Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
BIOCOMPATIBLE MEMBRANES OF BLOCK COPOLYMERS
AND FUEL CELLS PRODUCED THEREWITH
BACKGROUND ART
In one series of articles by Meier et al., various
constructions were proposed for polymer based membranes, which
included functional proteins. While such membranes had been the
subject of speculation in the past, this is believed to be the
first successful biological protein containing polymer based
membrane that included an imbedded enzyme that retained its
functionality. See 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
a functionalized poly(2-methyloxazoline)-block-poly
(dimethylsiloxane)-block-poly(2-methyloxazoline) triblock
copolymer in which a protein (a "porin" -- a non-selective,
passive pore-forming molecule) is embedded.
The Meier et a1. work is unique and limited in scope. It
does not broadly discuss the use of polymers, nor suggest that
even the polymer membrane disclosed could be used in conjunction
with even other enzymes. Certainly nothing in these articles
suggests the possibility of creating a synthetic membrane
containing an embedded biological species capable of
participating in oxidation or reduction, or "polypeptide mediated
transporting of active molecules, atoms, protons or electrons
across the membrane." Indeed, the narrowness of the disclosure
and the lack of other successes offer little reason for optimism
that other biological materials could be successfully embedded
into polymer membranes.
The creation of membranes for the study of membrane
associated proteins has long been known. See Functional Assembly
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of Membrane Proteins in Planar Lipid Bilayers, 14 Quart. Rev.
Biophys. 1-79 (1981). Indeed, transmembrane and redox proteins
were embedded in biologically based membranes, e.g., membranes
produced of molecules found in living cells or organisms, for
purposes of studying their structure and mechanism. The use of
lipid bilayers containing embedded enzyme complexes such as NADH
dehydrogenase from E. coli, which can transport protons across
the membrane and/or participate in redox reactions has also been
described. See Liberatore et al., U.S. Patent Application
Publication No. US 2002/0001739 A1, published January 3, 2002.
Indeed, Liberatore et al. described the use of such membranes as
part of a battery.
Neither the existence of biological membranes containing
enzyme complexes nor the discovery of a single combination of a
polymer membrane and a specific enzyme offers much hope for the
development of a broad class of synthetic, biocompatible polymer
membranes, which are stable and functional. See also G. Tayhas
et al. "A Methanol/Dioxogen Biofuel Cell That Uses NAD+ Dependent
Dehydrogenases as Catalysts: Application of an Electro-Enzymatic
Method to Regenerate Nicotinamide Adenine Dinucleotide at Low
Overpotentials," 43 J. Electroanalytical Chem. 155-161 (1998).
SUMMARY OF THE INVENTION
The present invention relates to a biocompatible membrane
including at least one layer of a synthetic polymer material
having a first side and a second side. The biocompatible
membrane includes at least one polypeptide associated therewith.
In a preferred aspect, the present invention relates to a
biocompatible membrane wherein the polypeptide is capable of
participating in a chemical reaction, participating in the
transporting of molecules, atoms, protons or electrons from the
first side of the at least one layer to the second side of the
same layer or participating in the formation of molecular
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structures that facilitate such reactions or transport. In an
even more particularly preferred aspect of the invention, the
polypeptide is a redox enzyme and/or is an enzyme capable of
participating in the transmembrane transport of protons. Indeed,
the polypeptide may have both the ability to cause the liberation
of electrons and to participate in transporting protons across
the membrane. When discussing transporting protons across a
biocompatible membrane, it will be appreciated that neither the
exact mechanism, nor the exact species transferred is known. The
transferred species might be a proton per se, a positively
charged hydrogen, a hydronium ion, H30+ or indeed some other
charged species. For convenience, however, these will be
collectively discussed herein as "protons."
Any synthetic polymer material which is a block copolymer,
copolymer or polymer or mixtures thereof may be used in
accordance with the present invention so long as they are capable
of forming biocompatible membrane. In one preferred aspect of
the present invention, the synthetic polymer material consists
exclusively of at least one block copolymer. Mixtures of block
copolymers are also contemplated. Optionally, the synthetic
polymer material will include at least one additive. In a second
preferred embodiment in accordance with the present invention,
the synthetic polymer material includes at least one polymer,
copolymer or block copolymer. However, if a block copolymer is
present, the synthetic polymer material also includes at least
one polymer or copolymer. An optional additive is also
contemplated. In another preferred embodiment of the present
invention, the synthetic polymer material can be any polymer
material capable of forming a biocompatible membrane and includes
in addition thereto at least one stabilizing polymer.
In another aspect of the present invention, the synthetic
polymer material is a block copolymer, a mixture of block
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copolymers or a mixture of one or more block copolymers and a
hydrogen bonding rich stabilizing polymer. Preferably, the
polypeptide is embedded in the synthetic polymer material so as
to form a biocompatible membrane.
"Biocompatible membrane" as used herein is one or more
layers of a synthetic polymeric material forming a sheet, plug or
other structure that can be used as a membrane and is associated
with a polypeptide or other molecule, often of biological origin.
By "biocompatible, " it is meant that the membrane itself is made
of synthetic polymer materials that will not incapacitate or
otherwise block all of the functionality of a polypeptide when
they are associated with one another. A "membrane" as used herein
is a structure such as a sheet, layer or plug of a material that
includes, at least as its major structural component, synthetic
polymer materials and can be used to selectively segregate space,
fluids (liquids or gases), solids and the like. A membrane as
used herein may include permeable materials that allow the
passage or diffusion of some species from one side to the other.
A membrane used in a fuel cell, for example, prevents the passage
of some components from within a cathode compartment into the
anode compartment and/or prevents some components within the
anode compartment from passing into the cathode compartment.
Other components, however, may pass freely. At the same time, as
exemplified in one embodiment of a membrane in accordance with
the present invention, it will permit and indeed facilitate the
passage of protons from the anode compartment to the cathode
compartment.
"Associated" in accordance with the present invention can
mean a number of things depending on the circumstances. A
polypeptide can be associated with a biocompatible membrane by
being bound to one or more of the surfaces thereof, and/or by
being wedged or bound within one or more of the surfaces of the
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membrane (such as in recesses or pores). The "associated"
polypeptide can be disposed within the interior of the membrane
or in a vesicle or lumen contained within the membrane.
Polypeptides could also be disposed between successive layers.
5 Polypeptides may be embedded in the membrane as well. Indeed, in
a particularly preferred embodiment, the polypeptide is embedded
or integrated in the membrane in such a way so that it is at
least partially exposed through at least one surface of the
membrane and/or can participate in a redox reaction or in the
polypeptide mediated transporting of a molecule, atom, proton or
electron from one side of the membrane to the other.
The term "participate" in the context of transporting a
molecule, atom, proton or electron, from one side of the membrane
to the other includes active transport where, for example, the
polypeptide physically or chemically "pumps" the molecule, atom,
proton or electron across the membrane, usually, but not
exclusively, against a pH, concentration or charge gradient or
any other active transport mechanism. However, participation
need not be so limited. The mere presence of the polypeptide in
the membrane may alter the structure or properties of the
membrane sufficiently to allow a proton, for example, to be
transported from a relatively high proton concentration to a
relatively low proton concentration on the other side of the
membrane. This is not exclusively a passive, non-selective
process such as might result from the use of non-selective,
passive pore formers or from simple diffusion. Indeed, in some
cases, inactivation of the polypeptides in a membrane provides
results that are inferior to similar membranes made without
polypeptides at all. These processes (excluding passive
diffusion) are collectively referred to as "polypeptide mediated
transport" where the presence of the polypeptide plays a role in
the transporting of a species across the membrane, in ways other
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than merely structurally providing a static channel. Stated
another way, "polypeptide mediated transport" means that the
presence of the polypeptide results in effective transport from
one side of the membrane to the other in response to something
other than just concentration. "Participate," in the context of
a redox reaction, means that the polypeptide causes or
facilitates the oxidation and/or reduction of a species, or
conveys to or from that reaction protons, electrons or oxidized
or reduced species.
"Polypeptide(s)" includes at least one molecule composed of
four or more amino acids that is capable of participating in a
chemical reaction, often as a catalyst, or participating in the
transporting of a molecule, atom, proton or electron from one
side of a membrane to another, or participating in the formation
of molecular structures that facilitate or enable such reactions
or transport. The polypeptide can be single stranded, multiple
stranded, can exist in a single subunit or multiple subunits. It
can be made up of exclusively amino acids or combinations of
amino acids and other molecules. This can include, for example,
pegalated peptides, peptide nucleic acids, peptide mimetics,
neucleoprotein complexes. Strands of amino acids that include
such modifications as glycosolation are also contemplated.
Polypeptides in accordance with the present invention are
generally biological molecules or derivatives or conjugates of
biological molecules. Polypeptides can therefore include
molecules that can be isolated, as well as molecules that can be
produced by recombinant technology or which must be, in whole or
in part, chemically synthesized. The term therefore encompasses
naturally occurring proteins and enzymes, mutants of same,
derivatives and conjugates of same, as well as wholly synthetic
amino acid sequences and derivatives and conjugates thereof. In
one preferred embodiment, polypeptides in accordance with the
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present invention can participate in the transporting of
molecules, atoms, protons and/or electrons from one side of a
membrane to another side thereof, can participate in oxidation or
reduction, or are charge driven proton pumping polypeptides such
as DH- Complex I (also referred to as "Complex 1").
The present invention stems from the recognition that it is
possible to create biocompatible membranes using a wide range of
synthetic polymer materials and polypeptides. Biocompatible
membranes, when produced in accordance with the present
invention, can have advantages over their completely biological
counterparts in that they can be more stable, longer lived, more
durable and able to be placed in a wider range of useful
environments. Indeed, some of the biocompatible membranes of the
invention can operate when in contact with solutions having very
different and very extreme pHs on either side. They can also be
formulated to be stable in the presence of certain oxidizing
and/or reducing agents and useful at relative extremes of
temperature or other operating and storage conditions. They will
facilitate the passage of current to a degree at least greater
than that which would occur using the identical membrane without
a polypeptide. Preferably, the biocompatible membranes of the
present invention will provide at least about 10 picoamps/cm~
(such as when the biocompatible membrane is used in a sensor)
more preferably at least about 10 milliamps/cm2 and even more
preferably about 100 milliamps/cm2 or more.
These biocompatible membranes are also generally, but not
exclusively, free-standing as a membrane in air and thus can be
at least partially desolvated. When used in a fuel cell, these
biocompatible membranes will have a useful operating life of,
preferably, at least 8 hours, more preferably, at least 3 days,
and even more preferably one month or more, and still more
preferably, six months or more.
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Synthetic polymer membranes that are biocompatible and
contain polypeptides capable of participating in a redox reaction
and/or participating in the transport of a molecule, atom, proton
or electron from one side of the membrane to the other are
particularly advantageous because they can be used in the
creation of a wide range of batteries or fuel cells. These
include batteries that are environmentally friendly, light,
compact and easily transportable. It is also possible to produce
fuel cells that are very high in terms of power output.
Preferably, a fuel cell produced in accordance with the present
invention can generate at least 10 milliwatts/cm2, preferably at
least about 50 milliwatts/cmz and most preferably at least about
100 milliwatts/cm2 when a circuit, usually with a load or
resistance, is created between the anode and cathode. This is
also referred to as being in electrical contact.
Accordingly, 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, which can be
disposed within the anode compartment, within the cathode
compartment or between the anode and cathode compartments. The
biocompatible membrane, as previously discussed, can include at
least one layer of a synthetic polymer material and at least one
polypeptide associated therewith. Preferably, the polypeptide
has the ability to participate in a redox reaction and/or to
participate in the transporting of molecules, atoms, protons or
electrons from one side of the membrane to the other. In a
particularly preferred embodiment, the polypeptide can
participate in both a redox reaction and in transportation of a
molecule, atom, proton or electron. Such a fuel cell may also
include an electron carrier and a second polypeptide, both of
which are disposed within the anode compartment.
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Another aspect of the present invention is the creation of
solutions which are themselves useful for producing biocompatible
membranes in accordance with the present invention. The
solutions include at least one synthetic polymer material and at
least one polypeptide in a solvent system which often includes
both organic' solvents and water. Preferable, the synthetic
polymer materials is present in an amount of between about 1 and
about 30% w/v, and more preferably, and between about 2 and
about 20% w/v and most preferably between about 2 and about
10% w/v. Similarly, the polypeptide is present in the solution
in an amount of between about 0.001 and about 10.0% w/v, more
preferably between about 0.01 and about 7.0% w/v, and even more
preferably between about 0.1 and about 5.0~ w/v. The solution
may also include a solubilizing detergent additives and other
materials as desirable. The synthetic polymer material, in a
preferred embodiment, consists of at least one block copolymer.
In another preferred embodiment, the synthetic polymer material
includes at least one polymer, copolymer or block copolymer with
the proviso that when the synthetic copolymer materials includes
at least one block copolymer, the synthetic polymer material also
includes at least one polymer or copolymer. In another
particularly preferred embodiment, the synthetic polymer material
includes at least one stabilizing polymer.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a fuel cell in accordance with the
present invention.
Figure 2 is a schematic representation of the transfer of
electrons and protons in an anode compartment of a fuel cell in
one embodiment of the present invention.
Figure 3a illustrates an anode and cathode disposed on
opposite sides of a dielectric barrier.
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Figure 3b illustrates, in cross section, an arrangement of a
dielectric barrier, an anode, a cathode and a biocompatible
membrane in accordance with the present invention.
Figure 3c shows schematically a view of a fuel cell
5 including the barrier, anode, cathode and membrane of Figure 3b.
Figure 3d is a second embodiment of a membrane in accordance
with the present invention illustrated schematically as disposed
within perforations contained in a dielectric substrate.
Figure 3e is a second embodiment of a membrane in accordance
10 with the present invention illustrated schematically as disposed
within perforations contained in a dielectric substrate.
Figure 4a is a cross-sectional view of an aperture having a
beveled edge and a biocompatible membrane
Figure 4b is a cross-sectional view of an aperture having a
beveled edge and a biocompatible membrane
Figure 4c is a cross-sectional view of an aperture having a
beveled edge and a biocompatible membrane
BEST MODE OF CARRYING OUT THE INVENTION
Biocompatible membranes in accordance with the present
invention can be formed from any synthetic polymer material that,
when associated with one or more polypeptides as described
herein, meet the objectives of the present invention.
Synthetic polymer materials can include polymers, copolymers
and block copolymers and mixtures of same. These can be bound,
crosslinked, functionalized or otherwise associated with one
another. "Functionalized" means that the polymers, copolymers
and/or block copolymers have been modified with end groups that
are selected to perform a specific function, whether that be
polymerization (crosslinking of blocks, for example), anchoring
to a particular surface chemistry (use of, for example, certain
sulfur linkages), facilitated electron transport via covalently
linking an electron carrier or electron transfer mediator, and
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the like known to, the art. Typically, these end groups are not
considered a constituent of the polymer or block itself and are
often added at the end of or after synthesis. Synthetic polymer
materials are generally present on the finished membrane (the
membrane in condition for use) in an amount of at least about 50%
by weight of the finished membrane, more typically at least about
60% by weight of the finished membrane and often between about 70
and about as much as 99% by weight thereof. A portion of the
total amount of the synthetic polymer material may be a
stabilizing polymer, generally up to about a third, by weight
based on the weight of the total synthetic polymer material in
the finished biocompatible membrane.
The biocompatible membranes of the invention are preferably
produced from one or more block copolymers such as A-B, A-B-A or
A-B-C block copolymers, with or without other synthetic polymer
materials such as polymers or copolymers, and with or without
additives.
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:
O
O N
EGO-nP ~ i Si nPr-O-E~-C
O
x
0~~~
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In the above chemical formula, the average x value is 68,
and the average y value is 15. This is an A-B-A block copolymer
in which the "C" recited in the formula does not necessarily
equate with the "C" designation of an A-B-C block copolymer.
The polymer illustrated above can provide relatively large
membranes that can incorporate functional proteins. The
methacrylate moieties at the ends of the polymer molecules allow
for free-radical mediated crosslinking after incorporating
protein to add greater mechanical stability. Biocompatible
membranes such as this, particularly those that are nonionic,
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 discussed above is one example of a synthetic polymer
material that can be used. Other exemplary block copolymers
include, without limitation: Amphiphilic block copolymers [The
triblock copolymer shells of the vesicles can be regarded as a
mimetic of biological membranes although they are 2 to 3 times
thicker than a conventional lipid bilayer. Nevertheless, they can
serve as a matrix for membrane-integral proteins. Surprisingly,
the proteins remain functional despite the extreme thickness of
the membranes and even after polymerization of the reactive
triblock copolymers.]; Triblock copolyampholytes from 5-(N,N-
dimethylamino)isoprene, styrene, and methacrylic acid [Bieringer
et al., Eur. Phys. J.E. 5:5-12, 2001. Among such polymers are
A114S63A23 ~ A131S23A46 i A~-42S23A35 i A~-56S23A21 i A157S11A32] i Styrene-
ethylene/butylene-styrene triblock copolymer [(KRATON) G 1650, a
29% styrene, 8000 solution viscosity (25 wt-% polymer), 100%
triblock styrene-ethylene/butylene-styrene (S-EB-S) block
copolymer; (KRATON) G 1652, a 29% styrene, 1350 solution
viscosity (25 wt-% polymer), 100% triblock S-EB-S block
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copolymer; (KRATON) G 1657, a 4200 solution viscosity (25 wt-
polymer), 35% diblock S-EB-S block copolymer; all available from
the Shell Chemical Company. The preferred block copolymers are of
the styrene-ethylene/propylene (S-EP) types and are commercially
available under the tradenames (KRATON) G 1726, a 28a styrene,
200 solution viscosity (25 wt-% polymer), 70% diblock S-EB-S
block copolymer; (KRATON) G-1701X a 37% styrene,>50,000 solution
viscosity, 100% diblock S-EP block copolymer; and (KRATON) G-
1702X, a 28a styrene,>50,000 solution viscosity, 100a diblock S-
EP block copolmyer also available from the Shell Chemical
Company, Houston, Texas, USA]; Siloxane triblock copolymer
[Nitrile containing siloxane block copolymers were developed as
stabilizers for siloxane magnetic fluids. The siloxane magnetic
fluids have been recently proposed as internal tamponades for
retinal detachment surgery. PDMS-b-PCPMS-b-PDMSs (PDMS -
polydimethylsiloxane, PCPMS - poly(3-cyanopropylmethyl-
cyclosiloxane) were successfully prepared through kinetically
controlled polymerization of hexamethylcyclotrisiloxane initiated
by lithium silanolate endcapped PCPMS macroinitiators. The
macroinitiators were prepared by equilibrating mixtures of 3-
cyanopropylmethylcyclosiloxanes (DxCN) and dilithium
diphenylsilanediolate (DLDPS). DxCNs were synthesized by
hydrolysis of 3-cyanopropylmethyldichlorosilane, followed by
cyclization and equilibration of the resultant hydrolysates.
DLDPS was prepared by deprotonation of diphenylsilanediol with
diphenylmethyllithium. It was found that mixtures of DxCN and
DLDPS could be equilibrated at 100°C within 5-10 hours. By
controlling the DxCN-to-DLDPS ratio, macroinitiators of different
molecular weights could be obtained. The major cyclics in the
macroinitiator equilibrate 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 were prepared and
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characterized. These triblock copolymers are transparent,
microphase separated and highly viscous liquids. It was found
that these triblock copolymers can stabilize nanometer gamma-
Fe203 and cobalt particles in octamethylcyclotetrasiloxane or
hexane. Hence PDMS-b-PCPMS -b-PDMSs represent a class of
promising steric stabilizers for silicone magnetic fluids.]; DEO-
CPPO-CPEO triblock copolymer; PEO-PDMS-PEO triblock copolymer
[Polyethylene oxide (PEO) is soluble in the aqueous phase, while
the poly-dimethyl siloxane (PDMS) is soluble in oil phase]; PLA-
PEG-PLA triblock copolymer; Poly(styrene-b-butadiene-b-styrene)
triblock copolymer [Commonly used thermoplastic elastomers,
includes Styrolux from BASF, Ludwigshafen, Germany];
Polyethylene oxide)/poly(propylene oxide) triblock copolymer
films [Pluronic F127, Pluronic P105, or Pluronic L44 from BASF,
Ludwigshafen, Germany]; Poly(ethylene glycol)-polypropylene
glycol) triblock copolymer; PDMS-PCPMS-PDMS
(polydimethylsiloxane-polycyanopropylmethylsiloxane) triblock
copolymer [A series of epoxy and vinyl endcapped polysiloxane
triblock copolymers with systematically varied molecular weights
were synthesized via anionic polymerization using LiOH as an
initiator. The nitrile groups on the central copolymer block are
thought to adsorb onto the particle surfaces, while the PDMS
endblocks protrude into the reaction medium.]; Azo-functional
styrene-butadiene-HEMA triblock copolymer, Amphiphilic triblock
copolymer carrying polymerizable end groups; Syndiotactic
polymethylmethacrylate (sPMMA)-polybutadiene (PBD)- sPMMA
triblock copolymer, Tertiary amine methacrylate triblock [AB
diblock copolymer which can form 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 [Same as Pluronic from BASF];
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Poly(isoprene-block-styrene-block-dimethylsiloxane) triblock
copolymer; Polyethylene oxide)-block-polystyrene-block-
poly(ethylene oxide) triblock copolymer; Polyethylene oxide)-
poly(THF)-polyethylene oxide) triblock copolymer; Ethylene oxide
5 triblock; Poly E-caprolactone [Birmingham Polymers]; Poly(DL-
lactide-co-glycolide) [Birmingham Polymers]; Poly(DL-lactide)
[Birmingham Polymers]; Poly(L-lactide) [Birmingham Polymers];
Poly(glycolide) [Birmingham Polymers]; Poly(DL-lactide-co-
caprolactone) [Birmingham Polymers]; Styrene-Isoprene-styrene
10 triblock copolymer [Japan Synthetic Rubber Co., Mw= 140kg/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 membrane. Available as
15 Protolyte A700 from Dais Analytic, Odessa FL]; Poly(1-lactide)-
block-polyethylene oxide)-block-poly(1-lactide) triblock
copolymer; Poly-ester-ester-ester triblock copolymer; PLA/PEO/PLA
triblock copolymer [The synthesis of the triblock copolymers will
be prepared by ring-opening polymerization of DL-lactide or e-
caprolactone in the presence of polyethylene glycol), using non-
toxic Zn metal or calcium hydride as co-initiator instead of the
stannous octoate. The composition of the copolymers will be
varied by adjusting the polyester/polyether ratio.]; PCC/PEO/PCC
triblock copolymer [The above polymers can be used in mixtures of
two or more. For example, in two polymer mixtures measured in
weight percent of the first polymer, such mixtures can comprise
20-25%, 25-30%, 30-350, 35-40%, 40-45% or 45-500.]; Poly(t-butyl
acrylate-b-methyl methacrylate-b-t-butyl acrylate) [Polymer
Source, Inc., Dorval, Quebec, Canada]; Poly(t-butyl acrylate-b-
styrene-b-t-butyl acrylate) [Polymer Source, Inc.]; Poly(t-butyl
methacrylate-b-t-butyl acrylate-b-t-butyl methacrylate) [Polymer
Source, Inc.]; Poly(t-butyl methacrylate-b-methyl methacrylate-b-
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t-butyl methacrylate) [Polymer Source, Inc.]; Poly(t-butyl
methacrylate-b-styrene-b-t-butyl methacrylate) [Polymer Source,
Inc.]; Poly(methyl methacrylate-b-butadiene(1,4 addition)-b-
methyl methacrylate) [Polymer Source, Inc.]; Poly(methyl
methacrylate-b-n-butyl acrylate-b-methyl methacrylate) [Polymer
Source, Inc.]; Poly(methyl methacrylate-b-t-butyl acrylate-b-
methyl methacrylate) [Polymer Source, Inc.]; Poly(methyl
methacrylate-b-t-butyl methacrylate-b-methyl methacrylate)
[Polymer Source, Inc.]; Poly(methyl methacrylate-b-
dimethylsiloxane-b-methyl methacrylate) [Polymer Source, Inc.];
Poly(methyl methacrylate-b-styrene-b-methyl methacrylate)
[Polymer Source, Inc.]; Poly(methyl methacrylate-b-2-vinyl
pyridine-b-methyl methacrylate) [Polymer Source, Inc.];
Poly(butadiene(1,2 addition)-b-styrene-b-butadiene(1,2 addition))
[Polymer Source, Inc.]; Poly(butadiene(1,4 addition)-b-styrene-b-
butadiene(1,4 addition)) [Polymer Source, Inc.]; Poly(ethylene
oxide-b-propylene oxide-b-ethylene oxide) [Polymer Source, Inc.];
Polyethylene oxide-b-styrene-b-ethylene oxide) [Polymer Source,
Inc.]; Poly(lactide-b-ethylene oxide-b-lactide) [Polymer Source,
Inc.]; Poly(lactone-b-ethylene oxide-b-lactone) [Polymer Source,
Inc.]; a, w-Diacrylonyl Terminated poly(lactide-b-ethylene oxide-
b-lactide) [Polymer Source, Inc.]; Poly(styrene-b-acrylic acid-b-
styrene) [Polymer Source, Inc.]; Poly(styrene-b-butadiene (1,4
addition) -b-styrene) [Polymer Source, Inc.]; Poly(styrene-b-
butylene-b-styrene) [Polymer Source, Inc.]; Poly(styrene-b-n-
butyl acrylate-b-styrene) [Polymer Source, Inc.]; Poly(styrene-b-
t-butyl acrylate-b-styrene) [Polymer Source, Inc.]; Poly(styrene-
b-ethyl acrylate-b-styrene) [Polymer Source, Inc.]; Poly(styrene-
b-ethylene-b-styrene) [Polymer Source, Inc.]; Poly(styrene-b-
isoprene-b-styrene) [Polymer Source, Inc.]; Poly(styrene-b-
ethylene oxide-b-styrene) [Polymer Source, Inc.]; Poly(2-vinyl
pyridine-b-t-butyl acrylate-b-2-vinyl pyridine) [Polymer Source,
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Inc.]; Poly(2-vinyl pyridine-b-butadiene(1,2 addition)-b-2-vinyl
pyridine) [Polymer Source, Inc.]; Poly(2-vinyl pyridine-b-
styrene-b-2-vinyl pyridine) [Polymer Source, Inc.]; Poly(4-vinyl
pyridine-b-t-butyl acrylate-b-4-vinyl pyridine) [Polymer Source,
Inc.]; Poly(4-vinyl pyridine-b-methyl methacrylate-b-4-vinyl
pyridine) [Polymer Source, Inc.]; Poly(4-vinyl pyridine-b-
styrene-b-4-vinyl pyridine) [Polymer Source, Inc.];
Poly(butadiene-b-styrene-b-methyl methacrylate) [Polymer Source,
Inc.]; Poly(styrene-b-acrylic acid-b-methyl methacrylate)
[Polymer Source, Inc.]; Poly(styrene-b-butadiene-b-methyl
methacrylate) [Polymer Source, Inc.]; Poly(styrene-b-butadiene-b-
2-vinyl pyridine) [Polymer Source, Inc.]; Poly(styrene-b-
butadiene-b-4-vinyl pyridine) [Polymer Source, Inc.];
Polystyrene-b-t-butyl methacrylate-b-2-vinyl pyridine) [Polymer
Source, Inc.]; Poly(styrene-b-t-butyl methacrylate-b-4-vinyl
pyridine) [Polymer Source, Inc.]; Poly(styrene-b-isoprene-b-
glycidyl methacrylate) [Polymer Source, Inc.]; Poly(styrene-b-a-
methyl styrene-b-t-butyl acrylate) [Polymer Source, Inc.];
Polystyrene-b-a-methyl styrene-b-methyl methacrylate) [Polymer
Source, Inc.]; Poly(styrene-b-2-vinyl pyridine-b-ethylene oxide)
[Polymer Source, Inc.]; Poly(styrene-b-2-vinyl pyridine-b-4-vinyl
pyridine) [Polymer Source, Inc.].
The above block copolymers can be used alone or in mixtures
of two or more in the same or different classes. For example, in
mixtures of two block copolymers measured in weight percent of
the first polymer, such mixtures can comprise 10-15%, 15-20%, 20-
25%, 25-300, 30-350, 35-40%, 40-45% or 45-50%. Where three
polymers are used, the first can comprise 10-15%, 15-20%, 20-25%,
25-30%, 30-35%, 35-40%, 40-45% or 45-50% of the whole of the
polymer components, and the second can 10-150, 15-20%, 20-25%,
25-30%, 30-35%, 35-40%, 40-45% or 45-500 of the remainder.
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Stated another way, the amount of each block copolymer in a
mixture can vary considerably with the nature and number of the
block copolymers used and the desired properties to be obtained.
However, generally, each block copolymer of a mixture in
accordance with the present invention will be present in an
amount of at least about 10% based on weight of total polymers in
the membrane or solution. These same general ranges would apply
to membranes produced from one or more polymers, copolymers
and/or mixtures with block copolymers. There may also be
instances where a single polymer, copolymer or block copolymer
may be "doped" with a small amount of a distinct polymer,
copolymer or block copolymer, even as little as 1.0% by weight of
the membrane to adjust the membrane's specific properties.
Embodiments of the invention include, without limitation, A
B, A-B-A or A-B-C block copolymers. The average molecular weight
for triblock copolymers 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 between about 1,000 to 20,000 Daltons, more preferably,
about 2,000-15,000 Daltons. The average molecular weight of B is
between about 1,000 to 20,000 Daltons, more preferably about
2,000 to 15,000 Daltons.
Preferably, the block copolymer will have a
hydrophobic/hydrophilic balance that is selected to (i) provide a
solid at the anticipated operating and storage temperature and
(ii) promote the formation of biomembrane-like structures rather
than micelles. More preferably, the hydrophobic content (or
block) shall exceed the hydrophilic content (or block). Thus, at
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least one block of the diblock or triblock copolymers is
preferably hydrophobic. While wettable membranes are possible,
preferably the content of hydrophobic and hydrophilic synthetic
polymeric materials will render the membrane sparingly wettable.
As described above, in one preferred embodiment of the
present invention, there is provided a biocompatible membrane
produced using a mixture of synthetic polymer materials. Such
mixtures can be a mixture of two or more block copolymers that
are identical but for the molecular weight of their respective
blocks. For example, a biocompatible membrane can be produced
using a mixture of two block copolymers, both of which are
poly(2-methloxazoline)-polydimethylsiloxane-poly(2-
methloxazoline), one of which having an average molecular weight
of 2kD-5kD-2kD and the other 3kD-7kD-3kD and the ratio of the
first block copolymer to the second is about 67% to 33% of the
total synthetic polymer material used w/w. This, of course means
that the majority block copolymer's first block has a molecular
weight of about 2 thousand Daltons, the second block has a
molecular weight of 5 thousand Daltons and the third block has a
molecular weight of 2 thousand Daltons. The minority block
copolymer has blocks of about 3 thousand, 7 thousand and 3
thousand Daltons respectively.
Of course, two or more entirely 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 contemplated. But mixtures are not
limited to block copolymers.
Polymers and copolymers can be used, alone, in combination,
and in combination with block copolymers in accordance with the
present invention to produce biocompatible membranes having the
properties described herein. Polymers and copolymers useful are
preferably solid at room temperature (25°C). They can be
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dissolved in solvents or solvent systems that can accommodate any
other synthetic polymer material used, any additive used, and the
polypeptide used. Polymers and copolymers useful in producing
biocompatible membranes can include, without limitation
5 polystyrenes, polyalkyl and polydialkyl siloxanes such as
polydimethylsiloxane, polyacrylates such as
polymethylmethacrylate, polyalkenes such as polybutadiene,
polyalkylenes and polyalkylene glycols, sulfonated polystyrene,
polydienes, polyoxiranes, polyvinyl pyridines), polyolefins,
10 polyolefin/alkylene vinyl alcohol copolymers, ethylene propylene
copolymers, ethylene-butene-propylene copolymers, ethyl vinyl
alcohol copolymers, perfluorinated sulfonic acids, vinyl halogen
polymers and copolymers such as copolymers of vinyl chloride and
acrylonitrile, methacrylicJethylene copolymers and other soluble
15 but generally hydrophobic polymers and copolymers all in a
molecular weight of between about 5,000 and about 500,000.
Particularly preferred polymers include: Poly(n-butyl acrylate);
Poly(t-butyl acrylate); Poly(ethyl acrylate); Poly(2-ethyl hexyl
acrylate); Poly(hydroxy propyl acrylate); Poly(methyl acrylate);
20 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 1,4 addition); Polyethylene; Poly(dimethyl
siloxane); Poly(ethyl methyl siloxane); Poly(phenyl methyl
siloxane); Polypropylene; Polypropylene oxide); Poly(4-acetoxy
styrene); Poly(4-bromo styrene); Poly(4-t-butyl styrene); Poly(4-
chloro styrene); Poly(4-hydroxyl styrene); Poly(a-methyl
styrene); Poly(4-methyl styrene); Poly(4-methoxy styrene);
Polystyrene; Isotactic Polystyrene; Syndiotactic Polystyrene;
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Poly(2-vinyl pyridine); Poly(4-vinyl pyridine); Poly(2,6-
dimethyl-p-phenylene oxide); Poly(3-(hexafluoro-2-hydroxypropyl)-
styrene); Polyisobutylene; Poly(9-vinyl anthracene); Poly(4-vinyl
benzoic acid); Poly(4-vinyl benzoic acid sodium salt) ;
Polyvinyl benzyl chloride); Poly(3(4)-vinyl benzyl
tetrahydrofurfuryl ether); Poly(N-vinyl carbazole); Poly(2-vinyl
naphthalene) and Poly(9-vinyl phenanthrene). Since polymers and
copolymers are generally synthetic polymer materials, they may be
used in the same amounts described previously for block
copolymers and mixtures.
In a particularly preferred aspect of the present invention,
the biocompatible membrane includes a synthetic polymer material,
preferably at least one block copolymer (most preferably one that
is, at least in part, amphiphilic) and a synthetic polymer
material that can stabilize the biocompatible membrane. It has
been discovered that certain polymers, most notably, hydrophilic
polymers and copolymers capable of forming a plurality of
hydrogen-bonds ("hydrogen bonding rich") can stabilize the
membrane. In the context of stabilizing polymers, the term
"polymer" includes monomers, polymers and copolymers.
"Hydrophilic" in this context means that the stabilizing polymer
will dissolve or be solubilized in water or water miscible
solvents. Without wishing to be bound to any particular theory of
operation, it is believed that the use of such polymers can
assist in functionally integrating polypeptides into the
biocompatible membrane's structure. A stabilizing polymer imparts
to a biocompatible membrane greater operating life and/or greater
resistance to mechanical failure when compared to an identical
biocompatible membrane produced without the stabilizing polymer
when exposed to the same conditions. A stabilized biocompatible
membrane wherein the synthetic polymer material includes a
stabilizing polymer, used in a fuel cell, for example, can have
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22
an increased operating life of at least about 100, more
preferably at least about 50%, most preferably at least about
100%.
Particularly preferred polymers capable of stabilizing the
polypeptides in the biocompatible membranes of the present
invention include: dextrans, polyalkylene glycols, polyalkylene
oxides, polyacrylamides, and polyalkyleneamines. These
stabilized polymers (again including copolymers) have an average
molecular weight which is generally lower than polymers and
copolymers used as synthetic polymer materials. Their molecular
weight generally ranges from about 1,000 daltons to about 15,000
daltons. Particularly preferred polymers capable of stabilizing
biocompatible membranes include, without limitation, polyethylene
glycol having an average molecular weight of between about 2,000
and about 10, 000, polyethylene oxide having an average molecular
weight of between about 2,000 and about 10,000, poly acrylamide
having an average molecular weight of between about 5,000 and
15,000 daltons. Other stabilizing polymers include:
polypropylene, Poly(n-butyl acrylate); Poly(t-butyl acrylate);
Poly(ethyl acrylate); Poly(2-ethyl hexyl acrylate); Poly(hydroxy
propyl 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 stabilizing polymers) used in the
biocompatible membranes is not critical so long as some
measurable improvement in properties is realized and the
functionality of the biocompatible membrane is not unduly
hampered. Some trade of functionality and longevity is to be
expected. However, generally, the amount of stabilizing polymer
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23
used, as a function of the total amount of synthetic polymer
material found in the finished biocompatible membrane (by weight)
is generally not more than one-third, and typically 30% by weight
or less. Preferably, the amount used is between 5 and about 30%,
more preferably between about 5 and about 15% by weight of the
synthetic polymer material in the finished membrane is used.
In addition to one or more polymers, copolymers and/or block
copolymers, and/or stabilized polymers, the synthetic polymer
material of the invention can include at least one additive.
Additives can include crosslinking agents and lipids, fatty
acids, sterols and other natural biological membrane components
and their synthetic analogs. These are generally added to the
synthetic polymer material when in solution. These additives, if
present at all, generally would be found in an amount of between
about 0.500 and about 300, preferably between about 1.0% and
about 15%, based on the weight of the synthetic polymer material.
Where the biocompatible membrane incorporates cross-linking
moieties, procedures useful for polymerization include chemical
polymerization with radical-forming or propagating agents and
polymerization via photochemical radical generation with or
without further radical propagating agents. Parameters can be
adjusted depending on such conditions as the membrane material,
the size of biocompatible membrane segments, the structure of the
support, and the like. Care should be taken to minimize the
damage to the polypeptide. One particularly useful method
involves using peroxide at a neutral pH, followed by
acidification.
Examples of useful polypeptides that can be associated with
a synthetic polymer material, so as to form a biocompatible
membrane in accordance with the present invention, and that can
participate in one or both of the oxidation/reduction and
transmembrane transport functions (molecules, atoms, protons,
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24
electrons) include, for example, NADH dehydrogenase ("complex I")
(e. g., from E. coli. Tran et al., "Requirement for the proton
pumping NADH dehydrogenase I of Escherichia coli in respiration
of NADH to fumarate and its bioenergetic implications," Eur. J.
Biochem. 244: 155, 1997), NADPH transhydrogenase, proton ATPase,
and cytochrome oxidase and its various forms. Further
polypeptides include: glucose oxidase (using NADH, available from
several sources, including number of types of this enzyme
available from Sigma Chemical), glucose-6-phosphate dehydrogenase
(NADPH, Boehringer Mannheim, Indianapolis, IN), 6-
phosphogluconate dehydrogenase (NADPH, Boehringer Mannheim),
malate dehydrogenase (NADH, Boehringer Mannheim), glyceraldehyde-
3-phosphate dehydrogenase (NADH, Sigma, Boehringer Mannheim),
isocitrate dehydrogenase (NADH, Boehringer Mannheim; NADPH,
Sigma), a-ketoglutarate dehydrogenase complex (NADH, Sigma) and
proton-translocating pyrophosphates. Also included are
succinate:quinone oxidoreductase, also referred to as "Complex
II," "A structural model for the membrane-integral domain of
succinate:quinone oxidoreductases" Hagerhall, C. and Hederstedt,
L., FEBS Letters 389; 25-31 (1996) and "Purification,
crystallisation and preliminary crystallographic studies of
succinate:ubiquinone oxidoreductase from Escherichia coli."
Tornroth, S., et al., Biochim. Biophys. Acta 1553; 171-176
(2002), heterodisulfide reductases, F(420)H(2) dehydrogenase,
(Baumer et al., "The F420H2 dehydrogenase from Methanosarcina
mazei is a Redox-driven proton pump closely related to NADH
dehydrogenases." 275 J. Biol. Chem. 17968 (2000)) or a formate
hydrogenlyase (Andrews, et al., A 12-cistron Escherichia coli
operon (hyf) encoding a putative proton-translocating formate
hydrogenlyase system." 143 Microbiology 3633 (1997)),
Nicotinamide nucleotide transhydrogenases: "Nicotinamide
nucleotide transhydrogenase: a model for utilization of substrate
CA 02470125 2004-06-11
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binding energy for proton translocation." Hatefi, Y. and
Yamaguchi, M., Faseb J., 10; 444-452 (1996), Proline
Dehydrogenase: "Proline Dehydrogenase from Escherichia coli K12."
Graham, S., et al., J. Biol. Chem. 259; 2656-2661 (1984), and
5 Cytochromes including, without limitation, cytochrome C oxidase
(crystallized with either undecyl-(3-D-maltoside or cyclohexyl-
hexyl-(3-D-maltoside), Cytochrome bcl: "Ubiquinone at Center N is
responsible for triphasic reduction of cytochrome bc1 complex."
Snyder, C.H., and Trumpower, B.L., J. Biol. Chem. 274; 31209-16
10 (1999), Cytochrome boa: "Oxygen reaction and proton uptake in
helix VIII mutants of cytochrome bo3." Svensson, M., et al.,
Biochemistry 34; 5252-58 (1995), "Thermodynamics of electron
transfer in Escherichia coli cytochrome bo3." Schultz, B.E., and
Chan, S.I., Proc. Natl. Acad. Sci. USA 95; 11643-48 (1998), and
15 Cytochrome d: "Reconstitution of the Membrane-bound, ubiquinone-
dependent pyruvate oxidase respiratory chain of Escherichia coli
with the cytochrome d terminal oxidase." Koland, J.G., et al.,
Biochemistry 23; 445-453 (1984), Joost and Thorens, "The extended
GLUT-family of sugar/polyol transport facilitators: nomenclature,
20 sequence characteristics, and potential function of its novel
members (review)" 18 Mol. Membr. Biol. 247-56 (2001), and
selective channel proteins including those disclosed in Goldin,
A.L., "Evolution of voltage-gated Na(+) channels." J. Exp. Biol.
205; 575-84 (2002), Choe, S., "Potassium channel structures."
25 Nat. Rev. Neurosci. 3;115-21 (2002), Dimroth, P., "Bacterial
sodium ion-coupled energetics." Antonie Van Leeuwenhoek 65; 381-
95 (1994), and Park, J.H. and Saier, M.H.Jr., "Phylogenetic,
structural and functional characteristics of the Na-K-C1
cotransporter family." J. Membr. Biol. 149; 161-8 (1996). All of
the foregoing are hereby incorporated by reference. Methods of
isolating such an NADH dehydrogenase enzyme are described in
detail, for example, in Braun et al., Biochemistry 37: 1861-1867,
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26
1998; and Bergsma et al., "Purification and characterization of
NADH dehydrogenase from Bacillus subtilis," Eur. J. Biochem. 128:
151-157, 1982. As described by Spehr et al., Biochemistry
38:16261-16267, 1999, the complex I NADH dehydrogenase (or,
NADH:ubiquinone oxidoreductase), which is expressed from a
operon, can be overexpressed in E. coli by substituting a T7
promoter in the operon to provide useful quantities for use in
the invention. Complex I can be isolated from over-expressing E.
coli by the method described by Spehr et al. using solubilization
with dodecyl maltoside.
Complex I can be handled such that NADH dehydrogenase
activity is eliminated or greatly reduced. As described in
Bottcher et al., "A Novel, Enzymatically Active Conformation of
the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I),"
web published as accepted for publication at www.jbc.org, 2002
(Manuscript M112357200), in high salt or high pH solution Complex
I changes conformation such that proton transport is uncoupled
from NADH dehydrogenase activity, creating DH- form. Applicants
have used these conditions and combinations of these conditions
to show that the fuel cell of the invention can operate without
NADH dehydrogenase activity in the anode/cathode barrier. Such
conditions include anolyte or anode salt concentrations of 200 mM
to 2M, and pH of 8.0 or above. Transporter activity is believed
to function against a countering [H+] gradient, due to the charge
imbalance between the anode and cathode sides. Proton
transporter activity of the DH- form has been confirmed from the
maintenance of current generation in fuel cells in which
biocompatible membranes gated by this form provided the only
avenue to relieve charge imbalance. (Note that with complex I
reverse transport of protons has been further controlled against
by using conditions on the cathode side that maintain the NADH
dehydrogenase coupling of any inversely oriented complex
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27
I - thereby blocking reverse transport due to lack of NADH
substrate.)
It will be recognized that the source of any enzyme used in
the invention can be a thermophilic organism providing a more
temperature stabile enzyme. For example, complex I can be
isolated from Aquifex aeolicus in a form that operates optimally
at 90 °C, as described in Scheide et al., FEBS Letters 512: 80-84,
2002 (describing a preliminary isolation using the type of
detergent extraction used elsewhere for complex I).
Additionally, it is contemplated that genetically modified
polypeptides, such as modified enzymes, can be used. One
commonly applied technique for genetically modifying an enzyme is
to use recombinant tools (e.g., exonucleases) to delete N-
terminal, C-terminal or internal sequence. These deletion
products are created and tested systematically using ordinary
experimentation. As is often the case, significant portions of
the gene product can be found to have little effect on the
commercial function of interest. More focused deletions and
substitutions can increase stability, operating temperature,
catalytic rate and/or solvent compatibility providing enzymes
that can be used in the invention. Of course it is possible to
use mixtures of various polypeptides described herein as may be
desirable.
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 it will be used, etc. The
amount of polypeptide may be important to certain applications
such as fuel cells where, in general, the higher the
concentration of polypeptide per square centimeter of surface
area, the higher the rate of proton transfer per unit area (in
terms of current). In general, however, as long as some
polypeptide is present and functional, and as long as the amount
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28
of polypeptide used does not prevent membrane formation or render
the membrane unstable, then any amount of polypeptide is
possible. Generally, the amount of polypeptide will be at least
about 0.01%, more preferably about 5%, even more preferably 100,
and still more preferably at least about 20o and most preferably
30% or more by weight based on the final weight of the
biocompatible membrane. The amount of polypeptide to solvent can
be as low as 0.001% w/v and as high as 50.00 w/v. Preferably,
the concentration is from about 0.5% to about 5.0% w/v. More
preferably the concentration is from about 1.0% to about 3.0%
w/v.
Suitable solubilizing and/or stabilizing agents such as
cosolvents, detergents and the like may also be needed,
particularly in connection with the polypeptide solution.
Solubilizing detergents are commonly found at the 0.010 to 1.0%
concentration level, and more preferably up to about 0.5% is
contemplated. Such detergents include ionic detergents: Sodium
dodecyl sulfate, Sodium N-dodecyl sarcosinate, N-dodecyl Beta-D-
glucopyranoside, Octyl-Beta-D-glucopyranoside, dodecyl-maltoside,
decyl, undecyl, tetradecyl-maltoside (in general, an alkyl chain
of about 8 carbons or more bonded to a sugar as a general form of
an ionic detergent) octyl-beta-D-glucoside and polyoxytheylane
(9) dodecyl-ether, C12E9, as well as non-ionic detergents, such as
triton X-100, or Nonidet P-40. Also useful are certain polymers,
typically diblock copolymers which exhibit surfactant properties,
such as BASF's Pluronic series, or Disperplast (BYK-Chemie).
The solvent used in producing the synthetic polymer material
solution is preferably selected to be miscible with both the
water used (the polypeptide solution often includes water) and at
least one of the synthetic polymer materials (polymer, copolymer
and/or block copolymer). However, as described above, it is
possible to form membranes using solvents or mixtures which are
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29
not water miscible. Note that while the use of solvents to
produce solutions is preferred, the term "solution" as used
herein generally encompasses suspensions as well.
When a block copolymer is used, the solvent should
solubilize these synthetic polymer materials. While the
synthetic polymer material may be relatively sparingly soluble in
the solvent (less than 5o w/v), it is preferably more soluble
than 5 o w/v and generally, solubility is at least 5 to 10 o w/v,
preferably greater than 10o w/v synthetic polymer material to
solvent.
Appropriate solvents may include, without limitation, low
molecular weight aliphatic alcohols and diols of between 1 and 12
carbons such as methanol, ethanol, 2-propanol, isopropanol,
1-propanol, aryl alcohols such as phenols, benzyl alcohols, low
molecular weight aldehydes and ketones such as acetone, methyl
ethyl ketone, cyclic compounds such as benzene, cyclohexane,
toluene and tetrahydrofuran, halogenated solvents such as
dichloromethane and chloroform, and common solvent materials such
as 1,4-dioxane, normal alkanes (Cz-C1~) and water. Solvent
mixtures are also possible as long as the mixture has the
appropriate miscibility, rate of evaporation and the other
criteria described for individual solvents. (Solvent components
that have any tendency to form protein-destructive contaminants
such as peroxides can be used as long as they can be
appropriately purified and handled.) Solvent typically comprises
30o v/v or more of the polypeptide/synthetic polymer material
solution, preferably 20% v/v or more, and usefully 10% v/v or
more.
If the membranes are to include "other materials" such as
detergents, lipids (e. g. cardiolipin), sterols (e. g. cholesterol)
or buffers and/or salts, those too would be added prior to
formation of the membrane and they would be present in an amount
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of between about 0.01 and about 30%, preferably between about .01
and about 15% based on the weight of the finished biocompatible
membrane. Other materials, as opposed to additives, are most
often mixed with the polypeptide solutions, not the synthetic
5 polymer solutions.
Biocompatible membranes in accordance with the present
invention can be produced using any one of a number of
conventional techniques used in the production of membranes from
synthetic polymer materials and even lipid bilayers, as long as
10 the resulting biocompatible membranes are useful as described
herein. One method of forming a biocompatible membrane, which is
preferred for use with block copolymer-based membrane, is as
follows:
1. Form a solution or suspension of synthetic polymer
15 material in a solvent or mixed solvent system. The solution or
suspension can be a mixture of two or more block copolymers,
although it may contain one or more polymers and/or copolymers.
The solution or suspension preferably contains 1 to 90% w/v
synthetic polymer material, more preferably 2 to 70%, or yet more
20 preferably 3 to 20% w/v. Seven % w/v is particularly preferred.
2. One or more polypeptides (typically with solubilizing
detergent) are placed in solution or suspension, either
separately or by being added to the existing polymer solution or
suspension. Where the solvent used to solubilize the synthetic
25 polymer materials is the same, or of similar characteristics and
solubility to that which can solubilize the polypeptide, it is
usually more convenient to add the polypeptide to the polymer
solution or suspension directly. Otherwise, the two or more
solutions or suspensions containing the synthetic polymer
30 materials and the polypeptide must be mixed, possibly with an
additional cosolvent or solubilizer. Most often, the solvent
used for the polypeptide is aqueous.
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Mixing of these solutions and/or suspensions is often a
relatively simple matter and can be accomplished by hand or with
automated mixing tools. Heating or cooling may also be useful in
membrane formation depending on the solvents and polymers used.
In general, rapidly evaporating solvents tend to form membranes
better with cooling while extremely slowly evaporating solvents
would most likely benefit from a slight degree of heating. One
can examine the boiling point of solvents used to select those
with the most favorable characteristics provided they are
appropriate for the polymer used. One must, of course, however
consider also the need to incorporate the polypeptide into the
solvent polymer mixture, which can be a nontrivial matter. It is
possible, for example, to mix 5 microliters of a detergent
solubilized Complex I (0.15 o w/v dodecyl maltoside) having 10
mg/ml of Complex I into 95 microliters of a mixture of a 3.2%
w/v polystyrene-polybutadiene-polystyrene triblock copolymer (a
completely hydrophobic triblock Sold under the trademark STYROLUX
3655, Lot No. 7453064P, available from BASF in a 50/50 mixture
of acetone and hexane and to deposit same in a manner that will
allow for membrane formation. In this case, the final mixture
included about 5% v/v of water, and 0 . 75 o w/w Complex I relative
to the weight of the synthetic polymer material. Generally, the
solutions are sufficiently stable at room temperature to be
useful for at least about 30 minutes, provided that the solvents
do not evaporate during that time. They also can be stored
overnight, or longer, generally under refrigerated conditions.
3. A volume of the final solution or suspension including
both the polypeptide(s) and the synthetic polymer materials is
formed into a membrane and allowed to at least partially dry,
thereby removing at least a portion of the solvent. It is
possible to completely dry some of the membranes produced in
accordance with the invention or to substantially dry same. By
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substantially dry it is meant that there may be some residual
solvent,. up to about 150, which is often retained even if left
out at room temperature for several hours.
In a particularly preferred embodiment, substantially all of
the weight of the finished membrane will be either polypeptide or
synthetic polymer material. In this case, the amount of
synthetic polymer material, including additives and stabilizing
polymers ranges from about 70% to about 99% by weight of the
finished membrane. However, it may be desirable to have an even
greater polypeptide content or it may be necessary to retain some
solvent, so the amount of synthetic polymer material may be
reduced accordingly. Generally, however, at least about 50% by
weight of the finished biocompatible membrane will be synthetic
polymer material. When the synthetic polymer material is a
mixture that includes a block copolymer and a polymer or
copolymer, other than a stabilizing polymer, the block copolymer
can be present in an amount of at least about 35o by weight of
the biocompatible membrane. Up to about 30% by weight of the
biocompatible membrane can 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 polymer material can be stabilizing polymer. Generally
the stabilizing polymer will be present in an amount of between
about 5 and about 20% of the weight of the synthetic polymer
material used.
Identifying which solvents are particularly useful in
accordance with the present invention and which combination of
polymers and polypeptides and solvents should be used depends on
a number of factors, some of which have already been discussed in
terms of miscibility, evaporation and the like. The polymer and
protein constituents must be able to be completely dissolved in
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the solvent or solvent mixture. Evaporation rate must be
sufficiently long to allow one time to produce a membrane.
However, the amount of time should not be so long as to render
manufacturing impractical. While apolar solvents may be useful,
generally more apolar solvents may not be useful in certain
circumstances as ionic or hydroxyl components of the polymer may
be poorly soluble in completely apolar solvents. Thus one may be
able to dissolve a highly rigid, hydrophobic component such as
polystyrene and be unable to simultaneously dissolve a highly
ionic component such as an acrylic acid. However, with polymers
of completely hydrophobic character, then apolar solvents are
preferred. The solvents should generally be, in part, nonaqueous
as the polymer should be at least in part nonwater dissolvable.
And while water-miscibility is most desired for membrane protein
reconstitution, it is not a rigidly limiting factor. Thus,
preferably, all solvents are nonaqueous. The solvent for the
polypeptide and stabilizing polymers, however, is predominantly
water or at least water miscible.
Preferred methods of forming biocompatible membranes
including both at least one synthetic polymer material and a
stabilizing polymer include the step of making an appropriate
solution of block copolymer and, usually separately stabilizing
polymer and polypeptide. As described elsewhere, the polypeptide
may include one or more detergents or surfactants and is
typically in an aqueous solution. Once the appropriate solutions
are made and mixed, membranes can be made by any of the
techniques disclosed herein or known to the art including, for
example, coating a perforated dielectric substrate with the
solution followed by at least partial evaporation of solvents.
Such evaporation can be facilitated in a vacuum.
One method of forming a biocompatible membrane, including a
hydrogen-bonding rich stabilizing polymer, is as follows:
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1. A solution or suspension of Protolyte A700 block
copolymer in a solvent as supplied is diluted with an equal
volume of ethanol (5a water w/v). The solution contains about 5%
w/v of block copolymer.
2. Separately, an aqueous solution or suspension of the
stabilizing agent 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 the stabilizing agent in
solution is near the saturation limit.
3. Next, 4 microliters of a solution including 10 mg/ml of
E. coli derived Complex I along with 0.15% w/v of dodecyl
maltoside is added to 6 microliters of the PEG solution and mixed
them to generate a solution or suspension.
4. The 10 microliters of the solution is then mixed with 10
microliters of the solution including the block copolymer.
5. A small volume (e. g., 4 microliters) resulting solution
is dropped onto the apertures of a subset of apertures (holes
drilled through the support) of a perforated substrate of 1 mil
(25.4 microns) thick KAPTON, a brand of polyimide, having
apertures that are 100 micrometers in diameter and 1 mil deep.
6. The solution is allowed to air dry in a hood thereby
removing the solvent.
7. Steps 5 and 6 are repeated as needed to cover all
apertures.
The above-described method of introducing polypeptide to a
solution containing a stabilizing polymer prior to mixing with
non-aqueous solvents) in the presence of block copolymers is
believed to stabilize the function of polypeptides used in the
biocompatible membrane. However, the polymer and block copolymer
could also be mixed and the resulting solution mixed with a
generally aqueous polypeptide solution. Optionally one would
check each aperture to ensure membrane formation, or check at
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least a statistically relevant number of apertures
microscopically. If apertures do not contain a membrane, repair
holes using additional solution and a micropipette-scaled
pipetting device. It typically requires only a very small volume
5 of solution to repair such holes. The membranes can be
completely or substantially completely dried in a vacuum
apparatus, or desiccator. Membranes so formed may be stored dried
in vacuum or desiccated, if desired.
Where the biocompatible membrane incorporates cross-linking
10 moieties, such as methacrylates, and will be used in a fuel cell,
the following procedure can be used:
Prepare biocompatible membrane in a support that will form
the cathode/anode barrier.
Assemble a cell with biocompatible membrane on anode/cathode
15 barrier support, electrodes and buffers only.
Connect the two electrodes to a high load, such as
approximately 150 kilo-Ohms.
Add hydrogen peroxide to cathode side to initiate cross
linking process, for example such that the concentration of the
20 peroxide will be 1% by volume.
Let fuel cell stand under load for a period of time, for
example 1 hour (~10%).
Adjust pH of the cathode side to below pH 5 to stop the
crosslinking.
25 Parameters can be adjusted depending on such conditions as
the membrane material, the size of biocompatible membrane, the
thickness of the biocompatible membrane, the structure of the
support, and the like.
Once the polypeptide/synthetic polymer material solution has
30 been produced, it can be formed into a membrane. Biocompatible
membranes in accordance with the present invention can be free
standing membranes. Such membranes can be formed by pouring the
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solution into a pan or onto a sheet such that they achieve the
desired thickness. Once the solution has been dried and the
solvent dried off, the dry membrane may be removed from the pan
or peeled from the backing layer. Suitable antitack agents may
be used to assist in this process. Biocompatible membranes can
be formed against a solid material, such as by coating onto
glass, carbon that is surface modified to increase
hydrophobicity, or a polymer (such as polyvinyl acetate, PDMS,
Kapton°, a perfluorinated polymer, PVDF, PEEK, polyester, or
UHMWPE, polypropylene or polysulfone). Polymers such as PDMS
provide an excellent support that can be used to establish
openings on which biocompatible membranes can be formed.
The membrane may then be cut or shaped as needed or used as
is. Furthermore, to facilitate use of the membrane, it may be
attached physically or through some sort of fastening device or
adhesive to a holder if desired. This can be conceptualized as
stretching a canvas over a frame prior to painting a picture when
the frame is the support and the membrane is the canvas.
Alternatively, the membrane may be formed with such a structure.
A suitable analogy would be taking a child's bubble wand, used
for blowing bubbles, and dipping it into a solution of soap and
water. A film of soap and water forms across the opening of the
wand. The structural material used at the periphery allows the
film to be handled and manipulated and provides rigidity and
strength. It also helps provide the desired shape of the film.
The same sort of process can be employed using a physical
structure and the membrane forming solutions of the present
invention.
In one preferred embodiment in accordance with the present
invention, a biocompatible membrane may be disposed and/or formed
within or across apertures of various perforated substrates
including preferably dielectric substrates. ~~Perforated
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substrates° means that it has at least one hole, aperture
(synonymous with hole as used herein) or pore into which, or over
which, a biocompatible membrane could be disposed. For example,
Figure 3b shows an embodiment of a membrane construction useful
in a fuel cell. A perforated substrate 42, which defines various
perforations 49, has its surfaces metalized to form a perforated
anode 44 and a perforated cathode 45. Note also that perforated
substrate 42 can be a porous substrate without, for example,
drilled holes. In such instances, perforations 49 are to be
understood as being pores. A biocompatible membrane 61 in
accordance with the present invention is formed across the
apertures or perforations 49 of perforated substrate 42, and is
attached directly to the surface of the anode. Biocompatible
membrane 61 can also be disposed within the perforation and flush
with anode 44 or can be attached to or adj acent cathode 45 . Two
membranes 61 can be provided, one, for example, disposed across
the anode as illustrated and one within the perforation 49 of the
substrate 42, flush with anode 45, etc. (not shown). The
membranes 61 may be the same or different in terms of the
synthetic polymer materials used, the polypeptides used or both.
Indeed, a plurality of such membrane 61 and indeed, layers of
biocompatible membrane 61 can be used in conjunction with other
types of membranes, diffusive barriers and the like. While the
foregoing has been explained in the context of Figure 3b, it is
equally applicable to other constructions and, in particular, any
type of fuel cell construction. Biocompatible membrane 61 can
include one or more polypeptides 62 and 63 as illustrated.
Coating methods which can be used to form electrodes (44,
45) on a substrate include a first coating or lamination of
conductor, followed by plating, sputtering or using another
coating procedure to coat with titanium or a noble conductor such
as gold or platinum. Another method is directly sputtering an
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attachment layer, such as chromium or titanium onto the support,
followed by plating, sputtering or other coating procedure to
attach a noble conductor. The outer metal layer can be favorably
treated to increase its hydrophobicity, such as with dodecane
thiol.
Supports or substrates with high natural surface charge
densities, such as Kapton and Teflon, are in some embodiments
preferred. As noted above, these can be used to form the
anode/cathode barrier without the use of surface electrodes.
Substrate 42 is often preferably dielectric.
The perforations or pores 49 and metallized surfaces (anode
44 and cathode 45 (for embodiments that use so-located
electrodes)) of the substrate 42 can be constructed, for example,
with masking and etching techniques of photolithography well
known in the art. Perforations can also be formed, for example,
by punching, drilling, laser drilling, stretching, and the like.
Alternatively, the metallized surfaces (electrodes) can be formed
for example by (1) thin film deposition through a mask, (2)
applying a blanket coat of metallization by thin film then photo-
defining, selectively etching a pattern into the metallization,
or (3) photo-defining the metallization pattern directly without
etching using a metal impregnated resist (DuPont Fodel process,
Drozdyk et al., "Photopatternable Conductor Tapes for PDP
Applications," Society for Information Display 1999 Digest, 1044-
1047; Nebe et al., US Patent 5,049,480). In one embodiment, the
perforated or porous substrate is a film. For example, the
dielectric can be a porous film that is rendered non-permeable
outside the "perforations" by the metallizations. The surfaces
of the metal layers can be modified with other metals, for
instance by electroplating. Such electroplatings are,. for
example, with titanium, gold, silver, platinum, palladium,
mixtures thereof, or the like. In addition to metallized
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surfaces, the electrodes can be formed by other appropriate
conductive .materials, which materials can be surface modified.
For example, the electrodes can be formed of carbon (graphite),
including graphite fiber, which can be applied to the dielectric
substrate by, for example, electron beam evaporation, chemical
vapor deposition or pyrolysis. Surfaces to be metallized can be
solvent cleaned and oxygen plasma etched. Useful means of
forming hydrophilic electrodes are described for example in
Surampudi, US Patent 5,773,162, Surampudi, US Patent 5,599,638,
Narayanan, US Patent 5,945,231, Kindler, US Patent 5,992,008,
Surampudi, WO 96/12317, Surampudi, WO 97/21256 and Narayanan,
WO 99/16137.
Biocompatible membranes used in the invention are optionally
stabilized against a solid support. One method for accomplishing
such stabilization uses sulfur-mediated linkages of lipid-related
molecules to glue, tether or bond metal surfaces or surfaces of
another solid support to biocompatible membranes. For example, a
porous support can be coated with a sacrificial or removable
filler layer, and the coated surface smoothed by, for example,
polishing. Such a porous support can include any of the proton-
conductive polymeric membranes discussed, typically so long as
the proton-conductive polymeric membrane can be smoothed
following coating, and is stable to the processing described
below. One useful porous support is glass frit. The smoothed
surface is then coated (with prior cleaning as necessary) with
metal, such as with a first layer of chrome and an overcoat of
gold. The sacrificial material is then removed, such as by
dissolution, taking with it the metallization over the pores but
leaving a metallized surface surrounding the pores. The
sacrificial layer can comprise photoresist, paraffin, cellulose
resins (such as ethyl cellulose), and the like.
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The tether or glue comprises alkyl thiol, alkyl disulfides;
thiolipids and the like adapted to tether a biocompatible
membrane as illustrated if Figures 7A and 7B. Such tethers are
described for example in Lang et al., Langmuir 10: 197-210, 1994.
5 Additional tethers of this type are described in Lang et al., US
Patent 5,756,355 and Hui et al., US Patent 5,919,576.
Figure 3d includes a similar arrangement. However, unlike
Figure 3b, where biocompatible membrane 61 is actually attached
to the metalized surface of anode 44, in Figure 3d, membrane 61
10 is formed within aperture 49 in perforated substrate 42, such
that it does not necessarily contact either anode 44 or cathode
45. Note that these figures are not to scale and that the
membrane may be thicker or thinner than the electrode and may be
thicker or thinner than the perforated substrate 42. Note also
15 that in Figure 3b, biocompatible membrane 61 is not disposed
between the anode 44 and cathode 45. However, biocompatible
membrane 61 is disposed between anode 44 and cathode 45 in Figure
3d. In each of Figures 3b and 3d, the combination of the
substrate 42 and biocompatible membrane 61 (along with the anode
20 44 in Figure 3b) form a structure that can also be referred to as
a barrier.
Figure 3e illustrates a preferred embodiment for fuel cells.
In this figure, the arrangement of the membrane 61 and the
perforated substrate 42 (a substrate containing pores,
25 perforations or apertures) is as described previously in
connection with Figure 3d. However, the cathode and anode are
spaced apart from the perforated substrate. They can be plate
electrodes, but they are not plated onto the surface, or even in
contact with substrate 42 or membrane 61. In this case, the
30 membrane 61 and substrate 42 are the barrier.
The biocompatible membrane can be formed across the pores,
perforations or apertures 49 and enzyme incorporated therein by,
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41
for example, the methods described in detail in Niki et al., US
Patent 4,541,908 (annealing cytochrome C to an electrode) and
Persson et al., J. Electroanalytical Chem. 292: 115, 1990. Such
methods can comprise the steps of: making an appropriate solution
of polypeptide and synthetic polymer material as previously
discussed, the perforated substrate 49 preferably a dielectric
substrate is dipped into the solution to form the
enzyme-containing biocompatible membranes. Sonication or
detergent dilution may be required to facilitate enzyme
incorporation into a 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
reassembly of membrane proteins in planar lipid bilayers," wart.
Rev. Biophys. 14: 1-79, 1981; Helenius et al., "Asymmetric and
symmetric membrane reconstitution by detergent elimination," Eur.
J. Biochem. 11',6: 27-31, 1981; Volumes on biomembranes (e. g.,
Fleischer and Packer (eds.)), in Methods in Enzymology series,
Academic Press.
Alternatively, a thin partition made (preferably but not
necessarily) of a hydrophobic material such as Teflon with a
small aperture has a small amount of amphiphile introduced. The
coated aperture is immersed in a dilute electrolyte solution upon
which the droplet will thin and spontaneously self-orient
spanning the aperture. Biocompatible membranes of substantial
area have been prepared using this general technique. Two common
methods for formation of the biocompatible membranes themselves
are the Langmuir-Blodgett technique and the injection technique.
The Langmuir-Blodgett technique involves the use of a
Langmuir-Blodgett trough with a partition, such as a TeflonTM
polymer partition at the center. The trough is filled with
aqueous solution. The aperture of the polymer partition is
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42
placed above the water level. The polypeptide and synthetic
polymer material containing solution is spread over the surface
and the polymer partition is lowered slowly into the aqueous
solution forming a biocompatible membrane over the aperture. The
injection method is similar except the polymer partition is kept
fixed. In this method the aqueous phase is filled to just under
the aperture, the solution is introduced over the surface and
then the liquid level is raised over the partition by injecting
additional electrolyte solution from underneath.
Another method for forming biocompatible membranes is using
the technique of self-assembly. This is a variation from the
above two described techniques and was in fact the first
technique to be successfully employed to fabricate synthetic
lipid membranes. The technique involves the preparation of a
membrane forming solution as described above. A drop of the
solution is introduced into a perforated substrate 42, often a
hydrophobic substrate. The substrate 42 is then immersed in a
dilute aqueous electrolyte solution whereupon the droplet will
spontaneously thin and orient. The remaining material migrates
to the perimeter of the layer where it forms a reservoir called
the Plateau-Gibbs border.
The thickness of substrate 42, be it a perforated substrate
having apertures or a porous material, is for example between
about 15 micrometer (~,m) to about 5 millimeters, preferably from
about 15 to about 1,000 micrometers, and more preferably, from
about 15 micrometer to about 30 micrometers. The width of the
perforations or pores is, for example, from about 1 micrometer to
about 1,500 micrometers, more preferably about 20 to about 200
micrometers, and even more preferably, about 60 to about 140
micrometers. About 100 micrometers is particularly preferred.
Preferably, perforations or pores comprise in excess of about 30%
of the area of any area of the dielectric substrate involved in
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transport between the chambers, such as from about 50 to about
75% of the area.
In certain preferred embodiments, the substrate is glass or
a polymer (such as polyvinyl acetate, polydimethylsiloxane
(PDMS), Kapton~ (polyimide film, Dupont de Nemours, Wilmington,
DE), a perfluorinated polymer (such as Teflon, from DuPont de
Nemours, Wilmington, DE), polyvinylidene fluoride (PVDF, e.g., a
semi-crystalline polymer containing approximately 59% fluorine
sold as KynarTM by Atofina, Philadelphia, PA), PEEK (defined
below), polyester, UHMWPE (described below), polypropylene or
polysulfone), soda lime glass or borosilicate glass, or any of
the foregoing coated with metal. The metal can be used to anchor
biocompatible membrane (such as a monolayer or bilayer of
amphiphilic molecules). The metal coating can be receded from
any junctions in which they provide too likely an electrically
conductive pathway for a short between the anode and cathode
compartments. In a particularly preferred aspect of the present
invention, perforated substrate 42 is made of a dielectric
material.
The polypeptide 62 can be immobilized in the biocompatible
membrane with the appropriate orientation to allow access of the
catalytic site for the oxidative reaction to the anode
compartment and asymmetric pumping of protons. However, if the
polypeptide is not asymmetrically oriented, the reverse oriented
polypeptide is not detrimental for a variety of reasons depending
on the context. First, the charge imbalance created by the fuel
cell on the anode side drives proton transport to the cathode
side even against a proton concentration gradient. In situations
where the pumping is tied to the use of a reduced electron
carrier, the reverse pumping has no such carrier since the
electron carrier is substantially isolated in the anode
compartment 41. (By "substantially isolated" those of ordinary
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skill will recognize sufficiently isolated to allow the fuel cell
to operate.)
In one embodiment, as shown in Figures 4a to 4c, the
biocompatible membrane 61 contains cross-linking moieties and is
formed across an aperture with beveled edges to the substrate 42.
The degree of beveling can be any degree that increases the
stability of the biocompatible membrane. Where the cross-linked
block copolymer is relatively less rigid, greater beveling can be
used to increase stability, while a lesser amount of beveling can
be appropriate for more rigid cross-linked block copolymer. As
illustrated, numerous beveling shapes can contribute to
increasing stability.
In another alternate embodiment in accordance with the
present invention, the solution containing the polypeptide and
the synthetic polymer material can be laid across a surface of a
porous supporting material, rather than a perforated material as
illustrated in Figure 3b. Once protons, for example, were pumped
across the membrane, they could migrate through the pores of the
supporting barrier material.
Whether a substrate is perforated or porous, it is not
necessary that a membrane be formed across its entire surface.
For example, while it may be convenient to form a membrane across
the entire surface of a perforated substrate, it may be preferred
merely to selectively introduce a solution containing polypeptide
and synthetic polymer material into the perforations or merely
across the perforations.
The thickness of the biocompatible membrane in accordance
with the present invention can be adjusted by known techniques
such as controlling the volume introduced to a particular size
pore, perforation, pan or tray, etc. The thickness of the
membrane will be dictated largely by its composition and
function. A membrane intended to include a transmembrane proton
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transporting complex such as complex I must be thick enough to
provide sufficient support and orientation to the enzyme complex.
It should not, however, be so thick as to prevent effective
transportation of the proton across the membrane. For an
5 aperture or perforation of about 100 microns in diameter in an
array of about 100 apertures and a solution including complex I
in an amount of about 4 microliters in a copolymer solution
containing about 7% w/v of the poly(2-methyloxazoline)-block-
poly(dimethylsiloxane)-block-poly(2-methyloxazoline)-triblock
10 copolymer described in one of the previously identified Meier et
a1. articles, a membrane of suitable thickness can be obtained.
The thickness of the membrane can vary widely depending upon its
needed longevity, its function, etc. Membranes that are designed
to transport protons for example are often thinner than membranes
15 to which are attached an enzyme which can oxidize something.
However, in general, the membranes will range from between about
10 nanometers to 100 micrometers or even thicker. Indeed,
biocompatible membranes useful for transporting protons in a fuel
cell have been successful at thicknesses of 10 nanometers up to
20 10 micrometers. Again, thicker membranes are possible.
In certain embodiments of the present invention,
particularly useful in the creation of fuel cells, the
biocompatible membranes of the present invention are capable of
transporting protons against a pH gradient. This concept will be
25 discussed further herein. However, conceptually, on the cathode
side of the biocompatible membrane, the pH of any medium,
electrolyte or the like is acidic while on the anode side of the
membrane, the pH is basic. This is opposite of what is found in
most fuel cells. Generally, such conditions would favor the
30 transfer of protons from the proton rich acidic side to the
relatively proton poor basic side. The use of membranes in
accordance with the present invention can, however, pump upstream
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from the proton poor side to the proton rich side. This
highlights another aspect of the present invention which can be
particularly useful. The membranes of the present invention can
also be active and functional despite relatively large variations
in pH conditions on opposite sides thereof. For example,
membranes in accordance with the present invention can catalyze
proton transfer where the pH in an 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 greater
concentration of protons. However, fuel cells produced in
accordance with the present invention do not need to rely on
proton concentration differences to drive protons across the
membrane by diffusion. This can be a particularly important
advantage because the species used as electron carriers and/or
electron transfer mediators often work more efficiently in
relatively alkaline pH. Fuel oxidation reactions may also be
more efficient under such pH conditions. The pH differential,
based on the electrolytes used, etc. may not need to be adjusted
during the useful life of a fuel cell. Alternatively, a
buffering system can be added, and additional buffer added as
needed, to the anode and/or cathode compartment during operation.
Preferably, the anode compartment will have a pH that is at least
about 1 pH unit higher than the pH in the cathode compartment,
more preferably 2 pH units higher. In a particularly preferred
embodiment, the pH of the anode compartment is 8 or higher and
the pH in a cathode compartment is 5 or lower. See Example
No. 59.
Another aspect of the present invention is a fuel cell
produced using a biocompatible membrane as described herein.
Without limitation to other appropriate definitions known in the
art, a fuel cell is a device that generates electrical energy by
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the chemical conversion of a fuel. The specific type of fuel
cell, in terms of the type of fuel used, the type of electron
transporting species (electron carriers, soluble enzymes,
transfer mediators and the like) or electrolytes used, the types
of electrodes used and the like are subject to wide variation and
all are contemplated so long as they are capable of meeting the
appropriate criteria. For example, the systems used must be
compatible with the biocompatible membrane. If they are, for
example, corrosive to the membrane, then the life of the fuel
cell may be unusually short (less than 8 hours useful life) . If
the materials used cause sufficient instability, then that too
may be reason why particular fuel, for example, may not be useful
in accordance with the invention. Particularly preferred are
fuel cells which are small and light enough to be used in
portable electronic devices such as computers, PDAs, cell phones,
beepers, personal entertainment systems, PlayStation 2, Game Boy,
portable DvD players, power tools, toys, stereo equipment,
radios, cameras and video recorders, digital recorders and
cameras, flashlights, cars, trucks, boats, planes, etc. The fuel
cells are preferably "green," which is to say that they can be
disposed of readily because they do not contain corrosive or
dangerous chemicals, either as fuels or waste. In addition,
these fuel cells may be refillable (adding additional fuel, etc.)
or may be single use/disposable.
As illustrated in Figure 1, a fuel cell in accordance with
the present invention can include an anode compartment 1 having
an anode 4 and a cathode compartment 3 having a cathode 5. The
assembly also includes a dielectric perforated or porous
substrate 2. The fuel cell also includes at least one
biocompatible membrane 61 as previously described (not shown in
Figure 1) . The anode 4 has an electrical lead or contact 6 and
the cathode 5 has an electrical lead or contact 7 which can be
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electrically linked, or linked in an electrical circuit through a
load or resistance so as to place them in electrical contact.
The biocompatible membrane 61 can be disposed within the anode
compartment, as is the case in Figure 3b, within the cathode
compartment or between the anode and cathode compartments as
shown in Figures 3d and 3e. The biocompatible membrane is also
disposed between the anode and cathode in Figures 3d and 3e and
can be thought of as defining the boundary between the anode
compartment and the cathode compartment in that illustration.
The fuel cell normally includes electrical contacts which allow a
circuit to be formed between the two electrodes. Anodes and
cathodes can be made of any electrically conductive material
which is otherwise generally unreactive with the elements of the
fuel cell. The anode and cathode are preferably made of metals
or carbon as previously described. The size and shape of the
anode and cathode can be made to fit the necessary dimensions of
a fuel cell and allow for passage of various chemical species.
Figure 3c illustrates a fuel cell produced in accordance with the
present invention comprising a cell housing 51 and a perforated
substrate 42 including within its perforations a biocompatible
membrane as described herein 61. The anode and cathode are
plated onto the substrate as previously described and are not
individually shown. However, as shown in Figure 3c, the anode
contact 54 and the cathode contact 55 are attached to the
relevant electrodes within the fuel cell.
If an electrode is used as part of the support system for a
biocompatible membrane, as illustrated in Figure 3b, then the
electrode must have sufficient perforations or other means of
providing access to allow molecules, atoms, protons or electrons
to flow therethrough. When the fuel cell has a configuration
similar to Figure 3e, however, it is possible that the electrodes
be completely solid. However, it still may be desirable to have
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fuel or other components of a fuel cell able to pass through and
around the electrode and therefore, it is possible to provide
perforations in any event.
The biocompatible membrane useful in the fuel cell according
to the present invention has already been discussed. The
biocompatible membrane preferably will facilitate the passage of
current in an amount that is greater than that which would result
from the use of the same membrane without the polypeptide. More
preferably, the biocompatible membrane will facilitate the flow
of at least about 10 milliamps/cmz, more preferably at least about
50 milliamps/cmz, and most preferably at least about
100 milliamps/cm2. In the simplest embodiment, the biocompatible
membrane is itself free standing and able to support itself or to
be supported by a peripheral structure and is disposed across an
aperture or is disposed between an anode and cathode. It is also
important in this instance that the biocompatible membrane itself
be dielectric and that it prevent the free flow of certain
components between the anode and cathode compartments such as
catholyte, electrolyte, cathode fuel, analyte anode fuel, other
ions, etc.
The next least complicated embodiment would involve the use
of a similar biocompatible membrane, but one that is either
incapable of preventing the complete intermixing of the necessary
species or which is not dielectric. In such an instance, an
additional barrier may be necessary. Such barriers can be made
of the same materials used to produce the substrate 42 described
earlier or, in the alternative, as illustrated in Figures 3d and
3e, the membrane can be disposed either in or covering
perforations or pores in a substrate 42. Materials useful for
the substrate and the methods of preparing same have already been
discussed.
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The anode electrode can be coated with an electron transfer
mediator such as an organometallic compound which functions as a
substitute electron recipient for the biological substrate of the
redox enzyme. Similarly, the biocompatible membrane of the
5 embodiment of Fig. 3 or structures adjacent to the biocompatible
membrane can incorporate such electron transfer mediators, or the
electron transfer mediator can be more generally available in the
anode chamber. Such organometallic compounds can include,
without limitation, dicyclopentadienyliron (CloHloFe, ferrocene,
10 available along with analogs that can be substituted, from
Aldrich, Milwaukee, WI), platinum on carbon, and' palladium on
carbon. Further examples include ferredoxin molecules of
appropriate oxidation/reduction potential, such as the ferredoxin
formed of rubredoxin and other ferredoxins available from Sigma
15 Chemical. Other electron transfer mediators include organic
compounds such as quinone and related compounds. Still further
electron transfer mediators are methylviologen, ethylviologen or
benzylviologen (CAS 1102-19-8; 1,1'-bis(phenylmethyl)-4,4'-
bipyridinium, N,N'-y,y'-dipyridylium), and any listed below in the
20 definition of electron transfer mediator.
The anode electrode (as opposed to the biocompatible
membrane) can be impregnated with an enzyme, which can be
applied before or after the electron transfer mediator. One way
to assure the association of the enzyme with the electrode is
25 simply to incubate a solution of the enzyme with electrode for
sufficient time to allow associations between the electrode and
the enzyme, such as Van der Waals associations, to mature.
Alternatively, a first binding moiety, such as biotin or its
binding complement avidin/streptavidin, can be attached to the
30 electrode and the enzyme bound to the first binding moiety
through an attached molecule of the binding complement.
Additional methods of attaching enzyme to electrodes or other
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materials, and additional electron transfer mediators are
described in Willner and Katz, Angew. Chem. Int. Ed. 39:1181-
1218, 2000. The anode chamber can include enzymes adjacent to or
associated with the anode electrode, or separate therefrom. For
example, a redox enzyme can be attached to the anode chamber side
of a polymer forming a proton conductive anode/cathode barrier,
with a layer of conductive material on the anode side providing
the anode electrode. In some embodiments of the invention, it is
anticipated that the electron carrier will be effective to
transfer electrons to the anode electrode in the absence of the
redox enzyme.
Some embodiments of the invention can, in addition, use a
traditional form of anode/cathode barrier: a polymeric membrane
selected for it ability to passively conduct protons in
conjunction with the biocompatible membrane of the invention.
The former anode/cathode barrier is useful since it is effective
to pump against a proton gradient.
Dual membranes can be disposed across and/or within the
perforations or pores of an anode/cathode barrier or can be
placed between the anode and cathode compartments. These
membranes can be of the traditional composition or biocompatible
membranes. One context in which such dual membranes are observed
are those in which the pores are of relatively narrow diameter.
Another context is one in which the anode cathode barrier is
formed of sandwiched materials such that separate junctions
between differing materials nucleate the formation of separate
biocompatible membranes across the pore.
Without limitation to theory, it is believed that the
second, more cathode proximate biocompatible membrane, operates
to some degree passively, as the pumping from the first
biocompatible membrane creates a high proton concentration,
driving passive transport to the cathode compartment. Thus, to
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the extent the cathode compartment contains peroxide that could
prospectively damage the transport protein, the active transport
function can be damaged, while the second biocompatible membrane
insulates the first from higher concentrations of the peroxide.
In one embodiment, the dual membrane benefit is obtained
with one or more biocompatible membranes, the first of which (at
the anode side) incorporates the polypeptide and a proton-
conductive polymeric membrane fitted at the cathode chamber side
to limit peroxide transit towards the biocompatible membranes.
Again, an intermediate zone between the biocompatible membranes)
and the proton-conductive polymeric membrane gains a high proton
concentration due to active transport, driving further transit
along a concentration gradient into the cathode compartment.
In one embodiment, the substrate in which the pores are
formed is a sandwich of dielectric Kapton, and conductive Kapton
(conductive through the presence of incorporated graphite). The
conductive Kapton can form the anode electrode, or be
appropriately metallized to form the anode electrode. The three
layers are relatively hydrophilic, relatively hydrophobic, then
relatively hydrophilic.
Figure 2 shows a schematic block diagram representation of
one embodiment of the anode side of a fuel cell in accordance
with the present invention. The anode compartment contains the
fuel. Fuel is an organic molecule that is depleted in accordance
with the present invention and consumed. However, its
consumption generates protons and electrons. Preferred fuels in
accordance with the present invention are compounds that are, or
can be transformed into, single carbon compounds. Preferred
amongst these is methanol. However, fuels can include, without
limitation, oxidizable sugars and sugar alcohols, alcohols,
organic acids such as pyruvates, succinates, etc., fatty acids,
lactic acids, citric acid, etc., amino acids and short
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53
polypeptides, aldehydes, ketones, etc. The fuel in this
embodiment is first acted upon by soluble enzymes that, in the
case of methanol, can be an alcohol dehydrogenase. As will be
seen below, other dehydrogenases such as aldehyde dehydrogenase
and formate dehydrogenase can also be used or can be used in
conjunction with one another. These soluble enzymes are capable
of acting upon the fuel to generate electrons and protons.
Soluble enzymes are preferably present in a range of between
about 0.001 to about 25,000 units, more preferably about 0.1
units to about 12,500 units, and most preferably from about 1
unit to about 12,5000 units. Units, in this context, refers to
the specific activity of the soluble enzyme and one unit of
activity is the amount required to convert 1 micromole of fuel
(or enzyme substitution in other contexts) per minute at 25°C.
"Soluble enzymes" is a bit of a misnomer as these compounds need
neither be soluble nor enzymes. These compounds may be suspended
or emulsified and/or immobilized on beads or some other solid
support. It really does not matter as long as they are
functional, can act upon the fuel, and allow electron carriers
and/or transfer mediators to carry protons to the biocompatible
membrane and electrons to the anode.
The protons and electrons are transferred by the coordinated
action of the soluble enzymes on the fuel to an electron carrier
also referred to as a cofactor. One such electron carrier is
NAD+/NADH. Electron carriers can include, without limitation,
reduced nicotinamide adenine dinucleotide (denoted NADH;
oxidized form denoted NAD or NAD+), reduced nicotinamide adenine
dinucleotide phosphate (denoted NADPH; oxidized form denoted
NADP or NADP+), reduced nicotinamide mononucleotide (NMNH;
oxidized form NMN), reduced flavin adenine dinucleotide (FADHz;
oxidized form FAD), reduced flavin mononucleotide (FMNHa;
oxidized form FMN), reduced coenzyme A, and the like. Electron
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54
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
the electrons and .protons that can be generated by the action of
the soluble .enzymes on the fuel. However, not all electron
carriers will convey protons. It will be recognized that C1
compounds comprising carbon oxygen and hydrogen are electron
carriers. However, these are also fuels. Also within the
definition of electron carrier are electron transfer mediators,
as specified below. Electron carriers, when present, generally
are provided in concentrations of between about 1 microMolar to
about 2 Molar, more preferably about 10 microMolar to about 1
Molar, and most preferably about 100 microMolar to about 500
milliMolar.
Under the influence of the soluble enzymes, protons and
electrons, NAD+ is converted to NADH. From this point, electrons
and/or protons can be handed off and traded between a number of
additional cofactors and/or transfer mediators. An electron
transfer mediator is a composition which facilitates transfer of
electrons released from an electron carrier to another molecule,
typically an electrode or another electron transfer mediator with
an equal or lower reduction potential. Examples, in addition to
those previously identified, include phenazine methosulfate
(PMS), pyrroloquinoline quinone (PQQ, also called 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 dicyclopentadienyliron
(ClOHIOFe, ferrocene). Electron transfer mediators, when
present, generally are provided in concentrations of between
about 1 microMolar to abut 2 Molar, more preferably between about
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10 microMolar and about 2 Molar, and even more preferably between
but 100 microMolar and about 2 Molar.
For simplicity, however, and as illustrated in Figure 2, the
reduced cofactor or electron carrier can next interact with the
5 polypeptides, in this case, the dehydrogenase function of Complex
I embedded in a biocompatible membrane in accordance with the
present invention. The Complex I liberates protons from the NADH
molecule, as well as electrons. The electrons might flow
directly to the anode. However, more often, they are taken up by
10 a transfer mediator, which then transports the electrons to the
anode.
NADH dehydrogenase Complex I is an interesting polypeptide
in that it also can participate in transporting protons across
the biocompatible membrane. What is particularly interesting,
15 however, is that the protons transferred are not necessarily the
protons liberated by the action of the dehydrogenase portion of
Complex I. Therefore, to be most successful, a fuel cell in
accordance with this particular aspect of the invention will
contain additional proton species in the anode compartment. The
20 proton transporting function of the Complex I is illustrated in
Figure 2, as the redox function.
When the transfer mediator gives up its electrons to the
anode, it has been oxidized, allowing it to be capable of
obtaining additional electrons liberated by oxidizing other
25 electron carriers. Oxidized cofactor (NAD+) is also now ready to
receive protons and electrons under the influence of fuel and the
soluble enzymes. The reactions just described occur at the anode
electrode and in the anode compartment and can be exemplified
chemically as follows.
30 Had + NADH NAD+ + H30+ + 2e
This reaction can be fed by the following reactions:
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ADH
CH30H + NAD+ ~ HCHO + H+ + NADH ( 4 )
ALD
HCHO + H20 + NAD+ ~ HCOZH + H+ + NADH (
FDH
HCOZH + NAD+ ~ COZ + H+ + NADH (
Total:
CH30H+ H20 +3 NAD+ ~ COZ + 3H+ + 3NADH ( 7 )
Thus, the reactions and the electron-generating reaction sum
as follows:
3NADH ~ 3NAD+ + 3H+ + 6e- ( 3 * )
CH30H+ H20 +3 NAD+ ~ COZ + 3H+ + 3NADH
(8)
Total
CH30H + H2O ~-~C20 + 6H+ + 6e- (
The soluble enzymes that can be used to generate a reduced
electron carrier (such as NADH as illustrated above) from an
organic molecule such as methanol can start with a form of
alcohol dehydrogenase (ADH). Suitable ADH enzymes are described
for example in Ammendola et al., "Thermostable NAD(+)-dependent
alcohol dehydrogenase from Sulfolobus solfataricus: gene and
protein sequence determination and relationship to other alcohol
dehydrogenases," Biochemistry 31: 12514-23, 1992; Cannio et al.,
"Cloning and overexpression in Escherichia coli of the genes
encoding NAD-dependent alcohol dehydrogenase from two Sulfolobus
species," J. Bacteriol. 178: 301-5, 1996; Saliola et al., "Two
genes encoding putative mitochondrial alcohol dehydrogenases are
present in the yeast Kluyveromyces lactis," Yeast 7: 391-400,
1991; and Young et al., "Isolation and DNA sequence of ADH3, a
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57
nuclear gene encoding the mitochondrial isozyme of alcohol
dehydrogenase in Saccharomyces cerevisiae," Mol. Cell Biol. 5:
3024-34, 1985. If the resulting formaldehyde is oxidized, an
aldehyde dehydrogenase (ALD) is used. Suitable ALD enzymes are
described for example in Peng et al., "cDNA cloning and
characterization of a rice aldehyde dehydrogenase induced by
incompatible blast fungus," GeneBank Accession AF323586; Sakano
et al., "Arabidopsis thaliana [thale cress] aldehyde
dehydrogenase (NAD+)-like protein" GeneBank Accession AF327426.
If the further resulting formic acid is oxidized, a formats
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 nonphotosynthetic
tissues as NAD-dependent formats dehydrogenase [from potato],"
Plant Physiol. 102(4): 1171-1177, 1993; Hourton-Cabassa,
"Evidence for multiple copies of formats dehydrogenase genes in
plants: isolation of three potato fdh genes, fdhl, fdh2, and
fdh3," Plant Physiol. 117: 719-719, 1998.
For reasons discussed below, it can be useful to use soluble
enzymes that are adapted to use or otherwise can accommodate
quinone-based electron carriers. Such enzymes are, for example,
described in: Pommier et al., "A second phenazine methosulphate
linked formats dehydrogenase isoenzyme in Escherichia coli,"
Biochim Biophys Acta. 1107(2):305-13, 1992. ("The diversity of
reactions involving formats dehydrogenases is apparent in the
structures of electron acceptors which include pyridine
nucleotides, 5-deazaflavin, quinones, and ferredoxin"); Ferry,
J.G. "Formats dehydrogenase" FEMS Microbiol. Rev. 7(3-4):377-82,
1990. (formaldehyde dehydrogenase with quinone activity); Klein
et al., "A novel dye-linked formaldehyde dehydrogenase with some
properties indicating the presence of a protein-bound redox-
active quinone cofactor" Biochem J. 301 ( Pt 1):289-95, 1994.
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58
(representative of a number of articles on dehydrogenases with
bound quinone cofactors); Goodwin et al., "The biochemistry,
physiology and genetics of PQQ and PQQ-containing enzymes" Adv.
Microb. Physiol. 40:1-80, 1998. (on alcohol dehydrogenases that
utilize quinones); Maskos et al., "Mechanism of p-nitrosophenol
reduction catalyzed by horse liver and human pi-alcohol
dehydrogenase (ADH)" J. Biol. Chem. 269(50):31579-84, 1994
(example of mediator-catalyzed transfer of electrons from NADH to
an electrode following NADH reduction by an enzyme); and Pandey,
"Tetracyanoquinodimethane-mediated flow injection analysis
electrochemical sensor for NADH coupled with dehydrogenase
enzymes" Anal. Biochem. 221(2):392-6, 1994.
The corresponding reaction at the cathode in the cathode
compartment can be any reaction that consumes the produced
electrons with a useful redox potential. Using oxygen, for
example, the reaction can be:
2H30+ + 1/2 OZ + 2e -~3H20 ( 2 )
Using reaction 2, the catholyte solution (an electrolyte
used in the cathode compartment) can be buffered to account for
the consumption of hydrogen ions, hydrogen ion donating compounds
can be supplied during operation of the fuel cell, or more
preferably, the barrier between the anode and cathode
compartments is sufficiently effective to deliver the
neutralizing hydrogen ions (hydrogen ion or proton).
In one embodiment, the corresponding reaction at the cathode
is:
H202 + 2H+ + 2e- H 2H~0 (10)
The cathode reactions result in a net production of water,
which, if significant, can be dealt with by, for example,
providing for space for overflow liquid, or providing for vapor-
phase exhaust. A number of electron acceptor molecules are often
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solids at operating temperatures or solutes in a carrier liquid,
in which case the cathode chamber should be adapted to carry such
non-gaseous material.
Where, as possibly the case with hydrogen peroxide as the
electron acceptor molecule, the electron acceptor molecule can
damage the polypeptides of the biocompatible membrane and any
other species in the anode chamber, and a scavenger for such
electron acceptor molecules can be used in the fuel cell to
prevent peroxide or damaging electron acceptor molecules from
entering the anode chamber. Such a scavenger can be, for
example, the enzyme catalase (2H2O2 TM 2HZO + Oz) , especially where
conditions at the anode electrode are not effective to catalyze
electron transfer to O~. Alternatively, the scavenger can be any
noble metal, such as gold or platinum. Such a scavenger, where
an enzyme, can be covalently linked to a solid support material.
Alternatively, a barrier between the anode chamber and the
cathode chamber is provided and has at most limited permeability
to hydrogen peroxide.
Solid oxidants, such as calcium peroxide, potassium
perchlorite (KC104) or potassium permanganate (KMn04), can be used
as the electron acceptor.
The fuel cell operates within a temperature range
appropriate for the operation of the redox enzyme or proton
transporter. This temperature range typically varies with the
stability of the enzyme, and the source of the enzyme. To
increase the appropriate temperature range, one can select the
appropriate redox enzyme from a thermophilic organism, such as a
microorganism isolated from a volcanic vent or hot spring.
Additionally genetically modified enzymes can be used.
Nonetheless, preferred temperatures of operation of at least the
first electrode are about 80°C or less, preferably 60°C or less.
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Preferred fuel cells in accordance with the present
invention include an anode compartment, a cathode compartment, an
anode and a cathode, as well as a biocompatible membrane
described herein including a polypeptide capable of participating
5 in the transfer of protons from one side of the membrane to the
other. At least one of an electron transfer mediator and an
electrode carrier are also found in the anode compartment. The
fuel cells of the present invention can preferably generate at
least about 10 milliwatts/cm2, more preferably at least about 50
10 milliwatts/cmz and most preferably at least about 100
milliwatts/cm~ over its useful life (there will be some diminished
output toward the end of its life). The fuel. cell will
preferably generate such power density until its fuel ultimately
runs out (unless they are refillable), but generally at least
15 eight hours, preferably one week, more preferably a month and
most preferably six months or more.
nvTner~r nc~
Example No. 1
A solution useful for producing a biocompatible membrane in
20 accordance with the present invention was produced as follows:
7% w/v (70 mg) of a block copolymer (poly (2-methyloxazoline)
polydimethyl siloxane-poly(2-methyl(oxazoline) having an average
molecular weight of 2KD-5KI7-2KD was dissolved in an 95%v/v /
5%v/v ethanol/water solvent mixture with stirring using a
25 magnetic stirrer. Six microliters of this solution was removed
and mixed with four microliters of a solution containing 0.015%
w/v dodecyl maltoside, 40 micrograms of Complex I (10 mg/ml) in
water. This is then mixed. The resulting solution contains 4.2%
w/v polymer, 55% EtOH v/v, 45% HZO v/v, 0.06o w/v dodecyl
30 maltoside and protein/polymer ratio is 6% w/w.
Example No. 2
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A solution useful for producing a biocompatible membrane
in
accordance with the present invention was prepared generally
as
described in Example No. 1 with the following changes: less
polypeptide solution was used so as provide a final solution
to
including 0.015% w/v dodecyl maltoside and 1.5o w/w polypeptide
relative to synthetic polymer materials.
Example No. 3
A solution useful for producing a biocompatible membrane
in
accordance with the present invention
was prepared generally as
described in Example No. 1 with the following changes: less
polypeptide solution was used so as provide a final solution
to
including 0.03% w/v dodecyl maltoside and the final solution
contained 3.0% w/w polypeptide relative
to synthetic polymer
materials.
Example No. 4
A solution useful for producing a biocompatible membrane
in
accordance with the present invention
was prepared generally as
described in Example No. 1 with the following changes: less
polypeptide solution was used so as provide a final solution
to
including 0.045w/v dodecyl maltoside and the final solution
contained 4.5% w/w polypeptide relati ve to synthetic polymer
materials.
Example No. 5
A solution useful for producing a biocompatible membrane
in
accordance with the present invention
was prepared generally as
described in Example No. 1 with the following changes: less
polypeptide solution was used so as provide a final solution
to
including 0.0075w/v dodecyl maltoside and the final solution
contained 0.75% w/w polypeptide relative
to synthetic polymer
materials.
Example No. 6
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A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generallyas
described in Example No. 5 with the following changes: the
synthetic polymer material was originally present in a solution
of 5.0% w/v. Sufficient polypeptide solution of the t ype
described in Example 1 was added so as to produce a fi nal
solution including 0.0075% w/v dodecyl maltoside and 0.75a w/w
polypeptide relative to synthetic polymer materials.
Example No. 7
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generallyof
the type described in Example No. 6 with the following changes:
sufficient polypeptide solution as described in Example 1 was
included so as to produce a final solution including 0.015% w/v
dodecyl maltoside and the final solution contained 1.5% w/w
polypeptide relative to synthetic polymer materials.
Example No. 8
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generallyof
the type described in Example No. 6 with the following changes:
sufficient polypeptide solution as described in Example 1 was
included so as to produce a final solution including 0.030 w/v
dodecyl maltoside and the final solution contained 3% w/w
polypeptide relative to synthetic polymer materials.
Example No. 9
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generallyof
the type described in Example No. 6 with the following changes:
sufficient polypeptide solution as described in Example 1 was
included so as to produce a final solution including 0.045% w/v
dodecyl maltoside and the final solution contained 4.5% w/w
polypeptide relative to synthetic polymer materials.
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63
Example No. 10
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally of
the type described in Example No . 6 with the following changes
sufficient polypeptide solution as described in Example 1 was
included so as to produce a final solution including 0.06% w/v
dodecyl maltoside and the final solution contained 6.Oa w/w
polypeptide relative to synthetic polymer materials.
Examples Nos. 11-15
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Example Nos. 1-5 respectively except that the amount
of the synthetic polymer material used in each solution was
originally 10% w/v. When 6 microliters of that solution was
mixed with sufficient polypeptide solution of the type described
in Example 1 a final solution was produced including .06, 0.15,
.03, .045 and .0075% w/v dodecyl maltoside and 6.0, 1.5, 3.0, 4.5
and 0.750 w/w polypeptide relative to synthetic polymer
materials, respectively.
Example No. 16
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally as
described in Example No. 3, however, the solvent used to dissolve
the synthetic polymer material included ethanol, 25% methanol v/v
and the amount of water indicated in Example No. 3. Sufficient
polypeptide solution was used so as to provide a final solution
including 0.03a w/v dodecyl maltoside and 3.Oo w/w polypeptide
relative to synthetic polymer materials.
Example No. 17
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally as
described in Example No. 2, however, the solvent used to dissolve
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64
the synthetic polymer material included 47.5% v/v ethanol, 2.5%
v/v water, 25o v/v Tetrahydrofuran ("THF"), 25% v/v
dichloromethane. Sufficient polypeptide solution was used so as
to provide a final solution including 0.015.% w/v dodecyl
maltoside and 1.5% w/w polypeptide relative to synthetic polymer
materials.
Example No. 18
A solution useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Example No. 6, however, the solvent used to
dissolve the synthetic polymer material included 9.5o v/v
ethanol, 0.5o v/v water, 40% v/v acetone, and 40% v/v hexane.
Examples Nos. 19-24
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Example Nos. 11-15 above, however, the final
concentration of dodecyl maltoside was 0.15% w/v. Solutions
useful for producing a biocompatible membrane in accordance with
the present invention can be prepared generally as described in
Example No. 4 above, however, the balance of the surfactant used
in the polypeptide solution is dodecyl (3-D-glucopyranoside and
the final concentration of the surfactants is 0.15% w/v.
Example No. 26
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally as
described in Example No. 9 above, however, the surfactant used in
the polypeptide solution included a mixture of a polymeric
surfactant sold under the trademark PLURONIC L101, lot WPDX-522B
from BASF, Ludwigshafen Germany and the same concentration of
dodecyl maltoside specified in Example No. 9. The polymeric
surfactant was diluted to 0.1%v/v of its supplied concentration
in the final solution.
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Example No. 27
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally as
described in Example No. 2 above, however the surfactant used in
5 the polypeptide solution included a mixture of a polymeric
surfactant sold under the trademark DISPERPLAST, lot no. 31J022
from BYK Chemie, Wallingford CT and the same concentration of
dodecyl maltoside specified in Example No. 2. The polymeric
surfactant was diluted to 0.135%v/v of the supplied concentration
10 in the final solution.
Examples Nos. 28-32
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Example Nos. 6-10 respectively, however, the
15 synthetic polymer material used can be a poly(2-methyloxazoline)-
polydimethylsiloxane-poly(2-methyloxazoline) (5o w/v) having an
average molecular weight of 3kD-7kD-3kD. When 6 microliters of
that solution is mixed with sufficient polypeptide solution of
the type described in Example 1 a final solution is produced
20 including 0.0075, 0.015, 0.030, 0.045 and 0.0600 w/v dodecyl
maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w polypeptide
relative to synthetic polymer materials respectively.
Examples Nos. 33-38
Solutions useful for producing a biocompatible membrane in
25 accordance with the present invention were prepared generally as
described in Example Nos. 1-5 respectively, however, the
synthetic polymer material used was a mixture of two block
copolymers, both of which were poly(2-methyloxazoline)
polydimethylsiloxane-poly(2-methyloxazoline), (total 7% w/v) one
30 of which having an average molecular weight of 2kD-5kD-2kD and
the other 1kD-2kD-1kD and the ratio of the first block copolymer
to the second was about 67% to 33% of the total polymer used w/w.
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When 6 microliters of that solution was mixed with sufficient
polypeptide solution of the type described in Example 1 a final
solution was produced including 0.06, 0.015, 0.030, 0.045 and
0.00750 w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.Oa
w/w polypeptide relative to synthetic polymer materials
respectively.
Examples Nos. 39-43
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Example Nos. 11-15 respectively, however, the
synthetic polymer material used can be a mixture of two block
copolymers, both of which are poly(2-methyloxazoline)-
polydimethylsiloxane-poly(2-methyloxazoline), (10% w/v) one of
which having an average molecular weight of 1kD-2kD-1kD and the
other 3kD-7kD-3kD and the ratio of the first block copolymer to
the second being about 330 to 67% of the total polymer used w/w.
When 6 microliters of that solution is mixed with sufficient
polypeptide solution of the type described in Example 1 a final
solution is produced including 0.075, 0.15, 0.30, 0.45 and 0.60%
w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w
polypeptide relative to synthetic polymer materials respectively.
Examples Nos. 44-48
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 6-10 respectively, however, the
synthetic polymer material used can be a mixture of two block
copolymers, both of which are poly(2-methyloxazoline)-
polydimethylsiloxane-poly(2-methyloxazoline), (5% w/v) one of
which having an average molecular weight of 2kD-5kD-2kD and the
other 3kD-7kD-3kD and the ratio of the first block copolymer to
the second being about 330 to 67% of the total polymer used w/w.
When 6 microliters of that solution is mixed with sufficient
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polypeptide solution of the type described in Example 1 a final
solution is produced including 0.0075, 0.015, 0.030, 0.045 and
0.060% w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w
polypeptide relative to synthetic polymer materials respectively.
Examples Nos. 49-53
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Example Nos. 1-5 respectively, however, the
synthetic polymer material used can be a mixture of two block
copolymers, both of which are poly(2-methyloxazoline)-
polydimethylsiloxane-poly(2-methyloxazoline), (7% w/v) one of
which having an average molecular weight of 2kD-5kD-2kD and the
other 3kD-7kD-3kD and the ratio of the first block copolymer to
the second being about 67% to 33% of the total polymer used w/w.
When 6 microliters of that solution is mixed with sufficient
polypeptide solution of the type described in Example 1 a final
solution is produced including 0.06, 0.015, 0.030, 0.045 and
0.0075% w/v dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and .025%
w/w polypeptide relative to synthetic polymer materials
respectively.
Examples Nos. 54-58
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 1-5 respectively, however, the
synthetic polymer used can be a mixture of poly(2-
methyloxazoline)-polydimethylsiloxane- poly(2-methyloxazoline)
(7% w/v) having an average molecular weight of 2kD-5kD-2kD in a
solvent of 95o ethanol, 5% water mixed with a solution of 23.50
w/v polyethyleneglycol with an average molecular weight of
approximately 3,300 Daltons in water in the proportion of 85%
triblock copolymer solution, 15% polyethyleneglycol solution v/v.
When 6 microliters of that solution is mixed with sufficient
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polypeptide solution of the type described in Example 1 a final
solution is produced including 0.06, 0.015, 0.030, 0.045 and
0.0075% w/v dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and .75% w/w
polypeptide relative to synthetic polymer materials respectively.
Examples Nos. 59-63
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally as
described in Example No. 12, however, the synthetic polymer used
was a mixture of loo w/v of poly(2-methyloxazoline)-
polydimethylsiloxane- poly(2-methyloxazoline) having an average
molecular weight of 2kD-5kD-2kD in a solvent of 95% ethanol, 50
water mixed with a solution of 23.5% w/v polyethyleneglycol with
an average molecular weight of approximately 8,000 Daltons in
water in the proportion of 85o triblock copolymer solution, 15%
polyethyleneglycol solution v/v. When 6 microliters of that
solution was mixed with sufficient polypeptide solution of the
type described in Example 1 a final solution was produced
including 0.15% w/v dodecyl maltoside and 1.5% w/w polypeptide
relative to synthetic polymer materials. Similar solutions can
be made using the procedures of examples 11 and 13-15.
Examples Nos. 64-68
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 28-32 respectively, however, the
synthetic polymer used can be a mixture of 5% w/v of poly(2-
methyloxazoline)-polydimethylsiloxane- poly(2-methyloxazoline)
having an average molecular weight of 3kD-7kD-3kD in a solvent of
95o ethanol, 5% water mixed with a solution of 23.5% w/v
polyethyleneglycol with an average molecular weight of
approximately 3,300 Daltons in water in the proportion of 85%
triblock copolymer solution, 15% polyethyleneglycol solution v/v.
When 6. microliters of that solution is mixed with sufficient
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polypeptide solution of the type described in Example 1 a final
solution is produced including 0.0075, 0.015, 0.030, 0.045 and
0.060% w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w
polypeptide relative to synthetic polymer materials respectively.
Examples Nos. 69-73
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 1-5 respectively, however, the
synthetic polymer used can be a mixture of 7% w/v of poly(2-
methyloxazoline)-polydimethylsiloxane- poly(2-methyloxazoline)
having an average molecular weight of 3kD-7kD-3kD in a solvent of
95% ethanol, 5o water mixed with a solution of 23.5% w/v
polyethyleneglycol with an average molecular weight of
approximately 8,000 Daltons in water in the proportion of 850
triblock copolymer solution, 15% polyethyleneglycol solution v/v.
When 6 microliters of that solution is mixed with sufficient
polypeptide solution of the type described in Example 1 a final
solution is produced including 0.060, 0.015, 0.030, 0.045 and
0.00750 w/v dodecyl maltoside and 6.0, 1.5, 3.0, 4.5 and 0.75%
w/w polypeptide relative to synthetic polymer materials
respectively.
Examples Nos. 74-78
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 6-10 respectively, however the
synthetic polymer used can be a mixture of 5% w/v of poly(2-
methyloxazoline)-polydimethylsiloxane- poly(2-methyloxazoline)
having an average molecular weight of 2kD-5kD-2kD in a solvent of
50%v/v acetone, 50ov/v heptane mixed with a solution of 5o w/v
polystyrene of about 250,000 in molecular weight in 50%v/v
acetone, 50av/v octane in the proportion of 80%v/v block
copolymer, 20% v/v polystyrene. When 6 microliters of that
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solution is mixed with sufficient polypeptide solution of the
type described in Example 1 a final solution is produced
including 0.0075, 0.015, 0.030, 0.045 and 0.060% w/v dodecyl
maltoside and 0.75, 1.5, 3.0, 4.5 and 6.Oo w/w polypeptide
5 relative to synthetic polymer materials respectively.
Examples Nos. 79-83
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 1-5 respectively, however, the
10 synthetic polymer used can be a mixture of 7o w/v of poly(2-
methyloxazoline)-polydimethylsiloxane- poly(2-methyloxazoline)
having an average molecular weight of 2kD-5kD-2kD in a solvent of
95% ethanol, 5% water mixed with a solution of 5% w/v of
polymethylmethacrylate-polydimethylsiloxane-
15 polymethylmethacrylate having an average molecular weight of ,4kD-
8kD-4kD in a solvent of 50%v/v THF, 50%v/v dichloromethane in the
proportion of 66%v/v to 33%v/v, respectively. When 6 microliters
of that solution is mixed with sufficient polypeptide solution of
the type described in Example 1 a final solution is produced
20 including 0.06, 0.015, 0.030, 0.045 and 0.00750 w/v dodecyl
maltoside and 6.0, 1.5, 3.0, 4.5 and 0.075% w/w polypeptide
relative to synthetic polymer materials respectively.
Examples Nos. 84-88
Solutions useful for producing a biocompatible membrane in
25 accordance with the present invention were prepared generally as
described in Examples Nos. 11-15 respectively, however, the
synthetic polymer material used was 10% w/v of sulfonated
styrene/ethylene-butylene/sulfonated styrene, supplied as
Protolyte° A700, lot number LC-29/60-011 by Dais Analytic,
30 Odessa, FL in solvent as supplied, diluted 50%v/v with ethanol
containing 5%v/v water. When 6 microliters of that solution was
mixed with Buff icient polypeptide solution of the type described
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in Example 1 a final solution was produced including 0.0075,
0.015, 0.030, 0.045 and 0.060% w/v dodecyl maltoside and 0.75,
1.5, 3.0, 4.5 and 6.0% w/w polypeptide relative to synthetic
polymer materials respectively.
Example No. 89
A solution useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Example No. 84 however, the solvent used to
dilute the synthetic polymer material can include 50% v/v
Tetrahydrofuran (°THF~~), 50o v/v dichloromethane.
Examples Nos. 90-94
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Examples Nos. 84-88 above, however, the final
concentration of dodecyl maltoside was 0.15% w/v.
Example No. 95
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Example No. 85 above, however, the surfactant
used in the polypeptide solution can include a mixture of dodecyl
(3-D-glucopyranoside and dodecyl maltoside and the final
concentration of the surfactants is 0.15% w/v.
Example No. 96
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally as
described in Example No. 87 above, however, the surfactant used
in the polypeptide solution included a mixture of a polymeric
surfactant sold under the trademark PLURONIC L101, lot WPDX-522B
from BASF, Ludwigshafen Germany and the same concentration of
dodecyl maltoside specified in Example No. 87. The polymeric
surfactant was diluted to 0.1%v/v of its supplied concentration
in the final solution.
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Example No. 97
A solution useful for producing a biocompatible membrane in
accordance with the present invention was prepared generally as
described in Example No. 88 above, however, the surfactant used
in the polypeptide solution included a mixture of a polymeric
surfactant sold under the trademark DISPERPLAST, lot no. 31J022
from BYK Chemie, Wallingford CT and the same concentration of
dodecyl maltoside specified in Example No. 88. The final
concentration of the polymeric surfactant was diluted to
0.135ov/v of the supplied concentration in the final solution.
Examples No. 98-102
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Examples Nos. 84-88 respectively, however, the
synthetic polymer material used was a mixture of two block
copolymers, one of which was 10% w/v of sulfonated
styrene/ethylene-butylene/sulfonated styrene, supplied as
Protolyte~ A700, lot number LC-29/60-011 by Dais Analytic,
Odessa, FL in solvent as supplied, diluted 50%v/v with ethanol
containing 5%v/v water, the other of which was 5% w/v of poly(2-
methyloxazoline)-polydimethylsiloxane-poly(2-methyloxazoline)
having an average molecular weight of 2kD-5kD-2kD and the ratio
of the first block copolymer to the second was about 67% to 330
of the total polymer used w/w. When 6 microliters of that
solution was mixed with sufficient polypeptide solution as
described in Example 1 a final solution was produced including
0.0075, 0.015, 0.030, 0.045 and 0.0600 w/v dodecyl maltoside and
0.75, 1.5, 3.0, 4.5 and 6.0% w/w polypeptide relative to
synthetic polymer materials respectively.
Examples Nos. 103-107
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
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described in Examples Nos. 84-88 respectively, however, the
synthetic polymer material used was a mixture of two block
copolymers, one of which was 10% w/v of sulfonated
styrene/ethylene-butylene/sulfonated styrene, supplied as
Protolyte~ A700, lot number LC-29/60-011 by Dais Analytic,
Odessa, FL in solvent as supplied, diluted 50%v/v with ethanol
containing 5%v/v water, the other of which was 5% w/v of poly(2-
methyloxazoline)-polydimethylsiloxane-poly(2-methyloxazoline)
having an average molecular weight of 2kD-5kD-2kD and the ratio
of the first block copolymer to the second was about 33% to 67 0
of the total polymer used w/w. When 6 microliters of that
solution was mixed with sufficient polypeptide solution as
described in Example 1 a final solution was produced including
0.0075, 0.015, 0.030, 0.045 and 0.060a w/v dodecyl maltoside and
0.75, 1.5, 3.0, 4.5 and 6.0% w/w polypeptide relative to
synthetic polymer materials respectively.
Examples Nos. 108-112
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 103-107 respectively, however, the
synthetic polymer material used can be a mixture of two block
copolymers, one of which is 10% w/v of sulfonated
styrene/ethylene-butylene/sulfonated styrene, supplied as
Protolyte~ A700, lot number LC-29/60-011 by Dais Analytic,
Odessa, FL in solvent as supplied, diluted 50%v/v with ethanol
containing 5%v/v water, the other of which is 5% w/v
of polymethylmethacrylate-polydimethylsiloxane-
polymethylmethacrylate having an average molecular weight of 4kD-
8kD-4kD in a solvent mixture of 50%v/v THF, 50% v/v
dichloromethane, the ratio of the first block copolymer to the
second being about 67% to 33% of the total polymer used w/w. When
6 microliters of that solution is mixed with sufficient
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polypeptide solution of the type described in Example 1 a final
solution is produced including 0.0075, 0.015, 0.030, 0.045 and
0.060% w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w
polypeptide relative to synthetic polymer materials respectively.
Examples Nos. 113-117
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 103-107 respectively, however, the
synthetic polymer material used can be a mixture of two block
copolymers, one of which is 10% w/v of sulfonated
styrene/ethylene-butylene/sulfonated styrene, supplied as
Protolyte~ A700, lot number LC-29/60-011 by Dais Analytic,
Odessa, FL in solvent as supplied, diluted 50ov/v with ethanol
containing 5%v/v water, the other of which is 5% w/v of
polymethylmethacrylate-polydimethylsiloxane-
polymethylmethacrylate having an average molecular weight of 4kD-
8kD-4kD in a solvent mixture of 50%v/v THF, 50% v/v
dichloromethane, the ratio of the first block copolymer to the
second being about 33o to 67% of the total polymer used w/w. When
6 microliters of that solution is mixed with sufficient
polypeptide solution of the type described in Example 1 a final
solution is produced including 0.0075, 0.015, 0.030, 0.045 and
0.060% w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w
polypeptide relative to synthetic polymer materials respectively.
Examples Nos. 118-122
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Examples Nos. 84-88 respectively, however, the
synthetic polymer material used was a mixture of 10a w/v of
sulfonated styrene/ethylene-butylene/sulfonated styrene, supplied
as Protolyte~ A700, lot number LC-29/60-011 by Dais Analytic,
Odessa, FL in solvent as supplied, diluted 50%v/v with ethanol
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containing 5%v/v water mixed with a solution of 23.5% w/v
polyethyleneglycol with an average molecular weight of
approximately 3,300 Daltons in water in the proportion of 85a
triblock copolymer solution, 15% polyethyleneglycol solution v/v.
5 When 6 microliters of that solution was mixed with sufficient
polypeptide solution of the type described in Example 1 a final
solution was produced including 0.0075, 0.015, 0.030, 0.045 and
0.060% w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w
polypeptide relative to synthetic polymer materials respectively.
10 Examples Nos. 123-127
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Examples Nos. 84-88 respectively, however, the
synthetic polymer material used was a mixture of 100 w/v of
15 sulfonated styrene/ethylene-butylene/sulfonated styrene, supplied
as Protolyte° A700, lot number LC-29/60-O11 by Dais Analytic,
Odessa, FL in solvent as supplied, diluted 50%v/v with ethanol
containing 5%v/v water mixed with a solution of 23.5% w/v
polyethyleneglycol with an average molecular weight of
20 approximately 8,000 Daltons in water in the proportion of 85%
triblock copolymer solution, 15% polyethyleneglycol solution v/v.
When 6 microliters of that solution was mixed with sufficient
polypeptide solution of the type described in Example 1 a final
solution was produced including 0.0075, 0.015, 0.030, 0.045 and
25 0.0600 w/v dodecyl maltoside and 0.75, 1.5, 3.0, 4.5 and 6.0% w/w
polypeptide relative to synthetic polymer materials respectively.
Examples Nos. 128-132
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
30 as described in Examples Nos. 6-10 respectively, however, the
synthetic polymer material used can be 5% w/v of
polymethylmethacrylate-polydimethylsiloxane-
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polymethylmethacrylate having an average molecular weight of 4kD-
8kD-4kD in a solvent mixture of 50ov/v THF, 50% v/v
dichloromethane. When 6 microliters of that solution is mixed
with sufficient polypeptide solution of the type described in
Example 1 a final solution is produced including 0.0075, 0.015,
0.030, 0.045 and 0.060% w/v dodecyl maltoside and 0.75, 1.5, 3.0,
4.5 and 6.0% w/w polypeptide relative to synthetic polymer
materials respectively.
Examples Nos. 133-134
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Examples Nos. 6 and 7 respectively, however, the
synthetic polymer material used was 3.2o w/v of polystyrene-
polybutadiene-polystyrene, supplied as Stryolux° 3655, lot
7453064P by BASF, Ludwigshafen Germany in a 50%/50%v/v mixture of
acetone and hexane. When 6 microliters of that solution was mixed
with sufficient polypeptide solution of the type described in
Example 1 a final solution was produced including 0.0075 and
0.015% w/v dodecyl maltoside and 0.75 and 1.5% w/w polypeptide
relative to synthetic polymer materials respectively.
Examples Nos. 135-136
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Examples Nos. 6 and 7 respectively, however, the
synthetic polymer material used was 3.2% w/v of polystyrene-
polybutadiene-polystyrene, supplied as Stryolux~ 3655, lot
7453064P by BASF, Ludwigshafen Germany in a 50%/50%v/v mixture of
acetone and heptane. When 6 microliters of that solution was
mixed with sufficient polypeptide solution of the type described
in Example 1 a final solution was produced including 0.0075 and
0.015% w/v dodecyl maltoside and 0.75 and 1.5% w/w polypeptide
relative to synthetic polymer materials respectively.
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Example Nos. 137-138
Solutions useful for producing a biocompatible membrane in
accordance with the present invention were prepared generally as
described in Examples Nos. 135 and 136 respectively, however, the
synthetic polymer material used was 5% w/v of polystyrene-
polybutadiene-polystyrene, supplied as Stryolux~ 3655, lot
7453064P by BASF, Ludwigshafen Germany in a 50x/50%v/v mixture of
acetone and heptane. When 6 microliters of that solution was
mixed with sufficient polypeptide solution of the type described
in Example 1 a final solution was produced including 0.0075 and
0.0150 w/v dodecyl maltoside and 0.75 and 1.5% w/w polypeptide
relative to synthetic polymer materials respectively.
Examples Nos. 139-141
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 6-8 respectively, however, the
synthetic polymer material used can be a mixture of 5% w/v of
polystyrene-polybutadiene-polystyrene, supplied as Stryolux°
3655, lot 7453064P by BASF, Ludwigshafen Germany in a 50%/50%v/v
mixture of acetone and hexane and 5% w/v poly(2-methyloxazoline)-
polydimethylsiloxane-poly(2-methyloxazoline) having an average
molecular weight of 2kD-5kD-2kD in the same solvent in the
proportion of about 80%v/v to 20% v/v, respectively. When 6
microliters of that solution is mixed with sufficient polypeptide
solution of the type described in Example 1 a final solution is
produced including 0.0075, 0.015, and 0.030% w/v dodecyl
maltoside and 0.75, 1.5 and 3.0% w/w polypeptide relative to
synthetic polymer materials respectively.
Examples Nos. 142-145
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 139-141 respectively, however, the
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synthetic polymer material used can be a mixture of 5o w/v of
polystyrene-polybutadiene-polystyrene, supplied as Stryolux°
3655, lot 7453064P by BASF, Ludwigshafen Germany in a 50%/50%v/v
mixture of acetone and hexane and 5% w/v poly(2-methyloxazoline)-
polydimethylsiloxane-poly(2-methyloxazoline) having an average
molecular weight of 3kD-7kD-3kD in the same solvent in the
proportion of about 80%v/v to 20% v/v, respectively. When 6
microliters of that solution is mixed with sufficient polypeptide
solution of the type described in Example 1 a final solution is
produced including 0.0075, 0.015, and 0.030% w/v dodecyl
maltoside and 0.75, 1.5 and 3.0% w/w polypeptide relative to
synthetic polymer materials respectively.
Examples Nos. 146-290
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 1-145, respectively, however, the
polypeptide solution mixed with the synthetic polymer can be a
solution of lOmg/ml of Succinate:ubiquinone oxidoreductase
(Complex II) in water which also can include 0.15% Thesit
(polyoxyethylene(9)dodecyl ether, ClzE9) available from Roche,
Indianapolis, IN. This surfactant replaces, in general, the
dodecyl maltoside in examples 1-145 in similar concentration.
Examples Nos. 291-435
Solutions useful for producing a biocompatible membrane in
accordance with the present invention can be prepared generally
as described in Examples Nos. 1-145, respectively, however, the
polypeptide solution used to dilute the synthetic polymer can be
a solution of lOmg/ml of Nicotinamide Nucleotide Transhydrogenase
in water which also can include 0.15% Triton X-100. This
surfactant replaces, in general, the dodecyl maltoside in
examples 1-145 in similar concentration. Furthermore, in examples
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1-145 which include dodecyl (3-D-glucopyranoside, this detergent
can be substituted with Nonidet P-40 in similar concentration.
Example 436
Membranes are formed on a dielectric perforated support.
The support is made of KAPTON available from DuPont (1 mil thick)
and is laser-drilled with apertures of 100 micrometers in
diameter and 1 mil deep. The array of apertures can have a
density as high as 1,700 apertures/cm2. A biocompatible membrane
is formed across the apertures using the PEG 8000/PROTOLYTE A700
membrane described in detail previously. The resulting final
solution containing the block copolymer, stabilizing polymer and
polypeptide is then deposited onto the substrate in a manner that
completely covered the apertures, dropwise by pipet, 4
microliters at a time. The solvent was allowed to evaporate at
room temperature under a hood. The membrane-support assembly was
stored in a vacuum chamber prior to use.
A test device, in this case a fuel cell, was constructed
from DELRAN plastic. The membrane-support assembly produced as
described above was sealed in place within the fuel cell with
rubber gaskets to form two chambers, an anode compartment and a
cathode compartment. The anode and cathode compartments were then
filled (20 ml in each) with an aqueous electrolyte (1M TMA-
formate pH 10 in the anode compartment and 100 mM TMA-sulfate, pH
2.0, containing 1% hydrogen peroxide in the cathode compartment).
A titanium foil anode was connected in parallel to an
electronically varied load. A computer with an analog/digital
board was used to measure current and voltage output. The circuit
was completed by wiring these elements to a graphite cathode
electrode in the cathode compartment.
The titanium foil anode was immersed in the anolyte. Also
contained in the anode compartment was 5% v/v methanol as fuel,
12.5mM NAD+ was used as electron carrier, 1M hydroquinone was
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used as 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 produced consistent with the function of Complex
5 I embedded in the biocompatible membrane in translocating protons
from the anode compartment to the cathode compartment, even
against the proton concentration gradient. Peak current density
was 158 mA/cm2. The membrane was stable for approximately 3 days.
By comparison, in another cell formed using the same
10 components and concentrations as above, with the exception that
the membrane-forming solution did not include PEG 8000, the peak
current density was similar. However, the membrane integrity was
limited to 10-12 hours. Membrane failure was assessed via visible
flow of the mediator into the cathode compartment.
15 In the absence of Complex I in the membrane, the Protolyte
block copolymer nonetheless forms membranes which are modestly
permeable to protons. The use of such membranes formed without
Complex I in a fuel cell, constructed similarly to those above,
with the exception that 300 mM PMS was present in the anode as
20 the electron transfer mediator instead of hydroxyquinone,
produced maximally 4mA/cm~ for only a matter of approximately 5
minutes before decreasing in output rapidly.
Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
25 these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be
devised without departing from the spirit and scope of the
30 present invention as defined by the appended claims.
INDUSTRIAL APPLICABILITY
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81
The invention relates to the industries that produce and use
membranes including, without limitation, the fuel cell industry.
The invention is also applicable to the industries that use fuel
cells such as the automotive and electronic industries.
