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WO2017212304A1 - Bioelectrode coated with a gel of modified polysaccharide - Google Patents

Bioelectrode coated with a gel of modified polysaccharide Download PDF

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Publication number
WO2017212304A1
WO2017212304A1 PCT/IB2016/000918 IB2016000918W WO2017212304A1 WO 2017212304 A1 WO2017212304 A1 WO 2017212304A1 IB 2016000918 W IB2016000918 W IB 2016000918W WO 2017212304 A1 WO2017212304 A1 WO 2017212304A1
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WO
WIPO (PCT)
Prior art keywords
bioelectrode
polysaccharide
conjugated system
modified
alginate
Prior art date
Application number
PCT/IB2016/000918
Other languages
French (fr)
Inventor
Serge Cosnier
Robert Marks
Kamal ELOUARZAKI
Original Assignee
Universite Grenoble Alpes
Nanyang Technological University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universite Grenoble Alpes, Nanyang Technological University filed Critical Universite Grenoble Alpes
Priority to PCT/IB2016/000918 priority Critical patent/WO2017212304A1/en
Publication of WO2017212304A1 publication Critical patent/WO2017212304A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G01N2400/44—Guluromannuronans, e.g. alginic acid

Definitions

  • the invention is in the technical field of bioelectrodes.
  • the present invention relates to a bioelectrode coated with a gel of modified polysaccharide comprising a conjugated system.
  • the invention is directed to a bioelectrode coated with a gel of modified alginate.
  • Bioelectrodes are electrodes that can be implanted in the body of animals or humans. As bioelectrodes lead to the generation of an electric potential, they can be used in biofuel cells or in biosensors. Bioelectrodes are also named bioreactors as they are the seats of (bio)chemical reactions. For example, they can be used in glucose biosensors in order to monitor the level of glucose in the blood. Because of their in vivo use, bioelectrodes must be biocompatible: they should be inert (nontoxic) towards the organism in which they are implanted. In particular, no inflammatory reaction should be seen after implantation of the bioelectrode. Different electrical conductor materials can be used in bioelectrodes.
  • FR2963989 discloses a bioelectrode, made by compression of carbon nanotubes and an enzyme, encapsulated by a semi-permeable membrane such as a dialysis membrane. This semi-permeable membrane allows the glucose and the oxygen to go through, but not the enzyme or the carbon nanotubes.
  • WO2015145054 discloses a bioreactor obtained by compression of a mixture of enzyme, chitosan and a conductor which can be carbon nanotubes. This document also discloses a chitosan membrane coating a part of the electrode. This membrane ensures a good biocompatibility of the electrode and prevents diffusion of the constituents of the bioreactor.
  • these known types of membrane used to coat or encapsulate the bioelectrodes are not stable over time. Disbonding of the membrane or loss of sealing are seen when these types of membrane are used on bioelectrodes.
  • the present invention aims to meet at least one of the objectives stated below.
  • One of the essential objectives of the present invention is to provide a novel bioelectrode coated with a membrane, the membrane being stable and bound to the rest of the bioelectrode over time.
  • Another essential objective of the present invention is to provide a novel bioelectrode coated with a membrane, the membrane being biocompatible and being capable to make the bioelectrode biocompatible.
  • Another essential objective of the present invention is to provide a bioelectrode coated with a membrane, the membrane preventing diffusion of the constituents of the in vivo implanted bioelectrode into the body.
  • Another essential objective of the present invention is to provide a bioelectrode coated with a membrane, the membrane showing no disbonding from the bioelectrode, even after a long period of time, particularly when the bioelectrode is implanted in vivo .
  • Another essential objective of the present invention is to provide a method for manufacturing a bioelectrode coated with a membrane, having all or part of the above characteristics, which is easy to implement and cost effective.
  • a bioelectrode coated with a gel of modified polysaccharide comprising a conjugated system This particular material, preferably based on alginate, allows good membrane properties and prevents diffusion of the constituents of the in vivo implanted bioelectrode into the body.
  • the coating is made from polysaccharide makes the bioelectrode biocompatible and the conjugated system attached to the polysaccharide makes good interaction possible, especially by pi-stacking, between an electron conductor material of the bioelectrode and the membrane, therefore preventing disbonding of the membrane over time.
  • the invention also relates to a method for producing such a bioelectrode and a specific hydrogel used to coat the electrode.
  • the invention relates to a bioelectrode coated with a gel of modified polysaccharide comprising a conjugated system.
  • "Coated” means that a part or all of the bioelectrode is coated.
  • the gel of modified polysaccharide comprising a conjugated system forms a coating, in other words a film, covering at least a part of the bioelectrode.
  • This coating acts as a membrane and enables certain components to go through, for example glucose or oxygen, but not the constituents of the bioelectrode.
  • the coating has a thickness between 100 nm and several micrometers. This very thin coating prevents the generation of steric hindrances towards the diffusion of substrates.
  • Gel is intended to mean e.g. a nonfluid polymer network that is expanded throughout its whole volume by a fluid.
  • the fluid is water, and therefore the gel is a hydrogel.
  • the polysaccharide can be chosen from alginates, agaroses, carrageenans, cellulose, dextrans, amidons, hyaluronic acid, chitosans and mixtures thereof.
  • the polysaccharide is a thermally gellable polysaccharide or a chemically gellable polysaccharide.
  • Thermally gellable is intended to mean, for example, the ability of a composition to form a gel via a decrease of temperature.
  • “Chemically gellable” is intended to mean, for example, the ability of a composition to form a gel via a reaction, for example, with metal cations which cause crosslinking and in so doing gel formation, or by modification of the pH of the polysaccharide solution.
  • the polysaccharide is a chemically gellable polysaccharide, and even more preferably gellable via a reaction with metal cations.
  • the coating of the bioelectrode is a hydrogel comprising:
  • the divalent or polyvalent cations can be Ca 2+ or Ba 2+ .
  • the polysaccharide is alginate.
  • Alginates are polysaccharides derived from algine, a polysaccharide found in the cellular wall of a brown algae. Alginates are linear unbranched polymers constituted of polymer blocks of mannuronic acid residues and polymer blocks of guluronic acid residues. This polysaccharide is known to form gels in the presence of divalent or polyvalent cations, like Ca 2+ or Ba 2+ .
  • sodium alginate can be crosslinked with calcium ion in order to form a hydrogel.
  • the hydrogels made from alginate are composed of 97.5 to 99% of water and 0.3 to 2.5% of crosslinked alginate.
  • the bioelectrode is coated with a hydrogel of modified alginate comprising a conjugated system and divalent or polyvalent cations, like Ca 2+ .
  • This hydrogel is particularly interesting as its pore size can be controlled. The pore size is adjustable by varying the concentration and the type of alginates, the concentration of gelation cation and the gelation time.
  • Conjugated system is intended to mean, for example, a molecular moiety whose structure may be represented as a system of alternating single and multiple bonds. It is a system of connected p-orbitals with delocalized electrons in molecules with alternating single and multiple bonds, which in general may lower the overall energy of the molecule and increase stability.
  • the conjugated system comprises at least one aromatic ring.
  • the conjugated system can be a polycyclic aromatic hydrocarbon.
  • the conjugated system is selected from the group comprising, preferably consisting of, porphyrins, phthalocyanines, pyrene, benzene, indole, azulene, phenothiazines, naphthalene, perylene, anthracene, phenanthrene, corene, corannulene, ovalene, other molecular-benzene-based structures, cyclic aromatic hydrocarbons, graphene-like structures, and mixtures thereof, preferably pyrene.
  • the modified polysaccharide comprising a conjugated system is a pyrene modified alginate.
  • Modified polysaccharide comprising a conjugated system is intended to mean, for example, that a polysaccharide was chemically modified by reacting it with a molecule or a metal complex comprising a conjugated system. In other words, a molecule or a metal complex comprising conjugated system was bound to the polysaccharide via a chemical reaction.
  • the modified polysaccharide is obtained by amidation, esterification and/or thioesterification of carboxylic acid groups of the polysaccharide, respectively, with at least one amine, at least one alcohol or at least one thiol comprising the conjugated system.
  • the amidation, esterification and thioesterification can be done using standard protocols, for example, via activation of the carboxylic acid groups of the polysaccharide.
  • the amidation can be done using N-hydroxysulfosuccinimide and l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide as reagents in order to activate the carboxylic acid groups.
  • the amount of modified carboxylic group of the polysaccharide is between 15 and 30%, preferably between 20 and 25%, of the total amount of carboxylic groups of the polysaccharide.
  • Bioelectrode is intended to mean, for example, an electrode that can be implanted in the body of animals or humans.
  • the bioelectrode is an electrode comprising, functionalized or coated with a macromolecular biomolecule like enzyme, coenzyme, DNA, antibody, proteins, antigens, aptamers and even micro-organisms.
  • the bioelectrode according to the invention comprises an electrical conductor, for example a carbon-based material, and, preferably, at least one macromolecular biomolecule, like an enzyme.
  • the bioelectrode is obtained by compression of a mixture of a macromolecular biomolecule and an electrical conductor.
  • the bioelectrode can be partly coated with an insulating water repellant layer, like silicone gel.
  • the bioelectrode is a disk which back side and edges are coated with an insulating water repellant layer and which top side is coated with a gel of modified polysaccharide comprising a conjugated system.
  • the electrical conductor material is selected from the group comprising, preferably consisting of, carbon fibers, carbon nanotubes, graphene, graphene derivatives, graphite and mixtures thereof.
  • the carbon nanotubes can be, for example, single-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes or carbon nanotubes arrays.
  • These electrical conductor materials are especially suitable as they make it possible for a good chemical interaction between the bioelectrode and the coating. Indeed, these materials have aromatic rings that can interact with the conjugated system of the modified polysaccharide.
  • pi-stacking interaction can occur between the aromatic rings of the coating and the electrical conductor material of the bioelectrode.
  • the pi-stacking interaction is a non- covalent attractive force between aromatic rings. This interaction contributes to the good stability of the membrane over time as it is strong enough to prevent disbonding of the coating from the bioelectrode.
  • the bioelectrode comprises
  • the bioelectrode is a bioanode.
  • the invention also relates to biofuel cells or biosensors comprising at least one bioelectrode as described above.
  • the biosensor can be a glucose biosensor.
  • the invention also pertains to a method for producing a bioelectrode as described above, said method comprising:
  • a bioelectrode preferably comprising a material selected from the group comprising, preferably consisting of, carbon nanotubes, graphite and graphene,
  • the bioelectrode is a disk of compressed enzyme and carbon nanotubes.
  • step b) is done by
  • step b) is done by
  • the invention also relates to a hydrogel of modified alginate, the modified alginate comprising a conjugated system.
  • the conjugated system comprises at least one aromatic ring.
  • the conjugated system can be a polycyclic aromatic hydrocarbon.
  • the conjugated system is selected from the group comprising, preferably consisting of, porphyrins, phthalocyanines, pyrene, benzene, indole, azulene, phenothiazines, naphthalene, perylene, anthracene, phenanthrene, corene, corannulene, ovalene, other molecular-benzene-based structures, cyclic aromatic hydrocarbons, graphene-like structures, and mixtures thereof, preferably pyrene.
  • the modified alginate is synthesized by amidation of the carboxylic groups with an amine comprising a conjugated system.
  • the amidation is undergone using standard amidation procedures, for example, by using N-hydroxysulfosuccinimide and l-Ethyl-3-(3-dimethyl- aminopropyl)carbodiimide as reagents.
  • the amount of modified carboxylic group of the alginate is between 15 and 30%, preferably between 20 and 25% of the total amount of carboxylic groups of the alginate.
  • the hydrogel of modified alginate comprising a conjugated system is obtained by crosslinking using divalent or polyvalent cations, like Ca 2+ or Ba 2+ .
  • divalent or polyvalent cations like Ca 2+ or Ba 2+ .
  • a solution of divalent or polyvalent cations is added to a solution of modified alginate comprising a conjugated system, this induces the crosslinking and gelation occurs rapidly.
  • the hydrogel of modified alginate is a hydrogel of pyrene modified alginate and Ca 2+ .
  • the schematic structure of such a hydrogel is shown in figure 1.
  • This hydrogel is particularly suitable for coating bioelectrodes, especially made from carbon nanotubes as the pyrene moiety allows good pi- stacking interaction with the carbon nanotubes.
  • Figure 1 shows a schematic representation of a gel of pyrene modified alginate crosslinked by Ca 2+ ions.
  • FIG. 2 shows photographs of carbon nanotube (CNT) pellet electrodes: (A) unmodified (according to example 1), (B) modified with pyrene-alginate film after addition of 0.1 M CaCl 2 (according to example 4) and (C) modified with alginate film after addition of 0.1 M CaCl 2 (according to example 2).
  • Figure 3 shows optical microscopy images recorded using lOx objective of CNT pellets (A) before (according to example 1) and (B) after modification with alginate and addition of 0.1 M CaCl 2 (according to example 2); (C) before (according to example 1) and (D) after modification with pyrene alginate and addition of 0.1 M CaCl 2 (according to example 4) .
  • Figure 6 shows five consecutive cyclic voltammograms recorded in 1 mM K 3 FeCN 6 in 0.1 M KCl of CNT pellets (A, i-v) after modification with pyrene alginate and addition of 0.1 M CaCl 2 (according to example 4) ; (B, i-v) after modification with alginate and addition of 0.1 M CaCl 2 (according to example 2).
  • Scan rate 5 mV/s.
  • Figure 7 shows cyclic voltammograms recorded in 0.5 mM FcMeOH in PB pH 7 of CNT pellets (A) after modification with pyrene alginate and addition of 0.1 M CaCl 2 (according to example 4), after immersion in the solution for (i) 0 min, (ii) 120 min and (iii) 1200 min.
  • Electrodes Materials used for the preparation of the electrodes The electrodes were synthesized using commercial products. Mono-sodium phosphate monohydrate (98-102%), di-sodium hydrogen phosphate heptahydrate (98-102%), potassium ferricyanide (K 3 FeCN 6 , 99%), potassium chloride (KC1, > 99%), calcium chloride (meets USP testing specifications), ferrocene methanol (FcMeOH, 97%), ruthenium hexamine (Ru(NH 3 ) 6 , 98%), alginic acid sodium salt, TRIZMA ® base hydrochloride (Tris HC1, > 99%), sodium hydroxide (98-100.5%) and potassium chloride (99-100.5%) were purchased from Sigma-Aldrich and used without further purification.
  • Aqueous solutions were prepared from ultrapure water at 25°C (resistivity > 18.2 MQcm).
  • Commercial grade multi-walled carbon nanotubes (MWCNTs, 9.5 nm diameter, > 95% purity) were obtained from Nanocyl and used as received without purification.
  • Example 1 Preparation of carbon nanotube pellet electrode (CNT electrode)
  • MWCNTs multi-walled carbon nanotubes
  • aqueous alginate (2 % w/v) solution was prepared in 0.1 M Tris HCL buffer pH 7 at room temperature then left stirring overnight at 200 rpm to aid solubilization.
  • 150 of the alginate solution was delicately added to the surface of the electrode.
  • 100 of 0.1 M CaCl 2 was dispersed on the surface. The gel formed rapidly within seconds. The electrode was left overnight to dry then rinsed with 0.1 M Tris HC1 buffer pH 7.
  • Example 3 preparation of pyrene modified alginate (pyrene- alginate)
  • Pyrenemethylammonium hydrochloride (540 mg, 2.0 mmol) was added to a solution of NaOH (120 mg, 3 mmol) in water (60 mL). The resulting suspension was extracted with CH 2 C1 2 (3x40 mL). Removal of the solvent under vacuum yields a white oil of pyrenemethylamine (410 mg, 1.8 mmol, 90% yield).
  • the pyrene-alginate conjugate was prepared by adding 214 mg (2.5 mmol) of pyrenemethylamine to a solution of alginate (2.5 mmol of alginate monomer, 500 mg) in 100 mL of 0.1 M MES buffer, pH 6.0.
  • the reaction mixture was stirred at room temperature for 10 min to facilitate a homogeneous dispersion of the pyrenemethylamine reagent in the reaction solution. Then 135 mg of NHSS (N- hydroxysulfosuccinimide, 0.625 mmol) and 240 mg of EDC (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, 1.25 mmol) were added (ratios of reagents were calculated for a theoretical 20-25% molar modification of the number of carboxylic groups of alginate). The reaction was stirred for 20 h, and modified alginate was precipitated by adding NaCl in order to have a concentration of 1 M, and two volumes of ethanol.
  • NHSS N- hydroxysulfosuccinimide, 0.625 mmol
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, 1.25 mmol
  • modified alginate In order to remove unreacted alginate, precipitation was repeated three times using the same procedure after dissolving modified alginate at 1% (w/v) concentration in water. Modified alginate was dried over night at 40 °C in the dark and stored at -20 °C.
  • Example 4 Preparation of pyrene-alginate film-modified carbon nanotube pellet electrodes
  • a pyrene-alginate film-modified carbon nanotube pellet electrodes was prepared according to example 2 using pyrene-alginate obtained in example 3 instead of alginate.
  • Figure 2 shows pictures of the different electrodes according to example 1, 2 and 4. These photographs indicate a change before and after coating.
  • Figure 3 shows optical microscopy images of the different electrodes according to example 1, 2 and 4. Optical microscopy images were obtained using a Keyence VK X200 Series Microscope with VK 2.5 software. These images indicate a change in surface topography following the coating of the CNT pellet.
  • R a roughness values were estimated from the -1.5 mm images using VK analyzer software. The average roughness values reveal a decrease in surface roughness on modification, consistent with the formation of a film. Indeed, the roughness decreases from image A (carbon nanotube pellet) to image B (carbon nanotube pellet + alginate gel) due to the presence of an alginate film on the carbon nanotube pellet. The roughness exhibits an even more important decrease from image C (carbon nanotube pellet) to image D (carbon nanotube pellet + pyrene alginate gel) due to the presence of a pyrene alginate film interacting with the carbon nanotube pellet.
  • Electrochemical measurements were performed at room temperature using an Eco Chemie Autloab PGSTAT 100 potentiostat running GPES 4.9 software.
  • a conventional three- electrode cell set-up was used for all electrochemical experiments comprising a CNT pellet working electrode, a saturated calomel (SCE) reference electrode and a Pt wire counter electrode.
  • SCE saturated calomel
  • the cyclic voltammogram of the CNT pellet modified with pyrene-alginate did not change over 5 scans, consistent with the formation of a stable film.
  • CNT pellet modified with alginate (example 2: Fig.6B) changed considerably with each of the 5 scans, becoming increasingly less blocking towards K 3 FeCN 6 ions in solution.
  • the cyclic voltammogram response after 5 scans of the alginate modified CNT pellet is similar to that of the unmodified surface.
  • the new peak is most likely ferrocene related and may be attributed to ferrocene groups trapped within the film.

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Abstract

The invention is in the technical field of bioelectrodes. The objective of the présent invention is to provide a novel bioelectrode coated with a membrane, the membrane being capable to make the bioelectrode biocompatible and being stable and bound to the rest of the bioelectrode over time. This novel bioelectrode is coated with a gel of modified polysaccharide comprising a conjugated System. The fact that the coating is made from polysaccharide makes the bioelectrode biocompatible and the conjugated System attached to the polysaccharide makes good interaction possible, especially by pi-stacking, between an électron conductor material of the bioelectrode and the membrane, therefore preventing disbonding of the membrane over time. The invention also relates to a method for producing such a bioelectrode and a spécifie hydrogel used to coat the électrode.

Description

BIOELECTRODE COATED WITH A GEL OF MODIFIED POLYSACCHARIDE
Technical field The invention is in the technical field of bioelectrodes. The present invention relates to a bioelectrode coated with a gel of modified polysaccharide comprising a conjugated system. In particular, the invention is directed to a bioelectrode coated with a gel of modified alginate.
Background Art
Bioelectrodes are electrodes that can be implanted in the body of animals or humans. As bioelectrodes lead to the generation of an electric potential, they can be used in biofuel cells or in biosensors. Bioelectrodes are also named bioreactors as they are the seats of (bio)chemical reactions. For example, they can be used in glucose biosensors in order to monitor the level of glucose in the blood. Because of their in vivo use, bioelectrodes must be biocompatible: they should be inert (nontoxic) towards the organism in which they are implanted. In particular, no inflammatory reaction should be seen after implantation of the bioelectrode. Different electrical conductor materials can be used in bioelectrodes. For example, different metals like Pt or Pt-Ir are used in some commercial bioelectrodes. The use of carbon nanotubes as an electrical conductor material is also reported. Carbon nanotubes have very good electronic and physical properties and are therefore a very interesting material. However, the use of carbon nanotubes in bioelectrodes raises the issue of their possible toxicity as carbon nanotubes can accumulate in filtering organs like liver or kidneys. Therefore, diffusion of carbon nanotubes from bioelectrodes to the body should be avoided. Different methods can be used in order to prevent this migration and to make the bioelectrodes biocompatible. FR2963989 discloses a bioelectrode, made by compression of carbon nanotubes and an enzyme, encapsulated by a semi-permeable membrane such as a dialysis membrane. This semi-permeable membrane allows the glucose and the oxygen to go through, but not the enzyme or the carbon nanotubes. WO2015145054 discloses a bioreactor obtained by compression of a mixture of enzyme, chitosan and a conductor which can be carbon nanotubes. This document also discloses a chitosan membrane coating a part of the electrode. This membrane ensures a good biocompatibility of the electrode and prevents diffusion of the constituents of the bioreactor. However these known types of membrane used to coat or encapsulate the bioelectrodes are not stable over time. Disbonding of the membrane or loss of sealing are seen when these types of membrane are used on bioelectrodes.
Technical problem and objectives to achieve
In this context, the present invention aims to meet at least one of the objectives stated below. One of the essential objectives of the present invention is to provide a novel bioelectrode coated with a membrane, the membrane being stable and bound to the rest of the bioelectrode over time.
Another essential objective of the present invention is to provide a novel bioelectrode coated with a membrane, the membrane being biocompatible and being capable to make the bioelectrode biocompatible.
Another essential objective of the present invention is to provide a bioelectrode coated with a membrane, the membrane preventing diffusion of the constituents of the in vivo implanted bioelectrode into the body.
Another essential objective of the present invention is to provide a bioelectrode coated with a membrane, the membrane showing no disbonding from the bioelectrode, even after a long period of time, particularly when the bioelectrode is implanted in vivo .
Another essential objective of the present invention is to provide a method for manufacturing a bioelectrode coated with a membrane, having all or part of the above characteristics, which is easy to implement and cost effective. Summary
These objectives, among others, are reached by the present invention which first of all relates to a bioelectrode coated with a gel of modified polysaccharide comprising a conjugated system. This particular material, preferably based on alginate, allows good membrane properties and prevents diffusion of the constituents of the in vivo implanted bioelectrode into the body.
The fact that the coating is made from polysaccharide makes the bioelectrode biocompatible and the conjugated system attached to the polysaccharide makes good interaction possible, especially by pi-stacking, between an electron conductor material of the bioelectrode and the membrane, therefore preventing disbonding of the membrane over time.
The invention also relates to a method for producing such a bioelectrode and a specific hydrogel used to coat the electrode.
Definitions
In the present description, any singular designates equally a singular and a plural and vice versa, unless otherwise stated.
The expression "between x and y" is understood to mean a range or ranges of values the limits whereof are closed: [x,y]. Detailed description
The invention relates to a bioelectrode coated with a gel of modified polysaccharide comprising a conjugated system. "Coated" means that a part or all of the bioelectrode is coated.
The gel of modified polysaccharide comprising a conjugated system forms a coating, in other words a film, covering at least a part of the bioelectrode. This coating acts as a membrane and enables certain components to go through, for example glucose or oxygen, but not the constituents of the bioelectrode. According to a preferred embodiment, the coating has a thickness between 100 nm and several micrometers. This very thin coating prevents the generation of steric hindrances towards the diffusion of substrates.
"Gel" is intended to mean e.g. a nonfluid polymer network that is expanded throughout its whole volume by a fluid. Preferably, the fluid is water, and therefore the gel is a hydrogel.
For example, the polysaccharide can be chosen from alginates, agaroses, carrageenans, cellulose, dextrans, amidons, hyaluronic acid, chitosans and mixtures thereof.
In one embodiment of the invention, the polysaccharide is a thermally gellable polysaccharide or a chemically gellable polysaccharide. "Thermally gellable" is intended to mean, for example, the ability of a composition to form a gel via a decrease of temperature. "Chemically gellable" is intended to mean, for example, the ability of a composition to form a gel via a reaction, for example, with metal cations which cause crosslinking and in so doing gel formation, or by modification of the pH of the polysaccharide solution. Preferably, the polysaccharide is a chemically gellable polysaccharide, and even more preferably gellable via a reaction with metal cations.
In one embodiment of the invention, the coating of the bioelectrode is a hydrogel comprising:
- a modified polysaccharide comprising a conjugated system,
divalent or polyvalent cations,
and water.
For example, the divalent or polyvalent cations can be Ca2+ or Ba2+. According to a preferred embodiment, the polysaccharide is alginate. Alginates are polysaccharides derived from algine, a polysaccharide found in the cellular wall of a brown algae. Alginates are linear unbranched polymers constituted of polymer blocks of mannuronic acid residues and polymer blocks of guluronic acid residues. This polysaccharide is known to form gels in the presence of divalent or polyvalent cations, like Ca2+ or Ba2+. For example sodium alginate can be crosslinked with calcium ion in order to form a hydrogel. In general, the hydrogels made from alginate are composed of 97.5 to 99% of water and 0.3 to 2.5% of crosslinked alginate.
According to a preferred embodiment, the bioelectrode is coated with a hydrogel of modified alginate comprising a conjugated system and divalent or polyvalent cations, like Ca2+. This hydrogel is particularly interesting as its pore size can be controlled. The pore size is adjustable by varying the concentration and the type of alginates, the concentration of gelation cation and the gelation time. "Conjugated system" is intended to mean, for example, a molecular moiety whose structure may be represented as a system of alternating single and multiple bonds. It is a system of connected p-orbitals with delocalized electrons in molecules with alternating single and multiple bonds, which in general may lower the overall energy of the molecule and increase stability. According to one embodiment, the conjugated system comprises at least one aromatic ring. The conjugated system can be a polycyclic aromatic hydrocarbon.
According to a preferred embodiment of the present invention, the conjugated system is selected from the group comprising, preferably consisting of, porphyrins, phthalocyanines, pyrene, benzene, indole, azulene, phenothiazines, naphthalene, perylene, anthracene, phenanthrene, corene, corannulene, ovalene, other molecular-benzene-based structures, cyclic aromatic hydrocarbons, graphene-like structures, and mixtures thereof, preferably pyrene. Preferably, the modified polysaccharide comprising a conjugated system is a pyrene modified alginate.
"Modified polysaccharide comprising a conjugated system" is intended to mean, for example, that a polysaccharide was chemically modified by reacting it with a molecule or a metal complex comprising a conjugated system. In other words, a molecule or a metal complex comprising conjugated system was bound to the polysaccharide via a chemical reaction.
According to a preferred aspect of the invention, the modified polysaccharide is obtained by amidation, esterification and/or thioesterification of carboxylic acid groups of the polysaccharide, respectively, with at least one amine, at least one alcohol or at least one thiol comprising the conjugated system. The amidation, esterification and thioesterification can be done using standard protocols, for example, via activation of the carboxylic acid groups of the polysaccharide. For example, the amidation can be done using N-hydroxysulfosuccinimide and l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide as reagents in order to activate the carboxylic acid groups.
In particular, the amount of modified carboxylic group of the polysaccharide is between 15 and 30%, preferably between 20 and 25%, of the total amount of carboxylic groups of the polysaccharide.
"Bioelectrode" is intended to mean, for example, an electrode that can be implanted in the body of animals or humans. Preferably, the bioelectrode is an electrode comprising, functionalized or coated with a macromolecular biomolecule like enzyme, coenzyme, DNA, antibody, proteins, antigens, aptamers and even micro-organisms. The bioelectrode according to the invention comprises an electrical conductor, for example a carbon-based material, and, preferably, at least one macromolecular biomolecule, like an enzyme. For example, the bioelectrode is obtained by compression of a mixture of a macromolecular biomolecule and an electrical conductor. The bioelectrode can be partly coated with an insulating water repellant layer, like silicone gel. According to a preferred embodiment, the bioelectrode is a disk which back side and edges are coated with an insulating water repellant layer and which top side is coated with a gel of modified polysaccharide comprising a conjugated system.
According to a preferred aspect of the invention, the electrical conductor material is selected from the group comprising, preferably consisting of, carbon fibers, carbon nanotubes, graphene, graphene derivatives, graphite and mixtures thereof. The carbon nanotubes can be, for example, single-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes or carbon nanotubes arrays. These electrical conductor materials are especially suitable as they make it possible for a good chemical interaction between the bioelectrode and the coating. Indeed, these materials have aromatic rings that can interact with the conjugated system of the modified polysaccharide. In particular, when the conjugated system of the modified polysaccharide comprises at least an aromatic ring, pi-stacking interaction can occur between the aromatic rings of the coating and the electrical conductor material of the bioelectrode. The pi-stacking interaction is a non- covalent attractive force between aromatic rings. This interaction contributes to the good stability of the membrane over time as it is strong enough to prevent disbonding of the coating from the bioelectrode.
According to one embodiment, the bioelectrode comprises
a material selected from the group comprising, preferably consisting of, carbon nanotubes, graphite and graphene,
- and at least an enzyme.
In one embodiment of the invention, the bioelectrode is a bioanode.
According to a further aspect, the invention also relates to biofuel cells or biosensors comprising at least one bioelectrode as described above. For example, the biosensor can be a glucose biosensor.
From another aspect thereof, the invention also pertains to a method for producing a bioelectrode as described above, said method comprising:
- a) implementation of a bioelectrode, preferably comprising a material selected from the group comprising, preferably consisting of, carbon nanotubes, graphite and graphene,
b) coating said bioelectrode with a gel of modified polysaccharide comprising a conjugated system.
In one embodiment, the bioelectrode is a disk of compressed enzyme and carbon nanotubes.
In a preferred embodiment of the invention, step b) is done by
applying a solution or a suspension of modified polysaccharide comprising a conjugated system onto the surface of the bioelectrode,
crosslinking this solution by applying a solution of divalent or polyvalent cations onto the solution of modified polysaccharide on the surface of the bioelectrode, in order to form a gel. In another embodiment of the invention, step b) is done by
mixing a solution or a suspension of modified polysaccharide comprising a conjugated system and a solution of divalent or polyvalent cations, and, before crosslinking and formation of a gel,
applying the resulting solution or suspension onto the surface of the electrode.
According to a further aspect, the invention also relates to a hydrogel of modified alginate, the modified alginate comprising a conjugated system. According to one embodiment, the conjugated system comprises at least one aromatic ring. The conjugated system can be a polycyclic aromatic hydrocarbon. According to a preferred embodiment of the present invention, the conjugated system is selected from the group comprising, preferably consisting of, porphyrins, phthalocyanines, pyrene, benzene, indole, azulene, phenothiazines, naphthalene, perylene, anthracene, phenanthrene, corene, corannulene, ovalene, other molecular-benzene-based structures, cyclic aromatic hydrocarbons, graphene-like structures, and mixtures thereof, preferably pyrene.
The modified alginate is synthesized by amidation of the carboxylic groups with an amine comprising a conjugated system. The amidation is undergone using standard amidation procedures, for example, by using N-hydroxysulfosuccinimide and l-Ethyl-3-(3-dimethyl- aminopropyl)carbodiimide as reagents.
In particular, the amount of modified carboxylic group of the alginate is between 15 and 30%, preferably between 20 and 25% of the total amount of carboxylic groups of the alginate.
The hydrogel of modified alginate comprising a conjugated system is obtained by crosslinking using divalent or polyvalent cations, like Ca2+ or Ba2+. In particular, a solution of divalent or polyvalent cations is added to a solution of modified alginate comprising a conjugated system, this induces the crosslinking and gelation occurs rapidly.
In a preferred embodiment, the hydrogel of modified alginate is a hydrogel of pyrene modified alginate and Ca2+. The schematic structure of such a hydrogel is shown in figure 1. This hydrogel is particularly suitable for coating bioelectrodes, especially made from carbon nanotubes as the pyrene moiety allows good pi- stacking interaction with the carbon nanotubes. Brief description of the figures
Figure 1 shows a schematic representation of a gel of pyrene modified alginate crosslinked by Ca2+ ions.
Figure 2 shows photographs of carbon nanotube (CNT) pellet electrodes: (A) unmodified (according to example 1), (B) modified with pyrene-alginate film after addition of 0.1 M CaCl2 (according to example 4) and (C) modified with alginate film after addition of 0.1 M CaCl2 (according to example 2).
Figure 3 shows optical microscopy images recorded using lOx objective of CNT pellets (A) before (according to example 1) and (B) after modification with alginate and addition of 0.1 M CaCl2 (according to example 2); (C) before (according to example 1) and (D) after modification with pyrene alginate and addition of 0.1 M CaCl2 (according to example 4).
Figure 4 shows cyclic voltammograms recorded in 1 mM K3FeCN6 in 0.1 M KCl of CNT pellets (A, Pill) before (according to example 1) and (A, Pill-pyAlginate) after modification with pyrene alginate and addition of 0.1 M CaCl2 (according to example 4); (B, Pill) before (according to example 1) and (B, Pill-pyAlginate) after modification with alginate and addition of 0.1 M CaCl2 (according to example 2). Scan rate = 5 mV/s.
Figure 5 shows cyclic voltammograms recorded in phosphate buffer pH 7 of CNT pellet (Pill) before (according to example 1) and (Pill-pyAlginate) after modification with pyrene alginate and addition of 0.1 M CaCl2 (according to example 4). Scan rate = 5 mV/s.
Figure 6 shows five consecutive cyclic voltammograms recorded in 1 mM K3FeCN6 in 0.1 M KCl of CNT pellets (A, i-v) after modification with pyrene alginate and addition of 0.1 M CaCl2 (according to example 4) ; (B, i-v) after modification with alginate and addition of 0.1 M CaCl2 (according to example 2). Scan rate = 5 mV/s.
Figure 7 shows cyclic voltammograms recorded in 0.5 mM FcMeOH in PB pH 7 of CNT pellets (A) after modification with pyrene alginate and addition of 0.1 M CaCl2 (according to example 4), after immersion in the solution for (i) 0 min, (ii) 120 min and (iii) 1200 min. Examples
Materials used for the preparation of the electrodes The electrodes were synthesized using commercial products. Mono-sodium phosphate monohydrate (98-102%), di-sodium hydrogen phosphate heptahydrate (98-102%), potassium ferricyanide (K3FeCN6, 99%), potassium chloride (KC1, > 99%), calcium chloride (meets USP testing specifications), ferrocene methanol (FcMeOH, 97%), ruthenium hexamine (Ru(NH3)6, 98%), alginic acid sodium salt, TRIZMA® base hydrochloride (Tris HC1, > 99%), sodium hydroxide (98-100.5%) and potassium chloride (99-100.5%) were purchased from Sigma-Aldrich and used without further purification. Aqueous solutions were prepared from ultrapure water at 25°C (resistivity > 18.2 MQcm). Commercial grade multi-walled carbon nanotubes (MWCNTs, 9.5 nm diameter, > 95% purity) were obtained from Nanocyl and used as received without purification.
Example 1 : Preparation of carbon nanotube pellet electrode (CNT electrode)
First 35 mg of multi-walled carbon nanotubes (MWCNTs) was mixed with 300 of water using a spatula. Next, the wet CNTs mixture was placed inside a metal cell having an internal diameter of 13 mm then compacted with an applied force of 1.5 tons in a hydraulic press (Perkin Elmer). The resulting disks have a diameter of 13 mm and a width of 2 mm. A metal wire was attached to the back side of the electrode using carbon paste. Silicone gel was subsequently applied as an insulating water repellent layer to cover the back side and the edges of the electrode, leaving the top surface area exposed (area = 1.3 cm ).
Example 2: Preparation of alginate film-modified carbon nanotube pellet electrodes
An aqueous alginate (2 % w/v) solution was prepared in 0.1 M Tris HCL buffer pH 7 at room temperature then left stirring overnight at 200 rpm to aid solubilization. Next, 150 of the alginate solution was delicately added to the surface of the electrode. To obtain the alginate gel film on the electrode surface, 100 of 0.1 M CaCl2 was dispersed on the surface. The gel formed rapidly within seconds. The electrode was left overnight to dry then rinsed with 0.1 M Tris HC1 buffer pH 7.
Example 3: preparation of pyrene modified alginate (pyrene- alginate)
Pyrenemethylammonium hydrochloride (540 mg, 2.0 mmol) was added to a solution of NaOH (120 mg, 3 mmol) in water (60 mL). The resulting suspension was extracted with CH2C12 (3x40 mL). Removal of the solvent under vacuum yields a white oil of pyrenemethylamine (410 mg, 1.8 mmol, 90% yield). The pyrene-alginate conjugate was prepared by adding 214 mg (2.5 mmol) of pyrenemethylamine to a solution of alginate (2.5 mmol of alginate monomer, 500 mg) in 100 mL of 0.1 M MES buffer, pH 6.0. The reaction mixture was stirred at room temperature for 10 min to facilitate a homogeneous dispersion of the pyrenemethylamine reagent in the reaction solution. Then 135 mg of NHSS (N- hydroxysulfosuccinimide, 0.625 mmol) and 240 mg of EDC (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, 1.25 mmol) were added (ratios of reagents were calculated for a theoretical 20-25% molar modification of the number of carboxylic groups of alginate). The reaction was stirred for 20 h, and modified alginate was precipitated by adding NaCl in order to have a concentration of 1 M, and two volumes of ethanol. In order to remove unreacted alginate, precipitation was repeated three times using the same procedure after dissolving modified alginate at 1% (w/v) concentration in water. Modified alginate was dried over night at 40 °C in the dark and stored at -20 °C.
Example 4: Preparation of pyrene-alginate film-modified carbon nanotube pellet electrodes
A pyrene-alginate film-modified carbon nanotube pellet electrodes was prepared according to example 2 using pyrene-alginate obtained in example 3 instead of alginate.
Figure 2 shows pictures of the different electrodes according to example 1, 2 and 4. These photographs indicate a change before and after coating. Figure 3 shows optical microscopy images of the different electrodes according to example 1, 2 and 4. Optical microscopy images were obtained using a Keyence VK X200 Series Microscope with VK 2.5 software. These images indicate a change in surface topography following the coating of the CNT pellet. Ra roughness values were estimated from the -1.5 mm images using VK analyzer software. The average roughness values reveal a decrease in surface roughness on modification, consistent with the formation of a film. Indeed, the roughness decreases from image A (carbon nanotube pellet) to image B (carbon nanotube pellet + alginate gel) due to the presence of an alginate film on the carbon nanotube pellet. The roughness exhibits an even more important decrease from image C (carbon nanotube pellet) to image D (carbon nanotube pellet + pyrene alginate gel) due to the presence of a pyrene alginate film interacting with the carbon nanotube pellet.
Cyclic voltammetry
Electrochemical measurements were performed at room temperature using an Eco Chemie Autloab PGSTAT 100 potentiostat running GPES 4.9 software. A conventional three- electrode cell set-up was used for all electrochemical experiments comprising a CNT pellet working electrode, a saturated calomel (SCE) reference electrode and a Pt wire counter electrode. Example 5
Cyclic voltammetry experiments were recorded to compare CNT electrodes before and after coating. The experiments were conducted in K3FeCN6, using the different electrodes according to examples 1, 2 and 4, using a negatively-charged surface-sensitive redox probe, and with KC1 as supporting electrolyte. The results are shown in figure 4.
For the pyrene-alginate film, no significant change was observed, with or without the coating. This is consistent with the presence of a highly permeable film on the surface. Under these conditions, this indicates a fast electron transfer. In contrast, the alginate coated electrode exhibited a blocking behavior towards the potassium ferricyanide redox probe consistent with the presence of a less permeable film. This is shown by the increase in peak- to peak separation and current decrease. Example 6
Cyclic voltammetry experiments were recorded in phosphate buffer pH 7 without the addition of KC1 as supporting electrolyte, using the electrodes according to examples 1 and 4. The results are shown in figure 5.
These results revealed a significant increase in the capacitive current for the electrode according to example 4 (CNT pellet coated with a pyrene-alginate film), consistent with the formation of an organic film on the surface. Due to the large negative reduction at -0.2 V, the potential window may be restricted for cathode applications.
Example 7
Cyclic voltammetry experiments in K3FeCN6 were recorded to assess the stability of CNT pellets after coating with a pyrene-alginate or an alginate film. The experiments were conducted using the different electrodes according to examples 1, 2 and 4, using a negatively- charged surface- sensitive redox probe and with KC1 as supporting electrolyte. The results are shown in figure 6.
The cyclic voltammogram of the CNT pellet modified with pyrene-alginate (according to example 4: Fig.6A) did not change over 5 scans, consistent with the formation of a stable film. In contrast, CNT pellet modified with alginate (example 2: Fig.6B) changed considerably with each of the 5 scans, becoming increasingly less blocking towards K3FeCN6 ions in solution. The cyclic voltammogram response after 5 scans of the alginate modified CNT pellet is similar to that of the unmodified surface.
Example 8
Cyclic voltammetry experiments were recorded to assess the stability of CNT pellets over time after coating with a pyrene-alginate or an alginate film. The experiments were conducted in FcOH (Fc=ferrocene), using the different electrodes according to example 1, 2 and 4, using a neutral surface-sensitive redox probe and with KC1 as supporting electrolyte. The results are shown in figure 7.
The CNT pellet modified with pyrene-alginate showed time dependant behaviour whereby the reduction and oxidation peak currents increased after 120 min immersion in the redox probe solution, consistent with an improvement in the permeability of the film with soaking in aqueous solution. Further immersion of the electrode resulted in a slight decrease in the anodic current at Ep= 0.3 V and the emergence of a new peak at Ep = 0.45. The new peak is most likely ferrocene related and may be attributed to ferrocene groups trapped within the film.

Claims

Claims
1. Bioelectrode coated with a gel of modified polysaccharide comprising a conjugated system.
2. Bioelectrode according to claim 1 wherein the polysaccharide is alginate.
3. Bioelectrode according to claims 1 or 2 wherein the conjugated system comprises at least one aromatic ring.
4. Bioelectrode according to any of claims 1 to 3 wherein the conjugated system is selected from the system comprising, preferably consisting of, porphyrins, phthalocyanines, pyrene, benzene, indole, azulene, phenothiazines, naphthalene, perylene, anthracene, phenanthrene, corene, corannulene, ovalene, other molecular- benzene-based structures, cyclic aromatic hydrocarbons, graphene-like structures, and mixtures thereof, preferably pyrene.
5. Bioelectrode according to any of claims 1 to 4 wherein the modified polysaccharide comprising a conjugated system is obtainable by amidation, esterification and/or thioesterification of carboxylic acid groups of the polysaccharide, respectively, with at least one amine, at least one alcohol or at least one thiol comprising the conjugated system.
6. Bioelectrode according to claim 5 wherein the amount of modified carboxylic group of the polysaccharide is between 15 and 30%, preferably between 20 and 25% of the total amount of carboxylic groups of the polysaccharide.
7. Bioelectrode according to any of claims 1 to 6 wherein the gel of modified polysaccharide comprising a conjugated system is a hydrogel comprising
modified polysaccharide comprising conjugated systems,
divalent or polyvalent cations,
and water.
8. Bioelectrode according to any of claims 1 to 7 wherein said bioelectrode comprises a material selected from the group comprising, preferably consisting of, carbon nanotubes, graphite and graphene,
and at least an enzyme.
9. Bioelectrode according to any of claims 1 to 8 wherein said bioelectrode is a bioanode.
10. Biofuel cell comprising a bioelectrode according to any of claims 1 to 9.
11. Biosensor comprising a bioelectrode according to any of claims 1 to 9.
12. A method for producing a bioelectrode according to claim 1 to 9 comprising:
a) implementation of an bioelectrode, preferably comprising a material selected from the group comprising, preferably consisting of, carbon nanotubes, graphite and graphene,
b) coating said bioelectrode with a hydrogel of modified polysaccharide comprising a conjugated system.
13. Method according to claim 12 wherein step b) is done by
applying a solution or a suspension of modified polysaccharide comprising a conjugated system onto the surface of the bioelectrode,
crosslinking this solution by applying a solution of divalent or polyvalent cations onto the solution of modified polysaccharide on the surface of the bioelectrode, in order to form a gel.
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