Method for preparing a working electrode comprising laser irradiation of a sensing material and corresponding analyte sensor

Abstract

A method for preparing a working electrode (122) on a sensor substrate (114) is disclosed, the method comprising the steps of: a) providing at least one sensor substrate (114) including at least a first side (120), the first side (120) including at least one conductive trace (111), b) applying at least one layer of at least one sensing material (118) onto the first side (120) of the sensor substrate (114), the sensing material (118) covering at least a portion of the at least one conductive trace (111), and c) irradiating the layer of sensing material (118) with at least one laser beam, whereby at least the first portion of the layer of sensing material (118) is at least partially removed. a step of irradiating the layer of sensing material (118) such that at least a second portion of the sensing material (118) covering at least one conductive trace (111) is maintained on the first surface (120) of the sensor substrate (114) to obtain at least one working electrode (122) on the sensor substrate (114); and e) a step of applying at least one membrane layer, the membrane layer at least partially covering the working electrode and the membrane layer including at least one cross-linking agent for cross-linking at least a portion of the sensing material, the method further including at least one diffusion step, in which the cross-linking agent included in the membrane layer at least partially diffuses into the sensing material.

Description

Technical Field

[0001] The present invention generally relates to a method for preparing a working electrode, an analyte sensor comprising the working electrode, and the use of the analyte sensor for detecting at least one analyte in a sample. In particular, the present invention relates to a method for preparing a working electrode, which method includes laser irradiation.

Background Art

[0002] Monitoring of specific body functions, more specifically monitoring of the concentration of one or more specific analytes, plays an important role in the prevention and treatment of various diseases.

[0003] In addition to so-called point measurements in which a sample of body fluid is specifically taken from a user and investigated for analyte concentration, continuous measurements are becoming increasingly available. Therefore, there is a growing need for accurate analyte sensors that enable reliable and cost-effective analyte detection from body fluids or other samples. Analyte sensors for determining the concentration of analytes under in vivo conditions are known from WO 2010 / 028708 pamphlet. Another example of such a sensor is disclosed in WO 2012 / 130841 pamphlet. Further, WO 2007 / 147475 pamphlet discloses an amperometric sensor configured to be implanted in a living body and measure the concentration of an analyte in a body fluid. Alternative sensor elements are disclosed in WO 2014 / 001382 pamphlet.

[0004] In the manufacture of commercially available analyte sensors, a structuring step is required to form a layer of sensing material on the substrate of the sensor, such as a continuous or long-term glucose monitoring sensor. Current methods for performing this structuring step include screen printing, dip coating, and dispensing methods.

[0005] These methods are disadvantageous in that the sensing material cannot be applied sufficiently uniformly on the sensor substrate, especially such that non-uniformities occur at the edges of the sensor substrate. These so-called edge effects vary the sensor sensitivity across the charge. To reduce the variation of the sensor signal sensitivity across different charges, detailed monitoring of manufacturing parameters and precise adaptation are required. This can be time-consuming and costly.

[0006] WO 2006 / 018447 discloses a method for preparing an electrode assembly for use in an electrochemical sensor. In this method, a conductive layer is applied to a dielectric substrate and conductive traces are formed by laser ablation. Details regarding the addition of a layer of sensing material to the working electrode are not shown.

[0007] U.S. Pat. No. 9,880,126 discloses a biosensor and a method for its manufacture. The sensor includes a carbon layer that can be prepared by laser ablation. Details regarding the addition of a layer of sensing material to the working electrode are not shown.

[0008] WO 2010 / 099507 discloses a method and system for providing continuous analyte monitoring, including an in vivo sensor that does not require any user calibration during in vivo use. Additionally, the present invention discloses a method for manufacturing a calibration-free sensor as well as post-manufacture packaging and storage techniques.

[0009] JP 2019078573 discloses a method for manufacturing a biosensor that reduces the sensitivity during manufacture of the biosensor, independent of the concentration of the component to be measured. In this method for manufacturing a biosensor, an electrode layer of a biosensor including an electrode system including at least a working electrode and a counter electrode is formed on an insulating substrate, and a detection layer including a component that reacts with the component to be measured is formed on the electrode layer. After the detection layer is formed, at least the detection layer is scraped off.

[0010] U.S. Patent Application Publication No. 2014 / 0054171 discloses an analyte sensor configured to utilize oxygen as an oxidizing agent, as well as methods for manufacturing and using the same. The analyte sensor includes a catalyst to facilitate the use of oxygen as an oxidizing agent. The catalyst may be provided on the electrodes of the analyte sensor.

[0011] Feldmann et al., Diabetes Technology & Therapeutics, Vol. 5, No. 5, 2003, 769 - 780 disclose a study using 48 sensors implanted for three days. Forward calibration was performed using capillary blood.

[0012] European Patent Application Publication No. 2 119 795 relates to a disposable electrode strip for attachment to a signal readout circuit of a sensor system for detecting a current representative of an analyte in an aqueous medium.

[0013] The problem to be solved by the present invention is to provide a method for preparing an analyte sensor that avoids the above - mentioned disadvantages. In particular, the present invention aims to provide a preparation method that results in a homogeneous sensing material structure on the working electrode such that the need for detailed monitoring and precise adaptation of manufacturing parameters is avoided or at least significantly reduced.

[0014] Therefore, it is desirable to provide a method for preparing an analyte sensor that addresses the above - described technical problems. It is further desirable to provide an analyte sensor that has high reproducible sensitivity over charge and can be manufactured at low cost, for example, by using a preparation process that avoids detailed monitoring and precise adaptation of manufacturing parameters. SUMMARY OF THE INVENTION

[0015] This problem is addressed by a method for preparing a working electrode having the features of the independent claims and an analyte sensor comprising the working electrode. Advantageous embodiments, which can be implemented alone or in any combination, are listed in the dependent claims and throughout this specification.

[0016] The method according to the invention is advantageous for enabling the production of a working electrode that can be included in an analyte sensor having a highly reproducible sensor sensitivity over charge. Furthermore, the sensitivity can be selected and precisely adjusted during production. Since detailed monitoring and precise adaptation of production parameters can be avoided, costs can be reduced and factory calibration of the sensor is possible. Furthermore, sensor drift can be reduced.

[0017] When used hereinafter, the terms "having", "comprising" or "including" or any grammatical variations thereof are used in a non-exclusive manner. Thus, these terms may refer to both situations where there are no additional features in the entity being described in this context in addition to the features introduced by these terms, and situations where one or more additional features are present. By way of example, the expressions "A has B", "A comprises B" and "A includes B" all refer to both situations where there are no other elements in A other than B (i.e., the situation where A consists solely and exclusively of B), and situations where one or more additional elements such as element C, elements C and D, or even further elements are present in entity A in addition to B.

[0018] Furthermore, note that the terms "at least one", "one or more" or similar expressions indicating that a feature or element can be present one or more times are usually only used once when introducing each respective feature or element. In the following, in most cases, when referring to each respective feature or element, the expressions "at least one" or "one or more" are not repeated, despite the fact that each respective feature or element can be present one or more times.

[0019] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "in particular", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with any feature without limiting the possibility of alternatives. Thus, the features introduced by these terms are any features and are not intended to limit the scope of the claims in any way. The present invention may be practiced, as will be recognized by those skilled in the art, by using alternative features. Similarly, features introduced by "in an embodiment of the present invention" or similar expressions are without limitation as to alternative embodiments of the present invention, without limitation as to the scope of the present invention, and are intended to be any features without limitation as to the possibility of combining the features introduced in such a way with any other optional or non-optional features of the present invention.

[0020] According to the present invention, a method for preparing a working electrode on a sensor substrate is disclosed. The working electrode may be part of an analyte sensor.

[0021] The method specifically includes the following steps that can be executed in a given order. Further, two or more process steps may be executed simultaneously or partially simultaneously. Further, one or more, or even all, of the method steps may be executed one or more times, or even repeatedly or continuously. The method may further include additional method steps not described. The method includes the following steps: a) Providing at least one sensor substrate including at least a first surface, the first surface including at least one conductive trace, providing one sensor substrate; b) Adding at least one layer of at least one sensing material on the first surface of the sensor substrate, the sensing material covering at least a part of at least one conductive trace, adding at least one layer; and c) irradiating a layer of a sensing material with at least one laser beam, wherein at least a first portion of the layer of the sensing material is at least partially removed, and at least a second portion of the sensing material covering at least one conductive trace is maintained on a first surface of the sensor substrate to obtain at least one working electrode on the sensor substrate, the step of irradiating the layer of the sensing material.

[0022] A further object of the present invention is a method for preparing a working electrode (122) on a sensor substrate (114), comprising: a) providing at least one sensor substrate (114) including at least a first surface (120), the first surface (120) including at least one conductive trace (111), the step of providing at least one sensor substrate (114); b) adding at least one layer of at least one sensing material (118) on the first surface (120) of the sensor substrate (114), the sensing material (118) covering at least a part of at least one conductive trace (111), the step of adding at least one layer; c) irradiating the layer of the sensing material with at least one laser beam, wherein at least a first portion of the layer of the sensing material (118) is at least partially removed, and at least a second portion of the sensing material (118) covering at least one conductive trace (111) is maintained on the first surface (120) of the sensor substrate (114) to obtain at least one working electrode (122) on the sensor substrate (114), the step of irradiating the layer of the sensing material; e) adding at least one film layer, the film layer at least partially covering the working electrode (122), the film layer including at least one crosslinking agent for crosslinking at least a part of the sensing material (118), the step of adding at least one film layer, and the method further comprises: f) at least one diffusion step, in which the crosslinking agent contained in the film layer diffuses at least partially into the sensing material (118), the method including at least one diffusion step.

[0023] The embodiments and preferences described below apply to both of the methods described above.

[0024] As used herein, the term "working electrode" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, without limitation, this term can refer to the electrode of an analyte sensor that is sensitive to an analyte. The working electrode may be disposed on at least one sensor substrate including at least one first surface. In particular, the working electrode includes at least one conductive trace and at least one layer of at least one sensing material disposed on the conductive trace on the first surface of the sensor substrate. The sensing material can cover at least a portion of at least one conductive trace. The first portion of the layer of sensing material may be laser irradiated by at least one laser beam such that the second portion of the sensing material covering the conductive trace is maintained on the first surface (120) of the sensor substrate (114), and the second portion of the sensing material together with the conductive trace forms at least one working electrode. The working electrode may be included in an analyte sensor. The analyte sensor typically further comprises additional electrodes such as, for example, a counter electrode and / or a reference electrode. The layer of sensing material may be present only on the working electrode and typically may be absent of any additional electrodes. For example, the counter electrode and / or the reference electrode may not include the layer of sensing material.

[0025] Furthermore, the present invention discloses a method for preparing an analyte sensor. The method for preparing an analyte sensor includes a method for preparing a working electrode on a substrate disclosed herein and a step of providing at least one additional electrode.

[0026] The analyte sensor may be configured for at least partial implantation into a user's body tissue, specifically for percutaneous insertion. More specifically, the analyte sensor may be configured for continuous monitoring of an analyte. Even more specifically, the analyte sensor may be configured for continuous glucose monitoring.

[0027] The terms "user" and "subject" are used interchangeably herein. These terms can specifically relate to humans.

[0028] As used herein, the term "analyte sensor" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically refer to, but is not limited to, any element or device configured to detect or measure the concentration of at least one analyte. The analyte sensor can specifically be an analyte sensor suitable for at least partial implantation into a user's body tissue, more specifically, an analyte sensor for continuous monitoring of an analyte.

[0029] The analyte sensor of the present invention is an electrochemical sensor comprising a working electrode that can be obtained according to the method of the present invention, at least one additional electrode and respective circuitry. More particularly, the sensor is an amperometric electrochemical sensor comprising at least one working electrode. Typically, the analyte sensor comprises at least one additional electrode, particularly a counter electrode and / or a reference electrode or a combination of counter / reference electrodes. The working electrode is sensitive to an analyte that is measured at a polarization voltage that can be applied between the working electrode and the reference electrode and can be adjusted by a potentiostat. The measurement signal can be provided as a current between the counter electrode and the working electrode. There may not be a separate counter electrode, and there may be a pseudo-reference electrode, which can also function as a counter electrode. Thus, the analyte sensor can typically comprise a set of at least two electrodes, in embodiments a set of three electrodes. In an embodiment, the analyte sensor comprises exactly two electrodes, particularly exactly one working electrode and one combined counter / reference electrode. In particular, the sensing material is present only at the working electrode.

[0030] In particular, the analyte sensor according to the present invention can be fully or partially implantable and can thus be adapted to perform the detection of analytes in body fluids in subcutaneous tissue, in particular interstitial fluid. Other parts or components may remain outside the body tissue. For example, as used herein, the terms "implantable" or "subcutaneous" refer to being placed fully or at least partially within the body tissue of the user. For this purpose, the analyte sensor can comprise an insertable part, and the term "insertable part" generally refers to a part or component of an element configured to be insertable into any body tissue while the counter electrode and / or reference electrode or a combined counter / reference electrode can remain outside the body tissue. Preferably, the insertable part can be fully or partially provided with a biocompatible surface, and the biocompatible surface can be made to have as little harmful effect as possible on the user or body tissue, at least during a typical period of use. For this purpose, the insertable part may be fully or partially covered by at least one polymer membrane, for example at least one biocompatible membrane layer such as a gel membrane, which on the one hand may be permeable to body fluids or at least analytes contained therein and on the other hand may be impermeable to compounds contained in the analyte sensor, in particular the working electrode, thus preventing its migration into the body tissue. Further details regarding the biocompatible membrane layer are disclosed elsewhere in this specification.

[0031] Furthermore, the term "analyte" as used herein is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not exclusively, refer to any element, component, or compound that can be present in a body fluid and whose concentration can be of interest to a user. Specifically, an analyte can be or can include any chemical substance or chemical compound that can be involved in a user's metabolism, such as at least one metabolite. By way of example, the at least one metabolite can be selected from the group consisting of glucose, cholesterol, triglyceride, lactate, ketone, urea, creatinine, glutamate, ethanol, ascorbic acid, choline, acetylcholine, polyphenol, monophenol, dihydroxyphenol, bisphenol A (BPA), and hydrochlorothiazide (Hct). More specifically, the analyte can be glucose. However, additionally or alternatively, other types of analytes and / or any combination of analytes can be determined, such as the concentration of electrolytes, or pO2, pCO2, pH, or Hb values indicating the concentration of oxygen, carbon dioxide, hydrogen, or hemoglobin, respectively.

[0032] Furthermore, the term "sensor substrate" as used herein is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not exclusively, refer to any type of material or combination of materials suitable for forming a carrier layer for supporting the conductive traces and / or layers of sensing materials described herein. In particular, the "sensor substrate" as understood herein can include an electrically insulating material.

[0033] As used herein, the term "layer" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, but not limited thereto, this term can refer to an element of the layer configuration of an analyte sensor. Specifically, the term "layer" can refer to any coating of any substrate, specifically a flat substrate. A layer can specifically have a lateral extension that exceeds a thickness of at least 2 times, at least 5 times, at least 10 times, and even at least 20 times or more. Specifically, an analyte sensor can have a layer configuration. An analyte sensor can include a plurality of layers such as at least one conductive trace, at least one layer of at least one sensing material, and at least one film layer. One or more layers of an analyte sensor can include sublayers. For example, a layer including a conductive trace may include at least one additional layer.

[0034] As used herein, the term "electrically insulating material" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. "Electrically insulating material" can also refer to a dielectric material. Specifically, but not limited thereto, this term can refer to a material or combination of materials that prevents the movement of electric charges and does not sustain a large current. Specifically, without limiting other possibilities, at least one electrically insulating material can be or can include at least one insulating resin such as an insulating epoxy resin used in the manufacture of an electronic printed circuit board. In particular, it can include or be those thermoplastic materials such as polycarbonate, polyesters such as polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or their copolymers such as glycol-modified polyethylene terephthalate, polyethylene naphthalate, polytetrafluoroethylene, or alumina.

[0035] In the method and analyte sensor according to the present invention, the sensor substrate can include two opposing surfaces, at least a first surface and at least a second surface opposing the first surface.

[0036] Specifically, the analyte sensor, more specifically the sensor substrate, can further include at least one additional electrode, and the at least one additional electrode can include at least one of a reference electrode and a counter electrode. In an embodiment, the at least one additional electrode includes a combined counter / reference electrode. In particular, the reference electrode may include at least one reference electrode conductive trace. And / or the counter electrode may include at least one counter electrode conductive trace. When the additional electrode includes a combined counter / reference electrode, it should be understood that the embodiments and preferences described with respect to the reference electrode conductive trace and the counter electrode conductive trace apply mutatis mutandis to the conductive trace of the combined counter / reference electrode. More specifically, the at least one additional electrode may be disposed on at least one of the first surface of the sensor substrate and the second surface opposing the first surface.

[0037] As used herein, the term "conductive trace" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, without limitation, this term can refer to a conductive strip, layer, wire, or other type of elongated conductor. More specifically, the term "conductive trace" can refer, without limitation, to a material that is conductive and thus can sustain an electric current. For example, a conductive trace can include at least one material selected from the group consisting of: carbon, carbon paste, gold, copper, silver, nickel, platinum, palladium. Specifically, a conductive trace can be or include at least one metal such as one or more of gold, copper, silver, nickel, palladium, or platinum. Additionally or alternatively, at least one conductive trace can be or include at least one conductive compound such as at least one conductive organic or inorganic compound. Additionally or alternatively, at least one conductive trace can be or include at least one non-metallic conductive material, such as polyaniline, poly-3,4-ethylenedioxythiophene (PEDOT), carbon, or carbon paste. Carbon paste can specifically relate to a material that includes carbon, a solvent such as diethylene glycol butyl ether, and at least a binder such as a vinyl chloride copolymer and a terpolymer. Preferably, the conductive trace according to the present invention can include gold and / or carbon. More preferably, the conductive trace can consist of gold and / or carbon. Specifically, the conductive trace can include gold and an additional material, such as carbon.

[0038] Furthermore, the conductive trace may include at least one additional layer of at least one additional material. Specifically, the additional layer may include an additional conductive material. More specifically, the additional layer of the conductive trace may include carbon or may consist of carbon. The additional layer may be disposed on the first surface. Using an additional layer that includes carbon or consists of carbon within the conductive trace on the sensor substrate can be advantageous because it can improve the adhesion of the layer of sensing material on the conductive trace. Further, in the case of carbon, the additional material can contribute to efficient electron transfer by the conductive trace.

[0039] The conductive trace can have a thickness of at least 0.1 μm, preferably at least 0.5 μm, more preferably at least 5 μm, specifically at least 7 μm, or at least 10 μm. When the conductive trace includes carbon or is carbon, the conductive trace can have a thickness of specifically at least 7 μm, more specifically at least 10 μm. Specifically, when the conductive trace is gold, the conductive trace can have a thickness of at least 100 nm, more specifically at least 500 nm.

[0040] The minimum thickness as described above can be advantageous for ensuring proper electron transport. A thickness below the specified value is usually not sufficient for reliable electron transport. Even more specifically, the thickness should not exceed a value of 30 μm in the case of carbon and should not exceed a value of 5 μm in the case of gold. If the thickness is too large, the overall thickness and thus the size of the analyte sensor may increase. A larger analyte sensor size is generally not desirable because it can cause difficulties during implantation.

[0041] As used herein, the terms "reference electrode conductive trace" and "counter electrode conductive trace" are broad terms and should be given their ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. These terms can specifically, but not limited to, refer to conductive strips, layers, wires or other types of elongated conductors present on the reference electrode or the counter electrode respectively. More specifically, these terms can, but not limited to, refer to materials that are conductive and thus can sustain an electric current. For example, the reference electrode conductive trace and / or the counter electrode conductive trace can include at least one of the materials described hereinabove with respect to the conductive trace. In addition to the above materials, the reference electrode conductive trace and / or the counter electrode conductive trace can specifically include Ag / AgCl.

[0042] As used herein, the term "detection material" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, without limitation, this term can refer to at least one polymer material or a material that may include at least one polymer material. Specifically, it can be at least one polymer material and at least one metal-containing complex, or may include these. The metal-containing complex can be selected from the group of transition metal element complexes. Specifically, the metal-containing complex can be selected from osmium complexes, ruthenium complexes, vanadium complexes, cobalt complexes, and iron complexes, such as ferrocene, such as 2-aminoethylferrocene. Even more specifically, the detection material can be a polymer transition metal complex as described, for example, in WO 01 / 36660 pamphlet, the content of which is incorporated by reference. In particular, the detection material can include a modified poly(vinylpyridine) backbone carrying a poly(bisimidazolyl)Os complex covalently bonded via a bidentate bond. The detection material is further described in Feldmann et al., Diabetes Technology & Therapeutics, 5(5), 2003, 769-779, the content of which is incorporated by reference. Suitable detection materials may further include ferrocene-containing polyacrylamide-based biologen-modified redox polymers, pyrrole-2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-pyrene, naphthoquinone-LPEI. The polymer transition metal complex can represent a redox mediator incorporated into a cross-linked redox polymer network. This is advantageous because it can facilitate electron transfer between at least one enzyme or analyte and the conductive trace. To avoid sensor drift, the redox mediator and enzyme may be covalently incorporated into the polymer structure.

[0043] In an embodiment, the detection material may include a polymer material and MnO2 particles.

[0044] Furthermore, the sensing material may include at least one enzyme. Specifically, the enzyme can catalyze a chemical reaction that consumes at least the analyte. Specifically, the enzyme is H 2 O 2 generating and / or consuming enzymes, and more specifically, glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), (S)-2-hydroxy acid oxidase (EC 1.1.3.15), cholesterol oxidase (EC 1.1.3.6), glucose dehydrogenase, galactose oxidase (EC 1.1.3.9), alcohol oxidase (EC 1.1.3.13), L-glutamate oxidase (EC 1.4.3.11) or L-aspartate oxidase (EC 1.4.3.16), and even more specifically, glucose oxidase (GOx) and / or its mutants can be used.

[0045] Furthermore, the sensing material may further include at least one cross-linking agent. The cross-linking agent can cross-link at least a part of the sensing material, for example. Specifically, the sensing material may include at least one cross-linking agent selected from a UV curable cross-linking agent and a chemical cross-linking agent. More specifically, the sensing material includes a chemical cross-linking agent. Alternatively, the sensing material may not include a cross-linking agent. As used herein, "not including any cross-linking agent" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term can specifically, but not limited to, refer to a situation where no cross-linking agent is present in the sensing material during step b) of applying to the first surface of the sensor substrate. More specifically, "not including a cross-linking agent" can refer to a concentration of 0 to 0.5% by weight based on the dry weight of the sensing material. As used herein, the term "dry weight" refers to the dried product of each material, for example, a material without adding water or other solvents. Specifically, the sensing material may not include a cross-linking agent during step b), but after the manufacturing process, for example, by diffusion from the film layer as described below herein, a cross-linking agent may be added to the sensing material.Suitable chemical crosslinking agents according to the present invention include epoxy-based crosslinking agents such as diglycidyl ethers like poly(ethylene glycol) diglycidyl ether (PEG-DGE) and poly(propylene glycol) diglycidyl ether; trifunctional short-chain epoxides; anhydrides; resorcinol diglycidyl ether, bisphenol A diglycidyl ether, diglycidyl 1,2-cyclohexanedicarboxylate, poly(ethylene glycol) diglycidyl ether, glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, poly(propylene glycol) diglycidyl ether, bisphenol diglycidyl ether, poly(dimethylsiloxane), diglycidyl ether, neopentyl glycol diglycidyl ether, 1,2,7,8-diepoxyoctane, 1,3-glycidoxypropyl-1,1,3,3-tetramethyldisiloxane and other diglycidyl ethers; triglycidyl ethers such as N,N-diglycidyl-4-glycidyloxyaniline, trimethylolpropane triglycidyl ether; tetraglycidyl ethers, for example, selected from tetrakisepoxysiloxane, pentaerythritol tetraglycidyl ether, tetraglycidyl-4,4'-methylenebisbenzeneamine.

[0046] As used herein, the term "chemical crosslinking agent" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, but not limited to, this term can refer to a crosslinking agent capable of initiating a chemical reaction that produces a crosslinked molecular network and / or a crosslinked polymer when exposed to heat. "Exposed to heat" can refer to being exposed to a temperature above 15°C, specifically above 20°C, more specifically in the range of 20°C to 50°C, and even more specifically in the range of 20°C to 25°C. More specifically, the chemical crosslinking agent can initiate crosslinking of the layer of the sensing material when exposed to heat.

[0047] As used herein, the term "UV curable" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, but not limited to, this term can refer to the ability of a chemical substance, such as a crosslinking agent, to initiate a photochemical reaction that produces a crosslinked molecular network and / or a crosslinked polymer when irradiated with light in the UV spectral range. More specifically, a UV curable crosslinking agent can initiate crosslinking of a layer of a sensing material when irradiated with UV light. Crosslinking can be specifically initiated as shown below in this specification.

[0048] Suitable UV curable crosslinking agents according to the present invention include benzophenone, diazirine and azide. Particularly suitable UV curable crosslinking agents are, for example, benzophenone containing a crosslinking agent, poly(di(2-hydroxy 3-aminobenzophenone propylene) glycol), dibenzophenone 1,2-cyclohexanedicarboxylate, bis[2-(4-azidosalicylamido)ethyl] disulfide, 4-aminobenzophenone and any one of the above-mentioned diglycidyl crosslinking agents, triglycidyl crosslinking agents and tetraglycidyl crosslinking agents, and are selected from the group consisting of reaction products of such reactions, and examples of such reaction products are 2,4,6,8-tetramethyl-2,4,6,8-tetrakis(2-hydroxy 3-aminopropylbenzophenone)-cyclotetrasiloxane, reaction products of the reaction of 4-benzoylbenzoic acid N-succinimidyl ester with a diamine or a Jeffamine.

[0049] The UV-curing crosslinking agent is particularly useful and advantageous according to the present invention because it does not change the viscosity characteristics of the sensing material during step b) of adding at least one layer of the sensing material. Similarly, when the crosslinking agent is not present in the sensing material, the viscosity characteristics remain unchanged. The consistent viscosity characteristics of the sensing material enable the layers of the sensing material to be added more evenly and uniformly during manufacture, thus reducing non-uniformities and edge effects on the sensor surface that might otherwise occur. Reducing non-uniformities and edge effects can result in a reproducible sensor sensitivity across the charge. Thus, the use of a sensing material that does not contain a crosslinking agent or a UV-curing crosslinking agent can contribute to reducing the need for detailed monitoring and precise adaptation of manufacturing parameters during manufacture.

[0050] The sensing material can comprise at least a polymeric transition metal complex, an enzyme capable of catalyzing a chemical reaction that consumes at least an analyte, in particular H 2 O 2 generating and / or consuming enzymes, and optionally a crosslinking agent. Specifically, the sensing material may comprise at least a polymeric transition metal complex, GOx, and optionally a chemical crosslinking agent. More specifically, the sensing material can comprise a modified poly(vinylpyridine) backbone carrying a poly(bisimidazyl)Os complex covalently bonded via a bidentate linkage, GOx, and a chemical crosslinking agent such as poly(ethylene glycol) diglycidyl ether (PEG-DGE). Suitable additional sensing materials are known to those skilled in the art and are specified above herein.

[0051] The sensing material according to the present invention can comprise, for example, 60% by weight of a polymeric transition metal complex, 30 - 40% by weight of an enzyme capable of catalyzing a chemical reaction that consumes at least an analyte, in particular H 2 O 2 generating and / or consuming enzymes, and 0 - 10% by weight of a crosslinking agent based on the total weight of the sensing material. The enzyme can be present, for example, at a concentration of 50 mg / ml in water.

[0052] The method according to the invention can in particular further comprise at least one curing step d), in which at least a part of the detection material is crosslinked. The terms "crosslinking" and "curing" are used interchangeably herein. Specifically, the curing step d) may be carried out before the laser irradiation in step c), or may be carried out at least partially after step c) has been carried out.

[0053] Suitable methods for initiating crosslinking depend on the type of crosslinking agent and are known to those skilled in the art. Curing using a UV curable crosslinking agent is generally induced by irradiation with UV light. As used herein, the term "UV light" generally refers to electromagnetic radiation in the ultraviolet spectral range. The term "ultraviolet spectral range" generally refers to electromagnetic radiation in the range from 1 nm to 380 nm, preferably light in the range from 100 nm to 380 nm. Curing can usually be carried out at room temperature.

[0054] The application of the sensing material in step b) may include using at least one coating process. Further, as used herein, the term "coating process" can refer to any process for adding at least one layer to at least one surface of any object. The coating layer may completely cover an object, such as a conductive trace and / or a sensor substrate, or may cover only a part of the object. The layer can be applied by a coating process in which the material can be provided, for example, in liquid form, illustratively as a suspension or a solution, and distributed over the surface. Specifically, the coating process can include a wet coating process selected from the group consisting of: spin coating, spray coating, doctor blade, printing, dispensing, slot coating, dip coating. Specifically, step b) may include using a doctor blade or slot coating. The sensing material in step b) may be further applied such that at least 5%, at least 10%, at least 30% of the surface of the conductive trace is covered. Specifically, 5 to 100%, more specifically 5% to 40% of the surface of the conductive trace is covered.

[0055] The method according to the present invention includes step c) of irradiating a sensing material with at least one laser beam, at least a first portion of the layer of the sensing material is at least partially removed, and at least a second portion of the sensing material covering at least one conductive trace is maintained on the first surface of the sensor substrate to obtain at least one working electrode of the analyte sensor. As used herein, the term "irradiating" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term can specifically refer to, but is not limited to, the process of exposing the layer of the sensing material to laser light. In particular, it is preferred that at least one laser beam irradiates the first portion and at least one laser beam does not irradiate the second portion. Specifically, step c) can include at least one ablation process, particularly at least one laser ablation process. As used herein, the term "first portion" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term can specifically refer to, but is not limited to, a part of the layer of the sensing material within the outer region. More specifically, the first portion may be a part of the layer of the sensing material that contributes to edge effects and / or non-uniformities. In particular, the first portion is a part of the layer of the sensing material that is irradiated with at least one laser beam. As used herein, the term "second portion of the layer of the sensing material" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term can specifically refer to, but is not limited to, the central portion of the layer of the sensing material. In particular, at least one laser beam does not irradiate the second portion.

[0056] As used herein, the term "removed" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not exclusively, refer to the ablated portion of the layer of sensing material, specifically the first portion. The removed portion can typically be aspirated by techniques known to those of ordinary skill in the art. As used herein, the term "partially remove" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. This term can specifically, but not exclusively, refer to the complete removal of the first portion, and embodiments are possible where 70% or more, preferably 80% or more, more preferably 90% or more of the first portion is removed. Partially removing the first portion may include removing a portion of the conductive trace. Partially removing the first portion may also include removing a portion of the second portion. Preferably, only the first portion is at least partially removed.

[0057] If the removal of the first portion of the layer of sensing material includes removing a portion of the conductive trace, it is possible to completely or partially, preferably completely, remove the conductive trace within the region of the first portion of the layer of sensing material. Thus, in embodiments where the conductive trace is completely removed within the region of the first portion of the layer of sensing material after removal of the first portion, the second portion of the layer of sensing material and the conductive trace can have the same size. In embodiments where the conductive trace includes at least one additional layer, for example, the conductive trace includes gold and the additional layer includes carbon, it is possible to further remove only the first portion of the additional layer and not other portions of the conductive trace while removing the first portion of the layer of sensing material.

[0058] Thus, in embodiments of the present invention, a method for preparing a working electrode on a sensor substrate includes the following steps: a1) Providing at least one sensor substrate comprising at least a first surface, the first surface comprising at least one conductive trace, in particular the conductive trace comprising gold and / or carbon, in particular the conductive trace comprising at least one further layer of at least one further material, in particular at least one further layer comprising carbon, the step of providing at least one sensor substrate; b1) Applying at least one layer of at least one sensing material onto the first surface of the sensor substrate, the sensing material covering at least a part of at least one conductive trace, in particular at least one further layer, the step of applying onto the first surface of the sensor substrate; and c1) Irradiating the layer of sensing material with at least one laser beam, at least a first part of the layer of sensing material and the conductive trace, in particular at least a first part of the further layer being at least partially removed, at least a second part of the sensing material covering the at least one conductive trace, in particular the further layer and a second part of the conductive trace, in particular the further layer being maintained on the first surface of the sensor substrate to obtain at least one working electrode, the step of irradiating the layer of sensing material.

[0059] For the first and second parts of the conductive trace, in particular the further layer, the embodiments and preferences described above for the first and second parts of the layer of sensing material apply. Furthermore, the embodiments and preferences described below and above for the method according to the invention also apply to a method in which the conductive trace is also removed.

[0060] Also removing the first part of the conductive trace, in particular the first part of the further layer, is advantageous for reducing the background current of a sensor comprising a working electrode.

[0061] As used herein, the term "maintained" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. Specifically, but not limited thereto, this term can refer to a maintained second portion and / or a remaining second portion of the layer of the sensing material. The second portion may be or may include a patterned layer. As used herein, the term "patterned layer" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. Specifically, but not limited thereto, this term can refer to at least one conductive trace patterned by irradiation with a laser beam or to the surface of one or more additional layers included in at least one layer of at least one sensing material. Specifically, a laser beam is used under the conditions specified herein. The terms "patterned layer" and "laser-patterned layer" are used interchangeably herein. In particular, the term "patterned layer" refers to at least a portion of the surface of a layer of sensing material and / or an additional layer of conductive traces patterned using a laser beam. More specifically, the "patterned layer" corresponds to the second portion. Thus, the patterned layer is the portion of the sensing material that is not removed and preferably not irradiated by the laser beam. Thus, the patterned layer can be obtained by irradiating the layer of sensing material and / or the sensor substrate with a laser beam under the conditions specified herein. An ablation-patterned layer having any kind of shape and / or structure can be realized. It can also be possible to have a patterned layer including letters or a checkerboard-like structure.

[0062] For example, the layer of sensing material can have a thickness of from 1 to 4 μm prior to step c). By irradiating the sensing material with a laser beam, a first portion can be ablated from the layer of sensing material, thereby creating or forming a patterned layer on the sensor substrate. The patterned layer can then correspond to the second portion.

[0063] As used herein, the term "obtaining at least one working electrode" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or customized meaning. Specifically, without limitation, this term can refer to the formation and / or manufacture of a working electrode.

[0064] The term "laser ablation" generally can refer to any process of removing at least a portion of a material from a surface by irradiating the surface with a laser beam in a point-like manner. Thereby, the material can be removed. The material can be at least partially destroyed, for example, by sublimation due to the transfer of energy. The transfer of energy, such as heat, can be locally restricted. As an example, a pulsed laser may be used for the purpose of laser ablation. The energy of the pulse can be in the range of less than 40 μJ. Specifically, the laser can be configured to irradiate a layer of the sensing material using an ultrashort laser pulse such that the first portion is removed within a very short time so that the diffusion of heat to a maintained second portion is minimized. The pulse length can be restricted to less than 12 ps. For example, the laser can be configured to generate a laser beam within the UV spectral range. Specifically, a laser with a wavelength of 355 nm can be used. As an example, for the purpose of laser ablation, a frequency-tripled solid-state laser emitting at a wavelength of 355 nm with a pulse duration of less than 12 ps and a pulse repetition rate of 400 kHz can be used. In step c), at least one ablation pattern can be used. The ablation pattern may be provided as a mask image projected onto the layer of the sensing material. The ablation pattern may alternatively be drawn by scanning a laser beam across the layer of the sensing material. For example, the layer of the sensing material may be irradiated using at least one scanning process. The term "scanning" as used herein is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term can specifically, but not limited to, refer to the continuous or pulsed irradiation of the layer of the sensing material during the generation of a patterned layer at a specific scanning speed.The ablation rate can depend on pulse energy, laser wavelength, pulse length, pulse repetition rate, beam diameter on the irradiated surface, scanning speed, pulse overlap, overlap of adjacent scan lines, spectral absorption coefficient of the irradiated material, and ablation threshold of the irradiated material. Specifically, the term "ablation rate" can refer to the thickness of the ablated material. For example, in the case of laser ablation using a mask, the ablation rate can refer to the entire mask image. In this case, the ablation rate can particularly depend on pulse energy, irradiated surface area, and / or the ablation threshold of the material, e.g., the energy required to remove the material. In the case of laser ablation by a scanning process, the ablation rate of the entire image depends on the characteristics of the laser, including laser wavelength, pulse energy, pulse rate; in combination with the characteristics of the optical system, such as the beam diameter on the surface of the material; in combination with the laser settings, including pulse overlap, e.g., pulse overlap along the scan line and line overlap, e.g., overlap of adjacent scan lines; and, the characteristics of the irradiated material, e.g., the sensing material, including the absorption coefficient and ablation threshold.

[0065] During or after the ablation process, the ablated material can be removed by any process known in the art, such as by using at least one dust extractor, etc.

[0066] Specifically, the layer of the second portion of the sensing material has a thickness of at least 0.5 μm. More specifically, it is from 0.5 to 5 μm, even more specifically from 1 to 4 μm. More specifically, the value refers to the thickness of the dried product after partially removing by irradiating with a laser beam. The specified thickness can be advantageous as it can ensure sufficient electron transfer over a wide range of analyte concentrations. Thicknesses below and / or above the described values can be disadvantageous as they can result in sub-optimal electron transfer and glucose conversion.

[0067] The method according to the invention can further comprise an additional step bb) of drying at least one layer of at least one sensing material. Specifically, the layer of the sensing material is dried before the step c) of irradiating the layer of the sensing material with a laser beam. The drying step before step c) is advantageous in order to enable an accurate removal of at least a part of the layer of the sensing material in step c). If the layer of the sensing material is not sufficiently dried, the partial removal of the sensing material by laser irradiation is not efficient and not very accurate. The drying step bb) may be carried out at ambient temperature. Specifically, the sensing material can be dried at ambient temperature for 0.5 to 15 minutes. The term "ambient temperature" as used herein is specifically understood as a temperature of 15°C to 30°C, more specifically 20°C to 25°C.

[0068] The method according to the invention may further comprise an additional step e) of adding at least one membrane layer, which at least partially covers the working electrode.

[0069] The term "membrane layer" as used herein is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term can specifically refer to, but is not limited to, a layer of at least one material that provides a selective barrier. Thus, a membrane layer can generally allow one or more of a molecule and / or a compound to pass through, while other molecules and / or compounds are stopped by the membrane layer. Thus, as outlined above, the membrane layer is permeable to at least one analyte to be detected. Thus, by way of example, the membrane layer can allow one or more of glucose, lactate, cholesterol or other types of analytes to pass through. Thus, at least one membrane layer can function as a diffusion barrier that controls the diffusion of the analyte from the outside, for example the body fluid surrounding the analyte sensor, to the sensing material, i.e., the enzyme molecules in the sensing material. Further, at least one membrane layer can function as a biocompatible membrane layer as mentioned elsewhere herein.

[0070] The film layer can, for example, have a thickness sufficient to provide mechanical stability. At least one film layer can specifically have a thickness from 1 μm to 150 μm. For at least one film layer, as outlined herein, several materials can be used alone or in combination. Thus, for example, the film layer can specifically include one or more of a polymer material, specifically a polyvinylpyridine-based copolymer, polyurethane, hydrogel, polyacrylate, methacrylate-acrylate copolymer or block copolymer, among which polyvinylpyridine-based copolymers are particularly suitable. Films of these types are generally known in the art. For example, films such as those disclosed in, for example, European Patent Application Publication No. 2697388, International Publication No. 2007 / 071562 pamphlet and / or International Publication No. 2005 / 078424 pamphlet can be used. Specifically, the polymer material can have a weight average molecular weight (MW) exceeding 10,000 kDa. More specifically, the polymer material can have a weight average molecular weight (MW) exceeding 50,000 kDa or even exceeding 100,000 kDa. A polymer material having a weight average molecular weight (MW) from 10,000 to 500,000 kDa is particularly suitable. The polymer material of the film may be the same as or different from the polymer material of the sensing material.

[0071] Furthermore, the membrane layer can contain a crosslinking agent, specifically a chemical crosslinking agent or a UV curable crosslinking agent. The crosslinking agent may be suitable for crosslinking at least a part of the sensing material. Crosslinking agents as described hereinabove with respect to the sensing material are generally suitable for use in the membrane layer. Divalent and / or polyvalent epoxide-based crosslinking agents, such as bifunctional and / or trifunctional short-chain epoxides, are particularly suitable. Suitable crosslinking agents can have a molar mass in the range from 150 g / mol to 10,000 g / mol. The molar mass of the crosslinking agent as described above can be beneficial for enabling efficient diffusion into the sensing material. Specific examples of suitable crosslinking agents contained in the membrane layer include the following: diglycidyl ethers such as resorcinol diglycidyl ether, bisphenol A diglycidyl ether, diglycidyl 1,2-cyclohexanedicarboxylate, poly(ethylene glycol) diglycidyl ether, glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, poly(propylene glycol) diglycidyl ether, bisphenol diglycidyl ether, poly(dimethylsiloxane), diglycidyl ether, neopentyl glycol diglycidyl ether, 1,2,7,8-diepoxyoctane, 1,3-glycidoxypropyl-1,1,3,3-tetramethyldisiloxane, N,N-diglycidyl-4-glycidyloxyaniline, triglycidyl ethers such as trimethylolpropane triglycidyl ether, tetrakisepoxysiloxane, pentaerythritol tetraglycidyl ether, tetraglycidyl-4,4'-methylenebisbenzeneamine and other tetraglycidyl ethers, in particular, poly(ethylene glycol) diglycidyl ether, N,N-diglycidyl-4-glycidyloxyaniline.

[0072] Specifically, in step e) of the method according to the invention, in addition to at least one membrane layer, at least a second membrane layer can be added. The second membrane layer may be a biocompatible membrane layer.

[0073] As used herein, the term "biocompatible membrane layer", also referred to as a biocompatible layer, relates to a layer made of a biocompatible material, particularly the outermost layer or a part thereof of an analyte sensor. Specifically, the biocompatible layer has a thickness of from 1 μm to 10 μm, and in embodiments from 3 μm to 6 μm. More specifically, the biocompatible layer at least partially or completely covers the analyte sensor. Even more specifically, the biocompatible layer may be the outermost layer of the analyte sensor. Thus, more specifically, at least a part of the biocompatible layer is in contact with the subject's body fluid. For example, the biocompatible layer may not be diffusion limiting for an analyte, as specified elsewhere herein. For example, the biocompatible layer may not be diffusion limiting for small molecules having a molecular weight of less than 2,000 Da, and in embodiments less than 1,000 Da. For example, the biocompatible layer may not contain added enzymes. For example, the biocompatible layer may not contain added polypeptides. As will be understood by those skilled in the art, this does not preclude the diffusion of enzyme or polypeptide molecules from an adjacent layer, tissue or body fluid into the biocompatible layer. As used herein, the term "biocompatible material" relates to a material that is not toxic, injurious, or physiologically reactive, or that is toxic, injurious, or physiologically reactive and / or causes a reduced degree or does not cause immune rejection, suitable for use in a biological tissue or biological system. In embodiments, the biocompatible material is a material that does not elicit a body response, such as an inert material, or a material containing a chemical compound that prevents a body response from occurring in the vicinity of the biocompatible layer. In another embodiment, the biocompatible material is a material that prevents cells from adhering to the biocompatible layer. The biocompatible membrane layer may be or may include the following materials: methacrylate-based polymers and copolymers, acrylamide-methacrylate-based copolymers, biodegradable polysaccharides such as hyaluronic acid (HA), agarose, dextran, and chitosan.Additional biocompatible materials are disclosed in WO 2019 / 166394 Pamphlet, including hydrogels prepared from the copolymerization of non-biodegradable synthetic hydrogels such as 2-hydroxyethyl methacrylate (HEMA), 2-hydroxypropyl methacrylate (HPMA), acrylamide (AAm), acrylic acid (AAc), N-isopropylacrylamide (NIPAm) and methoxypoly(ethylene glycol) (PEG) monoacrylate (mPEGMA or PEGMA) with crosslinking agents such as N,N'-methylenebis(acrylamide) (MBA), ethylene glycol diacrylate (EGDA) and PEG diacrylate (PEGDA), Pluronic® polymers having a poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO structure, modified poly(vinyl alcohol) (PVA), poly(4-vinylpyridine), and PEG.

[0074] At least one membrane layer and / or biocompatible membrane layer can be applied by techniques known to those skilled in the art using at least one coating process selected from the group consisting of, specifically, wet coating processes: for example, spin coating, spray coating, doctor blade, printing, dispensing, slot coating, dip coating. Preferred wet coating processes are dip coating or spray coating.

[0075] The method according to the invention can further comprise at least one diffusion step f), in which the crosslinking agent comprised in the membrane layer can diffuse at least partially into the sensing material. The diffusion can occur during the application of the membrane layer to the sensing material. The diffusion of the crosslinking agent into the sensing material can enable at least partial crosslinking of the sensing material, independently of the presence of the crosslinking agent in the sensing material, during step b) of applying the sensing material to the substrate. This can be advantageous since the presence of the crosslinking agent in the sensing material during step b) of applying the sensing material to the sensor substrate is not required and can be avoided. Avoiding the presence of the crosslinking agent in the sensing material during the application of the sensing material to the substrate can, as specified above herein, have the advantages, i.e., can have certain viscosity characteristics of the sensing material, for example, during application to the sensor substrate.

[0076] In the method according to the invention, the diffusion step can further comprise swelling of at least a part of the sensing material.

[0077] As used herein, the term "swelling" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term can specifically, but not exclusively, refer to the binding of water and / or water-soluble solvents such as ethanol, methanol, acetone, etc. to the material, specifically the binding of water and / or water-soluble solvents to the sensing material. The uptake of water and / or water-soluble solvents into the sensing material can advantageously enable the diffusion of the crosslinking agent into the sensing material, which can be required for efficient crosslinking. Swelling can further refer to the uptake of water from the membrane layer.

[0078] In the method according to the present invention, in order to allow sufficient swelling, the polymer material in the sensing material is at least 10% by weight, more specifically at least 20% by weight, even more specifically at least 30% by weight, and even more specifically up to 90% by weight of water and / or solvent from the film layer based on the dry weight of the polymer material within a time frame of several minutes, for example 1 to 15 minutes.

[0079] This swelling and / or uptake of water and / or solvent is advantageous because it can enable the diffusion of the crosslinking agent from the film layer into the sensing material.

[0080] Furthermore, the present invention relates to an analyte sensor. The analyte sensor comprises at least one sensor substrate including at least one first surface. The first surface comprises at least one conductive trace.

[0081] The analyte sensor comprises at least one layer of at least one sensing material disposed on the first surface of the sensor substrate, the sensing material comprising at least one layer that covers at least a portion of at least one conductive trace. The layer of sensing material is irradiated by at least one laser beam, and a first portion of the layer of sensing material is at least partially removed such that a second portion of the sensing material that covers the conductive trace is maintained on the first surface of the sensor substrate. The said second portion of the sensing material, together with the conductive trace, forms at least one working electrode of the analyte sensor.

[0082] In particular, the working electrode of the analyte sensor can comprise at least one patterned layer of at least one sensing material disposed on the first surface of the sensor substrate. The patterned layer can be obtained specifically by irradiating the layer of sensing material and / or the sensor substrate with a laser beam. The patterned layer is specifically the second portion of the sensing layer.

[0083] The analyte sensor described in this specification can be obtained, in particular, by the method according to the invention for preparing a working electrode on a sensor substrate and by providing at least one further electrode, for example a counter electrode or a reference electrode or a combined counter / reference electrode.

[0084] Furthermore, the invention relates to the use of an analyte sensor for detecting at least one analyte in a sample, specifically a sample of a body fluid. More particularly, the analyte sensor is a sensor for continuous glucose measurement.

[0085] As used herein, the term "body fluid" relates to all body fluids of a subject known or suspected to contain an analyte of the invention, including interstitial fluid, blood, plasma, tears, urine, lymph, cerebrospinal fluid, bile, feces, sweat and saliva. In general, any kind of body fluid can be used. Preferably, the body fluid is a body fluid present in the user's body tissue such as interstitial tissue. Thus, by way of example, the body fluid can be selected from the group consisting of blood and interstitial fluid. However, additionally or alternatively, one or more other types of body fluids may be used. The body fluid can generally be contained in body tissue. Thus, generally, the detection of at least one analyte in the body fluid can preferably be determined in vivo.

[0086] The term "sample" is understood by those skilled in the art and relates to any part of a body fluid. The sample can be obtained, for example, by well-known techniques including venous or arterial puncture, epidermal puncture and the like.

[0087] As used herein, the term "subject" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to a special or customized meaning. Specifically, but not limited to, this term can refer to a human or an animal, regardless of whether the human or animal is in a healthy state or can suffer from one or more diseases. By way of example, the subject can be a human or an animal suffering from diabetes. However, additionally or alternatively, the present invention may be applied to other types of subjects.

[0088] Furthermore, the present invention relates to a method for measuring an analyte in a sample comprising the analyte sensor described above herein.

[0089] The method for measuring an analyte of the present invention can in particular be an in vivo method. Alternatively, the method of the present invention can also include the measurement of an analyte in a sample of a body fluid obtained under in vitro conditions, for example from a subject, in particular a human subject. Specifically, the method may not include the diagnosis of a disease based on the measurement.

[0090] In summary, without excluding further possible embodiments, the following embodiments can be envisaged.

[0091] Embodiment 1: A method for preparing a working electrode on a sensor substrate, comprising: a) providing at least one sensor substrate comprising at least a first surface, the first surface comprising at least one conductive trace; b) adding at least one layer of at least one sensing material on the first surface of the sensor substrate, the sensing material covering at least a part of at least one conductive trace. c) irradiating a layer of the sensing material with at least one laser beam, wherein at least a first portion of the layer of the sensing material is at least partially removed and at least a second portion of the sensing material covering at least one conductive trace is maintained on a first surface of the sensor substrate to obtain at least one working electrode on the sensor substrate, the step of irradiating the layer of the sensing material.

[0092] Embodiment 2: The method according to embodiment 1, wherein the sensing material comprises at least a polymer material, specifically a metal-containing complex selected from the group consisting of at least one polymer material and at least one transition metal element complex, more specifically an osmium complex, a ruthenium complex, a vanadium complex, a cobalt complex, and an iron complex such as ferrocene, especially 2-aminoethylferrocene.

[0093] Embodiment 3: The method according to embodiment 1 or 2, further comprising an additional step bb) of drying at least one layer of at least one sensing material, specifically, drying is performed at ambient temperature.

[0094] Embodiment 4: The method according to any one of embodiments 1 to 3, wherein the sensing material comprises at least one crosslinking agent.

[0095] Embodiment 5: The method according to embodiment 4, wherein the crosslinking agent is a chemical crosslinking agent, specifically an epoxide-based crosslinking agent, such as poly(ethylene glycol) diglycidyl ether (PEG-DGE) and poly(propylene glycol) diglycidyl ether, trifunctional short-chain epoxide.

[0096] Embodiment 6: The method according to any one of embodiments 1 to 5, wherein step b) comprises using at least one coating process selected from the group consisting of spin coating, spray coating, doctor blade, printing, dispensing, slot coating, dip coating, specifically a wet coating process.

[0097] Embodiment 7: d) The method according to any one of Embodiments 1 to 6, further comprising at least one curing step, wherein in the curing step, at least a part of the sensing material is crosslinked.

[0098] Embodiment 8: The method according to Embodiment 7, wherein step d) is at least partially performed after step c).

[0099] Embodiment 9: e) The method according to any one of Embodiments 1 to 8, further comprising the step of adding at least one film layer, wherein the film layer at least partially covers the working electrode, specifically the second part of the sensing material.

[0100] Embodiment 10: The method according to Embodiment 9, wherein the film layer comprises one or more of a polymer material, specifically a polyvinyl pyridine copolymer, polyurethane, hydrogel, polyacrylate, methacrylate-acrylate copolymer, or block copolymer.

[0101] Embodiment 11: The method according to Embodiment 9 or 10, wherein the film layer comprises at least one crosslinking agent for crosslinking at least a part of the sensing material.

[0102] Embodiment 12: f) The method according to Embodiment 11, further comprising at least one diffusion step, wherein in the diffusion step, the crosslinking agent contained in the film layer at least partially diffuses into the sensing material.

[0103] Embodiment 13: The method according to Embodiment 12, wherein the diffusion step comprises swelling of at least a part of the sensing material.

[0104] Embodiment 14: The method according to any one of Embodiments 11 to 13, wherein the crosslinking agent contained in the film layer comprises at least one low molecular weight divalent and / or polyvalent epoxide-based crosslinking agent, such as a trifunctional short-chain epoxide.

[0105] Embodiment 15: The method according to any one of Embodiments 10 to 14, wherein the polymeric material in the membrane layer has a molecular weight exceeding 10,000 kDa.

[0106] Embodiment 16: The method according to any one of Embodiments 9 to 15, wherein the membrane layer is added by dip coating.

[0107] Embodiment 17: The sensing material contains at least one enzyme. Specifically, the enzyme is an enzyme capable of catalyzing a chemical reaction that consumes at least the analyte. More specifically, H 2 O 2 generating and / or consuming enzymes, even more specifically glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), (S)-2-hydroxyacid oxidase (EC 1.1.3.15), cholesterol oxidase (EC 1.1.3.6), galactose oxidase (EC 1.1.3.9), glucose dehydrogenase, alcohol oxidase (EC 1.1.3.13), L-glutamate oxidase (EC 1.4.3.11) or L-aspartate oxidase (EC 1.4.3.16), even more specifically glucose oxidase and / or its mutants. The method according to any one of Embodiments 1 to 16.

[0108] Embodiment 18: The method according to any one of Embodiments 1 to 17, wherein the conductive trace contains at least one conductive material selected from the group consisting of carbon, gold, platinum, and palladium.

[0109] Embodiment 19: The method according to any one of Embodiments 1 to 18, wherein the sensor substrate includes two opposing surfaces, a first surface and at least a second surface opposing the first surface.

[0110] Embodiment 20: The method according to Embodiment 18, wherein the conductive trace includes at least one additional layer of at least one additional material. Specifically, the additional layer contains a conductive material, such as carbon.

[0111] Embodiment 21: The method according to any one of Embodiments 1 to 20, wherein the conductive trace, specifically the conductive trace containing carbon, has a thickness of at least 7 μm, specifically at least 10 μm.

[0112] Embodiment 22: The method according to any one of Embodiments 1 to 21, wherein the layer of the sensing material has a thickness of 0.5 to 5 μm, specifically 1 to 4 μm, specifically the dry thickness.

[0113] Embodiment 23: The method according to any one of Embodiments 1 to 22, wherein step c) includes laser ablation.

[0114] Embodiment 24: A method for preparing an analyte sensor, comprising a method for preparing a working electrode on a substrate according to any one of Embodiments 1 to 23 and a step of providing at least one additional electrode.

[0115] Embodiment 25: The method according to Embodiment 24, wherein the at least one additional electrode includes at least one of a reference electrode, a counter electrode, and a counter electrode / reference electrode.

[0116] Embodiment 26: The method according to Embodiment 25, wherein the reference electrode comprises at least one reference electrode conductive trace.

[0117] Embodiment 27: The method according to Embodiment 25 or 26, wherein the counter electrode comprises at least one counter electrode conductive trace.

[0118] Embodiment 28: The method according to any one of Embodiments 25 to 27, wherein the at least one additional electrode is disposed on at least one of a first surface and a second surface opposite the first surface.

[0119] Embodiment 29: An analyte sensor comprising at least one working electrode obtainable by the method according to any one of Embodiments 1 to 24 and at least one additional electrode.

[0120] Embodiment 30: An analyte sensor, A) At least one sensor substrate including at least one first surface, wherein the first surface includes at least one conductive trace, and at least one sensor substrate; B) At least one layer of at least one sensing material disposed on the first surface of the sensor substrate, wherein the sensing material covers at least a portion of at least one conductive trace, the layer of sensing material is irradiated by at least one laser beam, and a first portion of the layer of sensing material is at least partially removed such that a second portion of the sensing material covering the conductive trace is maintained on the first surface of the sensor substrate, and the second portion of the sensing material forms at least one working electrode of an analyte sensor together with the conductive trace, and at least one layer; An analyte sensor comprising:

[0121] Embodiment 31: The analyte sensor according to Embodiment 30, wherein the working electrode can be obtained by the method according to Embodiments 1 to 23.

[0122] Embodiment 32: The analyte sensor according to Embodiment 30 or 31, wherein the working electrode includes at least one patterned layer of at least one sensing material disposed on the first surface of the sensor substrate.

[0123] Embodiment 33: Use of the analyte sensor according to Embodiments 29 to 32 for detecting at least one analyte in a sample, specifically in a sample of body fluid.

[0124] Embodiment 34: A method for measuring an analyte in a sample comprising the analyte sensor according to Embodiments 29 to 32.

[0125] Embodiment 35: The analyte sensor obtained by the method according to Embodiments 24 to 28.

Brief Description of the Drawings

[0126] Any further optional features and embodiments are preferably disclosed in more detail in the following description of the embodiments, in conjunction with the dependent claims. Here, each optional feature may be implemented in an independent manner and in any feasible combination, as will be understood by those skilled in the art. The scope of the present invention is not limited by the preferred embodiments. The embodiments are schematically shown in the figures. Here, the same reference signs in these figures refer to the same or functionally equivalent elements. The figures are as follows.

[0127]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

DETAILED DESCRIPTION OF THE INVENTION

[0128] Figures 1 to 3 show intermediate products in the method for preparing the analyte sensor 124. Figure 4 gives a schematic view of the steps of the method according to the present invention. These figures will be described together below.

[0129] FIG. 1 shows at least one sensor substrate 114 of at least one analyte sensor 124 including at least one first surface 120. The first surface 120 comprises at least one conductive trace 111. The analyte sensor 124 includes at least one layer of at least one sensing material 118 disposed on the first surface 120 of the sensor substrate 114, as shown in FIGS. 2 and 3. The sensing material 118 covers at least a portion of at least one conductive trace 111. As shown in FIG. 3, at least one laser beam 126 is laser-irradiated onto the layer of the sensing material 118 such that a portion of the sensing material 118 covering the conductive trace 111 is maintained on the first surface of the sensor substrate. The said portion of the sensing material, together with the conductive trace 111, forms at least one working electrode 122 of the analyte sensor 124. In particular, the working electrode 122 of the analyte sensor can include at least one patterned layer 128 of at least one sensing material disposed on the first surface of the sensor substrate. The patterned layer 128 can be obtained specifically by irradiating the layer of the sensing material 118 and / or the sensor substrate 114 with the laser beam 126. The patterned layer 128 is then the second portion that was not irradiated.

[0130] The analyte sensor 124 can specifically be an analyte sensor 124 suitable for at least partial implantation into a user's body tissue, and more specifically, an analyte sensor for continuously monitoring an analyte. The analyte sensor 124 can in particular be obtained by the method according to the present invention.

[0131] Furthermore, the analyte sensor 124 is an electrochemical sensor comprising at least one electrode and respective circuitry. More particularly, the analyte sensor 124 is an amperometric electrochemical sensor comprising at least one working electrode. Typically, the analyte sensor 124 comprises at least one additional electrode, particularly a counter electrode and / or a reference electrode. The working electrode 122 can be sensitive to an analyte that is measured at a polarization voltage that can be applied between the working electrode and the reference electrode and can be adjusted by a potentiostat. The measurement signal can be provided as a current between the counter electrode and the working electrode. There may not be a separate counter electrode, and there may be a pseudo-reference electrode, which can also function as a counter electrode. Thus, the analyte sensor 124 can typically comprise a set of at least two electrodes or a set of three electrodes. Specifically, the sensing material 118 is present only on the working electrode 122.

[0132] In particular, the working electrode 122 may be disposed on at least one sensor substrate 114 including at least one first surface 120. The first surface 120 may comprise at least one conductive trace 111 and at least one layer of at least one sensing material 118 disposed on the first surface of the sensor substrate 114. The sensing material 118 may cover at least a portion of at least one conductive trace 111. The layer of sensing material 118 may be laser irradiated by at least one laser beam, and a first portion of the layer of sensing material 118 is at least partially removed such that a second portion of the sensing material 118 covering the conductive trace 111 is maintained on the first surface of the sensor substrate. The second portion of the sensing material together with the conductive trace 111 forms at least one working electrode 122 of the analyte sensor 124. The layer of sensing material 118 may be present only on the working electrode and typically may not be present from any additional electrodes, for example, the counter electrode and / or the reference electrode may not include the layer of sensing material 118.

[0133] Specifically, the analyte sensor 124, and more specifically the sensor substrate 114, can further include at least one additional electrode, and the at least one additional electrode can include at least one of a reference electrode and a counter electrode. In particular, the reference electrode may comprise at least one reference electrode conductive trace. And / or the counter electrode may comprise at least one counter electrode conductive trace.

[0134] The at least one conductive trace 111 may comprise at least one material selected from the group consisting of: carbon, carbon paste, gold, copper, silver, nickel, platinum, palladium. Specifically, the conductive trace 111 may be or comprise at least one metal such as one or more of gold, copper, silver, nickel, palladium or platinum. Additionally or alternatively, the at least one conductive trace 111 may be or comprise at least one conductive compound such as at least one conductive organic or inorganic compound. Additionally or alternatively, the conductive trace 111 may be or comprise at least one non-metallic conductive material, such as carbon or carbon paste, or may comprise them. Preferably, the conductive trace 111 according to the invention can comprise gold and / or carbon. More preferably, the conductive trace may consist of gold 112 and / or carbon 110. Specifically, the conductive trace may comprise gold 112 and a further material, such as carbon 110.

[0135] Furthermore, the conductive trace 111 may include at least one additional layer of at least one additional material, and specifically, the additional layer may include a further conductive material. More specifically, the additional layer of the conductive trace may include or consist of carbon 110. Using an additional layer including or consisting of carbon 110 within the conductive trace 111 on the sensor substrate can be advantageous because it can enhance the adhesion of the layer of the sensing material 118 on the conductive trace 111.

[0136] The sensing material 118 may be at least a polymer material or may include a polymer material. Specifically, it may be at least one polymer material and at least one metal-containing complex, or may include these. The metal-containing complex can be selected from the group of transition metal element complexes. Specifically, the metal-containing complex can be selected from osmium complexes, ruthenium complexes, vanadium complexes, cobalt complexes, and iron complexes, such as ferrocene, particularly 2-aminoethylferrocene. In particular, the sensing material 118 can include a modified poly(vinylpyridine) backbone carrying a poly(biiimidyl)Os complex covalently bonded via a bidentate bond.

[0137] The sensing material 118 can be at least a polymer transition metal complex, at least an enzyme capable of catalyzing a chemical reaction that consumes an analyte, particularly H 2 O 2 generating and / or consuming enzyme 116, and optionally a crosslinking agent. Specifically, the sensing material 118 may include at least a polymer transition metal complex, GOx, and optionally a chemical crosslinking agent. More specifically, the sensing material 118 may include a modified poly(vinylpyridine) backbone carrying a poly(biiimidyl)Os complex covalently bonded via a bidentate bond, GOx, and optionally a chemical crosslinking agent.

[0138] Furthermore, the sensing material 118 may include at least one enzyme 116. Specifically, the enzyme can catalyze at least a chemical reaction that consumes an analyte. Specifically, the enzyme is H 2 O 2Generating and / or consuming enzymes, more specifically, glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), (S)-2-hydroxy acid oxidase (EC 1.1.3.15), cholesterol oxidase (EC 1.1.3.6), glucose dehydrogenase, galactose oxidase (EC 1.1.3.9), alcohol oxidase (EC 1.1.3.13), L-glutamate oxidase (EC 1.4.3.11) or L-aspartate oxidase (EC 1.4.3.16), even more specifically, glucose oxidase (GOx) and / or its mutants can be used.

[0139] Furthermore, the sensing material 118 may further include at least one crosslinking agent. The crosslinking agent may crosslink at least a part of the sensing material 118, for example. Specifically, the sensing material 118 may include at least one crosslinking agent selected from a UV curable crosslinking agent and a chemical crosslinking agent. Even more specifically, the sensing material 118 includes a chemical crosslinking agent. Alternatively, the sensing material 118 may not include any crosslinking agent.

[0140] A flowchart of the method according to the present invention is shown in FIG. 4. The method includes the following steps: a) Providing at least one sensor substrate 114 including at least a first surface 120, the first surface 120 including at least one conductive trace 111 (step 130); b) Adding at least one layer of at least one sensing material 118 onto the first surface 120 of the sensor substrate 114, the sensing material 118 covering at least a part of at least one conductive trace 111 (step 132); and c) irradiating at least one laser beam 126 onto the layer of the sensing material 118, wherein at least a first portion of the layer of the sensing material is at least partially removed, and at least a second portion of the sensing material covering the at least one conductive trace is maintained on the first surface of the sensor substrate, to obtain at least one working electrode 122 on the sensor substrate 114, the irradiating step.

[0141] By irradiating the sensing material 118 with the laser beam 126 (134), a first portion can be ablated from the layer of the sensing material 118, thereby generating or forming a patterned layer 128 on the sensor substrate 114.

[0142] The present invention is not limited to one of the above-described embodiments and can be modified in various ways. Those skilled in the art will recognize that the embodiments according to the present invention can be easily adapted without departing from the scope of the present invention. Therefore, simple adaptations are conceivable for the preparation of the analyte sensor. The present invention enables the preparation of an analyte with reduced production costs and reproducible sensor sensitivity. Further features, details and advantages of the present invention can be obtained from the following description of the examples based on the language of the claims and the drawings.

[0143] The contents of all references cited in this patent application are incorporated herein by reference to their respective specific disclosures and in their entirety. Examples

[0144] The following examples serve to illustrate the present invention. They should not be construed as limiting with respect to the scope of protection. Example 1: Preparation of the layer of the sensing material of the working electrode

[0145] A sensor substrate as schematically shown in FIG. 1 based on polyethylene terephthalate and a thin layer of gold was coated with a carbon paste by the doctor blade method. Suitable carbon conductive inks are available from Ercon, Inc. (Wareham, Massachusetts), E.I. du Pont de Nemours and Co. (Wilmington, Delaware), Emca-Remex Products (Montgomeryville, Pennsylvania), or TEKRA, A Division of EIS, Inc (New Berlin, Wisconsin).

[0146] Thereafter, the carbon paste was dried at 50 ° C for 12 hours. Enzyme- and mediator-containing formulations that can be used in the sensing layer are known in the art, for example, from "A Continuous Glucose Sensor Based on Wired Enzyme™ Technology - Results from a 3-Day Trial in Patients with Type 1 Diabetes". DIABETES TECHNOLOGY & THERAPEUTICS Volume 5, Number 5, 2003 Approximately 35% (by weight) redox polymer, 40% GOx and 25% crosslinker were used, or approximately 50% (by weight) redox polymer, 50% GOx were used.

[0147] A layer of the sensing material was applied onto the sensor substrate by cannula coating (PTFE cannula 1.6 mm, flow rate 0.09 ml / min, speed 8 mm / s). The sensing material was dried at room temperature for 10 minutes. Thereby, a layer configured as schematically shown in FIG. 2 was obtained. Example 2: Structuring of the layer of the sensing material using laser ablation

[0148] After drying, the sensor element obtained in Example 1 was further structured and cut using a laser beam. The conditions shown in Table 1 were used for the laser system 3D Micromac microCut TMS; UKP-Laser Hyper Rapid 50-SW 355.

[0149] A schematic diagram of the layer of the analyte after laser ablation is shown in FIG. 3.

Table 1

[0150] The membrane polymer 15% (w / v) poly(4-(N-(3-sulfonatopropyl)-pyridinium)-co-pyridine-co-styrene) was dissolved in ethanol / water (80 / 20) and mixed until a brown solution was obtained. The crosslinking agent 3.75% (w / v) glycerol triglycidyl ether crosslinking agent solution was dissolved in ethanol / water (80 / 20) and mixed until a clear solution was obtained. The membrane polymer and the crosslinking agent solution were mixed (4:1). The laser-ablated sensor was dip-coated three times in the membrane polymer / crosslinking agent solution as described in "Miniature Amperometric Self-Powered Continuous Glucose Sensor with Linear Response" Anal.Chem. 2012, 84, 7, 3403-3409, publication date: March 14, 2012. Example 4: Determination of the sensor performance

[0151] Both sensor types (with structuring by laser ablation according to Example 2 and without structuring according to Example 1) were analyzed using a potentiostat in a chronoamperometric setup. The potential was 50 mV with respect to Ag / AgCl as the reference electrode. The measurements were carried out over 14 days at different glucose levels c(glucose) (glucose concentration) (mg / dl: 0; 14, 4; 36; 54; 72; 90; 108; 126; 144; 180; 216; 270; 306; 360; 414; 468). Each step lasted approximately 90 minutes, and after 1 day, the glucose level measurement was repeated.

[0152] The sensor performance of sensors prepared without laser ablation (5 sensors; n = 5) is shown in FIG. 5 and Table 2. The sensor performance of sensors prepared using laser ablation (7 sensors; n = 7) is shown in FIG. 6 and Table 3. [Table 2]

[0153] [Table 3]

[0154] Sensors with laser ablation have reduced sensitivity compared to sensors without ablation due to the ablated sensing material. However, the ablated sensors have significantly reduced relative standard deviation and significantly reduced drift due to the uniform thickness and area of the sensing material. [Description of Reference Signs]

[0155] 110 Carbon 111 Conductive Trace 112 Gold 114 Sensor Substrate 116 Enzyme 118 Sensing Material 120 First Surface 122 Working Electrode 124 Analyte Sensor 126 Laser Beam 128 Patterned Layer 130 Step a) Provide at least one sensor substrate 132 Step b) Add at least one layer of at least one sensing material 134 Step c) Irradiate at least one laser beam onto the layer of sensing material

Claims

1. A method for preparing a working electrode (122) on a sensor substrate (114), comprising: a) providing at least one sensor substrate (114) comprising at least a first surface (120), wherein the first surface (120) comprises at least one conductive trace (111); b) adding at least one layer of at least one sensing material (118) onto the first surface (120) of the sensor substrate (114), wherein the sensing material (118) covers at least a portion of the at least one conductive trace (111); c) irradiating the layer of the sensing material with at least one laser beam, wherein at least a first portion of the layer of the sensing material (118) is at least partially removed, and at least a second portion of the sensing material (118) covering the at least one conductive trace (111) is maintained on the first surface (120) of the sensor substrate (114) to obtain at least one working electrode (122) on the sensor substrate (114); e) adding at least one film layer, wherein the film layer at least partially covers the working electrode (122) and comprises at least one crosslinking agent for crosslinking at least a portion of the sensing material (118); and further comprising: f) at least one diffusion step, wherein in the diffusion step, the crosslinking agent comprised in the film layer at least partially diffuses into the sensing material (118). A method as described above.

2. The method according to claim 1, further comprising an additional step bb) of drying the at least one layer of the at least one sensing material before step c).

3. The method according to claim 1 or 2, further comprising: d) at least one curing step, wherein in the curing step, at least a portion of the sensing material (118) is crosslinked.

4. The method according to claim 3, wherein step d) is at least partially performed after step c).

5. The method according to any one of claims 1 to 4, wherein the crosslinking agent contained in the film layer comprises at least one low molecular weight divalent and / or polyvalent epoxy-based crosslinking agent.

6. The method according to any one of claims 1 to 5, wherein the conductive trace (111) comprises at least one conductive material selected from the group consisting of carbon, gold, copper, silver, nickel, platinum, and palladium.

7. The method according to claim 6, wherein the conductive trace (111) comprises at least one additional layer of at least one additional material.

8. The method according to any one of claims 1 to 7, wherein the conductive trace (111) has a thickness of at least 7 μm.

9. An analyte sensor (124) comprising at least one working electrode (122) obtainable by the method according to any one of claims 1 to 8 and at least one additional electrode, wherein the at least one working electrode (122) comprises a sensing material (118), and at least a part of the sensing material (118) is crosslinked.

10. An analyte sensor (124), A) at least one sensor substrate (114) having at least one first surface (120), the first surface (120) comprising at least one conductive trace (111), at least one sensor substrate (114); B) at least one layer of at least one sensing material (118) disposed on the first surface (120) of the sensor substrate (114), the sensing material (118) covering at least a part of the at least one conductive trace (111), the layer of the sensing material (118) being irradiated by at least one laser beam, and a first portion of the layer of the sensing material being at least partially removed such that a second portion of the sensing material (118) covering the conductive trace (111) is maintained on the first surface (120) of the sensor substrate (114), the second portion of the sensing material forming at least one working electrode (122) of the analyte sensor (124) together with the conductive trace (111), at least one layer; comprising at least a part of the sensing material (118) maintained on the first surface (120) is crosslinked by a crosslinking agent, The analyte sensor (124) further comprises at least one membrane layer, the membrane layer at least partially covering the working electrode (122), the analyte sensor comprising the crosslinking agent. **Claim 11** Use of the analyte sensor (124) according to claim 9 or 10 for detecting at least one analyte in a sample. **Claim 12** A method for measuring an analyte in a sample, comprising using the analyte sensor (124) according to claim 9 or 10.

Images