes ⁇ 1 to ⁇ 3 are connected to a potentiostat ⁇ 4.
• the electrodes ⁇ 1 to ⁇ 3 can be immersed in a glucose solution to apply a voltage to the glucose solution, and the response current value at the time of voltage application can be measured.
• working electrode Y1 is the proposed electrode or comparative electrode formed by the method described above
• reference electrode ⁇ 2 is a silver ⁇ silver chloride electrode (trade name “RE-1B”; manufactured by BAS).
• the counter electrode Y3 is a platinum electrode.
• the current value described above was used with the proposed electrode as the working electrode Y1 for each of a plurality of types of glucose solutions having different concentrations.
• the sweep voltage is lOOmVZsec, 400mV to + 700mV
• the response current value was measured in the range.
• the glucose solution OmgZdL, 50 mgZdL, 1 OOmg / dL, 200 mgZdL, 400 mg / dL, and 600 mgZdL were used.
• the voltage applied to the glucose solution was set to +600 mV in the response current value measurement to be performed below.
• the responsiveness of the proposed electrode and the reference electrode is determined by using the current value measuring device Y described above for each of a plurality of types of glucose solutions having different concentrations as the working electrode Y1. This was done by measuring the course.
• the applied voltage at the time of response current measurement was +600 mV as described above, and glucose solutions of 0 mgZdL, 50 mgZdL, lOOmg / dL, 200 mg / dL, 400 mgZdL, and 600 mg ZdL were used.
• Figure 10 shows the time course of the response current value for each glucose solution.
• the response current value 1 second after the start of measurement is shown in Fig. 11 as the relationship of glucose concentration.
• the proposed electrode in which CyGDH is immobilized via a phospholipid polymer has sufficient response (sensitivity) to measure the concentration of dalcose without using an electron transfer material such as a metal complex. have. For this reason, the proposed electrode does not use an electron transfer material and is suitable for use.
• lucose concentration for example, blood glucose level
• the present invention can be applied without problems to a biosensor that is embedded in a human body and used to continuously measure blood glucose levels.
• the CyGDH fixation method used in the proposed electrode that is, the phospholipid polymer solution spotting and the CyGDH solution immersion is an extremely simple task.
• the phospholipid polymer layer and the CyGDH layer formed in the fine channel are very thin layers, even if these layers are formed, the movement of the sample in the fine channel is not significantly hindered. Therefore, in most of the microchannels, no problem occurs even if the reagent part composed of the phospholipid polymer layer and the CyGDH layer is formed. Therefore, by forming the reagent part over a wide range with respect to the fine channel, it is possible to improve the low sensitivity, which was a drawback of ⁇ TAS, and to provide a highly sensitive TAS.

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 2005
  • 2005-05-20 JP JP2005148253A patent/JP5021183B2/ja not_active Expired - Fee Related
• 2006
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• 2015
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