Active phospholipid membranes and related methods of production

Abstract

An activated phospholipid membrane (200) comprising: a double phospholipid layer; at least one support (201) for supporting the double phospholipid layer and thereby improving the resistance of the activated phospholipid membrane (200); a plurality of monoclonal antibodies (202) bound to the support (201); a plurality of predetermined molecules (203) bound to the monoclonal antibodies (202) at the transmembrane level. The at least one support (201) comprises a first substrate containing the monoclonal antibodies (202) and a second substrate containing the double phospholipid layer.

Description

[Technical field]

[0001] The present invention relates to active phospholipid membranes.

[0002] In addition, the present invention relates to a method for producing active phospholipid membranes.

[0003] In particular, the present invention relates to a type of membrane having a bilayer of activated phospholipid membranes, activated by the insertion of specific transmembrane molecules, and related methods of production. [Background technology]

[0004] As is known, activated membranes are currently used in many technical fields. Some of the main fields of application are, for example, the energy sector, or the biomedical sector, where semipermeable membranes are generated that are activated by specific molecules.

[0005] In the technical field of accumulators, chemical accumulators have been known for some time, such as lithium-ion batteries, which have a high density of charge and do not suffer from memory effects, or silver-zinc batteries, which have a higher energy density but have excessive production costs. Microbial generators that use cell cultures to generate electricity have been tested in recent years.

[0006] The technology of adenosine triphosphate-dependent generators / accumulators is based on the idea of ​​using the potential difference extracted from the molecular activity of cell membrane proteins. To develop an adenosine triphosphate-dependent generator / accumulator, it is necessary to build a series of substructures or cells, enclosed in a double phospholipid membrane or a substance of the same effect, that allows the localization of the cells and the development of said molecular activity.

[0007] An example of an electrochemical cell utilizing the molecular activity of a particular cell culture is described in US Patent Application Publication No. 2010 / 178592, which relates to a device comprising an outer shell and an artificial biomimetic membrane arranged inside the outer shell to form two separate chambers, each chamber enclosing a liquid of a particular composition, the biomimetic artificial membrane comprising a semipermeable membrane for supporting the lipid membrane, a plurality of lipid molecules arranged in a layer, and at least one transport protein, suitable for transporting ions and / or liquid molecules between the two chambers.

[0008] Another known membrane is described in US Patent Publication No. 2007116610. In particular, a biofunctional synthetic composition membrane is described that includes phospholipids, proteins, and a porous substrate or membrane. Lipid bilayers are formed on porous polycarbonate membranes, polyethylene terephthalate acid, and polylactic acid (PLLA), as well as in holes drilled with a laser in plates made of plastic materials.

[0009] Among the currently known membrane production methods, the following are most used: - fusion of vesicles; -Combination of Langmuir-Blodgett technique with vesicle fusion technique.

[0010] In the case of membranes equipped with a substrate acting as a support material, some known supports are: -Quartz glass -Borosilicate glass -Not at all -Silicon oxide -Thin film TiO2 -Indium Tin Oxide -gold -silver -Platinum.

[0011] Methods for producing active films, such as dip pen nanolithography or DPN, are also known.

[0012] A known solution reported in GB 2477158 describes a photovoltaic device with a charging station comprising first and second passages for passing positive and negative electrolytes, a membrane separating the electrolytes in the passages, and a discharge station including first and second passages in communication with the respective passages of the charging station. The discharge station comprises electrodes adapted to connect to an external load, first and second reservoirs connected to the discharge station passages for storing the electrolytes, and one or more pumps for pumping the electrolyte between the reservoirs and the charging and discharging stations. The charging station is arranged to be illuminated by incident solar radiation, and the membrane comprises photosynthetic biological material attached to a support structure.

[0013] Another solution reported in US 2003 / 100019 describes new artificial supported membranes and methods for the preparation of such membranes or for the reconstruction of cell membranes from their constituent proteins and lipids. Such membranes comprise a lipid bilayer attached to a support by phospholipid chains (P) that create a space between the bilayer and the support, and one or more protein-specific ligands (L) that are covalently bound to the support and exposed in said space. The invention also describes the use of the membranes to allow the purification and / or reversible capture of membrane proteins, or even to block components that interact with some of these proteins. The membranes according to the invention can further be useful for assessing toxicity or, conversely, for evaluating the therapeutic effect of test compounds. The invention is particularly useful for analyzing protein-protein interactions within the membrane and can therefore be used in the pharmaceutical industry to analyze the toxicity or beneficial properties of molecules that are candidates for drug development and / or for inclusion in drug compositions. In the field of biotechnology or medicine, such supported membranes can also be used for the production of more efficient biomaterials.

[0014] Another solution described in US Patent Application Publication No. 2011 / 136022 describes a fuel cell and a method for its production, in which one or more types of enzymes, or another coenzyme, are enclosed in a microspace, whereby electrons are efficiently extracted from a fuel, such as glucose, by an enzymatic reaction using the microspace as a reaction field to generate electrical energy, where the enzyme or another coenzyme can be easily immobilized on an electrode. Enzymes 13 and 14, as well as a coenzyme 15 required for the enzymatic reaction, are enclosed in a liposome 12. The liposome 12 is immobilized on the surface of an electrode made of porous carbon or the like to form an enzyme-immobilized electrode. An antibiotic 16 is bound to the bimolecular lipid membrane that constitutes the liposome 12, forming one or more pores 17 that are permeable to glucose. The enzyme-immobilized electrode is used, for example, as a negative electrode of a biofuel cell.

[0015] Finally, the article "Part 3 Systematic Bioelectronic strategies" by Ashutosh Tiwari et al. describes several assembly techniques for bilayer membranes (BLMs), specifically constrained bilayer membranes (tBLMs) with a protein tBLM(ii) containing a protein bound to a support and a lipid bilayer. D4 also discloses how to form a bilayer membrane on an agarose gel and sandwich this membrane between two supports. The mechanical stability of the support and the gel cushion is disclosed.

[0016] However, although useful for the synthesis of active membranes, these methods have significant limitations, including the cost of materials and the complexity of the production procedures.

[0017] Furthermore, one of the problems with known production techniques is that it is difficult to ensure maximum density of active molecules on the phospholipid surface.

[0018] Furthermore, currently known active membranes and associated production methods are unable to predict and determine the selectivity or density of molecules associated with the active membrane. Indeed, currently known active membranes and associated production methods are unable to determine the presence or absence of a particular transmembrane molecule, or the representativeness of a particular area in terms of density per square unit for a particular molecule. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] US Patent Application Publication No. 2010 / 178592 [Patent Document 2] US Patent Publication No. 2007116610 [Patent Document 3] GB 2477158 [Patent Document 4] US Patent Application Publication No. 2003 / 100019 [Patent Document 5] US Patent Application Publication No. 2011 / 136022 [Non-patent literature]

[0020] [Non-Patent Document 1] "Part 3 Systematic Bioelectronic strategies" by Ashutosh Tiwari et al. Summary of the Invention [Problem to be solved by the invention]

[0021] The object of the present invention is to provide active membranes of phospholipids and related production methods that guarantee a specific density per unit area in the transmembrane.

[0022] Another goal of the present invention is to provide a method for producing bilayer active phospholipid membranes, characterized in that it is technically easy, effective and efficient, thereby overcoming the limitations that also affect current methods for producing active membranes as referred to in the prior art. [Means for solving the problem]

[0023] According to the present invention there is provided an active phospholipid membrane as defined in claim 1.

[0024] According to the present invention there is provided a method for producing an active phospholipid membrane, as defined in claim 4.

[0025] In order that the invention may be better understood, preferred embodiments will now be described, purely by way of non-limiting example, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 illustrates the organization of active phospholipid membranes according to the present invention. [Diagram 2] FIG. 2 shows an alternative organization of an active phospholipid membrane according to the present invention. [Diagram 3] FIG. 1 illustrates a method for producing an active phospholipid membrane according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] With reference to these drawings, and in particular to FIG. 1, there is shown an activated phospholipid membrane in accordance with the present invention.

[0028] In the following, an activated membrane is meant a membrane that is activated, for example by biomolecules, which are capable of generating electricity via alternating polarization and depolarization.

[0029] In particular, the activated phospholipid membrane 200 according to the present invention comprises: - with a double phospholipid layer; - at least one support 201 or substrate that improves the resistance of the active membrane and supports the double phospholipid layer; a plurality of monoclonal antibodies 202 bound to the support 201 and selected according to the molecule that needs to be inserted into the membrane 200; a predetermined molecule 203 bound to a monoclonal antibody; Equipped with.

[0030] According to an embodiment of the present invention, the activated phospholipid membrane 200 is inserted into a support matrix, preferably constructed with a gelling agent such as agar. The activated phospholipid membrane 200 is immersed in a liquid that contains, in this case, agar, which provides mechanical support to the structure of the membrane itself at the end of the gelling process.

[0031] Thus, advantageously, the active phospholipid membrane is stabilized and easily transportable.

[0032] According to this embodiment of the invention, the support to which the antibody is attached may preferably be made of polyvinyl chloride, cellulose nitrate, or polycarbonate.

[0033] The activated phospholipid membrane 200 comprises a plurality of supports 201 or substrates, preferably a first substrate and a second substrate. The following steps are performed: - immobilizing the monoclonal antibody on a first substrate; - creating a link between the molecule to be inserted at the transmembrane level and a monoclonal antibody immobilized on a first substrate; -Phospholipids on the second substrate Calm the step of: - The first substrate to which the monoclonal antibody is bound and the molecule that inserts at the transmembrane level are anchored onto a second substrate, forming a bilayer or phospholipid layer in which a series of transmembrane molecules are bound in turn to the monoclonal antibody. This structure results in the permeable support being present at two outer levels.

[0034] As shown in FIG. 3, a method 100 for producing an activated phospholipid membrane comprises the following steps: - providing a double phospholipid layer; - providing at least one support for supporting the bilayer of phospholipids; - selecting 101 a monoclonal antibody specific for a molecule that inserts into the phospholipid bilayer; - a step 102 of attaching the monoclonal antibody selected in the previous step to a support or substrate; - a step 103 of promoting binding between the monoclonal antibodies immobilized on the support using a predetermined molecule that has a specific affinity for the monoclonal antibodies; a step 104 of inserting into the system obtained in the previous step and constituted by the monoclonal antibody and the antigenic substrate a quantity of a polar liquid which allows the phospholipids to assemble in a bilayer containing the molecules bound by the antibody in a subsequent step 105; - a step 105 of adding phospholipids which, thanks to the presence of the polar liquid inserted in step 104, collect in the membrane at the level of the molecules bound by the antibodies.

[0035] The monoclonal antibody is selected such that it binds to the molecule but does not functionally interfere with the activity of the molecule.

[0036] According to an embodiment of the invention, the support, or substrate, to which the monoclonal antibodies are immobilized in step 102 is constructed of a layer of polyvinyl chloride or cellulose nitrate.

[0037] According to an embodiment of the invention, in step 105 a double phospholipid layer is formed on top of the polar liquid, where its level is precisely predetermined and the height of the molecules immobilized by the monoclonal antibody is then included at the transmembrane level.

[0038] Advantageously, the method of producing activated phospholipid membranes according to the invention makes it possible to obtain membranes that are activated by including molecules that perform a desired function, and that are easily manipulable thanks to the mechanical substrate support.

[0039] According to an embodiment of the invention, step 101 is preceded by a selection step and a step of synthesis, by recombinant DNA techniques, of the molecule to be inserted at the transmembrane level.

[0040] The monoclonal antibodies selected in step 101 will bind the molecule that needs to be inserted at the transmembrane level, but advantageously they do not affect the function of the same molecule, so the link between the monoclonal antibody and the molecule must not be made at the level of the active site of the molecule or at the level of a moiety that may alter its functionality.

[0041] According to an aspect of the invention, at the end of the synthesis of the active membrane by the steps mentioned, it is possible to maintain or to destroy the link between the antibody and the last subunit inserted at the transmembrane level.

[0042] Industrial applications of the active phospholipid membranes and associated production methods according to the present invention are, for example, in generators and energy use in vehicles and everyday electrical systems, or in biomedical applications such as filter systems used in the field of dialysis, pacemaker devices, aortic counter-pusters, etc.

[0043] Another industrial application of the phospholipid membranes according to the invention is the extraction of adenosine triphosphate from organic waste.

[0044] The active phospholipid membrane according to the invention makes it possible to obtain a maximum density of active molecules and their precise orientation per unit in the phospholipid surface.

[0045] The activated phospholipid membrane according to the invention is, thanks to its activation by using specific molecules: -Based on voltage-sensitive sodium channels; -Based on voltage-sensitive potassium channels; -Based on the adenosine diphosphate translocase channel; -Sodium-potassium / pump based; -Based on the funny channel For example, this allows for use in generating electricity in the system.

[0046] In addition to the molecules listed above, the present invention is applicable to additional specific molecules for preferred industrial applications.

[0047] Advantageously, the production method according to the invention makes it possible to obtain, in an efficient and practical manner, active phospholipid membranes that are easily manageable and mechanically resistant.

[0048] More advantageously, the production method according to the invention makes it possible to obtain active membranes with maximum density per unit of phospholipid surface.

[0049] Furthermore, the production method according to the invention is advantageously versatile.

[0050] The production method according to the invention is therefore simple and easy to use.

[0051] Finally, it will be apparent that the active phospholipid membranes and related production methods described and illustrated herein can be subject to modification and variation without abandoning the scope of the invention as defined in the appended claims.

Claims

1. An activated phospholipid membrane (200) comprising: A double phospholipid layer, At least first and second substrates for supporting the double phospholipid layer and thereby improving the resistance of the active phospholipid membrane (200); a plurality of monoclonal antibodies (202) bound to the first substrate; a plurality of pre-determined active phospholipid transmembrane molecules (203) bound to said monoclonal antibody (202); A plurality of the predetermined molecules (203) are linked to the monoclonal antibody, and the plurality of the predetermined molecules (203) are anchored to the second substrate, creating a double phospholipid layer with a series of predetermined activated phospholipid membrane-spanning molecules (203) which are sequentially linked to monoclonal antibodies, the substrate being permeable to glucose, and an activated phospholipid membrane (200) present at two outer levels in the activated phospholipid membrane.

2. The active phospholipid membrane (200) of claim 1, characterized in that the substrate is made of cellulose nitrate or polycarbonate and the active phospholipid membrane (200) is inserted into a support matrix containing a gelling agent.

3. 2. The active phospholipid membrane (200) of claim 1, characterized in that the substrate is made of cellulose nitrate or polycarbonate.

4. A method (100) for producing an active phospholipid membrane according to any one of claims 1 to 3, comprising: providing phospholipids to form a bilayer of phospholipids; providing at least first and second substrates onto which said double phospholipid layer can be deposited; selecting and synthesizing, using recombinant DNA techniques, a predetermined transmembrane molecule to be inserted into the bilayer phospholipid; Selecting (101) a plurality of predetermined monoclonal antibodies for the predetermined molecule that will be inserted into the bilayer of phospholipids deposited on the second substrate; binding (102) the selected monoclonal antibody to the first substrate; (103) promoting binding between the monoclonal antibodies immobilized on the substrate using a predetermined molecule that has a specific affinity for the monoclonal antibodies; In a subsequent step (105), a predetermined amount of a polar liquid is inserted (104) that allows phospholipids to assemble into a bilayer containing the predetermined transmembrane molecule bound by the antibody; adding (105) the phospholipids collected by the polar liquid introduced in step (104) to the predetermined membrane bound by the antibody; and immersing the activated phospholipid membrane (200) in a liquid containing gellable agar to provide mechanical support to the structure of the activated phospholipid membrane (200).

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