JP2012146460A - Fuel cell, fuel cell manufacturing method, electronics equipment, enzyme immobilized electrode, biosensor, energy conversion element, cell, cell organelle, and bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fuel cell and its manufacturing method which can confine one or more kinds of enzymes or further coenzymes in a minute space and, by carrying out enzymatic reaction using this space as a reaction field, can extract electrons efficiently from glucose, etc. to generate electric energy, and also can easily get these kinds of enzymes or further coenzymes stuck to an electrode.SOLUTION: Enzymes 13, 14 and coenzymes 15 which are needed for enzymatic reaction are sealed in a liposome 12, and an antibiotic 16 is bound to a lipid bilayer membrane constituting the liposome 12 to form one or more holes 17 through which glucose can pass, and further a sterol 18 is bound to the lipid bilayer membrane. The liposome 12 is gotten stuck to the surface of an electrode consisting of porous carbon, etc. to form an enzyme immobilized electrode. The enzyme immobilized electrode is used, for example, as an anode of a biofuel cell.

Description

  The present invention relates to a fuel cell, a method for producing a fuel cell, an electronic device, an enzyme-immobilized electrode, a biosensor, an energy conversion element, a cell, an organelle, and a bacterium. More specifically, the present invention is suitable for application to, for example, a biofuel cell using glucose as a fuel or a substrate, a biosensor and an energy conversion element, and various electronic devices using the biofuel cell as a power source. .

In recent years, a fuel cell (biofuel cell) using an enzyme has attracted attention (see, for example, Patent Documents 1 to 12). In this biofuel cell, fuel is decomposed by enzymes and separated into protons (H ⁺ ) and electrons. Alcohols such as methanol and ethanol, monosaccharides such as glucose, and polysaccharides such as starch are used as fuel. What has been developed.

  In this biofuel cell, it has been found that immobilization / arrangement of the enzyme with respect to the electrode is very important. It has also been found that there is a need for an electron mediator that plays a role in transferring electrons to be present effectively with the enzyme. There are various conventional methods for immobilizing an enzyme. Among them, the present inventors mixed a positively charged polymer and a negatively charged polymer with an enzyme in an appropriate ratio to obtain a porous material. The polyion complex method and the glutaraldehyde method have been developed mainly to stabilize the fixed film while maintaining the adhesion to the electrode by applying it on an electrode made of carbonaceous material.

  However, the immobilization method using the polyion complex described above relies heavily on the physicochemical properties of the enzyme, especially the charge, and the immobilization state constantly changes due to changes in the external solution and environmental changes during use. The immobilized enzyme is likely to elute. In general, enzymes have low heat resistance. However, as the enzymes are modified for practical use of biofuel cells, the physicochemical properties of the enzymes themselves change each time. It is necessary to aim for optimization, and is complicated. Furthermore, when it is desired to extract more electrons from the fuel, more enzymes are required. However, when these enzymes are immobilized, a great deal of effort is required to optimize the immobilization conditions.

  Under these circumstances, the present inventors have proposed that an enzyme or a coenzyme is encapsulated in a liposome and an enzyme reaction is performed using this space as a reaction field to efficiently extract electrons from fuel and generate electric energy ( (See Patent Document 13). Thus, by immobilizing the liposome encapsulating the enzyme or coenzyme on the electrode, the enzyme or coenzyme can be easily immobilized on the electrode.

JP 2000-133297 A JP 2003-282124 A JP 2004-71559 A JP 2005-13210 A JP 2005-310613 A JP 2006-24555 A JP 2006-49215 A JP 2006-93090 A JP 2006-127957 A JP 2006-156354 A JP 2007-12281 A JP 2007-35437 A JP 2009-158458 A

Biophys. J., 71, pp. 2984-2995 (1996)

  However, in the method of Patent Document 13 in which an enzyme or coenzyme is confined inside the liposome, when glucose is used as a fuel, the lipid bimolecular membrane constituting the liposome does not permeate glucose. Must be incorporated into a lipid bilayer. In addition, since organic acids such as lactic acid do not permeate the lipid bilayer membrane constituting the liposome, these organic acids cannot be used as fuel when the liposome is used as it is.

  Therefore, the problem to be solved by the present invention is that one or more kinds of enzymes or further coenzymes are confined in a minute space inside the liposome, and fuel such as glucose and organic acid is easily permeated into the liposome. It is possible to control the permeation rate of this fuel, and it is possible to efficiently extract electrons from the fuel and generate electric energy by performing an enzyme reaction using the internal space of the liposome as a reaction field, It is an object of the present invention to provide a fuel cell capable of easily immobilizing these enzymes or further coenzymes on electrodes, and a method for producing the same.

  Another problem to be solved by the present invention is to provide a high-performance electronic device using the above-described excellent fuel cell.

  Still another problem to be solved by the present invention is to provide an enzyme-immobilized electrode, a high-efficiency biosensor and an energy conversion element suitable for application to the fuel cell.

  Still another problem to be solved by the present invention is that a substrate such as glucose or an organic acid can be easily permeated into the interior, and the permeation rate of the substrate can be controlled. It is to provide cells, organelles, and bacteria that can efficiently extract electrons from a substrate and generate electric energy by performing an enzymatic reaction.

  The inventors of the present invention have intensively studied to solve the above problems. As a result, in a biofuel cell, when an enzyme required for an enzymatic reaction or further coenzyme is encapsulated in a liposome, which is an artificial cell, compared to the case where the same amount of enzyme or further coenzyme is not encapsulated in the liposome. It was found that the enzyme reaction can be carried out much more efficiently to obtain a very high catalyst current or to be easily immobilized on the electrode.

  On the other hand, when glucose is used as a fuel in a biofuel cell, as is well known, glucose has a remarkably low permeability to the lipid bilayer membrane constituting the liposome. Therefore, even if the liposome is placed in a fuel solution containing glucose, It is very difficult to take in glucose from the fuel solution inside (see FIG. 58). As a result of intensive studies on this point, the present inventors solved this problem by binding an antibiotic having a hole (channel) capable of permeating glucose to the lipid bilayer membrane constituting the liposome. Found that you can. Antibiotics having holes that allow glucose to pass through can also pass organic acids. Furthermore, the present inventors have found that the permeation rate of organic acids and glucose through the holes of antibiotics can be controlled by binding sterols in addition to antibiotics to lipid bilayer membranes constituting liposomes.

  The present invention has been devised by examining the range in which the above-described method can be applied from various viewpoints based on the above-mentioned knowledge obtained independently by the present inventors.

That is, in order to solve the above problems, the present invention provides:
The positive electrode and the negative electrode have a structure facing each other through a proton conductor, and are configured to extract electrons from the fuel using an enzyme,
At least one of the above enzymes is encapsulated in the liposome,
This is a fuel cell in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to the lipid bimolecular membrane constituting the liposome.

In addition, this invention
When manufacturing a fuel cell having a structure in which a positive electrode and a negative electrode are opposed to each other through a proton conductor and taking out electrons from fuel using an enzyme, after encapsulating at least one kind of the enzyme in the liposome, It is a method for producing a fuel cell, comprising a step of binding an antibiotic having a hole capable of permeating glucose and a sterol to a lipid bimolecular membrane constituting the same.

  In each of the above inventions, typically, the fuel cell is configured to extract electrons from the fuel using an enzyme and a coenzyme, and at least one enzyme and at least one coenzyme are encapsulated in the liposome.

  Liposomes are closed vesicles formed by a lipid bimolecular membrane made of phospholipid or the like, and the inside is an aqueous phase. In this liposome, not only monolayer liposomes (SUV: small unilamellar liposomes, GUV: giant unilamellar liposomes) consisting of a single lipid bilayer, but also small liposomes (SUV) are incorporated into large liposomes (GUV). Also included are nested multilamellar liposomes (MUVs). Liposomes can be produced, for example, from those having a diameter of about 100 nm to a large one having a diameter of 10 μm, and the diameter is selected as necessary. Basically, any phospholipid may be used, and either glycerophospholipid or sphingophospholipid may be used. Examples of glycerophospholipid include, but are not limited to, phosphatidic acid, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and diphosphatidylglycerol (cardiolipin). Sphingophospholipids include, but are not limited to, sphingomyelin. A representative example of phosphatidylcholine is dimyristoyl phosphatidylcholine (DMPC). Conventionally known methods can be used to form liposomes and encapsulate enzymes and coenzymes inside the liposomes, and are selected as necessary.

  By binding an antibiotic having a hole capable of permeating glucose to the lipid bimolecular membrane constituting the liposome, a hole penetrating the lipid bimolecular membrane is formed in a form bordered by the antibiotic. Various conventionally known antibiotics can be used as the antibiotic, and are selected as necessary. This hole has a size that prevents an enzyme or even a coenzyme enclosed inside the liposome from penetrating at least easily. Antibiotics typically consist of multiple molecules in which a plurality of molecules are arranged in a ring.

  As the sterol to be bound to the lipid bilayer membrane constituting the liposome, various conventionally known sterols can be used and selected as necessary. Representative examples of sterols include animal sterols such as cholesterol and coprostanol, sitosterol and stigmasterol in plants, and ergosterol produced by yeasts and ergots. The weight ratio of sterol to the lipid bilayer membrane constituting the liposome is preferably ¼ or more and ３ or less, but is not limited thereto.

  The liposome encapsulates at least one enzyme or at least one coenzyme among enzymes or further coenzymes necessary for the enzyme reaction, but encapsulates all the enzymes necessary for the enzyme reaction or further coenzymes in the liposome. Alternatively, a part of the enzyme or coenzyme may not be encapsulated in the liposome, but may be incorporated into the lipid bilayer membrane constituting the liposome, immobilized, or present outside the liposome. When the enzyme or coenzyme is immobilized on the lipid bilayer membrane constituting the liposome, for example, an anchor such as a polyethylene glycol chain can be used.

  The liposome is preferably immobilized on the negative electrode, but it is not always necessary to immobilize the liposome. In the case where an electrolyte containing a buffer solution (buffer material) is used as the proton conductor, the liposome is included in the buffer solution. It may be. For fixing the liposome, various conventionally known immobilization methods used for cell immobilization, for example, can be used. In this case, an intermediate layer may be formed between the negative electrode and the liposome in order to stabilize the fixation of the liposome to the negative electrode. As this intermediate layer, not only biopolymers such as proteins and DNA, but also polymer electrolytes having both hydrophilic and hydrophobic properties, and structures such as micelles, reverse micelles, and lamellae can be formed. A compound having a nanometer structure, a compound having one or more properties, and high biocompatibility can be used. As the protein, for example, acidic proteins such as alcohol dehydrogenase, lactate dehydrogenase, ovalbumin, and myokinase represented by albumin, as well as lysotium, cytochrome c, myoglobin, trypsinogen, etc. having an isoelectric point on the alkali side may be used. it can. It is possible to stably immobilize the liposome with respect to the negative electrode by physically adsorbing such an intermediate layer made of protein or the like on the electrode surface and immobilizing the liposome on the intermediate layer.

  As a method for stably immobilizing liposomes with respect to the negative electrode, it is effective to use the following method. That is, the first substance is fixed to the negative electrode, the second substance is bonded to the liposome, and the first substance fixed to the negative electrode and the second substance bonded to the liposome are bonded to each other. The liposome can be stably immobilized on the negative electrode. As such a combination of the first substance and the second substance, a binding pair that specifically binds is preferably used, but is not necessarily limited thereto. A specific example of such a combination of the first substance and the second substance is as follows.

・ Avidin and biotin
Avidin is a glycoprotein that specifically binds to biotin. For example, it is possible to immobilize avidin on the negative electrode, bind biotin to the liposome, and bind the avidin immobilized on the negative electrode to biotin bound to the liposome to immobilize the liposome on the negative electrode. Biotin may be immobilized on the negative electrode, and avidin may be bound to the liposome. As avidin, various kinds such as streptavidin (StreptAvidin) and neutroavidin (NeutroAvidin) can be used, and these mutants can also be used.

-Antigens and antibodies Antibodies are immunoglobulins that specifically bind to antigens. For example, while preparing a liposome using an antigen-modified lipid, the antibody can be immobilized on the negative electrode, and the liposome immobilized on the negative electrode can be immobilized by binding the antibody immobilized on the negative electrode and the antigen bound to the liposome. An antigen may be immobilized on the negative electrode, and an antibody may be bound to the liposome.

・ Protein A or Protein G and immunoglobulin _Ig G
Protein A or protein G is a protein having a strong affinity for immunoglobulin I _g G. For example, immunoglobulin I _g G is immobilized on the negative electrode, and protein A or protein G is bound to the liposome. The liposome can be immobilized on the negative electrode by binding immunoglobulin I _g G immobilized on the negative electrode to protein A or protein G bound to the liposome. Protein A or protein G may be immobilized on the negative electrode, and immunoglobulin I _g G may be bound to the liposome.

-Sugar molecule (or sugar chain-containing compound) and lectin Lectin is a general term for sugar-binding proteins. For example, a transmembrane lectin is mixed to produce a liposome, and a sugar molecule (or a sugar chain-containing compound) is immobilized on the negative electrode. The liposome can be immobilized on the negative electrode by binding the sugar chain of the sugar molecule (or sugar chain-containing compound) immobilized on the negative electrode and the lectin bound to the liposome. Lectins may be immobilized on the negative electrode, and sugar molecules (or sugar chain-containing compounds) may be bound to the liposomes.

・ DNA and complementary DNA
For example, a liposome is prepared using water-soluble lipid bound with DNA, and complementary strand DNA is immobilized on the negative electrode. Then, the liposome can be immobilized on the negative electrode by bonding the complementary strand DNA immobilized on the negative electrode and the DNA bonded to the liposome by hybridization.

Glutathione and glutathione S-transferase (GST)
For example, a GST fusion transmembrane protein is mixed to produce a liposome, and glutathione is immobilized on the negative electrode. Then, the liposome can be immobilized on the negative electrode by combining glutathione immobilized on the negative electrode and GST bonded to the liposome.

-Heparin and heparin binding molecule For example, the lipid bilayer membrane constituting the liposome is modified with heparin binding molecule, and the negative electrode is modified with heparin. The liposome can be immobilized on the negative electrode by binding the heparin of the heparin-modified negative electrode and the heparin-binding molecule bound to the liposome.

Hormones and hormone receptors Hormone receptors are protein molecules or molecular complexes that specifically accept hormone molecules. For example, it is possible to immobilize the hormone receptor on the negative electrode, bind the hormone to the liposome, and immobilize the liposome to the negative electrode by binding the hormone receptor immobilized on the negative electrode and the hormone bound to the liposome. .

Carboxylic acid and imide For example, carboxylic acid is bonded to the negative electrode and amine is bonded to the liposome. And a liposome can be fix | immobilized by the negative electrode by couple | bonding the carboxylic acid couple | bonded with the negative electrode and the amine couple | bonded with the liposome by amide coupling using imides, such as carbodiimide and N-hydroxysuccinimide. An amine may be bonded to the negative electrode, and a carboxylic acid may be bonded to the liposome.

  In addition, when the electrode material of the negative electrode is carbon (carbon), as a method for bonding carboxylic acid to the negative electrode, for example, a diazonium salt is refluxed in a solution containing nitric acid, sulfuric acid, hydrogen peroxide, or the like. A method of performing local oxidation can be used.

  Various fuels such as glucose can be used as the fuel, and are selected as necessary. Examples of fuels other than glucose include various organic acids involved in the citric acid cycle, sugars and organic acids involved in the pentose phosphate cycle and glycolysis. Various organic acids involved in the citric acid cycle are lactic acid, pyruvic acid, acetyl CoA, citric acid, isocitric acid, α-ketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid and the like. Sugars and organic acids involved in the pentose phosphate cycle and glycolysis are glucose 6-phosphate, 6-phosphogluconolactone, 6-phosphogluconoic acid, ribulose 5-phosphate, glyceraldehyde 3-phosphate, fructose 6-phosphate, xylulose 5-phosphate, sedheptulose 7-phosphate, erythrose 4-phosphate, phosphoenolpyruvate, 1,3-bisphosphoglycerate, ribose 5-phosphate and the like. Any of these fuels can permeate through a hole that is capable of permeating glucose, which is possessed by an antibiotic bonded to a lipid bilayer membrane constituting a liposome.

  These fuels are typically used in the form of a fuel solution obtained by dissolving these fuels in a conventionally known buffer solution such as a phosphate buffer solution or a Tris buffer solution.

The enzyme encapsulated in the liposome typically contains an oxidase that promotes and decomposes the oxidation of fuel such as glucose, and further returns the coenzyme that has been reduced along with the oxidation of the fuel to the oxidant and an electron mediator. Including a coenzyme oxidase that passes electrons to the negative electrode. Specifically, the enzyme encapsulated in the liposome is preferably an oxidase that promotes and decomposes oxidation of fuel such as glucose, and a coenzyme oxidase that returns a coenzyme reduced by the oxidase to an oxidant. including. By the action of the coenzyme oxidase, electrons are generated when the coenzyme returns to the oxidized form, and the electrons are transferred from the coenzyme oxidase to the negative electrode via the electron mediator. For example, when glucose is used as a fuel, for example, glucose dehydrogenase (GDH) (particularly NAD-dependent glucose dehydrogenase) is used as an oxidase, NAD ⁺ or NADP ⁺ is used as a coenzyme, and diaphorase (eg, a coenzyme oxidase is used). DI) is used.

  Basically, any electron mediator may be used, but a compound having a quinone skeleton is preferably used. Specifically, for example, 2,3-dimethoxy-5-methyl-1 is used. , 4-benzoquinone (Q0) or a compound having a naphthoquinone skeleton, such as 2-amino-1,4-naphthoquinone (ANQ), 2-amino-3-methyl-1,4-naphthoquinone (AMNQ), 2-methyl Various naphthoquinone derivatives such as -1,4-naphthoquinone (VK3), 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) and vitamin K1 are used. As a compound having a quinone skeleton, for example, anthraquinone or a derivative thereof can be used. In addition to the compound having a quinone skeleton, the electron mediator may contain one or two or more other compounds that function as an electron mediator, if necessary. This electron mediator may be immobilized on the negative electrode together with the liposome in which the enzyme and coenzyme are encapsulated, encapsulated inside the liposome, immobilized on the liposome, or contained in the fuel solution. You may make it let.

When an enzyme is immobilized on the positive electrode, this enzyme typically includes an enzyme that reduces oxygen. Examples of the enzyme that reduces oxygen include bilirubin oxidase, laccase, and ascorbate oxidase. In this case, an electron mediator is preferably immobilized on the positive electrode in addition to the enzyme. As the electron mediator, for example, potassium hexacyanoferrate or potassium octacyanotungstate is used. The electron mediator is preferably immobilized at a sufficiently high concentration, for example, 0.64 × 10 ⁻⁶ mol / mm ² or more on average.

  Various proton conductors can be used and are selected as necessary. Specifically, for example, cellophane, perfluorocarbon sulfonic acid (PFS) -based resin film, and trifluorostyrene derivative can be used together. Polymer film, polybenzimidazole film impregnated with phosphoric acid, aromatic polyetherketone sulfonic acid film, PSSA-PVA (polystyrene sulfonate polyvinyl alcohol copolymer), PSSA-EVOH (polystyrene sulfonate ethylene vinyl alcohol copolymer) And an ion exchange resin having a fluorine-containing carbon sulfonic acid group (Nafion (trade name, DuPont, USA), etc.).

When an electrolyte containing a buffer solution (buffer substance) is used as the proton conductor, sufficient buffer capacity can be obtained during high output operation, and the ability inherent to the enzyme can be fully exhibited. It is desirable to do. For this reason, it is effective that the concentration of the buffer substance contained in the electrolyte is 0.2 M or more and 2.5 M or less, preferably 0.2 M or more and 2 M or less, more preferably 0.4 M or more and 2 M or less, More preferably, it is 0.8M or more and 1.2M or less. Buffer substances, in general, as long as _a pK _a of 6 to 9, may it be used What Specific examples and, dihydrogen phosphate ion (H ₂ PO ₄ ^- ), 2-amino-2-hydroxymethyl-1,3-propanediol (abbreviated to Tris), 2- (N-morpholino) ethanesulfonic acid (MES), cacodylic acid, carbonic acid (H ₂ CO ₃ ), hydrogen citrate Ions, N- (2-acetamido) iminodiacetic acid (ADA), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid ( ACES), 3- (N-morpholino) propanesulfonic acid (MOPS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), N-2-hydroxyethylpiperazine- '-3-propanesulfonic acid (HEPPS), N- [tris (hydroxymethyl) methyl] glycine (abbreviation tricine), glycylglycine, N, N-bis (2-hydroxyethyl) glycine (abbreviation bicine), etc. . Examples of the substance that generates dihydrogen phosphate ions (H ₂ PO ₄ ⁻ ) include sodium dihydrogen phosphate (NaH ₂ PO ₄ ) and potassium dihydrogen phosphate (KH ₂ PO ₄ ). As the buffer substance, a compound containing an imidazole ring is also preferable. Specifically, the compound containing an imidazole ring includes imidazole, triazole, pyridine derivative, bipyridine derivative, imidazole derivative (histidine, 1-methylimidazole, 2-methylimidazole, 4-methylimidazole, 2-ethylimidazole, imidazole-2 -Ethyl carboxylate, imidazole-2-carboxaldehyde, imidazole-4-carboxylic acid, imidazole-4,5-dicarboxylic acid, imidazol-1-yl-acetic acid, 2-acetylbenzimidazole, 1-acetylimidazole, N-acetyl Imidazole, 2-aminobenzimidazole, N- (3-aminopropyl) imidazole, 5-amino-2- (trifluoromethyl) benzimidazole, 4-azabenzimidazole, 4-aza-2-mercapto Benzimidazole, benzimidazole, 1-benzylimidazole, 1-butylimidazole) and the like. As needed, in addition to these buffer substances, for example, selected from the group consisting of hydrochloric acid (HCl), acetic acid (CH ₃ COOH), phosphoric acid (H ₃ PO ₄ ) and sulfuric acid (H ₂ SO ₄ ) At least one acid may be added as a neutralizing agent. By doing so, the activity of the enzyme can be maintained higher. The pH of the electrolyte containing the buffer substance is preferably around 7, but may generally be any of 1-14.

  Various materials can be used as the positive electrode and negative electrode materials. For example, carbon-based materials such as porous carbon, carbon pellets, carbon felt, and carbon paper are used. As the electrode material, a porous conductive material including a skeleton made of a porous material and a material mainly composed of a carbon-based material covering at least a part of the surface of the skeleton can be used (Patent Document). 12).

  This fuel cell can be used for almost anything that requires electric power, and can be of any size. For example, electronic devices, mobile objects (automobiles, motorcycles, aircraft, rockets, spacecrafts, etc.), power units, construction machinery, It can be used for machine tools, power generation systems, cogeneration systems, etc. The output, size, shape, type of fuel, etc. are determined depending on the application.

In addition, this invention
Using one or more fuel cells,
At least one of the fuel cells is
The positive electrode and the negative electrode have a structure facing each other through a proton conductor, and are configured to extract electrons from the fuel using an enzyme,
At least one of the above enzymes is encapsulated in the liposome,
It is an electronic device in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to the lipid bimolecular membrane constituting the liposome.

  This electronic device may basically be any type, and includes both portable and stationary devices. Specific examples include cell phones, mobile devices (portable information terminals). Machine (PDA), etc.), robot, personal computer (including both desktop type and notebook type), game machine, camera-integrated VTR (video tape recorder), in-vehicle device, home appliance, industrial product and the like.

  In the invention of the electronic device, what has been described in relation to the invention of the fuel cell and the method of manufacturing the fuel cell is valid as long as it is not contrary to the nature.

In addition, this invention
A liposome encapsulating at least one enzyme is immobilized,
It is an enzyme-immobilized electrode in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the liposome.

  In the invention of the enzyme-immobilized electrode, what has been described in relation to the above-described invention of the fuel cell and the method of manufacturing the fuel cell is valid as long as it does not contradict its properties.

In addition, this invention
Using enzymes,
At least one of the above enzymes is encapsulated in the liposome,
It is a biosensor in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the liposome.

  In the invention of the biosensor, what has been described in relation to the invention of the fuel cell and the method of manufacturing the fuel cell is valid as long as the nature of the biosensor is not violated.

In addition, this invention
It is an energy conversion element using a liposome in which at least one enzyme is encapsulated, and an antibiotic having a hole capable of permeating glucose and a sterol bound to a lipid bimolecular membrane constituting the liposome.

  Here, this energy conversion element is an element that converts chemical energy of a fuel or a substrate into electric energy by an enzyme reaction, and the above-described fuel cell, that is, a biofuel cell is a kind of this energy conversion element.

  In the invention of the energy conversion element, what has been described in relation to the invention of the fuel cell and the method of manufacturing the fuel cell is valid as long as it is not contrary to the nature thereof.

In addition, this invention
It is a cell in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the cell membrane.

In addition, this invention
It is an organelle in which an antibiotic having a hole that allows glucose to permeate and a sterol are bound to a lipid bimolecular membrane constituting the cell membrane.

In addition, this invention
It is a bacterium in which an antibiotic and a sterol having a hole capable of permeating glucose are bound to a lipid bimolecular membrane constituting a cell membrane.

  The cells, organelles and bacteria are not particularly limited as long as they have a fuel or substrate metabolic system such as glucose, and include various conventionally known ones. If necessary, these cells, organelles, and bacteria may be encapsulated with an enzyme or further a coenzyme necessary for decomposing a fuel such as glucose or a substrate or the like, similar to the fuel cell described above.

  In the present invention configured as described above, at least one enzyme involved in an enzymatic reaction or at least one coenzyme can be encapsulated in liposomes while maintaining high activity. The liposome can be easily permeated with a fuel or a substrate such as glucose or organic acid from the hole of the antibiotic bound to the lipid bimolecular membrane constituting the liposome, and the lipid two molecules constituting the liposome. This fuel permeation rate can be controlled by the sterols attached to the membrane. Then, an enzymatic reaction is efficiently performed using the minute space inside the liposome as a reaction field, and electrons can be efficiently extracted from a fuel or a substrate such as glucose. In this case, the concentration of the enzyme and the coenzyme encapsulated inside the liposome is extremely high, and therefore the distance between the enzyme and the coenzyme is extremely small, so that the catalytic cycle of these enzymes and coenzyme proceeds very fast. The enzyme reaction proceeds at high speed. Then, by immobilizing the liposome on the negative electrode or electrode, the enzyme or further coenzyme enclosed in the liposome can be easily immobilized on the negative electrode or electrode via the liposome. For this reason, the electrons extracted from the fuel or substrate such as glucose can be reliably transferred to the negative electrode or the electrode. In this case, the liposome can be fixed more easily than when the enzyme or coenzyme is immobilized by a polyion complex or the like.

  In addition, in the cells, organelles and bacteria described above, an enzymatic reaction is efficiently carried out using a minute space inside these cells, organelles and bacteria as a reaction field, and fuel such as glucose and organic acids or Electrons can be efficiently extracted from the substrate. In this case, the enzymatic reaction is carried out by the metabolic system of these cells, organelles, and bacteria. However, the enzymes or enzymes necessary for decomposing fuel or substrates in these cells, organelles, and bacteria are not used. Furthermore, when encapsulating a coenzyme, these enzymes and coenzymes can be used.

According to the present invention, an enzyme or further a coenzyme can be confined in a minute space inside the liposome, and fuel such as glucose and organic acid can be easily permeated into the liposome, and the permeation rate of this fuel can be increased. Electrons can be efficiently extracted from the fuel by generating an enzymatic reaction using the internal space of the liposome as a reaction field, and electric energy can be generated. A highly efficient fuel cell that can be easily fixed can be realized. By using such a highly efficient fuel cell, a high-performance electronic device can be realized. Similarly, a highly efficient biosensor and energy conversion element can be realized.
In addition, cells, organelles, and bacteria that can efficiently extract electrons from a fuel or substrate such as glucose or an organic acid to generate electric energy can be realized.

It is a basic diagram which shows the enzyme fixed electrode by 1st Embodiment of this invention. It is a basic diagram which shows the liposome with which the enzyme and coenzyme which are used in the enzyme fixed electrode by 1st Embodiment of this invention were enclosed. It is a basic diagram which shows typically the electron transfer reaction by the enzyme group enclosed by the liposome and the coenzyme in the enzyme fixed electrode by 1st Embodiment of this invention. It is a basic diagram for demonstrating the advantage by enclosing an enzyme group and a coenzyme in a liposome, and performing an enzyme reaction. 1 is a drawing-substituting photograph showing a fluorescence microscopic image of a liposome encapsulating fluorescently labeled alcohol dehydrogenase, fluorescently labeled diaphorase and NADH. 1 is a drawing-substituting photograph showing a fluorescence microscopic image of a liposome encapsulating fluorescently labeled alcohol dehydrogenase, fluorescently labeled diaphorase and NADH. 1 is a drawing-substituting photograph showing a fluorescence microscopic image of a liposome encapsulating fluorescently labeled alcohol dehydrogenase, fluorescently labeled diaphorase and NADH. It is a basic diagram which shows the result of having carried out the fluorescence monitoring of the liposome which enclosed fluorescent delabelled alcohol dehydrogenase, the fluorescent labeled diaphorase, and NADH. It is a basic diagram which shows the result of having carried out the fluorescence monitoring of the liposome which enclosed fluorescent delabelled alcohol dehydrogenase, the fluorescent labeled diaphorase, and NADH. It is a basic diagram which shows the result of having carried out the fluorescence monitoring of the liposome which enclosed fluorescent delabelled alcohol dehydrogenase, the fluorescent labeled diaphorase, and NADH. It is a basic diagram for demonstrating stability of a liposome. An abbreviation showing the results of chronoamperometry carried out when liposomes encapsulating alcohol dehydrogenase, diaphorase and NADH were dispersed in a predetermined solution and when alcohol dehydrogenase, diaphorase and NADH were simply dispersed in a predetermined solution. FIG. It is a basic diagram which shows the state which disperse | distributed the liposome which enclosed alcohol dehydrogenase, diaphorase, and NADH in the buffer solution. FIG. 4 is a schematic diagram showing a state where alcohol dehydrogenase, diaphorase and NADH are simply dispersed in a buffer solution. It is a basic diagram which shows the result of the chronoamperometry performed when the liposome which enclosed glucose dehydrogenase, diaphorase, and NADH was disperse | distributed in the measurement solution. It is a basic diagram which shows the result of the chronoamperometry performed when the liposome which enclosed glucose dehydrogenase, diaphorase, and NADH was disperse | distributed in the measurement solution. It is a basic diagram which shows the structural formula of amphotericin B used as an antibiotic in Example 1 of this invention. It is sectional drawing which shows the state by which amphotericin B couple | bonds with the lipid bilayer membrane which comprises a liposome in Example 1 of this invention, and the hole is formed. It is a top view which shows the state by which amphotericin B couple | bonds with the lipid bilayer membrane which comprises a liposome in Example 1 of this invention, and the hole is formed. It is a drawing substitute photograph which shows the scanning electron microscope image which image | photographed by immersing the liposome which couple | bonded amphotericin B to the lipid bilayer membrane in the ionic liquid. It is a drawing substitute photograph which shows the scanning electron microscope image which image | photographed by immersing the liposome which has not couple | bonded the amphotericin B with the lipid bilayer membrane in the ionic liquid. It is a basic diagram which shows the result of the chronoamperometry performed when the liposome which enclosed glucose dehydrogenase, diaphorase, and NADH in Example 1 of this invention was disperse | distributed in the measurement solution. FIG. 6 is a schematic diagram showing the results of chronoamperometry performed for comparison with Example 1. FIG. 6 is a schematic diagram showing the results of chronoamperometry performed for comparison with Example 1. It is a basic diagram which shows the electrochemical measuring apparatus used for the electrochemical analysis. It is a basic diagram which shows the measurement conditions of an electrochemical analysis. It is a basic diagram which shows the measurement result of an electrochemical analysis. It is a basic diagram which shows ion distribution of the both sides of a lipid bilayer membrane. It is a basic diagram which shows the ion concentration inside and outside a lipid bimolecular membrane. It is a basic diagram which shows the ion concentration inside and outside a lipid bimolecular membrane. It is a basic diagram which shows the measurement result of an electrochemical analysis. It is a basic diagram which shows the relationship between the diffusion coefficient ratio of various organic acids, and the molecular weight of an organic acid. It is a basic diagram which shows the measurement result of the electrochemical analysis at the time of using cholesterol. It is a basic diagram which shows the measurement result of the electrochemical analysis at the time of using ergosterol. It is a basic diagram which shows the relationship between the diffusion coefficient ratio of various organic acids at the time of using cholesterol or ergosterol, and the molecular weight of an organic acid. It is a basic diagram which shows an electrochemical measurement system. It is a basic diagram which shows the measurement result of an electrochemical analysis. It is a basic diagram for demonstrating the advantage at the time of using ergosterol. It is a basic diagram which shows the measurement result of an electrochemical analysis. It is a basic diagram which shows the measurement result of an electrochemical analysis. It is a basic diagram which shows the measurement result of an electrochemical analysis. It is a basic diagram which shows the biofuel cell by the 2nd Embodiment of this invention. Details of the configuration of the negative electrode of the biofuel cell according to the second embodiment of the present invention, an example of an enzyme group and a coenzyme encapsulated in a liposome immobilized on the negative electrode, and delivery of electrons by the enzyme group and the coenzyme It is a basic diagram which shows reaction typically. It is a basic diagram which shows the specific structural example of the biofuel cell by the 2nd Embodiment of this invention. It is the basic diagram and sectional drawing for demonstrating the structure of the porous electroconductive material used for the electrode material of a negative electrode in the biofuel cell by the 3rd Embodiment of this invention. It is a basic diagram for demonstrating the manufacturing method of the porous electroconductive material used for the electrode material of a negative electrode in the biofuel cell by the 3rd Embodiment of this invention. It is a basic diagram which shows the principal part of the cell by the 4th Embodiment of this invention. It is a basic diagram which shows the mitochondria by 5th Embodiment of this invention. It is a basic diagram which shows the bacteria by 6th Embodiment of this invention. It is the perspective view and sectional drawing which show the porous electrode used for the electrode of a negative electrode in the biofuel cell by the 7th Embodiment of this invention. It is a basic diagram which shows the enzyme fixed electrode by 8th Embodiment of this invention. It is a basic diagram which shows the sample produced in order to perform a chronoamperometry measurement in Example 2 of this invention. It is a basic diagram which shows the 1st sample produced for the comparison with Example 2 of this invention. It is a basic diagram which shows the 2nd sample produced for the comparison with Example 2 of this invention. It is a basic diagram which shows the 3rd sample produced for the comparison with Example 2 of this invention. It is a basic diagram which shows the result of the chronoamperometry performed in Example 2 of this invention with the result of a comparative example. It is a basic diagram which shows the enzyme fixed electrode by 9th Embodiment of this invention. It is an approximate line figure showing permeability of various molecules to a lipid bimolecular membrane.

Hereinafter, modes for carrying out the invention (hereinafter referred to as “embodiments”) will be described. The description will be given in the following order.
1. 1st Embodiment (Enzyme fixed electrode and its manufacturing method)
2. Second embodiment (biofuel cell)
3. Third embodiment (biofuel cell)
4). Fourth embodiment (cell)
5. Fifth embodiment (mitochondrion)
6). Sixth embodiment (bacteria)
7). Seventh embodiment (biofuel cell)
8). Eighth embodiment (enzyme-immobilized electrode and manufacturing method thereof)
9. Ninth embodiment (enzyme-immobilized electrode and manufacturing method thereof)

<1. First Embodiment>
[Enzyme immobilized electrode]
FIG. 1 shows an enzyme-immobilized electrode according to a first embodiment of the present invention. In this enzyme-immobilized electrode, glucose, organic acid, or the like is used as a substrate.

  As shown in FIG. 1, in this enzyme-immobilized electrode, liposomes 12 made of a lipid bimolecular film such as phospholipid are immobilized on the surface of an electrode 11 made of porous carbon or the like by physical adsorption or the like. At least one type of enzyme and at least one type of coenzyme involved in the target enzyme reaction are encapsulated in the aqueous phase inside the liposome 12.

  FIG. 2 shows details of the structure of the liposome 12. In FIG. 2, two types of enzymes 13 and 14 and one type of coenzyme 15 are enclosed in the aqueous phase 12 a inside the liposome 12. In addition to these enzymes 13 and 14 and coenzyme 15, for example, an electron mediator may be enclosed in the aqueous phase 12 a inside the liposome 12. This electron mediator may be immobilized on the electrode 11 together with the liposome 12. For example, the enzyme 13 is an oxidase that promotes and decomposes the oxidation of glucose used as a substrate, and the enzyme 14 returns the coenzyme 15 that has been reduced along with the oxidation of glucose to an oxidant, and an electron via an electron mediator. 11 is a coenzyme oxidase to be passed to

  As shown in FIG. 2, one or a plurality of antibiotics 16 are bonded to the lipid bilayer membrane constituting the liposome 12, and a hole 17 penetrating the lipid bilayer membrane in a form bordered by the antibiotic 16. Is formed. Although only one hole 17 is shown in FIG. 2, the number and arrangement of the holes 17 are arbitrary. The size of the hole 17 is selected so that glucose can be permeated but the permeation of the enzymes 13 and 14 and the coenzyme 15 enclosed in the liposome 12 is impossible or difficult.

  As the antibiotic 16, for example, amphotericin B which is a polyene antibiotic, ionophore and the like can be used, but the antibiotic 16 is not limited thereto. Examples of the ionophore include valinomycin, ionomycin, nigericin, gramicidin A, monensin, carbonyl cyanide-m-chlorophenylhydrazone, carbonylcyanide-p-fluoromethoxyphenylhydrazone, and the like.

  In addition to the antibiotic 16, one or more sterols 18 are bound to the lipid bilayer membrane constituting the liposome 12. Although five sterols 18 are shown in FIG. 2, the number and arrangement of the sterols 18 are arbitrary and are selected as necessary. Preferably, the weight ratio of the sterol 18 to the lipid bilayer membrane constituting the liposome 12 is selected in the range from 1/4 to 1/3.

やエルゴステロール

などである。 Specific examples of sterol 18 include cholesterol

Or ergosterol

Etc.

[Method for producing enzyme-immobilized electrode]
This enzyme-immobilized electrode can be produced, for example, as follows. First, the liposome 12 in which the enzymes 13 and 14 and the coenzyme 15 are encapsulated is prepared. Next, an antibiotic 16 is bound to the lipid bilayer membrane constituting the liposome 12 to form a hole 17, and a sterol 18 is bound to the lipid bilayer membrane. Next, the liposome 12 to which the antibiotic 16 and the sterol 18 are bound in this way is immobilized on the electrode 11. As a result, an enzyme-immobilized electrode is produced. Antibiotic 16 and sterol 18 may be bound to the lipid bilayer membrane constituting liposome 12 before encapsulating enzymes 13 and 14 and coenzyme 15. Alternatively, after the liposome 12 is immobilized on the electrode 11, the antibiotic 16 and the sterol 18 may be bound to the lipid bilayer membrane constituting the liposome 12.

  More specifically, the enzyme-immobilized electrode is manufactured as follows, for example. First, a buffer solution containing the enzyme 13, a buffer solution containing the enzyme 14, a buffer solution containing the coenzyme 15 and the liposome 12 to which the sterol 18 is bound (in which the enzymes 13, 14 and the coenzyme 15 are not enclosed) ) Are prepared. Next, after mixing these buffers, the enzymes 13 and 14 and the coenzyme 15 outside the liposome 12 are removed by, for example, passing the mixed solution through a gel filtration column. Next, the liposome 12 in which the enzymes 13 and 14 and the coenzyme 15 are encapsulated in this way is placed in a buffer solution, and an antibiotic 16 is added to the buffer solution so that the lipid bilayer membrane constituting the liposome 12 has antibiotics. The substance 16 is bonded to form the hole 17.

FIG. 3 schematically shows an example of an electron transfer reaction by an enzyme, a coenzyme, and an electron mediator in the enzyme-immobilized electrode. In this example, the enzyme involved in glucose degradation is glucose dehydrogenase (GDH). In addition, NAD ⁺ is a coenzyme that produces a reductant with an oxidation reaction in the glucose decomposition process. A coenzyme oxidase that oxidizes NADH, which is a reduced form of the coenzyme, is diaphorase (DI). An electron mediator receives electrons generated from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes them to the electrode 11. In this case, glucose permeates through the holes 17 formed in the lipid bilayer membrane constituting the liposome 12 and enters the inside of the liposome 12, and gluconolactone is produced by the decomposition of the glucose by glucose dehydrogenase. The lactone passes through the hole 17 and comes out of the liposome 12. The electron mediator enters and exits the lipid bilayer membrane constituting the liposome 12 and performs electron transfer.

  Before describing the examples, the results of detailed studies on the effectiveness of using liposomes encapsulating an enzyme and a coenzyme as a reaction field for an enzyme reaction will be described (see Patent Document 13). However, here, a case where a liposome in which a hole capable of permeating glucose is not formed is used as a liposome and ethyl alcohol is used as a substrate for an enzyme reaction is considered.

As shown in FIG. 4, an enzyme and a coenzyme involved in the decomposition of ethyl alcohol (EtOH) are encapsulated in the electrode 19 in the liposome 20 in which a hole capable of permeating glucose is not formed in the lipid bilayer membrane. An enzyme-immobilized electrode was prepared by immobilizing the sample. The electrode 19 was the same as the electrode 11. FIG. 4 schematically shows an example of an electron transfer reaction by an enzyme, a coenzyme, and an electron mediator in the enzyme-immobilized electrode. In this example, the enzyme involved in the decomposition of ethyl alcohol (EtOH) is alcohol dehydrogenase (ADH). Further, NAD ⁺ is a coenzyme that generates a reductant with an oxidation reaction in the decomposition process of ethyl alcohol. A coenzyme oxidase that oxidizes NADH, which is a reduced form of the coenzyme, is diaphorase (DI). Then, the electron mediator receives electrons generated from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes them to the electrode 19. In this case, ethyl alcohol permeates the lipid bimolecular membrane constituting the liposome 20 and enters the inside of the liposome 20, and acetaldehyde (CH ₃ CHO) is generated by the decomposition of the ethyl alcohol by alcohol dehydrogenase. Go outside 20 The electron mediator enters and exits the lipid bilayer membrane constituting the liposome 20 and performs electron transfer.

The enzyme-immobilized electrode shown in FIG. 4 was actually produced and tested.
First, an enzyme-immobilized electrode was produced as follows.
5 mg of diaphorase (DI) (EC 1.8.1.4, manufactured by Amano Enzyme) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a DI enzyme buffer solution (1).

  5 mg of alcohol dehydrogenase (ADH) (NAD-dependent, EC 1.1.1.1, manufactured by Amano Enzyme) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7). ).

  The buffer solution for dissolving the enzyme is preferably refrigerated until just before, and the enzyme buffer solution is preferably stored as refrigerated as much as possible.

  35 mg of NADH (manufactured by Sigma Aldrich, N-8129) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a NADH buffer solution (3).

  15-300 mg of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a Q0 buffer solution (4).

  100 mg of egg yolk lecithin (manufactured by Wako) was weighed and dissolved in 10 mL of a buffer solution (10 mM phosphate buffer, pH 7) and homogenized with a homogenizer to obtain a liposome buffer solution (5).

The various solutions prepared as described above were collected and mixed in the amounts shown below, and freeze-thaw was repeated three times.
DI enzyme buffer solution (1): 50 μL
ADH enzyme buffer solution (2): 50 μL
NADH buffer solution (3): 50 μL
Liposome buffer solution (5): 50 μL

  The mixed solution is passed through a gel filtration column to remove extraliposomal enzymes and NADH. The liposome solution obtained here was used as an ADH, DI, NADH-encapsulated liposome solution (6).

5, 6 and 7 are fluorescence micrographs of liposomes encapsulating ADH fluorescently labeled with the cyanine dye Cy2 and DI and NADH fluorescently labeled with the cyanine dye Cy3. 5, 6, and 7, Ex (Excitation) indicates excitation light, has a wavelength described on the right of Ex, DM (Dichroic mirror) indicates a mirror that separates excitation light and fluorescence, Transmits only light above the wavelength indicated on the right side of DM, BA (Barrier filter)
Indicates a filter that separates fluorescence and scattered light, and transmits light having a wavelength longer than that indicated on the right side of BA. In this fluorescence microscope, the dye is excited by excitation light, and light obtained thereby is sequentially passed through DM and BA to remove unnecessary light, and only the fluorescence from the dye is detected. FIG. 5 shows the ADH distribution in which fluorescence is generated by exciting Cy2 with excitation light having a wavelength of 450 to 490 nm. FIG. 6 shows the distribution of DI in which fluorescence is generated by exciting Cy3 with excitation light having a wavelength of 510 to 560 nm. FIG. 7 shows NADH distribution in which NADH is excited by excitation light having a wavelength of 380 to 420 nm to generate fluorescence.

  From FIG. 5, FIG. 6, and FIG. 7, the average diameter of the liposomes was 4.6 μm, and the standard deviation was 2.0 μm. However, the average diameter of the liposome was determined by measuring the diameters of 30 liposomes in FIGS. 5, 6 and 7 and taking the average of them.

  8, 9 and 10 are graphs showing the results of fluorescence monitoring of liposomes encapsulating ADH fluorescently labeled with Cy2 and DI and NADH fluorescently labeled with Cy3. FIG. 8 shows the fluorescence intensity from ADH fluorescently labeled with Cy2, FIG. 9 shows the fluorescence intensity from DI fluorescently labeled with Cy3, and FIG. 10 shows the fluorescence intensity from NADH. 8, 9, and 10, Em (Emission) indicates the light emitted when the dye is excited by the excitation light Ex, and has the wavelength described on the right side of Em. The numerical value in the parenthesis on the right side of the wavelength is the full width at half maximum. As can be seen from FIGS. 8, 9 and 10, in any case, the fluorescence intensity did not change up to an ethyl alcohol concentration of 100 mM. That is, it was found that the liposome was stable at least up to an ethyl alcohol concentration of 100 mM, and ADH, DI, and NADH were reliably encapsulated inside the liposome. Further, when 0.3% Triton X as a surfactant was added, the fluorescence intensity increased. This means that the liposome was destroyed by 0.3% Triton X, and internal ADH, DI, and NADH were released outside the liposome, and the fluorescence intensity increased. The state at this time is shown in FIG. 11 taking NADH as an example. As shown in FIG. 11, the fluorescence intensity due to NADH is low when NADH is encapsulated inside the liposome, but when NADH is destroyed and the NADH encapsulated inside is released to the outside of the liposome, it is caused by NADH. The fluorescence intensity increases.

  The ADH, DI and NADH encapsulated liposome solution (6) thus obtained and the Q0 buffer solution (4) were mixed to prepare a measurement solution having a total volume of 100 μL. The carbon felt was used as the working electrode, and the reference electrode Ag | AgCl Thus, 0.3 V was set to a potential sufficiently higher than the oxidation-reduction potential of the electron mediator, and chronoamperometry was performed while stirring the solution. During chronoamperometry, ethyl alcohol was added to the measurement solution in such a way that final concentrations of 1, 10, and 100 mM were added.

  The results of chronoamperometry when ethyl alcohol is added to the measurement solution containing ADH, DI, and NADH-encapsulated liposomes as described above are shown in curve (a) of FIG. As is apparent from this curve (a), when ADH, DI and NADH are encapsulated in liposomes, a catalyst current derived from ethyl alcohol is observed, and the catalyst current increases as the concentration of ethyl alcohol increases. That is, the electrochemical catalytic activity by the artificial cells ADH, DI and NADH encapsulated liposomes was observed. On the other hand, the same amount of ADH, DI and NADH as encapsulated in liposomes but not encapsulated in liposomes but simply dispersed in Q0 buffer solution (4) was treated in the same manner as described above. Amperometry was performed. The result is shown in the curve (b) of FIG. As is clear from this curve (b), when ADH, DI and NADH are not encapsulated in the liposome and are simply dispersed in the Q0 buffer solution (4), a catalyst current derived from ethyl alcohol is hardly observed. For example, when compared with the case where the concentration of ethyl alcohol is 100 mM, the catalyst current obtained when ADH, DI and NADH are not encapsulated in liposomes but simply dispersed in the Q0 buffer solution (4) is ADH, DI and This is only about 1 / 30th of the catalyst current obtained when NADH is encapsulated in liposomes. From this, it is clear that when ADH, DI, and NADH are encapsulated in liposomes, a much higher catalyst current can be obtained than when they are not encapsulated.

Thus, the reason why a much higher catalyst current is obtained when ADH, DI, and NADH are encapsulated in liposomes than when they are not encapsulated will be described. As shown in FIG. 13, the liposome 20 encapsulates ADH (indicated by a circle with a horizontal line), DI (indicated by a vertical line with a circle), and NADH (indicated by a blank circle). Consider a case in which a square lattice is arranged inside. On the other hand, as shown in FIG. 14, in the same volume of buffer S as shown in FIG. 13, the same amount of ADH (shown as a circle with a horizontal line) and DI (shown with a vertical line) as shown in FIG. Let us consider a case in which NADH (indicated by blank circles) and NADH (indicated by blank circles) are arranged in a hexagonal lattice pattern. The volume of the buffer S is, for example, 100 μL, and the internal volume of the liposome 20 is, for example, about 0.17 μL. However, 5.5 × 10 ⁻³ μmol of phospholipid constituting the lipid bilayer membrane of liposome 20 is present in buffer S, and the total volume inside liposome 20 is 30 μL / μmol when converted per 1 μmol of liposome. is there. Compared to the concentrations of ADH, DI, and NADH when ADH, DI, and NADH are simply dispersed in buffer S as shown in FIG. 14, ADH, DI, and NADH are present in liposome 20 as shown in FIG. These local concentrations of ADH, DI, and NADH when encapsulated are about 600 times higher. That is, by encapsulating ADH, DI, and NADH in the liposome 20, the concentration of these ADH, DI, and NADH can be remarkably increased, and the distance between these ADH, DI, and NADH can be made extremely small. Can do. For this reason, in the liposome 20, the catalytic cycle by ADH, DI, and NADH advances very rapidly, and a result as shown in FIG. 12 is obtained.

  From the above, by encapsulating the enzymes 13 and 14 and the coenzyme 15 necessary for the enzyme reaction inside the liposome 12, the enzyme reaction can be efficiently performed using the minute space inside the liposome 12 as a reaction field. It can be seen that electrons can be efficiently extracted from the substrate.

  Next, the results of detailed studies on the effectiveness of binding the antibiotic 16 to the lipid bilayer membrane constituting the liposome 12 will be described. Amphotericin B was used as antibiotic 16.

An enzyme-immobilized electrode was produced as follows.
1 to 10 mg of diaphorase (DI) (EC 1.8.1.4, manufactured by Amano Enzyme) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a DI enzyme buffer solution (11). .

  Glucose dehydrogenase (GDH) (NAD-dependent, EC 1.1.1.17, manufactured by Amano Enzyme) was weighed 10 to 50 mg, dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7), and GDH enzyme buffer solution (12).

  The buffer solution for dissolving the enzyme is preferably refrigerated until just before, and the enzyme buffer solution is preferably stored as refrigerated as much as possible.

  10 to 50 mg of NADH (manufactured by Sigma Aldrich, N-8129) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a NADH buffer solution (13).

  100-200 mg of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a Q0 buffer solution (14).

  10 to 100 mg of egg yolk lecithin (manufactured by Wako) was weighed and dissolved in 10 mL of a buffer solution (10 mM phosphate buffer, pH 7) and homogenized with a homogenizer to obtain a liposome buffer solution (15).

  The various solutions prepared as described above were collected and mixed in the amounts shown below, and freeze-thaw was repeated three times.

DI enzyme buffer solution (11): 50 μL
GDH enzyme buffer solution (12): 50 μL
NADH buffer solution (13): 50 μL
Liposome buffer solution (15): 50 μL

  The mixed solution is passed through a gel filtration column to remove extraliposomal enzymes and NADH. The liposome solution obtained here was used as a GDH, DI, NADH-encapsulated liposome solution (16).

  The GDH, DI and NADH encapsulated liposome solution (16) thus obtained and the Q0 buffer solution (14) were mixed to prepare a measurement solution having a total volume of 100 μL. Then, carbon felt is used as the working electrode, Ag | AgCl as the reference electrode, platinum wire as the counter electrode, the potential is set to +0.1 V with respect to Ag | AgCl, glucose is used as fuel, and chronoamperometry is performed under solution stirring. Went. The results of chronoamperometry are shown in FIG.

  As shown in FIG. 15, first, when glucose was added to the measurement solution so as to have a final concentration of 100 mM during chronoamperometry, the reaction hardly occurred and the obtained current value hardly changed. This is because glucose has a remarkably low rate of permeation through the lipid bilayer membrane constituting the liposome and does not substantially permeate.

  Next, amphotericin B (AmB) as antibiotic 16 was added to the measurement solution so as to have a final concentration of 100 μM. Then, a catalytic current derived from glucose was observed. That is, it was found that glucose permeated through the lipid bilayer membrane constituting the liposome and a series of enzyme reactions proceeded inside the liposome. As far as the present inventors know, this is the first time that a catalytic current derived from glucose incorporated into the liposome is observed.

  Next, when 0.3% Triton X, which is a surfactant capable of breaking liposomes, was added to the measurement solution, the catalyst current was significantly reduced. This means that the liposome was destroyed by 0.3% Triton X, and internal GDH, DI, and NADH were released out of the liposome.

  FIG. 16 shows the results of chronoamperometry when the final concentration of amphotericin B added to a measurement solution containing 100 mM glucose similar to that described above was changed to 1, 10, and 100 μM. As shown in FIG. 16, within this concentration range of 1 to 100 μM, the catalyst current increases as the final concentration of amphotericin B added increases. However, when the final concentration of amphotericin B is 100 μM, the increase in the catalyst current peaked out. Show the trend.

  FIG. 17 shows the structural formula of amphotericin B. FIG. 18 is a cross-sectional view showing a state where amphotericin B is bound to the lipid bimolecular membrane constituting the liposome 12. FIG. 19 is a plan view showing a state in which amphotericin B is bound to a lipid bilayer membrane (not shown). As shown in FIG. 19, a plurality of amphotericin B cyclically bonded to the lipid bilayer membrane form a hole penetrating the lipid bilayer membrane in a form bordered by these amphotericin B. It has been reported that amphotericin B can form holes in liposomes (see, for example, Non-Patent Document 1). This hole is permeable to glucose, and has a size that prevents the enzymes GDH and DI and the coenzyme NADH from permeating.

を用いた。試料溶液は、リポソーム懸濁液１０μＬとＢＭＩＢＦ₄ １０μＬとを混合することにより調製した。このように試料をイオン液体中に浸漬して電子顕微鏡により撮影する手法は、本発明者らが知る限り、これまで全く用いられておらず、本発明者らが初めて用いたものである。 FIG. 20 is a scanning electron microscope (SEM) photograph of a liposome in which amphotericin B is bound to the lipid bilayer membrane constituting the liposome. FIG. 21 is an SEM photograph of liposomes to which amphotericin B is not bound. However, photographing was performed in a state where a container in which the liposome was immersed in an ionic liquid was placed in a sample chamber of a scanning electron microscope and the sample chamber was evacuated to a high vacuum of 1 Pa or less. As the ionic liquid, BMIBF ₄ : 1-butyl-3-methylimidazolium tetrafluoroborate

Was used. A sample solution was prepared by mixing 10 μL of liposome suspension and 10 μL of BMIBF ₄ . Thus, as far as the present inventors know, the method of immersing a sample in an ionic liquid and photographing with an electron microscope has not been used at all so far, and is the first method used by the present inventors.

  As shown in FIG. 21, the liposome to which amphotericin B is not bound has a spherical shape. On the other hand, as shown in FIG. 20, the liposome to which amphotericin B is bonded has a spherical shape with a small protrusion around it. This protrusion corresponds to the portion where amphotericin B is bound. Thus, by observing the liposome in an ionic liquid in an immersed state, the liposome can be photographed very clearly, and the shape of the liposome can be accurately known.

<Example 1>
Next, the results of a detailed study of the effectiveness of binding sterol 18 to the lipid bilayer membrane constituting liposome 12 will be described. Cholesterol was used as sterol 18. Antibiotic 16 is also used for this verification.

An enzyme-immobilized electrode was produced as follows.
1 to 10 mg of diaphorase (DI) (EC 1.8.1.4, manufactured by Amano Enzyme) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a DI enzyme buffer solution (17). .

  Glucose dehydrogenase (GDH) (NAD-dependent, EC 1.1.1.17, manufactured by Amano Enzyme) was weighed 10 to 50 mg, dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7), and GDH enzyme buffer solution (18).

  The buffer solution for dissolving the enzyme is preferably refrigerated until just before, and the enzyme buffer solution is preferably stored as refrigerated as much as possible.

  10 to 50 mg of NADH (manufactured by Sigma Aldrich, N-8129) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a NADH buffer solution (19).

  100-200 mg of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0) was weighed and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain a Q0 buffer solution (20).

  10 to 100 mg of egg yolk lecithin (manufactured by Wako) and 1 to 50 mg of cholesterol are weighed, dissolved in 10 mL of ethanol, ethanol is volatilized by a rotary evaporator, and a film in which lecithin and cholesterol are uniformly mixed is formed. The film was suspended in 10 mL of a buffer solution (10 mM phosphate buffer, pH 7) and homogenized with a homogenizer to obtain a liposome buffer solution (21).

  The various solutions prepared as described above were collected and mixed in the amounts shown below, and freeze-thaw was repeated three times.

DI enzyme buffer solution (17): 50 μL
GDH enzyme buffer solution (18): 50 μL
NADH buffer solution (19): 50 μL
Liposome buffer solution (21): 50 μL

  The mixed solution is passed through a gel filtration column to remove extraliposomal enzymes and NADH. The liposome solution obtained here was used as a GDH, DI, and NADH encapsulated liposome solution (22).

  The GDH, DI and NADH encapsulated liposome solution (22) thus obtained and the Q0 buffer solution (20) were mixed to prepare a measurement solution having a total volume of 100 μL. Then, carbon felt is used as the working electrode, Ag | AgCl as the reference electrode, platinum wire as the counter electrode, the potential is set to +0.1 V with respect to Ag | AgCl, glucose is used as fuel, and chronoamperometry is performed under solution stirring. Went. During chronoamperometry, glucose was added to the measurement solution so that the final concentration was 100 mM, and amphotericin B was further added so that the final concentration was 100 μM. The results of chronoamperometry are shown in FIG. For comparison, FIGS. 23 and 24 show the results of chronoamperometry of two examples performed under the same conditions except that cholesterol is not bound. As shown in FIGS. 23 and 24, a decrease in current is observed in liposomes not bound with cholesterol, whereas liposomes bound with cholesterol show a steady current as shown in FIG. From this, it was confirmed that by binding sterol, the amphotericin B-bound liposome was structurally stabilized, glucose permeation was improved, and a more stable catalytic current was exhibited. That is, it can be seen that binding of sterol is a very useful substance for using liposome as an electrochemical catalyst.

  The verification so far confirmed that sterols are useful for improving the permeation of organic acids to lipid bilayers. Next, we examined whether permeation changes depending on the type of sterol. The permeability of organic acid to amphotericin B was examined in a state where amphotericin B and cholesterol or ergosterol as sterol were bound to the lipid bilayer membrane constituting the liposome. Organic acids are ionized in aqueous solutions and have a charge. Therefore, the movement of organic acid ions was analyzed electrochemically.

  However, the electrochemical analysis was performed by using amphotericin B and cholesterol or ergosterol bound as sterol to a flat lipid bilayer membrane instead of liposome.

  FIG. 25 shows an electrochemical measurement apparatus used for electrochemical analysis. As shown in FIG. 25, in this electrochemical measuring apparatus, two containers W1 and W2 are connected so that a hole H1 and a hole H2 formed in their walls coincide with each other. A lipid bimolecular membrane BLM is provided between the hole H1 and the hole H2 so as to partition the inside of the container W1 and the inside of the container W2. Amphotericin B and cholesterol were bound to the lipid bilayer membrane BLM. As the lipid bilayer membrane BLM, one made of phosphatidylcholine was used. A supporting electrolyte SE made of a buffer solution is placed in each of the containers W1 and W2. The counter electrode CE and the reference electrode RE are immersed in the supporting electrolyte SE in the container W1, the working electrode WE is immersed in the supporting electrolyte SE in the container W2, and the potentiostat P is placed in the counter electrode CE, the reference electrode RE, and the working electrode WE. Connected. Platinum (Pt) was used as the counter electrode CE, and Ag | AgCl was used as the reference electrode RE and the working electrode WE. The value of current that flows between the working electrode WE and the counter electrode CE to permeate the organic acid through the lipid bilayer membrane BLM by applying a voltage ΔE to the supporting electrolyte SE in the container W2 with reference to the supporting electrolyte SE in the container W1. (Current density j) was determined.

Sodium lactate was used as the organic acid, and the permeation of lactate ion and sodium ion (Na ⁺ ) generated by ionization of sodium lactate to amphotericin B was observed. As shown in FIG. 26, the buffer solution used as the supporting electrolyte SE in the containers W1 and W2 is 10 ⁻² M BisTris-HEPES (pH 7.4). The amount of amphotericin B is 5 × 10 ⁻⁷ M. 0.01M sodium lactate was added to the supporting electrolyte SE in the container W1, and c times, that is, 0.01 × cM sodium lactate was added to the supporting electrolyte SE in the container W2.

FIG. 27 shows the results of electrochemical measurement. As shown in FIG. 27, as c increases, the current j becomes a large negative value, and the potential at which the current becomes zero (zero current potential) shifts positively. FIG. 28 schematically shows the distribution of lactate ions and Na ⁺ in the supporting electrolyte SE in the containers W1 and W2. As shown in FIG. 28, in order to make the movement of lactate ions and Na ⁺ due to the concentration gradient zero, a positive voltage E must be applied. This shows that amphotericin B has anion selectivity.

Here, the ion distribution inside and outside the lipid bilayer membrane BLM is evaluated.
FIG. 29 schematically shows the distribution of Na ⁺ and lactate ions (Org ⁻ ) in the supporting electrolyte SE in the containers W1 and W2. The concentration of Na ⁺ in the supporting electrolyte SE in the container W1 is [Na ⁺ ] _W1 , the concentration of lactate ion (Org ⁻ ) is [Org ⁻ ] _W1 , and the concentration of Na ⁺ in the supporting electrolyte SE in the container W2 is [ The concentration of Na ⁺ ] _W2 and lactate ions (Org ⁻ ) is expressed as [Org ⁻ ] _W2 . In addition, the concentration of Na ^{+ in} the portion of the lipid bilayer membrane BLM in contact with the supporting electrolyte SE in the container W1 is expressed as [Na ⁺ ] _M1 , and the concentration of lactate ion (Org ⁻ ) is expressed as [Org ⁻ ] _M1 . In the lipid bilayer membrane BLM, the Na ⁺ concentration in the supporting electrolyte SE in the container W2 is represented as [Na ⁺ ] _M2 , and the lactate ion (Org ⁻ ) concentration is represented as [Org ⁻ ] _M2 .

と表される。 The distribution equilibrium of ions inside and outside the lipid bilayer membrane BLM is

It is expressed.

である。また、電気的中性則より

となる。 Experimental conditions are

It is. From the electrical neutral law

It becomes.

が成立する。 From the above,

Is established.

Next, the zero current potential is evaluated by a theoretical formula.
[Na ^+] _W2 and in Figure 29 [Org ^-] _W2, respectively [Na ^+] _W1 and ^- expressed in _W1, [Na ^+] _M2 and [Org] [Org ^-] _M2, respectively [Na ^+] _M1 and [ Org ⁻ ] _M1 is shown in FIG. The interface potential between the supporting electrolyte SE and the lipid bilayer membrane BLM in the container _{W1 is expressed as ES: W1 / BLM} , and the interface potential between the supporting electrolyte SE and the lipid bilayer membrane BLM in the container W2 is _{expressed as ES: BLM / W2} , The potential of the lipid bilayer membrane BLM is represented as E _M.

が成立する。 From Goldman formula and Nernst formula

Is established.

とすると、

と表すことができる。ただし、Ｒは気体定数、Ｆはファラデー定数、Ｔは絶対温度である。 The diffusion coefficient of Na ⁺ and Org ⁻ is expressed as D _{Na +} and D _Org− , respectively, and the diffusion coefficient ratio

Then,

It can be expressed as. However, R is a gas constant, F is a Faraday constant, and T is an absolute temperature.

Next, a method for calculating the diffusion coefficient ratio α will be described.
FIG. 31 is a diagram in which the zero current potential E _obs is plotted with respect to the actually measured value of c _ratio . As shown in FIG. 31, α = 2.0 is obtained from the E _obs equation by fitting to the actual measurement value.
FIG. 32 shows α values of various organic acid ions with respect to the molecular weight of the organic acid.

  FIGS. 33 and 34 show the results of electrochemical measurements when cholesterol and ergosterol were used as sterols, respectively. The measurement conditions are set to c = 1 in FIG. From FIG. 33 and FIG. 34, when cholesterol is used, the current density j with lactic acid 0.01M increases about 200 times compared with the case without lactic acid. On the other hand, when ergosterol is used, the current density j at lactic acid 0.01M increases about 30 times compared with the case without lactic acid. Thus, when ergosterol is used, the increase in current density j is less than when cholesterol is used. This means that the pore diameter of amphotericin B was affected and the permeation of sodium lactate was controlled by binding sterol in addition to amphotericin B to the lipid bilayer membrane constituting the liposome. Alternatively, it is considered that the anion selectivity of amphotericin B is increased by binding sterol to the lipid bilayer membrane BLM in addition to amphotericin B, and sodium lactate is easily permeated. That is, by using sterols, the permeation of organic acids and anion selectivity can be controlled.

  FIG. 35 shows the diffusion coefficient ratio α of various organic acids with respect to the molecular weight of the organic acid when cholesterol or ergosterol is used.

As shown in FIG. 35, with any organic acid, the diffusion coefficient ratio α is greater when cholesterol is used than when ergosterol is used. This is considered to be due to the reduced permeability of all organic acid ions to Na ⁺ when ergosterol is used compared to when cholesterol is used.

  Next, the result of confirming that glucose permeates through the hole of amphotericin B will be described. Since glucose has no charge, it cannot perform electrochemical measurements directly. Therefore, glucose is oxidized with an enzyme, and electron transfer is observed with an electrode.

  FIG. 36 shows the electrochemical measurement system used. As shown in FIG. 36, a biosensor in which glucose oxidase (GOD), peroxidase (POD) and ferrocene were immobilized on a carbon paste electrode CP was produced. And glucose was enclosed in a liposome and the elution of glucose by addition of amphotericin B was measured.

  FIG. 37 shows the measurement results. In FIG. 37, the vertical axis represents glucose concentration c, and the horizontal axis represents time t. As shown in FIG. 37, it can be seen that simultaneously with the addition of amphotericin B, the glucose concentration rose and glucose was eluted.

The glucose elution rate is theoretically determined as follows.
When the glucose elution rate is expressed as V, the initial glucose concentration as c _in , and the glucose concentration outside the liposome as c _out , the glucose elution rate V is expressed by the following equation.

ただし、ｋはアムホテリシンＢの穴の透過性を示す定数である。

Here, k is a constant indicating the permeability of the amphotericin B hole.

と求められる。ただし、

である。 Solving this differential equation, the glucose concentration c _out outside the liposome is

Is required. However,

It is.

k can be calculated by fitting the expression of c _{out with} the experimental result shown in FIG. As a result, k when using cholesterol was 6.1 ± 1.8 × 10 ⁻⁵ s ⁻¹ and k when using ergosterol was 6.3 ± 0.3 × 10 ⁻⁵ s ^−1. It was almost the same. This shows that the diameter of the hole of amphotericin B does not change for both cholesterol and ergosterol.

  With reference to FIG. 38, the advantage at the time of making an ergosterol couple | bond with a liposome is demonstrated. As shown in FIG. 38, in this case, when a neutral substance such as glucose is used as a substrate, and an anion such as an organic acid is generated as an intermediate, the substrate uptake from the amphotericin B hole is reduced. Without any problem, leakage of the intermediate to the outside of the liposome can be suppressed by reducing the anion selectivity of amphotericin B by ergosterol.

  Next, the result of confirming the outflow of glucose from the liposome when the lipid bilayer membrane constituting the liposome is a monolayer and when it is a multilayer will be described.

  FIG. 39A shows the time change of glucose concentration when using a monolayer liposome having a diameter of 50 nm combined with cholesterol, and FIG. 39B shows the time change of glucose concentration when using a monolayer liposome having a diameter of 100 nm combined with cholesterol. Show. FIG. 40 shows the change in glucose concentration over time when multilamellar liposomes bound with cholesterol are used. From FIGS. 39A and B and FIG. 40, no elution of glucose is observed until amphotericin B is added in both unilamellar liposomes and multilamellar liposomes.

  FIG. 41A shows the time change of the glucose concentration when using a monolayer liposome with a diameter of 100 nm combined with ergosterol, and FIG. 41B shows the time change of the glucose concentration when using a multilayer liposome combined with ergosterol.

  As described above, according to the first embodiment, the antibiotic 16 is bonded to the lipid bilayer membrane to form the hole 17, and the sterol 18 is further bonded to the inside of the liposome 12, for example, a substrate such as glucose. Enzymes 13 and 14 and coenzyme 15 necessary for an enzymatic reaction for decomposing organic acids and organic acids are enclosed. The liposome 12 is immobilized on the electrode 11. For this reason, the enzyme reaction can be efficiently performed using the minute space inside the liposome 12 as a reaction field, and electrons can be efficiently extracted from glucose as a substrate and transferred to the electrode 11. Further, the immobilization can be easily performed as compared with the conventional case in which an enzyme or the like is directly immobilized on the electrode 11 by a polyion complex or the like.

<2. Second Embodiment>
[Bio fuel cell]
Next explained is the second embodiment of the invention. In the second embodiment, the enzyme-immobilized electrode according to the first embodiment is used as the negative electrode of the biofuel cell.

  FIG. 42 schematically shows this biofuel cell. In this biofuel cell, glucose is used as a fuel. FIG. 43 schematically shows details of the configuration of the negative electrode of this biofuel cell, an example of an enzyme group and a coenzyme enclosed in the liposome 12 immobilized on the negative electrode, and an electron transfer reaction by the enzyme group and the coenzyme. Shown in

As shown in FIGS. 42 and 43, this biofuel cell has a structure in which the negative electrode 21 and the positive electrode 22 face each other with the electrolyte layer 23 therebetween. The negative electrode 21 decomposes glucose supplied as fuel with an enzyme to extract electrons and generate protons (H ⁺ ). The positive electrode 22 generates water by protons transported from the negative electrode 21 through the electrolyte layer 23, electrons sent from the negative electrode 21 through an external circuit, and oxygen in the air, for example.

  As the negative electrode 21, the enzyme-immobilized electrode according to the first embodiment is used. Specifically, the enzyme-immobilized electrode is obtained by immobilizing an antibiotic 11 having a hole 17 capable of permeating glucose and a sterol 18 bound to a lipid bimolecular membrane on the electrode 11. It is. Encapsulated in the liposome 12 are enzymes involved in the degradation of glucose, a coenzyme that produces a reductant during the oxidation process in the glucose degradation process, and a coenzyme oxidase that oxidizes the reductant of the coenzyme. ing. On the electrode 11, if necessary, in addition to the liposome 12, an electron mediator that receives electrons generated from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes it to the electrode 11 is also immobilized.

  As an enzyme involved in the degradation of glucose, for example, glucose dehydrogenase (GDH), preferably NAD-dependent glucose dehydrogenase can be used. In the presence of this oxidase, for example, β-D-glucose can be oxidized to D-glucono-δ-lactone.

  Furthermore, this D-glucono-δ-lactone can be decomposed into 2-keto-6-phospho-D-gluconate by the presence of two enzymes, gluconokinase and phosphogluconate dehydrogenase (PhGDH). Can do. That is, D-glucono-δ-lactone is converted to D-gluconate by hydrolysis, and D-gluconate is converted to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in the presence of gluconokinase. And then phosphorylated to 6-phospho-D-gluconate. This 6-phospho-D-gluconate is oxidized to 2-keto-6-phospho-D-gluconate by the action of the oxidase PhGDH.

In addition to the above decomposition process, glucose can also be decomposed to CO ₂ by utilizing sugar metabolism. The decomposition process utilizing sugar metabolism is roughly divided into glucose decomposition and pyruvic acid generation by a glycolysis system and a TCA cycle, which are widely known reaction systems.

The oxidation reaction in the monosaccharide decomposition process is accompanied by a coenzyme reduction reaction. This coenzyme is almost determined by the acting enzyme. In the case of GDH, NAD ⁺ is used as the coenzyme. That is, when β-D-glucose is oxidized to D-glucono-δ-lactone by the action of GDH, NAD ⁺ is reduced to NADH to generate H ⁺ .

The produced NADH is immediately oxidized to NAD ⁺ in the presence of diaphorase (DI), generating two electrons and H ⁺ . Therefore, two electrons and two H ⁺ are generated by one-step oxidation reaction per glucose molecule. In the two-stage oxidation reaction, a total of four electrons and four H ⁺ are generated.

The electrons generated in the above process are transferred from diaphorase to the electrode 11 via the electron mediator, and H ⁺ is transported to the positive electrode 22 through the electrolyte layer 23.

The liposome 12 encapsulating the enzyme and coenzyme and the electron mediator are provided with a buffer such as a phosphate buffer or a Tris buffer contained in the electrolyte layer 23 so that the electrode reaction can be performed efficiently and constantly. The solution is preferably maintained at an optimum pH for the enzyme, for example, around pH 7. As the phosphate buffer, for example, NaH ₂ PO ₄ or KH ₂ PO ₄ is used. Further, the ionic strength (IS) is too large or too small to adversely affect the enzyme activity, but considering the electrochemical response, it should be an appropriate ionic strength, for example, about 0.3. Is preferred. However, pH and ionic strength have optimum values for each enzyme used, and are not limited to the values described above.

In FIG. 43, for example, glucose dehydrogenase (GDH) is an enzyme involved in the degradation of glucose, NAD ⁺ is a coenzyme that produces a reductant in the oxidation reaction in the glucose degradation process, and a reductant of coenzyme. The case where the coenzyme oxidase that oxidizes a certain NADH is diaphorase (DI), and the electron mediator that receives electrons from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes it to the electrode 11 is ACNQ is shown.

  The positive electrode 22 is obtained by immobilizing an enzyme that decomposes oxygen, such as bilirubin oxidase, laccase, and ascorbate oxidase, on an electrode made of an electrode material such as porous carbon. The outer part of the positive electrode 22 (the part opposite to the electrolyte layer 23) is usually formed by a gas diffusion layer made of porous carbon, but is not limited thereto. Preferably, in addition to the enzyme, an electron mediator that transfers electrons to and from the positive electrode 22 is also immobilized on the positive electrode 22.

In the positive electrode 22, in the presence of the enzyme that decomposes oxygen, oxygen in the air is reduced by H ⁺ from the electrolyte layer 23 and electrons from the negative electrode 21 to generate water.

The electrolyte layer 23 is for transporting H ⁺ generated in the negative electrode 21 to the positive electrode 22, and is made of a material that does not have electron conductivity and can transport H ⁺ . Specific examples of the electrolyte layer 23 include those already mentioned, such as cellophane.

In the biofuel cell configured as described above, when glucose is supplied to the negative electrode 21 side, the glucose is decomposed by a decomposing enzyme including an oxidase. Since the oxidase is involved in the monosaccharide decomposition process, electrons and H ⁺ can be generated on the negative electrode 21 side, and a current can be generated between the negative electrode 21 and the positive electrode 22.

Next, a specific structural example of the biofuel cell will be described.
As shown in FIGS. 44A and B, this biofuel cell has a configuration in which the negative electrode 21 and the positive electrode 22 face each other with the electrolyte layer 23 therebetween. In this case, Ti current collectors 41 and 42 are placed under the positive electrode 22 and the negative electrode 21, respectively, so that current collection can be performed easily. Reference numerals 43 and 44 denote fixed plates. The fixing plates 43 and 44 are fastened to each other by screws 45, and the positive electrode 22, the negative electrode 21, the electrolyte layer 23, and the Ti current collectors 41 and 42 are sandwiched between them. One surface (outer surface) of the fixing plate 43 is provided with a circular recess 43a for taking in air, and a plurality of holes 43b penetrating to the other surface are provided in the bottom surface of the recess 43a. These holes 43 b serve as air supply paths to the positive electrode 22. On the other hand, a circular recess 44a for fuel loading is provided on one surface (outer surface) of the fixing plate 44, and a number of holes 44b penetrating to the other surface are provided on the bottom surface of the recess 44a. These holes 44 b serve as a fuel supply path to the negative electrode 21. A spacer 46 is provided on the periphery of the other surface of the fixing plate 44, and when the fixing plates 43 and 44 are fastened to each other with screws 45, the interval between them becomes a predetermined interval. .

  As shown in FIG. 44B, a load 47 is connected between the Ti current collectors 41 and 42, and a glucose solution in which glucose is dissolved in a phosphate buffer, for example, is put in the recess 44a of the fixed plate 44 to generate power. .

  According to the second embodiment, the liposomes 12 on which the antibiotics 16 having the holes 17 and the sterols 18 are bound to the lipid bimolecular membrane are encapsulated on the electrode 11. The enzyme-immobilized electrode immobilized on is used as the negative electrode 21. For this reason, the enzyme reaction can be efficiently performed using the minute space inside the liposome 12 as a reaction field, and electrons can be efficiently extracted from the fuel glucose and transferred to the electrode 11, as well as enzymes, etc. Can be immobilized more easily than the case of directly immobilizing on the electrode 11 with a polyion complex or the like. As described above, since the enzyme-immobilized electrode used as the negative electrode 21 is highly efficient, a highly efficient biofuel cell can be realized. In addition, in order to increase the output of a biofuel cell, it is necessary to extract more than two electrons from glucose as a fuel. For this purpose, an enzyme in which three or more enzymes are immobilized at appropriate positions. Although it is necessary to use an immobilized electrode for the negative electrode 21, such a requirement can be satisfied by encapsulating three or more kinds of enzymes inside the liposome 12. In addition, by mixing a plurality of types of liposomes in which three or more different types of enzymes are encapsulated, it becomes easy to handle various types of fuels. Furthermore, it is possible to realize a micro biofuel cell in which negative and positive liposomes are arranged.

<3. Third Embodiment>
[Bio fuel cell]
The biofuel cell according to the third embodiment is the same as the biofuel cell according to the second embodiment except that a porous conductive material as shown in FIGS. 45A and 45B is used for the electrode material of the negative electrode 21. It has a configuration.

  FIG. 45A schematically shows the structure of the porous conductive material, and FIG. 45B is a cross-sectional view of the skeleton of the porous conductive material. As shown in FIGS. 45A and B, this porous conductive material includes a skeleton 79a made of a porous material having a three-dimensional network structure and a carbon-based material 79b covering the surface of the skeleton 79a. Liposomes 12 similar to those of the second embodiment are immobilized on the surface of the material 79b. This porous conductive material has a three-dimensional network structure in which a large number of holes 80 surrounded by the carbon-based material 79b correspond to a network. In this case, these holes 80 communicate with each other. The form of the carbon-based material 79b is not limited and may be any of fibrous (needle-like) or granular.

  As the skeleton 79a made of the porous material, foam metal or foam alloy, for example, foam nickel is used. The porosity of the skeleton 79a is generally 85% or more, or 90% or more, and the pore diameter is generally, for example, 10 nm to 1 mm, alternatively 10 nm to 600 μm, alternatively 1 to 600 μm, typically 50 ˜300 μm, more typically 100 to 250 μm. The carbon-based material 79b is preferably a highly conductive material such as ketjen black, but a functional carbon material such as carbon nanotube or fullerene may be used.

  The porosity of the porous conductive material is generally 80% or more, or 90% or more, and the diameter of the hole 80 is typically 9 nm to 1 mm, alternatively 9 nm to 600 μm, alternatively 1 to 600 μm, typically Specifically, the thickness is 30 to 400 μm, more typically 80 to 230 μm.

Next, a method for producing this porous conductive material will be described.
As shown in FIG. 46A, first, a skeleton 79a made of a foam metal or a foam alloy (for example, foam nickel) is prepared.

  Next, as shown in FIG. 46B, a carbon-based material 79b is coated on the surface of the skeleton 79a made of the foam metal or foam alloy. As this coating method, a conventionally known method can be used. For example, the carbon-based material 79b is coated by spraying an emulsion containing carbon powder, a suitable binder, or the like onto the surface of the skeleton 79a by spraying. The coating thickness of the carbon-based material 79b is determined in accordance with the porosity and pore size required for the porous conductive material in consideration of the porosity and pore size of the skeleton 79a made of foam metal or foam alloy. In this coating, a large number of holes 80 surrounded by the carbon-based material 79b communicate with each other.

In this way, the target porous conductive material is manufactured. Thereafter, the liposome 12 is immobilized on the surface of the carbon-based material 79b of the porous conductive material.
The other than the above is the same as that of the second embodiment.

  According to the third embodiment, in addition to the same advantages as those of the second embodiment, the following advantages can be obtained. That is, the porous conductive material in which the surface of the skeleton 79a made of a foam metal or a foam alloy is coated with the carbon-based material 79b has a sufficiently large hole 80 diameter and a rough three-dimensional network structure, while having a high strength. In addition, it has high conductivity and can provide a necessary and sufficient surface area. For this reason, the negative electrode 21 composed of an enzyme / coenzyme / electron mediator immobilized electrode obtained by immobilizing an enzyme, a coenzyme and an electron mediator on the porous conductive material is used as an electrode material. The enzyme metabolic reaction can be performed with high efficiency, or the enzyme reaction phenomenon occurring in the vicinity of the electrode can be efficiently captured as an electrical signal, and is stable regardless of the use environment, It is possible to realize a high-performance biofuel cell.

<4. Fourth Embodiment>
[cell]
In the fourth embodiment, an antibiotic having a hole capable of permeating glucose in a lipid bimolecular film constituting a cell membrane of a cell obtained by culturing a cell collected from a living organism or a cell collected from an organism. And binds sterols.

  FIG. 47 shows a part of a cell membrane 81 composed of a lipid bimolecular membrane on the outer periphery of the cell. Here, although the case where this cell is an animal cell is considered, it may be a plant cell. These cells mainly include the nucleus, organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, lysosome, and water in which various ions, nutrients, enzymes, and the like are dissolved. As shown in FIG. 47, various proteins 82, 83, 84, etc. are embedded in the cell membrane 81 of this cell. For example, the protein 82 is a pump protein that transports ions and the like from the outside to the inside of the cell, the protein 83 is an intramembrane protein, and the protein 84 is a channel protein that has a function of transporting a specific molecule into and out of the cell. In addition to the above-mentioned various proteins 82, 83, and 84, the antibiotic 16 is bound to the cell membrane 81, and one or a plurality of holes 17 capable of permeating glucose are formed, and further sterol 18 is bound. is doing.

  The cells configured as described above are immobilized on an electrode made of porous carbon or the like by a conventionally known method.

  According to the fourth embodiment, when the cells are placed in a buffer solution or the like containing glucose, for example, glucose can be taken into the cell membrane 81 through the holes 17. The glucose thus taken into the cell is decomposed by a metabolic system such as a citrate circuit, a glycolysis system, and a pentose phosphate circuit of the cell, and electrons are taken out. The electrons taken out in this way are finally delivered to the electrode on which the cells are immobilized via the electron mediator or directly without the electron mediator and taken out to the outside. Thus, electric energy can be extracted from glucose.

<5. Fifth Embodiment>
[Mitochondria]
In the fifth embodiment, it is possible to permeate glucose into the lipid bilayer membrane constituting mitochondria, which is an organelle contained in a cell collected from a cell or a cell obtained by culturing a cell collected from a living organism. Forming one or more holes. Mitochondria are the place of eukaryotic energy production, and ATP is produced by the energy obtained during the oxidation reaction of food molecules.

  FIG. 48 shows mitochondria. As shown in FIG. 48, the mitochondria have an outer membrane 85 and an inner membrane 86. These outer membrane 85 and inner membrane 86 are cell membranes composed of lipid bimolecular membranes. The inner membrane 86 forms a pleated crystal. Various enzymes are concentrated in the matrix cavity 87 inside the inner membrane 86. The final stage of molecular oxidation occurs at the inner membrane 86. Antibiotics 16 are bound to the cell membranes constituting the mitochondrial outer membrane 85 and inner membrane 86, one or more holes 17 capable of permeating glucose are formed, and sterols 18 are further bound. .

  The mitochondria configured as described above are immobilized on an electrode made of porous carbon or the like by a conventionally known method.

  According to the fifth embodiment, when the mitochondria are placed in a buffer solution or the like containing glucose, glucose can be taken in through the holes 17 of the outer membrane 85 and the inner membrane 86. The glucose thus taken into the mitochondria is decomposed by the oxidation system of the molecules of the mitochondria, that is, the citrate circuit, glycolysis system, pentose phosphate circuit, etc., and electrons are taken out. The electrons taken out in this way are finally transferred to the electrode on which the mitochondria are immobilized via the electron mediator or directly without the electron mediator and taken out to the outside. Thus, electric energy can be extracted from glucose.

<6. Sixth Embodiment>
[Bacteria]
In the sixth embodiment, one or a plurality of holes capable of glucose permeation are formed in the lipid bimolecular membrane constituting the bacterial cell membrane.

  FIG. 49 shows bacteria. Here, consider a case where this bacterium is an eubacteria. As shown in FIG. 49, this bacterium is surrounded by a capsule 88, a cell wall 89, and a cell membrane 90 in order from the outside, and contains a cytoplasm 91 therein. Flagella 92 and cilia 93 are bound to the outside of this bacterium. However, some bacteria lack the capsule 88, flagella 92, and cilia 93. For example, E. coli does not have these capsules 88, flagella 92, and cilia 93. In the cytoplasm 91, although not shown, nucleoids, ribosomes and the like are present. Proteins such as an electron transport system and various transporters are distributed in the cell membrane 90. Antibiotic 16 is bound to cell membrane 90 made of this bacterial lipid bilayer, one or a plurality of holes 17 capable of permeating glucose are formed, and sterol 18 is further bound.

  The bacterium configured as described above is immobilized on an electrode made of porous carbon or the like by a conventionally known method.

  According to the sixth embodiment, when this bacterium is placed in a buffer solution or the like containing glucose, glucose can be taken in through the hole 17 of the cell membrane 90 of this bacterium. The glucose thus taken into the inside of the bacterium is decomposed by a metabolic system such as a citrate circuit, a glycolysis system, and a pentose phosphate circuit of the bacterium, and an electron is taken out. The electrons thus extracted are finally transferred to the electrode on which the bacteria are immobilized via the electron mediator or directly without the electron mediator, and taken out to the outside. Thus, electric energy can be extracted from glucose.

<7. Seventh Embodiment>
[Bio fuel cell]
The biofuel cell according to the seventh embodiment is the same as the biofuel cell according to the second embodiment except that a porous electrode as shown in FIGS. 50A and B is used as the electrode 11 of the negative electrode 21. It has a configuration.

  FIG. 50A shows the overall configuration of the electrode 11 made of this porous electrode, and FIG. 50B schematically shows the cross-sectional structure of the electrode 11. As shown in FIG. 50B, the electrode 11 made of this porous electrode has a large number of pores 11a. On the inner surface of the hole 11a, the same liposome 12 as in the second embodiment (enzymes and coenzymes enclosed in the internal aqueous phase, antibiotics bound to the lipid bilayer, holes bordered by the antibiotics, etc.) Is omitted). In this case, these holes 11a communicate with each other. As the material for the porous electrode, a carbon-based material is preferably used, but the material is not limited to this. This porous electrode is typically a carbon paste electrode.

The liposome 12 can be fixed to the inner surface of the hole 11a of the electrode 11 made of this porous electrode by allowing a solution containing the liposome 12 to penetrate into the electrode 11.
The other than the above is the same as that of the second embodiment.
According to the seventh embodiment, the same advantages as those of the third embodiment can be obtained.

<8. Eighth Embodiment>
[Enzyme immobilized electrode]
FIG. 51 shows an enzyme-immobilized electrode according to the eighth embodiment. In this enzyme-immobilized electrode, glucose is used as a substrate.

  As shown in FIG. 51, in this enzyme-immobilized electrode, avidin 101 is immobilized on the surface of electrode 11 made of porous carbon or the like (including the inner surface of the hole inside electrode 11). On the other hand, biotin 102 is bound to the same liposome 12 as in the first embodiment. Then, avidin 101 immobilized on the surface of electrode 11 and biotin 102 bound to liposome 12 are irreversibly specifically bound. In FIG. 51, a case where one biotin 102 is bound to one liposome 12 is shown, but a plurality of biotins 102 may be bound to one liposome 12.

  Enzymes 13 and 14 and coenzyme 15 involved in the target enzyme reaction are enclosed in the aqueous phase 12a inside the liposome 12. In addition to these enzymes 13 and 14 and coenzyme 15, for example, an electron mediator may be enclosed in the aqueous phase 12 a inside the liposome 12. This electron mediator may be immobilized on the electrode 11 together with the liposome 12. Enzyme 13 is an oxidase that promotes and decomposes the oxidation of glucose used as a substrate, and enzyme 14 returns coenzyme 15 that has been reduced due to the oxidation of glucose to an oxidant and also sends electrons to electrode 11 via an electron mediator. It is a coenzyme oxidase that passes.

  Although illustration is omitted, like the liposome 12 shown in FIG. 2, one or a plurality of antibiotics and sterols are bound to the lipid bilayer membrane constituting the liposome 12, and this antibiotic is surrounded by this antibiotic. A hole penetrating the lipid bilayer membrane is formed.

[Method for producing enzyme-immobilized electrode]
This enzyme-immobilized electrode can be produced, for example, as follows. First, the liposomes 12 in which the enzymes 13 and 14 and the coenzyme 15 are enclosed and the biotin 102 is bound to the outer peripheral surface are prepared. In order to bind the biotin 102 to the liposome 12, for example, biotin 102 modified with NHS ester is used for dehydration condensation with a phospholipid such as phosphatidylethanolamine to form an amide bond. Next, an antibiotic is bound to the lipid bilayer membrane constituting the liposome 12 to form a hole, and sterol is bound to the lipid bilayer membrane. On the other hand, avidin 101 is immobilized on the surface of the electrode 11. In order to immobilize the avidin 101 on the electrode 11, for example, a carbon paste electrode mixed with agarose beads to which the avidin 101 is bonded is used as the electrode 11, or the avidin 101 is poly-L-lysine on the surface of the electrode 11. Or embedded in a polymer such as (PLL). Next, the liposome 12 is immobilized on the electrode 11 by binding the avidin 101 thus immobilized on the surface of the electrode 11 and the biotin 102 bound to the liposome 12 having the holes 17 formed therein. As a result, an enzyme-immobilized electrode is produced. Antibiotics may be bound to the lipid bimolecular membrane constituting the liposome 12 before encapsulating the enzymes 13 and 14 and the coenzyme 15 to form holes, and sterols may be bound. Further, after immobilizing the liposome 12 on the electrode 11, an antibiotic may be bound to the lipid bimolecular membrane constituting the liposome 12 to form a hole, and a sterol may be bound.

<Example 2>
An enzyme-immobilized electrode was produced as follows. Here, however, sterol is not bound to the liposome in order to exclude the effect of sterol.

  Diaphorase (DI) (EC 1.8.1.4, manufactured by Amano Enzyme) was weighed in 5 mg and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to prepare a 5 mg / mL DI enzyme buffer solution. .

  Glucose dehydrogenase (GDH) (NAD-dependent, EC 1.1.1.47, manufactured by Amano Enzyme) was weighed in 25 mg and dissolved in 1 mL of a buffer solution (10 mM phosphate buffer, pH 7) to obtain 25 mg / mL GDH enzyme. A buffer solution was prepared.

  The buffer solution for dissolving the enzyme is preferably refrigerated until just before, and the enzyme buffer solution is preferably stored as refrigerated as much as possible.

  NADH (manufactured by Sigma) was dissolved in a buffer solution (10 mM phosphate buffer, pH 7) to prepare a 50 mM NADH buffer solution.

  2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0) was dissolved in a buffer solution (10 mM phosphate buffer, pH 7) to prepare a 10 mM Q0 buffer solution.

  Egg yolk lecithin (manufactured by Wako) and biotin-modified phosphatidylethanolamine (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (cap biotinyl)) were used as phospholipids.

Biotin-modified liposomes were prepared as follows.
Lecithin is dissolved in ethanol to prepare a 100 mg / mL lecithin solution. In addition, biotin-modified phosphatidylethanolamine is dissolved in a mixed solution of CHCl ₃ , methanol and water (CHCl ₃ : methanol: water = 65: 35: 8) to prepare a 10 mg / mL biotin-modified phosphatidylethanolamine solution.

  Add 1 mL of the above lecithin solution and 50 μL of the above biotin-modified phosphatidylethanolamine solution to 20 mL of ethanol and mix well. The mixed solution thus obtained is treated with a rotary evaporator in a 40 ° C. bath under vacuum for 1 hour or longer to remove the solvent under reduced pressure.

  Next, 50 μL of 25 mg / mL GDH enzyme buffer solution and 50 μL of 5 mg / mL DI enzyme buffer solution and 50 μL of 50 mM NADH buffer solution were added to the obtained product, and added for 30 to 60 seconds. Put on the bus. At that time, the phospholipid film is scraped off with a polytetrafluoroethylene scraper.

  Next, the liposome solution obtained as described above is transferred to a 1.5 mL tube, and the remaining liposome solution is also collected with 50 μL of 100 mM phosphate buffer, and 1500 g using 1 mL of 100 mM phosphate buffer, Wash twice by centrifugation at 4 min.

  GDH, DI and NADH encapsulated biotin-modified liposomes thus obtained are suspended in 600 μL of 0.1 M phosphate buffer, amphotericin B is added to a final concentration of 1 mM, and mixed by vortexing.

  As described above, biotin-modified liposomes in which GDH, DI, and NADH are encapsulated and amphotericin B is bound are prepared.

An avidin modified electrode was produced as follows.
Add 100 μL of avidin agarose bead suspension to 200 mg of carbon paste (manufactured by BAS Co., Ltd.) mixed with graphite powder of uniform particle size and paraffin oil used as an adhesive, and knead with light force on an agate mortar. Include. Next, the avidin-containing carbon paste thus obtained is dried in a desiccator for 1 hour or more, and then kneaded into a pocket portion of an electrode having a pocket portion (concave portion) on the upper surface. Thereafter, the upper surface side of the electrode was pressed in a circle on a copy paper to flatten the surface.
An avidin modified electrode is produced as described above.

  Next, GDH, DI, and NADH were encapsulated and biotin-modified liposomes bound with amphotericin B were immobilized on an avidin-modified electrode as follows.

  First, the avidin-modified electrode prepared as described above is immersed in 300 μL of the biotin-modified liposome prepared as described above. Then, after incubation at room temperature for 1 hour, the cells are washed twice with 500 μL of 0.1 M phosphate buffer.

  FIG. 52 shows a biotin-modified liposome in which GDH, DI, and NADH are encapsulated and amphotericin B is bound to an avidin-modified electrode. As shown in FIG. 52, a carbon paste electrode 104 is embedded in a pocket portion 103 a provided on the upper surface of the electrode 103, and the avidin 101 is fixed to the carbon paste electrode 104. The avidin 101 and the biotin 102 of the liposome 12 are bonded to each other, so that the liposome 12 is bonded to the carbon paste electrode 104 and immobilized. Enzymes 13 and 14 and coenzyme 15 enclosed in aqueous phase 12a inside liposome 12 are GDH, DI and NADH, respectively.

  The electrochemical response of the liposome-immobilized electrode prepared as described above (hereinafter abbreviated as AviCPE-BioLipo if necessary) was measured. As a first comparative example, as shown in FIG. 53, a liposome-immobilized electrode similar to the above except that the avidin 101 is not immobilized on the carbon paste electrode 104 (hereinafter referred to as CPE-BioLipo as necessary). (Abbreviation) was prepared. As a second comparative example, as shown in FIG. 54, a liposome-immobilized electrode similar to the above except that biotin 102 is not bound to the liposome 12 (hereinafter abbreviated as AviCPE-Lipo as necessary). Produced. As a third comparative example, as shown in FIG. 55, a liposome similar to the above except that avidin 101 is not immobilized on carbon paste electrode 104 and biotin 102 is not bound to liposome 12. A fixed electrode (hereinafter abbreviated as CPE-Lipo as needed) was produced.

  Add 180 μL of 0.1 M phosphate buffer and 20 μL of 10 mM Q0 buffer solution to a final concentration of 1 mM into a 2 mL tube, and add platinum wire as a counter electrode, Ag | AgCl as a reference electrode, and the above four types as a working electrode. The liposome-immobilized electrode (AviCPE-BioLipo, CPE-BioLipo, AviCPE-Lipo, CPE-Lipo) was dipped, the potential was set to +0.3 V with respect to Ag | AgCl, and chronoamperometry was performed. After the start of measurement, 20 μL of 1M glucose solution was added so that the final concentration was 100 mM. The results of chronoamperometry are shown in FIG. Each current (I) -time curve in FIG. 56 is shown with an offset of 0.03 μA added, but the base current values are comparable. From FIG. 56, when using a liposome-immobilized electrode (AviCPE-BioLipo) in which avidin 101 is immobilized on the carbon paste electrode 104 and biotin 102 is bound to the liposome 12, 0.015 μA → It turns out that a comparatively high electric current value response is seen compared with 0.023 microampere and other liposome fixed electrodes (CPE-BioLipo, AviCPE-Lipo, CPE-Lipo).

  As described above, according to the eighth embodiment, the antibiotic 16 is bonded to the lipid bilayer membrane to form the hole 17, and glucose is decomposed inside the liposome 12 to which the sterol 18 is bonded. Enzymes 13 and 14 and a coenzyme 15 necessary for the enzyme reaction to enclose are encapsulated. The avidin 101 is immobilized on the electrode 11, the biotin 102 is bound to the liposome 12, and the avidin 101 immobilized on the electrode 11 and the biotin 102 bound to the liposome 12 are bound, whereby the liposome 12 is bound to the electrode 11. Is fixed. For this reason, in addition to the advantage similar to 1st Embodiment, the advantage that the liposome 12 can be fixed to the electrode 11 easily and reliably and firmly can be acquired. This liposome-immobilized electrode is suitable for use as, for example, a negative electrode of a biofuel cell.

<9. Ninth Embodiment>
[Enzyme immobilized electrode]
FIG. 57 shows an enzyme-immobilized electrode according to the ninth embodiment. In this enzyme-immobilized electrode, glucose is used as a substrate.

  As shown in FIG. 57, in this enzyme-immobilized electrode, multilayer liposome 12 is immobilized on the surface of electrode 11 by utilizing the fact that avidin 101 has four biotin binding pockets. That is, as in the eighth embodiment, the avidin 101 immobilized on the surface of the electrode 11 and the biotin 102 bound to the liposome 12 are irreversibly and specifically bound to each other, thereby causing the liposome on the surface of the electrode 11. 12 is fixed. In addition, in this case, a plurality of (two in the example shown in FIG. 57) biotins 102 are bound to the liposome 12 immobilized on the surface of the electrode 11. Biotin 102 that is not used for immobilizing liposome 12 immobilized on the surface of electrode 11 on the surface of electrode 11 is bonded to avidin 101 that is not immobilized on the surface of electrode 11. And this avidin 101 and the one or two or more biotin 102 couple | bonded with the other liposome 12 couple | bond together, and the liposomes 12 mutually couple | bond together. Thus, the multilayer liposome 12 is immobilized on the surface of the electrode 11. Others are the same as in the eighth embodiment.

[Method for producing enzyme-immobilized electrode]
In order to manufacture this enzyme-immobilized electrode, after the liposome 12 is immobilized on the electrode 11 in the same manner as the enzyme-immobilized electrode according to the eighth embodiment, for example, avidin 101 or avidin agarose beads are added. Then, the liposome 12 is laminated. In this case, by adjusting the amount of avidin to be added and the ratio of biotin-modified lipid in the lipid bilayer membrane constituting the liposome 12, the layer thickness of the liposome 12 to be laminated, the density of the liposome 12 and the like can be controlled. .
According to the ninth embodiment, the same advantages as in the eighth embodiment can be obtained.

  Although the embodiments and examples of the present invention have been specifically described above, the present invention is not limited to the above-described embodiments and examples, and various modifications based on the technical idea of the present invention. Is possible.

  For example, the numerical values, structures, configurations, shapes, materials, and the like given in the above-described embodiments and examples are merely examples, and different numerical values, structures, configurations, shapes, materials, etc. are used as necessary. Also good.

  The method of immobilizing the liposomes on the electrode according to the present invention using specific binding between avidin and biotin, etc. is also used when the lipid bilayer membrane constituting the liposome does not have a hole through which glucose can permeate. It is an effective method. For example, when this electrode is used for the negative electrode of a fuel cell, the fuel is not limited to glucose, and various fuels including alcohol can be used.

  DESCRIPTION OF SYMBOLS 11 ... Electrode, 12 ... Liposome, 13, 14 ... Enzyme, 15 ... Coenzyme, 16 ... Antibiotic, 17 ... Hole, 18 ... Sterol, 21 ... Negative electrode, 22 ... Positive electrode, 23 ... Electrolyte layer, 41, 42 ... Ti Current collector, 43, 44 ... Fixing plate, 47 ... Load, 81, 90 ... Cell membrane, 85 ... Outer membrane, 86 ... Inner membrane, 101 ... Avidin, 102 ... Biotin

Claims (20)

The positive electrode and the negative electrode have a structure facing each other through a proton conductor, and are configured to extract electrons from the fuel using an enzyme,
At least one of the above enzymes is encapsulated in the liposome,
A fuel cell in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the liposome.   The fuel cell according to claim 1, wherein the fuel cell is configured to take out electrons from the fuel using the enzyme and the coenzyme, and at least one kind of the enzyme and at least one coenzyme are encapsulated in the liposome.   The fuel cell according to claim 2, wherein the weight ratio of the sterol to the lipid bilayer membrane constituting the liposome is from 1/4 to 1/3.   4. The fuel cell according to claim 3, wherein the antibiotic is amphotericin B or ionophore.   The fuel cell according to claim 4, wherein the sterol is at least one selected from the group consisting of cholesterol, ergosterol, coprostanol, sitosterol and stigmasterol.   The fuel cell according to claim 5, wherein the liposome is immobilized on the negative electrode.   The fuel cell according to claim 6, wherein the first substance immobilized on the negative electrode and the second substance bonded to the liposome are bonded to each other. Combination of the first substance and the second substance, avidin and biotin, antigen and antibody, protein A and immunoglobulin I _g G, protein G and immunoglobulin I _g G, sugar molecule and a lectin, a DNA complementary The fuel cell according to claim 7, which is a strand DNA, glutathione and glutathione S-transferase, heparin and heparin-binding molecule, hormone and hormone receptor or carboxylic acid and imide.   The fuel cell according to claim 7, wherein at least two liposomes are bonded to each other.   When manufacturing a fuel cell having a structure in which a positive electrode and a negative electrode are opposed to each other through a proton conductor and taking out electrons from fuel using an enzyme, after encapsulating at least one kind of the enzyme in the liposome, A method for producing a fuel cell, comprising a step of binding an antibiotic having a hole capable of permeating glucose and a sterol to a lipid bimolecular membrane constituting the same.   The method for producing a fuel cell according to claim 10, wherein the fuel cell takes out electrons from the fuel using the enzyme and the coenzyme, and encapsulates at least one type of the enzyme and at least one type of coenzyme in the liposome. Using one or more fuel cells,
At least one of the fuel cells is
The positive electrode and the negative electrode have a structure facing each other through a proton conductor, and are configured to extract electrons from the fuel using an enzyme,
At least one of the above enzymes is encapsulated in the liposome,
An electronic device that is a fuel cell in which an antibiotic having a hole capable of permeating glucose and a sterol are bonded to a lipid bimolecular membrane constituting the liposome.   13. The electronic device according to claim 12, wherein the electronic device is configured to take out electrons from the fuel using the enzyme and the coenzyme, and at least one kind of the enzyme and at least one coenzyme are encapsulated in the liposome. A liposome encapsulating at least one enzyme is immobilized,
An enzyme-immobilized electrode in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the liposome.   The enzyme-immobilized electrode according to claim 14, wherein at least one enzyme and at least one coenzyme are encapsulated in the liposome. Using enzymes,
At least one of the above enzymes is encapsulated in the liposome,
A biosensor in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the liposome.   An energy conversion element, wherein a liposome encapsulating at least one enzyme is used, and an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the liposome.   A cell in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting the cell membrane.   An organelle in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting a cell membrane.   A bacterium in which an antibiotic having a hole capable of permeating glucose and a sterol are bound to a lipid bimolecular membrane constituting a cell membrane.

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