An electrode for an enzyme biosensor using a fiber material, a method for preparing the same, and the biosensor

Abstract

To provide an enzymatic biosensor, and notably the enzymatic biosensor for an electrode which can be easily produced and thus is inexpensive, and which allows a liquid sample that can contain an analyte to be assayed to be present in an adjustable quantity and to be agitated and expelled easily.SOLUTION: The present invention relates to an enzymatic biosensor electrode formed of a fibrous material and comprised of electrically conductive fiber and electrically non-conductive fiber functionalized in whole or part by identical or different enzymes. The present invention also relates to a method of preparing such an electrode and an electrochemical detection enzymatic biosensor including the same.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention belongs to the field of electrochemical devices, and more particularly to enzyme biosensors or bioreactors that implement electrochemical detection.

[0002] More specifically, the present invention relates to an electrode for an enzyme biosensor, comprising, in particular, different fibrous materials, wherein some of the different fibrous materials are suitable for ensuring electrical conductivity, and others are suitable for immobilizing enzymes and enabling the conversion of assay analytes into measurable reaction products.

[0003] The present invention also relates to a method for preparing such electrodes and the use thereof. [Background technology]

[0004] Enzyme biosensors or enzymatic bioreactors enable the transformation of biological or chemical analytes by one or more enzymes. In the case of enzyme biosensors, the reaction products or enzyme cascades of the analyte with the enzyme are measured, and the initial undetectable analytes can be studied retrospectively. Glucose sensors are the best known example of enzyme sensors, enabling the monitoring of individuals with diabetes. Detection of the enzyme reaction products may be optical (colorimetric or fluorescent) or electrochemical if the products can exchange electrons with a conductive material.

[0005] Whatever type of biosensor may be conceived, it will contain two distinct elements: 1) one or more enzymes that enable the conversion of the assay analyte (in this case, glucose) into a measurable reaction product through an electrochemical reaction; and 2) conductive electrodes that enable the transfer of electrons to the electrochemical reaction and / or to the electron measurement.

[0006] There are three main types of electrochemical detection enzyme biosensors.

[0007] These types of first-generation enzymatic biosensors, or "first-generation biosensors," utilize enzymes that produce products that can be directly reduced or oxidized on an electrode. For example, such biosensors implement the reduction or oxidation of hydrogen peroxide (H2O2) produced from glucose by an enzymatic reaction on an electrode in the presence of oxygen.

[0008] The second type of enzyme biosensor, known as the "second-generation biosensor," uses a redox mediator that exchanges electrons with enzyme reaction products in solution. Once modified, the mediator exchanges electrons with the conductive electrode of the biosensor when it is its turn. This type of biosensor allows for the elimination of the oxygen input required for the operation of first-generation bioreactors, if the mediator directly interacts with the active center of the enzyme, or for lowering the redox potential through the use of a so-called "fast" mediator (which has a high rate of electron transfer with and from solution) that acts as a redox shuttle between the electrode and one of the enzyme reaction products with a low electron transfer rate, such as H2O2.

[0009] Furthermore, first- and second-generation biosensors include a metal electrode covered with a biofilm that contains enzymes trapped in a porous or diffusing material and allows the analyte to pass through. The analyte must penetrate the biofilm to come into contact with the enzyme. Once the enzymatic reaction is carried out, the reaction products must reach the electrode by diffusion for detection. Since not all reaction products diffuse towards the electrode, this diffusion reduces the efficiency of the sensor. It is possible to reduce the thickness of the enzyme-containing layer to statistically increase contact between the reaction products and the electrode. In this case, the amount of available enzyme is limited, and the usable range and / or sensitivity of the sensor decreases. In fact, the substrate rapidly becomes in excess compared to the available enzyme, and the sensor rapidly saturates. In addition, the sensitivity of the sensor is fixed by the amount of active enzyme immobilized on the electrode.

[0010] Third-generation biosensors utilize enzymes that have the ability to directly couple to conductive electrodes and directly exchange electrons. Several materials have been used to produce these electrodes using various packaging techniques. For example, one can cite nanometer metal beads, particularly gold, platinum, or carbon-based electrodes; zinc oxide (ZnO) nanowires on silver electrodes; and even nanowires produced by electrospinning. A list of materials is given by Mahbur Rahman et al., 2010 [1]. Electrodes for third-generation biosensors are difficult to produce and require expensive packaging techniques, and therefore they are not widely used. Furthermore, the chemical reaction of enzyme immobilization is determined by the properties of the electrode being packaged.

[0011] One advantage of using a three-dimensional electrode is that, unlike a flat electrode where the electrode is immersed in solution, the electrode partially contains a liquid sample capable of containing the analyte for assay. Therefore, this advantage allows for quantitative reactions of the entire substrate contained within the electrode (the concept of a bioreactor), which is not possible with a flat electrode and, moreover, takes a very long time. This functionality is interesting not only in the case of electroenzymatic bioproduction of chemical products, but also in the context of biosensors for embedded measurement of substrate quantities, enabling better detection sensitivity.

[0012] Another weakness of these three-dimensional electrodes is their rigid nature. In fact, often based on metal, electrodes are rigid and do not deform. The volume of liquid sample that can "enter" a three-dimensional electrode, containing an analyte for assay, is determined by the free volume between the nanowire or nanobead-type nanostructures that make up the electrode. This liquid is not only difficult to agitate but also difficult to drain. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Mahbur Rahman et al, 2010, “A comprehensive review of glucose biosensors based on nanostructured metal-oxide”, Sensor (Basel), vol. 10, pages 4855-4886. [Overview of the project] [Problems that the invention aims to solve]

[0014] Therefore, the inventors set the goal of proposing an enzyme biosensor for electrodes, in particular, one that is easy to prepare, and therefore inexpensive, and in which a liquid sample capable of containing an assay analyte is present in an adjustable amount and can be easily stirred and discharged. [Means for solving the problem]

[0015] This invention makes it possible to achieve the objectives set by the inventors and to solve all or part of the technical problems of prior art enzyme biosensors.

[0016] Therefore, the inventors propose a three-dimensional electrode type enzyme biosensor electrode that has the advantage of being able to contain a liquid sample that can contain an analyte for assay.

[0017] The unique feature of the electrode according to the present invention is that, because the electrode is in the form of a compressible and elastic fibrous material, it can adapt to the amount of liquid sample being measured. This adaptation offers two advantages. This adaptation makes it possible to measure an adjustable amount of liquid sample within a range corresponding to its deformability. Furthermore, by simply applying pressure, it is possible to agitate the liquid sample contained within the structure and promote the encounter between the enzyme and the analyte by alternately applying positive and negative pressure to the electrode. Finally, the liquid sample contained within the three-dimensional electrode can be discharged by pressure at the end of the measurement. Due to the compressibility and elasticity of the electrode, it is possible to periodically fill and empty the electrode at the beginning and end of the measurement. This capability enables continuous measurements (filling and discharging the liquid before another measurement).

[0018] The "compressible material" shall mean a porous fibrous material having the ability to increase or decrease its volume when absorbing or discharging a liquid by mechanical compression or extension or by capillary action. The deformation of the electrode according to the present invention is included between 5% and 200%, more specifically between 30% and 90%. This deformation can be obtained directly, for example, using a traction table or any other device that allows bringing a distance between two points or two surfaces while enabling applying a given pressure to the zone where measurement is performed using a thickness gauge, calipers, microscope observation, traction table. In either case, the compression value is obtained from the difference between the first thickness obtained without applying pressure or under a pressure of 1 kPa or less and the second thickness obtained under a pressure exceeding 1 kPa, especially between 1 kPa (the boundary is not included) and 50 kPa, considering differences in the thickness of the material, differences in dryness or wetness. The "wet sample" shall mean a sample that has been placed in an aqueous solution for at least overnight so that it is completely filled with the aqueous solution, and this sample is then placed on the measuring device without drying or washing it. The measurement is usually performed using a traction table, which enables measurement of displacement. Thus, without pre-filling, as a function of the force (F) applied to a sample of defined dimensions (S) at an average test speed of 10 mm / min, a pressure value (P = F / S) can be obtained, and it becomes possible to measure the deformation under a pressure in the range of 100 Pa to 50 kPa.

[0019] "Elasticity" shall mean the ability of a material to recover to its original shape when no mechanical force is applied.

[0020] More specifically, the present invention relates to an electrode for an enzyme biosensor in the form of a fibrous material, comprising conductive fibers and fibers that are the same as or different from the conductive fibers and are functionalized by the same or different enzymes. Several different embodiments can be envisaged for the electrode according to the present invention.

[0021] Thus, in a first embodiment, the electrode according to the present invention comprises or consists of conductive fibers, and all or part of these conductive fibers are functionalized by the same or different enzymes.

[0022] In this first embodiment, the electrode comprises or consists only of conductive fibers on all or part of which the same or different enzymes are chemically immobilized.

[0023] In the second embodiment, the electrode according to the invention comprises or consists of conductive and non-conductive fibers, all or part of which are functionalized with the same or different enzymes.

[0024] In this second embodiment, the electrode comprises or consists only of two types of fibers: 1) conductive fibers on all or part of which the same or different enzymes are chemically immobilized, and 2) non-conductive fibers. When only part of the conductive fibers is functionalized with enzymes, the non-conductive fibers, and optionally the non-functionalized conductive fibers, serve as elastic fibers to double the elastic properties of the fiber material constituting the electrode according to the invention.

[0025] In the third embodiment, the electrode according to the invention comprises or consists of conductive fibers and non-conductive fibers, all or part of which are functionalized with the same or different enzymes.

[0026] In this third embodiment, the electrode comprises or consists only of two types of fibers: 1) conductive fibers, and 2) non-conductive fibers on all or part of which the same or different enzymes are chemically immobilized. When only part of the non-conductive fibers is functionalized with enzymes, the other non-conductive and non-functionalized fibers serve as elastic fibers to double the elastic properties of the fiber material constituting the electrode according to the invention.

[0027] In the fourth embodiment, the electrode according to the invention comprises or consists of conductive fibers, a first type of non-conductive fibers not functionalized with enzymes, and at least one second type of non-conductive fibers different from the first type of non-conductive fibers, all or part of which are functionalized with the same or different enzymes.

[0028] This fourth embodiment is a specific implementation of the third embodiment.

[0029] In this fourth embodiment, the electrode includes or is composed of at least three types of fibers: 1) conductive fibers, 2) first type nonconductive fibers that are not functionalized with enzymes, and 3) at least one second type nonconductive fiber different from the first type, on which the same or different enzymes are chemically immobilized in all or part thereof. The first type nonconductive fibers, and optionally at least one second type nonconductive fiber that is not functionalized with enzymes, serve as elastic fibers.

[0030] The phrase "at least one second type of nonconductive fiber" means that in this fourth embodiment, there may be not only a second type of nonconductive fiber different from the first type, but also up to two, three, four, or five different types of nonconductive fibers, all or some of which are functionalized by enzymes, and that these different types of fibers are not only different from the first type of nonconductive fiber, but also different from each other. The different types of nonconductive fibers implemented in this embodiment, in particular the first and second types of nonconductive fibers, are distinguished from each other, in particular at the level of their chemical composition and / or their yarn count.

[0031] In this fourth embodiment, it is possible to have different types of nonconductive fibers, for each type, in which at least one portion of the fiber is functionalized with a specific enzyme. To achieve this objective, each type of fiber may retain chemical properties suitable for the fixation of a specific enzyme. This embodiment makes it possible to obtain electrodes arranged on different fibers and containing several enzymes, thus enabling the production of composite electrodes composed of specifically mixed fibers coupled to different enzymes, for example, for sequence cascade enzyme reactions. Furthermore, it should be noted that since all embodiments of the electrodes according to the present invention assume the functionalization of fibers to be implemented with different enzymes, these electrodes can be used to carry out cascade enzyme reactions.

[0032] Regardless of the embodiment, the electrode according to the present invention is in the form of a compressible and elastic fibrous material.

[0033] The third and fourth embodiments of the electrode according to the present invention offer the advantage of distinguishing conductive fibers from enzyme-retaining fibers. In fact, it is possible to prepare the three-dimensional electrode according to the present invention using fibers with different properties, thereby enabling localized concentration of enzyme-retaining fibers relative to conductive fibers.

[0034] This offers a dual advantage: it ensures that the conductive fibers do not retain chemical properties that could alter the conductivity or electron transfer capability between the enzyme reaction products and these electrochemical reactants on the conductive fibers. This separation between the enzyme-retaining fibers and the conductive fibers also allows the enzyme to select the properties of the immobilized fiber, thereby enabling the selection of simpler chemical reactions or higher chemical yields. The chemical reaction of enzyme immobilization is not determined by the properties of the electrodes on which it is implemented.

[0035] Finally, another advantage of the electrodes according to the present invention, particularly the advantages of the electrodes according to the third and fourth embodiments, is that, if desired, the ratio of conductive fibers to enzyme-retaining fibers can be modified to produce different bioreactors with the same fibers, and because only these ratios differ, it is possible to adapt them to the concentration of the medium being tested.

[0036] In the third and fourth embodiments of the electrode according to the present invention, such as those defined above, the ratio of fibers to be mounted is 5% to 90% conductive fibers, 10% to 80% non-conductive and enzyme-retaining fibers, and 0% to 80% elastic fibers. More specifically, these ratios are 50% to 70% conductive fibers, 10% to 50% non-conductive and enzyme-retaining fibers, and 0% to 30% elastic fibers.

[0037] In addition, in the third and fourth embodiments of the electrode according to the present invention, such as those defined above, the average distance between the conductive fiber and the enzyme-retaining fiber is 100 nm to 200 μm.

[0038] The electrode according to the present invention is in the form of a fibrous material, that is, a material whose main component is fiber. This material may be in the form of a woven fabric, a knitted fabric, or a nonwoven fabric.

[0039] Within the scope of the present invention, "fiber" means a one-dimensional or substantially one-dimensional structure having a thickness or diameter that varies between 500 nm and 100 μm, particularly between 1 μm and 50 μm, and especially between 4 μm and 30 μm.

[0040] The length of the fibers implemented in this invention is selected as a function of the technique used to prepare the fibrous material of the electrodes. Typically, the length of the implemented fibers is greater than 4 mm. This length may be between 5 mm and 120 mm, particularly between 6 mm and 20 mm, especially when these fibers are used to prepare nonwoven fabrics via the airlaid method. Alternatively, the length of the implemented fibers may be between 15 mm and 180 mm, particularly between 30 mm and 120 mm, especially when these fibers are used to prepare carded nonwoven fabrics. In a further alternative, the fibers implemented in this invention may have a length greater than 180 mm, particularly greater than 200 mm, especially when these fibers are used to prepare woven or knitted fabrics. In this alternative, the fibers may be in the form of wires or filaments, and may be obtained by processing shorter fibers, for example, by spinning.

[0041] Advantageously, the fibers implemented in the present invention have yarn counts ranging from 0.2 dTex to 30 dTex, particularly from 0.3 dTex to 20 dTex.

[0042] Within the scope of the present invention, "conductive fiber" means a fiber that is conductive either originally or after treatment, such as those previously defined. Such fibers are selected from the group consisting of metal fibers, carbon fibers, fibers made of conductive polymers or copolymers, fibers made of conductive polymer composites (CPCs), and fibers made conductive through coating or metallization.

[0043] Examples of metal fibers include fibers made from metals, metal oxides, metal nitrides, or metal sulfides, where the metal is selected from the group consisting of one of gold, copper, stainless steel, silver, nickel, aluminum, platinum, palladium, molybdenum, or alloys thereof.

[0044] Carbon fibers that can be used within the scope of the present invention may belong to any of three main families of carbon fibers: ex-cellulose fibers obtained by carbonizing materials such as paper or viscose; ex-PAN fibers produced using polyacrylonitrile (PAN) as a precursor; and ex-bitumen fibers produced from aromatic residues of petroleum or coal distillation. Specific examples of fibers made from conductive polymers or copolymers include fibers made from polyaniline (PANI), poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) coupled to sodium poly(styrenesulfonate), polypyrrole, or polyacetylene. It should be noted that these conductive polymers or copolymers are also designated as "inherently conductive polymers (ICPs)."

[0045] Fibers made from filled polymer composites (CPCs) are, for example, fibers made from polymers or copolymers containing particles such as carbon nanotubes or carbon black, metal particles, or intrinsically conductive polymers (ICPs), such as those previously cited, or mixtures of these different types of particles.

[0046] Conductive fibers that can be used within the scope of the present invention also form a group with fibers that have been made conductive by coating, for example, through the deposition of a mixture of polymers or copolymers having conductive particles, or by metallization using techniques such as electroplating, plasma, or CVD (chemical vapor deposition). A further specific example of fibers made conductive by coating is gold-coated fibers.

[0047] In certain embodiments, the conductive fibers implemented within the scope of the present invention are carbon fibers, and more specifically, ex-PAN fibers.

[0048] Within the scope of the present invention, "non-conductive fiber" means a fiber previously selected from non-conductive natural fibers and non-conductive chemical fibers, such as those previously defined.

[0049] Specific examples of non-conductive natural fibers include materials selected from the group consisting of cotton, wool, linen, jute, cellulose, hemp, raffia, sisal, silk, tussah, byssal, alginate, polysaccharides, and mixtures thereof, or fibers made from these materials.

[0050] Chemical fibers include synthetic fibers and artificial fibers. Specific examples of non-conductive chemical fibers include materials selected from the group consisting of glass, rayon, chitosan, polyolefins such as polyethylene (PE) or polypropylene (PP); polytetrafluoroethylene (PTFE); polyesters such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN); polyamides; polyimides; polycarbonates (PC) and mixtures thereof, or fibers made from these materials.

[0051] In certain embodiments, non-conductive fibers implemented within the scope of the present invention include low-melting-point fibers, i.e., fibers whose melting point is less than 190°C, particularly between 105°C and 170°C. Such low-melting-point fibers are fibers that can be used as fibers in which enzymes are immobilized by covalent bonds, and / or as elastic fibers. A specific example of such low-melting-point fibers corresponds to the PET / coPET fibers sold by Fiber Partner (registered trademark). In the second, third, and fourth embodiments of the electrodes according to the present invention, such as those defined above, the proportion of non-conductive low-melting-point fibers is typically 0-50%, particularly 10-30%. The use of low-melting-point fibers is interesting because it is possible to bond the fibers together by appropriate heat treatment that allows for partial melting of these fibers.

[0052] The enzyme implemented in the electrode according to the present invention is selected from the group consisting of oxidoreductase, oxygenase, peroxidase, catalase, transhydrogenase, dehydrogenase, transferase, hydrolase, lyase, and ligase. Advantageously, the enzyme implemented in the electrode according to the present invention is oxidoreductase.

[0053] More specifically, the enzymes implemented in the electrode according to the present invention include creatinase, creatinamide hydrolase, reductase, esterase, catalase, uric acid oxidase, galactose oxidase, histamine oxidase, choline oxidase, glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, gluconate dehydrogenase, glutamate oxidase, glutamate dehydrogenase, cholesterol oxidase, cholesterol esterase, lactate oxidase, lactate dehydrogenase, ascorbic acid oxidase, and pirubin. The enzymes selected are from the group consisting of oxidase, alcohol oxidase, alcohol dehydrogenase, aldehyde dehydrogenase, bilirubin oxidase, choline oxidase, xanthine oxidase, amino acid oxidase, peroxidase, urease, formate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, methylamine dehydrogenase, succinate dehydrogenase, fumarate reductase, p-cresol methyl hydroxylase, glutamate oxaloacetate transaminase, and glutamate pyrubate transaminase.

[0054] Within the scope of the electrode according to the present invention, all or part of the fibers are functionalized with the same or different enzymes, such as those previously defined. This functionalization implements at least one chemical bond. "Chemical bond" means not only covalent chemical bonds but also non-covalent chemical bonds, such as ionic bonds, hydrogen bonds, hydrophobic bonds, or van der Waals bonds. Thus, the enzyme is immobilized, grafted, or fixed to the lateral portion of the conductive or non-conductive fiber by covalent or non-covalent bonds. This immobilization, grafting, or fixation may be localized to a limited and defined zone on the surface, or conversely, the enzyme may be distributed across the entire surface.

[0055] Within the scope of the present invention, the immobilization, grafting, or fixation of the enzyme onto conductive or non-conductive fibers may be direct or indirect, i.e., the functionalization of conductive or non-conductive fibers with enzymes may be direct or indirect.

[0056] In a direct manner, the chemical bonds implemented involve atoms present on the surface of the fiber and atoms of enzymes.

[0057] When fixation is indirect, fixation, grafting, or immobilization involves a spacer arm (or binder) on which one end is bonded to the surface of the fiber and the other end is bonded to the enzyme. The bond implemented may be covalent or non-covalent. The spacer arm creates two functionalities: one is to impart mobility to the structure (e.g., carbon-chemical type polyethylene glycol (PEG), alkyl groups, polyethylene terephthalate (PET)), and the other is to bind the enzyme to the electrode. This binding function may be symmetrical (same at each end of the spacer arm) or asymmetrical (e.g., silane, thiol, aldehyde, epoxy functional group).

[0058] To promote chemical bonding between the enzyme and the surface of the electrode fibers according to the present invention, both the enzyme and the fiber surface retain or are replaced by at least one reactive functional group, either the same or different. If a spacer arm is implemented, the spacer arm retains two types of reactive functional groups, either the same or different.

[0059] "Reactive functional group" refers to, within the scope of the present invention, a carboxyl functional group (reactive with amine or alcohol functional groups), an aryl group (e.g., pyrene, naphthalene, or polycyclic aromatic), a radical, a hydroxyl functional group or alcohol functional group (reactive with carboxyl or isocyanate), an amine functional group (reactive with ester or carboxylic acid functional groups), an ester functional group (reactive with amine functional groups), an aldehyde functional group (reactive with hydrazide functional groups), and a hydrazide. Zide functional group (reactive with aldehyde functional group), ketone functional group (reactive with two alcohol functional groups for acetalization), epoxy functional group (reactive with amine functional group), isocyanate functional group (reactive with hydroxyl functional group), maleimide functional group (reactive with thiol functional group, amine functional group, or diene functional group), diene functional group (reactive with maleimide functional group), thiol functional group (reactive with maleimide or another thiol functional group), phosphonate functional group (zirconium (Zr 4+ ) or titanium (Ti 4+ (Ion chelation is possible), functional group chelated zirconium ion (Zr 4+ ), titanium ions (Ti 4+ ), iron ions (Fe 3+ ) and / or gallium ions (Ga 3+ ) (Phosphopeptide immobilization is possible), biotin (can bind to avidin or streptavidin), avidin or streptavidin (can bind to biotin) and polyhistidine tag (metal ion, e.g., nickel ion (Ni 2+ ) or cobalt ion (Co 2+ This refers to a functional group selected from among those that can be bonded to ).

[0060] These reactive functional groups may naturally be present at the enzyme level, on the fiber surface, or on the spacer arm. For example, an enzyme containing at least one cysteine ​​in its amino acid sequence naturally possesses a thiol functional group. Similarly, a spacer arm naturally holds two types of reactive functional groups, one or different.

[0061] Alternatively, this reactive functional group may need to be introduced at the enzyme level and / or on the surface of the fiber.

[0062] With respect to enzymes, reactive functional groups can be introduced by functionalizing the amino acids at the C-terminal position, N-terminal position, and / or side chains of the amino acid sequence. Any functionalization technique known to those skilled in the art can be used for this purpose. For example, thiol functional groups can be introduced into enzymes using isocyanates, isothiocyanates, or succinate ester type reagents. It is clear that the introduction of reactive functional groups into enzymes should not substantially alter the enzyme's activity.

[0063] With respect to the surface of fibers, it is possible to introduce reactive functional groups, particularly via oxidation treatment, by techniques known to those skilled in the art for functionalizing metal, carbon, or polymer surfaces. In fact, oxidation treatment aims to oxidize the surface of fibers by immobilizing and / or introducing oxygen-rich, identical or different groups, i.e., identical or different groups containing at least one oxygen atom, particularly groups selected from the group consisting of carboxyl groups (-C(=O)OH), hydroxyl groups (-OH), alkoxyl groups (-OX, where X represents an alkyl, acyl, or aryl group), carbonyl groups (-C(=O)-), percarbonate groups (-C(=O)-O-OH), and amide groups (-C(=O)NH2).

[0064] Such oxidation treatments are based on two main types of surface modification: - Physical treatments, such as plasma treatments, especially oxygen treatments, UV treatments, gamma or X-ray treatments, and irradiation treatments using electrons and heavy ions. - Chemical treatments, such as treatment with alcoholic potassium carbonate, treatment with a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) (also known as the "piranha mixture"), treatment with strong acids (HCl, H2SO4, HNO3, HClO4), treatment with soda, treatment with strong oxidizing agents (KMnO4 in hydrochloric acid, K2Cr2O7, KClO3 or CrO3, sulfuric acid or nitric acid), treatment with ozone, and heat treatment under oxygenated air (O2, H2O, etc.).

[0065] The present invention relates to a method for preparing electrodes for enzyme biosensors, such as those previously defined.

[0066] The method for preparing electrodes according to the third and fourth embodiments comprises at least the following two steps: - A step of mixing conductive fibers with non-conductive fibers, and - A process of functionalizing certain non-conductive fibers with enzymes. These two steps are carried out sequentially in any order.

[0067] In the first alternative, this method is a1) A process of functionalizing conductive or non-conductive fibers with enzymes, b1) A step of mixing an enzyme-functionalized conductive or nonconductive fiber with another fiber selected from the group consisting of conductive fibers, nonconductive fibers, and mixtures thereof, wherein if the enzyme is immobilized on the conductive fiber in step a1), this mixing step is optional, and c1) If step b1) is optional for obtaining a fiber material, then a step to form a mixture of fibers obtained in step b1) or step a1). Includes.

[0068] More specifically, the first alternative method is: a1) A process of functionalizing non-conductive fibers with enzymes, b1) A step of mixing an enzyme-functionalized non-conductive fiber with another fiber selected from the group consisting of conductive fibers, non-conductive fibers, and mixtures thereof, and c1) A process of molding the fiber mixture obtained in step b1) to obtain a fiber material. Includes.

[0069] This alternative method, without step b1), corresponds in particular to a method for preparing an electrode for an enzyme biosensor according to the first embodiment, such as those previously described, wherein the electrode comprises conductive fibers covalently immobilized on the enzyme throughout.

[0070] Prior to step a1), conductive or non-conductive fibers can be subjected to oxidation treatment, such as those previously described.

[0071] "Mixture" means not only a mixture of different types of conductive fibers, a mixture of different types of non-conductive fibers, but also a mixture of at least one type of conductive fiber and at least one type of non-conductive fiber.

[0072] In the second alternative method, this method is a2) A step of functionalizing conductive or non-conductive fibers with reactive functional groups, b2) A step of mixing conductive or nonconductive fibers functionalized with reactive functional groups with other fibers selected from the group consisting of conductive fibers, nonconductive fibers, and mixtures thereof, and c2) A process to obtain a fiber material by molding the fiber mixture obtained in step b2). The enzymatic functionalization of conductive or non-conductive fibers, which include and have been pre-functionalized with reactive functional groups, is carried out either after step b2) and before step c2), or after step c2).

[0073] More specifically, this second alternative method is: a2) A process of functionalizing non-conductive fibers with reactive functional groups, b2) A step of mixing a non-conductive fiber functionalized with a reactive functional group with another fiber selected from the group consisting of conductive fibers, non-conductive fibers, and mixtures thereof, and c2) A process to obtain a fiber material by molding the fiber mixture obtained in step b2). The enzymatic functionalization of non-conductive fibers, which include and have been pre-functionalized with reactive functional groups, is carried out either after step b2) and before step c2), or after step c2).

[0074] This second alternative method has two embodiments, in which the immobilization, grafting, or immobilization of enzymes onto conductive or non-conductive fibers functionalized with reactive functional groups is performed on the mixture of step b2) (first embodiment) or after the fibers have been formed (second embodiment).

[0075] During step a2), the functionalization of conductive or nonconductive fibers with reactive functional groups comprises (i) a step of subjecting the fibers to an oxidation treatment, (ii) a step of fixing, grafting, or immobilizing reactive functional groups, such as those defined herein, onto the fibers, and / or (iii) a step of fixing, grafting, or immobilizing spacer arms that hold the reactive functional groups onto the fibers.

[0076] In the third alternative method, this method is a3) A step of mixing conductive fibers and non-conductive fibers, b3) A process of molding the fiber mixture obtained in step a3) to obtain a fiber material. The functionalization of conductive or non-conductive fibers by enzyme is carried out either after step a3) and before step b3), or after step b3).

[0077] More specifically, this third alternative method is: a3) A step of mixing conductive fibers and non-conductive fibers, b3) A process of molding the fiber mixture obtained in step a3) to obtain a fiber material. The enzymatic functionalization of the non-conductive fibers is carried out either after step a3) and before step b3), or after step b3).

[0078] This third alternative method also has two embodiments, according to which the immobilization, grafting or fixing of the enzyme onto the conductive or non-conductive fibers is carried out in the mixture of step a3) (first embodiment) or is carried out after the fiber shaping has been carried out once (second embodiment).

[0079] One skilled in the art will know, without making inventive efforts, the method of selecting the reactive functional groups that must hold the enzyme and what must necessarily hold the conductive or non-conductive fibers in order to obtain on these fibers a specific immobilization, grafting or fixing of the enzyme that is present only in the mixture of fibers being implemented.

[0080] Regardless of what is assumed for the alternative method, any technique for shaping the fibers to produce a woven or non-woven fiber material can be used during steps c1), c2) and b3).

[0081] Examples of techniques that can be used during steps c1), c2) and b3) of the method according to the invention for producing a non-woven fiber material include dry techniques using carding or airlaid methods, spunbonded, meltblown, melt techniques using electrospinning methods, or alternatively wet techniques. Advantageously, the shaping during said steps c1), c2) and b3) is carried out using a dry technique, for example a carding method or an airlaid method.

[0082] Examples of techniques that can be used during steps c1), c2) and b3) of the method according to the invention for producing a woven or knitted fiber material include weaving or knitting techniques.

[0083] The woven, knitted or non-woven fiber material obtained following steps c1), c2) and b3) has a mass between 100 g / m 2 and 800 g / m 2 , especially between 200 g / m 2 and 300 g / m 2 per unit area.

[0084] The fibrous material obtained following steps c1), c2), and b3) can be subjected to solidification. In other words, the method according to the present invention may include a solidification step after steps c1), c2), and b3). Any technique for solidifying nonwoven, woven, or knitted fibrous materials can be used for this purpose. Thus, the fibrous material obtained following steps c1), c2), and b3), particularly the nonwoven fibrous material obtained following steps c1), c2), and b3), can be solidified by different bonding methods, particularly needling, hydrobonding, thermal bonding, calendering, or alternatively by chemical bonding, stitch bonding, or a combination of these different bonding methods. Typically, the fibrous material obtained following steps c1), c2), and b3), particularly the nonwoven fibrous material obtained following steps c1), c2), and b3), can be solidified by mechanical bonding after thermal bonding.

[0085] The present invention also relates to an electrochemical detection type enzyme biosensor, including electrodes, such as those defined herein. Such a biosensor may also be referred to as an "enzymatic bioreactor with electrochemical detection."

[0086] In this biosensor, electrodes, such as those previously defined, function as the working electrode (WE) thanks to the conductive fibers they contain. For the counter electrode (CE), a platinum grid, platinum wire, titanium-platinum plate, a paste of carbon black and activated carbon encapsulated in a stainless steel grid, or a paste of carbon black, activated carbon and Teflon® encapsulated in a stainless steel grid can be used. For the reference electrode (RE), a saturated calomel electrode, such as a calomel electrode saturated with potassium chloride or sodium chloride, an Ag / AgCl electrode, or a platinum wire can be used instead.

[0087] Such biosensors are particularly useful for the detection and potential quantification of analytes of interest, selected from the group consisting of, in particular, ethanol, glucose, skin microorganisms, toxins, chemical or biological compounds related to medical conditions, nutrients, metabolic by-products, e.g., urea or cholesterol, hormones, environmental ligands, or combinations thereof. Such applications are found in the fields of medicine, agrofood, or the environment. [Brief explanation of the drawing]

[0088] [Figure 1] This is a schematic representation of a method for preparing an electrode, for example, an electrode as defined in a fourth embodiment of the present invention, the method being, for example, the method defined in a first alternative method of the present invention. [Figure 2] This is a schematic representation of a method for preparing an electrode, for example, an electrode as defined in a fourth embodiment of the present invention, the method being, for example, the method defined in a second alternative method of the present invention. [Figure 3] This is a schematic representation of a method for preparing an electrode, for example, an electrode as defined in a fourth embodiment of the present invention, the method being, for example, a method defined in a third alternative method of the present invention. [Modes for carrying out the invention]

[0089] I. Electrode for enzyme biosensor according to the present invention. The electrode according to the present invention is made of a nonwoven material. - Conductive fibers, which are carbon fibers with a diameter between 4 μm and 8 μm, typically derived from polyacrylonitrile (PAN) based multifilaments. - Non-conductive and enzyme-retaining PET fibers of counts between 0.3dTex and 17dTex, particularly between 0.5dTex and 2dTex, wherein oxidoreductase and glucose oxidase type enzymes are covalently immobilized in these non-conductive and enzyme-retaining fibers, and - Elastic fibers with a low melting point, such as LowMelt 2-component PET / coPET fibers with a count in the range of 2dTex to 12dTex. Includes.

[0090] In this electrode, the ratio of fibers implemented is between 50% and 80% carbon fibers, between 10% and 50% enzyme-retaining fibers, and between 10% and 30% elastic fibers.

[0091] The fibers are interwoven in contact zones, where conductive fibers are in contact with each other and with enzyme-retaining fibers, while in other zones the fibers are separated from each other.

[0092] II. A method for preparing electrodes by a first alternative method. In this method, whose principle is schematically shown in Figure 1, the non-conductive PET fibers are pre-functionalized with a glucose oxidase-type enzyme.

[0093] This functionalization process involves first subjecting the fibers to surface oxidation to generate -OH or -COOH polar functional groups on the fiber surface, and then chemically grafting glucose oxidase-type enzymes onto the fibers via peptide bonds (e.g., generation of activated esters and amide bonds, esterification, etc.).

[0094] The functionalized fibers are then mixed with carbon fibers and LowMelt 2-component PET / coPET elastic fibers.

[0095] Airlaid technology is used to produce nonwoven materials. To do this, the fibers of a fiber mixture are released and then inserted into a device, where they are mixed by airflow to obtain a nonwoven with a homogeneous distribution of fibers. Upon discharge from the device, a controlled portion of the fiber mixture is deposited onto the carpet by air, thus achieving a density of 100 g / m² per unit area. 2 From 800g / m 2 During that period, especially 200g / m 2 From 300g / m 2 It forms a web of mass between them.

[0096] This web is then solidified. The solidification technique used is preferably thermal bonding in an oven. The resulting fibrous material acts as the working electrode (WE) thanks to the conductive fibers it contains. As the counter electrode (CE), a platinum grid, platinum wire, titanium platinum plate, a paste of carbon black and activated carbon sealed in a stainless steel grid, or a paste of carbon black, activated carbon and Teflon® sealed in a stainless steel grid can be used. As the reference electrode (RE), a saturated calomel electrode, for example, a calomel electrode saturated with potassium chloride or sodium chloride, an Ag / AgCl electrode, or a platinum wire can be used instead.

[0097] III. A method for preparing electrodes according to a second alternative method. In this method, whose principle is schematically shown in Figure 2, PET fibers are subjected to surface oxidation to generate -OH or -COOH polar functional groups on the surface of the fibers, and then the fibers, i.e., the pre-functionalized PET fibers, are mixed with carbon fibers and elastic LowMelt 2-component PET / coPET fibers.

[0098] The mixture thus obtained is subjected to the airlaid technique described for the method in item II above.

[0099] Once the web is obtained, it is brought into contact with glucose oxidase-type enzymes and placed under conditions that allow for the chemical immobilization of these enzymes onto the pre-functionalized PET fibers contained within the web via peptide bonds (e.g., formation of activated ester and amide bonds, esterification).

[0100] The remainder of this method, namely coagulation, is the same as that described for the method in item II above.

[0101] IV. A method for preparing electrodes according to a third alternative method. In this third alternative method, the principle is schematically shown in Figure 3, PET fibers, carbon fibers, and elastic LowMelt 2-component PET / coPET fibers are all mixed together.

[0102] The mixture thus obtained is subjected to the airlaid technique described for the method in item II above.

[0103] Once the web is obtained, it is brought into contact with glucose oxidase-type enzymes and placed under conditions that allow for the chemical immobilization of these enzymes on the PET fibers contained in the web via covalent bonding.

[0104] The remainder of this method, namely coagulation, is the same as that described for the method in item II above.

Claims

1. It is a form of fibrous material, comprising conductive fibers and non-conductive fibers. All or part of the aforementioned non-conductive fibers are functionalized with the same or different enzymes. The aforementioned conductive fibers are not functionalized with enzymes. An electrode for an enzyme biosensor, wherein the enzyme is distributed throughout the entire lateral portion of the non-conductive fiber.

2. The electrode according to claim 1, characterized by comprising a conductive fiber, a first type of nonconductive fiber not functionalized with an enzyme, and at least one second type of nonconductive fiber different from the first type of nonconductive fiber, all or part of which is functionalized with the same or different enzymes.

3. The electrode according to claim 1 or 2, characterized in that it is in the form of a compressible and elastic fibrous material.

4. The electrode according to any one of claims 1 to 3, characterized in that the fibrous material is in the form of a woven fabric, a knitted fabric, or a nonwoven fabric.

5. The electrode according to any one of claims 1 to 4, characterized in that the conductive fiber is selected from the group consisting of metal fibers, carbon fibers, fibers made of conductive polymers or copolymers, fibers made of conductive polymer composites (CPCs), and fibers made conductive through coating or metallization.

6. The electrode according to any one of claims 1 to 5, characterized in that the enzyme is selected from the group consisting of oxidoreductase, oxygenase, peroxidase, catalase, transhydrogenase, dehydrogenase, transferase, hydrolase, lyase, and ligase.

7. The electrode according to any one of claims 1 to 6, characterized in that the enzymatic functionalization of the non-conductive fiber is direct or indirect.

8. A method for preparing an electrode for an enzyme biosensor according to any one of claims 1 to 7, a1) A step of functionalizing the non-conductive fiber with an enzyme in the entire side portion of the non-conductive fiber, b1) A step of mixing enzyme-functionalized non-conductive fibers with other fibers selected from the group consisting of enzyme-unfunctionalized conductive fibers, non-conductive fibers, and mixtures thereof, and c1) A process of molding the fiber mixture obtained in step b1) to obtain a fiber material. Methods that include...

9. A method for preparing an electrode for an enzyme biosensor according to any one of claims 1 to 7, a2) A step of functionalizing the non-conductive fiber with a reactive functional group on the entire side surface portion of the non-conductive fiber, b2) A step of mixing non-conductive fibers functionalized with reactive functional groups with other fibers selected from the group consisting of conductive fibers, non-conductive fibers, and mixtures thereof that have not been functionalized with enzymes, and c2) A process to obtain a fiber material by molding the fiber mixture obtained in step b2). A method comprising a nonconductive fiber that has been pre-functionalized with a reactive functional group, wherein the enzymatic functionalization of the nonconductive fiber is carried out after step b2) and before step c2), or after step c2).

10. A method for preparing an electrode for an enzyme biosensor according to any one of claims 1 to 7, a3) A step of mixing conductive fibers and non-conductive fibers that have not been functionalized with enzymes, and b3) A process of molding the fiber mixture obtained in step a3) to obtain a fiber material. A method comprising the enzymatic functionalization of the nonconductive fiber in the entire lateral portion of the nonconductive fiber, which is performed after step a3) and before step b3), or after step b3).

11. The method according to claim 8, characterized in that the fibrous material is in the form of a nonwoven fabric, and the molding in step c1) is carried out using a dry technique.

12. The method according to claim 9, characterized in that the fiber material is in the form of a nonwoven fabric, and the molding in step c2) is carried out using a dry technique.

13. The method according to claim 10, characterized in that the fiber material is in the form of a nonwoven fabric, and the molding in step b3) is carried out using a dry technique.

14. The method according to claim 8 or 11, characterized in that the fibrous material obtained after step c1) is subjected to solidification.

15. The method according to claim 9 or 12, characterized in that the fibrous material obtained after step c2) is subjected to solidification.

16. The method according to claim 10 or 13, characterized in that the fibrous material obtained after step b3) is subjected to solidification.

17. An electrochemical detection type enzyme biosensor comprising the electrode described in any one of claims 1 to 7.

Images