CN100377766C - Bionic membrane - Google Patents

Abstract

Biomembrane proteins are incorporated into the copolymer matrix to create membranes with alternative functions. In one embodiment of the invention, the composite membrane incorporates two different proteins that work together to generate electricity from light. In another embodiment, water transport proteins are embedded in the membrane to purify water.

Description

Biomimetic membrane

This application claims the benefit of US Provisional Application No. 60/398784, filed July 29, 2002, and U.S. Provisional Application No. 60/438784, filed January 9, 2003, the contents of which are incorporated herein by reference.

Background of the invention

The present invention relates to methods of producing artificial devices having the properties and functions of biological membranes and membrane proteins; and also to the structure of such devices.

Biomembrane proteins have a variety of functions, including as pumps, channels, valves, transducers, and mechanical, thermal, and electrical sensors, among others. Because of their nanoscale size and high efficiency, these proteins are attractive for use in artificial devices. However, their natural lipid membrane environment has disadvantages such as low strength, requires an aqueous environment, and is sensitive to chemical or microbial degradation.

Summary of the invention

Briefly, one aspect of the present invention incorporates native or genetically engineered membrane proteins into block copolymer matrices, resulting in membranes with various genetic functions, including the ability to selectively transport and/or filter compounds between fluids. By selecting proteins with specific properties, membranes can be made with specific functionality, including molecular-scale addressability through directed electrostatic, electromagnetic, and chemical forces.

The block copolymers of the present invention can be designed and produced such that they have the following properties as required: the ability to form films of desired thickness; the ability to form films of desired chemical composition; the ability to form high-strength films; The ability of the strength of the film that has been formed. One of the most important properties of these membranes is their ability to accommodate native biomembrane proteins in a functional state. properties and functions of the device. Suitable polymers need only form membranes that divide membrane proteins into upper and lower halves that sufficiently resemble natural lipid membranes to easily insert proteins when they are properly oriented, and they do not accommodate the protein's natural function. Polymers meeting these criteria include triblock copolymers, which have the general nature of a hydrophilic outer block and a hydrophobic inner block.

One aspect of the present invention involves the creation of composite membranes containing two different proteins that, when acting in unison, result in photoelectrically generated devices - "biosolar cells". Another aspect of the invention utilizes water transport proteins to allow water purification from any water source. These aspects will be described in detail below.

As technological innovations in device miniaturization have made electronic devices smaller, lighter and more efficient, progress in the power supplies used by these devices has been relatively slow. 21st century power supplies face the challenge of powering an increasing number of devices while reducing size and weight. Furthermore, the power requirements for tomorrow's nanotechnology and biotechnology products will not even resemble in form or function the power supplies used today.

There is an urgent need for lighter and smaller power supplies to meet the needs of new applications where conditions are constantly emerging. Such power sources may be able to achieve a wider range of functional goals than contemporary battery technologies, maximizing power density, minimizing weight, and implementing specific power requirements. The weight requirement is critical because with traditional fuel sources the fuel source must be close to the device and, if movable, transport the device. Fuel can also run out, so supplies must be replenished. This limits the range and mobility of users.

Contemporary science shows that the development of nanobiotechnology has exciting potential. Fabricating devices from components that don't waste atoms holds promise in terms of efficiency and the highest levels of miniaturization. While recent advances in power supply technology are exciting, they are incremental improvements over existing technologies. Power supplies ideally suited for next-generation devices will employ nanotechnology to enhance their functionality and also be able to drive current-generation devices at the highest levels of performance.

Only recently has the technology and knowledge required to develop the first nanobiotechnological devices become available, and the engineering and construction of nanoscale organic/inorganic hybrid devices driven by the biochemical fuel ATP has been reported (Soong, R.K., Bachand, G.D., Neves, H.P. , Olkhovets, A.G., Craighead, H.G. and Montemagno, C.D. (2000), Science, 290, pp. 1555-1558). The generation of ATP for these devices, and the use of these devices to drive other machinery, is of interest in the transfer of energy between the macroscale and the nanoscale and in the interchange of different types of energy.

In another aspect of the invention, other proteins with varying degrees of functionality can be used to transport electrons/protons, enable electrical and chemical energy transduction, and function as mechanical valves and sensors.

In a preferred embodiment of the present invention, biosolar materials and fabrics are provided by membranes, which consist of thin fabrics incorporating biocompatible polymer membranes that entrap two energy-converting proteins, bacterial Rhodopsin and cytochrome oxidase, which convert light energy into electrical energy and transfer this energy to an external load. Significant weight savings are achieved due to the use of thin (less than 1 μm) polymer membranes, and the need not to carry fuel with a power source. Therefore, a system can be developed that can be integrated into cloth and the surface of most materials, providing a light weight (less than 1 kg/m ² ) energy source with an efficiency equal to or greater than that achieved with solar cells. In this way, biosolar materials form a hybrid organic/inorganic power source that harvests energy from light.

The process of the present invention involves the production of a thin fabric consisting of a biocompatible polymer film embedding two energy-converting proteins, bacteriorhodopsin and cytochrome oxidase, which convert light energy into electrical energy and This energy is transferred to an external load. Through millions of years of natural selection, these proteins have been isolated and optimized to convert light and electricity into electrochemical energy. Packed into devices, they can deliver useful amounts of power indefinitely, and are light, small, and robust enough for applications requiring high mobility in both favorable and adverse environments.

Bacteriorhodopsin is a bacterial protein that transports protons across the cell membrane when light is absorbed. Cytochrome oxidase is a membrane protein that transports protons with energetic electrons. Using these proteins in concert, light energy is converted into an electrochemical proton gradient, which is then converted into electromotive force that can be used for external work. Because the device is a biological "version" of a conventional solar cell, the power source does not need to carry "fuel," significantly increasing power density. In addition, the theoretically usable maximum energy deduced from this device is infinite, as long as the sun or the device exists, it can work. The final device has an estimated areal mass density of ~100 g/m ² , providing light energy with an efficiency equal to or greater than that of solar cells. The material composition and size of this bio-solar cell allow it to finally achieve high power density (>250W/kg) and high energy density (800Whr/kg for 3 hours, 9500Whr/kg for 3 days, 32000Whr/kg for 10 days), enough to drive a large number of devices , while the effective occupied volume and weight are 0. Furthermore, it operates with negligible acoustic, thermal and electrical signatures.

There are important differences between the power supply of the present invention and conventional solar cells. Because the power source of the present invention is composed of mass-produced proteins and common polymers, it is lightweight, flexible, and strong, and can be mass-produced at low cost. The relative length of this device is the thickness of the package, which is less than 1 μm. Membranes containing these enzymes are typically 5 nm thick. Layered panels of bio-solar cells could be incorporated into cloth and other surfaces without adding cost in terms of weight, since they would have to be worn anyway. Appropriate modular design of the power generating cells in the fabric will allow the power fabric to retain significant damage prevention and functional maintenance capabilities. Using the mutual conversion ability between electrical energy and biochemical energy can construct electrokinetic biodevices and convert biochemical fuels into electricity. Harnessing the ability to convert energy between electrical, biochemical, and optical forms allows the design and production of nanobiotechnological devices independent of the type of input energy.

Brief description of the drawings

The above and other objects, features and advantages of this invention will be best understood with the aid of the following drawings and detailed description of preferred embodiments, in which:

Figure 1 is a schematic diagram of the simplified backbone belt structure of bacteriorhodopsin, with protons transported across the membrane via a central inner channel.

Figure 2 illustrates a process in which, in Halobacteriam salinarium, the BR pump draws protons out of the bacterium upon absorption of photons of green light, creating an electrochemical gradient, and ATP synthase brings these protons back into the cell , using their electrochemical energy to prepare ATP from ADP, providing a net conversion from light energy to chemical energy.

Figures 3A and 3b illustrate the main chain belt structure of COX, where Figure 3A is the membrane, Figure 3B is the cytoplasm, and the three regions marked with asterisks are "pores" that are recognized as proton transport channels.

Figure 4a illustrates the incorporation of liposomes into a planar solid-supported lipid bilayer; Figure 4b illustrates the incorporation of vesicles with COX into a planar membrane.

Fig. 5 is a schematic diagram of a biological solar cell of the present invention.

Figure 6 is a chromatogram of purified bacteriorhodopsin.

Fig. 7 is a pH gradient graph formed within 30 minutes by liposomes containing bacteriorhodopsin () and without bacteriorhodopsin (□).

Figure 8 illustrates fluorescein fluorescence due to the presence of ATP produced by liposomes containing varying amounts of bacteriorhodopsin and F ₀ F ₁ -ATPase.

Figure 9 is a SEM micrograph of an array of silicon tips (tips smaller than 1 nm, axes about 1 μm).

Figure 10 is a schematic diagram of the overexpression and purification method of COX.

Figure 11 is a schematic partial cross-sectional view of a device for measurable and controlled proton and electron transport through COX.

Figure 12 is a schematic diagram of aquaporins.

Fig. 13 is an enlarged view of some proteins in Fig. 12 .

Figure 14 illustrates the use of membranes incorporating aquaporins for water purification ponds.

Figure 15 is a schematic diagram of a conventional water purification system.

Description of the preferred embodiment

In one embodiment of the invention, bacteriorhodopsin and cytochrome oxidase are integrated into a biocompatible polymer membrane in contact with microfabricated electrodes. The operation of the proposed device is best understood following the incorporation of bacteriorhodopsin, cytochrome oxidase, as well as their incorporation into lipid and polymer membranes. All three have been extensively studied and there are many literatures on their synthesis and function.

Bacteriorhodopsin (BR), as the most widely studied ion transporter, is a proton transporter with a molecular weight of 26kD, as shown by number 10 in Figure 1. It is found in the cell membrane of halobacterium halobacterium. Halophilic archaea that thrive in sunlit saltwater and swamps. BR allows Halobacterium halobacterium to survive in an anaerobic environment; BR comes into play when there is not enough oxygen for the respiration process. As shown in Figure 2, when BR 12 cells absorb green light photons 18 (λ=500-600nm), protons 14 are transported through the cell membrane 16 and come out of the cells. Each BR molecule undergoes various electronic intermediate states during the process of being excited by light and transporting protons, and the total time for BR to return to its initial state is on the order of 3 ms. This is the shortest timescale for energy transfer.

As protons 14 are pumped from the cell 12, a charge and pH gradient (low H ⁺ - high H ⁺ ) is formed across the cell membrane 16, creating an electrochemical potential. This electrochemical potential provides the energy to drive ATP synthase (ATPase), as indicated at 20 . ATPase uses its electrochemical energy to generate adenosine triphosphate (ATP) in the transfer of protons 14 back across the membrane, as indicated by number 22 in FIG. 2 . ATP is the ubiquitous biofuel that drives most of the cellular processes essential to life. This natural biological system has been replicated in the laboratory by constructing a system consisting of BR and ATPase in lipid vesicles (Pitard, B., Richard, P., Dunarch, M., & Riguard, J. (1996), Eur. J. Biochem., 235, pp. 769-788), wherein BR can form and maintain a pH difference of 1.25 at 40° C. over the vesicle boundary. At 20°C, ΔpH=2 can be obtained. Higher pH differences cannot be obtained because BR is inhibited by the proton gradient. A proton gradient was used to generate ATP and the performance of the two enzymes in the system was determined. This work also shows that adding negatively charged phospholipids to the liposome membrane can increase the coupling efficiency between BR and ATPase.

BRs are ideal candidates for device integration because proteins, as two-dimensional crystals, can exist in high concentrations in cell membranes. In this respect, it is unique among retinal proteins. In this form it is called "purple membrane" and has a mass proportion of 7% protein and 25% lipid (-10 lipid molecules/protein). It was observed that these purple membranes had a patch size of 0.5 μm or larger. Because these protein aggregates are stable (in their natural state) at such high concentrations, they enhance energy yield, providing a redundant, engineered element of safety for any manufactured device. In addition, the evolution of Halobacterium haloides optimized the function of BR because it can operate at high temperature and high luminous flux for a longer period of time.

The work of Pitard et al. mentioned above utilized highly diluted BR in small (150 nm) lipid films and showed that the yield of ATP was inversely proportional to the BR/lipid mass ratio. Extrapolating their data for the BR/lipid mass ratio of purple membranes, they found that 320 nmol ATP was produced per mg of BR per minute. ATPase synthesizes ATP with ADP indicated by number 24 in Figure 2 and inorganic phosphoric acid, increasing energy by 35kJ/mol. Considering only the mass of purple membrane and lipids, the power density provided by this light-driven ATP synthesis system is 140W/kg. If ATP synthase pumped protons out as fast as BR pumped them in, the power produced would increase to 180-280W/kg.

Due to the vast body of knowledge about BRs and their strength and lifetime, there has been considerable interest and research in developing BRs as active optical elements in optical devices and computer storage applications. It has been shown that purple membranes are active in light for several years and are stable when completely dehydrated in polymer matrices up to 180 °C between pH 0-12 in the presence of organic solvents (Vsevolodov , N (1998), "Biomolecular Electronics: An Introduction via Photosensitive Proteins", 125 pages, Birkhauser, Boston). Due to the interest of the scientific and engineering communities, a protocol was developed for the production and isolation of BR overproducing strains of Halobacterium halobacterium (Lorber, B. and DeLucas, L.J. (1990) FEBS Lett. 261, pp. 14-18). Numerous methods of extraction and purification, as well as processing and handling, of purple membranes are well known (e.g. Stuart, J.A.; Vought, B.W., Schmidt, E.J.; Ross, R.B.; Tallent, J.R.; Dewey, T.G.; Birge, R.R., IEEE EMBS, in press). Experiments on its combination with non-biological materials have shown that BR can be used in conjunction with common polymers such as poly(vinyl alcohol) and poly(acrylamide) (Birge, R., Gillespie, N., Izaguirre, E., Kusnetzow, A., Lawrence, A., Singh, D., Song, W., Schmidt, E., Stuart, J., Seetharaman, S., Wise, K. (1999), J. Phys. Chem. B 103, 10746-10766).

The second enzyme, cytochrome oxidase (COX), is an electron and proton transporter, the last of the four enzymes that enable respiration to occur. The membrane map of the COX main chain belt shown at 30 in Figure 3A, and the cytoplasmic map shown at 32 in Figure 3B, the region 34 marked with an asterisk represents a "pore", or proton transport channel. In respiration, energetic electrons from NADH (initially produced by the oxidation of glucose) are transferred to _O2 as it is reduced to produce _H2O .

COX accepts electrons from an earlier stage of the respiratory process, carried by cytochrome c, and transferred to two internal heme groups containing iron and copper ions. These heme groups are reduced by electrons accepted from cytochrome c, which are deoxygenated after electron transfer to molecular _O2 docked with one of the hemes. O ₂ acquires additional electrons and becomes the target of the oxidation reaction with peripheral protons. After the reaction, O ₂ is detached from the heme. With the transfer of these high-energy electrons, the energy obtained from its 460mV voltage drop (Nicholls, D. (1982), "Bioenergetics: An Introduction to the Chemiosmotic Theory", 123 pages, Academic Press, London) for the transfer of protons to the mitochondrial space, usually at a ratio of 1:1 (Lee, H., Das T., Rousseau, D., Mills D., Ferguson-Miller, S. , Gennis, R. (2000), Biochemistry 29, 2989-2996), although other ratios have been discussed (Papa, S., Lorusso, M., and Capitanio, N. (1994), J.Bioenerg.Biomembr.26 , pp. 609-617, Michel, H., Behr, J., Harrenga, A., and Kannt, A., (1998), Ann.Rev.Biophys.Biomol.Struct.27, pp. 329-356), the The ratio is generally a function of the membrane potential (Murphy, M., and Brand, M. (1998), Eur. J. Biochem. 173, pp. 645-651).

When protons are pumped from the mitochondrial matrix, an electrochemical proton gradient is created. ATPase generates ATP with the aid of this proton gradient. BR and COX are very similar in generating proton gradients; the difference is that BR is driven by light, while COX is driven by chemical energy. This can be seen in Figure 2, replacing BR12 with COX, replacing green photon 18 with energetic electrons from cytochrome c, plus reducing oxygen to water. In fact, both BR and COX are used in Halobacter halobacterium and serve the same purpose: BR is used in Halobacter halobacterium and COX is used in the organism when there is not enough oxygen supply to breathe.

The incorporation of COX into a solid-matrix-supported lipid membrane 40, as shown in Figures 4A and 4B, suggests that it is possible to measure electron transport as well as control proton transport electrically (Naumann, R., Schmidt, E., Jonesyk, A. ., Fendler, K., Kadenbach, B., Liebermann, T., Offenhausser, A., Knoll (1999), Biosensors & Bioelectronics 14, pp. 651-662). These experiments used sulfur-functionalized peptide chains 42 attached to gold membranes 44 as lipid membrane monolayers 46 of dimyristoylphosphatidylethanolamine (DMPE). COX and cytochrome c are incorporated into liposomal vesicles of DMPE 48, which are fused to the peptide surface, as indicated at 50 in Figure 4A. Electrical measurements revealed the transfer of electrons into and out of the gold-based matrix via COX, as well as the controlled transport of protons by the application of an electric current.

While in vitro experiments provide the most accurate reproduction of the natural environment of membrane-bound proteins, these conditions are not the most conducive to measuring phenomena that are obscured by other cellular processes, or that occur infrequently on the experimental timescale. Furthermore, the use of proteins as active elements to produce useful devices requires a support that is easy to produce and maintain, does not denature the protein, and preserves the protein's function as closely as possible in vivo, while being easy to use and fabricate. A large number of biological enzymes have been incorporated into artificial lipid membranes in experiments, and they remain functional for an effective experimental time; the present invention relates to the production of BR/COX light-driven devices using lipid and polymer membranes.

Artificial lipid membranes prepared from lecithin or DMPE replicate the amphiphilic components of natural cell membranes. Membrane proteins are solubilized by addition of detergents such as Triton-X or sodium lauryl sulfate and incorporated into liposomes by gentle sonication of the protein/lipid solution. Liposomes can remain vesicle-like (Pitard et al., 1996) or can form planes in the presence of a flattened matrix (Naumann et al., 1999 and Steinber-Yfrach, G., Rigaud, J., Durantini, E., Moore, A. ., Gust, D., Moore, T. (1998), Nature, 392, pp. 479-482). To maintain the biological function of the protein, the concentration of the protein can be thousands of times higher than that in the body, making the experiment highly sensitive and precise. High protein concentrations are also necessary for building power sources and biosensors, since they exploit the collective effects of individual molecules.

Given the properties of BR, it is possible to predict the power that a device may generate using BR and COX. The incident solar photons in the green range per second are about 7.5 x 10 ²⁰ per square meter of area on the Earth's surface, or 1.9 x 10 ⁴ over a BR molecular area (25 nm ² ). The absorption coefficient of BR is 66000/mol/cm (Vsevolodov, N. (1998), "Biomolecular Electronics: An Introduction via Photosensitive Proteins", 125 pages, Birkhauser, Boston), or about 4.4 x ^10-4 /single BR. The quantum efficiency of BR is 0.7, and the proton transport probability is 3.08×10 ^-4 . Therefore, approximately 5.8 transport events per second in sunlight can be expected to occur per BR molecule.

On an area of 1 m ² , when BR:COX is 57:1, a steady-state proton transport rate is obtained, and every time COX transports a proton, BR transports a proton. At this ratio, there are 3.9×10 ¹⁶ BR per square meter of single floor. Since each proton in COX transports one electron, a current of 37 mA/m ² /monolayer is obtained. 1 thousand stacked monolayers utilize only 36% of the light, but the current increases to 37A/ ^m2 at 760mV, giving 28W/ ^m2 . While these currents and voltages are not available for all devices, the current output of the device is highly configurable, and a wide range of voltage and current combinations are possible for any power output. The mass of protein and lipid (or equivalent) in this system is 2.3 g/m ² . Due to the same polymer layer thickness (5nm) of the gold electrode and polyvinyl alcohol, an areal mass density of 105.3 g/ ^m2 was obtained, resulting in a power density of over 265W/kg. The available energy of this device is 795Whr/kg for 3 hours, 9540Whr/kg for 3 days, and 31800Whr/kg for 10 days. Since energy is derived from the sun, the energy gained increases directly with the duration of the sun exposure

These power and energy densities can increase with alternating selection of electrode and polymer materials, but can also decrease with different selections of layer thickness due to additional weight gain and light scattering. Light-scattering effects of electrodes and polymeric materials are negligible for the following reasons: (1) the polymers used are minimally active in the range λ = 500–650 nm; (2) metal electrodes do not absorb part of the non-transmitted light passing through the device’s initial layer; If light interacts with the electrodes, it is simply reflected within the device and eventually absorbed by the BR.

As mentioned above, both BR and COX are proton transporters that convert the energy of light and high-energy electrons into proton gradients that drive ATPase and generate ATP, respectively. By reversing COX, the proton gradient can be converted into an electromotive force (EMF), which transfers energy to electrons. Eliminate oxygen and cytochrome c, replace with an electrode connected to an external load, and then the EMF can function. The combination of BR and COX in the electrode overloaded film culminates in the process and structure shown in Figure 5. The figure is a schematic illustration of a biological solar cell 60 in which bacteriorhodopsin 62 transports protons 64 across a polymer membrane 66 upon absorption of photons 68 of green light. This increases the proton concentration on the upper side 70 of the membrane, causing cytochrome oxidase (yellow) 72 to act in reverse. As a result, the electrochemical energy obtained from the protons 64 is transported to the underside 74 of the membrane, this energy is used to transport the protons 76 from the upper electrode 78 on the upper side of the membrane to the lower electrode 80 on the lower side of the membrane, generating an electromotive force across the electrodes , this electromotive force is used to do external work.

At the end of the electron transfer, the system returns to its original state: COX has been reduced and reoxidized, the proton concentration on both sides of the polymer film 66 is unchanged, and the electrodes have not acquired or consumed any net charge. EMF does external work, has absorbed photons. The system is ready to convert the light energy of the next photon into electricity.

The core process of converting proton power to electric power is through the COX 72 reverse action. There are many examples of reversible energy conversion proteins in the literature, such as F ₀ F ₁ -ATPase (Hammes.G. (1983), TrendsBiochem.Sci.8.131-134 pages) and ion transporters (Nicholls, D., (1982) , "Bioenergetics: An Introduction to the Chemiosmotic Theory" (Bioenergetics: An Introduction to the Chemiosmotic Theory, 123 pages, Academic Press, London). However, there are also energy conversion proteins that cannot act in reverse, such as the response of bacteriorhodopsin to a proton gradient Does not emit green light.

In work done by Wikstrom (Wikstrom, (1981), Proc. Natl. Acad. Sci. USA 78, pp. 4051-4054), a partial reversal of electron flow in COX was observed in ATP-incorporated mitochondria. F ₀ F ₁ -ATPases are reversible proton pumps with the following function: ATPases transfer external protons into the mitochondrial matrix to produce ATP. In turn, it can consume ATP and pump protons out. As described by Wikstrom, ATPases transport protons across the membrane, adding ATP in parallel to COX. This reaction produces a high proton concentration on the outside of the membrane, which in turn creates an electroosmotic proton pressure gradient across the COX. When this condition was created, a shift in the absorption spectrum was observed, indicating electron transfer from water to heme, a reversal of the typical process. The following analyzes why this happens.

In an electrochemical reaction, the excess or deficiency of energy during the reaction is given by: (see for example, De Vault, D., (1971), Biochim. Biophys. Acta 226, pp. 193-199):

-ΔE=ΔG/(nF)

where ΔE is the change in redox potential before and after electron donation, ΔG is the free energy change in the reaction, n is the number of electrons transferred, and F is Faraday's constant. During electron transfer from cytochrome c (reduction potential = +220mV) through COX, the free energy of the electrons decreases continuously until they finally reduce _O2 (reduction potential = +860mV) to _H2O . The free energy change is -14.8 kcal/mol per electron transferred. This energy is used to transport protons and create an electrochemical gradient. Wikstrom raises the external proton concentration sufficiently that the normal effect of ΔG on COX is positive so that pumping protons forward requires more energy than _O2 reduction can provide. By utilizing a "redox buffer", the redox potential of cytochrome c is kept constant; this means that the redox potential of _H2O / _O2 changes with a change in ΔG. Due to the creation of a sufficiently high external proton concentration, electrons are reversely transferred, accepting electrons from water and donating them to COX. However, the complete reverse transfer of electrons is not complete because _O2 has not yet been generated.

In the system of the present invention, as shown in Figure 5, COX has neither the initial electron donor—cytochrome c, nor the final electron acceptor—O ₂ in the system described by Wikstrom. Because the source of electrons is electrode 78 rather than water, electron acquisition costs are the lowest compared to 820 mV for _H2O . When the electrons reach the heme a ₃ , they are subjected to a positive redox potential of +380mV, and the heme can be used for external work. This potential drops by 140 to +240 mV by heme a ₁ , which requires an energy input from the proton gradient. At Cua, the potential drops a further 50 to +190 mV and finally transfers to electrode 80 at 0 V, requiring input energy. This reaction occurs spontaneously due to the reduction potential increase (free energy drop) due to electron transfer from the originating electrode 78 to the heme a ₃ . The transfer of electrons from a3 to the counter electrode is a reduction in the reduction potential of 380 mV, which requires an external energy input (proton motive force) to occur. With BR 62 and appropriate doping of membrane 66, the proton drive can be increased. Since the electrodes are electron donors, obtaining electrons from them is easier than from _H2O and avoids any chemical intermediates between water and oxygen.

Since the ion diffusion on the membrane surface is large, and can be even greater with a suitable choice of membrane composition, the membrane surface itself is required for successful functioning of biosolar cells (Pitard et al., 1996). Lipid membranes, such as membrane 40 (FIG. 4A) or any of a number of biocompatible polymer matrices contain proteins and act as proton carriers. These polymer matrices are very general, requiring only that (a) they form membranes that divide proteins into upper and lower halves, (b) that they form an environment sufficiently similar to natural lipid membranes to allow easy insertion of proteins into membranes with proper orientation,( c) The local chemical environment of the polymer membrane to which the protein is exposed does not unfold or deform the protein in such a way as to constitute the protein's natural function. Polymers meeting these criteria include, but are not limited to, triblock copolymers, which have the general nature of a hydrophilic outer block and a hydrophobic inner block. BR 62 and COX 72 are oriented and joined in polymer film 66 which is covered with electrodes 78 and 80.

Figure 2 shows the construction, implementation, and evaluation of a light-driven ATP production system that can continuously power F1-ATPase-driven nanochemical devices. As shown in Figure 5, the present invention involves the construction of liposomal vesicles 66 comprising BR and ATPase oriented in such a way that energy from green light is used to continuously generate ATP from ADP. A system has been established for the large-scale production and purification of bacteriorhodopsin (BR), which was isolated from the hyperproducer Halobacterium and purified by gel filtration chromatography (curve 90 in Figure 6).

According to the present invention, purified lecithin, phosphatidic acid and cholesterol are used to reconstitute liposomes as described previously (Pitard et al., 1996). Liposomes were sequentially size-selected with 0.45 and 0.2 μm filters, leaving liposomes smaller than 200 nm in solution. Incorporation of F ₀ F ₁ -ATPase and BR was performed in the presence of Triton X-100. To ensure liposome formation, Pyranine was added as a pH sensitive indicator, which was assessed visually using a fluorescence microscope. This work has shown that pH gradients as large as 1.5 can be obtained at 20°C, as shown by curves 92 and 94 in Figure 7, for liposomes with (curve 92) and without (curve 94) bacteriorhodopsin A pH gradient formed within 30 minutes.

The analysis demonstrated that ATP was generated using liposomes, as shown at 96 in FIG. 8 . This figure shows luciferin fluorescence due to the presence of ATP generated by liposomes containing varying amounts of BR and F ₀ F ₁ -ATPase. Liposomes were incubated in the light for 2.5 hours before ADP was added to the solution. The goal is to optimize the electrochemical gradient to increase the rate of steady-state ATP production to levels previously described (Pitard et al., 1996).

The nanoscale composite molecular devices described above were injected into living cells using arrays of hollow cylinders with atomic-scale spires -- "nanoinjectors." The first step in this process is to construct a non-hollow version of the nanosyringe. FIG. 9 is a micrograph of a portion of a silicon tip array 100 comprising tips 102 with diameters less than 10 nm and shafts 104 with diameters of approximately 1 μm. Using these arrays 100 as electrodes, arrays of nanoscale nickel dots were electrochemically deposited as supports for various molecular devices. These arrays also allow the deposition of microscale or nanoscale electrodes directly on top of protein-laden membranes.

The production and synthesis of BR is routine. Halobacterium was fermented in 50L batches, and after fermentation treatment and purification, more than 100 mg of purple membranes were obtained. This is consistent with a protein monolayer area of about 60 m ² , which is sufficient for prototype devices.

Zhen, Y., Qian, J., Follmann, K., Hayward, T., Nilsson, T., Dahn, M., Hilmi, Y., Hamer, A., Hosler, J., Ferguson-Miller can be used , S. (1998), Prot. Expr. & Pur. 13, 326-336 described method produces COX (Zhen et al., 1998). This method involves the overexpression and purification of COX from Rhodobacter sphaeroides, as indicated at 90 in FIG. 10 . The process of constructing the overexpression plasmid pRK-pYJ123H includes subcloning the subunit I gene (coxI) of cytochrome c oxidase into pUC19 by Rhodobacter sphaeroides, and using the SmaI site in pJS2-X6H2 to generate pJS3-SH, such as As shown in the figure. The six histidine sequence that marks the His-tag is located at the C-terminus of coxI, and pYJ124H was generated by ligating the PstI/PstI fragment from pYJ100 into the unique PstI site on pJS3-SH. Next, the three subunit genes were put into expression vector Prk415-1 using EcoRI and HindIII sites. This process yielded 61 mg/10L of broth, which is a large amount relative to the large monolayer area.

The generation and synthesis of BR and liposomal vesicles incorporating BR are routine. Orientation of BR is most easily facilitated through membranes with planar symmetry, although it is problematic in terms of spherical vesicles before lipid insertion with a uniform vesicle plus electric field. The apparatus shown at 120 in Figure 11 is an electrochemical cell for measuring impedance spectroscopy, and is designed to measure electron transport and potential caused by proton transport, and is used for planar membranes. Furthermore, it is easily adapted to apply electric fields across membrane/protein complexes. The cell included a stopper 121, an Ag:AgCl saturated KCl reference electrode 122, a liquid outlet 123, a Teflon spacer 124, a golf ball support 125, a liquid inlet 126 and a platinum counter electrode 127 as described by Naumann.

Regarding the orientation of violet membranes and BR, a number of techniques have been reported in the literature: electrostatic layer-layer assembly; electric-field-enhanced Langmuir-Blodgett film formation; orientation of BR in the electric field of an air condenser; direct application of an electric field on a suspension; and A combination of electric and magnetic fields. Most techniques exploit the large natural permanent electric dipole moment of PM (~ ¹⁰⁶ Debye for 1 μm diameter particles). This large electric dipole moment originates from the additivity of the individual BR dipole moments (570 Debye estimated from theoretical calculations and 55 Debye experimentally). Only an electric field of ~20 V/cm perpendicular to the film is sufficient to orient the BR, which is readily available.

The dipole moment of the localized COX needs to be large enough to enhance the docking of cytochrome c. COX uses its internal dipole to attract and push away across the membrane, just like BR does. Because the cytoplasmic portion of COX is hydrophilic, the enzyme has a barrier to rotation in lipids. Oval proteins also prevent this movement. Therefore, it is necessary to orient COX before its incorporation into lipid membranes. Adding PM after COX is incorporated into the lipid membrane requires a significantly lower electric field for orientation, so as not to disturb the alignment of COX. If the arrangement of the COX requires a voltage that is incompatible with other parts of the experiment, such as lipid membranes, BR, or hydrolysis of water, the counter electrode is moved closer and the applied voltage is kept at a safe level, ensuring both the desired electric field is generated and the The remainder of the process is left undisturbed.

The apparatus above for orienting PM and COX, respectively, in lipid membranes can also be used to measure proton transport in BR and transport of electrons through COX.

Preliminary experiments with only oriented BR incorporated into lipid membranes were performed using the structure of Figure 5 above. Proton pumping across the membrane gives an easily detectable signal. These experiments were repeated with COX.

In the device shown in Figure 5, electrodes 78 and 80 function to donate and accept electrons as a substitute for cytochrome c and _O2 which are not present in the device. Because pumping protons through COX 72 with current would complicate the measurement of proton flux, a pH-sensitive fluorescent indicator similar to that used in previous experiments with liposome BR was employed. The ability of the electrodes to associate with COX was determined by placing the protein alone in the lipid membrane. The ability to pump protons using COX transferred from the attached electrode was investigated using the same method described above (Naumann et al., 1999) to monitor proton pumping in BR. Experiments performed with and without the application of an orienting electric field showed that the orientation was successful. A major consequence of proton pumping guided by an electric field is that ATPases can be incorporated into lipid membranes with COX. Activation of COX creates a proton gradient, which can then be used by ATPase to make ATP. This demonstrates that ATP can be synthesized electrically, which is a major milestone in biology and has important implications for the further development of ATP-driven nanodevices.

To test the reverse function of the enzyme, an artificial pH gradient was generated, whereby one side of the membrane space was more acidic than the other. After determining COX orientation by measuring proton transport, the voltage due to the reverse flow of protons and the current through COX are measured. If the electrodes are configured as shown in Figure 11, electron flow does not occur. In this case, the upper electrode can be brought closer to the top surface of the membrane.

There are many ways to increase the proximity of the electrode to the protein. An electrode grid can be placed directly on top of the lipid in the form of a fine wire mesh that can be externally connected for electrical measurements. After removing the liquid from the top surface, a thin transparent layer of aluminum or nickel can be sprayed directly on the membrane to form the counter electrode. Alternatively, electrodes are deposited electrochemically on the lipid surface by grating the tip array shown in Figure 9. This deposition can form millions of nanowires on the top surface of the film. Repeating and combining the above steps resulted in oriented COX and BR gadolinium contained in the lipid film.

There are two possible schemes for the orientation of BR and COX: parallel and antiparallel dipole orientations. If the dipoles are parallel, applying an electric field achieves the alignment of both. If they are antiparallel, use a large PM to aggregate the dipole moment. By initially orienting the COX in a high electric field, followed by orienting the PM in an electric field small enough to avoid disturbing the COX and large enough to operate fully, the PM fragments thus acquire proper orientation.

Initially, measuring the voltage should indicate that the BR is functioning properly and that a proton gradient is formed. The transmembrane voltage from this gradient is expected to be in the hundreds of mV. Subsequent measurements of the current indicated success and it was estimated that the partially functioning COX protein could be accessed by the electrodes in the membrane.

Because protons are back-transferred by COX, and thus the current depends on the proton concentration provided by BR, the current should be proportional to the light intensity. The total current is also proportional to the number of COX molecules contributing, due to the parallel configuration of COX molecules in lipid membranes. As mentioned above, the voltage generated should be constant, >200mV, and the power generated is also determined by the light intensity and the net COX oriented in the appropriate direction.

Approaches to maximize power have focused on optimizing COX orientation and proper selection and modification of film layers. The device can deliver power under various irradiation conditions, such as continuous irradiation at high and low intensities, periodic irradiation, etc.

Polymeric membranes are preferred for the following reasons: they have a longer lifetime than lipid membranes; they are more robust; and they have properties that are easier to prepare, such as electronic and ionic conductivity and permeability. The interior of these membranes must be hydrophobic and elastic so that the native protein environment can be mimicked as closely as possible.

Many biocompatible membranes have a wide range of properties, such as light absorption, polarity, electronic and ionic conductivity, and more. Polymers that enhance the properties of the solar cells of the present invention must be compatible with proteins and electrodes. The rejection of protons is also important. The doping ability of the polymer surface is also important because it plays a major role in proton conductivity and transmembrane conductivity. The lifetime of the polymer and its effect on the lifetime of the protein contained within it is also relevant and is a factor when choosing a polymer. Selecting short-lived but high-performance polymers can be used for special applications.

The method for preparing high-efficiency and high-yield solar power sources from biological components was introduced earlier, showing the integration of energy-converting biological proteins with external devices; method.

On the other hand, by incorporation of triblock copolymer membranes using aquaporin family proteins, stable membranes are created that only allow water to pass through, thus facilitating water purification, desalination, and molecular concentration by dialysis. Aquaporins block the passage of any contaminants, including bacteria, viruses, minerals, proteins, DNA, salts, detergents, dissolved gases, and even protons from aqueous solutions, but aquaporin molecules are able to transport water due to their structure. As shown in Figure 12, each aquaporin 130 contains six transmembrane alpha-helical regions 132-137, which anchor the protein in the membrane, and two highly conserved NPA loops 138 and 140, which apex the central protein until the vertices come together to form a sort of hourglass shape. The narrow part of the hourglass is where the water molecules pass through the membrane in a single row, as indicated by reference numeral 142 in FIG. 13 . It has been shown that the movement of water is symmetrical and can take place in both directions; this fact is important because this process consumes no energy. Water moves through the membrane in a specific direction because of hydraulic or osmotic pressure. As shown in Figure 14, water purification and desalination can be achieved with a dual chamber device 150 having chambers 152 and 154 separated in the center by a rigid membrane 156 filled with aquaporins. The membrane itself is impervious to water and it separates the dirty water 158 in chamber 152 from the purified water 160 in chamber 154. Only pure water can flow between the two chambers. Thus, pure water flows naturally into the other chamber 154 when seawater or other sewage 158 on one side of the membrane is put under the proper pressure. Thus, pure water can be obtained from a non-potable source, or, if the source contains the chemical of interest, the water can be selectively removed, leaving the desired high concentration of the chemical in the input chamber. Importantly, however, aquaporins are also suitable for use in the present invention, not because of their specific selectivity for water. Many members of this protein family, such as aquaporin Z (AqpZ), are very stable and can withstand harsh conditions in sewage sources without loss of function. AqpZ also does not denature or decompose in the presence of acid, voltage, detergent and heat. Therefore, the device can be used to purify water sources contaminated by substances that may stain or damage other membranes, and it can also be used in areas where high temperatures are constantly present.

AqpZ is also variable. Since the protein is specifically expressed in the host bacterium according to the gene sequence, which affects its final shape and function, it is easy for a skilled person to change its genetic code in order to change the properties of the protein. Thus, proteins can be processed for desired applications, which may differ from the protein's original function. For example, by simply exchanging a specific amino acid residue near the center of the water channel 142 in Figure 13 with cysteine, the resulting aquaporin may bind any free mercury in solution and stop water transport due to blockage. Thus, these mutant proteins used in the membrane device can detect mercury contamination in water samples, and when the concentration of the toxic substance rises too high, the water simply stops flowing.

The preferred embodiment of the invention is in the form of a traditional filter disc, as it is the easiest to determine functionality. To prepare such discs, a 5 nm thick monolayer of synthetic triblock copolymer and protein was deposited on the surface of a 25 mm commercial ultrafiltration disc using a Langmuir-Blodgett cell. The monolayer on the disc is then irradiated with 254nm UV light to crosslink the polymer and increase its resistance. Finally, a PVDF membrane with a 220nm pore size was epoxy bonded to the surface of the disc to ensure safe handling and prevent leakage at the edges.

To test the device, it was held in a chamber as shown in Figure 14 and a source of pressurized water was forced through the membrane. The device was considered functional when only pure water passed through the other side of the membrane and contaminating solutes remained concentrated in the original chamber. Contaminated solutions must be pressurized to overcome the natural flow of pure water into the chamber where large amounts of dissolved particles are present. The purpose of the aquaporin Z membrane is to reverse osmosis and separate pure water from contaminating solutes. This tendency, or osmotic pressure, of the system can be expressed in pounds per square inch (psi). For example, seawater has an osmotic pressure of approximately 360 psi.

There are several methods that can be used to subject devices to these types of stresses. Some polymers are inherently more resistant than others and can be cross-linked with UV light to provide ultra-rigidity. Another approach is to add high concentrations of nontoxic, easily removable solutes to the freshwater chamber to promote normal osmosis across the membrane, while back osmosis also occurs due to chamber pressurization. Finally, a way to reduce the pressure required for back osmosis could be to use multiple AqpZ devices in a series of sealed, connected chambers containing sequentially decreasing concentrations of contaminants. The resulting pressure is the pressure required to purge each pair of chambers as a fraction of the total pressure required for reverse osmosis. Therefore, each chamber only needs to experience a small pressure, with a high probability of remaining intact. Therefore, if the concentration difference between each pair of chambers is only 10%, instead of 100%, only 10% of the above high pressure is required to purify the water source at each connection. Pure water can be produced continuously at constant pressure and flow rate in the final chamber.

Aquaporin reverse osmosis membranes can purify water with several different types of pollutants in just one step. Conventional high-purity systems, shown at 170 in Figure 15, require several components, including water softener 172, carbon filter, ion exchanger, UV or chemical sterilization 174, and dual-pass reverse osmosis filter unit 176 to produce water (that is, not as clear as aquaporin purified water) can be used in combination before. This complex device cannot remove dissolved gases or substances smaller than 150 Daltons from water sources like aquaporin membranes can. Additionally, all of these components require maintenance. UV bulbs require replacement and energy. Chemical regeneration is required after the ion exchanger has filled the leaves. Softeners need salt. Carbon and reverse osmosis cartridges must be replaced when clogged. Finally, the one-step device requires much less space and is much lighter than typical purification systems, an advantage that makes the aquaporin water purification device of the present invention portable.

Aquaporin membranes are also faster than conventional systems. A conventional high-speed reverse osmosis (RO) unit can produce approximately 28.4 liters (7.5 gallons) of clean water per minute. Current studies show that water molecules pass through the AqpZ saturated lipid membrane (0.0177 mm ² ) at a rate of 54 μmol/s (Pohl, P., Saparov, SM, Borgnia, MJ, and Agre.P. (2001) Proceedings of the National Academy of Science 98, pp. 9624-9629). Therefore, an aquaporin Z reverse osmosis membrane with a surface area of 1.0 square meters can filter 3295 liters of purified water per minute. This rate is 116 times faster than conventional water purifiers.

Finally, it is also cheap to produce new protein-based membranes. Milligram quantities of the core of this process, AqpZ, are readily available from engineered E. coli strains. An average of 2.5 mg of pure protein can be obtained from each liter of culture medium. 10 mg of protein can be produced from about $5 in growth medium. This is enough protein for several full-scale devices. The polymers that entrap AqpZ can be prepared in the same laboratory, using only a few cents of chemistry per device. Aquaporin Z reverse osmosis membrane is a novel, effective and inexpensive water purification tool.

Accordingly, methods and apparatus have been disclosed for the efficient production of perfectly pure water from dirty, salt water, or other contaminated water using biological components. The present invention demonstrates the integration of water transport biological proteins and external devices, and points to a fabrication route for large-scale production of water purification devices.

Although the present invention has been described above in connection with preferred embodiments, it should be understood that various changes and modifications may be made to the methods and devices described without departing from the spirit and scope of the present invention, as set forth in the following claims.

Claims (28)

1. bionical film, it comprises:

The triblock copolymer matrix of simulation natural biological film and native protein environment; With

Mix the memebrane protein that described matrix forms film/protein complex.

2. the described film of claim 1 is characterized in that film/protein complex composition device, and this device has the function of the memebrane protein that mixes.

3. the described film of claim 2 is characterized in that the function of protein comprises valve, passage, sensor, detector, pump and transducer.

4. the described film of claim 1 it is characterized in that the described memebrane protein of selecting only transports hydrone, and described bionical film is a water filter.

5. the described film of claim 4 is characterized in that described memebrane protein is selected from the aquaporin family of protein.

6. the described film of claim 5 is characterized in that described matrix does not seep water, and the described memebrane protein of selection allows hydrone to pass through under pressure.

7. the described film of claim 6 is characterized in that described matrix is fixed in the water purifier spare, and described device is separated into first Room and second Room, and described memebrane protein only allows water to flow between described chamber.

8. the described film of claim 7 is characterized in that described matrix is biocompatible polymer, is selected from poly-(vinyl alcohol), poly-(acrylamide) and colloidal sol.

9. the described film of claim 1 is characterized in that described memebrane protein is a natural biological albumen.

10. the described film of claim 9 is characterized in that two different memebrane proteins are mixed described matrix.

11. the described film of claim 10 is characterized in that described memebrane protein is a power conversion protein.

12. the described film of claim 1 is characterized in that described matrix is mixed flimsy material.

13. the described film of claim 12 is characterized in that described memebrane protein comprises bacterium rhodopsin and the cytochrome oxidase that is embedded in the described matrix, is used for luminous energy is converted into electric energy.

14. the described film of claim 13 is characterized in that described matrix is biocompatible polymer, is selected from poly-(vinyl alcohol), poly-(acrylamide) and colloidal sol.

15. the described film of claim 13 is characterized in that also being included in first and second electrodes on the opposed surface of described fabric, is used to receive described electric energy.

16. the described film of claim 1 is characterized in that described matrix receives directed bacterium rhodopsin and cytochrome oxidase, produces biologic solar cell.

17. the described film of claim 16 is characterized in that also being included in the electrode on the opposite sides of described matrix.

18. the described film of claim 16 is characterized in that described matrix is the biocompatible polymer that can not see through proton.

19. a compound organic/inorganic power supply, it comprises:

Triblock copolymer matrix;

Be embedded in the first and second different memebrane proteins in the described matrix.

20. the described power supply of claim 19 is characterized in that also comprising sheer fabric material, described matrix is embedded in the described fabric.

21. the described power supply of claim 20 is characterized in that described memebrane protein is a natural biological albumen.

22. the described power supply of claim 21 is characterized in that described protein comprises bacterium rhodopsin and cytochrome oxidase, is used for luminous energy is converted into electric energy.

23. the described power supply of claim 22 is characterized in that also being included in the electrode on the opposed surface of described fabric, is used to receive described electric energy.

24. prepare biomembranous method, it comprises:

Preparation triblock copolymer matrix; With

In described matrix, insert natural or genetic engineering memebrane protein.

25. the described method of claim 24 is characterized in that also comprising that to make described memebrane protein directed in described matrix.

26. the described method of claim 24 is characterized in that also comprising and selects described protein to produce corresponding film function.

27. the described method of claim 24 is characterized in that also being included in and inserts two kinds of different memebrane proteins in the described matrix.

28. the described method of claim 27 is characterized in that also comprising described matrix is exposed to light, passes the electric energy of described matrix with generation.

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