CN101301583A

CN101301583A - Biomimetic membranes

Info

Publication number: CN101301583A
Application number: CNA2008100097938A
Authority: CN
Inventors: C·D·蒙特马格诺; J·J·施米特; S·P·托齐
Original assignee: MT Technologies Inc
Current assignee: MT Technologies Inc
Priority date: 2002-07-29
Filing date: 2003-07-28
Publication date: 2008-11-12
Also published as: CN100377766C; CN1703268A

Abstract

The present invention relates to a biomimetic membrane. Biological membrane proteins are incorporated into a co-polymer matrix to produce membranes with a wide variety of functionalities. In one form of the invention, a composite membrane incorporates two different proteins which cooperate to produce electricity from light. In another form, water transport proteins are embedded in a membrane to enable water purification.

Description

Bionical film

Patent application of the present invention is that international application no is PCT/US2003/019879, and international filing date is on July 28th, 2003, and the application number that enters the China national stage is 03817994.6, and name is called the dividing an application of application for a patent for invention of " bionical film ".

The application requires to be filed in the U.S. Provisional Application number 60/398784 on July 29th, 2002 and is filed in the right of the provisional application number 60/438784 on January 9th, 2003, fits into this paper as a reference in these two applications.

Technical field

The present invention relates to produce the method for artificial device, described artificial device has the character and the function of biomembrane and memebrane protein; The structure that also relates to this device.

Background technology

Biomembrane albumen has various functions, comprises as pump, passage, valve, transducer and machinery, heat and electric transducer, or the like.Because these protein sizes are nanoscales, and the efficient height, so they are being used for having much attraction aspect the artificial device.But their natural grease plasma membrane environment has shortcoming, and is low as intensity, needs water environment, to chemistry or microbial degradation sensitivity.

Summary of the invention

In brief, one aspect of the present invention is mixed the block copolymer matrix with natural or genetic engineering memebrane protein, produces the film with various hereditary functions, is included in the ability that selectivity between the fluid transported and/or filtered compound.By selecting to have the protein of special properties, can be made into film with specific functionalities, comprise molecule scale addressing by directed electrostatic force, electromagnetic force and chemical force.

Can design and produce block copolymer of the present invention, make them have following character on request: the ability that forms film with desired thickness; Form the ability of the film of required chemical composition; Form the ability of high-strength membrane; Improve the ability of the film strength that has formed on demand.One of most important character of these films is that they can hold the natural biological memebrane protein that is in a kind of functional status, these composite membranes are firm and the life-span is long, because by inserting biomembrane albumen in this polymer film, can produce the character with protein and the device of function.Suitable polymers only need form such film, is about to memebrane protein and is divided into two halves up and down, fully is similar to the natural grease plasma membrane, insert protein easily when they are suitably directed, and they does not take into account the natural function of protein.The polymer that satisfies these conditions comprises triblock copolymer, and they have the general aspects of hydrophilic outside block and hydrophobic interior block.

One aspect of the present invention relates to the generation of the composite membrane that contains two kinds of different proteins, does the time spent when consistent, obtains device---" biologic solar cell " of photoproduction electricity.The present invention utilizes the water transport protein that the water at any water source is purified on the other hand.Below will describe these aspects in detail.

Because the technological innovation of device miniature aspect makes electronic device littler, lighter and higher effectively, but the progress of the used power supply of these devices aspect is then slow relatively.The power supply of 21 century faces to increasing device energy supply, but size and weight are wanted ever-reduced challenge.In addition, tomorrow, nanometer technology and biological technology products even were not similar to the power supply that uses today on form or function to the requirement of power supply.

Currently press for lighter, more small-sized power supply, with the new demands of applications that satisfies condition and constantly occur.This power supply perhaps can be realized wider functional objective than the battery technology in the present age, makes the power density maximum, and weight is the lightest, needs to carry out specific power requirement.It is crucial that weight requires, because must if movably talk about, also want transport means near device to traditional fuel source fuel source.Fuel also may exhaust, and therefore essential replenishing supplied with.This scope and flowability to the user causes certain limitation.

Contemporary science shows that the development nanometer biotechnology has breathtaking potentiality.Utilizing the component do not waste atom to make device is likely aspect efficient and highest level microminiaturized.Though the progress that is nearly relevant power technology is exciting, the improvement that they just obtain increasing progressively in prior art.The power supply that is ideally suited device of future generation will adopt nanometer technology to promote their function, also can be with current this generation device of the performance driving of highest level.

Only just possessed exploitation needed technology of first nanometer biotechnology device and knowledge recently, reported the engineering and structure (Soong, the R.K. that drive nanoscale organic/inorganic hybrid device by biochemical fuel ATP, Bachand, G.D., Neves, H.P., Olkhovets, A.G., Craighead, H.G. and Montemagno, C.D. (2000), Science, 290, the 1555-1558 page or leaf).Be used for these devices ATP generation and with other machineries of these device drive, between macro-scale and nanoscale, appeal to aspect the exchange of the transfer of energy and dissimilar energy.

In another aspect of this invention, other protein with difference in functionality degree can be used for conveying electronic/proton, make the transduction of electric energy and chemical energy, and play mechanical valve and sensor.

In a preferred embodiment of this invention, available film provides biological solar energy materials and fabric, it is made of flimsy material, wherein be mixed with and have the biocompatible polymer film, described polymer film embedding two kinds of power conversion protein, bacterium rhodopsin and cytochrome oxidase, they can be converted into electric energy with luminous energy, and this energy is delivered to outer load.Owing to use thin (less than 1 μ m) polymer film, and do not need to carry fuel, so saved weight greatly with power supply.Therefore, can develop a kind of system, it can be integrated into the surface of cloth and most materials, provide in light weight (less than 1kg/m ²) the energy, its efficient is equal to or greater than the efficient that reaches with solar cell.Like this, biological solar energy materials has formed mixing organic/inorganic power supply, and it can obtain energy from light.

Technology of the present invention relates to a kind of production of flimsy material, and this fabric is by two kinds of power conversion protein of embedding, and the biocompatible polymer film of bacterium rhodopsin and cytochrome oxidase is formed, and they can be electric energy with transform light energy and this energy is delivered to outer load.Through the natural selection in millions of years, separated and optimized these protein, light and electric energy can be converted to electrochemical energy.The device of packing into, they can provide the electric power of consumption indefinitely, and enough light, small-sized and stable, under favourable and adverse environment, all are fit to require mobile high application.

The bacterium rhodopsin is a kind of bacterioprotein, and when absorbing light, it can cross cell membrane with proton transport.Cytochrome oxidase is a kind of memebrane protein, can use the high energy electron proton delivery.Collaborative these protein that use can be electro chemical proton gradient with transform light energy, and this gradient is converted into the electric power that can be used for operate outside subsequently.Because this device is the biology " version " of conventional solar cell, power supply does not need to carry " fuel ", has improved power density significantly.In addition, from this device in theory the ceiling capacity of available deduction be unlimited, as long as have the sun or device to exist, it just can be worked.The estimated area metric density of resulting devices is～100g/m ², provide the light energy, and efficient is equal to or greater than the efficient of solar cell.The material composition of this biologic solar cell and size make it finally obtain high power density (＞250W/kg) and macro-energy density (800Whr/kg 3 hours, 9500Whr/kg 3 days, 32000Whr/kg 10 days), be enough to drive large number quipments, and effective possessive volume and weight are 0.In addition, produce insignificant sound, heat and electronic characteristic during its work.

Important difference is arranged between power supply of the present invention and the conventional solar cell.Because power supply of the present invention is made of the protein and the general polymer of large-scale production, it is in light weight, flexible, firm, can be low-cost a large amount of.The correlation length of this device is the thickness of packing, less than 1 μ m.Usually the film thickness that contains these enzymes is 5nm.The lamellated plate of biologic solar cell can mix cloth and other surfaces, and can not increase cost aspect weight, in any case because they must dress.Power in the suitable modular design fabric produces battery, will make the electric power fabric keep tangible loss prevention and keep the ability of function.Utilize the mutual transfer capability between electric energy and the biochemical energy can construct the electrically and biologically device, and biochemical fuel is converted into.Utilize the ability of the conversion energy between electricity, biochemistry and the light form, can design and produce the nanometer biotechnology device, and do not imported the restriction of kind of energy.

Description of drawings

By following accompanying drawing with to the detailed description of preferred implementation, can understand above-mentioned and other purpose of the present invention, characteristics and advantage best, wherein:

Fig. 1 is the schematic diagram of the simplification main chain belt structure of bacterium rhodopsin, and wherein proton is carried by the center internal channel and passed film.

Fig. 2 illustrates a kind of process, in salt product salt bacillus (Halobacteriam salinarium), the BR pump is extracted proton out from bacterium when absorbing the photon of green glow, produce electrochemical gradient, and atp synthase is got back in the cell these protons, utilize their electrochemical energy to prepare ATP, the clean conversion from luminous energy to the chemical energy is provided by ADP.

The main chain belt structure of Fig. 3 A and 3B explanation COX, wherein Fig. 3 A is a film, and Fig. 3 B is a cytosome, and three zones beating asterisk are " holes " that are known as the proton transport passage.

Fig. 4 A explanation liposome mixes the double-layer of lipoid that planar solid is supported; Fig. 4 B explanation vesica combines with COX to be integrated with in the planar film.

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

Fig. 6 is the tomographic map of the bacterium rhodopsin of purifying.

Fig. 7 be contain the bacterium rhodopsin (

) and do not contain the pH gradient map that the liposome of bacterium rhodopsin () formed in 30 minutes.

The fluorescein fluorescence of Fig. 8 explanation because of existing ATP to send, described ATP is by the bacterium rhodopsin and the F that contain different amounts ₀F ₁The liposome of-ATP enzyme produces.

Fig. 9 is the SEM micrograph (sharp less than 1nm, the about 1 μ m of axle) of silicon tip array.

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

Figure 11 is the proton that can measure and control and the electron transport partial cross section schematic diagram by the equipment of COX.

Figure 12 is the schematic diagram of aquaporin.

Figure 13 is the enlarged drawing of the Partial Protein of Figure 12.

Figure 14 explanation is mixed aquaporin with film and is carried out water purifying tank.

Figure 15 is the schematic diagram of traditional water cleaning system.

The specific embodiment

In one embodiment of the invention, bacterium rhodopsin and cytochrome oxidase are integrated into the biocompatible polymer film, contact with the electrode of little manufacturing.Operating in of the device that is proposed is integrated into the group bacteria rhodopsin, cytochrome oxidase can get the best understanding afterwards, also understands them simultaneously and is integrated into lipid and polymer film.All these threes are broad research, and the document of many synthetic and functions about them is arranged.

Bacterium rhodopsin (BR), as the ion transporter of broad research, be that molecular weight is the proton transport protein of 26kD, shown in label among Fig. 1 10, it finds in the cell membrane of salt product salt bacillus, be a kind of at the bright light photograph salt solution and the marsh in multiply have a liking for the salt archeobacteria.BR allows salt product salt bacillus to survive in anaerobic environment; When not having enough oxygen to carry out respiratory, BR just has an effect.As shown in Figure 2, BR 12 cells absorb green glow photon 18 and (during λ=500-600nm), proton 14 are carried passed cell membrane 16, and come out from cell.Each BR molecule has experienced various electronics intermediatenesses in the process that is subjected to optical excitation and proton delivery, the total time that BR is returned to its original state is the level of 3ms.This is the short time scale that energy shifts.

Along with proton 14 pumps from cell 12, the electric charge of formation and pH gradient (low H ⁺-Gao H ⁺) pass cell membrane 16, form electrochemical potential.This electrochemical potential provides energy for driving atp synthase (ATP enzyme), as shown in the reference numeral 20.The ATP enzyme passes in the film in that proton 14 is gone back to, and utilizes its electrochemical energy to produce atriphos (ATP), shown in Fig. 2 label 22.ATP is ubiquitous bio-fuel, and it has promoted most of to teleorganic cell processes.This natural biosystem is duplicated in the laboratory, and concrete grammar is to make up system (Pitard, B., Richard, P., Dunarch, the M. ， ﹠amp that is made up of BR and ATP enzyme in lipid vesicle; And Riguard, J. (1996), Eur.J.Biochem., 235,769-788 page or leaf), wherein BR can form and keep 1.25 pH difference on the vesicle border over there down at 40 ℃.At 20 ℃, can obtain Δ pH=2.Higher pH difference then can't obtain, and suppresses because BR is subjected to the feedback that reaches of proton gradient.Proton gradient is used for producing ATP, and the performance of two kinds of enzymes in the mensuration system.This work also shows, adds electronegative phosphatide in liposome membrane, can improve the coupling efficiency between BR and the ATP enzyme.

BR is the integrated ideal candidates person of device, because protein as two dimensional crystal, can high concentration be present in the cell membrane.Thus, in retina protein, it is unique.It is called " purple membrane " under this form, protein accounts for 7% in the mass ratio, and lipid accounts for 25% (～10 lipid molecular/protein).Observe, the diaphragm size of these purple membranes is 0.5 μ m or bigger.Because these protein aggregates are stable (existing with nature) under so high concentration, they have improved energy yield, and the element of redundancy, engineering safety is provided for the device of any manufacturing.In addition, the evolutionary optimization of salt product salt bacillus the function of BR because it can be under high temperature, big luminous flux the longer time of operation.

The BR of the work utilization of Pitard above-mentioned etc. high dilution in little (150nm) lipid film, result show that the productive rate of ATP and BR/ lipid mass ratio are inversely proportional to.The data of the BR/ lipid mass ratio of the relevant purple membrane that they are obtained are extrapolated, and find that every milligram of BR per minute produces 320nmol ATP.The ATP enzyme increases the 35kJ/mol energy with ADP shown in the label 24 among Fig. 2 and the synthetic ATP of inorganic phosphate.Only consider the quality of purple membrane and lipid, the power density that the ATP synthesis system of this optical drive provides is 140W/kg.If atp synthase is the same fast with the speed that they pump into BR with the speed that proton pumps, then the power of Chan Shenging will be increased to 180-280W/kg.

Since a large amount of about the BR and the knowledge in intensity and life-span thereof, aspect the active optical element in optics and the Computer Storage application, caused extensive interest and research at exploitation BR.Show, purple membrane has the activity of several years under illumination, in up to 180 ℃ polymer substrate, between pH value 0-12, in the presence of organic solvent, when dewatering fully, purple membrane is stable (Vsevolodov, N (1998), " biomolecular electronics: photosensitive protein matter outline " (Biomolecular Electronics:AnIntroduction via Photosensitive Proteins), 125 pages, Birkhauser, Boston).Because the concern of scientific circles and engineering circle, the BR that has formulated salt product salt bacillus the overfulfil a production target production of bacterial strain and the scheme of separating (Lorber, B. and DeLucas, L.J. (1990) FEBS Lett.261,14-18 page or leaf).Purple membrane is extracted and purification process in a large number, and processing and carrying (Stuart for example, the J.A. that are to know; Vought, B.W., Schmidt, E.J.; Gross, R.B.; Tallent, J.R.; Dewey, T.G.; Birge, R.R., IEEE EMBS is in sending to press).The experiment that combines with non-biological material about it shows that BR can be used with general polymer, 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).

Second kind of enzyme---cytochrome oxidase (COX) is electronics and proton transport protein, makes a kind of at last in four kinds of enzymes that breathe to take place.The film figure of COX main chain belt shown in the label 30 among Fig. 3 A, and the figure of its cytosome shown in the label 32 among Fig. 3 B, zone 34 expressions " hole " of beating asterisk, perhaps proton transhipment passage.In breathing, the high energy electron of NADH (oxidation by glucose at the beginning produces) is transferred to O ₂, because its reduction produces H ₂O.

COX accepts electronics from the last stage of respiratory, and electronics is carried by cytochrome c, and transfers on two inside heme group that contain iron ion and copper ion.These heme group are accepted the electron reduction from cytochrome c, transfer at electronics and stop the molecule O that one of ferroheme is arranged ₂Afterwards, these heme group are by deoxidation.O ₂Obtain additional additional electronics, become the target with peripheral proton oxidation reaction, after the reaction, O ₂Just break away from from ferroheme.Along with the transfer of these high energy electrons, the energy (Nicholls, the D. (1982) that obtain from the voltage drop of its 460mV, " bioenergetics: chemiosmotic theory introduction " (Bioenergetics:An Introduction to the Chemiosmotic Theory), 123 pages, Academic Press, London) be used for proton translocation to the mitochondria space transfer ratio of proton and electronics is generally 1: 1 (Lee, H., Das T., Rousseau, D., Mills D., Ferguson-Miller, S., Gennis, R. (2000), Biochemistry 29,2989-2996), though other ratios (Papa had been discussed, S., Lorusso, M., and Capitanio, N. (1994), J.Bioenerg.Biomembr.26,609-617 page or leaf, Michel, H., Behr, J., Harrenga, A., and Kannt, A., (1998), Ann.Rev.Biophys.Biomol.Struct.27, the 329-356 page or leaf), this ratio function of membrane potential (Murphy, M. normally, and Brand, M. (1998), Eur.J.Biochem.173,645-651 page or leaf).

When proton pumps, produced electro chemical proton gradient from mitochondrial matrix.The ATP enzyme produces ATP by this proton gradient.BR and COX are closely similar aspect the generation proton gradient; Difference be BR by optical drive, and COX is driven by chemical energy.This can as can be seen from Figure 2 replace BR12 with COX, replaces green glow 18 with the high energy electron from cytochrome c, and adds hydrogen reduction Cheng Shui.In fact, BR and COX all are used for salt product salt bacillus, and play identical purpose: BR is not used for salt product salt bacillus when having enough oxygen supplies to breathe, and COX then is used for organism.

COX is integrated into the lipid film 40 that solid matrix is supported, shown in Fig. 4 A and 4B, shows and to utilize electrical method to measure electron transport and control proton transport (Naumann, R., Schmidt, E., Jonezyk, A., Fendler, K., Kadenbach, B., Liebermann, T., Offenhausser, A., Knoll (1999), Biosensors ﹠amp; Bioelectronics 14, the 651-662 page or leaf).These experiments sulfur functional peptide chain 42 that links to each other with golden film 44 lipid film individual layer 46 as two myristoyl phosphatidyl-ethanolamines (DMPE).COX and cytochrome c are mixed the liposome vesicle 48 of DMPE, and the latter is fused on the peptide surface, shown in label 50 among Fig. 4 A.Electricity is measured and is shown, electronics shifts turnover auri matrix by COX, and by applying transporting of Current Control proton.

Though experiment in vitro provides the natural environment of the most accurate reproduction membrane bound protein, these conditions are not to help to measure the phenomenon of being blured by other cell processes most, perhaps these phenomenons that frequently do not take place at the experimental period yardstick.In addition, producing useful device with protein as activeleg needs to produce easily and the maintenance support body, and it can not make protein denaturation, and to make the function of protein keep as far as possible with body in the same close, the while uses easily and makes.The large number of biological enzyme is mixed artificial lipid film in experiment, they are effectively keeping function in the experimental period; The present invention relates to use lipid and manufacture of polymeric films BR/COX optical drive device.

The amphiphilic component that the artificial lipid film that is prepared by lecithin or DMPE duplicates the natural fine after birth.Add that washing agent such as Triton-X or lauryl sodium sulfate make the memebrane protein dissolving and to protein/lipid soln slight ultrasonic processing mix liposome.Liposome can keep little blister (Pitard etc., 1996), also can form plane (Naumann etc. in the presence of smooth matrix, 1999 and Steinber-Yfrach, G., Rigaud, J., Durantini, E., Moore, A., Gust, D., Moore, T. (1998), Nature, 392, the 479-482 page or leaf).The biological function that keeps protein can reach high thousands of times of concentration in the concentration ratio body of protein, makes experiment highly sensitive and accurately.The protein concentration height also is necessary for making up power supply and biology sensor, because they utilize the collective effect of each molecule.

Fix the character of BR, just can estimate the contingent power of devices use BR and COX.On the earth surface on every square metre the area solar photon in the incident each second green range be about 7.5 * 10 ²⁰, perhaps at BR molecular area (25nm ²) on have 1.9 * 10 ⁴Individual.The absorption coefficient of BR is 66000/mol/cm (Vsevolodov, N. (1998), " biomolecular electronics: photosensitive protein matter outline " (Biomolecular Electronics:An Introduction via PhotosensitiveProteins), 125 pages, Birkhauser, or about 4.4 * 10 Boston), ^-4/ individual layer BR.The quantum efficiency of BR is 0.7, and the proton transport probability is 3.08 * 10 ^-4Therefore, can expect that each BR molecule takes place to transport incident 5.8 times each second approximately in sunlight.

At 1m ²Area on, BR: COX is 57: 1 o'clock, obtains the proton transport rate of stable state, COX whenever transports a proton, BR just transports a proton.Under this ratio, every square metre of individual layer has 3.9 * 10 ¹⁶BR.Because therefore electronics of each proton transport among the COX obtains 37mA/m ²The electric current of/individual layer.The individual layer of 1,000 accumulations only utilizes 36% light, but is increased to 37A/m at the electric current of 760mV ², obtain 28W/m ²Though these electric currents and voltage are not suitable for all devices, the output of the electric current of device is highly configurable, and the voltage and current combination is possible to any power output on a large scale.The quality of protein and lipid (or equivalent) is 2.3g/m in this system ²Because the polymer layer of thickness identical (5nm) of gold electrode and polyvinyl alcohol obtains 105.3g/m ²Regional quality density, the power density of generation surpasses 265W/kg.But 3 hours energizing quantity of this device is 795Whr/kg, but 3 days energizing quantity is 9540Whr/kg, but 10 days energizing quantity is 31800Whr/kg.Because energy obtains from the sun, the energy of acquisition directly increases along with the sun-exposed duration

These power and energy density can increase along with alternate selection electrode and polymeric material, but because additional weight increases and light scattering, also can reduce along with the layer thickness of different choice.The light scattering effect of electrode and polymeric material can be ignored, and reason is as follows: (1) used polymer is active minimum in λ=500-650nm scope; (2) metal electrode does not absorb by the non-transmitted light of the part of device initial layers; If light and electrode interact, it only in the device internal reflection, is finally absorbed by BR.

As mentioned above, BR and COX are the proton transport proteins, and they are driving ATP enzyme and the proton gradient that produces ATP with the power conversion of light and high energy electron respectively.Make the COX acting in opposition, proton gradient can be converted into electromotive force (EMF), energy is passed to electronics.Eliminate oxygen and cromoci, replace with the electrode that is connected to external loading, EMF can work then.Electrode crosses that BR and being combined in process shown in Figure 5 and the structure of COX culminate in the film carrier.This figure is the schematic diagram of biologic solar cell 60, when wherein bacterium rhodopsin 62 absorbs the photon 68 of green glow, proton 64 is carried polymer film 66.The proton concentration that this has increased on the upside 70 of film makes cytochrome oxidase (yellow) 72 that acting in oppositions take place.As a result, obtain to be transported to from the electrochemical energy of proton 64 downside 74 of film, this energy is used for proton 76 is transported to bottom electrode 80 on the film downside from the top electrode 78 on the film upside, produces electromotive force and passes electrode, and this electronic force is used for doing operate outside.

When electronics shift to finish, this system was returned to its original state: COX and has reduced and reoxidized, and the proton concentration on the both sides of polymer film 66 is constant, and electrode does not acquire or do not consume any net charge.EMF does operate outside, has absorbed photon.This system is prepared the luminous energy of next photon is converted into electric energy.

It is by COX 72 acting in oppositions that proton motive force is converted to electrodynamic core process.The example that many reversible power conversion protein are arranged in the document is as F ₀F ₁-ATP enzyme (Hammes.G. (1983), the TrendsBiochem.Sci.8.131-134 page or leaf) and ion transporter (Nicholls, D., (1982), " bioenergetics: chemiosmotic theory introduction " (Bioenergetics:An Introduction to theChemiosmotic Theory, 123 pages, Academic Press, London).Yet, also have can not acting in opposition power conversion protein, for example the bacterium rhodopsin does not send green glow to the reaction of proton gradient.

In the work that Wikstrom did (Proc.Natl.Acad.Sci.USA 78 for Wikstrom, (1981), the 4051-4054 page or leaf), in the mitochondria that adds ATP, it is reverse to observe the part that electronics flows among the COX.F ₀F ₁-ATP enzyme is the reversible proton pump with following function: the ATP enzyme is produced ATP with outside proton translocation in mitochondrial matrix.Conversely, it can consume ATP and proton is pumped.As described in Wikstrom, the ATP enzyme passes film with the proton commentaries on classics, and is parallel with COX when adding ATP.This is reflected at and produces high proton concentration on the outside of film, oppositely forms electric osmose proton barometric gradient on COX.When producing this condition, observe the skew in the absorption spectrum, show that electronics transfers to the ferroheme from water, this is the reverse of canonical process.Why below analyzing can this thing happens.

In electrochemical reaction, energy surplus or deficiency are provided by following formula in the course of reaction: (for example, referring to example, De Vault, D., (1971), Biochim.Biophys.Acta 226, the 193-199 page or leaf):

-ΔE＝ΔG/(nF)

Wherein, Δ E is the variation in the redox potential before and after power supply, and Δ G is the Gibbs free in the reaction, and n is the quantity that electronics shifts, and F is a Faraday constant.Electronics from cytochrome c (reduction potential=+ process that 220mV) shifts by COX, the free energy of electronics descends continuously, up to they O the most at last ₂(reduction potential=+ 860mV) be reduced to H ₂O.The electronics that revolution moves, free energy change turns to 14.8kcal/mol.This energy is used to transport proton, and produces electrochemical gradient.Wikstrom improves outside proton concentration fully, make Δ G to COX normally act as the positive, making forward, the required energy of pumping proton compares O ₂The energy that reduction can provide is more.By utilizing " potential buffer solution ", it is constant that the redox potential of cytochrome c keeps; This means H ₂O/O ₂Redox potential change with the change of Δ G.Owing to produced sufficiently high outside proton concentration, electronics generation reverse transition receives electronics and supplies with COX from water.Yet the complete reverse transfer of electronics is not finished, because O ₂Also do not produce.

In system of the present invention, as shown in Figure 5, COX had not both had initial electron donor---the cytochrome c of the described system of Wikstrom, did not have final electron acceptor---O yet ₂Because electron source is electrode 78 rather than water, with H ₂The 820mV of O compares, and it is minimum that electronics obtains cost.Electronics arrives Cytoheme ₃When last, be subjected to positive redox potential+380mV, ferroheme can be used for doing operate outside.To Cytoheme ₁The time, this electromotive force has descended 140 to+240mV, at this moment needs from proton gradient input energy.To Cu _aThe time, electromotive force further descends 50 to+190mV, finally transfers on the electrode 80 at 0V, requires the input energy.Because electronics is transferred to Cytoheme from initial electrode 78 ₃Be that reduction potential increases (free energy decline), this reaction spontaneously takes place.It is reduction potential decline 380mV that electronics is transferred to counterelectrode from a3, needs external energy input (proton motive force) to take place.Utilize BR 62 and film 66 is suitably mixed, can increase proton motive force.Because electrode is an electron donor, compare from H from their electron gains ₂O obtains easily, and avoids any chemical intermediate between water and oxygen.

Because the lip-deep ions diffusion of film is big, and can be bigger by the film composition of suitable selection, the film surface itself just needs the function (Pitard etc., 1996) of the achieving success of biologic solar cell.Lipid film, as any protein that comprises of film 40 (Fig. 4 A) perhaps many biocompatible polymer matrixs matter, and as proton carrier.These polymer substrates are very common, only require that (a) their formation can be divided into protein the film of half up and down, (b) they form the environment that fully is similar to the natural grease plasma membrane, make protein insert film easily with suitable orientation, (c) the topochemistry environment of the suffered polymer film of protein can not make protein launch or distortion, constitutes the natural function of protein by this way.The polymer that satisfies these conditions includes but not limited to triblock copolymer, and they have the general aspects of hydrophilic outer block and hydrophobic interior block.BR 62 and COX 72 be directed and knot in polymer film 66, and film is to cover with

electrode

78 and 80.

Figure 2 shows that structure, implementation and the evaluation of optical drive ATP production system, the nanochemistry device that this system can drive for the F1-ATP enzyme continuously provides energy.As shown in Figure 5, the present invention relates to comprise the structure of the liposome vesicle 66 of BR and ATP enzyme, the ATP enzyme is directed by this way, promptly is used to produce ATP from ADP continuously from the energy of green glow.Set up a cover system, be used for large-scale production and purification of bacterial rhodopsin (BR), from the salt bacillus of overfulfiling a production target, separated, with gel filtration chromatography purifying (curve 90 among Fig. 6).

According to the present invention, with lecithin, phosphatidic acid and the cholesterol of purifying, according to preceding method (Pitard etc., 1996) Structured Lipids body.Liposome is selected size with 0.45 and 0.2 μ m filter successively, makes the liposome that stays in the solution less than 200nm.F ₀F ₁Carry out under the existence that is incorporated in Triton X-100 of-ATP enzyme and BR.For guaranteeing the formation of liposome, add Pyranine as the pH sensitive indicators, this indicator utilizes fluorescence microscope by visual appraisal.This work shows, greatly can be 20 ℃ of acquisition to 1.5 pH gradient, and shown in curve among Fig. 7 92 and 94, contain bacterium rhodopsin (curve 92) and do not contain the pH gradient that the liposome of bacterium rhodopsin (curve 94) formed in 30 minutes.

Analytical proof produces ATP with liposome, shown in label among Fig. 8 96.This figure has shown the fluorescein fluorescence that takes place owing to the ATP that exists liposome to produce, and liposome comprises the BR and the F of different amounts ₀F ₁-ATP enzyme.ADP is added before the solution, and liposome was cultivated 2.5 hours under illumination.Target is to optimize electrochemical gradient, makes the generation speed of stable state ATP be increased to foregoing level (Pitard etc., 1996).

---" nano injection device "---is expelled to above-mentioned nanoscale compound molecule device in the living cells with the hollow cylinder array that has the atom level pinnacle.The first step of this process is the non-hollow pattern that makes up the nano injection device.Fig. 9 is the part micrograph of silicon tip array 100, and it comprises that diameter is less than the point 102 of 10nm and the axle 104 of the about 1 μ m of diameter.As electrode, use the array of electrochemical method depositing nano level nickel point, with these arrays 100 as the carrier of various molecular devices.These arrays also allow directly be covered with the film top deposition micron order or the nanoscale electrode of protein.

The production of BR and synthetic be conventional.The salt bacillus after fermentation process and the purifying, obtains the above purple membrane of 100mg with the 50L batch fermentation.This and about 60m ²The monolayer of protein area be consistent, it is enough to deal with antetype device.

Can adopt Zhen, Y., Qian, J., Follmann, K., Hayward, T., Nilsson, T., Dahn, M., Hilmi, Y., Hamer, A., Hosler, J., Ferguson-Miller, S. (1998), Prot.Expr.﹠amp; Pur.13, the described method of 326-336 is produced COX (Zhen etc., 1998).The method relates to by the red bacterium of class ball (Rhodobacter sphaeroides) overexpression and purifying COX, shown in label among Figure 10 90.The process that makes up overexpression plasmid pRK-pYJ123H comprises by the red bacterium of class ball goes into PUC19 with subunit I gene (coxI) subclone of cytochrome c oxidase, utilizes the SmaI site among the pJS2-X6H2, produces pJS3-SH, as shown in the figure.The 61 histidine sequences of mark His-tag are positioned at the C-end of coxI, produce pYJ124H by connecting into from the PstI/PstI fragment of pYJ100 in the unique PstI site on the pJS3-SH.Then, with EcoRI and HindIII site three subunit genes are put into expression vector Prk415-1.This process produces the 61mg/10L nutrient solution, and for big individual layer area, this is a big amount.

Generation and synthetic BR are conventional with the liposome vesicle that mixes BR.Before uniform bubble added electric field insertion lipid, the orientation of BR is that the easiest promotion is by having the film of plane symmetry, though it is the problem of spherical vesicle aspect.Label 120 apparatus shown are the electrochemical cells that are used to measure impedance spectra among Figure 11, and design to be used for measuring by proton and transport electron transport and the electromotive force that causes, and are used for planar film.In addition, be suitable on film/protein complex, applying electric field easily.This pond comprises stopper 121, the saturated KCl reference electrode 122 of Ag:AgCl, and liquid outlet 123, Teflon sept 124, golf support 125, liquid-inlet 126 and platinum counterelectrode 127 are as described in Naumann.

About the orientation of purple membrane and BR, many technology have been reported in the document: electrostatic layer-layer assembling; The Langmuir-Blodgett film that electric field strengthens forms; The orientation of BR in the electric field of aerial condenser; On suspended substance, directly apply electric field; Combination with electric field and magnetic field.Most technology are utilized the big natural permanent electric dipole moment of PM, and (diameter is 1 μ m particle～10 ⁶The debye).This big electric dipole moment is derived from the additivity (calculate to be estimated as 570 debye and experiment is 55 debye from theory) of single BR dipole moment.Only need to costal fold directly apply～electric field of 20V/cm just is enough to make the BR orientation, this electric field obtains easily.

It is enough big that the dipole moment of oriented COX is wanted, to strengthen the stop of cytochrome c.COX utilizes its internal dipole to pass film attraction and pushes film open, just as BR.Because the cytosome of COX partly is hydrophilic, enzyme has barrier one to rotation in the lipid.Oval protein also stops this motion.Therefore, be necessary before COX mixes in the lipid film, just to make its orientation.COX adds PM after mixing lipid film, and the directed significantly lower electric field that needs can not disturb the arrangement of COX like this.If the arrangement of COX needs other parts of voltage and experiment incompatible, as the hydrolysis of lipid film, BR or water, counterelectrode moves nearer, and the voltage that applies is remained on level of security, guarantees both to produce required electric field, again the remainder of this process of not disturbance.

Be used for making in each comfortable lipid film the equipment of PM and COX orientation also to can be used to measure transporting and electronics transporting of proton among the BR above by COX.

Only be to use the structure of above-mentioned Fig. 5 to carry out with the preliminary experiment of mixing the directed BR in the lipid film.The signal that film is easily detected is passed in the proton pumping.Repeat these experiments with COX.

In equipment shown in Figure 5,

electrode

78 and 80 effect provide electronics and receive electronics, as non-existent cytochrome c and O in the device ₂Substitute.Because electric current can make the mensuration complexity of proton flow by COX 72 pumping protons, thereby adopted pH sensitive fluorescence indicator used in the experiment that is similar to front liposome BR.The ability that electrode and COX link is measured by protein is placed in the lipid film separately.With the proton pump of above-mentioned (Naumann etc., 1999) the identical method monitoring BR condition of giving a present, studied the ability that shifts from connect electrode COX pumping proton of utilizing.With with need not apply the experiment that directed electric field carries out and show that orientation is successful.A main result by the pumping of electric field leading proton is that the ATP enzyme can mix in the lipid film with COX.Activation COX produces proton gradient, and the ATP enzyme can be used to prepare ATP then.This explanation can be synthesized ATP with method for electrically, and this is a main milestone biologically, and is significant for the nano-device that further exploitation ATP drives.

Be the inverting function of tested enzyme, produced an artificial pH gradient, even the acidity of film space one side is greater than opposite side.By after measuring proton transport and determining the COX orientation, measure the voltage that produces because of the proton reverse flow and the electric current by COX.If the structure of electrode as shown in figure 11, then electronics flows and does not take place.In this case, can make the top surface of the more close film of top electrode.

Can adopt many methods to improve the degree of approach of electrode and protein.Electrode grid can be placed directly in the lipid top, its form is a filamentary webs, can connect outward and carry out electrical measurement.After removing liquid from top surface, can be directly on film the thin hyaline layer of aluminium-plated or nickel, form counterelectrode.Perhaps, by grating tip array shown in Figure 9 electrode is deposited on the lipid surface with electrochemical method.This deposition can form nano wire up to a million on the top surface of film.Repeat and make up above step, cause directed COX and BR gadolinium to be contained in the lipid film.

The orientation of BR and COX has two kinds of possible schemes: parallel and antiparallel dipole orientation.If dipole is parallel, applies an electric field and just obtain both arrangements simultaneously.If they are antiparallel, will gather dipole moment with big PM.Make the COX initial orientation in high electric field, then make the PM orientation in an electric field, this electric field is enough little, and big as enough to operate fully to avoid interference COX, the PM fragment has so just obtained the orientation that is fit to.

At first, measure voltage and should show that BR suitably works, and formed proton gradient.Coming since then, the transmembrane voltage of gradient is expected to be hundreds of mV.Mensuration with after-current shows success, and can estimate the COX protein that can work near part by the electrode in the film.

Because proton moves by COX counter-rotating, so electric current depends on the proton concentration that BR provides, and electric current should be directly proportional with luminous intensity.Total current also is directly proportional with the COX molecular number that works, because the parallel configuration of COX molecule in the lipid film.As mentioned above, the voltage that is produced should be constant,＞200mV, and the same power that produces is determined by luminous intensity with the net amount COX of suitable direction orientation.

Make the maximized method of power concentrate on the orientation of optimizing COX and suitably select and improve rete.This device can be under various illuminate conditions transmitted power, Continuous irradiation under high strength and low-intensity for example, periodically irradiation etc.

Should adopt polymer film, the reasons are as follows: they have the longer life-span than lipid film; They are firmer; They have the character of easier preparation, as electronics and ionic conductivity and permeability.The inside of these films must be hydrophobic and resilient, so that can simulate as far as possible near natural protein environment.

Many biocompatible membranes have character widely, as light absorptive, polarity, electronics and ionic conductivity, or the like.The polymer that can improve solar cell character of the present invention must be compatible with protein and electrode.To proton not by also being important.The doping ability of polymer surfaces is also very important, because it is played a leading role with striding in the film conductibility at proton conductive.The factor when being selective polymer also is correlated with to the influence in the life-span of contained protein wherein in the life-span of polymer and it.The selection life-span is short but the high polymer of performance can be used for special applications.

Method efficient with the biological components preparation, the high yield sun-generated electric power that the front has been introduced shows that power conversion bioprotein and external devices integrate; And having pointed out to help can the large-scale production biologic solar cell, can drive the method for the manufacturing approach of various devices.

On the other hand, mix the triblock copolymer film, produced the stabilising membrane that only allows water to pass through, therefore promote water purification, desalination and carry out molecule to concentrate by dialysis by using aquaporin family protein.Aquaporin cuts off passing through of any pollutant, comprises bacterium, virus, mineral, protein, DNA, salt, washing agent, dissolved gas, even from the proton of the aqueous solution, but the aquaporin molecule can transport water because of its structure.As shown in figure 12, each aquaporin 130 comprises 6 and strides film alpha helical region territory 132-137, and they are anchored on protein in the film and the NPA of two high conservatives ring 138 and 140, they flock together to the limit at the protein culminating point, form a kind of hourglass shape.The stenosis of this hourglass is that hydrone is with the place of delegation by film, shown in label among Figure 13 142.Show that the mobile of water is symmetrical, and can in both direction, carry out; This fact is important, because this process consumed energy not.Water is because the cause of hydraulic pressure or osmotic pressure by film with specific direction.As shown in figure 14, water purification and desalination can realize that this device has family 152 and 154 with two chambers device 150, and central authorities are separated by rigid film 156, have filled up aquaporin in the film.This film itself does not seep water, and it separates the pure water 160 in sewage in the chamber 152 158 and the chamber 154.Have only pure water between two chambers, to flow.Therefore, and the seawater on film one side or other sewage 158 are placed suitable following time of pressure, pure water just flows to another chamber 154 naturally.Therefore, pure water can obtain from the undrinkable water source, perhaps, if contain interested chemical substance in the water source, then can selectively remove and anhydrate, and required high concentration chemical substance is stayed in the input chamber.But importantly, aquaporin also is applicable to the present invention, and its reason is not that it has single-minded selectivity to water.Many members of this protein families (as aquaporin Z (AqpZ)) are highly stable, can stand the mal-condition in the source of sewage, and not loss of function.AqpZ can sex change or decomposition in the presence of acid, voltage, washing agent and heat yet.Therefore, this device can be used for purifying the water source that is polluted by material, and other film may be made or destroy to these materials dirty, and it can also be used to continue to have the area of high temperature.

AqpZ also is variable.Because this protein is expressed according to gene order in host bacteria single-mindedly, this gene order influences its net shape and function, so the technical staff changes its gene-code easily, so that change the characteristic of protein.Therefore, can process protein, to satisfy required application, this application may be different from the original function of protein.For example, will change cysteine near the particular amino acid residue at aquaporin 142 centers among Figure 13 simply, the aquaporin that is produced is any free mercury in the binding soln perhaps, and owing to block and stop water delivery.Therefore, these used muteins can detect the mercury pollution in the water sample in the membrane module, and when the concentration rising of Toxic matter was too high, water stopped to flow simply.

Preferred implementation of the present invention is the form of traditional filter disc, because its easiest measurement function.For preparing this dish, available Langmuir-Blodgett groove synthetic triblock copolymer and the protein of individual layer that deposition 5nm is thick on the surface of the commercial ultrafiltration dish of 25mm.Penetrate individual layer on the dish with 254nm UV illumination then, make crosslinked polymer, increase its tolerance.At last, be that the pvdf membrane of 220nm adheres on the panel surface with epoxides with the aperture, carry and prevent edge's seepage to guarantee safety.

Test is during device, it is fixed on indoor as shown in figure 14, forces pressurized water source to pass film.When the opposite side that has only pure water by film with when polluting solute and still concentrating in the chamber of beginning, think that this device has function.Must be to the solution pressurization of polluting, to overcome in the chamber that pure water flows to a large amount of dissolved particles naturally.The purpose of aquaporin Z film is that counter-infiltration takes place, and pure water is separated from pollute solute.This trend of system, perhaps osmotic pressure can be used pound/square inch (psi) expression.For example, the osmotic pressure of seawater is about 360psi.

There is Several Methods to can be used to make device to bear the pressure of these types.Some polymer innately more tolerate than other polymer, and available UV photo-crosslinking, so that super rigidity to be provided.Another kind method is nontoxic, the solute easily removed that adds high concentration in freshwater room, passes film with short normal infiltration, simultaneously because reverse osmosis also takes place in the chamber supercharging.At last, the method that reduces the required pressure of reverse osmosis can be to use a plurality of AqpZ devices in containing a string sealing that pollutant levels reduce successively, continuous chamber.Gained pressure is to purify every pair of pressure that the chamber is required, and it is the part of the required gross pressure of counter-infiltration.Therefore, little pressure only need be stood in each chamber, and the possibility that is kept perfectly is very big.Therefore, if the concentration difference between the every pair of chamber is 10%, rather than 100%, only need 10% above-mentioned high pressure can purify the water source of each junction.Can constant compression force in the last chamber and flow velocity produce pure water continuously.

The aquaporin reverse osmosis membrane only needs a step to purify to have the water of several dissimilar pollutants.Traditional high-purity system needs several components shown in label among Figure 15 170, comprises that water softener 172, carbon filter, ion-exchanger, UV or chemical sterilization 174 and dual channel reverse permeation filtration apparatus 176 can unite use before producing water (promptly do not have aquaporin purified water clear like that).This complicated apparatus can not be removed dissolved gas or less than 150 daltonian materials from the water source as the aquaporin film.In addition, all these assemblies all need to safeguard.The UV bulb need be changed and energy.After being full of leaf, ion-exchanger needs chemical regeneration.Softening agent needs salt.Essential replacing when carbon and reverse osmosis cartridge are stopped up.At last, system compares with typical decontamination, and single step device requisite space is much smaller, and light, and this advantage is easy to carry aquaporin water purification device of the present invention.

The aquaporin film is also fast than conventional system.Routine high speed counter-infiltration (R.O.) unit per minute can produce about 28.4 liters (7.5 gallons) water purification.Current studies show that, hydrone is by AqpZ saturated fat plasma membrane (0.0177mm ²) speed be 54 μ mol/s (Pohl, P., Saparov, S.M., Borgnia, M.J., and Agre.P. (2001) Proceedings of the National Academy of Science 98,9624-9629 pages or leaves).Therefore, surface area is 295 liters of pure water of aquaporin Z reverse osmosis membrane per minute energy filter 23 of 1.0 square metres.Fast 116 times of the conventional water purifier of this speed ratio.

At last, it is also very cheap to produce the new film based on protein.From the colibacillus engineering strain, obtain core---the AqpZ of this process of milligram quantities easily.From every liter of nutrient solution, on average can obtain the 2.5mg true protein.From about 5 dollars growth medium, can produce 10mg protein.This is enough protein for several full scale devices.The polymer of embedding AqpZ can prepare in same laboratory, and the used chemical reagent of each device only is worth several cents.Aquaporin Z reverse osmosis membrane is novel, effective, cheap water cleaning of tool.

Therefore, disclose with biological components method and apparatus with the complete pure water of high efficiency production from the water of addle, salt solution or other pollutions.The present invention illustrated that integration water transports bioprotein and external devices, and the manufacturing approach of the device of having pointed out can large-scale production water to purify waste water.

Though in conjunction with preferred implementation the present invention is described above, should be appreciated that and can carry out various changes and modification described method and device, only otherwise deviate from the spirit and scope of the present invention, as described in following claims.

Claims

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;

Wherein, some protein can be in conjunction with the material that is not water.

2. the described film of claim 1 is characterized in that the function of protein comprises valve, passage, sensor, detector, pump and transducer.

3. the described film of claim 1 is characterized in that the described memebrane protein of selecting only transports hydrone, and described bionical film is the water filter that filters aqueous solution, and described material is not a water.

4. the described film of claim 3 is characterized in that when the concentration of material that is not water is too high, describedly can stop transport of water owing to stopping up in conjunction with the protein of dissociant.

5. the described film of claim 3 is characterized in that described can be engineered protein in conjunction with the protein that is not the dissociant of water.

6. the described film of claim 5 is characterized in that described engineered protein makes a variation and becomes to have function in conjunction with specific dissociant.

7. the described film of claim 6 is characterized in that described memebrane protein comprises the protein that is selected from aquaporin family.

8. the described film of claim 7 is characterized in that described matrix does not seep water, and the described memebrane protein of selection allows hydrone to pass through under pressure.

9. the described film of claim 8 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.

10. the described film of claim 9 is characterized in that the solution that will filter is exerted pressure, and thus, described filtration is counter-infiltration.

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