JP2005511098A - Biocompatible membrane of block copolymer and fuel cell produced using the same - Google Patents

Abstract

  The present invention is made from biocompatible membranes, solutions useful for producing biocompatible membranes, and synthetic polymeric materials comprising at least one block copolymer and optionally at least one additive and a polypeptide. The present invention relates to a fuel cell that can use the biocompatible membrane.

Description

  The present invention relates to a biocompatible membrane of a block copolymer and a fuel cell produced using the same.

  In a series of papers by Meier et al., Various structures were proposed for polymer-based membranes containing functional proteins. Such membranes were previously only the subject of reasoning, but are now considered to be the first successful biological protein containing polymer-based membranes containing embedded enzymes that retain their functionality. It has been. See, for example, Corinne Nardin, Wolfgang Meier et al. , 39 Angew Chem. Int. Ed. 4599-602 (2000), Langmuir, 16 1035-41 (2000) and Langmuir, 16 7708-12 (2000). These articles describe functionalized poly (2-methyloxazoline) -block-poly (dimethylsiloxane) -block-poly (in which a protein ("porin" -non-selective passive pore-forming molecule) is incorporated. 2-methyloxazoline) triblock copolymers are described.

  The work of Meier et al. Is original but limited in scope. Their work does not consider the use of polymers extensively and does not even suggest that the disclosed polymer membrane can be used in conjunction with other enzymes. These papers discuss the possibility of creating synthetic membranes containing embedded biological species that can participate in oxidation or reduction, or "polypeptide-mediated transport of active molecules, atoms, protons or electrons across the membrane". Certainly not suggest anything. Indeed, the narrow scope of disclosure and other lack of success provide little reason for optimistic maneuvers that other biological materials can be successfully incorporated into polymer membranes.

  The creation of membranes for testing membrane-bound proteins has been known for many years. “Functional Assembly of Membrane Proteins in Planar Lipid Bilayers”, 14 Quart. Rev. Biophys. 1-79 (1981). In fact, transmembrane proteins and redox proteins are membranes of biological systems, such as membranes made from molecules found in living cells or organisms, for the purpose of studying their structure and mechanism. Built in. Mention is also made of the use of lipid bilayers containing integral enzyme complexes that can transport protons across the membrane and / or participate in redox reactions, such as NADH dehydrogenase from E. coli. . See US Patent Application Publication No. 2002/0001739 to Liberatore et al., Published January 3, 2002. Indeed, Liberatore et al. Mention the use of such a membrane as part of a battery.

  Whether there is a biological membrane containing an enzyme complex or the discovery of a single combination of a polymer membrane and a specific enzyme, develop a wide variety of synthetic biocompatible polymer membranes that are stable and functional Give little expectation to. G. Tayhas et al. , “A Methanol / Dioxogen Biofuel Cell That Uses NAD + Dependent tide in Ode ed e ent e ed e ent e ed e m e ent e ed e ent e ed e ent e m e ent e ed e ent e ed e ent e ed e ent e ed e ent e ed e ent e ed e ent e ed e ent e ed e ed e ed e ed e ed e ed e ed e ed e ed e ed e ed e ed e ed e ed e ed e ed e ent Electroanalytical Chem. See also 155-161 (1998).

  The present invention relates to a biocompatible membrane comprising at least one layer of a synthetic polymeric material having a first side and a second side. The biocompatible membrane contains at least one polypeptide associated therewith.

In one preferred embodiment, the present invention relates to the transport of molecules, atoms, protons or electrons from which the polypeptide can participate in a chemical reaction or from the first side of at least one layer to the second side of the same layer. It relates to biocompatible membranes that can participate or participate in the formation of molecular structures that facilitate such reactions or transport. In a more particularly preferred embodiment of the invention, the polypeptide is an oxidoreductase and / or an enzyme capable of participating in transmembrane transport of protons. Furthermore, the polypeptide may have the ability to both cause the release of electrons and participate in proton transport across the membrane. When considering transporting protons across a biocompatible membrane, it will be understood that neither the exact mechanism nor the exact chemical species being transmitted is known. The species to be transmitted will probably be the proton itself, positively charged hydrogen, hydronium ion, H ₃ O ⁺ or other charged species. For convenience, however, these are collectively referred to herein as “protons”.

  According to the invention, any synthetic polymeric material that is a block copolymer, copolymer or polymer or mixtures thereof can be used as long as it can form a biocompatible membrane. In one preferred embodiment of the present invention, the synthetic polymeric material consists exclusively of at least one block copolymer. Mixtures of block copolymers are also anticipated. Optionally, the synthetic polymeric material will contain at least one additive. In a second preferred embodiment according to the present invention, the synthetic polymeric material contains at least one polymer, copolymer or block copolymer. However, if a block copolymer is present, the synthetic polymeric material also contains at least one polymer or copolymer. One optional additive is also contemplated. In yet another preferred embodiment of the present invention, the synthetic polymeric material can be any polymeric material capable of forming a biocompatible membrane, and additionally contains at least one stabilizing polymer.

  In yet another aspect of the present invention, the synthetic polymeric material is a block copolymer, a mixture of block copolymers, or a mixture of one or more block copolymers and a stabilizing polymer rich in hydrogen bonds. Preferably, the polypeptide is incorporated into a synthetic polymeric material so that a biocompatible membrane can be formed.

  As used herein, a “biocompatible membrane” is a composition that can be used as a membrane to form sheets, plugs or other structures that are frequently associated with polypeptides or other molecules of biological origin. One or more layers of polymeric material. “Biocompatibility” means a synthetic polymeric material that does not disable or otherwise block all of the functionality of the polypeptide when the synthetic polymeric material and the polypeptide are bound to each other. This means that the film itself is made. As used herein, a “membrane” contains a synthetic polymeric material as at least its main structural element and can be used to selectively separate spaces, fluids (liquid or gas), solids, etc. A structure such as a sheet of material, a layer, or a plug. As used herein, a membrane may include a permeable material that allows passage or diffusion of some species from one side to the other. Membranes used in fuel cells, for example, prevent passage of some components from the cathode compartment into the anode compartment and / or prevent passage of some components from the anode compartment into the cathode compartment. However, other components can pass freely. At the same time, as illustrated in one embodiment of the membrane according to the present invention, the membrane will allow and further facilitate the passage of protons from the anode compartment to the cathode compartment.

  “Connected” according to the present invention can mean a number of things depending on the situation. The polypeptide associates with the biocompatible membrane by binding to one or more surfaces of the membrane and / or by packing or binding within one or more surfaces of the membrane (eg, within a recess or pore). be able to. A “linked” polypeptide can be placed within a membrane or within a vesicle or cavity contained within the membrane. The polypeptide could also be placed between successive layers. The polypeptide can also be incorporated into the membrane. Indeed, in a particularly preferred embodiment, the polypeptide is at least partially exposed from at least one side of the membrane and / or molecules, atoms from one side to the other side of the redox reaction or membrane. Embedded in or integrated into the membrane in such a way that it can participate in polypeptide-mediated transport of protons or electrons.

  The term “involved” in the context of transporting molecules, atoms, protons or electrons from one side of the membrane to the other, eg, polypeptides are not exclusive, but usually pH, concentration across the membrane Or active transport that physically or chemically “pumps” molecules, atoms, protons or electrons against the charge gradient, or other active transport mechanisms. However, “involvement” need not be so limited. The mere presence of the polypeptide in the membrane can change the structure or properties of the membrane, for example, to permit sufficiently that protons are transported from a fairly high proton concentration to a fairly low proton concentration on the other side of the membrane. Also good. This is not limited to passive non-selective processes, such as those resulting from the use of non-selective passive pore formers or simple diffusion. In particular, in some cases, inactivation of polypeptides within the membrane provides inferior results to similar membranes made without any polypeptide. These processes (excluding passive diffusion) are collectively referred to as “polypeptide-mediated transport,” where the presence of the polypeptide is a chemistry that transcends membranes in ways other than simply providing structural static channels. It plays an important role in the transport of species. In other words, “polypeptide-mediated transport” means that the presence of a polypeptide responds to something other than just concentration, resulting in effective transport from one side of the membrane to the other. . “Involved” in the context of a redox reaction means that the polypeptide initiates or promotes the oxidation and / or reduction of a chemical species, or a proton, an electron or an oxidized or reduced species into or from the reaction. Means carrying.

  "One or more polypeptides" often involves a chemical reaction as a catalyst, participates in the transport of molecules, atoms, protons or electrons from one side of the membrane to the other, or such Included are at least one molecule composed of four or more amino acids that can participate in the formation of a molecular structure that facilitates or enables reaction or transport. A polypeptide may be single chain, multiple chain, and may be present in a single subunit or multiple subunits. Polypeptides can be generated from amino acids alone or from combinations of amino acids with other molecules. This can include, for example, PEGylated peptides, peptide nucleic acids, peptide mimetics, nucleoprotein complexes. Also contemplated are strands of amino acids that contain modifications such as glycosylation. The polypeptides according to the invention are generally biological molecules or derivatives or conjugates of biological molecules. Thus, a polypeptide can include molecules that can be isolated, as well as molecules that can be produced by recombinant techniques, or molecules that must be chemically synthesized in whole or in part. The term thus includes native proteins and enzymes, variants thereof, derivatives and conjugates thereof, as well as all synthetic amino acid sequences and derivatives and conjugates thereof. In one preferred embodiment, the polypeptide according to the invention can participate in the transport of molecules, atoms, protons and / or electrons from one side of the membrane to the other, or participate in oxidation or reduction. Or a charge-driven proton pump polypeptide such as DH-complex I (also referred to as “complex 1”).

The present invention stems from the recognition that a wide range of synthetic polymeric materials and polypeptides can be used to make biocompatible membranes. Biocompatible membranes, when manufactured according to the present invention, are more stable, have a longer life, are more durable, and can be deployed in a wide range of useful environments compared to their full biological counterparts. It can have advantages in that respect. Also, some of the biocompatible membranes of the present invention can operate upon contact on one side with a solution that is very different and has a very extreme pH. They can also be prepared to be stable in the presence of certain oxidizing and / or reducing agents and to be useful at fairly extreme temperatures or other operating and storage conditions. They will facilitate the passage of current to at least a greater extent than the passage of current that would occur using the same membrane except for the polypeptide. Preferably, the biocompatible membrane of the present invention is at least 10 pA (picoampere) / cm ² (such as when a biocompatible membrane is used in the sensor), more preferably at least about 10 mA (milliampere) / cm ² , and more More preferably it will produce about 100 mA / cm ² .

  These biocompatible membranes generally stand, but are not limited to, stand alone as membranes in air and can therefore be at least partially desolvated. When used in fuel cells, these biocompatible membranes preferably have a useful operating period of at least 8 hours, more preferably at least 3 days, even more preferably 1 month or more, and even more preferably 6 months or more. Would have.

A biocompatible synthetic polymer membrane containing a polypeptide capable of participating in redox reactions and / or in transporting molecules, atoms, protons or electrons from one side of the membrane to the other, This is particularly advantageous because it can be used to make a wide range of batteries or fuel cells. These include batteries that are environmentally friendly, lightweight, compact and easy to carry. It is also possible to produce a fuel cell with a very high output. Preferably, a fuel cell made in accordance with the present invention has at least 10 mW (milliwatts) / cm ² , preferably at least about 50 mW / cm ² when a circuit with a load or resistance is created between the anode and cathode. ² and most preferably at least about 100 mW / cm ² can be generated. This is also said to be electrically connected.

  Therefore, another aspect of the present invention is a fuel cell. The fuel cell includes an anode compartment having an anode and a cathode compartment having a cathode. The fuel cell also includes at least one biocompatible membrane that can be disposed in the anode compartment, in the cathode compartment, or between the anode compartment and the cathode compartment. The biocompatible membrane can include at least one layer of synthetic polymeric material and at least one polypeptide associated therewith, as discussed above. Preferably, the polypeptide has the ability to participate in redox reactions and / or participate in the transport of molecules, atoms, protons or electrons from one side of the membrane to the other. In particularly preferred embodiments, the polypeptide can participate in both redox reactions and transport of molecules, atoms, protons or electrons. Such a fuel cell may include an electron carrier and a second polypeptide that are both disposed within the anode compartment.

  Yet another aspect of the present invention is the creation of a solution that is itself useful for producing a biocompatible membrane according to the present invention. The solution contains at least one synthetic polymeric material and at least one polypeptide in a solvent system that typically includes both an organic solvent and water. Preferably, the synthetic polymeric material is about 1 to about 30% (w / v), and more preferably about 2 to about 20% (w / v), and most preferably about 2 to about 10% (w / v). ). Similarly, the polypeptide is about 0.001 to about 10.0% (w / v) in solution, more preferably about 0.01 to about 7.0% (w / v), and even more preferably about Present in an amount of 0.1 to about 5.0% (w / v). The solution may also contain solubilized detergent additives and other materials if desired. In one preferred embodiment, the synthetic polymeric material consists of at least one block copolymer. In another preferred embodiment, the synthetic polymeric material comprises at least one polymer, provided that the synthetic polymeric material also comprises at least one polymer or copolymer when the synthetic copolymer material comprises at least one block copolymer. Including polymers, copolymers or block copolymers. In another preferred embodiment, the synthetic polymeric material contains at least one stabilizing polymer.

  A biocompatible membrane according to the present invention can be formed from any synthetic polymeric material that meets the objectives of the present invention when combined with one or more polypeptides as described herein.

  Synthetic polymeric materials can include polymers, copolymers and block copolymers and mixtures thereof. They can be linked together, crosslinked, functionalized, or otherwise bound. “Functionalization” refers to polymer, copolymer and / or block copolymer polymerization known in the art (eg, cross-linking of blocks), anchoring to specific surface chemistry (eg, certain Whether using sulfur linkages), electron transport facilitated through covalent bonds such as electron carriers or electron transfer mediators, modified with end groups selected to perform a specific function Means that. Typically, these end groups are not considered components of the polymer or block itself, and are often added at the end of synthesis or after synthesis. Synthetic polymeric materials generally are at least about 50% by weight of the final membrane on the final membrane (a membrane ready for use), more typically at least about 60% by weight of the final membrane, and often about 70-99% by weight. Present in amounts up to. A portion of the total amount of synthetic polymeric material may be a stabilizing polymer, generally up to about one third the weight based on the weight of the total synthetic polymeric material in the final biocompatible membrane.

  The biocompatible membranes of the present invention preferably include AB, ABA, with or without other synthetic polymeric materials such as polymers or copolymers, and with or without additives. Or it is produced from one or more block copolymers such as ABC block copolymers.

One suitable block copolymer is described in a series of articles by Corinne Nardin, Wolfgang Meier and others. Angew Chem Int. Ed. 39: 4599-4602, 2000, Langmuir 16: 1035-1041, 2000, Langmuir 16: 7708-7712, 2000. The functionalized poly (2-methyloxazoline) -block-poly (dimethylsiloxane) -block-poly (2-methyloxazoline) triblock copolymer described is as follows.

  In the above chemical formula, the average value of x is 68 and the average value of y is 15. This is an ABA block copolymer in which the “C” listed in the formula does not necessarily match the “C” name of the ABC block copolymer.

  The polymers exemplified above can provide a reasonably large membrane that can incorporate functional proteins. The methacrylate component at the end of the polymer molecule allows free radical mediated crosslinking after incorporation of the protein to add greater mechanical stability. Biocompatible membranes such as this, especially those that are non-ionic, have greater stability to higher voltage differences between the anode and cathode.

The functionalized poly (2-methyloxazoline) -block-poly (dimethylsiloxane) -block-poly (2-methyloxazoline) triblock copolymer is one example of a synthetic polymeric material that can be used. Other representative block copolymers include, without limitation, the following block copolymers. Amphiphilic block copolymers [vesicular triblock copolymer shells can be considered biological membrane mimics, but they are 2-3 times thicker than conventional lipid bilayers. Nevertheless, they can function as a matrix for intramembrane proteins. Surprisingly, these proteins remain functional even though the membrane is very thick and even after polymerization of the reactive triblock copolymer. ], A triblock copolymer ampholyte from 5- (N, N-dimethylamino) isoprene, styrene, and methacrylic acid [Bieringer et al. , Eur. Phys. J. et al. E. 5: 5-12, 2001. Such polymers are particularly _{Ai 14 S 63 A 23, Ai} 31 S 23 A 46, Ai 42 S 23 A 35, Ai 56 S 23 A 21, Ai 57 S 11 A 32], styrene - ethylene / butylene - Styrene triblock copolymer [(KRATON) G 1650, 29% styrene, 8000 solution viscosity (25 wt% polymer), 100% triblock styrene-ethylene / butylene-styrene (S-EB-S) block copolymer, (KRATON) G 1652, 29% styrene, 1350 solution viscosity (25 wt% polymer), 100% triblock S-EB-S block copolymer, (KRATON) G 1657, 4200 solution viscosity (25 wt% polymer), 35% diblock S -EB-S block copolymer, all available from Shell Chemical . A preferred block copolymer is a block copolymer of the styrene-ethylene / propylene (S-EP) type, trademark (KRATON) G 1726 (28% styrene, 200 solution viscosity (25 wt% polymer), 70% diblock S-EB. -S block copolymer), (KRATON) G-1701X (37% styrene,> 50,000 solution viscosity, 100% diblock S-EP block copolymer), and (KRATON) G-1702X (28% styrene,> 50). And a 100% diblock S-EP block copolymer is also commercially available from Shell Chemical (Houston, Texas, USA)], a siloxane triblock copolymer [siloxane Trib Nitriles containing lacquer copolymers have been developed as stabilizers for siloxane ferrofluids, which have recently been proposed as intraocular tamponade for retinal detachment surgery: PDMS-b-PCPMS-b-PDMSs ( PDMS = polydimethylsiloxane, PCPMS = poly (3-cyanopropylmethyl-cyclosiloxane) was kinetically controlled for hexamethylcyclotrisiloxane initiated by a PCPMS macroinitiator endcapped with lithium silanolate Prepared very well through polymerization, the macroinitiator was prepared by equilibrating a mixture of 3-cyanopropylmethylcyclosiloxane (DxCN) and dilithium diphenylsilanediolate (DLDPS). - Synthesized by hydrolysis of anopropylmethyldichlorosilane followed by cyclization and equilibration of the resulting hydrolyzate DDLPS was prepared by deprotonation of diphenylsilanediol with diphenylmethyllithium. A mixture of DxCN and DLDPS was found to equilibrate within 5-10 hours at 100 ° C. By controlling the ratio of DxCN to DLDPS, different molecular weight macroinitiators could be obtained. The main cyclic compounds in the agent equilibrium are tetramer (8.6 ± 0.7 wt%), pentamer (6.3 ± 0.8 wt%) and hexamer (2.1 ± 0.5 wt%). 2.5k-2.5k-2.5k, 4k-4k-4k, and 8k-8k-8k triblock copolymers Are manufactured, it was characterized. These triblock copolymers are highly viscous liquids with a transparent microphase separation structure. It has been found that these triblock copolymers can stabilize nanometer sized γ-Fe ₂ O ₃ and cobalt particles in octamethylcyclotetrasiloxane or hexane. Thus, PDMS-b-PCPMS-b-PDMS represents a class of promising steric stabilizers for silicone ferrofluids. ], DEO-CPPO-CPEO triblock copolymer, PEO-PDMS-PEO triblock copolymer [polyethylene oxide (PEO) is soluble in water phase, but polydimethylsiloxane (PDMS) is soluble in oil phase] , PLA-PEG-PLA triblock copolymer, poly (styrene-b-butadiene-b-styrene) triblock copolymer [typically used thermoplastic elastomers include Styrolux from BASF (Ludwigshafen, West Germany) Poly (ethylene oxide) / poly (propylene oxide) triblock copolymer film [Pluronic F127, Pluronic P105 or Pluronic L44 manufactured by BASF (Ludwigshafen, West Germany)], PO (Ethylene glycol) -poly (propylene glycol) triblock copolymer, PDMS-PCPMS-PDMS (polydimethylsiloxane-polycyanopropylmethylsiloxane) triblock copolymer [a series of epoxy and vinyl endcaps with systematically varied molecular weights The resulting polysiloxane triblock copolymer was synthesized by anionic polymerization using LiOH as an initiator. Nitrile groups on the central copolymer block are believed to adsorb on the particle surface, while PDMS end blocks protrude into the reaction solvent. ], Azo functional styrene-butadiene-HEMA triblock copolymer, amphiphilic triblock copolymer with polymerizable end groups, syndiotactic polymethyl methacrylate (sPMMA) -polybutadiene (PBD) -sPMMA triblock copolymer, third Amine methacrylate triblock [AB diblock copolymer capable of forming both micelles (B block in the core) and reverse micelles (A block in the core) in water at 20 ° C.], biodegradable PLGA-b-PEO-b— PLGA triblock copolymer, polyactide-b-polyisoprene-b-polyactide triblock copolymer, PEO-PPO-PEO triblock copolymer (similar to Pluronic manufactured by BASF), poly (isoprene-block- Tylene-block-dimethylsiloxane) triblock copolymer, poly (ethylene oxide) -block-polystyrene-block-poly (ethylene oxide) triblock copolymer, poly (ethylene oxide) -poly (THF) -poly (ethylene oxide) triblock copolymer, ethylene oxide tri Block, poly E-caprolactone [manufactured by Birmingham Polymers], poly (DL-lactide-co-glycolide) [manufactured by Birmingham Polymers], poly (DL-lactide) [manufactured by Birmingham Polymers], poly (L-lactide) Birmingham Polymers], poly (glycolide) [Birmingham Polymers], poly (DL-lactide) -Co-caprolactone) [manufactured by Birmingham Polymers], styrene-isoprene-styrene triblock copolymer [manufactured by Japan Synthetic Rubber, molecular weight = 140 kg / mol, block ratio of PS / PI = 15/85], PEO / PPO triblock Copolymer, PMMA-b-PIB-b-PMMA [linear triblock TPE], PLGA-block-PEO-block-PLGA triblock copolymer [sulfonated styrene / ethylene-butylene / styrene (S-SEBS) TBC polymer proton conducting Sex membrane. Available as Protolyte A700 from Dais Analytic (Odessa, FL)], poly (l-lactide) -block-poly (ethylene oxide) -block-poly (1-lactide) triblock copolymer, poly-ester-ester-ester Triblock copolymers, PLA / PEO / PLA triblock copolymers [Synthesis of triblock copolymers using poly (ethylene glycol) using non-toxic zinc metal or calcium hydride as a co-initiator instead of stannous octoate Will be prepared by ring-opening polymerization of DL-lactide or e-caprolactone. The composition of the copolymer will vary by adjusting the polyester / polyether ratio. ], PCC / PEO / PCC triblock copolymer [The above polymers can be used in a mixture of two or more. For example, in two polymer blends measured in weight percent of the first polymer, such blends are 20-25%, 25-30%, 30-35%, 35-40%, 40-45% or 45 It can contain ~ 50%. ], Poly (t-butyl acrylate-b-methyl methacrylate-bt-butyl acrylate) [manufactured by Polymer Source (Doval, Quebec, Canada)], poly (t-butyl acrylate-b-styrene-bt- Butyl acrylate) [manufactured by Polymer Source], poly (t-butyl methacrylate-bt-butyl acrylate-bt-butyl methacrylate) [manufactured by Polymer Source], poly (t-butyl methacrylate-b-methyl methacrylate) b-t-butyl methacrylate) [manufactured by Polymer Source], poly (t-butyl methacrylate-b-styrene-bt-butyl methacrylate) [manufactured by Polymer Source], poly (methyl methacrylate-b-butadiene) (1,4-added) -b-methyl methacrylate) (manufactured by Polymer Source), poly (methyl methacrylate-bn-butyl acrylate-b-methyl methacrylate) [manufactured by Polymer Source], poly (methyl methacrylate-b -T-butyl acrylate-b-methyl methacrylate) (manufactured by Polymer Source), poly (methyl methacrylate-bt-butyl methacrylate-b-methyl methacrylate) [manufactured by Polymer Source], poly (methyl methacrylate-b-dimethyl) Siloxane-b-methyl methacrylate) (manufactured by Polymer Source), poly (methyl methacrylate-b-styrene-b-methyl methacrylate) [manufactured by Polymer Source], poly (meth) Tylmethacrylate-b-2-vinylpyridine-b-methylmethacrylate) [manufactured by Polymer Source], poly (butadiene (1,2 addition) -b-styrene-b-butadiene (1,2 addition)) [Polymer Source Made by], poly (butadiene (1,4 addition) -b-styrene-b-butadiene (1,4 addition)) [manufactured by Polymer Source], poly (ethylene oxide-b-propylene oxide-b-ethylene oxide) [Polymer Source] Company], poly (ethylene oxide-b-styrene-b-ethylene oxide) [manufactured by Polymer Source], poly (lactide-b-ethylene oxide-b-lactide) [manufactured by Polymer Source], poly (lactone-b-ethylene oxide) b- Lactone) [Polymer Source, Inc.], a, w- Jiakurironiru terminated poly (lactide -b- ethylene oxide -b- lactide) [Polymer Source Inc., poly (styrene -
b-acrylic acid-b-styrene) [manufactured by Polymer Source], poly (styrene-b-butadiene (1,4 addition) -b-styrene) [manufactured by Polymer Source], poly (styrene-b-butylene-b) -Styrene) [manufactured by Polymer Source], poly (styrene-bn-butyl acrylate-b-styrene) [manufactured by Polymer Source], poly (styrene-bt-butyl acrylate-b-styrene) [Polymer Source] Company], poly (styrene-b-ethyl acrylate-b-styrene) [manufactured by Polymer Source], poly (styrene-b-ethylene-b-styrene) [manufactured by Polymer Source], poly (styrene-b-isoprene) -B-styrene) [Polymer Manufactured by Source], poly (styrene-b-ethylene oxide-b-styrene) [manufactured by Polymer Source], poly (2-vinylpyridine-bt-butylacrylate-b-2-vinylpyridine) [manufactured by Polymer Source ], Poly (2-vinylpyridine-b-butadiene (1,2 addition) -b-2-vinylpyridine) [manufactured by Polymer Source], poly (2-vinylpyridine-b-styrene-b-2-vinylpyridine) ) [Manufactured by Polymer Source], poly (4-vinylpyridine-bt-butyl acrylate-b-4-vinylpyridine) [manufactured by Polymer Source], poly (4-vinylpyridine-b-methylmethacrylate-b- 4-vinylpyridine) [manufactured by Polymer Source] , Poly (4-vinylpyridine-b-styrene-b-4-vinylpyridine) [manufactured by Polymer Source], poly (butadiene-b-styrene-b-methyl methacrylate) [manufactured by Polymer Source], poly (styrene- b-acrylic acid-b-methyl methacrylate) [manufactured by Polymer Source], poly (styrene-b-butadiene-b-methyl methacrylate) [manufactured by Polymer Source], poly (styrene-b-butadiene-b-2-vinyl) Pyridine) [manufactured by Polymer Source], poly (styrene-b-butadiene-b-4-vinylpyridine) [manufactured by Polymer Source], poly (styrene-bt-butylmethacrylate-b-2-vinylpyridine) [ Polymer Source manufactured by ce Co.], poly (styrene-bt-butyl methacrylate-b-4-vinylpyridine) [manufactured by Polymer Source], poly (styrene-b-isoprene-b-glycidyl methacrylate) [manufactured by Polymer Source], Poly (styrene-ba-methylstyrene-bt-butyl acrylate) [manufactured by Polymer Source], poly (styrene-ba-methylstyrene-b-methyl methacrylate) [manufactured by Polymer Source], poly ( Styrene-b-2-vinylpyridine-b-ethylene oxide) [manufactured by Polymer Source], poly (styrene-b-2-vinylpyridine-b-4-vinylpyridine) [manufactured by Polymer Source].

  The above block copolymers can be used alone or in a mixture of two or more of the same or different classes. For example, in a mixture of two block copolymers measured in weight percent of the first polymer, such a mixture is 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45% or 45-50% can be included. When three polymers are used, the first polymer is 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40% of the total polymer component. -45% or 45-50% and the second polymer is the remaining 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40% , 40-45% or 45-50%.

  In other words, the amount of each block copolymer in the mixture can vary considerably with the nature and number of block copolymers used and the desired properties to be obtained. In general, however, each block copolymer of the mixture according to the invention will be present in an amount of at least about 10% by weight, based on the weight of the total polymer in the membrane or solution. These same general ranges will apply to membranes made from mixtures with one or more polymers, copolymers and / or block copolymers. Furthermore, a single polymer, copolymer or block copolymer can be “doped” with a small amount of a separate polymer, copolymer or block copolymer, even as small as 1.0% by weight of the membrane, to adjust the special properties of the membrane. It will also apply if possible.

  Embodiments of the present invention include, without limitation, A-B, A-B-A or A-B-C block copolymers. The average molecular weight for a triblock copolymer of A (or C) is, for example, 1,000 to 15,000 daltons, and the average molecular weight of B is 1,000 to 20,000 daltons. More preferably, block A and / or C will have an average molecular weight of about 2,000-10,000 daltons and block B will have an average molecular weight of about 2,000-10,000 daltons.

  If a diblock copolymer is used, the average molecular weight for A is about 1,000 to 20,000 daltons, more preferably about 2,000 to 15,000 daltons. The average molecular weight of B is about 1,000 to 20,000 daltons, more preferably about 2,000 to 15,000 daltons.

  Preferably, the block copolymer has a hydrophobic / hydrophilic equilibrium selected to (i) provide a solid at the expected operating and storage temperatures and (ii) promote the formation of biofilm-like structures rather than micelles. Would have. More preferably, the hydrophobic content (or block) should exceed the hydrophilic content (or block). Thus, at least one block of the diblock or triblock copolymer is preferably hydrophobic. Although wettable membranes are contemplated, preferably the content of hydrophobic and hydrophilic synthetic polymeric materials will make the membrane low wettable.

  As noted above, in one preferred embodiment of the present invention, a biocompatible membrane is provided that is manufactured using a mixture of synthetic polymeric materials. Such a mixture may be a mixture of two or more block copolymers that are identical except for the molecular weight of their respective blocks. For example, biocompatible membranes can be made using a mixture of two block copolymers, both of which are poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline) One has an average molecular weight of 2 kD-5 kD-2 kD and the other 3 kD-7 kD-3 kD, and the ratio of the first block copolymer to the second block copolymer is about 67 pairs of the total synthetic polymeric material used. 33% (w / w). Of course, the first block of most block copolymers has a molecular weight of about 2,000 daltons, the second block has a molecular weight of 5,000 daltons, and the third block has a molecular weight of 2,000 daltons. It means having a molecular weight. A small number of block copolymers each have blocks of about 3,000, 7,000 and 3,000 daltons.

  Of course, two or more completely different block copolymers can be used, and mixtures of different block copolymers and identical block copolymers that differ only in the size of their respective blocks are also envisaged. However, the mixture is not limited to block copolymers.

  The polymers and copolymers can be used alone, in combination, and in combination with block copolymers to produce a biocompatible membrane having the properties described herein according to the present invention. Useful polymers and copolymers are preferably solid at room temperature (25 ° C.). They can be dissolved in any other synthetic polymeric material used, any additive used, and a solvent or solvent system compatible with the polypeptide used. Polymers and copolymers useful in producing biocompatible membranes include, without limitation, polydialkylsiloxanes such as polystyrene, polyalkyl and polydimethylsiloxane, polyacrylates such as polymethylmethacrylate, polybutadiene, polyalkylenes and polyalkylene glycols. Such as polyalkene, sulfonated polystyrene, polydiene, polyoxirane, poly (vinyl pyridine), polyolefin, polyolefin / alkylene vinyl alcohol copolymer, ethylene propylene copolymer, ethylene-butene-propylene copolymer, ethyl vinyl alcohol copolymer, perfluorinated sulfonic acid, Vinyl halogen polymers and copolymers such as vinyl chloride and acrylonitrile copolymers, methacryl / ethylene copolymers And it is a more soluble generally may include polymers and copolymers with a molecular weight of all of the hydrophobic about 5,000 to about 500,000. Particularly preferred polymers include poly (n-butyl acrylate), poly (t-butyl acrylate), poly (ethyl acrylate), poly (2-ethylhexyl acrylate), poly (hydroxypropyl acrylate), poly (methyl acrylate), Poly (n-butyl methacrylate), poly (s-butyl methacrylate), poly (t-butyl methacrylate), poly (ethyl methacrylate), poly (glycidyl methacrylate), poly (2-hydroxypropyl methacrylate), poly (methyl methacrylate) , Poly (n-nonyl methacrylate), poly (octadecyl methacrylate), polybutadiene (1,4 addition), polybutadiene (1,2 addition), polyisoprene (1,4 addition), polyisoprene (1,2 addition and 1, Addition), polyethylene, poly (dimethylsiloxane), poly (ethylmethylsiloxane), poly (phenylmethylsiloxane), polypropylene, poly (propylene oxide), poly (4-acetoxystyrene), poly (4-bromostyrene), poly (4-t-butylstyrene), poly (4-chlorostyrene), poly (4-hydroxylstyrene), poly (a-methylstyrene), poly (4-methylstyrene), poly (4-methoxystyrene), polystyrene , Isotactic polystyrene, syndiotactic polystyrene, poly (2-vinylpyridine), poly (4-vinylpyridine), poly (2,6-dimethyl-p-phenylene oxide), poly (3- (hexafluoro-2) -Hydroxypropyl) -styrene), polyisobutylene Poly (9-vinylanthracene), poly (4-vinylbenzoic acid), poly (4-vinylbenzoic acid sodium salt), poly (vinylbenzyl chloride), poly (3 (4) -vinylbenzyltetrahydrofurfuryl ether), poly (N-vinylcarbazole), poly (2-vinylnaphthalene) and poly (9-vinylphenanthrene) are included. Since polymers and copolymers are generally synthetic polymeric materials, they can be used in the same amounts previously described for block copolymers and mixtures.

  In a particularly preferred embodiment of the present invention, the biocompatible membrane comprises a synthetic polymeric material, preferably at least one block copolymer (most preferably a block copolymer that is at least partially amphiphilic) and a biocompatible membrane. Contains a synthetic polymer material capable of stabilizing It has been found that certain polymers, most notably hydrophilic polymers and copolymers capable of forming multiple hydrogen bonds ("rich in hydrogen bonds") can stabilize the membrane. In the context of stabilizing polymers, the term “polymer” includes one or more monomers, polymers and copolymers. “Hydrophilic” in this context means that the stabilizing polymer is dissolved or solubilized in water or a water-miscible solvent. Without trying to be bound by any particular theory of operation, it is believed that the use of such polymers can help in functionally integrating the polypeptide into the structure of the biocompatible membrane. The stabilized polymer has a much longer useful life and / or machine compared to the same biocompatible membrane produced without the use of the stabilizing polymer when exposed to the same conditions. Great resistance to mechanical failure. Stabilized biocompatible membranes used in fuel cells where the synthetic polymeric material includes a stabilizing polymer, for example, are at least about 10%, more preferably at least about 50%, and most preferably at least about 100%. Can have an increased useful life.

  Particularly preferred polymers that can stabilize polypeptides within the biocompatible membrane of the present invention include dextrans, polyalkylene glycols, polyalkylene oxides, polyacrylamides, and polyalkyleneamines. These stabilized polymers (also containing copolymers) generally have a lower average molecular weight than the polymers and copolymers used as synthetic polymeric materials. Their molecular weights generally range from about 1,000 daltons to about 15,000 daltons. Particularly preferred polymers that can stabilize the biocompatible membrane include, without limitation, polyethylene glycol having an average molecular weight of about 2,000 to about 10,000 daltons, an average of about 2,000 to about 10,000 daltons Polyethylene oxide having a molecular weight, polyacrylamide having an average molecular weight of about 5,000 to 15,000 daltons are included. Other stabilizing polymers include polypropylene, poly (n-butyl acrylate), poly (t-butyl acrylate), poly (ethyl acrylate), poly (2-ethylhexyl acrylate), poly (hydroxypropyl acrylate), poly (methyl Acrylate), poly (n-butyl methacrylate), poly (s-butyl methacrylate), poly (t-butyl methacrylate), poly (ethyl methacrylate), poly (glycidyl methacrylate), poly (2-hydroxypropyl methacrylate), poly ( Methyl methacrylate), poly (n-nonyl methacrylate), and poly (octadecyl methacrylate).

  The amount of one or more stabilizing polymers used in the biocompatible membrane is not critical as long as some measurable improvement in properties is realized and the functionality of the biocompatible membrane is not unduly disturbed. Some sacrifices in functionality and lifetime must be anticipated. However, generally, based on the total amount of synthetic polymeric material found in the final biocompatible membrane, the amount of stabilizing polymer used (by weight) is generally less than one-third, and typically Is 30% by weight or less. Preferably, the amount used is from 5 to about 30% by weight of the synthetic polymeric material in the final membrane, more preferably from about 5 to about 15% by weight.

  In addition to one or more polymers, copolymers and / or block copolymers, and / or stabilizing polymers, the synthetic polymeric materials of the present invention can include at least one additive. Additives can include cross-linking agents and lipids, fatty acids, sterols and other natural biological membrane components and their synthetic analogs. These are generally added to the synthetic polymeric material at some point in solution. These additives, if present at all, will be found in an amount of about 0.50 to about 30%, preferably about 1.0 to about 15% by weight of the synthetic polymeric material.

  If the biocompatible membrane incorporates a cross-linking component, useful techniques for polymerization include chemical polymerization with a radical former or radical growth agent, and also photochemical radicals with or without another radical growth agent. Polymerization by formation is included. The parameters can be adjusted depending on conditions such as membrane material, size of biocompatible membrane section, support structure, and the like. Care must be taken to minimize damage to the polypeptide. One particularly useful method involves using a peroxide at a neutral pH followed by acidification.

Can be combined with synthetic polymeric materials to form biocompatible membranes according to the present invention and be involved in one or both of oxidation / reduction and transmembrane transport functions (molecules, atoms, protons, electrons) Examples of useful polypeptides that can be used include, for example, NADH dehydrogenase (“complex I”) (eg, E. coli-derived, Tran et al., “Requirement for the proton pumping NADH dehydrogenase I of Escherichia coli N. and it bioenergetic implications ", Eur. J. Biochem. 244: 155, 1997), NADPH transhydrogenase, proton ATPa. se and cytochrome oxidase and its various forms are included. Other polypeptides include glucose oxidase (available from several sources, including many types of this enzyme available from Sigma Chemical using NADH), glucose-6-phosphate dehydrogenase (NADPH). Boehringer Mannheim, Indianapolis, IN), 6-phosphogluconate dehydrogenase (NADPH, Boehringer Mannheim), malate dehydrogenase (NADH, Boehringer Mannheim), glyceraldehyde 3-N , Manufactured by Sigma, Boehringer Mannheim), isocitrate dehydrogenase (NADH, Boehringer Mannhei) Company Ltd., NADPH, manufactured by Sigma Co.), alpha-ketoglutarate dehydrogenase complex (NADH, include Sigma) and proton metastatic pyrophosphate. Also included are succinic acid: quinone oxidoreductase, also referred to as “complex II”, “A structural model for the membrane-integrated domain of succinate: quinone oxidoreductases”, Hage. and Hederstedt L. , FEBS Letters 389; 25-31 (1996) and “Purification, crystallisation and preliminary crystallographic studies of succinate: ubiquinone oxidoresductor. et al. , Biochim. Biophys. Acta 1553; 171-176 (2002), heterodisulfide reductase, F (420) H (2) dehydrogenase, (Baumer, et al., “The F420H2 dehydrogenase from Methylenesarcina mazei redox-en-redox-and , 275 J. Biol. Chem. 17968 (2000)) or hydrogenate formate (Andrews, et al., “A 12-citron Escherichia coli operon (hyf) encoding a putative proton-toning proton proton. "enilase system", 143 Microbiology 3633 (1997)), nicotinamide nucleotide transhydrogenase: "model fortension ration". and Yamaguchi, M .; , Faceb J. et al. , 10; 444-452 (1996), proline dehydrogenase: “Proline Dehydrogenase from Escherichia coli K12”, Graham S. et al. et al. , J .; Biol. Chem. 259; 2656-2661 (1984) and, without limitation, cytochrome C oxidase (crystallized using either undecyl- (either β-D-maltoside or cyclohexyl-hexyl-β-D-maltoside), cytochrome bc _1: "Ubiquinone at Center N is responsible for triphasic reduction of cytochrome bc 1 complex ", Snyder C.H., and Trumpower B.L., J.Biol.Chem.274; 31209-16 (1999), cytochrome bo ₃ : "Oxygen reaction and proton uptake in helix VIII mutants of cytochrome bo 3 ", Sven . son M.et al, Biochemistry 34; 5252-58 (1995), "Thermodynamics of electron transfer in Escherichia coli cytochrome bo 3 ", Schultz B.E., and Chan S.I., Proc.Natl.Acad.Sci USA 95; 11643-48 (1998), and cytochrome d: “Reconfiguration of the Membrane-bound, ubiquinone-dependent pyrethate oxidase respiratory chain of chemistry”. . J.G.et al, Biochemistry 23; 445-453 (1984), Joost and Thorens, "The extended GLUT-family of sugar / polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review) ", 18 Mol. Membr. Biol. 247-56 (2001), and Goldin AL," Evolution of voltage-gated Na (+) channels ", J. Chem. Exp. Biol. 205; 575-84 (2002), Choe, S .; "Potassium channel structures", Nat. Rev. Neurosci. 3; 115-21 (2002); , “Bacterial sodium ion-coupled energy”, Antonio Van Leeuwenhoek 65; 381-95 (1994), and Park J. et al. H. and Saier M. et al. H. Jr. "Phylogenetic, structural and functional charactaristics of the Na-K-Cl cotransporter family". Membr. Biol. 149; 161-8 (1996). All of the above are intended to form part of this specification. Methods for isolating such NADH dehydrogenase enzymes are described, for example, in Braun et al. Biochemistry 37: 1861-1867, 1998, and Bergsma et al. , "Purification and characterisation of NADH dehydrogenase from Bacillus subtilis", Eur. J. et al. Biochem. 128: 151-157, 1982. Spehr et al. , Biochemistry 38: 16261-16267, 1999, complex I NADH dehydrogenase (or NADH: ubiquinone oxidoreductase) expressed from the operon provides a useful amount for use in the present invention. In addition, it can be overexpressed in E. coli by replacing the T7 promoter. Complex I is described in Spehr et al. Can be isolated from overexpressed E. coli using solubilization with dodecyl maltoside.

Complex I can be processed such that NADH dehydrogenase is eliminated or greatly reduced. www. jbc. org, 2002 (Manusscript M112357200) was accepted and published on the web by Boettcher et al. , "A Novel, Enzymatically Active Conformation of the Escherichia coli NADH: Ubiquinone Oxidoreductase (Complex I) from high-salt or high-pHN in a high-salt or high-pHN solution. The conformation is changed to release to create the DH-form. Applicants have used these conditions and combinations of these conditions to demonstrate that the fuel cells of the present invention can operate without NADH dehydrogenase activity at the anode / cathode barrier. Such conditions include an anolyte or anodic salt concentration of 200 mM to 2 M and a pH of 8.0 or higher. Transporter activity is thought to function against the opposite [H ⁺ ] gradient due to charge imbalance between the anode and cathode sides. The DH form of proton transporter activity has been confirmed from the maintenance of current generation in the fuel cell where the biocompatible membrane gated by this configuration provides the only means for reducing charge imbalance. (With complex I, reverse transport further maintains the condition that either reverse-directed NADH dehydrogenase binding of complex I is maintained on the cathode side, thereby blocking reverse transport due to lack of NADH substrate. (Note that it was further controlled by use.)

  It will be appreciated that the enzyme source used in the present invention may be a thermophilic organism that provides a more temperature stable enzyme. For example, complex I can be synthesized according to Scheide et al. , FEBS Letters 512: 80-84, 2002 (described for preliminary isolation using the type of detergent extraction used elsewhere for Complex I) at 90 ° C. And can be isolated from Aquifex aeolicus in a form that performs optimally.

  It is also anticipated that recombinant polypeptides such as modifying enzymes can be used. One commonly applied technique for engineering enzymes by genetic recombination is to use genetic recombination tools (eg, exonucleases) to delete the N-terminus, C-terminus or internal sequences. is there. These deletion products are systematically created and tested using routine experimentation. In most cases, it can be found that an important part of the gene product has little effect on the industrial function in question. More intensive deletions and substitutions can increase the stability, working temperature, catalytic rate and / or solvent compatibility to provide enzymes that can be used in the present invention. Of course, if desired, mixtures of the various polypeptides described herein can be used.

The amount of polypeptide used will vary with the type of polypeptide used, the nature and function of the biocompatible membrane, the environment in which the biocompatible membrane is used, and the like. The amount of polypeptide may be important for certain applications, such as fuel cells, where generally the higher the concentration of polypeptide per cm ² of surface area, the faster the proton transfer rate (in terms of current) per unit area. In general, however, any amount of polypeptide will be present as long as some polypeptide is present and functional, and the amount of polypeptide used does not interfere with membrane formation or destabilize the membrane. Is possible. Generally, the amount of polypeptide is at least 0.01 wt%, more preferably about 5 wt%, even more preferably 10 wt%, even more preferably at least about 20 wt%, based on the final weight of the biocompatible membrane. And most preferably will be 30% by weight or more. The amount of polypeptide relative to the solvent can be as low as 0.001% (w / v) and as high as 50.0% (w / v). Preferably, the concentration is about 0.5 to about 5.0% (w / v). More preferably, the concentration is from about 1.0 to about 3.0% (w / v).

Appropriate solubilizers and / or stabilizers such as co-solvents, detergents, etc. may also be required, particularly in conjunction with polypeptide solutions. Solubilized detergents are generally found at a concentration level of 0.01-1.0%, but more preferably up to about 0.5% is expected. Such detergents include ionic detergents: sodium dodecyl sulfate, sodium N-dodecyl sarcosinate, N-dodecyl-β-D-glucopyranoside, octyl-β-D-glucopyranoside, dodecyl maltoside, decyl, undecyl, tetradecyl Maltoside (generally an alkyl chain of about 8 or more carbons attached to a sugar as a general form of an ionic detergent), octyl-β-D-glucoside and polyoxytheylane (9) dodecyl ether, C ₁₂ E ₉ and triton X-100 Or non-ionic detergents such as Nonidet P-40. Also useful are diblock copolymers that exhibit surface active properties such as certain polymers, typically Pluronic series from BASF, or Disperplast (BYK-Chemie).

  The solvent used in preparing the synthetic polymeric material solution is preferably water used (polypeptide solutions often contain water) and at least one synthetic polymeric material (polymer, copolymer and / or block copolymer). ) Are selected to be miscible with both. However, as noted above, it is possible to form a film using a solvent or mixture that is not water miscible. Although the use of a solvent to form a solution is preferred, the term “solution” as used herein generally includes suspensions as well.

  If block copolymers are used, the solvent must solubilize these synthetic polymeric materials. Synthetic polymeric materials can be fairly poorly soluble in solvents (5% (w / v)), but synthetic polymeric materials are preferably more soluble than 5% (w / v) and generally Specifically, the solubility is preferably such that the ratio of synthetic polymeric material to solvent is at least 5-10% (w / v), preferably greater than 10% (w / v).

Suitable solvents include, but are not limited to, 1-12 carbon low molecular weight aliphatic alcohols such as methanol, ethanol, 2-propanol, isopropanol, 1-propanol, and aryl alcohols such as diol, phenol, benzyl alcohol, acetone, methyl ethyl ketone, etc. Low molecular weight aldehydes and ketones, cyclic compounds such as benzene, cyclohexane, toluene and tetrahydrofuran, halogenated solvents such as dichloromethane and chloroform, and general such as 1,4-dioxane, normal alkanes (C ₂ -C ₁₂ ) and water Solvent materials can be included. Solvent mixtures are also possible as long as the mixtures have the appropriate miscibility, evaporation rate and other criteria described for the individual solvents. (Solvent components that tend to form protein destructive contaminants such as peroxides can be used as long as they can be properly purified and processed.) Solvents are typically 30% (v / v) or higher, Preferably it contains 20% (v / v) or more, and usefully 10% (v / v) or more of the polypeptide / synthetic polymer material.

  If the membrane must contain detergents, lipids (eg, cardiolipin), sterols (eg, cholesterol) or “other materials” such as buffers and / or salts, they must also be added prior to membrane formation Rather, they will be present in an amount of about 0.01 to about 30%, preferably about 0.01 to about 15% by weight of the final biocompatible membrane. In contrast to the additive, the other materials are not the synthetic polymeric material, but are most often mixed with the polypeptide solution.

  The biocompatible membrane according to the present invention is used for the production of membranes from synthetic polymeric materials and further lipid bilayers, so long as the resulting biocompatible membrane is useful as described herein. It can be manufactured using any one of a number of conventional techniques. One method of forming a preferred biocompatible membrane for use with a block copolymer-based membrane is as follows.

  1. A solution or suspension of the synthetic polymer material is prepared in a solvent or mixed solvent system. The solution or suspension may be a mixture of two or more block copolymers, but may contain one or more polymers and / or copolymers. The solution or suspension is preferably 1-90% (w / v), more preferably 2-70% (w / v), or even more preferably 3-20% (w / v) synthetic polymer Contains material. 7% (w / v) is particularly preferred.

  2. One or more polypeptides (typically with a solubilizing detergent) are placed in the solution or suspension either individually or by addition to an existing polymer solution or suspension. If the solvent used to solubilize the synthetic polymeric material is the same, or if it has similar characteristics and solubility as the solvent that can solubilize the polypeptide, it will usually be in the polymer solution or suspension It is more expedient to add directly. Otherwise, the two or more solutions or suspensions containing the synthetic polymeric material and the polypeptide must possibly be mixed with an additional co-solvent or solubilizer. Most often, the solvent used for the polypeptide is aqueous.

  Mixing these solutions and / or suspensions is often quite simple and can be accomplished manually or using automatic mixing equipment. Depending on the solvent and polymer used, heating or cooling may also be useful in forming the film. In general, rapidly evaporating solvents tend to form films better than with cooling, while a slight degree of heating may be most beneficial for very slowly evaporating solvents. Given that it is appropriate for the polymer used, the boiling point of the solvent used to select the solvent with the most favorable characteristics can be investigated. However, of course, the need to incorporate the polypeptide into the solvent polymer mixture must also be considered, but this may not be important. For example, 5 μL of detergent solubilized complex I (0.15% (w / v) dodecyl maltoside) with 10 mg / mL complex I is 3.2% in a 50/50 mixture of acetone and hexane. (W / v) Polystyrene-polybutadiene-polystyrene triblock copolymer (fully hydrophobic triblock (lot no. 7453064P) sold under the trademark STYROLUX 3G55 available from BASF) mixed in 95 μL of membrane It is possible to deposit the same thing in a way that allows formation. In this case, the final mixture contained about 5% (v / v) water and 0.75% (w / w) Complex I compared to the weight of the synthetic polymeric material. In general, the solution is sufficiently stable at room temperature to be useful for at least about 30 minutes, provided that the solvent does not evaporate during that time. They can be stored overnight or longer, generally under refrigerated conditions.

  3. An amount of a final solution or suspension containing both one or more polypeptides and a synthetic polymeric material is formed into a membrane and at least partially allowed to dry, thereby leaving at least a portion of the solvent . It is possible to completely dry a part of the membrane produced according to the invention or to substantially dry the same. “Substantially dry” means that there may be up to about 15% of some residual solvent, which is often retained even when left at room temperature for several hours. .

  In particularly preferred embodiments, the substantially total weight of the final membrane will be either a polypeptide or a synthetic polymeric material. In this case, the amount of synthetic polymeric material including additives and stabilizing polymer ranges from about 70 to about 99% by weight of the final membrane. However, it may be desirable to have a higher polypeptide content, or it may be necessary to retain some solvent, and the amount of synthetic polymeric material may be reduced accordingly. In general, however, at least about 50% by weight of the final biocompatible membrane will be a synthetic polymeric material. When the synthetic polymeric material is a block copolymer and a mixture containing a polymer or copolymer other than a stabilizing polymer, the block copolymer may be present in an amount of at least about 35% by weight of the biocompatible membrane. Up to about 30% by weight of the biocompatible membrane may be “additives” and “other materials” (collectively) as defined herein. More preferably, the amount of additives and other materials is up to about 15% by weight of the biocompatible membrane. Up to about 30% by weight of the synthetic polymeric material may be a stabilizing polymer. Generally, the stabilizing polymer is present in an amount from about 5 to about 20% by weight of the synthetic polymeric material.

  The identification of which solvents are particularly useful according to the invention and which combination of polymers and polypeptides and solvents should be used depends on a number of factors, some of which are miscible, evaporate Has already been considered as a problem. The polymer and protein components must be able to be completely dissolved in the solvent or solvent mixture. The evaporation rate must be long enough so that the film can be produced in one go. However, the amount of time must not be so long as to make the production infeasible. Nonpolar solvents may be useful, but the ionic or hydroxyl component of the polymer may be poorly soluble in completely nonpolar solvents, so in general, nonpolar solvents are less useful in certain situations There is. Thus, highly rigid hydrophobic components such as polystyrene can be dissolved, and highly ionic components such as acrylic acid cannot be dissolved simultaneously. However, when a polymer having a completely hydrophobic property is used, a nonpolar solvent is preferred. The solvent generally must be partly non-aqueous because the polymer must be at least partly water-insoluble. And water miscibility is most desirable for membrane protein reconstitution but is not a strict limiting factor. Thus, preferably all solvents are non-aqueous. However, solvents for polypeptides and stabilizing polymers are primarily water, or at least water miscible.

  A preferred method of forming a biocompatible membrane comprising at least one synthetic polymeric material and a stabilizing polymer includes the steps of making a suitable solution of a block copolymer, and usually stabilizing the polymer and polypeptide individually. Is included. As described elsewhere, the polypeptide may include one or more detergents or surfactants, typically an aqueous solution. Making and mixing an appropriate solution, for example, as disclosed herein or known in the art, including coating a perforated dielectric substrate with the solution, followed by at least partial evaporation of the solvent. It can be produced by any of the known techniques. Such evaporation can be accelerated in a vacuum.

  One method of forming a biocompatible membrane containing a stabilizing polymer rich in hydrogen bonds is as follows.

1. A solution or suspension of Protolyte A700 block copolymer in the supplied solvent is diluted with an equal volume of ethanol (5% water (w / v)). This solution contains about 5% (w / v) block copolymer.
2. Separately, an aqueous solution or suspension of stabilizer is made by mixing 943 mg of polyethylene glycol (PEG) 8000 to produce a solution having a concentration of about 2.3% (w / v). The concentration of stabilizer in the solution is close to the saturation limit.
3. Next, 4 μL of a solution containing 10 mg / mL E. coli-derived complex I together with 0.15% (w / v) dodecyl maltoside is added to 6 μL of PEG solution, and they are mixed to form a solution or suspension. A turbid liquid is produced.
4). Thereafter, 10 μL of this solution and 10 μL of the solution containing the block copolymer are mixed.
5. A small amount of the resulting solution (eg, 4 μL) is placed on a small population of apertures (holes drilled through the support) of a 1 mil (25.4 μ) thick KAPTON perforated substrate, one brand of polyimide. The perforated substrate has a plurality of apertures with a diameter of 100 μm and a depth of 1 mil.
6). The solution is air dried in the hood, thereby removing the solvent.
7). Repeat steps 5 and 6 as necessary to cover the entire aperture.

  The method described above for introducing a polypeptide into a solution containing a stabilizing polymer prior to mixing with one or more non-aqueous solvents in the presence of a block copolymer is a method of introducing a polypeptide for use in a biocompatible membrane. It is thought to stabilize the function. However, polymers and block copolymers can also be mixed, and the resulting solution could generally be mixed with an aqueous polypeptide solution. Optionally, each aperture is inspected to ensure film formation, or at least a statistically appropriate number of apertures are inspected using a microscope. If the aperture does not contain a membrane, use an additional solution and micropipette scale pipetting device to repair the hole. To repair such holes, typically only a very small amount of solution is required. The membrane can be completely or substantially completely dried in a vacuum apparatus or in a dryer. The film thus formed can be stored and dried in vacuum or, if desired, dehydrated and dried.

  If the biocompatible membrane incorporates a crosslinking component such as methacrylate and is used in a fuel cell, the following approach can be used.

A biocompatible membrane is prepared in a support that forms the cathode / anode barrier.
Assemble the cell with the biocompatible membrane on the anode / cathode barrier support, electrode and buffer only.
Connect the two electrodes to a high load, such as approximately 150 kilohms.
In order to start the cross-linking process, hydrogen peroxide is added to the cathode side, for example, so that the concentration of peroxide is 1% by volume.
The fuel cell is placed under load for a time such as 1 hour (± 10%).
In order to stop cross-linking, the pH on the cathode side is adjusted to less than pH 5.

  The parameters can be adjusted depending on conditions such as membrane material, biocompatible membrane size, biocompatible membrane thickness, support structure, and the like.

  Once the polypeptide / synthetic polymer material solution is produced, it can be formed into a membrane. The biocompatible membrane according to the present invention may be an independently standing membrane. Such membranes can be formed by pouring the solution into a dish or onto a sheet so that they achieve the desired thickness. When the solution is dried and the solvent is evaporated, the dried membrane can be removed from the dish or peeled away from the backing layer. Any suitable antiblocking agent can be used to assist in this process. Biocompatible membranes can be glass, carbon that has been surface modified to increase hydrophobicity, or polymers (polyvinyl acetate, PDMS, Kapton®, perfluorinated polymers, PVDF, PEEK, polyester, or UHMWPE, A solid material can be formed in the background, for example by applying it onto a polypropylene or polysulfone). Polymers such as PDMS provide an excellent support that can be used to establish an opening over which a biocompatible membrane can be formed.

  The membrane can be cut or shaped as needed, or can be used as is. Further, to facilitate the use of the membrane, the membrane can be attached to the holder, if desired, either physically or through some type of coupling device or adhesive. Conceptually, assuming that the frame is a support and the membrane is a canvas, it can be described as being similar to the step of placing a canvas on the frame before drawing a picture. Alternatively, the membrane can be formed with such a structure. A suitable illustration would be to take a children's bubble wand used to make soap bubbles and immerse it in a soapy water solution. A soapy water film is formed over the entire opening of the wand. The structural material used in the periphery allows the film to be handled and manipulated and provides rigidity and strength. This also helps to give the film the desired shape. Similar types of processes can be used with physical structures and membranes that form the solutions of the present invention.

  In one preferred embodiment according to the present invention, the biocompatible membrane can be placed and / or formed within the aperture of various perforated substrates, preferably including a dielectric substrate, or across the aperture. “Perforated substrate” means that the substrate has at least one hole, aperture (synonymous with a hole as used herein) or a hole in which a biocompatible membrane can be placed. means. For example, FIG. 3b illustrates an embodiment of a membrane structure useful in a fuel cell. A perforated substrate 42 defining various perforations 49 has a metalized surface to form a perforated anode 44 and a perforated cathode 45. It should also be noted that the perforated substrate 42 may be a porous substrate having no drill holes, for example. In such an example, it should be understood that the perforation 49 is a hole. The biocompatible membrane 61 according to the present invention is formed over the entire area of the aperture 49 of the perforated substrate 42 or the perforated substrate 42 and is directly attached to the surface of the anode. The biocompatible membrane 61 can also be coplanar with the anode 44 into the perforation, or can be attached to or adjacent to the cathode 45. For example, as illustrated, one film is disposed over the entire area of the anode, and the other film is disposed in the perforated portion 49 of the substrate 42 in the same plane as the anode 45 and the like, thereby providing two films 61. (Not shown). The membrane 61 may be the same or different with respect to the synthetic polymeric material used, the polypeptide used, or both. Indeed, a plurality of such membranes 61, and indeed layers of biocompatible membranes 61, can be used in conjunction with other types of membranes, diffusive barriers, and the like. Although the above has been described in the context of FIG. 3b, it is equally applicable to other structures, and particularly any type of fuel cell structure. The biocompatible membrane 61 can include one or more polypeptides 62 and 63 as illustrated.

  Coating methods that can be used to form the electrodes (44, 45) on the substrate include a first coating or lamination of a conductor that is subsequently plated, sputtered, or a noble metal conductor such as titanium or gold or platinum. Use of other coating methods to coat is included. Another method is to sputter an adhesion layer such as chromium or titanium directly onto the support, followed by plating, sputtering or other coating methods to attach to the noble metal conductor. The outer metal layer can be conveniently treated to increase its hydrophobicity using dodecanethiol or the like.

  In some embodiments, a support or substrate with a high natural surface charge density such as Kapton and Teflon is preferred. As described above, they can be used to form an anode / cathode barrier without the use of surface electrodes. The substrate 42 is preferably dielectric in many cases.

  Perforations or holes 49 and metallized surfaces (anode 44 and cathode 45 (for embodiments using so-called electrodes)) in substrate 42 are, for example, photolithography masking and etching techniques well known in the art. Can be used. The perforated part can also be formed by, for example, punching, drilling, laser drilling, stretching or the like. Alternatively, the metallized surface (electrode) provides, for example, (1) a thin film deposition through a mask, (2) a blanket coating of the metallization with a thin film, and then sensitizes and selectively etches the pattern into the metallization. Or (3) using a metal-impregnated resist to form the metallization pattern directly without etching (DuPont Fedel process, Droddyk et al., “Photopatternable Conductor Tapes for PDP). Applications ", Society for Information Display 1999 Digest, 1044-1047, Nebe et al., US Pat. No. 5,049,480). In one embodiment, the perforated substrate, or porous substrate, is a film. For example, the dielectric may be a porous film that is rendered impermeable outside the “perforations” by metallization. The surface of the metal layer can be modified with other metals, for example by electroplating. Such electroplating is performed using, for example, titanium, gold, silver, platinum, palladium, a mixture thereof, or the like. In addition to the metallized surface, the electrodes can be formed by other suitable conductor materials, which can be surface modified. For example, the electrodes can be formed from carbon containing graphite fibers (graphite), which can be applied to a dielectric substrate by, for example, electron beam evaporation, chemical vapor deposition, or thermal decomposition. The surface to be metallized can be solvent cleaned and oxygen plasma etched. Useful means for forming the hydrophilic electrode include, for example, Surampuddi US Pat. No. 5,773,162, Surampudi US Pat. No. 5,599,638, Narayanan US Pat. No. 5,945,231, Kindler, US Pat. No. 5,992,008, Surampudi International Patent Application No. 96/12317, Surampudi International Patent Application No. 97/12256, and Narayanan International Patent Application No. 99/16137.

  The biocompatible membrane used in the present invention is optionally stabilized by contact with a solid support. One way of performing such stabilization uses sulfur-mediated binding of lipid-related molecules to adhere, restrain or anchor the surface of a metal surface or another solid support to a biocompatible membrane. . For example, the porous support can be coated with a sacrificial or removable filler layer, and the coated surface can be smoothed, for example, by polishing. Such porous supports are typically proton conductive polymers discussed above as long as the proton conductive polymer membrane can be smoothed following coating and is stable to the processing described below. Any of the membranes can be included. One useful porous support is a glass frit. The smoothed surface is then coated with metal to provide a first layer of chromium and a gold overcoat (previously cleaned if necessary). The sacrificial material is then removed, such as by dissolving, removing the metallization above the holes, leaving the metallized surface surrounding the holes. The sacrificial layer can include photoresist, paraffin, cellulose resin (such as ethyl cellulose), and the like.

  Tethers or adhesives include alkyl thiols, alkyl disulfides, thiolipids, and the like that are suitable for attaching biocompatible membranes as illustrated in FIGS. 7A and 7B. Such tethers are described, for example, in Lang et al. , Langmuir 10: 197-210, 1994. Additional tethers of this type are described in Lang et al. US Pat. No. 5,756,355 and Hui et al. US Pat. No. 5,919,576.

  FIG. 3d includes a similar arrangement. However, unlike FIG. 3b, where the biocompatible membrane 61 is actually attached to the metallized surface of the anode 44, in FIG. 3d the membrane 61 is not necessarily in contact with the anode 44 or the cathode 45. It is formed in the aperture 49 of the perforated substrate 42. Note that these figures are not drawn to scale, the membrane may be thicker or thinner than the electrodes, and may be thicker or thinner than the perforated substrate 42. Furthermore, it should be noted that in FIG. 3 b, the biocompatible membrane 61 is not disposed between the anode 44 and the cathode 45. However, in FIG. 3 d, the biocompatible membrane 61 is disposed between the anode 44 and the cathode 45. In each of FIGS. 3b and 3d, the combination of substrate 42 and biocompatible membrane 61 forms a structure that can also be referred to as a barrier (along with anode 44 in FIG. 3b).

  FIG. 3e illustrates a preferred embodiment for a fuel cell. In this figure, the arrangement of the membrane 61 and the perforated substrate 42 (substrate containing holes, perforations or apertures) is as previously described in connection with FIG. 3d. However, the cathode and anode are spaced from the perforated substrate. They may be plate electrodes, but are not placed on the surface or even in contact with the substrate 42 or film 61. In this case, the film 61 and the substrate 42 are barriers.

  Biocompatible membranes are described, for example, by Niki et al., US Pat. No. 4,541,908 (annealing cytochrome C to an electrode) and Personson et al. , J .; It can be formed across the pores, perforations or apertures 49 and the enzyme incorporated therein by the method described in detail in Electroanalytical Chem 292: 115, 1990. Such a method can include making a suitable solution of the polypeptide and synthetic polymeric material as previously discussed, wherein the perforated substrate 49, preferably a dielectric substrate, forms an enzyme-containing biocompatible membrane. Soaked in the solution. Sonication or detergent dilution may be required to facilitate enzyme uptake into the biocompatible membrane. See, for example, Singer, Biochemical Pharmacology 31: 527-534, 1982, Madden, “Current concepts in membrane protein reconstitution”, Chem. Phys. Lipids 40: 207-222, 1986, Montal et al. , “Functional residuals of membrane proteins in planar lipid bilayers”, Quart. Rev. Biophys. 14: 1-79, 1981, Helenius et al. , Et al. , “Asymmetric and symbolic membrane reconstitution by target elimination”, Eur. J. et al. Biochem. 116: 27-31, 1981, Methods in Enzymology series, Academic Press, see “Volumes on biomembranes” (eg, Fleischer and Packer (edit)).

  Alternatively, a thin compartment (preferably, but not necessarily) made from a hydrophobic material with a small aperture such as Teflon has a small amount of amphiphile introduced. When the coated aperture is immersed in a thin electrolyte solution, the droplets are thinned across the aperture and at the same time spread in a self-oriented manner. Substantial area biocompatible membranes have been prepared using this general technique. Two common methods for forming the biocompatible membrane itself are the Langmuir-Blodgett technique and the injection technique.

  The Langmuir-Blodgett technology involves the use of a Langmuir-Blodgett trough with a partition such as a Teflon ™ polymer partition in the center. This trough is filled with an aqueous solution. The polymer partition aperture is placed above the water level. A solution containing the polypeptide and the synthetic polymeric material is spread over the entire surface, and the polymer partition is slowly lowered into an aqueous solution that forms a biocompatible membrane above the aperture. The injection method is similar except that the polymer partition is held fixed. In this method, when the aqueous phase is filled just below the aperture and the solution is introduced above the surface, the liquid level is raised above the partition by injecting additional electrolyte solution from below.

  Another method of forming a biocompatible membrane is to use self-assembly techniques. This is a variation on the above two techniques and in fact is the first technique that has been used successfully to produce synthetic lipid membranes. This technique involves the preparation of the film-forming solution described above. Droplets of the solution are introduced into the perforated substrate 42, which is often a hydrophobic film. If the substrate 42 is then immersed in a thin aqueous electrolyte solution, then the droplet will naturally thin and will point in a particular direction. The remaining material moves around the layer where a reservoir called the Plateau-Gibbs boundary is formed.

  The thickness of the substrate 42 is, for example, from about 15 μm to about 5 mm, preferably from about 15 μm to about 1,000 μm, and more preferably from about 15 μm to about 30 μm when it is a perforated substrate having an aperture or porous material. . The width of the perforations or holes is, for example, from about 1 μm to about 1,500 μm, more preferably from about 20 μm to about 200 μm, and even more preferably from about 60 μm to about 140 μm. About 100 μm is particularly preferred. Preferably, the perforations or holes comprise an area that is about 30% greater than any area of the dielectric substrate involved in transport between chambers, such as about 50 to about 75% of the area.

  In certain preferred embodiments, the substrate is glass or polymer (eg, polyvinyl acetate, polydimethylsiloxane (PDMS), Kapton® (polyimide film, Dupont de Nemours, Wilmington, Del.), Perfluorinated. Contains about 59% fluorine sold as a polymer (Teflon, manufactured by DuPont de Nemours, Wilmington, Del.), Polyvinylidene fluoride (PVDF, for example, Kynar (TM) manufactured by Atofina (Philadelphia, PA). Semi-crystalline polymer), PEEK (defined below), polyester, UHMWPE (defined below), polypropylene or polysulfone, etc.), soda lime glass or borosilicate Glass, or have been either of the coating with a metal. Metals can be used to immobilize a biocompatible membrane (such as a monolayer or bilayer of amphiphilic molecules). Metal coatings can be repelled from any junction where they appear to provide a short conductive path between the anode and cathode compartments. In a particularly preferred embodiment of the invention, the perforated substrate 42 is manufactured from a dielectric material.

  Polypeptide 62 can be immobilized on the biocompatible membrane with proper orientation to allow access to the catalytic site for oxidation reaction to the anode compartment and asymmetric pumping of protons. However, if the polypeptide is not asymmetrically oriented, the reverse-oriented polypeptide is not harmful for a variety of reasons depending on the situation. First, the charge imbalance created by the fuel cell on the anode side drives proton transport to the cathode side even against the proton concentration gradient. In situations where pumping is associated with the use of reduced electron carriers, reverse pumping does not have such a transmitter because the electron carrier is substantially insulated within the anode compartment 41. ("Substantially insulated" will be recognized by those skilled in the art as being sufficiently insulated to allow fuel cell operation.)

  In the embodiment as shown in FIGS. 4a-4c, the biocompatible membrane 61 contains a cross-linking component and is formed on the substrate 42 beyond the aperture with a chamfered edge. The chamfer angle may be any angle that increases the stability of the biocompatible membrane. If the cross-linked block copolymer is significantly less rigid, use of a larger chamfer angle can increase stability, while a smaller chamfer angle is more appropriate for a more rigid cross-linked block copolymer. Sometimes. As illustrated, multiple chamfer shapes may contribute to increase stability.

  In yet another alternative embodiment of the present invention, the solution containing the polypeptide and the synthetic polymeric material is placed over the entire surface of the porous support material rather than the perforated material as illustrated in FIG. 3b. be able to. For example, if protons are pumped across the membrane, they will be able to move through the pores of the support barrier material.

  Whether the substrate is a perforated substrate or a porous substrate, it is not necessary to form the film over its entire surface. For example, it may be convenient to form a film over the entire surface of the perforated substrate, but a solution containing a polypeptide and a synthetic polymer material may be selectively introduced into the perforated part, or the perforated part may be It may be preferable to simply cross.

  The thickness of the biocompatible membrane according to the present invention can be adjusted by known techniques such as controlling the volume introduced into a specific size hole, perforation, dish or tray or the like. The thickness of the film will be largely determined by its composition and function. Membranes intended to contain a transmembrane proton transport complex such as Complex I must be thick enough to provide sufficient support and orientation to the enzyme complex. However, it must be thick enough to prevent effective transport of protons across the membrane. Previously confirmed Meier et al. In an array with about 100 apertures and a poly (2-methyloxazoline) -block-poly (dimethylsiloxane) -block-poly ( For a solution containing Complex I in an amount of about 4 μL in a 7% (w / v) copolymer solution containing 2-methyloxazoline) -triblock copolymer, a film of appropriate thickness is available. The thickness of the membrane can vary widely depending on its required lifetime, its function, etc. For example, membranes designed to transport protons are often thinner than membranes on which enzymes capable of oxidizing something are attached. In general, however, the thickness of the film will range from about 10 nm to even over 100 nm. In fact, biocompatible membranes useful for transporting protons in fuel cells have yielded good results with thicknesses from 10 nm to 10 μm. In addition, thicker films are possible.

  In certain embodiments of the invention that are particularly useful in the creation of fuel cells, the biocompatible membrane of the invention can transport protons against a pH gradient. This concept is discussed in more detail herein. However, theoretically, on the cathode side of the biocompatible membrane, the pH of any medium, electrolyte, etc. is acidic, but the pH on the anode side of the membrane is basic. This is the opposite of what is found in the majority of fuel cells. In general, such conditions will favor the transfer of protons from the acidic side rich in protons to the basic side where there are considerably fewer protons. However, the use of the membrane according to the present invention allows pumping upward from the proton-poor side to the proton-rich side. This highlights yet another aspect of the invention that may be particularly useful. The membrane of the present invention may be active and functional despite the considerable variation in pH conditions on the opposite side. For example, a membrane according to the present invention can catalyze proton transfer when the pH in the anode compartment is at least 0.5 pH units higher than the pH in the cathode compartment.

  In many fuel cells, the pH in the anode compartment is lower than the pH found in the cathode compartment due to the high proton concentration. However, fuel cells made according to the present invention do not need to rely on proton concentration differences to drive protons by diffusion across the membrane. This can be a particularly important advantage because the species used as electron carriers and / or electron transfer mediators often function more efficiently at fairly alkaline pH. The fuel oxidation reaction may also be more efficient under such pH conditions. Based on the electrolyte used, it is not necessary to adjust the pH difference during the useful life of the fuel cell. Alternatively, buffer systems can be added to the anode and / or cathode compartment during operation, and additional buffers can be added as needed. Preferably, the anode compartment will have a pH that is at least about 1 pH unit higher, more preferably 2 pH units higher than the pH in the cathode compartment. In a particularly preferred embodiment, the pH of the anode compartment is 8 or higher and the pH in the cathode compartment is 5 or lower. See, for example, Example 59.

  Another aspect of the present invention is a fuel cell manufactured using a biocompatible membrane as described herein. Without being limited to other suitable definitions known in the art, a fuel cell is a device that generates electrical energy by chemical conversion of fuel. The specific types of fuel cells vary widely with respect to the type of fuel used, the electron transport species (electron carrier, soluble enzyme, transfer mediator, etc.), or the type of electrolyte used, the type of electrode used, etc. As long as they meet the appropriate criteria, they are all expected. For example, the system used must be compatible with the biocompatible membrane. For example, if they are corrosive to the membrane, the lifetime of the fuel cells can be significantly shortened (effective lifetime of less than 8 hours). If the materials used cause great instability, this may be the reason why certain fuels are not useful, for example according to the present invention. Particularly preferred are computers, PDAs, mobile phones, pagers, personal entertainment systems, PlayStation 2, Game Boy, portable DVD players, electric tools, toys, stereo devices, radios, cameras and video recorders, digital recorders and digital cameras, flashlights It is a fuel cell that is sufficiently small and lightweight for use in portable electronic devices such as automobiles, trucks, ships, and airplanes. The fuel cells are preferably "green", i.e. they do not contain corrosive or hazardous chemicals, both as fuel and waste, so they can be easily disposed of. Further, these fuel cells may be refillable (such as adding additional fuel) or disposable / disposable.

  As illustrated in FIG. 1, the fuel cell according to the present invention may include an anode compartment 1 having an anode 4 and a cathode compartment 3 having a cathode 5. This assembly also includes a dielectric perforated substrate or porous substrate 2. The fuel cell further includes at least one biocompatible membrane 61 as previously described (not shown in FIG. 1). The anode 4 has a conductor or electrical contact 6 and the cathode 5 has a conductor or electrical contact 7 that can be electrically connected or electrically connected with an electrical circuit through a load or resistor so as to place them in the electrical contact. Have. The biocompatible membrane 61 can be placed in the anode compartment as in FIG. 3b, in the cathode compartment, or between the anode and cathode compartments as shown in FIGS. 3d and 3e. A biocompatible membrane can also be placed between the anode and cathode in FIGS. 3d and 3e, which can be considered to define the boundary between the anode and cathode compartments. Fuel cells typically include electrical contacts that allow an electrical circuit to be formed between the two electrodes. The anode and cathode can be made from any conductive material that otherwise would not generally react with the fuel cell elements. The anode and cathode are preferably made from the previously described metals or carbon. The size and shape of the anode and cathode can be made to match the required dimensions of the fuel cell and to allow the passage of various chemical species. FIG. 3c illustrates a fuel cell made in accordance with the present invention that includes a battery housing 51 and a perforated substrate 42 containing the biocompatible membrane 61 described herein within the perforated portion thereof. The anode and cathode are plated on the substrate as previously described, but are not separately illustrated. However, as shown in FIG. 3c, anode contact 54 and cathode contact 55 are attached to appropriate electrodes in the fuel cell.

  When an electrode is used as part of a support system for a biocompatible membrane as illustrated in FIG. 3b, the electrode is sufficient to provide a passage through which molecules, atoms, protons or electrons can flow. Must have an appropriate perforation or other means. However, if the fuel cell has a configuration similar to that of FIG. 3e, the electrodes can be completely solid. However, it may also be desirable to allow fuel or other components of the fuel cell to pass through and around the electrodes, so that it is possible to provide a perforation anyway.

Biocompatible membranes useful in the fuel cell according to the present invention have already been discussed. The biocompatible membrane will preferably facilitate the passage of current in an amount greater than that resulting from the use of the same membrane without the polypeptide. More preferably, the biocompatible membrane will promote a current of at least about 10 mA / cm ² , more preferably at least about 50 mA / cm ² , and most preferably at least about 100 mA / cm ² . In the simplest embodiment, the biocompatible membrane can stand independently, or be supported by surrounding structures, and is placed across the aperture, or between the anode and cathode. Be placed. Furthermore, in this case, the biocompatible membrane itself is dielectric, which is a certain component between the anode and cathode compartments such as catholyte, electrolyte, cathode fuel, analyte anode fuel and other ions. It is important to disturb the free flow of

  A less complex embodiment would then include the use of a biocompatible membrane that is similar, but cannot interfere with thorough mixing of the required species, or is not dielectric. In such cases, an additional barrier may be required. Such a barrier can be manufactured from the same material used to manufacture the previously described substrate 42, or alternatively, the membrane can be perforated in the substrate 42, as shown in FIGS. 3d and 3e. Or it can be placed in the holes or to cover them. Materials useful for the substrate and methods of preparing the same have already been discussed.

The anode electrode can be coated with an electron transfer mediator such as an organometallic compound that functions as a surrogate electron acceptor for the biological substrate of the oxidoreductase. Similarly, the biocompatible membrane of the embodiment of FIG. 3 or the structure adjacent to the biocompatible membrane can incorporate such an electron transfer mediator, or more generally an anode chamber. Available within. Such organometallic compounds include, without limitation, dicyclopentadienyl iron (available with analogs that can be substituted from C ₁₀ H ₁₀ Fe, ferrocene, Aldrich (Milwaukee, Wis.)), Platinum on carbon , And palladium on carbon. Other examples include ferredoxin molecules with appropriate oxidation / reduction potentials, such as ferrodoxin formed from rubredoxin and other ferredoxins available from Sigma Chemical. Other electron transfer mediators include organic compounds such as quinones and related compounds. Yet another electron transfer mediator is methyl viologen, ethyl viologen, or benzyl viologen (CAS 1102-19-8, 1,1′-bis (phenylmethyl) -4,4′-bipyridinium, N, N′-γ). , Γ'-dipyridyllium) and any of those listed below in the definition of electron transfer mediators.

  The anode electrode (relative to the biocompatible membrane) can be impregnated with an enzyme, which can be applied before or before the electron transfer mediator. One way to ensure enzyme-electrode binding is to simply incubate the enzyme solution with the electrode for a sufficient amount of time to complete the electrode-enzyme binding, such as van der Waals binding. . Alternatively, a first binding component, such as biotin or its binding complement, avidin / streptavidin, can be attached to the electrode and to the enzyme bound to the first binding component through the binding complement attachment molecule. Additional methods for attaching enzymes to electrodes or other materials and additional electron transfer mediators are described in Willner and Katz, Angew. Chem. Int. Ed. 39: 1181-1218,2000. The anode chamber can contain an enzyme adjacent to or bound to the anode electrode or away from the anode electrode. For example, the oxidoreductase can be attached to the anode chamber side of the polymer that forms the proton conducting anode / cathode barrier, where a layer of conductive material on the anode side provides the anode electrode. In some embodiments of the invention, the electron carrier is expected to be effective for transferring electrons to the anode electrode in the absence of oxidoreductase.

  Some embodiments of the present invention can further use the traditional form of anode / cathode barrier: polymer membranes selected for their ability to bind the biocompatible membrane of the present invention and passively transfer protons. The former anode / cathode barrier is useful because it is effective for pumping against the proton gradient.

  The bilayer membrane can be disposed across and / or within the perforations or holes in the anode / cathode barrier, or can be disposed between the anode and cathode compartments. These membranes may be traditional composition membranes or biocompatible membranes. One situation where such a bilayer is observed is where the pore diameter is fairly narrow. Another situation is that the anode / cathode barrier is formed from a sandwich-type material so that separate joints between different materials are the core of the formation of separate biocompatible membranes across the pores. Is the situation.

  Without being limited to theory, the second more biocompatible membrane closer to the cathode functions passively to some extent as pumping from the first biocompatible membrane creates a higher proton concentration, and passive to the cathode compartment. It is thought that it drives dynamic transportation. Thus, the active transport function may be compromised to the extent that the cathode compartment contains a peroxide that can be expected to damage the transport protein, while the second biocompatible membrane has a higher concentration of peroxide. 1 Isolate the biocompatible membrane.

  In one embodiment, the benefit of a bimembrane is that the first membrane (on the anode side) has a polypeptide and proton attached to the cathode chamber side to limit the migration of peroxide toward the biocompatible membrane. Obtained by one or more biocompatible membranes incorporating a conductive polymer membrane. Furthermore, the intermediate zone between the one or more biocompatible membranes and the proton conducting polymer membrane acquires a high proton concentration due to active transport, and further migration along a long concentration gradient into the cathode compartment Drive.

  In one embodiment, the substrate in which the holes are formed is a sandwich of dielectric Kapton and conductive Kapton (conductive through the presence of incorporated graphite). The conductive Kapton can form an anode electrode or can be appropriately metallized to form the anode electrode. The three layers are relative hydrophilic, relative hydrophobic, and relative hydrophilic.

  FIG. 2 is a schematic block diagram of one embodiment on the anode side of a fuel cell according to the present invention. The anode compartment contains fuel. A fuel is an organic molecule that is depleted and consumed according to the present invention. However, its consumption generates protons and electrons. Preferred fuels according to the present invention are monocarbon compounds or can be converted to monocarbon compounds. Of these, methanol is preferred. However, fuels include, without limitation, oxidizable sugars and sugar alcohols, alcohols, pyruvates, succinates, etc., fatty acids, organic acids such as lactic acid, citric acid, amino acids and short polypeptides, aldehydes, ketones, etc. be able to. The fuel in this embodiment is first acted on by a soluble enzyme that may be an alcohol dehydrogenase in the case of methanol. As will become apparent below, other dehydrogenases such as aldehyde dehydrogenase and formate dehydrogenase can also be used or used in combination with each other. These soluble enzymes can act on the fuel to generate electrons and protons.

  The soluble enzyme is preferably present in the range of about 0.001 to about 25,000 units, more preferably about 0.1 units to about 12,500 units, and most preferably about 1 unit to about 12,5000 units. To do. The unit in this context means the specific activity of the soluble enzyme, and one unit of activity is the amount required to convert 1 μM fuel per minute at 25 ° C. (or enzyme replacement in other situations). . Since these compounds do not need to be soluble or enzymes, “soluble enzyme” is somewhat misleading. These compounds can be suspended or emulsified and / or immobilized on beads or some other solid support. That is true as long as they are functional, can act on fuel, and the electron mediator and / or mediator allows the protons to be transported to the biocompatible membrane and the electrons to the anode. Is not a problem.

Protons and electrons are transferred to an electron carrier, also called a cofactor, by the coordinated action of soluble enzymes on the fuel. One such electron carrier is NAD + / NADH. The electron carrier is not limited and is reduced nicotinamide adenine dinucleotide (NADH. The oxidized form is indicated as NAD or NAD ⁺ ), and reduced nicotinamide adenine dinucleotide phosphate (NADPH is indicated. The type is indicated as NADP or NADP ⁺ ), reduced nicotinamide mononucleotide (NMNH, oxidized form is NMN), reduced flavin adenine dinucleotide (FADH _2, oxidized form is FAD), reduced flavin mononucleotide (FMNH ₂ The oxidized form may include FMN), reduced coenzyme A, and the like. Electron carriers include proteins with incorporated electron donating prosthetic groups such as coenzyme A, protoporphyrin IX, vitamin B12, and the like. All of the above are believed to carry both electrons and protons that can be generated by the action of soluble enzymes on the fuel. However, not all electron carriers carry protons. It will be appreciated that C ₁ compounds containing carbon, oxygen and hydrogen are electron carriers. But these are also fuels. Similarly, the definition of an electron carrier includes an electron transfer mediator as described in detail below. When present, the electron carrier is generally provided at a concentration of about 1 μM to about 2 μM, more preferably about 10 μM to about 1 M, and most preferably about 100 μM to about 500 mM.

Under the influence of soluble enzymes, protons and electrons, NAD ⁺ is converted to NADH. From this point, electrons and / or protons can be removed and replaced between a number of additional cofactors and / or transfer mediators. An electron transfer mediator is a composition that facilitates the transfer of electrons released from an electron carrier to another molecule, typically an electrode with a reduction potential equal to or less than that, or another electron transfer mediator. In addition to those identified above, examples include phenazine methosulfate (PMS), pyrroloquinoline quinone (also called PQQ, methoxatin), hydroquinone, methoxyphenol, ethoxyphenol, or other typical quinone molecules, Methyl viologen, 1,1′-dibenzyl-4,4′-dipyridinium dichloride (benzyl viologen), N, N, N ′, N′-tetramethylphenylenediamine (TMPD) and dicyclopentadienyl iron (C ₁₀ H ₁₀ Fe, ferrocene). Electron transfer mediators, when present, are generally provided at a concentration of about 1 μM to about 2M, more preferably about 10 μM to about 2M, and even more preferably about 100 μM to about 2M.

  However, for simplicity and as illustrated in FIG. 2, the reduced cofactor or electron carrier is then combined with the polypeptide, in this case the biocompatible membrane according to the invention. It can interact with dehydrogenase function of body I. Complex I liberates protons and further electrons from the NADH molecule. The electrons may flow directly to the anode. More often, however, the electrons are taken up by the transfer mediator, which transports the electrons to the anode.

  NADH dehydrogenase complex I is an interesting polypeptide in that it can also participate in proton transport across biocompatible membranes. Of particular interest, however, is that the transferred proton is not necessarily a proton released by the action of the dehydrogenase portion of complex I. Thus, to obtain the best results, the fuel cell according to this particular embodiment of the invention will contain additional proton species in the anode compartment. The proton transport function of Complex I is illustrated in FIG. 2 as a redox function.

When the transfer mediator delivers the electrons to the anode, the transfer mediator is oxidized and it becomes possible to obtain additional electrons liberated by oxidizing other electron mediators. The oxidized cofactor (NAD ⁺ ) is now ready to accept protons and electrons under the influence of fuel and soluble enzymes. The reaction just described takes place in the anode electrode and anode compartment and can be chemically illustrated as follows.

これをまとめると、以下となる。

This reaction can be supplied by the following reaction.

This is summarized as follows.

これをまとめると、以下となる。

Therefore, the sum of these reactions and the electron generation reaction is as follows.

This is summarized as follows.

Soluble enzymes that can be used to generate electron carriers (such as NADH exemplified above) from organic molecules such as methanol can start in the form of alcohol dehydrogenase (ADH). For suitable ADH enzymes, see, for example, Amendola et al. , "Thermotable NAD (+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus3: gene and protein sequence dehydration and rehabilitation. , "Cloning and overexpression in Escherichia coli of the genes encoding NAD-dependent alcohol dehydrogenase from two types of Sulfobus specialties." Bacteriol. 178: 301-5, 1996, Saliola et al. , “Two genes encoding putative mitochondral alcohol dehydrogenases are present in the yeast Kluyveromyces lactis”, Yeast 7: 391-400, un 1991, et al. , "Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondral isozyme of alcoholic dehydrogenase in Saccharomyces cerevisiae." Cell Biol. 5: 3024-34, 1985. When the resulting formaldehyde is oxidized, aldehyde logase (ALD) is used. Suitable ALD enzymes are described, for example, in Peng et al. , “CDNA cloning and charac- terization of a aldehyde dehydrated induced by incompatible blast fungus”, GeneBank accession number AF323586, Sakano et al. , “Arabidopsis thaliana [thal cress] aldehyde dehydrogenase (NAD ⁺ ) -like protein”, GeneBank Accession No. AF327426. In addition, when the resulting formic acid is oxidized, formate dehydrogenase (FDH) is used. Suitable FDH enzymes are described, for example, in Colas des Francs-Small et al. , “Identification of a major soluble protein in mitochondria from nonphosphotetic tissues as NAD-dependent format dehydration. 102 (4): 1171-1177, 1993, Hourton-Cabassa, “Evidence for multiple copies of formative genes, in fandh, p3. 117: 719-719, 1998.

  For reasons discussed below, it is useful to adapt to use quinone-based electron carriers, or to use soluble enzymes that are compatible with quinone-based electron carriers. There is. Such enzymes are described, for example, by Pommier et al. "A second phenazine method-linked form dehydrogenase isoenzyme in Escherichia coli", Biochim Biophys Acta. 1107 (2): 305-13,1992 "The diversity of reactions involving formate dehydrogenases is apparent in the structures of electron acceptors which include pyridine nucleotides, 5-deazaflavin, quinones, and ferredoxin", Ferry, J. G. “Formate dehydrogenase”, FEMS Microbiol. Rev. 7 (3-4): 377-82, 1990 (formaldehyde dehydrogenase with quinone activity), Klein et al. , "A novel dye-linked formaldehyde withogeneous some properties indicating the presence of the protein-bound redox-active cofactor." 301 (Pt 1): 289-95, 1994 (representative among many papers on dehydrogenase with bound quinone cofactor), Goodwin et al. , "The biochemistry, physology and genetics of PQQ and PQQ-containing enzymes", Adv. Microb. Physiol. 40: 1-80, 1998 (for alcohol dehydrogenases utilizing quinones), Maskos et al. , “Mechanism of p-nitrosophenol reduction catalyzed by horse liber and human pi-alcohol dehydrogenase (ADH)”, J. et al. Biol. Chem. 269 (50): 31579-84, 1994 (example of mediator-catalyzed transfer of electrons from NADH to electrode following NADH reduction by enzyme), and Pandey, "Tetracyanoquinodiene methane hydrate dehydrogenation enzyme chemistry?" , Anal. Biochem. 221 (2): 392-6, 1994.

The corresponding reaction at the cathode in the cathode compartment may be any reaction that consumes the generated electrons with a useful redox potential. For example, using oxygen, the reaction may be:

  Using Scheme 2, the catholyte (electrolyte used in the cathode compartment) can be buffered to account for hydrogen ion consumption, and the hydrogen ion donating compound can be supplied during fuel cell operation. Or more preferably, the barrier between the anode and cathode compartments is sufficiently effective to deliver neutralized hydrogen ions (hydrogen ions or protons).

In one embodiment, the corresponding reaction at the cathode is:

  This cathodic reaction results in a net production of water, but if this is significant, it can be processed, for example, by providing space for the overflow liquid or by providing a gas phase outlet. Many electron acceptor molecules are often solid at operating temperatures, or solutes in the carrier fluid, but in the latter case, the cathode chamber must be adapted to carry such non-gaseous materials. .

As is likely the case with hydrogen peroxide as the electron acceptor molecule, if the electron acceptor molecule can damage the biocompatible membrane polypeptide and any other species in the anode chamber, Such electron acceptor molecule scavengers can be used in fuel cells to prevent oxides or damaging electron acceptor molecules from entering the anode chamber. Such scavengers, especially if the condition of the anode electrode is not effective to catalyze the transfer of electrons to O _2, may be, for example, enzyme catalase _{(2H 2 O 2 ⇔2H 2 O} + O 2). Alternatively, the scavenger may be any noble metal such as gold or platinum. Such scavengers can be covalently bound to the solid support material if they are enzymes. Alternatively, a barrier having the most limited permeability to hydrogen peroxide is provided between the anode chamber and the cathode chamber.

Solid oxidants such as calcium peroxide, potassium perchlorate (KClO ₄ ) or potassium permanganate (KMnO ₄ ) can be used as electron acceptors.

  Fuel cells operate within a temperature range appropriate for oxidoreductase or proton transporters to operate. This temperature range typically varies with the stability of the enzyme and the source of the enzyme. In order to increase the appropriate temperature range, an appropriate oxidoreductase derived from a thermophilic organism such as a microorganism isolated from a conduit or hot spring can be selected. In addition, genetically modified enzymes can be used. Nevertheless, at least the preferred temperature for the operation of the first electrode is about 80 ° C. or less, preferably 60 ° C. or less.

Preferred fuel cells in accordance with the present invention are described herein comprising an anode compartment, a cathode compartment, an anode and a cathode, and a polypeptide that can participate in proton transfer from one side of the membrane to the other. A biocompatible membrane is included. Within the anode compartment, at least one electron transfer mediator and electrode carrier are also found. The fuel cell of the present invention is preferably capable of generating at least about 10 mW / cm ² , more preferably at least about 50 mW / cm ² , and most preferably at least about 100 mW / cm ² over its useful life ( Some power reduction will occur towards the end of its life). The fuel cells are preferably generally at least 8 hours, preferably 1 week, more preferably 1 month, and most preferably 6 until the fuel is eventually exhausted (unless they are refillable). Generate such a current density for more than a month.

(Example 1)
A solution useful for producing a biocompatible membrane according to the present invention was prepared as follows. 7% (w / v) (70 mg) block copolymer (poly (2-methylxazoline) -polydimethylsiloxane-poly (2-) having an average molecular weight of 2 kD-5 kD-2 kD while stirring using a magnetic stirrer Methyl (oxazoline) was dissolved in a 95% (v / v) / 5% (v / v) ethanol / water solvent mixture, 6 μL of this solution was removed and 0.015% (w / v) in water. ) Dodecylmaltoside, 4 μL of a solution containing 40 μg of complex I (10 mg / mL), which was further mixed.The resulting solution was 4.2% (w / v) polymer, Contains 55% (v / v) EtOH, 45% (v / v) H ₂ O, 0.06% (w / v) dodecyl maltoside, and a protein / polymer ratio of 6% (w / W) There was.

(Example 2)
Solutions useful for producing biocompatible membranes according to the present invention were prepared as generally described in Example 1, with the following modifications. Less polypeptide solution to provide a final solution containing 0.015% (w / v) dodecyl maltoside and 1.5% (w / w) polypeptide compared to the synthetic polymeric material. used.

(Example 3)
Solutions useful for producing biocompatible membranes according to the present invention were prepared as generally described in Example 1, with the following modifications. Less polypeptide solution is used to provide a final solution containing 0.03% (w / v) dodecyl maltoside, the final solution being 3.0% (w / w) compared to the synthetic polymeric material. ) Polypeptide.

(Example 4)
Solutions useful for producing biocompatible membranes according to the present invention were prepared as generally described in Example 1, with the following modifications. Less polypeptide solution was used to provide a final solution containing 0.045% (w / v) dodecyl maltoside, which was 4.5% (w / w) compared to the synthetic polymeric material. ) Polypeptide.

(Example 5)
Solutions useful for producing biocompatible membranes according to the present invention were prepared as generally described in Example 1, with the following modifications. Less polypeptide solution is used to provide a final solution containing 0.0075% (w / v) dodecyl maltoside, the final solution being 0.75% (w / w) compared to the synthetic polymeric material. ) Polypeptide.

(Example 6)
Solutions useful for producing biocompatible membranes according to the present invention were prepared with the following modifications, generally as described in Example 5. The synthetic polymeric material was initially present in a 5.0% (w / v) solution. Described in Example 1 so that a final solution containing 0.0075% (w / v) dodecyl maltoside and 0.75% (w / w) polypeptide compared to synthetic polymeric material can be produced. Sufficient polypeptide solution of the type indicated was added.

(Example 7)
Solutions useful for producing biocompatible membranes according to the present invention were prepared as generally described in Example 6 with the following modifications. Sufficient polypeptide solution as described in Example 1 is included so that a final solution containing 0.015% (w / v) dodecyl maltoside can be prepared, the final solution being 1. It contained 5% (w / w) polypeptide.

(Example 8)
Solutions useful for producing biocompatible membranes according to the present invention were prepared as generally described in Example 6 with the following modifications. Sufficient polypeptide solution as described in Example 1 was included so that a final solution containing 0.03% (w / v) dodecyl maltoside could be prepared, the final solution being 3% compared to the synthetic polymeric material (W / w) polypeptide.

Example 9
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in Example 6 with the following modifications. 3. Sufficient polypeptide solution as described in Example 1 was included so that a final solution containing 0.045% (w / v) dodecyl maltoside could be prepared, the final solution being 4. It contained 5% (w / w) polypeptide.

(Example 10)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in Example 6 with the following modifications. Sufficient polypeptide solution as described in Example 1 was included so that a final solution containing 0.06% (w / v) dodecyl maltoside could be prepared, the final solution being 6. It contained 0% (w / w) polypeptide.

(Examples 11 to 15)
Solutions useful for producing biocompatible membranes according to the present invention are generally described in Example 1 except that the synthetic polymeric material used in each solution was initially 10% (w / v). Prepared as described in each of -5. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each 0.06, 0.15, 0.03, 0.045 and 0.005 compared to the synthetic polymeric material. Final solutions containing 0075% (w / v) dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and 0.75% (w / w) polypeptide were produced.

(Example 16)
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 3, except that the solvent used to dissolve the synthetic polymeric material was ethanol, 25% (v / v) methanol and the amount of water indicated in Example 3 were contained. Sufficient polypeptide solution to produce a final solution containing 0.03% (w / v) dodecyl maltoside and 3.0% (w / w) polypeptide compared to synthetic polymeric material It was used.

(Example 17)
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 2, but the solvent used to dissolve the synthetic polymeric material was 47.5% (v / v) ethanol, 2.5% (v / v) water, 25% (v / v) tetrahydrofuran (THF), 25% (v / v) dichloromethane. Sufficient polypeptide solution so that a final solution containing 0.015% (w / v) dodecyl maltoside and 1.5% (w / w) polypeptide compared to synthetic polymeric material can be produced. Was added.

(Example 18)
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 6, except that the solvent used to dissolve the synthetic polymeric material was 9.5% (v / v) ethanol, 0.5% (v / v) water, 40% (v / v) acetone, and 40% (v / v) hexane.

(Examples 19 to 24)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in each of Examples 11-15, with a final concentration of dodecyl maltoside of 0.15% (w / v). Met. A solution useful for producing a biocompatible membrane according to the present invention can be prepared generally as described in Example 4, but the remainder of the surfactant used in the polypeptide solution is dodecyl β-D-glucopyranoside. The final concentration of surfactant is 0.15% (w / v).

(Example 26)
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 9, but the surfactant used in the polypeptide solution was manufactured by BASF (Ludwigshafen, West Germany). Of PLURONIC L101 (lot no. WPDX-522B) and a mixture of a polymeric surfactant sold under the trademark PLURONIC L101 (lot number WPDX-522B) and the same concentration of dodecyl maltoside as defined in Example 9. The polymeric surfactant was diluted to 0.1% (v / v) of the feed concentration in the final solution.

(Example 27)
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 2, but the surfactant used in the polypeptide solution was BYK Chemie (Warringford, Conn.). It contained a mixture of a polymeric surfactant sold under the trademark DISPERPLAST (lot number 31J022) and the same concentration of dodecyl maltoside as defined in Example 2. The polymeric surfactant was diluted to 0.135% (v / v) of the feed concentration in the final solution.

(Examples 28 to 32)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 6-10, but the synthetic polymeric material used has an average molecular weight of 3 kD-7 kD-3 kD. It may be poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline) (5% (w / v)). When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. A final solution containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 33-38)
Solutions useful for producing biocompatible membranes according to the present invention were generally prepared as described in each of Examples 1-5, but the synthetic polymeric materials used were both poly (2-methyloxazoline). -Polydimethylsiloxane-poly (2-methyloxazoline), one of them (total 7% (w / v)) has an average molecular weight of 2 kD-5 kD-2 kD and the other 1 kD-2 kD-1 kD, A mixture of two block copolymers in which the ratio of the first block copolymer to the second block copolymer is about 67% to 33% (w / w) of the total synthetic polymeric material used. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.06, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. Final solutions containing 0075% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide were produced.

(Examples 39 to 43)
While solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 11-15, the synthetic polymeric materials used are both poly (2-methyloxazoline). -Polydimethylsiloxane-poly (2-methyloxazoline), one of them (10% (w / v)) has an average molecular weight of 1 kD-2 kD-1 kD and the other 3 kD-7 kD-3 kD, It may be a mixture of two block copolymers where the ratio of one block copolymer to second block copolymer is about 33% to 67% (w / w) of the total polymer used. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.075, 0.15, 0.30, 0.45 and 0.005 compared to the synthetic polymeric material. A final solution containing 60% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 44 to 48)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 6-10, although the synthetic polymeric materials used are both poly (2-methyloxazoline). -Polydimethylsiloxane-poly (2-methyloxazoline), one of them (5% (w / v)) has an average molecular weight of 2 kD-5 kD-2 kD and the other 3 kD-7 kD-3 kD, It may be a mixture of two block copolymers where the ratio of one block copolymer to second block copolymer is about 33% to 67% (w / w) of the total polymer used. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. A final solution containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 49-53)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 1-5, although the synthetic polymeric materials used are both poly (2-methyloxazoline). -Polydimethylsiloxane-poly (2-methyloxazoline), one of them (7% (w / v)) has an average molecular weight of 2 kD-5 kD-2 kD and the other 3 kD-7 kD-3 kD, It may be a mixture of two block copolymers where the ratio of one block copolymer to second block copolymer is about 67% to 33% (w / w) of the total polymer used. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.06, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. Final solutions containing 0075% (w / v) dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and 0.025% (w / w) polypeptide are produced.

(Examples 54-58)
Although solutions useful for producing biocompatible membranes according to the present invention can be prepared generally as described in each of Examples 1-5, the synthetic polymeric material used is a 85% triblock copolymer solution (v / 95% ethanol mixed with 23.5% (w / v) polyethylene glycol solution having an average molecular weight of about 3,300 daltons in water at a ratio of v) to 15% (v / v) polyethylene glycol solution And a mixture of poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline) (7% (w / v)) having an average molecular weight of 2 kD-5 kD-2 kD in 5% water solvent It may be. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.06, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. Final solutions containing 0075% (w / v) dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and 0.75% (w / w) polypeptide are produced.

(Examples 59 to 63)
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 12, except that the synthetic polymeric material used was 85% triblock copolymer solution and 15% polyethylene glycol solution. In a solvent of 95% ethanol and 5% water mixed with 23.5% (w / v) polyethylene glycol solution having an average molecular weight of about 8,000 Daltons in water at a ratio of (v / v) 10% (w / v) poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline) mixture having an average molecular weight of 2 kD-5 kD-2 kD. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, 0.15% (w / v) dodecyl maltoside and 1.5% (w / v) compared to the synthetic polymeric material. A final solution containing the polypeptide of w) was produced. Similar solutions can be made using the procedures of Examples 11 and 13-15.

(Examples 64-68)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 28-32, but the synthetic polymeric materials used are 85% triblock copolymer solution and polyethylene glycol. 95% ethanol and 5% water mixed with 23.5% (w / v) polyethylene glycol solution having an average molecular weight of about 3,300 Daltons in water at a ratio of 15% (v / v) solution 5% (w / v)) poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline) mixture having an average molecular weight of 3 kD-7 kD-3 kD in the solvent. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. A final solution containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 69 to 73)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 1-5, but the synthetic polymeric materials used are 85% triblock copolymer solution and polyethylene glycol. 95% ethanol and 5% water mixed with 23.5% (w / v) polyethylene glycol solution having an average molecular weight of about 8,000 Daltons in water at a ratio of 15% (v / v) solution 7% (w / v)) poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline) mixture having an average molecular weight of 3 kD-7 kD-3 kD in the solvent. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.060, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. Final solutions containing 0075% (w / v) dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and 0.75% (w / w) polypeptide are produced.

(Examples 74 to 78)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 6-10, except that the synthetic polymeric material used is 80% triblock copolymer and 20% 50% (v / v) acetone mixed with 5% (w / v) polystyrene solution having a molecular weight of about 250,000 Daltons in 50% (v / v) acetone 5% (w / v)) poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline) with an average molecular weight of 2 kD-5 kD-2 kD in a solvent of 50% (v / v) heptane ). When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. A final solution containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 79 to 83)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 1-5, but the synthetic polymeric material used is 66% (v / v) each. 5% (w / v) having an average molecular weight of 4 kD-8 kD-4 kD in a solvent of 50% (v / v) THF and 50% (v / v) dichloromethane in a ratio of 33% (v / v) 7% (w / v) having an average molecular weight of 2 kD-5 kD-2 kD in a solvent of 95% ethanol and 5% water mixed with a solution of polymethyl methacrylate-polydimethylsiloxane-polymethyl methacrylate) It may be a mixture of poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline). When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.06, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. Final solutions containing 0075% (w / v) dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and 0.075% (w / w) polypeptide are produced.

(Examples 84 to 88)
Solutions useful for producing biocompatible membranes according to the present invention were generally prepared as described in each of Examples 11-15, but the synthetic polymeric material used was 5% (v / v). Protolyte (R) A700 (Lot No. LC-29 / 60-) from Dais Analytic (Odessa, Florida) in a solvent supplied diluted with ethanol containing water to 50% (v / v) 10% (w / v) sulfonated styrene / ethylene-butylene / sulfonated styrene supplied as 011). When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. Final solutions containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide were produced.

Example 89
Solutions useful for producing a biocompatible membrane according to the present invention can generally be prepared as described in Example 84, but the solvent used to dilute the synthetic polymeric material is 50% (v / v). Of tetrahydrofuran (THF) and 50% (v / v) dichloromethane.

(Examples 90 to 94)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in each of Examples 84-88, but the final concentration of dodecyl maltoside was 0.15% (w / v). Met.

(Example 95)
While solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in Example 85 above, the surfactants used in the polypeptide solution are dodecyl β-D-glucopyranoside and dodecyl. It may contain a mixture with maltoside and the final concentration of the surfactant is 0.15% (w / v).

Example 96
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 87, but the surfactant used in the polypeptide solution was manufactured by BASF (Ludwigshafen, West Germany). Contained a mixture of a polymeric surfactant sold under the trademark PLURONIC L101 (lot number WPDX-522B) and the same concentration of dodecyl maltoside as defined in Example 87. The polymeric surfactant was diluted to 0.1% (v / v) of the feed concentration in the final solution.

(Example 97)
A solution useful for producing a biocompatible membrane according to the present invention was prepared generally as described in Example 88 above, but the surfactant used in the polypeptide solution was BYK Chemie (Warlin, Conn.). Ford) contained a mixture of a polymeric surfactant sold under the trademark DISPERPLAST (Lot No. 31J022) and the same concentration of dodecyl maltoside as defined in Example 88. The final concentration of the polymeric surfactant was diluted to 0.135% (v / v) of the feed concentration in the final solution.

(Examples 98-102)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in each of Examples 84-88, one of which was 5% (v / v) Protolyte® A700 (Lot No. LC-29) manufactured by Dais Analytic (Odessa, Florida) in a solvent supplied diluted to 50% (v / v) with ethanol containing water. 10% (w / v) sulfonated styrene / ethylene-butylene / sulfonated styrene supplied as / 60-011), the other 5% (w / v) with an average molecular weight of 2 kD-5 kD-2 kD v) poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline), a first block copolymer versus a second block copolymer A mixture of two block copolymers with a ratio of about 67% to 33% of the total polymer used. When 6 μL of the solution is mixed with sufficient polypeptide solution as described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.060% compared to the synthetic polymeric material. Final solutions containing (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide were produced.

(Examples 103 to 107)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in each of Examples 84-88, one of which was 5% (v / v) Protolyte® A700 (Lot No. LC-29) manufactured by Dais Analytic (Odessa, Florida) in a solvent supplied diluted to 50% (v / v) with ethanol containing water. 10% (w / v) sulfonated styrene / ethylene-butylene / sulfonated styrene supplied as / 60-011), the other 5% (w / v) with an average molecular weight of 2 kD-5 kD-2 kD v) poly (2-methyloxazoline) -polydimethylsiloxane-poly (2-methyloxazoline), a first block copolymer versus a second block copolymer A mixture of two block copolymers with a ratio of about 33% to 67% of the total polymer used. When 6 μL of the solution is mixed with sufficient polypeptide solution as described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.060% compared to the synthetic polymeric material. Final solutions containing (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide were produced.

(Examples 108 to 112)
While solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 103-107, one of the synthetic polymeric materials used is 5% (v / v) Protolyte® A700 (Lot No. LC-29) manufactured by Dais Analytic (Odessa, Florida) in a solvent supplied diluted to 50% (v / v) with ethanol containing water. / 60-011) 10% (w / v) sulfonated styrene / ethylene-butylene / sulfonated styrene, the other being 50% (v / v) THF, 50% (v / v) 5% (w / v) polymethylmethacrylate-polydimethylsiloxane-polymethylmeta having an average molecular weight of 4 kD-8 kD-4 kD in the solvent of dichloromethane of v) It may be a mixture of two block copolymers that are acrylate and the ratio of the first block copolymer to the second block copolymer is about 67% to 33% of the total polymer used. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. A final solution containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 113 to 117)
While solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 103-107, one of the synthetic polymeric materials used is 5% (v / v) Protolyte® A700 (Lot No. LC-29) manufactured by Dais Analytic (Odessa, Florida) in a solvent supplied diluted to 50% (v / v) with ethanol containing water. / 60-011) 10% (w / v) sulfonated styrene / ethylene-butylene / sulfonated styrene, the other being 50% (v / v) THF, 50% (v / v) 5% (w / v) polymethylmethacrylate-polydimethylsiloxane-polymethylmeta having an average molecular weight of 4 kD-8 kD-4 kD in the solvent of dichloromethane of v) It may be a mixture of two block copolymers that are acrylate and the ratio of the first block copolymer to the second block copolymer is about 33% to 67% of the total polymer used. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. A final solution containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 118 to 122)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in each of Examples 84-88, but the synthetic polymeric material used was an 85% triblock copolymer solution and 5% (v / v) mixed with 23.5% (w / v) polyethylene glycol solution having an average molecular weight of about 3,300 Daltons in water at a ratio of 15% polyethylene glycol solution (v / v) ) Protolyte (R) A700 (lot number LC) from Dais Analytic (Odessa, Florida) in a solvent supplied diluted 50% (v / v) with ethanol containing water It was a mixture of 10% (w / v) sulfonated styrene / ethylene-butylene / sulfonated styrene supplied as -29 / 60-011. When 6 μL of the solution was mixed with sufficient polypeptide solution of the type described in Example 1, each 0.0075, 0.015, 0.030, 0.045 and 0 compared to the synthetic polymeric material. Final solutions containing 0.060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide were produced.

(Examples 123-127)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in each of Examples 84-88, but the synthetic polymeric material used was an 85% triblock copolymer solution and 5% (v / v) mixed with 23.5% (w / v) polyethylene glycol solution having an average molecular weight of about 8,000 daltons in water at a ratio of 15% polyethylene glycol solution (v / v) ) Protolyte (R) A700 (lot number LC) from Dais Analytic (Odessa, Florida) in a solvent supplied diluted 50% (v / v) with ethanol containing water It was a mixture of 10% (w / v) sulfonated styrene / ethylene-butylene / sulfonated styrene supplied as -29 / 60-011. When 6 μL of the solution was mixed with sufficient polypeptide solution of the type described in Example 1, each 0.0075, 0.015, 0.030, 0.045 and 0 compared to the synthetic polymeric material. Final solutions containing 0.060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide were produced.

(Examples 128 to 132)
Solutions useful for producing a biocompatible membrane according to the present invention can generally be prepared as described in each of Examples 6-10, but the synthetic polymeric material used is 50% (v / v) THF. , 5% (w / v) polymethyl methacrylate-polydimethylsiloxane-polymethyl methacrylate having an average molecular weight of 4 kD-8 kD-4 kD in a solvent of 50% (v / v) dichloromethane. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, each is 0.0075, 0.015, 0.030, 0.045 and 0.005 compared to the synthetic polymeric material. A final solution containing 060% (w / v) dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% (w / w) polypeptide is produced.

(Examples 133-134)
Solutions useful for producing biocompatible membranes according to the present invention were generally prepared as described in each of Examples 6 and 7, but the synthetic polymeric material used was 50% / 50% of acetone and hexane. (V / v) with 3.2% (w / v) polystyrene-polybutadiene-polystyrene supplied as Styrolux® 3G55 (lot number 74553064P) manufactured by BASF (Ludwigshafen, West Germany) in the mixture. there were. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, 0.0075% and 0.015% (w / v) dodecyl maltoside and 0. Final solutions containing 75% and 1.5% (w / w) polypeptide were produced.

(Examples 135-136)
Solutions useful for producing biocompatible membranes according to the present invention were prepared generally as described in each of Examples 6 and 7, but the synthetic polymeric material used was 50% / 50% of acetone and heptane. (V / v) with 3.2% (w / v) polystyrene-polybutadiene-polystyrene supplied as Styrolux® 3G55 (Lot No. 7453064P) from BASF (Ludwigshafen, West Germany) in the mixture. there were. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, 0.0075% and 0.015% (w / v) dodecyl maltoside and 0. Final solutions containing 75% and 1.5% (w / w) polypeptide were produced.

(Examples 137 to 138)
Solutions useful for producing biocompatible membranes according to the present invention were generally prepared as described in each of Examples 135 and 136, but the synthetic polymeric materials used were 50% / 50% of acetone and heptane. (V / v) 5% (w / v) polystyrene-polybutadiene-polystyrene supplied as Stryolux 3G55 (Lot No. 7453064P) manufactured by BASF (Ludwigshafen, West Germany) in the mixture. When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, 0.0075% and 0.015% (w / v) dodecyl maltoside and 0. Final solutions containing 75% and 1.5% (w / w) polypeptide were produced.

(Examples 139 to 141)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 6-8, although the synthetic polymeric materials used are each about 80% (v / v) Strylux® 3G55 (lot number) manufactured by BASF (Ludwigshafen, West Germany) in a mixture of acetone and hexane at a ratio of 20% (v / v) to 50% / 50% (v / v) 5% (w / v) polystyrene-polybutadiene-polystyrene supplied as 74553064P) and 5% (w / v)) poly (2-methyl) having an average molecular weight of 2 kD-5 kD-2 kD in the same solvent It may be a mixture of (oxazoline) -polydimethylsiloxane-poly (2-methyloxazoline). When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, 0.0075%, 0.015, and 0.030% (w / v) dodecyl compared to the synthetic polymeric material. Final solutions containing maltoside and 0.75%, 1.5 and 3.0% (w / w) polypeptide are produced.

(Examples 142-145)
Solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 139-141, but the synthetic polymeric materials used are each about 80% (v / v) Strylux® 3G55 (lot number) manufactured by BASF (Ludwigshafen, West Germany) in a mixture of acetone and hexane at a ratio of 20% (v / v) to 50% / 50% (v / v) 5% (w / v) polystyrene-polybutadiene-polystyrene supplied as 74553064P) and 5% (w / v) poly (2-methyloxazoline) having an average molecular weight of 3 kD-7 kD-3 kD in the same solvent ) -Polydimethylsiloxane-poly (2-methyloxazoline). When 6 μL of the solution is mixed with sufficient polypeptide solution of the type described in Example 1, 0.0075%, 0.015, and 0.030% (w / v) dodecyl compared to the synthetic polymeric material. Final solutions containing maltoside and 0.75%, 1.5 and 3.0% (w / w) polypeptide are produced.

(Examples 146 to 290)
While solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 1-145, polypeptide solutions mixed with synthetic polymers can be obtained from Roche (Indiana). In 10 mg / mL succinic acid: ubiquinone oxidoreductase (complex II) solution, which may also contain 0.15% Thesit (polyoxyethylene (9) dodecyl ether, C ₁₂ E ₉ ) available from Indianapolis, State of California It may be. This surfactant is generally substituted at a similar concentration to the dodecyl maltoside of Examples 1-145.

(Examples 291 to 435)
While solutions useful for producing biocompatible membranes according to the present invention can generally be prepared as described in each of Examples 1-145, the polypeptide solution used to dilute the synthetic polymer is 0 It may be a 10 mg / mL nicotinamide nucleotide transhydrogenase aqueous solution that may also contain 15% Triton X-100. This surfactant is generally substituted at a similar concentration to the dodecyl maltoside of Examples 1-145. Furthermore, in Examples 1-145 containing dodecyl β-D-glucopyranoside, this surfactant can generally be replaced at a similar concentration as Nonidet P-45.

(Example 436)
A film is formed on the dielectric perforated support. The support is made from KAPTON (1 mil thick) available from DuPont and laser drilled with an aperture of 100 μm diameter and 1 mil depth. The array of apertures may have a high density of 1,700 apertures / cm ² . The biocompatible membrane is formed across the aperture using the PEG8000 / PROTOLYTE A700 membrane previously described in detail. The final solution containing the resulting block copolymer, stabilizing polymer and polypeptide is deposited on the substrate in a manner that completely coats the aperture by a drop method with a pipette 4 μl at a time. The solvent was left to evaporate under a room temperature hood. The membrane-support assembly was stored in a vacuum chamber until use.

  In this case, the test device which is a fuel cell was made of DELRAN plastic. The membrane-support assembly produced as described above was sealed in place in the fuel cell using rubber gaskets to form two chambers, an anode compartment and a cathode compartment. The anode and cathode compartments were then filled with aqueous electrolyte (20 mL each) (100 mM TMA- containing 1 M TMA-formate (pH 10) in the anode compartment and 1% hydrogen peroxide in the cathode compartment). Sulfate (pH 2.0)). The titanium foil anode was connected in parallel with an electronically varied load. Current and output voltage were measured using a computer equipped with an analog / digital board. The circuit was completed by wiring these elements to the graphite cathode electrode in the cathode compartment.

A titanium foil anode was immersed in the anolyte. Furthermore, the anode compartment contains 5% (v / v) methanol as fuel, 12.5 mM NAD ⁺ is used as the electron carrier, 1M hydroquinone is used as the electron transfer mediator, Yeast alcohol dehydrogenase (5,000 units), aldehyde dehydrogenase (10 units) and formate dehydrogenase (100 units) were used as soluble enzymes. Current and voltage were generated consistent with the function of complex I incorporated within the biocompatible membrane in transferring protons from the anode compartment to the cathode compartment even against the proton concentration gradient. The peak current density was 158 mA / cm ² . The membrane was stable for about 3 days.

  In comparison, the peak current density was similar for another battery formed using the same components and concentrations as described above, except that the film-forming solution did not contain PEG 8000. However, membrane integrity was limited to 10-12 hours. Membrane failure was assessed by the visible flow of mediator into the cathode compartment.

Even in the absence of Complex I in the membrane, the Protolyte block copolymer still forms a slightly impervious membrane for protons. Of such a membrane formed without the use of Complex I in a fuel cell constructed similar to the above except that 300 mM PMS was present as an electron transfer mediator in place of hydroxyquinone in the anode. Use only generated up to 4 mM / cm ² for approximately approximately 5 minutes before the output dropped rapidly.

  Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Thus, it will be understood that numerous modifications may be made to the exemplary embodiments and other arrangements may be devised without departing from the spirit and scope of the invention as defined by the appended claims. It must be.

  The present invention relates to industries that make and use membranes, including without limitation, the fuel cell industry. The present invention can also be applied to industries using fuel cells such as the automobile industry and the electronics industry.

1 is a schematic view showing a fuel cell according to the present invention. FIG. 3 is a schematic diagram showing electron and proton transfer in the anode compartment of a fuel cell in an embodiment of the present invention. It is a schematic diagram which shows the anode and cathode which are arrange | positioned at the both sides of a dielectric barrier. FIG. 3 is a cross-sectional view showing the arrangement of a dielectric barrier, an anode, a cathode and a biocompatible membrane according to the present invention. Fig. 3b is a schematic diagram showing a fuel cell including the barrier, anode, cathode and membrane of Fig. 3b. It is a schematic diagram which shows 2nd Embodiment which has arrange | positioned the film | membrane which concerns on this invention in the perforation part contained in a dielectric substrate. It is a schematic diagram which shows 2nd Embodiment which has arrange | positioned the film | membrane which concerns on this invention in the perforation part contained in a dielectric substrate. FIG. 6 is a cross-sectional view of an aperture having a chamfered edge and a biocompatible membrane. FIG. 6 is a cross-sectional view of an aperture having a chamfered edge and a biocompatible membrane. FIG. 6 is a cross-sectional view of an aperture having a chamfered edge and a biocompatible membrane.

Claims (58)

  A biocompatible membrane comprising at least one layer of synthetic polymeric material having a first side and a second side and at least one polypeptide associated therewith, wherein the polypeptide participates in a chemical reaction Or participate in the transport of molecules, atoms, protons or electrons from the first side of the at least one layer to the second side of the at least one layer, or facilitate such a reaction or transport A biocompatible membrane capable of participating in the formation of a molecular structure, wherein the synthetic polymeric material comprises at least one block copolymer and optionally at least one additive.   The biocompatible membrane of claim 1, wherein the at least one polypeptide is capable of participating in proton transport from the first side of the at least one layer to the second side of the at least one layer.   The at least one polypeptide can participate in proton transport from the first side of the at least one layer to the second side of the at least one layer, and is identical except for the polypeptide 3. The biocompatible membrane of claim 2 that can facilitate the passage of current through the layer to a degree that is at least greater than when a biocompatible membrane is used. Protons from the first side of the at least one layer to the second side of the at least one layer so that the at least one polypeptide can provide a current density of at least about 10 pA (picoamperes) / cm ^2. The biocompatible membrane according to claim 3, which can participate in the transport of the blood. Protons from the first side of the at least one layer to the second side of the at least one layer so that the at least one polypeptide can provide a current density of at least about 10 mA (milliamperes) / cm ^2. The biocompatible membrane according to claim 4, which can participate in the transport of the blood.   The biocompatible membrane of claim 1, wherein said at least one polypeptide is incorporated within said at least one layer.   The biocompatible membrane of claim 1, wherein said at least one polypeptide is present in an amount of at least about 0.01% by weight of said biocompatible membrane.   8. The biocompatible membrane of claim 7, wherein the at least one polypeptide is present in an amount of at least about 5% by weight of the biocompatible membrane.   9. The biocompatible membrane of claim 8, wherein the at least one polypeptide is present in an amount of at least about 10% by weight of the biocompatible membrane.   The biocompatible membrane of claim 1, wherein the at least one block copolymer has a hydrophobic content that exceeds the hydrophilic content.   The biocompatible membrane of claim 1, wherein the at least one block copolymer has at least one block having an average molecular weight of about 1,000 to 15,000 daltons.   12. The biocompatible membrane of claim 11, wherein the at least one block copolymer has at least one second block having an average molecular weight of about 1,000 to 20,000 daltons.   The biocompatible membrane of claim 1, wherein the at least one block copolymer is provided in an amount of at least about 50 significant percent of the biocompatible membrane.   14. The biocompatible membrane of claim 13, wherein the at least one block copolymer is provided in an amount of about 50 to about 99% by weight of the biocompatible membrane.   The biocompatible membrane of claim 1, wherein the synthetic polymeric material is a mixture of a plurality of block copolymers.   The biocompatible membrane of claim 1, wherein said at least one polypeptide is capable of participating in a redox reaction.   The at least one polypeptide participates in a redox reaction or transports molecules, atoms, protons or electrons from the first side of the at least one layer to the second side of the at least one layer; The biocompatible membrane of claim 1 having the ability to participate.   The at least one polypeptide participates in a redox reaction and both transports molecules, atoms, protons or electrons from the first side of the at least one layer to the second side of the at least one layer. 18. The biocompatible membrane of claim 17 having the ability to participate in. A biocompatible membrane comprising at least one layer of a synthetic polymeric material having a first side and a second side, wherein the synthetic polymeric material is in an amount of at least about 5% by weight, based on the weight of the final membrane. Wherein at least one polypeptide incorporated therein is present in an amount of at least about 10% by weight of the biocompatible membrane, wherein the polypeptide is in a redox reaction or the at least one polypeptide. Having the ability to participate in proton transport from the first side of the layer to the second side of the at least one layer and providing a current density of at least about 10 mA / cm ² , A biocompatible membrane wherein the material comprises at least one block copolymer and optionally at least one additive.   Synthetic polymer material comprising an anode compartment comprising an anode, a cathode compartment comprising a cathode, and within the anode compartment, within the cathode compartment, or between the anode compartment and the cathode compartment and comprising an anode side and a cathode side And at least one biocompatible membrane having at least one polypeptide associated therewith, wherein said polypeptide is involved in a chemical reaction or said at least one Involved in the transport of protons from the first anode side of the layer to the first cathode side of the at least one layer, or involved in the formation of a molecular structure that facilitates such a reaction or transport; The molecular material comprises at least one block copolymer and optionally at least one additive A fuel cell that. 21. The fuel cell according to claim 20, wherein the anode and the cathode are electrically connected through a circuit, and 10 mW (milliwatt) / cm ² is generated. The fuel cell according to claim 21, wherein the anode and the cathode are electrically connected through a circuit, and 50 mW / cm ² is generated. 23. The fuel cell according to claim 22, wherein the anode and the cathode are electrically connected via a circuit, and 100 mW / cm < ² > is generated.   21. The fuel cell of claim 20, further comprising at least one electron mediator in the anode compartment.   21. The fuel cell of claim 20, further comprising at least one second polypeptide capable of releasing protons or electrons from the electron carrier in the anode compartment.   21. The fuel cell of claim 20, further comprising at least one transmission mediator capable of transmitting electrons to the anode within the anode compartment.   21. The fuel cell of claim 20, wherein the at least one biocompatible membrane is disposed between the anode and the cathode.   21. The fuel cell of claim 20, further comprising a dielectric material disposed between the anode and the cathode and allowing proton flow from the anode compartment to the cathode compartment.   A first electron transfer mediator disposed in the anode compartment receives at least one electron from an electron carrier disposed in the anode compartment, and receives the at least one electron from a second electron carrier, 21. The fuel cell of claim 20, wherein the fuel cell can be transmitted to two electron transfer mediators or to the anode.   21. The fuel cell of claim 20, further comprising at least one fuel disposed within the anode compartment.   21. The fuel cell assembly of claim 20, wherein the fuel is an organic compound.   21. The fuel cell of claim 20, wherein the polypeptide has the ability to participate in a redox reaction or proton transport from the anode side of the at least one layer to the cathode side of the at least one layer.   35. The fuel cell of claim 32, wherein the polypeptide has the ability to participate in redox reactions and proton transport from the first anode side of the at least one layer to the cathode side of the at least one layer.   A solution useful for producing a biocompatible membrane comprising at least one synthetic polymer material comprising at least one block copolymer and optionally at least one additive, comprising an organic solvent and water In a solvent system containing both, the synthetic polymeric material is present in an amount of about 1-30% (w / v) and at least one polypeptide is at least about 0.001 to about 10.0% ( present in the amount of w / v) to form a molecular structure in which the polypeptide is involved in a chemical reaction, is involved in the transport of molecules, atoms, protons or electrons, or facilitates such a reaction or transport. Solutions that can be involved.   35. The solution of claim 34, further comprising at least one detergent in an amount of about 0.01 to about 1.0% (v / v).   35. The solution of claim 34, wherein said at least one polypeptide has the ability to participate in redox reactions or proton transport.   38. The solution of claim 36, wherein the at least one polypeptide has the ability to participate in both redox reactions and proton transport.   35. The solution of claim 34, wherein said at least one polypeptide has the ability to participate in proton transport.   35. The solution of claim 34, wherein the synthetic polymeric material is a mixture of a plurality of block copolymers.   An anode compartment comprising an anode; a cathode compartment comprising a cathode; and a composite height disposed within said anode compartment, within said cathode compartment, or between said anode compartment and said cathode compartment, comprising an anode side and a cathode side A fuel cell comprising at least one layer of molecular material and at least one biocompatible membrane having at least one polypeptide associated therewith, wherein said polypeptide participates in a chemical reaction or said at least one A fuel cell capable of participating in the transport of protons from the anode side of one layer to the cathode side of the at least one layer or in the formation of a molecular structure that facilitates such a reaction or transport . 41. The fuel cell according to claim 40, wherein the anode and the cathode are electrically connected via a circuit to generate 10 mW / cm < ² >. 42. The fuel cell according to claim 41, wherein the anode and the cathode are electrically connected via a circuit to generate 50 mW / cm < ² >. 43. The fuel cell according to claim 42, wherein the anode and the cathode are electrically connected via a circuit to generate 100 mW / cm < ² >.   41. The fuel cell of claim 40, further comprising at least one electron carrier in the anode compartment.   41. The fuel cell of claim 40, further comprising at least one second polypeptide capable of releasing protons or electrons from the electron carrier in the anode compartment.   42. The fuel cell of claim 40, further comprising at least one transfer mediator capable of transferring electrons to the anode within the anode compartment.   41. The fuel cell of claim 40, wherein the at least one biocompatible membrane is disposed between the anode and the cathode.   41. The fuel cell of claim 40, further comprising a dielectric material disposed between the anode and the cathode and allowing proton flow from the anode compartment to the cathode compartment.   A second electron carrier, a second electron transfer mediator that is disposed in the anode compartment, receives at least one electron from an electron carrier disposed in the anode compartment, and receives the at least one electron as a second electron carrier; 41. The fuel cell of claim 40, further comprising a first electron transfer mediator capable of being transmitted to the anode.   41. The fuel cell of claim 40, further comprising at least one fuel disposed within the anode compartment.   51. The fuel cell according to claim 50, wherein the fuel is an organic compound.   41. The fuel cell of claim 40, wherein the polypeptide has the ability to participate in a redox reaction or proton transport from the anode side of the at least one layer to the cathode side of the at least one layer.   53. The fuel cell of claim 52, wherein the polypeptide has the ability to participate in both a redox reaction and proton transport from the anode side of the at least one layer to the cathode side of the at least one layer. .   41. The fuel cell of claim 40, wherein the synthetic polymeric material is at least one polymer, copolymer, block copolymer, or a mixture thereof.   41. The fuel cell of claim 40, wherein the pH in the anode compartment is at least about 0.5 pH higher than the pH in the cathode compartment.   56. The fuel cell of claim 55, wherein the pH in the anode compartment is at least about 1.0 pH higher than the pH in the cathode compartment.   57. The fuel cell of claim 56, wherein the pH in the anode compartment is at least about 2.0 pH higher than the pH in the cathode compartment.   58. The fuel cell of claim 57, wherein the pH in the anode compartment is about 8 or higher and the pH in the cathode compartment is about 5 or lower.

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