WO2011084540A1 - Bubble architectures and methods of making and using thereof - Google Patents

Bubble architectures and methods of making and using thereof Download PDF

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Publication number
WO2011084540A1
WO2011084540A1 PCT/US2010/060610 US2010060610W WO2011084540A1 WO 2011084540 A1 WO2011084540 A1 WO 2011084540A1 US 2010060610 W US2010060610 W US 2010060610W WO 2011084540 A1 WO2011084540 A1 WO 2011084540A1
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Prior art keywords
bubble
μηι
sol
gel
secondary component
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PCT/US2010/060610
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French (fr)
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Carlo Montemagno
David Wendell
Hyo-Jick Choi
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Heliotronic Energy Systems, Llc
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Publication of WO2011084540A1 publication Critical patent/WO2011084540A1/en
Priority to US13/446,880 priority Critical patent/US8647853B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/16—Enzymes or microbial cells immobilised on or in a biological cell
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group

Definitions

  • the present invention relates to bubble architectures and methods of making and using such bubble architectures, wherein the bubble architectures are formed using biologically derived surfactant, for example, the protein Ranaspumin-2 and other biologically derived surfactants to creation functional materials that mimic cellular physiological processes.
  • the bubble architecture is used to form an artificial photosynthesis platform for converting light and CO2 to a value-added product, for example, simple sugar.
  • DMF 2,5- dimethylfuran
  • Engineered biological solar energy conversion has produced a variety of electrical and chemical energy storage strategies.
  • ATP serves as the most important natural energy molecule and has been formed artificially by coupling F 0 F1 ATP synthase to a photon induced proton motive force.
  • photo synthetic organisms long term energy storage is accomplished through biomass synthesis through ATP dependent carbon-fixation providing a foundation for liquid biofuel production.
  • CBB cycle intermediates and cell extracts using radiometric and spectrophotometric techniques Photosynthesis, carbon sequestration and carbohydrate generation involve several complex and well-studied processes; among these, a suite of 8 enzymes make up the portion of the CBB cycle responsible for converting the energy of ATP into 6-carbon sugars such as glucose and fructose.
  • the chief products of the light-dependent reaction of photosynthesis are NADPH and ATP.
  • the thermophilic FoFi ATP synthase has been purified and reconstituted in both liposomes and ABA triblock polymersomes, along with the photoactivated proton pump bacteriorhodopsin (BR) to form ATP producing vesicles.
  • BR photoactivated proton pump bacteriorhodopsin
  • Bubbles are natural structures that are encountered in everyday life such as dishwashing foam or beer foam. While bubbles are common, they are deceptively complex structures, typically composed of a water layer sandwiched between two- surfactant monolayers. Despite their everyday appearance, bubbles and foams have been interesting research topics to scientists for the past several centuries, where many have sought to understand and utilize the chemical, physical, and mechanical properties of bubbles. Their applications, however, have been limited by their innate properties of drainage and uncontrollable size distributions. Recently, it has become possible to produce a microfoam, having no vertical drainage, from monodisperse stable microbubbles (Garstecki et al, Appl Phys Lett 2004, 85 :2649).
  • MEMS semiconductor device technology
  • foam nests are used by a variety of organisms (e.g., fish, amphibians and insects) is quite impressive, given the biological necessities required to persist in the environment. These include resistance to microbial and insect assault, resilience to changes in heat, humidity and desiccation, but continue to be compatible with exposed eggs and sperm. The latter requirement presents a remarkable paradox, since surfactant used to produce stable bubble films would by its very nature also destabilize and destroy the cell membranes and proteins necessary for reproduction.
  • the foam nest produced by the Tungara frog is one of the largest found in nature. It is used to protect developing tadpoles in terrestrial areas of tropical and subtropical Central America, until maturation or greater water availability.
  • the creation and maintenance of the Tungara frog's foam nest can be attributable to a small but astonishing suite of six proteins called ranaspumins (Rsnl-6). Of these, it is Rsn2 which is responsible for the reduction in water surface tension allowing foam creation upon liquid agitations.
  • the other ranaspumins offer an arsenal of microbio and insecticides, as well as carbohydrate binding proteins which help stabilize the foams to drainage and desiccation.
  • Rsn-2 plays the surfactant role very economically at 0.1 g/ml, but also has the ability to exist in two conformational states (see Mackenzie, CD., et al., Ranaspumin-2 ; Structure and Function of a Surfactant Protein from the Foam Nests of a Tropical Frog. Biophysical Journal, 2009. 96(12); p.4984-4992, the contents of which are expressly incorporated herein in its entirety). When agitated, the protein denatures slightly, allowing the single hydrophobic alpha helix to extend into the air while the hydrophilic beta sheet remains in the water phase.
  • the blue foam nests of the Tungara frog offer an excellent example of a protein based foam, which is compatible with lipid membranes, yet resistant to environmental factors and can persist for the time required for tadpole maturation, usually three days or more (see Downie, J.R., Functions of the foam in foam-nesting Leptodactylids: the nest as a posthatching refuge in Physalaemus pustulosus. Herperol, J 1993. 3 : p. 35-42.)
  • the disclosed subject matter in one aspect, relates to bubble and anti-bubble compounds and compositions and methods for preparing and using such compounds and compositions wherein the surfactant used includes biologically derived surfactant, for example, the protein Ranaspumin-2 (Rsn2).
  • the term "bubble” as used herein also expressly refers to "anti-bubble” compounds.
  • the bubble compound is used as an artificial photosynthesis platform combining two technology platforms to yield value-added products: bubble/foam architecture and proteopolymersomes.
  • value-added product is simple sugar prepared by carbon fixation accomplished by integrating biosolar
  • BR-ATPase polymersomes may be used to convert light into ATP, which powers a rubisco substrate-enzyme reaction of carbon synthesis, and eventually the formation of hydrocarbon for bio fuels.
  • This artificial photosynthesis platform produces glyceraldehyde-3 -phosphate (G3P) and/or simple sugars that can be used to make a variety of useful organic compounds like HMF, DMF, methanol, ethanol or even sugars for human consumption.
  • G3P glyceraldehyde-3 -phosphate
  • simple sugars can be used to make a variety of useful organic compounds like HMF, DMF, methanol, ethanol or even sugars for human consumption.
  • FIG. 1 is a schematic of a portion of a bubble wall where a secondary component (3) is incorporated in the aqueous layer (2) between two surfactant monolayers (1 and 4).
  • FIG. 2(a) is a photograph of a foam; the bubbles are polyhedral.
  • FIG 2(b) is a schematic showing the geometry of a single foam polyhedron. Almost all liquid is concentrated in the Plateau borders, shown in the expanded view.
  • FIG. 3(a) is a schematic of a bubble and an antibubble.
  • FIG. 3(b) is a magnified structure of a portion of an antibubble wall where two surfactant monolayers (62 and 63) define an air layer (61) between two aqueous layers (64 and 65).
  • FIG. 4(a) is a schematic of a single bubble structure.
  • FIG. 4(b) is a schematic of a cross-sectional view of a bubble.
  • FIG. 5 is an illustration of the BR/FoFi ATP synthase vesicle solar conversion system coupled to the RuBisCO CBB cycle enzymes and trapped within the foam channels in accordance with one embodiment of the present invention.
  • FIG. 6 is a schematic of the encapsulating method from a mixture of bubble solution and secondary component.
  • FIG. 6 (a) is a schematic showing a bubble solution containing surfactant (71) and a secondary component (70) (shown here as already-made functional polymersomes).
  • FIG. 6(b) is a schematic showing a cross-sectional view of bubble containing secondary component (70) inside the water channel (72) after the blowing process.
  • FIG. 7 is a schematic of the encapsulating method using coalescence between bubbles; (a) preparation of bubbles (one with polymersomes (shown as dots), the other without) under different conditions, (b) coalescence process by contacting bubbles, and (c) after coalescence process.
  • FIG. 8 is an illustration of a sol-gel design for a foam encasement in accordance with one embodiment of the present invention.
  • FIG. 9(a) is a synthetic scheme of PEtOz-PDMS-PEtOz triblock copolymer.
  • FIG. 9(b) is a *H NMR spectrum of PEtOz-PDMS-PEtOz in DMSO-d 6 .
  • FIG. 10 is a graph showing Control Group for ATP synthesis in Bulk Solution.
  • FIG. 11 is a graph showing Control Group for ATP synthesis in Foam Architecture.
  • FIG. 12 is a graph showing Control Group for ATP synthesis in Deflated Foam Solution.
  • FIG. 13 is a graph showing G3P Production with RuBisCO and ATP stock in bulk solution.
  • FIG. 14 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay with Artificial ATP Source and various components removed.
  • FIG. 15 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control with Bulk Vesicle ATP Source and various components removed.
  • FIG. 16 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control Group with Foam Vesicle ATP Source.
  • FIG. 17 is a graph showing an Absorbance Plot for glucose oxidase assay showing the oxidation of o-dianisidine to a pink colored product absorbing at 540 nm from various glucose concentrations.
  • FIG. 18 is a graph showing DLS Size Distribution Plot for polymer vesicles.
  • FIG. 19 is a graph showing DLS Size Distribution Plot for lipid vesicles.
  • FIG. 20 shows TEM Micrographs of BR/FoFi ATP Synthase Proteopolymersomes.
  • FIG. 21 shows TEM Micrographs of BR/FoFi ATP Synthase Liposomes.
  • FIG. 22 shows fluorescent images of foam vesicle solutions.
  • FIG. 23 is a graph showing the production of ATP with BR/ ATP synthase lipid vesicles in Rsn-2 foam (A ), in bulk ( ⁇ ), in deflated Rsn-2 foam ( -4) in T20 foam ( T ) and a control experiment in the dark ( ⁇ ) for comparison.
  • Inset is the light intensity standard curve created with ATP stock dilutions.
  • FIG. 24 is a graph showing BR/ATP synthase function in a lipid membrane was limited to the Rsn-2 based foam since the T20 adversely affected coupled FiFo-Atpase/BR vesicle function.
  • FIG. 25 is a graph showing ATP synthesis using BR/ATP synthase polymersomes in
  • Inset is the light intensity standard curve created with ATP stock dilutions.
  • FIG. 27(a) is a TEM image of polymersomes after bacteriorhodopsin/ATP synthase incorporation.
  • FIG. 27(b) is a size distribution histogram derived from direct measurement of polymersome sizes by TEM micrographs.
  • FIG. 28(a) is a graph showing internal pH change for bacteriorhodopsin
  • FIG. 28(b) is a graph showing photo- induced ATP synthesis in bacteriorhodopsin- ATP synthase-polymersomes in a foam.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations by use of the antecedent "about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • references in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • compositions are based on the total weight of the formulation or composition in which the component is included.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
  • Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art.
  • the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St.
  • a bubble comprising a wall, wherein the wall comprises a liquid layer between two layers of surfactant; and at least one secondary component, wherein the at least one secondary component is substantially present or completely present in the liquid layer.
  • the liquid layer is an aqueous layer.
  • Figure 1 provides a general structure of a portion of a wall of the bubbles described herein (further examples are shown in Figures 2-6).
  • the wall of the bubble is composed of one or more surfactants, where the surfactant(s) forms a layer defining the outer wall (1) and a layer defining the inner wall (4) of the bubble.
  • the wall structure created by surfactant layers (1 and 4) creates a channel, which is depicted as (2) in Figure 1.
  • the channel can be filled with a liquid.
  • the channel can be filled with water alone or water in combination with one or more liquid solvents such as, for example, an organic solvent.
  • the channel with and without organic solvent is referred to herein as the "aqueous layer.”
  • the bubbles described herein can be any shape such as, for example, spherical, elliptical, or polyhedral.
  • the bubbles can be a thin film with an aqueous layer sandwiched between two layers of surfactant.
  • the bubbles can exist as a foam. Foam formation takes place when bubbles come together and they share the same water layer to form a polyhedron. As shown in Figure 2, the edges of the polyhedron are connected to form channel-like structures known as Plateau borders. The froth of bubbles begins to drain under gravity, removing much of the water between the bubbles. Most of the water resides in the Plateau borders.
  • bubbles merge into larger bubbles, which is called coarsening (Aubert et al, Scientific American 1986, 254:74-82; Isenberg, The science of soap films and soap bubbles. Dover, New York, 1992, pp. 17-21 ; Weaire and Hutzler, The physics of foams. Oxford, 2000, pp. 6-12; Stone et al, J Phys Condens Matter 2003, 15:S283- S290; Hilgenfeldt et al, Europhys Lett, 2004, 67(3):484-90, which are each incorporated by reference herein at least for their teachings of bubbles and bubble structures).
  • the width of the channel created by the surfactant (i.e., the thickness of the bubble wall; e.g., as shown as (2) in Figure 1 , (61) in Figure 3, and (72) in Figure 4) can typically be from about 1 nm to about 10 ⁇ (for spherical bubbles) and from about 10 nm to about 600 ⁇ (for foams).
  • the width of the channel distance can be about 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 7
  • substantially present is meant that the secondary component is mostly present in the aqueous layer; however, it is contemplated that some amount of the secondary component can also be present, either entirely or partially, in either or both of the surfactant layers (1 or 4 in Figure 1). It is also contemplated that the secondary component can partially extend out from either or both surfactant layers into the gas (e.g., air) space.
  • the phrase "incorporated into the bubble wall” is also used synonymously herein with the phrase “substantially present.”
  • a bubble comprising a wall, wherein the wall comprises an inner wall and an outer wall, wherein the inner wall comprises an inner surface and an outer surface and the outer wall comprises an inner surface and an outer surface, wherein the inner wall and the outer wall comprises a surfactant, wherein the inner wall and the outer wall comprises a gas between two layers of surfactant; an aqueous layer, wherein the aqueous layer is adjacent to the outer surface of the inner wall of the bubble; and a secondary component, wherein the secondary component is substantially present in the aqueous layer.
  • bubble is also referred to herein as "an anti-bubble.”
  • bubble as used herein includes the bubbles described above in section (a) and anti- bubbles. Techniques for producing anti-bubbles are known (Hughes and Hughes, Nature 1932,
  • the anti-bubble can have a spherical air shell surrounding a liquid.
  • a gas layer (61) e.g., air
  • an aqueous layer (64) is adjacent to the outer surface of inner wall (62).
  • a second aqueous layer (65) can be adjacent to the outer surface of the outer wall (63).
  • the term "adjacent" is defined herein as any solvent (e.g., water) that is in contact with the surfactant, which also includes penetration of the solvent into the surfactant layer.
  • the secondary component can be substantially present in the aqueous layer.
  • the secondary component can be present in the aqueous layers (64) and/or (65).
  • the dimensions, shapes, and sizes of the anti-bubbles can be the same as those described above as for the bubbles described in section (a).
  • a “surfactant” as used herein is a molecule composed of hydrophilic and hydrophobic groups (i.e., an amphiphile). Because of solubility differences in water, when a bubble is formed, the hydrophobic ends of the surfactant molecules accumulate at an air/water interface, thereby reducing the surface tension (Weaire and Hutzler, The physics of foams, Oxford, 2000, Ch. 1-2). Thus, the surfactant forms a monolayer on the inside and a monolayer on the outside of the water.
  • a schematic of a surfactant bubble composed of a several micrometer- thick water layer sandwiched between two surfactant monolayers is shown in Figure 4 (a close up of a portion of the bubble wall is shown in Figure 1).
  • Bubbles suitable for the compositions and methods disclosed herein can be made from biologically-derived surfactants.
  • the present invention the present invention
  • biologically-derived surfactant may be a natural protein surfactant.
  • the natural protein surfactant is Ranaspumin (Rsn) protein surfactant, such as Rsn-2.
  • a bubble can be prepared from mixtures of two or more surfactants.
  • the expression of the Rsn2 gene factor may be accomplished using a variety of known techniques.
  • the expression of the Rsn2 gene has been demonstrated in bacteria (see Mackenzie, CD., et al., Ranaspumin-2 ; Structure and Function of a Surfactant Protein from the Foam Nests of a Tropical Frog. Biophysical Journal, 2009. 96(12); p.4984- 4992, the contents of which are expressly incorporated herein in its entirety).
  • a gene containing idealized E.Coli codon usage can be constructed for more efficient bacterial expression.
  • the next step is to transform the gene into an inducible expression host and purify the protein.
  • the Rsn2 gene may include two affinity tags.
  • a Langmuir Blodgett film may be used to acquire a more precise quantification of the protein's surfactant properties.
  • Foam topology arises from surprisingly uniform physical principles and structural elements. The surface of the bubbles of aqueous foams are mediated by surfactants which are necessary to stabilize the air-water interface and provide an energetic (both electrostatic and steric) barrier to rupture and collapse. These form tetrahedral structures commonly referred to as Plateau junctions. The legs and nodes of the junction contain the trapped liquid phase typically 0.01-1 mm wide. The drainage of these channels is a primary concern for the foam stability and functionality. ii. Secondary Component
  • the secondary component can be anything (e.g., molecule,
  • compositions, device that can be substantially present in the channel (e.g., aqueous layer) of the bubble wall.
  • the bubble can comprise two or more different secondary components.
  • the secondary component can have a width greater than, equal to, or less than the width of the bubble wall, as described herein.
  • the secondary component can have a width greater, equal to, or less than about 600 ⁇ , 500 ⁇ , 400 ⁇ , 300 ⁇ , 200 ⁇ , 100 ⁇ , 90 ⁇ , 80 ⁇ , 70 ⁇ , 60 ⁇ , 50 ⁇ , 40 ⁇ , 30 ⁇ , 20 ⁇ , 10 ⁇ , 9 ⁇ , 8 ⁇ , 7 ⁇ , 6 ⁇ , 5 ⁇ , 4 ⁇ , 3 ⁇ , 2 ⁇ , 1 ⁇ , 500 ⁇ , 100 nm, or lnm.
  • the secondary component can be a biomolecule.
  • biomolecules include, but are not limited to, a small molecule (e.g., a drag), a peptide, a protein, an enzyme (e.g., a kinase, a phosphatase, a methylating agent, a protease, a transcriptase, an enzyme (e.g., a kinase, a phosphatase, a methylating agent, a protease, a transcriptase, an enzyme (e.g., a kinase, a phosphatase, a methylating agent, a protease, a transcriptase, an enzyme (e.g., a kinase, a phosphatase, a methylating agent, a protease, a transcriptase, an enzyme (e.g., a kinase, a phosphatase, a methylating agent, a protease,
  • nucleic acid e.g., an oligonucleotide, a prime, a probe, an aptamer, a ribozyme, etc.
  • lipid e.g., a carbohydrate, a steroid, a hormone, a vitamin, a potential therapeutic agent.
  • Small molecule as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD, for example, less than about 4 kD.
  • Small molecules can be nucleic acids (e.g., DNA, RNA), peptides, polypeptides, peptidomimetics, carbohydrates, lipids, factors, cofactors, hormones, vitamins, steroids, trace elements, pharmaceutical drugs, or other organic (carbon containing) or inorganic molecules.
  • nucleic acids e.g., DNA, RNA
  • peptides e.g., DNA, RNA
  • polypeptides e.g., polypeptides, peptidomimetics
  • carbohydrates e.g., lipids, factors, cofactors, hormones, vitamins, steroids, trace elements, pharmaceutical drugs, or other organic (carbon containing) or inorganic molecules.
  • the secondary component can also be a macromolecule such as a polymer, a vesicle, or a dendrimer, or a cell or a microbe (e.g., a detoxifying organism), including mixtures thereof.
  • a macromolecule such as a polymer, a vesicle, or a dendrimer, or a cell or a microbe (e.g., a detoxifying organism), including mixtures thereof.
  • compositions disclosed herein where the secondary component can comprise an amino acid based molecule, including for example enzymes and antibodies.
  • amino acid means the typically encountered twenty amino acids which make up polypeptides.
  • it further includes less typical constituents which are both naturally occurring, such as, but not limited to formylmethionine and selenocysteine, analogs of typically found amino acids, and mimetics of amino acids or amino acid functionalities.
  • amino acids include glutas, glut
  • peptide and protein refer to a class of compounds composed of amino acids chemically bound together. Non-limiting examples of these and other molecules are discussed herein. In general, the amino acids are chemically bound together via amide linkages (CONH); however, the amino acids can be bound together by other chemical bonds known in the art. For example, the amino acids can be bound by amine linkages.
  • Peptide as used herein includes oligomers of amino acids and small and large peptides, including naturally occurring or engineered polypeptides and proteins. It is understood that the terms “peptide” and “protein” can be used interchangeably herein.
  • peptide analogs can have more than one atom between the bond atoms, such as beta-alanine, gama- aminobutyric acid, and the like. Such analogs are contemplated within the meaning of the terms peptide and protein.
  • peptides and proteins contemplated herein as biomolecules can be derivatives and variants of the disclosed peptides and proteins that also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes:
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final construct.
  • certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide.
  • Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions.
  • Other post- translational modifications include hydro xylation of proline and lysine, phosphorylation of hydroxy! groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains (T .E. Creighton, Proteins: Structure and Molecular Properties, W. H.
  • peptides and proteins can be linked to other molecules (e.g., to form conjugates).
  • carbohydrates e.g., glycoproteins
  • Such derivatives, variants, and analogs of peptides and proteins are contemplated herein within the meaning of the terms peptide and protein.
  • One method of producing the disclosed proteins is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9- fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied
  • a peptide or polypeptide corresponding to the disclosed proteins can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • a peptide or polypeptide can be independently synthesized in vivo.
  • advances in recombinant glycoprotein production methods which allow more cost effective production of human glycoproteins by colonies of transgenic rabbits or by yeast strains carrying human N-glycosylation system enzymes can be used (Hamilton et ah, Science 2003, 301 : 1244-6; Gerngross, Nature Biotechnology 2004, 22: 1409, which are incorporated by reference herein at least for their teachings of peptide and protein synthesis).
  • independent peptides or polypeptides may be linked, if needed, to form a peptide or fragment thereof via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et ah, Biochemistry 1991, 30:4151, which is incorporated by reference herein at least for its teachings of peptide and protein synthesis).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments.
  • unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton, et al, (1992) Techniques in Protein Chemistry IV. Academic Press, N. Y., pp. 257-67 ' , which is incorporated by reference herein at least for its teachings of peptide and protein synthesis).
  • the secondary component can comprise an antibody.
  • antibody encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class.
  • Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains.
  • L light
  • H heavy
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes.
  • IgA human immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • IgG-I immunoglobulins
  • IgG-3 immunoglobulin-3
  • IgG-4 immunoglobulins
  • IgA-I and IgA-2 immunoglobulins
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • variable is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • variable domains of antibodies typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A.
  • antibody as used herein is meant to include intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding the epitopic determinant.
  • antibody also includes monoclonal and polyclonal antibodies, anti-idiopathic, and humanized antibodies.
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab and the like, including hybrid fragments.
  • Such antibodies and fragments can be made by techniques known in the art (see Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, N. Y., 1988). Such antibodies and fragments thereof can be screened for specificity and activity according to the methods disclosed herein.
  • antibody or fragments thereof conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Patent No. 4,704,692, the contents of which are hereby incorporated by reference for at least its teaching of antibody conjugates.
  • the fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues.
  • Methods of producing and/or isolating antibodies as disclosed herein are well known.
  • compositions disclosed herein where the secondary component can comprise a nucleic acid based molecule.
  • nucleic acid means a molecule made up of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein.
  • a nucleic acid can be double stranded or single stranded. Nucleic acid is also meant to include oliognucleotides.
  • nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage.
  • the base moiety of a nucleotide can be adenine-9-yl (A), cytosine-l-yl (C), guanine-9-yl (G), uracil- 1 -yl (U), and thymin-l-yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • a non- limiting example of a nucleotide would be 3'- AMP (3 '-adenosine monophosphate) or 5'- GMP (5'-guanosine monophosphate).
  • Nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • nucleic acid includes such conjugates, analogs, and variants of nucleic acids.
  • Nucleic acids such as those described herein, can be made using standard chemical synthetic methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001, Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B).
  • a Milligen or Beckman System lPlus DNA synthesizer for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. "Primers” are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • aptamers are also contemplated herein and are molecules that interact with a target molecule, preferably in a specific way.
  • aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
  • Aptamers can bind small molecules, such as ATP (U.S. Patent No. 5,631, 146) and theophiline (U.S. Patent No. 5,580,737), as well as large molecules, such as reverse transcriptase (U.S. Patent No. 5,786,462) and thrombin (U.S. Patent No. 5,543,293).
  • Rabozymes are also contemplated herein and are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly.
  • Ribozymes are thus catalytic nucleic acid.
  • ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes (for example, but not limited to the following U.S.
  • Patents 5,334,711 , 5,436,330, 5,616,466, 5,633, 133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621 , 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following U.S.
  • ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following U.S. Patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408).
  • Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non- canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non- limiting list of U.S. Patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855,
  • the secondary component can be an artificial or natural organelle (e.g., chlorop lasts, mitochondria for energy production, etc.), including mixtures thereof.
  • an artificial organelle e.g., chlorop lasts, mitochondria for energy production, etc.
  • An example of an artificial organelle that can be incorporated into the bubble wall is disclosed in U.S. Application Publication No. 2004-0049230, which is incorporated by reference herein for its teachings of artificial organelles.
  • the biomolecule can be a protein, such as a membrane protein or enzyme.
  • the biomolecule can be a receptor, a channel, a signal transducer, or an ion pump.
  • the biomolecule can be an energy converting protein (e.g., bacteriorhodopsin), an aquaporin, MscL, a cytochrome oxidase, hemoglobin, hemerythrin, hemocyanin, GutR, VR15 CMR1, connexin,calreticulin, microtubule, S 100 proteins, heat shock proteins (hsps), OmpA, Omp F, FhuA, FecA, BtuB, OMPLA, OpcA, FadL, NspA, light-harvesting complex (LHC) proteins, fumarate reductase, succinate dehydrogenase, formate dehydrogenase, nitrate reductase, or an ATPase.
  • LHC light-harvesting
  • the secondary component can be an indicator (e.g., pH, fluorescence, etc.), a carbon based nanostructure (e.g., buckyballs and nano tubes), a dendrimer, a nanoscale device, a microelectric machine (MEMs), an organic or inorganic compound, a non- water liquid, a gas (e.g., hydrogen), and mixtures thereof.
  • an indicator e.g., pH, fluorescence, etc.
  • a carbon based nanostructure e.g., buckyballs and nano tubes
  • a dendrimer e.g., buckyballs and nano tubes
  • a dendrimer e.g., buckyballs and nano tubes
  • a dendrimer e.g., buckyballs and nano tubes
  • a dendrimer e.g., buckyballs and nano tubes
  • a dendrimer e.g., buckyballs and nano tubes
  • any of the secondary components described herein can be imbedded into a polymer matrix prior to bubble formation.
  • imbedded into a polymer matrix is meant that the secondary component is chemically attached (e.g., covalently, ionically, electrostatically, or by hydrogen bonding) to the polymer matrix or physically attached with the polymer matrix (e.g. , wholly or partially encapsulated within the matrix). This is also referred to herein as a "polymersome.”
  • the secondary component comprises a biomolecule imbedded into a polymer matrix.
  • the polymer matrix can comprise any polymer. Suitable polymers include, but are not limited to, homopolymers or copolymers. In some examples, the polymer can be a block, random, or graft copolymer. Suitable polymers for the polymer matrix are readily available from commercial sources and/or can be prepared by methods known to those of ordinary skill in the art.
  • polymers suitable for use in the polymeric matrix include, but are not limited to, modified or unmodified polyolefms, polyethers, and polyalkylene oxides. More specific examples of suitable polymers can include, but are not limited to, modified or unmodified polyethylene, polypropylene, polystyrene, polybutylene, poly(meth)acrylate, polymethylmethacrylate, polyacrylonitrile, ABS, polyethylene oxide, polypropylene oxide, polybutylene oxide, polyterephthalate, polyamide, nylon, polysiloxane, polyvinylacetate, polyvinylethers, polyoxazoline, polyacrylic acid, polyacyl alkylene imine,
  • the term "modified" is used herein to describe polymers and means that a particular monomelic unit that would typically make up the pure polymer has been replaced by another monomelic unit that shares a common polymerization capacity with the replaced monomelic unit.
  • the polymer used to prepare the polymer matrix comprises a polymer produced by the ring-opening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride- terminated PDMS.
  • secondary component comprises a protein such as, for example, bacteriorhodopsin, imbedded in a polymer matrix comprising a polymer produced by the ring-opening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride- terminated PDMS.
  • a protein such as, for example, bacteriorhodopsin
  • the secondary component may be a polymer vesicle, or polymersome, embedded with biomolecules, such as proteins, in a manner which retains the functionality of the biomolecule.
  • the biomolecule is embedded within the wall of the polymersome such that a portion of the biomolecule extends outside the polymersome and a portion of the biomolecule extends inside the polymersome.
  • proteopolymersomes are referred to as "proteopolymersomes”.
  • block copolymer BR/ATPase polymersomes may be used as the secondary component.
  • Such nanoscale polymesomes can produce ATP from light while in foam scaffold which contains moderate levels of detergent.
  • a diblock copolymer poly(ethylene oxide-b- polyethylene) (OE) of a particular molecular weight and composition
  • OE poly(ethylene oxide-b- polyethylene)
  • a diblock copolymer poly(ethylene oxide-b- polyethylene) (OE) of a particular molecular weight and composition
  • OE poly(ethylene oxide-b- polyethylene)
  • OB poly(l,2 butadiene-b-polyethylene oxide) polymers
  • One strategy to modulate the toughness and stability of polymersomes is to cross-link the membrane to form a robust polymer network (Discher, B.M., et al., Cross- linked polymersome membranes: Vesicles with broadly adjustable properties. Journal of Physical Chemistry B, 2002. 106(1 1); p.
  • polymer vesicles can be crosslinked to form solid networks that, if the concentration of crosslinkable polymer is sufficiently high, can greatly increase the critical tension required to cause the vesicle to fail.
  • Crosslinking may be achieved using a chemical electron donor that facilitates the saturation of opposing unsaturated side chains, and the degree of crosslinking is adjusted using mixtures of crosslinkable (OB) and non-crosslinkable (OE) polymers.
  • the degree of crosslinking may be adjusted by mixing fully crosslinkably polymers, but adjusting the extent of crosslinking by using a solute that can crosslink pendant unsaturated side chains using UV radiation of tunable duration and intensity.
  • a vesicle made with 100% crosslinkable OB polymer has a 100- fold greater critical tension than an OE-21 fluid vesicle.
  • the strength of the membrane can be tuned by changing the percentage of crosslinkable polymer in the membrane, with the strength increasing monotonically with % crosslinkable polymer beyond 10%.
  • crosslinked vesicles can be either air dried of lyophilized, are stable, and can be rehydrated later.
  • a broad family of polymers may be used to make proteopolymersomes.
  • a functional BR-containing proteopolymersome membrane may be formed. Foams may be formed using biodegradable BR-PEO-PCL vesicles or nonbiodegradable BR-PEO-PB vesicles. iii. Additional components
  • the bubbles disclosed herein can also comprise additional components.
  • additional components can be added to make the bubble more stable.
  • Suitable additional components can include, but are not limited to, preservatives, antioxidants, stabilizers, and the like.
  • preservatives antioxidants
  • stabilizers stabilizers
  • by adding glycerine long-lasting bubbles can be made.
  • the present invention may be used to create an artificial photosynthesis platform for converting light into value-added products.
  • light energy is converted to chemical energy by providing the microchannels of the bubble and/or foam with at least one biosolar component that is capable of converting light energy to chemical energy.
  • the chemical energy is then converted to a value-added product using a plurality of enzymes selected from the Calvin cycle enzymes, or CBB enzymes, such enzymes are also provided within the microchannels of the bubble and/or foam.
  • the secondary components include proteopolymersomes replete with biomolecules capable of producing ATP, and several plant and yeast enzymes representing the requisite components of the Calvin cycle.
  • the biomolecules may include a biological proton pump and an ATP generator.
  • the ATP generator produces ATP when activated by protons produced by the biological proton pump.
  • the proton pump may be a photoactivated proton pump which produces protons when subject to light energy.
  • the biomolecules may be bacteriorhodopsin (BR) and FoFi-ATP synthase (herein referred to as "ATP synthase") entirely embedded in the wall of a polymer vesicle to form the proteopolymersomes in a manner which retains their biological functionality ( Choi, H-J.; Montemagno, C. Nano techno logy, 2006, 17, 2198-2202, the content of which is expressly incorporated herein by reference in its entirety).
  • BR bacteriorhodopsin
  • ATP synthase FoFi-ATP synthase
  • Fig 5. illustrates one embodiment of the artificial photosynthesis platform.
  • Foam 20 is fabricated such that the secondary components are retained in microchannels 22 formed from the aqueous layer provided between the two layers of surfactant.
  • One of the secondary components is a proteopolymersome 24 including bacteriorhodopsin (BR) 26 and ATP synthase 28 entirely embedded in the wall 30 of the polymer vesicle.
  • BR utilizes light to create a proton gradient, which is subsequently used by ATP synthase to produce chemical energy. It is desirable to widen the excitation bandwidth of BR and further increase the efficiency of BR.
  • a purified form of BR is attached to quantum dots (QD's).
  • Quantum dots are UV sensitive semiconductor nanoparticles which emit visible and IR wavelength photons in a size dependent manner.
  • QD's can be attached to BR via nickel nitrilotriacetic acid (Ni-NTA) using the c-terminal Histidine (His) tag engineered onto the BR.
  • Ni-NTA nickel nitrilotriacetic acid
  • His Histidine
  • the chelated Ni ion of Ni-NTA is capable of orthogonal attachment to the His-tag on the c- terminus, which in the correct orientation, would be on the outside of the vesicles.
  • BR absorption spectra
  • Other proteins may be used instead of or in conjunction with BR as a proton pump for the conversion of light to ATP as described herein.
  • the proton pump for the conversion of light to ATP as described herein.
  • Xanthorhodopsin may also be used (Balashov, S.P., et al., Xanthorhodopsin: A Proton Pump with a Light-Harvesting Carotenoid Antenna, Science, 2005. 309 (5743): p. 2061-2064, the contents of which are expressly incorporated herein in its entirety).
  • the other important secondary components of the artificial photosynthesis platform are the enzymes 32 required to convert ATP to glyceraldehyde-3 - phosphate (G3P) and/or sugar using ribulose 1,5-biphosphate (RuBP) and CO2 from the air.
  • G3P glyceraldehyde-3 - phosphate
  • RuBP ribulose 1,5-biphosphate
  • CO2 CO2 from the air.
  • RuBisCO phosphoglycerate kinase (PGK) and
  • GPDH glyceraldehyde phosphate dehydrogenase
  • G3P glyceraldehyde-3 -phosphate
  • the conversion of G3P to hexose is accomplished using triosephosphate isomerase, fructose- 1,6-biphosphate aldolase, fructose- 1 ,6-bisphosphatase, phosphoglucose isomerase and glucose-6-phosphatase.
  • the three necessary substrates for conversion of light and CO 2 to glyceraldehyde-3 -phosphate (G3P) and/or hexose are ATP, NADH, and ribulose- 1, 5 -bisphosphate (RuBP).
  • Bisphosphate (RuBP) and carbon dioxide (CO 2 ) are catalysed by the enzyme ribulose- 1 ,5-bisphosphate carboxylase oxygenase (RuBisCo) using the energy in ATP.
  • ATP is provided by the BR/ATP synthase proteopolymersome.
  • NADH can be provided to the artificial photosynthesis platform in a variety of ways.
  • NADH is oxidized into NAD + by the enzyme GAPDH. Since the described embodiment synthesizes simple sugars, NADH is used up and must be replenished.
  • the NADH may be photocatalytically regenerated. NAD + can readily be converted back into NADH via P-doped Titanium Oxide nanoparticles using light and a Ru charge carrier. (Shi, Q., Yang, D., Jiang, Z. and Jian Li (2006). Visible-ligth photocatalytic regeneration of NADH using P- Doped T1-O 2 nanoparticles. J. Mol. Cat. B, 43 : 44-48, the entire contents of which are expressly incorporated herein by reference).
  • the nanoparticles are available form Reade Inc. (Providence, RI)_and could be added to the foam's aqueous phase and inflated alongside the ATP synthesizing vesicles.
  • RuBP is regenerated within the system as well.
  • the shortest path to recycle a portion of the fixed carbon back to RuBP involves three enzymes: transketolase, phosphopentose epimerase and phosphoribulose kinase at a cost of 6 ATP molecules. This will convert fructose back into RuBP with an intermediate product either in the form of ribose-5-phosphate or xylulose-5-phosphate by way of the versatility of transketolase, and adolase already present.
  • NADH and RuBP may be added to the artificial photosynthesis platform using a variety of known processes or each may be regenerated within the system using known techniques.
  • NADH may be regenerated while RuBP is added or RuBP may be regenerated while NADH is added.
  • the bubbles disclosed can be prepared by various methods.
  • the bubbles can be prepared by admixing an aqueous solution comprising one or more secondary components and one or more biologically-derived surfactants, and blowing a gas into the mixture.
  • admixing is defined as mixing two or more components together. Depending upon the components to be admixed, there may or may not be a chemical or physical interaction between two or more components.
  • Figure 6 shows a schematic of one possible process for constructing a bubble as disclosed herein.
  • the secondary component (70) as shown is encapsulated within a polymer matrix.
  • the bubble solution can be admixed with pre- formed functional polymersomes comprising the secondary component and polymer matrix, as shown in Figure 6(a). While being blown with gas, the biologically- derived surfactant molecules (71) can self-assemble to form monolayers on the inside and outside surface of the water channel (72) (see Figure 6(b)). As a result, biologically-derived surfactant molecules can form two layers that sandwich a layer of water-containing secondary component (e.g., in Figure 6, a polymersome) in between. It is also contemplated to form two or more bubble compositions comprising different secondary components and admixing the bubble compositions.
  • a layer of water-containing secondary component e.g., in Figure 6, a polymersome
  • the bubbles described herein can be prepared by admixing an aqueous solution comprising one or more secondary components and one or more surfactants, and blowing a gas into the mixture.
  • Figure 7 shows a schematic of a procedure of
  • bubbles are blown with bubble solutions containing no or small amounts of secondary components (e.g., vesicles containing secondary components). Also, other bubbles blown from bubble solutions containing polymersomes can be prepared. This bubble solution can have a different composition (different pH, temperature, additives, surfactant molecules) compared with the first one. When these two different kinds of bubbles come in contact, this can lead to the growth of some bubbles at the expense of others.
  • the effect of biologically-derived surfactant molecules on the components can be minimized during the mixing process between the bubble solution and the polymersome solution.
  • this method can be used.
  • bubbles using amphiphilic block copolymers as bubble surfactant can be made.
  • these bubbles are typically not stable.
  • bubbles blown from the bubble solution by admixing a high concentration of the same amphiphilic copolymer with BR/ ATP synthase reconstituted polymersomes can be merged with longer lasting surfactant bubbles.
  • biologically functional polymersomes can be incorporated inside strong biologically-derived surfactant bubbles without the side effects of detergent molecules.
  • compositions disclosed herein can be used for many varied uses.
  • the disclosed bubbles can be used for chemical and biochemical syntheses, chemical and biological assays, as biochemical sensors, drug delivery, purification in biology, specific gas filters, environmental hazard monitoring systems, cosmetics, gas or liquid transporters, fluidic channels, fuel cells, to measure various properties, conditions, and/or interactions, and the like. It is contemplated that any molecular, nanoscale, or microscale chemical or biochemical analysis can be performed within the bubbles disclosed herein.
  • a detectable interaction can indicate that the first compound has an activity or specific affinity for the second compound or vice- versa, a.
  • reaction means and is meant to include any measurable physical, chemical, or biological affinity between two or more molecules or between two or more moieties on the same or different molecules.
  • any measurable interaction between molecules can be involved in and are suitable for the methods and compositions disclosed herein.
  • General examples include interactions between small molecules, between proteins, between nucleic acids, between small molecules and proteins, between small molecules and nucleic acids, between proteins and nucleic acids, and the like.
  • An interaction can be characterized by a dissociation constant of at least about lxlO "6 M, generally at least about lxlO "7 M, usually at least about lxlO "8 M, or at least about lxlO "9 M, or at least about lxlO "10 M or greater.
  • An interaction generally is stable under physiological conditions, including, for example, conditions that occur in a living individual such as a human or other vertebrate or invertebrate, as well as conditions that occur in a cell culture such as used for maintaining mammalian cells or cells from another vertebrate organism or an invertebrate organism.
  • interactions that can be involved in and/or determined by the compositions and methods disclosed herein include, but are not limited to, an attraction, affinity, a binding specificity, an electrostatic interaction, a van der Waals interaction, a hydrogen bonding interaction, and the like.
  • One specific type of interaction that can be involved in and/or determined by the methods and compositions disclosed herein is an interaction between a ligand (e.g., a potential therapeutic agent, a small molecule, an agonist, an antagonist, an inhibitor, an activator, a suppressor, a stimulator, and the like) and a protein (e.g., a receptor, a channel, a signal transducer, an enzyme, and the like).
  • a ligand e.g., a potential therapeutic agent, a small molecule, an agonist, an antagonist, an inhibitor, an activator, a suppressor, a stimulator, and the like
  • a protein e.g., a receptor, a channel, a signal transducer, an enzyme, and the like.
  • an interaction between a potential therapeutic agent and a target protein can indicate a potential therapeutic activity for the agent.
  • an interaction between a small molecule e.g., a lipid, a carbohydrate, etc.
  • an enzyme e.g., a kinase, a phosphatase, a reductase, an oxidase, and the like
  • an interaction between two proteins or fragments thereof e.g., an enzyme and a protein substrate or an antibody and an antigen or an epitope of an antigen).
  • An example of this interaction can include, but is not limited to, the binding of a kinase, a protease, a phosphatase, and the like to a substrate protein. Such interactions can, but need not, result in a reaction or chemical transformation (e.g., phosphorylation, cleavage, or dephosphorylation).
  • Another example of an interaction includes the binding or affinity of an antibody for an antigen or epitope of an antigen.
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide substitute in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions.
  • sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
  • a Watson-Crick interaction i.e., at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute.
  • the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
  • a Hoogsteen interaction is another example and is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups (NH 2 or O) at the C6 position of purine nucleotides.
  • compositions and methods disclosed herein includes an interaction between a protein ⁇ e.g., a polymerase, endonuclease, or ligase) and a nucleic acid.
  • a protein e.g., a polymerase, endonuclease, or ligase
  • Detecting an interaction in the methods disclosed herein can be performed by any method, but will usually depend on the particular interaction being detected.
  • the first compound and/or second compound may contain a fluorescent marker, and detection of an interaction can be made by measuring fluorescence or changes in fluorescence.
  • detecting an interaction can involve identifying a particular product.
  • the first and second compound interact in such a way as to produce a reaction product (e.g., a kinase phosphorylating a substrate protein, a protease cleaving a particular protein, an endonuclease cleaving a particular nucleic acid, a ligase ligating nucleic acids, and the like), detection can be accomplished by identifying a particular product (e.g., the phosphorylated or cleaved product). Identifying a product can be done by known methods such as
  • chromatography e.g., retention times or Rf
  • fluorescence detection e.g., ionization
  • mass spectral analysis nuclear magnetic resonance imaging
  • immunohistological techniques e.g., microscopy (e.g., TEM, SEM, optical microscope, or AFM), XRD, XPS, AES, infrared spectroscopy, kinetic analysis, circular dichroism
  • electrochemical analysis e.g., cyclic voltametry or impedance spectroscopy
  • dynamic light scattering e.g., static light scattering, and the like.
  • the first compound can be any molecule that one may desire to measure a potential interaction with any other desired molecule.
  • the first compound can be any of the secondary components disclosed herein, for example, amino acid based molecules (e.g., peptide, proteins, enzymes, or antibodies, including variants, derivatives, and analogs thereof), nucleic acid based molecules (e.g., primers, probes, aptamers, or ribozymes, including variants, derivatives, and analogs thereof), small molecules (e.g., biomolecules, drugs, potential therapeutics, or organic and inorganic compounds), macromolecules (e.g., carbon based nanostructures, dendrimers, or polymers), cell, or organelle (natural or artificial).
  • amino acid based molecules e.g., peptide, proteins, enzymes, or antibodies, including variants, derivatives, and analogs thereof
  • nucleic acid based molecules e.g., primers, probes, aptamers, or ribozymes, including variant
  • the second compound which is present in the secondary component, can also be any molecule as described above for the first compound. It is contemplated that the disclosed methods are not limited by the particular order, identity or priority of the first or second component; the identifiers "first” and “second” are merely arbitrary and are used herein to simply distinguish one compound from the other; no connotation of order of addition is intended as any order of the compounds is contemplated and can be used in the methods disclosed herein,
  • the second compound can be a protein and the first compound can be a small molecule such as a potential therapeutic agent, a kinase, a phosphatase, a protease, a methylating agent, an antibody, or fragments thereof.
  • the second compound can be a small molecule, a kinase, a protease, a methylating agent, an antibody, or fragment thereof and the first compound can be a target protein.
  • the detectable interaction can indicate a potential therapeutic activity.
  • the method can be used to screen for potential drugs against a particular protein.
  • the detectable interaction can indicate enzymatic activity.
  • the detectable interaction can indicate enzymatic activity.
  • phosphorylate a particular protein or the ability of a protein to be dephosphorylated by a particular phosphatase, and the like.
  • the second compound can be a protein, antigen, or epitope
  • the first compound can be an antibody or fragment thereof, or vice-versa.
  • the method can be used to detect an interaction that indicates binding activity. Thus, one can use this method to screen antibodies to find those that bind to a particular antigen or epitope.
  • the disclosed method can find particular antigens or epitopes recognized by a particular antibody. It can also be possible, when the first compound is a cell or microorganism and the second compound is an antibody or fragment thereof, to screen for particular surface antigens on the cell surface, or to screen for antibodies that recognize a given organism. These and other uses are contemplated herein.
  • compositions can be used to detect a particular infection in a subject.
  • a bubble as disclosed herein, wherein the secondary component comprises second compound that is a particular antigen can be contacted with an antibody- containing sample from a subject. Detecting an interaction of the antigen and the antibody specifically reactive therewith can indicate the presence of the antigen or previous infection in the subject.
  • the second compound can be a nucleic acid and the first compound can be a primer, a probe, a ligase, an endonuclease, a transcriptase, a ribozyme, or fragment thereof, or vice- versa, that is the second compound can be a primer, a probe, a ligase, an endonuclease, a transcriptase, a ribozyme, or fragment thereof and the first compound can be a target nucleic acid.
  • the interaction can indicate enzymatic activity.
  • the interaction can indicate hybridization.
  • the methods can further comprise contacting the bubble with a third compound. This can be done to, for example, evaluate or analyze a particular interaction between a first compound and a second compound while a third compound is present. Also, it is contemplated that the methods disclosed herein can further comprise contacting the bubble with a fourth, fifth, six, etc. compound. Any number of additional compounds can be used in the methods and compositions disclosed herein.
  • the third compound can be any molecule or group of molecules.
  • any of the molecules disclosed herein such as amino acid based molecules, nucleic acid based molecules, small molecules, macromolecules, cells, etc.
  • suitable third compounds include, but are not limited to, an antagonist, an agonist, a ligand, an inhibitor, an activator, a primer, a promoter, a transcription factor, an endonuclease, a ligase, a transcriptase, a protease, a kinase, a phosphatase, a methylating agent, or mixtures thereof.
  • a condition comprising subjecting a bubble as disclosed herein, wherein the secondary component comprises an indicator to a condition to be assayed, and detecting the indicator.
  • indicator any molecule, compound, or composition, which when contacted with or subjected to a particular condition (e.g., pH, light intensity, temperature, ionic strength, electrochemical potential), provides a detectable signal.
  • a detectable signal that a suitable indicator can provide can be, for example, a color change, fluorescence, phosphorescence, magnetic resonance, electric potential, and the like.
  • an indicator can provide a change in color or emit light in response to being subjected to a particular pH condition. e. Chemical and biochemical synthesis
  • the disclosed bubbles can be used to form a hybrid ATP generating bubble device.
  • the protein bacteriorhodopsin (BR) and F 0 Fi-ATP synthase were reconstituted into 4 nm thick polymersome membranes that can convert optical energy to electrochemical energy.
  • BR transports protons across the cell membrane upon the absorption of a photon of green light. Because of the pumping of protons, a pH gradient forms across the cell membrane, forming an electrochemical potential.
  • F 0 Fi-ATP synthase this proton gradient drives the synthesis of ATP from ADP and inorganic phosphate (Pi).
  • these biologically active polymersomes were packaged into the thin water channel of the surfactant bubbles.
  • BR-ATP synthase-polymersomes The ATP production by BR-ATP synthase-polymersomes was demonstrated in the bubble architecture. This has significance both in the development of a hybrid organic/inorganic power source obtaining its energy from light and in using surfactant bubbles for packaging structures. Functional polymersomes incorporated into the water channel of bubble walls were able to provide useful amounts of electrochemical energy which can be used for other nano- bio applications.
  • the bubble architecture is used to form an artificial photosynthesis platform for converting light and CO 2 to a value-added product, for example, simple sugar.
  • Carbon fixation for the production of sugar is achieved by incorporating biosolar proteo- polymersomes and a plurality of enzymes into the microchannels of an inflatable foam provided by the present invention.
  • BR-ATPase polymersomes may be used to convert light into ATP, which powers a rubisco substrate-enzyme reaction of carbon synthesis, and eventually the formation of hydrocarbon for biofuels.
  • This artificial photosynthesis platform produces simple sugars that can be used to make a variety of useful organic compounds like HMF, DMF, methanol, ethanol or even sugars for human consumption.
  • DMF can be generated in low boiling point solvents, which are also good sources of energy rich compounds that can be used in fuel.
  • the combination of free carbohydrates, exothermic reactions, and the absence of distillation would make energy rich fuels in an extremely efficient and inexpensive manner.
  • the artificial photosynthesis platform is encased in a multifunctional material for the production of solar derived liquid fuels.
  • the encasement provides a robust environment for the foam without compromising optical transparency and enables the separation of the liquid fuel (G3P) from the other constituents, while providing antifouling and antimicrobial protection.
  • the encasement will be synthesized via a sol-gel process which enables control of materials chemistry and micro structure.
  • the sol-gel process is a chemical synthesis technique for preparing amorphous inorganic solids.
  • the most common synthetic route involves the use of metal alkoxides which undergo hydrolysis and condensation polymerization reactions to give rigid solids (gels) of metal oxides such as Si0 2 , Ti0 2 , A1 2 0 3 , Zr0 2 , etc. (Brinker, C.J. and G.W. Scherer, Sol-Gel Science 1990, New York: Academic Press, the contents of which are expressly incorporated herein by reference in its entirety).
  • the micro structure of the resulting gel is determined by the synthesis conditions (pH, starting alkoxide, ratio of alkoxide: water, type of catalyst, etc.).
  • the material is technically defined as a gel, it is a nanoporous glass that is rigid and dimensionally stable.
  • sol-gel processing Another important advantage of sol-gel processing is that the solution nature of the synthesis enables one to cast or form the sol-gel derived materials into a wide variety of shapes and sizes, including thin films or fibers. It has been shown that it is possible to immobilize biomolecules which retain their characteristic reactivities and spectroscopic properties in the pores of the sol-gel glass. (Rolison, D.R. and B. Dunn, Electrically conductive oxide aerogels: new material in electrochemistry. Journal of Materials Chemistry, 2001. 11(4): p.963 -980, the contents of which are expressly incorporated herein in its entirety). As a result, a new generation of bioactive materials was created.
  • the sol-gel encasement includes the following material requirements: 1) optical transparency in the 350-700 nm wavelength range; 2) good diffusion of CO2 through the encasement; 3) antifouling and antimicrobial properties; 4) good compatibility with the foam; and 5) ability to separate the G3P liquid fuel from the other constituents.
  • FIG. 8 shows one design of the sol-gel encasement in accordance with the present invention. Due to the inherent optical transparency of S1O2 and high porosity of sol-gel derived materials, requirements 1) and 2) are fulfilled. In one aspect, to become antifouling/antimicrobial, S1O2 is functionalized with nanoparticle Ag and/or MgO.
  • Nanoparticles MgO are known to be antibacterial, and nanoparticles Ag have demonstrated to be both antibacterial and antifungal.
  • S1O2 is able to prevent the protein protease from entering the sol-gel, unfunctionalized S1O2 will exclude bacteria and microbes from contact with the foam since bacteria and other microbes are larger than protease.
  • Increased antifouling protection can be afforded by addition of nanoparticles Ag and/or MgO. It is understood that other antimicrobial and/or antifouling materials may also be used subject the material requirements provided herein.
  • the matrix and/or surface of the sol-gel may be functionalized with lipids.
  • Another issue is to minimize water evaporation from the foam, which can be accomplished by incorporating lipids into the sol-gel matrix.
  • lipids into the sol-gel matrix.
  • sol-gel derived S1O2 By incorporating, for example, short chain diacylphosphotidylcholine into sol-gel derived S1O2, the hygroscopic nature of the lipid and their organization into the uniform SiC -lipid structure suppresses overall water loss so that a water-rich
  • the generated G3P can be captured by the foam by incorporating a "capture chamber" specifically designed to capture G3P.
  • This capture chamber may also be sol-gel derived inorganic, porous matrix involving the use of metal alkoxides which undergo hydrolysis and condensation
  • the G3P therefore, will be temporarily captured in the imprinted sol-gel but not covalently attached. Since the G3P is trapped in the matrix without covalent attachment or electrostatic interaction, it can be subsequently eluted from the capture chamber. Moreover, post-treatment on the MIP-sol-gel matrix can be performed to enlarge the imprinted size to more easily elute the G3P.
  • An interesting phenomenon has been observed when working with the enzyme creatine kinase (CK), where mild heat treatment (only - 15° above room temperature) was able to slightly enlarge the pores of the sol-gel matrix.
  • the sol-gel network typically forms around the biomolecule (CK in this case), creating a site-specific pore around the enzyme.
  • the ability to enlarge the pores can be utilized in MIP sol-gel, so that the G3P can be more readily eluted from the capture chamber.
  • the G3P to sugar conversion can be accomplished by adding a "conversion chamber" to the encasement.
  • the conversion chamber can be a sol-gel derived inorganic, porous matrix involving the use of metal alkoxides which undergo hydrolysis and condensation polymerization reactions to give rigid solids (gels) of metal oxides such as S1O2, Ti0 2 , AI2O3, ⁇ (3 ⁇ 4, etc.
  • G3P Upon entering the conversion chamber, G3P will be converted to sugar due to the immobilized aldolase and isomerase, and the sugar can be subsequently eluted from the conversion chamber.
  • the encasement will be fabricated with an inlet and outlet to facilitate the addition of foam and separation of the biofuel (G3P or sugar). There will be a semi-permeable membrane dividing the encasement from the sol-gel derived S1O2 capture or conversion chamber, which will permit the diffusion of G3P. Once inside the capture or conversion chamber, the G3P or sugar can be eluted from the chamber by application of a gentle vacuum.
  • PDMS bis(hydroxyalkyl) terminated polydimethylsiloxane
  • bis(hydroxyalkyl) terminated PDMS was dehydrated under vacuum at 80-90 °C for 24 h and freeze-dried. After this drying process, cyclohexane (10 mL) (Aldrich; anhydrous) was added to 3.308 g of PDMS, and the mixture was stirred for 6 hours. To this reaction mixture, two molar excess volume of sec-butyl lithium (Aldrich; 1.4 M in cyclohexane) was added dropwise with a syringe at -20 °C, and the resulting solution was kept stirring until the temperature increased to room temperature under a nitrogen atmosphere.
  • EtOz (Aldrich; purity >99%) was dried over calcium hydride (Aldrich; powder 99.99%) followed by double distillation under a nitrogen atmosphere.
  • the reaction mixture was stirred under reflux for 2 hours at room temperature and next heated to 100 °C. The reaction was allowed to proceed until all the monomer was depleted, as monitored by ⁇ - MR.
  • the end-capping of triblock copolymers by hydroxyl terminal groups was carried out by adding 2.5 mL of potassium hydroxide solution (Aldrich; 0.1N in methanol) to the system at room temperature, yielding a solution color change from light yellow to colorless.
  • the solution was diluted with chloroform (Aldrich; anhydrous) and washed with a 10% Na 2 S 2 O3 (Aldrich; >99.99%) solution, followed by a washing with water. After evaporation of the solvent to remove any unreacted PDMS oligomers, the products were dissolved in hexane supplemented with charcoal and MgSC , then filtered.
  • Example 2 TEM sample preparation
  • the polymersome solution was dropped onto a 3 mm amorphous carbon coated Cu-grid by pipette.
  • copper grids were placed on KIMWIPESTM, and, after 1 minute, excess solution was removed by blotting.
  • the samples were transferred to the transmission electron microscope using a liquid-nitrogen cooled specimen stage, designed to maintain a temperature from about
  • Elevated temperatures that could cause structural changes of the specimen due to long electron beam exposure were minimized by performing TEM analysis under low electron beam density and also, by using the cooling stage during TEM
  • BR Bacteriorhodopsin
  • PM purple membrane
  • F 0 Fi-ATP synthase was purified from Bacillus PS3 cells as described in Hazard et al., Arch Biochem
  • EPPENDORFTM 10-100 adjustable- volume pipette
  • Blown bubbles were transferred to fill a UV cuvette (12.5 mm x 12.5 mm x 45 mm). Before any measurements, the cuvette was kept inverted on top of KIMWIPESTM in the dark for 20 to 30 minutes in order to remove the excess polymersome solution not incorporated in the water channels. The cuvette entrance was sealed to prevent the liquid in the bubbles' aqueous channels from evaporating and to increase the stability of the foam structure. Before taking any measurements, samples having similar density of bubbles (bubble size: 3.5 to 4 mm) were chosen; also the formation of dry foam where bubbles take the form of polyhedra with nanoscale liquid films and Plateau borders were confirmed (see Figure 2). During measurements, special care was taken not to break the bubbles. During incubations both in the dark and in light, cuvettes were rotated every 3 seconds to minimize the destabilization of the bubble architecture due to gravity-induced drainage.
  • BR was obtained from the purple membrane of Halobacterium and was purified using the method adapted from Pitard. (see Pitard, B.; Richard, P.; Dunach, M.; Girault, G.; Rigaud, J-L. Eur. J. Biochem. 1996, 235, 769-778, the entire contents of which are expressly incorporated herein by reference.)
  • FoFi-ATP synthase was purified from Bacillus PS3 cells and photophosphorylating lipid vesicles were made as described in Hazard, (see Hazard, A; Montemagno, C. Arch. Biochem. Biophys.
  • the Rsn-2 gene was synthesized using PCR since the length was relatively short (338 bp).
  • the sequence was designed for E.coli codon usage with a 6xHis c-terminal tag. Purification was performed with Ni-NTA resin as described by Mackenzie, (see Mackenzie, CD.; Smith, B.O.; Meister, A.; Blume, A.; Zhau, X.; Lu, J.R.; Kennedy, M.W.; Cooper, A. Biophysical Journal.
  • This solution contained 2 ml of Tween-20 ® (non-ionic acid detergent), 1 ml of glycerin, and 2 ml of deionized water. The solution was vortexed for 2 minutes and stored at 4 °C. Rsn-2 foam solutions were created by diluting a 1 mg/ml stock solution 1 : 10 into the vesicle mixture and aspirating the liquid.
  • Photo-derived ATP synthesis activity was measured in bulk-vesicle solution, as well as in both inflated and deflated foam-vesicle solutions.
  • a bioluminescence assay FLAA Lucifer in-Luciferase, Sigma-Aldrich, USA was used for ATP measurement. The procedure used here was adapted from previous methods, (see Choi, H-J,; Montemagno, C.
  • the reaction mixture in bulk-vesicle solutions contained 200 ⁇ BR-ATP synthase proteopolymersome solution, 20 ⁇ of 0.2 M ADP, 20 ⁇ of 0.5 M KH 2 PO 4 , and 60 ⁇ deionized water.
  • 60 ⁇ of foam stock solution was substituted for the deionized water. All solutions were illuminated using a Fostec xenon lamp using a yellow filter with maximum emission at a wavelength of 572 nm for a total time of one hour unless otherwise noted. Initially before exposure and at subsequent time points 10 ⁇ aliquots were removed from the reaction to quantify the rate and amount of ATP production.
  • the volume of the bubble solution was calculated using the weight of each sample with the density values. In this calculation, an assumption was made that the density does not change before and after blowing bubbles. Foams were then broken and ATP was measured by recording the intensity of light produced by the sample and comparing that with a standard calibration curve. All experiments were performed at room temperature.
  • the final assay mix contained 625 ⁇ g of RuBisCO, 5 units of GAPDH, 10 units of PGK, 250 nmol of NADH, 375 nmol of RuBP, and 750 nmol of ATP in a volume of 750 of reaction buffer (pH 7.8) containing 0.1 M Tris-HCL, 5 mM MgCl 2 , 66 mM KHC0 3 , and 5 mM DTT.
  • the assay was allowed to react for 20 minutes at room temperature with absorbance being measured at 10 second intervals.
  • glucose was assayed using a colorimetric assay kit (GAGO20, Sigma-Aldrich, USA).
  • Glucose oxidase forms gluconic acid and hydrogen peroxide from glucose.
  • Hydrogen peroxide reacts with the o-dianisidine in the assay mixture forming a colored product.
  • the oxidized o-dianisidine then reacts with sulfuric acid to form a more stable pink colored product.
  • the intensity of the pink color measured at 540 nm is proportional to the original glucose concentration.
  • the amount of glucose present was quantified by comparison to a standard calibration curve obtained from samples of glucose of known concentration.
  • Glucose was formed by the addition of 12.5 units each of TPI, fructose- 1,6-biphosphatase, and PGI, 0.2 units of fructose- 1 ,6-biphosphate aldolase, and 0.1 units of glucose-6-phosphatase, diluting the total volume to 1 ml using water and incubating for 4 hours.
  • Glucose was formed from starting components RuBP, ATP and NADH using the following CBB enzymes (Sigma-Aldrich, USA): RuBisCO, GAPDH, PGK, triose phosphate isomerase (TPI), fructose 1,6-biphosphate aldolase, fructose- 1,6-biphosphatase, phospho- glucose isomerase (PGI), and glucose 6-phosphatase, during a 4 hour incubation.
  • CBB enzymes Sigma-Aldrich, USA
  • a typical reaction mixture contained 250 ⁇ g of RuBisCO, 2 units of GAPDH, 4 units of PGK, 125 nmol of NADH, 200 nmol of RuBP, 500 nmol of ATP, 5 units each of TPI, fructose-1 ,6- biphosphatase, and PGI, 0.1 units of fructose- 1,6-biphosphate aldolase, and 0.05 units of glucose-6-phosphatase in a volume of 260 ⁇ g of reaction buffer (pH 7.8) containing 0.1 M Tris-HCL, 5 mM MgCi 2 , 66 mM KHCO 3 , and 5 mM DTT was prepared and incorporated in a bubble architecture by the addition of 65 ⁇ of foam stock solution. Comparative samples with 65 ⁇ of water in place of foam stock solution in order to give bulk vesicle solution results were also prepared. The samples were then exposed to light for one hour and monitored for absorbance at 350 and 540 nm.
  • FIG. 10 is a graph showing Control Group for ATP synthesis in Bulk Solution. Blue triangles are samples without vesicles, Black squares are samples with no ADP, Red circles are samples with no KH2PO4.
  • FIG. 11 is a graph showing Control Group for ATP synthesis in Foam Architecture. Blue triangles are samples without vesicles, Black squares are samples with no ADP, Red circles are samples with no KH2PO4.
  • FIG. 12 is a graph showing Control Group for ATP synthesis in Deflated Foam Solution. Blue triangles are samples without vesicles, Black squares are samples with no ADP, Red circles are samples with no KH2PO4.
  • FIG. 14 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay with Artificial ATP Source and various components removed. Lines overlap at 0, extended y- axis scale is provided for direct comparison to non-control results.
  • FIG. 15 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control with Bulk Vesicle ATP Source and various components removed. Lines overlap at 0, extended y-axis scale is provided for direct comparison to non-control results.
  • FIG. 16 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control Group with Foam Vesicle ATP Source. Lines overlap at 0, extended y-axis scale is provided for direct comparison to non-control results.
  • FIG. 17 is a graph showing an Absorbance Plot for glucose oxidase assay showing the oxidation of o-dianisidine to a pink colored product absorbing at 540 nm from various glucose concentrations.
  • FIG. 18 is a graph showing DLS Size Distribution Plot for polymer vesicles. Graph displays particle size relative to particle number. TEM and Weighted DLS data was used to determine percentage of functional proteopolymersomes with diameter ranging from 69-1 10 nm. This range was determined by directly observing vesicles with TEM.
  • FIG. 19 is a graph showing DLS Size Distribution Plot for lipid vesicles. Lipid vesicles were relatively abundant compared to polymersomes with a peak readily observed and close to 100 nm. Particle size versus particle number was not investigated because lipid vesicle appeared to form functional hollow vesicles at all sizes as observed under TEM.
  • FIG. 22 shows fluorescent images of foam vesicle solutions. Liposomes containing 1 nM quantum dots, 0QD565 (Quantum Dot Corporation, USA.) were added to 0.1 mg/mL Rsn-2 foam after size exclusion chromatography on a S-200 column (GE Biosciences) and observed to flow between nodes in the foam channels as the foam drained. Rapid
  • Figure 23 is a graph showing the production of ATP with BR/ATP synthase lipid vesicles in Rsn-2 foam (A ), in bulk ( ⁇ ), in deflated Rsn-2 foam ( -4) in T20 foam ( T ) and a control experiment in the dark ( ⁇ ) for comparison.
  • Inset is the light intensity standard curve created with ATP stock dilutions.
  • Inset is the light intensity standard curve created with ATP stock dilutions.
  • Figure 26 is a graph showing the foam system containing BR/ATP synthase vesicles
  • Spectrometer (LS 50B Perkin Elmer) was performed from 350 nm to 475 nm at an emission wavelength of 511 nm. Small shifts in the excitation spectrum were corrected and the conversion from fluorescence to pH was performed as described in Hazard et al, Arch Biochem Biophys 2002, 407: 117-24.
  • FIG. 20 The bright-field TEM images of BR-reconstituted polymersomes are shown in Figure 20. As seen in Figure 27(a), spherical polymersomes were observed distributed throughout the sample. Figure 27(b) shows the size distribution histogram derived from direct measurement of polymersome sizes by TEM micrographs. The size distributions with a mean polymersome diameter of 270 ⁇ 156 nm are based on an analysis of 135 polymersomes from TEM images.
  • Example 8 Bubble water channel thickness measurement using IR
  • planar bubble film thickness was measured following the procedures described in Wu et al, Review of Scientific Instruments 2001, 72(5):2467-71. Using IR, the thickness of the bubble wall was measured to be 1.23 ⁇ .
  • Example 9 Proton pumping activity of hybrid BR/ATP synthase incorporated polymersome system in buffer solution
  • Figure 28(a) shows ⁇ as a function of time together with a control.
  • Intravesicular pH measurements were performed in buffer solution using BR- polymersomes and BR-ATP synthase-polymersomes. Both systems in buffer solution showed an increase in the internal pH with illumination. That is, the generation of a photo-induced proton gradient resulted in alkalinization of the protein-incorporated polymer vesicles. This pH change over time indicates that more than 50% of BR is selectively oriented, allowing protons to be pumped primarily outward.
  • the kinetics of light- induced proton transport were affected by the presence of ATP synthase, which can be seen in the slower and slightly smaller pH change in the presence of ATP synthase.
  • Example 10 ATP synthesis activity of hybrid BR/ATP synthase incorporated polymersome system within bubble architecture

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Abstract

Bubble architectures are formed using biologically-derived surfactant, for example, the protein Ranaspumin-2 and other biologically derived surfactants, to create functional materials that mimic cellular physiological processes. In one embodiment, the bubble architecture is used to form an artificial photosynthesis platform for converting light and CO2 to a value-added product, for example, simple sugar.

Description

BUBBLE ARCHITECTURES AND METHODS OF MAKING AND USING THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Application No. 61/286,578, filed 12/15/2009, which is expressly incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to bubble architectures and methods of making and using such bubble architectures, wherein the bubble architectures are formed using biologically derived surfactant, for example, the protein Ranaspumin-2 and other biologically derived surfactants to creation functional materials that mimic cellular physiological processes. In one embodiment, the bubble architecture is used to form an artificial photosynthesis platform for converting light and CO2 to a value-added product, for example, simple sugar.
2. Related Art
Harvesting and converting biomass to combustible fuel has been suggested as a renewable energy solution to the ongoing depletion of fossil fuels. Nature has acquired means for solar energy capture in the photo-reactive centers of plants for the purpose of synthesizing and storing such biomass. However, the near 100 percent quantum efficiency of the photo -reactive centers is reduced to approximately 5 percent of that value in usable energy due to limited wavelength sensitivities and necessary cellular processes, including growth, repair, and maintenance. Converting plant sugars to ethanol has been proposed as a renewable energy source, particularly from sugar cane, corn stover and switchgrass; however, this requires that limited land and water resources be diverted, in part, to biomass production.
Processes which evolve liquid fuels, such as ethanol, from biomass have been widely developed. Due to only marginal energy yield from the production of bio-ethanol, 2,5- dimethylfuran (DMF) is considered a prudent alternative to ethanol given its higher energy density, boiling point, and insolubility in water. In addition to attractive liquid fuel properties, DMF has been synthesized from simple carbohydrates, such as glucose or fructose, harvested from biomass.
Engineered biological solar energy conversion has produced a variety of electrical and chemical energy storage strategies. Of the latter, ATP serves as the most important natural energy molecule and has been formed artificially by coupling F0F1 ATP synthase to a photon induced proton motive force. In photo synthetic organisms, long term energy storage is accomplished through biomass synthesis through ATP dependent carbon-fixation providing a foundation for liquid biofuel production.
To date, in vitro carbon fixation experiments have been limited to the examination of
CBB cycle intermediates and cell extracts using radiometric and spectrophotometric techniques. Photosynthesis, carbon sequestration and carbohydrate generation involve several complex and well-studied processes; among these, a suite of 8 enzymes make up the portion of the CBB cycle responsible for converting the energy of ATP into 6-carbon sugars such as glucose and fructose. The chief products of the light-dependent reaction of photosynthesis are NADPH and ATP. The thermophilic FoFi ATP synthase has been purified and reconstituted in both liposomes and ABA triblock polymersomes, along with the photoactivated proton pump bacteriorhodopsin (BR) to form ATP producing vesicles.
Recently, an improvement in the artificial synthesis of ATP was demonstrated using Tween- 20 (T20)-based bubbles/foam and polymersomes.
Bubbles are natural structures that are encountered in everyday life such as dishwashing foam or beer foam. While bubbles are common, they are deceptively complex structures, typically composed of a water layer sandwiched between two- surfactant monolayers. Despite their everyday appearance, bubbles and foams have been interesting research topics to scientists for the past several centuries, where many have sought to understand and utilize the chemical, physical, and mechanical properties of bubbles. Their applications, however, have been limited by their innate properties of drainage and uncontrollable size distributions. Recently, it has become possible to produce a microfoam, having no vertical drainage, from monodisperse stable microbubbles (Garstecki et al, Appl Phys Lett 2004, 85 :2649). And several techniques have been proposed for formation of micro-scale droplets (Sugiura et al. , Langmuir 2001, 17:5562; Thorsen et ah, Phys Rev Lett 2001, 86:4163 ; Anna et ah, Appl Phys Lett 2003, 82:364). Engineering complex biochemical cascades in vitro can be difficult because of an inability to locally contain chemical distributions within a defining nanostructure. Therefore, producing locally high
concentrations of biochemicals in vitro is regarded as a major challenge in creating "life-like" function in engineered systems. Further, the recent technical developments in semiconductor device technology (MEMS) have been promising for use in nano-packaging. However, considering the time, cost, complexity, and biocompatibility of silicon technology, MEMS are not expected to serve equally well for all kinds of hybrid organic/inorganic bioelectronic devices and sensors.
The International Published Application WO 2006/089245 is directed to a bubble architecture and method of making such a bubble, the contents of which are expressly incorporated herein by reference in its entirety. Although this document lists a variety of surfactants that may be used in making the bubble, the Examples are primarily directed to the use of TWEEN-20™. As explained in detail below, bubbles using such a surfactant have various drawbacks.
Because of drainage, evaporation, and hydrophilicity water-based foams are inherently fragile and relatively short-lived. The fact that foam nests are used by a variety of organisms (e.g., fish, amphibians and insects) is quite impressive, given the biological necessities required to persist in the environment. These include resistance to microbial and insect assault, resilience to changes in heat, humidity and desiccation, but continue to be compatible with exposed eggs and sperm. The latter requirement presents a remarkable paradox, since surfactant used to produce stable bubble films would by its very nature also destabilize and destroy the cell membranes and proteins necessary for reproduction.
The foam nest produced by the Tungara frog is one of the largest found in nature. It is used to protect developing tadpoles in terrestrial areas of tropical and subtropical Central America, until maturation or greater water availability. The creation and maintenance of the Tungara frog's foam nest can be attributable to a small but astonishing suite of six proteins called ranaspumins (Rsnl-6). Of these, it is Rsn2 which is responsible for the reduction in water surface tension allowing foam creation upon liquid agitations. The other ranaspumins offer an arsenal of microbio and insecticides, as well as carbohydrate binding proteins which help stabilize the foams to drainage and desiccation. Rsn-2 plays the surfactant role very economically at 0.1 g/ml, but also has the ability to exist in two conformational states (see Mackenzie, CD., et al., Ranaspumin-2 ; Structure and Function of a Surfactant Protein from the Foam Nests of a Tropical Frog. Biophysical Journal, 2009. 96(12); p.4984-4992, the contents of which are expressly incorporated herein in its entirety). When agitated, the protein denatures slightly, allowing the single hydrophobic alpha helix to extend into the air while the hydrophilic beta sheet remains in the water phase. Normally these two regions are folded onto each other, so without agitation or continued bridging of the air-water interface, the protein is most likely to exist as an invert water soluble protein. The blue foam nests of the Tungara frog offer an excellent example of a protein based foam, which is compatible with lipid membranes, yet resistant to environmental factors and can persist for the time required for tadpole maturation, usually three days or more (see Downie, J.R., Functions of the foam in foam-nesting Leptodactylids: the nest as a posthatching refuge in Physalaemus pustulosus. Herperol, J 1993. 3 : p. 35-42.)
SUMMARY OF THE INVENTION
In accordance with the purposes of the disclosed materials, compositions, articles, devices, and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to bubble and anti-bubble compounds and compositions and methods for preparing and using such compounds and compositions wherein the surfactant used includes biologically derived surfactant, for example, the protein Ranaspumin-2 (Rsn2). The term "bubble" as used herein also expressly refers to "anti-bubble" compounds.
In one embodiment of the present invention, the bubble compound is used as an artificial photosynthesis platform combining two technology platforms to yield value-added products: bubble/foam architecture and proteopolymersomes. One such value-added product is simple sugar prepared by carbon fixation accomplished by integrating biosolar
proteopolymersomes and a plurality of enzymes into the microchannels of inflatable foam. BR-ATPase polymersomes may be used to convert light into ATP, which powers a rubisco substrate-enzyme reaction of carbon synthesis, and eventually the formation of hydrocarbon for bio fuels. This artificial photosynthesis platform produces glyceraldehyde-3 -phosphate (G3P) and/or simple sugars that can be used to make a variety of useful organic compounds like HMF, DMF, methanol, ethanol or even sugars for human consumption.
Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.
BRIEF DESCRIPTION OF THE FIGURES
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.
FIG. 1 is a schematic of a portion of a bubble wall where a secondary component (3) is incorporated in the aqueous layer (2) between two surfactant monolayers (1 and 4).
FIG. 2(a) is a photograph of a foam; the bubbles are polyhedral. FIG 2(b) is a schematic showing the geometry of a single foam polyhedron. Almost all liquid is concentrated in the Plateau borders, shown in the expanded view.
FIG. 3(a) is a schematic of a bubble and an antibubble.
FIG. 3(b) is a magnified structure of a portion of an antibubble wall where two surfactant monolayers (62 and 63) define an air layer (61) between two aqueous layers (64 and 65).
FIG. 4(a) is a schematic of a single bubble structure.
FIG. 4(b) is a schematic of a cross-sectional view of a bubble.
FIG. 5 is an illustration of the BR/FoFi ATP synthase vesicle solar conversion system coupled to the RuBisCO CBB cycle enzymes and trapped within the foam channels in accordance with one embodiment of the present invention.
FIG. 6 is a schematic of the encapsulating method from a mixture of bubble solution and secondary component.
FIG. 6 (a) is a schematic showing a bubble solution containing surfactant (71) and a secondary component (70) (shown here as already-made functional polymersomes).
FIG. 6(b) is a schematic showing a cross-sectional view of bubble containing secondary component (70) inside the water channel (72) after the blowing process.
FIG. 7 is a schematic of the encapsulating method using coalescence between bubbles; (a) preparation of bubbles (one with polymersomes (shown as dots), the other without) under different conditions, (b) coalescence process by contacting bubbles, and (c) after coalescence process.
FIG. 8 is an illustration of a sol-gel design for a foam encasement in accordance with one embodiment of the present invention.
FIG. 9(a) is a synthetic scheme of PEtOz-PDMS-PEtOz triblock copolymer.
FIG. 9(b) is a *H NMR spectrum of PEtOz-PDMS-PEtOz in DMSO-d6.
FIG. 10 is a graph showing Control Group for ATP synthesis in Bulk Solution.
FIG. 11 is a graph showing Control Group for ATP synthesis in Foam Architecture.
FIG. 12 is a graph showing Control Group for ATP synthesis in Deflated Foam Solution.
FIG. 13 is a graph showing G3P Production with RuBisCO and ATP stock in bulk solution.
FIG. 14 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay with Artificial ATP Source and various components removed. FIG. 15 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control with Bulk Vesicle ATP Source and various components removed.
FIG. 16 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control Group with Foam Vesicle ATP Source.
FIG. 17 is a graph showing an Absorbance Plot for glucose oxidase assay showing the oxidation of o-dianisidine to a pink colored product absorbing at 540 nm from various glucose concentrations.
FIG. 18 is a graph showing DLS Size Distribution Plot for polymer vesicles.
FIG. 19 is a graph showing DLS Size Distribution Plot for lipid vesicles.
FIG. 20 shows TEM Micrographs of BR/FoFi ATP Synthase Proteopolymersomes.
FIG. 21 shows TEM Micrographs of BR/FoFi ATP Synthase Liposomes.
FIG. 22 shows fluorescent images of foam vesicle solutions.
FIG. 23 is a graph showing the production of ATP with BR/ ATP synthase lipid vesicles in Rsn-2 foam (A ), in bulk (■), in deflated Rsn-2 foam ( -4) in T20 foam ( T ) and a control experiment in the dark (·) for comparison. Inset is the light intensity standard curve created with ATP stock dilutions.
FIG. 24 is a graph showing BR/ATP synthase function in a lipid membrane was limited to the Rsn-2 based foam since the T20 adversely affected coupled FiFo-Atpase/BR vesicle function.
FIG. 25 is a graph showing ATP synthesis using BR/ATP synthase polymersomes in
T20 foam (■), in bulk (·), deflated T20 foam ( T ), and a control experiment in the dark ( A ) for comparison (n=3 for each). Inset is the light intensity standard curve created with ATP stock dilutions.
FIG. 26 is a graph showing the foam system containing BR/ATP synthase vesicles, RuBisCO, PGK, GAPDH, NADH, which is converting C02 and RuBP to G3P using photoderived ATP wherein the RuBisCO dependent carbon fixation reaction is fueled by lipid photophosphorylation vesicles fuels within the Rsn-2 foam (black, n=3), and a in bulk (brown, n=3); and the proteopolymersomes within Two foam (red, n=3), and in bulk (blue, n=3).
FIG. 27(a) is a TEM image of polymersomes after bacteriorhodopsin/ATP synthase incorporation.
FIG. 27(b) is a size distribution histogram derived from direct measurement of polymersome sizes by TEM micrographs. FIG. 28(a) is a graph showing internal pH change for bacteriorhodopsin
polymersomes (·) and bacteriorhodopsin- ATP synthase-polymersomes (■) together with a dark-incubated control (o) in buffer solution.
FIG. 28(b) is a graph showing photo- induced ATP synthesis in bacteriorhodopsin- ATP synthase-polymersomes in a foam.
DETAILED DESCRIPTION OF THE INVENTION
The materials, compounds, compositions, articles, and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein and to the Figures.
Before the present materials, compounds, compositions, articles, and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:
Throughout the description and claims of this specification the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.
As used in the description and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes mixtures of two or more such compounds, reference to "an agent" includes mixtures of two or more such agents, reference to "the moiety" includes mixtures of two or more such moieties, and the like. "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed, then "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the application data is provided in a number of different formats and that these data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed, as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 1 1, 12, 13, and 14 are also disclosed.
References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. I. Compositions
Disclosed herein are materials, compounds, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a compound is disclosed and a number of modifications that can be made to a number of components or residues of the compound are discussed, each and every combination and permutation that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of components or residues A, B, and C are disclosed as well as a class of components or residues D, E, and F, and an example of a combination compound A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C- F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1 -5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1 -40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
a. Bubbles
In one aspect, described herein is a bubble, comprising a wall, wherein the wall comprises a liquid layer between two layers of surfactant; and at least one secondary component, wherein the at least one secondary component is substantially present or completely present in the liquid layer. In one embodiment, the liquid layer is an aqueous layer. Such bubbles can be used as a biological system that serves as a synthesis chamber to produce biological products.
Figure 1 provides a general structure of a portion of a wall of the bubbles described herein (further examples are shown in Figures 2-6). Referring to Figure 1, the wall of the bubble is composed of one or more surfactants, where the surfactant(s) forms a layer defining the outer wall (1) and a layer defining the inner wall (4) of the bubble. The wall structure created by surfactant layers (1 and 4) creates a channel, which is depicted as (2) in Figure 1. The channel can be filled with a liquid. In one aspect, the channel can be filled with water alone or water in combination with one or more liquid solvents such as, for example, an organic solvent. The channel with and without organic solvent is referred to herein as the "aqueous layer." The bubbles described herein can be any shape such as, for example, spherical, elliptical, or polyhedral. In other aspects, the bubbles can be a thin film with an aqueous layer sandwiched between two layers of surfactant. Alternatively, the bubbles can exist as a foam. Foam formation takes place when bubbles come together and they share the same water layer to form a polyhedron. As shown in Figure 2, the edges of the polyhedron are connected to form channel-like structures known as Plateau borders. The froth of bubbles begins to drain under gravity, removing much of the water between the bubbles. Most of the water resides in the Plateau borders. Some of the bubbles merge into larger bubbles, which is called coarsening (Aubert et al, Scientific American 1986, 254:74-82; Isenberg, The science of soap films and soap bubbles. Dover, New York, 1992, pp. 17-21 ; Weaire and Hutzler, The physics of foams. Oxford, 2000, pp. 6-12; Stone et al, J Phys Condens Matter 2003, 15:S283- S290; Hilgenfeldt et al, Europhys Lett, 2004, 67(3):484-90, which are each incorporated by reference herein at least for their teachings of bubbles and bubble structures). The width of the channel created by the surfactant (i.e., the thickness of the bubble wall; e.g., as shown as (2) in Figure 1 , (61) in Figure 3, and (72) in Figure 4) can typically be from about 1 nm to about 10 μηι (for spherical bubbles) and from about 10 nm to about 600 μιη (for foams). In still other examples, the width of the channel distance can be about 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, 900 nm, 925 nm, 950 nm, 975 nm, lOOOnm (1.0 μιη), 1.1 μιη, 1.2 μιη, 1.3 μιη, 1.4 μιη, 1.5 μιη, 1.6 μιη, 1.7 μιη, 1.8 μιη, 1.9 μιη, 2.0 μιη, 2.1 μηι, 2.2 μιη, 2.3 μιη, 2.4 μηι, 2.5 μιη, 2.6 μιη, 2.7 μιη, 2.8 μιη, 2.9 μιη, 3.0 μιη, 3.1 μιη, 3.2 μηι, 3.3 μιη, 3.4 μηι, 3.5 μιη, 3.6 μιη, 3.7 μιη, 3.8 μιη, 3.9 μιη, 4.0 μιη, 4.1 μηι, 4.2 μηι, 4.3 μιη, 4.4 μιη, 4.5 μιη, 4.6 μιη, 4.7 μιη, 4.8 μιη, 4.9 μιη, 5.0 μιη, 5.1 μιη, 5.2 μηι, 5.3 μιη, 5.4 μηι, 5.5 μιη, 5.6 μιη, 5.7 μm, 5.8 μιη, 5.9 μm, 6.0 μm, 6.1 μιη, 6.2 μm, 6.3 μιη, 6.4 μm, 6.5 μm, 6.6 μιη, 6.7 μm, 6.8 μιη, 6.9 μm, 7.0 μm, 7.1 μιη, 7.2 μm, 7.3 μιη, 7.4 μm, 7.5 μm, 7.6 μιη, 7.7 μm, 7.8 μιη, 7.9 μm, 8.0 μm, 8.1 μιη, 8.2 μm, 8.3 μιη, 8.4 μm, 8.5 μm, 8.6 μιη, 8.7 μm, 8.8 μιη, 8.9 μm, 9.0 μιη, 9.1 μm, 9.2 μm, 9.3 μιη, 9.4 μm, 9.5 μιη, 9.6 μm, 9.7 μm, 9.8 μιη, 9.9 μm, 10 μιη, 15 μm, 20 μm, 25 μιη, 30 μm, 35 μιη, 40 μm, 45 μm, 50 μιη, 55 μm, 60 μιη, 65 μm, 70 μm, 75 μιη, 80 μm, 85 μιη, 90 μm, 95 μm, 100 μιη, 101 μm, 102 μηι, 103 μm, 104 μm, 105 μιη, 106 μm, 107 μιη, 108 μm, 109 μm, 1 10 μιη, 1 11 μm, 1 12 μηι, 1 13 μm, 114 μm, 1 15 μιη, 1 16 μm, 1 17 μιη, 118 μm, 119 μm, 120 μιη, 121 μm, 122 μηι, 123 μm, 124 μm, 125 μιη, 126 μm, 127 μιη, 128 μm, 129 μιη, 130 μm, 131 μm, 132 μηι, 133 μm, 134 μηι, 135 μm, 136 μm, 137 μιη, 138 μm, 139 μιη, 140 μm, 141 μm, 142 μηι, 143 μm, 144 μηι, 145 μm, 146 μm, 147 μιη, 148 μm, 149 μιη, 150 μm, 151 μm, 152 μηι, 153 μm, 154 μηι, 155 μm, 156 μm, 157 μιη, 158 μm, 159 μιη, 160 μm, 161 μm, 162 μηι, 163 μm, 164 μηι, 165 μm, 166 μm, 167 μιη, 168 μm, 169 μιη, 170 μm, 171 μm, 172 μηι, 173 μm, 174 μηι, 175 μm, 176 μm, 177 μιη, 178 μm, 179 μιη, 180 μm, 181 μιη, 182 μm, 183 μm, 184 μηι, 185 μm, 186 μιη, 187 μm, 188 μm, 189 μιη, 190 μm, 191 μιη, 192 μm, 193 μm, 194 μηι, 195 μm, 196 μιη, 197 μm, 198 μm, 199 μιη, 200 μm, 201 μιη, 202 μm, 203 μm, 204 μηι, 205 μm, 206 μιη, 207 μm, 208 μm, 209 μιη, 210 μm, 21 1 μηι, 212 μm, 213 μm, 214 μηι, 215 μm, 216 μιη, 217 μm, 218 μm, 219 μιη, 220 μm, 221 μηι, 222 μm, 223 μm, 224 μηι, 225 μm, 226 μιη, 227 μm, 228 μm, 229 μιη, 230 μm, 231 μιη, 232 μm, 233 μιη, 234 μm, 235 μm, 236 μιη, 237 μm, 238 μιη, 239 μm, 240 μm, 241 μηι, 242 μm, 243 μιη, 244 μm, 245 μm, 246 μιη, 247 μm, 248 μιη, 249 μm, 250 μm, 251 μιη, 252 μm, 253 μιη, 254 μm, 255 μm, 256 μιη, 257 μm, 258 μιη, 259 μm, 260 μm, 261 μιη, 262 μm, 263 μιη, 264 μm, 265 μm, 266 μιη, 267 μm, 268 μιη, 269 μm, 270 μm, 271 μιη, 272 μm, 273 μιη, 274 μm, 275 μm, 276 μιη, 277 μm, 278 μιη, 279 μm, 280 μm, 281 μιη, 282 μm, 283 μηι, 284 μηι, 285 μηι, 286 μηι, 287 μηι, 288 μηι, 289 μηι, 290 μηι, 291 μηι, 292 μηι, 293 μηι, 294 μηι, 295 μηι, 296 μηι, 297 μηι, 298 μηι, 299 μηι, 300 μηι, 301 μηι, 302 μηι, 303 μηι, 304 μηι, 305 μηι, 306 μηι, 307 μηι, 308 μηι, 309 μηι, 310 μηι, 311 μηι, 312 μηι, 313 μηι, 314 μηι, 315 μηι, 316 μηι, 317 μηι, 318 μηι, 319 μηι, 320 μηι, 321 μηι, 322 μηι, 323 μηι, 324 μηι, 325 μηι, 326 μηι, 327 μηι, 328 μηι, 329 μηι, 330 μηι, 331 μηι, 332 μηι, 333 μηι, 334 μηι, 335 μηι, 336 μηι, 337 μηι, 338 μηι, 339 μηι, 340 μηι, 341 μηι, 342 μηι, 343 μηι, 344 μηι, 345 μηι, 346 μηι, 347 μηι, 348 μηι, 349 μηι, 350 μηι, 351 μηι, 352 μηι, 353 μηι, 354 μηι, 355 μηι, 356 μηι, 357 μηι, 358 μηι, 359 μηι, 360 μηι, 361 μηι, 362 μηι, 363 μηι, 364 μηι, 365 μηι, 366 μηι, 367 μηι, 368 μηι, 369 μηι, 370 μηι, 371 μηι, 372 μηι, 373 μηι, 374 μηι, 375 μηι, 376 μηι, 377 μηι, 378 μηι, 379 μηι, 380 μηι, 381 μηι, 382 μηι, 383 μηι, 384 μηι, 385 μηι, 386 μηι, 387 μηι, 388 μηι, 389 μηι, 390 μηι, 391 μηι, 392 μηι, 393 μηι, 394 μηι, 395 μηι, 396 μηι, 397 μηι, 398 μηι, 399 μηι, 400 μηι, 401 μηι, 402 μηι, 403 μηι, 404 μηι, 405 μηι, 406 μηι, 407 μηι, 408 μηι, 409 μηι, 410 μηι, 411 μηι, 412 μηι, 413 μηι, 414 μηι, 415 μηι, 416 μηι, 417 μηι, 418 μηι, 419 μηι, 420 μηι, 421 μηι, 422 μηι, 423 μηι, 424 μηι, 425 μηι, 426 μηι, 427 μηι, 428 μηι, 429 μηι, 430 μηι, 431 μηι, 432 μηι, 433 μηι, 434 μηι, 435 μηι, 436 μηι, 437 μηι, 438 μηι, 439 μηι, 440 μηι, 441 μηι, 442 μηι, 443 μηι, 444 μηι, 445 μηι, 446 μηι, 447 μηι, 448 μηι, 449 μηι, 450 μηι, 451 μηι, 452 μηι, 453 μηι, 454 μηι, 455 μηι, 456 μηι, 457 μηι, 458 μηι, 459 μηι, 460 μηι, 461 μηι, 462 μηι, 463 μηι, 464 μηι, 465 μηι, 466 μηι, 467 μηι, 468 μηι, 469 μηι, 470 μηι, 471 μηι, 472 μηι, 473 μηι, 474 μηι, 475 μηι, 476 μηι, 477 μηι, 478 μηι, 479 μηι, 480 μηι, 481 μηι, 482 μηι, 483 μηι, 484 μηι, 485 μηι, 486 μηι, 487 μηι, 488 μηι, 489 μηι, 490 μηι, 491 μηι, 492 μηι, 493 μηι, 494 μηι, 495 μηι, 496 μηι, 497 μηι, 498 μηι, 499 μηι, 500 μηι, 501 μηι, 502 μηι, 503 μηι, 504 μηι, 505 μηι, 506 μηι, 507 μηι, 508 μηι, 509 μηι, 510 μηι, 511 μηι, 512 μηι, 513 μηι, 514 μηι, 515 μηι, 516 μηι, 517 μηι, 518 μηι, 519 μηι, 520 μηι, 521 μηι, 522 μηι, 523 μηι, 524 μηι, 525 μηι, 526 μηι, 527 μηι, 528 μηι, 529 μηι, 530 μηι, 531 μηι, 532 μηι, 533 μηι, 534 μηι, 535 μηι, 536 μηι, 537 μηι, 538 μηι, 539 μηι, 540 μηι, 541 μηι, 542 μηι, 543 μηι, 544 μηι, 545 μηι, 546 μηι, 547 μηι, 548 μηι, 549 μηι, 550 μηι, 551 μηι, 552 μηι, 553 μηι, 554 μηι, 555 μηι, 556 μηι, 557 μηι, 558 μηι, 559 μηι, 560 μηι, 561 μηι, 562 μηι, 563 μηι, 564 μηι, 565 μηι, 566 μηι, 567 μηι, 568 μηι, 569 μηι, 570 μηι, 571 μηι, 572 μηι, 573 μηι, 574 μηι, 575 μηι, 576 μηι, 577 μηι, 578 μηι, 579 μηι, 580 μηι, 581 μηι, 582 μηι, 583 μηι, 584 μηι, 585 μηι, 586 μηι, 587 μηι, 588 μηι, 589 μηι, 590 μηι, 591 μηι, 592 μηι, 593 μηι, 594 μηι, 595 μηι, 596 μηι, 597 μηι, 598 μηι, 599 μηι, or 600 μηι, where any of the stated values can form an upper or lower endpoint when appropriate.
Referring to Figure 1 (and also Figure 2), the secondary component (3) is
substantially or completely present in the channel (2) created by the surfactant layers (1 and 4). By "substantially present" is meant that the secondary component is mostly present in the aqueous layer; however, it is contemplated that some amount of the secondary component can also be present, either entirely or partially, in either or both of the surfactant layers (1 or 4 in Figure 1). It is also contemplated that the secondary component can partially extend out from either or both surfactant layers into the gas (e.g., air) space. The phrase "incorporated into the bubble wall" is also used synonymously herein with the phrase "substantially present." b. Anti-bubbles
In another aspect, described herein is a bubble comprising a wall, wherein the wall comprises an inner wall and an outer wall, wherein the inner wall comprises an inner surface and an outer surface and the outer wall comprises an inner surface and an outer surface, wherein the inner wall and the outer wall comprises a surfactant, wherein the inner wall and the outer wall comprises a gas between two layers of surfactant; an aqueous layer, wherein the aqueous layer is adjacent to the outer surface of the inner wall of the bubble; and a secondary component, wherein the secondary component is substantially present in the aqueous layer.
In this aspect, the bubble is also referred to herein as "an anti-bubble." The term bubble as used herein includes the bubbles described above in section (a) and anti- bubbles. Techniques for producing anti-bubbles are known (Hughes and Hughes, Nature 1932,
129:599). In one aspect, the anti-bubble can have a spherical air shell surrounding a liquid. This aspect is depicted in Figure 3, wherein a gas layer (61) (e.g., air) is sandwiched between two surfactant layers (62 and 63). In one aspect, an aqueous layer (64) is adjacent to the outer surface of inner wall (62). It is also contemplated that a second aqueous layer (65) can be adjacent to the outer surface of the outer wall (63). The term "adjacent" is defined herein as any solvent (e.g., water) that is in contact with the surfactant, which also includes penetration of the solvent into the surfactant layer. Similar to the bubbles described above, the secondary component can be substantially present in the aqueous layer. For example, referring to Figure 3, the secondary component can be present in the aqueous layers (64) and/or (65).
Additionally, the dimensions, shapes, and sizes of the anti-bubbles can be the same as those described above as for the bubbles described in section (a).
Described below are the different surfactants and secondary components useful in producing the bubbles described herein. i. Surfactant
A "surfactant" as used herein is a molecule composed of hydrophilic and hydrophobic groups (i.e., an amphiphile). Because of solubility differences in water, when a bubble is formed, the hydrophobic ends of the surfactant molecules accumulate at an air/water interface, thereby reducing the surface tension (Weaire and Hutzler, The physics of foams, Oxford, 2000, Ch. 1-2). Thus, the surfactant forms a monolayer on the inside and a monolayer on the outside of the water. A schematic of a surfactant bubble composed of a several micrometer- thick water layer sandwiched between two surfactant monolayers is shown in Figure 4 (a close up of a portion of the bubble wall is shown in Figure 1). Because the hydrophobic end of the surfactant molecule sticks out from the surface of the bubble, the surfactant film is somewhat protected from evaporation which can prolong the life of the bubble. A closed container saturated with water vapor also slows evaporation and can allow surfactant films to last even longer.
Bubbles suitable for the compositions and methods disclosed herein can be made from biologically-derived surfactants. In one embodiment of the present invention, the
biologically-derived surfactant may be a natural protein surfactant. In yet another embodiment of the present invention, the natural protein surfactant is Ranaspumin (Rsn) protein surfactant, such as Rsn-2. In one aspect, a bubble can be prepared from mixtures of two or more surfactants.
The expression of the Rsn2 gene factor may be accomplished using a variety of known techniques. For example, the expression of the Rsn2 gene has been demonstrated in bacteria (see Mackenzie, CD., et al., Ranaspumin-2 ; Structure and Function of a Surfactant Protein from the Foam Nests of a Tropical Frog. Biophysical Journal, 2009. 96(12); p.4984- 4992, the contents of which are expressly incorporated herein in its entirety). As a result, for example, a gene containing idealized E.Coli codon usage can be constructed for more efficient bacterial expression. Using this example, once the synthetic gene has been completed, the next step is to transform the gene into an inducible expression host and purify the protein. In a preferred embodiment, the Rsn2 gene may include two affinity tags.
However, one must ensure that the protein retains its natural surfactant and foam forming capabilities. As a rudimentary test, one may vary Rsn2 concentration in an aqueous solution and measure the resulting contact angle of the water droplet. In one aspect, a Langmuir Blodgett film may be used to acquire a more precise quantification of the protein's surfactant properties. Foam topology arises from surprisingly uniform physical principles and structural elements. The surface of the bubbles of aqueous foams are mediated by surfactants which are necessary to stabilize the air-water interface and provide an energetic (both electrostatic and steric) barrier to rupture and collapse. These form tetrahedral structures commonly referred to as Plateau junctions. The legs and nodes of the junction contain the trapped liquid phase typically 0.01-1 mm wide. The drainage of these channels is a primary concern for the foam stability and functionality. ii. Secondary Component
As used herein the secondary component can be anything (e.g., molecule,
compositions, device) that can be substantially present in the channel (e.g., aqueous layer) of the bubble wall. In one aspect, the bubble can comprise two or more different secondary components. In another aspect, the secondary component can have a width greater than, equal to, or less than the width of the bubble wall, as described herein. For example, the secondary component can have a width greater, equal to, or less than about 600 μιη, 500 μιη, 400 μιη, 300 μιη, 200 μιη, 100 μιη, 90 μιη, 80 μιη, 70 μιη, 60 μιη, 50 μιη, 40 μιη, 30 μιη, 20 μιη, 10 μιη, 9 μιη, 8 μιη, 7 μιη, 6 μιη, 5 μιη, 4 μηι, 3 μιη, 2 μηι, 1 μηι, 500 ηηι, 100 nm, or lnm. In one aspect, the secondary component can be a biomolecule. Examples of biomolecules include, but are not limited to, a small molecule (e.g., a drag), a peptide, a protein, an enzyme (e.g., a kinase, a phosphatase, a methylating agent, a protease, a transcriptase, an
endonuclease, a ligase, and the like), an antibody and/or fragment thereof, a nucleic acid (e.g., an oligonucleotide, a prime, a probe, an aptamer, a ribozyme, etc.), a lipid, a carbohydrate, a steroid, a hormone, a vitamin, a potential therapeutic agent. "Small molecule" as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD, for example, less than about 4 kD. Small molecules can be nucleic acids (e.g., DNA, RNA), peptides, polypeptides, peptidomimetics, carbohydrates, lipids, factors, cofactors, hormones, vitamins, steroids, trace elements, pharmaceutical drugs, or other organic (carbon containing) or inorganic molecules.
The secondary component can also be a macromolecule such as a polymer, a vesicle, or a dendrimer, or a cell or a microbe (e.g., a detoxifying organism), including mixtures thereof.
There are a variety of compositions disclosed herein where the secondary component (e.g., biomolecule) can comprise an amino acid based molecule, including for example enzymes and antibodies. Thus, as used herein, "amino acid," means the typically encountered twenty amino acids which make up polypeptides. In addition, it further includes less typical constituents which are both naturally occurring, such as, but not limited to formylmethionine and selenocysteine, analogs of typically found amino acids, and mimetics of amino acids or amino acid functionalities. Non-limiting examples of these and other molecules are discussed herein.
As used herein, the terms "peptide" and "protein" refer to a class of compounds composed of amino acids chemically bound together. Non-limiting examples of these and other molecules are discussed herein. In general, the amino acids are chemically bound together via amide linkages (CONH); however, the amino acids can be bound together by other chemical bonds known in the art. For example, the amino acids can be bound by amine linkages. "Peptide" as used herein includes oligomers of amino acids and small and large peptides, including naturally occurring or engineered polypeptides and proteins. It is understood that the terms "peptide" and "protein" can be used interchangeably herein.
It is also understood that there are numerous amino acid and peptide analogs that can be used as the secondary component. For example, there are numerous D amino acids or amino acids which have a different functional substituent than the typically encountered amino acids. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. Additionally, molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include - CH2NH-, -CH2S-, -CH2CH2-, - CH=CH- (cis and trans), -COCH2-, - CH(OH)CH2-, and -CHH2SO-. These and others can be found in Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, 1983, p. 267; Spatola, Vega Data 1983, Vol. 1 , Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci, 1980, pp. 463-68; Hudson et al, Int J Pept Prot Res 1979, 14: 177-85 (-CH2NH-, - CH2CH2-);
Spatola et at, Life Sci 1986, 38: 1243-9 (-CH H2-S); Hann, J Chem Soc Perkin Trans 1 1982, 307-14 (-CH=CH- cis and trans); Almquist et al, J Med Chem 1980, 23 : 1392-8 (-COCH2-); Jennings- White et al, Tetrahedron Lett 1982, 23 :2533 (-COCH2-); Szelke et al, European Appln, EP 45665 CA (1982): 97:39405 (-CH(OH)CH2-); Holladay et al, Tetrahedron Lett 1983, 24:4401-4 (-C(OH)CH2-); and Hruby, Life Sci 1982, 31 : 189-99 ( - CH2S - ) each of which is incorporated herein by reference herein for at least their teachings of amino acid analogs. It is understood that peptide analogs can have more than one atom between the bond atoms, such as beta-alanine, gama- aminobutyric acid, and the like. Such analogs are contemplated within the meaning of the terms peptide and protein. In addition, peptides and proteins contemplated herein as biomolecules can be derivatives and variants of the disclosed peptides and proteins that also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes:
substitutional, insertional, and deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final construct.
Also, certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydro xylation of proline and lysine, phosphorylation of hydroxy! groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains (T .E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco 1983, pp. 79-86, which is incorporated herein at least for its teachings of peptide and protein modifications), acetylation of the N- terminal amine and, in some instances, amidation of the C-terminal carboxyl. It is also possible to link peptides and proteins to other molecules (e.g., to form conjugates). For example, carbohydrates (e.g., glycoproteins) can be linked to a protein or peptide. Such derivatives, variants, and analogs of peptides and proteins are contemplated herein within the meaning of the terms peptide and protein.
Methods for producing such peptides and proteins are well known. One method of producing the disclosed proteins is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9- fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied
Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant, Synthetic Peptides: A User Guide. W.H. Freeman and Co., N. Y. 1992; Bodansky and Trost, Ed. Principles of Peptide Synthesis. Springer- Verlag Inc., N. Y., 1993, which are incorporated by reference herein at least for their teachings of peptide synthesis).
Alternatively, a peptide or polypeptide can be independently synthesized in vivo. For example, advances in recombinant glycoprotein production methods, which allow more cost effective production of human glycoproteins by colonies of transgenic rabbits or by yeast strains carrying human N-glycosylation system enzymes can be used (Hamilton et ah, Science 2003, 301 : 1244-6; Gerngross, Nature Biotechnology 2004, 22: 1409, which are incorporated by reference herein at least for their teachings of peptide and protein synthesis).
Once isolated, independent peptides or polypeptides may be linked, if needed, to form a peptide or fragment thereof via similar peptide condensation reactions. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et ah, Biochemistry 1991, 30:4151, which is incorporated by reference herein at least for its teachings of peptide and protein synthesis). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. (See e.g., Dawson et ah, Science 1994, 266:776-9; Baggiolini et al, FEBS Lett 1992, 307:97-101 ; Clark-Lewis et al, J Biol Chem 1994, 269: 16075; Clark-Lewis et al, Biochemistry 1991, 30:3128; Rajarathnam et al, Biochemistry 1994, 33 :6623-30, which are incorporated by reference herein at least for their teachings of peptide and protein synthesis). Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer et al, Science 1992, 256:221 , which is incorporated by reference herein at least for its teachings of peptide and protein synthesis). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton, et al, (1992) Techniques in Protein Chemistry IV. Academic Press, N. Y., pp. 257-67 ', which is incorporated by reference herein at least for its teachings of peptide and protein synthesis).
In another aspect, the secondary component (e.g., bio molecule) can comprise an antibody. As used herein, the term "antibody" encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-I, IgG-2, IgG-3, and IgG-4; IgA-I and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The term "variable" is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1987)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. The term "antibody" as used herein is meant to include intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding the epitopic determinant. The term "antibody" also includes monoclonal and polyclonal antibodies, anti-idiopathic, and humanized antibodies.
As used herein, the term "antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab and the like, including hybrid fragments. Such antibodies and fragments can be made by techniques known in the art (see Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, N. Y., 1988). Such antibodies and fragments thereof can be screened for specificity and activity according to the methods disclosed herein.
Also included within the meaning of "antibody or fragments thereof are conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Patent No. 4,704,692, the contents of which are hereby incorporated by reference for at least its teaching of antibody conjugates. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues. Methods of producing and/or isolating antibodies as disclosed herein are well known. There are also a variety of compositions disclosed herein where the secondary component can comprise a nucleic acid based molecule. Thus, as used herein, "nucleic acid" means a molecule made up of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. A nucleic acid can be double stranded or single stranded. Nucleic acid is also meant to include oliognucleotides.
As used herein, "nucleotide" is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-l-yl (C), guanine-9-yl (G), uracil- 1 -yl (U), and thymin-l-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non- limiting example of a nucleotide would be 3'- AMP (3 '-adenosine monophosphate) or 5'- GMP (5'-guanosine monophosphate).
"Nucleotide analog," as used herein, is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as
modifications at the sugar or phosphate moieties.
"Nucleotide substitutes," as used herein, are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
It is also possible to link other types of molecules to nucleotides or nucleotide analogs to make conjugates that can enhance, for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc Natl Acad Sd USA, 1989, 86:6553-6, which is incorporated by reference herein at least for its teachings of nucleic acid conjugates). As used herein, the term nucleic acid includes such conjugates, analogs, and variants of nucleic acids.
Nucleic acids, such as those described herein, can be made using standard chemical synthetic methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001, Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B).
Synthetic methods useful for making oligonucleotides are also described by Ikuta et al, Ann Rev Biochem 1984, 53 :323-56 (phosphotriester and phosphite-triester methods), and Narang et al, Methods Enzymol 1980, 65 :610-20 (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug Chem 1994, 5:3-7. Each of these references is incorporated by reference herein at least for their teachings of nucleic acid synthesis.
"Probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. "Primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
"Aptamers" are also contemplated herein and are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP (U.S. Patent No. 5,631, 146) and theophiline (U.S. Patent No. 5,580,737), as well as large molecules, such as reverse transcriptase (U.S. Patent No. 5,786,462) and thrombin (U.S. Patent No. 5,543,293). Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of U.S. Patents: 5,476,766, 5,503,978, 5,631 ,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721 , 5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028, 186, 6,030,776, and 6,051,698, which are incorporated by reference herein for at least their teachings of aptamers.
"Ribozymes" are also contemplated herein and are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly.
Ribozymes are thus catalytic nucleic acid. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes (for example, but not limited to the following U.S. Patents: 5,334,711 , 5,436,330, 5,616,466, 5,633, 133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621 , 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following U.S. Patent Nos.: 5,631 ,1 15, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, but not limited to the following U.S. Patents: 5,595,873 and 5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following U.S. Patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non- canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non- limiting list of U.S. Patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855,
5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756. These patents are all incorporated by reference herein at least for their teachings of ribozymes.
In another aspect, the secondary component can be an artificial or natural organelle (e.g., chlorop lasts, mitochondria for energy production, etc.), including mixtures thereof. An example of an artificial organelle that can be incorporated into the bubble wall is disclosed in U.S. Application Publication No. 2004-0049230, which is incorporated by reference herein for its teachings of artificial organelles.
In one aspect, the biomolecule can be a protein, such as a membrane protein or enzyme. In other specific examples, the biomolecule can be a receptor, a channel, a signal transducer, or an ion pump. In still other example, the biomolecule can be an energy converting protein (e.g., bacteriorhodopsin), an aquaporin, MscL, a cytochrome oxidase, hemoglobin, hemerythrin, hemocyanin, GutR, VR15 CMR1, connexin,calreticulin, microtubule, S 100 proteins, heat shock proteins (hsps), OmpA, Omp F, FhuA, FecA, BtuB, OMPLA, OpcA, FadL, NspA, light-harvesting complex (LHC) proteins, fumarate reductase, succinate dehydrogenase, formate dehydrogenase, nitrate reductase, or an ATPase.
In alternative aspects, the secondary component can be an indicator (e.g., pH, fluorescence, etc.), a carbon based nanostructure (e.g., buckyballs and nano tubes), a dendrimer, a nanoscale device, a microelectric machine (MEMs), an organic or inorganic compound, a non- water liquid, a gas (e.g., hydrogen), and mixtures thereof.
It is contemplated that any of the secondary components described herein can be imbedded into a polymer matrix prior to bubble formation. By "imbedded into a polymer matrix" is meant that the secondary component is chemically attached (e.g., covalently, ionically, electrostatically, or by hydrogen bonding) to the polymer matrix or physically attached with the polymer matrix (e.g. , wholly or partially encapsulated within the matrix). This is also referred to herein as a "polymersome." In one aspect, the secondary component comprises a biomolecule imbedded into a polymer matrix.
The polymer matrix can comprise any polymer. Suitable polymers include, but are not limited to, homopolymers or copolymers. In some examples, the polymer can be a block, random, or graft copolymer. Suitable polymers for the polymer matrix are readily available from commercial sources and/or can be prepared by methods known to those of ordinary skill in the art.
Specific examples of polymers suitable for use in the polymeric matrix include, but are not limited to, modified or unmodified polyolefms, polyethers, and polyalkylene oxides. More specific examples of suitable polymers can include, but are not limited to, modified or unmodified polyethylene, polypropylene, polystyrene, polybutylene, poly(meth)acrylate, polymethylmethacrylate, polyacrylonitrile, ABS, polyethylene oxide, polypropylene oxide, polybutylene oxide, polyterephthalate, polyamide, nylon, polysiloxane, polyvinylacetate, polyvinylethers, polyoxazoline, polyacrylic acid, polyacyl alkylene imine,
polyhydroxyalkylacrylates, copolymers, and mixtures thereof.
The term "modified" is used herein to describe polymers and means that a particular monomelic unit that would typically make up the pure polymer has been replaced by another monomelic unit that shares a common polymerization capacity with the replaced monomelic unit. Thus, for example, it is possible to substitute diol residues for glycol in poly(ethylene glycol), in which case the polyethylene glycol) will be "modified" with the diol. In one aspect, the polymer used to prepare the polymer matrix comprises a polymer produced by the ring-opening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride- terminated PDMS.
In one aspect, secondary component comprises a protein such as, for example, bacteriorhodopsin, imbedded in a polymer matrix comprising a polymer produced by the ring-opening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride- terminated PDMS.
In one aspect, the secondary component may be a polymer vesicle, or polymersome, embedded with biomolecules, such as proteins, in a manner which retains the functionality of the biomolecule. In one aspect, the biomolecule is embedded within the wall of the polymersome such that a portion of the biomolecule extends outside the polymersome and a portion of the biomolecule extends inside the polymersome. These types of polymer vesicles, or polymersomes, are referred to as "proteopolymersomes". In one embodiment, block copolymer BR/ATPase polymersomes may be used as the secondary component. Such nanoscale polymesomes can produce ATP from light while in foam scaffold which contains moderate levels of detergent. It is known that a diblock copolymer, poly(ethylene oxide-b- polyethylene) (OE) of a particular molecular weight and composition, can form bilayer membranes and enclosed vesicles can range in size from hundreds of nanometers to tens of microns in diameter. (Discher, B.M., et al., Polymersomes: tough, giant vesicles made from diblock copolymers. Science, 1999. 284: p.1143-1 146 the entire contents of which are expressly incorporated herein by reference.) The family of di-block copolymers that can be used to make polymersomes is known to those of skill in the art. A series of poly(l,2 butadiene-b-polyethylene oxide) polymers (OB), most notably OB-2 (MW= 3600 g/mol, ethylene oxide block fraction (fEO)=0.28); OB-29 (MW=3800 g/mol, fEO=0.34); OB-9
(MW=5200 g/mol, fEO=0.37); and )B-18 (MW=10,400 g/mol, fEO=0.39). (Bermudez, H. et al., Molecular weight dependence of polymersome membrane elasticity and stability.
Macromolecules, 2002. 35: p. 8203-8202, the entire contents of which are expressly incorporated herein by reference. ) These polymers have pendant side unsaturation that can be used for crosslinking and further stabilization. Further increases in stability can be achieved by crosslinking polymer vesicles if there is a pendant side group that allows linking among vesicles. One strategy to modulate the toughness and stability of polymersomes is to cross-link the membrane to form a robust polymer network (Discher, B.M., et al., Cross- linked polymersome membranes: Vesicles with broadly adjustable properties. Journal of Physical Chemistry B, 2002. 106(1 1); p. 2848-2854, the entire contents of which are expressly incorporated herein by reference.) Using OB polymers with unsaturated side groups, polymer vesicles can be crosslinked to form solid networks that, if the concentration of crosslinkable polymer is sufficiently high, can greatly increase the critical tension required to cause the vesicle to fail. Crosslinking may be achieved using a chemical electron donor that facilitates the saturation of opposing unsaturated side chains, and the degree of crosslinking is adjusted using mixtures of crosslinkable (OB) and non-crosslinkable (OE) polymers. Alternatively, the degree of crosslinking may be adjusted by mixing fully crosslinkably polymers, but adjusting the extent of crosslinking by using a solute that can crosslink pendant unsaturated side chains using UV radiation of tunable duration and intensity. Once crosslinkied, a vesicle made with 100% crosslinkable OB polymer has a 100- fold greater critical tension than an OE-21 fluid vesicle. The strength of the membrane can be tuned by changing the percentage of crosslinkable polymer in the membrane, with the strength increasing monotonically with % crosslinkable polymer beyond 10%. Furthermore, crosslinked vesicles can be either air dried of lyophilized, are stable, and can be rehydrated later. A broad family of polymers may be used to make proteopolymersomes. In one aspect, as described above, a functional BR-containing proteopolymersome membrane may be formed. Foams may be formed using biodegradable BR-PEO-PCL vesicles or nonbiodegradable BR-PEO-PB vesicles. iii. Additional components
The bubbles disclosed herein can also comprise additional components. For example, additional components can be added to make the bubble more stable. Suitable additional components can include, but are not limited to, preservatives, antioxidants, stabilizers, and the like. For example, by adding glycerine, long-lasting bubbles can be made. c. Artificial Photosynthesis Platform
In one aspect, the present invention may be used to create an artificial photosynthesis platform for converting light into value-added products. In one embodiment, light energy is converted to chemical energy by providing the microchannels of the bubble and/or foam with at least one biosolar component that is capable of converting light energy to chemical energy. The chemical energy is then converted to a value-added product using a plurality of enzymes selected from the Calvin cycle enzymes, or CBB enzymes, such enzymes are also provided within the microchannels of the bubble and/or foam. In one embodiment whereby the bubble and/or foam provides an artificial photosynthesis platform, the secondary components include proteopolymersomes replete with biomolecules capable of producing ATP, and several plant and yeast enzymes representing the requisite components of the Calvin cycle. In one aspect, the biomolecules may include a biological proton pump and an ATP generator. The ATP generator produces ATP when activated by protons produced by the biological proton pump. The proton pump may be a photoactivated proton pump which produces protons when subject to light energy. For example, the biomolecules may be bacteriorhodopsin (BR) and FoFi-ATP synthase (herein referred to as "ATP synthase") entirely embedded in the wall of a polymer vesicle to form the proteopolymersomes in a manner which retains their biological functionality ( Choi, H-J.; Montemagno, C. Nano techno logy, 2006, 17, 2198-2202, the content of which is expressly incorporated herein by reference in its entirety).
Fig 5. illustrates one embodiment of the artificial photosynthesis platform. Foam 20 is fabricated such that the secondary components are retained in microchannels 22 formed from the aqueous layer provided between the two layers of surfactant. One of the secondary components is a proteopolymersome 24 including bacteriorhodopsin (BR) 26 and ATP synthase 28 entirely embedded in the wall 30 of the polymer vesicle. BR utilizes light to create a proton gradient, which is subsequently used by ATP synthase to produce chemical energy. It is desirable to widen the excitation bandwidth of BR and further increase the efficiency of BR. In one embodiment, a purified form of BR is attached to quantum dots (QD's). Quantum dots are UV sensitive semiconductor nanoparticles which emit visible and IR wavelength photons in a size dependent manner. QD's can be attached to BR via nickel nitrilotriacetic acid (Ni-NTA) using the c-terminal Histidine (His) tag engineered onto the BR. The chelated Ni ion of Ni-NTA is capable of orthogonal attachment to the His-tag on the c- terminus, which in the correct orientation, would be on the outside of the vesicles. Once the QD is attached, the protein is separated from the unbound QD's using gel-filtration. By widening the absorption spectra of BR to include a greater portion of solar radiation, the photoconversion efficiency and overall output of the system may be improved. Other proteins may be used instead of or in conjunction with BR as a proton pump for the conversion of light to ATP as described herein. For example, the proton pump
Xanthorhodopsin may also be used (Balashov, S.P., et al., Xanthorhodopsin: A Proton Pump with a Light-Harvesting Carotenoid Antenna, Science, 2005. 309 (5743): p. 2061-2064, the contents of which are expressly incorporated herein in its entirety).
Referring again to Fig. 5, the other important secondary components of the artificial photosynthesis platform are the enzymes 32 required to convert ATP to glyceraldehyde-3 - phosphate (G3P) and/or sugar using ribulose 1,5-biphosphate (RuBP) and CO2 from the air. In the conversion to sugar, there are eight enzymes that are used for the conversion to hexose, such as fructose and/or glucose. RuBisCO, phosphoglycerate kinase (PGK) and
glyceraldehyde phosphate dehydrogenase (GAPDH) are enzymes used to form
glyceraldehyde-3 -phosphate (G3P). The conversion of G3P to hexose is accomplished using triosephosphate isomerase, fructose- 1,6-biphosphate aldolase, fructose- 1 ,6-bisphosphatase, phosphoglucose isomerase and glucose-6-phosphatase.
In order to reduce the cost of the artificial photosynthesis platform, an inexpensive source of a majority of the enzymes is desired. All the proteins except the solar conversion system proteins (BR and ATP synthase) can be engineered to coexpress in an Algae based biofuel system, which the former could be cheaply obtained from bacteria. If a commercial Algae biofuel system like Algaenol is used, where the product is excreted, the algae biomass could then be used to supply the proteins. It is understood that those of skill in the art would understand alternate means for obtaining the listed enzymes, proteins and bacteria.
In the above-described embodiment, the three necessary substrates for conversion of light and CO2 to glyceraldehyde-3 -phosphate (G3P) and/or hexose are ATP, NADH, and ribulose- 1, 5 -bisphosphate (RuBP). Bisphosphate (RuBP) and carbon dioxide (CO2) are catalysed by the enzyme ribulose- 1 ,5-bisphosphate carboxylase oxygenase (RuBisCo) using the energy in ATP. As described above, in a preferred embodiment ATP is provided by the BR/ATP synthase proteopolymersome. NADH can be provided to the artificial photosynthesis platform in a variety of ways. During the process of carbon fixation, NADH is oxidized into NAD+ by the enzyme GAPDH. Since the described embodiment synthesizes simple sugars, NADH is used up and must be replenished. In one embodiment, the NADH may be photocatalytically regenerated. NAD+ can readily be converted back into NADH via P-doped Titanium Oxide nanoparticles using light and a Ru charge carrier. (Shi, Q., Yang, D., Jiang, Z. and Jian Li (2006). Visible-ligth photocatalytic regeneration of NADH using P- Doped T1-O2 nanoparticles. J. Mol. Cat. B, 43 : 44-48, the entire contents of which are expressly incorporated herein by reference). The nanoparticles are available form Reade Inc. (Providence, RI)_and could be added to the foam's aqueous phase and inflated alongside the ATP synthesizing vesicles. In one embodiment, RuBP is regenerated within the system as well. The shortest path to recycle a portion of the fixed carbon back to RuBP involves three enzymes: transketolase, phosphopentose epimerase and phosphoribulose kinase at a cost of 6 ATP molecules. This will convert fructose back into RuBP with an intermediate product either in the form of ribose-5-phosphate or xylulose-5-phosphate by way of the versatility of transketolase, and adolase already present. Since there is a net gain from the atmosphere, this regeneration cycle could be balanced with sugar output to produce a completely renewable system while still maximizing yield. One of skill in the art will appreciate that NADH and RuBP may be added to the artificial photosynthesis platform using a variety of known processes or each may be regenerated within the system using known techniques.
Additionally, one of skill in the art will understand the NADH may be regenerated while RuBP is added or RuBP may be regenerated while NADH is added.
II. Methods of Making
In one aspect, the bubbles disclosed can be prepared by various methods. In one aspect, the bubbles can be prepared by admixing an aqueous solution comprising one or more secondary components and one or more biologically-derived surfactants, and blowing a gas into the mixture. The term "admixing" is defined as mixing two or more components together. Depending upon the components to be admixed, there may or may not be a chemical or physical interaction between two or more components. Figure 6 shows a schematic of one possible process for constructing a bubble as disclosed herein. In this aspect, the secondary component (70) as shown is encapsulated within a polymer matrix. The bubble solution can be admixed with pre- formed functional polymersomes comprising the secondary component and polymer matrix, as shown in Figure 6(a). While being blown with gas, the biologically- derived surfactant molecules (71) can self-assemble to form monolayers on the inside and outside surface of the water channel (72) (see Figure 6(b)). As a result, biologically-derived surfactant molecules can form two layers that sandwich a layer of water-containing secondary component (e.g., in Figure 6, a polymersome) in between. It is also contemplated to form two or more bubble compositions comprising different secondary components and admixing the bubble compositions.
In another aspect, the bubbles described herein can be prepared by admixing an aqueous solution comprising one or more secondary components and one or more surfactants, and blowing a gas into the mixture. Figure 7 shows a schematic of a procedure of
constructing the polymersome-incorporated bubbles using a coalescence process that occurs between bubbles. First, bubbles are blown with bubble solutions containing no or small amounts of secondary components (e.g., vesicles containing secondary components). Also, other bubbles blown from bubble solutions containing polymersomes can be prepared. This bubble solution can have a different composition (different pH, temperature, additives, surfactant molecules) compared with the first one. When these two different kinds of bubbles come in contact, this can lead to the growth of some bubbles at the expense of others.
Eventually, all the bubbles merge into a single one to reduce the surface energy of the system. Using this method, the effect of biologically-derived surfactant molecules on the components (such as protein in polymersomes) can be minimized during the mixing process between the bubble solution and the polymersome solution. Especially, when it is desired to incorporate components incompatible with bubble solution, this method can be used. For example, bubbles using amphiphilic block copolymers as bubble surfactant can be made. However, these bubbles are typically not stable. Thus, for example, bubbles blown from the bubble solution by admixing a high concentration of the same amphiphilic copolymer with BR/ ATP synthase reconstituted polymersomes can be merged with longer lasting surfactant bubbles. As a result, biologically functional polymersomes can be incorporated inside strong biologically-derived surfactant bubbles without the side effects of detergent molecules.
It is also possible to prepare the bubbles disclosed herein with gases generated from chemical reactions. In this method, a manual bubble blowing process is not needed. The gases coming from various experimental conditions can automatically blow the bubbles with the presence of surfactant molecules.
III. Methods of Using
The compositions disclosed herein can be used for many varied uses. For example, the disclosed bubbles can be used for chemical and biochemical syntheses, chemical and biological assays, as biochemical sensors, drug delivery, purification in biology, specific gas filters, environmental hazard monitoring systems, cosmetics, gas or liquid transporters, fluidic channels, fuel cells, to measure various properties, conditions, and/or interactions, and the like. It is contemplated that any molecular, nanoscale, or microscale chemical or biochemical analysis can be performed within the bubbles disclosed herein.
In one aspect, disclosed herein are methods of assaying an interaction between a first compound and a second compound, wherein the method comprises providing a bubble as disclosed herein, wherein the secondary component of the bubble comprises the second compound; contacting the bubble with the first compound; and detecting an interaction between the first compound and the second compound. A detectable interaction can indicate that the first compound has an activity or specific affinity for the second compound or vice- versa, a. Interaction
The term "interaction" means and is meant to include any measurable physical, chemical, or biological affinity between two or more molecules or between two or more moieties on the same or different molecules. As will be understood from the compositions and methods disclosed herein, any measurable interaction between molecules can be involved in and are suitable for the methods and compositions disclosed herein. General examples include interactions between small molecules, between proteins, between nucleic acids, between small molecules and proteins, between small molecules and nucleic acids, between proteins and nucleic acids, and the like.
An interaction can be characterized by a dissociation constant of at least about lxlO"6 M, generally at least about lxlO"7 M, usually at least about lxlO"8 M, or at least about lxlO"9 M, or at least about lxlO"10 M or greater. An interaction generally is stable under physiological conditions, including, for example, conditions that occur in a living individual such as a human or other vertebrate or invertebrate, as well as conditions that occur in a cell culture such as used for maintaining mammalian cells or cells from another vertebrate organism or an invertebrate organism.
Examples of interactions that can be involved in and/or determined by the compositions and methods disclosed herein include, but are not limited to, an attraction, affinity, a binding specificity, an electrostatic interaction, a van der Waals interaction, a hydrogen bonding interaction, and the like.
One specific type of interaction that can be involved in and/or determined by the methods and compositions disclosed herein is an interaction between a ligand (e.g., a potential therapeutic agent, a small molecule, an agonist, an antagonist, an inhibitor, an activator, a suppressor, a stimulator, and the like) and a protein (e.g., a receptor, a channel, a signal transducer, an enzyme, and the like). For example, an interaction between a potential therapeutic agent and a target protein can indicate a potential therapeutic activity for the agent. In another example, an interaction between a small molecule (e.g., a lipid, a carbohydrate, etc.) and an enzyme (e.g., a kinase, a phosphatase, a reductase, an oxidase, and the like) can indicate enzymatic activity or substrate specificity. In another example of a type of interaction that can be involved in and/or determined by the methods and compositions disclosed herein is an interaction between two proteins or fragments thereof (e.g., an enzyme and a protein substrate or an antibody and an antigen or an epitope of an antigen). An example of this interaction can include, but is not limited to, the binding of a kinase, a protease, a phosphatase, and the like to a substrate protein. Such interactions can, but need not, result in a reaction or chemical transformation (e.g., phosphorylation, cleavage, or dephosphorylation). Another example of an interaction includes the binding or affinity of an antibody for an antigen or epitope of an antigen.
Another type of interaction that can be involved in and/or determined by the methods and compositions disclosed herein is hybridization between two nucleic acid sequences (e.g., a prime, probe, aptamer, ribozyme, and the like hybridizing to a target sequence of a nucleic acid). The term "hybridization" typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. "Sequence driven interaction" means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide substitute in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
Another type of interaction that can be involved in and/or determined by the compositions and methods disclosed herein includes a Watson-Crick interaction, i.e., at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
A Hoogsteen interaction is another example and is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.
Yet another type of interaction that can be involved in and/or detected by the compositions and methods disclosed herein includes an interaction between a protein {e.g., a polymerase, endonuclease, or ligase) and a nucleic acid.
b. Detection
Detecting an interaction in the methods disclosed herein can be performed by any method, but will usually depend on the particular interaction being detected. For example, the first compound and/or second compound may contain a fluorescent marker, and detection of an interaction can be made by measuring fluorescence or changes in fluorescence. In another aspect, detecting an interaction can involve identifying a particular product. For example, if the first and second compound interact in such a way as to produce a reaction product (e.g., a kinase phosphorylating a substrate protein, a protease cleaving a particular protein, an endonuclease cleaving a particular nucleic acid, a ligase ligating nucleic acids, and the like), detection can be accomplished by identifying a particular product (e.g., the phosphorylated or cleaved product). Identifying a product can be done by known methods such as
chromatography (e.g., retention times or Rf), fluorescence detection, ionization, mass spectral analysis, nuclear magnetic resonance imaging, immunohistological techniques, microscopy (e.g., TEM, SEM, optical microscope, or AFM), XRD, XPS, AES, infrared spectroscopy, kinetic analysis, circular dichroism, electrochemical analysis (e.g., cyclic voltametry or impedance spectroscopy), dynamic light scattering, static light scattering, and the like.
c. First and Second Compounds
In the disclosed methods the first compound can be any molecule that one may desire to measure a potential interaction with any other desired molecule. For example, the first compound can be any of the secondary components disclosed herein, for example, amino acid based molecules (e.g., peptide, proteins, enzymes, or antibodies, including variants, derivatives, and analogs thereof), nucleic acid based molecules (e.g., primers, probes, aptamers, or ribozymes, including variants, derivatives, and analogs thereof), small molecules (e.g., biomolecules, drugs, potential therapeutics, or organic and inorganic compounds), macromolecules (e.g., carbon based nanostructures, dendrimers, or polymers), cell, or organelle (natural or artificial). The second compound, which is present in the secondary component, can also be any molecule as described above for the first compound. It is contemplated that the disclosed methods are not limited by the particular order, identity or priority of the first or second component; the identifiers "first" and "second" are merely arbitrary and are used herein to simply distinguish one compound from the other; no connotation of order of addition is intended as any order of the compounds is contemplated and can be used in the methods disclosed herein,
d. Exemplary Assays
In one example, the second compound can be a protein and the first compound can be a small molecule such as a potential therapeutic agent, a kinase, a phosphatase, a protease, a methylating agent, an antibody, or fragments thereof. Alternatively, the second compound can be a small molecule, a kinase, a protease, a methylating agent, an antibody, or fragment thereof and the first compound can be a target protein. When the second compound is a protein and the first compound is a potential therapeutic or vice- versa, the detectable interaction can indicate a potential therapeutic activity. In this example, the method can be used to screen for potential drugs against a particular protein.
When the second compound is a protein and the first compound is a kinase, a phosphatase, a protease, a methylating agent, or a fragment thereof, or vice-versa, the detectable interaction can indicate enzymatic activity. Thus, in this example, one can analyze the ability of a protease to cleave a particular protein, or the ability of a kinase to
phosphorylate a particular protein, or the ability of a protein to be dephosphorylated by a particular phosphatase, and the like.
In another example, the second compound can be a protein, antigen, or epitope, and the first compound can be an antibody or fragment thereof, or vice-versa. Here, the method can be used to detect an interaction that indicates binding activity. Thus, one can use this method to screen antibodies to find those that bind to a particular antigen or epitope.
Conversely, one can use the disclosed method to find particular antigens or epitopes recognized by a particular antibody. It can also be possible, when the first compound is a cell or microorganism and the second compound is an antibody or fragment thereof, to screen for particular surface antigens on the cell surface, or to screen for antibodies that recognize a given organism. These and other uses are contemplated herein.
Still further, the disclosed compositions can be used to detect a particular infection in a subject. For example, a bubble as disclosed herein, wherein the secondary component comprises second compound that is a particular antigen, can be contacted with an antibody- containing sample from a subject. Detecting an interaction of the antigen and the antibody specifically reactive therewith can indicate the presence of the antigen or previous infection in the subject. In another example, the second compound can be a nucleic acid and the first compound can be a primer, a probe, a ligase, an endonuclease, a transcriptase, a ribozyme, or fragment thereof, or vice- versa, that is the second compound can be a primer, a probe, a ligase, an endonuclease, a transcriptase, a ribozyme, or fragment thereof and the first compound can be a target nucleic acid. When the second compound is a nucleic acid and the first compound is a ligase, an endonuclease, a transcriptase, a ribozyme, or a fragment thereof, or vice-versa, the interaction can indicate enzymatic activity. For example, one can use the disclosed method to analyze the ability of an endonuclease to recognize and/or cleave a particular nucleic acid sequence, or the ability of a particular nucleic acid {e.g., a primer) to initiate transcription with a particular transcriptase.
When the second compound is a nucleic acid and the first compound is a primer, probe, or aptamer, or vice-versa, the interaction can indicate hybridization. In this example, one can use the disclosed methods to analyze the ability of a primer or probe sequence to hybridize to a particular nucleic acid sequence.
In the methods disclosed herein, the methods can further comprise contacting the bubble with a third compound. This can be done to, for example, evaluate or analyze a particular interaction between a first compound and a second compound while a third compound is present. Also, it is contemplated that the methods disclosed herein can further comprise contacting the bubble with a fourth, fifth, six, etc. compound. Any number of additional compounds can be used in the methods and compositions disclosed herein.
In the methods disclosed herein, the third compound can be any molecule or group of molecules. For example, any of the molecules disclosed herein, such as amino acid based molecules, nucleic acid based molecules, small molecules, macromolecules, cells, etc.
Specific examples of suitable third compounds include, but are not limited to, an antagonist, an agonist, a ligand, an inhibitor, an activator, a primer, a promoter, a transcription factor, an endonuclease, a ligase, a transcriptase, a protease, a kinase, a phosphatase, a methylating agent, or mixtures thereof.
In another aspect, disclosed herein are methods of assaying a condition, comprising subjecting a bubble as disclosed herein, wherein the secondary component comprises an indicator to a condition to be assayed, and detecting the indicator. By indicator is meant any molecule, compound, or composition, which when contacted with or subjected to a particular condition (e.g., pH, light intensity, temperature, ionic strength, electrochemical potential), provides a detectable signal. The detectable signal that a suitable indicator can provide can be, for example, a color change, fluorescence, phosphorescence, magnetic resonance, electric potential, and the like. For example, an indicator can provide a change in color or emit light in response to being subjected to a particular pH condition. e. Chemical and biochemical synthesis
In one specific aspect, the disclosed bubbles can be used to form a hybrid ATP generating bubble device. The protein bacteriorhodopsin (BR) and F0Fi-ATP synthase were reconstituted into 4 nm thick polymersome membranes that can convert optical energy to electrochemical energy. BR transports protons across the cell membrane upon the absorption of a photon of green light. Because of the pumping of protons, a pH gradient forms across the cell membrane, forming an electrochemical potential. When coupled with F0Fi-ATP synthase, this proton gradient drives the synthesis of ATP from ADP and inorganic phosphate (Pi). Next, these biologically active polymersomes were packaged into the thin water channel of the surfactant bubbles. The ATP production by BR-ATP synthase-polymersomes was demonstrated in the bubble architecture. This has significance both in the development of a hybrid organic/inorganic power source obtaining its energy from light and in using surfactant bubbles for packaging structures. Functional polymersomes incorporated into the water channel of bubble walls were able to provide useful amounts of electrochemical energy which can be used for other nano- bio applications.
In one aspect, the bubble architecture is used to form an artificial photosynthesis platform for converting light and CO2 to a value-added product, for example, simple sugar. Carbon fixation for the production of sugar is achieved by incorporating biosolar proteo- polymersomes and a plurality of enzymes into the microchannels of an inflatable foam provided by the present invention. BR-ATPase polymersomes may be used to convert light into ATP, which powers a rubisco substrate-enzyme reaction of carbon synthesis, and eventually the formation of hydrocarbon for biofuels. This artificial photosynthesis platform produces simple sugars that can be used to make a variety of useful organic compounds like HMF, DMF, methanol, ethanol or even sugars for human consumption.
There are a handful of technologies available that can be used for the production of liquid fuel from biomass. Recently, a 4-phase acid catalyzed dehydration/hydration of sugars for the production of long chained alkanes has been developed. (Blommel, P. and R. Cortright. Production of Conventional Liquid Fuels from Sugars. 2008: available from:
http://www. virent.com/BioBorming/Virent_T echnology_Whitepaper.pdf.. the contents of which are expressly incorporated herein by reference in its entirety.) A similar 2-phase can be used to convert biomass sugars into 2,5-dimethylfuran (DMF). (Huber, G.W., et al., Production of Liquid Alkanes by Aqueous-Phase Processing of Biomass-Derived
Carbohydrates. Science, 2005. 308 (5727): p. 1446-1450., the contents of which are expressly incorporated herein by reference in its entirety.) Similarly, DMF can be generated in low boiling point solvents, which are also good sources of energy rich compounds that can be used in fuel. The combination of free carbohydrates, exothermic reactions, and the absence of distillation would make energy rich fuels in an extremely efficient and inexpensive manner.
In one aspect, the artificial photosynthesis platform is encased in a multifunctional material for the production of solar derived liquid fuels. The encasement provides a robust environment for the foam without compromising optical transparency and enables the separation of the liquid fuel (G3P) from the other constituents, while providing antifouling and antimicrobial protection. In one embodiment, the encasement will be synthesized via a sol-gel process which enables control of materials chemistry and micro structure. The sol-gel process is a chemical synthesis technique for preparing amorphous inorganic solids. The most common synthetic route involves the use of metal alkoxides which undergo hydrolysis and condensation polymerization reactions to give rigid solids (gels) of metal oxides such as Si02, Ti02, A1203, Zr02, etc. (Brinker, C.J. and G.W. Scherer, Sol-Gel Science 1990, New York: Academic Press, the contents of which are expressly incorporated herein by reference in its entirety). The micro structure of the resulting gel is determined by the synthesis conditions (pH, starting alkoxide, ratio of alkoxide: water, type of catalyst, etc.). Although the material is technically defined as a gel, it is a nanoporous glass that is rigid and dimensionally stable. Another important advantage of sol-gel processing is that the solution nature of the synthesis enables one to cast or form the sol-gel derived materials into a wide variety of shapes and sizes, including thin films or fibers. It has been shown that it is possible to immobilize biomolecules which retain their characteristic reactivities and spectroscopic properties in the pores of the sol-gel glass. (Rolison, D.R. and B. Dunn, Electrically conductive oxide aerogels: new material in electrochemistry. Journal of Materials Chemistry, 2001. 11(4): p.963 -980, the contents of which are expressly incorporated herein in its entirety). As a result, a new generation of bioactive materials was created. (Ellerby, L.M., et al., Encapsulation of Proteins in Transparent Porous Silicate-Glasses Prepared by the Sol- Gel Method., Science, 1992. 555(5048): p. 1 113-1 115., the contents of which are expressly incorporated herein in its entirety). Relatively large biomolecules such as proteins and enzymes are trapped inside the pores of the inorganic matrix while small analytes can diffuse in and out. Important benefits of sol-gel technology include a marked improvement in the stability of the biomolecules as well as protection from protease and microorgansims. In one aspect, the sol-gel encasement includes the following material requirements: 1) optical transparency in the 350-700 nm wavelength range; 2) good diffusion of CO2 through the encasement; 3) antifouling and antimicrobial properties; 4) good compatibility with the foam; and 5) ability to separate the G3P liquid fuel from the other constituents. FIG. 8 shows one design of the sol-gel encasement in accordance with the present invention. Due to the inherent optical transparency of S1O2 and high porosity of sol-gel derived materials, requirements 1) and 2) are fulfilled. In one aspect, to become antifouling/antimicrobial, S1O2 is functionalized with nanoparticle Ag and/or MgO. Nanoparticles MgO are known to be antibacterial, and nanoparticles Ag have demonstrated to be both antibacterial and antifungal. However, since S1O2 is able to prevent the protein protease from entering the sol-gel, unfunctionalized S1O2 will exclude bacteria and microbes from contact with the foam since bacteria and other microbes are larger than protease. Increased antifouling protection, however, can be afforded by addition of nanoparticles Ag and/or MgO. It is understood that other antimicrobial and/or antifouling materials may also be used subject the material requirements provided herein. To optimize the compatibility with the foam, the matrix and/or surface of the sol-gel may be functionalized with lipids. Another issue is to minimize water evaporation from the foam, which can be accomplished by incorporating lipids into the sol-gel matrix. By incorporating, for example, short chain diacylphosphotidylcholine into sol-gel derived S1O2, the hygroscopic nature of the lipid and their organization into the uniform SiC -lipid structure suppresses overall water loss so that a water-rich
microenvironment is retained. Water evaporation through the porous sol-gel encasement can also be minimized by incorporating polyethylene glycol (PEG) into the sol-gel starting solution to retain a water-rich environment in the foam. Another important function of the encasement is to separate the G3P or sugar from the rest of the constituents. The generated G3P can be captured by the foam by incorporating a "capture chamber" specifically designed to capture G3P. This capture chamber may also be sol-gel derived inorganic, porous matrix involving the use of metal alkoxides which undergo hydrolysis and condensation
polymerization reactions to give rigid solids (gels) of metal oxides such as S1O2, Ti02, AI2O3, ZrC>2, etc., but molecularly imprinted with G3P. Molecular imprinting (MIP) allows the polymerization/cross-linking of the alkoxide monomers around template molecules, whereby removal of the template leaves behind a tailored pocked (an imprint) with greater affinity for the template over other structurally related compounds. (Diaz-Garcia, M.E. and R.B. Laino, Molecular imprinting in sol-gel materials: Recent developments and applications. Microchimica Acta, 2005. 149 (1-2): p. 19-36, the contents of which are expressly
incorporated herein by reference in its entirety.) The G3P, therefore, will be temporarily captured in the imprinted sol-gel but not covalently attached. Since the G3P is trapped in the matrix without covalent attachment or electrostatic interaction, it can be subsequently eluted from the capture chamber. Moreover, post-treatment on the MIP-sol-gel matrix can be performed to enlarge the imprinted size to more easily elute the G3P. An interesting phenomenon has been observed when working with the enzyme creatine kinase (CK), where mild heat treatment (only - 15° above room temperature) was able to slightly enlarge the pores of the sol-gel matrix. The sol-gel network typically forms around the biomolecule (CK in this case), creating a site-specific pore around the enzyme. With mild heat treatment, the pores enlarge -10%, providing more space around the enzyme. The ability to enlarge the pores can be utilized in MIP sol-gel, so that the G3P can be more readily eluted from the capture chamber. Alternatively, we can convert the G3P to sugar using isomerase and aldolase enzymes and elute the sugar. The G3P to sugar conversion can be accomplished by adding a "conversion chamber" to the encasement. The conversion chamber can be a sol-gel derived inorganic, porous matrix involving the use of metal alkoxides which undergo hydrolysis and condensation polymerization reactions to give rigid solids (gels) of metal oxides such as S1O2, Ti02, AI2O3, ΖΓ(¾, etc. with immobilized aldolase and isomerase. Upon entering the conversion chamber, G3P will be converted to sugar due to the immobilized aldolase and isomerase, and the sugar can be subsequently eluted from the conversion chamber. The encasement will be fabricated with an inlet and outlet to facilitate the addition of foam and separation of the biofuel (G3P or sugar). There will be a semi-permeable membrane dividing the encasement from the sol-gel derived S1O2 capture or conversion chamber, which will permit the diffusion of G3P. Once inside the capture or conversion chamber, the G3P or sugar can be eluted from the chamber by application of a gentle vacuum.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the methods described herein. Only reasonable and routine experimentation will be required to optimize such process conditions.
Example 1: Triblock copolymer synthesis
PEtOz-PDMS-PEtOz triblock copolymer (Mn = 7800, polydispersity index = 1.48) was synthesized by ring-opening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride-terminated PDMS in the presence of Nal (see Figure 9(a)). To utilize bis(hydroxyalkyl) terminated polydimethylsiloxane (PDMS) (Aldrich; Mn = 5600 gmol"1) as a macroinitiator for oxazoline polymerization, the hydroxyl group must be converted to a functional group that can initiate the polymerization of oxazoline. First, bis(hydroxyalkyl) terminated PDMS was dehydrated under vacuum at 80-90 °C for 24 h and freeze-dried. After this drying process, cyclohexane (10 mL) (Aldrich; anhydrous) was added to 3.308 g of PDMS, and the mixture was stirred for 6 hours. To this reaction mixture, two molar excess volume of sec-butyl lithium (Aldrich; 1.4 M in cyclohexane) was added dropwise with a syringe at -20 °C, and the resulting solution was kept stirring until the temperature increased to room temperature under a nitrogen atmosphere. Then 0.58 g of ((chloromethyl) phenylethyl)dimethylchlorosilane (Gelest) was added, and the mixture was stirred for approximately 1 hour at room temperature to prepare bifunctional benzyl chloride-terminated PDMS. The resulting suspension was washed with methanol and sodium thiosulfate solution, then filtered under vacuum using a separatory funnel to remove LiCl salt. The solvent was evaporated at about 60 °C under high vacuum. The resulting product was dissolved in 40 ml of hexane (anhydrous; Aldrich), supplemented with activated charcoal, and then filtered again. After that, the solvent was removed in a vacuum evaporator a final time. EtOz (Aldrich; purity >99%) was dried over calcium hydride (Aldrich; powder 99.99%) followed by double distillation under a nitrogen atmosphere. To a solution of room temperature bifunctional PDMS in 30 mL of chlorobenzene, freshly distilled EtOz (2.4 g) and 0.408 g of Nal (Aldrich; >99.99%) were added successively. The reaction mixture was stirred under reflux for 2 hours at room temperature and next heated to 100 °C. The reaction was allowed to proceed until all the monomer was depleted, as monitored by ^- MR. The end-capping of triblock copolymers by hydroxyl terminal groups was carried out by adding 2.5 mL of potassium hydroxide solution (Aldrich; 0.1N in methanol) to the system at room temperature, yielding a solution color change from light yellow to colorless. The solution was diluted with chloroform (Aldrich; anhydrous) and washed with a 10% Na2S2O3 (Aldrich; >99.99%) solution, followed by a washing with water. After evaporation of the solvent to remove any unreacted PDMS oligomers, the products were dissolved in hexane supplemented with charcoal and MgSC , then filtered. The hexane was evaporated under high vacuum and the remaining material was finally dehydrated using a freeze-dryer. The final product was a yellowish, fine powder. ABA triblock copolymer with hydroxyl terminal groups on the polyamide ends was confirmed by the ^- MR spectrum. Figure 9(b) shows the XH NMR spectrum of the obtained PEtOz-PDMS-PEtOz measured in DMSO-d6. It shows a sharp peak at 5=3.3 ppm (N-CH2-CH2-N) due to the PEtOz backbone and two broad peaks at δ =2.25 ppm (- C(0)C]¾-) and δ =0.93 ppm (CH3-CH2-), which represent the successful formation of PEtOz blocks. Gel permeation chromatography (GPC) analysis in THF revealed a molecular weight of Mn = 7800 g/mol and a polydispersity of MJMn = 1.48.
Example 2: TEM sample preparation
For the TEM observation, the polymersome solution was dropped onto a 3 mm amorphous carbon coated Cu-grid by pipette. For faster drying, copper grids were placed on KIMWIPES™, and, after 1 minute, excess solution was removed by blotting. The samples were transferred to the transmission electron microscope using a liquid-nitrogen cooled specimen stage, designed to maintain a temperature from about
-160 °C to about -185 °C. Elevated temperatures that could cause structural changes of the specimen due to long electron beam exposure were minimized by performing TEM analysis under low electron beam density and also, by using the cooling stage during TEM
observation.
Example 3: Purple membrane and F„Fi-ATP synthase preparation and it's incorporation into polymersomes
Bacteriorhodopsin (BR) was incorporated into the polymersomes in the form of purple membrane (PM). Purple membrane was obtained from Halobacterium Salinarium grown in high volume. The bacterial culture conditions and the procedure for isolation of PM mainly followed those described in Heyn et al., Methods Enzymol, 1982, 88:5-10. F0Fi-ATP synthase was purified from Bacillus PS3 cells as described in Hazard et al., Arch Biochem
Biophys 2002, 407: 1 17-24). All samples were stored and prepared in the dark to preserve the maximum proton pumping activity during assays. To form protein- incorporated
polymersomes, 3 mg of the polymer powder was first added to 68.5 μΕ of the PM (BR concentration: 4.8 mg/mL) with vigorous mixing for 1.5 hours. Then, 27.7 of FoFi-ATP synthase (2.6 mg/niL) was added to the polymer/BR mixture. After stirring for 30 minutes, this protein-polymer mixture was added drop-wise to buffer solution (20 mM MOPS-Sigma, 50 mM Na2S04, 50 mM K2S04, 2.5 mM MgS04, 0.25 mM DTT-Fluka, 0.2 mM EDTA- Sigma, pH = 7.20-7.25) at the rate of 10 μΐ, every 30 seconds. Syringe filtration through a membrane with a pore size of 0.2 μιη was used to remove non-functional multi-lamellar vesicles and tube-like structures. This yielded functional BR/ATP synthase-reconstituted polymersomes (BR-ATP synthase- polymersomes) after overnight dialysis.
Comparative Example 4 using TWEEN-20 Surfactant: Bubble solution preparation and polymersome incorporation into the water channel of bubble wall A bubble stock solution (pH = 6.5) was prepared by mixing glycerin, TWEEN- 20™, and deionized water with a volume ratio of 2: 1 :2, respectively. To prepare samples with protein-incorporated polymersomes in bubbles and in detergent solution, bubble stock solution and polymersome solution were mixed with a 1 :4 (bubble solution:polymersome solution) volume ratio for 30 seconds. Bubbles were blown outside the mixture solution using a 10-100 adjustable- volume pipette (EPPENDORF™) after dipping the tip into the solution by expelling air. Blown bubbles were transferred to fill a UV cuvette (12.5 mm x 12.5 mm x 45 mm). Before any measurements, the cuvette was kept inverted on top of KIMWIPES™ in the dark for 20 to 30 minutes in order to remove the excess polymersome solution not incorporated in the water channels. The cuvette entrance was sealed to prevent the liquid in the bubbles' aqueous channels from evaporating and to increase the stability of the foam structure. Before taking any measurements, samples having similar density of bubbles (bubble size: 3.5 to 4 mm) were chosen; also the formation of dry foam where bubbles take the form of polyhedra with nanoscale liquid films and Plateau borders were confirmed (see Figure 2). During measurements, special care was taken not to break the bubbles. During incubations both in the dark and in light, cuvettes were rotated every 3 seconds to minimize the destabilization of the bubble architecture due to gravity-induced drainage.
Example 5 using Ranaspumin-2 (Rsn-2) Surfactant:
a. Preparation of BR-ATP synthase vesicles and foam solution
BR was obtained from the purple membrane of Halobacterium and was purified using the method adapted from Pitard. (see Pitard, B.; Richard, P.; Dunach, M.; Girault, G.; Rigaud, J-L. Eur. J. Biochem. 1996, 235, 769-778, the entire contents of which are expressly incorporated herein by reference.) FoFi-ATP synthase was purified from Bacillus PS3 cells and photophosphorylating lipid vesicles were made as described in Hazard, (see Hazard, A; Montemagno, C. Arch. Biochem. Biophys. 2002, 407, 177-124, the entire contents of which are expressly incorporated herein by reference.) The Rsn-2 gene was synthesized using PCR since the length was relatively short (338 bp). The sequence was designed for E.coli codon usage with a 6xHis c-terminal tag. Purification was performed with Ni-NTA resin as described by Mackenzie, (see Mackenzie, CD.; Smith, B.O.; Meister, A.; Blume, A.; Zhau, X.; Lu, J.R.; Kennedy, M.W.; Cooper, A. Biophysical Journal. 2009, 96(12), 4984-4992, the entire contents of which are expressly incorporated herein by reference.) Surfactant measurements were done with capillary tube rise (0.1 cm diameter) and a rame-hart goniometer with water droplets placed on a flat Teflon surface. The custom made ABA triblockpolymer 12:33 : 12 polydimethylsiloxane-methyloxazoline-polydimethylsioloxane
(PMOXA-PDMS-PMOXA, -5200 MW) was purchased from Polymer Source Inc. (Montreal, Canada). BR-ATP synthase proteopolymersomes were formed and injected into a Tween-20 foam solution as described in Choi, (see Choi, H-J,; Montemagno, C. Nano techno logy.
2006,17, 2198-2202, the entire contents of which are expressly incorporated by reference.) This solution contained 2 ml of Tween-20® (non-ionic acid detergent), 1 ml of glycerin, and 2 ml of deionized water. The solution was vortexed for 2 minutes and stored at 4 °C. Rsn-2 foam solutions were created by diluting a 1 mg/ml stock solution 1 : 10 into the vesicle mixture and aspirating the liquid.
b. ATP synthesis activity assays
Photo-derived ATP synthesis activity was measured in bulk-vesicle solution, as well as in both inflated and deflated foam-vesicle solutions. A bioluminescence assay (FLAA Lucifer in-Luciferase, Sigma-Aldrich, USA) was used for ATP measurement. The procedure used here was adapted from previous methods, (see Choi, H-J,; Montemagno, C.
Nanotechnology. 2006,17, 2198-2202). The reaction mixture in bulk-vesicle solutions contained 200 μΐ BR-ATP synthase proteopolymersome solution, 20 μΐ of 0.2 M ADP, 20 μΐ of 0.5 M KH2PO4, and 60 μΐ deionized water. In the foam-vesicle solutions, 60 μΐ of foam stock solution was substituted for the deionized water. All solutions were illuminated using a Fostec xenon lamp using a yellow filter with maximum emission at a wavelength of 572 nm for a total time of one hour unless otherwise noted. Initially before exposure and at subsequent time points 10 μΐ aliquots were removed from the reaction to quantify the rate and amount of ATP production. After light incubation, the volume of the bubble solution was calculated using the weight of each sample with the density values. In this calculation, an assumption was made that the density does not change before and after blowing bubbles. Foams were then broken and ATP was measured by recording the intensity of light produced by the sample and comparing that with a standard calibration curve. All experiments were performed at room temperature.
c. RuBisCO carbon-fixation Assay
Carbon- fixing activity and subsequent formation of G3P was assayed using an adapted protocol based on the method of Racker. (see Wu, R.; Racker, E. J. Biol. Chem. 1958, 234, 1029-1035, the entire contents of which are expressly incorporated herein by reference.) The change in absorbance at 340 nm due to the oxidation of NADH by GAPDH was monitored using a Beckman Coulter® DU® 720 spectrophotometer. The amount of G3P was calculated using the extinction coefficient of 6220 M"1 for NADH. The final assay mix contained 625 μg of RuBisCO, 5 units of GAPDH, 10 units of PGK, 250 nmol of NADH, 375 nmol of RuBP, and 750 nmol of ATP in a volume of 750 of reaction buffer (pH 7.8) containing 0.1 M Tris-HCL, 5 mM MgCl2, 66 mM KHC03, and 5 mM DTT. The assay was allowed to react for 20 minutes at room temperature with absorbance being measured at 10 second intervals.
d. Glucose Assay
The formation of glucose was assayed using a colorimetric assay kit (GAGO20, Sigma-Aldrich, USA). Glucose oxidase forms gluconic acid and hydrogen peroxide from glucose. Hydrogen peroxide reacts with the o-dianisidine in the assay mixture forming a colored product. The oxidized o-dianisidine then reacts with sulfuric acid to form a more stable pink colored product. The intensity of the pink color measured at 540 nm is proportional to the original glucose concentration. The amount of glucose present was quantified by comparison to a standard calibration curve obtained from samples of glucose of known concentration. Glucose was formed by the addition of 12.5 units each of TPI, fructose- 1,6-biphosphatase, and PGI, 0.2 units of fructose- 1 ,6-biphosphate aldolase, and 0.1 units of glucose-6-phosphatase, diluting the total volume to 1 ml using water and incubating for 4 hours.
e. Full Sugar Synthesis Process and Assay
Glucose was formed from starting components RuBP, ATP and NADH using the following CBB enzymes (Sigma-Aldrich, USA): RuBisCO, GAPDH, PGK, triose phosphate isomerase (TPI), fructose 1,6-biphosphate aldolase, fructose- 1,6-biphosphatase, phospho- glucose isomerase (PGI), and glucose 6-phosphatase, during a 4 hour incubation. A typical reaction mixture contained 250 μg of RuBisCO, 2 units of GAPDH, 4 units of PGK, 125 nmol of NADH, 200 nmol of RuBP, 500 nmol of ATP, 5 units each of TPI, fructose-1 ,6- biphosphatase, and PGI, 0.1 units of fructose- 1,6-biphosphate aldolase, and 0.05 units of glucose-6-phosphatase in a volume of 260 μg of reaction buffer (pH 7.8) containing 0.1 M Tris-HCL, 5 mM MgCi2, 66 mM KHCO3, and 5 mM DTT was prepared and incorporated in a bubble architecture by the addition of 65 μΐ of foam stock solution. Comparative samples with 65 μΐ of water in place of foam stock solution in order to give bulk vesicle solution results were also prepared. The samples were then exposed to light for one hour and monitored for absorbance at 350 and 540 nm.
f. Results
FIG. 10 is a graph showing Control Group for ATP synthesis in Bulk Solution. Blue triangles are samples without vesicles, Black squares are samples with no ADP, Red circles are samples with no KH2PO4.
FIG. 11 is a graph showing Control Group for ATP synthesis in Foam Architecture. Blue triangles are samples without vesicles, Black squares are samples with no ADP, Red circles are samples with no KH2PO4.
FIG. 12 is a graph showing Control Group for ATP synthesis in Deflated Foam Solution. Blue triangles are samples without vesicles, Black squares are samples with no ADP, Red circles are samples with no KH2PO4.
FIG. 13 is a graph showing G3P Production with RuBisCO and ATP stock in bulk solution. The data shows that functional RubisCO reaction using RuBP to form G3P from NADH, ATP and dissolved carbonate (red. n=3) and without ATP (black, n=3).
FIG. 14 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay with Artificial ATP Source and various components removed. Lines overlap at 0, extended y- axis scale is provided for direct comparison to non-control results.
FIG. 15 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control with Bulk Vesicle ATP Source and various components removed. Lines overlap at 0, extended y-axis scale is provided for direct comparison to non-control results.
FIG. 16 is a graph showing an Absorbance Plot for RuBisCO Carbon Fixation Assay Control Group with Foam Vesicle ATP Source. Lines overlap at 0, extended y-axis scale is provided for direct comparison to non-control results.
FIG. 17 is a graph showing an Absorbance Plot for glucose oxidase assay showing the oxidation of o-dianisidine to a pink colored product absorbing at 540 nm from various glucose concentrations.
FIG. 18 is a graph showing DLS Size Distribution Plot for polymer vesicles. Graph displays particle size relative to particle number. TEM and Weighted DLS data was used to determine percentage of functional proteopolymersomes with diameter ranging from 69-1 10 nm. This range was determined by directly observing vesicles with TEM.
FIG. 19 is a graph showing DLS Size Distribution Plot for lipid vesicles. Lipid vesicles were relatively abundant compared to polymersomes with a peak readily observed and close to 100 nm. Particle size versus particle number was not investigated because lipid vesicle appeared to form functional hollow vesicles at all sizes as observed under TEM.
FIG. 20 shows TEM Micrographs of BR/FoFi ATP Synthase Proteopolymersomes (average diameter = 91 nm ± 64, n= 38). Proteopolymer vesicle solutions were taken from working stocks and dried for analysis. Salts from the buffer can be seen as dark crystals in the background.
FIG. 21 shows TEM Micrographs of BR/FoFi ATP Synthase Liposomes (average diameter = 102 nm ± 108 nm, n = 69). Liposomes were diluted from working stocks 1 : 100 and placed on TEM grid.
FIG. 22 shows fluorescent images of foam vesicle solutions. Liposomes containing 1 nM quantum dots, 0QD565 (Quantum Dot Corporation, USA.) were added to 0.1 mg/mL Rsn-2 foam after size exclusion chromatography on a S-200 column (GE Biosciences) and observed to flow between nodes in the foam channels as the foam drained. Rapid
reorganization and collection at foam nodes was observed throughout the imaging process.
Figure 23 is a graph showing the production of ATP with BR/ATP synthase lipid vesicles in Rsn-2 foam (A ), in bulk (■), in deflated Rsn-2 foam ( -4) in T20 foam ( T ) and a control experiment in the dark (·) for comparison. Inset is the light intensity standard curve created with ATP stock dilutions.
Figure 24 is a graph showing BR/ ATP synthase function in a lipid membrane was limited to the Rsn-2 based foam since the T20 adversely affected coupled FiFo-Atpase/BR vesicle function. All error bars refer to standard deviation (n=3).
Figure 25 is a graph showing ATP synthesis using BR/ATP synthase polymersomes in T20 foam (■), in bulk (·), deflated T20 foam (T ), and a control experiment in the dark ( A ) for comparison (n=3 for each). Inset is the light intensity standard curve created with ATP stock dilutions.
Figure 26 is a graph showing the foam system containing BR/ATP synthase vesicles,
RuBisCO, PGK, GAPDH, NADH, which is converting C02 and RuBP to G3P using photoderived ATP wherein the RuBisCO dependent carbon fixation reaction is fueled by lipid photophosphorylation vesicles fuels within the Rsn-2 foam (black, n=3), and a in bulk (brown, n=3); and the proteopolymersomes within Two foam (red, n=3), and in bulk (blue, n=3). Control experiments were also performed in the dark (green and purple overlapping at 0, n=3 for each) and with selectively removed individual components (S5-S7_ without detectable G3P production).
Proton pumping activity assays
The generation of a photo-induced electrochemical proton gradient was measured by trapping the fluorescent probe, 8-hydroxyprene-l ,3,6-trisulphonic acid (pyranine) outside the polymersomes. When the pyranine was trapped inside polymersomes, the relatively small concentration of polymersomes resulted in low fluorescence intensity. Therefore, in these experiments, pyranine was trapped outside polymersomes (inside bubble aqueous channels) allowing the monitoring of external pH. An excitation scan with a Luminescence
Spectrometer (LS 50B Perkin Elmer) was performed from 350 nm to 475 nm at an emission wavelength of 511 nm. Small shifts in the excitation spectrum were corrected and the conversion from fluorescence to pH was performed as described in Hazard et al, Arch Biochem Biophys 2002, 407: 117-24.
Morphology and size distribution of polymersomes after BR- incorporation
The bright-field TEM images of BR-reconstituted polymersomes are shown in Figure 20. As seen in Figure 27(a), spherical polymersomes were observed distributed throughout the sample. Figure 27(b) shows the size distribution histogram derived from direct measurement of polymersome sizes by TEM micrographs. The size distributions with a mean polymersome diameter of 270 ± 156 nm are based on an analysis of 135 polymersomes from TEM images.
Example 8: Bubble water channel thickness measurement using IR
The planar bubble film thickness was measured following the procedures described in Wu et al, Review of Scientific Instruments 2001, 72(5):2467-71. Using IR, the thickness of the bubble wall was measured to be 1.23 μιη.
Example 9: Proton pumping activity of hybrid BR/ATP synthase incorporated polymersome system in buffer solution
Figure 28(a) shows ΔρΗ as a function of time together with a control. Intravesicular pH measurements were performed in buffer solution using BR- polymersomes and BR-ATP synthase-polymersomes. Both systems in buffer solution showed an increase in the internal pH with illumination. That is, the generation of a photo-induced proton gradient resulted in alkalinization of the protein-incorporated polymer vesicles. This pH change over time indicates that more than 50% of BR is selectively oriented, allowing protons to be pumped primarily outward. The kinetics of light- induced proton transport were affected by the presence of ATP synthase, which can be seen in the slower and slightly smaller pH change in the presence of ATP synthase. While the BR-ATP synthase-polymersome system showed a smaller increase in pH at the initial stages (first 20 minutes: 3.5xl0~3 ΔρΗ mm 1), ultimately a level of photo- induced basicity was similar to that of the BR-polymersome system. All of these effects indicate the light-driven generation of a proton gradient. In other words, upon illumination, BR undergoes a series of conformational changes, resulting in the transfer of protons across the membrane. For both systems, a light-driven pH change occurred rapidly for the initial 20 minutes and then saturated to a ΔρΗ of about 0.08 units. Proton permeability through the polymer membrane as well as the backpressure effect experienced by BR account for the limitation on the maximum obtainable pH changes from both systems.
Example 10: ATP synthesis activity of hybrid BR/ATP synthase incorporated polymersome system within bubble architecture
ATP production, normalized to the amount of ATP synthase present in the polymersomes was plotted as a function of light incubation time (Figure 28(b)).
Polymersomes in the bubble architecture showed stable light-driven ATP synthesis. Initially, the ATP synthesis rate was small then, increased rapidly to 1800 nmol/mg of ATP synthase after 60 minutes. Considering the fact that electrochemical proton gradient drives the synthesis of ATP from ADP and inorganic phosphate (Pi) with FoFi-ATP synthase, these measurements demonstrate that both BR and ATP synthase did not denature and retained their biological functionality in the PEtOz-PEMS-PEtOz polymersomes inside the bubble water channel.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

What is claimed is: 1. A bubble, comprising:
a. a wall, wherein the wall comprises an aqueous layer between two layers of a biologically-derived surfactant; and
b. at least one secondary component, wherein said at least one secondary
component is substantially or completely present in the aqueous layer.
2. A bubble, comprising:
a. a wall, wherein the wall comprises an inner wall and an outer wall,
wherein the inner wall comprises an inner surface and an outer surface and the outer wall comprises an inner surface and an outer surface, wherein the inner wall and the outer wall comprises a biologically-derived surfactant, wherein the inner wall and the outer wall comprises a gas between two layers of the biologically-derived surfactant;
b. an aqueous layer, wherein the aqueous layer is adjacent to the outer
surface of the inner wall of the bubble; and
c. a secondary component, wherein the secondary component is
substantially or completely present in the aqueous layer.
3. The bubble of any one of claim 1 or 2, wherein said biologically-derived surfactant is a natural protein surfactant.
4. The bubble of claim 3, wherein said natural protein surfactant is a Ranaspumin protein surfactant.
5. The bubble of claim 4, wherein the Ranaspumin protein surfactant is a Ranaspumin-2 protein surfactant.
6. The bubble of any one of claims 1-5, wherein the bubble comprises two or more different secondary components.
7. The bubble of any one of claims 1-6, wherein the secondary component comprises a biomolecule.
8. The bubble of claim 7, wherein the biomolecule is selected from the group comprising a small molecule, a peptide, a protein, an enzyme, an antibody, a nucleic acid, a lipid, a carbohydrate, a steroid, a hormone, a vitamin, a potential therapeutic agent, a polymer, a vesicle, a cell, a microbe, a drug, an organelle, and mixtures thereof.
9. The bubble of claim 8, wherein said enzymes are Calvin cycle enzymes.
10. The bubble of claim 9, wherein the Calvin cycle enzymes are selected from the group comprising ribulose-l ,5-bisphosphate carboxylase oxygenase (RuBisCO), phosphoglycerate kinase (PGK), glyceraldehyde phosphate dehydrogenase (GAPDH), triosephosphate isomerase, fructose- 1,6-biphosphate aldolase, fructose- 1,6-bisphosphatase, phosphoglucose isomerase and glucose-6-phosphatase.
1 1. The bubble of any one of the claims 8-10, wherein said enzymes are transketolase, phosphopentose epimerase and phosphoribulose kinase.
12. The bubble of any one of claims 1-1 1 , wherein the secondary component comprises a components selected from the group comprising an indicator, a carbon based nanostructure, a dendrimer, a nanoscale device, microelectric machine (MEMs), a microbe, a non- water liquid, a gas, and mixtures thereof.
13. The bubble of any one of claims 1-12, wherein the secondary component comprises a protein.
14. The bubble of any one of claims 1-13, wherein the secondary component comprises a membrane protein.
15. The bubble of any one of claims 1-14, wherein the secondary component comprises a receptor, a channel, a signal transducer, or an ion pump.
16. The bubble of any one of claims 1-15 wherein the secondary component comprises bacteriorodopsin, cytochrome oxidase, aquaporin, or ATPase.
17. The bubble in claim 16, wherein the secondary component comprises bacteriorodopsin.
18. The bubble of any one of claims 1-17, wherein the secondary component comprises a polymer matrix and a biomolecule, wherein the biomolecule is imbedded in the polymer matrix.
19. The bubble of claim 18, wherein the polymer matrix comprises a homopolymer.
20. The bubble of claim 18, wherein the polymer matrix comprises a copolymer.
21. The bubble of claim 18, wherein the polymer matrix comprises polyvinyl alcohol, polyacrylamide, a sol-gel, or mixture thereof.
22. The bubble of claim 18, wherein the polymer matrix comprises modified or unmodified polyethylene, polypropylene, polystyrene, polybutylene, poly(meth)acrylate,
polymethylmethacrylate, polyacrylonitrile, ABS, polyethylene oxide, polypropylene oxide, polybutylene oxide, polyterephthalate, polyamide, nylon, or a mixture thereof.
23. The bubble of claim 18, wherein the polymer matrix comprises a polymer produced by the ring-opening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride-terminated PDMS.
24. The bubble in any one of claims 1 -23 , wherein the secondary component comprises bacteriorhodopsin imbedded in a polymer matrix comprising a polymer produced by the ring- opening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride- terminated PDMS.
25. The bubble in any one of claims 1 -24, wherein the secondary component is a polymersome embedded with functional biomolecules.
26. The bubble of claim 25, wherein the functional biomolecules are selected from the group including a biological proton pump and an ATP generator.
27. The bubble of claim 26, wherein the biological proton pump is selected from the family of rhodopsin proteins.
28. The bubble of claim 27, wherein the family of rhodopsin proteins is selected from the group comprising bacteriorhodopsin and xanthorhodopsin.
29. The bubble of any one of claims 26-28, wherein ATP generator is FoFi-ATP synthase.
30. The bubble of any one of claims 26-29, wherein a quantum dot is attached to said biological proton pump.
31. The bubble of claim 30, wherein said quantum dot is attached to bacteriorhodopsin.
32. The bubble of any one of claims 25-31, wherein said polymersome is a di-block copolymer.
33. The bubble of claim 32, wherein said di-block copolymer is selected from the group comprising poly(ethylene oxide-b-polyethylethylene) and poly(l,2 butadiene-b-polyethylene oxide).
34. The bubble of any one of claims 1 -33, wherein the secondary component is ribulose 1 ,5- biphosphate or NADH.
35. The bubble of any one of claims 1 -34, wherein said secondary component is a P-doped titanium oxide.
36. A sol-gel encasement comprising an inorganic matrix having a plurality of pores for retaining the bubble of any one of claims 1 -35.
37. The sol-gel encasement of claim 36 wherein said matrix is selected from the group comprising at least one metal oxide matrix.
38. The sol-gel encasement of claim 37, wherein said at least one metal oxide matrix is selected from the group comprising S1O2, Ti02, AI2O3, and Zr02.
39. The sol-gel encasement of any one of claims 36-38, wherein said matrix is functionalized with an antimicrobial agent or an antifouling agent or both.
40. The sol-gel encasement of claim 39, wherein said matrix is functionalized with a nanoparticle including silver or magnesium oxide or a combination thereof.
41. The sol-gel encasement of claim 39 or 40, wherein said matrix is functionalized with a lipid.
42. The sol-gel encasement of any one of claims 41, wherein said lipid is
diacylphosphotidylcholine.
43. The sol-gel encasement of any one of claims 36-42, further comprising a capture chamber for capturing glyceraldehyde-3 -phosphate (G3P).
44 The sol-gel encasement of claim 43, wherein the capture chamber is a sol-gel having an inorganic matrix.
45. The sol-gel encasement of claim 44, wherein said inorganic matrix is selected from the group comprising at least one metal oxide matrix.
46. The sol-gel encasement of claim 45, wherein said at least one metal oxide matrix is selected from the group comprising S1O2, Ti02, AI2O3, and ZrC .
47. The sol-gel encasement of any one of claims 43-46, wherein said capture chamber is imprinted with glyceraldehyde-3 -phosphate (G3P).
48. The sol-gel encasement of any one of claims 36-47, further including the enzyme creatine kinase.
49. The sol-gel encasement of any one of claims 36-48, further comprising a conversion chamber for converting glyceraldehyde-3 -phosphate (G3P) to sugar.
50. The sol-gel encasement of claim 49, wherein the conversion chamber is a sol-gel having an inorganic matrix.
51. The sol-gel encasement of claim 50, wherein said inorganic matrix is selected from the group comprising at least one metal oxide matrix.
52. The sol-gel encasement of claim 51, wherein said at least one metal oxide matrixis selected from the group comprising S1O2, T1O2, AI2O3, and ZrC .
53. The sol-gel encasement of any one of claims 50-52, wherein said sol-gel includes immobilized aldolase and isomerase.
54. A method for converting light and carbon dioxide to glyceraldehyde-3 -phosphate (G3P) or hexose comprising,
a. providing a bubble of any one of claims 1 -5 including a first secondary components include at least one polymersome embedded with at least one functional biomolecule and a second secondary component including a plurality of Calvin cycle enzymes.
b. subjecting said bubble to light and carbon dioxide to produce glyceraldehyde-3 - phosphate (G3P) or hexose.
55. The method of claim 54, wherein the Calvin cycle enzymes are selected from the group comprising ribulose-l ,5-bisphosphate carboxylase oxygenase (RuBisCO), phosphoglycerate kinase (PGK), glyceraldehyde phosphate dehydrogenase (GAPDH), triosephosphate isomerase, fructose- 1,6-biphosphate aldolase, fructose- 1,6-bisphosphatase, phosphoglucose isomerase and glucose-6-phosphatase.
56. The method of claim 54 or 55, wherein said bubble further includes enzymes selected from the group comprising transketolase, phosphopentose epimerase and phosphoribulose kinase.
57. The method of any one of claims 54-56, wherein the functional biomolecules are selected from the group including a biological proton pump and an ATP generator.
58. The method of claim 57, wherein the biological proton pump is selected from the family of rhodopsin proteins.
59. The method of claim 58, wherein the family of rhodopsin proteins is selected from the group comprising bacteriorhodopsin and xanthorhodopsin.
60. The method of any one of claims 57-59 , wherein ATP generator is FoFi-ATP synthase.
61. The method of any one of claims 57-60, wherein a quantum dot is attached to said biological proton pump.
62. The method of claim 61, wherein said quantum dot is attached to bacteriorhodopsin.
63. The method of any one of claims 54-62, wherein said polymersome is a di-block copolymer.
64. The method of claim 63, wherein said di-block copolymer is selected from the group comprising poly(ethylene oxide-b-polyethylethylene) and poly(l,2 butadiene-b-polyethylene oxide).
65. The method of any one of claims 54-64, wherein another secondary component is provided to include ribulose 1,5-biphosphate or NADH.
66. The method of any one of claims 54-65, wherein another secondary component is provided including P-doped titanium oxide.
67. The method of any of claims 54-66, wherein said hexose is fructose or glucose.
68. A method for harvesting and converting light energy to chemical energy comprising, a) providing the bubble of any one of claims 1-5 including a first secondary components include at least one biosolar component and a second secondary component including a plurality of Calvin cycle enzymes.
b. subjecting said biosolar component in said bubble to light energy to produce chemical energy;
c. subjecting said plurality of Calvin cycle enzymes in said bubble to carbon dioxide and the chemical energy of step b) to produce glyceraldehyde-3 -phosphate (G3P) or hexose; d. harvesting said G3P or hexose to produce a biomass.
69. The method of claim 68, wherein said biosolar component is a polymersome embedded with at least one biological proton pump and at least one ATP generator.
70. The method of claim 69, wherein said biological proton pump is selected from the family of rhodopsin proteins.
71. The method of claim 70, wherein the family of rhodopsin proteins is selected from the group comprising bacteriorhodopsin and xanthorhodopsin.
72. The method of any one of claims 69-71 , wherein ATP generator is FoFi-ATP synthase.
73. The method of any one of claims 69-72, wherein a quantum dot is attached to said biological proton pump.
74. The method of claim 73, wherein said quantum dot is attached to bacteriorhodopsin.
75. The method of any one of claims 69-74, wherein said polymersome is a di-block copolymer.
76. The method of claim 75, wherein said di-block copolymer is selected from the group comprising poly(ethylene oxide-b-polyethylethylene) and poly(l,2 butadiene-b-polyethylene oxide).
77. The method of any one of claims 68-76, wherein another secondary component is provided to include ribulose 1,5-biphosphate or NADH.
78. The method of any one of claims 68-77, wherein another secondary component is provided including P-doped titanium oxide.
79. The method of any one of claims 68-78, wherein said hexose is fructose or glucose.
80. The method of any one of claims 68-79, wherein said biomass is selected from the group comprising HMF, DMF, methanol, ethanol, and sugar.
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