EP2606527A1

EP2606527A1 - Direct-transfer biopile

Info

Publication number: EP2606527A1
Application number: EP11758521.6A
Authority: EP
Inventors: Serge Cosnier; Michael Holzinger; Alan Le Goff; Abdelkader Zebda
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite Joseph Fourier Grenoble 1
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Joseph Fourier Grenoble 1
Priority date: 2010-08-19
Filing date: 2011-08-18
Publication date: 2013-06-26
Also published as: US20130284596A1; CA2808869A1; JP5833123B2; FR2963989A1; JP2013541132A; FR2963989B1; WO2012022921A1

Abstract

The invention relates to a biopile electrode or biosensor electrode intended to be immersed in a liquid medium containing a target and an oxidizer, respectively a reducer, in which the anode comprises an enzyme able to catalyse the oxidation of a target, and the cathode comprises an enzyme able to catalyse the reduction of the oxidizer, and in which each of the anode electrode and cathode electrode consists of a solid agglomeration of carbon nanotubes mixed with the enzyme, and is secured to an electrode wire.

Description

BIOPILE WITH DIRECT TRANSFER

Field of the invention

The present invention relates to bioelectrodes adapted to biofuels (in English biofuel cells) or biosensors, for example of the sugar-oxygen type, for example glucose-oxygen.

Hereinafter will be described essentially biopiles. It will be understood that a biosensor has the same structure as a biopile but is used to detect the content of one of the components of the enzymatic reaction, for example glucose.

Presentation of the prior art

Various types of glucose-oxygen biopiles are described in the prior art. For example, in patent application PCT / FR2009 / 050639 (B8606) each electrode, anode and cathode, of the biopile corresponds to an enclosure containing a liquid medium into which an electrode wire is immersed. The anode and cathode enclosures are delimited by membranes that can be traversed by hydrogen and oxygen but avoiding the circulation of other heavier elements.

The anode comprises in a solution an enzyme and a redox mediator. The enzyme is capable of catalyzing the oxidation of sugar and is for example glucose oxidase if the sugar is glucose. The redox mediator has a low redox potential tible to exchange electrons with the anode enzyme and is for example ubiquinone (UQ).

The cathode also comprises in a solution an enzyme and a redox mediator. The enzyme is capable of catalyzing the reduction of oxygen and is for example polyphenol oxidase (PPO). The redox mediator has a high redox potential capable of exchanging electrons with the cathode enzyme and is, for example, hydroquinone (QHD).

At the level of the anode and the cathode, reactions of the following type occur:

OPP

Cathode: QH ₂ + 1/2 0 ₂ Q + H ₂ 0

GOX

Anode: glucose + UQ> gluconolactone + UQH2

Cathode: Q + 2H + + 2e ^~ → QH ₂

Anode: UQH ₂ → UQ + 2H + + 2e ^~

20 mV an anode potential is then obtained and a cathode potential of 250 mV, which leads to a difference of poten tial ^¬ current zero of the biofuel cell of 230 mV.

Such biocells function well but require anode and cathode conductors to be immersed in enclosures containing appropriate liquids, which is a practical disadvantage in many cases and makes it particularly difficult or impossible to implant such biocells into living beings, in particular to feed various actuators, such as pacemakers, artificial sphincters, or even artificial hearts.

Solid electrode biopiles have been proposed. The unpublished French patent application 10/52657 of April 8, 2010 describes such a biopile. As illustrated in FIG. 1, this biopile comprises an anode body A and a cathode body K. The anode body consists of a solid body comprising a conductive material associated with an enzyme and a redox mediator. anode appropriate. The anode body is secured to anode wire 1. Similarly, the cathode consists of a solid body formed of a conductor associated with an enzyme and a mediator of cathode appropriate. The cathode body is secured to a cathode wire 3. The anode and cathode wires, for example platinum, are shown as penetrating into the anode and cathode bodies; they can simply be stuck to these bodies. For example, the anode body and the cathode body are formed by compressing powdered graphite mixed with the appropriate redox enzyme and mediator.

Redox chemical mediators provide an electrical connection between the enzyme and the electrode by electron jumps between the redox mediators positioned between the surface of the electrode and the prosthetic center or active center of the enzyme.

In addition to a complexification of the construction of the bioelectrodes (the redox mediators are generally soluble in aqueous medium, it is necessary to fix them on the surface of the electrode), a main disadvantage of the use of these mediators is the fact that they greatly reduce poten ^¬ tiel provided by the biopile. By definition, these mediators must have a potential greater than that of the redox center of the enzyme catalyzing the oxidation of glucose in order to react with it, in particular with its reduced form in order to oxidize it. Similarly, the redox mediators dedicated to the connection of the oxygen-reducing enzyme must have a lower potential ^¬ than the active center of this enzyme in order to react with its oxidized form. As a result, the potential difference between the active sites of the enzyme catalyzing the oxidation of glucose and that catalyzing the reduction of oxygen is necessarily greater than the potential difference between the redox mediators involved in these two reactions. E.g., theoretically, a biofuel cell glucose / oxygen should provide a potential of 1 V, or the use of media ^¬ tors redox leads to biofuel cells having significantly lower potentials. It should be noted that the potential of the battery is also limited by kinetic limitation problems and ohmic drops. A direct electrical connection was so tempted to do (without using mediators) enzymes to elec ^¬ trodes. However, the electron transfer remains very weak and sometimes requires modification of the enzyme. In addition, the performance of these batteries remains low.

There is therefore a need for high performance implantable biopiles.

summary

Thus, according to one embodiment of the present invention, attempts are made to produce bioelectrodes that are simple to handle for applications in the field of biopiles and biosensors, and in particular that can be implanted in a living animal or human being.

More particularly, an embodiment of the present invention provides a biopile or biosensor electrode to be immersed in a liquid medium containing a target and an oxidant, respectively a reducing agent, wherein the anode comprises an enzyme capable of catalyzing the oxidation of a target, and the cathode comprises an enzyme capable of catalyzing the reduction of the oxidant, and wherein each of the anode and cathode electrodes is made of a chipboard ^¬ solid carbon nanotubes mixed with Mérat the enzyme, and is integral with an electrode wire.

According to one embodiment of the present invention, the electrode is surrounded by a semipermeable membrane allowing the oxidant and the target to pass and not allowing the enzyme to pass.

According to one embodiment of the present invention, the membrane is of the dialysis membrane type.

According to one embodiment of the present invention, the target is glucose.

One embodiment of the present invention provides a method of manufacturing a biopile or biosensor, wherein the anode and the cathode are formed by compressing a solution mixture comprising carbon nanotubes and an enzyme. According to one embodiment of the present invention, the carbon nanotubes are of the multi-sheet type.

Brief description of the drawings

These and other objects, features, and advantages will be set forth in detail in the following description of particular embodiments in a non-limitative manner with reference to the accompanying figures in which:

FIG. 1 very schematically represents a biopile with solid electrodes;

FIG. 2 illustrates the current and power performance as a function of the voltage of a glucose / oxygen biofuel made of bioelectrodes manufactured according to the present invention; and

FIGS. 3A and 3B respectively illustrate the electrochemical response of a biosensor to glucose injections and the current response of this biosensor as a function of the glucose concentration.

detailed description

In general, the present invention relates to a new type of solid electrolyte containing electri cally connected ^¬ enzymes. The invention provides for the electrical connection of a large density of enzymes by compression in the form of a compact block, for example a disk, a mixture of carbon nanotubes, enzymes, water required for solubilization enzymes and glycerol as a binder between the different constituents. Carbon nanotubes are of the mono- or multi-layer type.

The use of very thin and highly conductive carbon nanotubes makes it possible to immobilize the enzymes and to connect them, because the carbon nanotubes penetrate the large enzyme molecules which consist of a protein envelope protecting their active redox center. .

The conductivity of the nanotubes and their very small diameter (of the order of a nanometer) allows electrical communication with the enzyme which retains its catalytic activity. We can exploit the catalytic properties of the enzyme or for the bioelectrochemical detection or for power conversion and more particularly the production of electric energy ^¬ stick. These bioelectrodes can be used in the fields of biopiles and biosensors.

The anode consists, for example, of a compression of carbon nanotubes containing an oxidase such as glucose oxidase (GOX) capable of catalyzing the oxidation of a fuel, for example sugar.

The cathode is for example constituted by a compres ^¬ carbon nanotube sion containing an enzyme such as laccase or bilirubin oxidase capable of catalyzing the reduction ^¬ an oxidant such as oxygen.

Cathode: 1/2 0 ₂ ^Laccase > 2e + H ₂ 0

GOX

Anode: - 2nd - 2H ⁺ gluconolactone

These reactions are given in the particular case where the fuel is glucose, the anode enzyme is glucose oxidase (GOX), and the oxidant is oxygen. The cathode enzyme is laccase.

For example, an anode was prepared by Mélan ^¬ giant 150 mg of carbon nanotubes, 30 mg of glucose oxidase and 30 mg of catalase (the role of the catalase is to eliminate ¾02 (a noxious product) formed by glucose oxidase in the presence of

O2: Η ₂ θ2 → ½02 + H ₂ 0), 0.6 ml of water and glycerol (50 μΐ) in a ceramic mortar. A cathode was prepared in a similar manner: 150 mg of carbon nanotubes, 30 mg of laccase, 0.6 ml of water and 25 μl of glycerol were mixed in a ceramic mortar. The resulting pastes consisting of carbon nanotubes and enzymes were pressed at a pressure of 1500 kg / cm 2 to form discs. The area and thickness of the disks were 1.33 cm 2 and 0.1 cm, respectively. A platinum wire was fixed by a conductive glue to the carbon nanotubes compacted on one side of each disk and covered with a silicon film to enhance the mechanical strength of the biomaterial and the electrical contact.

To operate in a stack, these anode and cathode bodies are arranged in a fluid containing oxygen and a sugar, for example glucose.

This biopile has a zero current potential of 1 V, a maximum power of 1800 and a maximum current of 8 mA. These performances are far superior to those obtained for known biocells with direct enzyme connection (maximum power 5 and maximum potential at zero current 0.73 V). In addition, this biopile gives the possibility of having a high power at a high enough potential to operate devices: 800 μίί 0.8 V.

FIG. 2 represents power and electric current curves as a function of the potential of a biopile as described by way of example above.

Figure 3A illustrates the electrochemical response of a biosensor constituted as the cell described above to the presence of glucose and Figure 3B shows the measured current as a function of glucose concentration.

To use this biosensor, the electrode is immersed in an aqueous liquid and glucose is added. An electric potential, for example 0.1 V, is applied between the bioelectrode and a reference electrode both immersed in the liquid analysis medium and the electric current is measured between the bioelectrode and an auxiliary electrode also immersed in this medium. The detection and quantification of glucose present in the liquid is done by measuring the glucose oxidation current catalyzed by the enzyme.

The performances of the biosensor maintained at the potential of 0.1 V are 17 mA / M / cm 2 and 685 AU / cm 2 for sensitivity and maximum current density, respectively. In addition to working potential to overcome the anodic interfer ^¬ ences, this system has the highest density maximum current so far described even for conventional glucose biosensors.

However, it has been found that biopiles using such anode and cathode bodies have a short life. The inventors have attributed this problem to the fact that enzyme leaks over time outside the anode body and the cathode body. To solve this problem, each of the anode and cathode bodies may be surrounded by a microperforated membrane such as membranes commonly used in dialysis, which allows glucose and oxygen to pass and prohibits the passage of the enzyme. and carbon nanotubes of higher molecular weight. The set of anode and cathode electrodes may be surrounded by a semipermeable membrane permitting glucose and oxygen to pass through and impervious to enzymes and carbon nanotubes, in particular to allow their implantation in an animal or human body. .

An example of a glucose-oxygen biopile has been given above. Any sugar-oxygen biopile can be modi ^¬ fied according to the present invention, and more generally any biopile whose anode comprises an enzyme capable of catalyzing the oxidation of a target, and whose cathode comprises an enzyme capable of catalyzing the oxidation of a target. reduction of the oxidant.

Various embodiments with various variants have been described above. It will be appreciated that those skilled in the art may combine various elements of these various exemplary embodiments and variants without demonstrating inventive step.

Claims

A biopile or biosensor electrode intended to be immersed in a liquid medium containing a target and an oxidant, respectively a reducing agent, in which the anode comprises an enzyme capable of catalyzing the oxidation of a target, and the cathode comprises an enzyme capable of catalyzing the reduction of the oxidant, and wherein each of the anode and cathode electrodes consists of a solid block of carbon nanotubes mixed with the enzyme, and is integral with a conductive wire.

2. Electrode according to claim 1, surrounded by a semipermeable membrane passing the oxidant and the target and not passing the enzyme.

The electrode of claim 2, wherein said membrane is of the dialysis membrane type.

The electrode of claim 1 or 2, wherein the target is glucose.

A method of manufacturing a biopile or biosensor, wherein the anode and the cathode are formed by compressing a solution mixture comprising carbon nanotubes and an enzyme to the exclusion of any redox mediator.

6. Process according to claim 5, in which the carbon nanotubes are of the multi-sheet type.