US20090101499A1 - Protein-Immobilized membrane, method for immobilization of protein, enzyme-immobilized electrode, and biosensor - Google Patents

Protein-Immobilized membrane, method for immobilization of protein, enzyme-immobilized electrode, and biosensor Download PDF

Info

Publication number
US20090101499A1
US20090101499A1 US11/920,782 US92078206A US2009101499A1 US 20090101499 A1 US20090101499 A1 US 20090101499A1 US 92078206 A US92078206 A US 92078206A US 2009101499 A1 US2009101499 A1 US 2009101499A1
Authority
US
United States
Prior art keywords
protein
immobilized
enzyme
cell membrane
layer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/920,782
Other languages
English (en)
Inventor
Koji Katsuki
Hideaki Yamaoka
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arkray Inc
Original Assignee
Arkray Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Arkray Inc filed Critical Arkray Inc
Assigned to ARKRAY, INC. reassignment ARKRAY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KATSUKI, KOJI, YAMAOKA, HIDEAKI
Publication of US20090101499A1 publication Critical patent/US20090101499A1/en
Abandoned legal-status Critical Current

Links

Images

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

  • the present invention relates to a technique for immobilizing a protein containing cytochrome to an immobilization target material.
  • Biosensors designed to analyze a sample by an electrochemical or optical method are widely used.
  • An example of biosensors designed to analyze a sample by an electrochemical method is a biosensor 9 shown in FIG. 12 of the present application.
  • the illustrated biosensor 9 includes a substrate 92 formed with a working electrode 90 and a counter electrode 91 , and a cover 94 bonded to the substrate via a spacer 93 .
  • the biosensor 9 further includes a flow path 95 defined by the substrate 92 , the spacer 93 and the cover 94 .
  • the flow path 95 is used for moving a sample by capillary force and formed with a reagent portion 96 .
  • the reagent portion 96 connects the ends of the working electrode 90 and the counter electrode 91 and contains oxidoreductase.
  • the oxidoreductase catalyzes the reaction of taking electrons from glucose, for example.
  • the electrons taken from the glucose are supplied to the working electrode 90 .
  • the amount of electrons supplied to the working electrode 90 is measured as the responsive current by utilizing the working electrode 90 and the counter electrode 91 .
  • the four methods described below are typical methods for forming a reagent portion 96 , i.e., the methods for immobilizing oxidoreductase (see Non-patent document 1, for example).
  • a material liquid containing oxidoreductase is applied to an intended portion of a target, and then the material liquid is dried. In this way, the oxidoreductase is immobilized to the intended portion of the target.
  • oxidoreductase is immobilized to an intended portion of a target by using a cross-linker such as glutaraldehyde.
  • oxidoreductase is contained in a polymer such carboxymethylcellulose (CMC), and then the oxidoreductase is immobilized together with the polymer.
  • CMC carboxymethylcellulose
  • oxidoreductase is dispersed in a conductive material such as a carbon paste, and the resultant paste is applied to an intended portion of a target, to immobilize the oxidoreductase.
  • oxidoreductase fails to be immobilized in a manner such that the active sites are oriented (located) to exhibit efficient activity of the oxidoreductase.
  • the conventional methods have a drawback that the immobilization is not performed with the orientation of the oxidoreductase being controlled.
  • active sites of oxidoreductase existing adjacent to each other may face each other or proteins may aggregate each other so that the active site exists within the aggregate.
  • the ratio of the oxidoreductase (active site) which can be utilized efficiently is relatively low.
  • the probability that oxidoreductase comes into contact with a substrate is relatively low, so that the activity of the immobilized oxidoreductase as a whole is low.
  • the amount of oxidoreductase to be loaded needs to be increased, which is disadvantageous in terms of cost.
  • oxidoreductases are generally expensive, the increase in the amount of oxidoreductase to be loaded leads to a considerably disadvantageous cost increase.
  • the biosensor 9 provided by immobilizing oxidoreductase by a conventional method is disadvantageous in terms of cost, because it requires an electron mediator.
  • the electron mediator metal complexes such as potassium ferrocyanide are used some of which have an adverse effect on the human body. Thus, it is not desirable to use an electron mediator for such an analytical tool as the biosensor 9 .
  • Patent document 1 JP-B-H08-10208
  • Non-patent document 1 MIZUTANI Fumio, “Application of enzyme-modified electrodes to biosensors,” BUNSEKI KAGAKU, Vol. 48, No. 9 pp. 809-821, The Japan Society for Analytical Chemistry, September, 1999.
  • An object of the present invention is to immobilize a protein such as oxidoreductase with good orientation and to cause the activity to be exhibited efficiently and advantageously in terms of cost with the use of a small amount of enzyme.
  • Another object of the present invention is to provide a biosensor which is capable of properly measuring the concentration of a substrate such as glucose without using an electron mediator.
  • a protein-immobilized membrane comprising a cell membrane homologous layer, and a protein immobilized to the cell membrane homologous layer, where the protein contains cytochrome or a cytochrome complex.
  • a method for immobilizing a protein comprises a first step of forming a cell membrane homologous layer at an intended portion of an immobilization target member, and a second step of causing self organization of a protein with respect to the cell membrane homologous layer, the protein containing cytochrome or a cytochrome complex.
  • the protein immobilization method according to the present invention further comprises a third step of subjecting the intended portion to hydrophilic treatment before the first step.
  • an enzyme-immobilized electrode comprising a substrate, and an enzyme-containing layer immobilized to the substrate.
  • the enzyme-containing layer includes a cell membrane homologous layer and an enzyme.
  • the enzyme contains, as a subunit, cytochrome C immobilized to the cell membrane homologous layer by self organization.
  • a biosensor comprising a substrate, and an enzyme-containing layer immobilized to the substrate.
  • the enzyme-containing layer includes a cell membrane homologous layer and an enzyme.
  • the enzyme contains, as a subunit, cytochrome C immobilized to the cell membrane homologous layer by self organization.
  • the biosensor according to the present invention may further comprise a flow path for moving a sample, and a reagent portion provided in the flow path.
  • the biosensor according to the present invention may further comprise a working electrode and a counter electrode which are partially exposed at the flow path and utilized for applying a voltage to a sample.
  • a working electrode and a counter electrode which are partially exposed at the flow path and utilized for applying a voltage to a sample.
  • at least part of the cell membrane homologous layer is formed on the working electrode.
  • the reagent portion may contain a color former.
  • the reagent portion may include a chromogenic layer containing a color former, a cell membrane homologous layer, and a layer containing an enzyme.
  • the cell membrane homologous layer in the present invention may contain a phospholipid polymer.
  • a phospholipid polymer it is preferable to use 2-methacryloyloxyethyl phosphorylcholine polymer.
  • the cell membrane homologous layer in the present invention contains a silane coupling agent.
  • a silane coupling agent it is preferable to use tetraethoxysilane.
  • the protein such as an enzyme in the present invention is CyGDH containing an ⁇ subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.
  • 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. 1 .
  • FIG. 3 is a sectional view taken along lines III-III in FIG. 1 , a principal portion of which is shown as enlarged.
  • FIG. 4 is an overall perspective view showing a biosensor according to a second embodiment of the present invention.
  • FIG. 5 is a sectional view taken along lines V-V in FIG. 4 , a principal portion of which is shown as enlarged.
  • FIG. 6 is an AFM image showing the observation results of the surface condition of a carbon electrode using an AFM in Example 1.
  • FIG. 7 is an AFM image showing the observation results of the condition of a phospholipid polymer layer formed on the carbon electrode surface using an AFM in Example 1.
  • FIG. 8 is an AFM image showing the observation results of CyGDH immobilized to the surface of the phospholipid polymer layer using an AFM in Example 1.
  • FIG. 9 is a schematic view showing the structure of a current measuring apparatus used in Example 2.
  • FIG. 10 is a graph showing the time-course measurements of the responsive current in Example 2.
  • FIG. 11 is a graph showing the measurements of the responsive current in Example 2 in relation to glucose level.
  • FIG. 12 is a sectional view showing a principal portion of an example of conventional biosensor.
  • the biosensor X 1 shown in FIGS. 1-3 is a disposable sensor to be mounted to a concentration measuring apparatus (not shown) to measure a blood glucose level.
  • the biosensor X 1 is adapted to measure the blood glucose level by an electrochemical method and includes a substrate 1 , which is in the form of an elongated rectangle, and a cover 3 laminated on the substrate via a spacer 2 .
  • a capillary 4 extending in the longitudinal direction of the substrate 1 (N 1 , N 2 directions in the figures) is defined by the elements 1 - 3 .
  • the capillary 4 is utilized for moving the blood introduced from an introduction port 40 in the longitudinal direction of the substrate 1 (N 1 , N 2 directions in the figures) utilizing capillary action and retaining the introduced blood.
  • the spacer 2 defines the distance from the upper surface 10 of the substrate 1 to the lower surface 30 of the cover 3 , i.e., the height of the capillary 4 and may comprise a double-sided tape.
  • the spacer 2 is formed with a slit 20 having an open end.
  • the slit 20 defines the width of the capillary 4 .
  • the open end of the slid 20 serves as the introduction port 40 for introducing blood into the capillary 4 .
  • the cover 3 includes an exhaust port 30 for discharging gas from the capillary 4 .
  • the cover 3 is made of a thermoplastic resin having a high wettability, such as Vinylon or highly crystalline PVA.
  • the upper surface 10 of the substrate 1 which is made of an insulating resin such as PET, is formed with a working electrode 11 , a counter electrode 12 , an insulating film 13 and a reagent portion 14 .
  • Each of the working electrode 11 and the counter electrode 12 is L-shaped as a whole. Specifically, the working electrode 11 and the counter electrode 12 mostly extend in the longitudinal direction of the substrate 1 (N 1 , N 2 directions in the figures) and respectively include ends 11 a and 12 a extending in the width direction (N 3 , N 4 directions in the figures). The working electrode 11 and the counter electrode 12 further include ends 11 b and 12 b , respectively, which provide terminals for coming into contact with the terminals of the concentration measuring apparatus (not shown).
  • the working electrode 11 and the counter electrode 12 may be formed by screen printing using carbon paste.
  • the working electrode 11 and the counter electrode 12 may be made of a conductive material other than carbon by spin coating, thermal transfer, carbon rod slice, vapor deposition, sputtering or CVD.
  • the insulating film 13 covers most part of the working electrode 11 and the counter electrode 12 while exposing the ends 11 a , 12 a , 11 b and 12 b of the working electrode 11 and the counter electrode 12 .
  • the insulating film 13 includes an opening 13 a for exposing the ends 11 a and 12 a of the working electrode 11 and the counter electrode 12 .
  • the opening 13 a defines the region for forming the reagent portion 14 and is in the form of a rectangle elongated in the longitudinal direction of the substrate 1 (N 1 , N 2 directions in the figures).
  • the insulating film 13 may be formed by screen printing using ink containing a material having high water repellency or photolithography using a photosensitive resin.
  • the reagent portion 14 is arranged to bridge the ends 11 a and 12 a of the working electrode 11 and the counter electrode 12 at the opening 13 a of the insulating film 13 .
  • the reagent portion 14 includes a cell membrane homologous layer 14 A and a CyGDH layer 14 B.
  • the cell membrane homologous layer 14 A is utilized for immobilizing CyGDH with controlled orientation.
  • the cell membrane homologous layer 14 A may be formed by applying a solution containing phospholipid polymer to the portion 14 ′ of the working electrode 11 and the counter electrode 12 which is exposed through the opening 13 a of the insulating film 13 (hereinafter, the portion 14 ′ is referred to as “exposed portion 14 ′”) and then drying the solution.
  • phospholipid polymer use may be made of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, for example.
  • MPC 2-methacryloyloxyethyl phosphorylcholine
  • polymers other than MPC polymer may be used as long as the polymer contains a monomeric unit having a structure similar to phospholipid forming a cell membrane in the molecules.
  • the phospholipid polymer it is preferable to use one to which a silane coupling agent is added. In this case, the phospholipid polymer is reliably bonded to the exposed portion 14 ′.
  • the exposed portion 14 ′ it is preferable to subject the exposed portion 14 ′ to hydrophilic treatment in advance.
  • hydrophilic groups such as a hydroxyl group or a carboxyl group enters the exposed portion 14 ′ and is bonded to the silane coupling agent.
  • phospholipid polymer is more strongly fixed to the exposed portion 14 ′.
  • the amount of the silane coupling agent in the polymer may be set to 10 to 500 parts by weight relative to 100 parts by weight of the polymer component.
  • silane coupling agent include: tetraethoxysilane; vinyltrichlorosilane; vinyl-tris(2-methoxyethoxy)silane; ⁇ -methacryloxypropyltrimethoxysilane; ⁇ -methacryloxypropyltriethoxysilane; ⁇ -(3,4-epoxycyclohexyl)ethyltrimethoxysilane; ⁇ -glycidoxypropyltriethoxysilane; ⁇ -aminopropyltriethoxysilane; N-phenyl- ⁇ -aminopropyltrimethoxysilane; ⁇ -chloropropyltrimethoxysilane; and ⁇ -mercaptopropyltrimethoxysilane. These silane coupling agents may be used solely or in combination.
  • hydrophilic treatment of the exposed portion 14 ′ can be performed by various known techniques.
  • hydrophilic treatment which can be employed in the present invention include VUV treatment, UV treatment, corona discharge and plasma treatment.
  • the CyGDH layer 14 B is provided by immobilizing CyGDH self-organizingly to the cell membrane homologous layer 14 A.
  • FIG. 3 shows the state in which CyGDH is immobilized to the surface of the cell membrane homologous layer 14 A, this figure is a schematic view for describing the present invention.
  • CyGDH is self-organizingly immobilized to the cell membrane homologous layer 14 A
  • the inventors have not yet found out how CyGDH is immobilized to the cell membrane homologous layer 14 A.
  • CyGDH derived from a microorganism belonging to the burkhorderia cepacia which will be described later, is a transmembrane protein.
  • CyGDH may not be immobilized only at the surface of the cell membrane homologous layer 14 A, as shown in FIG. 3 , but may be immobilized to the cell membrane homologous layer 14 A while penetrating the cell membrane homologous layer 14 A.
  • the self-organizing immobilization of CyGDH to the cell membrane homologous layer 14 A may be performed by immersing the substrate 1 provided with the cell membrane homologous layer 14 A at the exposed portion 14 ′ into an enzyme solution containing CyGDH or spraying the enzyme solution to the cell membrane homologous layer 14 A and then drying the solution.
  • CyGDH is immobilized with controlled orientation. Specifically, CyGDH is so immobilized to the cell membrane homologous layer 14 A that the active site of the ⁇ subunit is positioned at the surface of the reagent portion 14 , whereas cytochrome C is positioned close to or in contact with the exposed portion 14 ′ (working electrode 11 ).
  • CyGDH use is made of those which at least contain an ⁇ subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.
  • CyGDH further containing a subunit other than ⁇ subunit and cytochrome C may be used. Examples of such CyGDH are disclosed in international publication WO02/36779.
  • the CyGDH disclosed in this international publication is derived from a microorganism belonging to the burkholderia cepacia and includes an ⁇ subunit having a molecular weight of about 60 kDa in SDS-polyacrylamide gel electrophoresis under a reduced condition, including FAD as a cofactor and having a glucose dehydrogenase activity, and cytochrome C having a molecular weight of about 43 kDa in SDS-polyacrylamide gel electrophoresis under a reduced condition and having a function of electron transfer.
  • the CyGDH in the present invention further includes one prepared by utilizing a transformant to which a gene encoding CyGDH taken from a microorganism belonging to the burkholderia cepacia is transferred.
  • the CyGDH derived from a microorganism belonging to the burkhorderia cepacia is a transmembrane protein. That is, the CyGDH derived from this microorganism originally exists in a cell membrane.
  • CyGDH is immobilized to the cell membrane homologous layer 14 A by self organization with controlled orientation similarly to that in existing in a cell membrane.
  • Such self-organizing immobilization of CyGDH is possible not only when CyGDH derived from a microorganism belonging to the burkhorderia cepacia is used but also when CyGDH originally existing in a cell membrane is used.
  • the biosensor X 1 having the above-described structure is mounted to a concentration measuring apparatus (not shown) and blood is introduced to the capillary 4 through the introduction port 40 of the biosensor X 1 , the blood glucose level is measured automatically at the concentration measuring apparatus (not shown).
  • the introduction of blood to the biosensor X 1 may be performed either before or after the biosensor is mounted to the concentration measuring apparatus (not shown). Generally, blood is introduced by cutting the skin of the person to be tested to cause bleeding and then applying the blood to the introduction port 40 of the biosensor X 1 .
  • the working electrode 11 and the counter electrode 12 of the biosensor X 1 come into contact with the terminals (not shown) of the concentration measuring apparatus.
  • the blood applied to the introduction port 40 moves toward the exhaust port 30 due to capillary action at the capillary 4 and fills the capillary 4 .
  • CyGDH reacts specifically with the glucose in the blood to take electrons from the glucose.
  • the electrons taken out by the CyGDH are transferred to the working electrode 11 .
  • the concentration measuring apparatus (not shown), when a voltage is applied to the working electrode 11 and the counter electrode 12 , the amount of electrons transferred to the working electrode 11 , for example, is measured as the responsive current. Based on the responsive current, the blood glucose level is computed.
  • CyGDH is immobilized with controlled orientation, the amount of CyGDH contained in the reagent portion 14 and the orientation (position) of the active site do not vary among biosensors X 1 . Thus, variation in sensitivity among the biosensors X 1 does not occur, so that the blood glucose level measurement is performed properly.
  • CyGDH is immobilized with controlled orientation in the biosensor X 1 , cytochrome C exists close to or in contact with the exposed portion 14 ′ (working electrode 11 ). Thus, in the reagent portion 14 , electrons taken from the glucose are efficiently transferred to the working electrode 11 . Thus, in the biosensor X 1 , proper responsive current is obtained without using an electron mediator such as a metal complex.
  • the biosensor X 2 shown in FIGS. 4 and 5 is adapted to measure the blood glucose level by an optical method.
  • the biosensor X 2 includes a substrate 5 , which is in the form of an elongated rectangle, and a cover 7 laminated on the substrate via a pair of spacers 6 .
  • a capillary 8 extending in the longitudinal direction of the substrate 5 (N 1 , N 2 directions in the figures) is defined by the elements 5 - 7 .
  • the capillary 8 is used for moving the blood introduced from an introduction port 80 in the longitudinal direction of the substrate 5 (N 1 , N 2 directions in the figures) utilizing capillary action and retaining the introduced blood.
  • a reagent portion 51 is provided in the capillary 8 .
  • the reagent portion 51 includes a chromogenic layer 51 A, and a cell membrane homologous layer 51 B and a CyGDH layer 51 C which are formed on the chromogenic layer 51 A.
  • the chromogenic layer 51 A includes a color former and may be formed by applying a solution containing a color former to an intended portion of the substrate 5 and then drying the solution.
  • MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide); INT(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazoli um chloride); WST-4(2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfop henyl)-2H-tetrazolium,monosodium salt); and
  • the cell membrane homologous layer 51 B and the CyGDH layer 51 C can be formed similarly to those of the foregoing biosensor X 1 (see FIGS. 1-3 ).
  • the reagent portion 51 includes a cell membrane homologous layer 51 B and a CyGDH layer 51 C, similarly to the biosensor X 1 (see FIGS. 1-3 ). Further, the cell membrane homologous layer 51 B is held in contact with the chromogenic layer 51 A. Thus, in the reagent portion 51 , CyGDH is immobilized with controlled orientation, i.e., with the active site of the ⁇ subunit positioned at the surface whereas cytochrome C is positioned in contact with or close to chromogenic layer 51 A. Thus, the biosensor X 2 has the same advantages as those of the biosensor X 1 (see FIGS. 1-3 ).
  • the present invention is not limited to the foregoing embodiments and may be modified in various ways.
  • the present invention is not limited to a disposable biosensor and is also applicable to a biosensor used for monitoring the blood glucose level with at least the electrode portion embedded in the human body.
  • the invention is also applicable to a biosensor for measuring the concentration of a substrate other than glucose or to an enzyme electrode for measuring the concentration of a substrate such as glucose.
  • a carbon electrode, a phospholipid polymer layer and a CyGDH layer were formed on a surface of a PET substrate.
  • the conditions of the surface before and after the formation of these layers were observed using an atomic force microscope (AFM) (Tradename “D-3100” available from Digital Instruments).
  • the carbon electrode was formed by screen printing using a carbon ink available from Acheson Japan Ltd.
  • the AFM image of the carbon electrode is shown in FIG. 6 .
  • the surface of the carbon electrode had relatively large irregularities, with carbon particles (having average particle size of about 100 nm) appearing on the surface.
  • the surface of the carbon electrode was first subjected to VUV treatment (hydrophilic treatment). Then, MPC polymer solution was applied to the surface of the carbon electrode and then dried, whereby the phospholipid polymer layer was formed.
  • the VUV treatment was performed by irradiating the surface of the carbon electrode with excimer laser having a wavelength of 172 nm in the atmosphere for 180 seconds with the irradiation distance of 1 mm by using “MECL-M3-750” (available from M.D. Excimer Inc.).
  • MPC polymer solution use was made of a solution of MPC polymer containing tetraethoxysilane as a silane coupling agent (Tradename “LIPIDURER” available from NOF CORPORATION).
  • FIG. 7 The AFM image after the formation of the phospholipid polymer layer was shown in FIG. 7 .
  • the phospholipid portion of the polymer appeared on the surface of the phospholipid polymer layer, the surface of the phospholipid polymer was smooth as compared with that of the carbon electrode layer (see FIG. 6 ), because the diameter of the phospholipid portion was about 2 to 3 nm which was smaller than that of carbon particles.
  • the CyGDH layer was formed by immersing the carbon electrode formed, with the phospholipid polymer layer in a CyGDH solution for ten minutes.
  • the concentration of CyGDH in the CyGDH solution was 100 U/ ⁇ L on the activity basis.
  • the AFM image after the formation of the CyGDH layer is shown in FIG. 8 .
  • the surface of the phospholipid polymer layer was formed with regularly arranged clusters (CyGDH) each having a diameter of about 6 to 30 nm. That is, CyGDH was immobilized to the phospholipid polymer in such a manner that at least part of CyGDH appeared on the surface. From the fact that the clusters are arranged regularly, it is presumed that CyGDH is immobilized to the phospholipid polymer layer with controlled orientation.
  • CyGDH regularly arranged clusters
  • responsiveness was examined with respect to an electrode (inventive electrode) to which CyGDH is immobilized via a phospholipid polymer layer and to an electrode (comparative electrode) to which CyGDH is immobilized without the intervention of a phospholipid polymer layer.
  • the inventive electrode was prepared by forming a phospholipid polymer layer on a carbon electrode and then immobilizing CyGDH, similarly to Example 1.
  • the comparative electrode was prepared similarly to the inventive electrode except that a phospholipid polymer layer was not formed.
  • the responsiveness of the inventive electrode and the comparative electrode was evaluated as the responsive current obtained when a voltage was applied to a glucose solution using a current measuring apparatus Y prepared as shown in FIG. 9 .
  • the current measuring apparatus Y includes a working electrode Y 1 , a reference electrode Y 2 and a counter electrode Y 2 , which are connected to a potentiostat Y 4 .
  • the current measuring apparatus Y is designed to measure the responsive current by immersing the electrodes Y 1 -Y 3 in a glucose solution and applying a voltage to the glucose solution.
  • the working electrode Y 1 is the inventive electrode or the comparative electrode prepared in the above-described manner.
  • the reference electrode Y 2 is a silver-silver chloride electrode (Tradename “RE-1B”; available from BAS Inc.).
  • the counter electrode Y 3 is a platinum electrode.
  • the sweep voltage was 100 mV/sec, and the responsive current was measured with respect to the range of ⁇ 400 mV to +700 mV.
  • the glucose solutions had the concentrations of 0 mg/dL, 50 mg/dL, 100 mg/dL, 200 mg/dL, 400 mg/dL and 600 mg/dL, respectively.
  • the voltage to be applied to the glucose solutions was set to +600 mV.
  • the responsiveness of the inventive electrode and the comparative electrode was evaluated by measuring the time course of the responsive current with respect to each of the glucose solutions of different concentrations.
  • the measurement was performed using the above-described current measuring apparatus Y employing the inventive electrode or the comparative electrode as the working electrode Y 1 .
  • the voltage of +600 mV was applied in measuring the responsive current.
  • the concentrations of the used glucose solutions were 0 mg/dL, 50 mg/dL, 100 mg/dL, 200 mg/dL, 400 mg/dL and 600 mg/dL, respectively.
  • the time course of the responsive current with respect to each of the glucose solutions is shown in FIG. 10 .
  • the responsive current one second after the start of the measurement is shown in FIG. 11 in relation to the glucose level.
  • the inventive electrode when the inventive electrode was used, the responsive current in the p order was measured. However, when the comparative electrode was used, merely the responsive current in the n order was measured. Specifically, the results obtained when the comparative electrode was used were similar to the conventionally reported measurement results (n order) of the responsive current obtained when use was made of a system which does not include an electron mediator such as a metal complex. When the inventive electrode was used, on the other hand, the responsive current in the u order which was much higher than the conventionally reported level was measured. Thus, it is demonstrated that the inventive electrode has high responsiveness (sensitivity).
  • the inventive electrode when the inventive electrode is used, the difference in glucose level is properly reflected as the difference in responsive current.
  • the glucose level is measured properly at least in the glucose level range (0 to 600 mg/dL) with respect to which the responsive current was measured in this example.
  • the inventive electrode in which CyGDH is immobilized via a phospholipid polymer layer has sufficient responsiveness (sensitivity) to properly measure the glucose level without using an electron mediator such as a metal complex.
  • an electron mediator such as a metal complex.
  • the inventive electrode proper measurement of the glucose level (e.g. blood glucose level) without using an electron mediator is possible. Since an electron mediator is not used, to embed the inventive electrode in the human body for use causes no harm to the human body.
  • the present invention is applicable to a biosensor to be embedded in the human body to monitor the blood glucose level.
  • the method for immobilizing CyGDH which is employed for the inventive electrode i.e., the application of a phospholipid polymer solution and the immersion in a CyGDH solution is a very easy work.
  • this method is applicable to a biosensor including minute paths such as ⁇ TAS. Since the phospholipid polymer layer and the CyGDH layer formed at the minute paths are extremely thin, the formation of these layers does not considerably hinder the movement of a sample in the minute paths. Thus, the provision of a reagent portion, which is made up of a phospholipid polymer layer and a CyGDH layer, at most part of the minute paths does not cause any problems.
  • the sensitivity of the ⁇ TAS which has been disadvantageously low, is improved. In this way, a ⁇ TAS having a high sensitivity can be provided.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Emergency Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US11/920,782 2005-05-20 2006-05-18 Protein-Immobilized membrane, method for immobilization of protein, enzyme-immobilized electrode, and biosensor Abandoned US20090101499A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2005148253A JP5021183B2 (ja) 2005-05-20 2005-05-20 タンパク質固定化膜および固定化方法、ならびにバイオセンサ
JP2005-148253 2005-05-20
PCT/JP2006/309906 WO2006123730A1 (ja) 2005-05-20 2006-05-18 タンパク質固定膜、タンパク質の固定化方法、酵素固定化電極、およびバイオセンサ

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/309906 A-371-Of-International WO2006123730A1 (ja) 2005-05-20 2006-05-18 タンパク質固定膜、タンパク質の固定化方法、酵素固定化電極、およびバイオセンサ

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/850,576 Division US9702843B2 (en) 2005-05-20 2015-09-10 Biosensor incorporating protein-immobilized membrane and method of immobilizing protein in biosensor

Publications (1)

Publication Number Publication Date
US20090101499A1 true US20090101499A1 (en) 2009-04-23

Family

ID=37431302

Family Applications (2)

Application Number Title Priority Date Filing Date
US11/920,782 Abandoned US20090101499A1 (en) 2005-05-20 2006-05-18 Protein-Immobilized membrane, method for immobilization of protein, enzyme-immobilized electrode, and biosensor
US14/850,576 Expired - Fee Related US9702843B2 (en) 2005-05-20 2015-09-10 Biosensor incorporating protein-immobilized membrane and method of immobilizing protein in biosensor

Family Applications After (1)

Application Number Title Priority Date Filing Date
US14/850,576 Expired - Fee Related US9702843B2 (en) 2005-05-20 2015-09-10 Biosensor incorporating protein-immobilized membrane and method of immobilizing protein in biosensor

Country Status (5)

Country Link
US (2) US20090101499A1 (enExample)
EP (1) EP1884771B1 (enExample)
JP (1) JP5021183B2 (enExample)
CN (1) CN101203748B (enExample)
WO (1) WO2006123730A1 (enExample)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090177067A1 (en) * 2003-09-02 2009-07-09 Arkray, Inc. Glucose Sensor and Glucose Level Measuring Apparatus
US20110136158A1 (en) * 2009-04-30 2011-06-09 Takenaka Ryo Protein electron mediator
USD665278S1 (en) * 1999-11-04 2012-08-14 Abbott Diabetes Care Inc. Analyte sensor
US8858768B2 (en) 2009-02-09 2014-10-14 Arkray, Inc. Electrochemical sensor and method for manufacturing same
US10974246B2 (en) 2015-06-08 2021-04-13 Japan Science And Technology Agency High-density micro-chamber array and measurement method using same

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008243380A (ja) * 2007-03-23 2008-10-09 Sony Corp 酵素固定化電極、燃料電池、電子機器、酵素反応利用装置および酵素固定化基体
US8268604B2 (en) * 2007-12-20 2012-09-18 Abbott Point Of Care Inc. Compositions for forming immobilized biological layers for sensing
US9653006B2 (en) 2008-09-17 2017-05-16 Avery Dennison Corporation Activatable adhesive, labels, and related methods
JP5432575B2 (ja) * 2009-04-21 2014-03-05 グンゼ株式会社 バイオセンサ及びその製造方法
CN102449086B (zh) 2009-09-17 2014-08-13 艾利丹尼森公司 可活化粘合剂、标签及相关方法
JP5665070B2 (ja) * 2009-09-25 2015-02-04 独立行政法人産業技術総合研究所 酸化還元タンパク質固定化ナノ構造電極
CN103344639B (zh) * 2013-07-11 2015-10-28 山东大学 一种快速检测尿中吡咯加合物的检测管
CN105492902B (zh) * 2013-08-07 2020-07-24 爱科来株式会社 使用电化学式生物传感器的物质测量方法和测量装置
CN105355530B (zh) * 2015-10-30 2017-08-11 三诺生物传感股份有限公司 一种用于试条电极预处理的等离子处理装置和方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6773563B2 (en) * 2000-08-09 2004-08-10 Nec Corporation Electrochemical sensor having a reference electrode
US20060035300A1 (en) * 2002-06-17 2006-02-16 Arkray, Inc Method of measuring glucose concentration and glucose sensor with the use of glucose dehydrogenase
US20060258959A1 (en) * 2003-09-02 2006-11-16 Koji Sode Glucose sensor and glucose level measuring apparatus
US7604887B2 (en) * 2002-07-25 2009-10-20 Panasonic Corporation Electrolyte membrane, membrane electrode assembly using this and fuel cell
US7713544B2 (en) * 2000-07-28 2010-05-11 Emory University Biological component comprising artificial membrane
US7741090B2 (en) * 2000-10-31 2010-06-22 Koji Sode Glucose dehydrogenase and process for producing the dehydrogenase

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0810208A (ja) 1994-06-30 1996-01-16 Toshiba Corp 食器洗浄機
US5650062A (en) * 1995-03-17 1997-07-22 Matsushita Electric Industrial Co., Ltd. Biosensor, and a method and a device for quantifying a substrate in a sample liquid using the same
CN1321245A (zh) * 1999-09-13 2001-11-07 松下电器产业株式会社 脂质修饰酶的制备方法及生物传感器
JP2003525320A (ja) * 2000-02-18 2003-08-26 アスピラ バイオシステムズ，インコーポレイティド 分子インプリンティング組成物、並びに高分子の結合及び分析方法
US6458599B1 (en) 2000-02-18 2002-10-01 Aspira Biosystems, Inc. Compositions and methods for capturing, isolating, detecting, analyzing and quantifying macromolecules
JP4385219B2 (ja) * 2001-09-14 2009-12-16 アークレイ株式会社 濃度測定方法
JP4250481B2 (ja) * 2003-08-11 2009-04-08 キヤノン株式会社 多孔質構造体
WO2005030807A1 (ja) * 2003-09-30 2005-04-07 Koji Sode グルコース脱水素酵素とシトクロームとの融合蛋白質

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7713544B2 (en) * 2000-07-28 2010-05-11 Emory University Biological component comprising artificial membrane
US6773563B2 (en) * 2000-08-09 2004-08-10 Nec Corporation Electrochemical sensor having a reference electrode
US7741090B2 (en) * 2000-10-31 2010-06-22 Koji Sode Glucose dehydrogenase and process for producing the dehydrogenase
US20060035300A1 (en) * 2002-06-17 2006-02-16 Arkray, Inc Method of measuring glucose concentration and glucose sensor with the use of glucose dehydrogenase
US7604887B2 (en) * 2002-07-25 2009-10-20 Panasonic Corporation Electrolyte membrane, membrane electrode assembly using this and fuel cell
US20060258959A1 (en) * 2003-09-02 2006-11-16 Koji Sode Glucose sensor and glucose level measuring apparatus

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USD665278S1 (en) * 1999-11-04 2012-08-14 Abbott Diabetes Care Inc. Analyte sensor
USD665279S1 (en) * 1999-11-04 2012-08-14 Abbott Diabetes Care Inc. Analyte sensor
US20090177067A1 (en) * 2003-09-02 2009-07-09 Arkray, Inc. Glucose Sensor and Glucose Level Measuring Apparatus
US8277636B2 (en) 2003-09-02 2012-10-02 Koji Sode Glucose sensor and glucose level measuring apparatus
US8858768B2 (en) 2009-02-09 2014-10-14 Arkray, Inc. Electrochemical sensor and method for manufacturing same
US20110136158A1 (en) * 2009-04-30 2011-06-09 Takenaka Ryo Protein electron mediator
US8969025B2 (en) 2009-04-30 2015-03-03 Panasonic Healthcare Holdings Co., Ltd. Protein electron mediator
US10974246B2 (en) 2015-06-08 2021-04-13 Japan Science And Technology Agency High-density micro-chamber array and measurement method using same

Also Published As

Publication number Publication date
US9702843B2 (en) 2017-07-11
EP1884771B1 (en) 2017-12-13
EP1884771A4 (en) 2011-10-19
CN101203748B (zh) 2011-09-14
JP5021183B2 (ja) 2012-09-05
WO2006123730A1 (ja) 2006-11-23
CN101203748A (zh) 2008-06-18
JP2006322889A (ja) 2006-11-30
EP1884771A1 (en) 2008-02-06
US20150377818A1 (en) 2015-12-31

Similar Documents

Publication Publication Date Title
US9702843B2 (en) Biosensor incorporating protein-immobilized membrane and method of immobilizing protein in biosensor
US8758591B2 (en) Electrochemical nanocomposite biosensor system
Emr et al. Use of polymer films in amperometric biosensors
US5795774A (en) Biosensor
CN103328650B (zh) 用于分析物传感器的层状酶组合物
EP0585113B1 (en) Detecting an analyte in the gaseous or vapour phase by bioelectrochemical reactions and media therefor
CN110514704B (zh) 新型生物传感方法
EP1678492A1 (en) Electrochemical test strip for reducing the effect of direct and mediated interference current
CA2375302A1 (en) Electrochemical sensor for analysis of liquid samples
Deng et al. New glucose biosensor based on a poly (o-phenylendiamine)/glucose oxidase-glutaraldehyde/Prussian blue/Au electrode with QCM monitoring of various electrode-surface modifications
US8858768B2 (en) Electrochemical sensor and method for manufacturing same
JP2006322889A5 (enExample)
Pedrosa et al. Acetylcholinesterase Immobilization on 3‐Mercaptopropionic Acid Self Assembled Monolayer for Determination of Pesticides
JP2009500601A (ja) 電極プリコンディショニング
JP5164656B2 (ja) センサ及びバイオセンサ
Pariente et al. Enzyme support systems for biosensor applications based on gold-coated nylon meshes
JP4627912B2 (ja) バイオセンサ
Navera et al. Nafion‐coated carbon fiber for acetylcholine and choline sensors
Navera et al. Micro-choline sensor for acetylcholinesterase determination
Okawa et al. Glucose sensor carrying monomolecular layer of glucose oxidase covalently bound to tin (IV) oxide electrode
Genshaw Enzyme electrode for determining glucose in whole blood.
US7794778B2 (en) Amperometric sensor for uric acid and method for the same
Xie et al. An interference-free implantable glucose microbiosensor based on use of a polymeric analyte-regulating membrane
JPH0750063B2 (ja) バイオセンサ
Tamiya et al. Micro-biosensors for Clinical and Food Analyses

Legal Events

Date Code Title Description
AS Assignment

Owner name: ARKRAY, INC., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KATSUKI, KOJI;YAMAOKA, HIDEAKI;REEL/FRAME:020192/0201

Effective date: 20071112

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION