"Biochemical electrical generator with electronic transfer mediator" * * *

The present invention relates to a biochemical electrical generator with an electronic transfer mediator.

As is known, there are microbiological fuel cells (MFCs) or biological fuel cells which are bio electrochemical systems that generate current by mimicking bacterial interactions found in nature. Micro-organisms catabolize compounds such as glucose, acetate, butyrate or wastewater . The electrons obtained with this oxidation are transferred to an anode, from which they pass through an electrical circuit before reaching the cathode. From here they are transferred to a high potential electron acceptor such as oxygen. As the current flows due to the potential difference, the power is generated directly from the biofuel via the catalytic activity of the bacteria.

A microbiological fuel cell converts chemical energy into electrical energy by the catalytic reaction of microorganisms. A typical microbiological stack consists of two compartments, one enclosing the anode and the other the cathode, joined by a semipermeable membrane that only allows the passage of cations.

In the oxygen-free anode compartment, the fuel is oxidized by microorganisms, releasing electrons and cations.

The cations travel to the cathode through the membrane, while the electrons reach it through an electric circuit outside the cell; electrons and cations are then recombined with oxygen in the cathode compartment, forming water.

Bacteria in biological fuel cells feed on glucose and methanol obtained from food waste and convert them to hydrogen (waste water and even pure urine can also be used in this regard).

In general, there are two types of microbiological fuel cells, without electron transfer mediator which use electrochemically active bacteria to transfer electrons to the electrode and with electron transfer mediator. With particular reference to microbiological fuel cells with electron transfer mediator, it is known that most microbiological cells are electrochemically inactive.

Therefore, the transfer of electrons from such cells to the anode electrode is facilitated by mediators such as thionine, paraquat, methylene blue, humic acid, toluylene red and other substances, most of which are, however, expensive and toxic.

Furthermore, when microorganisms consume a substrate such as sugar under aerobic conditions, they produce carbon dioxide and water. However, when oxygen is not present, they produce carbon dioxide, protons and electrons.

Microbiological cells use inorganic mediators to enter the electron transport chain between cells and to "steal" those that are produced. The mediator crosses the external lipid membranes and the plasma wall; it then begins to free the electrons from their transport chain which would normally be occupied by oxygen or other intermediaries . The mediator, now reduced, leaves the cell charged with electrons which are carried to an electrode where they are deposited; this electrode becomes the electro-generator anode (the negative electrode).

The release of the electrons brings the mediator back to its previous oxidized state, ready to repeat the process; it is important to note that the process can only take place under anaerobic conditions since, if oxygen were present, it would collect all the electrons due to its greater electronegativity than the mediator. This is the principle for generating a flow of electrons from most microorganisms. To turn this flow into a usable electrical generator, the process must be housed in a fuel cell. Furthermore, it is necessary to create a complete electrical circuit, it is not enough to bring the electrons to a single point.

In the second compartment of the battery there is another solution and another electrode. This electrode, called the cathode, is positively charged and is equivalent to the oxygen well at the end of the electron transport chain, only it is external to the biological cell.

The solution is an oxidizing agent that collects electrons at the cathode. To connect the two electrodes there is a wire (or any other conductive path that could include some electrical instrument, such as a light bulb), and to complete the circuit by connecting the two compartments there is a salt bridge or a membrane that allows the exchange of ions. This last characteristic allows the protons produced, as described in the equation above, to pass from the anode compartment to that of the cathode.

The reduced mediator carries the electrons from the cells to the electrode; hence the mediator oxidizes as it deposits electrons. These then flow through the wire to the second electrode, which functions as a ground connection; from here they then pass into an oxidizing material.

A known solution is reported in patent application US2011136022A1, also published as EP2442394A1, which describes a fuel cell and a related production method in which one or more types of enzymes or further coenzymes are enclosed in a micro space so that electrons can be extracted efficiently from a fuel such as glucose, or the like, by an enzymatic reaction using the micro space as a reaction field, thus producing electrical energy, and in which the enzyme, or further the coenzyme, can be easily immobilized on an electrode.

Two enzymes and a coenzyme required for an enzymatic reaction are encased in a liposome which is immobilized on a surface of a porous carbon composite electrode or the like to form an enzyme-immobilized electrode. An antibiotic is bound to a bimolecular lipid membrane that makes up the liposome to form one or more glucose-permeable pores.

The enzyme immobilized electrode is used, for example, as a negative electrode of a biofuel cell. Specifically, the solution features a complex poly-ion method in which a positively charged polymer and a negatively charged polymer are mixed with an enzyme in an appropriate ratio and applied to a porous carbon compound electrode to stabilize an immobilization membrane while maintaining the adhesion to the electrode.

However, the method of immobilization described above using a poly-ion complex largely depends on the physicochemical properties of an enzyme, in particular on the electrical charge, and therefore suffers from the problem that a state of immobilization changes with changes in an external solution or in the operating environment, thus easily causing the avoidance of the immobilized enzyme and the like.

When an enzyme enclosed in the liposome is considered a biocatalyst, the reaction rate is low because the permeation rate of a substrate to a bimolecular lipid membrane (lipid bilayer) that makes up the liposome is limited. This problem is solved by forming a glucose permeable pore in the bimolecular lipid membrane that constitutes the liposome.

This cell and method of energy production, however, suffer from the limitation of having to manage the synthesis of micelles, a process that is difficult to insert within industrial production lines as this is a process whose product is not completely predictable in quantity and in the quality of the same (micelles) synthesized. Furthermore, by using micelles, it is not possible to finely establish the quantity of enzymes, coenzymes and reagents that will be present. The impossibility of establishing how many components of a reaction are present in a micelle (and therefore in their plurality) can make the reaction inefficient and difficult to control. The difficulties of producing the cells described in patent application US2011136022A1 at an industrial level derives from the obvious impossibility of producing micelles that contain the same molecules within them in the same quantity, this ineluctable fact will lead to cells whose yield cannot be standardized. The patent document EP2442394A1 and US2011/076736A1 describe inventions in which the enzymes and / or coenzymes are enclosed in a microspace. Said documents describe an insertion of enzymes and / or coenzymes within microspaces, and this synthesis process does not allow a fine control of the quality and quantity of the molecular species that will be included in each microspace. Through the synthesis processes described in EP2442394A1 and US2011/076736A1 some microspaces may be empty, some presenting a single molecular species, some comprising another molecular species, some comprising several species but in unknown or unfathomable proportions. In fact, the different microspaces may present variable quantities of molecular species, when all are present. As illustrated, the synthesis processes described in EP2442394A1 and

US2011/076736A1 are unusable within large-scale industrial production systems so they would not allow predictable production in terms of pure functionality, efficiency, effectiveness .

The patent document W02009039136A2, in the name of Siometrix Corporation, describes an analysis system of great sensitivity and portability in which the presence of a series of enzymes and / or coenzymes are immobilized on a phospholipid bilayer by means of an anchoring system that could include a chain of polyethylene glycol.The use of an anchoring system that includes, for example, a polyethylene glycol chain does not allow to achieve a quantitatively and qualitatively estimating the presence or absence of enzymes and / or coenzymes. The anchoring system that provides for example a polyethylene glycol chain also does not allow the estimation of how many molecules are actually active since the bond being "random" could involve the active site of the enzymes, inactivating them.

Other known systems are described in EP2672559A1, that describes a fuel cell capable of preventing elution of nicotinamide adenine dinucleotide and/or a derivative thereof immobilized on an electrode, and W02009/039136A2 that describes an assay system of great sensitivity and portability where the reaction is produced by binding a capture moiety to an enzymatic redox reaction partner, allowing the capture moiety to bind to any target in the sample, and washing any such bound target.

The purpose of the present invention is to provide a biochemical electrical generator with electronic transfer mediator capable of establishing the precise concentration of each element participating in the reaction and making the reaction itself efficient, thus having characteristics such as to overcome the limits of current electrical generators .

According to the present invention, a biochemical electrical generator with an electronic transfer mediator is realized, as defined in claim 1.

For a better understanding of the present invention, a preferred embodiment is now described, purely by way of non-limiting example, with reference to the attached drawings, in which: - Figure 1 shows a chemical reaction on which a biochemical electrical generator with electronic transfer mediator is based, according to the invention; - Figure 2 shows a schematic view of a biochemical electrical generator with electronic transfer mediator, according to the invention.

With reference to this figure 1, the reaction on which a biochemical electrical generator with electronic transfer mediator is based, according to the invention, is shown.

The biochemical electrical generator with electronic transfer mediator according to the invention consists of a fuel cell with one or more types of enzymes or further coenzymes interacting so that electrons can be efficiently extracted from a molecule of nicotinamide adenine dinucleotide (NADH), a biomolecule whose biological role consists in transferring electrons, thus allowing the oxidation-reductions, thus producing electrical energy. According to an aspect of the invention, the NADH molecule is synthesized from a fuel such as glucose or the like by an enzymatic reaction, in a generic oxidase (variable according to the molecule used as fuel to produce NADH) which decomposes a fuel molecule, for example the molecule glucose 1-dehydrogenase (ADH), placing it at the interior of bioreactors under controlled conditions.

According to another aspect of the invention, the diaphorase enzyme (Di - NAD dehydrogenase) is made to bind to monoclonal antibodies which are themselves immobilized on a microperforated substrate. In particular, the enzymatic components of the generator, according to the invention, can be linked to monoclonal antibodies in the vicinity of the electrode. According to a further aspect of the invention, the generator provides that an extremely small, or reduced, virtual space is created between the electrode and the diaphorase enzyme (Di - NAD dehydrogenase) bound to the microperforated substrate by means a monoclonal antibodies. According to one aspect of the invention, the substrate consists of microperforated PVC (polyvinyl chloride) or cellulose nitrate or any material that allows the immobilization of monoclonal antibodies.

Advantageously according to the invention, the fact that the enzymes are bound guarantees not only an excellent interaction (due to the proximity to which the molecules are placed) also the possibility of accurately determining the amount of enzymes and coenzymes that take part in it. Furthermore, the synthesis process lends itself to insertion within an industrial production system since the yield in terms of quantity and quality of both the intermediate processing products and the finished product is predictable in all phases, a further advantage is the possibility of obtain through the synthesis process products (cells) that can be standardized in terms of electrical yield.

To clarify the operation of the biochemical electrical generator according to the invention, it must be remembered that, starting from a molecule, called antigen, it is almost always possible to produce specific antibodies that recognize it by binding to it.

Polyclonal antibodies are a set of antibodies, produced by different clones of B cells, which each recognize an epitope (a piece) of a different given molecule. Monoclonal antibodies, on the other hand, all recognize a single epitope or piece of a given molecule.

The advantage of this behavior is that it will make it possible to achieve predictable behavior. In fact, since monoclonal antibodies all bind a single epitope, it allows to determine a single binding site and to direct the molecule in the insertion phase in the membrane.

In the electrical generator according to the invention, the monoclonal antibodies will bind in a specific and precise way a predetermined epitope thus allowing the molecule / enzyme to be able to carry out its activity, while if the antibody binds the enzyme molecule in a portion called the active site it would make the same non-functional . In this first component of the hybrid system the reactions that take place are the following: an oxidase which determines the oxidation of a fuel, such as glucose or similar molecules, and which produces NADH starting from NAD + (nicotinamide adenine dinucleotide). The NADH thus obtained from the previous step is returned to its original form by the diaphorase enzyme (DI) (NAD dehydrogenase) which picks up the electrons which are transferred to a molecule defined as an electron mediator which transfers them to the electrode.

The use of a previously synthesized NADH molecule allow a spatial and temporal separation of the phases that is an advantage. In fact, it shifts a problematic phase of glucose manipulation, subject to a risk of bacterial contamination, in a controlled environment and leaves the use of NADH which is not problematic to the end user, effectively reducing the risk of loss of performance.

According to one aspect of the invention, in case of use of glucose it will be possible to use glucose 1- dehydrogenase (ADH).

Advantageously according to the invention, the fact that the NADH molecule is produced separately reduces the problems inherent in any bacterial contamination / infections or by microorganisms.

According to one aspect of the invention, any electron mediator (EM) can be employed, and preferably a compound having a quinone skeleton e.g. 2,3-dimethoxy-5-methyl-l,4- benzoquinone (Q0) and compounds having a naphthoquinone skeleton, e.g. various naphthoquinone derivatives such as 1-amino-l,4-naphthoquinone (ANQ), 2-amino-3-methyl-l,4- naphthoquinone (AMNQ), 2-methyl-l, 4-naphthoquinone (VK3), 2-amino-3-carboxy-l,4-naphthoquinone (ACNQ), vitamin Kl, and the like. As a compound having a quinone skeleton, for example, anthraquinone and its derivatives can also be used. If necessary, the electron mediator may contain one or two or more types of other compounds which serve as electron mediators, other than the compound having a quinone skeleton.

Figure 1 shows the scheme of the reaction that takes place.

On the positive electrode (anode) of the cell, saturated with the oxygen present in the air, the oxygen itself is reduced while the hydrogen oxidation takes place on the cathode. As a result of the reaction, in addition to electricity and a certain amount of heat generated, water is produced; the two electrodes are submerged in an electrolyte, a concentrated solution of potassium hydroxide (KOH), and coated with catalysts, based on a molecule of nicotinamide adenine dinucleotide (NADH) and on at least one enzyme or coenzyme suitable for extracting electrons from the molecule of nicotinamide adenine dinucleotide (NADH), to increase the speed of the electrode reactions.

According to an aspect of the invention, the fuel cell used uses a polymeric membrane with high proton conductivity as an electrolyte and operates at temperatures between 70 and 100 ° C. For the conversion of hydrogen into energy, a proton exchange membrane will be required for the fuel cell. Proton exchange membranes are known by the acronym PEM (from the English Proton Exchange Membrane) or PEMFC (Proton Exchange Membrane Fuel Cell). Compared to other types of fuel cells, those based on this membrane have the advantage of being light and not bulky. One of their peculiar characteristics is the possibility of operating at low values of temperature (50-100 ° C) and pressure (0.3 MPa).

According to one aspect of the invention, the biochemical electrical generator does not use liposomes but monoclonal antibodies immobilized on a substrate. The substrate is microperforated and is placed near an electrode, surrounding it. Advantageously, the electric generator is configured to work with both the negative electrode and the positive electrode. The preferred substrate is microperforated PVC or cellulose nitrate. Figure 2 shows a diagram of the electric generator in an extended configuration, while in use the substrate is rolled up on itself to surround the electrode. Therefore, the biochemical electrical generator with electronic transfer mediator according to the invention allows to establish in advance the number and type of enzymes or molecules and therefore to standardize energy production by having total control of the reaction stoichiometry.

The main advantage of the invention is the possibility of implementation on a large scale, the possibility of producing the idea in sequence and at an industrial level, the possibility of industrializing production by means of a methodology / synthesis process that is flexible and easily adaptable to the insertion into production lines. Advantageously according to the invention, enzymes and / or coenzymes are immobilized on a support through a monoclinal antibody, immobilized on the support in a specific way, enzyme and/or immobilized enzyme on support specifically, allowing a finely determination of the presence of molecular species (enzymes and / or coenzymes); quantitative estimate of each molecular species present (enzymes and / or coenzymes); it guarantee the functionality of each molecular species present. This qualitative and quantitative fine control (of enzymes and / or coenzymes) of the synthesis process according to the invention advantageously allow a synthesis process designed for massive industrial production.

Furthermore, the invention advantageously allows a control of the efficiency and effectiveness of the systems produced. Another advantage of the biochemical electrical generator with electronic transfer mediator according to the invention is that it can be advantageously industrialized by being able to determine the amount of energy that can be produced based on the number and type of linked molecules.

Finally, it is clear that modifications and variations may be made to the biochemical electrical generator with electronic transfer mediator according to the invention described and illustrated here without thereby departing from the protective scope of the present invention, as defined in the attached claims.